To ensure rapid and efficient impulse conduction, myelinated axons establish and maintain specific protein domains. For instance, sodium (Na+) channels accumulate in the node of Ranvier; potassium (K+) channels aggregate in the juxtaparanode and neurexin/caspr/paranodin clusters in the paranode. Our understanding of the mechanisms that control the initial clustering of these proteins is limited and less is known about domain maintenance. Correlative data indicate that myelin formation and/or mature myelin-forming cells mediate formation of all three domains. Here, we test whether myelin is required for maintaining Na+ channel domains in the nodal gap by employing two demyelinating murine models: (1) cuprizone ingestion, which induces complete demyelination through oligodendrocyte toxicity; and (2) ceramide galactosyltransferase deficient mice, which undergo spontaneous adult-onset demyelination without oligodendrocyte death. Our data indicate that the myelin sheath is essential for long-term maintenance of sodium channel domains; however, oligodendrocytes, independent of myelin, provide a partial protective influence on the maintenance of nodal Na+ channel clusters. Thus, we propose that multiple mechanisms regulate the maintenance of nodal protein organization. Finally, we present evidence that following the loss of Na+ channel clusters the chronological progression of expression and reclustering of Na+ channel isoforms during the course of CNS remyelination recapitulates development.

Abundant recent evidence favors a neurotransmitter/neuromodulator role for D-serine. D-serine is synthesized from L-serine by serine racemase in astrocytic glia that ensheath synapses, especially in regions of the brain that are enriched in NMDA-glutamate receptors. D-serine is more potent than glycine at activating the ‘glycine’ site of these receptors. Moreover, selective degradation of D-serine but not glycine by D-amino acid oxidase markedly reduces NMDA neurotransmission. D-serine appears to be released physiologically in response to activation by glutamate of AMPA-glutamate receptors on D-serine-containing glia. This causes glutamate-receptor-interacting protein, which binds serine racemase, to stimulate enzyme activity and D-serine release. Thus, glutamate triggers the release of D-serine so that the two amino acids can act together on postsynaptic NMDA receptors. D-serine also plays a role in neural development, being released from Bergmann glia to chemokinetically enhance the migration of granule cell cerebellar neurons from the external to the internal granular layer.

There is increasing evidence that severe mood disorders are associated with impairment of structural plasticity and cellular resilience. Cumulative data demonstrate that mood stabilizers regulate intracellular signaling cascades, including protein kinase C (PKC), PKA, mitogen-activated protein (MAP) kinase, glycogen synthase kinase 3-β (GSK3-β) and intracellular calcium, which are signaling pathways that regulate synaptic plasticity. In this context, it is noteworthy that a growing body of data indicates that the glutamatergic system, has a major role in neuronal plasticity and cellular resilience, might be involved in the pathophysiology and treatment of mood disorders. AMPA glutamate-receptor trafficking is important in synaptic plasticity and might play crucial roles in maintaining critical neuronal circuits associated with mood. Two clinically effective, structurally dissimilar, antimanic agents, lithium and valproate (VPA), down-regulate synaptic expression of AMPA receptor subunit GluR1 in hippocampus in chronically treated rats. This reduction in synaptic GluR1 by lithium and VPA is due to attenuated phosphorylation of GluR1 at a specific PKA site (residue 845 of GluR1), which is crucial for AMPA receptor insertion. By contrast, imipramine, which can provoke mania, increases synaptic expression of GluR1 in the hippocampus in vivo. Furthermore, there is ample evidence from preclinical and clinical research that the glutamatergic system is involved in the pathophysiology of mood disorders and that many of the somatic treatments used for mood disorders including antidepressants, mood stabilizers, atypical antipsychotic drugs and electroconvulsive therapy have both direct and indirect effects on the glutamatergic system. Given these findings, further research with medications that specifically affect the glutamatergic system is warranted. Recent studies in our lab have shown that riluzole, a FDA approved medicine that regulates the glutamatergic system, shows antidepressant efficacy in unipolar and bipolar depression. These studies indicate that regulation of glutamate-mediated synaptic plasticity might play a role in the treatment of mood disorders, and raise new avenues for novel therapies for this devastating illness.

Transplantation of olfactory ensheathing cells (OECs) into the injured spinal cord has been shown to exert neuroprotective effects and promote functional recovery. In the present study, we investigated the potential modulatory effects of OECs on the inflammatory reaction developed after photochemical injury to the spinal cord. OEC cultures were obtained from olfactory bulbs of adult Sprague-Dawley rats. Photochemical spinal cord injury was induced in adult rats at T8. Thirty minutes after the insult, either a suspension of OECs (180 000 cells in 12 µl DMEM) or DMEM alone was injected into the lesioned spinal cord. At 3, 7 and 14 days post-operation (dpo), five animals from each group were processed for histology. Double-fluorescent labeling of transverse sections of the cord were made by combination of immunohistochemistry for inflammatory markers, interleukin 1β (IL-1β) and inducible nitric oxide synthase (iNOS), and for selective markers of astrocytes (glial fibrillar acidic protein; GFAP) and microglia/macrophages (tomato lectin; LEC). Differences in the intensity and time course of glial response, and IL-1β and iNOS expression were found between the two groups of rats. The reactivity grade against IL-1β, iNOS, GFAP and LEC in OEC-transplanted rats was higher at 7 dpo and lower at 14 dpo compared with DMEM-injected rats. These results indicate that the mechanisms underlying neuroprotection by OECs might be caused by earlier, higher and shorter duration of microglia/macrophage and astrocyte responses after injury.

Bidirectional signaling between neurons and glial cells has been demonstrated in brain slices and is believed to mediate glial modulation of synaptic transmission in the CNS. Our laboratory has characterized similar neuron–glia signaling in the mammalian retina. We find that light-evoked neuronal activity elicits Ca2+ increases in Müller cells, which are specialized retinal glial cells. Neuron to glia signaling is likely mediated by the release of ATP from neurons and is potentiated by adenosine. Glia to neuron signaling has also been observed and is mediated by several mechanisms. Stimulation of glial cells can result in either facilitation or depression of synaptic transmission. Release of D-serine from Müller cells might also potentiate NMDA receptor transmission. Müller cells directly inhibit ganglion cells by releasing ATP, which, following hydrolysis to adenosine, activates neuronal A1 receptors. The existence of bidirectional signaling mechanisms indicates that glial cells participate in information processing in the retina.

Minocycline, a tetracycline derivative with pleiotropic biological effects, exhibits anti-inflammatory properties in several models of CNS disease. In addition to reducing production of inflammatory mediators, it has been postulated that minocycline might also be directly neuroprotective under these circumstances. Therefore, we investigated the effect of minocycline on primary cortical neuronal cultures exposed to a nitric oxide (NO)-donor. Cultures were assessed for neuronal survival, axon survival and markers of intracellular signaling pathways. The NO donor significantly increased neuronal death and minocycline was protective under these conditions. Furthermore NO-induced reductions in axonal length were significantly attenuated by minocycline. Improvements in axonal length were dependent on mitogen-activated protein kinase (MAP kinase)/extracellular signal-related kinase (Erk) signaling, whereas phosphatidylinositol 3-kinase (PI 3-kinase)/Akt signaling was important in neuronal survival. Further investigation into MAP kinase signaling pathways revealed inhibition of p38 MAP kinase and c-jun N-terminal kinase (JNK) signaling by minocycline. JNK pathways were activated by trophic factor-withdrawal and minocycline attenuated neuronal death induced by trophic withdrawal. These results indicate that, in addition to anti-inflammatory properties, minocycline has direct protective effects on neurons and provides further evidence for its use in disorders of the CNS.

Injury and disease in the CNS increases the amount of tumor necrosis factor α (TNFα) that neurons are exposed to. This cytokine is central to the inflammatory response that occurs after injury and during prolonged CNS disease, and contributes to the process of neuronal cell death. Previous studies have addressed how long-term apoptotic-signaling pathways that are initiated by TNFα might influence these processes, but the effects of inflammation on neurons and synaptic function in the timescale of minutes after exposure are largely unexplored. Our published studies examining the effect of TNFα on trafficking of AMPA-type glutamate receptors (AMPARs) in hippocampal neurons demonstrate that glial-derived TNFα causes a rapid (<15 minute) increase in the number of neuronal, surface-localized, synaptic AMPARs leading to an increase in synaptic strength. This indicates that TNFα-signal transduction acts to facilitate increased surface localization of AMPARs from internal postsynaptic stores. Importantly, an excess of surface localized AMPARs might predispose the neuron to glutamate-mediated excitotoxicity and excessive intracellular calcium concentrations, leading to cell death. This suggests a new mechanism for excitotoxic TNFα-induced neuronal death that is initiated minutes after neurons are exposed to the products of the inflammatory response.

Here we review the importance of AMPAR trafficking in normal neuronal function and how abnormalities that are mediated by glial-derived cytokines such as TNFα can be central in causing neuronal disorders. We have further investigated the effects of TNFα on different neuronal cell types and present new data from cortical and hippocampal neurons in culture. Finally, we have expanded our investigation of the temporal profile of the action of this cytokine relevant to neuronal damage. We conclude that TNFα-mediated effects on AMPAR trafficking are common in diverse neuronal cell types and very rapid in their onset. The abnormal AMPAR trafficking elicited by TNFα might present a novel target to aid the development of new neuroprotective drugs.

In earlier studies we have shown that a protein-synthesis-independent, early, long-term potentiaton (early-LTP) that lasts up to 4–5 hours can be transformed (reinforced) into a protein-synthesis-dependent late-LTP that lasts ≥8 hours by either an emotional challenge (e.g. swim stress) or mastering a cognitive task (e.g. spatial learning). In the present study we show that LTP-reinforcement by spatial training depends on the specific constraints of the learning paradigm. In a holeboard paradigm, LTP-reinforcement is related to the formation of a lasting reference memory whereas water-maze training gives more heterogenous results. Thus, cognitive aspects interfere with emotionally challenging components of the latter paradigm. These data indicate that different spatial-learning tasks are weighted distinctly by the animal. Thus, we show that aspects of specific spatial-learning paradigms such as shifts of attention and emotional content directly influence functional plasticity and memory formation.

Based on studies of the molecular and cellular cascades that occur during memory consolidation for a one-trial passive-avoidance learning task in the young chick, I review the evidence that memory is encoded in permanent changes in synaptic connectivity in a specific brain region, the Hebb hypothesis. I conclude that despite the fact that such a cascade occurs, culminating in the synthesis of cell-adhesion molecules that are involved in synaptic remodelling, synaptic events are not in themselves sufficient to account for the phenomena of memory. Both whole brain (neuromodulator) and whole body (hormonal) processes are engaged. Memories are labile, disarticulated and stored in a distributed manner; how the mind/brain recreates coherent memories from this pattern is a mystery.

Activity-dependent neuroprotective protein (ADNP, ∼123562.8 Da), is synthesized in astrocytes and expression of ADNP mRNA is regulated by the neuroprotective peptide vasoactive intestinal peptide (VIP). The gene that encodes ADNP is conserved in human, rat and mouse, and contains a homeobox domain profile that includes a nuclear-export signal and a nuclear-localization signal. ADNP is essential for embryonic brain development, and NAP, an eight-amino acid peptide that is derived from ADNP, confers potent neuroprotection. Here, we investigate the subcellular localization of ADNP through cell fractionation, gel electrophoresis, immunoblotting and immunocytochemistry using α-CNAP, an antibody directed to the neuroprotective NAP fragment that constitutes part of an N-terminal epitope of ADNP. Recombinant ADNP was used as a competitive ligand to measure antibody specificity. ADNP-like immunoreactivity was found in the nuclear cell fraction of astrocytes and in the cytoplasm. In the cytoplasm, ADNP-like immunoreactivity colocalized with tubulin-like immunoreactivity and with microtubular structures, but not with actin microfilaments. Because microtubules are key components of developing neurons and brain, possible interaction between tubulin and ADNP might indicate a functional correlate to the role of ADNP in the brain. In addition, ADNP-like immunoreactivity in the extracellular milieu of astrocytes increased by ∼1.4 fold after incubation of the astrocytes with VIP. VIP is known to cause astrocytes to secrete neuroprotective/neurotrophic factors, and we suggest that ADNP constitutes part of this VIP-stimulated protective milieu.

Early in development, steroid hormones structurally organize various regions of the CNS. However, steroid hormones continue to affect the structure and function of the CNS throughout the life of the individual. In this review, we discuss sex differences and similarities in steroid-induced synaptic plasticity in the adult brain. Particular emphasis is placed on steroid-induced plasticity in the hippocampus, a brain region important in learning and memory. This topic is relevant to the growing evidence for the actions of sex hormones outside of the reproductive neuroendocrine axis. It also tells an important and emerging story about non-genomic and genomic actions of steroids at the cellular and molecular levels. Specifically, the effects of estrogen and progesterone as well as the androgens and glucocorticoids are discussed. The influence of steroids on hippocampal structure and function can differ vastly between the sexes. However, there are certain similarities that might aid in our understanding of how steroids affect CNS plasticity in general. Although future studies will undoubtedly lead us to a greater understanding of these phenomena, the data reviewed indicate that when studying synaptic plasticity, the sex and hormonal milieu of the individual might significantly influence the outcome and interpretation of the research.

Recent evidence argues that the oncogenesis and growth of CNS tumors occurs through dysregulated molecular and cellular mechanisms of neural development. New insights have emerged that have had a significant impact on both research and treatment of these cancers.

The diffuse, extensive infiltration of malignant gliomas into the surrounding normal brain is believed to rely on modification of the proteolysis of extracellular matrix components. Our previous results clearly demonstrate that uPA, uPAR and MMP-9 concentrations increase significantly during tumor progression and that tumor growth can be inhibited with antisense stable clones of these molecules. Because antisense-mediated gene silencing does not completely inhibit the translation of target mRNA and high concentrations of antisense molecules are required to achieve gene silencing, we used the RNAi approach to silence uPA, uPAR and MMP-9 in this study. We examined a cytomegalovirus (CMV) promoter-driven DNA-template approach to induce hairpin RNA (hpRNA)-triggered RNAi to inhibit uPA, uPAR and MMP-9 gene expression with a single construct. uPAR protein levels and enzymatic activity of uPA and MMP-9 were found to significantly decrease in cells transfected with a plasmid expressing hairpin siRNA for uPAR, uPA and MMP-9. pU2M-transfected SNB19 cells significantly decreased uPA, uPAR and MMP-9 expression compared to mock and EV/SV-transfected cells, determined by immunohistochemical analysis. Furthermore, the effect of the single constructs for these molecules was a specific inhibition of their respective protein levels, as demonstrated by immunohistochemical analysis. After transfection with a plasmid vector expressing dsRNA for uPA, uPAR and MMP-9, glioma-cell invasion was retarded compared with mock and EV/SV-treated groups, demonstrated by Matrigel-invasion assay and spheroid-invasion assay. Downregulation of uPA, uPAR and MMP-9 using RNAi inhibited angiogenesis in an in vitro (co-culture) model. Direct intratumoral injections of plasmid DNA expressing hpRNA for uPA, uPAR and MMP-9 significantly regressed pre-established intracranial tumors in nude mice. In addition, cells treated with RNAi for uPAR, uPA and MMP-9 showed reduced pERK levels compared with parental and EV/SV-treated SNB19 cells. Our results support the therapeutic potential of RNAi as a method for gene therapy in treating gliomas.

Malignant peripheral nerve sheath tumors (MPNSTs) are characteristic of Neurofibromatosis type 1 (NF1), a human genetic disorder affecting approximately 1 in 3000 individuals. The absence of neurofibromin in Schwann cells results in hyperactivation of Ras, which contributes to Schwann cell hyperplasia. However, additional intracellular abnormalities in Schwann cells might contribute to the malignancy. We now report that cell lines derived from MPNSTs secrete elevated levels of prostaglandin E2 (PGE2), express higher levels of phosphorylated mitogen-activated protein kinase (MAPK), phosphorylated cytosolic phospholipase A2 (cPLA2) and cyclooxygenase 2 (COX-2) when compared to normal adult human Schwann cells (nhSCs). PCR analysis reveals that NF1 MPNST cell lines express mRNA for both EP2 and EP4 prostaglandin E2 receptors, whereas nhSCs express only the EP4 receptor. COX-2 inhibitors and PGE2 receptor antagonists decrease the proliferation of MPNST cell lines. These results indicate that prostaglandin metabolism is activated in MPNSTs and might contribute to tumor growth in NF1.

Glia are required for axon pathfinding along longitudinal trajectories, but it is unknown how this relates to the molecular paradigm of axon guidance across the midline. Most interneuron axons in bilateral organisms cross the midline only once. Preventing them from recrossing the midline requires the expression of Robo receptors on the axons. These sense the repulsive signal Slit, which is produced by the midline. The lateral positioning of longitudinal axons depends on the response to Slit by the combination of Robo receptors expressed by the axons, on selective fasciculation, and on longitudinal (lateral) glia. Here, we analyse how longitudinal glia influence reading of the ‘Robo code’ by axons. We show that whereas loss of robo1 alone only affects the most medial axons, loss of both glial cells missing (gcm) and robo1 causes a severe midline collapse of longitudinal axons, similar to that caused by the loss of multiple Robo receptors. Furthermore, whereas ectopic expression of robo2 is sufficient to displace the medial MP2 axons along a more lateral trajectory, this does not occur in gcm–robo1 double-mutant embryos, where axons either do not extend at all or they misroute exiting the CNS. Hence, lateral neuron–glia interactions steer the response of axons to the Robo code.

cDNA microarrays were utilized to identify abnormally expressed genes in a malignant peripheral nerve sheath tumor (MPNST)-derived cell line, T265, by comparing the mRNA abundance profiles with that of normal human Schwann cells (nhSCs). The findings characterize the molecular phenotype of this important cell-line model of MPNSTs, and elucidate the contribution of Schwann cells in MPNSTs. In total, 4608 cDNA sequences were screened and hybridizations replicated on custom cDNA microarrays. In order to verify the microarray data, a large selection of differentially expressed mRNA transcripts were subjected to semi-quantitative reverse transcription PCR (LightCycler). Western blotting was performed to investigate a selection of genes and signal transduction pathways, as a further validation of the microarray data. The data generated from multiple microarray screens, semi-quantitative RT–PCR and Western blotting are in broad agreement. This study represents a comprehensive gene-expression analysis of an MPNST-derived cell line and the first comprehensive global mRNA profile of nhSCs in culture. This study has identified ∼900 genes that are expressed abnormally in the T265 cell line and detected many genes not previously reported to be expressed in nhSCs. The results provide crucial information on the T265 cells that is essential for investigation using this cell line in experimental studies in neurofibromatosis type I (NF1), and important information on normal human Schwann cells that is applicable to a wide range of studies on Schwann cells in cell culture.

The c-MYC protooncogene is overexpressed in the most malignant primary brain tumor, glioblastoma multiforme (GBM), and has been correlated with the undifferentiated character of several cell types. However, the role of Myc activity in the generation of GBMs is not known. In this report, we show that gene transfer of c-MYC to GFAP-expressing astrocytes in vitro promotes the outgrowth of GFAP-negative, nestin-expressing cells with progenitor-like morphology, growth characteristics and gene-expression pattern. In addition, gene transfer of c-MYC to GFAP-expressing astrocytes in vivo induces GBMs when co-expressed with activated Ras and Akt. Without c-MYC, Ras+Akt induces GBMs from nestin-expressing CNS progenitors but is insufficient in GFAP-expressing differentiated astrocytes. The ability of Myc activity to enhance the oncogenic effects of Ras+Akt appears to be limited to GFAP-expressing astrocytes because nestin-expressing progenitors show no increase in GBM formation with the addition of MYC to Ras+Akt. These studies indicate that one role of MYC activity in the formation of gliomas might be to either promote or reinforce an undifferentiated phenotype required for glioma cells to respond to the oncogenic effects of elevated Ras and Akt activity.

Neuroglia, which are the non-neuronal cells of the nervous system, except for meninges and vasculature with its endothelium, blood vessel walls and relatively rare smooth muscle cells, have received much attention in mammalian investigations and occupy a voluminous literature.

Multiple classes of precursor cells have been isolated and characterized from the developing spinal cord including multipotent neuroepithelial (NEP) stem cells and lineage-restricted precursors for neurons (NRPs) and glia (GRPs). We have compared the survival, differentiation and integration of multipotent NEP cells with lineage-restricted NRPs and GRPs using cells isolated from transgenic rats that express the human placental alkaline phosphatase gene. Our results demonstrate that grafted NEP cells survive poorly, with no cells observed 3 days after transplant in the adult hippocampus, striatum and spinal cord, indicating that most CNS regions are not compatible with transplants of multipotent cells derived from fetal CNS. By contrast, at 3 weeks and 5 weeks post-engraftment, lineage-restricted precursors showed selective migration along white-matter tracts and robust survival in all three CNS regions. The grafted precursors expressed the mature neuronal markers NeuN and MAP2, the astrocytic marker GFAP, the oligodendrocytic markers RIP, NG2 and Sox-10, and the synaptic marker synaptophysin. Similar behavior was observed when these precursors were transplanted into the injured spinal cord. Predifferentiated, multipotent NEP cells also survive and integrate, which indicates that lineage-restricted CNS precursors are well suited for transplantation into the adult CNS and provide a promising cellular replacement candidate.