Tabla espec. clave
|Species Reactivity||Key Applications||Host||Format||Antibody Type|
|B, Ch, H, M, Po, R, Rb||ICC, IHC, IH(P), WB||M||Ascites||Monoclonal Antibody|
|Presentation||Unpurified mouse monoclonal IgG1 liquid in buffer containing 15 mM sodium azide.|
|Safety Information according to GHS|
|Material Size||100 µL|
Ficha datos de seguridad (MSDS)
Referencias bibliográficas | 236 Disponible | Ver todas las referencias
|Visión general referencias||Aplicación||Especie||Pub Med ID|
|Expression of progerin in aging mouse brains reveals structural nuclear abnormalities without detectible significant alterations in gene expression, hippocampal stem cells or behavior. |
Baek, JH; Schmidt, E; Viceconte, N; Strandgren, C; Pernold, K; Richard, TJ; Van Leeuwen, FW; Dantuma, NP; Damberg, P; Hultenby, K; Ulfhake, B; Mugnaini, E; Rozell, B; Eriksson, M
Human molecular genetics 24 1305-21 2015
Hutchinson-Gilford progeria syndrome (HGPS) is a segmental progeroid syndrome with multiple features suggestive of premature accelerated aging. Accumulation of progerin is thought to underlie the pathophysiology of HGPS. However, despite ubiquitous expression of lamin A in all differentiated cells, the HGPS mutation results in organ-specific defects. For example, bone and skin are strongly affected by HGPS, while the brain appears to be unaffected. There are no definite explanations as to the variable sensitivity to progeria disease among different organs. In addition, low levels of progerin have also been found in several tissues from normal individuals, but it is not clear if low levels of progerin contribute to the aging of the brain. In an attempt to clarify the origin of this phenomenon, we have developed an inducible transgenic mouse model with expression of the most common HGPS mutation in brain, skin, bone and heart to investigate how the mutation affects these organs. Ultrastructural analysis of neuronal nuclei after 70 weeks of expression of the LMNA c.1824Cgreater than T mutation showed severe distortion with multiple lobulations and irregular extensions. Despite severe distortions in the nuclei of hippocampal neurons of HGPS animals, there were only negligible changes in gene expression after 63 weeks of transgenic expression. Behavioral analysis and neurogenesis assays, following long-term expression of the HGPS mutation, did not reveal significant pathology. Our results suggest that certain tissues are protected from functional deleterious effects of progerin.
|Dysregulation of astrocyte extracellular signaling in Costello syndrome. |
Krencik, R; Hokanson, KC; Narayan, AR; Dvornik, J; Rooney, GE; Rauen, KA; Weiss, LA; Rowitch, DH; Ullian, EM
Science translational medicine 7 286ra66 2015
Astrocytes produce an assortment of signals that promote neuronal maturation according to a precise developmental timeline. Is this orchestrated timing and signaling altered in human neurodevelopmental disorders? To address this question, the astroglial lineage was investigated in two model systems of a developmental disorder with intellectual disability caused by mutant Harvey rat sarcoma viral oncogene homolog (HRAS) termed Costello syndrome: mutant HRAS human induced pluripotent stem cells (iPSCs) and transgenic mice. Human iPSCs derived from patients with Costello syndrome differentiated to astroglia more rapidly in vitro than those derived from wild-type cell lines with normal HRAS, exhibited hyperplasia, and also generated an abundance of extracellular matrix remodeling factors and proteoglycans. Acute treatment with a farnesyl transferase inhibitor and knockdown of the transcription factor SNAI2 reduced expression of several proteoglycans in Costello syndrome iPSC-derived astrocytes. Similarly, mice in which mutant HRAS was expressed selectively in astrocytes exhibited experience-independent increased accumulation of perineuronal net proteoglycans in cortex, as well as increased parvalbumin expression in interneurons, when compared to wild-type mice. Our data indicate that astrocytes expressing mutant HRAS dysregulate cortical maturation during development as shown by abnormal extracellular matrix remodeling and implicate excessive astrocyte-to-neuron signaling as a possible drug target for treating mental impairment and enhancing neuroplasticity.
|Characterization of glioma stem-like cells from human glioblastomas. |
Yamamuro, S; Okamoto, Y; Sano, E; Ochiai, Y; Ogino, A; Ohta, T; Hara, H; Ueda, T; Nakayama, T; Yoshino, A; Katayama, Y
International journal of oncology 47 91-6 2015
Glioma stem-like cells (GSCs) could have potential for tumorigenesis, treatment resistance, and tumor recurrence (GSC hypothesis). However, the mechanisms underlying such potential has remained elusive and few ultrastructural features of the cells have been reported in detail. We therefore undertook observations of the antigenic characteristics and ultrastructural features of GSCs isolated from human glioblastomas. Tumor spheres formed by variable numbers of cells, exhibiting a variable appearance in both their size and shape, were frequently seen in GSCs expressing the stem cell surface markers CD133 and CD15. Increased cell nucleus atypia, mitochondria, rough endoplasmic reticulum, coated vesicles, and microvilli, were noted in the GSCs. Furthermore, cells at division phases and different phases of the apoptotic process were occasionally observed. These findings could imply that GSCs have certain relations with human neural stem cells (NSCs) but are primitively different from undifferentiated NSCs. The data may provide support for the GSC hypothesis, and also facilitate the establishment of future glioblastoma treatments targeting GSCs.
|Cuprizone-induced demyelination and demyelination-associated inflammation result in different proton magnetic resonance metabolite spectra. |
Praet, J; Orije, J; Kara, F; Guglielmetti, C; Santermans, E; Daans, J; Hens, N; Verhoye, M; Berneman, Z; Ponsaerts, P; Van der Linden, A
NMR in biomedicine 28 505-13 2015
Conventional MRI is frequently used during the diagnosis of multiple sclerosis but provides only little additional pathological information. Proton MRS ((1) H-MRS), however, provides biochemical information on the lesion pathology by visualization of a spectrum of metabolites. In this study we aimed to better understand the changes in metabolite concentrations following demyelination of the white matter. Therefore, we used the cuprizone model, a well-established mouse model to mimic type III human multiple sclerosis demyelinating lesions. First, we identified CX3 CL1/CX3 CR1 signaling as a major regulator of microglial activity in the cuprizone mouse model. Compared with control groups (heterozygous CX3 CR1(+/-) C57BL/6 mice and wild type CX3 CR1(+/+) C57BL/6 mice), microgliosis, astrogliosis, oligodendrocyte cell death and demyelination were shown to be highly reduced or absent in CX3 CR1(-/-) C57BL/6 mice. Second, we show that (1) H-MRS metabolite spectra are different when comparing cuprizone-treated CX3 CR1(-/-) mice showing mild demyelination with cuprizone-treated CX3 CR1(+/+) mice showing severe demyelination and demyelination-associated inflammation. Following cuprizone treatment, CX3 CR1(+/+) mice show a decrease in the Glu, tCho and tNAA concentrations as well as an increased Tau concentration. In contrast, following cuprizone treatment CX3 CR1(-/-) mice only showed a decrease in tCho and tNAA concentrations. Therefore, (1) H-MRS might possibly allow us to discriminate demyelination from demyelination-associated inflammation via changes in Tau and Glu concentration. In addition, the observed decrease in tCho concentration in cuprizone-induced demyelinating lesions should be further explored as a possible diagnostic tool for the early identification of human MS type III lesions.
|The clinical heterogeneity of coenzyme Q10 deficiency results from genotypic differences in the Coq9 gene. |
Luna-Sánchez, M; Díaz-Casado, E; Barca, E; Tejada, MÁ; Montilla-García, Á; Cobos, EJ; Escames, G; Acuña-Castroviejo, D; Quinzii, CM; López, LC
EMBO molecular medicine 7 670-87 2015
Primary coenzyme Q10 (CoQ10) deficiency is due to mutations in genes involved in CoQ biosynthesis. The disease has been associated with five major phenotypes, but a genotype-phenotype correlation is unclear. Here, we compare two mouse models with a genetic modification in Coq9 gene (Coq9(Q95X) and Coq9(R239X)), and their responses to 2,4-dihydroxybenzoic acid (2,4-diHB). Coq9(R239X) mice manifest severe widespread CoQ deficiency associated with fatal encephalomyopathy and respond to 2,4-diHB increasing CoQ levels. In contrast, Coq9(Q95X) mice exhibit mild CoQ deficiency manifesting with reduction in CI+III activity and mitochondrial respiration in skeletal muscle, and late-onset mild mitochondrial myopathy, which does not respond to 2,4-diHB. We show that these differences are due to the levels of COQ biosynthetic proteins, suggesting that the presence of a truncated version of COQ9 protein in Coq9(R239X) mice destabilizes the CoQ multiprotein complex. Our study points out the importance of the multiprotein complex for CoQ biosynthesis in mammals, which may provide new insights to understand the genotype-phenotype heterogeneity associated with human CoQ deficiency and may have a potential impact on the treatment of this mitochondrial disorder.
|Identification of Spinal Cord MicroRNA and Gene Signatures in a Model of Chronic Stress-Induced Visceral Hyperalgesia in Rat. |
Bradesi, S; Karagiannides, I; Bakirtzi, K; Joshi, SM; Koukos, G; Iliopoulos, D; Pothoulakis, C; Mayer, EA
PloS one 10 e0130938 2015
Animal studies have shown that stress could induce epigenetic and transcriptomic alterations essential in determining the balance between adaptive or maladaptive responses to stress. We tested the hypothesis that chronic stress in rats deregulates coding and non-coding gene expression in the spinal cord, which may underline neuroinflammation and nociceptive changes previously observed in this model.Male Wistar rats were exposed to daily stress or handled, for 10 days. At day 11, lumbar spinal segments were collected and processed for mRNA/miRNA isolation followed by expression profiling using Agilent SurePrint Rat Exon and Rat miRNA Microarray platforms. Differentially expressed gene lists were generated using the dChip program. Microarrays were analyzed using the Ingenuity Pathways Analysis (IPA) tool from Ingenuity Systems. Multiple methods were used for the analysis of miRNA-mRNA functional modules. Quantitative real time RT-PCR for Interleukin 6 signal transducer (gp130), the Signal Transducer And Activator Of Transcription 3 (STAT3), glial fibrillary acidic protein and mir-17-5p were performed to confirm levels of expression.Gene network analysis revealed that stress deregulated different inflammatory (IL-6, JAK/STAT, TNF) and metabolic (PI3K/AKT) signaling pathways. MicroRNA array analysis revealed a signature of 39 deregulated microRNAs in stressed rats. MicroRNA-gene network analysis showed that microRNAs are regulators of two gene networks relevant to inflammatory processes. Specifically, our analysis of miRNA-mRNA functional modules identified miR-17-5p as an important regulator in our model. We verified miR-17-5p increased expression in stress using qPCR and in situ hybridization. In addition, we observed changes in the expression of gp130 and STAT3 (involved in intracellular signaling cascades in response to gp130 activation), both predicted targets for miR-17-5p. A modulatory role of spinal mir17-5p in the modulation of visceral sensitivity was confirmed in vivo.Using an integrative high throughput approach, our findings suggest a link between miR-17-5p increased expression and gp130/STAT3 activation providing new insight into the possible mechanisms mediating the effect of chronic stress on neuroinflammation in the spinal cord.
|Aberrant hippocampal neurogenesis contributes to epilepsy and associated cognitive decline. |
Cho, KO; Lybrand, ZR; Ito, N; Brulet, R; Tafacory, F; Zhang, L; Good, L; Ure, K; Kernie, SG; Birnbaum, SG; Scharfman, HE; Eisch, AJ; Hsieh, J
Nature communications 6 6606 2015
Acute seizures after a severe brain insult can often lead to epilepsy and cognitive impairment. Aberrant hippocampal neurogenesis follows the insult but the role of adult-generated neurons in the development of chronic seizures or associated cognitive deficits remains to be determined. Here we show that the ablation of adult neurogenesis before pilocarpine-induced acute seizures in mice leads to a reduction in chronic seizure frequency. We also show that ablation of neurogenesis normalizes epilepsy-associated cognitive deficits. Remarkably, the effect of ablating adult neurogenesis before acute seizures is long lasting as it suppresses chronic seizure frequency for nearly 1 year. These findings establish a key role of neurogenesis in chronic seizure development and associated memory impairment and suggest that targeting aberrant hippocampal neurogenesis may reduce recurrent seizures and restore cognitive function following a pro-epileptic brain insult.
|miR-26a and miR-384-5p are required for LTP maintenance and spine enlargement. |
Gu, QH; Yu, D; Hu, Z; Liu, X; Yang, Y; Luo, Y; Zhu, J; Li, Z
Nature communications 6 6789 2015
Long-term potentiation (LTP) is a form of synaptic plasticity that results in enhanced synaptic strength. It is associated with the formation and enlargement of dendritic spines-tiny protrusions accommodating excitatory synapses. Both LTP and spine remodelling are crucial for brain development, cognition and the pathophysiology of neurological disorders. The role of microRNAs (miRNAs) in the maintenance of LTP, however, is not well understood. Using next-generation sequencing to profile miRNA transcriptomes, we demonstrate that miR-26a and miR-384-5p specifically affect the maintenance, but not induction, of LTP and different stages of spine enlargement by regulating the expression of RSK3. Using bioinformatics, we also examine the global effects of miRNA transcriptome changes during LTP on gene expression and cellular activities. This study reveals a novel miRNA-mediated mechanism for gene-specific regulation of translation in LTP, identifies two miRNAs required for long-lasting synaptic and spine plasticity and presents a catalogue of candidate 'LTP miRNAs'.
|Protein carbonylation after traumatic brain injury: cell specificity, regional susceptibility, and gender differences. |
Lazarus, RC; Buonora, JE; Jacobowitz, DM; Mueller, GP
Free radical biology & medicine 78 89-100 2015
Protein carbonylation is a well-documented and quantifiable consequence of oxidative stress in several neuropathologies, including multiple sclerosis, Alzheimer׳s disease, and Parkinson׳s disease. Although oxidative stress is a hallmark of traumatic brain injury (TBI), little work has explored the specific neural regions and cell types in which protein carbonylation occurs. Furthermore, the effect of gender on protein carbonylation after TBI has not been studied. The present investigation was designed to determine the regional and cell specificity of TBI-induced protein carbonylation and how this response to injury is affected by gender. Immunohistochemistry was used to visualize protein carbonylation in the brains of adult male and female Sprague-Dawley rats subjected to controlled cortical impact (CCI) as an injury model of TBI. Cell-specific markers were used to colocalize the presence of carbonylated proteins in specific cell types, including astrocytes, neurons, microglia, and oligodendrocytes. Results also indicated that the injury lesion site, ventral portion of the dorsal third ventricle, and ventricular lining above the median eminence showed dramatic increases in protein carbonylation after injury. Specifically, astrocytes and limited regions of ependymal cells adjacent to the dorsal third ventricle and the median eminence were most susceptible to postinjury protein carbonylation. However, these patterns of differential susceptibility to protein carbonylation were gender dependent, with males showing significantly greater protein carbonylation at sites distant from the lesion. Proteomic analyses were also conducted and determined that the proteins most affected by carbonylation in response to TBI include glial fibrillary acidic protein, dihydropyrimidase-related protein 2, fructose-bisphosphate aldolase C, and fructose-bisphosphate aldolase A. Many other proteins, however, were not carbonylated by CCI. These findings indicate that there is both regional and protein specificity in protein carbonylation after TBI. The marked increase in carbonylation seen in ependymal layers distant from the lesion suggests a mechanism involving the transmission of a cerebral spinal fluid-borne factor to these sites. Furthermore, this process is affected by gender, suggesting that hormonal mechanisms may serve a protective role against oxidative stress.
|Spinal autophagy is differently modulated in distinct mouse models of neuropathic pain. |
Berliocchi, L; Maiarù, M; Varano, GP; Russo, R; Corasaniti, MT; Bagetta, G; Tassorelli, C
Molecular pain 11 3 2015
Autophagy is a homeostatic degradative process essential for basal turnover of long-lived proteins and organelles as well as for removal of dysfunctional cellular components. Dysregulation of the autophagic machinery has been recently associated to several conditions including neurodegenerative diseases and cancer, but only very few studies have investigated its role in pain processing.We previously described autophagy impairment at the spinal cord in the experimental model of neuropathic pain induced by spinal nerve ligation (SNL). In this study, we characterized the main autophagic markers in two other common experimental models of neuropathic pain, the chronic constriction injury (CCI) and the spared nerve injury (SNI). The different modulation of LC3-I, Beclin 1 and p62 suggested that autophagy is differentially affected in the spinal dorsal horn depending on the type of peripheral injury. Confocal analysis of p62 distribution in the spinal dorsal horn indicated its presence mainly in NeuN-positive cell bodies and occasionally in glial processes, thus suggesting a predominant expression in the neuronal compartment. Finally, we investigated the consequences of autophagy impairment on pain behaviour by using the autophagy blocker cloroquine. Intrathecal chloroquine injection in naïve mice induced spinal accumulation of LC3 and p62 paralleled by significant mechanical hypersensitivity thus confirming the block in autophagosome clearance and suggesting the participation of the autophagic process in spinal mechanisms of pain processing. Altogether, our data indicate that spinal autophagy is differentially altered in different experimental pain models of neuropathic pain and that this process may be relevant for pain control.
|Connexin 43 stabilizes astrocytes in a stroke-like milieu to facilitate neuronal recovery. |
Wu, LY; Yu, XL; Feng, LY
Acta pharmacologica Sinica 36 928-38 2015
Connexin 43 (Cx43) is a member of connexin family mainly expressed in astrocytes, which forms gap junctions and hemichannels and maintains the normal shape and function of astrocytes. In this study we investigated the role of Cx43 in astrocytes in facilitating neuronal recovery during ischemic stroke.Primary culture of astrocytes or a mixed culture of astrocytes and cortical neurons was subjected to oxygen glucose deprivation and reperfusion (OGD/R). The expression of Cx43 and Ephrin-A4 in astrocytes was detected using immunocytochemical staining and Western blot assays. Intercellular Ca(2+) concentration was determined with Fluo-4 AM fluorescent staining. Middle cerebral artery occlusion (MCAO) model rats were used for in vivo studies.OGD/R treatment of cultured astrocytes caused a decrement of Cx43 expression and translocation of Cx43 from cell membrane to cytoplasm, accompanied by cell retraction. Furthermore, OGD/R increased intracellular Ca(2+) concentration, activated CaMKII/CREB pathways and upregulated expression of Ephrin-A4 in the astrocytes. All these changes in OGD/R-treated astrocytes were alleviated by overexpression of Cx43. In the cortical neurons cultured with astrocytes, OGD/R inhibited the neurite growth, whereas overexpression of Cx43 or knockdown of Ephrin-A4 in astrocytes restored the neurite growth. In MCAO model rats, neuronal recovery was found to be correlated with the recuperation of Cx43 and Ephrin-A4 in astrocytes.Cx43 can stabilize astrocytes and facilitate the resistance to the deleterious effects of a stroke-like milieu and promote neuronal recovery.
|Early pro-inflammatory cytokine elevations in the DBA/2J mouse model of glaucoma. |
Wilson, GN; Inman, DM; Dengler Crish, CM; Denger-Crish, CM; Smith, MA; Crish, SD
Journal of neuroinflammation 12 176 2015
Neuroinflammation-astrogliosis, microglial activation, and changes in cytokine signaling-is a prominent feature of neurodegenerative disorders. Glaucoma is a group of chronic neurodegenerative conditions that make up the leading cause of irreversible blindness worldwide. Neuroinflammation has been postulated to play a significant role in the pathogenesis and progression of glaucomatous neurodegeneration. Though much is known regarding inflammation in the eye in glaucoma, little is known about cytokine activity outside of the retina where pathologies develop early.We traced the primary visual projection from the eye to the superior colliculus (SC) in DBA/2J and DBA/2J.Gpnmb (+) (control) mice using the anterograde tracer cholera toxin-B (CTB) to assay axonal transport deficits. Forty-eight hours later, visual structures were microdissected from fresh tissue based on transport outcome. Using magnetic bead multiplexing assays, we measured levels of 20 cytokines in the retina, proximal and distal optic nerves, CTB-positive and negative SC subdivisions, cerebellum, and serum at different ages representing different stages of pathology.Pro- and anti-inflammatory cytokine levels in mice often changed in the same direction based on strain, age, and tissue. Significant elevations in retinal pro-inflammatory cytokines were observed in young DBA/2J mice compared to controls, followed by an age-dependent decrease in the DBA/2J mice. Proximal optic nerve of young DBA/2J mice showed a 50 % or greater decrease in levels of certain cytokines compared to older DBA/2J cohorts and controls, while both proximal and distal optic nerve of DBA/2Js showed elevations in IL-1β at all ages compared to controls. Pro-inflammatory cytokine IL-6 levels varied in accordance with transport outcome in the SC: IL-6 was elevated 44-80 % in glaucomatous DBA/2J collicular regions deficient in anterograde transport from retinal ganglion cells (RGCs) compared to areas with intact transport.Dysregulation of cytokine signaling in the RGC projection of DBA/2J mice was evident early in distal retinal targets, well before intraocular pressure elevation or axonal degeneration begins.
|Evidence for a novel functional role of astrocytes in the acute homeostatic response to high-fat diet intake in mice. |
Buckman, LB; Thompson, MM; Lippert, RN; Blackwell, TS; Yull, FE; Ellacott, KL
Molecular metabolism 4 58-63 2015
Introduction of a high-fat diet to mice results in a period of voracious feeding, known as hyperphagia, before homeostatic mechanisms prevail to restore energy intake to an isocaloric level. Acute high-fat diet hyperphagia induces astrocyte activation in the rodent hypothalamus, suggesting a potential role of these cells in the homeostatic response to the diet. The objective of this study was to determine physiologic role of astrocytes in the acute homeostatic response to high-fat feeding.We bred a transgenic mouse model with doxycycline-inducible inhibition of NFkappaB (NFκB) signaling in astrocytes to determine the effect of loss of NFκB-mediated astrocyte activation on acute high-fat hyperphagia. ELISA was used to measure the levels of markers of astrocyte activation, glial-fibrillary acidic protein (GFAP) and S100B, in the medial basal hypothalamus.Inhibition of NFκB signaling in astrocytes prevented acute high-fat diet-induced astrocyte activation and resulted in a 15% increase in caloric intake (P less than 0.01) in the first 24 h after introduction of the diet.These data reveal a novel homeostatic role for astrocytes in the acute physiologic regulation of food intake in response to high-fat feeding.
|Dopamine D1 receptor activation regulates the expression of the estrogen synthesis gene aromatase B in radial glial cells. |
Xing, L; McDonald, H; Da Fonte, DF; Gutierrez-Villagomez, JM; Trudeau, VL
Frontiers in neuroscience 9 310 2015
Radial glial cells (RGCs) are abundant stem-like non-neuronal progenitors that are important for adult neurogenesis and brain repair, yet little is known about their regulation by neurotransmitters. Here we provide evidence for neuronal-glial interactions via a novel role for dopamine to stimulate RGC function. Goldfish were chosen as the model organism due to the abundance of RGCs and regenerative abilities of the adult central nervous system. A close anatomical relationship was observed between tyrosine hydroxylase-positive catecholaminergic cell bodies and axons and dopamine-D1 receptor expressing RGCs along the ventricular surface of telencephalon, a site of active neurogenesis. A primary cell culture model was established and immunofluorescence analysis indicates that in vitro RGCs from female goldfish retain their major characteristics in vivo, including expression of glial fibrillary acidic protein and brain lipid binding protein. The estrogen synthesis enzyme aromatase B is exclusively found in RGCs, but this is lost as cells differentiate to neurons and other glial types in adult teleost brain. Pharmacological experiments using the cultured RGCs established that specific activation of dopamine D1 receptors up-regulates aromatase B mRNA through a cyclic adenosine monophosphate-dependent molecular mechanism. These data indicate that dopamine enhances the steroidogenic function of this neuronal progenitor cell.
|Effects of dextromethorphan and oxycodone on treatment of neuropathic pain in mice. |
Yang, PP; Yeh, GC; Huang, EY; Law, PY; Loh, HH; Tao, PL
Journal of biomedical science 22 81 2015
Neuropathic pain is a very troublesome and difficult pain to treat. Although opioids are the best analgesics for cancer and surgical pain in clinic, only oxycodone among opioids shows better efficacy to alleviate neuropathic pain. However, many side effects associated with the use of oxycodone render the continued use of it in neuropathic pain treatment undesirable. Hence, we explored whether dextromethorphan (DM, a known N-methyl-D-aspartate receptor antagonist with neuroprotective properties) could potentiate the anti-allodynic effect of oxycodone and underlying mechanisms regarding to glial cells (astrocytes and microglia) activation and proinflammatory cytokines release in a spinal nerve injury (SNL) mice model.Oxycodone produced a dose-dependent anti-allodynic effect. Co-administration of DM at a dose of 10 mg/kg (i.p.) (DM10) which had no anti-allodynic effect by itself enhanced the acute oxycodone (1 mg/kg, s.c.) effect. When the chronic anti-allodynic effects were examined, co-administration of DM10 also significantly enhanced the oxycodone effect at 3 mg/kg. Furthermore, oxycodone decreased SNL-induced activation of glial cells (astrocytes and microglia) and plasma levels of proinflammatory cytokines (IL-6, IL-1β and TNF-α). Co-administration of DM10 potentiated these effects of oxycodone.The combined use of DM with oxycodone may have therapeutic potential for decreasing the effective dose of oxycodone on the treatment of neuropathic pain. Attenuation of the glial activation and proinflammatory cytokines in the spinal cord may be important mechanisms for these effects of DM.
|Effect of thymic stimulation of CD4+ T cell expansion on disease onset and progression in mutant SOD1 mice. |
Sheean, RK; Weston, RH; Perera, ND; D'Amico, A; Nutt, SL; Turner, BJ
Journal of neuroinflammation 12 40 2015
The peripheral immune system is implicated in modulating microglial activation, neurodegeneration and disease progression in amyotrophic lateral sclerosis (ALS). Specifically, there is reduced thymic function and regulatory T cell (Treg) number in ALS patients and mutant superoxide dismutase 1 (SOD1) mice, while passive transfer of Tregs ameliorates disease in mutant SOD1 mice. Here, we assessed the effects of augmenting endogenous CD4+ T cell number by stimulating the thymus using surgical castration on the phenotype of transgenic SOD1(G93A) mice.Male SOD1(G93A) mice were castrated or sham operated, and weight loss, disease onset and progression were examined. Thymus atrophy and blood CD4+, CD8+ and CD4+ FoxP3+ T cell numbers were determined by fluorescence activated cell sorting (FACS). Motor neuron counts, glial cell activation and androgen receptor (AR) expression in the spinal cord were investigated using immunohistochemistry and Western blotting. Differences between castrated and sham mice were analysed using an unpaired t test or one-way ANOVA.Castration significantly increased thymus weight and total CD4+ T cell numbers in SOD1(G93A) mice, although Tregs levels were not affected. Despite this, disease onset and progression were similar in castrated and sham SOD1(G93A) mice. Castration did not affect motor neuron loss or astrocytic activation in spinal cords of SOD1(G93A) mice; however, microglial activation was reduced, specifically M1 microglia. We also show that AR is principally expressed in spinal motor neurons and progressively downregulated in spinal cords of SOD1(G93A) mice from disease onset which is further enhanced by castration.These results demonstrate that increasing thymic function and CD4+ T cell number by castration confers no clinical benefit in mutant SOD1 mice, which may reflect an inability to stimulate neuroprotective Tregs. Nonetheless, castration decreases M1 microglial activation in the spinal cord without any clinical improvement and motor neuron rescue, in contrast to other approaches to suppress microglia in mutant SOD1 mice. Lastly, diminished AR expression in spinal motor neurons, which links to another motor neuron disorder, spinal bulbar muscular atrophy (SBMA), may contribute to ALS pathogenesis and suggests a common disease pathway in ALS and SBMA mediated by disruption of AR signalling in motor neurons.
|Curcumin attenuates brain edema in mice with intracerebral hemorrhage through inhibition of AQP4 and AQP9 expression. |
Wang, BF; Cui, ZW; Zhong, ZH; Sun, YH; Sun, QF; Yang, GY; Bian, LG
Acta pharmacologica Sinica 36 939-48 2015
Aquaporins (AQPs) are the water-channels that play important roles in brain water homeostasis and in cerebral edema induced by brain injury. In this study we investigated the relationship between AQPs and a neuroprotective agent curcumin that was effective in the treatment of brain edema in mice with intracerebral hemorrhage (ICH).ICH was induced in mice by autologous blood infusion. The mice immediately received curcumin (75, 150, 300 mg/kg, ip). The Rotarod test scores, brain water content and brain expression of AQPs were measured post ICH. Cultured primary mouse astrocytes were used for in vitro experiments. The expression of AQP1, AQP4 and AQP9 and NF-κB p65 were detected using Western blotting or immunochemistry staining.Curcumin administration dose-dependently reduced the cerebral edema at d 3 post ICH, and significantly attenuated the neurological deficits at d 5 post ICH. Furthermore, curcumin dose-dependently decreased the gene and protein expression of AQP4 and AQP9, but not AQP1 post ICH. Treatment of the cultured astrocytes with Fe(2+) (10-100 μmol/L) dose-dependently increased the expression and nuclear translocation of NF-κB p65 and the expression of AQP4 and AQP9, which were partly blocked by co-treatment with curcumin (20 μmol/L) or the NF-κB inhibitor PDTC (10 μmol/L).Curcumin effectively attenuates brain edema in mice with ICH through inhibition of the NF-κB pathway and subsequently the expression of AQP4 and AQP9. Curcumin may serve as a potential therapeutic agent for ICH.
|Buyang Huanwu decoction increases the expression of glutamate transporter-1 and glutamate synthetase in association with PACAP-38 following focal ischemia. |
Ding, W; Yu, P; Liu, W; Zhou, L; Guan, LI; Lin, R
Biomedical reports 3 651-656 2015
The neuroprotective role of Buyang Huanwu decoction (BYHWD) in focal ischemia is associated with decreasing glutamate concentration. However, the mechanisms are not fully understood. The present study aimed to explore whether glutamate transporter-1 (GLT-1) and glutamine synthetase (GS) participated in the decreased level of glutamate and whether pituitary adenylate cyclase-activating polypeptide-38 (PACAP-38) was involved in this process. BYHWD was found to significantly upregulate the expression of GLT-1 and GS in the hippocampal CA1 area compared to the ischemia group, with the difference on day 3 being most significant. BYHWD increased the level of PACAP-38, and PACAP-(6-38) (PACAP receptor antagonist) significantly attenuated the effect of BYHWD on GLT-1 and GS, suggesting that PACAP-38 was involved in the upregulation of GLT-1 and GS induced by BYHWD. In addition, as GLT-1 and GS are mainly located in astrocytes, the changes of astrocytes were detected by glial fibrillary acidic protein (GFAP; an astrocytic marker) immunostaining. The results showed that BYHWD inhibited the expression of GFAP compared with the ischemia group, however, co-administration with PACAP-(6-38), which inhibited the effect of BYHWD on GLT-1 and GS in astrocytes, attenuated this effect, indicating that astrocytes participated in the protective role of BYHWD following focal ischemia. These results provided the evidence for the first time that not only neurons but also astrocytes contribute to the protective role of BYHWD, which opposes previous studies and may be a starting point for traditional medicine.
|NFκB signaling drives pro-granulocytic astroglial responses to neuromyelitis optica patient IgG. |
Walker-Caulfield, ME; Guo, Y; Johnson, RK; McCarthy, CB; Fitz-Gibbon, PD; Lucchinetti, CF; Howe, CL
Journal of neuroinflammation 12 185 2015
Astrocytes expressing the aquaporin-4 water channel are a primary target of pathogenic, disease-specific immunoglobulins (IgG) found in patients with neuromyelitis optica (NMO). Immunopathological analyses of active NMO lesions highlight a unique inflammatory phenotype marked by infiltration of granulocytes. Previous studies characterized this granulocytic infiltrate as a response to vasculocentric complement activation and localized tissue destruction. In contrast, we observe that granulocytic infiltration in NMO lesions occurs independently of complement-mediated tissue destruction or active demyelination. These immunopathological findings led to the hypothesis that NMO IgG stimulates astrocyte signaling that is responsible for granulocytic recruitment in NMO.Histopathology was performed on archival formalin-fixed paraffin-embedded autopsy-derived CNS tissue from 23 patients clinically and pathologically diagnosed with NMO or NMO spectrum disorder. Primary murine astroglial cultures were stimulated with IgG isolated from NMO patients or control IgG from healthy donors. Transcriptional responses were assessed by microarray, and translational responses were measured by ELISA. Signaling through the NFκB pathway was measured by western blotting and immunostaining.Stimulation of primary murine astroglial cultures with NMO IgG elicited a reactive and inflammatory transcriptional response that involved signaling through the canonical NFκB pathway. This signaling resulted in the release of pro-granulocytic chemokines and was inhibited by the clinically relevant proteasome inhibitors bortezomib and PR-957.We propose that the astrocytic NFκB-dependent inflammatory response to stimulation by NMO IgG represents one of the earliest events in NMO pathogenesis, providing a target for therapeutic intervention upstream of irreversible cell death and tissue damage.
|Translation of the prion protein mRNA is robust in astrocytes but does not amplify during reactive astrocytosis in the mouse brain. |
Jackson, WS; Krost, C; Borkowski, AW; Kaczmarczyk, L
PloS one 9 e95958 2014
Prion diseases induce neurodegeneration in specific brain areas for undetermined reasons. A thorough understanding of the localization of the disease-causing molecule, the prion protein (PrP), could inform on this issue but previous studies have generated conflicting conclusions. One of the more intriguing disagreements is whether PrP is synthesized by astrocytes. We developed a knock-in reporter mouse line in which the coding sequence of the PrP expressing gene (Prnp), was replaced with that for green fluorescent protein (GFP). Native GFP fluorescence intensity varied between and within brain regions. GFP was present in astrocytes but did not increase during reactive gliosis induced by scrapie prion infection. Therefore, reactive gliosis associated with prion diseases does not cause an acceleration of local PrP production. In addition to aiding in Prnp gene activity studies, this reporter mouse line will likely prove useful for analysis of chimeric animals produced by stem cell and tissue transplantation experiments.
|Abnormal serine phosphorylation of insulin receptor substrate 1 is associated with tau pathology in Alzheimer's disease and tauopathies. |
Yarchoan, M; Toledo, JB; Lee, EB; Arvanitakis, Z; Kazi, H; Han, LY; Louneva, N; Lee, VM; Kim, SF; Trojanowski, JQ; Arnold, SE
Acta neuropathologica 128 679-89 2014
Neuronal insulin signaling abnormalities have been associated with Alzheimer's disease (AD). However, the specificity of this association and its underlying mechanisms have been unclear. This study investigated the expression of abnormal serine phosphorylation of insulin receptor substrate 1 (IRS1) in 157 human brain autopsy cases that included AD, tauopathies, α-synucleinopathies, TDP-43 proteinopathies, and normal aging. IRS1-pS(616), IRS1-pS(312) and downstream target Akt-pS(473) measures were most elevated in AD but were also significantly increased in the tauopathies: Pick's disease, corticobasal degeneration and progressive supranuclear palsy. Double immunofluorescence labeling showed frequent co-expression of IRS1-pS(616) with pathologic tau in neurons and dystrophic neurites. To further investigate an association between tau and abnormal serine phosphorylation of IRS1, we examined the presence of abnormal IRS1-pS(616) expression in pathological tau-expressing transgenic mice and demonstrated that abnormal IRS1-pS(616) frequently co-localizes in tangle-bearing neurons. Conversely, we observed increased levels of hyperphosphorylated tau in the high-fat diet-fed mouse, a model of insulin resistance. These results provide confirmation and specificity that abnormal phosphorylation of IRS1 is a pathological feature of AD and other tauopathies, and provide support for an association between insulin resistance and abnormal tau as well as amyloid-β.
|Expression of Nampt in hippocampal and cortical excitatory neurons is critical for cognitive function. |
Stein, LR; Wozniak, DF; Dearborn, JT; Kubota, S; Apte, RS; Izumi, Y; Zorumski, CF; Imai, S
The Journal of neuroscience : the official journal of the Society for Neuroscience 34 5800-15 2014
Nicotinamide adenine dinucleotide (NAD(+)) is an enzyme cofactor or cosubstrate in many essential biological pathways. To date, the primary source of neuronal NAD(+) has been unclear. NAD(+) can be synthesized from several different precursors, among which nicotinamide is the substrate predominantly used in mammals. The rate-limiting step in the NAD(+) biosynthetic pathway from nicotinamide is performed by nicotinamide phosphoribosyltransferase (Nampt). Here, we tested the hypothesis that neurons use intracellular Nampt-mediated NAD(+) biosynthesis by generating and evaluating mice lacking Nampt in forebrain excitatory neurons (CaMKIIαNampt(-/-) mice). CaMKIIαNampt(-/-) mice showed hippocampal and cortical atrophy, astrogliosis, microgliosis, and abnormal CA1 dendritic morphology by 2-3 months of age. Importantly, these histological changes occurred with altered intrahippocampal connectivity and abnormal behavior; including hyperactivity, some defects in motor skills, memory impairment, and reduced anxiety, but in the absence of impaired sensory processes or long-term potentiation of the Schaffer collateral pathway. These results clearly demonstrate that forebrain excitatory neurons mainly use intracellular Nampt-mediated NAD(+) biosynthesis to mediate their survival and function. Studying this particular NAD(+) biosynthetic pathway in these neurons provides critical insight into their vulnerability to pathophysiological stimuli and the development of therapeutic and preventive interventions for their preservation.
|Glia-related mechanisms in the anteroventral cochlear nucleus of the adult rat in response to unilateral conductive hearing loss. |
Fuentes-Santamaría, V; Alvarado, JC; López-Muñoz, DF; Melgar-Rojas, P; Gabaldón-Ull, MC; Juiz, JM
Frontiers in neuroscience 8 319 2014
Conductive hearing loss causes a progressive decline in cochlear activity that may result in functional and structural modifications in auditory neurons. However, whether these activity-dependent changes are accompanied by a glial response involving microglia, astrocytes, or both has not yet been fully elucidated. Accordingly, the present study was designed to determine the involvement of glial related mechanisms in the anteroventral cochlear nucleus (AVCN) of adult rats at 1, 4, 7, and 15 d after removing middle ear ossicles. Quantitative immunohistochemistry analyses at light microscopy with specific markers of microglia or astroglia along with immunocytochemistry at the electron microscopy level were used. Also, in order to test whether trophic support by neurotrophins is modulated in glial cells by auditory activity, the expression and distribution of neurotrophin-3 (NT-3) and its colocalization with microglial or astroglial markers was investigated. Diminished cochlear activity after middle ear ossicle removal leads to a significant ipsilateral increase in the mean gray levels and stained area of microglial cells but not astrocytes in the AVCN at 1 and 4 d post-lesion as compared to the contralateral side and control animals. These results suggest that microglial cells but not astrocytes may act as dynamic modulators of synaptic transmission in the cochlear nucleus immediately following unilateral hearing loss. On the other hand, NT-3 immunostaining was localized mainly in neuronal cell bodies and axons and was upregulated at 1, 4 and 7 d post-lesion. Very few glial cells expressed this neurotrophin in both control and experimental rats, suggesting that NT-3 is primarily activated in neurons and not as much in glia after limiting auditory activity in the AVCN by conductive hearing loss.
|CCL2-ethanol interactions and hippocampal synaptic protein expression in a transgenic mouse model. |
Gruol, DL; Vo, K; Bray, JG; Roberts, AJ
Frontiers in integrative neuroscience 8 29 2014
Chronic exposure to ethanol produces a number of detrimental effects on behavior. Neuroadaptive changes in brain structure or function underlie these behavioral effects and may be transient or persistent in nature. Central to the functional changes are alterations in the biology of neuronal and glial cells of the brain. Recent data show that ethanol induces glial cells of the brain to produce elevated levels of neuroimmune factors including CCL2, a key innate immune chemokine. Depending on the conditions of ethanol exposure, the upregulated levels of CCL2 can be transient or persistent and outlast the period of ethanol exposure. Importantly, results indicate that the upregulated levels of CCL2 may lead to CCL2-ethanol interactions that mediate or regulate the effects of ethanol on the brain. Glial cells are in close association with neurons and regulate many neuronal functions. Therefore, effects of ethanol on glial cells may underlie some of the effects of ethanol on neurons. To investigate this possibility, we are studying effects of chronic ethanol on hippocampal synaptic function in a transgenic mouse model that expresses elevated levels of CCL2 in the brain through enhanced glial expression, a situation know to occur in alcoholics. Both CCL2 and ethanol have been reported to alter synaptic function in the hippocampus. In the current study, we determined if interactions are evident between CCL2 and ethanol at the level of hippocampal synaptic proteins. Two ethanol exposure paradigms were used; the first involved ethanol exposure by drinking and the second involved ethanol exposure in a paradigm that combines drinking plus ethanol vapor. The first paradigm does not produce dependence on ethanol, whereas the second paradigm is commonly used to produce ethanol dependence. Results show modest effects of both ethanol exposure paradigms on the level of synaptic proteins in the hippocampus of CCL2 transgenic mice compared with their non-transgenic littermate controls, consistent with ethanol-CCL2 interactions. No evidence of toxic effects of CCL2 or CCL2-ethanol interactions was observed. Taken together, these results support the idea that ethanol induced astrocyte production of CCL2 can result in neuroadaptive changes that interact with the actions of ethanol.
|GluA2 mRNA distribution and regulation by miR-124 in hippocampal neurons. |
Ho, VM; Dallalzadeh, LO; Karathanasis, N; Keles, MF; Vangala, S; Grogan, T; Poirazi, P; Martin, KC
Molecular and cellular neurosciences 61 1-12 2014
AMPA-type glutamate receptors mediate fast, excitatory neurotransmission in the brain, and their concentrations at synapses are important determinants of synaptic strength. We investigated the post-transcriptional regulation of GluA2, the calcium-impermeable AMPA receptor subunit, by examining the subcellular distribution of its mRNA and evaluating its translational regulation by microRNA in cultured mouse hippocampal neurons. Using computational approaches, we identified a conserved microRNA-124 (miR-124) binding site in the 3'UTR of GluA2 and demonstrated that miR-124 regulated the translation of GluA2 mRNA reporters in a sequence-specific manner in luciferase assays. While we hypothesized that this regulation might occur in dendrites, our biochemical and fluorescent in situ hybridization (FISH) data indicate that GluA2 mRNA does not localize to dendrites or synapses of mouse hippocampal neurons. In contrast, we detected significant concentrations of miR-124 in dendrites. Overexpression of miR-124 in dissociated neurons results in a 30% knockdown of GluA2 protein, as measured by immunoblot and quantitative immunocytochemistry, without producing any changes in GluA2 mRNA concentrations. While total GluA2 concentrations are reduced, we did not detect any changes in the concentration of synaptic GluA2. We conclude from these results that miR-124 interacts with GluA2 mRNA in the cell body to downregulate translation. Our data support a model in which GluA2 is translated in the cell body and subsequently transported to neuronal dendrites and synapses, and suggest that synaptic GluA2 concentrations are modified primarily by regulated protein trafficking rather than by regulated local translation.
|Molecular and cellular features of murine craniofacial and trunk neural crest cells as stem cell-like cells. |
Hagiwara, K; Obayashi, T; Sakayori, N; Yamanishi, E; Hayashi, R; Osumi, N; Nakazawa, T; Nishida, K
PloS one 9 e84072 2014
The outstanding differentiation capacities and easier access from adult tissues, cells derived from neural crest cells (NCCs) have fascinated scientists in developmental biology and regenerative medicine. Differentiation potentials of NCCs are known to depend on their originating regions. Here, we report differential molecular features between craniofacial (cNCCs) and trunk (tNCCs) NCCs by analyzing transcription profiles and sphere forming assays of NCCs from P0-Cre/floxed-EGFP mouse embryos. We identified up-regulation of genes linked to carcinogenesis in cNCCs that were not previously reported to be related to NCCs, which was considered to be, an interesting feature in regard with carcinogenic potentials of NCCs such as melanoma and neuroblastoma. Wnt signal related genes were statistically up-regulated in cNCCs, also suggesting potential involvement of cNCCs in carcinogenesis. We also noticed intense expression of mesenchymal and neuronal markers in cNCCs and tNCCs, respectively. Consistent results were obtained from in vitro sphere-forming and differentiation assays. These results were in accordance with previous notion about differential potentials of cNCCs and tNCCs. We thus propose that sorting NCCs from P0-Cre/floxed-EGFP mice might be useful for the basic and translational research of NCCs. Furthermore, these newly-identified genes up-regulated in cNCC would provide helpful information on NC-originating tumors, developmental disorders in NCC derivatives, and potential applications of NCCs in regenerative medicine.
|Transglutaminase 2 exacerbates α-synuclein toxicity in mice and yeast. |
Grosso, H; Woo, JM; Lee, KW; Im, JY; Masliah, E; Junn, E; Mouradian, MM
FASEB journal : official publication of the Federation of American Societies for Experimental Biology 28 4280-91 2014
α-Synuclein is a key pathogenic protein that aggregates in hallmark lesions in Parkinson's disease and other α-synucleinopathies. Prior in vitro studies demonstrated that it is a substrate for cross-linking by transglutaminase 2 (TG2) into higher-order species. Here we investigated whether this increased aggregation occurs in vivo and whether TG2 exacerbates α-synuclein toxicity in Mus musculus and Saccharomyces cerevisiae. Compared with α-synuclein transgenic (Syn(Tg)) mice, animals double transgenic for human α-synuclein and TG2 (TG2(Tg)/Syn(Tg)) manifested greater high-molecular-weight insoluble species of α-synuclein in brain lysates and developed α-synuclein aggregates in the synaptic vesicle fraction. In addition, larger proteinase K-resistant aggregates developed, along with increased thioflavin-S-positive amyloid fibrils. This correlated with an exaggerated neuroinflammatory response, as seen with more astrocytes and microglia. Further neuronal damage was suggested by greater morphological disruption of nerve fibers and a trend toward decreased c-Fos immunoreactive neurons. Finally, the performance of TG2(Tg)/Syn(Tg) animals on motor behavioral tasks was worse relative to Syn(Tg) mice. Greater toxicity of α-synuclein was also demonstrated in yeast cells coexpressing TG2. Our findings demonstrate that TG2 promotes the aggregation of α-synuclein in vivo and that this is associated with aggravated toxicity of α-synuclein and its downstream neuropathologic consequences.
|Astrocyte Reactivity: A Biomarker for Retinal Ganglion Cell Health in Retinal Neurodegeneration. |
Formichella, CR; Abella, SK; Sims, SM; Cathcart, HM; Sappington, RM
Journal of clinical & cellular immunology 5 2014
Retinal ganglion cell (RGC) loss in glaucoma is sectorial in nature and preceded by deficits in axonal transport. Neuroinflammation plays an important role in the pathophysiology of glaucoma in the retina, optic nerve and visual centers of the brain, where it similarly appears to be regulated spatially. In a murine model, we examined the spatial characteristics of astrocyte reactivity (migration/proliferation, hypertrophy and GFAP expression) in healthy retina, retina with two glaucoma-related risk factors (aging and genetic predisposition) and glaucomatous retina and established relationships between these reactivity indices and the spatial organization of astrocytes as well as RGC health. Astrocyte reactivity was quantified by morphological techniques and RGC health was determined by uptake and transport of the neural tracer cholera toxin beta subunit (CTB). We found that: (1) astrocyte reactivity occurs in microdomains throughout glaucomatous retina as well as retina with risk factors for glaucoma, (2) these astrocyte microdomains are primarily differentiated by the degree of retinal area covered by the astrocytes within them and (3) percent retinal area covered by astrocytes is highly predictive of RGC health. Our findings suggest that microdomains of astrocyte reactivity are biomarkers for functional decline of RGCs. Based on current and emerging imaging technologies, diagnostic assessment of astrocytes in the nerve fiber layer could succeed in translating axonal transport deficits to a feasible clinical application.
|Suppression of spinal connexin 43 expression attenuates mechanical hypersensitivity in rats after an L5 spinal nerve injury. |
Xu, Q; Cheong, YK; He, SQ; Tiwari, V; Liu, J; Wang, Y; Raja, SN; Li, J; Guan, Y; Li, W
Neuroscience letters 566 194-9 2014
Activation of spinal astrocytes may contribute to neuropathic pain. Adjacent astrocytes can make direct communication through gap junctions formed by connexin 43 (Cx43) in the central nervous system. Yet, the role of spinal astroglial gap junctions in neuropathic pain is not fully understood. Since Cx43 is the connexin isoform expressed preferentially in astrocytes in the spinal cord, we used a small interfering RNA (siRNA) approach to examine whether suppression of spinal Cx43 expression inhibits mechanical hypersensitivity in rats after an L5 spinal nerve ligation (SNL). SNL rats were administered intrathecal Cx43 siRNA (3μg/15μl, twice/day) or an equal amount of mismatch siRNA (control) on days 14-17 post-SNL. Cx43 siRNA, but not mismatch siRNA, alleviated mechanical hypersensitivity in SNL rats. Furthermore, Western blot analysis showed that the pain inhibition induced by Cx43 siRNA correlated with downregulation of Cx43 expression, but not that of Cx36 (the neuronal gap junction protein) or glial fibrillary acidic protein (GFAP, a marker for reactive astrocytes) in the spinal cord of SNL rats. Western blot analysis and immunohistochemistry also showed that SNL increased GFAP expression, but decreased Cx43 expression, in spinal cord. Our results provide direct evidence that selective suppression of spinal Cx43 after nerve injury alleviates neuropathic mechanical hypersensitivity. These findings suggest that in the spinal cord, the enhanced function of astroglial gap junctions, especially those formed by Cx43, may be important to neuropathic pain in SNL rats.
|Progressive neurodegeneration after experimental brain trauma: association with chronic microglial activation. |
Loane, DJ; Kumar, A; Stoica, BA; Cabatbat, R; Faden, AI
Journal of neuropathology and experimental neurology 73 14-29 2014
Recent clinical studies indicate that traumatic brain injury (TBI) produces chronic and progressive neurodegenerative changes leading to late neurologic dysfunction, but little is known about the mechanisms underlying such changes. Microglial-mediated neuroinflammationis an important secondary injury mechanism after TBI. In human studies, microglial activation has been found to persist for many years after the initial brain trauma, particularly after moderate to severe TBI. In the present study, adult C57Bl/6 mice were subjected to single moderate-level controlled cortical impact and were followed up by longitudinal T2-weighted magnetic resonance imaging in combination with stereologic histologic assessment of lesion volume expansion, neuronal loss, and microglial activation for up to 1 year after TBI. Persistent microglial activation was observed in the injured cortex through 1 year after injury and was associated with progressive lesion expansion, hippocampal neurodegeneration, and loss of myelin. Notably, highly activated microglia that expressed major histocompatibility complex class II (CR3/43), CD68, and NADPH oxidase (NOX2) were detected at the margins of the expanding lesion at 1 year after injury; biochemical markers of neuroinflammation and oxidative stress were significantly elevated at this time point. These data support emerging clinical TBI findings and provide a mechanistic link between TBI-induced chronic microglial activation and progressive neurodegeneration.
|Prolonged neuroinflammation after lipopolysaccharide exposure in aged rats. |
Fu, HQ; Yang, T; Xiao, W; Fan, L; Wu, Y; Terrando, N; Wang, TL
PloS one 9 e106331 2014
Inflammation is a hallmark of several disease states ranging from neurodegeneration to sepsis but is also implicated in physiological processes like ageing. Non-resolving inflammation and prolonged neuroinflammation are unclear processes implicated in several conditions, including ageing. In this study we studied the long-term effects of endotoxemia, as systemic lipopolysaccharide (LPS) injection, focusing on the role of astrocyte activation and cytokine release in the brain of aged rats. A single dose of LPS (2 mg/kg) or 0.9% saline was injected intraperitoneally in aged rats. Levels of pro-inflammatory cytokines (TNFα and IL-1β) and NF-κB p65 activation were measured systemically and in hippocampal tissue. Astrocytes and cytokines release in the CNS were detected via double immunofluorescence staining at different time-points up to day 30. Serum levels of TNFα and IL-1β were significantly increased acutely after 30 minutes (pless than 0.001) and up to 6 hours (pless than 0.001) following LPS-injection. Centrally, LPS-treated rats showed up-regulated mRNA expression and protein levels of pro-inflammatory cytokines in the hippocampus. These changes associated with astrogliosis in the hippocampus dentate gyrus (DG), IL-1β immunoreactivity and elevated NF-κB p65 expression up to day 30 post LPS exposure. Overall, these data demonstrate that LPS induces prolonged neuroinflammation and astrocyte activation in the hippocampus of aged rats. Hippocampal NF-κB p65 and excessive astrocytes-derived IL-1β release may play a pivotal role in regulating long-lasting neuroinflammation.
|Increased astrocyte expression of IL-6 or CCL2 in transgenic mice alters levels of hippocampal and cerebellar proteins. |
Gruol, DL; Vo, K; Bray, JG
Frontiers in cellular neuroscience 8 234 2014
Emerging research has identified that neuroimmune factors are produced by cells of the central nervous system (CNS) and play critical roles as regulators of CNS function, directors of neurodevelopment and responders to pathological processes. A wide range of neuroimmune factors are produced by CNS cells, primarily the glial cells, but the role of specific neuroimmune factors and their glial cell sources in CNS biology and pathology have yet to be fully elucidated. We have used transgenic mice that express elevated levels of a specific neuroimmune factor, the cytokine IL-6 or the chemokine CCL2, through genetic modification of astrocyte expression to identify targets of astrocyte produced IL-6 or CCL2 at the protein level. We found that in non-transgenic mice constitutive expression of IL-6 and CCL2 occurs in the two CNS regions studied, the hippocampus and cerebellum, as measured by ELISA. In the CCL2 transgenic mice elevated levels of CCL2 were evident in the hippocampus and cerebellum, whereas in the IL-6 transgenic mice, elevated levels of IL-6 were only evident in the cerebellum. Western blot analysis of the cellular and synaptic proteins in the hippocampus and cerebellum of the transgenic mice showed that the elevated levels of CCL2 or IL-6 resulted in alterations in the levels of specific proteins and that these actions differed for the two neuroimmune factors and for the two brain regions. These results are consistent with cell specific profiles of action for IL-6 and CCL2, actions that may be an important aspect of their respective roles in CNS physiology and pathophysiology.
|The failure in the stabilization of glioblastoma-derived cell lines: spontaneous in vitro senescence as the main culprit. |
Stoczynska-Fidelus, E; Piaskowski, S; Bienkowski, M; Banaszczyk, M; Hulas-Bigoszewska, K; Winiecka-Klimek, M; Radomiak-Zaluska, A; Och, W; Borowiec, M; Zieba, J; Treda, C; Rieske, P
PloS one 9 e87136 2014
Cell line analysis is an important element of cancer research. Despite the progress in glioblastoma cell culturing, the cells isolated from the majority of specimens cannot be propagated infinitely in vitro. The aim of this study was to identify the processes responsible for the stabilization failure. Therefore, we analyzed 56 primary GB cultures, 7 of which were stabilized. Our results indicate that senescence is primarily responsible for the glioblastoma cell line stabilization failure, while mitotic catastrophe and apoptosis play a minor role. Moreover, a new technical approach allowed for a more profound analysis of the senescent cells in primary cultures, including the distinction between tumor and normal cells. In addition, we observed that glioblastoma cells in primary cultures have a varied potential to undergo spontaneous in vitro senescence, which is often higher than that of the normal cells infiltrating the tumor. Thus, this is the first report of GB cells in primary cell cultures (including both monolayer and spheroid conditions) rapidly and spontaneously becoming senescent. Intriguingly, our data also suggest that nearly half of GB cell lines have a combination of TP53 mutation and CDKN2A homozygous deletion, which are considered as mutually exclusive in glioblastoma. Moreover, recognition of the mechanisms of senescence and mitotic catastrophe in glioblastoma cells may be a step towards a potential new therapeutic approach.
|Astrocyte-derived proinflammatory cytokines induce hypomyelination in the periventricular white matter in the hypoxic neonatal brain. |
Deng, Y; Xie, D; Fang, M; Zhu, G; Chen, C; Zeng, H; Lu, J; Charanjit, K
PloS one 9 e87420 2014
Hypoxic exposure in the perinatal period causes periventricular white matter damage (PWMD), a condition associated with myelination abnormalities. Under hypoxic conditions, glial cells were activated and released a large number of inflammatory mediators in the PWM in neonatal brain, which may result in oligodendrocyte (OL) loss and axonal injury. This study aims to determine if astrocytes are activated and generate proinflammatory cytokines that may be coupled with the oligodendroglial loss and hypomyelination observed in hypoxic PWMD. Twenty-four 1-day-old Wistar rats were exposed to hypoxia for 2 h. The rats were then allowed to recover under normoxic conditions for 7 or 28 days before being killed. Another group of 24 rats kept outside the chamber was used as age-matched controls. Upregulated expression of TNF-α and IL-1β was observed in astrocytes in the PWM of P7 hypoxic rats by double immunofluorescence, western blotting and real time RT-PCR. This was linked to apoptosis and enhanced expression of TNF-R1 and IL-1R1 in APC(+) OLs. PLP expression was decreased significantly in the PWM of P28d hypoxic rats. The proportion of myelinated axons was markedly reduced by electron microscopy (EM) and the average g-ratios were higher in P28d hypoxic rats. Upregulated expression of TNF-α and IL-1β in primary cultured astrocytes as well as their corresponding receptors in primary culture APC(+) oligodendrocytes were detected under hypoxic conditions. Our results suggest that following a hypoxic insult, astrocytes in the PWM of neonatal rats produce inflammatory cytokines such as TNF-α and IL-1β, which induce apoptosis of OLs via their corresponding receptors associated with them. This results in hypomyelination in the PWM of hypoxic rats.
|Striatal astrocytes act as a reservoir for L-DOPA. |
Asanuma, M; Miyazaki, I; Murakami, S; Diaz-Corrales, FJ; Ogawa, N
PloS one 9 e106362 2014
L-DOPA is therapeutically efficacious in patients with Parkinson's disease (PD), although dopamine (DA) neurons are severely degenerated. Since cortical astrocytes express neutral amino acid transporter (LAT) and DA transporter (DAT), the uptake and metabolism of L-DOPA and DA in striatal astrocytes may influence their availability in the dopaminergic system of PD. To assess possible L-DOPA- and DA-uptake and metabolic properties of striatal astrocytes, we examined the expression of L-DOPA, DA and DAT in striatal astrocytes of hemi-parkinsonian model rats after repeated L-DOPA administration, and measured the contents of L-DOPA, DA and their metabolite in primary cultured striatal astrocytes after L-DOPA/DA treatment. Repeated injections of L-DOPA induced apparent L-DOPA- and DA-immunoreactivities and marked expression of DAT in reactive astrocytes on the lesioned side of the striatum in hemi-parkinsonian rats. Exposure to DA for 4h significantly increased the levels of DA and its metabolite DOPAC in cultured striatal astrocytes. L-DOPA was also markedly increased in cultured striatal astrocytes after 4-h L-DOPA exposure, but DA was not detected 4 or 8h after L-DOPA treatment, despite the expression of aromatic amino acid decarboxylase in astrocytes. Furthermore, the intracellular level of L-DOPA in cultured striatal astrocytes decreased rapidly after removal of extracellular L-DOPA. The results suggest that DA uptaken into striatal astrocytes is rapidly metabolized and that striatal astrocytes act as a reservoir of L-DOPA that govern the uptake or release of L-DOPA depending on extracellular L-DOPA concentration, but are less capable of converting L-DOPA to DA.
|Specific retrograde transduction of spinal motor neurons using lentiviral vectors targeted to presynaptic NMJ receptors. |
Eleftheriadou, I; Trabalza, A; Ellison, S; Gharun, K; Mazarakis, N
Molecular therapy : the journal of the American Society of Gene Therapy 22 1285-98 2014
To understand how receptors are involved in neuronal trafficking and to be able to utilize them for specific targeting via the peripheral route would be of great benefit. Here, we describe the generation of novel lentiviral vectors with tropism to motor neurons that were made by coexpressing onto the lentiviral surface a fusogenic glycoprotein (mutated sindbis G) and an antibody against a cell-surface receptor (Thy1.1, p75(NTR), or coxsackievirus and adenovirus receptor) on the presynaptic terminal of the neuromuscular junction. These vectors exhibit binding specificity and efficient transduction of receptor positive cell lines and primary motor neurons in vitro. Targeting of each of these receptors conferred to these vectors the capability of being transported retrogradely from the axonal tip, leading to transduction of motor neurons in vitro in compartmented microfluidic cultures. In vivo delivery of coxsackievirus and adenovirus receptor-targeted vectors in leg muscles of mice resulted in predicted patterns of motor neuron labeling in lumbar spinal cord. This opens up the clinical potential of these vectors for minimally invasive administration of central nervous system-targeted therapeutics in motor neuron diseases.
|Morphine dependence in single enteric neurons from the mouse colon requires deletion of β-arrestin2. |
Smith, TH; Ngwainmbi, J; Hashimoto, A; Dewey, WL; Akbarali, HI
Physiological reports 2 2014
Chronic administration of morphine results in the development of tolerance to the analgesic effects and to inhibition of upper gastrointestinal motility but not to colonic motility, resulting in persistent constipation. In this study we examined the effect of chronic morphine in myenteric neurons from the adult mouse colon. Similar to the ileum, distinct neuronal populations exhibiting afterhyperpolarization (AHP)-positive and AHP-negative neurons were identified in the colon. Acute morphine (3 μM) decreased the number of action potentials, and increased the threshold for action potential generation indicative of reduced excitability in AHP-positive neurons. In neurons from the ileum of mice that were rendered antinociceptive tolerant by morphine-pellet implantation for 5 days, the opioid antagonist naloxone precipitated withdrawal as evidenced by increased neuronal excitability. Overnight incubation of ileum neurons with morphine also resulted in enhanced excitability to naloxone. Colonic neurons exposed to long-term morphine, remained unresponsive to naloxone suggesting that precipitated withdrawal does not occur in colonic neurons. However, morphine-treated colonic neurons from β-arrestin2 knockout mice demonstrated increased excitability upon treatment with naloxone as assessed by change in rheobase, number of action potentials and input resistance. These data suggest that similar to the ileum, acute exposure to morphine in colonic neurons results in reduced excitability due to inhibition of sodium currents. However, unlike the ileum, dependence to chronic exposure of morphine develops in colonic neurons from the β-arrestin2 knockout mice. These studies corroborate the in-vivo findings of the differential role of neuronal β-arrestin2 in the development of morphine tolerance/dependence in the ileum and colon.
|New α- and γ-synuclein immunopathological lesions in human brain. |
Surgucheva, I; Newell, KL; Burns, J; Surguchov, A
Acta neuropathologica communications 2 132 2014
Several neurodegenerative diseases are classified as proteopathies as they are associated with the aggregation of misfolded proteins. Synucleinopathies are a group of neurodegenerative disorders associated with abnormal deposition of synucleins. α-Synucleinopathies include Parkinson's disease, dementia with Lewy bodies, and multiple system atrophy. Recently accumulation of another member of the synuclein family- γ-synuclein in neurodegenerative diseases compelled the introduction of the term γ-synucleinopathy. The formation of aggregates and deposits of γ-synuclein is facilitated after its oxidation at methionine 38 (Met38).Several types of intracytoplasmic inclusions containing post-translationally modified α- and γ-synucleins are detected. Oxidized Met38-γ-synuclein forms aberrant inclusions in amygdala and substantia nigra. Double staining revealed colocalization of oxidized-γ-synuclein with α-synuclein in the cytoplasm of neurons. Another type of synuclein positive inclusions in the amygdala of dementia with Lewy bodies patients has the appearance of Lewy bodies. These inclusions are immunoreactive when analyzed with antibodies to α-synuclein phosphorylated on serine 129, as well as with antibodies to oxidized-γ-synuclein. Some of these Lewy bodies have doughnut-like shape with round or elongated shape. The separate immunofluorescent images obtained with individual antibodies specific to oxidized-γ-synuclein and phospho-α-synuclein clearly shows the colocalization of these synuclein isoforms in substantia nigra inclusions. Phospho-α-synuclein is present almost exclusively at the periphery of these structures, whereas oxidized-γ-syn immunoreactivity is also located in the internal parts forming dot-like pattern of staining.These results reveal new γ-synuclein positive lesions in human brain. Oxidized-γ-synuclein is colocalized with phospho-α-synuclein in doughnut-like inclusions. Several types of astrocytes with different morphology are immunopositive for oxidized-γ-synuclein.
|Enhanced central nervous system transduction with lentiviral vectors pseudotyped with RVG/HIV-1gp41 chimeric envelope glycoproteins. |
Trabalza, A; Eleftheriadou, I; Sgourou, A; Liao, TY; Patsali, P; Lee, H; Mazarakis, ND
Journal of virology 88 2877-90 2014
To investigate the potential benefits which may arise from pseudotyping the HIV-1 lentiviral vector with its homologous gp41 envelope glycoprotein (GP) cytoplasmic tail (CT), we created chimeric RVG/HIV-1gp41 GPs composed of the extracellular and transmembrane sequences of RVG and either the full-length gp41 CT or C terminus gp41 truncations sequentially removing existing conserved motifs. Lentiviruses (LVs) pseudotyped with the chimeric GPs were evaluated in terms of particle release (physical titer), biological titers, infectivity, and in vivo central nervous system (CNS) transduction. We report here that LVs carrying shorter CTs expressed higher levels of envelope GP and showed a higher average infectivity than those bearing full-length GPs. Interestingly, complete removal of GP CT led to vectors with the highest transduction efficiency. Removal of all C-terminal gp41 CT conserved motifs, leaving just 17 amino acids (aa), appeared to preserve infectivity and resulted in a significantly increased physical titer. Furthermore, incorporation of these 17 aa in the RVG CT notably enhanced the physical titer. In vivo stereotaxic delivery of LV vectors exhibiting the best in vitro titers into rodent striatum facilitated efficient transduction of the CNS at the site of injection. A particular observation was the improved retrograde transduction of neurons in connected distal sites that resulted from the chimeric envelope R5 which included the "Kennedy" sequence (Ken) and lentivirus lytic peptide 2 (LLP2) conserved motifs in the CT, and although it did not exhibit a comparable high titer upon pseudotyping, it led to a significant increase in distal retrograde transduction of neurons.In this study, we have produced novel chimeric envelopes bearing the extracellular domain of rabies fused to the cytoplasmic tail (CT) of gp41 and pseudotyped lentiviral vectors with them. Here we report novel effects on the transduction efficiency and physical titer of these vectors, depending on CT length and context. We also managed to achieve increased neuronal transduction in vivo in the rodent CNS, thus demonstrating that the efficiency of these vectors can be enhanced following merely CT manipulation. We believe that this paper is a novel contribution to the field and opens the way for further attempts to surface engineer lentiviral vectors and make them more amenable for applications in human disease.
|Application of apolipoprotein E-modified liposomal nanoparticles as a carrier for delivering DNA and nucleic acid in the brain. |
Tamaru, M; Akita, H; Nakatani, T; Kajimoto, K; Sato, Y; Hatakeyama, H; Harashima, H
International journal of nanomedicine 9 4267-76 2014
An innovative drug delivery technology is urgently needed to satisfy unmet medical needs in treating various brain disorders. As a fundamental carrier for plasmid DNA or nucleic acids, we developed a liposomal nanoparticle (multifunctional envelope-type nano device [MEND]) containing a proton-ionizable amino lipid (YSK-MEND). Here we report on the impact of apolipoprotein E (ApoE) modification on the function of YSK-MEND in terms of targeting brain cells. The cellular uptake and function of YSK-MEND encapsulating short interference RNA or plasmid DNA were significantly improved as a result of ApoE modification in mouse neuron-derived cell lines (Neuro-2a and CAD). Intracerebroventricular administration of ApoE-modified YSK-MEND (ApoE/YSK-MEND) encapsulating plasmid DNA also resulted in higher transgene expression in comparison with YSK-MEND that was not modified with ApoE. Moreover, observation of fluorescence-labeled ApoE/YSK-MEND and expression of mCherry (fluorescence protein) derived from plasmid DNA indicated that this carrier might be useful for delivering and conferring transgene expression in neural stem cells and/or neural progenitor cells. Thus, this system may be a useful tool for the treatment of neurodegenerative disease.
|Complete morphologies of basal forebrain cholinergic neurons in the mouse. |
Wu, H; Williams, J; Nathans, J
eLife 3 e02444 2014
The basal forebrain cholinergic system modulates neuronal excitability and vascular tone throughout the cerebral cortex and hippocampus. This system is severely affected in Alzheimer's disease (AD), and drug treatment to enhance cholinergic signaling is widely used as symptomatic therapy in AD. Defining the full morphologies of individual basal forebrain cholinergic neurons has, until now, been technically beyond reach due to their large axon arbor sizes. Using genetically-directed sparse labeling, we have characterized the complete morphologies of basal forebrain cholinergic neurons in the mouse. Individual arbors were observed to span multiple cortical columns, and to have greater than 1000 branch points and total axon lengths up to 50 cm. In an AD model, cholinergic axons were slowly lost and there was an accumulation of axon-derived material in discrete puncta. Calculations based on published morphometric data indicate that basal forebrain cholinergic neurons in humans have a mean axon length of ∼100 meters.DOI: http://dx.doi.org/10.7554/eLife.02444.001.
|Neuronal glycogen synthesis contributes to physiological aging. |
Sinadinos, C; Valles-Ortega, J; Boulan, L; Solsona, E; Tevy, MF; Marquez, M; Duran, J; Lopez-Iglesias, C; Calbó, J; Blasco, E; Pumarola, M; Milán, M; Guinovart, JJ
Aging cell 13 935-45 2014
Glycogen is a branched polymer of glucose and the carbohydrate energy store for animal cells. In the brain, it is essentially found in glial cells, although it is also present in minute amounts in neurons. In humans, loss-of-function mutations in laforin and malin, proteins involved in suppressing glycogen synthesis, induce the presence of high numbers of insoluble polyglucosan bodies in neuronal cells. Known as Lafora bodies (LBs), these deposits result in the aggressive neurodegeneration seen in Lafora's disease. Polysaccharide-based aggregates, called corpora amylacea (CA), are also present in the neurons of aged human brains. Despite the similarity of CA to LBs, the mechanisms and functional consequences of CA formation are yet unknown. Here, we show that wild-type laboratory mice also accumulate glycogen-based aggregates in the brain as they age. These structures are immunopositive for an array of metabolic and stress-response proteins, some of which were previously shown to aggregate in correlation with age in the human brain and are also present in LBs. Remarkably, these structures and their associated protein aggregates are not present in the aged mouse brain upon genetic ablation of glycogen synthase. Similar genetic intervention in Drosophila prevents the accumulation of glycogen clusters in the neuronal processes of aged flies. Most interestingly, targeted reduction of Drosophila glycogen synthase in neurons improves neurological function with age and extends lifespan. These results demonstrate that neuronal glycogen accumulation contributes to physiological aging and may therefore constitute a key factor regulating age-related neurological decline in humans.
|Myelin basic protein induces neuron-specific toxicity by directly damaging the neuronal plasma membrane. |
Zhang, J; Sun, X; Zheng, S; Liu, X; Jin, J; Ren, Y; Luo, J
PloS one 9 e108646 2014
The central nervous system (CNS) insults may cause massive demyelination and lead to the release of myelin-associated proteins including its major component myelin basic protein (MBP). MBP is reported to induce glial activation but its effect on neurons is still little known. Here we found that MBP specifically bound to the extracellular surface of the neuronal plasma membrane and induced neurotoxicity in vitro. This effect of MBP on neurons was basicity-dependent because the binding was blocked by acidic lipids and competed by other basic proteins. Further studies revealed that MBP induced damage to neuronal membrane integrity and function by depolarizing the resting membrane potential, increasing the permeability to cations and other molecules, and decreasing the membrane fluidity. At last, artificial liposome vesicle assay showed that MBP directly disturbed acidic lipid bilayer and resulted in increased membrane permeability. These results revealed that MBP induces neurotoxicity through its direct interaction with acidic components on the extracellular surface of neuronal membrane, which may suggest a possible contribution of MBP to the pathogenesis in the CNS disorders with myelin damage.
|Hypertonic saline alleviates cerebral edema by inhibiting microglia-derived TNF-α and IL-1β-induced Na-K-Cl Cotransporter up-regulation. |
Huang, LQ; Zhu, GF; Deng, YY; Jiang, WQ; Fang, M; Chen, CB; Cao, W; Wen, MY; Han, YL; Zeng, HK
Journal of neuroinflammation 11 102 2014
Hypertonic saline (HS) has been successfully used clinically for treatment of various forms of cerebral edema. Up-regulated expression of Na-K-Cl Cotransporter 1 (NKCC1) and inflammatory mediators such as tumor necrosis factor alpha (TNF-α) and interleukin-1 beta (IL-1β) has been demonstrated to be closely associated with the pathogenesis of cerebral edema resulting from a variety of brain injuries. This study aimed to explore if alleviation of cerebral edema by 10% HS might be effected through down-regulation of inflammatory mediator expression in the microglia, and thus result in decreased NKCC1 expression in astrocytes in the cerebral cortex bordering the ischemic core.The Sprague-Dawley (SD) rats that underwent right-sided middle cerebral artery occlusion (MCAO) were used for assessment of NKCC1, TNF-α and IL-1β expression using Western blotting, double immunofluorescence and real time RT-PCR, and the model also was used for evaluation of brain water content (BWC) and infarct size. SB203580 and SP600125, specific inhibitors of the p38 and JNK signaling pathways, were used to treat primary microglia cultures to determine whether the two signaling pathways were required for the inhibition of HS on microglia expressing and secreting TNF-α and IL-1β using Western blotting, double immunofluorescence and enzyme-linked immunosorbent assay (ELISA). The effect of TNF-α and IL-1β on NKCC1 expression in primary astrocyte cultures was determined. In addition, the direct inhibitory effect of HS on NKCC1 expression in primary astrocytes was also investigated by Western blotting, double immunofluorescence and real time RT-PCR.BWC and infarct size decreased significantly after 10% HS treatment. TNF-α and IL-1β immunoexpression in microglia was noticeably decreased. Concomitantly, NKCC1 expression in astrocytes was down-regulated. TNF-α and IL-1β released from the primary microglia subjected to hypoxic exposure and treatment with 100 mM HS were decreased. NKCC1 expression in primary astrocytes was concurrently and progressively down-regulated with decreasing concentration of exogenous TNF-α and IL-1β. Additionally, 100 mM HS directly inhibited NKCC1 up-regulation in astrocytes under hypoxic condition.The results suggest that 10% HS alleviates cerebral edema through inhibition of the NKCC1 Cotransporter, which is mediated by attenuation of TNF-α and IL-1β stimulation on NKCC1.
|Genetic labeling reveals novel cellular targets of schizophrenia susceptibility gene: distribution of GABA and non-GABA ErbB4-positive cells in adult mouse brain. |
Bean, JC; Lin, TW; Sathyamurthy, A; Liu, F; Yin, DM; Xiong, WC; Mei, L
The Journal of neuroscience : the official journal of the Society for Neuroscience 34 13549-66 2014
Neuregulin 1 (NRG1) and its receptor ErbB4 are schizophrenia risk genes. NRG1-ErbB4 signaling plays a critical role in neural development and regulates neurotransmission and synaptic plasticity. Nevertheless, its cellular targets remain controversial. ErbB4 was thought to express in excitatory neurons, although recent studies disputed this view. Using mice that express a fluorescent protein under the promoter of the ErbB4 gene, we determined in what cells ErbB4 is expressed and their identity. ErbB4 was widely expressed in the mouse brain, being highest in amygdala and cortex. Almost all ErbB4-positive cells were GABAergic in cortex, hippocampus, basal ganglia, and most of amygdala in neonatal and adult mice, suggesting GABAergic transmission as a major target of NRG1-ErbB4 signaling in these regions. Non-GABAergic, ErbB4-positive cells were present in thalamus, hypothalamus, midbrain, and hindbrain. In particular, ErbB4 is expressed in serotoninergic neurons of raphe nuclei but not in norepinephrinergic neurons of the locus ceruleus. In hypothalamus, ErbB4 is present in neurons that express oxytocin. Finally, ErbB4 is expressed in a group of cells in the subcortical areas that are positive for S100 calcium binding protein β. These results identify novel cellular targets of NRG1-ErbB4 signaling.
|Overexpression of adenosine kinase in cortical astrocytes and focal neocortical epilepsy in mice. |
Shen, HY; Sun, H; Hanthorn, MM; Zhi, Z; Lan, JQ; Poulsen, DJ; Wang, RK; Boison, D
Journal of neurosurgery 120 628-38 2014
New experimental models and diagnostic methods are needed to better understand the pathophysiology of focal neocortical epilepsies in a search for improved epilepsy treatment options. The authors hypothesized that a focal disruption of adenosine homeostasis in the neocortex might be sufficient to trigger electrographic seizures. They further hypothesized that a focal disruption of adenosine homeostasis might affect microcirculation and thus offer a diagnostic opportunity for the detection of a seizure focus located in the neocortex.Focal disruption of adenosine homeostasis was achieved by injecting an adeno-associated virus (AAV) engineered to overexpress adenosine kinase (ADK), the major metabolic clearance enzyme for the brain's endogenous anticonvulsant adenosine, into the neocortex of mice. Eight weeks following virus injection, the affected brain area was imaged via optical microangiography (OMAG) to detect changes in microcirculation. After completion of imaging, cortical electroencephalography (EEG) recordings were obtained from the imaged brain area.Viral expression of the Adk cDNA in astrocytes generated a focal area (~ 2 mm in diameter) of ADK overexpression within the neocortex. OMAG scanning revealed a reduction in vessel density within the affected brain area of approximately 23% and 29% compared with control animals and the contralateral hemisphere, respectively. EEG recordings revealed electrographic seizures within the focal area of ADK overexpression at a rate of 1.3 ± 0.2 seizures per hour (mean ± SEM).The findings of this study suggest that focal adenosine deficiency is sufficient to generate a neocortical focus of hyperexcitability, which is also characterized by reduced vessel density. The authors conclude that their model constitutes a useful tool to study neocortical epilepsies and that OMAG constitutes a noninvasive diagnostic tool for the imaging of seizure foci with disrupted adenosine homeostasis.
|The cancer drug tamoxifen: a potential therapeutic treatment for spinal cord injury. |
Guptarak, J; Wiktorowicz, JE; Sadygov, RG; Zivadinovic, D; Paulucci-Holthauzen, AA; Vergara, L; Nesic, O
Journal of neurotrauma 31 268-83 2014
Tamoxifen (TMX) is a selective estrogen receptor modulator that can mimic the neuroprotective effects of estrogen but lacks its systemic adverse effects. We found that TMX (1 mg/day) significantly improved the motor recovery of partially paralyzed hind limbs of male adult rats with thoracic spinal cord injury (SCI), thus indicating a translational potential for this cancer medication given its clinical safety and applicability and the lack of currently available treatments for SCI. To shed light on the mechanisms underlying the beneficial effects of TMX for SCI, we used proteomic analyses, Western blots and histological assays, which showed that TMX treatment spared mature oligodendrocytes/increased myelin levels and altered reactive astrocytes, including the upregulation of the water channels aquaporin 4 (AQP4), a novel finding. AQP4 increases in TMX-treated SCI rats were associated with smaller fluid-filled cavities with borders consisting of densely packed AQP4-expressing astrocytes that closely resemble the organization of normal glia limitans externa (in contrast to large cavities in control SCI rats that lacked glia limitans-like borders and contained reactive glial cells). Based on our findings, we propose that TMX is a promising candidate for the therapeutic treatment of SCI and a possible intervention for other neuropathological conditions associated with demyelination and AQP4 dysfunction.
|Overexpression of survival motor neuron improves neuromuscular function and motor neuron survival in mutant SOD1 mice. |
Turner, BJ; Alfazema, N; Sheean, RK; Sleigh, JN; Davies, KE; Horne, MK; Talbot, K
Neurobiology of aging 35 906-15 2014
Spinal muscular atrophy results from diminished levels of survival motor neuron (SMN) protein in spinal motor neurons. Low levels of SMN also occur in models of amyotrophic lateral sclerosis (ALS) caused by mutant superoxide dismutase 1 (SOD1) and genetic reduction of SMN levels exacerbates the phenotype of transgenic SOD1(G93A) mice. Here, we demonstrate that SMN protein is significantly reduced in the spinal cords of patients with sporadic ALS. To test the potential of SMN as a modifier of ALS, we overexpressed SMN in 2 different strains of SOD1(G93A) mice. Neuronal overexpression of SMN significantly preserved locomotor function, rescued motor neurons, and attenuated astrogliosis in spinal cords of SOD1(G93A) mice. Despite this, survival was not prolonged, most likely resulting from SMN mislocalization and depletion of gems in motor neurons of symptomatic mice. Our results reveal that SMN upregulation slows locomotor deficit onset and motor neuron loss in this mouse model of ALS. However, disruption of SMN nuclear complexes by high levels of mutant SOD1, even in the presence of SMN overexpression, might limit its survival promoting effects in this specific mouse model. Studies in emerging mouse models of ALS are therefore warranted to further explore the potential of SMN as a modifier of ALS.
|Synthesis, transport, and metabolism of serotonin formed from exogenously applied 5-HTP after spinal cord injury in rats. |
Li, Y; Li, L; Stephens, MJ; Zenner, D; Murray, KC; Winship, IR; Vavrek, R; Baker, GB; Fouad, K; Bennett, DJ
Journal of neurophysiology 111 145-63 2014
Spinal cord transection leads to elimination of brain stem-derived monoamine fibers that normally synthesize most of the monoamines in the spinal cord, including serotonin (5-hydroxytryptamine, 5-HT) synthesized from tryptophan by enzymes tryptophan hydroxylase (TPH, synthesizing 5-hydroxytryptophan, 5-HTP) and aromatic l-amino acid decarboxylase (AADC, synthesizing 5-HT from 5-HTP). Here we examine whether spinal cord caudal to transection remains able to manufacture and metabolize 5-HT. Immunolabeling for AADC reveals that, while most AADC is confined to brain stem-derived monoamine fibers in spinal cords from normal rats, caudal to transection AADC is primarily found in blood vessel endothelial cells and pericytes as well as a novel group of neurons (NeuN positive and GFAP negative), all of which strongly upregulate AADC with injury. However, immunolabeling for 5-HT reveals that there is no detectable endogenous 5-HT synthesis in any structure in the spinal cord caudal to a chronic transection, including in AADC-containing vessels and neurons, consistent with a lack of TPH. In contrast, when we applied exogenous 5-HTP (in vitro or in vivo), AADC-containing vessels and neurons synthesized 5-HT, which contributed to increased motoneuron activity and muscle spasms (long-lasting reflexes, LLRs), by acting on 5-HT2 receptors (SB206553 sensitive) located on motoneurons (TTX resistant). Blocking monoamine oxidase (MAO) markedly increased the sensitivity of the motoneurons (LLR) to 5-HTP, more than it increased the sensitivity of motoneurons to 5-HT, suggesting that 5-HT synthesized from AADC is largely metabolized in AADC-containing neurons and vessels. In summary, after spinal cord injury AADC is upregulated in vessels, pericytes, and neurons but does not endogenously produce 5-HT, whereas when exogenous 5-HTP is provided AADC does produce functional amounts of 5-HT, some of which is able to escape metabolism by MAO, diffuse out of these AADC-containing cells, and ultimately act on 5-HT receptors on motoneurons.
|Isoflurane-induced apoptosis of neurons and oligodendrocytes in the fetal rhesus macaque brain. |
Creeley, CE; Dikranian, KT; Dissen, GA; Back, SA; Olney, JW; Brambrink, AM
Anesthesiology 120 626-38 2014
The authors have previously shown that exposure of the neonatal nonhuman primate (NHP) brain to isoflurane for 5 h causes widespread acute apoptotic degeneration of neurons and oligodendrocyte. The current study explored the potential apoptogenic action of isoflurane in the fetal NHP brain.Fetal rhesus macaques at gestational age of 120 days (G120) were exposed in utero for 5 h to isoflurane anesthesia (n = 5) or to no anesthesia (control condition; n = 4), and all regions of the brain were systematically evaluated 3 h later for evidence of apoptotic degeneration of neurons or glia.Exposure of the G120 fetal NHP brain to isoflurane caused a significant increase in apoptosis of neurons and of oligodendrocytes at a stage when oligodendrocytes were just beginning to myelinate axons. The neuroapoptosis response was most prominent in the cerebellum, caudate, putamen, amygdala, and several cerebrocortical regions. Oligodendrocyte apoptosis was diffusely distributed over many white matter regions. The total number of apoptotic profiles (neurons + oligodendrocytes) in the isoflurane-exposed brains was increased 4.1-fold, compared with the brains from drug-naive controls. The total number of oligodendrocytes deleted by isoflurane was higher than the number of neurons deleted.Isoflurane anesthesia for 5 h causes death of neurons and oligodendrocytes in the G120 fetal NHP brain. In the fetal brain, as the authors previously found in the neonatal NHP brain, oligodendrocytes become vulnerable when they are just achieving myelination competence. The neurotoxic potential of isoflurane increases between the third trimester (G120) and the neonatal period in the NHP brain.
|Tau reduction diminishes spatial learning and memory deficits after mild repetitive traumatic brain injury in mice. |
Cheng, JS; Craft, R; Yu, GQ; Ho, K; Wang, X; Mohan, G; Mangnitsky, S; Ponnusamy, R; Mucke, L
PloS one 9 e115765 2014
Because reduction of the microtubule-associated protein Tau has beneficial effects in mouse models of Alzheimer's disease and epilepsy, we wanted to determine whether this strategy can also improve the outcome of mild traumatic brain injury (TBI).We adapted a mild frontal impact model of TBI for wildtype C57Bl/6J mice and characterized the behavioral deficits it causes in these animals. The Barnes maze, Y maze, contextual and cued fear conditioning, elevated plus maze, open field, balance beam, and forced swim test were used to assess different behavioral functions. Magnetic resonance imaging (MRI, 7 Tesla) and histological analysis of brain sections were used to look for neuropathological alterations. We also compared the functional effects of this TBI model and of controlled cortical impact in mice with two, one or no Tau alleles.Repeated (2-hit), but not single (1-hit), mild frontal impact impaired spatial learning and memory in wildtype mice as determined by testing of mice in the Barnes maze one month after the injury. Locomotor activity, anxiety, depression and fear related behaviors did not differ between injured and sham-injured mice. MRI imaging did not reveal focal injury or mass lesions shortly after the injury. Complete ablation or partial reduction of tau prevented deficits in spatial learning and memory after repeated mild frontal impact. Complete tau ablation also showed a trend towards protection after a single controlled cortical impact. Complete or partial reduction of tau also reduced the level of axonopathy in the corpus callosum after repeated mild frontal impact.Tau promotes or enables the development of learning and memory deficits and of axonopathy after mild TBI, and tau reduction counteracts these adverse effects.
|Influence of pigment epithelium-derived factor on outcome after striatal cerebral ischemia in the mouse. |
Zille, M; Riabinska, A; Terzi, MY; Balkaya, M; Prinz, V; Schmerl, B; Nieminen-Kelhä, M; Endres, M; Vajkoczy, P; Pina, AL
PloS one 9 e114595 2014
We here suggest that pigment epithelium-derived factor (PEDF) does not have an effect on lesion size, behavioral outcome, cell proliferation, or cell death after striatal ischemia in the mouse. PEDF is a neurotrophic factor with neuroprotective, antiangiogenic, and antipermeability effects. It influences self-renewal of neural stem cells and proliferation of microglia. We investigated whether intraventricular infusion of PEDF reduces infarct size and cell death, ameliorates behavioral outcome, and influences cell proliferation in the one-hour middle cerebral artery occlusion (MCAO) mouse model of focal cerebral ischemia. C57Bl6/N mice were implanted with PEDF or artificial cerebrospinal fluid (control) osmotic pumps and subjected to 60-minute MCAO 48 hours after pump implantation. They received daily BrdU injections for 7 days after MCAO in order to investigate cell proliferation. Infarct volumes were determined 24 hours after reperfusion using magnetic resonance imaging. We removed the pumps on day 5 and performed behavioral testing between day 7 and 21. Immunohistochemical staining was performed to determine the effect of PEDF on cell proliferation and cell death. Our model produced an ischemic injury confined solely to striatal damage. We detected no reduction in infarct sizes and cell death in PEDF- vs. CSF-infused MCAO mice. Behavioral outcome and cell proliferation did not differ between the groups. However, we cannot exclude that PEDF might work under different conditions in stroke. Further studies will elucidate the effect of PEDF treatment on cell proliferation and behavioral outcome in moderate to severe ischemic injury in the brain.
|Activation of type-2 cannabinoid receptor inhibits neuroprotective and antiinflammatory actions of glucocorticoid receptor α: when one is better than two. |
Bisicchia, Elisa, et al.
Cell. Mol. Life Sci., (2013) 2013
Endocannabinoids (eCBs) and glucocorticoids (GCs) are two distinct classes of signaling lipids that exert both neuroprotective and immunosuppressive effects; however, the possibility of an actual interaction of their receptors [i.e., type-2 cannabinoid (CB(2)) and glucocorticoid receptor α (GRα), respectively] remains unexplored. Here, we demonstrate that the concomitant activation of CB(2) and GRα abolishes the neuroprotective effects induced by each receptor on central neurons and on glial cells in animal models of remote cell death. We also show that the ability of eCBs and GCs, used individually, to inhibit tumour necrosis factor-α (TNF-α) and interferon-γ (IFN-γ) production from activated human T lymphocytes is lost when CB(2) and GRα are activated simultaneously. In addition, signal transduction pathways triggered by concomitant activation of both receptors led to increased levels of GRβ, heat-shock proteins-70 and -90, and p-JNK, as well as to reduced levels of p-STAT6. These effects were reversed only by selectively antagonizing CB(2), but not GRα. Overall, our study demonstrates for the first time the existence of a CB(2)-driven negative cross-talk between eCB and GC signaling in both rats and humans, thus paving the way to the possible therapeutic exploitation of CB(2) as a new target for chronic inflammatory and neurodegenerative diseases.
|Regulation of Microglial Proliferation during Chronic Neurodegeneration. |
Gómez-Nicola, Diego, et al.
J. Neurosci., 33: 2481-93 (2013) 2013
An important component of chronic neurodegenerative diseases is the generation of an innate inflammatory response within the CNS. Microglial and astroglial cells play a key role in the development and maintenance of this inflammatory response, showing enhanced proliferation and activation. We studied the time course and regulation of microglial proliferation, using a mouse model of prion disease. Our results show that the proliferation of resident microglial cells accounts for the expansion of the population during the development of the disease. We identify the pathway regulated by the activation of CSF1R and the transcription factors PU.1 and C/EBPα as the molecular regulators of the proliferative response, correlating with the chronic human neurodegenerative conditions variant Creutzfeldt-Jakob disease and Alzheimer's disease. We show that targeting the activity of CSF1R inhibits microglial proliferation and slows neuronal damage and disease progression. Our results demonstrate that microglial proliferation is a major component in the evolution of chronic neurodegeneration, with direct implications for understanding the contribution of the CNS innate immune response to disease progression.
|Germ-line recombination activity of the widely used hGFAP-Cre and nestin-Cre transgenes. |
Zhang, J; Dublin, P; Griemsmann, S; Klein, A; Brehm, R; Bedner, P; Fleischmann, BK; Steinhäuser, C; Theis, M
PloS one 8 e82818 2013
Herein we demonstrate with PCR, immunodetection and reporter gene approaches that the widely used human Glial Fibrillary Acidic Protein (hGFAP)-Cre transgene exhibits spontaneous germ-line recombination activity in leading to deletion in brain, heart and tail tissue with high frequency. The ectopic activity of hGFAP-Cre requires a rigorous control. We likewise observed that a second widely used nestin-Cre transgene shows germ-line deletion. Here we describe procedures to identify mice with germ-line recombination mediated by the hGFAP-Cre and nestin-Cre transgenes. Such control is essential to avoid pleiotropic effects due to germ-line deletion of loxP-flanked target genes and to maintain the CNS-restricted deletion status in transgenic mouse colonies.
|Alcohol-induced apoptosis of oligodendrocytes in the fetal macaque brain. |
Creeley, CE; Dikranian, KT; Johnson, SA; Farber, NB; Olney, JW
Acta neuropathologica communications 1 23 2013
In utero exposure of the fetal non-human primate (NHP) brain to alcohol on a single occasion during early or late third-trimester gestation triggers widespread acute apoptotic death of cells in both gray and white matter (WM) regions of the fetal brain. In a prior publication, we documented that the dying gray matter cells are neurons, and described the regional distribution and magnitude of this cell death response. Here, we present new findings regarding the magnitude, identity and maturational status of the dying WM cells in these alcohol-exposed fetal NHP brains.Our findings document that the dying WM cells belong to the oligodendrocyte (OL) lineage. OLs become vulnerable when they are just beginning to generate myelin basic protein in preparation for myelinating axons, and they remain vulnerable throughout later stages of myelination. We found no evidence linking astrocytes, microglia or OL progenitors to this WM cell death response. The mean density (profiles per mm3) of dying WM cells in alcohol-exposed brains was 12.7 times higher than the mean density of WM cells dying by natural apoptosis in drug-naive control brains.In utero exposure of the fetal NHP brain to alcohol on a single occasion triggers widespread acute apoptotic death of neurons (previous study) and of OLs (present study) throughout WM regions of the developing brain. The rate of OL apoptosis in alcohol-exposed brains was 12.7 times higher than the natural OL apoptosis rate. OLs become sensitive to the apoptogenic action of alcohol when they are just beginning to generate constituents of myelin in their cytoplasm, and they remain vulnerable throughout later stages of myelination. There is growing evidence for a similar apoptotic response of both neurons and OLs following exposure of the developing brain to anesthetic and anticonvulsant drugs. Collectively, this body of evidence raises important questions regarding the role that neuro and oligo apoptosis may play in the human condition known as fetal alcohol spectrum disorder (FASD), and also poses a question whether other apoptogenic drugs, although long considered safe for pediatric/obstetric use, may have the potential to cause iatrogenic FASD-like developmental disability syndromes.
|Dysfunctional Coq9 protein causes predominant encephalomyopathy associated with CoQ deficiency. |
García-Corzo, L; Luna-Sánchez, M; Doerrier, C; García, JA; Guarás, A; Acín-Pérez, R; Bullejos-Peregrín, J; López, A; Escames, G; Enríquez, JA; Acuña-Castroviejo, D; López, LC
Human molecular genetics 22 1233-48 2013
Coenzyme Q10 (CoQ(10)) or ubiquinone is a well-known component of the mitochondrial respiratory chain. In humans, CoQ(10) deficiency causes a mitochondrial syndrome with an unexplained variability in the clinical presentations. To try to understand this heterogeneity in the clinical phenotypes, we have generated a Coq9 Knockin (R239X) mouse model. The lack of a functional Coq9 protein in homozygous Coq9 mutant (Coq9(X/X)) mice causes a severe reduction in the Coq7 protein and, as consequence, a widespread CoQ deficiency and accumulation of demethoxyubiquinone. The deficit in CoQ induces a brain-specific impairment of mitochondrial bioenergetics performance, a reduction in respiratory control ratio, ATP levels and ATP/ADP ratio and specific loss of respiratory complex I. These effects lead to neuronal death and demyelinization with severe vacuolization and astrogliosis in the brain of Coq9(X/X) mice that consequently die between 3 and 6 months of age. These results suggest that the instability of mitochondrial complex I in the brain, as a primary event, triggers the development of mitochondrial encephalomyopathy associated with CoQ deficiency.
|TGF-β superfamily gene expression and induction of the Runx1 transcription factor in adult neurogenic regions after brain injury. |
Logan, TT; Villapol, S; Symes, AJ
PloS one 8 e59250 2013
Traumatic brain injury (TBI) increases neurogenesis in the forebrain subventricular zone (SVZ) and the hippocampal dentate gyrus (DG). Transforming growth factor-β (TGF-β) superfamily cytokines are important regulators of adult neurogenesis, but their involvement in the regulation of this process after brain injury is unclear. We subjected adult mice to controlled cortical impact (CCI) injury, and isolated RNA from the SVZ and DG at different post-injury time points. qPCR array analysis showed that cortical injury caused significant alterations in the mRNA expression of components and targets of the TGF-β, BMP, and activin signaling pathways in the SVZ and DG after injury, suggesting that these pathways could regulate post-injury neurogenesis. In both neurogenic regions, the injury also induced expression of Runt-related transcription factor-1 (Runx1), which can interact with intracellular TGF-β Smad signaling pathways. CCI injury strongly induced Runx1 expression in activated and proliferating microglial cells throughout the neurogenic regions. Runx1 protein was also expressed in a subset of Nestin- and GFAP-expressing putative neural stem or progenitor cells in the DG and SVZ after injury. In the DG only, these Runx1+ progenitors proliferated. Our data suggest potential roles for Runx1 in the processes of microglial cell activation and proliferation and in neural stem cell proliferation after TBI.
|Traumatic brain injury in aged animals increases lesion size and chronically alters microglial/macrophage classical and alternative activation states. |
Kumar, A; Stoica, BA; Sabirzhanov, B; Burns, MP; Faden, AI; Loane, DJ
Neurobiology of aging 34 1397-411 2013
Traumatic brain injury (TBI) causes chronic microglial activation that contributes to subsequent neurodegeneration, with clinical outcomes declining as a function of aging. Microglia/macrophages (MG/Mɸ) have multiple phenotypes, including a classically activated, proinflammatory (M1) state that might contribute to neurotoxicity, and an alternatively activated (M2) state that might promote repair. In this study we used gene expression, immunohistochemical, and stereological analyses to show that TBI in aged versus young mice caused larger lesions associated with an M1/M2 balance switch and increased numbers of reactive (bushy and hypertrophic) MG/Mɸ in the cortex, hippocampus, and thalamus. Chitinase3-like 3 (Ym1), an M2 phenotype marker, displayed heterogeneous expression after TBI with amoeboid-like Ym1-positive MG/Mɸ at the contusion site and ramified Ym1-positive MG/Mɸ at distant sites; this distribution was age-related. Aged-injured mice also showed increased MG/Mɸ expression of major histocompatibility complex II and NADPH oxidase, and reduced antioxidant enzyme expression which was associated with lesion size and neurodegeneration. Thus, altered relative M1/M2 activation and an nicotinamide adenine dinucleotide phosphate oxidase (NADPH oxidase)-mediated shift in redox state might contribute to worse outcomes observed in older TBI animals by creating a more proinflammatory M1 MG/Mɸ activation state.
|Nf1 loss and Ras hyperactivation in oligodendrocytes induce NOS-driven defects in myelin and vasculature. |
Mayes, DA; Rizvi, TA; Titus-Mitchell, H; Oberst, R; Ciraolo, GM; Vorhees, CV; Robinson, AP; Miller, SD; Cancelas, JA; Stemmer-Rachamimov, AO; Ratner, N
Cell reports 4 1197-212 2013
Patients with neurofibromatosis type 1 (NF1) and Costello syndrome Rasopathy have behavioral deficits. In NF1 patients, these may correlate with white matter enlargement and aberrant myelin. To model these features, we induced Nf1 loss or HRas hyperactivation in mouse oligodendrocytes. Enlarged brain white matter tracts correlated with myelin decompaction, downregulation of claudin-11, and mislocalization of connexin-32. Surprisingly, non-cell-autonomous defects in perivascular astrocytes and the blood-brain barrier (BBB) developed, implicating a soluble mediator. Nitric oxide (NO) can disrupt tight junctions and gap junctions, and NO and NO synthases (NOS1-NOS3) were upregulated in mutant white matter. Treating mice with the NOS inhibitor NG-nitro-L-arginine methyl ester or the antioxidant N-acetyl cysteine corrected cellular phenotypes. CNP-HRasG12V mice also displayed locomotor hyperactivity, which could be rescued by antioxidant treatment. We conclude that Nf1/Ras regulates oligodendrocyte NOS and that dysregulated NO signaling in oligodendrocytes can alter the surrounding vasculature. The data suggest that antioxidants may improve some behavioral deficits in Rasopathy patients.
|Evidence for NG2-glia derived, adult-born functional neurons in the hypothalamus. |
Robins, SC; Trudel, E; Rotondi, O; Liu, X; Djogo, T; Kryzskaya, D; Bourque, CW; Kokoeva, MV
PloS one 8 e78236 2013
Accumulating evidence suggests that the adult murine hypothalamus, a control site of several fundamental homeostatic processes, has neurogenic capacity. Correspondingly, the adult hypothalamus exhibits considerable cell proliferation that is ongoing even in the absence of external stimuli, and some of the newborn cells have been shown to mature into cells that express neuronal fate markers. However, the identity and characteristics of proliferating cells within the hypothalamic parenchyma have yet to be thoroughly investigated. Here we show that a subset of NG2-glia distributed throughout the mediobasal hypothalamus are proliferative and express the stem cell marker Sox2. We tracked the constitutive differentiation of hypothalamic NG2-glia by employing genetic fate mapping based on inducible Cre recombinase expression under the control of the NG2 promoter, demonstrating that adult hypothalamic NG2-glia give rise to substantial numbers of APC+ oligodendrocytes and a smaller population of HuC/D+ or NeuN+ neurons. Labelling with the cell proliferation marker BrdU confirmed that some NG2-derived neurons have proliferated shortly before differentiation. Furthermore, patch-clamp electrophysiology revealed that some NG2-derived cells display an immature neuronal phenotype and appear to receive synaptic input indicative of their electrical integration in local hypothalamic circuits. Together, our studies show that hypothalamic NG2-glia are able to take on neuronal fates and mature into functional neurons, indicating that NG2-glia contribute to the neurogenic capacity of the adult hypothalamus.
|Use of a synthetic xeno-free culture substrate for induced pluripotent stem cell induction and retinal differentiation. |
Tucker, BA; Anfinson, KR; Mullins, RF; Stone, EM; Young, MJ
Stem cells translational medicine 2 16-24 2013
The purpose of this study was to determine whether a proprietary xeno-free synthetic culture surface could be used to aid in the production and subsequent retinal-specific differentiation of clinical-grade induced pluripotent stem cells (iPSCs). iPSCs were generated using adult somatic cells via infection with either a single cre-excisable lentiviral vector or four separate nonintegrating Sendai viruses driving expression of the transcription factors OCT4, SOX2, KLF4, and c-MYC. Retinal precursor cells were derived via targeted differentiation of iPSCs with exogenous delivery of dkk-1, noggin, insulin-like growth factor-1, basic fibroblast growth factor, acidic fibroblast growth factor, and DAPT. Phase contrast microscopy, immunocytochemistry, hematoxylin and eosin staining, and reverse transcription-polymerase chain reaction were used to determine reprogramming efficiency, pluripotency, and fate of undifferentiated and differentiated iPSCs. Following viral transduction, cells underwent prototypical morphological changes resulting in the formation of iPSC colonies large enough for manual isolation/passage at 3-4 weeks postinfection. Both normal and disease-specific iPSCs expressed markers of pluripotency and, following transplantation into immune-compromised mice, formed teratomas containing tissue comprising all three germ layers. When subjected to our established retinal differentiation protocol, a significant proportion of the xeno-free substrate-derived cells expressed retinal cell markers, the number of which did not significantly differ from that derived on traditional extracellular matrix-coated dishes. Synthetic cell culture substrates provide a useful surface for the xeno-free production, culture, and differentiation of adult somatic cell-derived iPSCs. These findings demonstrate the potential utility of these surfaces for the production of clinical-grade retinal neurons for transplantation and induction of retinal regeneration.
|Treating brain tumor-initiating cells using a combination of myxoma virus and rapamycin. |
Zemp, FJ; Lun, X; McKenzie, BA; Zhou, H; Maxwell, L; Sun, B; Kelly, JJ; Stechishin, O; Luchman, A; Weiss, S; Cairncross, JG; Hamilton, MG; Rabinovich, BA; Rahman, MM; Mohamed, MR; Smallwood, S; Senger, DL; Bell, J; McFadden, G; Forsyth, PA
Neuro-oncology 15 904-20 2013
Intratumoral heterogeneity in glioblastoma multiforme (GBM) poses a significant barrier to therapy in certain subpopulation such as the tumor-initiating cell population, being shown to be refractory to conventional therapies. Oncolytic virotherapy has the potential to target multiple compartments within the tumor and thus circumvent some of the barriers facing conventional therapies. In this study, we investigate the oncolytic potential of myxoma virus (MYXV) alone and in combination with rapamycin in vitro and in vivo using human brain tumor-initiating cells (BTICs).We cultured fresh GBM specimens as neurospheres and assayed their growth characteristics in vivo. We then tested the susceptibility of BTICs to MYXV infection with or without rapamycin in vitro and assessed viral biodistribution/survival in vivo in orthotopic xenografts.The cultured neurospheres were found to retain stem cell markers in vivo, and they closely resembled human infiltrative GBM. In this study we determined that (i) all patient-derived BTICs tested, including those resistant to temozolomide, were susceptible to MYXV replication and killing in vitro; (ii) MYXV replicated within BTICs in vivo, and intratumoral administration of MYXV significantly prolonged survival of BTIC-bearing mice; (iii) combination therapy with MYXV and rapamycin improved antitumor activity, even in mice bearing "advanced" BTIC tumors; (iv) MYXV treatment decreased expression of stem cell markers in vitro and in vivo.Our study suggests that MYXV in combination with rapamycin infects and kills both the BTICs and the differentiated compartments of GBM and may be an effective treatment even in TMZ-resistant patients.
|Rac1 selective activation improves retina ganglion cell survival and regeneration. |
Lorenzetto, E; Ettorre, M; Pontelli, V; Bolomini-Vittori, M; Bolognin, S; Zorzan, S; Laudanna, C; Buffelli, M
PloS one 8 e64350 2013
In adult mammals, after optic nerve injury, retinal ganglion cells (RGCs) do not regenerate their axons and most of them die by apoptosis within a few days. Recently, several strategies that activate neuronal intracellular pathways were proposed to prevent such degenerative processes. The rho-related small GTPase Rac1 is part of a complex, still not fully understood, intracellular signaling network, mediating in neurons many effects, including axon growth and cell survival. However, its role in neuronal survival and regeneration in vivo has not yet been properly investigated. To address this point we intravitreally injected selective cell-penetrating Rac1 mutants after optic nerve crush and studied the effect on RGC survival and axonal regeneration. We injected two well-characterized L61 constitutively active Tat-Rac1 fusion protein mutants, in which a second F37A or Y40C mutation confers selectivity in downstream signaling pathways. Results showed that, 15 days after crush, both mutants were able to improve survival and to prevent dendrite degeneration, while the one harboring the F37A mutation also improved axonal regeneration. The treatment with F37A mutant for one month did not improve the axonal elongation respect to 15 days. Furthermore, we found an increase of Pak1 T212 phosphorylation and ERK1/2 expression in RGCs after F37A treatment, whereas ERK1/2 was more activated in glial cells after Y40C administration. Our data suggest that the selective activation of distinct Rac1-dependent pathways could represent a therapeutic strategy to counteract neuronal degenerative processes in the retina.
|The absence of myelin basic protein promotes neuroinflammation and reduces amyloid β-protein accumulation in Tg-5xFAD mice. |
Ou-Yang, MH; Van Nostrand, WE
Journal of neuroinflammation 10 134 2013
Abnormal accumulation of amyloid β-protein (Aβ) in the brain plays an important role in the pathogenesis \of Alzheimer's disease (AD). Aβ monomers assemble into oligomers and fibrils that promote neuronal dysfunction. This assembly pathway is influenced by naturally occurring brain molecules, the Aβ chaperone proteins, which bind to Aβ and modulate its aggregation. Myelin basic protein (MBP) was previously identified as a novel Aβ chaperone protein and a potent inhibitor for Aβ fibril assembly in vitro.In this study, we determined whether the absence of MBP would influence Aβ pathology in vivo by breeding MBP knockout mice (MBP-/-) with Tg-5xFAD mice, a model of AD-like parenchymal Aβ pathology.Through biochemical and immunohistochemical experiments, we found that bigenic Tg-5xFAD/MBP-/- mice had a significant decrease of insoluble Aβ and parenchymal plaque deposition at an early age. The expression of transgene encoded human AβPP, the levels of C-terminal fragments generated during Aβ production and the intracellular Aβ were unaffected in the absence of MBP. Likewise, we did not find a significant difference in plasma Aβ or cerebrospinal fluid Aβ, suggesting these clearance routes were unaltered in bigenic Tg-5xFAD/MBP-/- mice. However, MBP-/- mice and bigenic Tg-5xFAD/MBP-/- mice exhibited elevated reactive astrocytes and activated microglia compared with Tg-5xFAD mice. The Aβ degrading enzyme matrix metalloproteinase 9 (MMP-9), which is expressed by activated glial cells, was significantly increased in the Tg-5xFAD/MBP-/- mice.These findings indicate that the absence of MBP decreases Aβ deposition in transgenic mice and that this consequence may result from increased glial activation and expression of MMP-9, an Aβ degrading enzyme.
|Adeno-associated virus type 6 is retrogradely transported in the non-human primate brain. |
San Sebastian, W; Samaranch, L; Heller, G; Kells, AP; Bringas, J; Pivirotto, P; Forsayeth, J; Bankiewicz, KS
Gene therapy 20 1178-83 2013
We recently demonstrated that axonal transport of adeno-associated virus (AAV) is serotype-dependent. Thus, AAV serotype 2 (AAV2) is anterogradely transported (e.g., from cell bodies to nerve terminals) in both rat and non-human primate (NHP) brain. In contrast, AAV serotype 6 (AAV6) is retrogradely transported from terminals to neuronal cell bodies in the rat brain. However, the directionality of axonal transport of AAV6 in the NHP brain has not been determined. In this study, two Cynomolgus macaques received an infusion of AAV6 harboring green fluorescent protein (GFP) into the striatum (caudate and putamen) by magnetic resonance (MR)-guided convection-enhanced delivery. One month after infusion, immunohistochemical staining of brain sections revealed a striatal GFP expression that corresponded well with MR signal observed during gene delivery. As shown previously in rats, GFP expression was detected throughout the prefrontal, frontal and parietal cortex, as well as the substantia nigra pars compacta and thalamus, indicating retrograde transport of the vector in NHP. AAV6-GFP preferentially transduced neurons, although a few astrocytes were also transduced. Transduction of non-neuronal cells in the brain was associated with the upregulation of the major histocompatibility complex-II and lymphocytic infiltration as previously observed with AAV1 and AAV9. This contrasts with highly specific neuronal transduction in the rat brain. Retrograde axonal transport of AAV6 from a single striatal infusion permits efficient transduction of cortical neurons in significant tissue volumes that otherwise would be difficult to achieve.
|Aquaporin-4 expression in post-traumatic syringomyelia. |
Hemley, SJ; Bilston, LE; Cheng, S; Chan, JN; Stoodley, MA
Journal of neurotrauma 30 1457-67 2013
Aquaporin-4 (AQP4) is an astroglial water channel protein that plays an important role in the transmembrane movement of water within the central nervous system. AQP4 has been implicated in numerous pathological conditions involving abnormal fluid accumulation, including spinal cord edema following traumatic injury. AQP4 has not been studied in post-traumatic syringomyelia, a condition that cannot be completely explained by current theories of cerebrospinal fluid dynamics. Alterations of AQP4 expression or function may contribute to the fluid imbalance leading to syrinx formation or enlargement. The aim of this study was to examine AQP4 expression levels and distribution in an animal model of post-traumatic syringomyelia. Immunofluorescence and western blotting were used to assess AQP4 and glial fibrillary acidic protein (GFAP) expression in an excitotoxic amino acid/arachnoiditis model of post-traumatic syringomyelia in Sprague-Dawley rats. At all time-points, GFAP-positive astrocytes were observed in tissue surrounding syrinx cavities, although western blot analysis demonstrated an overall decrease in GFAP expression, except at the latest stage investigated. AQP4 expression was significantly higher at the level of syrinx at three and six weeks following the initial syrinx induction surgery. Significant increases in AQP4 expression also were observed in the upper cervical cord, rostral to the syrinx except in the acute stage of the condition at the three-day time-point. Immunostaining showed that AQP4 was expressed around all syrinx cavities, most notably adjacent to a mature syrinx (six- and 12-week time-point). This suggests a relationship between AQP4 and fluid accumulation in post-traumatic syringomyelia. However, whether this is a causal relationship or occurs in response to an increase in fluid needs to be established.
|Failure of axonal transport induces a spatially coincident increase in astrocyte BDNF prior to synapse loss in a central target. |
Crish, SD; Dapper, JD; MacNamee, SE; Balaram, P; Sidorova, TN; Lambert, WS; Calkins, DJ
Neuroscience 229 55-70 2013
Failure of anterograde transport to distal targets in the brain is a common feature of neurodegenerative diseases. We have demonstrated in rodent models of glaucoma, the most common optic neuropathy, early loss of anterograde transport along the retinal ganglion cell (RGC) projection to the superior colliculus (SC) is retinotopic and followed by a period of persistence of RGC axon terminals and synapses through unknown molecular pathways. Here we use the DBA/2J mouse model of hereditary glaucoma and an acute rat model to demonstrate that retinotopically focal transport deficits in the SC are accompanied by a spatially coincident increase in brain-derived neurotrophic factor (BDNF), especially in hypertrophic astrocytes. These neurochemical changes occur prior to loss of RGC synapses in the DBA/2J SC. In contrast to BDNF protein, levels of Bdnf mRNA decreased with transport failure, even as mRNA encoding synaptic structures remained unchanged. In situ hybridization signal for Bdnf mRNA was the strongest in SC neurons, and labeling for the immature precursor pro-BDNF was very limited. Subcellular fractionation of SC indicated that membrane-bound BDNF decreased with age in the DBA/2J, while BDNF released from vesicles remained high. These results suggest that in response to diminished axonal function, activated astrocytes in the brain may sequester mature BDNF released from target neurons to counter stressors that otherwise would challenge survival of projection synapses.
|RING finger protein 11 (RNF11) modulates susceptibility to 6-OHDA-induced nigral degeneration and behavioral deficits through NF-κB signaling in dopaminergic cells. |
Pranski, EL; Dalal, NV; Sanford, CV; Herskowitz, JH; Gearing, M; Lazo, C; Miller, GW; Lah, JJ; Levey, AI; Betarbet, RS
Neurobiology of disease 54 264-79 2013
Chronic activation of the NF-κB pathway is associated with progressive neurodegeneration in Parkinson's disease (PD). Given the role of neuronal RING finger protein 11 (RNF11) as a negative regulator of the NF-κB pathway, in this report we investigated the function of RNF11 in dopaminergic cells in PD-associated neurodegeneration. We found that RNF11 knockdown in an in vitro model of PD mediated protection against 6-OHDA-induced toxicity. In converse, over-expression of RNF11 enhanced 6-OHDA-induced dopaminergic cell death. Furthermore, by directly manipulating NF-κB signaling, we showed that the observed RNF11-enhanced 6-OHDA toxicity is mediated through inhibition of NF-κB-dependent transcription of TNF-α, antioxidants GSS and SOD1, and anti-apoptotic factor BCL2. Experiments in an in vivo 6-OHDA rat model of PD recapitulated the in vitro results. In vivo targeted RNF11 over-expression in nigral neurons enhanced 6-OHDA toxicity, as evident by increased amphetamine-induced rotations and loss of nigral dopaminergic neurons as compared to controls. This enhanced toxicity was coupled with the downregulation of NF-κB transcribed GSS, SOD1, BCL2, and neurotrophic factor BDNF mRNA levels, in addition to decreased TNF-α mRNA levels in ventral mesenchephalon samples. In converse, knockdown of RNF11 was associated with protective phenotypes and increased expression of above-mentioned NF-κB transcribed genes. Collectively, our in vitro and in vivo data suggest that RNF11-mediated inhibition of NF-κB in dopaminergic cells exaggerates 6-OHDA toxicity by inhibiting neuroprotective responses while loss of RNF11 inhibition on NF-κB activity promotes neuronal survival. The decreased expression of RNF11 in surviving cortical and nigral tissue detected in PD patients, thus implies a compensatory response in the diseased brain to PD-associated insults. In summary, our findings demonstrate that RNF11 in neurons can modulate susceptibility to 6-OHDA toxicity through NF-κB mediated responses. This neuron-specific role of RNF11 in the brain has important implications for targeted therapeutics aimed at preventing neurodegeneration.
|Resistance to oncolytic myxoma virus therapy in nf1(-/-)/trp53(-/-) syngeneic mouse glioma models is independent of anti-viral type-I interferon. |
Zemp, FJ; McKenzie, BA; Lun, X; Maxwell, L; Reilly, KM; McFadden, G; Yong, VW; Forsyth, PA
PloS one 8 e65801 2013
Despite promising preclinical studies, oncolytic viral therapy for malignant gliomas has resulted in variable, but underwhelming results in clinical evaluations. Of concern are the low levels of tumour infection and viral replication within the tumour. This discrepancy between the laboratory and the clinic could result from the disparity of xenograft versus syngeneic models in determining in vivo viral infection, replication and treatment efficacy. Here we describe a panel of primary mouse glioma lines derived from Nf1 (+/-) Trp53 (+/-) mice in the C57Bl/6J background for use in the preclinical testing of the oncolytic virus Myxoma (MYXV). These lines show a range of susceptibility to MYXV replication in vitro, but all succumb to viral-mediated cell death. Two of these lines orthotopically grafted produced aggressive gliomas. Intracranial injection of MYXV failed to result in sustained viral replication or treatment efficacy, with minimal tumour infection that was completely resolved by 7 days post-infection. We hypothesized that the stromal production of Type-I interferons (IFNα/β) could explain the resistance seen in these models; however, we found that neither the cell lines in vitro nor the tumours in vivo produce any IFNα/β in response to MYXV infection. To confirm IFNα/β did not play a role in this resistance, we ablated the ability of tumours to respond to IFNα/β via IRF9 knockdown, and generated identical results. Our studies demonstrate that these syngeneic cell lines are relevant preclinical models for testing experimental glioma treatments, and show that IFNα/β is not responsible for the MYXV treatment resistance seen in syngeneic glioma models.
|Localization and divergent profiles of estrogen receptors and aromatase in the vocal and auditory networks of a fish with alternative mating tactics. |
Fergus, DJ; Bass, AH
The Journal of comparative neurology 521 2850-69 2013
Estrogens play a salient role in the development and maintenance of both male and female nervous systems and behaviors. The plainfin midshipman (Porichthys notatus), a teleost fish, has two male reproductive morphs that follow alternative mating tactics and diverge in multiple somatic, hormonal, and neural traits, including the central control of morph-specific vocal behaviors. After we identified duplicate estrogen receptors (ERβ1 and ERβ2) in midshipman, we developed antibodies to localize protein expression in the central vocal-acoustic networks and saccule, the auditory division of the inner ear. As in other teleost species, ERβ1 and ERβ2 were robustly expressed in the telencephalon and hypothalamus in vocal-acoustic and other brain regions shown previously to exhibit strong expression of ERα and aromatase (estrogen synthetase, CYP19) in midshipman. Like aromatase, ERβ1 label colocalized with glial fibrillary acidic protein (GFAP) in telencephalic radial glial cells. Quantitative polymerase chain reaction revealed similar patterns of transcript abundance across reproductive morphs for ERβ1, ERβ2, ERα, and aromatase in the forebrain and saccule. In contrast, transcript abundance for ERs and aromatase varied significantly between morphs in and around the sexually polymorphic vocal motor nucleus (VMN). Together, the results suggest that VMN is the major estrogen target within the estrogen-sensitive hindbrain vocal network that directly determines the duration, frequency, and amplitude of morph-specific vocalizations. Comparable regional differences in steroid receptor abundances likely regulate morph-specific behaviors in males and females of other species exhibiting alternative reproductive tactics.
|Lack of evidence for vesicular glutamate transporter expression in mouse astrocytes. |
Li, D; Hérault, K; Silm, K; Evrard, A; Wojcik, S; Oheim, M; Herzog, E; Ropert, N
The Journal of neuroscience : the official journal of the Society for Neuroscience 33 4434-55 2013
The concept of a tripartite synapse including a presynaptic terminal, a postsynaptic spine, and an astrocytic process that responds to neuronal activity by fast gliotransmitter release, confers to the electrically silent astrocytes an active role in information processing. However, the mechanisms of gliotransmitter release are still highly controversial. The reported expression of all three vesicular glutamate transporters (VGLUT1-3) by astrocytes suggests that astrocytes, like neurons, may release glutamate by exocytosis. However, the demonstration of astrocytic VGLUT expression is largely based on immunostaining, and the possibility of nonspecific labeling needs to be systematically addressed. We therefore examined the expression of VGLUT1-3 in astrocytes, both in culture and in situ. We used Western blots and single-vesicle imaging by total internal reflection fluorescence microscopy in live cultured astrocytes, and confocal microscopy, at the cellular level in cortical, hippocampal, and cerebellar brain slices, combined with quantitative image analysis. Control experiments were systematically performed in cultured astrocytes using wild-type, VGLUT1-3 knock-out, VGLUT1(Venus) knock-in, and VGLUT2-EGFP transgenic mice. In fixed brain slices, we quantified the degree of overlap between VGLUT1-3 and neuronal or astrocytic markers, both in an object-based manner using fluorescence line profiles, and in a pixel-based manner using dual-color scatter plots followed by the calculation of Pearson's correlation coefficient over all pixels with intensities significantly different from background. Our data provide no evidence in favor of the expression of any of the three VGLUTs by gray matter protoplasmic astrocytes of the primary somatosensory cortex, the thalamic ventrobasal nucleus, the hippocampus, and the cerebellum.
|Inhibition of the group I mGluRs reduces acute brain damage and improves long-term histological outcomes after photothrombosis-induced ischaemia. |
Li, H; Zhang, N; Sun, G; Ding, S
ASN neuro 5 195-207 2013
Group I mGluRs (metabotropic glutamate receptors), including mGluR1 and mGluR5, are GPCRs (G-protein coupled receptors) and play important roles in physiology and pathology. Studies on their role in cerebral ischaemia have provided controversial results. In this study, we used a PT (photothrombosis)-induced ischaemia model to investigate whether antagonists to the group I mGluRs may offer acute and long-term protective effects in adult mice. Our results demonstrated that administration with mGluR5 antagonist MPEP [2-methyl-6-(phenylethynyl)-pyridine] or mGluR1 antagonist LY367385 by intraperitoneal injection at 3 h after PT decreased brain infarct volume evaluated one day after ischaemia. Additive effects on infarct volume were observed upon co-injection with MPEP and LY367385. These antagonists also significantly alleviated neurodegeneration and apoptosis in the penumbra. In addition, when evaluated 2 weeks after PT, they reduced infarct volume and tissue loss, attenuated glial scar formation, and inhibited cell proliferation in the penumbra. Importantly, co-injection with MPEP and LY367385 reduced the expression levels of calpain, a Ca2+-activated protease known to mediate ischaemia-induced neuronal death. Injection of calpeptin, a calpain inhibitor, could inhibit neuronal death and brain damage after PT but injection of calpeptin together with MPEP and LY367385 did not further improve the protective effects mediated by MPEP and LY367385. These results suggest that inhibition of group I mGluRs is sufficient to protect ischaemic damage through the calpain pathway. Taken together, our results demonstrate that inhibition of group I mGluRs can mitigate PT-induced brain damage through attenuating the effects of calpain, and improve long-term histological outcomes.
|SnoN facilitates axonal regeneration after spinal cord injury. |
Do, JL; Bonni, A; Tuszynski, MH
PloS one 8 e71906 2013
Adult CNS neurons exhibit a reduced capacity for growth compared to developing neurons, due in part to downregulation of growth-associated genes as development is completed. We tested the hypothesis that SnoN, an embryonically regulated transcription factor that specifies growth of the axonal compartment, can enhance growth in injured adult neurons. In vitro, SnoN overexpression in dissociated adult DRG neuronal cultures significantly enhanced neurite outgrowth. Moreover, TGF-β1, a negative regulator of SnoN, inhibited neurite outgrowth, and SnoN over-expression overcame this inhibition. We then examined whether SnoN influenced axonal regeneration in vivo: indeed, expression of a mutant form of SnoN resistant to degradation significantly enhanced axonal regeneration following cervical spinal cord injury, despite peri-lesional upregulation of TGF-β1. Thus, a developmental mechanism that specifies extension of the axonal compartment also promotes axonal regeneration after adult CNS injury.
|Monocyte chemoattractant protein-1 affects migration of hippocampal neural progenitors following status epilepticus in rats. |
Hung, YW; Lai, MT; Tseng, YJ; Chou, CC; Lin, YY
Journal of neuroinflammation 10 11 2013
Epilepsy is a common brain disorder characterized by a chronic predisposition to generate spontaneous seizures. The mechanisms for epilepsy formation remain unknown. A growing body of evidence suggests the involvement of inflammatory processes in epileptogenesis. In the present study, we investigated the involvement of monocyte chemoattractant protein-1 (MCP-1) in aberrant migration of hippocampal progenitors in rats after the insult of status epilepticus (SE).SE was induced with pilocarpine in Sprague-Dawley rats. Transcriptional expression of MCP-1 in the dentate gyrus (DG) was measured using quantitative real-time PCR. From 1 to 28 days after SE, the temporal profiles of MCP-1 protein expression in DG were evaluated using enzyme-linked immunosorbent assay. Chemokine (C-C motif) receptor 2 (CCR2) expression in doublecortin-positive neuronal progenitors was examined using double-labeling immunohistochemistry. The involvement of MCP-1/CCR2 signaling in aberrant neuronal progenitor migration in the epileptic hippocampus was assessed in the SE rats using a CCR2 antagonist, RS102895, and the ectopic migration of neuronal progenitors was determined using Prox1/doublecortin double immunostaining.After SE, MCP-1 gene was significantly upregulated and its corresponding protein expression in the DG was significantly increased on days 1 and 3. Some hilar ectopic progenitor cells of SE rats expressed the MCP-1 receptor, CCR2. Notably, the ectopic migration of neuronal progenitors into hilus was attenuated by a blockade of the MCP-1/CCR2 interaction with a selective CCR2 inhibitor, RS102895.An increase in dentate MCP-1 is associated with seizure-induced aberrant migration of neuronal progenitors through the interaction with CCR2. The upregulation of MCP-1 after an insult of SE may play a role in the generation of epilepsy.
|A novel activator of CBP/p300 acetyltransferases promotes neurogenesis and extends memory duration in adult mice. |
Chatterjee, S; Mizar, P; Cassel, R; Neidl, R; Selvi, BR; Mohankrishna, DV; Vedamurthy, BM; Schneider, A; Bousiges, O; Mathis, C; Cassel, JC; Eswaramoorthy, M; Kundu, TK; Boutillier, AL
The Journal of neuroscience : the official journal of the Society for Neuroscience 33 10698-712 2013
Although the brain functions of specific acetyltransferases such as the CREB-binding protein (CBP) and p300 have been well documented using mutant transgenic mice models, studies based on their direct pharmacological activation are still missing due to the lack of cell-permeable activators. Here we present a small-molecule (TTK21) activator of the histone acetyltransferases CBP/p300, which, when conjugated to glucose-based carbon nanosphere (CSP), passed the blood-brain barrier, induced no toxicity, and reached different parts of the brain. After intraperitoneal administration in mice, CSP-TTK21 significantly acetylated histones in the hippocampus and frontal cortex. Remarkably, CSP-TTK21 treatment promoted the formation of long and highly branched doublecortin-positive neurons in the subgranular zone of the dentate gyrus and reduced BrdU incorporation, suggesting that CBP/p300 activation favors maturation and differentiation of adult neuronal progenitors. In addition, mRNA levels of the neuroD1 differentiation marker and BDNF, a neurotrophin required for the terminal differentiation of newly generated neurons, were both increased in the hippocampus concomitantly with an enrichment of acetylated-histone on their proximal promoter. Finally, we found that CBP/p300 activation during a spatial training, while not improving retention of a recent memory, resulted in a significant extension of memory duration. This report is the first evidence for CBP/p300-mediated histone acetylation in the brain by an activator molecule, which has beneficial implications for the brain functions of adult neurogenesis and long-term memory. We propose that direct stimulation of acetyltransferase function could be useful in terms of therapeutic options for brain diseases.
|Association of HK2 and NCK2 with normal tension glaucoma in the Japanese population. |
Shi, D; Funayama, T; Mashima, Y; Takano, Y; Shimizu, A; Yamamoto, K; Mengkegale, M; Miyazawa, A; Yasuda, N; Fukuchi, T; Abe, H; Ideta, H; Nishida, K; Nakazawa, T; Richards, JE; Fuse, N
PloS one 8 e54115 2013
Although family studies and genome-wide association studies have shown that genetic factors play a role in glaucoma, it has been difficult to identify the specific genetic variants involved. We tested 669 single nucleotide polymorphisms (SNPs) from the region of chromosome 2 that includes the GLC1B glaucoma locus for association with primary open-angle glaucoma (POAG) and normal tension glaucoma (NTG) in the Japanese population. We performed a two-stage case-control study. The first cohort consisted of 123 POAG cases, 121 NTG cases and 120 controls: the second cohort consisted of 187 POAG cases, 286 NTG cases, and 271 controls. Out of six SNPs showing significant association with POAG in the first round screening, seven SNPs were tested in the second round. Rs678350 in the HK2 gene coding sequence showed significant allelic (p=0.0027 in Stage Two, 2.7XE-4 in meta-analysis) association with POAG, and significant allelic (p=4.7XE-4 in Stage Two, 1.0XE-5 in meta-analysis) association with NTG. Although alleles in the TMEM182 gene did not show significant association with glaucoma in the second round, subjects with the A/A allele in TMEM182 rs869833 showed worse visual field mean deviation (p=0.01). Even though rs2033008 in the NCK2 gene coding sequence did not show significant association in the first round, it had previously shown association with NTG so it was tested for association with NTG in round 2 (p=0.0053 in Stage Two). Immunohistochemistry showed that both HK2 and NCK2 are expressed in the retinal ganglion cell layer. Once multi-testing was taken into account, only HK2 showed significant association with POAG and NTG in Stage Two. Our data also support previous reports of NCK2 association with NTG, and raise questions about what role TMEM182 might play in phenotypic variability. Our data suggest that HK2 may play an important role in NTG in the Japanese population.
|Strong cortical and spinal cord transduction after AAV7 and AAV9 delivery into the cerebrospinal fluid of nonhuman primates. |
Samaranch, L; Salegio, EA; San Sebastian, W; Kells, AP; Bringas, JR; Forsayeth, J; Bankiewicz, KS
Human gene therapy 24 526-32 2013
The present study builds on previous work showing that infusion of adeno-associated virus type 9 (AAV9) into the cisterna magna (CM) of nonhuman primates resulted in widespread transduction throughout cortex and spinal cord. Transduction efficiency was severely limited, however, by the presence of circulating anti-AAV antibodies. Accordingly, we compared AAV9 to a related serotype, AAV7, which has a high capsid homology. CM infusion of either AAV7 or AAV9 directed high level of cell transduction with similar patterns of distribution throughout brain cortex and along the spinal cord. Dorsal root ganglia and corticospinal tracts were also transduced. Both astrocytes and neurons were transduced. Interestingly, little transduction was observed in peripheral organs. Our results indicate that intrathecal delivery of either AAV7 or AAV9 directs a robust and widespread cellular transduction in the central nervous system and other peripheral neural structures.
|Vasculogenesis in experimental stroke after human cerebral endothelial cell transplantation. |
Ishikawa, H; Tajiri, N; Shinozuka, K; Vasconcellos, J; Kaneko, Y; Lee, HJ; Mimura, O; Dezawa, M; Kim, SU; Borlongan, CV
Stroke; a journal of cerebral circulation 44 3473-81 2013
Despite the reported functional recovery in transplanted stroke models and patients, the mechanism of action underlying stem cell therapy remains not well understood. Here, we examined the role of stem cell-mediated vascular repair in stroke.Adult rats were exposed to transient occlusion of the middle cerebral artery and 3 hours later randomly stereotaxically transplantated with 100K, 200K, or 400K human cerebral endothelial cell 6 viable cells or vehicle. Animals underwent neurological examination and motor test up to day 7 after transplantation then euthanized for immunostaining against neuronal, vascular, and specific human antigens. A parallel in vitro study cocultured rat primary neuronal cells with human cerebral endothelial cell 6 under oxygen-glucose deprivation and treated with vascular endothelial growth factor (VEGF) and anti-VEGF.Stroke animals that received vehicle infusion displayed typical occlusion of the middle cerebral artery-induced behavioral impairments that were dose-dependently reduced in transplanted stroke animals at days 3 and 7 after transplantation and accompanied by increased expression of host neuronal and vascular markers adjacent to the transplanted cells. Some transplanted cells showed a microvascular phenotype and juxtaposed to the host vasculature. Infarct volume in transplanted stroke animals was significantly smaller than vehicle-infused stroke animals. Moreover, rat neurons cocultured with human cerebral endothelial cell 6 or treated with VEGF exhibited significantly less oxygen-glucose deprivation-induced cell death that was blocked by anti-VEGF treatment.We found attenuation of behavioral and histological deficits coupled with robust vasculogenesis and neurogenesis in endothelial cell-transplanted stroke animals, suggesting that targeting vascular repair sets in motion a regenerative process in experimental stroke possibly via the VEGF pathway.
|Regional astrogliosis in the mouse hypothalamus in response to obesity. |
Buckman, LB; Thompson, MM; Moreno, HN; Ellacott, KL
The Journal of comparative neurology 521 1322-33 2013
Obesity is associated with chronic low-grade inflammation in peripheral tissues, which contributes to the development of comorbidities such as insulin resistance and cardiovascular disease. While less extensively characterized, obesity also promotes inflammation in the central nervous system (CNS) and the consequences of this inflammation for CNS function are only beginning to be examined. In response to CNS insults such as inflammation, astrocytes undergo a process of hypertrophy and hyperplasia known as reactive astrogliosis. We used immunohistochemistry to examine the differential distribution of the astrocyte marker glial-fibrillary acidic protein (GFAP) in the brains of diet-induced or genetically obese mice compared with their respective lean controls to determine whether different nuclei of the hypothalamus showed comparable astrogliosis in response to obesity. The areas that showed the highest differential GFAP immunoreactivity between lean and obese animals include the medial preoptic, paraventricular, and dorsomedial nuclei. Comparatively, little astrogliosis was seen in the ventromedial nucleus, lateral hypothalamus, or anterior hypothalamic area. In obese animals high levels of GFAP immunoreactivity were often associated with the microvasculature. There were no differences in the differential distribution of GFAP staining between obese animals and their lean controls in the diet-induced compared with the genetic model of obesity. The exact cause(s) of the astrogliosis in obesity is not known. The finding that obesity causes a distinct pattern of elevated GFAP immunoreactivity associated with microvessels suggests that the astrogliosis may be occurring as a response to changes at the blood-brain barrier and/or in the peripheral circulation.
|Coordination of hypothalamic and pituitary T3 production regulates TSH expression. |
Fonseca, TL; Correa-Medina, M; Campos, MP; Wittmann, G; Werneck-de-Castro, JP; Arrojo e Drigo, R; Mora-Garzon, M; Ueta, CB; Caicedo, A; Fekete, C; Gereben, B; Lechan, RM; Bianco, AC
The Journal of clinical investigation 123 1492-500 2013
Type II deiodinase (D2) activates thyroid hormone by converting thyroxine (T4) to 3,5,3'-triiodothyronine (T3). This allows plasma T4 to signal a negative feedback loop that inhibits production of thyrotropin-releasing hormone (TRH) in the mediobasal hypothalamus (MBH) and thyroid-stimulating hormone (TSH) in the pituitary. To determine the relative contributions of these D2 pathways in the feedback loop, we developed 2 mouse strains with pituitary- and astrocyte-specific D2 knockdown (pit-D2 KO and astro-D2 KO mice, respectively). The pit-D2 KO mice had normal serum T3 and were systemically euthyroid, but exhibited an approximately 3-fold elevation in serum TSH levels and a 40% reduction in biological activity. This was the result of elevated serum T4 that increased D2-mediated T3 production in the MBH, thus decreasing Trh mRNA. That tanycytes, not astrocytes, are the cells within the MBH that mediate T4-to-T3 conversion was defined by studies using the astro-D2 KO mice. Despite near-complete loss of brain D2, tanycyte D2 was preserved in astro-D2 KO mice at levels that were sufficient to maintain both the T4-dependent negative feedback loop and thyroid economy. Taken together, these data demonstrated that the hypothalamic-thyroid axis is wired to maintain normal plasma T3 levels, which is achieved through coordination of T4-to-T3 conversion between thyrotrophs and tanycytes.
|Mitral and tufted cells are potential cellular targets of nitration in the olfactory bulb of aged mice. |
Yang, MJ; Sim, S; Jeon, JH; Jeong, E; Kim, HC; Park, YJ; Kim, IB
PloS one 8 e59673 2013
Olfactory sensory function declines with age; though, the underlying molecular changes that occur in the olfactory bulb (OB) are relatively unknown. An important cellular signaling molecule involved in the processing, modulation, and formation of olfactory memories is nitric oxide (NO). However, excess NO can result in the production of peroxynitrite to cause oxidative and nitrosative stress. In this study, we assessed whether changes in the expression of 3-nitrotyrosine (3-NT), a neurochemical marker of peroxynitrite and thus oxidative damage, exists in the OB of young, adult, middle-aged, and aged mice. Our results demonstrate that OB 3-NT levels increase with age in normal C57BL/6 mice. Moreover, in aged mice, 3-NT immunoreactivity was found in some blood vessels and microglia throughout the OB. Notably, large and strongly immunoreactive puncta were found in mitral and tufted cells, and these were identified as lipofuscin granules. Additionally, we found many small-labeled puncta within the glomeruli of the glomerular layer and in the external plexiform layer, and these were localized to mitochondria and discrete segments of mitral and tufted dendritic plasma membranes. These results suggest that mitral and tufted cells are potential cellular targets of nitration, along with microglia and blood vessels, in the OB during aging.
|Assessment of adult neurogenesis in mice. |
Pan, YW; Wang, W; Xia, Z
Current protocols in toxicology / editorial board, Mahin D. Maines (editor-in-chief) ... [et al.] Chapter 12 Unit12.20 2013
Adult neurogenesis is a lifelong developmental process that occurs in two discrete regions in the adult mammalian brain: the subgranular zone of the dentate gyrus (DG) and the subventricular zone (SVZ) along the lateral ventricles. Despite immense interest in the therapeutic potential of adult neural stem cells (aNSCs) residing along these two neurogenic regions, molecular and cellular mechanisms regulating this process are not fully defined. Defining the regulatory mechanisms responsible for the genesis of new neurons in the adult brain is integral to understanding the basic biology of aNSCs. The techniques described here provide a basic blueprint to isolate, culture, and perform experiments using aNSCs in vitro as well as providing methods to perform immunohistochemistry on brain sections. Curr. Protoc. Toxicol. 56:12.20.1-12.20.16. © 2013 by John Wiley & Sons, Inc.
|Primary blast injury-induced lesions in the retina of adult rats. |
Zou, YY; Kan, EM; Lu, J; Ng, KC; Tan, MH; Yao, L; Ling, EA
Journal of neuroinflammation 10 79 2013
The effect of primary blast exposure on the brain is widely reported but its effects on the eye remains unclear. Here, we aim to examine the effects of primary blast exposure on the retina.Adult male Sprague-Dawley rats were exposed to primary blast high and low injury and sacrificed at 24 h, 72 h, and 2 weeks post injury. The retina was subjected to western analysis for vascular endothelial growth factor (VEGF), aquaporin-4 (AQP4), glutamine synthethase (GS), inducible nitric oxide synthase (NOS), endothelial NOS, neuronal NOS and nestin expression; ELISA analysis for cytokines and chemokines; and immunofluorescence for glial fibrillary acidic protein (GFAP)/VEGF, GFAP/AQP4, GFAP/nestin, GS/AQP4, lectin/iNOS, and TUNEL.The retina showed a blast severity-dependent increase in VEGF, iNOS, eNOS, nNOS, and nestin expression with corresponding increases in inflammatory cytokines and chemokines. There was also increased AQP4 expression and retinal thickness after primary blast exposure that was severity-dependent. Finally, a significant increase in TUNEL+ and Caspase-3+ cells was observed. These changes were observed at 24 h post-injury and sustained up to 2 weeks post injury.Primary blast resulted in severity-dependent pathological changes in the retina, manifested by the increased expression of a variety of proteins involved in inflammation, edema, and apoptosis. These changes were observed immediately after blast exposure and sustained up to 2 weeks suggesting acute and chronic injury mechanisms. These changes were most obvious in the astrocytes and Müller cells and suggest important roles for these cells in retina pathophysiology after blast.
|Expression of angiotensinogen and receptors for angiotensin and prorenin in the monkey and human substantia nigra: an intracellular renin-angiotensin system in the nigra. |
Garrido-Gil, P; Valenzuela, R; Villar-Cheda, B; Lanciego, JL; Labandeira-Garcia, JL
Brain structure & function 218 373-88 2013
We have previously obtained in rodents a considerable amount of data suggesting a major role for the brain renin-angiotensin system (RAS) in dopaminergic neuron degeneration and potentially in Parkinson's disease. However, the presence of a local RAS has not been demonstrated in the monkey or the human substantia nigra compacta (SNc). The present study demonstrates the presence of major RAS components in dopaminergic neurons, astrocytes and microglia in both the monkey and the human SNc. Angiotensin type 1 and 2 and renin-prorenin receptors were located at the surface of dopaminergic neurons and glial cells, as expected for a tissular RAS. However, angiotensinogen and receptors for angiotensin and renin-prorenin were also observed at the cytoplasm and nuclear level, which suggests the presence of an intracrine or intracellular RAS in monkey and human SNc. Although astrocytes and microglia were labeled for angiotensin and prorenin receptors in the normal SNc, most glial cells appeared less immunoreactive than the dopaminergic neurons. However, our previous studies in rodent models of PD and studies in other animal models of brain diseases suggest that the RAS activity is significantly upregulated in glial cells in pathological conditions. The present results together with our previous findings in rodents suggest a major role for the nigral RAS in the normal functioning of the dopaminergic neurons, and in the progression of the dopaminergic degeneration.
|Androgen receptors mediate masculinization of astrocytes in the rat posterodorsal medial amygdala during puberty. |
Johnson, RT; Breedlove, SM; Jordan, CL
The Journal of comparative neurology 521 2298-309 2013
Astrocytes in the posterodorsal portion of the medial amygdala (MePD) are sexually dimorphic in adult rats: males have more astrocytes in the right MePD and more elaborate processes in the left MePD than do females. Functional androgen receptors (ARs) are required for masculinization of MePD astrocytes, as these measures are demasculinized in adult males carrying the testicular feminization mutation (Tfm) of the AR gene, which renders AR dysfunctional. We now report that the number of astrocytes is already sexually dimorphic in the right MePD of juvenile 25-day-old (P25) rats. Because Tfm males have as many astrocytes as wild-type males at this age, this prepubertal sexual dimorphism is independent of ARs. After P25, astrocyte number increases in the MePD of all groups, but activation of ARs augments this increase in the right MePD, where more astrocytes are added in males than in Tfm males. Consequently, by adulthood, females and Tfm males have equivalent numbers of astrocytes in the right MePD. Sexual dimorphism in astrocyte arbor complexity in the left MePD arises after P25, and is entirely AR-dependent. Thus, masculinization of MePD astrocytes is a result of both AR-independent processes before the juvenile period and AR-dependent processes afterward.
|Wingless-type mammary tumor virus integration site family, member 5A (Wnt5a) regulates human immunodeficiency virus type 1 (HIV-1) envelope glycoprotein 120 (gp120)-induced expression of pro-inflammatory cytokines via the Ca2+/calmodulin-dependent protein kinase II (CaMKII) and c-Jun N-terminal kinase (JNK) signaling pathways. |
Li, B; Shi, Y; Shu, J; Gao, J; Wu, P; Tang, SJ
The Journal of biological chemistry 288 13610-9 2013
HIV-1 infection causes chronic neuroinflammation in the central nervous system (CNS).The spinal cytokine up-regulation induced by HIV-1 gp120 protein depends on Wnt5a/CaMKII and/or Wnt5a/JNK pathways.gp120 stimulates cytokine expression in the spinal cord dorsal horn by activating Wnt5a signaling.The finding reveals Wnt signaling-mediated novel mechanisms by which HIV-1 may cause neuroinflammation. Chronic expression of pro-inflammatory cytokines critically contributes to the pathogenesis of HIV-associated neurological disorders (HANDs), but the host mechanism that regulates the HIV-induced cytokine expression in the CNS remains elusive. Here, we present evidence for a crucial role of Wnt5a signaling in the expression of pro-inflammatory cytokines in the spinal cord induced by a major HIV-envelope protein, gp120. Wnt5a is mainly expressed in spinal neurons, and rapidly up-regulated by intrathecal injection (i.t.) of gp120. We show that inhibition of Wnt5a by specific antagonists blocks gp120-induced up-regulation of IL-1β, IL-6, and TNF-α in the spinal cord. Conversely, injection (i.t.) of purified recombinant Wnt5a stimulates the expression of these cytokines. To elucidate the role of the Wnt5a-regulated signaling pathways in gp120-induced cytokine expression, we have focused on CaMKII and JNKs, the well characterized down-stream targets of Wnt5a signaling. We find that Wnt5a is required for gp120 to activate CaMKII and JNK signaling. Furthermore, we demonstrate that the Wnt5a/CaMKII pathway is critical for the gp120-induced expression of IL-1β, whereas the Wnt5a/JNK pathway is for TNF-α expression. Meanwhile, the expression of IL-6 is co-regulated by both pathways. These results collectively suggest that Wnt5a signaling cascades play a crucial role in the regulation of gp120-induced expression of pro-inflammatory cytokines in the CNS.
|Brain tissue interaction with three-dimensional, honeycomb polycaprolactone-based scaffolds designed for cranial reconstruction following traumatic brain injury. |
Choy, DK; Nga, VD; Lim, J; Lu, J; Chou, N; Yeo, TT; Teoh, SH
Tissue engineering. Part A 19 2382-9 2013
Following traumatic brain injury (TBI), resultant voids are unable to support injections of suspension treatments, leading to ineffective healing. Moreover, without a structure to support the large defect, the defect site suffers from mechanical instability, which may impair the healing process. Therefore, having a delivery vehicle that can temporarily fill and provide mechanical support to the defect site may alleviate the healing process. In this work, we reported for the first time, the inflammatory response of brain tissue with polycaprolactone (PCL) and PCL-tricalcium phosphate (TCP) scaffolds designed and fabricated for cranial reconstruction. After cranial defects were created in Sprague-Dawley rats, PCL and PCL-TCP scaffolds were implanted for a period of 1 week and 1 month. Following histology and immunofluorescence staining with the ionized calcium binding adaptor molecule-1 (IBA-1), glial fibrillary acidic protein (GFAP), nestin, and neuronal nuclei (NeuN), results indicated that IBA-1-positive activated microglia were observed across all groups, and declined significantly by 1 month (pless than 0.05). Interestingly, IBA-1-positive microglia were significantly fewer in the PCL-TCP group (pless than 0.05), suggesting a relatively milder inflammatory response. A decrease in the number of GFAP-positive cells among all groups over time (greater than 29%) was also observed. Initially, astrocyte hypertrophy was observed proximal to the TBI site (55% in PCL and PCL-TCP groups, 75% in control groups), but it subsided by 1 month. Proximal to the TBI site, nestin immunoreactivity was intense during week 1, and which reduced by 1 month across all groups. NeuN-positive neurons were shrunken proximal to the TBI site (less than 0.9 mm), 32% smaller in the PCL-TCP group and 27% smaller in the PCL group. Based on above data indicating the comparatively milder, initial inflammatory response of brain tissue to PCL-TCP scaffolds, it is suggested that PCL-TCP scaffolds have notable clinical advantages as compared to PCL scaffolds.
|Neuropathic pain in rats with a partial sciatic nerve ligation is alleviated by intravenous injection of monoclonal antibody to high mobility group box-1. |
Nakamura, Y; Morioka, N; Abe, H; Zhang, FF; Hisaoka-Nakashima, K; Liu, K; Nishibori, M; Nakata, Y
PloS one 8 e73640 2013
High mobility group box-1 (HMGB1) is associated with the pathogenesis of inflammatory diseases. A previous study reported that intravenous injection of anti-HMGB1 monoclonal antibody significantly attenuated brain edema in a rat model of stroke, possibly by attenuating glial activation. Peripheral nerve injury leads to increased activity of glia in the spinal cord dorsal horn. Thus, it is possible that the anti-HMGB1 antibody could also be efficacious in attenuating peripheral nerve injury-induced pain. Following partial sciatic nerve ligation (PSNL), rats were treated with either anti-HMGB1 or control IgG. Intravenous treatment with anti-HMGB1 monoclonal antibody (2 mg/kg) significantly ameliorated PSNL-induced hind paw tactile hypersensitivity at 7, 14 and 21 days, but not 3 days, after ligation, whereas control IgG had no effect on tactile hypersensitivity. The expression of HMGB1 protein in the spinal dorsal horn was significantly increased 7, 14 and 21 days after PSNL; the efficacy of the anti-HMGB1 antibody is likely related to the presence of HMGB1 protein. Also, the injury-induced translocation of HMGB1 from the nucleus to the cytosol occurred mainly in dorsal horn neurons and not in astrocytes and microglia, indicating a neuronal source of HMGB1. Markers of astrocyte (glial fibrillary acidic protein (GFAP)), microglia (ionized calcium binding adaptor molecule 1 (Iba1)) and spinal neuron (cFos) activity were greatly increased in the ipsilateral dorsal horn side compared to the sham-operated side 21 days after PSNL. Anti-HMGB1 monoclonal antibody treatment significantly decreased the injury-induced expression of cFos and Iba1, but not GFAP. The results demonstrate that nerve injury evokes the synthesis and release of HMGB1 from spinal neurons, facilitating the activity of both microglia and neurons, which in turn leads to symptoms of neuropathic pain. Thus, the targeting of HMGB1 could be a useful therapeutic strategy in the treatment of chronic pain.
|Patient-specific iPSC-derived photoreceptor precursor cells as a means to investigate retinitis pigmentosa. |
Tucker, BA; Mullins, RF; Streb, LM; Anfinson, K; Eyestone, ME; Kaalberg, E; Riker, MJ; Drack, AV; Braun, TA; Stone, EM
eLife 2 e00824 2013
Next-generation and Sanger sequencing were combined to identify disease-causing USH2A mutations in an adult patient with autosomal recessive RP. Induced pluripotent stem cells (iPSCs), generated from the patient's keratinocytes, were differentiated into multi-layer eyecup-like structures with features of human retinal precursor cells. The inner layer of the eyecups contained photoreceptor precursor cells that expressed photoreceptor markers and exhibited axonemes and basal bodies characteristic of outer segments. Analysis of the USH2A transcripts of these cells revealed that one of the patient's mutations causes exonification of intron 40, a translation frameshift and a premature stop codon. Western blotting revealed upregulation of GRP78 and GRP94, suggesting that the patient's other USH2A variant (Arg4192His) causes disease through protein misfolding and ER stress. Transplantation into 4-day-old immunodeficient Crb1 (-/-) mice resulted in the formation of morphologically and immunohistochemically recognizable photoreceptor cells, suggesting that the mutations in this patient act via post-developmental photoreceptor degeneration. DOI:http://dx.doi.org/10.7554/eLife.00824.001.
|Altered synaptic transmission in the hippocampus of transgenic mice with enhanced central nervous systems expression of interleukin-6. |
Nelson, TE; Olde Engberink, A; Hernandez, R; Puro, A; Huitron-Resendiz, S; Hao, C; De Graan, PN; Gruol, DL
Brain, behavior, and immunity 26 959-71 2012
Elevated levels of the inflammatory cytokine interleukin-6 (IL-6) occur in a number of CNS disorders. However, little is known about how this condition affects CNS neuronal function. Transgenic mice that express elevated levels of IL-6 in the CNS show cognitive changes, increased propensity for hippocampal seizures and reduced number of inhibitory interneurons, suggesting that elevated levels of IL-6 can cause neuroadaptive changes that alter hippocampal function. To identify these neuroadaptive changes, we measured the levels of protein expression using Western blot analysis and synaptic function using field potential recordings in hippocampus from IL-6 transgenic mice (IL-6 tg) and their non-transgenic (non-tg) littermates. Western blot analysis showed enhanced levels of the GFAP and STAT3 in the IL-6 tg hippocampus compared with the non-tg hippocampus, but no difference for several other proteins. Field potential recordings of synaptic transmission at the Schaffer collateral to CA1 synapse showed enhanced dendritic excitatory postsynaptic potentials and somatic population spikes in the CA1 region of hippocampal slices from IL-6 tg mice compared with slices from non-tg littermate controls. No differences were observed for several forms of short-term and long-term synaptic plasticity between hippocampal slices from IL-6 tg and non-tg mice. These results demonstrate that elevated levels of IL-6 can alter mechanisms involved in the excitability of hippocampal neurons and synapses, an effect consistent with recent evidence indicating that elevated production of IL-6 plays an important role in conditions associated with seizure activity and in other impairments observed in CNS disorders with a neuroinflammatory component.
|Chondroitinase and growth factors enhance activation and oligodendrocyte differentiation of endogenous neural precursor cells after spinal cord injury. |
Karimi-Abdolrezaee, S; Schut, D; Wang, J; Fehlings, MG
PloS one 7 e37589 2012
The adult spinal cord harbours a population of multipotent neural precursor cells (NPCs) with the ability to replace oligodendrocytes. However, despite this capacity, proliferation and endogenous remyelination is severely limited after spinal cord injury (SCI). In the post-traumatic microenvironment following SCI, endogenous spinal NPCs mainly differentiate into astrocytes which could contribute to astrogliosis that exacerbate the outcomes of SCI. These findings emphasize a key role for the post-SCI niche in modulating the behaviour of spinal NPCs after SCI. We recently reported that chondroitin sulphate proteoglycans (CSPGs) in the glial scar restrict the outcomes of NPC transplantation in SCI by reducing the survival, migration and integration of engrafted NPCs within the injured spinal cord. These inhibitory effects were attenuated by administration of chondroitinase (ChABC) prior to NPC transplantation. Here, in a rat model of compressive SCI, we show that perturbing CSPGs by ChABC in combination with sustained infusion of growth factors (EGF, bFGF and PDGF-AA) optimize the activation and oligodendroglial differentiation of spinal NPCs after injury. Four days following SCI, we intrathecally delivered ChABC and/or GFs for seven days. We performed BrdU incorporation to label proliferating cells during the treatment period after SCI. This strategy increased the proliferation of spinal NPCs, reduced the generation of new astrocytes and promoted their differentiation along an oligodendroglial lineage, a prerequisite for remyelination. Furthermore, ChABC and GF treatments enhanced the response of non-neural cells by increasing the generation of new vascular endothelial cells and decreasing the number of proliferating macrophages/microglia after SCI. In conclusions, our data strongly suggest that optimization of the behaviour of endogenous spinal NPCs after SCI is critical not only to promote endogenous oligodendrocyte replacement, but also to reverse the otherwise detrimental effects of their activation into astrocytes which could negatively influence the repair process after SCI.
|Comparative distribution of protein components of the A20 ubiquitin-editing complex in normal human brain. |
Pranski, EL; Van Sanford, CD; Dalal, NV; Orr, AL; Karmali, D; Cooper, DS; Costa, N; Heilman, CJ; Gearing, M; Lah, JJ; Levey, AI; Betarbet, RS
Neuroscience letters 520 104-9 2012
Activation of innate and adaptive immune responses is tightly regulated, as insufficient activation could result in defective clearance of pathogens, while excessive activation might lead to lethal systemic inflammation or autoimmunity. A20 functions as a negative regulator of innate and adaptive immunity by inhibiting NF-κB activation. A20 mediates its inhibitory function in a complex with other proteins including RNF11 and Itch, both E3 ubiquitin ligases and TAX1BP1, an adaptor protein. Since NF-κB has been strongly implicated in various neuronal functions, we predict that its inhibitor, the A20 complex, is also present in the nervous system. In efforts to better understand the role of A20 complex and NF-κB signaling pathway, we determined regional distribution of A20 mRNA as well as protein expression levels and distribution of RNF11, TAX1BP1 and Itch, in different brain regions. The distribution of TRAF6 was also investigated since TRAF6, also an E3 ligase, has an important role in NF-κB signaling pathway. Our investigations, for the first time, describe and demonstrate that the essential components of the A20 ubiquitin-editing complex are present and mainly expressed in neurons. The A20 complex components are also differentially expressed throughout the human brain. This study provides useful information about region specific expression of the A20 complex components that will be invaluable while determining the role of NF-κB signaling pathway in neuronal development and degeneration.
|Thrombin activity associated with neuronal damage during acute focal ischemia. |
Chen, B; Friedman, B; Whitney, MA; Winkle, JA; Lei, IF; Olson, ES; Cheng, Q; Pereira, B; Zhao, L; Tsien, RY; Lyden, PD
The Journal of neuroscience : the official journal of the Society for Neuroscience 32 7622-31 2012
Mechanisms of ischemic neuronal and vascular injury remain obscure. Here we test the hypothesis that thrombin, a blood-borne coagulation factor, contributes to neurovascular injury during acute focal ischemia. Stroke was induced in adult Sprague Dawley rats by occluding the middle cerebral artery. Intra-arterial thrombin infusion during ischemia significantly increased vascular disruption and cellular injury. Intravenous infusion of argatroban, a direct thrombin inhibitor, alleviated neurovascular injury. Immunostaining showed thrombin on neurons in the ischemic core. Using an activatable cell-penetrating peptide engineered to detect thrombin activity, we discovered that thrombin proteolytic activity was specifically associated with neuronal damage during ischemia. Protease activated receptor-1, the presumptive thrombin receptor, appeared to mediate ischemic neurovascular injury. Furthermore, rats receiving thrombin during ischemia showed cognitive deficit, whereas rats receiving argatroban retained intact learning and memory. These results suggest a potential role for thrombin contributing to neurovascular injury and several potential avenues for neuroprotection.
|Morphine decreases enteric neuron excitability via inhibition of sodium channels. |
Smith, TH; Grider, JR; Dewey, WL; Akbarali, HI
PloS one 7 e45251 2012
Gastrointestinal peristalsis is significantly dependent on the enteric nervous system. Constipation due to reduced peristalsis is a major side-effect of morphine, which limits the chronic usefulness of this excellent pain reliever in man. The ionic basis for the inhibition of enteric neuron excitability by morphine is not well characterized as previous studies have mainly utilized microelectrode recordings from whole mount myenteric plexus preparations in guinea pigs. Here we have developed a Swiss-Webster mouse myenteric neuron culture and examined their electrophysiological properties by patch-clamp techniques and determined the mechanism for morphine-induced decrease in neuronal excitability. Isolated neurons in culture were confirmed by immunostaining with pan-neuronal marker, β-III tubulin and two populations were identified by calbindin and calretinin staining. Distinct neuronal populations were further identified based on the presence and absence of an afterhyperpolarization (AHP). Cells with AHP expressed greater density of sodium currents. Morphine (3 µM) significantly reduced the amplitude of the action potential, increased the threshold for spike generation but did not alter the resting membrane potential. The decrease in excitability resulted from inhibition of sodium currents. In the presence of morphine, the steady-state voltage dependence of Na channels was shifted to the left with almost 50% of channels unavailable for activation from hyperpolarized potentials. During prolonged exposure to morphine (two hours), action potentials recovered, indicative of the development of tolerance in single enteric neurons. These results demonstrate the feasibility of isolating mouse myenteric neurons and establish sodium channel inhibition as a mechanism for morphine-induced decrease in neuronal excitability.
|Glial activation in a pilocarpine rat model for epileptogenesis: a morphometric and quantitative analysis. |
Felipe S Estrada,Vito S Hernández,Estela López-Hernández,Aleph A Corona-Morales,Hugo Solís,Alfonso Escobar,Limei Zhang
Neuroscience letters 514 2012
In this work we examined the correlation between long-term glial resilience and slow epileptogenesis using the pilocarpine-insult rat model. We assessed, quantitatively and morphometrically, glial fibrillary acidic protein (GFAP) expression and cell densities in hippocampus in a dose-response manner 2, 4 and 8 weeks after the pilocarpine insult. GFAP changes were correlated with observations on microglial activation. We used a commonly applied epileptogenic pilocarpine dose (380mg/kg) and its fractions of 1/10, 1/4 and 1/2. GFAP expression evaluated at 2 weeks revealed dose-dependent cytoskeletal hypertrophy and loss of GFAP+ cell densities in hippocampus. At 4-week timepoint, recoveries of the above mentioned parameters were observed in all groups, except for the full dose group in which the astrocytic hypertrophy reached the highest level, while its density dropped to the lowest level. Strong and localized microgliosis revealed by CD11b immunoreactivity was observed in hilus in the full dose group at 2- and 4-, persisting at 8-week timepoints. Through changing pattern analysis, we conclude that the loss of astroglial resilience is likely to be a determining factor for spontaneous recurrent seizure onset.
|Differential expression and HIV-1 regulation of μ-opioid receptor splice variants across human central nervous system cell types. |
Dever, SM; Xu, R; Fitting, S; Knapp, PE; Hauser, KF
Journal of neurovirology 18 181-90 2012
The μ-opioid receptor (MOR) is known to undergo extensive alternative splicing as numerous splice variants of MOR have been identified. However, the functional significance of MOR variants, as well as how splice variants other than MOR-1 might differentially regulate human immunodeficiency virus type-1 (HIV-1) pathogenesis in the central nervous system (CNS), or elsewhere, has largely been ignored. Our findings suggest that there are specific differences in the MOR variant expression profile among CNS cell types, and that the expression levels of these variants are differentially regulated by HIV-1. While MOR-1A mRNA was detected in astroglia, microglia, and neurons, MOR-1 and MOR-1X were only found in astroglia. Expression of the various forms of MOR along with the chimeric G protein qi5 in HEK-293T cells resulted in differences in calcium/NFAT signaling with morphine treatment, suggesting that MOR variant expression might underlie functional differences in MOR-effector coupling and intracellular signaling across different cell types. Furthermore, the data suggest that the expression of MOR-1 and other MOR variants may also be differentially regulated in the brains of HIV-infected subjects with varying levels of neurocognitive impairment. Overall, the results reveal an unexpected finding that MOR-1 may not be the predominant form of MOR expressed by some CNS cell types and that other splice variants of MOR-1, with possible differing functions, may contribute to the diversity of MOR-related processes in the CNS.
|Minocycline-preconditioned neural stem cells enhance neuroprotection after ischemic stroke in rats. |
Sakata, H; Niizuma, K; Yoshioka, H; Kim, GS; Jung, JE; Katsu, M; Narasimhan, P; Maier, CM; Nishiyama, Y; Chan, PH
The Journal of neuroscience : the official journal of the Society for Neuroscience 32 3462-73 2012
Transplantation of neural stem cells (NSCs) offers a novel therapeutic strategy for stroke; however, massive grafted cell death following transplantation, possibly due to a hostile host brain environment, lessens the effectiveness of this approach. Here, we have investigated whether reprogramming NSCs with minocycline, a broadly used antibiotic also known to possess cytoprotective properties, enhances survival of grafted cells and promotes neuroprotection in ischemic stroke. NSCs harvested from the subventricular zone of fetal rats were preconditioned with minocycline in vitro and transplanted into rat brains 6 h after transient middle cerebral artery occlusion. Histological and behavioral tests were examined from days 0-28 after stroke. For in vitro experiments, NSCs were subjected to oxygen-glucose deprivation and reoxygenation. Cell viability and antioxidant gene expression were analyzed. Minocycline preconditioning protected the grafted NSCs from ischemic reperfusion injury via upregulation of Nrf2 and Nrf2-regulated antioxidant genes. Additionally, preconditioning with minocycline induced the NSCs to release paracrine factors, including brain-derived neurotrophic factor, nerve growth factor, glial cell-derived neurotrophic factor, and vascular endothelial growth factor. Moreover, transplantation of the minocycline-preconditioned NSCs significantly attenuated infarct size and improved neurological performance, compared with non-preconditioned NSCs. Minocycline-induced neuroprotection was abolished by transfecting the NSCs with Nrf2-small interfering RNA before transplantation. Thus, preconditioning with minocycline, which reprograms NSCs to tolerate oxidative stress after ischemic reperfusion injury and express higher levels of paracrine factors through Nrf2 up-regulation, is a simple and safe approach to enhance the effectiveness of transplantation therapy in ischemic stroke.
|Ceramide Levels Regulated by Carnitine Palmitoyltransferase 1C Control Dendritic Spine Maturation and Cognition. |
Patricia Carrasco,Ignasi SahÃºn,Jerome McDonald,Sara RamÃrez,Jordi Jacas,Esther GratacÃ³s,Adriana Y Sierra,Dolors Serra,Laura Herrero,Amparo Acker-Palmer,Fausto G Hegardt,Mara Dierssen,NÃºria Casals
The Journal of biological chemistry 287 2012
The brain-specific isoform carnitine palmitoyltransferase 1C (CPT1C) has been implicated in the hypothalamic regulation of food intake and energy homeostasis. Nevertheless, its molecular function is not completely understood, and its role in other brain areas is unknown. We demonstrate that CPT1C is expressed in pyramidal neurons of the hippocampus and is located in the endoplasmic reticulum throughout the neuron, even inside dendritic spines. We used molecular, cellular, and behavioral approaches to determine CPT1C function. First, we analyzed the implication of CPT1C in ceramide metabolism. CPT1C overexpression in primary hippocampal cultured neurons increased ceramide levels, whereas in CPT1C-deficient neurons, ceramide levels were diminished. Correspondingly, CPT1C knock-out (KO) mice showed reduced ceramide levels in the hippocampus. At the cellular level, CPT1C deficiency altered dendritic spine morphology by increasing immature filopodia and reducing mature mushroom and stubby spines. Total protrusion density and spine head area in mature spines were unaffected. Treatment of cultured neurons with exogenous ceramide reverted the KO phenotype, as did ectopic overexpression of CPT1C, indicating that CPT1C regulation of spine maturation is mediated by ceramide. To study the repercussions of the KO phenotype on cognition, we performed the hippocampus-dependent Morris water maze test on mice. Results show that CPT1C deficiency strongly impairs spatial learning. All of these results demonstrate that CPT1C regulates the levels of ceramide in the endoplasmic reticulum of hippocampal neurons, and this is a relevant mechanism for the correct maturation of dendritic spines and for proper spatial learning.
|Maternal vitamin C deficiency during pregnancy persistently impairs hippocampal neurogenesis in offspring of guinea pigs. |
Tveden-Nyborg, P; Vogt, L; Schjoldager, JG; Jeannet, N; Hasselholt, S; Paidi, MD; Christen, S; Lykkesfeldt, J
PloS one 7 e48488 2012
While having the highest vitamin C (VitC) concentrations in the body, specific functions of VitC in the brain have only recently been acknowledged. We have shown that postnatal VitC deficiency in guinea pigs causes impairment of hippocampal memory function and leads to 30% less neurons. This study investigates how prenatal VitC deficiency affects postnatal hippocampal development and if any such effect can be reversed by postnatal VitC repletion. Eighty pregnant Dunkin Hartley guinea pig dams were randomized into weight stratified groups receiving High (900 mg) or Low (100 mg) VitC per kg diet. Newborn pups (n = 157) were randomized into a total of four postnatal feeding regimens: High/High (Control); High/Low (Depleted), Low/Low (Deficient); and Low/High (Repleted). Proliferation and migration of newborn cells in the dentate gyrus was assessed by BrdU labeling and hippocampal volumes were determined by stereology. Prenatal VitC deficiency resulted in a significant reduction in postnatal hippocampal volume (Pless than 0.001) which was not reversed by postnatal repletion. There was no difference in postnatal cellular proliferation and survival rates in the hippocampus between dietary groups, however, migration of newborn cells into the granular layer of the hippocampus dentate gyrus was significantly reduced in prenatally deficient animals (Pless than 0.01). We conclude that a prenatal VitC deficiency in guinea pigs leads to persistent impairment of postnatal hippocampal development which is not alleviated by postnatal repletion. Our findings place attention on a yet unrecognized consequence of marginal VitC deficiency during pregnancy.
|Regulation of Wnt signaling by nociceptive input in animal models. |
Shi, Y; Yuan, S; Li, B; Wang, J; Carlton, SM; Chung, K; Chung, JM; Tang, SJ
Molecular pain 8 47 2012
Central sensitization-associated synaptic plasticity in the spinal cord dorsal horn (SCDH) critically contributes to the development of chronic pain, but understanding of the underlying molecular pathways is still incomplete. Emerging evidence suggests that Wnt signaling plays a crucial role in regulation of synaptic plasticity. Little is known about the potential function of the Wnt signaling cascades in chronic pain development.Fluorescent immunostaining results indicate that β-catenin, an essential protein in the canonical Wnt signaling pathway, is expressed in the superficial layers of the mouse SCDH with enrichment at synapses in lamina II. In addition, Wnt3a, a prototypic Wnt ligand that activates the canonical pathway, is also enriched in the superficial layers. Immunoblotting analysis indicates that both Wnt3a a β-catenin are up-regulated in the SCDH of various mouse pain models created by hind-paw injection of capsaicin, intrathecal (i.t.) injection of HIV-gp120 protein or spinal nerve ligation (SNL). Furthermore, Wnt5a, a prototypic Wnt ligand for non-canonical pathways, and its receptor Ror2 are also up-regulated in the SCDH of these models.Our results suggest that Wnt signaling pathways are regulated by nociceptive input. The activation of Wnt signaling may regulate the expression of spinal central sensitization during the development of acute and chronic pain.
|Venezuelan equine encephalitis virus glycoprotein pseudotyping confers neurotropism to lentiviral vectors. |
Trabalza, A, et al.
Gene Ther., (2012) 2012
We have produced high-titre HIV-1 green fluorescent protein-expressing lentiviral (LV) vectors pseudotyped with strain 3908 Venezuelan equine encephalitis virus glycoprotein (VEEV-G) and used them to study transduction of: (1) rat embryonic motor neuron (MN) and striatal neuron primary cultures, (2) differentiated MN cell line NSC-34 and (3) adult rat striatum. In primary neuronal cultures, transduction with VEEV-G-pseudotyped LV was more efficient and more neuronal than with vesicular stomatitis virus glycoprotein (VSV-G)-pseudotyped LV. In NSC-34 cells clear retrograde transport of VEEV-G vector particles was observed. In the striatum at the injection site, transduction with the VEEV-G vectors driven by cytomegalovirus or phosphoglycerate kinase promoters exhibited a distinct neuronal tropism with no microglial and only a minor astroglial component, superior to that obtained with VSV-G-pseudotyped LV, irrespective of the promoter used. Neuronal transduction efficiency increased over time. Distal to the injection site transduction of mitral cells in the olfactory bulb, thalamic neurons and dopaminergic neurons in the substantia nigra pars compacta was detected. This, together with observations of retrograde axonal trafficking in vitro indicates that these vectors also possess low level of retrograde neuronal transduction capability in vivo. In this study, we demonstrate both strong neurotropism as well as sustainability of expression and minimal host immune response in vivo, making the VEEV-G-pseudotyped LV vectors potentially useful for gene therapy of neurodegenerative diseases.Gene Therapy advance online publication, 22 November 2012; doi:10.1038/gt.2012.85.
|Expression of nuclear factor one A and -B in the olfactory bulb. |
Céline Plachez,Kathleen Cato,Robert C McLeay,Yee Hsieh Evelyn Heng,Timothy L Bailey,Richard M Gronostasjki,Linda J Richards,Adam C Puche,Michael Piper
The Journal of comparative neurology 520 2012
The nuclear factor one (NFI) family of transcription factors consists of four members in vertebrates, NFIA, NFIB, NFIC, and NFIX, which share a highly conserved N-terminal DNA-binding domain. NFI genes are widely expressed in the developing mouse brain, and mouse mutants lacking NFIA, NFIB, or NFIX exhibit developmental deficits in several areas, including the cortex, hippocampus, pons, and cerebellum. Here we analyzed the expression of NFIA and NFIB in the developing and adult olfactory bulb (OB), rostral migratory stream (RMS), and subventricular zone (SVZ). We found that NFIA and NFIB are expressed within these regions during embryonic and postnatal development and in the adult. Immunohistochemical analysis using cell-type-specific markers revealed that migrating neuroblasts in the adult brain express NFI transcription factors, as do astrocytes within the RMS and progenitor cells within the SVZ. Moreover, astrocytes within the OB express NFIA, whereas mitral cells within the OB express NFIB. Taken together these data show that NFIA and NFIB are expressed in both the developing and the adult OB and in the RMS and SVZ, indicative of a regulatory role for these transcription factors in the development of this facet of the olfactory system.
|Proteasome Inhibition Alleviates SNARE-Dependent Neurodegeneration. |
Sharma, Manu, et al.
Sci Transl Med, 4: 147ra113 (2012) 2012
Activation of the proteasomal degradation of misfolded proteins has been proposed as a therapeutic strategy for treating neurodegenerative diseases, but it is unclear whether proteasome dysfunction contributes to neurodegeneration. We tested the role of proteasome activity in neurodegeneration developed by mice lacking cysteine string protein-α (CSPα). Unexpectedly, we found that proteasome inhibitors alleviated neurodegeneration in CSPα-deficient mice, reversing impairment of SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor)-complex assembly and extending life span. We tested whether dysfunctional SNARE-complex assembly could contribute to neurodegeneration in Alzheimer's and Parkinson's disease by analyzing postmortem brain tissue from these patients; we found reduced SNARE-complex assembly in the brain tissue samples. Our results suggest that proteasomal activation may not always be beneficial for alleviating neurodegeneration and that blocking the proteasome may represent a potential therapeutic avenue for treating some forms of neurodegenerative disease.
|Effects of chemostimuli on [Ca2+]i responses of rat aortic body type I cells and endogenous local neurons: comparison with carotid body cells. |
Piskuric, NA; Nurse, CA
The Journal of physiology 590 2121-35 2012
Mammalian aortic bodies (ABs) are putative peripheral arterial chemoreceptors whose function remains controversial, partly because information on their cellular physiology is lacking. In this study, we used ratiometric Ca2+ imaging to investigate for the first time chemosensitivity in short-term cultures of dissociated cells of juvenile rat ABs, located near the junction of the left vagus and recurrent laryngeal nerves. Among the surviving cell population were glomus or type I cell clusters, endogenous local neurons and glia-like cells. A variety of chemostimuli, including hypoxia, isohydric or acidic hypercapnia, and isocapnic acidosis, caused a rise in intracellular [Ca2+] in AB type I cells. The [Ca2+]i responses were indistinguishable from those in carotid body (CB) type I cells grown in parallel cultures from the same animals, and responses to acidic hypercapnia were prevented by the non-specific voltage-gated Ca2+ channel antagonist, 2mM Ni2+. Furthermore, we identified a subpopulation (∼40%) of glia-like cells in AB cultures that resembled CB type II cells based on their approximately equal sensitivity to ATP and UTP, consistent with the expression of purinergic P2Y2 receptors. Finally, we showed that some local neurons, known to be uniquely associated with these AB paraganglia in situ, generated robust [Ca2+]i responses to these chemostimuli. Thus, these AB type I cells and associated putative type II cells resemble those from the well-studied CB. Unlike the CB, however, they also associate with a special group of endogenous neurons which we propose may subserve a sensory function in local cardiovascular reflexes.
|Astrocytes in the rat medial amygdala are responsive to adult androgens. |
Ryan T Johnson,Amanda Schneider,Lydia L Doncarlos,S Marc Breedlove,Cynthia L Jordan
The Journal of comparative neurology 520 2012
The posterodorsal medial amygdala (MePD) exhibits numerous sex differences including differences in volume and in the number and morphology of neurons and astroctyes. In adulthood, gonadal hormones, including both androgens and estrogens, have been shown to play a role in maintaining the masculine character of many of these sex differences, but whether adult gonadal hormones maintain the increased number and complexity of astrocytes in the male MePD was unknown. To answer this question we examined astrocytes in the MePD of male and female Long Evans rats that were gonadectomized as adults and treated for 30 days with either testosterone or a control treatment. At the end of treatment brains were collected and immunostained for glial fibrillary acidic protein. Stereological analysis revealed that adult androgen levels influenced the number and complexity of astrocytes in the MePD of both sexes, but the specific effects of androgens were different in males and females. However, sex differences in the number and complexity of adult astrocytes persisted even in the absence of gonadal hormones in adulthood, suggesting that androgens also act earlier in life to determine these adult sex differences. Using immunofluorescence and confocal microscopy, we found robust androgen receptor immunostaining in a subpopulation of MePD astrocytes, suggesting that testosterone may act directly on MePD astrocytes to influence their structure and function.
|Widespread suppression of huntingtin with convection-enhanced delivery of siRNA. |
David K Stiles,Zhiming Zhang,Pei Ge,Brian Nelson,Richard Grondin,Yi Ai,Peter Hardy,Peter T Nelson,Andrei P Guzaev,Mark T Butt,Klaus Charisse,Verbena Kosovrasti,Lubomir Tchangov,Michael Meys,Martin Maier,Lubomir Nechev,Muthiah Manoharan,William F Kaemmerer,Douglas Gwost,Gregory R Stewart,Don M Gash,Dinah W Y Sah
Experimental neurology 233 2012
Huntington's disease is an autosomal dominant neurodegenerative disease caused by a toxic gain of function mutation in the huntingtin gene (Htt). Silencing of Htt with RNA interference using direct CNS delivery in rodent models of Huntington's disease has been shown to reduce pathology and promote neuronal recovery. A key translational step for this approach is extension to the larger non-human primate brain, achieving sufficient distribution of small interfering RNA targeting Htt (siHtt) and levels of Htt suppression that may have therapeutic benefit. We evaluated the potential for convection enhanced delivery (CED) of siHtt to provide widespread and robust suppression of Htt in nonhuman primates. siHtt was infused continuously for 7 or 28 days into the nonhuman primate putamen to analyze effects of infusion rate and drug concentration on the volume of effective suppression. Distribution of radiolabeled siHtt and Htt suppression were quantified by autoradiography and PCR, respectively, in tissue punches. Histopathology was evaluated and Htt suppression was also visualized in animals treated for 28 days. Seven days of CED led to widespread distribution of siHtt and significant Htt silencing throughout the nonhuman primate striatum in an infusion rate and dose dependent manner. Htt suppression at therapeutic dose levels was well tolerated by the brain. A model developed from these results predicts that continuous CED of siHtt can achieve significant coverage of the striatum of Huntington's disease patients. These findings suggest that this approach may provide an important therapeutic strategy for treating Huntington's disease.
|Deletion of aquaporin-4 changes the perivascular glial protein scaffold without disrupting the brain endothelial barrier. |
Martine Eilert-Olsen,Nadia Nabil Haj-Yasein,Gry Fluge Vindedal,Rune Enger,Georg Andreas Gundersen,Eystein Hellstrøm Hoddevik,Pétur Henry Petersen,Finn-Mogens S Haug,Oivind Skare,Marvin E Adams,Stanley C Froehner,John Michael Burkhardt,Anna E Thoren,Erlend A Nagelhus,Øivind Skare
Glia 60 2012
Expression of the water channel aquaporin-4 (AQP4) at the blood-brain interface is dependent upon the dystrophin associated protein complex. Here we investigated whether deletion of the Aqp4 gene affects the molecular composition of this protein scaffold and the integrity of the blood-brain barrier. High-resolution immunogold cytochemistry revealed that perivascular expression of α-syntrophin was reduced by 60% in Aqp4(-/-) mice. Additionally, perivascular AQP4 expression was reduced by 88% in α-syn(-/-) mice, in accordance with earlier reports. Immunofluorescence showed that Aqp4 deletion also caused a modest reduction in perivascular dystrophin, whereas β-dystroglycan labeling was unaltered. Perivascular microglia were devoid of AQP4 immunoreactivity. Deletion of Aqp4 did not alter the ultrastructure of capillary endothelial cells, the expression of tight junction proteins (claudin-5, occludin, and zonula occludens 1), or the vascular permeability to horseradish peroxidase and Evans blue albumin dye. We conclude that Aqp4 deletion reduces the expression of perivascular glial scaffolding proteins without affecting the endothelial barrier. Our data also indicate that AQP4 and α-syntrophin are mutually dependent upon each other for proper perivascular expression.
|Multipotent stem cells from trabecular meshwork become phagocytic TM cells. |
Du, Y; Roh, DS; Mann, MM; Funderburgh, ML; Funderburgh, JL; Schuman, JS
Investigative ophthalmology & visual science 53 1566-75 2012
To isolate and characterize stem cells from human trabecular meshwork (TM) and to investigate the potential of these stem cells to differentiate into TM cells.Human trabecular meshwork stem cells (TMSCs) were isolated as side population cells by fluorescence-activated cell sorting or isolated by clonal cultures. Passaged TMSCs were compared with primary TM cells by immunostaining and quantitative RT-PCR. TMSC purity was assessed by flow cytometry and TMSC multipotency was examined by induction of neural cells, adipocytes, keratocytes, or TM cells. Differential gene expression was detected by quantitative RT-PCR, immunostaining, and immunoblotting. TM cell function was evaluated by phagocytic assay using inactivated Staphylococcus aureus bioparticles.Side population and clonal isolated cells expressed stem cell markers ABCG2, Notch1, OCT-3/4, AnkG, and MUC1 but not TM markers AQP1, MGP, CHI3L1, or TIMP3. Passaged TMSCs are a homogeneous population with greater than 95% cells positive to CD73, CD90, CD166, or Bmi1. TMSCs exhibited multipotent ability of differentiation into a variety of cell types with expression of neural markers neurofilament, β-tubulin III, GFAP; or keratocyte-specific markers keratan sulfate and keratocan; or adipocyte markers ap2 and leptin. TMSC readily differentiated into TM cells with phagocytic function and expression of TM markers AQP1, CHI3L1, and TIMP3.TMSCs, isolated as side population or as clones, express specific stem cell markers, are homogeneous and multipotent, with the ability to differentiate into phagocytic TM cells. These cells offer a potential for development of a novel stem cell-based therapy for glaucoma.
|Western Blotting, Fluorescence Activated Cell Sorting (FACS)||Human||22297497|
|An alteration in the lateral geniculate nucleus of experimental glaucoma monkeys: in vivo positron emission tomography imaging of glial activation. |
Shimazawa, M; Ito, Y; Inokuchi, Y; Yamanaka, H; Nakanishi, T; Hayashi, T; Ji, B; Higuchi, M; Suhara, T; Imamura, K; Araie, M; Watanabe, Y; Onoe, H; Hara, H
PloS one 7 e30526 2012
We examined lateral geniculate nucleus (LGN) degeneration as an indicator for possible diagnosis of glaucoma in experimental glaucoma monkeys using positron emission tomography (PET). Chronic intraocular pressure (IOP) elevation was induced by laser trabeculoplasty in the left eyes of 5 cynomolgus monkeys. Glial cell activation was detected by PET imaging with [(11)C]PK11195, a PET ligand for peripheral-type benzodiazepine receptor (PBR), before and at 4 weeks after laser treatment (moderate glaucoma stage). At mild, moderate, and advanced experimental glaucoma stages (classified by histological changes based on the extent of axonal loss), brains were stained with cresyl violet, or antibodies against PBR, Iba-1 (a microglial marker), and GFAP (an activated astrocyte marker). In laser-treated eyes, IOP was persistently elevated throughout all observation periods. PET imaging showed increased [(11)C]PK11195 binding potential in the bilateral LGN at 4 weeks after laser treatment; the increase in the ipsilateral LGN was statistically significant (Pless than 0.05, n = 4). Immunostaining showed bilateral activations of microglia and astrocytes in LGN layers receiving input from the laser-treated eye. PBR-positive cells were observed in LGN layers receiving input from laser-treated eye at all experimental glaucoma stages including the mild glaucoma stage and their localization coincided with Iba-1 positive microglia and GFAP-positive astrocytes. These data suggest that glial activation occurs in the LGN at a mild glaucoma stage, and that the LGN degeneration could be detected by a PET imaging with [(11)C]PK11195 during the moderate experimental glaucoma stage after unilateral ocular hypertension. Therefore, activated glial markers such as PBR in the LGN may be useful in noninvasive molecular imaging for diagnosis of glaucoma.
|Safety and tolerability of magnetic resonance imaging-guided convection-enhanced delivery of AAV2-hAADC with a novel delivery platform in nonhuman primate striatum. |
San Sebastian, W; Richardson, RM; Kells, AP; Lamarre, C; Bringas, J; Pivirotto, P; Salegio, EA; Dearmond, SJ; Forsayeth, J; Bankiewicz, KS
Human gene therapy 23 210-7 2012
Degeneration of nigrostriatal neurons in Parkinson's disease (PD) causes progressive loss of aromatic l-amino acid decarboxylase (AADC), the enzyme that converts levodopa (l-DOPA) into dopamine in the striatum. Because loss of this enzyme appears to be a major driver of progressive impairment of response to the mainstay drug, l-DOPA, one promising approach has been to use gene therapy to restore AADC activity in the human putamen and thereby restore normal l-DOPA response in patients with PD. An open-label phase I clinical trial of this approach in patients with PD provided encouraging signs of improvement in Unified Parkinson's Disease Rating Scale scores and reductions in antiparkinsonian medications. However, such improvement was modest compared with the results previously reported in parkinsonian rhesus macaques. The reason for this discrepancy may have been that the relatively small volume of vector infused in the clinical study restricted the distribution of AADC expression, such that only about 20% of the postcommissural putamen was covered, as revealed by l-[3-(18)F]-α-methyltyrosine-positron emission tomography. To achieve more quantitative distribution of vector, we have developed a visual guidance system for parenchymal infusion of AAV2. The purpose of the present study was to evaluate the combined magnetic resonance imaging-guided delivery system with AAV2-hAADC under conditions that approximate the intended clinical protocol. Our data indicate that this approach directed accurate cannula placement and effective vector distribution without inducing any untoward effects in nonhuman primates infused with a high dose of AAV2-hAADC.
|Clinical case of cerebral amebiasis caused by E. histolytica. |
Cinthya A Maldonado-Barrera,Maria Del Rosario Campos-Esparza,Luis Muñoz-Fernández,Joaquin A Victoria-Hernández,Rafael Campos-Rodríguez,Patricia Talamás-Rohana,Javier Ventura-Juárez
Parasitology research 110 2012
Although amebic brain abscess is a rare form of invasive amebiasis, when present, it is frequently lethal. This disorder always begins with the infection of the colon by Entamoeba histolytica trophozoites, which then travel to extra-intestinal tissues through the bloodstream. Amebic brain abscesses are produced when trophozoites invade the central nervous system. Computerized axial tomography scans can be used to diagnose the presence or absence of a brain abscess with a certainty of 100%. However, this diagnostic tool does not reveal the etiological agent of disease. By analyzing the clinical case of a patient that died due to untimely treatment of this malady, the present study aims to identify a diagnostic tool that can give a precise determination of the etiological agent and therefore permit adequate and opportune treatment. Currently, diagnosis of amebic brain abscess is often done by identification of the ameba in a biopsy or autopsy. By immunohistochemistry and immunofluorescence with specific antibodies, we identified the existence of E. histolytica, which presents proteins similar to Naegleria fowleri in its membrane.
|CSPα knockout causes neurodegeneration by impairing SNAP-25 function. |
Sharma, M; Burré, J; Bronk, P; Zhang, Y; Xu, W; Südhof, TC
The EMBO journal 31 829-41 2012
At a synapse, the synaptic vesicle protein cysteine-string protein-α (CSPα) functions as a co-chaperone for the SNARE protein SNAP-25. Knockout (KO) of CSPα causes fulminant neurodegeneration that is rescued by α-synuclein overexpression. The CSPα KO decreases SNAP-25 levels and impairs SNARE-complex assembly; only the latter but not the former is reversed by α-synuclein. Thus, the question arises whether the CSPα KO phenotype is due to decreased SNAP-25 function that then causes neurodegeneration, or due to the dysfunction of multiple as-yet uncharacterized CSPα targets. Here, we demonstrate that decreasing SNAP-25 levels in CSPα KO mice by either KO or knockdown of SNAP-25 aggravated their phenotype. Conversely, increasing SNAP-25 levels by overexpression rescued their phenotype. Inactive SNAP-25 mutants were unable to rescue, showing that the rescue was specific. Under all conditions, the neurodegenerative phenotype precisely correlated with SNARE-complex assembly, indicating that impaired SNARE-complex assembly due to decreased SNAP-25 levels is the ultimate correlate of neurodegeneration. Our findings suggest that the neurodegeneration in CSPα KO mice is primarily produced by defective SNAP-25 function, which causes neurodegeneration by impairing SNARE-complex assembly.
|Increased cone sensitivity to ABCA4 deficiency provides insight into macular vision loss in Stargardt's dystrophy. |
Conley, SM; Cai, X; Makkia, R; Wu, Y; Sparrow, JR; Naash, MI
Biochimica et biophysica acta 1822 1169-79 2012
Autosomal recessive Stargardt macular dystrophy is caused by mutations in the photoreceptor disc rim protein ABCA4/ABCR. Key clinical features of Stargardt disease include relatively mild rod defects such as delayed dark adaptation, coupled with severe cone defects reflected in macular atrophy and central vision loss. In spite of this clinical divergence, there has been no biochemical study of the effects of ABCA4 deficiency on cones vs. rods. Here we utilize the cone-dominant Abca4(-/-)/Nrl(-/-) double knockout mouse to study this issue. We show that as early as post-natal day (P) 30, Abca4(-/-)/Nrl(-/-) retinas have significantly fewer rosettes than Abca4(+/+)/Nrl(-/-) retinas, a phenotype often associated with accelerated degeneration. Abca4-deficient mice in both the wild-type and cone-dominant background accumulate more of the toxic bisretinoid A2E than their ABCA4-competent counterparts, but Abca4(-/-)/Nrl(-/-) eyes generate significantly more A2E per mole of 11-cis-retinal (11-cisRAL) than Abca4(-/-) eyes. At P120, Abca4(-/-)/Nrl(-/-) produced 340 ± 121 pmoles A2E/nmol 11-cisRAL while Abca4(-/-) produced 50.4 ± 8.05 pmoles A2E/nmol 11-cisRAL. Nevertheless, the retinal pigment epithelium (RPE) of Abca4(-/-)/Nrl(-/-) eyes exhibits fewer lipofuscin granules than the RPE of Abca4(-/-) eyes; at P120: Abca4(-/-)/Nrl(-/-) exhibit 0.045 ± 0.013 lipofuscingranules/μm² of RPE vs. Abca4(-/-) 0.17 ± 0.030 lipofuscingranules/μm² of RPE. These data indicate that ABCA4-deficient cones simultaneously generate more A2E than rods and are less able to effectively clear it, and suggest that primary cone toxicity may contribute to Stargardt's-associated macular vision loss in addition to cone death secondary to RPE atrophy.
|Spatial memory decline after masticatory deprivation and aging is associated with altered laminar distribution of CA1 astrocytes. |
Frota de Almeida, MN; de Siqueira Mendes, Fde C; Gurgel Felício, AP; Falsoni, M; Ferreira de Andrade, ML; Bento-Torres, J; da Costa Vasconcelos, PF; Perry, VH; Picanço-Diniz, CW; Kronka Sosthenes, MC
BMC neuroscience 13 23 2012
Chewing imbalances are associated with neurodegeneration and are risk factors for senile dementia in humans and memory deficits in experimental animals. We investigated the impact of long-term reduced mastication on spatial memory in young, mature and aged female albino Swiss mice by stereological analysis of the laminar distribution of CA1 astrocytes. A soft diet (SD) was used to reduce mastication in the experimental group, whereas the control group was fed a hard diet (HD). Assays were performed in 3-, 6- and 18-month-old SD and HD mice.Eating a SD variably affected the number of astrocytes in the CA1 hippocampal field, and SD mice performed worse on water maze memory tests than HD mice. Three-month-old mice in both groups could remember/find a hidden platform in the water maze. However, 6-month-old SD mice, but not HD mice, exhibited significant spatial memory dysfunction. Both SD and HD 18-month-old mice showed spatial memory decline. Older SD mice had astrocyte hyperplasia in the strata pyramidale and oriens compared to 6-month-old mice. Aging induced astrocyte hypoplasia at 18 months in the lacunosum-moleculare layer of HD mice.Taken together, these results suggest that the impaired spatial learning and memory induced by masticatory deprivation and aging may be associated with altered astrocyte laminar distribution and number in the CA1 hippocampal field. The underlying molecular mechanisms are unknown and merit further investigation.
|Six-month partial suppression of Huntingtin is well tolerated in the adult rhesus striatum. |
Grondin, R; Kaytor, MD; Ai, Y; Nelson, PT; Thakker, DR; Heisel, J; Weatherspoon, MR; Blum, JL; Burright, EN; Zhang, Z; Kaemmerer, WF
Brain : a journal of neurology 135 1197-209 2012
Huntington's disease is caused by expression of a mutant form of Huntingtin protein containing an expanded polyglutamine repeat. One possible treatment for Huntington's disease may be to reduce expression of mutant Huntingtin in the brain via RNA interference. Unless the therapeutic molecule is designed to be allele-specific, both wild-type and mutant protein will be suppressed by an RNA interference treatment. A key question is whether suppression of wild-type as well as mutant Huntingtin in targeted brain regions can be tolerated and result in a net benefit to patients with Huntington's disease. Whether Huntingtin performs essential functions in the adult brain is unclear. Here, we tested the hypothesis that the adult primate brain can tolerate moderately reduced levels of wild-type Huntingtin protein for an extended period of time. A serotype 2 adeno-associated viral vector encoding for a short hairpin RNA targeting rhesus huntingtin messenger RNA (active vector) was bilaterally injected into the striatum of four adult rhesus monkeys. Four additional animals received a comparable vector encoding a scrambled control short hairpin RNA (control vector). General health and motor behaviour were monitored for 6 months. Upon termination, brain tissues were sampled and assessed blindly for (i) huntingtin messenger RNA knockdown; (ii) Huntingtin protein expression; and (iii) neuropathological changes. Reduction in wild-type huntingtin messenger RNA levels averaging ∼30% was measured in the striatum of active vector recipients 6 months post-injection. A widespread reduction in Huntingtin protein levels was also observed by immunohistochemistry in these animals, with an average protein reduction of ∼45% relative to controls measured by western blot analysis in the putamen of active vector recipients. As with control vector recipients, no adverse effects were observed behaviourally, and no neurodegeneration was found on histological examination of active vector recipients. Our results suggest that long-term partial suppression of wild-type Huntingtin may be safe, and thus if a comparable level of suppression of mutant Huntingtin is beneficial, then partial suppression of both wild-type and mutant Huntingtin may result in a net benefit in patients with heterozygous Huntington's disease.
|Foxg1 has an essential role in postnatal development of the dentate gyrus. |
Tian, C; Gong, Y; Yang, Y; Shen, W; Wang, K; Liu, J; Xu, B; Zhao, J; Zhao, C
The Journal of neuroscience : the official journal of the Society for Neuroscience 32 2931-49 2012
Foxg1, formerly BF-1, is expressed continuously in the postnatal and adult hippocampal dentate gyrus (DG). This transcription factor (TF) is thought to be involved in Rett syndrome, which is characterized by reduced hippocampus size, indicating its important role in hippocampal development. Due to the perinatal death of Foxg1(-/-) mice, the function of Foxg1 in postnatal DG neurogenesis remains to be explored. Here, we describe the generation of a Foxg1(fl/fl) mouse line. Foxg1 was conditionally ablated from the DG during prenatal and postnatal development by crossing this line with a Frizzled9-CreER(TM) line and inducing recombination with tamoxifen. In this study, we first show that disruption of Foxg1 results in the loss of the subgranular zone and a severely disrupted secondary radial glial scaffold, leading to the impaired migration of granule cells. Moreover, detailed analysis reveals that Foxg1 may be necessary for the maintenance of the DG progenitor pool and that the lack of Foxg1 promotes both gliogenesis and neurogenesis. We additionally show that Foxg1 may be required for the survival and maturation of postmitotic neurons and that Foxg1 may be involved in Reelin signaling in regulating postnatal DG development. Last, prenatal deletion of Foxg1 suggests that it is rarely involved in the migration of primordial granule cells. In summary, we report that Foxg1 is critical for DG formation, especially during early postnatal stage.
|CXCL16 orchestrates adenosine A3 receptor and MCP-1/CCL2 activity to protect neurons from excitotoxic cell death in the CNS. |
Rosito, M; Deflorio, C; Limatola, C; Trettel, F
The Journal of neuroscience : the official journal of the Society for Neuroscience 32 3154-63 2012
A role for chemokines as molecules mediating neuron-glia cross talk has emerged in recent years, both in physiological and pathological conditions. We demonstrate here for the first time that the chemokine CXCL16 and its unique receptor CXCR6 are functionally expressed in the CNS, and induce neuroprotection against excitotoxic damage due to excessive glutamate (Glu) exposure and oxygen glucose deprivation (OGD). In mice and rats we found that, to exert neuroprotection, CXCL16 requires the presence of extracellular adenosine (ADO), and that pharmacological or genetic inactivation of the ADO A(3) receptor, A(3)R, prevents CXCL16 effect. In experiments with astrocytes cocultured with cxcr6(gfp/gfp) hippocampal cells, we demonstrate that CXCL16 acts directly on astrocytes to release soluble factors that are essential to mediate neuroprotection. In particular, we report that (1) upon stimulation with CXCL16 astrocytes release monocyte chemoattractant protein-1/CCL2 and (2) the neuroprotective effect of CXCL16 is reduced in the presence of neutralizing CCL2 antibody. In conclusion, we found that chemokine CXCL16 is able to mediate cross talk between astrocytes and neighboring neurons and, in pathological conditions such as excessive Glu or OGD exposure, is able to counteract neuronal cell death through an ADO-dependent chemokine-induced chemokine-release mechanism.
|Isoflurane induces learning impairment that is mediated by interleukin 1β in rodents. |
Cao, L; Li, L; Lin, D; Zuo, Z
PloS one 7 e51431 2012
Postoperative cognitive decline is a clinical syndrome. Volatile anesthetics are commonly used during surgery. It is conceivable that volatile anesthetics may contribute to postoperative cognitive decline. Isoflurane can impair cognitive functions of animals under certain conditions. However, the mechanisms for this impairment are not clear. Here, male 18-month old Fisher 344 rats or 10-week old mice were exposed to 1.2 or 1.4% isoflurane for 2 h. Our studies showed that isoflurane impaired the cognitive functions of the rats in Barnes maze. Isoflurane-exposed rats had reduced freezing behavior during the training sessions in the fear conditioning test. This isoflurane effect was attenuated by lidocaine, a local anesthetic with anti-inflammatory property. Rats that had training sessions and were exposed to isoflurane 30 min later had freezing behavior similar to that of control animals. Isoflurane increased the expression of interleukin 1β (IL-1β), interleukin-6 and activated caspase 3 in the hippocampus of the 18-month old rats. IL-1β positive staining was co-localized with that of NeuN, a neuronal marker. The increase of IL-1β and activated caspase 3 but not interleukin-6 was attenuated by lidocaine. Isoflurane also impaired the cognitive functions of 10-week old C57BL/6J mice and increased IL-1β in their hippocampi. However, isoflurane did not affect the cognitive functions of IL-1β deficient mice. Our results suggest that isoflurane impairs the learning but may not affect the recall of the aged rats. IL-1β may play an important role in this isoflurane effect.
|Amyloid precursor protein revisited: neuron-specific expression and highly stable nature of soluble derivatives. |
Guo, Q; Li, H; Gaddam, SS; Justice, NJ; Robertson, CS; Zheng, H
The Journal of biological chemistry 287 2437-45 2012
APP processing and amyloid-β production play a central role in Alzheimer disease pathogenesis. APP has been considered a ubiquitously expressed protein. In addition to amyloid-β, α- or β-secretase-dependent cleavage of APP also generates soluble secreted APP (APPsα or APPsβ, respectively). Interestingly, APPsβ has been shown to be subject to further cleavage to create an N-APP fragment that binds to the DR6 death receptor and mediates axon pruning and degeneration under trophic factor withdrawal conditions. By performing APP immunocytochemical staining, we found that, unexpectedly, many antibodies yielded nonspecific staining in APP-null samples. Screening of a series of antibodies allowed us to identify a rabbit monoclonal antibody Y188 that is highly specific for APP and prompted us to re-examine the expression, localization, and stability of endogenous APP and APPsβ in wild-type and in APPsβ knock-in mice, respectively. In contrast to earlier studies, we found that APP is specifically expressed in neurons and that its expression cannot be detected in major types of glial cells under basal or neuroinflammatory conditions. Both APPsα and APPsβ are highly stable in the central nervous system (CNS) and do not undergo further cleavage with or without trophic factor support. Our results clarify several key questions with regard to the fundamental properties of APP and offer critical cellular insights into the pathophysiology of APP.
|Isoflurane-induced apoptosis of oligodendrocytes in the neonatal primate brain. |
Brambrink, AM; Back, SA; Riddle, A; Gong, X; Moravec, MD; Dissen, GA; Creeley, CE; Dikranian, KT; Olney, JW
Annals of neurology 72 525-35 2012
Previously we reported that exposure of 6-day-old (P6) rhesus macaques to isoflurane for 5 hours triggers a robust neuroapoptosis response in developing brain. We have also observed (unpublished data) that isoflurane causes apoptosis of cellular profiles in the white matter that resemble glia. We analyzed the cellular identity of the apoptotic white matter profiles and determined the magnitude of this cell death response to isoflurane.Neonatal (P6) rhesus macaques were exposed for 5 hours to isoflurane anesthesia according to current clinical standards in pediatric anesthesia. Brains were collected 3 hours later and examined immunohistochemically to analyze apoptotic neuronal and glial death.Brains exposed to isoflurane displayed significant apoptosis in both the white and gray matter throughout the central nervous system. Approximately 52% of the dying cells were glia, and 48% were neurons. Oligodendrocytes (OLs) engaged in myelinogenesis were selectively vulnerable, in contrast to OL progenitors, astrocytes, microglia, and interstitial neurons. When adjusted for control rates of OL apoptosis, the percentage of OLs that degenerated in the forebrain white matter of the isoflurane-treated group was 6.3% of the total population of myelinating OLs.Exposure of the infant rhesus macaque brain to isoflurane for 5 hours is sufficient to cause widespread apoptosis of neurons and OLs throughout the developing brain. Deletion of OLs at a stage when they are just beginning to myelinate axons could potentially have adverse long-term neurobehavioral consequences that might be additive to the potential consequences of isoflurane-induced neuroapoptosis.
|Rod microglia: elongation, alignment, and coupling to form trains across the somatosensory cortex after experimental diffuse brain injury. |
Ziebell, JM; Taylor, SE; Cao, T; Harrison, JL; Lifshitz, J
Journal of neuroinflammation 9 247 2012
Since their discovery, the morphology of microglia has been interpreted to mirror their function, with ramified microglia constantly surveying the micro-environment and rapidly activating when changes occur. In 1899, Franz Nissl discovered what we now recognize as a distinct microglial activation state, microglial rod cells (Stäbchenzellen), which he observed adjacent to neurons. These rod-shaped microglia are typically found in human autopsy cases of paralysis of the insane, a disease of the pre-penicillin era, and best known today from HIV-1-infected brains. Microglial rod cells have been implicated in cortical 'synaptic stripping' but their exact role has remained unclear. This is due at least in part to a scarcity of experimental models. Now we have noted these rod microglia after experimental diffuse brain injury in brain regions that have an associated sensory sensitivity. Here, we describe the time course, location, and surrounding architecture associated with rod microglia following experimental diffuse traumatic brain injury (TBI).Rats were subjected to a moderate midline fluid percussion injury (mFPI), which resulted in transient suppression of their righting reflex (6 to 10 min). Multiple immunohistochemistry protocols targeting microglia with Iba1 and other known microglia markers were undertaken to identify the morphological activation of microglia. Additionally, labeling with Iba1 and cell markers for neurons and astrocytes identified the architecture that surrounds these rod cells.We identified an abundance of Iba1-positive microglia with rod morphology in the primary sensory barrel fields (S1BF). Although present for at least 4 weeks post mFPI, they developed over the first week, peaking at 7 days post-injury. In the absence of contusion, Iba1-positive microglia appear to elongate with their processes extending from the apical and basal ends. These cells then abut one another and lay adjacent to cytoarchitecture of dendrites and axons, with no alignment with astrocytes and oligodendrocytes. Iba1-positive rod microglial cells differentially express other known markers for reactive microglia including OX-6 and CD68.Diffuse traumatic brain injury induces a distinct rod microglia morphology, unique phenotype, and novel association between cells; these observations entice further investigation for impact on neurological outcome.
|Effects of neonatal neural progenitor cell implantation on adult neuroanatomy and cognition in the Ts65Dn model of Down syndrome. |
Rachubinski, AL; Crowley, SK; Sladek, JR; Maclean, KN; Bjugstad, KB
PloS one 7 e36082 2012
As much of the aberrant neural development in Down syndrome (DS) occurs postnatally, an early opportunity exists to intervene and influence life-long cognitive development. Recent success using neural progenitor cells (NPC) in models of adult neurodegeneration indicate such therapy may be a viable option in diseases such as DS. Murine NPC (mNPC, C17.2 cell line) or saline were implanted bilaterally into the dorsal hippocampus of postnatal day 2 (PND 2) Ts65Dn pups to explore the feasibility of early postnatal treatment in this mouse model of DS. Disomic littermates provided karyotype controls for trisomic pups. Pups were monitored for developmental milestone achievement, and then underwent adult behavior testing at 14 weeks of age. We found that implanted mNPC survived into adulthood and migrated beyond the implant site in both karyotypes. The implantation of mNPC resulted in a significant increase in the density of dentate granule cells. However, mNPC implantation did not elicit cognitive changes in trisomic mice either neonatally or in adulthood. To the best of our knowledge, these results constitute the first assessment of mNPC as an early intervention on cognitive ability in a DS model.
|Optogenetic activation of LiGluR-expressing astrocytes evokes anion channel-mediated glutamate release. |
Li, D; Hérault, K; Isacoff, EY; Oheim, M; Ropert, N
The Journal of physiology 590 855-73 2012
Increases in astrocyte Ca(2+) have been suggested to evoke gliotransmitter release, however, the mechanism of release, the identity of such transmitter(s), and even whether and when such release occurs, are controversial, largely due to the lack of a method for selective and reproducible stimulation of electrically silent astrocytes. Here we show that photoactivation of the light-gated Ca(2+)-permeable ionotropic GluR6 glutamate receptor (LiGluR), and to a lesser extent the new Ca(2+)-translocating channelrhodopsin CatCh, evokes more reliable Ca(2+) elevation than the mutant channelrhodopsin 2, ChR2(H134R) in cultured cortical astrocytes. We used evanescent-field excitation for near-membrane Ca(2+) imaging, and epifluorescence to activate and inactivate LiGluR. By alternating activation and inactivation light pulses, the LiGluR-evoked Ca(2+) rises could be graded in amplitude and duration. The optical stimulation of LiGluR-expressing astrocytes evoked probabilistic glutamate-mediated signalling to adjacent LiGluR-non-expressing astrocytes. This astrocyte-to-astrocyte signalling was insensitive to the inactivation of vesicular release, hemichannels and glutamate-transporters, and sensitive to anion channel blockers. Our results show that LiGluR is a powerful tool to selectively and reproducibly activate astrocytes.
|Myelin gene regulatory factor is required for maintenance of myelin and mature oligodendrocyte identity in the adult CNS. |
Koenning, M; Jackson, S; Hay, CM; Faux, C; Kilpatrick, TJ; Willingham, M; Emery, B
The Journal of neuroscience : the official journal of the Society for Neuroscience 32 12528-42 2012
Although the transcription factors required for the generation of oligodendrocytes and CNS myelination during development have been relatively well established, it is not known whether continued expression of the same factors is required for the maintenance of myelin in the adult. Here, we use an inducible conditional knock-out strategy to investigate whether continued oligodendrocyte expression of the recently identified transcription factor myelin gene regulatory factor (MRF) is required to maintain the integrity of myelin in the adult CNS. Genetic ablation of MRF in mature oligodendrocytes within the adult CNS resulted in a delayed but severe CNS demyelination, with clinical symptoms beginning at 5 weeks and peaking at 8 weeks after ablation of MRF. This demyelination was accompanied by microglial/macrophage infiltration and axonal damage. Transcripts for myelin genes, such as proteolipid protein, MAG, MBP, and myelin oligodendrocyte glycoprotein, were rapidly downregulated after ablation of MRF, indicating an ongoing requirement for MRF in the expression of these genes. Subsequently, a proportion of the recombined oligodendrocytes undergo apoptosis over a period of weeks. Surviving oligodendrocytes gradually lose the expression of mature markers such as CC1 antigen and their association with myelin, without reexpressing oligodendrocyte progenitor markers or reentering the cell cycle. These results demonstrate that ongoing expression of MRF within the adult CNS is critical to maintain mature oligodendrocyte identity and the integrity of CNS myelin.
|Induction of amyloid-β(1-42) in the retina and optic nerve head of chronic ocular hypertensive monkeys. |
Ito, Y; Shimazawa, M; Tsuruma, K; Mayama, C; Ishii, K; Onoe, H; Aihara, M; Araie, M; Hara, H
Molecular vision 18 2647-57 2012
Recent studies have indicated that accumulation of amyloid β(1-42) (Aβ(1-42)), which is associated with the progression of Alzheimer disease, may also be responsible for retinal ganglion cell death in glaucoma. The purpose of this study was to investigate the expression and localization of Aβ(1-42) in the retina and the optic nerve head (ONH) of monkeys with experimental glaucoma.Five cynomolgus monkeys with a glaucomatous left eye at 4, 9, 11, 15, and 24 weeks after laser photocoagulation treatment were studied by immunohistochemical methods. Another two cynomolgus monkeys with a glaucomatous left eye at 133 weeks after laser photocoagulation treatment were used to measure Aβ(1-42) concentrations in the retina by enzyme-linked immunosorbent assay.At 11 to 24 weeks after the laser photocoagulation treatment, Aβ(1-42) was upregulated in the nerve fiber layer (NFL) and the ganglion cell layer (GCL) of the retina and the ONH, but the expression of amyloid precursor protein decreased in the NFL and ONH from levels at 9 weeks. The localizations of Aβ(1-42) were merged in glial fibrillary acidic protein-positive astroglial cells but not phosphorylated neurofilament heavy- or nonphosphorylated neurofilament heavy-positive axons in the retina and the ONH. Likewise, Aβ(1-42) concentrations in the retina of monkeys increased in the chronic stage of glaucoma.These findings indicate that the upregulation of Aβ(1-42) after an intraocular pressure elevation could apply to monkeys since the structure of the ONH is more similar to humans than that of rodents.
|Calcium/calmodulin dependent kinase II contributes to persistent central neuropathic pain following spinal cord injury. |
Crown, ED; Gwak, YS; Ye, Z; Yu Tan, H; Johnson, KM; Xu, GY; McAdoo, DJ; Hulsebosch, CE
Pain 153 710-21 2012
Chronic central neuropathic pain after central nervous system injuries remains refractory to therapeutic interventions. A novel approach would be to target key intracellular signaling proteins that are known to contribute to continued activation by phosphorylation of kinases, transcription factors, and/or receptors that contribute to changes in membrane excitability. We demonstrate that one signaling kinase, calcium/calmodulin-dependent kinase II (CaMKII), is critical in maintaining aberrant dorsal horn neuron hyperexcitability in the neuropathic pain condition after spinal cord injury (SCI). After contusion SCI at spinal level T10, activated CaMKII (phosphorylated, pCaMKII) expression is significantly upregulated in the T7/8 spinal dorsal horn in neurons, but not glial cells, and in oligodendrocytes in the dorsal column in the same rats that displayed at-level mechanical allodynia. Furthermore, identified spinothalamic neurons demonstrated significant increases of pCaMKII after SCI compared to sham-treated control animals. However, neither astrocytes nor microglia showed pCaMKII expression in either sham-treated or SCI rats. To demonstrate causality, treatment of SCI rats with KN-93, which prevents CaMKII activation, significantly attenuated at-level mechanical allodynia and aberrant wide dynamic range neuronal activity evoked by brush, pressure, and pinch stimuli and a graded series of von Frey stimuli, respectively. Persistent CaMKII activation contributes to chronic central neuropathic pain by mechanisms that involve maintained hyperexcitability of wide dynamic range dorsal horn neurons. Furthermore, targeting key signaling proteins is a novel, useful therapeutic strategy for treating chronic central neuropathic pain.
|Involvement of receptor tyrosine kinase Tyro3 in amyloidogenic APP processing and β-amyloid deposition in Alzheimer's disease models. |
Zheng, Y; Wang, Q; Xiao, B; Lu, Q; Wang, Y; Wang, X
PloS one 7 e39035 2012
Alzheimer's disease (AD) is the most common progressive neurodegenerative disease known to humankind. It is characterized by brain atrophy, extracellular amyloid plaques, and intracellular neurofibril tangles. β-Amyloid cascade is considered the major causative player in AD. Up until now, the mechanisms underlying the process of Aβ generation and accumulation in the brain have not been well understood. Tyro3 receptor belongs to the TAM receptor subfamily of receptor protein tyrosine kinases (RPTKs). It is specifically expressed in the neurons of the neocortex and hippocampus. In this study, we established a cell model stably expressing APPswe mutants and producing Aβ. We found that overexpression of Tyro3 receptor in the cell model significantly decreased Aβ generation and also down-regulated the expression of β-site amyloid precursor protein cleaving enzyme (BACE1). However, the effects of Tyro3 were inhibited by its natural ligand, Gas6, in a concentration-dependent manner. In order to confirm the role of Tyro3 in the progression of AD development, we generated an AD transgenic mouse model accompanied by Tyro3 knockdown. We observed a significant increase in the number of amyloid plaques in the hippocampus in the mouse model. More plaque-associated clusters of astroglia were also detected. The present study may help researchers determine the role of Tyro3 receptor in the neuropathology of AD.
|Induction of angiopoietin-2 after spinal cord injury. |
Durham-Lee, JC; Wu, Y; Mokkapati, VU; Paulucci-Holthauzen, AA; Nesic, O
Neuroscience 202 454-64 2012
Angiopoietin-1 (Ang-1) and angiopoietin-2 (Ang-2) have opposing effects on blood vessels, with Ang-2 being mainly induced during the endothelial barrier breakdown. It is known that spinal cord injury (SCI) induces lasting decreases in Ang-1 levels, underlying endothelial barrier disruption, but the expression of Ang-2 in spinal cord injury has not been studied. We characterized Ang-2 after SCI using a clinically relevant rat model of contusion SCI. We found that SCI induces marked and persistent upregulation of Ang-2 (up to 10 weeks after SCI), which does not reflect well-characterized temporal profile of the blood-spinal cord barrier (BSCB) breakdown after SCI, and thus suggests other role(s) for Ang-2 in injured spinal cords. Furthermore, we also found that higher Ang-2 levels were associated with more successful locomotor recovery after SCI, both in SCI rats with markedly better spontaneous motor recovery and in SCI rats receiving a neuroprotective pharmacological intervention (amiloride), suggesting a beneficial role for Ang-2 in injured spinal cords. Immunocytochemical analyses revealed that Ang-2 was not induced in endothelial cells, but in perivascular and non-vascular cells labeled with glial fibrillary acidic protein (GFAP) or with chondroitin sulfate proteoglycan (NG2). Therefore, it is unlikely that induction of Ang-2 contributes to vascular dysfunction underlying functional impairment after SCI, but rather that it contributes to the beneficial pro-angiogenic and/or gliogenic processes underlying recovery processes after SCI.
|Spatial regulation of interleukin-6 signaling in response to neurodegenerative stressors in the retina. |
Sims, SM; Holmgren, L; Cathcart, HM; Sappington, RM
American journal of neurodegenerative disease 1 168-79 2012
Neuroinflammation, defined as the induction of immune-related processes within the central nervous system, is recognized as a component of many neurodegenerative disorders, including glaucomatous degeneration of retinal ganglion cells (RGCs). Previous work in vitro identified IL-6 as a potential neuroprotective factor for RGCs, particularly those challenged by glaucoma-related stressors. Here we examined the temporal and spatial characteristics of IL-6 signaling in response to two stressors related to RGC neurodegeneration: age and elevated intraocular pressure (IOP). Using ELISA, immunoblotting, immunolabeling and quantitative microscopy, we measured and compared whole retina and RGC-related expression of IL-6 and IL-6Rα in normal retina (young C57), retina susceptible to glaucomatous neurodegeneration (young DBA/2), aging retina (aged C57) and aging retina challenged by elevated IOP (aged DBA/2). We found that: 1) neurodegenerative stressors induce alterations in whole retina expression of IL-6 and IL-6Rα, 2) these whole retina changes do not reflect the immediate milieu of RGCs, where IL-6 and IL-6Rα expression is spatially variable and 3) the extent and magnitude of this spatial variability is stressor-dependent. Our data provide the first evidence that neurodegenerative stressors produce microenvironments of IL-6 signaling in retina and that the nature and magnitude of spatial regulation is dependent on the identity of the stressor.
|Growth hormone responsive neural precursor cells reside within the adult mammalian brain. |
Blackmore, DG; Reynolds, BA; Golmohammadi, MG; Large, B; Aguilar, RM; Haro, L; Waters, MJ; Rietze, RL
Scientific reports 2 250 2012
The detection of growth hormone (GH) and its receptor in germinal regions of the mammalian brain prompted our investigation of GH and its role in the regulation of endogenous neural precursor cell activity. Here we report that the addition of exogenous GH significantly increased the expansion rate in long-term neurosphere cultures derived from wild-type mice, while neurospheres derived from GH null mice exhibited a reduced expansion rate. We also detected a doubling in the frequency of large (i.e. stem cell-derived) colonies for up to 120 days following a 7-day intracerebroventricular infusion of GH suggesting the activation of endogenous stem cells. Moreover, gamma irradiation induced the ablation of normally quiescent stem cells in GH-infused mice, resulting in a decline in olfactory bulb neurogenesis. These results suggest that GH activates populations of resident stem and progenitor cells, and therefore may represent a novel therapeutic target for age-related neurodegeneration and associated cognitive decline.
|Differential developmental deficits in retinal function in the absence of either protein tyrosine sulfotransferase-1 or -2. |
Sherry, DM; Kanan, Y; Hamilton, R; Hoffhines, A; Arbogast, KL; Fliesler, SJ; Naash, MI; Moore, KL; Al-Ubaidi, MR
PloS one 7 e39702 2012
To investigate the role(s) of protein-tyrosine sulfation in the retina and to determine the differential role(s) of tyrosylprotein sulfotransferases (TPST) 1 and 2 in vision, retinal function and structure were examined in mice lacking TPST-1 or TPST-2. Despite the normal histologic retinal appearance in both Tpst1(-/-) and Tpst2(-/-) mice, retinal function was compromised during early development. However, Tpst1(-/-) retinas became electrophysiologically normal by postnatal day 90 while Tpst2(-/-) mice did not functionally normalize with age. Ultrastructurally, the absence of TPST-1 or TPST-2 caused minor reductions in neuronal plexus. These results demonstrate the functional importance of protein-tyrosine sulfation for proper development of the retina and suggest that the different phenotypes resulting from elimination of either TPST-1 or -2 may reflect differential expression patterns or levels of the enzymes. Furthermore, single knock-out mice of either TPST-1 or -2 did not phenocopy mice with double-knockout of both TPSTs, suggesting that the functions of the TPSTs are at least partially redundant, which points to the functional importance of these enzymes in the retina.
|BACE2 expression increases in human neurodegenerative disease. |
Holler, CJ; Webb, RL; Laux, AL; Beckett, TL; Niedowicz, DM; Ahmed, RR; Liu, Y; Simmons, CR; Dowling, AL; Spinelli, A; Khurgel, M; Estus, S; Head, E; Hersh, LB; Murphy, MP
The American journal of pathology 180 337-50 2012
β-Secretase, the rate-limiting enzymatic activity in the production of the amyloid-β (Aβ) peptide, is a major target of Alzheimer's disease (AD) therapeutics. There are two forms of the enzyme: β-site Aβ precursor protein cleaving enzyme (BACE) 1 and BACE2. Although BACE1 increases in late-stage AD, little is known about BACE2. We conducted a detailed examination of BACE2 in patients with preclinical to late-stage AD, including amnestic mild cognitive impairment, and age-matched controls, cases of frontotemporal dementia, and Down's syndrome. BACE2 protein and enzymatic activity increased as early as preclinical AD and were found in neurons and astrocytes. Although the levels of total BACE2 mRNA were unchanged, the mRNA for BACE2 splice form C (missing exon 7) increased in parallel with BACE2 protein and activity. BACE1 and BACE2 were strongly correlated with each other at all levels, suggesting that their regulatory mechanisms may be largely shared. BACE2 was also elevated in frontotemporal dementia but not in Down's syndrome, even in patients with substantial Aβ deposition. Thus, expression of both forms of β-secretase are linked and may play a combined role in human neurologic disease. A better understanding of the normal functions of BACE1 and BACE2, and how these change in different disease states, is essential for the future development of AD therapeutics.
|In vivo optogenetic control of striatal and thalamic neurons in non-human primates. |
Galvan, A; Hu, X; Smith, Y; Wichmann, T
PloS one 7 e50808 2012
Electrical and pharmacological stimulation methods are commonly used to study neuronal brain circuits in vivo, but are problematic, because electrical stimulation has limited specificity, while pharmacological activation has low temporal resolution. A recently developed alternative to these methods is the use of optogenetic techniques, based on the expression of light sensitive channel proteins in neurons. While optogenetics have been applied in in vitro preparations and in in vivo studies in rodents, their use to study brain function in nonhuman primates has been limited to the cerebral cortex. Here, we characterize the effects of channelrhodopsin-2 (ChR2) transfection in subcortical areas, i.e., the putamen, the external globus pallidus (GPe) and the ventrolateral thalamus (VL) of rhesus monkeys. Lentiviral vectors containing the ChR2 sequence under control of the elongation factor 1α promoter (pLenti-EF1α -hChR2(H134R)-eYFP-WPRE, titer 10⁹ particles/ml) were deposited in GPe, putamen and VL. Four weeks later, a probe combining a conventional electrode and an optic fiber was introduced in the previously injected brain areas. We found light-evoked responses in 31.5% and 32.7% of all recorded neurons in the striatum and thalamus, respectively, but only in 2.5% of recorded GPe neurons. As expected, most responses were time-locked increases in firing, but decreases or mixed responses were also seen, presumably via ChR2-mediated activation of local inhibitory connections. Light and electron microscopic analyses revealed robust expression of ChR2 on the plasma membrane of cell somas, dendrites, spines and terminals in the striatum and VL. This study demonstrates that optogenetic experiments targeting the striatum and basal ganglia-related thalamic nuclei can be successfully achieved in monkeys. Our results indicate important differences of the type and magnitude of responses in each structure. Experimental conditions such as the vector used, the number and rate of injections, or the light stimulation conditions have to be optimized for each structure studied.
|In vivo gene knockdown in rat dorsal root ganglia mediated by self-complementary adeno-associated virus serotype 5 following intrathecal delivery. |
Xu, Q; Chou, B; Fitzsimmons, B; Miyanohara, A; Shubayev, V; Santucci, C; Hefferan, M; Marsala, M; Hua, XY
PloS one 7 e32581 2012
We report here in adult rat viral vector mediate-gene knockdown in the primary sensory neurons and the associated cellular and behavior consequences. Self-complementary adeno-associated virus serotype 5 (AAV5) was constructed to express green fluorescent protein (GFP) and a small interfering RNA (siRNA) targeting mammalian target of rapamycin (mTOR). The AAV vectors were injected via an intrathecal catheter. We observed profound GFP expression in lumbar DRG neurons beginning at 2-week post-injection. Of those neurons, over 85% were large to medium-diameter and co-labeled with NF200, a marker for myelinated fibers. Western blotting of mTOR revealed an 80% reduction in the lumbar DRGs (L4-L6) of rats treated with the active siRNA vectors compared to the control siRNA vector. Gene knockdown became apparent as early as 7-day post-injection and lasted for at least 5 weeks. Importantly, mTOR knockdown occurred in large (NF200) and small-diameter neurons (nociceptors). The viral administration induced an increase of Iba1 immunoreactivity in the DRGs, which was likely attributed to the expression of GFP but not siRNA. Rats with mTOR knockdown in DRG neurons showed normal general behavior and unaltered responses to noxious stimuli. In conclusion, intrathecal AAV5 is a highly efficient vehicle to deliver siRNA and generate gene knockdown in DRG neurons. This will be valuable for both basic research and clinic intervention of diseases involving primary sensory neurons.
|A quick and simple FISH protocol with hybridization-sensitive fluorescent linear oligodeoxynucleotide probes. |
Wang, DO; Matsuno, H; Ikeda, S; Nakamura, A; Yanagisawa, H; Hayashi, Y; Okamoto, A
RNA (New York, N.Y.) 18 166-75 2012
Fluorescence in situ hybridization (FISH) is a powerful tool used in karyotyping, cytogenotyping, cancer diagnosis, species specification, and gene-expression analysis. Although widely used, conventional FISH protocols are cumbersome and time consuming. We have now developed a FISH method using exciton-controlled hybridization-sensitive fluorescent oligodeoxynucleotide (ECHO) probes. ECHO-FISH uses a 25-min protocol from fixation to mounting that includes no stringency washing steps. We use ECHO-FISH to detect both specific DNA and RNA sequences with multicolor probes. ECHO-FISH is highly reproducible, stringent, and compatible with other fluorescent cellular labeling techniques. The resolution allows detection of intranuclear speckles of poly(A) RNA in HeLa cells and dissociated hippocampal primary cultures, and mRNAs in the distal dendrites of hippocampal neurons. We also demonstrate detection of telomeric and centromeric DNA on metaphase mouse chromosomes. The simplicity of the ECHO-FISH method will likely accelerate cytogenetic and gene-expression analysis with high resolution.
|Morphine and gp120 toxic interactions in striatal neurons are dependent on HIV-1 strain. |
Podhaizer, EM; Zou, S; Fitting, S; Samano, KL; El-Hage, N; Knapp, PE; Hauser, KF
Journal of neuroimmune pharmacology : the official journal of the Society on NeuroImmune Pharmacology 7 877-91 2012
A rigorously controlled, cell culture paradigm was used to assess the role of HIV-1 gp120 ± morphine in mediating opioid-HIV interactive toxicity in striatal neurons. Computerized time-lapse microscopy tracked the fate of individual neurons co-cultured with mixed-glia from mouse striata during opioid and gp120 exposure. Subpopulations of neurons and astroglia displayed μ-opioid receptor, CXCR4, and CCR5 immunoreactivity. While gp120 alone was or tended to be neurotoxic irrespective of whether X4-tropic gp120(IIIB), R5-tropic gp120(ADA), or dual-tropic gp120(MN) was administered, interactive toxicity with morphine differed depending on HIV-1 strain. For example, morphine only transiently exacerbated gp120(IIIB)-induced neuronal death; however, in combination with gp120(MN), morphine caused sustained increases in the rate of neuronal death compared to gp120(MN) alone that were prevented by naloxone. Alternatively, gp120(ADA) significantly increased the rate of neuron death, but gp120(ADA) toxicity was unaffected by morphine. The transient neurotoxic interactions between morphine and gp120(IIIB) were abrogated in the absence of glia suggesting that glia contribute significantly to the interactive pathology with chronic opiate abuse and neuroAIDS. To assess how mixed-glia might contribute to the neurotoxicity, the effects of morphine and/or gp120 on the production of reactive oxygen species (ROS) and on glutamate buffering were examined. All gp120 variants, and to a lesser extent morphine, increased ROS and/or decreased glutamate buffering, but together failed to show any interaction with morphine. Our findings indicate that HIV-1 strain-specific differences in gp120 are critical determinants in shaping both the timing and pattern of neurotoxic interactions with opioid drugs.
|Distinct regulatory functions of calpain 1 and 2 during neural stem cell self-renewal and differentiation. |
Santos, DM; Xavier, JM; Morgado, AL; Solá, S; Rodrigues, CM
PloS one 7 e33468 2012
Calpains are calcium regulated cysteine proteases that have been described in a wide range of cellular processes, including apoptosis, migration and cell cycle regulation. In addition, calpains have been implicated in differentiation, but their impact on neural differentiation requires further investigation. Here, we addressed the role of calpain 1 and calpain 2 in neural stem cell (NSC) self-renewal and differentiation. We found that calpain inhibition using either the chemical inhibitor calpeptin or the endogenous calpain inhibitor calpastatin favored differentiation of NSCs. This effect was associated with significant changes in cell cycle-related proteins and may be regulated by calcium. Interestingly, calpain 1 and calpain 2 were found to play distinct roles in NSC fate decision. Calpain 1 expression levels were higher in self-renewing NSC and decreased with differentiation, while calpain 2 increased throughout differentiation. In addition, calpain 1 silencing resulted in increased levels of both neuronal and glial markers, β-III Tubulin and glial fibrillary acidic protein (GFAP). Calpain 2 silencing elicited decreased levels of GFAP. These results support a role for calpain 1 in repressing differentiation, thus maintaining a proliferative NSC pool, and suggest that calpain 2 is involved in glial differentiation.
|Spatiotemporal evolution of early innate immune responses triggered by neural stem cell grafting. |
Reekmans, K; De Vocht, N; Praet, J; Fransen, E; Le Blon, D; Hoornaert, C; Daans, J; Goossens, H; Van der Linden, A; Berneman, Z; Ponsaerts, P
Stem cell research & therapy 3 56 2012
Transplantation of neural stem cells (NSCs) is increasingly suggested to become part of future therapeutic approaches to improve functional outcome of various central nervous system disorders. However, recently it has become clear that only a small fraction of grafted NSCs display long-term survival in the (injured) adult mouse brain. Given the clinical invasiveness of NSC grafting into brain tissue, profound characterisation and understanding of early post-transplantation events is imperative to claim safety and efficacy of cell-based interventions.Here, we applied in vivo bioluminescence imaging (BLI) and post-mortem quantitative histological analysis to determine the localisation and survival of grafted NSCs at early time points post-transplantation.An initial dramatic cell loss (up to 80% of grafted cells) due to apoptosis could be observed within the first 24 hours post-implantation, coinciding with a highly hypoxic NSC graft environment. Subsequently, strong spatiotemporal microglial and astroglial cell responses were initiated, which stabilised by day 5 post-implantation and remained present during the whole observation period. Moreover, the increase in astrocyte density was associated with a high degree of astroglial scarring within and surrounding the graft site. During the two-week follow up in this study, the NSC graft site underwent extensive remodelling with NSC graft survival further declining to around 1% of the initial number of grafted cells.The present study quantitatively describes the early post-transplantation events following NSC grafting in the adult mouse brain and warrants that such intervention is directly associated with a high degree of cell loss, subsequently followed by strong glial cell responses.
|Galanin transgenic mice with elevated circulating galanin levels alleviate demyelination in a cuprizone-induced MS mouse model. |
Zhang, L; Yu, W; Schroedter, I; Kong, J; Vrontakis, M
PloS one 7 e33901 2012
Multiple Sclerosis (MS) is a demyelinating autoimmune disease of the central nervous system (CNS) with a presumed autoimmune etiology. Approved treatments for MS are immunoregulatory and are able to reduce the inflammatory components of the disease. However, these treatments do not suppress progressive clinical disability. Approaches that directly protect myelin-producing oligodendrocytes and enhance remyelination are likely to improve long-term outcomes and reduce the rate of axonal damage. Galanin (GAL) is a bioactive neuropeptide that is widely distributed throughout the nervous system and has diverse neuromodulatory effects. In this study, using the cuprizone (CPZ) demyelination model of MS, we demonstrate that GAL has pronounced neuroprotective effects with respect to demyelination and remyelination. Using our GAL transgenic mouse (GAL-Tg), we identified a novel attenuation of OLs against CPZ induced demyelination, which was exerted independently of progenitor cells. Alleviation of myelin breakdown in the GAL-Tg mice was observed to be significant. Furthermore, we observed changes in the expression of the GAL receptor GalR1 during the demyelination and remyelination processes. Our data strongly indicate that GAL has the capacity to influence the outcome of primary insults that directly target OLs, as opposed to cases where immune activation is the primary pathogenic event. Taken together, these results suggest that GAL is a promising next-generation target for the treatment of MS.
|Altered expression of brain monocarboxylate transporter 1 in models of temporal lobe epilepsy. |
Lauritzen, F; Perez, EL; Melillo, ER; Roh, JM; Zaveri, HP; Lee, TS; Wang, Y; Bergersen, LH; Eid, T
Neurobiology of disease 45 165-76 2012
Monocarboxylate transporter 1 (MCT1) facilitates the transport of monocarboxylate fuels (lactate, pyruvate and ketone bodies) and acidic drugs, such as valproic acid, across cell membranes. We recently reported that MCT1 is deficient on microvessels in the epileptogenic hippocampal formation in patients with medication-refractory temporal lobe epilepsy (TLE). To further define the role of MCT1 in the pathophysiology of TLE, we used immunohistochemistry and stereological analysis to localize and quantify the transporter in the hippocampal formation in three novel and highly relevant rat models of TLE and in nonepileptic control animals. One model utilizes methionine sulfoximine to induce brain glutamine synthetase deficiency and recurrent limbic seizures, while two models employ an episode of perforant pathway stimulation to cause epilepsy. MCT1 was lost on microvessels and upregulated on astrocytes in the hippocampal formation in all models of TLE. Notably, the loss of MCT1 on microvessels was not due to a reduction in microvessel density. The similarities in MCT1 expression among human subjects with TLE and several animal models of the disease strongly suggest a critical role of this molecule in the pathogenesis of TLE. We hypothesize that the downregulation of MCT1 may promote seizures via impaired uptake of ketone bodies and antiepileptic drugs by the epileptogenic brain. We also propose that the overexpression of MCT1 on astrocytes may lead to increased uptake or release of monocarboxylates by these cells, with important implications for brain metabolism and excitability. These hypotheses can now be rigorously tested in several animal models that replicate key features of human TLE.
|Adenosine kinase as a target for therapeutic antisense strategies in epilepsy. |
Theofilas, P; Brar, S; Stewart, KA; Shen, HY; Sandau, US; Poulsen, D; Boison, D
Epilepsia 52 589-601 2011
Given the high incidence of refractory epilepsy, novel therapeutic approaches and concepts are urgently needed. To date, viral-mediated delivery and endogenous expression of antisense sequences as a strategy to prevent seizures have received little attention in epilepsy therapy development efforts. Here we validate adenosine kinase (ADK), the astrocyte-based key negative regulator of the brain's endogenous anticonvulsant adenosine, as a potential therapeutic target for antisense-mediated seizure suppression.We developed adenoassociated virus 8 (AAV8)-based gene therapy vectors to selectively modulate ADK expression in astrocytes. Cell type selectivity was achieved by expressing an Adk-cDNA in sense or antisense orientation under the control of an astrocyte-specific gfaABC1D promoter. Viral vectors where injected into the CA3 of wild-type mice or spontaneously epileptic Adk-tg transgenic mice that overexpress ADK in brain. After virus injection, ADK expression was assessed histologically and biochemically. In addition, intracranial electroencephalography (EEG) recordings were obtained.We demonstrate in wild-type mice that viral overexpression of ADK within astrocytes is sufficient to trigger spontaneous recurrent seizures in the absence of any other epileptogenic event, whereas ADK downregulation via AAV8-mediated RNA interference almost completely abolished spontaneous recurrent seizures in Adk-tg mice.Our data demonstrate that modulation of astrocytic ADK expression can trigger or prevent seizures, respectively. This is the first study to use an antisense approach to validate ADK as a rational therapeutic target for the treatment of epilepsy and suggests that gene therapies based on the knock down of ADK might be a feasible approach to control seizures in refractory epilepsy.
|Spontaneous reactive astrogliosis in the dentate gyrus of Bax-deficient mice. |
Kim TW, Kim H, Sun W.
Molecules and cells 31 379-83 2011
Astrocytes play critical roles in many aspects of brain functions via modulation of neurotransmission, metabolism, and structural remodeling in response to physiological or pathological stimuli. Activation of astrocytes is a common phenomenon in many brain pathologies such as stroke, trauma, and neurodegenerative diseases. In this study, we found that gene deletion of the pro-apoptotic gene Bax (Bax-knockout) resulted in a spontaneous reactive astrogliosis in the dentate gyrus, as evidenced by the increased number/volume of astrocytes and cytoplasmic localization of the Olig2 protein. On the other hand, there was no evidence for microglial activation in the dentate gyrus of Bax-knockout mice. Previously, we reported that Bax-knockout mice failed to execute programmed cell death of adult-produced neurons, but the surplus neurons eventually impaired normal synaptic connections and dendritic arborization of dentate gyrus neurons. Therefore, we propose that the reactive astrocytes in the Baxknockout mice may play a role in tissue remodeling of the dentate gyrus following a failure in the programmed cell death of adult-produced neurons.
|Enhanced neuronal Met signalling levels in ALS mice delay disease onset. |
Genestine, M; Caricati, E; Fico, A; Richelme, S; Hassani, H; Sunyach, C; Lamballe, F; Panzica, GC; Pettmann, B; Helmbacher, F; Raoul, C; Maina, F; Dono, R
Cell death & disease 2 e130 2011
Signalling by receptor tyrosine kinases (RTKs) coordinates basic cellular processes during development and in adulthood. Whereas aberrant RTK signalling can lead to cancer, reactivation of RTKs is often found following stress or cell damage. This has led to the common belief that RTKs can counteract degenerative processes and so strategies to exploit them for therapy have been extensively explored. An understanding of how RTK stimuli act at cellular levels is needed, however, to evaluate their mechanism of therapeutic action. In this study, we genetically explored the biological and functional significance of enhanced signalling by the Met RTK in neurons, in the context of a neurodegenerative disease. Conditional met-transgenic mice, namely Rosa26(LacZ-stop-Met), have been engineered to trigger increased Met signalling in a temporal and tissue-specific regulated manner. Enhancing Met levels in neurons does not affect either motor neuron (MN) development or maintenance. In contrast, increased neuronal Met in amyotrophic lateral sclerosis (ALS) mice prolongs life span, retards MN loss, and ameliorates motor performance, by selectively delaying disease onset. Thus, our studies highlight the properties of RTKs to counteract toxic signals in a disease characterized by dysfunction of multiple cell types by acting in MNs. Moreover, they emphasize the relevance of genetically assessing the effectiveness of agents targeting neurons during ALS evolution.
|Peripheral nerve pathology, including aberrant Schwann cell differentiation, is ameliorated by doxycycline in a laminin-α2-deficient mouse model of congenital muscular dystrophy. |
Homma, S; Beermann, ML; Miller, JB
Human molecular genetics 20 2662-72 2011
The most common form of childhood congenital muscular dystrophy, Type 1A (MDC1A), is caused by mutations in the human LAMA2 gene that encodes the laminin-α2 subunit. In addition to skeletal muscle deficits, MDC1A patients typically show a loss of peripheral nerve function. To identify the mechanisms underlying this loss of nerve function, we have examined pathology and cell differentiation in sciatic nerves and ventral roots of the laminin-α2-deficient (Lama2(-/-)) mice, which are models for MDC1A. We found that, compared with wild-type, sciatic nerves of Lama2(-/-) mice had a significant increase in both proliferating (Ki67+) cells and premyelinating (Oct6+) Schwann cells, but also had a significant decrease in both immature/non-myelinating [glial fibrillary acidic protein (GFAP)(+)] and myelinating (Krox20+) Schwann cells. To extend our previous work in which we found that doxycycline, which has multiple effects on mammalian cells, improves motor behavior and more than doubles the median life-span of Lama2(-/-) mice, we also determined how nerve pathology was affected by doxycycline treatment. We found that myelinating (Krox20+) Schwann cells were significantly increased in doxycycline-treated compared with untreated sciatic nerves. In addition, doxycycline-treated peripheral nerves had significantly less pathology as measured by assays such as amount of unmyelinated or disorganized axons. This study thus identified aberrant proliferation and differentiation of Schwann cells as key components of pathogenesis in peripheral nerves and provided proof-of-concept that pharmaceutical therapy can be of potential benefit for peripheral nerve dysfunction in MDC1A.
|IL-1β stimulates COX-2 dependent PGE₂ synthesis and CGRP release in rat trigeminal ganglia cells. |
Neeb, L; Hellen, P; Boehnke, C; Hoffmann, J; Schuh-Hofer, S; Dirnagl, U; Reuter, U
PloS one 6 e17360 2011
Pro-inflammatory cytokines like Interleukin-1 beta (IL-1β) have been implicated in the pathophysiology of migraine and inflammatory pain. The trigeminal ganglion and calcitonin gene-related peptide (CGRP) are crucial components in the pathophysiology of primary headaches. 5-HT1B/D receptor agonists, which reduce CGRP release, and cyclooxygenase (COX) inhibitors can abort trigeminally mediated pain. However, the cellular source of COX and the interplay between COX and CGRP within the trigeminal ganglion have not been clearly identified.1. We used primary cultured rat trigeminal ganglia cells to assess whether IL-1β can induce the expression of COX-2 and which cells express COX-2. Stimulation with IL-1β caused a dose and time dependent induction of COX-2 but not COX-1 mRNA. Immunohistochemistry revealed expression of COX-2 protein in neuronal and glial cells. 2. Functional significance was demonstrated by prostaglandin E2 (PGE(2)) release 4 hours after stimulation with IL-1β, which could be aborted by a selective COX-2 (parecoxib) and a non-selective COX-inhibitor (indomethacin). 3. Induction of CGRP release, indicating functional neuronal activation, was seen 1 hour after PGE(2) and 24 hours after IL-1β stimulation. Immunohistochemistry showed trigeminal neurons as the source of CGRP. IL-1β induced CGRP release was blocked by parecoxib and indomethacin, but the 5-HT1B/D receptor agonist sumatriptan had no effect.We identified a COX-2 dependent pathway of cytokine induced CGRP release in trigeminal ganglia neurons that is not affected by 5-HT1B/D receptor activation. Activation of neuronal and glial cells in the trigeminal ganglion by IL-β leads to an elevated expression of COX-2 in these cells. Newly synthesized PGE(2) (by COX-2) in turn activates trigeminal neurons to release CGRP. These findings support a glia-neuron interaction in the trigeminal ganglion and demonstrate a sequential link between COX-2 and CGRP. The results could help to explain the mechanism of action of COX-2 inhibitors in migraine.Artículo Texto completo
|Overweight worsens apoptosis, neuroinflammation and blood-brain barrier damage after hypoxic ischemia in neonatal brain through JNK hyperactivation. |
Tu, YF; Tsai, YS; Wang, LW; Wu, HC; Huang, CC; Ho, CJ
Journal of neuroinflammation 8 40 2011
Apoptosis, neuroinflammation and blood-brain barrier (BBB) damage affect the susceptibility of the developing brain to hypoxic-ischemic (HI) insults. c-Jun N-terminal kinase (JNK) is an important mediator of insulin resistance in obesity. We hypothesized that neonatal overweight aggravates HI brain damage through JNK hyperactivation-mediated upregulation of neuronal apoptosis, neuroinflammation and BBB leakage in rat pups.Overweight (OF) pups were established by reducing the litter size to 6, and control (NF) pups by keeping the litter size at 12 from postnatal (P) day 1 before HI on P7. Immunohistochemistry and immunoblotting were used to determine the TUNEL-(+) cells and BBB damage, cleaved caspase-3 and poly (ADP-ribose) polymerase (PARP), and phospho-JNK and phospho-BimEL levels. Immunofluorescence was performed to determine the cellular distribution of phospho-JNK.Compared with NF pups, OF pups had a significantly heavier body-weight and greater fat deposition on P7. Compared with the NF-HI group, the OF-HI group showed significant increases of TUNEL-(+) cells, cleaved levels of caspase-3 and PARP, and ED1-(+) activated microglia and BBB damage in the cortex 24 hours post-HI. Immunofluorescence of the OF-HI pups showed that activated-caspase 3 expression was found mainly in NeuN-(+) neurons and RECA1-(+) vascular endothelial cells 24 hours post-HI. The OF-HI group also had prolonged escape latency in the Morris water maze test and greater brain-volume loss compared with the NF-HI group when assessed at adulthood. Phospho-JNK and phospho-BimEL levels were higher in OF-HI pups than in NF-HI pups immediately post-HI. JNK activation in OF-HI pups was mainly expressed in neurons, microglia and vascular endothelial cells. Inhibiting JNK activity by AS601245 caused more attenuation of cleaved caspase-3 and PARP, a greater reduction of microglial activation and BBB damage post-HI, and significantly reduced brain damage in OF-HI than in NF-HI pups.Neonatal overweight increased HI-induced neuronal apoptosis, microglial activation and BBB damage, and aggravated HI brain damage in rat pups through JNK hyperactivation.
|Bone morphogenetic protein receptor expressions in the adult rat brain. |
M Miyagi,S Mikawa,T Hasegawa,S Kobayashi,K Sho,Y Matsuyama,K Sato
Neuroscience 176 2011
Bone morphogenetic proteins (BMP) are members of the transforming growth factor ? (TGF-?) superfamily. BMPs exert its biological functions by interacting with membrane bound receptors belonging to the serine/threonine kinase family including bone morphogenetic protein receptor I (BMPRIA, BMPRIB) and type II (BMPRII). Although BMPR expressions have been well described in the early development of the CNS, little information is available for their expressions in the adult CNS. We, thus, investigated BMPR expressions in the adult rat CNS using immunohistochemistry. Here, we show that BMPRIA, IB and II proteins are widely expressed throughout the adult CNS. Interestingly, we observed that BMPRIA, IB and II proteins are abundantly expressed in many kinds of axons. In addition, we found that BAMRIB-IR was preferentially expressed in dendrites of many neurons throughout the CNS, while BMPRIA was mainly expressed in cell bodies, showing that BMPRIA and BMPRIB are differentially targeted in a single neuron. In addition, besides abundant BMPR expressions in neurons, we exhibited BMPR expressions in astrocytes and ependymal cells. These data indicate that BMPRs are more widely expressed throughout the adult CNS than previously reported, and their continued abundant expressions in the adult brain strongly support the idea that BMPRs play pivotal roles also in the adult brain.
|Cellular organization of adult neurogenesis in the Common Marmoset. |
Bunk, Eva C, et al.
Aging Cell, 10: 28-38 (2011) 2011
Adult neurogenesis within the subgranular zone (SGZ) of the hippocampal dentate gyrus and the subventricular zone (SVZ) of the lateral ventricle (LV) has been most intensely studied within the brains of rodents such as mice and rats. However, little is known about the cell types and processes involved in adult neurogenesis within primates such as the common marmoset (Callithrix jacchus). Moreover, substantial differences seem to exist between the neurogenic niche of the LV between rodents and humans. Here, we set out to use immunohistochemical and autogradiographic analysis to characterize the anatomy of the neurogenic niches and the expression of cell type-specific markers in those niches in the adult common marmoset brain. Moreover, we demonstrate significant differences in the activity of neurogenesis in the adult marmoset brain compared to the adult mouse brain. Finally, we provide evidence for ongoing proliferation of neuroblasts within both the SGZ and SVZ of the adult brain and further show that the age-dependent decline of neurogenesis in the hippocampus is associated with a decrease in neuroblast cells.
|Expression of leptin and leptin receptor isoforms in the rat and human carotid body. |
Porzionato A, Rucinski M, Macchi V, Stecco C, Castagliuolo I, Malendowicz LK, De Caro R
Brain Res 2011
Leptin is known to play a role in the modulation of metabolism and control of breathing acting mainly on central nervous structures, although additional actions on peripheral arterial chemoreceptors have also been suggested in the literature. We therefore examined by means of immunohistochemistry the expression of leptin and leptin receptors in the carotid bodies of rats and humans. Leptin expression and relative expression of leptin receptor isoforms were also studied in rats by real-time PCR. No leptin or leptin receptor immunoreactivities were visible in the type II cells of either series. In rat carotid bodies, diffuse positive stainings for leptin and leptin receptors (both with antibody recognizing all receptor isoforms and antibody specific for Ob-Rb) were observed in type I cells. In human carotid bodies, the mean percentage (± Standard Error) of leptin immunoreactive type I cells was 39.4±5.1% and the percentages of leptin receptor immunoreactive type I cells were 57.3±3.9% with antibody recognizing all receptor isoforms and 33.3±4.2% with antibody specific for isoform Ob-Rb. Double immunofluorescences with anti-tyrosine hydroxylase (type I cell marker), and anti-Glial Fibrillary Acidic Protein (type II cell markers) confirmed the selective location of leptin and Ob-Rb in type I cells. Real time-PCR showed the expression of leptin and Ob-Ra, Ob-Rb, Ob-Rc and Ob-Rf isoform mRNA in the rat carotid body, levels of expression being Ob-Rf>Ob-Rc>Ob-Ra>Ob-Rb. Ob-Re mRNA was not detected. The above findings suggest a role of circulating or locally produced leptin in the regulation of chemoreceptor discharge and/or metabolic sensing function, by means of direct action on type I cells.Copyright © 2011. Published by Elsevier B.V.
|Calpain-mediated cleavage of Beclin-1 and autophagy deregulation following retinal ischemic injury in vivo. |
Russo, R; Berliocchi, L; Adornetto, A; Varano, GP; Cavaliere, F; Nucci, C; Rotiroti, D; Morrone, LA; Bagetta, G; Corasaniti, MT
Cell death & disease 2 e144 2011
Autophagy is the major intracellular degradation pathway that regulates long-lived proteins and organelles turnover. This process occurs at basal levels in all cells but it is rapidly upregulated in response to starvation and cellular stress. Although being recently implicated in neurodegeneration, it remains still unclear whether autophagy has a detrimental or protective role. In this study, we investigated the dynamics of the autophagic process in retinal tissue that has undergone transient ischemia, an experimental model that recapitulates features of ocular pathologies, including glaucoma, anterior ischemic optic neuropathy and retinal vessels occlusion. Retinal ischemia, induced in adult rats by increasing the intraocular pressure, was characterized by a reduction in the phosphatidylethanolamine-modified form of LC3 (LC3II) and by a significant decrease in Beclin-1. The latter event was associated with a proteolytic cleavage of Beclin-1, leading to the accumulation of a 50-kDa fragment. This event was prevented by intravitreal treatment with the non-competitive N-methyl-D-aspartate antagonist MK801 and calpain inhibitors or by calpain knockdown. Blockade of autophagy by pharmacological inhibition or Beclin-1 silencing in RGC-5 increased cell death, suggesting a pro-survival role of the autophagic process in this neuronal cell type. Altogether, our results provide original evidence for calpain-mediated cleavage of Beclin-1 and deregulation of basal autophagy in the rat retina that has undergone ocular ischemia/reperfusion injury.
|Neural stem cells reduce hippocampal tau and reelin accumulation in aged Ts65Dn Down syndrome mice. |
D S Kern,K N Maclean,H Jiang,E Y Synder,J R Sladek,K B Bjugstad
Cell transplantation 20 2011
Tau accumulation, in the form of neurofibrillary tangles (NFT), is an early neuropathological characteristic of Alzheimer's disease (AD) and early onset AD frequently seen in Down syndrome (DS). We investigated the presence of tau accumulation in the brains of aging DS mice using the Ts65Dn mouse model. All aged mice appeared to have substantial clusters of extracellular granules that were positive for tau and reelin, but not for amyloid-? or APP. These clusters were found primarily in CA1 of the hippocampus. In addition, the aged trisomic DS mice had a significantly greater accumulation of extracellular tau/reelin granular deposits compared to disomic littermates. These granules were similar to those described by others who also found extracellular proteinous granules in the brains of non-DS mice engineered to model aging and/or AD. When neural stem cells (NSC) were implanted unilaterally into the hippocampus of the Ts65Dn mice, the tau/reelin-positive granules were significantly reduced in both trisomic and disomic mice. Our findings indicate that changes in tau/reelin-positive granules could be used as an index for neuropathological assessment in aging DS and AD. Furthermore, changes in granule density could be used to test the efficacy of novel treatments, such as NSC implantation. Lastly, it is speculated that the unique abilities of NSC to migrate and express growth factors might be a contributing factor to reducing tau/reelin accumulation in aging DS and AD.
|Neuroadaptive changes in cerebellar neurons induced by chronic exposure to IL-6. |
Gruol, DL; Puro, A; Hao, C; Blakely, P; Janneke, E; Vo, K
Journal of neuroimmunology 239 28-36 2011
IL-6 is an important signaling molecule in the CNS. CNS neurons express IL-6 receptors and their signal transduction molecules, consistent with a role for IL-6 in neuronal physiology. Research indicates that IL-6 levels are low in the normal brain but can be significantly elevated in CNS injury and disease. Relatively little is known about how the elevated levels of IL-6 affect neurons. In the current study we show that under conditions of chronic exposure, IL-6 induces alterations in the level of protein expression in developing CNS cells. Such changes may play a role in the altered CNS function observed in CNS conditions associated with elevated levels of IL-6 in the CNS.
|The contribution of gliosis to diffusion tensor anisotropy and tractography following traumatic brain injury: validation in the rat using Fourier analysis of stained tissue sections. |
Budde, MD; Janes, L; Gold, E; Turtzo, LC; Frank, JA
Brain : a journal of neurology 134 2248-60 2011
Diffusion tensor imaging is highly sensitive to the microstructural integrity of the brain and has uncovered significant abnormalities following traumatic brain injury not appreciated through other methods. It is hoped that this increased sensitivity will aid in the detection and prognostication in patients with traumatic injury. However, the pathological substrates of such changes are poorly understood. Specifically, decreases in fractional anisotropy derived from diffusion tensor imaging are consistent with axonal injury, myelin injury or both in white matter fibres. In contrast, in both humans and animal models, increases in fractional anisotropy have been suggested to reflect axonal regeneration and plasticity, but the direct histological evidence for such changes remains tenuous. We developed a method to quantify the anisotropy of stained histological sections using Fourier analysis, and applied the method to a rat controlled cortical impact model to identify the specific pathological features that give rise to the diffusion tensor imaging changes in subacute to chronic traumatic brain injury. A multiple linear regression was performed to relate the histological measurements to the measured diffusion tensor changes. The results show that anisotropy was significantly increased (P less than 0.001) in the perilesioned cortex following injury. Cortical anisotropy was independently associated (standardized β = 0.62, P = 0.04) with the coherent organization of reactive astrocytes (i.e. gliosis) and was not attributed to axons. By comparison, a decrease in white matter anisotropy (P less than 0.001) was significantly related to demyelination (β = 0.75, P = 0.0015) and to a lesser extent, axonal degeneration (β = -0.48, P = 0.043). Gliosis within the lesioned cortex also influenced diffusion tensor tractography, highlighting the fact that spurious tracts in the injured brain may not necessarily reflect continuous axons and may instead depict glial scarring. The current study demonstrates a novel method to relate pathology to diffusion tensor imaging findings, elucidates the underlying mechanisms of anisotropy changes following traumatic brain injury and significantly impacts the clinical interpretation of diffusion tensor imaging findings in the injured brain.
|Lateral fluid percussion: model of traumatic brain injury in mice. |
Alder, J; Fujioka, W; Lifshitz, J; Crockett, DP; Thakker-Varia, S
Journal of visualized experiments : JoVE 2011
Traumatic brain injury (TBI) research has attained renewed momentum due to the increasing awareness of head injuries, which result in morbidity and mortality. Based on the nature of primary injury following TBI, complex and heterogeneous secondary consequences result, which are followed by regenerative processes (1,2). Primary injury can be induced by a direct contusion to the brain from skull fracture or from shearing and stretching of tissue causing displacement of brain due to movement (3,4). The resulting hematomas and lacerations cause a vascular response (3,5), and the morphological and functional damage of the white matter leads to diffuse axonal injury (6-8). Additional secondary changes commonly seen in the brain are edema and increased intracranial pressure (9). Following TBI there are microscopic alterations in biochemical and physiological pathways involving the release of excitotoxic neurotransmitters, immune mediators and oxygen radicals (10-12), which ultimately result in long-term neurological disabilities (13,14). Thus choosing appropriate animal models of TBI that present similar cellular and molecular events in human and rodent TBI is critical for studying the mechanisms underlying injury and repair. Various experimental models of TBI have been developed to reproduce aspects of TBI observed in humans, among them three specific models are widely adapted for rodents: fluid percussion, cortical impact and weight drop/impact acceleration (1). The fluid percussion device produces an injury through a craniectomy by applying a brief fluid pressure pulse on to the intact dura. The pulse is created by a pendulum striking the piston of a reservoir of fluid. The percussion produces brief displacement and deformation of neural tissue (1,15). Conversely, cortical impact injury delivers mechanical energy to the intact dura via a rigid impactor under pneumatic pressure (16,17). The weight drop/impact model is characterized by the fall of a rod with a specific mass on the closed skull (18). Among the TBI models, LFP is the most established and commonly used model to evaluate mixed focal and diffuse brain injury (19). It is reproducible and is standardized to allow for the manipulation of injury parameters. LFP recapitulates injuries observed in humans, thus rendering it clinically relevant, and allows for exploration of novel therapeutics for clinical translation (20). We describe the detailed protocol to perform LFP procedure in mice. The injury inflicted is mild to moderate, with brain regions such as cortex, hippocampus and corpus callosum being most vulnerable. Hippocampal and motor learning tasks are explored following LFP.
|Directed differentiation of functional astroglial subtypes from human pluripotent stem cells. |
Krencik, R; Zhang, SC
Nature protocols 6 1710-7 2011
Regionally and functionally diverse types of astrocytes exist throughout the central nervous system and participate in nearly every aspect of normal and abnormal neural function. Therefore, human astrocyte subtypes are useful tools for understanding brain function, modulating disease processes and promoting neural regeneration. Here we describe a protocol for directed differentiation and maintenance of functional astroglia from human pluripotent stem cells in a chemically defined system. Human stem cells are first differentiated into neuroepithelial cells with or without exogenous patterning molecules (days 0-21). Regular dissociation of the neuroepithelial clusters in suspension, and in the presence of mitogens, permits generation of astroglial subtypes over a long-term expansion (days 21-90). Finally, the astroglial progenitors are either amplified for an extended time or differentiated into functional astrocytes on removal of mitogens and the addition of ciliary neurotrophic factor (days greater than 90). This method generates robust populations of functionally diversified astrocytes with high efficiency.
|Laminar-specific and developmental expression of aquaporin-4 in the mouse hippocampus. |
Hsu, MS; Seldin, M; Lee, DJ; Seifert, G; Steinhäuser, C; Binder, DK
Neuroscience 178 21-32 2011
Mice deficient in the water channel aquaporin-4 (AQP4) demonstrate increased seizure duration in response to hippocampal stimulation as well as impaired extracellular K+ clearance. However, the expression of AQP4 in the hippocampus is not well described. In this study, we investigated (i) the developmental, laminar and cell-type specificity of AQP4 expression in the hippocampus; (ii) the effect of Kir4.1 deletion on AQP4 expression; and (iii) performed Western blot and RT-PCR analyses. AQP4 immunohistochemistry on coronal sections from wild-type (WT) or Kir4.1-/- mice revealed a developmentally-regulated and laminar-specific pattern, with highest expression in the CA1 stratum lacunosum-moleculare (SLM) and the molecular layer (ML) of the dentate gyrus (DG). AQP4 was colocalized with the glial markers glial fibrillary acidic protein (GFAP) and S100β in the hippocampus, and was also ubiquitously expressed on astrocytic endfeet around blood vessels. No difference in AQP4 immunoreactivity was observed in Kir4.1-/- mice. Electrophysiological and postrecording RT-PCR analyses of individual cells revealed that AQP4 and Kir4.1 were co-expressed in nearly all CA1 astrocytes. In NG2 cells, AQP4 was also expressed at the transcript level. This study is the first to examine subregional AQP4 expression during development of the hippocampus. The strikingly high expression of AQP4 in the CA1 SLM and DG ML identifies these regions as potential sites of astrocytic K+ and H2O regulation. These results begin to delineate the functional capabilities of hippocampal subregions and cell types for K+ and H2O homeostasis, which is critical to excitability and serves as a potential target for modulation in diverse diseases.
|Spinal phosphinositide 3-kinase-Akt-mammalian target of rapamycin signaling cascades in inflammation-induced hyperalgesia. |
Xu, Q; Fitzsimmons, B; Steinauer, J; O'Neill, A; Newton, AC; Hua, XY; Yaksh, TL
The Journal of neuroscience : the official journal of the Society for Neuroscience 31 2113-24 2011
Phosphinositide 3-kinase (PI3K), Akt, and their downstream kinase, mammalian target of rapamycin (mTOR), are implicated in neural plasticity. The functional linkages of this signaling cascade in spinal dorsal horn and their role in inflammatory hyperalgesia have not been elucidated. In the present work, we identified the following characteristics of this cascade. (1) Local inflammation led to increase in rat dorsal horn phosphorylation (activation) of Akt (pAkt) and mTOR (pmTOR), as assessed by Western blotting and immunocytochemistry. (2) Increased pAkt and pmTOR were prominent in neurons in laminae I, III, and IV, whereas pmTOR and its downstream targets (pS6, p4EBP) were also observed in glial cells. (3) Intrathecal treatment with inhibitors to PI3K or Akt attenuated Formalin-induced second-phase flinching behavior, as well as carrageenan-induced thermal hyperalgesia and tactile allodynia. (4) Intrathecal rapamycin (an mTORC1 inhibitor) displayed anti-hyperalgesic effect in both inflammatory pain models. Importantly, intrathecal wortmannin at anti-hyperalgesic doses reversed the evoked increase not only in Akt but also in mTORC1 signaling (pS6/p4EBP). (5) pAkt and pmTOR are expressed in neurokinin 1 receptor-positive neurons in laminae I-III after peripheral inflammation. Intrathecal injection of Substance P activated this cascade (increased phosphorylation) and resulted in hyperalgesia, both of which effects were blocked by intrathecal wortmannin and rapamycin. Together, these findings reveal that afferent inputs trigged by peripheral inflammation initiate spinal activation of PI3K-Akt-mTOR signaling pathway, a component of which participates in neuronal circuits of facilitated pain processing.
|miR-34a regulates mouse neural stem cell differentiation. |
Aranha, MM; Santos, DM; Solá, S; Steer, CJ; Rodrigues, CM
PloS one 6 e21396 2011
MicroRNAs (miRNAs or miRs) participate in the regulation of several biological processes, including cell differentiation. Recently, miR-34a has been implicated in the differentiation of monocyte-derived dendritic cells, human erythroleukemia cells, and mouse embryonic stem cells. In addition, members of the miR-34 family have been identified as direct p53 targets. However, the function of miR-34a in the control of the differentiation program of specific neural cell types remains largely unknown. Here, we investigated the role of miR-34a in regulating mouse neural stem (NS) cell differentiation.miR-34a overexpression increased postmitotic neurons and neurite elongation of mouse NS cells, whereas anti-miR-34a had the opposite effect. SIRT1 was identified as a target of miR-34a, which may mediate the effect of miR-34a on neurite elongation. In addition, acetylation of p53 (Lys 379) and p53-DNA binding activity were increased and cell death unchanged after miR-34a overexpression, thus reinforcing the role of p53 during neural differentiation. Interestingly, in conditions where SIRT1 was activated by pharmacologic treatment with resveratrol, miR-34a promoted astrocytic differentiation, through a SIRT1-independent mechanism.Our results provide new insight into the molecular mechanisms by which miR-34a modulates neural differentiation, suggesting that miR-34a is required for proper neuronal differentiation, in part, by targeting SIRT1 and modulating p53 activity.
|Histone deacetylase inhibitors preserve white matter structure and function during ischemia by conserving ATP and reducing excitotoxicity. |
Baltan, S; Murphy, SP; Danilov, CA; Bachleda, A; Morrison, RS
The Journal of neuroscience : the official journal of the Society for Neuroscience 31 3990-9 2011
The importance of white matter (WM) injury to stroke pathology has been underestimated in experimental animal models and this may have contributed to the failure to translate potential therapeutics into the stroke clinic. Histone deacetylase (HDAC) inhibitors are neuroprotective and also promote neurogenesis. These properties make them ideal candidates for stroke therapy. In a pure WM tract (isolated mouse optic nerve), we show that pan- and class I-specific HDAC inhibitors, administered before or after a period of oxygen and glucose deprivation (OGD), promote functional recovery of axons and preserve WM cellular architecture. This protection correlates with the upregulation of an astrocyte glutamate transporter, delayed and reduced glutamate accumulation during OGD, preservation of axonal mitochondria and oligodendrocytes, and maintenance of ATP levels. Interestingly, the expression of HDACs 1, 2, and 3 is localized to astrocytes, suggesting that changes in glial cell gene transcription and/or protein acetylation may confer protection to axons. Our findings suggest that a therapeutic opportunity exists for the use of HDAC inhibitors, targeting mitochondrial energy regulation and excitotoxicity in ischemic WM injury.
|IFNγ triggers a LIGHT-dependent selective death of motoneurons contributing to the non-cell-autonomous effects of mutant SOD1. |
Aebischer, J; Cassina, P; Otsmane, B; Moumen, A; Seilhean, D; Meininger, V; Barbeito, L; Pettmann, B; Raoul, C
Cell death and differentiation 18 754-68 2011
Amyotrophic lateral sclerosis (ALS) is an incurable neurodegenerative disease that primarily affects motoneurons in the brain and spinal cord. Dominant mutations in superoxide dismutase-1 (SOD1) cause a familial form of ALS. Mutant SOD1-damaged glial cells contribute to ALS pathogenesis by releasing neurotoxic factors, but the mechanistic basis of the motoneuron-specific elimination is poorly understood. Here, we describe a motoneuron-selective death pathway triggered by activation of lymphotoxin-β receptor (LT-βR) by LIGHT, and operating by a novel signaling scheme. We show that astrocytes expressing mutant SOD1 mediate the selective death of motoneurons through the proinflammatory cytokine interferon-γ (IFNγ), which activates the LIGHT-LT-βR death pathway. The expression of LIGHT and LT-βR by motoneurons in vivo correlates with the preferential expression of IFNγ by motoneurons and astrocytes at disease onset and symptomatic stage in ALS mice. Importantly, the genetic ablation of Light in an ALS mouse model retards progression, but not onset, of the disease and increases lifespan. We propose that IFNγ contributes to a cross-talk between motoneurons and astrocytes causing the selective loss of some motoneurons following activation of the LIGHT-induced death pathway.
|Biochip∕laser cell deposition system to assess polarized axonal growth from single neurons and neuron∕glia pairs in microchannels with novel asymmetrical geometries. |
Pirlo, RK; Sweeney, AJ; Ringeisen, BR; Kindy, M; Gao, BZ
Biomicrofluidics 5 13408 2011
Axon path-finding plays an important role in normal and pathogenic brain development as well as in neurological regenerative medicine. In both scenarios, axonal growth is influenced by the microenvironment including the soluble molecules and contact-mediated signaling from guiding cells and cellular matrix. Microfluidic devices are a powerful tool for creating a microenvironment at the single cell level. In this paper, an asymmetrical-channel-based biochip, which can be later incorporated into microfluidic devices for neuronal network study, was developed to investigate geometric as well as supporting cell control of polarized axonal growth in forming a defined neuronal circuitry. A laser cell deposition system was used to place single cells, including neuron-glia pairs, into specific microwells of the device, enabling axonal growth without the influence of cytophilic∕phobic surface patterns. Phase microscopy showed that a novel "snag" channel structure influenced axonal growth in the intended direction 4:1 over the opposite direction. In heterotypic experiments, glial cell influence over the axonal growth path was observed with time-lapse microscopy. Thus, it is shown that single cell and heterotypic neuronal path-finding models can be developed in laser patterned biochips.
|Glypican-1, phosphacan/receptor protein-tyrosine phosphatase-zeta/beta and its ligand, tenascin-C, are expressed by neural stem cells and neural cells derived from embryonic stem cells. |
Abaskharoun M, Bellemare M, Lau E, Margolis RU
ASN Neuro 2 e00039. 2010
The heparan sulfate proteoglycan glypican-1, the chondroitin sulfate proteoglycan phosphacan/RPTP (receptor protein-tyrosine phosphatase)-zeta/beta and the extracellular matrix protein tenascin-C were all found to be expressed by neural stem cells and by neural cells derived from them. Expression of proteoglycans and tenascin-C increased after retinoic acid induction of SSEA1-positive ES (embryonic stem) cells to nestin-positive neural stem cells, and after neural differentiation, proteoglycans and tenascin-C are expressed by both neurons and astrocytes, where they surround cell bodies and processes and in certain cases show distinctive expression patterns. With the exception of tenascin-C (whose expression may decrease somewhat), expression levels do not change noticeably during the following 2 weeks in culture. The significant expression, by neural stem cells and neurons and astrocytes derived from them, of two major heparan sulfate and chondroitin sulfate proteoglycans of nervous tissue and of tenascin-C, a high-affinity ligand of phosphacan/RPTP-zeta/beta, indicates that an understanding of their specific functional roles in stem cell neurobiology will be important for the therapeutic application of this new technology in facilitating nervous tissue repair and regeneration.Artículo Texto completo
|COL25A1 triggers and promotes Alzheimer's disease-like pathology in vivo. |
Tong, Y; Xu, Y; Scearce-Levie, K; Ptácek, LJ; Fu, YH
Neurogenetics 11 41-52 2010
Collagen XXV alpha 1 (COL25A1) is a collagenous type II transmembrane protein purified from senile plaques of Alzheimer's disease (AD) brains. COL25A1 alleles have been associated with increased risk for AD in a Swedish population. COL25A1 is specifically expressed in neurons and binds to aggregated Abeta in vitro. However, its contribution to the pathogenesis of AD and in vivo function are unknown. Here, we report that over-expression of COL25A1 in transgenic mice increases p35/p25 and beta-site APP-cleaving enzyme 1 (BACE1) levels, facilitates intracellular aggregation and extracellular matrix deposits of Abeta, and causes synaptophysin loss and astrocyte activation. COL25A1 mice displayed reduced anxiety-like behavior in elevated plus maze and open field tests and significantly slower swimming speed in Morris water maze. In stable cell lines, motifs in noncollagenous domains of COL25A1 were important for the induction of BACE1 expression. These findings demonstrate that COL25A1 leads to AD-like pathology in vivo. Modulation of COL25A1 function may represent an alternative therapeutic intervention for AD.Artículo Texto completo
|Locus ceruleus controls Alzheimer's disease pathology by modulating microglial functions through norepinephrine. |
Heneka, MT; Nadrigny, F; Regen, T; Martinez-Hernandez, A; Dumitrescu-Ozimek, L; Terwel, D; Jardanhazi-Kurutz, D; Walter, J; Kirchhoff, F; Hanisch, UK; Kummer, MP
Proceedings of the National Academy of Sciences of the United States of America 107 6058-63 2010
Locus ceruleus (LC)-supplied norepinephrine (NE) suppresses neuroinflammation in the brain. To elucidate the effect of LC degeneration and subsequent NE deficiency on Alzheimer's disease pathology, we evaluated NE effects on microglial key functions. NE stimulation of mouse microglia suppressed Abeta-induced cytokine and chemokine production and increased microglial migration and phagocytosis of Abeta. Induced degeneration of the locus ceruleus increased expression of inflammatory mediators in APP-transgenic mice and resulted in elevated Abeta deposition. In vivo laser microscopy confirmed a reduced recruitment of microglia to Abeta plaque sites and impaired microglial Abeta phagocytosis in NE-depleted APP-transgenic mice. Supplying the mice the norepinephrine precursor L-threo-DOPS restored microglial functions in NE-depleted mice. This indicates that decrease of NE in locus ceruleus projection areas facilitates the inflammatory reaction of microglial cells in AD and impairs microglial migration and phagocytosis, thereby contributing to reduced Abeta clearance. Consequently, therapies targeting microglial phagocytosis should be tested under NE depletion.
|Addition of glutamate to serum-free culture promotes recovery of electrical activity in adult hippocampal neurons in vitro. |
Edwards D, Das M, Molnar P, Hickman JJ
J Neurosci Methods 190 155-63. Epub 2010 May 7. 2010
A long-term cell culture system utilizing normal adult hippocampal neurons would represent an important tool that could be useful in research on the mature brain, neurological disorders and age-related neurological diseases. Historically, in vitro neuronal systems are derived from embryonic rather than mature brain tissue, a practice predicated upon difficulties in supporting regeneration, functional recovery and long-term survival of adult neurons in vitro. A few studies have shown that neurons derived from the hippocampal tissue of adult rats can survive and regenerate in vitro under serum-free conditions. However, while the adult neurons regenerated morphologically under these conditions, both the electrical activity characteristic of in vivo neurons as well as long-term neuronal survival was not consistently recovered in vitro. In this study, we report on the development of a defined culture system with the ability to support functional recovery and long-term survival of adult rat hippocampal neurons. In this system, the cell-adhesive substrate, N-1 [3-(trimethoxysilyl) propyl]-diethylenetriamine, supported neuronal attachment, regeneration, and long-term survival of adult neurons for more than 80 days in vitro. Additionally, the excitatory neurotransmitter glutamate, applied at 25muM for 1-7 days after morphological neuronal regeneration in vitro, enabled full recovery of neuronal electrical activity. This low concentration of glutamate promoted the recovery of neuronal electrical activity but with minimal excitotoxicity. These improvements allowed electrically active adult neurons to survive in vitro for several months, providing a stable test-bed for the long-term study of regeneration in adult-derived neuronal systems, especially for traumatic brain injury (TBI). Copyright 2010 Elsevier B.V. All rights reserved.
|Dynamics of neuroinflammation in the macrosphere model of arterio-arterial embolic focal ischemia: an approximation to human stroke patterns. |
Walberer, M; Rueger, MA; Simard, ML; Emig, B; Jander, S; Fink, GR; Schroeter, M
Experimental & translational stroke medicine 2 22 2010
Neuroinflammation evolves as a multi-facetted response to focal cerebral ischemia. It involves activation of resident glia cell populations, recruitment of blood-derived leucocytes as well as humoral responses. Among these processes, phagocyte accumulation has been suggested to be a surrogate marker of neuroinflammation. We previously assessed phagocyte accumulation in human stroke by MRI. We hypothesize that phagocyte accumulation in the macrosphere model may resemble the temporal and spatial patterns observed in human stroke.In a rat model of permanent focal ischemia by embolisation of TiO2-spheres we assessed key features of post-ischemic neuroinflammation by the means of histology, immunocytochemistry of glial activation and influx of hematogeneous cells, and quantitative PCR of TNF-α, IL-1, IL-18, and iNOS mRNA.In the boundary zone of the infarct, a transition of ramified microglia into ameboid phagocytic microglia was accompanied by an up-regulation of MHC class II on the cells after 3 days. By day 7, a hypercellular infiltrate consisting of activated microglia and phagocytic cells formed a thick rim around the ischemic infarct core. Interestingly, in the ischemic core microglia could only be observed at day 7. TNF-α was induced rapidly within hours, IL-1β and iNOS peaked within days, and IL-18 later at around 1 week after ischemia.The macrosphere model closely resembles the characteristical dynamics of postischemic inflammation previously observed in human stroke. We therefore suggest that the macrosphere model is highly appropriate for studying the pathophysiology of stroke in a translational approach from rodent to human.Artículo Texto completo
|BMP-induced REST regulates the establishment and maintenance of astrocytic identity. |
Kohyama J, Sanosaka T, Tokunaga A, Takatsuka E, Tsujimura K, Okano H, Nakashima K
J Cell Biol 189 159-70. Epub 2010 Mar 29. 2010
Once they have differentiated, cells retain their individual character and repress genes that are specifically expressed in other cell lineages, but how alternative fate choice is restricted during and/or after differentiation remains unclear. In the mammalian central nervous system, neurons, astrocytes, and oligodendrocytes are generated throughout life from common tripotent neural progenitor cells (NPCs). Bone morphogenetic proteins (BMPs) are well-known astrocyte-inducing cytokines. We show here that the expression of a transcriptional repressor, RE1 silencer of transcription (REST)/neuron-restrictive silencer factor (NRSF), is up-regulated and sustained by BMP signal activation in the course of astrocytic differentiation of NPCs, and restricts neuronal differentiation. We further show that, in differentiated astrocytes, endogenous REST/NRSF associates with various neuronal genes and that disruption of its function resulted in their derepression, thereby explaining how ectopic neuronal gene expression is prevented in cells with astrocytic traits. Collectively, our results suggest that REST/NRSF functions as a molecular regulator of the nonneuronal phenotype in astrocytes.Artículo Texto completo
|Sustained expression of vascular endothelial growth factor and angiopoietin-1 improves blood-spinal cord barrier integrity and functional recovery after spinal cord injury. |
Herrera, JJ; Sundberg, LM; Zentilin, L; Giacca, M; Narayana, PA
Journal of neurotrauma 27 2067-76 2010
Spinal cord injury (SCI) results in immediate disruption of the spinal vascular network, triggering an ischemic environment and initiating secondary degeneration. Promoting angiogenesis and vascular stability through the induction of vascular endothelial growth factor (VEGF) and angiopoietin-1 (Ang-1), respectively, provides a possible therapeutic approach in treating SCI. We examined whether supplementing the injured environment with these two factors, which are significantly reduced following injury, has an effect on lesion size and functional outcome. Sustained delivery of both VEGF(165) and Ang-1 was realized using viral vectors based on the adeno-associated virus (AAV), which were injected directly into the lesion epicenter immediately after injury. Our results indicate that the combined treatment with VEGF and Ang-1 resulted in both reduced hyperintense lesion volume and vascular stabilization, as determined by magnetic resonance imaging (MRI). Western blot analysis indicated that the viral vector expression was maintained into the chronic phase of injury, and that the use of the AAV vectors did not exacerbate infiltration of microglia into the lesion epicenter. The combined treatment with AAV-VEGF and AAV-Ang-1 improved locomotor recovery in the chronic phase of injury. These results indicate that combining angiogenesis with vascular stabilization may have potential therapeutic applications following SCI.Artículo Texto completo
|Nitric oxide and interleukin-1beta mediate noradrenergic induced corticotrophin-releasing hormone release in organotypic cultures of rat paraventricular nucleus. |
CH Hsieh, HY Li, JC Chen
Neuroscience 165 1191-202 2010
Noradrenergic inputs from the brainstem are critical for the central stress response. It has been suggested that endogenous interleukin-1beta (IL-1beta) is involved in norepinephrine (NE)-induced release of corticotropin-releasing hormone (CRH) from the paraventricular nucleus of the hypothalamus (PVN). However, no IL-1 receptor on PVN CRH neurons has been identified. Therefore we hypothesized that the action of IL-1beta in the PVN requires downstream modulators that eventually lead to CRH release by PVN neurons. In the current study, we used organotypic cultures from neonatal rat PVN which display neuroendocrine characteristics suitable for in vitro studies. Pharmacological treatments with NE or IL-1beta elicited nitric oxide (NO) release from the PVN cultures, implying that local NO might be a candidate for modulating the action of IL-1beta. In addition, NE treatments significantly increased IL-1beta and CRH release. Treatment with IL-1beta or sodium nitroprusside also induced CRH release. Next, we also showed that either an IL-1 receptor antagonist or NOS inhibitor Nomega-nitro-L-arginine (L-NNA) attenuated the NE-induced CRH release. These results suggest that IL-1beta and NO are involved in NE-induced CRH release. Moreover, we found that application of L-NNA attenuated IL-1beta-induced CRH release, indicating that NO likely mediates this process. In summary, the current study demonstrates that IL-1beta plays a significant role in NE-induced CRH release, and that neuroendocrine response in the PVN may depend on local NO action.
|Med12 is essential for early mouse development and for canonical Wnt and Wnt/PCP signaling. |
Rocha, PP; Scholze, M; Bleiss, W; Schrewe, H
Development (Cambridge, England) 137 2723-31 2010
The Mediator complex is commonly seen as a molecular bridge that connects DNA-bound transcription factors to the RNA polymerase II (Pol II) machinery. It is a large complex of 30 subunits that is present in all eukaryotes. The Med12 subunit has been implicated not only in the regulation of Pol II activity, but also in the binding of transcription factors to the bulk of the Mediator complex. We targeted Med12 in mouse embryonic stem cells to investigate the in vivo function of this subunit. We report here the developmental defects of Med12 hypomorphic mutants that have a drastic reduction in Med12 protein levels. These mutants fail to develop beyond embryonic day 10 and have severe defects in neural tube closure, axis elongation, somitogenesis and heart formation. We show that in Med12 hypomorphic embryos, the Wnt/planar cell polarity pathway is disrupted and that canonical Wnt/beta-catenin signaling is impaired. In agreement with this, embryos that are incapable of Med12 expression failed to establish the anterior visceral endoderm or activate brachyury expression, and did not complete gastrulation.
|CCR7 is expressed in astrocytes and upregulated after an inflammatory injury. |
Gomez-Nicola, Diego, et al.
J. Neuroimmunol., 227: 87-92 (2010) 2010
Neurodegenerative or autoimmune diseases are frequently regulated by chemokines and their receptors, controlling both glial activation and immune cell infiltration. CCL19 and CCL21 have been described to mediate crucial functions during CNS pathological states, regulating both immune cell traffic to the CNS and communication between glia and neurons. Here, we describe the expression pattern and cellular sources of CCR7, receptor of CCL19 and CCL21, in the normal mouse brain. Moreover, we found that CCR7 is upregulated in reactive astrocytes upon intracerebral LPS, regulating early glial reactivity through its ligands CCL19 and CCL21. Our results indicate that CCR7 is playing an important role for the intercellular communication during the inflammatory activation in the CNS.
|EphA4 deficient mice maintain astroglial-fibrotic scar formation after spinal cord injury. |
Herrmann JE, Shah RR, Chan AF, Zheng B
Experimental neurology 2010
One important aspect of recovery and repair after spinal cord injury (SCI) lies in the complex cellular interactions at the injury site that leads to the formation of a lesion scar. EphA4, a promiscuous member of the EphA family of repulsive axon guidance receptors, is expressed by multiple cell types in the injured spinal cord, including astrocytes and neurons. We hypothesized that EphA4 contributes to aspects of cell-cell interactions at the injury site after SCI, thus modulating the formation of the astroglial-fibrotic scar. To test this hypothesis, we studied tissue responses to a thoracic dorsal hemisection SCI in an EphA4 mutant mouse line. We found that EphA4 expression, as assessed by beta-galactosidase reporter gene activity, is associated primarily with astrocytes in the spinal cord, neurons in the cerebral cortex and, to a lesser extent, spinal neurons, before and after SCI. However, we did not observe any overt reduction of glial fibrillary acidic protein (GFAP) expression in the injured area of EphA4 mutants in comparison with controls following SCI. Furthermore, there was no evident disruption of the fibrotic scar, and the boundary between reactive astrocytes and meningeal fibroblasts appeared unaltered in the mutants, as were lesion size, neuronal survival and inflammation marker expression. Thus, genetic deletion of EphA4 does not significantly alter the astroglial response or the formation of the astroglial-fibrotic scar following a dorsal hemisection SCI in mice. In contrast to what has been proposed, these data do not support a major role for EphA4 in reactive astrogliosis following SCI.
|Expression of hyaluronan and the hyaluronan-binding proteoglycans neurocan, aggrecan, and versican by neural stem cells and neural cells derived from embryonic stem cells. |
Abaskharoun M, Bellemare M, Lau E, Margolis RU
Brain Res 1327 6-15. Epub 2010 Feb 20. 2010
We have examined the expression and localization patterns of hyaluronan and hyaluronan-binding chondroitin sulfate proteoglycans in neural stem cells and differentiated neural cells derived from mouse embryonic stem cells. Expression of proteoglycans and hyaluronan was weak in the SSEA1-positive embryonic stem cells but increased noticeably after retinoic acid induction to nestin-positive neural stem cells. After subsequent plating, the hyaluronan-binding chondroitin sulfate proteoglycans aggrecan, neurocan, and versican are expressed by cells in both the astrocytic and neuronal lineages. During the time period that hyaluronan was present, it co-localized with each of the hyaluronan-binding proteoglycans studied and was found to be clearly associated with beta-III tubulin-expressing neurons and oligodendrocytes expressing the O4 sulfatide marker. Although proteoglycan expression levels increased to varying degrees following neural differentiation, they did not change noticably during the following 2 weeks in culture, but there was a significant decrease in hyaluronan expression. Our studies therefore demonstrate the expression by neural stem cells and neural cells derived from them of hyaluronan and its associated proteoglycans, thereby providing a necessary foundation for integrating their specific properties into developing strategies for therapeutic applications.Artículo Texto completo
|Mammalian target of rapamycin in spinal cord neurons mediates hypersensitivity induced by peripheral inflammation. |
Norsted Gregory, E; Codeluppi, S; Gregory, JA; Steinauer, J; Svensson, CI
Neuroscience 169 1392-402 2010
mTOR, the mammalian target of rapamycin, is a serine-threonine kinase known to regulate cell proliferation and growth. mTOR has also been implicated in neuronal synaptic plasticity as well as in pain transmission in models of chemically induced and neuropathic pain. To date, the role of mTOR as a modulator of inflammatory pain has not been examined. In this study, we investigated the role of mTOR in Sprague-Dawley rats using the carrageenan model of inflammatory pain. mRNA of Ras homolog enriched in brain (Rheb), a GTPase that positively regulates mTOR activation, was significantly increased 2 h following carrageenan injection. Four hours after induction of inflammation phosphorylation (p) of p70S6 kinase (S6K), ribosomal protein S6 (S6) and eukaryotic initiation factor 4E-binding protein 1 (4E-BP1) was increased, indicating mTOR activation. Inhibition of spinal mTOR with intrathecal (i.t.) injection of rapamycin (0.1-3 microg) led to a dose-dependent decrease in carrageenan-induced thermal hyperalgesia and a reduction of mechanical allodynia. In vitro studies confirmed rapamycin inhibition of the mTOR pathway. Carrageenan-induced activation of the mTOR pathway in rats was localized predominantly to dorsal horn neurons in the superficial lamina. Taken together, these data show that the mTOR pathway is activated in dorsal horn neurons during inflammatory pain, and that inhibition of spinal mTOR attenuates inflammation-induced thermal and tactile hypersensitivity. Hence, our study indicates that spinal mTOR is an important regulator of spinal sensitization and suggests that targeting mTOR may provide a new avenue for pain therapy.
|M-tropic HIV envelope protein gp120 exhibits a different neuropathological profile than T-tropic gp120 in rat striatum. |
Bachis, A; Cruz, MI; Mocchetti, I
The European journal of neuroscience 32 570-8 2010
Most early human immunodeficiency virus type 1 (HIV-1) strains are macrophage (M)-tropic HIV variants and use the chemokine receptor CCR5 for infection. Neuronal loss and dementia are less severe among individuals infected with M-tropic strains. However, after several years, the T-cell (T)-tropic HIV strain, which uses the CXCR4 variant, can emerge in conjunction with brain abnormalities, suggesting strain-specific differences in neuropathogenicity. The molecular and cellular mechanisms of such diversity remain under investigation. We have previously demonstrated that HIV envelope protein gp120IIIB, which binds to CXCR4, causes neuronal apoptosis in rodents. Thus, we have used a similar experimental model to examine the neurotoxic effects of M-tropic gp120BaL. gp120BaL was microinjected in the rat striatum and neuronal apoptosis was examined in the striatum, as well as in anatomically connected areas, such as the somatosensory cortex and the substantia nigra. gp120BaL promoted neuronal apoptosis and tissue loss that were confined to the striatum. Apoptosis was associated with microglial activation and increased levels of interleukin-1beta. Intriguingly, gp120BaL increased brain-derived neurotrophic factor in the striatum. Overall, our data show that gp120BaL demonstrates a different neuropathological profile than gp120IIIB. A better understanding of the pathogenic mechanisms mediating HIV neurotoxicity is vital for developing effective neuroprotective therapies against AIDS-associated dementia complex.Artículo Texto completo
|Angiogenic factors stimulate growth of adult neural stem cells. |
Androutsellis-Theotokis, A; Rueger, MA; Park, DM; Boyd, JD; Padmanabhan, R; Campanati, L; Stewart, CV; LeFranc, Y; Plenz, D; Walbridge, S; Lonser, RR; McKay, RD
PloS one 5 e9414 2010
The ability to grow a uniform cell type from the adult central nervous system (CNS) is valuable for developing cell therapies and new strategies for drug discovery. The adult mammalian brain is a source of neural stem cells (NSC) found in both neurogenic and non-neurogenic zones but difficulties in culturing these hinders their use as research tools.Here we show that NSCs can be efficiently grown in adherent cell cultures when angiogenic signals are included in the medium. These signals include both anti-angiogenic factors (the soluble form of the Notch receptor ligand, Dll4) and pro-angiogenic factors (the Tie-2 receptor ligand, Angiopoietin 2). These treatments support the self renewal state of cultured NSCs and expression of the transcription factor Hes3, which also identifies the cancer stem cell population in human tumors. In an organotypic slice model, angiogenic factors maintain vascular structure and increase the density of dopamine neuron processes.We demonstrate new properties of adult NSCs and a method to generate efficient adult NSC cultures from various central nervous system areas. These findings will help establish cellular models relevant to cancer and regeneration.Artículo Texto completo
|Notch1 is required for maintenance of the reservoir of adult hippocampal stem cells. |
Ables, JL; Decarolis, NA; Johnson, MA; Rivera, PD; Gao, Z; Cooper, DC; Radtke, F; Hsieh, J; Eisch, AJ
The Journal of neuroscience : the official journal of the Society for Neuroscience 30 10484-92 2010
Notch1 regulates neural stem cell (NSC) number during development, but its role in adult neurogenesis is unclear. We generated nestin-CreER(T2)/R26R-YFP/Notch1(loxP/loxP) [Notch1inducible knock-out (iKO)] mice to allow tamoxifen (TAM)-inducible elimination of Notch1 and concomitant expression of yellow fluorescent protein (YFP) in nestin-expressing Type-1 NSCs and their progeny in the adult hippocampal subgranular zone (SGZ). Consistent with previous research, YFP+ cells in all stages of neurogenesis were evident in the subgranular zone (SGZ) of wild-type (WT) mice (nestin-CreER(T2)/R26R-YFP/Notch1(w/w)) after tamoxifen (post-TAM), producing adult-generated YFP+ dentate gyrus neurons. Compared with WT littermates, Notch1 iKO mice had similar numbers of total SGZ YFP+ cells 13 and 30 d post-TAM but had significantly fewer SGZ YFP+ cells 60 and 90 d post-TAM. Significantly fewer YFP+ Type-1 NSCs and transiently amplifying progenitors (TAPs) resulted in generation of fewer YFP+ granule neurons in Notch1 iKO mice. Strikingly, 30 d of running rescued this deficit, as the total YFP+ cell number in Notch iKO mice was equivalent to WT levels. This was even more notable given the persistent deficits in the Type-1 NSC and TAP reservoirs. Our data show that Notch1 signaling is required to maintain a reservoir of undifferentiated cells and ensure continuity of adult hippocampal neurogenesis, but that alternative Notch- and Type-1 NSC-independent pathways compensate in response to physical activity. These data shed light on the complex relationship between Type-1 NSCs, adult neurogenesis, the neurogenic niche, and environmental stimuli.
|Apoptosis-associated microRNAs are modulated in mouse, rat and human neural differentiation. |
Aranha, MM; Santos, DM; Xavier, JM; Low, WC; Steer, CJ; Solá, S; Rodrigues, CM
BMC genomics 11 514 2010
MicroRNAs (miRs or miRNAs) regulate several biological processes in the cell. However, evidence for miRNAs that control the differentiation program of specific neural cell types has been elusive. Recently, we have shown that apoptosis-associated factors, such as p53 and caspases participate in the differentiation process of mouse neural stem (NS) cells. To identify apoptosis-associated miRNAs that might play a role in neuronal development, we performed global miRNA expression profiling experiments in NS cells. Next, we characterized the expression of proapoptotic miRNAs, including miR-16, let-7a and miR-34a in distinct models of neural differentiation, including mouse embryonic stem cells, PC12 and NT2N cells. In addition, the expression of antiapoptotic miR-19a and 20a was also evaluated.The expression of miR-16, let-7a and miR-34a was consistently upregulated in neural differentiation models. In contrast, expression of miR-19a and miR-20a was downregulated in mouse NS cell differentiation. Importantly, differential expression of specific apoptosis-related miRNAs was not associated with increased cell death. Overexpression of miR-34a increased the proportion of postmitotic neurons of mouse NS cells.In conclusion, the identification of miR-16, let-7a and miR-34a, whose expression patterns are conserved in mouse, rat and human neural differentiation, implicates these specific miRNAs in mammalian neuronal development. The results provide new insights into the regulation of neuronal differentiation by apoptosis-associated miRNAs.
|Autism susceptibility candidate 2 (Auts2) encodes a nuclear protein expressed in developing brain regions implicated in autism neuropathology. |
Bedogni, F; Hodge, RD; Nelson, BR; Frederick, EA; Shiba, N; Daza, RA; Hevner, RF
Gene expression patterns : GEP 10 9-15 2010
Autism susceptibility candidate 2 (Auts2) is a gene associated with autism and mental retardation, whose function is unknown. Expression of Auts2 mRNA and protein were studied in the developing mouse brain by in situ hybridization, immunohistochemistry, and western blotting. Auts2 mRNA was highly expressed in the developing cerebral cortex and cerebellum, regions often affected by neuropathological changes in autism, and a few other brain regions. On embryonic day (E) 12, Auts2 mRNA was expressed in the cortical preplate, where it colocalized with Tbr1, a transcription factor specific for postmitotic projection neurons. From E16 to postnatal day 21, Auts2 was expressed most abundantly in frontal cortex, hippocampus and cerebellum, including Purkinje cells and deep nuclei. High levels of Auts2 were also detected in developing dorsal thalamus, olfactory bulb, inferior colliculus and substantia nigra. Auts2 protein showed similar regional expression patterns as the mRNA. At the cellular level, Auts2 protein was localized in the nuclei of neurons and some neuronal progenitors.
|Involvement of Notch1 inhibition in serum-stimulated glia and oligodendrocyte differentiation from human mesenchymal stem cells. |
Lee, YJ; Hung, SC; Chu, MS
Stem cells and cloning : advances and applications 3 165-73 2010
The use of in vitro oligodendrocyte differentiation for transplantation of stem cells to treat demyelinating diseases is an important consideration. In this study, we investigated the effects of serum on glia and oligodendrocyte differentiation from human mesenchymal stem cells (KP-hMSCs). We found that serum deprivation resulted in a reversible downregulation of glial- and oligodendrocyte-specific markers. Serum stimulated expression of oligodendrocyte markers, such as galactocerebroside, as well as Notch1 and JAK1 transcripts. Inhibition of Notch1 activation by the Notch inhibitor, MG132, led to enhanced expression of a serum-stimulated oligodendrocyte marker. This marker was undetectable in serum-deprived KP-hMSCs treated with MG132, suggesting that inhibition of Notch1 function is additive to serum-stimulated oligodendrocyte differentiation. Furthermore, a dominant-negative mutant RBP-J protein also inhibited Notch1 function and led to upregulation of oligodendrocyte-specific markers. Our results demonstrate that serum-stimulated oligodendrocyte differentiation is enhanced by the inhibition of Notch1-associated functions.
|Diabetes induces changes in ILK, PINCH and components of related pathways in the spinal cord of rats. |
Jiang, Y; Mizisin, AP; Rearden, A; Jolivalt, CG
Brain research 1332 100-9 2010
Recent work suggests that diabetes affects processing of peripheral, spinal and supraspinal signals in the spinal cord. However, there is little evidence for spinal cord lesions that would account for alterations in behavioral responses induced by experimental diabetes. Therefore, we assessed the expression of proteins that might affect neuronal cytoskeletal stability and thus promote dendritic and synaptic reorganization in diabetic rats. Expression of ILK, PINCH, PI3K, GSK-3beta, tau, MAP2, synaptophysin and drebrin in the lumbar spinal cord of non-diabetic and streptozotocin-diabetic rats was assessed by Western-blot analysis and immunocytochemistry after 8 and 20weeks of diabetes. The impact of diabetes on the proteins studied was duration-dependent with changes observed after 20 but not 8weeks of diabetes. ILK and PINCH proteins levels were significantly decreased and both colocalized to neurons and oligodendrocytes. PI3K protein levels were also significantly decreased, while GSK-3beta activity tended to be increased. Phosphorylation of tau and MAP2A/B protein expression were significantly increased, and expression of synaptophysin and drebrin were reduced in diabetic rats. Decreased ILK and PINCH as well as alterations of components of related signaling pathways are associated with tau hyperphosphorylation, MAP2 overexpression and reduction of synaptic proteins in the spinal cord of diabetic rats, suggesting that ILK and PINCH contribute to stabilization of axonal and dendritic structures. However, these changes are not likely the cause of altered behavioral responses in diabetic rats that occur after short-term diabetes, but may contribute to structural changes occurring in long-term diabetes.
|Nrf2 activity is lost in the spinal cord and its astrocytes of aged mice. |
Weisong Duan, Ruiyan Zhang, Yansu Guo, Yifang Jiang, Yanli Huang, Hong Jiang, Chunyan Li, Weisong Duan, Ruiyan Zhang, Yansu Guo, Yifang Jiang, Yanli Huang, Hong Jiang, Chunyan Li
In vitro cellular developmental biology. Animal 45 388-97 2009
We report herein a study of aging using in vitro and in vivo models. Glial fibrillary acidic protein and ferritin expression levels increased, and the levels of glutamate transporter 1 and transferrin receptor 1 decreased in aging mouse spinal cord and its astrocytes. Mitochondrial transmembrane potential in astrocytes decreased after 60 d of culture. Given the relationship between aging and loss of antioxidant tolerance capacity, we examined the expression of heme oxygenase 1 (HO1) and NAD(P)H/quinone oxidoreductase 1 (NQO1) in the old mouse astrocytes and spinal cord. Indeed, both antioxidant enzymes decreased there. Total nuclear factor E2-related factor 2, which governs basal and inducible expression of HO1 and NQO1, decreased significantly. Significantly, epigallocatechin gallate restored the Nrf2 activity.
|Lateral 2D-3D phase segregation in fatty acidfatty amine monolayers induced by langmuir-blodgett deposition. |
Lomova EM, Turygin DS, Ezhov AA, Arslanov VV, Kalinina MA.
The journal of physical chemistry. B 113 8581-7 2009
We describe the formation of lateral 2D-3D patterns in mixed multilayer LB films of stearic acid (SA) and octadecylamine (ODA) deposited from aqueous subphases at a basic pH. The 3D particles of SA constituting the micrometer-scale linear assemblies in the LB film are assumed to segregate at the three-phase contact line in the course of film deposition. This 2D-3D phase separation of the two-component system presumably originates from the substrate-induced lowering of the collapse point of SA that leads to spontaneous 3D condensation of an acid on a solid support. The morphology of SA/ODA LB patterns is sensitively influenced by the deposition speed and surface pressure, while the chemistry of the solid support does not affect the resulting structures. The possible mechanism that controls the specific orthogonal arrangement of the 3D phase of SA in the LB film through wettability oscillations is suggested.
|Uniquely hominid features of adult human astrocytes. |
Oberheim, NA; Takano, T; Han, X; He, W; Lin, JH; Wang, F; Xu, Q; Wyatt, JD; Pilcher, W; Ojemann, JG; Ransom, BR; Goldman, SA; Nedergaard, M
The Journal of neuroscience : the official journal of the Society for Neuroscience 29 3276-87 2009
Defining the microanatomic differences between the human brain and that of other mammals is key to understanding its unique computational power. Although much effort has been devoted to comparative studies of neurons, astrocytes have received far less attention. We report here that protoplasmic astrocytes in human neocortex are 2.6-fold larger in diameter and extend 10-fold more GFAP (glial fibrillary acidic protein)-positive primary processes than their rodent counterparts. In cortical slices prepared from acutely resected surgical tissue, protoplasmic astrocytes propagate Ca(2+) waves with a speed of 36 microm/s, approximately fourfold faster than rodent. Human astrocytes also transiently increase cystosolic Ca(2+) in response to glutamatergic and purinergic receptor agonists. The human neocortex also harbors several anatomically defined subclasses of astrocytes not represented in rodents. These include a population of astrocytes that reside in layers 5-6 and extend long fibers characterized by regularly spaced varicosities. Another specialized type of astrocyte, the interlaminar astrocyte, abundantly populates the superficial cortical layers and extends long processes without varicosities to cortical layers 3 and 4. Human fibrous astrocytes resemble their rodent counterpart but are larger in diameter. Thus, human cortical astrocytes are both larger, and structurally both more complex and more diverse, than those of rodents. On this basis, we posit that this astrocytic complexity has permitted the increased functional competence of the adult human brain.Artículo Texto completo
|Influenza virus- and cytokine-immunoreactive cells in the murine olfactory and central autonomic nervous systems before and after illness onset. |
Leyva-Grado, VH; Churchill, L; Wu, M; Williams, TJ; Taishi, P; Majde, JA; Krueger, JM
Journal of neuroimmunology 211 73-83 2009
Influenza virus invades the olfactory bulb (OB) and enhances cytokine mRNAs therein at the time of illness onset. Here we show that viral antigen immunoreactivity co-localized with glial markers in the OB but could not be detected in other brain areas. Interleukin 1beta- and tumor necrosis factor alpha-immunoreactivity co-localized with neuronal markers in olfactory and central autonomic systems, and the number of cytokine-immunoreactive neurons increased at the time of illness onset [15 h post-inoculation (PI)] but not before (10 h PI). These results suggest that the OB virus influences the brain cytokines and therefore the onset of illness.Artículo Texto completo
|Transmitochondrial embryonic stem cells containing pathogenic mtDNA mutations are compromised in neuronal differentiation. |
Kirby, DM; Rennie, KJ; Smulders-Srinivasan, TK; Acin-Perez, R; Whittington, M; Enriquez, JA; Trevelyan, AJ; Turnbull, DM; Lightowlers, RN
Cell proliferation 42 413-24 2009
Defects of the mitochondrial genome (mtDNA) cause a series of rare, mainly neurological disorders. In addition, they have been implicated in more common forms of movement disorders, dementia and the ageing process. In order to try to model neuronal dysfunction associated with mitochondrial disease, we have attempted to establish a series of transmitochondrial mouse embryonic stem cells harbouring pathogenic mtDNA mutations.Transmitochondrial embryonic stem cell cybrids were generated by fusion of cytoplasts carrying a variety of mtDNA mutations, into embryonic stem cells that had been pretreated with rhodamine 6G, to prevent transmission of endogenous mtDNA. Cybrids were differentiated into neurons and assessed for efficiency of differentiation and electrophysiological function.Neuronal differentiation could occur, as indicated by expression of neuronal markers. Differentiation was impaired in embryonic stem cells carrying mtDNA mutations that caused severe biochemical deficiency. Electrophysiological tests showed evidence of synaptic activity in differentiated neurons carrying non-pathogenic mtDNA mutations or in those that caused a mild defect of respiratory activity. Again, however, neurons carrying mtDNA mutations that resulted in severe biochemical deficiency had marked reduction in post-synaptic events.Differentiated neurons carrying severely pathogenic mtDNA defects can provide a useful model for understanding how such mutations can cause neuronal dysfunction.Artículo Texto completo
|Heterogeneity of Kir4.1 channel expression in glia revealed by mouse transgenesis. |
Tang X, Taniguchi K, Kofuji P
Glia 57 1706-15. 2009
The weakly inwardly rectifying K(+) channel Kir4.1 is found in many glial cells including astrocytes. However, questions remain regarding the relative contribution of Kir4.1 to the resting K(+) conductance of mature astrocytes in situ. We employed a bacterial artificial chromosome transgenic approach in mice to visualize Kir4.1 expression in vivo. These mice (Kir4.1-EGFP) express enhanced green fluorescent protein (EGFP) under the transcriptional control of the Kir4.1 promoter. The brains of adult Kir4.1-EGFP transgenic mice showed co-expression of EGFP and Kir4.1 in astrocytes. In addition, weaker expression of EGFP was detected in NG2+ glial cells when compared with EGFP expression in GFAP+ glial cells. Whole-cell voltage clamp recordings of EGFP+ glial cells in the CA1 area of the adult mouse hippocampus indicated astrocytes displaying properties consistent with both the "passive" and "complex" subpopulations. EGFP+ cells with bright fluorescence had the linear current-voltage (I-V) relationships and extensive gap junctional coupling characteristic of passive astrocytes. However, EGFP+ glia with weaker fluorescence displayed properties associated with complex astrocytes including nonlinear I-V relationships and lack of intercellular gap junctional coupling. Pharmacological blockade of inward currents implied that Kir4.1 channels constitute the dominant resting K(+) conductance in both glial cell types and are more highly expressed in passive astrocytes. These results suggest differential expression of Kir4.1 in glia and that this channel likely underlies the resting K(+) conductance in passive and complex astrocytes.Artículo Texto completo
|Brain injury does not alter the intrinsic differentiation potential of adult neuroblasts. |
Liu, F; You, Y; Li, X; Ma, T; Nie, Y; Wei, B; Li, T; Lin, H; Yang, Z
The Journal of neuroscience : the official journal of the Society for Neuroscience 29 5075-87 2009
Neuroblasts produced by the neural stem cells of the adult subventricular zone (SVZ) migrate into damaged brain areas after stroke or other brain injuries, and previous data have suggested that they generate regionally appropriate new neurons. To classify the types of neurons produced subsequent to ischemic injury, we combined BrdU or virus labeling with multiple neuronal markers to characterize new cells at different times after the induction of stroke. We show that SVZ neuroblasts give rise almost exclusively to calretinin-expressing cells in the damaged striatum, resulting in the accumulation of these cells during long term recovery after stroke. The vast majority of SVZ neuroblasts as well as newly born young and mature neurons in the damaged striatum constitutively express the transcription factor Sp8, but do not express transcription factors characteristic of medium-sized spiny neurons, the primary striatal projection neurons lost after stroke. Our results suggest that adult neuroblasts do not alter their intrinsic differentiation potential after brain injury.
|Spontaneous generation of prion infectivity in fatal familial insomnia knockin mice. |
Jackson WS, Borkowski AW, Faas H, Steele AD, King OD, Watson N, Jasanoff A, Lindquist S
Neuron 63 438-450 2009
A crucial tenet of the prion hypothesis is that misfolding of the prion protein (PrP) induced by mutations associated with familial prion disease is, in an otherwise normal mammalian brain, sufficient to generate the infectious agent. Yet this has never been demonstrated. We engineered knockin mice to express a PrP mutation associated with a distinct human prion disease, fatal familial insomnia (FFI). An additional substitution created a strong transmission barrier against pre-existing prions. The mice spontaneously developed a disease distinct from that of other mouse prion models and highly reminiscent of FFI. Unique pathology was transmitted from FFI mice to mice expressing wild-type PrP sharing the same transmission barrier. FFI mice were highly resistant to infection by pre-existing prions, confirming infectivity did not arise from contaminating agents. Thus, a single amino acid change in PrP is sufficient to induce a distinct neurodegenerative disease and the spontaneous generation of prion infectivity.
|Intermediate neuronal progenitors (basal progenitors) produce pyramidal-projection neurons for all layers of cerebral cortex. |
Kowalczyk, T; Pontious, A; Englund, C; Daza, RA; Bedogni, F; Hodge, R; Attardo, A; Bell, C; Huttner, WB; Hevner, RF
Cerebral cortex (New York, N.Y. : 1991) 19 2439-50 2009
The developing cerebral cortex contains apical and basal types of neurogenic progenitor cells. Here, we investigated the cellular properties and neurogenic output of basal progenitors, also called intermediate neuronal progenitors (INPs). We found that basal mitoses expressing transcription factor Tbr2 (an INP marker) were present throughout corticogenesis, from embryonic day 10.5 through birth. Postnatally, Tbr2(+) progenitors were present in the dentate gyrus, subventricular zone (SVZ), and posterior periventricle (pPV). Two morphological subtypes of INPs were distinguished in the embryonic cortex, "short radial" in the ventricular zone (VZ) and multipolar in the SVZ, probably corresponding to molecularly defined INP subtypes. Unexpectedly, many short radial INPs appeared to contact the apical (ventricular) surface and some divided there. Time-lapse video microscopy suggested that apical INP divisions produced daughter INPs. Analysis of neurogenic divisions (Tis21-green fluorescent protein [GFP](+)) indicated that INPs may produce the majority of projection neurons for preplate, deep, and superficial layers. Conversely, proliferative INP divisions (Tis21-GFP(-)) increased from early to middle corticogenesis, concomitant with SVZ growth. Our findings support the hypothesis that regulated amplification of INPs may be an important factor controlling the balance of neurogenesis among different cortical layers.Artículo Texto completo
|Arrested neural and advanced mesenchymal differentiation of glioblastoma cells-comparative study with neural progenitors. |
Rieske, P; Golanska, E; Zakrzewska, M; Piaskowski, S; Hulas-Bigoszewska, K; Wolańczyk, M; Szybka, M; Witusik-Perkowska, M; Jaskolski, DJ; Zakrzewski, K; Biernat, W; Krynska, B; Liberski, PP
BMC cancer 9 54 2009
Although features of variable differentiation in glioblastoma cell cultures have been reported, a comparative analysis of differentiation properties of normal neural GFAP positive progenitors, and those shown by glioblastoma cells, has not been performed.Following methods were used to compare glioblastoma cells and GFAP+NNP (NHA): exposure to neural differentiation medium, exposure to adipogenic and osteogenic medium, western blot analysis, immunocytochemistry, single cell assay, BrdU incorporation assay. To characterize glioblastoma cells EGFR amplification analysis, LOH/MSI analysis, and P53 nucleotide sequence analysis were performed.In vitro differentiation of cancer cells derived from eight glioblastomas was compared with GFAP-positive normal neural progenitors (GFAP+NNP). Prior to exposure to differentiation medium, both types of cells showed similar multilineage phenotype (CD44+/MAP2+/GFAP+/Vimentin+/Beta III-tubulin+/Fibronectin+) and were positive for SOX-2 and Nestin. In contrast to GFAP+NNP, an efficient differentiation arrest was observed in all cell lines isolated from glioblastomas. Nevertheless, a subpopulation of cells isolated from four glioblastomas differentiated after serum-starvation with varying efficiency into derivatives indistinguishable from the neural derivatives of GFAP+NNP. Moreover, the cells derived from a majority of glioblastomas (7 out of 8), as well as GFAP+NNP, showed features of mesenchymal differentiation when exposed to medium with serum.Our results showed that stable co-expression of multilineage markers by glioblastoma cells resulted from differentiation arrest. According to our data up to 95% of glioblastoma cells can present in vitro multilineage phenotype. The mesenchymal differentiation of glioblastoma cells is advanced and similar to mesenchymal differentiation of normal neural progenitors GFAP+NNP.
|Mannitol-facilitated CNS entry of rAAV2 vector significantly delayed the neurological disease progression in MPS IIIB mice. |
McCarty, DM; DiRosario, J; Gulaid, K; Muenzer, J; Fu, H
Gene therapy 16 1340-52 2009
The presence of the blood-brain barrier (BBB) presents the most critical challenge in therapeutic development for mucopolysaccharidosis (MPS) IIIB, a lysosomal storage disease with severe neurological manifestation, because of alpha-N-acetylglucosaminidase (NaGlu) deficiency. Earlier, we showed a global central nervous system (CNS) transduction in mice by mannitol-facilitated entry of intravenous (IV)-delivered recombinant adeno-associated viral serotype 2 (rAAV2) vector. In this study, we optimized the approach and showed that the maximal transduction in the CNS occurred when the rAAV2 vector was IV injected at 8 min after mannitol administration, and was approximately 10-fold more efficient than IV delivery of the vector at 5 or 10 min after mannitol infusion. Using this optimal (8 min) regimen, a single IV infusion of rAAV2-CMV-hNaGlu vector is therapeutically beneficial for treating the CNS disease of MPS IIIB in adult mice, with significantly extended survival, improved behavioral performance, and reduction of brain lysosomal storage pathology. The therapeutic benefit correlated with maximal delivery to the CNS, but not peripheral tissues. This milestone data shows the first effective gene delivery across the BBB to treat CNS disease. The critical timing of vector delivery and mannitol infusion highlights the important contribution of this pretreatment to successful intervention, and the long history of safe use of mannitol in patients bodes well for its application in CNS gene therapy.
|Chronic CXCL10 alters neuronal properties in rat hippocampal culture. |
Cho, J; Nelson, TE; Bajova, H; Gruol, DL
Journal of neuroimmunology 207 92-100 2009
The chemokine CXCL10 is expressed in the central nervous system (CNS) during neuroinflammatory conditions. Neurons express CXCR3, the receptor for CXCL10, and neuronal function has been shown to be altered by acute exposure to CXCL10. Little is known about the effects of chronic exposure to CXCL10 on neuronal function. Results from our studies show that chronic exposure of cultured rat hippocampal neurons to CXCL10 results in altered levels of protein for GABA and glutamate receptors and altered synaptic network activity. These effects of CXCL10 may contribute to altered CNS function that occurs in some chronic neuroinflammatory conditions.
|Loss of astrocytic domain organization in the epileptic brain. |
Oberheim, NA; Tian, GF; Han, X; Peng, W; Takano, T; Ransom, B; Nedergaard, M
The Journal of neuroscience : the official journal of the Society for Neuroscience 28 3264-76 2008
Gliosis is a pathological hallmark of posttraumatic epileptic foci, but little is known about these reactive astrocytes beyond their high glial fibrillary acidic protein (GFAP) expression. Using diolistic labeling, we show that cortical astrocytes lost their nonoverlapping domain organization in three mouse models of epilepsy: posttraumatic injury, genetic susceptibility, and systemic kainate exposure. Neighboring astrocytes in epileptic mice showed a 10-fold increase in overlap of processes. Concurrently, spine density was increased on dendrites of excitatory neurons. Suppression of seizures by the common antiepileptic, valproate, reduced the overlap of astrocytic processes. Astrocytic domain organization was also preserved in APP transgenic mice expressing a mutant variant of human amyloid precursor protein despite a marked upregulation of GFAP. Our data suggest that loss of astrocytic domains was not universally associated with gliosis, but restricted to seizure pathologies. Reorganization of astrocytes may, in concert with dendritic sprouting and new synapse formation, form the structural basis for recurrent excitation in the epileptic brain.
|Glia maturation factor modulates beta-amyloid-induced glial activation, inflammatory cytokine/chemokine production and neuronal damage. |
Asgar Zaheer, Smita Zaheer, Ramasamy Thangavel, Yanghong Wu, Shailendra K Sahu, Baoli Yang, Asgar Zaheer, Smita Zaheer, Ramasamy Thangavel, Yanghong Wu, Shailendra K Sahu, Baoli Yang, Asgar Zaheer, Smita Zaheer, Ramasamy Thangavel, Yanghong Wu, Shailendra K Sahu, Baoli Yang, Asgar Zaheer, Smita Zaheer, Ramasamy Thangavel, Yanghong Wu, Shailendra K Sahu, Baoli Yang
Brain research 1208 192-203 2008
Glia maturation factor (GMF), discovered and characterized in our laboratory, is a highly conserved protein primarily localized in mammalian central nervous system. Previously we demonstrated that GMF is required in the induced production of proinflammatory cytokines and chemokines in brain cells. We now report that ventricular infusion of human amyloid beta peptide1-42 (Abeta1-42) in mouse brain caused glial activation and large increases in the levels of GMF as well as induction of inflammatory cytokine/chemokine known for launching the neuro inflammatory cascade in Alzheimer's disease (AD). To test the hypothesis that GMF is involved in the pathogenesis of AD, we infused Abeta1-42 in the brain of GMF-deficient (GMF-KO) mice, recently prepared in our laboratory. GMF-deficient mice showed reduced glial activation and significantly suppressed proinflammatory cytokine/chemokine production following Abeta infusion compared to wild type (Wt) mice. The decrease in glial activation in the GMF-KO mice is also associated with significant reduction in Abeta induced loss of pre-synaptic marker, synaptophysin, and post-synaptic density protein-95 (PSD 95). We also examined the potential relationship between GMF or lack of it with learning and memory using the T-maze, Y-maze, and water maze, hippocampal-dependent spatial memory tasks. Our results show that memory retention was improved in GMF-KO mice compared to Wt controls following Abeta infusion. Diminution of these Abeta1-42 effects in primary cultures of GMF-KO astrocyte and microglia were reversed by reconstituted expression of GMF. Taken together, our results indicate a novel mediatory role of GMF in the neuro-inflammatory pathway of Abeta and its pro-inflammatory functions.Artículo Texto completo
|Local exposure of 849 MHz and 1763 MHz radiofrequency radiation to mouse heads does not induce cell death or cell proliferation in brain. |
Kim, TH; Kim, TH; Huang, TQ; Jang, JJ; Kim, MH; Kim, HJ; Lee, JS; Pack, JK; Seo, JS; Park, WY
Experimental & molecular medicine 40 294-303 2008
Even though there is no direct evidence to prove the cellular and molecular changes induced by radiofrequency (RF) radiation itself, we cannot completely exclude the possibility of any biological effect of mobile phone frequency radiation. We established a carousel-type exposure chamber for 849 MHz or 1763 MHz of mobile phone RF radiation to expose RF to the heads of C57BL mice. In this chamber, animals were irradiated intermittently at 7.8 W/kg for a maximum of 12 months. During this period, the body weights of 3 groups-sham, 849 MHz RF, and 1763 MHz RF-did not show any differences between groups. The brain tissues were obtained from 3 groups at 6 months and 12 months to examine the differences in histology and cell proliferation between control and RF exposure groups, but we could not find any change upon RF radiation. Likewise, we could not find changes in the expression and distribution of NeuN and GFAP in hippocampus and cerebellum, or in cell death by TUNEL assay in RF exposure groups. From these data, we conclude that the chronic exposure to 849 MHz and 1763 MHz RF radiation at a 7.8 W/kg specific absorption rate (SAR) could not induce cellular alterations such as proliferation, death, and reactive gliosis.Artículo Texto completo
|Use of a new polyclonal antibody to study the distribution and glycosylation of the sodium-coupled bicarbonate transporter NCBE in rodent brain. |
L-M Chen, M L Kelly, J D Rojas, M D Parker, H S Gill, B A Davis, W F Boron
Neuroscience 151 374-85 2008
NCBE (SLC4A10) is a member of the SLC4 family of bicarbonate transporters, several of which play important roles in intracellular-pH regulation and transepithelial HCO(3)(-) transport. Here we characterize a new antibody that was generated in rabbit against a fusion protein consisting of maltose-binding protein and the first 135 amino acids (aa) of the N-terminus of human NCBE. Western blotting--both of purified peptides representing the initial approximately 120 aa of the transporters and of full-length transporters expressed in Xenopus oocytes--demonstrated that the antibody is specific for NCBE versus the two most closely related proteins, NDCBE (SLC4A8) and NBCn1 (SLC4A7). Western blotting of tissue in four regions of adult mouse brain indicates that NCBE is expressed most abundantly in cerebral cortex (CX), cerebellum (CB) and hippocampus (HC), and less so in subcortex (SCX). NCBE protein was present in CX, CB, and HC microdissected to avoid choroid plexus. Immunocytochemistry shows that NCBE is present at the basolateral membrane of embryonic day 18 (E18) fetal and adult choroid plexus. NCBE protein is present by Western blot and immunocytochemistry in cultured and freshly dissociated HC neurons but not astrocytes. By Western blot, nearly all NCBE in mouse and rat brain is highly N-glycosylated (approximately 150 kDa). PNGase F reduces the molecular weight (MW) of natural NCBE in mouse brain or human NCBE expressed in oocytes to approximately the predicted MW of the unglycosylated protein. In oocytes, mutating any one of the three consensus N-glycosylation sites reduces glycosylation of the other two, and the triple mutant exhibits negligible functional expression.
|Transplantation of mouse embryonic stem cells into the cochlea of an auditory-neuropathy animal model: effects of timing after injury. |
Lang, H; Schulte, BA; Goddard, JC; Hedrick, M; Schulte, JB; Wei, L; Schmiedt, RA
Journal of the Association for Research in Otolaryngology : JARO 9 225-40 2008
Application of ouabain to the round window membrane of the gerbil selectively induces the death of most spiral ganglion neurons and thus provides an excellent model for investigating the survival and differentiation of embryonic stem cells (ESCs) introduced into the inner ear. In this study, mouse ESCs were pretreated with a neural-induction protocol and transplanted into Rosenthal's canal (RC), perilymph, or endolymph of Mongolian gerbils either 1-3 days (early post-injury transplant group) or 7 days or longer (late post-injury transplant group) after ouabain injury. Overall, ESC survival in RC and perilymphatic spaces was significantly greater in the early post-injury microenvironment as compared to the later post-injury condition. Viable clusters of ESCs within RC and perilymphatic spaces appeared to be associated with neovascularization in the early post-injury group. A small number of ESCs transplanted within RC stained for mature neuronal or glial cell markers. ESCs introduced into perilymph survived in several locations, but most differentiated into glia-like cells. ESCs transplanted into endolymph survived poorly if at all. These experiments demonstrate that there is an optimal time window for engraftment and survival of ESCs that occurs in the early post-injury period.Artículo Texto completo
|Establishment of autologous embryonic stem cells derived from preantral follicle culture and oocyte parthenogenesis. |
Seung Tae Lee, Mun Hwan Choi, Eun Ju Lee, Seung Pyo Gong, Mi Jang, Sang Hyun Park, Hyang Jee, Dae Yong Kim, Jae Yong Han, Jeong Mook Lim
Fertility and sterility 90 1910-20 2008
OBJECTIVE: To evaluate whether autologous embryonic stem cells can be established without generating clone embryos. DESIGN: Prospective model study. SETTING: Gamete and stem cell biotechnology laboratory in Seoul National University, Seoul, Korea. ANIMAL(S): F1 hybrid B6D2F1 mice. INTERVENTION(S): Preantral follicles were cultured, and oocytes matured in the follicles were parthenogenetically activated. MAIN OUTCOME MEASURE(S): Preimplantation development and stem cell characterization. RESULT(S): More intrafollicular oocytes that were retrieved from secondary follicles matured and developed into blastocysts after parthenogenesis than those that were retrieved from primary follicles. Of those 35 blastocysts derived from 193 parthenotes, one line of colony-forming cells was established from the culturing of early secondary follicles. The established cells were positive for embryonic stem cell-specific markers and had normal diploid karyotype and telomerase activity. They differentiated into embryoid bodies in vitro and teratomas in vivo. Inducible differentiation of the established cells into neuronal lineage cells also was possible. CONCLUSION(S): Autologous embryonic stem cells can be established by preantral follicle culture and oocyte parthenogenesis. A combined technique of follicle culture and oocyte parthenogenesis that does not use developmentally competent oocytes has the potential to replace somatic cell nuclear transfer for autologous cell therapy.
|Roscovitine reduces neuronal loss, glial activation, and neurologic deficits after brain trauma. |
Hilton, GD; Stoica, BA; Byrnes, KR; Faden, AI
Journal of cerebral blood flow and metabolism : official journal of the International Society of Cerebral Blood Flow and Metabolism 28 1845-59 2008
Traumatic brain injury (TBI) causes both direct and delayed tissue damage. The latter is associated with secondary biochemical changes such as cell cycle activation, which leads to neuronal death, inflammation, and glial scarring. Flavopiridol--a cyclin-dependent kinase (CDK) inhibitor that is neither specific nor selective--is neuroprotective. To examine the role of more specific CDK inhibitors as potential neuroprotective agents, we studied the effects of roscovitine in TBI. Central administration of roscovitine 30 mins after injury resulted in significantly decreased lesion volume, as well as improved motor and cognitive recovery. Roscovitine attenuated neuronal death and inhibited activation of cell cycle pathways in neurons after TBI, as indicated by attenuated cyclin G1 accumulation and phosphorylation of retinoblastoma protein. Treatment also decreased microglial activation after TBI, as reflected by reductions in ED1, galectin-3, p22(PHOX), and Iba-1 levels, and attenuated astrogliosis, as shown by decreased accumulation of glial fibrillary acidic protein. In primary cortical microglia and neuronal cultures, roscovitine and other selective CDK inhibitors attenuated neuronal cell death, as well as decreasing microglial activation and microglial-dependent neurotoxicity. These data support a multifactorial neuroprotective effect of cell cycle inhibition after TBI--likely related to inhibition of neuronal apoptosis, microglial-induced inflammation, and gliosis--and suggest that multiple CDKs are potentially involved in this process.Artículo Texto completo
|Lysophosphatidic acid inhibits neuronal differentiation of neural stem/progenitor cells derived from human embryonic stem cells. |
Dottori, Mirella, et al.
Stem Cells, 26: 1146-54 (2008) 2008
|The Angelman syndrome ubiquitin ligase localizes to the synapse and nucleus, and maternal deficiency results in abnormal dendritic spine morphology. |
Dindot, SV; Antalffy, BA; Bhattacharjee, MB; Beaudet, AL
Human molecular genetics 17 111-8 2008
Loss of function of the maternally inherited allele for the UBE3A ubiquitin ligase gene causes Angelman syndrome (AS), which is characterized by severe neurological impairment and motor dysfunction. In addition, UBE3A lies within chromosome 15q11-q13 region, where maternal, but not paternal, duplications cause autism. The UBE3A gene product, E6-AP, has been shown to function both as an E3 ligase in the ubiquitin proteasome pathway and as a transcriptional coactivator. However, the specific role of E6-AP in the brain, or how loss of function of E6-AP results in AS, is unclear. Herein, we show, using a recombinant transgenic mouse expressing a Ube3a(YFP) fusion gene, that the maternal Ube3a(YFP) allele is upregulated and preferentially expressed in neurons, and that the fusion protein, E6-AP:YFP, is enriched in the nucleus and dendrites in vivo. We also show that E6-AP:YFP localizes to the nucleus and to presynaptic and postsynaptic compartments in cultured hippocampal neurons. Furthermore, we show that cerebellar Purkinje cell number and dendritic branching are not affected in Ube3a maternal-deficient mice, but that dendritic spine development, including spine morphology, number and length, is affected on cerebellar Purkinje cells and on pyramidal neurons in the hippocampus and cortex. Collectively, these data suggest that the neurological deficits observed in AS patients and in AS mice may result from specific abnormalities in synaptic development and/or plasticity.
|Chronic CXCL10 alters the level of activated ERK1/2 and transcriptional factors CREB and NF-kappaB in hippocampal neuronal cell culture. |
Hilda Bajova, Thomas E Nelson, Donna L Gruol, Hilda Bajova, Thomas E Nelson, Donna L Gruol, Hilda Bajova, Thomas E Nelson, Donna L Gruol
Journal of neuroimmunology 195 36-46 2008
Signal transduction pathways may be important targets of chemokines during neuroinflammation. In the current study, Western blot analyses show that in rat hippocampal neuronal/glial cell cultures chronic CXCL10 increases the level of protein for ERK1/2 as well as for the transcriptional factors CREB and NF-kappaB. Bcl-2, an anti-apoptotic protein whose expression can be regulated by a pathway involving ERK1/2, CREB and NF-kappaB, was also increased in the CXCL10 treated cultures. These results implicate a role for ERK1/2, CREB and NF-kappaB in effects of CXCL10 on hippocampal cells and suggest that chronic CXCL10 may have a protective role during certain neuroinflammatory conditions.Artículo Texto completo
|Nuclear factor I gene expression in the developing forebrain. |
Céline Plachez, Charlotta Lindwall, Nana Sunn, Michael Piper, Randal X Moldrich, Christine E Campbell, Jason M Osinski, Richard M Gronostajski, Linda J Richards
The Journal of comparative neurology 508 385-401 2008
Three members of the Nuclear Factor I (Nfi) gene family of transcription factors; Nfia, Nfib, and Nfix are highly expressed in the developing mouse brain. Nfia and Nfib knockout mice display profound defects in the development of midline glial populations and the development of forebrain commissures (das Neves et al.  Proc Natl Acad Sci U S A 96:11946-11951; Shu et al.  J Neurosci 23:203-212; Steele-Perkins et al.  Mol Cell Biol 25:685-698). These findings suggest that Nfi genes may regulate the substrate over which the commissural axons grow to reach targets in the contralateral hemisphere. However, these genes are also expressed in the cerebral cortex and, thus, it is important to assess whether this expression correlates with a cell-autonomous role in cortical development. Here we detail the protein expression of NFIA and NFIB during embryonic and postnatal mouse forebrain development. We find that both NFIA and NFIB are expressed in the deep cortical layers and subplate prenatally and display dynamic expression patterns postnatally. Both genes are also highly expressed in the developing hippocampus and in the diencephalon. We also find that principally neither NFIA nor NFIB are expressed in callosally projecting neurons postnatally, emphasizing the role for midline glial cell populations in commissure formation. However, a large proportion of laterally projecting neurons express both NFIA and NFIB, indicating a possible cell-autonomous role for these transcription factors in corticospinal neuron development. Collectively, these data suggest that, in addition to regulating the formation of axon guidance substrates, these genes also have cell-autonomous roles in cortical development.
|Cell-specific actions of HIV-Tat and morphine on opioid receptor expression in glia. |
Turchan-Cholewo, J; Dimayuga, FO; Ding, Q; Keller, JN; Hauser, KF; Knapp, PE; Bruce-Keller, AJ
Journal of neuroscience research 86 2100-10 2008
HIV-1 patients who abuse opiate-based drugs, including heroin and morphine, are at a higher risk of developing HIV dementia. The effects of opiates are mediated predominantly through opioid receptors, which are expressed on glial cells. As HIV-1 infection in the CNS is restricted to glial cells, experiments were designed to measure the cell-specific effects of HIV Tat and morphine exposure on opioid receptor expression in both astrocytes and microglia. Specifically, the cell-type-specific pattern of mu opioid receptor (MOR), delta opioid receptor (DOR), and kappa opioid receptor (KOR) localization (surface vs. intracellular) and expression of opioid receptor mRNA were determined after exposure to morphine in the presence and the absence of Tat in primary cultured microglia and astrocytes. Data show that morphine treatment caused significantly decreased cell surface expression of opioid receptors in microglia but not in astrocytes. However, morphine treatment in the presence of Tat significantly increased intracellular expression of opioid receptors and prevented morphine-induced cell surface opioid receptor down-regulation in microglia. These findings document that cell surface opioid receptor expression is divergently regulated by morphine in microglia compared with in astrocytes, and further suggest that HIV-Tat could exacerbate opioid receptor signaling in microglia by increasing receptor expression and/or altering ligand-induced trafficking of opioid receptors.Artículo Texto completo
|Generation of Frizzled10-Cre transgenic mouse line: a useful tool for the study of dorsal telencephalic development. |
Xiaochun Gu, Dongyang He, Yiping Li, Chuanyin Hu, Yu-sheng Wei, Guang Liu, Depei Liu, Samuel J Pleasure, Wei Xie, Chunjie Zhao, Xiaochun Gu, Dongyang He, Yiping Li, Chuanyin Hu, Yu-sheng Wei, Guang Liu, Depei Liu, Samuel J Pleasure, Wei Xie, Chunjie Zhao
Genesis (New York, N.Y. : 2000) 46 523-9 2008
Wnt signaling plays an important role in regulating cortical and hippocampal development, but many of the other molecular mechanisms underlying dorsal telencephalic development are largely unknown. We are taking advantage of the highly regionalized expression patterns of signaling components of the Wnt pathway to generate new mouse lines that will be useful for studying forebrain development. Here, we describe a transgenic mouse line where Cre is driven by the promoter of the Wnt receptor, Frizzled10. In these mice, Cre activity is mainly detected in the dorsal telencephalon during development and is confined to the pyramidal cell fields in the adult hippocampus. The Cre recombinase has very high efficiency when assayed by crossing the transgenic line with the ROSA26 reporter line. Thus, this Cre line will be useful for the study of dorsal telencephalic development and conditional inactivation of target genes in the cortex and hippocampus.Artículo Texto completo
|Activation of p38 MAP kinase is involved in central neuropathic pain following spinal cord injury. |
Crown, ED; Gwak, YS; Ye, Z; Johnson, KM; Hulsebosch, CE
Experimental neurology 213 257-67 2008
Recent work regarding chronic central neuropathic pain (CNP) following spinal cord injury (SCI) suggests that activation of key signaling molecules such as members of the mitogen activated protein kinase (MAPK) family play a role in the expression of at-level mechanical allodynia. Previously, we have shown that the development of at-level CNP following moderate spinal cord injury is correlated with increased expression of the activated (and thus phosphorylated) forms of the MAPKs extracellular signal related kinase and p38 MAPK. The current study extends this work by directly examining the role of p38 MAPK in the maintenance of at-level CNP following spinal cord injury. Using a combination of behavioral, immunocytochemical, and electrophysiological measures we demonstrate that increased activation of p38 MAPK occurs in the spinal cord just rostral to the site of injury in rats that develop at-level mechanical allodynia after moderate SCI. Immunocytochemical analyses indicate that the increases in p38 MAPK activation occurred in astrocytes, microglia, and dorsal horn neurons in the spinal cord rostral to the site of injury. Inhibiting the enzymatic activity of p38 MAPK dose dependently reverses the behavioral expression of at-level mechanical allodynia and also decreases the hyperexcitability seen in thoracic dorsal horn neurons after moderate SCI. Taken together, these novel data are the first to demonstrate causality that increased activation of p38 MAPK in multiple cell types play an important role in the maintenance of at-level CNP following spinal cord injury.
|Successful elimination of non-neural cells and unachievable elimination of glial cells by means of commonly used cell culture manipulations during differentiation of GFAP and SOX2 positive neural progenitors (NHA) to neuronal cells. |
Witusik, M; Piaskowski, S; Hulas-Bigoszewska, K; Zakrzewska, M; Gresner, SM; Azizi, SA; Krynska, B; Liberski, PP; Rieske, P
BMC biotechnology 8 56 2008
Although extensive research has been performed to control differentiation of neural stem cells - still, the response of those cells to diverse cell culture conditions often appears to be random and difficult to predict. To this end, we strived to obtain stabilized protocol of NHA cells differentiation - allowing for an increase in percentage yield of neuronal cells.Uncommitted GFAP and SOX2 positive neural progenitors - so-called, Normal Human Astrocytes (NHA) were differentiated in different environmental conditions to: only neural cells consisted of neuronal [MAP2+, GFAP-] and glial [GFAP+, MAP2-] population, non-neural cells [CD44+, VIMENTIN+, FIBRONECTIN+, MAP2-, GFAP-, S100beta-, SOX2-], or mixture of neural and non-neural cells.In spite of successfully increasing the percentage yield of glial and neuronal vs. non-neural cells by means of environmental changes, we were not able to increase significantly the percentage of neuronal (GABA-ergic and catecholaminergic) over glial cells under several different cell culture testing conditions. Supplementing serum-free medium with several growth factors (SHH, bFGF, GDNF) did not radically change the ratio between neuronal and glial cells--i.e., 1,1:1 in medium without growth factors and 1,4:1 in medium with GDNF, respectively.We suggest that biotechnologists attempting to enrich in vitro neural cell cultures in one type of cells - such as that required for transplantology purposes, should consider the strong limiting influence of intrinsic factors upon extracellular factors commonly tested in cell culture conditions.
|In utero PCP exposure alters oligodendrocyte differentiation and myelination in developing rat frontal cortex. |
Lindahl, JS; Kjellsen, BR; Tigert, J; Miskimins, R
Brain research 1234 137-47 2008
Several neurodevelopmental disorders, including schizophrenia, autism, ADD/ADHD and dyslexia are believed to originate during gestation and involve white matter abnormalities. Modulation of glutamate environments and glutamate receptors has also been implicated in alteration of oligodendrocytes, the myelin forming cells of the CNS. To begin to understand how modulation of the glutamate system affects the maturation of oligodendrocytes, developing rats were subjected to prenatal blockade of the NMDA receptor with phencyclidine (PCP). Oligodendrocyte development and differentiation were then examined postnatally by measuring markers for early, middle and late stage cells. The results indicate that, while the level of marker proteins for neurons and astrocytes remains the same, early oligodendrocyte progenitor cell markers are decreased in rat brains prenatally exposed to PCP. Labeling of cells of intermediate, immature cell stages is elevated. Late stage markers for myelinating oligodendrocytes are subsequently decreased. These data suggest that prenatal NMDA receptor blockade reduces the level of progenitors and that the surviving cells are arrested at an immature stage. This premature arrest appears to result in fewer fully differentiated, mature oligodendrocytes that are capable of producing myelin. These results have interesting implications for the role of glutamate and glutamate receptors in white matter abnormalities in neurodevelopmental disorders.
|CPT1c is localized in endoplasmic reticulum of neurons and has carnitine palmitoyltransferase activity. |
Sierra, AY; Gratacós, E; Carrasco, P; Clotet, J; Ureña, J; Serra, D; Asins, G; Hegardt, FG; Casals, N
The Journal of biological chemistry 283 6878-85 2008
CPT1c is a carnitine palmitoyltransferase 1 (CPT1) isoform that is expressed only in the brain. The enzyme has recently been localized in neuron mitochondria. Although it has high sequence identity with the other two CPT1 isoenzymes (a and b), no CPT activity has been detected to date. Our results indicate that CPT1c is expressed in neurons but not in astrocytes of mouse brain sections. Overexpression of CPT1c fused to the green fluorescent protein in cultured cells demonstrates that CPT1c is localized in the endoplasmic reticulum rather than mitochondria and that the N-terminal region of CPT1c is responsible for endoplasmic reticulum protein localization. Western blot experiments with cell fractions from adult mouse brain corroborate these results. In addition, overexpression studies demonstrate that CPT1c does not participate in mitochondrial fatty acid oxidation, as would be expected from its subcellular localization. To identify the substrate of CPT1c enzyme, rat cDNA was overexpressed in neuronal PC-12 cells, and the levels of acylcarnitines were measured by high-performance liquid chromatography-mass spectrometry. Palmitoylcarnitine was the only acylcarnitine to increase in transfected cells, which indicates that palmitoyl-CoA is the enzyme substrate and that CPT1c has CPT1 activity. Microsomal fractions of PC-12 and HEK293T cells overexpressing CPT1c protein showed a significant increase in CPT1 activity of 0.57 and 0.13 nmol.mg(-1).min(-1), respectively, which is approximately 50% higher than endogenous CPT1 activity. Kinetic studies demonstrate that CPT1c has similar affinity to CPT1a for both substrates but 20-300 times lower catalytic efficiency.
|The role of thioredoxin reductases in brain development. |
Soerensen, J; Jakupoglu, C; Beck, H; Förster, H; Schmidt, J; Schmahl, W; Schweizer, U; Conrad, M; Brielmeier, M
PloS one 3 e1813 2008
The thioredoxin-dependent system is an essential regulator of cellular redox balance. Since oxidative stress has been linked with neurodegenerative disease, we studied the roles of thioredoxin reductases in brain using mice with nervous system (NS)-specific deletion of cytosolic (Txnrd1) and mitochondrial (Txnrd2) thioredoxin reductase. While NS-specific Txnrd2 null mice develop normally, mice lacking Txnrd1 in the NS were significantly smaller and displayed ataxia and tremor. A striking patterned cerebellar hypoplasia was observed. Proliferation of the external granular layer (EGL) was strongly reduced and fissure formation and laminar organisation of the cerebellar cortex was impaired in the rostral portion of the cerebellum. Purkinje cells were ectopically located and their dendrites stunted. The Bergmann glial network was disorganized and showed a pronounced reduction in fiber strength. Cerebellar hypoplasia did not result from increased apoptosis, but from decreased proliferation of granule cell precursors within the EGL. Of note, neuron-specific inactivation of Txnrd1 did not result in cerebellar hypoplasia, suggesting a vital role for Txnrd1 in Bergmann glia or neuronal precursor cells.
|Intermediate progenitors in adult hippocampal neurogenesis: Tbr2 expression and coordinate regulation of neuronal output. |
Hodge, RD; Kowalczyk, TD; Wolf, SA; Encinas, JM; Rippey, C; Enikolopov, G; Kempermann, G; Hevner, RF
The Journal of neuroscience : the official journal of the Society for Neuroscience 28 3707-17 2008
Neurogenesis in the adult hippocampus is a highly regulated process that originates from multipotent progenitors in the subgranular zone (SGZ). Currently, little is known about molecular mechanisms that regulate proliferation and differentiation in the SGZ. To study the role of transcription factors (TFs), we focused on Tbr2 (T-box brain gene 2), which has been implicated previously in developmental glutamatergic neurogenesis. In adult mouse hippocampus, Tbr2 protein and Tbr2-GFP (green fluorescent protein) transgene expression were specifically localized to intermediate-stage progenitor cells (IPCs), a type of transit amplifying cells. The Tbr2+ IPCs were highly responsive to neurogenic stimuli, more than doubling after voluntary wheel running. Notably, the Tbr2+ IPCs formed cellular clusters, the average size of which (Tbr2+ cells per cluster) likewise more than doubled in runners. Conversely, Tbr2+ IPCs were selectively depleted by antimitotic drugs, known to suppress neurogenesis. After cessation of antimitotic treatment, recovery of neurogenesis was paralleled by recovery of Tbr2+ IPCs, including a transient rebound above baseline numbers. Finally, Tbr2 was examined in the context of additional TFs that, together, define a TF cascade in embryonic neocortical neurogenesis (Pax6 --greater than Ngn2 --greater than Tbr2 --greater than NeuroD --greater than Tbr1). Remarkably, the same TF cascade was found to be linked to stages of neuronal lineage progression in adult SGZ. These results suggest that Tbr2+ IPCs play a major role in the regulation of adult hippocampal neurogenesis, and that a similar transcriptional program controls neurogenesis in adult SGZ as in embryonic cerebral cortex.
|Contribution of voltage-gated sodium channels to the b-wave of the mammalian flash electroretinogram. |
Mojumder, DK; Sherry, DM; Frishman, LJ
The Journal of physiology 586 2551-80 2008
Voltage-gated sodium channels (Na(v) channels) in retinal neurons are known to contribute to the mammalian flash electroretinogram (ERG) via activity of third-order retinal neurons, i.e. amacrine and ganglion cells. This study investigated the effects of tetrodotoxin (TTX) blockade of Na(v) channels on the b-wave, an ERG wave that originates mainly from activity of second-order retinal neurons. ERGs were recorded from anaesthetized Brown Norway rats in response to brief full-field flashes presented over a range of stimulus energies, under dark-adapted conditions and in the presence of steady mesopic and photopic backgrounds. Recordings were made before and after intravitreal injection of TTX (approximately 3 microm) alone, 3-6 weeks after optic nerve transection (ONTx) to induce ganglion cell degeneration, or in combination with an ionotropic glutamate receptor antagonist 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX, 200 microm) to block light-evoked activity of inner retinal, horizontal and OFF bipolar cells, or with the glutamate agonist N-methyl-D-aspartate (NMDA, 100-200 microm) to reduce light-evoked inner retinal activity. TTX reduced ERG amplitudes measured at fixed times corresponding to b-wave time to peak. Effects of TTX were seen under all background conditions, but were greatest for mesopic backgrounds. In dark-adapted retina, b-wave amplitudes were reduced only when very low stimulus energies affecting the inner retina, or very high stimulus energies were used. Loss of ganglion cells following ONTx did not affect b-wave amplitudes, and injection of TTX in eyes with ONTx reduced b-wave amplitudes by the same amount for each background condition as occurred when ganglion cells were intact, thereby eliminating a ganglion cell role in the TTX effects. Isolation of cone-driven responses by presenting test flashes after cessation of a rod-saturating conditioning flash indicated that the TTX effects were primarily on cone circuits contributing to the mixed rod-cone ERG. NMDA significantly reduced only the additional effects of TTX on the mixed rod-cone ERG observed under mesopic conditions, implicating inner retinal involvement in those effects. After pharmacological blockade with CNQX, TTX still reduced b-wave amplitudes in cone-isolated ERGs indicating Na(v) channels in ON cone bipolar cells themselves augment b-wave amplitude and sensitivity. This augmentation was largest under dark-adapted conditions, and decreased with increasing background illumination, indicating effects of background illumination on Na(v) channel function. These findings indicate that activation of Na(v) channels in ON cone bipolar cells affects the b-wave of the rat ERG and must be considered when analysing results of ERG studies of retinal function.
|Dynamic contribution of nestin-expressing stem cells to adult neurogenesis. |
Lagace, DC; Whitman, MC; Noonan, MA; Ables, JL; DeCarolis, NA; Arguello, AA; Donovan, MH; Fischer, SJ; Farnbauch, LA; Beech, RD; DiLeone, RJ; Greer, CA; Mandyam, CD; Eisch, AJ
The Journal of neuroscience : the official journal of the Society for Neuroscience 27 12623-9 2007
Understanding the fate of adult-generated neurons and the mechanisms that influence them requires consistent labeling and tracking of large numbers of stem cells. We generated a nestin-CreER(T2)/R26R-yellow fluorescent protein (YFP) mouse to inducibly label nestin-expressing stem cells and their progeny in the adult subventricular zone (SVZ) and subgranular zone (SGZ). Several findings show that the estrogen ligand tamoxifen (TAM) specifically induced recombination in stem cells and their progeny in nestin-CreER(T2)/R26R-YFP mice: 97% of SGZ stem-like cells (GFAP/Sox2 with radial glial morphology) expressed YFP; YFP+ neurospheres could be generated in vitro after recombination in vivo, and maturing YFP+ progeny were increasingly evident in the olfactory bulb (OB) and dentate gyrus (DG) granule cell layer. Revealing an unexpected regional dissimilarity in adult neurogenesis, YFP+ cells accumulated up to 100 d after TAM in the OB, but in the SGZ, YFP+ cells reached a plateau 30 d after TAM. In addition, most SVZ and SGZ YFP+ cells became neurons, underscoring a link between nestin and neuronal fate. Finally, quantification of YFP+ cells in nestin-CreER(T2)/R26R-YFP mice allowed us to estimate, for example, that stem cells and their progeny contribute to no more than 1% of the adult DG granule cell layer. In addition to revealing the dynamic contribution of nestin-expressing stem cells to adult neurogenesis, this work highlights the utility of the nestin-CreER(T2)/R26R-YFP mouse for inducible gene ablation in stem cells and their progeny in vivo in the two major regions of adult neurogenesis.
|Altered ATP7A expression and other compensatory responses in a murine model of Menkes disease. |
Niciu, MJ; Ma, XM; El Meskini, R; Pachter, JS; Mains, RE; Eipper, BA
Neurobiology of disease 27 278-91 2007
Mutations in the copper-transporter ATP7A lead to severe neurodegeneration in the mottled brindled hemizygous male (MoBr/y) mouse and human patients with Menkes disease. Our earlier studies demonstrated cell-type- and -stage-specific changes in ATP7A protein expression during postnatal neurodevelopment. Here we examined copper and cuproenzyme levels in MoBr/y mice to search for compensatory responses. While all MoBr/y neocortical subcellular fractions had decreased copper levels, the greatest decrease (8-fold) was observed in cytosol. Immunostaining for ATP7A revealed decreased levels in MoBr/y hippocampal pyramidal and cerebellar Purkinje neurons. In contrast, an upregulation of ATP7A protein occurred in MoBr/y endothelial cells, perhaps to compensate for a lack of copper in the neuropil. MoBr/y astrocytes and microglia increased their physical association with the blood-brain barrier. No alterations in ATP7A levels were observed in ependymal cells, arguing for specificity in the alteration observed at the blood-brain barrier. The decreased expression of ATP7A protein in MoBr/y Purkinje cells was associated with impaired synaptogenesis and dramatic cytoskeletal dysfunction. Immunoblotting failed to reveal any compensatory increase in levels of ATP7B. While total levels of several cuproenzymes (peptide-amidating monooxygenase, SOD1, and SOD3) were unaltered in the MoBr/y brain, levels of amidated cholecystokinin (CCK8) and amidated pituitary adenylate cyclase-activating polypeptide (PACAP38) were reduced in a tissue-specific fashion. The compensatory changes observed in the neurovascular unit provide insight into the success of copper injections within a defined neurodevelopmental period.
|Auto-catalytic ceria nanoparticles offer neuroprotection to adult rat spinal cord neurons. |
Das, M; Patil, S; Bhargava, N; Kang, JF; Riedel, LM; Seal, S; Hickman, JJ
Biomaterials 28 1918-25 2007
This paper describes the evaluation of the auto-catalytic anti-oxidant behavior and biocompatibility of cerium oxide nanoparticles for applications in spinal cord repair and other diseases of the central nervous system. The application of a single dose of nano-ceria at a nano-molar concentration is biocompatible, regenerative and provides a significant neuroprotective effect on adult rat spinal cord neurons. Retention of neuronal function is demonstrated from electrophysiological recordings and the possibility of its application to prevent ischemic insult is suggested from an oxidative injury assay. A mechanism is proposed to explain the auto-catalytic properties of these nanoparticles.
|Pathogenesis of spinally mediated hyperalgesia in diabetes. |
Ramos, KM; Jiang, Y; Svensson, CI; Calcutt, NA
Diabetes 56 1569-76 2007
Hyperalgesia to noxious stimuli is accompanied by increased spinal cyclooxygenase (COX)-2 protein in diabetic rats. The present studies were initiated to establish causality between increased spinal COX-2 activity and hyperalgesia during diabetes and to assess the potential involvement of polyol pathway activity in the pathogenesis of spinally mediated hyperalgesia. Rats with 1, 2, or 4 weeks of streptozotocin-induced diabetes exhibited significantly increased levels of spinal COX-2 protein and activity, along with exaggerated paw flinching in response to 0.5% paw formalin injection. Increased flinching of diabetic rats was attenuated by intrathecal pretreatment with a selective COX-2 inhibitor immediately before formalin injection, confirming the involvement of COX-2 activity in diabetic hyperalgesia. Chronic treatment with insulin or ICI222155, an aldose reductase inhibitor (ARI) previously shown to prevent spinal polyol accumulation and formalin-evoked hyperalgesia in diabetic rats, prevented elevated spinal COX-2 protein and activity in diabetic rats. In contrast, the ARI IDD676 had no effect on spinal polyol accumulation, elevated spinal COX-2, or hyperalgesia to paw formalin injection. In the spinal cord, aldose reductase immunoreactivity was present solely in oligodendrocytes, which also contained COX-2 immunoreactivity. Polyol pathway flux in spinal oligodendrocytes provides a pathogenic mechanism linking hyperglycemia to hyperalgesia in diabetic rats.
|Suppression of kindling epileptogenesis by adenosine releasing stem cell-derived brain implants. |
Li, T; Steinbeck, JA; Lusardi, T; Koch, P; Lan, JQ; Wilz, A; Segschneider, M; Simon, RP; Brüstle, O; Boison, D
Brain : a journal of neurology 130 1276-88 2007
Epilepsy therapy is largely symptomatic and no effective therapy is available to prevent epileptogenesis. We therefore analysed the potential of stem cell-derived brain implants and of paracrine adenosine release to suppress the progressive development of seizures in the rat kindling-model. Embryonic stem (ES) cells, engineered to release the inhibitory neuromodulator adenosine by biallelic genetic disruption of the adenosine kinase gene (Adk-/-), and respective wild-type (wt) cells, were differentiated into neural precursor cells (NPs) and injected into the hippocampus of rats prior to kindling. Therapeutic effects of NP-derived brain implants were compared with those of wt baby hamster kidney cells (BHK) and adenosine releasing BHK cell implants (BHK-AK2), which were previously shown to suppress seizures by paracrine adenosine release. Wild-type NP-graft recipients were characterized by an initial delay of seizure development, while recipients of adenosine releasing NPs displayed sustained protection from developing generalized seizures. In contrast, recipients of wt BHK cells failed to display any effects on kindling development, while recipients of BHK-AK2 cells were only moderately protected from seizure development. The therapeutic effect of Adk(-/-)-NPs was due to graft-mediated adenosine release, since seizures could transiently be provoked after blocking adenosine A1 receptors. Histological analysis of NP-implants at day 26 revealed cell clusters within the infrahippocampal cleft as well as intrahippocampal location of graft-derived cells expressing mature neuronal markers. In contrast, BHK and BHK-AK2 cell implants only formed cell clusters within the infrahippocampal cleft. We conclude that ES cell-derived adenosine releasing brain implants are superior to paracrine adenosine release from BHK-AK2 cell implants in suppressing seizure progression in the rat kindling-model. These findings may indicate a potential antiepileptogenic function of stem cell-mediated adenosine delivery.
|Prolonged glial expression of Sox4 in the CNS leads to architectural cerebellar defects and ataxia. |
Hoser, M; Baader, SL; Bösl, MR; Ihmer, A; Wegner, M; Sock, E
The Journal of neuroscience : the official journal of the Society for Neuroscience 27 5495-505 2007
Sox proteins of group C are strongly expressed in the developing nervous system and have been associated with maturation of neurons and glia. Here, we overexpressed the group C protein Sox4 in transgenic mice under the control of the human GFAP promoter. Transgene expression was detected in radial glia and astrocytes throughout the CNS. The transgenic mice were ataxic and exhibited hydrocephaly as well as cerebellar malformations. In the cerebellum, fissures were not formed and neuronal layering was dramatically disturbed. Nevertheless, all neuronal cell types of the cerebellum were present as well as cells with characteristics of early radial glia, astrocytes, and oligodendrocytes. However, radial glia failed to migrate into the position normally taken by Bergmann glia and did not extend radial fibers toward the pial surface. The cerebellar malformations can therefore be explained by the absence of functional Bergmann glia. We conclude that Sox4 expression counteracts differentiation of radial glia and has to be downregulated before full maturation can occur.
|Calsyntenin-1 docks vesicular cargo to kinesin-1. |
Konecna, A; Frischknecht, R; Kinter, J; Ludwig, A; Steuble, M; Meskenaite, V; Indermühle, M; Engel, M; Cen, C; Mateos, JM; Streit, P; Sonderegger, P
Molecular biology of the cell 17 3651-63 2006
We identified a direct interaction between the neuronal transmembrane protein calsyntenin-1 and the light chain of Kinesin-1 (KLC1). GST pulldowns demonstrated that two highly conserved segments in the cytoplasmic domain of calsyntenin-1 mediate binding to the tetratricopeptide repeats of KLC1. A complex containing calsyntenin-1 and the Kinesin-1 motor was isolated from developing mouse brain and immunoelectron microscopy located calsyntenin-1 in association with tubulovesicular organelles in axonal fiber tracts. In primary neuronal cultures, calsyntenin-1-containing organelles were aligned along microtubules and partially colocalized with Kinesin-1. Using live imaging, we showed that these organelles are transported along axons with a velocity and processivity typical for fast axonal transport. Point mutations in the two kinesin-binding segments of calsyntenin-1 significantly reduced binding to KLC1 in vitro, and vesicles bearing mutated calsyntenin-1 exhibited a markedly altered anterograde axonal transport. In summary, our results indicate that calsyntenin-1 links a certain type of vesicular and tubulovesicular organelles to the Kinesin-1 motor.
|Slow progressive degeneration of nigral dopaminergic neurons in postnatal Engrailed mutant mice. |
Sgadò, P; Albéri, L; Gherbassi, D; Galasso, SL; Ramakers, GM; Alavian, KN; Smidt, MP; Dyck, RH; Simon, HH
Proceedings of the National Academy of Sciences of the United States of America 103 15242-7 2006
The homeobox transcription factors Engrailed-1 and Engrailed-2 are required for the survival of mesencephalic dopaminergic neurons in a cell-autonomous and gene-dose-dependent manner. Because of this requirement, the cells die by apoptosis when all four alleles of the Engrailed genes are genetically ablated (En1-/-;En2-/-). In the present study, we show that viable and fertile mice, heterozygous null for Engrailed-1 and homozygous null for Engrailed-2 (En1+/-;En2-/-), have an adult phenotype that resembles key pathological features of Parkinson's disease. Specifically, postnatal mutant mice exhibit a progressive degeneration of dopaminergic neurons in the substantia nigra during the first 3 mo of their lives, leading to diminished storage and release of dopamine in the caudate putamen, motor deficits similar to akinesia and bradykinesia, and a lower body weight. This genetic model may provide access to the molecular etiology for Parkinson's disease and could assist in the development of novel treatments for this neurodegenerative disorder.
|Altered expression of alpha3-containing GABAA receptors in the neocortex of patients with focal epilepsy. |
Loup, F; Picard, F; André, VM; Kehrli, P; Yonekawa, Y; Wieser, HG; Fritschy, JM
Brain : a journal of neurology 129 3277-89 2006
Impaired transmission in GABAergic circuits is thought to contribute to the pathogenesis of epilepsy. Although it is well established that major reorganization of GABA(A) receptor subtypes occurs in the hippocampus of patients with medically refractory temporal lobe epilepsy (TLE), it is unclear whether this disorder is also associated with alterations in GABA(A) receptor subtypes in the neocortex. Here we have investigated immunohistochemically the subunit composition and neocortical distribution of three major GABA(A) receptor subtypes using antibodies specifically recognizing the subunits alpha1, alpha2, alpha3, beta2/3 and gamma2. Cortical tissue was obtained at surgery from patients with TLE and hippocampal sclerosis (HS; n = 9), TLE associated with neocortical lesions (non-HS; n = 12) and frontal lobe epilepsy (FLE; n = 5), with post-mortem samples serving as controls (n = 4). A distinct laminar and neuronal expression pattern of the alpha-subunit variants was found across the neocortical regions examined in the temporal and frontal lobes in both control and patient tissue samples. In the five patients with FLE, GABA(A) receptor subunit staining was unchanged as compared to controls. In patients with TLE we observed a marked decrease in alpha3-subunit staining in the superficial neocortical layers (I-III), but no change in the deep layers (V and VI) or in the expression pattern of the alpha1 and alpha2-subunits. Reduced expression in alpha3-containing GABA(A) receptors was detected in six out of nine patients of the HS group and four out of twelve patients of the non-HS group. Histopathological changes were present in eight out of the ten patients with decreased alpha3-subunit staining. The selective reduction in alpha3-containing GABA(A) receptors was confirmed using semiquantitative measurements of optical density (OD). The specific changes unique to alpha3-subunit expression in the superficial neocortical layers of patients with TLE suggest that this subtype is of particular significance in the reorganization of cortical GABAergic systems in focal epilepsy.
|Dopamine enhances motor and neuropathological consequences of polyglutamine expanded huntingtin. |
Cyr, M; Sotnikova, TD; Gainetdinov, RR; Caron, MG
FASEB journal : official publication of the Federation of American Societies for Experimental Biology 20 2541-3 2006
An expansion in the CAG repeat of the IT15 (huntingtin) gene underlies the development of Huntington's disease (HD), but the basis for the specific vulnerability of dopamine-receptive striatal neurons remains unclear. To examine the potential role of the dopamine system in the emergence of pathological conditions in HD, we generated a double mutant mouse strain with both enhanced dopamine transmission and endogenous expression of a mutant huntingtin gene. This strain was generated by crossing the dopamine transporter knock-out mouse, which exhibits a 5-fold elevation in extracellular dopamine levels in the striatum and locomotor hyperactivity, to a knock-in mouse model of HD containing 92 CAG repeats. These double mutant mice exhibited an increased stereotypic activity at 6 months of age, followed by a progressive decline of their locomotor hyperactivity. Expression of the mutated huntingtin did not alter dopamine or its metabolite levels in normal or dopamine transporter knock-out mice. However, the mutant huntingtin protein aggregated much earlier and to a greater extent in the striatum and other dopaminergic brain regions in the hyperdopaminergic mouse model of HD. Furthermore, the formation of neuropil aggregates in the striatum and other regions of hyperdopaminergic HD mice was observed at 4 months of age, well before similar events occurred in normal HD mice (12 months). These findings indicate that dopamine contributes to the deleterious effects of mutated huntingtin on striatal function, and this is accompanied by enhanced formation of huntingtin aggregates.
|Acute and chronic changes in aquaporin 4 expression after spinal cord injury. |
Nesic, O; Lee, J; Ye, Z; Unabia, GC; Rafati, D; Hulsebosch, CE; Perez-Polo, JR
Neuroscience 143 779-92 2006
The effect of spinal cord injury (SCI) on the expression levels and distribution of water channel aquaporin 4 (AQP4) has not been studied. We have found AQP4 in gray and white matter astrocytes in both uninjured and injured rat spinal cords. AQP4 was detected in astrocytic processes that were tightly surrounding neurons and blood vessels, but more robustly in glia limitans externa and interna, which were forming an interface between spinal cord parenchyma and cerebrospinal fluid (CSF). Such spatial distribution of AQP4 suggests a critical role that astrocytes expressing AQP4 play in the transport of water from blood/CSF to spinal cord parenchyma and vice versa. SCI induced biphasic changes in astrocytic AQP4 levels, including its early down-regulation and subsequent persistent up-regulation. However, changes in AQP4 expression did not correlate well with the onset and magnitude of astrocytic activation, when measured as changes in GFAP expression levels. It appears that reactive astrocytes began expressing increased levels of AQP4 after migrating to the wound area (thoracic region) two weeks after SCI, and AQP4 remained significantly elevated for months after SCI. We also showed that increased levels of AQP4 spread away from the lesion site to cervical and lumbar segments, but only in chronically injured spinal cords. Although overall AQP4 expression levels increased in chronically-injured spinal cords, AQP4 immunolabeling in astrocytic processes forming glia limitans externa was decreased, which may indicate impaired water transport through glia limitans externa. Finally, we also showed that SCI-induced changes in AQP4 protein levels correlate, both temporally and spatially, with persistent increases in water content in acutely and chronically injured spinal cords. Although correlative, this finding suggests a possible link between AQP4 and impaired water transport/edema/syringomyelia in contused spinal cords.
|Absence of tumor necrosis factor-alpha does not affect motor neuron disease caused by superoxide dismutase 1 mutations. |
Gowing, G; Dequen, F; Soucy, G; Julien, JP
The Journal of neuroscience : the official journal of the Society for Neuroscience 26 11397-402 2006
An increase in the expression of the proinflammatory cytokine tumor necrosis factor alpha (TNF-alpha) has been observed in patients with amyotrophic lateral sclerosis (ALS) and in the mice models of the disease. TNF-alpha is a potent activator of macrophages and microglia and, under certain conditions, can induce or exacerbate neuronal cell death. Here, we assessed the contribution of TNF-alpha in motor neuron disease in mice overexpressing mutant superoxide dismutase 1 (SOD1) genes linked to familial ALS. This was accomplished by the generation of mice expressing SOD1(G37R) or SOD1(G93A) mutants in the context of TNF-alpha gene knock out. Surprisingly, the absence of TNF-alpha did not affect the lifespan or the extent of motor neuron loss in SOD1 transgenic mice. These results provide compelling evidence indicating that TNF-alpha does not directly contribute to motor neuron degeneration caused by SOD1 mutations.
|Locus ceruleus degeneration promotes Alzheimer pathogenesis in amyloid precursor protein 23 transgenic mice. |
Heneka, MT; Ramanathan, M; Jacobs, AH; Dumitrescu-Ozimek, L; Bilkei-Gorzo, A; Debeir, T; Sastre, M; Galldiks, N; Zimmer, A; Hoehn, M; Heiss, WD; Klockgether, T; Staufenbiel, M
The Journal of neuroscience : the official journal of the Society for Neuroscience 26 1343-54 2006
Locus ceruleus (LC) degeneration and loss of cortical noradrenergic innervation occur early in Alzheimer's disease (AD). Although this has been known for several decades, the contribution of LC degeneration to AD pathogenesis remains unclear. We induced LC degeneration with N-(2-chloroethyl)-N-ethyl-bromo-benzylamine (dsp4) in amyloid precursor protein 23 (APP23) transgenic mice with a low amyloid load. Then 6 months later the LC projection areas showed a robust elevation of glial inflammation along with augmented amyloid plaque deposits. Moreover, neurodegeneration and neuronal loss significantly increased. Importantly, the paraventricular thalamus, a nonprojection area, remained unaffected. Radial arm maze and social partner recognition tests revealed increased memory deficits while high-resolution magnetic resonance imaging-guided micro-positron emission tomography demonstrated reduced cerebral glucose metabolism, disturbed neuronal integrity, and attenuated acetylcholinesterase activity. Nontransgenic mice with LC degeneration were devoid of these alterations. Our data demonstrate that the degeneration of LC affects morphology, metabolism, and function of amyloid plaque-containing higher brain regions in APP23 mice. We postulate that LC degeneration substantially contributes to AD development.
|Nitroxidergic neurons in rat nucleus tractus solitarii express vesicular glutamate transporter 3. |
Lin, L H and Talman, W T
J. Chem. Neuroanat., 29: 179-91 (2005) 2005
Earlier we reported that glutamate transporter (VGLUT) 2 and neuronal nitric oxide synthase (nNOS) are colocalized in some fibers and are present in apposing fibers in the nucleus tractus solitarii (NTS). Those findings provided anatomical support for a hypothesized physiological link between glutamate and nitric oxide (NO.) in the NTS. Recently a third class of VGLUT, VGLUT3, was identified, but its distribution in NTS and its anatomical relationship with nNOS have not been shown. In this study we tested the hypothesis that neurons and fibers containing VGLUT3 lie in close proximity to those containing nNOS and that both proteins colocalize in some neurons and fibers in the NTS. We perfused rats and obtained brain stem sections and nodose ganglion sections for immunofluorescent staining analyzed by confocal microscopy. The NTS contained moderate VGLUT3-immunoreactivity (IR), with the intermediate, medial and interstitial subnuclei containing higher VGLUT3-IR than other subnuclei. Although all three forms of VGLUT were present in the NTS, VGLUT3-IR was not colocalized with either VGLUT1-IR or VGLUT2-IR in either processes or cells in the brain stem. Cells and processes containing both VGLUT3-IR and nNOS-IR were noted in all NTS subnuclei and in the nodose ganglion. Triple immunofluorescent staining revealed that cells double-labeled for nNOS-IR and VGLUT3-IR were all additionally labeled for neuronal nuclear antigen (NeuN), a neuronal marker. These findings support our hypothesis that neurons and fibers containing VGLUT3 lie in close proximity to those containing nNOS and that both proteins colocalize in some neurons and fibers in the NTS.
|Viral induction of central nervous system innate immune responses. |
Rempel, JD; Quina, LA; Blakely-Gonzales, PK; Buchmeier, MJ; Gruol, DL
Journal of virology 79 4369-81 2005
The ability of the central nervous system (CNS) to generate innate immune responses was investigated in an in vitro model of CNS infection. Cultures containing CNS cells were infected with mouse hepatitis virus-JHM, which causes fatal encephalitis in mice. Immunostaining indicated that viral infection had a limited effect on culture characteristics, overall cell survival, or cell morphology at the early postinfection times studied. Results from Affymetrix gene array analysis, assessed on RNA isolated from virally and sham-infected cultures, were compared with parallel protein assays for cytokine, chemokine, and cell surface markers. Of the 126 transcripts found to be differentially expressed between viral and sham infections, the majority were related to immunological responses. Virally induced increases in interleukin-6 and tumor necrosis factor alpha mRNA and protein expression correlated with the genomic induction of acute-phase proteins. Genomic and protein analysis indicated that viral infection resulted in prominent expression of neutrophil and macrophage chemotactic proteins. In addition, mRNA expression of nonclassical class I molecules H2-T10, -T17, -M2, and -Q10, were enhanced three- to fivefold in virus-infected cells compared to sham-infected cells. Thus, upon infection, resident brain cells induced a breadth of innate immune responses that could be vital in directing the outcome of the infection and, in vivo, would provide signals which would summon the peripheral immune system to respond to the infection. Further understanding of how these innate responses participate in immune protection or immunopathology in the CNS will be critical in efforts to intervene in severe encephalitis.
|Astrogliosis in epilepsy leads to overexpression of adenosine kinase, resulting in seizure aggravation. |
Fedele, DE; Gouder, N; Güttinger, M; Gabernet, L; Scheurer, L; Rülicke, T; Crestani, F; Boison, D
Brain : a journal of neurology 128 2383-95 2005
Adenosine kinase (ADK) is considered to be the key regulator of the brain's endogenous anticonvulsant, adenosine. In adult brain, ADK is primarily expressed in a subpopulation of astrocytes and striking upregulation of ADK in these cells has been associated with astrogliosis after kainic acid-induced status epilepticus (KASE) in the kainic acid mouse model of temporal lobe epilepsy. To investigate the causal relationship between KASE-induced astrogliosis, upregulation of ADK and seizure activity, we have developed a novel mouse model [the Adktm1(-/-)-Tg(UbiAdk) mouse] lacking the endogenous astrocytic enzyme due to a targeted disruption of the endogenous gene, but containing an Adk transgene under the control of a human ubiquitin promoter. Mutant Adktm1(-/-)-Tg(UbiAdk) mice were characterized by increased brain ADK activity and constitutive overexpression of transgenic ADK throughout the brain, with particularly high levels in hippocampal pyramidal neurons. This ADK overexpression was associated with increased baseline levels of locomotion. Most importantly, two-thirds of the mutant mice analysed exhibited spontaneous seizure activity in the hippocampus and cortex. This was the direct consequence of transgene expression, since this seizure activity could be prevented by systemic application of the ADK inhibitor 5-iodotubercidin. Intrahippocampal injection of kainate in the mutant mice resulted in astrogliosis to the same extent as that observed in wild-type mice despite the absence of endogenous astrocytic ADK. Therefore, KASE-induced upregulation of endogenous ADK in wild-type mice is a consequence of astrogliosis. However, seizures in kainic acid-injected mutants displayed increased intra-ictal spike frequency compared with wild-type mice, indicating that, once epilepsy is established, increased levels of ADK aggravate seizure severity. We therefore conclude that therapeutic strategies that augment the adenosine system after astrogliosis-induced upregulation of ADK constitute a neurochemical rationale for the prevention of seizures in epilepsy.
|Focal glial activation coincides with increased BACE1 activation and precedes amyloid plaque deposition in APP[V717I] transgenic mice. |
Heneka, MT; Sastre, M; Dumitrescu-Ozimek, L; Dewachter, I; Walter, J; Klockgether, T; Van Leuven, F
Journal of neuroinflammation 2 22 2005
Inflammation is suspected to contribute to the progression and severity of neurodegeneration in Alzheimer's disease (AD). Transgenic mice overexpressing the london mutant of amyloid precursor protein, APP [V717I], robustly recapitulate the amyloid pathology of AD.Early and late, temporal and spatial characteristics of inflammation were studied in APP [V717I] mice at 3 and 16 month of age. Glial activation and expression of inflammatory markers were determined by immunohistochemistry and RT-PCR. Amyloid deposition was assessed by immunohistochemistry, thioflavine S staining and western blot experiments. BACE1 activity was detected in brain lysates and in situ using the BACE1 activity kit from R&D Systems, Wiesbaden, Germany.Foci of activated micro- and astroglia were already detected at age 3 months, before any amyloid deposition. Inflammation parameters comprised increased mRNA levels coding for interleukin-1beta, interleukin-6, major histocompatibility complex II and macrophage-colony-stimulating-factor-receptor. Foci of CD11b-positive microglia expressed these cytokines and were neighbored by activated astrocytes. Remarkably, beta-secretase (BACE1) mRNA, neuronal BACE1 protein at sites of focal inflammation and total BACE1 enzyme activity were increased in 3 month old APP transgenic mice, relative to age-matched non-transgenic mice. In aged APP transgenic mice, the mRNA of all inflammatory markers analysed was increased, accompanied by astroglial iNOS expression and NO-dependent peroxynitrite release, and with glial activation near almost all diffuse and senile Abeta deposits.The early and focal glial activation, in conjunction with upregulated BACE1 mRNA, protein and activity in the presence of its substrate APP, is proposed to represent the earliest sites of amyloid deposition, likely evolving into amyloid plaques.
|Neural activity triggers neuronal oxidative metabolism followed by astrocytic glycolysis. |
Kasischke, Karl A, et al.
Science, 305: 99-103 (2004) 2004
We have found that two-photon fluorescence imaging of nicotinamide adenine dinucleotide (NADH) provides the sensitivity and spatial three-dimensional resolution to resolve metabolic signatures in processes of astrocytes and neurons deep in highly scattering brain tissue slices. This functional imaging reveals spatiotemporal partitioning of glycolytic and oxidative metabolism between astrocytes and neurons during focal neural activity that establishes a unifying hypothesis for neurometabolic coupling in which early oxidative metabolism in neurons is eventually sustained by late activation of the astrocyte-neuron lactate shuttle. Our model integrates existing views of brain energy metabolism and is in accord with known macroscopic physiological changes in vivo.
|Synthetic hydrogel guidance channels facilitate regeneration of adult rat brainstem motor axons after complete spinal cord transection. |
Tsai, Eve C, et al.
J. Neurotrauma, 21: 789-804 (2004) 2004
Synthetic guidance channels or tubes have been shown to promote axonal regeneration within the spinal cord from brainstem motor nuclei with the inclusion of agents such as matrices, cells, or growth factors to the tube. We examined the biocompatibility and regenerative capacity of synthetic hydrogel tubular devices that were composed of poly(2-hydroxyethyl methacrylate-co-methyl methacrylate) (PHEMA-MMA). Two PHEMA-MMA channels, having a mean elastic modulus of either 177 or 311 kPa were implanted into T8-transected spinal cords of adult Sprague Dawley rats. The cord stumps were inserted into the channels and fibrin glue was applied to the cord-channel interface. An expanded polytetrafluoroethylene (ePTFE) membrane was used for duraplasty. Controls underwent cord transection alone. Gross and microscopic examination of the spinal cords showed continuity of tissue within the synthetic guidance channels between the cord stumps at 4 and 8 weeks. There was a trend towards an increased area and width of bridging neural tissue in the 311-kPa guidance channels compared to the 177-kPa channels. Neurofilament stained axons were visualized within the bridging tissue, and serotonergic axons were found to enter the 311-kPa channel. Retrograde axonal tracing revealed regeneration of axons from reticular, vestibular, and raphe brainstem motor nuclei. For both channels, there was minimal scarring at the channel-cord interface, and less scarring at the channel-dura interface compared to that observed next to the ePTFE. The present study is the first to show that axons from brainstem motor nuclei regenerated in unfilled synthetic hydrogel guidance channels after complete spinal cord transection.
|Localization and expression of the glutamate transporter, excitatory amino acid transporter 4, within astrocytes of the rat retina. |
Ward, Michelle M, et al.
Cell Tissue Res., 315: 305-10 (2004) 2004
Mechanisms for the removal of glutamate are vital for maintaining normal function of the retina. Five excitatory amino acid transporters have been characterized to date from neuronal tissue, all of which are expressed within the retina except excitatory amino acid transporter 4 (EAAT4). In this study we examined the expression and localization of the glutamate transporter EAAT4 in the rat retina using RT-PCR and immunocytochemistry. RT-PCR using rat EAAT4 specific primers revealed a prominent 296-bp product in the retina, cortex and cerebellum. The identity of the EAAT4 fragment was confirmed by DNA sequencing. We examined the tissue expression levels of EAAT4 in cortex, retina and cerebellum using real-time PCR. The highest expression level was found in the cerebellum. Expression in the cortex was approximately 3.1% that of the cerebellum and the retina was found to have approximately 0.8% the total cerebellar EAAT4 content. In order to examine the specific cell types within the retina that express EAAT4, we performed immunocytochemistry using a rat EAAT4 specific antiserum. Cellular processes within the nerve fibre layer of the retina were intensely labelled for EAAT4. Double labelling EAAT4 with glial fibrillary acidic protein (GFAP) revealed extensive colocalization indicating that EAAT4 is localized within astrocytes within the retina. Double labelling of EAAT4 and the glutamate transporter EAAT1 (GLAST) revealed extensive colocalization suggesting that astrocytes in the retina express at least two types of glutamate transporters. These results suggest that astrocytes within the retina are well placed to provide mechanisms for glutamate removal as well as controlling cellular excitability.
|Viral-induced spinal motor neuron death is non-cell-autonomous and involves glutamate excitotoxicity. |
Darman, J; Backovic, S; Dike, S; Maragakis, NJ; Krishnan, C; Rothstein, JD; Irani, DN; Kerr, DA
The Journal of neuroscience : the official journal of the Society for Neuroscience 24 7566-75 2004
Neuroadapted Sindbis virus (NSV) is a neurotropic virus capable of inducing the death of spinal motor neurons in mice and rats. In this study we investigated the mechanisms that underlie NSV-induced motor neuron death. We found that many degenerating spinal motor neurons were not infected directly with NSV, suggesting that bystander cell death occurs. An excitotoxic mechanism was confirmed when blockade of calcium-permeable AMPA receptors attenuated motor neuron death both in vitro and in vivo. Blockade of astroglial glutamate reuptake potentiated NSV-induced motor neuron loss in vivo, suggesting that astrocyte-mediated removal of perisynaptic glutamate is important in limiting NSV-induced excitotoxic injury. Astroglial glutamate transport was reduced markedly in the spinal cord during NSV infection, in advance of motor neuron injury in susceptible mice. In contrast, we found 5.6-fold elevated glutamate uptake in the spinal cords of mice resistant to NSV-induced paralysis. Likewise, minocycline markedly increased spinal cord glutamate transport and protected mice from NSV-induced motor neuron death. These studies suggest that NSV infection triggers a cascade of events in the spinal cord resulting in impaired astrocytic glutamate transport and excitotoxic injury of motor neurons mediated via calcium-permeable AMPA receptors. Similar changes may occur in other motor neuron disorders such as amyotrophic lateral sclerosis or West Nile Virus-induced poliomyelitis, suggesting a common tissue injury pathway.
|Overexpression of adenosine kinase in epileptic hippocampus contributes to epileptogenesis. |
Gouder, N; Scheurer, L; Fritschy, JM; Boison, D
The Journal of neuroscience : the official journal of the Society for Neuroscience 24 692-701 2004
Endogenous adenosine in the brain is thought to prevent the development and spread of seizures via a tonic anticonvulsant effect. Brain levels of adenosine are primarily regulated by the activity of adenosine kinase. To establish a link between adenosine kinase expression and seizure activity, we analyzed the expression of adenosine kinase in the brain of control mice and in a kainic acid-induced mouse model of mesial temporal lobe epilepsy. Immunohistochemical analysis of brain sections of control mice revealed intense staining for adenosine kinase, mainly in astrocytes, which were more or less evenly distributed throughout the brain, as well as in some neurons, particularly in olfactory bulb, striatum, and brainstem. In contrast, hippocampi lesioned by a unilateral kainic acid injection displayed profound astrogliosis and therefore a significant increase in adenosine kinase immunoreactivity accompanied by a corresponding increase of enzyme activity, which paralleled chronic recurrent seizure activity in this brain region. Accordingly, seizures and interictal spikes were suppressed by the injection of a low dose of the adenosine kinase inhibitor 5-iodotubercidin. We conclude that overexpression of adenosine kinase in discrete parts of the epileptic hippocampus may contribute to the development and progression of seizure activity.
|Anti-Glial Fibrillary Acidic Protein, clone GA5 - Data Sheet|