Tabela com principais espec.
|Species Reactivity||Key Applications||Host||Format||Antibody Type|
|Av, Ma, Rp||IH(P), WB, IHC||Rb||Serum||Polyclonal Antibody|
|Presentation||Neat rabbit antisera.|
|Safety Information according to GHS|
|Storage and Shipping Information|
|Storage Conditions||Maintain at -20°C in undiluted aliquots for up to 12 months. Avoid repeated freeze/thaw cycles.|
|Material Size||50 µL|
Referências | 28 Disponível | Ver todas as referências
|Visão geral das referências||Aplicação||Espécies||Pub Med ID|
|An NT4/TrkB-dependent increase in innervation links early-life allergen exposure to persistent airway hyperreactivity. |
Aven, L; Paez-Cortez, J; Achey, R; Krishnan, R; Ram-Mohan, S; Cruikshank, WW; Fine, A; Ai, X
FASEB journal : official publication of the Federation of American Societies for Experimental Biology 28 897-907 2014
Children who are exposed to environmental respiratory insults often develop asthma that persists into adulthood. In this study, we used a neonatal mouse model of ovalbumin (OVA)-induced allergic airway inflammation to understand the long-term effects of early childhood insults on airway structure and function. We showed that OVA sensitization and challenge in early life led to a 2-fold increase in airway smooth muscle (ASM) innervation (Pless than 0.05) and persistent airway hyperreactivity (AHR). In contrast, OVA exposure in adult life elicited short-term AHR without affecting innervation levels. We found that postnatal ASM innervation required neurotrophin (NT)-4 signaling through the TrkB receptor and that early-life OVA exposure significantly elevated NT4 levels and TrkB signaling by 5- and 2-fold, respectively, to increase innervation. Notably, blockade of NT4/TrkB signaling in OVA-exposed pups prevented both acute and persistent AHR without affecting baseline airway function or inflammation. Furthermore, biophysical assays using lung slices and isolated cells demonstrated that NT4 was necessary for hyperreactivity of ASM induced by early-life OVA exposure. Together, our findings show that the NT4/TrkB-dependent increase in innervation plays a critical role in the alteration of the ASM phenotype during postnatal growth, thereby linking early-life allergen exposure to persistent airway dysfunction.
|Antibodies against low-density lipoprotein receptor-related protein 4 induce myasthenia gravis. |
Shen, C; Lu, Y; Zhang, B; Figueiredo, D; Bean, J; Jung, J; Wu, H; Barik, A; Yin, DM; Xiong, WC; Mei, L
The Journal of clinical investigation 123 5190-202 2013
Myasthenia gravis (MG) is the most common disorder affecting the neuromuscular junction (NMJ). MG is frequently caused by autoantibodies against acetylcholine receptor (AChR) and a kinase critical for NMJ formation, MuSK; however, a proportion of MG patients are double-negative for anti-AChR and anti-MuSK antibodies. Recent studies in these subjects have identified autoantibodies against low-density lipoprotein receptor-related protein 4 (LRP4), an agrin receptor also critical for NMJ formation. LRP4 autoantibodies have not previously been implicated in MG pathogenesis. Here we demonstrate that mice immunized with the extracellular domain of LRP4 generated anti-LRP4 antibodies and exhibited MG-associated symptoms, including muscle weakness, reduced compound muscle action potentials (CMAPs), and compromised neuromuscular transmission. Additionally, fragmented and distorted NMJs were evident at both the light microscopic and electron microscopic levels. We found that anti-LRP4 sera decreased cell surface LRP4 levels, inhibited agrin-induced MuSK activation and AChR clustering, and activated complements, revealing potential pathophysiological mechanisms. To further confirm the pathogenicity of LRP4 antibodies, we transferred IgGs purified from LRP4-immunized rabbits into naive mice and found that they exhibited MG-like symptoms, including reduced CMAP and impaired neuromuscular transmission. Together, these data demonstrate that LRP4 autoantibodies induce MG and that LRP4 contributes to NMJ maintenance in adulthood.
|Rescue of peripheral and CNS axon defects in mice lacking NMNAT2. |
Gilley, J; Adalbert, R; Yu, G; Coleman, MP
The Journal of neuroscience : the official journal of the Society for Neuroscience 33 13410-24 2013
NMNAT2 is an NAD(+)-synthesizing enzyme with an essential axon maintenance role in primary culture neurons. We have generated an Nmnat2 gene trap mouse to examine the role of NMNAT2 in vivo. Homozygotes die perinatally with a severe peripheral nerve/axon defect and truncated axons in the optic nerve and other CNS regions. The cause appears to be limited axon extension, rather than dying-back degeneration of existing axons, which was previously proposed for the NMNAT2-deficient Blad mutant mouse. Neurite outgrowth in both PNS and CNS neuronal cultures consistently stalls at 1-2 mm, similar to the length of truncated axons in the embryos. Crucially, this suggests an essential role for NMNAT2 during axon growth. In addition, we show that the Wallerian degeneration slow protein (Wld(S)), a more stable, aberrant NMNAT that can substitute the axon maintenance function of NMNAT2 in primary cultures, can also correct developmental defects associated with NMNAT2 deficiency. This is dose-dependent, with extension of life span to at least 3 months by homozygous levels of Wld(S) the most obvious manifestation. Finally, we propose that endogenous mechanisms also compensate for otherwise limiting levels of NMNAT2. This could explain our finding that conditional silencing of a single Nmnat2 allele triggers substantial degeneration of established neurites, whereas similar, or greater, reduction of NMNAT2 in constitutively depleted neurons is compatible with normal axon growth and survival. A requirement for NMNAT2 for both axon growth and maintenance suggests that reduced levels could impair axon regeneration as well as axon survival in aging and disease.
|β-Catenin gain of function in muscles impairs neuromuscular junction formation. |
Wu, H; Lu, Y; Barik, A; Joseph, A; Taketo, MM; Xiong, WC; Mei, L
Development (Cambridge, England) 139 2392-404 2012
Neuromuscular junction (NMJ) formation requires proper interaction between motoneurons and muscle cells. β-Catenin is required in muscle cells for NMJ formation. To understand underlying mechanisms, we investigated the effect of β-catenin gain of function (GOF) on NMJ development. In HSA-β-cat(flox(ex3)/+) mice, which express stable β-catenin specifically in muscles, motor nerve terminals became extensively defasciculated and arborized. Ectopic muscles were observed in the diaphragm and were innervated by ectopic phrenic nerve branches. Moreover, extensive outgrowth and branching of spinal axons were evident in the GOF mice. These results indicate that increased β-catenin in muscles alters presynaptic differentiation. Postsynaptically, AChR clusters in HSA-β-cat(flox(ex3)/+) diaphragms were distributed in a wider region, suggesting that muscle β-catenin GOF disrupted the signal that restricts AChR clustering to the middle region of muscle fibers. Expression of stable β-catenin in motoneurons, however, had no effect on NMJ formation. These observations provide additional genetic evidence that pre- and postsynaptic development of the NMJ requires an intricate balance of β-catenin activity in muscles.
|Distinct roles of muscle and motoneuron LRP4 in neuromuscular junction formation. |
Wu, H; Lu, Y; Shen, C; Patel, N; Gan, L; Xiong, WC; Mei, L
Neuron 75 94-107 2012
Neuromuscular junction (NMJ) formation requires precise interaction between motoneurons and muscle fibers. LRP4 is a receptor of agrin that is thought to act in cis to stimulate MuSK in muscle fibers for postsynaptic differentiation. Here we dissected the roles of LRP4 in muscle fibers and motoneurons in NMJ formation by cell-specific mutation. Studies of muscle-specific mutants suggest that LRP4 is involved in deciding where to form AChR clusters in muscle fibers, postsynaptic differentiation, and axon terminal development. LRP4 in HEK293 cells increased synapsin or SV2 puncta in contacting axons of cocultured neurons, suggesting a synaptogenic function. Analysis of LRP4 muscle and motoneuron double mutants and mechanistic studies suggest that NMJ formation may also be regulated by LRP4 in motoneurons, which could serve as agrin's receptor in trans to induce AChR clusters. These observations uncovered distinct roles of LRP4 in motoneurons and muscles in NMJ development.
|VEGF-Induced Growth Cone Enhancement Is Diminished by Inhibiting Tyrosine-Residue 1214 of VEGFR-2. |
Daniel Foehring,Beate Brand-Saberi,Carsten Theiss
Cells, tissues, organs 196 2012
Axonal outgrowth is of paramount significance for establishing the intricate neuronal network both during embryogenesis and nerve regeneration. Vascular endothelial growth factor (VEGF), which is known for its essential role in vascular sprouting and its involvement in cancer, has recently been found to exert a trophic activity on neurons leading to an increased axonal outgrowth. Although two receptors, VEGFR-2 and neuropilin-1, were identified on neurons, the signaling pathways associated with them are not well understood. The aim of this study was to analyze the influence of VEGF on the growth cone morphology and motility of dorsal root ganglia (DRG) neurons. Moreover, we aimed for a deeper understanding of VEGFR-2 on growth cones that potentially mediates the stimulating and attractive effects. We cultivated chicken DRG in medium containing mouse VEGF and analyzed growth cone size. The data presented here show a positive effect of VEGF on growth cone size. Furthermore, we interrupted the activity of VEGFR-2 by either blocking the tyrosine residue 1214 (tyr1214) or by inhibiting the receptor phosphorylation with axitinib, a novel small molecule, which has recently entered phase III trials for cancer treatment. Disruption of the VEGFR-2 leads to a significantly diminished growth cone size. Based on these findings, we propose a positive effect of VEGF on peripheral nervous system growth cone size and show for the first time quantitative data to underline this hypothesis. Additionally, we propose that VEGFR-2 and especially the tyr1214-dependent pathway of VEGFR-2 are of importance in VEGF signaling in the growth cone of DRG neurons.
|Astrocyte-secreted factors modulate the developmental distribution of inhibitory synapses in nucleus laminaris of the avian auditory brainstem. |
Matthew J Korn,Scott J Koppel,Lan H Li,Divya Mehta,Sonia B Mehta,Armin H Seidl,Karina S Cramer
The Journal of comparative neurology 520 2012
Nucleus laminaris (NL) neurons in the avian auditory brainstem are coincidence detectors necessary for the computation of interaural time differences used in sound localization. In addition to their excitatory inputs from nucleus magnocellularis, NL neurons receive inhibitory inputs from the superior olivary nucleus (SON) that greatly improve coincidence detection in mature animals. The mechanisms that establish mature distributions of inhibitory inputs to NL are not known. We used the vesicular GABA transporter (VGAT) as a marker for inhibitory presynaptic terminals to study the development of inhibitory inputs to NL between embryonic day 9 (E9) and E17. VGAT immunofluorescent puncta were first seen sparsely in NL at E9. The density of VGAT puncta increased with development, first within the ventral NL neuropil region and subsequently throughout both the ventral and dorsal dendritic neuropil, with significantly fewer terminals in the cell body region. A large increase in density occurred between E13–15 and E16–17, at a developmental stage when astrocytes that express glial fibrillary acidic protein (GFAP) become mature. We cultured E13 brainstem slices together with astrocyte-conditioned medium (ACM) obtained from E16 brainstems and found that ACM, but not control medium, increased the density of VGAT puncta. This increase was similar to that observed during normal development. Astrocyte-secreted factors interact with the terminal ends of SON axons to increase the number of GABAergic terminals. These data suggest that factors secreted from GFAP-positive astrocytes promote maturation of inhibitory pathways in the auditory brainstem.
|Olig1 function is required for remyelination potential of transplanted neural progenitor cells in a model of viral-induced demyelination. |
Whitman, LM; Blanc, CA; Schaumburg, CS; Rowitch, DH; Lane, TE
Experimental neurology 235 380-7 2012
Multiple sclerosis (MS) is a chronic inflammatory disease of the central nervous system (CNS) resulting in cumulative neurologic deficits associated with progressive myelin loss. We have previously shown that transplantation of neural progenitor cells (NPCs) into mice persistently infected with the JHM strain of mouse hepatitis virus (JHMV) results in enhanced differentiation into oligodendrocyte progenitor cells (OPCs) that is associated with remyelination and axonal sparing. The current study examines the contributions of the transcription factor Olig1 on NPC differentiation and remyelination. Under defined conditions, NPCs preferentially differentiate into oligodendroglia whereas NPCs isolated from Olig1-deficient (Olig1-/-) mice exhibit enhanced differentiation into astrocytes. Transplantation of Olig1-/- and Olig1+/+ NPCs into JHMV-infected mice resulted in similar cell survival, proliferation, and selective migration to areas of demyelination. However, only recipients of wild type NPCs exhibited extensive remyelination compared to mice receiving Olig1-/- NPCs. In vivo characterization of NPCs revealed that Olig1+/+ NPCs preferentially differentiated into NG2-positive OPCs and formed processes expressing myelin basic protein that encircled axons. In contrast, the majority of transplanted Olig1-/- NPCs differentiated into GFAP-positive cells consistent with the astrocyte lineage. These results indicate that exogenous NPCs contribute to improved clinical and histological outcome and this is associated with remyelination by this donor population. Further, these findings reveal that Olig1function is required for the remyelination potential of NPCs after transplant, through specification and/or maintenance of oligodendroglial identity.
|A method to isolate and culture expand human dental pulp stem cells. |
Stan Gronthos,Agnieszka Arthur,P Mark Bartold,Songtao Shi
Methods in molecular biology (Clifton, N.J.) 698 2011
Dentinal repair in teeth occurs through the activity of specialized cells known as odontoblasts that are thought to be maintained by a precursor population associated with the perivascular cells within dental pulp tissue. We have previously isolated candidate dental pulp stem cells (DPSC) from adult human third molars, with the ability to generate clonogenic cell clusters (CFU-F: colony-forming units-fibroblastic), a high proliferation rate, and multi-potential differentiation in vitro. When cultured DPSC are transplanted into immunocompromised mice, they generated a dentin-like structure lined with human odontoblast-like cells that surrounded a pulp-like interstitial tissue, composed of collagen and a vascular network. The present protocol describes a methodology to generate highly purified preparations of human DPSC. This process involves the enzymatic digestion of fresh samples of human dental pulp tissue followed by the isolation of DPSC using magnetic bead cell separation, based on their expression of mesenchymal stem cell associated markers.
|Spreading of prions from the immune to the peripheral nervous system: a potential implication of dendritic cells. |
Dorban, Gauthier, et al.
Histochem. Cell Biol., 133: 493-504 (2010) 2010
The implication of dendritic cells (DCs) in the peripheral spreading of prions has increased in the last few years. It has been recently described that DCs can transmit prions to primary neurons from the central nervous system. In order to improve the understanding of the earliest steps of prion peripheral neuroinvasion, we studied, using an in vitro model, the effect of exposing primary peripheral neurons to scrapie-infected lymphoid cells. Thanks to this system, there is evidence that bone marrow dendritic cells (BMDCs) are in connection with neurites of peripheral neurons via cytoplasmic extensions. BMDCs are competent to internalize prions independently from the expression of cellular prion protein (PrP(C)) and have the capacity to transmit detergent-insoluble, relatively proteinase K-resistant prion protein (PrP(Sc)) to peripheral neurons after 96 h of coculture. Furthermore, we confirmed the special status of the peripheral nervous system in front of prion diseases. Contrary to central neurons, PrP(Sc) infection does not disturb survival and neurite outgrowth. Our model demonstrates that PrP(Sc)-loaded dendritic cells and peripheral nerve fibers that are included in neuroimmune interfaces can initiate and spread prion neuroinvasion.
|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.Texto completo do artigo
|Cadherin-7 and cadherin-6B differentially regulate the growth, branching and guidance of cranial motor axons. |
Sarah H Barnes,Stephen R Price,Corinna Wentzel,Sarah C Guthrie
Development (Cambridge, England) 137 2010
Cadherin-7 (Cad7) and cadherin-6B (Cad6B) are expressed in early and late phases of cranial motoneuron development, respectively. Cad7 is expressed by cranial motoneurons soon after they are generated, as well as in the environment through which their axons extend. By contrast, Cad6B is expressed by mature cranial motoneurons. We demonstrate in chick that these cadherins play distinct roles in cranial motor axon morphology, branching and projection. Using in vitro approaches, we show that Cad7 enhances motor axon outgrowth, suppresses the formation of multiple axons and restricts interstitial branching, thus promoting the development of a single unbranched axon characteristic of differentiating motoneurons. Conversely, Cad6B in vitro promotes motor axon branching, a characteristic of mature motoneurons. In vivo gain- and loss-of-function experiments for these cadherins yielded phenotypes consistent with this interpretation. In particular, a loss of cadherin-mediated interactions in vivo led to dysregulation of the cranial motoneuron normal branching programme and caused axon navigation defects. We also demonstrate that Cad6B functions via the phosphatidylinositol 3-kinase pathway. Together, these data show that Cad7 and Cad6B differentially regulate cranial motoneuron growth, branching and axon guidance.Texto completo do artigo
|Stromal cell-derived factor-1 and hepatocyte growth factor guide axon projections to the extraocular muscles. |
Lerner O, Davenport D, Patel P, Psatha M, Lieberam I, Guthrie S
Dev Neurobiol 70 549-64. 2010
Vertebrate eye movements depend on the co-ordinated function of six extraocular muscles that are innervated by the oculomotor, trochlear, and abducens nerves. Here, we show that the diffusible factors, stromal cell-derived factor-1 (SDF-1) and hepatocyte growth factor (HGF), guide the development of these axon projections. SDF-1 is expressed in the mesenchyme around the oculomotor nerve exit point, and oculomotor axons fail to exit the neuroepithelium in mice mutant for the SDF-1 receptor CXCR4. Both SDF-1 and HGF are expressed in or around the ventral and dorsal oblique muscles, which are distal targets for the oculomotor and trochlear nerves, respectively, as well as in the muscles which are later targets for oculomotor axon branches. We find that in vitro SDF-1 and HGF promote the growth of oculomotor and trochlear axons, whereas SDF-1 also chemoattracts oculomotor axons. Oculomotor neurons show increased branching in the presence of SDF-1 and HGF singly or together. HGF promotes the growth of trochlear axons more than that of oculomotor axons. Taken together, these data point to a role for both SDF-1 and HGF in extraocular nerve projections and indicate that SDF-1 functions specifically in the development of the oculomotor nerve, including oculomotor axon branch formation to secondary muscle targets. HGF shows some specificity in preferentially enhancing development of the trochlear nerve.
|Slit and Netrin-1 guide cranial motor axon pathfinding via Rho-kinase, myosin light chain kinase and myosin II. |
Murray, A; Naeem, A; Barnes, SH; Drescher, U; Guthrie, S
Neural development 5 16 2010
In the developing hindbrain, cranial motor axon guidance depends on diffusible repellent factors produced by the floor plate. Our previous studies have suggested that candidate molecules for mediating this effect are Slits, Netrin-1 and Semaphorin3A (Sema3A). It is unknown to what extent these factors contribute to floor plate-derived chemorepulsion of motor axons, and the downstream signalling pathways are largely unclear.In this study, we have used a combination of in vitro and in vivo approaches to identify the components of floor plate chemorepulsion and their downstream signalling pathways. Using in vitro motor axon deflection assays, we demonstrate that Slits and Netrin-1, but not Sema3A, contribute to floor plate repulsion. We also find that the axon pathways of dorsally projecting branchiomotor neurons are disrupted in Netrin-1 mutant mice and in chick embryos expressing dominant-negative Unc5a receptors, indicating an in vivo role for Netrin-1. We further demonstrate that Slit and Netrin-1 signalling are mediated by Rho-kinase (ROCK) and myosin light chain kinase (MLCK), which regulate myosin II activity, controlling actin retrograde flow in the growth cone. We show that MLCK, ROCK and myosin II are required for Slit and Netrin-1-mediated growth cone collapse of cranial motor axons. Inhibition of these molecules in explant cultures, or genetic manipulation of RhoA or myosin II function in vivo causes characteristic cranial motor axon pathfinding errors, including the inability to exit the midline, and loss of turning towards exit points.Our findings suggest that both Slits and Netrin-1 contribute to floor plate-derived chemorepulsion of cranial motor axons. They further indicate that RhoA/ROCK, MLCK and myosin II are components of Slit and Netrin-1 signalling pathways, and suggest that these pathways are of key importance in cranial motor axon navigation.
|Adult human dental pulp stem cells differentiate toward functionally active neurons under appropriate environmental cues. |
Agnes Arthur, Grigori Rychkov, Songtao Shi, Simon Andrea Koblar, Stan Gronthos
Stem cells (Dayton, Ohio) 26 1787-95 2008
Human adult dental pulp stem cells (DPSCs) reside within the perivascular niche of dental pulp and are thought to originate from migrating cranial neural crest (CNC) cells. During embryonic development, CNC cells differentiate into a wide variety of cell types, including neurons of the peripheral nervous system. Previously, we have demonstrated that DPSCs derived from adult human third molar teeth differentiate into cell types reminiscent of CNC embryonic ontology. We hypothesized that DPSCs exposed to the appropriate environmental cues would differentiate into functionally active neurons. The data demonstrated that ex vivo-expanded human adult DPSCs responded to neuronal inductive conditions both in vitro and in vivo. Human adult DPSCs, but not human foreskin fibroblasts (HFFs), acquired a neuronal morphology, and expressed neuronal-specific markers at both the gene and protein levels. Culture-expanded DPSCs also exhibited the capacity to produce a sodium current consistent with functional neuronal cells when exposed to neuronal inductive media. Furthermore, the response of human DPSCs and HFFs to endogenous neuronal environmental cues was determined in vivo using an avian xenotransplantation assay. DPSCs expressed neuronal markers and acquired a neuronal morphology following transplantation into the mesencephalon of embryonic day-2 chicken embryo, whereas HFFs maintained a thin spindle fibroblastic morphology. We propose that adult human DPSCs provide a readily accessible source of exogenous stem/precursor cells that have the potential for use in cell-therapeutic paradigms to treat neurological disease.
|Selective changes in nocifensive behavior despite normal cutaneous axon innervation in leptin receptor-null mutant (db/db) mice. |
Douglas E Wright, Megan S Johnson, M G Arnett, Susan E Smittkamp, Janelle M Ryals
Journal of the peripheral nervous system : JPNS 12 250-61 2007
Much of our understanding of the effects of diabetes on the peripheral nervous system is derived from models induced by streptozotocin in which hyperglycemia is rapidly caused by pancreatic beta-cell destruction. Here, we have quantified sensory impairments over time in leptin receptor (lepr)-null mutant -/- mice, a type 2 model of diabetes in which the absence of leptin receptor signaling leads to obesity and chronic hyperglycemia by 4 weeks of age. To assess these mice as a model for peripheral neuropathy, we quantified the responsiveness of lepr -/- mice to mechanical, thermal, and chemogenic stimuli, as well as epidermal and dermal innervation of the hind paw. Compared with wild-type +/+ and heterozygous +/- mice, lepr -/- mice displayed reduced sensitivity to mechanical stimuli by 6 weeks of age, and however, responses to noxious heat were normal. Lepr -/- mice also devoted less activity to their injected paw during the second phase following formalin administration. However, epidermal and dermal innervation of lepr -/- mice was not different from that of lepr +/+ and +/- mice even after 10 weeks of hyperglycemia, suggesting that cutaneous innervation is resistant to chronic hyperglycemia in these mice. These results suggest that certain rodent nocifensive behaviors may be linked to the abundance of cutaneous innervation, while others are not. Finally, these results reveal that the lepr -/- mice may not be useful to study neuropathy associated with distal axonal degeneration but may be better suited for studies of hyperglycemia-induced sensory neuron dysfunction without distal nerve loss.
|Hearing development and spiral ganglion neurite growth in VASP deficient mice. |
Stefan Dazert, Bernhard Schick, Rene Hartensuer, Stefan Volkenstein, Christoph Aletsee, Stefan Hansen, Wafaa E Shehata-Dieler, Martin Eigenthaler, Ulrich Walter, Allen F Ryan, Dominik Brors
Brain research 1178 73-82 2007
Vasodilator-stimulated phosphoprotein (VASP) has been found to be involved in intracellular signalling pathways and to play an important role in the actin associated organization and formation of the cytoskeleton. Since differential VASP expression was noted in inner ear tissues, the present study was performed to investigate the hearing development in VASP deficient mice. Hearing development in VASP-/- mice and wild type animals was investigated by auditory brain stem (ABR) measurements. In addition, inner ear tissues of wild type animals were tested for VASP expression using PCR, Western blot analysis, in situ hybridisation, and immunohistochemistry. To compare spiral ganglion (SG) neurite growth, SG explants from VASP-/- and wild type mice were analyzed under cell culture conditions. The electroacoustical results of the present study indicate that VASP deficient mice present with a later onset of hearing during postnatal development compared to wild type animals. Transient VASP expression was detected in neonatal SG of wild type mice. Tissue culture experiments with SG explants from VASP-/- animals revealed significant alterations in SG neurite extension compared to wild types. The present findings suggest a role for VASP during neonatal development of the mammalian cochlea and allow speculation on a possible delayed innervation of cochlear hair cells due to changes in SG neurite growth in VASP-deficient mice. Temporary VASP deficits in the neonatal inner ear may be compensated by related proteins like MENA leading to a delayed but complete development of hearing function in VASP-/- animals.
|Neurotrophic modulation of myelinated cutaneous innervation and mechanical sensory loss in diabetic mice. |
Christianson, JA; Ryals, JM; Johnson, MS; Dobrowsky, RT; Wright, DE
Neuroscience 145 303-13 2007
Human diabetic patients often lose touch and vibratory sensations, but to date, most studies on diabetes-induced sensory nerve degeneration have focused on epidermal C-fibers. Here, we explored the effects of diabetes on cutaneous myelinated fibers in relation to the behavioral responses to tactile stimuli from diabetic mice. Weekly behavioral testing began prior to streptozotocin (STZ) administration and continued until 8 weeks, at which time myelinated fiber innervation was examined in the footpad by immunohistochemistry using antiserum to neurofilament heavy chain (NF-H) and myelin basic protein (MBP). Diabetic mice developed reduced behavioral responses to non-noxious (monofilaments) and noxious (pinprick) stimuli. In addition, diabetic mice displayed a 50% reduction in NF-H-positive myelinated innervation of the dermal footpad compared with non-diabetic mice. To test whether two neurotrophins nerve growth factor (NGF) and/or neurotrophin-3 (NT-3) known to support myelinated cutaneous fibers could influence myelinated innervation, diabetic mice were treated intrathecally for 2 weeks with NGF, NT-3, NGF and NT-3. Neurotrophin-treated mice were then compared with diabetic mice treated with insulin for 2 weeks. NGF and insulin treatment both increased paw withdrawal to mechanical stimulation in diabetic mice, whereas NT-3 or a combination of NGF and NT-3 failed to alter paw withdrawal responses. Surprisingly, all treatments significantly increased myelinated innervation compared with control-treated diabetic mice, demonstrating that myelinated cutaneous fibers damaged by hyperglycemia respond to intrathecal administration of neurotrophins. Moreover, NT-3 treatment increased epidermal Merkel cell numbers associated with nerve fibers, consistent with increased numbers of NT-3-responsive slowly adapting A-fibers. These studies suggest that myelinated fiber loss may contribute as significantly as unmyelinated epidermal loss in diabetic neuropathy, and the contradiction between neurotrophin-induced increases in dermal innervation and behavior emphasizes the need for multiple approaches to accurately assess sensory improvements in diabetic neuropathy.
|Ephrin-As play a rhombomere-specific role in trigeminal motor axon projections in the chick embryo. |
Fabrice Prin, Keat-Eng Ng, Uma Thaker, Uwe Drescher, Sarah Guthrie
Developmental biology 279 402-19 2005
In this study, we investigate the possible role of ephrin-Eph signaling in trigeminal motor axon projections. We find that EphA receptors are expressed at higher levels by rhombomere 2 (r2) trigeminal motor neurons than by r3 trigeminal motor neurons in the chick embryo. Mapping of rhombomere-specific axon projections shows that r2 and r3 trigeminal motor neurons project to different muscle targets, including the mandibular adductor and the intermandibularis muscles respectively. Ephrin-A5 is expressed in these muscles, especially in some regions of the intermandibularis muscle, and can cause growth cone collapse of both r2 and r3 motor axons in vitro. We demonstrate that in vivo overexpression of ephrin-A5 in the intermandibularis muscle, or overexpression of dominant-negative EphA receptors in trigeminal motor neurons leads to a reduction in branching of r3-derived motor axons specifically. Overexpression of full-length EphA receptors impairs the formation of r3 projections to the intermandibularis muscle. These findings indicate that ephrins and their Eph receptors play a role in trigeminal motor axon topographic mapping and in rhombomere 3-derived projections in particular.
|Expression of Semaphorin-3A and its receptors in endochondral ossification: potential role in skeletal development and innervation. |
C Gomez, B Burt-Pichat, F Mallein-Gerin, B Merle, P D Delmas, T M Skerry, L Vico, L Malaval, C Chenu
Developmental dynamics : an official publication of the American Association of Anatomists 234 393-403 2005
Bone tissue is densely innervated, and there is increasing evidence for a neural control of bone metabolism. Semaphorin-3A is a very important regulator of neuronal targeting in the peripheral nervous system as well as in angiogenesis, and knockout of the Semaphorin-3A gene induces abnormal bone and cartilage development. We analyzed the spatial and temporal expression patterns of Semaphorin-3A signaling molecules during endochondral ossification, in parallel with the establishment of innervation. We show that osteoblasts and chondrocytes differentiated in vitro express most members of the Semaphorin-3A signaling system (Semaphorin-3A, Neuropilin-1, and Plexins-A1 and -A2). In vitro, osteoclasts express most receptor chains but not the ligand. In situ, these molecules are all expressed in the periosteum and by resting, prehypertrophic and hypertrophic chondrocytes in ossification centers before the onset of neurovascular invasion. They are detected later in osteoblasts and also osteoclasts, with differences in intensity and regional distribution. Semaphorin-3A and Neuropilin-1 are also expressed in the bone marrow. Plexin-A3 is not expressed by bone cell lineages in vitro. It is detected early in the periosteum and hypertrophic chondrocytes. After the onset of ossification, this chain is restricted to a network of cell processes in close vicinity to the cells lining the trabeculae, similar to the pattern observed for neural markers at the same stages. After birth, while the density of innervation decreases, Plexin-A3 is strongly expressed by blood vessels on the ossification front. In conclusion, Semaphorin-3A signaling is present in bone and seems to precede or coincide at the temporal but also spatial level with the invasion of bone by blood vessels and nerve fibers. Expression patterns suggest Plexin-A3/Neuropilin-1 as a candidate receptor in target cells for the regulation of bone innervation by Semaphorin-3A.
|Development of oculomotor axon projections in the chick embryo. |
John Kevin Chilton, Sarah Guthrie
The Journal of comparative neurology 472 308-17 2004
The pattern of innervation of the extraocular muscles is highly conserved across higher vertebrate species and mediates sophisticated visuomotor processes. Defects in oculomotor development often lead to strabismus, a misalignment of the eyes that can cause partial blindness. Although it has been intensively studied from a clinical perspective, relatively little is known about how the system develops embryonically. We have therefore mapped the development of the oculomotor nerve (OMN) in chick embryos by using confocal microscopy. We show that OMN development follows a series of stereotyped steps that are tightly regulated in space and time. The OMN initially grows past three of its targets to innervate its distal target, the ventral oblique muscle, only later forming branches to the more proximal muscles. We have also investigated spatiotemporal aspects of the unusual contralateral migration of a subpopulation of oculomotor neurons by using molecular markers and have found the semaphorin axon guidance molecules and their receptors, the neuropilins, to be expressed in discrete subnuclei during this migration. Finally, we have created an embryological model of Duane retraction syndrome (DRS), a form of strabismus in which the OMN is believed to innervate aberrantly the lateral rectus, the normal target of the abducens nerve. By ablating rhombomeres 5 and 6 and hence the abducens, we have mimicked a proposed oculomotor deficit occurring in DRS. We find that the absence of the abducens nerve is not sufficient to produce this inappropriate innervation, so other factors are required to explain DRS.
|Endogenous recovery of injured spinal cord: longitudinal in vivo magnetic resonance imaging. |
Ponnada A Narayana, Raymond J Grill, Tessy Chacko, Russell Vang
Journal of neuroscience research 78 749-59 2004
Pathological changes were followed longitudinally with in vivo magnetic resonance imaging (MRI) and behavioral studies in experimental spinal cord injury (SCI). MRI-observed pathology was correlated with histology. On MRI, the cavitated regions of the injured cord were gradually filled with viable tissue between two and 8 weeks postinjury, and a concomitant improvement was observed in the neurobehavioral scores. By weeks 3-6, on MRI, the gray matter (GM) returned in the segments caudal, but not rostral, to the injury site. The corresponding histological sections revealed motor neurons as well as other nuclei in the gray matter immediately caudal to the epicenter, but not at the site of injury, suggesting neuronal recovery in perilesioned areas. The neuronal and neurological recovery appeared to occur about the same time as neovasculature that was reported on the contrast-enhanced MRI, suggesting a role for angiogenesis in recovery from SCI. The role of angiogenesis in neuronal recovery is further supported by the immunohistochemical observation of greater bromodeoxyuridine uptake by blood vessels near the lesion site compared with uninjured cords.
|A syntaxin 1, Galpha(o), and N-type calcium channel complex at a presynaptic nerve terminal: analysis by quantitative immunocolocalization. |
Li, Q; Lau, A; Morris, TJ; Guo, L; Fordyce, CB; Stanley, EF
The Journal of neuroscience : the official journal of the Society for Neuroscience 24 4070-81 2004
Presynaptic Ca(V)2.2 (N-type) calcium channels are subject to modulation by interaction with syntaxin 1 and by a syntaxin 1-sensitive Galpha(O) G-protein pathway. We used biochemical analysis of neuronal tissue lysates and a new quantitative test of colocalization by intensity correlation analysis at the giant calyx-type presynaptic terminal of the chick ciliary ganglion to explore the association of Ca(V)2.2 with syntaxin 1 and Galpha(O). Ca(V)2.2 could be localized by immunocytochemistry (antibody Ab571) in puncta on the release site aspect of the presynaptic terminal and close to synaptic vesicle clouds. Syntaxin 1 coimmunoprecipitated with Ca(V)2.2 from chick brain and chick ciliary ganglia and was widely distributed on the presynaptic terminal membrane. A fraction of the total syntaxin 1 colocalized with the Ca(V)2.2 puncta, whereas the bulk colocalized with MUNC18-1. Galpha(O,) whether in its trimeric or monomeric state, did not coimmunoprecipitate with Ca(V)2.2, MUNC18-1, or syntaxin 1. However, the G-protein exhibited a punctate staining on the calyx membrane with an intensity that varied in synchrony with that for both Ca channels and syntaxin 1 but only weakly with MUNC18-1. Thus, syntaxin 1 appears to be a component of two separate complexes at the presynaptic terminal, a minor one at the transmitter release site with Ca(V)2.2 and Galpha(O), as well as in large clusters remote from the release site with MUNC18-1. These syntaxin 1 protein complexes may play distinct roles in presynaptic biology.
|A simple method for immunocytochemical staining with multiple rabbit polyclonal antibodies. |
Terence J Morris, Elise F Stanley
Journal of neuroscience methods 127 149-55 2003
We report a single step, simple, repeatable, rapid and reliable technique for simultaneous immunocytochemical staining with two or more rabbit polyclonal antibodies. This technique, which we have dubbed the Pretty Poly method, is based on conjugating the antibodies with commercially available, fluorophore-tagged Staphylococcal protein-A (SP-A). Staining is illustrated at the calyx type presynaptic nerve terminal of the chick ciliary ganglion with antibodies directed against three nerve terminal proteins: neurofilaments of the axonal cytoskeleton, and two secretory vesicle proteins, SV2 and cysteine string protein (CSP). Images were deblurred with an iterative deconvolution protocol. Staining with a single polyclonal antibody was bright and had a resolution approaching light microscope limit. Treatment with two different polyclonal antibodies conjugated with contrasting dye-tagged protein-A resulted in double staining without significant crossover that was fully equivalent to the standard primary/secondary technique. The same single step protocol was used to stain with all three rabbit polyclonal antibodies or to combine the technique with a standard monoclonal primary/secondary antibody stain. Thus, the Pretty Poly protocol is a highly flexible, simple and yet effective staining technique that essentially solves the problem of co-staining with multiple polyclonal rabbit antibodies.
|Somatic motoneurone specification in the hindbrain: the influence of somite-derived signals, retinoic acid and Hoxa3. |
Sonia Guidato, Fabrice Prin, Sarah Guthrie, Sonia Guidato, Fabrice Prin, Sarah Guthrie
Development (Cambridge, England) 130 2981-96 2003
We have investigated the mechanisms involved in generating hindbrain motoneurone subtypes, focusing on somatic motoneurones, which are confined to the caudal hindbrain within rhombomeres 5-8. Following heterotopic transplantation of rhombomeres along the rostrocaudal axis at various developmental stages, we have found that the capacity of rhombomeres to generate somatic motoneurones is labile at the neural plate stage but becomes fixed just after neural tube closure, at stage 10-11. Grafting of somites or retinoic acid-loaded beads beneath the rostral hindbrain induced the formation of somatic motoneurones in rhombomere 4 only, and Hox genes normally expressed more caudally (Hoxa3, Hoxd4) were induced in this region. Targeted overexpression of Hoxa3 in the rostral hindbrain led to the generation of ectopic somatic motoneurones in ventral rhombomeres 1-4, and was accompanied by the repression of the dorsoventral patterning gene Irx3. Taken together, these observations suggest that the somites, retinoic acid and Hox genes play a role in patterning somatic motoneurones in vivo.
|Compartmentation of alpha-internexin and neurofilament triplet proteins in cultured hippocampal neurons. |
Benson, D L, et al.
J. Neurocytol., 25: 181-96 (1996) 1996
Intermediate filaments comprise an integral part of the neuronal cytoskeleton. However, little is known about their function, and there remains some uncertainty about their precise subcellular localization. We examined the timing of expression and distribution of alpha-internexin, neurofilament triplet proteins and peripherin using immunocytochemistry in cultured hippocampal neurons. alpha-Internexin immunostaining was present in all neurons at all developmental stages. Immunostaining appeared as long filaments in axons and short fragments in dendrites which extended into dendritic spines. The presence of alpha-internexin in dendritic spines was confirmed in situ by electron microscopy of rat hippocampal tissue sections and suggests that this intermediate filament may serve as a link between cytoskeletal elements in dendritic shafts and spines. In culture, immunostaining using antibodies against individual triplet protein subunits indicated that light (NF-L) and middle (NF-M) subunits were first expressed in cells shortly after the initiation of axonal outgrowth. Expression of the heavy (NF-H) subunit occurred a few days later. Although timing and localization of expression did not correlate with the initiation of axonal or dendritic processes, it was coincident with periods of rapid outgrowth. Triplet proteins were more abundant in axons and appeared to be incorporated into lengthier filaments than in dendrites. Highly phosphorylated NFH/M immunoreactivity was polarized to axons after 6 days in culture. The distribution of one NF-H epitope was restricted to GABAergic neurons in mature cultures, suggesting a cell-type specific modification. Peripherin was not detectable at any time in hippocampal cultures. Our results show that intermediate filaments are integral components of the neuronal cytoskeleton of cultured hippocampal neurons throughout development. Furthermore, the localization of alpha-internexin suggests that it may be involved in the formation or maintenance of dendritic spines.
|Distribution of plectin, an intermediate filament-associated protein, in the adult rat central nervous system |
Errante, L D, et al
J Neurosci Res, 37:515-28 (1994) 1994
|A molecular dissection of the carboxyterminal tails of the major neurofilament subunits NF-M and NF-H. |
Harris, J, et al.
J. Neurosci. Res., 30: 47-62 (1991) 1991
We have initiated a multidisciplinary project that aims to dissect and ultimately define the functions of the long and unusual C-terminal "tail" sequences of the two high molecular weight neurofilament subunits, NF-M and NF-H. A series of recombinant fusion proteins containing selected NF-M and NF-H tail sequences were constructed using appropriate cDNAs. These fusion proteins were used to further define the epitopes for a variety of widely used neurofilament antibodies, including NN18 and N52, which are now available commercially from several companies. We also measured the SDS-PAGE mobility of the fusion proteins and found that, like the native neurofilament tails, the fusion proteins ran considerably slower than predicted from their molecular weight. Since all fusion proteins produced so far exhibit this characteristic we conclude that all segments of the NF-M and NF-H tail share this unusual property. Finally we were able to produce novel and potentially useful polyclonal and monoclonal antibodies to selected segments of NF-M and NF-H sequence. These antibody studies showed that the extreme C-termini of NF-M and NF-H are immunologically absolutely distinct from one another and also indicate that the extreme C-terminus of NF-M is immunologically much more conserved than the analogous region of NF-H. These findings are in complete agreement with our conclusions derived from amino acid sequence analysis, and further underline the possible functional importance of the extreme C-terminus of NF-M. We also show that the unusual immunological properties of the bovine NF-M tail we have previously observed do not extend to the extreme C-terminal region, which appears immunologically no different from the analogous region of other NF-M molecules. The peculiarities of bovine NF-M could be explained by the presence of a KSP motif that resembles the NF-H KSP prototype.
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