MAB365 Sigma-AldrichAnti-Nerve Growth Factor Receptor Antibody, extracellular, clone 192-IgG

Studies of central nervous system myelination lack defined in vitro models which would effectively dissect molecular mechanisms of myelination that contain cells of the correct phenotype. Here we describe a co-culture of purified motoneurons and oligodendrocyte progenitor cells, isolated from rat embryonic spinal cord using a combination of immunopanning techniques. This model illustrates differentiation of oligodendrocyte progenitors into fully functional mature oligodendrocytes that myelinate axons. It also illustrates a contribution of axons to the rate of oligodendrocyte maturation and myelin gene expression. The defined conditions used allow molecular analysis of distinct stages of myelination and precise manipulation of inductive cues affecting axonal-oligodendrocyte interactions. This phenotypic in vitro myelination model can provide valuable insight into our understanding of demyelinating disorders, such as multiple sclerosis and traumatic diseases such as spinal cord injury where demyelination represents a contributing factor to the pathology of the disorder.

Transplantation of bone marrow derived mesenchymal stromal cells (MSC) or olfactory ensheathing cells (OEC) have demonstrated beneficial effects after spinal cord injury (SCI), providing tissue protection and improving the functional recovery. However, the changes induced by these cells after their transplantation into the injured spinal cord remain largely unknown. We analyzed the changes in the spinal cord transcriptome after a contusion injury and MSC or OEC transplantation. The cells were injected immediately or 7 days after the injury. The mRNA of the spinal cord injured segment was extracted and analyzed by microarray at 2 and 7 days after cell grafting. The gene profiles were analyzed by clustering and functional enrichment analysis based on the Gene Ontology database. We found that both MSC and OEC transplanted acutely after injury induce an early up-regulation of genes related to tissue protection and regeneration. In contrast, cells transplanted at 7 days after injury down-regulate genes related to tissue regeneration. The most important change after MSC or OEC transplant was a marked increase in expression of genes associated with foreign body response and adaptive immune response. These data suggest a regulatory effect of MSC and OEC transplantation after SCI regarding tissue repair processes, but a fast rejection response to the grafted cells. Our results provide an initial step to determine the mechanisms of action and to optimize cell therapy for SCI.

Olfactory ensheathing cells (OECs) have neuro-restorative properties in animal models for spinal cord injury, stroke, and amyotrophic lateral sclerosis. Here we used a multistep screening approach to discover genes specifically contributing to the regeneration-promoting properties of OECs. Microarray screening of the injured olfactory pathway and of cultured OECs identified 102 genes that were subsequently functionally characterized in cocultures of OECs and primary dorsal root ganglion (DRG) neurons. Selective siRNA-mediated knockdown of 16 genes in OECs (ADAMTS1, BM385941, FZD1, GFRA1, LEPRE1, NCAM1, NID2, NRP1, MSLN, RND1, S100A9, SCARB2, SERPINI1, SERPINF1, TGFB2, and VAV1) significantly reduced outgrowth of cocultured DRG neurons, indicating that endogenous expression of these genes in OECs supports neurite extension of DRG neurons. In a gain-of-function screen for 18 genes, six (CX3CL1, FZD1, LEPRE1, S100A9, SCARB2, and SERPINI1) enhanced and one (TIMP2) inhibited neurite growth. The most potent hit in both the loss- and gain-of-function screens was SCARB2, a protein that promotes cholesterol secretion. Transplants of fibroblasts that were genetically modified to overexpress SCARB2 significantly increased the number of regenerating DRG axons that grew toward the center of a spinal cord lesion in rats. We conclude that expression of SCARB2 enhances regenerative sprouting and that SCARB2 contributes to OEC-mediated neuronal repair.

JC virus (JCV), a common human polyomavirus, is the etiological agent of the demyelinating disease, progressive multifocal leukoencephalopathy (PML). In addition to its role in PML, studies have demonstrated the transforming ability of the JCV early protein, T-antigen, and its association with some human cancers. JCV infection occurs in childhood and latent virus is thought to be maintained within the bone marrow, which harbors cells of hematopoietic and non-hematopoietic lineages. Here we show that non-hematopoietic mesenchymal stem cells (MSCs) isolated from the bone marrow of JCV T-antigen transgenic mice give rise to JCV T-antigen positive cells when cultured under neural conditions. JCV T-antigen positive cells exhibited neural crest characteristics and demonstrated p75, SOX-10 and nestin positivity. When cultured in conditions typical for mesenchymal cells, a population of T-antigen negative cells, which did not express neural crest markers arose from the MSCs. JCV T-antigen positive cells could be cultured long-term while maintaining their neural crest characteristics. When these cells were induced to differentiate into neural crest derivatives, JCV T-antigen was downregulated in cells differentiating into bone and maintained in glial cells expressing GFAP and S100. We conclude that JCV T-antigen can be stably expressed within a fraction of bone marrow cells differentiating along the neural crest/glial lineage when cultured in vitro. These findings identify a cell population within the bone marrow permissible for JCV early gene expression suggesting the possibility that these cells could support persistent viral infection and thus provide clues toward understanding the role of the bone marrow in JCV latency and reactivation. Further, our data provides an excellent experimental model system for studying the cell-type specificity of JCV T-antigen expression, the role of bone marrow-derived stem cells in the pathogenesis of JCV-related diseases and the opportunities for the use of this model in development of therapeutic strategies.

Copolymer networks from poly(ethylene glycol) methacrylate (PEGMA) and caprolactone 2-(methacryloyloxy) ethyl ester were synthesized and the resulting structure of the copolymer network was characterized by differential scanning calorimetry, thermogravimetry, Fourier transform infrared spectroscopy, equilibrium water gain and dynamic mechanical analysis, results which were employed to conclude about the network structure of the resulting copolymers. The new material is a random copolymer with a good miscibility and increasing hydrophilicity as the PEGMA content increases in the composition. Physical data suggest an excess free volume and synergistic interactions between the lateral chains of both comonomers. Olfactory ensheathing cells were cultured on the different networks, and cell viability and proliferation were assessed by MTS assay. The copolymers with a 30 wt% of PEGMA showed the best results compared with the other compositions in this respect, indicating the relevance for biological performance of a balance of hydrophilic and hydrophobic functionalities in the polymer chain.

Neural tube defects (NTDs) are complex congenital malformations resulting from incomplete neurulation in embryo. Despite surgical repair of the defect, most of the patients who survive with NTDs have a multiple system handicap due to neuron deficiency of the defective spinal cord. In this study, we successfully devised a prenatal surgical approach and transplanted mesenchymal stem cells (MSCs) to foetal rat spinal column to treat retinoic acid induced NTDs in rat. Transplanted MSCs survived, grew and expressed markers of neurons, glia and myoblasts in the defective spinal cord. MSCs expressed and perhaps induced the surrounding spinal tissue to express neurotrophic factors. In addition, MSC reduced spinal tissue apoptosis in NTD. Our results suggested that prenatal MSC transplantation could treat spinal neuron deficiency in NTDs by the regeneration of neurons and reduced spinal neuron death in the defective spinal cord.

Patterned neuronal activity such as spindle bursts in the neonatal cortex is likely to promote the maturation of cortical synapses and neuronal circuits. Previous work on cats has shown that removal of subplate neurons, a transient neuronal population in the immature cortex, prevents the functional maturation of thalamocortical and intracortical connectivity. Here we studied the effect of subplate removal in the neonatal rat primary somatosensory cortex (S1). Using intracortical EEG we show that after selective removal of subplate neurons in the limb region of S1, endogenous and sensory evoked spindle burst activity is largely abolished. Consistent with the reduced in vivo activity in the S1 limb region, we find by in vitro recordings that thalamocortical inputs to layer 4 neurons are weak. In addition, we find that removal of subplate neurons in the S1 barrel region prevents the development of the characteristic histological barrel-like appearance. Thus, subplate neurons are crucially involved in the generation of particular types of early network activity in the neonatal cortex, which are an important feature of cortical development. The altered EEG pattern following subplate damage could be applicable in the neurological assessment of human neonates.

Injured neurons become dependent on trophic factors for survival. However, application of trophic factors to the site of injury is technically extremely challenging. Novel approaches are needed to circumvent this problem. Here, we unravel the mechanism of the emergence of dependency of injured neurons on brain-derived neurotrophic factor (BDNF) for survival. Based on this mechanism, we propose the use of the diuretic bumetanide to prevent the requirement for BDNF and consequent neuronal death in the injured areas. Responses to the neurotransmitter GABA change from hyperpolarizing in intact neurons to depolarizing in injured neurons. We show in vivo in rats and ex vivo in mouse organotypic slice cultures that posttraumatic GABA(A)-mediated depolarization is a cause for the well known phenomenon of pathological upregulation of pan-neurotrophin receptor p75(NTR). The increase in intracellular Ca(2+) triggered by GABA-mediated depolarization activates ROCK (Rho kinase), which in turn leads to the upregulation of p75(NTR). We further show that high levels of p75(NTR) and its interaction with sortilin and proNGF set the dependency on BDNF for survival. Thus, application of bumetanide prevents p75(NTR) upregulation and neuronal death in the injured areas with reduced levels of endogenous BDNF.

The sensory circuit of the stretch reflex arc is composed of intrafusal muscle fibers and their innervating proprioceptive neurons that convert mechanical information regarding muscle length and tension into action potentials that synapse onto the homonymous motoneurons in the ventral spinal cord which innervate the extrafusal fibers of the same muscle. To date, the in vitro synaptic connection between proprioceptive sensory neurons and spinal motoneurons has not been demonstrated. A functional in vitro system demonstrating this connection would enable the understanding of feedback by the integration of sensory input into the spinal reflex arc. Here we report a co-culture of rat embryonic motoneurons and proprioceptive sensory neurons from dorsal root ganglia (DRG) in a defined serum-free medium on a synthetic silane substrate (DETA). Furthermore, we have demonstrated functional synapse formation in the co-culture by immunocytochemistry and electrophysiological analysis. This work will be valuable for enabling in vitro model systems for the study of spinal motor control and related pathologies such as spinal cord injury, muscular dystrophy and spasticity by improving our understanding of the integration of the mechanosensitive feedback mechanism.

Mechanisms that control the differentiation and function of oligodendrocytes in the central nervous system are complex and involve multiple inputs from the surrounding environment, including localized concentrations of growth factors and the extracellular matrix. Dissection and analysis of these inputs are key to understanding the pathology of central nervous system demyelinating diseases such as multiple sclerosis, where the differentiation of myelinating oligodendrocytes from their precursors underlies the remission phase of the disease. In vitro co-culture models provide a mechanism for the study of factors that regulate differentiation of oligodendrocyte precursors but have been difficult to develop due to the complex nature of central nervous system myelination. This study describes development of an in vitro model that merges a defined medium with a chemically modified substrate to study aspects of myelination in the central nervous system. We demonstrate that oligodendrocyte precursors co-cultured with rat embryonic motoneurons on non-biological substrate (diethylenetriamine trimethoxy-silylpropyldiethylenetriamine), can be induced to differentiate into mature oligodendrocytes that express myelin basic protein, using a serum-free medium. This defined and reproducible model of in vitro myelination could be a valuable tool for the development of treatments for demyelinating diseases such as multiple sclerosis.