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Progress in addressing the origins of intellectual and developmental disabilities accelerated with the establishment 50 years ago of the Eunice Kennedy Shriver National Institute of Child Health and Human Development of the National Institutes of Health and associated Intellectual and Developmental Disabilities Research Centers. Investigators at these Centers have made seminal contributions to understanding human brain and behavioral development and defining mechanisms and treatments of disorders of the developing brain. ANN NEUROL 2019;86:332-343.

The primary energy sources of mammalian cells are proteins, fats, and sugars that are processed by well-known biochemical mechanisms that have been discovered and studied in 1G (terrestrial gravity). Here we sought to determine how simulated microgravity (sim-µG) impacts both energy and lipid metabolism in oligodendrocytes (OLs), the myelin-forming cells in the central nervous system. We report increased mitochondrial respiration and increased glycolysis 24 hr after exposure to sim-µG. Moreover, examination of the secretome after 3 days' exposure of OLs to sim-µG increased the Krebs cycle (Krebs and Weitzman, 1987) flux in sim-µG. The secretome study also revealed a significant increase in the synthesis of fatty acids and complex lipids such as 1,2-dipalmitoyl-GPC (5.67); lysolipids like 1-oleoyl-GPE (4.48) were also increased by microgravity. Although longer-chain lipids were not observed in this study, it is possible that at longer time points OLs would have continued moving forward toward the synthesis of lipids that constitute myelin. For centuries, basic developmental biology research has been the pillar of an array of discoveries that have led to clinical applications; we believe that studies using microgravity will open new avenues to our understanding of the brain in health and disease—in particular, to the discovery of new molecules and mechanisms impossible to unveil while in 1G. © 2016 Wiley Periodicals, Inc.

Periventricular leukomalacia (PVL) is the most frequent cause of cerebral palsy and other intellectual disabilities, and currently there is no treatment. In PVL, glutamate excitotoxicity (GME) leads to abnormal oligodendrocytes (OLs), myelin deficiency, and ventriculomegaly. We have previously identified that the combination of transferrin and insulin growth factors (TSC1) promotes endogenous OL regeneration and remyelination in the postnatal and adult rodent brain. Here, we produced a periventricular white matter lesion with a single intracerebral injection of N-methyl-d-aspartate (NMDA). Comparing lesions produced by NMDA alone and those produced by NMDA + TSC1 we found that: NMDA affected survival and reduced migration of OL progenitors (OLPs). In contrast, mice injected with NMDA + TSC1 proliferated twice as much indicating that TSC1 supported regeneration of the OLP population after the insult. Olig2-mRNA expression showed 52% OLP survival in mice receiving a NMDA injection and increased to 78% when TSC1 + NMDA were injected simultaneously and ventricular size was reduced by TSC1. Furthermore, in striatal slices TSC1 reduced the inward currents induced by NMDA in medium-sized spiny neurons, demonstrating neuroprotection. Thus, white matter loss after excitotoxicity can be partially rescued as TSC1 conferred neuroprotection to preexisting OLP and regeneration via OLP proliferation. Furthermore, we showed that early TSC1 administration maximizes neuroprotection.

Here we document three highly reproducible protocols: 1) A culture system for the derivation of human oigodendrocites (OLs) from human induced pluripotent stem cells (hiPS) and their further maturation; Key features are the concomitant fate restriction, and lineage specification of hiPS towards the neutral and OL phenotypes. The use of very precise amounts of fresh additives and factors also contributes to the shorter periods of time (56days vs. /200days/75days) necessary to obtain hiPS-derived OLs. Our protocol generates viral and integration free OLs that efficiently commit and move forward in the OL lineage recapitulating all the steps known to occur during in vivo development. 2) Protocols for the isolation, propagation and maintenance of neural stem cells (NSCs); and 3) Protocols for the production, isolation, and maintenance of OLs from perinatal rodent and human brain-derived NSCs. Our unique culture systems rely on a series of chemically defined media, specifically designed and carefully characterized for each developmental stage of OL as they advance from OL progenitors to mature, myelinating cells. We are confident that these protocols bring our field a step closer to efficient autologous cell replacement therapies and disease modeling.

The primary energy sources of mammalian cells are proteins, fats, and sugars that are processed by well-known biochemical mechanisms that have been discovered and studied in 1G (terrestrial gravity). Here we sought to determine how simulated microgravity (sim-µG) impacts both energy and lipid metabolism in oligodendrocytes (OLs), the myelin-forming cells in the central nervous system. We report increased mitochondrial respiration and increased glycolysis 24 hr after exposure to sim-µG. Moreover, examination of the secretome after 3 days' exposure of OLs to sim-µG increased the Krebs cycle (Krebs and Weitzman, ) flux in sim-µG. The secretome study also revealed a significant increase in the synthesis of fatty acids and complex lipids such as 1,2-dipalmitoyl-GPC (5.67); lysolipids like 1-oleoyl-GPE (4.48) were also increased by microgravity. Although longer-chain lipids were not observed in this study, it is possible that at longer time points OLs would have continued moving forward toward the synthesis of lipids that constitute myelin. For centuries, basic developmental biology research has been the pillar of an array of discoveries that have led to clinical applications; we believe that studies using microgravity will open new avenues to our understanding of the brain in health and disease-in particular, to the discovery of new molecules and mechanisms impossible to unveil while in 1G. © 2016 Wiley Periodicals, Inc.

Trophic factor treatment has been shown to improve the recovery of brain and spinal cord injury (SCI). In this study, we examined the effects of TSC1 (a combination of insulin-like growth factor 1 and transferrin) 4 and 8 h after SCI at the thoracic segment level (T12) in nestin-GFP transgenic mice. TSC1 treatment for 4 and 8 h increased the number of nestin-expressing cells around the lesion site and prevented Wallerian degeneration. Treatment with TSC1 for 4 h significantly increased heat shock protein (HSP)-32 and HSP-70 expression 1 and 2 mm from lesion site (both, caudal and rostral). Conversely, the number of HSP-32 positive cells decreased after an 8-h TSC1 treatment, although it was still higher than in both, non-treated SCI and intact spinal cord animals. Furthermore, TSC1 increased NG2 expressing cell numbers and preserved most axons intact, facilitating remyelination and repair. These results support our hypothesis that TSC1 is an effective treatment for cell and tissue neuroprotection after SCI. An early intervention is crucial to prevent secondary damage of the injured SC and, in particular, to prevent Wallerian degeneration.

Human neurological disorders such as Parkinson's disease, Huntington's disease, amyotrophic lateral sclerosis (ALS), Alzheimer's disease, multiple sclerosis (MS), stroke, and spinal cord injury are caused by a loss of neurons and glial cells in the brain or spinal cord. Cell replacement therapy and gene transfer to the diseased or injured brain have provided the basis for the development of potentially powerful new therapeutic strategies for a broad spectrum of human neurological diseases. However, the paucity of suitable cell types for cell replacement therapy in patients suffering from neurological disorders has hampered the development of this promising therapeutic approach. In recent years, neurons and glial cells have successfully been generated from stem cells such as embryonic stem cells, mesenchymal stem cells, and neural stem cells, and extensive efforts by investigators to develop stem cell-based brain transplantation therapies have been carried out. We review here notable experimental and preclinical studies previously published involving stem cell-based cell and gene therapies for Parkinson's disease, Huntington's disease, ALS, Alzheimer's disease, MS, stroke, spinal cord injury, brain tumor, and lysosomal storage diseases and discuss the future prospects for stem cell therapy of neurological disorders in the clinical setting. There are still many obstacles to be overcome before clinical application of cell therapy in neurological disease patients is adopted: 1) it is still uncertain what kind of stem cells would be an ideal source for cellular grafts, and 2) the mechanism by which transplantation of stem cells leads to an enhanced functional recovery and structural reorganization must to be better understood. Steady and solid progress in stem cell research in both basic and preclinical settings should support the hope for development of stem cell-based cell therapies for neurological diseases.

Neuronal accumulation of oligomeric amyloid-beta (Alphabeta) is considered the proximal cause of neuronal demise in Alzheimer disease (AD) patients. Blood-borne macrophages might reduce Abeta stress to neurons by immigration into the brain and phagocytosis of Alphabeta. We tested migration and export across a blood-brain barrier model, and phagocytosis and clearance of Alphabeta by AD and normal subjects' macrophages. Both AD and normal macrophages were inhibited in Alphabeta export across the blood-brain barrier due to adherence of Abeta-engorged macrophages to the endothelial layer. In comparison to normal subjects' macrophages, AD macrophages ingested and cleared less Alphabeta, and underwent apoptosis upon exposure to soluble, protofibrillar, or fibrillar Alphabeta. Confocal microscopy of stained AD brain sections revealed oligomeric Abeta in neurons and apoptotic macrophages, which surrounded and infiltrated congophilic microvessels, and fibrillar Abeta in plaques and microvessel walls. After incubation with AD brain sections, normal subjects' monocytes intruded into neurons and uploaded oligomeric Abeta. In conclusion, in patients with AD, macrophages appear to shuttle Abeta from neurons to vessels where their apoptosis may release fibrillar Abeta, contributing to cerebral amyloid angiopathy.

To investigate the effect and molecular mechanism of Tiantai No.1 (天泰1号), a compound Chinese herbal preparation, for the prevention and reduction of neurotoxicity induced by beta-amyloid peptides (Abeta) in vitro and its effects on nuclear factor-κ B (NF-κ B) and cAMP responsive element-binding protein (CREB) pathways using the gene transfection technique. B104 neuronal cells were used to examine the effects of Tiantai No.1 on lowering the neurotoxicity induced by Abeta. The cells were pre-treated with Tiantai No.1 at doses of 50, 100, 150, or 200 μ g/mL respectively for 3 days and co-treated with Tiantai No.1 and beta-amyloid peptide1–40 (A β 1–40, 10 μ mol/L) for 48 h or post-treated with Tiantai No.1 for 48 h after the cells were exposed to beta-amyloid peptides25–35 (A β 25–35) for 8 h. In gene transfection assays, cells were treated with Tiantai No.1 at 50 μ g/mL and 150 μ g/mL for 5 days or co-treated with Tiantai No.1 and A β 1–40 (5 μ mo/L) for 3 days after electroporation for the evaluation of NF-κ B and CREB expression. Pre-treating and co-treating B104 neuronal cells with Tiantai No.1 lowered the neurotoxicity induced by Abeta, and post-treating with Tiantai No.1 reduced or blocked B104 neuronal apoptotic death induced by Abeta (P

Oligodendrocytes develop from oligodendrocyte progenitor cells (OPCs), which in turn arise from a subset of neuroepithelial precursor cells during midneurogenesis. Development of the oligodendrocyte lineage involves a plethora of cell-intrinsic and -extrinsic signals. A cell surface calcium-sensing receptor (CaR) has been shown to be functionally expressed in immature oligodendrocytes. Here, we investigated the expression and function of the CaR during oligodendrocyte development. We show that the order of CaR mRNA expression as assessed by quantitative polymerase chain reaction is mature oligodendrocyte > neuron > astrocyte. We next determined the rank order of CaR expression on inducing specification of neural stem cells to the neuronal, oligodendroglial, or astrocytic lineages and found that the relative levels of CaR mRNA expression are OPC > neuron > astrocytes. CaR mRNA expression in cells at various stages of development along the oligodendrocyte lineage revealed that its expression is robustly up-regulated during the OPC stage and remains high until the premyelinating stage, decreasing thereafter by severalfold in the mature oligodendrocyte. In OPCs, high Ca2+ acting via the CaR promotes cellular proliferation. We further observed that high Ca2+ stimulates the mRNA levels of myelin basic protein in preoligodendrocytes, which is also CaR mediated. Finally, myelin basic protein levels were significantly reduced in the cerebellum of CaR-null mice during development. Our results show that CaR expression is up-regulated when neural stem cells are specified to the oligodendrocyte lineage and that activation of the receptor results in OPC expansion and differentiation. We conclude that the CaR may be a novel regulator of oligodendroglial development and function. © 2008 Wiley-Liss, Inc.

The postnatal forebrain subventricular zone (SVZ) harbors stem cells that give rise to olfactory bulb interneurons throughout life. The identity of stem cells in the adult SVZ has been extensively debated. Although, ependymal cells were once suggested to have stem cell characteristics, subsequent studies have challenged the initial report and postulated that subependymal GFAP + cells were the stem cells. Here, we report that, in the adult mouse forebrain, immunoreactivity for a neural stem cell marker, prominin-1/CD133, is exclusively localized to the ependyma, although not all ependymal cells are CD133 + . Using transplantation and genetic lineage tracing approaches, we demonstrate that CD133 + ependymal cells continuously produce new neurons destined to olfactory bulb. Collectively, our data indicate that, compared with GFAP expressing adult neural stem cells, CD133 + ependymal cells represent an additional—perhaps more quiescent—stem cell population in the mammalian forebrain.

To successfully grow, neurons need to overcome the effects of hostile environments, such as the inhibitory action of myelin. We have evaluated the potential of exercise to overcome the intrinsic limitation of the central nervous system for axonal growth. In line with the demonstrated ability of exercise to increase the regenerative potential of neurons, here we show that exercise reduces the inhibitory capacity of myelin. Cortical neurons grown on myelin from exercised rats showed a more pronounced neurite extension compared with neurons grown on poly-D-lysine, or on myelin extracted from sedentary animals. The activity of cyclin-dependent kinase 5, a kinase involved in neurite outgrowth, was found to be increased in cortical neurons grown on exercise-myelin and in the lumbar spinal cord enlargement of exercised animals. Exercise significantly decreased the levels of myelin-associated glycoprotein (MAG), a potent axonal growth inhibitor, suggesting that downregulation of MAG is part of the mechanism through which exercise reduces growth inhibition. It is known that exercise elevates brain-derived neurotrophic factor (BDNF) spinal cord levels and that BDNF acts to overcome the inhibitory effects of myelin. Accordingly, we blocked the action of BDNF during exercise, which suppressed the exercise-related MAG decrease. Protein kinase A (PKA) has been related to the ability of BDNF to overcome growth inhibition; in agreement, we found that exercise increased PKA levels and this effect was reverted by blocking BDNF. Overall, these results show that exercise promotes a permissive cellular environment for axonal growth in the adult spinal cord requiring BDNF action.

Mitochondria show extensive movement along neuronal processes, but the mechanisms and function of this movement are not clearly understood. We have used high-resolution confocal microscopy to simultaneously monitor movement of mitochondria and changes in intracellular [Ca2+] ([Ca2+]i) in rat cortical neurons. A significant percentage (27%) of the total mitochondria in cortical neuronal processes showed movement over distances of >2 μM. The average velocity was 0.52 μm/s. The velocity, direction, and pattern of mitochondrial movement were not affected by transient increases in [Ca2+]i associated with spontaneous firing of action potentials. Stimulation of Ca2+ transients with forskolin (10 μM) or bicuculline (10 μM), or sustained elevations of [Ca2+]i evoked by glutamate (10 μM) also had no effect on mitochondrial transit. Neither removal of extracellular Ca2+, depletion of intracellular Ca2+ stores with thapsigargin, or inhibition of synaptic activity with TTX (1 μM) or a cocktail of CNQX (10 μM) and MK801 (10 μM) affected mitochondrial movement. These results indicate that movement of mitochondria along processes is a fundamental activity in neurons that occurs independently of physiological changes in [Ca2+]i associated with action potential firing, synaptic activity, or release of Ca2+ from intracellular stores.

We previously reported that rat oligodendrocyte progenitors (OLP) express receptors for the pituitary adenylyl cyclase-activating peptide (PACAP) in vivo and in vitro . Addition of PACAP to cultured OLP triggered a potent elevation in intracellular cAMP contents, a dose-dependent stimulation of proliferation, and a delay in myelinogenesis (Lee M, Lelievre V, Zhao P, Torres M, Rodriguez W, Byun JY, Doshi S, Ioffe Y, Gupta G, de los Monteros AE, de Vellis J, Waschek J. Pituitary adenylyl cyclase-activating polypeptide stimulates DNA synthesis but delays maturation of oligodendrocyte progenitors. J Neurosci. 2001 21:3849–59.). In an attempt to understand how PACAP might interact with growth factors known to stimulate OLP proliferation, we investigated PACAP actions on OLP proliferation in the presence of Fibroblast Growth Factor-2 (FGF-2) and PDGF. Multiple PACAP receptor subtype mRNAs and splice variants were detected in these cultures. PACAP by itself potently stimulated OLP proliferation and enhanced the ability of FGF-2 to stimulate DNA synthesis. In contrast, this peptide strongly antagonized the mitogenic effects of PDGF in association with a reduction of PDGFα receptor gene expression. Additionally, we investigated the interaction of PACAP with the morphogenetic factor sonic hedgehog (Shh), which recently was shown to be crucial for oligodendrocyte generation. OLP cultures were found to express mRNAs for both ptc1 (Shh receptor) and gli1 (Shh target gene) and responded to Shh treatment with an increase in proliferation. PACAP antagonized the ability of Shh to stimulate OLP proliferation. Moreover, transcriptional targets of Shh signaling were also reduced by this treatment, suggesting that PACAP directly antagonized Shh signaling. These studies reveal complex in vitro interactions of PACAP with other factors involved in OLP development.

Neurotransmitters and their receptors have been involved in both proper brain development and neurodevelopmental disorders. The role that nicotinic receptors play in immature cortical neurons was initially investigated by gene profiling using Affymetrix DNA arrays. Both short (15 min) and prolonged (18 h) treatments with nicotine did not induce modification in gene expression, whereas a significant down-regulation of c-fos protein levels was observed after 18 h treatment. Conversely, a brief treatment with the glutamatergic agonist NMDA triggered up-regulation of immediate early genes and transcription factors, which remained unaffected by pre-treatment for 18 h with nicotine. Calcium imaging studies revealed that NMDA activated a sustained increase in intracellular calcium concentration in the majority of neurons, whereas nicotine evoked only a transient calcium increase in a smaller percentage of neurons, suggesting that the calcium signalling response was correlated with activation of gene expression. Nicotine effects on immature cortical neurons perhaps do not require gene regulation but may be still acting on signalling pathways.

Loss of arginase I (AI) results in a metabolic disorder characterized by growth retardation, increased mental impairment and spasticity, and potentially fatal hyperammonemia. This syndrome plus a growing body of evidence supports a role for arginase and arginine metabolites in normal neuronal development and function. Here we report our initial observations of the effects of AI loss on proliferation and differentiation of neural stem cells (NSCs) isolated from the germinal zones of embryonic and newborn AI knockout (KO) mice compared with heterozygous (HET) and wild-type (WT) control animals. By using both short and long-term proliferation assays (3 and 10 days, respectively), we found a 1.5–2-fold increase in the number of KO cells compared with WT. FACS analysis showed an increase in KO cells in the synthesis phase of the cell cycle vs. WT cells. After NSC differentiation, AI-deficient cells expressed β-tubulin, SMI81 (SNAP25), glial fibrillary acidic protein, and CNPase, which are markers consistent with neurons, astrocytes, and oligodendrocytes. Many KO cells exhibited a more mature morphology and expressed mature neuronal markers that were decreased or not present in HET or WT cells. Limited, comparative expression array and quantitative RT-PCR analysis identified differences in the levels of several mRNAs encoding structural, signaling, and arginine metabolism proteins between KO and WT cells. The consequence of these changes may contribute to the differential phenotypes of KO vs. WT cells. It appears that AI may play an important and unanticipated role in growth and development of NSCs. © 2006 Wiley-Liss, Inc.

Microglia, one of three glial cell types in the central nervous system (CNS), play an important role as resident immunocompetent and phagocytic cells in the CNS in the event of injury and disease. It was del Rio Hortega in 1927 who determined that microglia belong a distinct glial cell type apart from astrocytes and oligodendrocytes, and since 1970s there has been wide recognition that microglia are immune effectors in the CNS that respond to pathological conditions and participate in initiation and progression of neurological disorders including Alzheimer's disease, Parkinson's disease, multiple sclerosis, and acquired immune deficiency syndrome dementia complex by releasing potentially cytotoxic molecules such as proinflammatory cytokines, reactive oxygen intermediates, proteinases and complement proteins. There is also evidence to suggest that microglia are capable of secreting neurotrophic or neuron survival factors upon activation via inflammation or injury. It is thus timely to review current status of knowledge on biology and immunology of microglia, and consider new directions of investigation on microglia in health and disease.

We recently showed a successful reconstruction of the cat spinal cord using NeuroGelTM a polymer hydrogel bridge between the two spinal stumps. The polymer graft supports axonal elongation, myelination and angiogenesis up to 21 months, Wallerian degeneration was diminished and gliotic scarring was prevented. In the present study, we report the expression patterns of two stress proteins, (HSPs) HSP-25 and HSP-32 after spinal cord hemisection with and without reparative surgery with NeuroGelTM. Double immunofluorescence using cell specific markers for neurons, astrocytes and oligodendrocytes (OL), in combination with antibodies for HSP-25 and 32 showed that mainly neurons express both proteins. Both HSPs displayed different temporal expression patterns in the reconstructed spinal cords with a concomitant reduction of secondary damage. In conclusion, Neurogel reconstruction of the spine during the acute phase considerably reduces secondary damage resulting in a rapid and stable regenerative response.

During development of the CNS, neurons and glia are generated in a sequential manner. The mechanism underlying the later onset of gliogenesis is poorly understood, although the cytokine-induced Jak-STAT pathway has been postulated to regulate astrogliogenesis. Here, we report that the overall activity of Jak-STAT signaling is dynamically regulated in mouse cortical germinal zone during development. As such, activated STAT1/3 and STAT-mediated transcription are negligible at early, neurogenic stages, when neurogenic factors are highly expressed. At later, gliogenic periods, decreased expression of neurogenic factors causes robust elevation of STAT activity. Our data demonstrate a positive autoregulatory loop whereby STAT1/3 directly induces the expression of various components of the Jak-STAT pathway to strengthen STAT signaling and trigger astrogliogenesis. Forced activation of Jak-STAT signaling leads to precocious astrogliogenesis, and inhibition of this pathway blocks astrocyte differentiation. These observations suggest that autoregulation of the Jak-STAT pathway controls the onset of astrogliogenesis.

Nicotine has been reported to be neuroprotective in experimental and epidemiological studies. In addition to nicotine, tobacco and cigarette smoke contain cembranoids, which are antagonists of neuronal nicotinic receptors (nAChR). Exposure of hippocampal slices to N-methyl-D-aspartate (NMDA) decreases the population spikes (PS). This parameter has been used as a measure of excitotoxicity. Surprisingly, both nicotine and tobacco cembranoids protected against NMDA and this neuroprotection was not blocked by methyllycaconitine (MLA), an antagonist of alpha7 nAChR. On the contrary, MLA had a neuroprotective effect of its own. We examined the effect of the tobacco cembranoid (1S,2E,4R,6R,7E,11E)-cembra-2,7,11-triene-4,6-diol (4R) on the neuroprotection against NMDA. DHbetaE, a selective antagonist of alpha4beta2 nAChR, inhibited the neuroprotection by nicotine, 4R, and MLA, suggesting the involvement of alpha4beta2 nAChRs in the neuroprotection. The cell-signaling pathways underlying the neuroprotection by 4R and by nicotine are different. The activity of phosphatidylinositol-3 kinase (PI3K) was required in both cases; however, 4R required the activity of L-type calcium channels and CAM kinase, whereas nicotine required the extracellular signal regulated kinase-1,2 (ERK) and protein kinase C (PKC). In addition, 4R did not enhance total phospho-ERK-1/2 but increased the amount of total Akt/PKB phosphorylated on the activation site and of glycogen synthase kinase 3-beta phosphorylated on the inhibitory site. Total levels of phosphoenzymes are presented instead of the ratio of phospho- over total enzyme because in preliminary experiments total ERK-1/2 levels were slightly increased by 4R. In conclusion, these findings demonstrate that there are two different nicotinic neuroprotective mechanisms mediated by alpha4beta2.

Nestin promoter-GFP (green fluorescent protein) transgenic mice were used to determine the presence of stem/progenitor cells in the mouse inner ear. We examined the inner ear of mice at the following postnatal days (P): P0, P4, P5, P15 and P60. Hair cells stereocilia were identified with the use of the histochemical marker phalloidin. Whole endorgans or cryosections were analyzed under epi-fluorescent or confocal microscopy. From P0 to P5, GFP expressing cells were found in the vestibular sensory epithelia of the macula utricle, but not in the crista ampullaris. Cells within the stroma (tissue underneath the sensory epithelia), utricle, and crista were also GFP-positive. Satellite cells in the vestibular ganglia were GFP-positive, while vestibular ganglia neurons were not. In the organ of Corti, GFP signal was found in inner border and inner phalangeal cells that surround the inner hair cells (GFP-negative), Dieters cells and cells in the great epithelial ridge. Outer hair cells were mildly positive for GFP. Satellite cells in the spiral ganglia were GFP-positive, while spiral ganglia neurons were not. Similar GFP expression was found in the vestibule and cochlea of animals at P15, however, outer hair cells showed no GFP expression. The inner ear of P60 animals contained moderate GFP expression in the stroma of the crista ampullaris and utricle, but not within the sensory epithelia. In the organ of Corti, moderate GFP expression was found in a few Deiters cells. The present data indicates that the expression of nestin in the mouse inner ear is developmentally regulated; yet in the adult inner ear there are some nestin expressing cells, suggesting an intrinsic repair potential, although to a more limited extent than during early post-natal life.

Myelin deficiency in the central nervous system (CNS) can cause severe disabling conditions. Most of the transgenic mice models overexpressing myelin components have limitations for investigators of myelin deficiency and myelin therapy as they severely alter CNS architecture. It has been postulated that transferrin (Tf) is involved in oligodendrocyte (OL) maturation and myelinogenesis. Because Tf is not an intrinsic myelin constituent, we decided to investigate if its overexpression could have an impact on the myelination process without affecting myelin integrity. We generated transgenic mice containing the complete human Tf gene specifically overexpressed in OLs. This overexpression leads to more than a 30% increase in myelin components, such as galactolipids, phospholipids, and proteins. Electron microscopy showed that myelin is structurally normal in terms of thickness and compaction. Behavior analysis showed that mice do not display significant modifications in their locomotion and cognitive and emotional abilities. Furthermore, in one of the genetic background, animals presented a significant increase in motor coordination. We did not find any modification in OL number during early postnatal development, suggesting that Tf does not act on OL proliferation. In addition, the levels of iron and ferritin remained unchanged in the brain of transgenic mice compared to control mice. Our findings indicate that, besides its known iron transport function, Tf is able to influence myelination process and induce behavioral improvements in mice.

Although in neuronal cultures nicotine was reported to prevent early and delayed excitotoxic death, no studies with nicotinic drugs have been done with acute hippocampal slices. We investigated the effect of nicotine and methyllycaconitine (MLA) on the toxicity of N-methyl-d-aspartate (NMDA) in the CA1 area of hippocampal slices. The excitotoxic effect of NMDA was assessed as decreased recovery of the capability to produce synaptically evoked population spikes (PSs). Application of nicotine or MLA before NMDA application increased the recovery of PSs. This electrophysiological recovery was used as a measure of the early neuroprotective events. The neuroprotection conferred by both nicotine and MLA was inhibited by dihydro-beta-erythroidine, showing mediation of neuroprotection by alpha 4 beta 2 neuronal nicotinic receptors (nAChRs). Because nicotine activates alpha 4 beta 2 and other nAChR subtypes, whereas 10 nM MLA inhibits the alpha 7 subtype, we propose the involvement of a neuronal circuitry-dependent mechanism for nicotinic neuroprotection. The effect of nicotine downstream from the receptors was investigated using inhibitors of cell signaling. The results suggest that the effect of nicotine is mediated by tyrosine receptor kinases, 1,2-phosphatidylinositol-3 kinase, and the mitogen-activated extracellular signal-regulated kinases. Although nicotine neuroprotection is Ca2+-dependent, neither L-type Ca2+ channels nor calmodulin-dependent protein kinase is involved in the effect of nicotine. In summary, these results suggest that in acute slices nicotinic protection is initiated either by direct activation of alpha 4 beta 2 or indirectly by inhibition of alpha 7 followed by signal transduction involving tyrosine kinases, phospholipid-dependent kinases, and mitogen-activated kinases.

The Notch1 pathway plays a fundamental role during the establishment of cell fates in the central nervous system (CNS) by regulating neural cell differentiation. In oligodendrocytes (OLs), Notch1 activity prevents these cells from becoming terminally mature, thereby influencing CNS myelination. Little is known of how OLs regulate the expression of this receptor at the gene level or if OLs have mechanisms to control the level of intracellular activity of the Notch1 pathway. In this study, we have found that Notch1 gene expression was higher in proliferative OL progenitor cells (OPCs) and was reduced when cells were forced to withdraw from the cell cycle and became mature, indicating that Notch1 gene expression is developmentally regulated in OLs. We observed that the blockade of terminal differentiation of OPCs by incubation with Delta1, an activator of Notch1, was a dominant process and OL-differentiating signals such as thyroid hormone could not overcome this inhibition in culture. This suggests that a downregulation of the Notch1 pathway might be required to allow OPCs to enter terminal differentiation. We also provide evidence that OPCs and OLs express the Numb gene, a known negative regulator of Notch1 activity. In vivo, Numb was found in postnatal OLs from cerebellar and cerebral white matter. In vitro, Numb expression showed to be inversely correlated to that of Notch1, with higher levels of Numb proteins in mature OLs, in association with myelin-like membranes.

Results of this study indicate a radioprotective effect of peroxiredoxin-I. Peroxiredoxin-I is an antioxidant that scavenges hydroperoxides, whereas reactive oxygen species are the main mediators of ionizing radiation toxicity. We hypothesized that peroxiredoxin-I might be induced by cellular exposure to radiation and act to protect them against its cytotoxic effects. Western blot and Northern blot analyses were used to assess peroxiredoxin-I protein and mRNA expression. Rat C6 glioma cells were engineered to overexpress sense or antisense human peroxiredoxin-I using retroviral vectors. Clonogenic cell survival was used to assess radiosensitivities of the engineered cells. Ionizing radiation induced peroxiredoxin-I protein and mRNA expression in human HT29 colon cancer and rat C6 glioma cells in a dose- and time-dependent manner over a 24 hr period. To determine the effect of peroxiredoxin-I on radiation responses, C6 glioma cells were engineered to overexpress sense or antisense human peroxiredoxin-I. In clonogenic assays, cells overexpressing peroxiredoxin-I were more radioresistant. Cells transduced with antisense peroxiredoxin-I were marginally more sensitive to radiation toxicity. Irradiation can induce peroxiredoxin-I expression, and the increased peroxiredoxin-I may protect cells from further radiation damage. These results suggest that protection by peroxiredoxin-I may play an important role in the survival of glioma and colon cancer cells in patients undergoing radiation therapy.

Neural stem cells (NSCs) were isolated from embryonic day 16 Sprague-Dawley rats and cultured in a novel serum-free stem cell medium that selected for the growth of NSCs and against the growth of GFAP(+) cells (astrocytes). NSCs maintained in culture for extended periods of time retained immunoreactivity for both nestin and PSA-NCAM, two markers characteristic of the stem cell phenotype. Moreover, using an oligodendrocyte (OL) specification medium, NSCs differentiated into OL as evidenced by their morphology and expression of multiple oligodendrocyte/myelin-specific markers. In addition, NSCs are capable of acquiring a neuronal phenotype as evidenced by expressing neuronal markers, such as neurofilament (NF) and NeuN when cultured in a defined medium for neurons indicating that these cells are also a good source of neuroblasts, which could be used to replace neuronal populations in the brain. We also showed successful propagation and differentiation of NSCs into OL after cryostorage, allowing for the later use of stored NSCs. The long-term goal of culturing NSCs and committed oligodendrocyte progenitors (OLP) is to obtain homogeneous populations for transplantation with the goal of remyelinating the myelin-deficient CNS. Our preliminary experiments carried out on normal and myelin deficient rats demonstrate that these cells survive and migrate extensively in both types of hosts. NSCs grafted as such, as well as cells derived from NSCs exposed to selective specification before grafting, are able to differentiate within the host brain. As expected, NSCs are capable of giving rise to astrocytes in a medium favoring this phenotype.

Tumor necrosis factor (TNF)-family cytokines induce reactive oxygen species (ROS) that injure vulnerable populations of brain cells. Among glia, oligodendrocytes are particularly susceptible to TNF-induced ROS whereas microglia are protected. We previously found that oligodendrocytes in vitro predominantly express the p55 type-1 TNF receptor, while microglial cells express both type-1 and p75 type-2 receptors. We hypothesized that differential TNF receptor expression and attendant signaling underlies the relative vulnerability of oligodendrocytes, versus microglia, to TNF-induced injury. To test this hypothesis, purified cultures of glial cells were incubated 0-48 hr with TNFalpha or lymphotoxin-alpha, following which levels of ROS, glutathione (GSH), nuclear factor kappa-B (NFkappaB) translocation, and anti-oxidant proteins and activity were measured. 48 hr exposure to TNF increased ROS levels 28% and decreased GSH levels 17% in oligodendrocytes, but decreased levels ROS levels 24% and increased GSH levels 112% increase in microglia. Thirty to 180 min exposure to TNF increased NFkappaB nuclear translocation to a greater extent and for a longer time in microglia versus oligodendrocytes, and this was followed 24-48 hr later with 3- to 13-fold increases in microglia manganese superoxide dismutase protein levels and 6-fold increases in enzyme activity. Collectively, these data suggest that signals transduced through the p75 receptor activate anti-oxidant mechanisms that protect microglia from TNF-induced injury. Lacking such signals, oligodendrocytes are considerably more vulnerable to the injurious effects of TNF.

The initial impact of spinal cord injury (SCI) often results in inflammation leading to irreversible damage with consequent loss of locomotor function. Minimal recovery is achieved once permanent damage has occurred. Using a mouse model of SCI we observed a transitory increase followed by a rapid decline in gene expression and protein levels of nuclear factor erythroid 2-related factor 2 (Nrf2), a master regulator of cellular anti-oxidative genes. Immediate treatment with diarylpropionitrile (DPN), a non-steroidal selective estrogen receptor ß ligand, resulted in a significant increase in Nrf2 levels, and reduction of inflammation and apoptosis compared to untreated SCI animals. Furthermore, DPN-treatment improved locomotor function within 7 days after induction of SCI. DPN acted through activation of PI3K/ Akt pathway, known to be involved in down-regulation of apoptosis and up-regulation of cell survival in injured tissues. These findings suggest that immediate activation of cellular anti-oxidative stress mechanisms should provide protection against irreversible tissue damage and its profound detrimental effect on locomotor function associated with SCI.

The studies on the exact lineage composition of NG2 expressing progenitors in the forebrain have been controversial. A number of studies have revealed the heterogeneous nature of postnatal NG2 cells. However, NG2 cells found in embryonic dates are far less understood. Our study indicates that early NG2 progenitors from a ventral origin (i.e., before embryonic day 16.5) tangentially migrate out of the medial ganglionic eminence and give rise to interneurons in deep layers of the dorsal cerebral cortex. The majority of myelinating oligodendrocytes found in both cortical gray and white matters are, in contrast, derived from NG2 progenitors with a neonatal subventricular zone origin. Our lineage tracing data reflect the heterogeneous nature of NG2 progenitor populations and define the relationship between lineage divergence and spatiotemporal origins. Beyond the typical lineage tracing studies of NG2(+) cells, by costaining with lineage-specific markers, our study addresses the origins of heterogeneity and its implications in the differentiation potentials of NG2(+) progenitors.

This chapter provides general information on the various types of cells that compose nervous tissues and serves as an introduction to cellular neuroscience. Different classes of neurons are presented in terms of their function, morphology, neurochemistry, and place in neural circuits. Cortical pyramidal neurons and inhibitory interneuron subtypes are discussed together with examples of specialized neurons from subcortical regions. The structure of excitatory and inhibitory synapses is also reviewed. Each type of glial cell (astrocytes, oligodendrocytes, as well as microglia) is introduced in separate sections that review their particular function, structure, and interactions with neurons. A final section presents brain endothelial cells and their role in maintenance of the blood-brain barrier.

Spinal cord injury (SCI) leads to a complex sequence of cellular responses, including astrocyte activation, oligodendrocyte death, and ependymal cell proliferation. Inhibitors of DNA binding (Id1, Id2, Id3) belong to a helix-loop-helix (HLH) gene family. Id genes have been implicated in playing a vital role in the proliferation of many cell types, including astrocytes and myoblasts. In the present study, the expression of Id family members in spinal cord after contusion injury was investigated by in situ hybridization. Id1, Id2, and Id3 mRNA expression was upregulated 5 mm rostral and caudal to the lesion center, and reached maximal levels 3 days after SCI. In addition, cell populations expressing Id1, Id2, and Id3 mRNA were maximally increased 3 days after SCI. The increase in Id2 and Id3 mRNA expression and Id2 and Id3 mRNA+ cells was still observed at 8 days. The Id mRNA expressing cells were phenotyped by combining immunostaining of cell-specific markers with in situ hybridization. Glial fibrillary acidic protein (GFAP)+ astrocytes were found to express all three Id mRNA, whereas S-100α+ astrocytes only expressed high levels of Id2 and Id3 mRNA. Cells having a neural progenitor morphology and the marker nestin appeared after SCI and they expressed Id1, Id2, and Id3 mRNA. Interestingly, some Rip+ oligodendrocytes located in the areas close to the central canal expressed Id3 mRNA after injury. In conclusion, Id genes are upregulated in a time-dependent manner in astrocytes, oligodendrocytes, and neural progenitor subpopulations after SCI, suggesting that they play major roles in cellular responses following SCI. J Neurosci. Res. 66:1161–1172, 2001. © 2001 Wiley-Liss, Inc.

Previous research has established that the development and function of oligodendrocytes are influenced by glucocorticoids. The enzyme glycerol phosphate dehydrogenase (E.C.1.1.1.8) has been used as a model to study glucocorticoid regulation of gene expression in oligodendrocytes and the C6 glial cell line. In the rat brain this enzyme is exclusively localized to oligodendrocytes. The sequence of the 5' flanking region for the rat gene encoding Glycerol Phosphate Dehydrogenase (GPDH; EC 1.1.1.8) was determined. 4 kb of sequence from the 5' flanking region, exon 1, and part of intron 1 of the rat GPDH gene was compared to the corresponding mouse sequence. Dotplot matrix comparison revealed that the rat sequence is more than 80% similar to the mouse sequence, but differs from the mouse sequence in two regions: the rat sequence is devoid of 200 bp of B1 repeat sequence that is present in the mouse, and the rat sequence has an excess 700 bp of B2 repeat sequence inserted between -0.7 kb and -1. 4 kb that is absent in the mouse. To determine the regulatory activity of the rat GPDH 5' flanking region, various portions of the rat GPDH 5' flanking region were placed in luciferase reporter constructs and tested for transcriptional activity. Transient transfection of reporter constructs into the C6 glial cell line revealed that the distal end of the 5' flanking region was glucocorticoid-inducible. A 385 bp Glucocorticoid Response Unit (GRU) was identified whose glucocorticoid induction was enhanced by dibutyryl-cAMP and reduced by phorbol esters. Sequence analysis of the GRU revealed the presence of four consensus GRE sequences and other putative consensus elements. Results here suggest that the 5' flanking region of the GPDH gene mediates the ligand-inducible regulation of GPDH, and that multiple signaling pathways converge at the 5' regulatory sequence to modulate GPDH gene expression in oligodendrocytes.

Communication between cells of the central nervous system (CNS) and of the immune system is accomplished by a network of cytokines and growth factors. Certain cytokines and growth factors cause activation of microglia, contributing to inflammatory states in the CNS. Granulocyte-macrophage colony-stimulating factor (GM-CSF) has numerous effects on microglia, ranging from induction of proliferation to changes in morphology. GM-CSF is also a growth factor for cells of the myeloid lineage, and the signal tranduction induced by GM-CSF in these cells has been extensively studied. Most notably, the importance of the Jak/STAT and MAP kinase pathways in mitogenesis has been shown in many different systems. We show here that primary microglia and a microglia cell line, BV-2, have a Jak/STAT expression pattern and GM-CSF inducibility similar to that of monocytes and macrophages. Primary microglia and BV-2 cells expressed identical Jak/STATs: Jakl, Jak2, Jak3, Tyk2, STAT1alpha/beta, STAT3, STAT5A, STAT5B, and STAT6. In addition, GM-CSF induced Jak2, STAT5A, and STAT5B in BV-2 cells, as it does in monocytes and macrophages. Immunocytochemical analysis showed that STAT5 translocates to the nucleus following GM-CSF stimulation of microglia. We also found the MAP kinases, ERK1 and ERK2, to be phosphorylated in microglia and BV-2 cells following induction by GM-CSF. Jak2, STAT5A, STAT5B, and ERKs are known to be important in controlling cellular proliferation. Drugs that block these pathways may become tools to control inflammation in the CNS by limiting microglial proliferation.

The inhibitors of DNA binding (Id) gene family is highly expressed during embryogenesis and throughout adulthood in the rat central nervous system (CNS). In vitro studies suggest that the Id gene family is involved in the regulation of cell proliferation and differentiation. Recently, Id gene expression was shown to be expressed in immature and mature astrocytes during development and upregulated in reactive astrocytes after spinal cord injury. These results suggest that the Id gene family may play an important role in regulating astrocyte development and reactivity; however, the factors regulating Id expression in astrocytes remain undefined. Tumor necrosis factor-alpha (TNF alpha), a proinflammatory cytokine, is thought to play a crucial role in astrocyte/microglia activation after injury to the CNS. To determine if TNF alpha plays a role in Id gene expression, we exogenously administered TNF alpha into developing postnatal rats. We report that TNF alpha injections resulted in a rapid and transient increase in both cell number and mRNA expression for Id2 and Id3 when compared to levels observed in noninjected or control-injected animals. Id1 mRNA levels were also upregulated after TNF alpha treatment, but to a lesser degree. Significant increases in TNF alpha-induced Id2 and Id3 mRNA were observed in the ventricular/subventricular zone, cingulum and corpus callosum. TNF alpha also increased Id2 mRNA expression in the caudate putamen and hippocampus at the injection site. Id2 and Id3 mRNA+ cells were identified as GFAP+ and S100 alpha + astrocytes as well as ED1+ microglia. This is the first report to show TNF-alpha-induced modulation of the Id gene family and suggests that Id may be involved in the formation of reactive astrocytes and activated microglia in the rodent brain. These results suggest a putative role for the Id family in the molecular mechanisms regulating cellular responsiveness to TNF alpha and CNS inflammation.

We have previously described the expression of a functional full-length trkC transcript for neurotrophin-3 (NT-3) receptor in oligodendroglia (OL) cells (Kumar and de Vellis, 1996). To date, the role of NT-3 and its signal transduction cascade in OL remains poorly defined. We report that the NT-3 responsive population of cells in the OL lineage are the progenitor cells and that the addition of NT-3 results in the autophosphorylation of p145TrkC. Furthermore, NT-3-mediated activation of p21ras and mitogen-activated protein kinase (MAPK), extracellular signal-regulated protein kinase2 (ERK2), were also observed in the progenitor OL cells. These protein tyrosine kinase (PTK)-induced responses were sensitive to the presence of K252a, an inhibitor for tyrosine kinase. We have determined that NT-3 promotes progenitor OL cell commitment to enter into S-phase of cell cycle to initiate DNA synthesis, in a manner similar to platelet-derived growth factor-AA (PDGF-AA). NT-3 thus plays a role in cell proliferation when present alone, while augmenting the proliferation capacity of PDGF-AA as indicated by the nuclear binding activity of the transcription factor, E2F-1. Both the initiation and progression of mitotic events were confirmed by the expression of c-myc and cdc2 in the presence of NT-3, PDGF-AA or NT-3 plus PDGF-AA. A cell survival assay examining interleukin 1-beta-converting enzyme (ICE)-like protease-mediated cleavage of poly (ADP-ribose) polymerase (PARP) revealed an increase in OL progenitor cell death in the absence of NT-3 or PDGF-AA. In corroboration with our in vitro studies, in vivo results show an increased expression of the progenitor OL cell marker, glycerol phosphate dehydrogenase (GPDH) within 48 hr following an intracranial injection of NT-3, PDGF-AA, or NT-3 plus PDGF-AA in PN4-5 rats. These novel findings suggest that PDGF-AA potentiates the OL progenitor cell's ability to enter into the S-phase of the cell cycle and that NT-3 can augment this activity. Furthermore, PDGF-AA and NT-3 can block ICE-like protease-mediated PARP fragmentation in progenitor OL cells. These results provide important information which further delineates the signal transduction cascades and the role of NT-3 and PDGF-AA on OL progenitor cells.

Id1, Id2, and Id3 mRNA are expressed mainly in the proliferating ependymal cell zone of the mouse brain during embryogenesis. In this study, the expression pattern and cell phenotypes of the Id family mRNA were examined in postnatal and adult rat brain. The expression of Idl and Id3 mRNA in rat brain was observed in the cortex layer 1, corpus callosum, ventricular/subventricular zone (VZ/ SVZ), and the CA1-4 layers of the hippocampus at postnatal day 1 (P1) through P14, whereby it declined at 2 months. In general, the developmental pattern of Idl mRNA coincided with the pattern observed for Id3 mRNA. Similar to Id1 and Id3, Id2 mRNA was highly expressed in the corpus callosum, VZ/SVZ, and the hippocampus. Examination of Id2 mRNA revealed high levels in the cortex and caudate putamen at P1 through P14, whereas a decline was observed in its expression in the adult cortex. In P5 rat cerebellum, all Id mRNA examined were found in the internal granular cell layers; however, at this time point, only Id2 mRNA expression was detected in the differentiating zone of the external granular cell layers, preferentially localizing to adult Purkinje cells. Furthermore, only Id2 mRNA expression in brain was observed in NF+ neurons at P5. Examination of S100alpha+ and GFAP+ astrocytes, revealed the presence of all three mRNAs, whereas the expression of Id2 and Id3 mRNA was absent in 04+ immature oligodendrocytes. These data suggest that the spatial and temporal kinetic patterns during development, as well as cellular specificity, of the Id gene family may play a critical role in neural precursor cell proliferation and cell divergence.

Thioredoxin peroxidase-1 (TxP-1), originally cloned as natural killer enhancing factor-B, belongs to a highly conserved antioxidant family. Tumor necrosis factor-α (TNF) activates the expression of activator protein-1 (AP-1) responsive genes. We show here that over-expression of TxP-1 blocks TNF-induced AP-1 activation in endothelial ECV304 cells, which was demonstrated by three independent experimental protocols: electromobility shift assay with AP-1 oligonucleotide probe; reporter gene expression with AP-1 binding site, and interleukin-8 production, which is dependent on AP-1. These results are consistent with the role of TxP-1 as an antioxidant and the previous reports that TNF-induced reactive oxygen species were responsible for AP-1 activation.

Free radicals and the oxidative stress they impose can cause serious injury in the nervous system and contribute to pathology associated with a wide variety of degenerative and traumatic disorders. In this study, we examined the expression of an antioxidant defense gene, nkef, in human tissue and isolated populations of rat brain cells using Western and Northern blot analysis. NKEF protein was expressed in human brain, liver, kidney, muscle, and lung. The human endothelial cell line ECV expressed a 25-kDa band in addition to the 22-kDa band normally observed. In the central nervous system, a 22-kDa NKEF band was present in cortical gray and white matter, hippocampus, cerebellum, and spinal cord in roughly similar amounts. Expression of NKEF-A and NKEF-B subtypes was evaluated by Northern analysis of cultured cell types from embryonic rat brain. Astrocyte and microglia expressed both 22- and 25-kDa bands, whereas cortical neurons and oligodendrocytes contained only the 22-kDa protein band. Northern blot analysis of these cell types revealed low levels of NKEF-A message in neurons and oligodendrocytes, and relatively low levels of NKEF-B in microglia. Differential expression of these antioxidant defense genes may contribute to the selective vulnerability of brain cell types to specific kinds of oxidative stress.

Although historically considered to be an “immunopriviledged” anatomical site, a diversity of events can initiate an inflammatory cascade within the central nervous system (CNS). Inflammatory cascades are characterized by the migration of activated leukocytes from the circulation into perivascular locations within CNS parenchyma and can be precipitated by events such as focal ischemia, viral infection, and demyelinating autoimmune diseases. Activated leukocytes can directly or indirectly disrupt tightly controlled CNS microenvironments, inducing the injury of brain cells and the attendant symptoms of neurological disease. From the onset of leukocyte migration into the CNS parenchyma, to the activation of indigenous CNS cells and subsequently infiltrating leukocytes, to the downregulation and exit of activated leukocytes from the CNS, each step in a CNS inflammatory cascade is exquisitely regulated by cytokines. The potential for blocking or downregulating cytokines or their receptors on specific types of cells at critical points during the course of an inflammatory cascade provides a promising approach to ameliorating the cellular pathology and diseases that result from unchecked inflammation in the CNS. Toward this end, numerous strategies have been developed, with some success, to block the actions of proinflammatory cytokines in the CNS. Such strategies include the systemic or intrathecal injection of antibodies against cytokines or their receptors, of receptor fusion proteins, or of cytokine antisense oligonucleotides. As techniques in molecular biology become increasingly sophisticated, a second generation of strategies has focused on manipulating the expression of cytokines or their receptors in specific types of brain cells at specific times during an inflammatory cascade. A conceptually related approach is to exploit the CNS sequestration of CNS-antigen–specific lymphocytes and use them as vehicles to deliver antiinflammatory cytokines into loci of CNS inflammation. Although technically challenging, the successful development of second-generation strategies holds tremendous therapeutic potential for CNS diseases involving acute and chronic inflammation. MRDD Research Reviews 1998;4:200–211. © 1998 Wiley-Liss, Inc.

The Id family of helix-loop-helix factors (Id1, Id2, and Id3) expressed in many types of cells has been reported to negatively regulate myoblast differentiation and is required for G1/S progression of arrested fibroblasts. Our previous studies have indicated that Id1, Id2, and Id3 mRNA expression appear in the subventricular zone of 1-day-old rat brains. At later ages, Id3 mRNA was only expressed in astrocytes. We now report that Id1 and Id3 mRNA expression increased in astrocytes during the first hour of serum stimulation. Subsequently, the Id1 and Id3 mRNA levels gradually declined to basal level as observed in cultures without serum stimulation. However, there was no significant difference in Id2 mRNA expression between serum-treated and control astrocyte cultures within 1 h of serum induction. In addition, a strong nuclear immunostaining for Id2 and Id3 proteins was observed 24 h after serum stimulation. This observation is consistent with our results that show an increase in Id2 and Id3 protein levels following 24 h serum induction. Furthermore, DNA synthesis in FCS-stimulated astrocytes was blocked by antisense oligonucleotides against Id3 mRNA. The addition of Id3 antisense oligonucleotides caused approximately 50% reduction in Id3 mRNA and protein levels when compared to that in sense-treated cultures. The results indicate that the inhibition of DNA synthesis in FCS-stimulated astrocytes is due to a decrease in Id3 levels by the antisense. These observations suggest that Id3 may play an important role in the regulation of astrocyte proliferation.

Injury to the spinal cord disrupts ascending and descending axonal pathways and causes tissue damage with a subsequent limited cellular regeneration. Successful treatment would encompass the restoration of the cytoarchitecture, homeostasis and function all in dear need. Transplantation-based treatments using exogenous cells are the most favoured approach. Yet, with the advent of the stem cell concept and continuous progress in the field it became clear that the endogenous potential for repair is greater than previously thought. As an alternative to neural grafting, we and other researchers have aimed at understanding what are the elements needed for a successful repair with self progenitors that would give rise to the cell types needed to restore function of the central nervous system. Some studies involve both scaffolds and cell grafts. Here we describe studies on spinal cord repair using what we call “endogenous tissue engineering for regenerative medicine”. The approach involves a hydrogel that mimics the natural milieu where endogenous pre-existing and newly formed cells populate the gel progressively allowing for the integration of CNS self populations leading to a successful recovery of function. Highlight aspects learned from this type of studies are that: Endogenous reconstruction of the injured spinal cord is possible by using the adequate support. The contribution of nestinexpressing progenitors to spinal cord regeneration is continuous and substantial both, in the reconstructed segment as well as, along the distal and caudal segments of the reconstructed spinal cord. Most of these cells appear to have been in a quiescent state until the injury occurred and only a small fraction of these neural progenitors was produced via cell proliferation. The hydrogel combined with exercise was necessary and sufficient to restore locomotor function in cats that underwent spinal transaction followed by reconstructive surgery. This recovery of function was first seen 28 days after surgery and continued to improve for at least 21 months. Therefore, endogenous pre-existing and newly formed cells populated the gel scaffold established contact with the non injured tissue and lead to recovery of function.