Lithium enhances the neuronal differentiation of neural progenitor cells in vitro and after transplantation into the avulsed ventral horn of adult rats through the secretion of brain-derived neurotrophic factor

This study was undertaken to elucidate the molecular mechanisms by which lithium regulates the development of spinal cord-derived neural progenitor cells (NPCs) in vitro and after transplanted in vivo . Our results show that lithium at the therapeutic concentration significantly increases the proliferation and neuronal differentiation of NPCs in vitro. Specific ELISAs, western blotting, and quantitative real-time RT-PCR assays demonstrate that lithium treatment significantly elevates the expression and production of brain-derived neurotrophic factor (BDNF) by NPCs in culture. Application of a BDNF neutralizing antibody in culture leads to a marked reduction in the neurogenesis of lithium-treated NPCs to the control level. However, it shows no effects on the proliferation of lithium-treated NPCs. These findings suggest that the BDNF pathway is possibly involved in the supportive role of lithium in inducing NPC neurogenesis but not proliferation. This study also provides evidence that lithium is able to elevate the neuronal generation and BDNF production of NPCs after transplantation into the adult rat ventral horn with motoneuron degeneration because of spinal root avulsion, which highlights the therapeutic potential of lithium in cell replacement strategies for spinal cord injury because of its ability to promote neuronal differentiation and BDNF production of grafted NPCs in the injured spinal cord.     

Immature spinal cord neurons are dynamic regulators of adult nociceptive sensitivity

Chronic pain is a debilitating condition with unknown mechanism. Nociceptive sensitivity may be regulated by genetic factors, some of which have been separately linked to neuronal progenitor cells and neuronal differentiation. This suggests that genetic factors that interfere with neuronal differentiation may contribute to a chronic increase in nociceptive sensitivity, by extending the immature, hyperexcitable stage of spinal cord neurons. Although adult rodent spinal cord neurogenesis was previously demonstrated, the fate of these progenitor cells is unknown. Here, we show that peripheral nerve injury in adult rats induces extensive spinal cord neurogenesis and a long‐term increase in the number of spinal cord laminae I–II neurons ipsilateral to injury. The production and maturation of these new neurons correlates with the time course and modulation of nociceptive behaviour, and transiently mimics the cellular and behavioural conditions present in genetically modified animal models of chronic pain. This suggests that the number of immature neurons present at any time in the spinal cord dorsal horns contributes to the regulation of nociceptive sensitivity. The continuous turnover of these neurons, which can fluctuate between normal and injured states, is a dynamic regulator of nociceptive sensitivity. In support of this hypothesis, we find that promoters of neuronal differentiation inhibit, while promoters of neurogenesis increase long‐term nociception. TrkB agonists, well‐known promoters of nociception in the short‐term, significantly inhibit long‐term nociception by promoting the differentiation of newly produced immature neurons. These findings suggest that promoters of neuronal differentiation may be used to alleviate chronic pain.     

Chondroitinase and growth factors enhance activation and oligodendrocyte differentiation of endogenous neural precursor cells after spinal cord injury

The adult spinal cord harbours a population of multipotent neural precursor cells (NPCs) with the ability to replace oligodendrocytes. However, despite this capacity, proliferation and endogenous remyelination is severely limited after spinal cord injury (SCI). In the post-traumatic microenvironment following SCI, endogenous spinal NPCs mainly differentiate into astrocytes which could contribute to astrogliosis that exacerbate the outcomes of SCI. These findings emphasize a key role for the post-SCI niche in modulating the behaviour of spinal NPCs after SCI. We recently reported that chondroitin sulphate proteoglycans (CSPGs) in the glial scar restrict the outcomes of NPC transplantation in SCI by reducing the survival, migration and integration of engrafted NPCs within the injured spinal cord. These inhibitory effects were attenuated by administration of chondroitinase (ChABC) prior to NPC transplantation. Here, in a rat model of compressive SCI, we show that perturbing CSPGs by ChABC in combination with sustained infusion of growth factors (EGF, bFGF and PDGF-AA) optimize the activation and oligodendroglial differentiation of spinal NPCs after injury. Four days following SCI, we intrathecally delivered ChABC and/or GFs for seven days. We performed BrdU incorporation to label proliferating cells during the treatment period after SCI. This strategy increased the proliferation of spinal NPCs, reduced the generation of new astrocytes and promoted their differentiation along an oligodendroglial lineage, a prerequisite for remyelination. Furthermore, ChABC and GF treatments enhanced the response of non-neural cells by increasing the generation of new vascular endothelial cells and decreasing the number of proliferating macrophages/microglia after SCI. In conclusions, our data strongly suggest that optimization of the behaviour of endogenous spinal NPCs after SCI is critical not only to promote endogenous oligodendrocyte replacement, but also to reverse the otherwise detrimental effects of their activation into astrocytes which could negatively influence the repair process after SCI.     

Nogo-66 promotes the differentiation of neural progenitors into astroglial lineage cells through mTOR-STAT3 pathway

Background

Neural stem/progenitor cells (NPCs) can differentiate into neurons, astrocytes and oligodendrocytes. NPCs are considered valuable for the cell therapy of injuries in the central nervous system (CNS). However, when NPCs are transplanted into the adult mammalian spinal cord, they mostly differentiate into glial lineage. The same results have been observed for endogenous NPCs during spinal cord injury. However, little is known about the mechanism of such fate decision of NPCs.

Methodology/Principal Findings

In the present study, we have found that myelin protein and Nogo-66 promoted the differentiation of NPCs into glial lineage. NgR and mTOR-Stat3 pathway were involved in this process. Releasing NgR from cell membranes or blocking mTOR-STAT3 could rescue the enhanced glial differentiation by Nogo-66.

Conclusions/Significance

These results revealed a novel function of Nogo-66 in the fate decision of NPCs. This discovery could have profound impact on the understanding of CNS development and could improve the therapy of CNS injuries.     

Neural stem cells for spinal cord repair

Spinal cord injury (SCI) causes the irreversible loss of spinal cord parenchyma including astroglia, oligodendroglia and neurons. In particular, severe injuries can lead to an almost complete neural cell loss at the lesion site and structural and functional recovery might only be accomplished by appropriate cell and tissue replacement. Stem cells have the capacity to differentiate into all relevant neural cell types necessary to replace degenerated spinal cord tissue and can now be obtained from virtually any stage of development. Within the last two decades, many in vivo studies in small animal models of SCI have demonstrated that stem cell transplantation can promote morphological and, in some cases, functional recovery via various mechanisms including remyelination, axon growth and regeneration, or neuronal replacement. However, only two well-documented neural-stem-cell-based transplantation strategies have moved to phase I clinical trials to date. This review aims to provide an overview about the current status of preclinical and clinical neural stem cell transplantation and discusses future perspectives in the field.     

Oligodendrocytes and radial glia derived from adult rat spinal cord progenitors: morphological and immunocytochemical characterization.

Self-renewing, multipotent neural progenitor cells (NPCs) reside in the adult mammalian spinal cord ependymal region. The current study characterized, in vitro, the native differentiation potential of spinal cord NPCs isolated from adult enhanced green fluorescence protein rats. Neurospheres were differentiated, immunocytochemistry (ICC) was performed, and the positive cells were counted as a percentage of Hoescht+ nuclei in 10 random fields. Oligodendrocytes constituted most of the NPC progeny (58.0% of differentiated cells; 23.4% in undifferentiated spheres). ICC and electron microscopy (EM) showed intense myelin production by neurospheres and progeny. The number of differentiated astrocytes was 18.0%, but only 2.8% in undifferentiated spheres. The number of differentiated neurons was 7.4%, but only 0.85% in undifferentiated spheres. The number of differentiated radial glia (RG) was 73.0% and in undifferentiated spheres 80.9%. EM showed an in vitro phagocytic capability of NPCs. The number of undifferentiated NPCs was 32.8% under differentiation conditions and 78.9% in undifferentiated spheres. Compared with ependymal region spheres, the spheres derived from the peripheral white matter of the spinal cord produced glial-restricted precursors. These findings indicate that adult rat spinal cord ependymal NPCs differentiate preferentially into oligodendrocytes and RG, which may support axonal regeneration in future trials of transplant therapy for spinal cord injury.     

Chondroitinase ABC treatment and the phenotype of neural progenitor cells isolated from injured rat spinal cord

The aim of the present study was to investigate whether enzyme chondroitinase ABC (ChABC) treatment influences the phenotype of neural progenitor cells (NPCs) derived from injured rat spinal cord. Adult as well as fetal spinal cords contain a pool of endogenous neural progenitors cells, which play a key role in the neuroregenerative processes following spinal cord injury (SCI) and hold particular promise for therapeutic approaches in CNS injury or neurodegenerative disorders. In our study we used in vitro model to demonstrate the differentiation potential of NPCs isolated from adult rat spinal cord after SCI, treated with ChABC. The intrathecal delivery of ChABC (10 U/ml) was performed at day 1 and 2 after SCI. The present findings indicate that the impact of SCI resulted in a decrease of all NPCs phenotypes and the ChABC treatment, on the contrary, caused an opposite effect.     

Endogenous neurogenesis in adult mammals after spinal cord injury

During the whole life cycle of mammals,new neurons are constantly regenerated in the subgranular zone of the dentate gyrus and in the subventricular zone of the lateral ventricles.Thanks to emerging methodologies,great progress has been made in the characterization of spinal cord endogenous neural stem cells(ependymal cells) and identification of their role in adult spinal cord development.As recently evidenced,both the intrinsic and extrinsic molecular mechanisms of ependymal cells control the sequential steps of the adult spinal cord neurogenesis.This review introduces the concept of adult endogenous neurogenesis,the reaction of ependymal cells after adult spinal cord injury(SCI),the heterogeneity and markers of ependymal cells,the factors that regulate ependymal cells,and the niches that impact the activation or differentiation of ependymal cells.     

Sox11 promotes endogenous neurogenesis and locomotor recovery in mice spinal cord injury

We introduced a lentiviral vector containing the Sox11 gene into injured spinal cords of mice to evaluate the therapeutic potential of Sox11 in spinal cord injury. Sox11 markedly improved locomotor recovery after spinal cord injury and this recovery was accompanied by an up-regulation of Nestin/Doublecortin expression in the injured spinal cord. Sox11 was mainly located in endogenous neural stem cells lining the central canal and in newly-generated neurons in the spinal cord. In addition, Sox 11 significantly induced expressions of BDNF in the spinal cords of LV-Sox11-treated mice. We concluded that Sox11 induced activation of endogenous neural stem cells into neuronal determination and migration within the injured spinal cord. The resultant increase of BDNF at the injured site might form a distinct neurogenic niche which induces a final neuronal differentiation of these neural stem cells. Enhancing Sox11 expression to induce neurogenic differentiation of endogenous neural stem cells after injury may be a promising strategy in restorative therapy after SCI in mammals.     

Glycerophospholipid metabolism in neuronal and glial cell-enriched fractions

The metabolism of phospholipids in separated glial and neuronal cells has been reviewed in this paper. Lipids are more abundant in glia; on the other hand, in vivo experiments performed with labeled precursors have indicated that lipid turnover is faster in neurons (with the possible exception of oligodendroglia). Biosynthetic and catabolic enzymes of lipid metabolism have been studied in separated cells (mainly in neurons and astroglia) and have been shown to be almost always more active in neurons. Also base exchange is probably more active in these cells. Therefore the results of in vitro and in vivo experiments indicate that neurons are more active than astroglia in metabolizing glycerophospholipids.     

Involvement of protein kinase C in the axonal growth-promoting effect on spinal cord neurons by target-derived astrocytes

Astroglial cells participate in a variety of developmental events during neuronal morphogenesis. We have shown that axonal, but not dendritic, outgrowth of spinal cord neurons can be promoted by a diffusible factor or factors secreted from target region-derived cerebellar astroglia in vitro in comparison with spinal astroglia. In the present study, we examined the involvement of protein kinase C (PKC) in the axon-promoting effect by astroglia. The inhibition of PKC by sphingosine or by the phorbol ester 12-O-tetradecanoylphorbol 13-acetate (TPA) at high concentration greatly reduced the mean axonal length of spinal neurons cultured in medium conditioned by cerebellar astroglia (SCn-CBg), while activation of PKC by TPA at low concentration, or by retinoic acid, was not additive to the glial effect. The activation of PKC by TPA or retinoic acid promoted axon growth of spinal neurons cultured in medium conditioned by spinal astroglia (SCn-SCg), which otherwise would not be as supportive for axon growth as cerebellar astroglia. Western blotting and PKC activity assays showed that there was a trend for increased PKC activity and protein levels (in particular, PKCβ) in SCn-CBg cultures, which correlated with enhanced axon growth. Inhibition of PKC by sphingosine appeared to decrease protein levels, especially PKCβ, which correlated with suppressed axon outgrowth. In SCn-SCg cultures, phorbol ester activation of PKC increased both activity and protein levels of both PKCα and PKCβ. This activation correlated with stimulated axonal outgrowth. These results suggest that the glial signaling that regulates specific axonal outgrowth by target astroglia is mediated in part by the PKC second messenger system. © 1994 John Wiley & Sons, Inc. 1994 John Wiley & Sons, Inc.     

Studies on expression of FSH and its anti-apoptotic effects on ischemia injury in rat spinal cord

Studies indicated that many tissues could express FSH. New functions of FSH have been recognized beyond reproduction regulation. However, no report has been made about the expression and function of FSH in rat spinal cord. Double-labeled immunofluorescence stain and in situ hybridization were used to study the co-localization of FSH with its receptor and co-localization of FSH with GnRH receptor in rat spinal cord. Spinal cord ischemia injury models were built, TUNEL stain and Fas immunostaining were made to observe the anti-apoptotic effects of FSH to neurons induced by spinal cord ischemia injury. The results found that some neurons and glias of rat spinal cord showed both FSH immunoreactivity and FSH mRNA positive signals; not only FSH and its receptor but also FSH and GnRH receptor co-located in cells of both gray matter and white matter; treatment with certain concentration of FSH before ischemia–reperfusion injury, less TUNEL positive cells and Fas positive cells were found in motor neurons of ventral gray matter in FSH experiment group than that in control group. These suggested that rat spinal cord could express FSH, it is also a target organ of FSH; FSH might exert functions through its receptor by paracrine or autocrine effects; GnRH in spinal cord might regulate FSH positive neurons through GnRH receptor; FSH might inhibit ischemia induced neuron apoptosis by down-regulating Fas expression in spinal cord.     

Ion channels in postnatal neurogenesis

Neural stem and progenitor cells (NSC/NPCs) are unspecialized cells found in the adult peri-ventricular and sub-granular zones that are capable of self-renewal, migration, and differentiation into new neurons through the remarkable process of postnatal neurogenesis. We are now beginning to understand that the concerted action of ion channels, multi-pass transmembrane proteins that allow passage of ions across otherwise impermeable cellular membranes tightly regulate this process. Specific ion channels control proliferation, differentiation and survival. Furthermore, they have the potential to be highly selective drug targets due to their complex structures. As such, these proteins represent intriguing prospects for control and optimization of postnatal neurogenesis for neural regeneration following brain injury or disease. Here, we concentrate on ion channels identified in adult ventricular zone NSC/NPCs that have been found to influence the stages of neurogenesis. Finally, we outline the potential of these channels to elicit repair, and highlight the outstanding challenges.     

Nitric oxide acts in a positive feedback loop with BDNF to regulate neural progenitor cell proliferation and differentiation in the mammalian brain

Nitric oxide (NO) is believed to act as an intercellular signal that regulates synaptic plasticity in mature neurons. We now report that NO also regulates the proliferation and differentiation of mouse brain neural progenitor cells (NPCs). Treatment of dissociated mouse cortical neuroepithelial cluster cell cultures with the NO synthase inhibitor L-NAME or the NO scavenger hemoglobin increased cell proliferation and decreased differentiation of the NPCs into neurons, whereas the NO donor sodium nitroprusside inhibited NPC proliferation and increased neuronal differentiation. Brain-derived neurotrophic factor (BDNF) reduced NPC proliferation and increased the expression of neuronal NO synthase (nNOS) in differentiating neurons. The stimulatory effect of BDNF on neuronal differentation of NPC was blocked by L-NAME and hemoglobin, suggesting that NO produced by the latter cells inhibited proliferation and induced neuronal differentiation of neighboring NPCs. A similar role for NO in regulating the switch of neural stem cells from proliferation to differentiation in the adult brain is suggested by data showing that NO synthase inhibition enhances NPC proliferation and inhibits neuronal differentiation in the subventricular zone of adult mice. These findings identify NO as a paracrine messenger stimulated by neurotrophin signaling in newly generated neurons to control the proliferation and differentiation of NPC, a novel mechanism for the regulation of developmental and adult neurogenesis.     

Ion channels in postnatal neurogenesis: potential targets for brain repair

Neural stem and progenitor cells (NSC/NPCs) are unspecialized cells found in the adult peri-ventricular and sub-granular zones that are capable of self-renewal, migration, and differentiation into new neurons through the remarkable process of postnatal neurogenesis. We are now beginning to understand that the concerted action of ion channels, multi-pass transmembrane proteins that allow passage of ions across otherwise impermeable cellular membranes tightly regulate this process. Specific ion channels control proliferation, differentiation and survival. Furthermore, they have the potential to be highly selective drug targets due to their complex structures. As such, these proteins represent intriguing prospects for control and optimization of postnatal neurogenesis for neural regeneration following brain injury or disease. Here, we concentrate on ion channels identified in adult ventricular zone NSC/NPCs that have been found to influence the stages of neurogenesis. Finally, we outline the potential of these channels to elicit repair, and highlight the outstanding challenges.     

Cytoprotective and anti-inflammatory effects of PAL31 overexpression in glial cells

Background

Acute spinal cord injury (SCI) leads to a series of reactive changes and causes severe neurological deficits. A pronounced inflammation contributes to secondary pathology after SCI. Astroglia respond to SCI by proliferating, migrating, and altering phenotype. The impact of reactive gliosis on the pathogenesis of SCI is not fully understood. Our previous study has identified an inflammatory modulating protein, proliferation related acidic leucine-rich protein (PAL31) which is upregulated in the microglia/macrophage of injured cords. Because PAL31 participates in cell cycle progression and reactive astroglia often appears in the injured cord, we aim to examine whether PAL31 is involved in glial modulation after injury.

Results

Enhanced PAL31 expression was shown not only in microglia/macrophages but also in spinal astroglia after SCI. Cell culture study reveal that overexpression of PAL31 in mixed glial cells or in C6 astroglia significantly reduced LPS/IFNγ stimulation. Further, enhanced PAL31 expression in C6 astroglia protected cells from H₂O₂ toxicity; however, this did not affect its proliferative activity. The inhibiting effect of PAL31 on LPS/IFNγ stimulation was observed in glia or C6 after co-culture with neuronal cells. The results demonstrated that the overexpressed PAL31 in glial cells protected neuronal damages through inhibiting NF-kB signaling and iNOS.

Conclusions

Our data suggest that PAL31upregulation might be beneficial after spinal cord injury. Reactive gliosis might become a good target for future therapeutic interventions.     

Regulation of neurogenesis by neurotrophins: implications in hippocampus-dependent memory

Neurogenesis, the generation of new neurons from neural precursor cells (NPCs), is a multi-step process that includes the proliferation of NPCs, fate determination, migration, and neuronal maturation. Neurogenesis is regulated by several extrinsic factors,such as enriched environment, physical exercise, hormones and stress, many of which also induce the expression of neurotrophins.In this review, we summarize studies on the role of neurotrophins in neurogenesis during development and in adults.We discuss the functional significance of neurogenesis in learning and memory, and how neurotrophins regulate this process.In this context, we describe recent experiments linking adult neurogenesis to long-term synaptic plasticity in the hippocampal dentate gyrus. Further study of the relationship between neurotrophins, adult neurogenesis and dentate synaptic plasticity might provide new insights into the mechanisms by which gene-environment interactions control cognition and brain plasticity.     

Transplanted neural progenitor cells expressing mutant NT3 promote myelination and partial hindlimb recovery in the chronic phase after spinal cord injury

Neutrotrophin-3 (NT3) plays a protective role in injured central nervous system tissues through interaction with trk receptors. To enhance the regeneration of damaged tissue, a combination therapy with cell transplantation and neurotrophins has been under development. We examined whether the transplantation of neural progenitor cells (NPCs) secreting NT3/D15A, a multi-neurotrophin with the capacity to bind both trkB and trkC, would enhance the repair of damaged tissues and the functional recovery in a chronic phase of spinal cord injury. The cultured NPCs with lentiviral vector containing either GFP or NT3/D15A were transplanted into the contused spinal cord at 6 weeks after the initial thoracic injury. Eight weeks after the transplantation, the NT3/D15A transplants displayed better survival than the GFP transplants, and they exhibited enhanced myelin formation and partial improvement of hindlimb function. Our study revealed that NT3/D15A produced positive effects in injured spinal cords even in the chronic phase. These effects suggest an enhanced neurotrophin-trk signaling by NT3/D15A.