mGluR5 is involved in proliferation of rat neural progenitor cells exposed to hypoxia with activation of mitogen-activated protein kinase signaling pathway

Hypoxia/ischemia induces proliferation of neural progenitor cells (NPCs) in rodent and human brain; however, the mechanisms remain unknown. We investigated the effects of metabotropic glutamate receptor 5 (mGluR5) on NPC proliferation under hypoxia, the expression of cyclin D1, and the activation of the mitogen-activated protein kinases (MAPKs) signaling pathway in cell culture. The results showed that hypoxia induced mGluR5 expression on NPCs in vitro. Under hypoxia, the mGluR5 agonists DHPG and CHPG significantly increased NPC proliferation in cell activity, diameter of neurospheres, bromodeoxyuridine (BrdU) incorporation and cell division, and expression of cyclin D1, with decreasing cell death. The mGluR5 siRNA and antagonist MPEP decreased the NPC proliferation and expression of cyclin D1, with increasing cell death. Phosphorylated JNK and ERK increased with the proliferation of NPCs after DHPG and CHPG treatment under hypoxia, while p-p38 level decreased. These results demonstrate that the expression of mGluR5 was upregulated during the proliferation of rat NPCs stimulated by hypoxia in vitro. The activation of the ERK and JNK signaling pathway and the expression of cyclin D1 were increased in this process. These finding suggest the involvement of mGluR5 in rat NPC proliferation and provide a target molecule in neural repair after ischemia/hypoxia injury of CNS.     

Single and local blockade of interleukin-6 signaling promotes neuronal differentiation from transplanted embryonic stem cell-derived neural precursor cells

Safe and efficient transplantation of embryonic stem (ES) cells to the brain requires that local inflammatory and immune responses to allogeneic grafts are inhibited. To investigate cytokines that affect graft cell survival and differentiation, we used stromal cell-derived inducing activity to induce the differentiation of neural progenitor cells (NPCs) from mouse ES cells and transplanted the NPCs into mouse brain. Examination of surrounding brain tissue revealed elevated expression levels of interleukin (IL)-1β, IL-4, and IL-6 in response to NPC transplantation. Among these, only IL-6 reduced neuronal differentiation and promoted glial differentiation in vitro. When we added anti-IL-6 receptor antibodies to NPCs during transplantation, this single and local blockade of IL-6 signaling reduced the accumulation of host-derived leukocytes, including microglia. Furthermore, it also promoted neuronal differentiation and reduced glial differentiation from the grafted NPCs to an extent similar to that with systemic and continuous administration of cyclosporine A. These results suggest that local administration of anti-IL-6 receptor antibodies with NPCs may promote neuronal differentiation during the treatment of neurological diseases with cell replacement therapy.     

Mammalian target of rapamycin (mTOR) is involved in the neuronal differentiation of neural progenitors induced by insulin

The fate of neural progenitor cells (NPCs) is determined by many extracellular cues. Among them, insulin and insulin-like growth factor (IGF) family are found to promote the neuronal differentiation of NPCs. Akt activation has been indicated to be responsible for the insulin/IGF-I induced neuronal differentiation. However, the mechanism by which insulin/IGF-I-PI3K-Akt pathway induces neurogenesis of NPCs is not clear. In this study, we have demonstrated that mTOR is involved in the insulin-induced neuronal differentiation. Insulin induces neurogenesis of NPCs in a dose-dependent manner. Phosphorylated mTOR has been up-regulated in a PI3K-Akt dependent manner during NPC differentiation induced by insulin. The specific inhibitor of mTOR, rapamycin, can abrogate the increase of differentiated neurons stimulated by insulin. In addition, this is not the result from the apoptosis of neurons or NPCs. This research has extended the understanding of functions of mTOR and the mechanism of NPC differentiation regulated by insulin.     

High-level expression of functional chemokine receptor CXCR4 on human neural precursor cells

Neural precursor cells (NPCs) are self-renewing, multipotent progenitors that give rise to neurons, astrocytes and oligodendrocytes in the central nervous system (CNS). Fetal NPCs have attracted attention for their potential use in studying normal CNS development. Several studies of rodent neural progenitors have suggested that chemokines and their receptors are involved in directing NPC migration during CNS development. In this study, we established a consistent system to culture human NPCs and examined the expression of chemokine receptors on these cells. NPCs were found to express the markers nestin and CD133 and to differentiate into neurons, astrocytes and oligodendrocytes at the clonal level. Flow cytometry and RNase protection assay (RPA) indicated that NPCs express high levels of CXCR4 and low levels of several other chemokine receptors. When examined using a chemotaxis assay, NPCs were able to respond to CXCL12/SDF-1alpha, a ligand of CXCR4. Treatment with anti-CXCR4 antibody or HIV-1 gp120 abolished the migratory response of NPCs towards CXCL12/SDF-1alpha. These findings suggest that CXCR4 may play a significant role in directing NPC migration during CNS development.     

Tumor necrosis factor-alpha and interleukin-18 modulate neuronal cell fate in embryonic neural progenitor culture

Neural progenitor cells (NPCs) in developing and adult CNS are capable of giving rise to various neuronal and glial cell populations. Neurogenesis in the adult hippocampus has been found to be inhibited by a proinflammatory cytokine, interleukin-6 (IL-6), suggesting that activated microglia in the inflamed brain may control neurogenesis. Yet, little is known about the effect of microglia-derived factors on the cell fate of embryonic NPCs. In this study, we show that neurons with betaIII-tubulin immunoreactivity in the NPC culture were reduced by the condition media collected from microglia treated with endotoxin lipopolysaccharide (LPS/M-CM). Treatment with pentoxifylline (PTX), an inhibitor for tumor necrosis factor-alpha (TNF-alpha) secretion from LPS-activated microglia, blocked the reduction of betaIII-tubulin+ cells in NPC culture. Furthermore, treatment of NPCs with interleukin-18 (IL-18), a recently discovered proinflammatory cytokine, also decreased the number of betaIII-tubulin+ cells in a dose- and time-dependent manner. Surprisingly, we also observed that the remaining betaIII-tubulin+ cells in the LPS/M-CM-treated culture exhibited more branching neurites. Thus, the activated microglia-derived cytokines, TNF-alpha and IL-18, may either inhibit the neuronal differentiation or induce neuronal cell death in the NPC culture, whereas these cells may also produce factors to improve the neurite branching in the NPC culture.     

Glucocorticoid signaling and exercise-induced downregulation of the mineralocorticoid receptor in the induction of adult mouse dentate neurogenesis by treadmill running

Physical exercise is known to promote adult neurogenesis, although the underlying mechanisms remain unclear. Glucocorticoid (corticosterone in rodents) is a factor that is known to affect neurogenesis. As physical exercise modulates corticosterone secretion, we hypothesized that corticosterone signaling is involved in exercise-induced adult neurogenesis. We chose treadmill running (TR) to accurately define the intensity and duration of exercise. Our results showed that 5 weeks of TR increased the doublecortin (DCX)-positive neuronal progenitor cells (NPCs) in adult hippocampus and transiently increased the serum corticosterone level at the end of the TR protocol. This protocol reduced the levels of hippocampal mineralocorticoid receptor (MR); however, glucocorticoid receptor levels were unaltered. We then investigated whether reducing corticosterone levels by bilateral adrenalectomy (ADX) attenuated the TR-enhanced adult neurogenesis. Our results showed that ADX not only blocked the TR-induced downregulation of MR, but also reduced the number of TR-enhanced NPCs. In order to examine the role of MR downregulation in TR-induced adult neurogenesis, animals were treated repeatedly with a selective MR antagonist, spironolactone, for 3 weeks. The results revealed that spironolactone increased the number of spontaneously occurring and TR-induced NPC in the dentate area. Further analysis revealed that spironolactone treatment did not alter precursor cell proliferation, but increased the number of DCX-positive NPCs, suggesting that blockage of MR signaling either facilitates the differentiation of progenitor cells towards neurons and/or enhances the survival of NPCs. Taken together, the data indicated that induction of NPCs in the dentate area of adult hippocampus by TR is partly due to the downregulation of glucocorticoid/MR signaling, which subsequently enhances differentiation along a neuronal lineage and/or NPC survival.     

Inhibition of glial maturation by bone morphogenetic protein 2 in a neural crest-derived cell line

Bone morphogenetic proteins (BMPs) regulate developmental decisions in many neural and nonneural lineages. BMPs influence both CNS neuronal and glial development and promote neuronal differentiation in neural crest derivatives. We investigated the actions of BMP2 on glial differentiation in the peripheral nervous system using NCM1 cells, a neural crest-derived cell line with the properties of peripheral glial precursor cells. BMP2 prevented the acquisition of a mature Schwann cell-like morphology, blocking the expression of mature genes and maintaining expression of several early glial markers. We provide evidence that BMP2 activates the GFAP promoter and define signaling pathways underlying this regulation. Our results demonstrate a novel role for BMPs as inhibitors of glial differentiation in the peripheral nervous system and suggest that BMPs may regulate the developmental timing of glial maturation.     

Pluripotent stem cell‐derived radial glia‐like cells as stable intermediate for efficient generation of human oligodendrocytes

Neural precursor cells (NPCs) derived from human pluripotent stem cells (hPSCs) represent an attractive tool for the in vitro generation of various neural cell types. However, the developmentally early NPCs emerging during hPSC differentiation typically show a strong propensity for neuronal differentiation, with more limited potential for generating astrocytes and, in particular, for generating oligodendrocytes. This phenomenon corresponds well to the consecutive and protracted generation of neurons and GLIA during normal human development. To obtain a more gliogenic NPC type, we combined growth factor‐mediated expansion with pre‐exposure to the differentiation‐inducing agent retinoic acid and subsequent immunoisolation of CD133‐positive cells. This protocol yields an adherent and self‐renewing population of hindbrain/spinal cord radial glia (RG)‐like neural precursor cells (RGL‐NPCs) expressing typical neural stem cell markers such as nestin, ASCL1, SOX2, and PAX6 as well as RG markers BLBP, GLAST, vimentin, and GFAP. While RGL‐NPCs maintain the ability for tripotential differentiation into neurons, astrocytes, and oligodendrocytes, they exhibit greatly enhanced propensity for oligodendrocyte generation. Under defined differentiation conditions promoting the expression of the major oligodendrocyte fate‐determinants OLIG1/2, NKX6.2, NKX2.2, and SOX10, RGL‐NPCs efficiently convert into NG2‐positive oligodendroglial progenitor cells (OPCs) and are subsequently capable of in vivo myelination. Representing a stable intermediate between PSCs and OPCs, RGL‐NPCs expedite the generation of PSC‐derived oligodendrocytes with O4‐, 4860‐, and myelin basic protein (MBP)‐positive cells that already appear within 7 weeks following growth factor withdrawal‐induced differentiation. Thus, RGL‐NPCs may serve as robust tool for time‐efficient generation of human oligodendrocytes from embryonic and induced pluripotent stem cells. GLIA 2015;63:2152–2167     

Neural progenitor cell apoptosis and differentiation were affected by activated microglia in spinal cord slice culture

Neural progenitor cell (NPC) transplantation offers great potential to treat spinal cord injury (SCI). NPCs may replace lost neurons or oligodendrocytes and act as a source of neurotrophic factors to support survival of remaining cells. However, their efficiency was limited by poor survival after transplantation, and they tended more to differentiate into astrocytes, but not neurons and oligodendrocytes. This study investigated whether activated microglia is a factor that contributes to this phenomenon. Organotypic spinal cord slice (SCS) culture was used to mimic the local environment after SCI, and NPCs were co-cultured with them to share the culture medium. After specific depletion of microglia in the SCSs with clodronate loaded liposome, the apoptotic rate of NPCs decreased, more NPCs differentiated into neurons, and glial differentiation was impaired. This suggested that microglia may impair NPC survival, and neuronal differentiation, but improve astrocyte differentiation. In NPC transplantation strategy for SCI, microglia would be manipulated to improve the survival and neuronal differentiation of NPCs.     

Enhanced neurogenesis from neural progenitor cells with G1/S-phase cell cycle arrest is mediated by transforming growth factor beta1

We have previously demonstrated that a G1/S-phase cell cycle blocker, deferoxamine (DFO), increased the number of new neurons from rat neurosphere cultures, which correlated with prolonged expression of cyclin-dependent kinase (cdk) inhibitor p27(kip1) [H. J. Kim et al. (2006)Brain Research, 1092, 1-15]. The present study focuses on neuronal differentiation mechanisms following treatment of neural stem/progenitor cells (NPCs) with a G1/S-phase cell cycle blocker. The addition of DFO (0.5 mm) or aphidicolin (Aph) (1.5 microm) to neurospheres for 8 h, followed by 3 days of differentiation, resulted in an increased number of neurons and neurite outgrowth. DFO induced enhanced expression of transforming growth factor (TGF)-beta1 and cdk5 at 24 h after differentiation, whereas Aph only increased TGF-beta1 expression. DFO-induced neurogenesis and neurite outgrowth were attenuated by administration of a cdk5 inhibitor, roscovitine, suggesting that the neurogenic mechanisms differ between DFO and Aph. TGF-beta1 (10 ng/mL) did not increase neurite outgrowth but rather the number of beta-tubulin III-positive cells, which was accompanied by enhanced p27(kip1) mRNA expression. In addition, TGF-beta receptor type II expression was observed in nestin-positive NPCs. Results indicated that DFO-induced TGF-beta1 signaling activated smad3 translocation from the cytoplasm to the nucleus. In contrast, TGF-beta1 signaling inhibition, via a TGF-beta receptor type I inhibitor (SB-505124), resulted in decreased DFO-induced neurogenesis, in conjunction with decreased p27(kip1) protein expression and smad3 translocation to the nucleus. These results suggest that cell cycle arrest during G1/S-phase induces TGF-beta1 expression. This, in turn, prompts enhanced neuronal differentiation via smad3 translocation to the nucleus and subsequent p27(kip1) activation in NPCs.     

Functional α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid receptors in differentiating embryonic neural progenitor cells

Glutamate-responsive α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) receptors are considered to play a significant role in neurogenesis. We have studied the functional expression of these receptors in migrating embryonic neural progenitor cells (NPCs). The majority of neurosphere-derived NPCs express AMPA receptors already during the first day of differentiation, based on mRNA quantification, immunocytochemistry, and Ca2+ imaging. The expression of GluR1 mRNA was significantly increased at 5 days of differentiation. The AMPA receptor subunits coexpressed with neuronal markers and were present in all cells at the outer periphery of the migration zone. In migrating NPCs, most of the AMPA receptors were philantotoxin sensitive and Ca2+-permeable, suggesting that in addition to their role in plasticity, the receptors are of importance in NPC differentiation.     

Neural precursor cell susceptibility to human cytomegalovirus diverges along glial or neuronal differentiation pathways

Cytomegalovirus (CMV) is a major cause of congenital brain disease, and its neuropathogenesis may be related to viral infection of rapidly dividing, susceptible neural precursor cells (NPCs). In the present study, we evaluated the susceptibility of human fetal brain-derived NPCs (nestin(+), A2B5(+), CD133(+)) to infection with CMV. Data derived from these studies demonstrated that undifferentiated NPCs supported productive viral replication. After differentiation in the presence of serum, a treatment that promotes development of an astroglial cell phenotype (GFAP(+), nestin(-), A2B5(-)), viral expression was retained. However, differentiation of NPCs in medium containing platelet-derived growth factor and brain-derived neurotropic factor, conditions that support the development of neurons (Tuj-1(+), nestin(-), A2B5(-)), resulted in reduced viral expression, with corresponding decreased CMV major immediate-early promoter (MIEP) activity relative to undifferentiated cells. Further experiments showed that cellular differentiation into a neuronal phenotype was associated with elevated levels of various CCAAT/enhancer binding protein beta (C/EBP)-beta isoforms, which suppressed MIEP activity in cotransfected NPCs. Taken together, these data demonstrate that the susceptibility of primary human NPCs to CMV is retained concomitantly with differentiation into glial cells but is actively repressed following differentiation into neurons.