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  • A neural extracellular matrix-based method for in vitro hippocampal neuron culture and dopaminergic differentiation of neural stem cells. 23594371

    The ability to recreate an optimal cellular microenvironment is critical to understand neuronal behavior and functionality in vitro. An organized neural extracellular matrix (nECM) promotes neural cell adhesion, proliferation and differentiation. Here, we expanded previous observations on the ability of nECM to support in vitro neuronal differentiation, with the following goals: (i) to recreate complex neuronal networks of embryonic rat hippocampal cells, and (ii) to achieve improved levels of dopaminergic differentiation of subventricular zone (SVZ) neural progenitor cells.Hippocampal cells from E18 rat embryos were seeded on PLL- and nECM-coated substrates. Neurosphere cultures were prepared from the SVZ of P4-P7 rat pups, and differentiation of neurospheres assayed on PLL- and nECM-coated substrates.When seeded on nECM-coated substrates, both hippocampal cells and SVZ progenitor cells showed neural expression patterns that were similar to their poly-L-lysine-seeded counterparts. However, nECM-based cultures of both hippocampal neurons and SVZ progenitor cells could be maintained for longer times as compared to poly-L-lysine-based cultures. As a result, nECM-based cultures gave rise to a more branched neurite arborization of hippocampal neurons. Interestingly, the prolonged differentiation time of SVZ progenitor cells in nECM allowed us to obtain a purer population of dopaminergic neurons.We conclude that nECM-based coating is an efficient substrate to culture neural cells at different stages of differentiation. In addition, neural ECM-coated substrates increased neuronal survival and neuronal differentiation efficiency as compared to cationic polymers such as poly-L-lysine.
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  • Crmp4 deletion promotes recovery from spinal cord injury by neuroprotection and limited scar formation. 25652774

    Axonal outgrowth inhibitors and scar formation are two major obstacles to central nervous system (CNS) repair. No target molecule that regulates both axonal growth and scarring has been identified. Here we identified collapsin response mediator protein 4 (CRMP4), a common mediator of inhibitory signals after neural injury, as a crucial factor that contributes to both axonal growth inhibition and scarring after spinal cord injury (SCI). We found increases in the inhibitory and toxic forms of CRMP4 in injured spinal cord. Notably, CRMP4 expression was evident in inflammatory cells as well as in neurons after spinal cord transection. Crmp4-/- mice displayed neuroprotection against SCI and reductions in inflammatory response and scar formation. This permissive environment for axonal growth due to CRMP4 deletion restored locomotor activity at an unusually early phase of healing. These results suggest that deletion of CRMP4 is a unique therapeutic strategy that overcomes two obstacles to CNS repair after SCI.
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  • Neuro-glial differentiation of human bone marrow stem cells in vitro. 15869934

    Bone marrow (BM) is a rich source of stem cells and may represent a valid alternative to neural or embryonic cells in replacing autologous damaged tissues for neurodegenerative diseases. The purpose of the present study is to identify human adult BM progenitor cells capable of neuro-glial differentiation and to develop effective protocols of trans-differentiation to surmount the hematopoietic commitment in vitro. Heterogeneous cell populations such as whole BM, low-density mononuclear and mesenchymal stem (MSCs), and several immunomagnetically separated cell populations were investigated. Among them, MSCs and CD90+ cells were demonstrated to express neuro-glial transcripts before any treatment. Several culture conditions with the addition of stem cell or astroblast conditioned media, different concentrations of serum, growth factors, and supplements, used alone or in combinations, were demonstrated to alter the cellular morphology in some cell subpopulations. In particular, MSCs and CD90+ cells acquired astrocytic and neuron-like morphologies in specific culture conditions. They expressed several neuro-glial specific markers by RT-PCR and glial fibrillary acid protein by immunocytochemistry after co-culture with astroblasts, both in the absence or presence of cell contact. In addition, floating neurosphere-like clones have been observed when CD90+ cells were grown in neural specific media. In conclusion, among the large variety of human adult BM cell populations analyzed, we demonstrated the in vitro neuro-glial potential of both the MSC and CD90+ subset of cells. Moreover, unidentified soluble factors provided by the conditioned media and cellular contacts in co-culture systems were effective in inducing the neuro-glial phenotype, further supporting the adult BM neural differentiative capability.
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  • An activated protein C analog stimulates neuronal production by human neural progenitor cells via a PAR1-PAR3-S1PR1-Akt pathway. 23554499

    Activated protein C (APC) is a protease with anticoagulant and cell-signaling activities. In the CNS, APC and its analogs with reduced anticoagulant activity but preserved cell signaling activities, such as 3K3A-APC, exert neuroprotective, vasculoprotective, and anti-inflammatory effects. Murine APC promotes subependymal neurogenesis in rodents in vivo after ischemic and traumatic brain injury. Whether human APC can influence neuronal production from resident progenitor cells in humans is unknown. Here we show that 3K3A-APC, but not S360A-APC (an enzymatically inactive analog of APC), stimulates neuronal mitogenesis and differentiation from fetal human neural stem and progenitor cells (NPCs). The effects of 3K3A-APC on proliferation and differentiation were comparable to those obtained with fibroblast growth factor and brain-derived growth factor, respectively. Its promoting effect on neuronal differentiation was accompanied by inhibition of astroglial differentiation. In addition, 3K3A-APC exerted modest anti-apoptotic effects during neuronal production. These effects appeared to be mediated through specific protease activated receptors (PARs) and sphingosine-1-phosphate receptors (S1PRs), in that siRNA-mediated inhibition of PARs 1-4 and S1PRs 1-5 revealed that PAR1, PAR3, and S1PR1 are required for the neurogenic effects of 3K3A-APC. 3K3A-APC activated Akt, a downstream target of S1PR1, which was inhibited by S1PR1, PAR1, and PAR3 silencing. Adenoviral transduction of NPCs with a kinase-defective Akt mutant abolished the effects of 3K3A-APC on NPCs, confirming a key role of Akt activation in 3K3A-APC-mediated neurogenesis. Therefore, APC and its pharmacological analogs, by influencing PAR and S1PR signals in resident neural progenitor cells, may be potent modulators of both development and repair in the human CNS.
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  • Isoflurane inhibits growth but does not cause cell death in hippocampal neural precursor cells grown in culture. 19293697

    Isoflurane causes long-term hippocampal-dependent learning deficits in rats despite limited isoflurane-induced hippocampal cell death, raising questions about the causality between isoflurane-induced cell death and isoflurane-induced cognitive function. Neurogenesis in the dentate gyrus is required for hippocampal-dependent learning and thus constitutes a potential alternative mechanism by which cognition can be altered after neonatal anesthesia. The authors tested the hypothesis that isoflurane alters proliferation and differentiation of hippocampal neural progenitor cells.Multipotent neural progenitor cells were isolated from pooled rat hippocampi (postnatal day 2) and grown in culture. These cells were exposed to isoflurane and evaluated for cell death using lactate dehydrogenase release, caspase activity, and immunocytochemistry for nuclear localization of cleaved caspase 3. Growth was assessed by cell counting and BrdU incorporation. Expression of markers of stemness (Sox2) and cell division (Ki67) were determined by quantitative polymerase chain reaction. Cell fate selection was assessed using immunocytochemistry to stain for neuronal and glial markers.Isoflurane did not change lactate dehydrogenase release, activity of caspase 3/7, or the amount of nuclear cleaved caspase 3. Isoflurane decreased caspase 9 activity, inhibited proliferation, and decreased the proportion of cells in s-phase. messenger ribonucleic acid expression of Sox2 (stem cells) and Ki67 (proliferation) were decreased. Differentiating neural progenitor cells more often select a neuronal fate after isoflurane exposure.The authors conclude that isoflurane does not cause cell death, but it does act directly on neural progenitor cells independently of effects on the surrounding brain to decrease proliferation and increase neuronal fate selection. These changes could adversely affect cognition after isoflurane anesthesia.
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  • Two classes of GABAergic neurons in the inferior colliculus. 19889997

    The inferior colliculus (IC) is unique, having both glutamatergic and GABAergic projections ascending to the thalamus. Although subpopulations of GABAergic neurons in the IC have been proposed, criteria to distinguish them have been elusive and specific types have not been associated with specific neural circuits. Recently, the largest IC neurons were found to be recipients of somatic terminals containing vesicular glutamate transporter 2 (VGLUT2). Here, we show with electron microscopy that VGLUT2-positive (VGLUT2(+)) axonal terminals make axosomatic synapses on IC neurons. These terminals contain only VGLUT2 even though others in the IC have VGLUT1 or both VGLUT1 and 2. We demonstrate that there are two types of GABAergic neurons: larger neurons with VGLUT2(+) axosomatic endings and smaller neurons without such endings. Both types are present in all subdivisions of the IC, but larger GABAergic neurons with VGLUT2(+) axosomatic terminals are most prevalent in the central nucleus. The GABAergic tectothalamic neurons consist almost entirely of the larger cells surrounded by VGLUT2(+) axosomatic endings. Thus, two types of GABAergic neurons in the IC are defined by different synaptic organization and neuronal connections. Larger tectothalamic GABAergic neurons are covered with glutamatergic axosomatic synapses that could allow them to fire rapidly and overcome a slow membrane time constant; their axons-05-be the largest in the brachium of the IC. Thus, large GABAergic neurons could deliver IPSPs to the medial geniculate body before EPSPs from glutamatergic IC neurons firing simultaneously.
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  • Tunicamycin produces TDP-43 cytoplasmic inclusions in cultured brain organotypic slices. 22459357

    The cellular distribution of TAR DNA binding protein (TDP-43) is disrupted in several neurodegenerative disorders, including frontotemporal lobar degeneration with ubiquitin-positive inclusions (FTLD-U subtype) and amyotrophic lateral sclerosis (ALS). In these conditions, TDP-43 is found in neuronal cytoplasmic inclusions, with loss of the normal nuclear expression. The mechanisms leading to TDP-43 redistribution and its role in disease pathophysiology remain unknown. We describe an in vitro neural tissue model that reproduces TDP-43 relocalization and inclusion formation. Two week-old coronal organotypic mouse brain slice cultures were treated with tunicamycin for 7 days. In cortical regions of treated slice cultures, cytoplasmic inclusions of TDP-43 immunoreactivity were observed, with loss of nuclear TDP-43 immunoreactivity. These inclusions were found in both astrocytes and neurons, and were of both skein-like and round morphologies. In contrast, TDP-43 cytoplasmic inclusions were not found in slices treated with staurosporine to induce apoptosis, or with trans-4-carboxy-l-proline (PDC) to induce chronic glutamate excitotoxicity. Furthermore, TDP-43 cytoplasmic inclusions did not co-localize with cleaved caspase-3, suggesting that TDP-43 mislocalization does not generally accompany caspase activation or apoptosis. The induction of TDP-43 cytoplasmic translocation in cerebrocortical slice cultures by tunicamycin provides a platform for further mechanistic investigations of pathological processing of TDP-43.
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  • Immunocytochemical localization of tubulin and microtubule-associated protein 2 during the development of hippocampal neurons in culture. 3514816

    In dissociated-cell cultures prepared from the embryonic rat hippocampus, neurons establish both axons and dendrites, which differ in geometry, in ultrastructure, and in synaptic polarity. We have used immunocytochemistry with monoclonal antibodies to study the regional distribution of beta-tubulin and micro-tubule-associated protein 2 (MAP2) in hippocampal cultures and their localization during early stages of axonal and dendritic development. After development for a week or more in culture, when axons and dendrites were well-differentiated, the distribution of these two proteins was quite different. Beta-tubulin was present throughout the nerve cell, in soma, dendrites, and axon. It was also present in all classes of non-neuronal cells, astrocytes, fibroblasts, and a presumptive glial progenitor cell. In contrast, MAP2 was preferentially localized to nerve cells; within neurons, MAP2 was present in soma and dendrites, but little or no immunostaining was detectable in axons. Both beta-tubulin and MAP2 were present in nerve cells at the time of plating. From the earliest stages of process extension, beta-tubulin was present in all neuronal processes, both axons and dendrites. Surprisingly, MAP2 was also initially present in both axons and dendrites, extending as far as the axonal growth cone. With subsequent development, MAP2 staining was selectively lost from the axon so that after 1 week in vitro little or no axonal staining remained. Taken together with earlier results (Cáceres et al., 1984a), these data indicate that the establishment of neuronal polarity, as manifested by the molecular differentiation of the axonal and dendritic cytoskeleton, occurs largely under endogenous control, even under culture conditions in which cell interactions are greatly restricted.(ABSTRACT TRUNCATED AT 250 WORDS)
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  • Loss of K-Cl co-transporter KCC3 causes deafness, neurodegeneration and reduced seizure threshold. 14532115

    K-Cl co-transporters are encoded by four homologous genes and may have roles in transepithelial transport and in the regulation of cell volume and cytoplasmic chloride. KCC3, an isoform mutated in the human Anderman syndrome, is expressed in brain, epithelia and other tissues. To investigate the physiological functions of KCC3, we disrupted its gene in mice. This severely impaired cell volume regulation as assessed in renal tubules and neurons, and moderately raised intraneuronal Cl(-) concentration. Kcc3(-/-) mice showed severe motor abnormalities correlating with a progressive neurodegeneration in the peripheral and CNS. Although no spontaneous seizures were observed, Kcc3(-/-) mice displayed reduced seizure threshold and spike-wave complexes on electrocorticograms. These resembled EEG abnormalities in patients with Anderman syndrome. Kcc3(-/-) mice also displayed arterial hypertension and a slowly progressive deafness. KCC3 was expressed in many, but not all cells of the inner ear K(+) recycling pathway. These cells slowly degenerated, as did sensory hair cells. The present mouse model has revealed important cellular and systemic functions of KCC3 and is highly relevant for Anderman syndrome.
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