Neural Stem Cell Death Research Articles

Statins impact primary embryonic mouse neural stem cell survival, cell death, and fate through distinct mechanisms.

Statins inhibit HMG-CoA reductase, the rate-limiting enzyme in the cholesterol biosynthesis pathway (CBP), and are used for the prevention of cardiovascular disease. The anti-inflammatory effects of statins may also provide therapeutic benefits and have led to their use in clinical trials for preeclampsia, a pregnancy-associated inflammatory condition, despite their current classification as category X (i.e. contraindicated during pregnancy). In the developing neocortex, products of the CBP play essential roles in proliferation and differentiation of neural stem-progenitor cells (NSPCs). To understand how statins could impact the developing brain, we studied effects of pravastatin and simvastatin on primary embryonic NSPC survival, proliferation, global transcription, and cell fate in vitro. We found that statins dose dependently decrease NSPC expansion by promoting cell death and autophagy of NSPCs progressing through the G1 phase of the cell cycle. Genome-wide transcriptome analysis demonstrates an increase in expression of CBP genes following pravastatin treatment, through activation of the SREBP2 transcription factor. Co-treatment with farnesyl pyrophosphate (FPP), a CBP metabolite downstream of HMG-CoA reductase, reduces SREBP2 activation and pravastatin-induced PARP cleavage. Finally, pravastatin and simvastatin differentially alter NSPC cell fate and mRNA expression during differentiation, through a non-CBP dependent pathway.

Phosphorylation of p62 by AMP-activated protein kinase mediates autophagic cell death in adult hippocampal neural stem cells

In the adult brain, programmed death of neural stem cells is considered to be critical for tissue homeostasis and cognitive function and is dysregulated in neurodegeneration. Previously, we have reported that adult rat hippocampal neural (HCN) stem cells undergo autophagic cell death (ACD) following insulin withdrawal. Because the apoptotic capability of the HCN cells was intact, our findings suggested activation of unique molecular mechanisms linking insulin withdrawal to ACD rather than apoptosis. Here, we report that phosphorylation of autophagy-associated protein p62 by AMP-activated protein kinase (AMPK) drives ACD and mitophagy in HCN cells. Pharmacological inhibition of AMPK or genetic ablation of the AMPK α2 subunit by clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 genome editing suppressed ACD, whereas AMPK activation promoted ACD in insulin-deprived HCN cells. We found that following insulin withdrawal AMPK phosphorylated p62 at a novel site, Ser-293/Ser-294 (in rat and human p62, respectively). Phosphorylated p62 translocated to mitochondria and induced mitophagy and ACD. Interestingly, p62 phosphorylation at Ser-293 was not required for staurosporine-induced apoptosis in HCN cells. To the best of our knowledge, this is the first report on the direct phosphorylation of p62 by AMPK. Our data suggest that AMPK-mediated p62 phosphorylation is an ACD-specific signaling event and provide novel mechanistic insight into the molecular mechanisms in ACD.

Proteolytic processed form of CXCL12 abolishes migration and induces apoptosis in neural stem cells in vitro

The subventricular zone (SVZ) of the adult mammalian brain hosts full potential neural stem cells (NSCs). NSCs are able to respond to extracellular signals in the brain, amplifying the pool of progenitor cells and giving rise to neuroblasts that show ability to migrate towards an injury site. These signals can come from vascular system, cerebrospinal fluid, glial cells, or projections of neurons in adjoining regions. CXCL12, a chemokine secreted after brain injury, reaches the SVZ in a gradient manner and drives neuroblasts towards the lesion area. Among many other molecules, matrix metalloproteinase 2 and 9 (MMP-2/9) are also released during brain injury. MMP-2/9 can cleave CXCL12 generating a new molecule, CXCL12(5-67), and its effects on NSCs viability is not well described. Here we produced recombinant CXCL12 and CXCL12(5-67) and evaluated their effect in murine adult NSCs migration and survival in vitro. We showed CXCL12(5-67) does not promote NSCs migration, but does induce cell death. The NSC death induced by CXCL12(5-67) involves caspases 9 and 3/7 activation, implying the intrinsic apoptotic pathway in this phenomenon. Our evidences in vitro make CXCL12(5-67) and its receptor potential candidates for brain injuries and neurodegeneration studies.

Inhibition of autophagy prevents irradiation-induced neural stem and progenitor cell death in the juvenile mouse brain

Radiotherapy is an effective tool in the treatment of malignant brain tumors. However, damage to brain stem and progenitor cells constitutes a major problem and is associated with long-term side effects. Autophagy has been shown to be involved in cell death, and the purpose of this study was to evaluate the effect of autophagy inhibition on neural stem and progenitor cell death in the juvenile brain. Ten-day-old selective Atg7 knockout (KO) mice and wild-type (WT) littermates were subjected to a single 6Gy dose of whole-brain irradiation. Cell death and proliferation as well as microglia activation and inflammation were evaluated in the dentate gyrus of the hippocampus and in the cerebellum at 6 h after irradiation. We found that cell death was reduced in Atg7 KO compared with WT mice at 6 h after irradiation. The number of activated microglia increased significantly in both the dentate gyrus and the cerebellum of WT mice after irradiation, but the increase was lower in the Atg7 KO mice. The levels of proinflammatory cytokines and chemokines decreased, especially in the cerebellum, in the Atg7 KO group. These results suggest that autophagy might be a potential target for preventing radiotherapy-induced neural stem and progenitor cell death and its associated long-term side effects.

Neural Stem Cell Death Mechanisms Induced by Amyloid Beta.

Background and PurposeAmyloid beta (Aβ) is the main component of amyloid plaques, which are deposited in the brains of patients with Alzheimer's disease (AD). Biochemical and animal studies support the central role of Aβ in AD pathogenesis. Despite several investigations focused on the pathogenic mechanisms of Aβ, it is still unclear how Aβ accumulates in the central nervous system and subsequently initiates the disease at the cellular level. In this study, we investigated the pathogenic mechanisms of Aβ using proteomics and antibody microarrays.MethodsTo evaluate the effect of Aβ on neural stem cells (NSCs), we treated primary cultured cortical NSCs with several doses of Aβ for 48 h. A 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide assay, trypan blue staining, and bromodeoxyuridine cell proliferation assay were performed. We detected several intracellular proteins that may be associated with Aβ by proteomics and Western blotting analysis.ResultsVarious viability tests showed that Aβ decreased NSCs viability and cell proliferation in a concentration-dependent manner. Aβ treatment significantly decreased lactate dehydrogenase B, high-mobility group box 1, aldolase C, Ezrin, and survival signals including phosphorylated phosphoinositide 3-kinase, Akt, and glycogen synthase kinase-3β.ConclusionsThese results suggest that several factors determined by proteomics and Western blot hold the clue to Aβ pathogenesis. Further studies are required to investigate the role of these factors.

Alterations in DNA and RNA profiles from alcohol exposed neuronal stem cells relates a possible mechanism for slowed proliferation and increased apoptosis.

Fetal alcohol spectrum disorders (FASD) are associated with a mother who consumed alcohol while pregnant. FASD symptoms in the fetus include physical effects, behavior abnormalities and learning problems. Phenotypes are associated with alterations in proliferation, differentiation, migration and increased cell death of neural crest stem cells during embryo development. Under normal growth conditions Cyclin D1 expression is transiently increased during late G1 phase and signals the cell to proceed to S-phase. CyclinD1 initiates a cascade of events necessary for transcription of genes required for proper DNA synthesis. It has been shown in mouse embryos that alcohol increases Cyclin D1 protein expression 2-fold, which in turn induces picnosis in 40 % of alcohol exposed neuronal stem cells. We suggest that alcohol has induced premature G1/S phase transition by Cyclin D1 overexpression, which, in turn, alters DNA synthesis and increases apoptosis.Through the use of flow cytometry we examined both RNA and DNA profiles with gating focuses on G1 and S phases respective. We then examined by antibody/fluorescent staining the overlapping expression of Cyclin D1 levels on these gated populations. Under normal growth conditions, Cyclin D1 expression is limited to the G1/S phase transition. Alcohol alters this expression pattern and suggests a continued expression in S phase cells. Profiles allowed an examination of apoptosis percentage as well. Understanding this mechanistic misregulation may eventually lead to a full understanding and improved treatment options for fetuses exposed to alcohol.

Mediation of Autophagic Cell Death by Type 3 Ryanodine Receptor (RyR3) in Adult Hippocampal Neural Stem Cells.

Cytoplasmic Ca2+ actively engages in diverse intracellular processes from protein synthesis, folding and trafficking to cell survival and death. Dysregulation of intracellular Ca2+ levels is observed in various neuropathological states including Alzheimer’s and Parkinson’s diseases. Ryanodine receptors (RyRs) and inositol 1,4,5-triphosphate receptors (IP3Rs), the main Ca2+ release channels located in endoplasmic reticulum (ER) membranes, are known to direct various cellular events such as autophagy and apoptosis. Here we investigated the intracellular Ca2+-mediated regulation of survival and death of adult hippocampal neural stem (HCN) cells utilizing an insulin withdrawal model of autophagic cell death (ACD). Despite comparable expression levels of RyR and IP3R transcripts in HCN cells at normal state, the expression levels of RyRs—especially RyR3—were markedly upregulated upon insulin withdrawal. While treatment with the RyR agonist caffeine significantly promoted the autophagic death of insulin-deficient HCN cells, treatment with its inhibitor dantrolene prevented the induction of autophagy following insulin withdrawal. Furthermore, CRISPR/Cas9-mediated knockout of the RyR3 gene abolished ACD of HCN cells. This study delineates a distinct, RyR3-mediated ER Ca2+ regulation of autophagy and programmed cell death in neural stem cells. Our findings provide novel insights into the critical, yet understudied mechanisms underlying the regulatory function of ER Ca2+ in neural stem cell biology.

Retinoic acid-loaded polymeric nanoparticles enhance vascular regulation of neural stem cell survival and differentiation after ischaemia.

Stroke is one of the leading causes of death and disability worldwide. However, current therapies only reach a small percentage of patients and may cause serious side effects. We propose the therapeutic use of retinoic acid-loaded nanoparticles (RA-NP) to safely and efficiently repair the ischaemic brain by creating a favourable pro-angiogenic environment that enhances neurogenesis and neuronal restitution. Our data showed that RA-NP enhanced endothelial cell proliferation and tubule network formation and protected against ischaemia-induced death. To evaluate the effect of RA-NP on vascular regulation of neural stem cell (NSC) survival and differentiation, endothelial cell-conditioned media (EC-CM) were collected. EC-CM from healthy RA-NP-treated cells reduced NSC death and promoted proliferation while EC-CM from ischaemic RA-NP-treated cells decreased cell death, increased proliferation and neuronal differentiation. In parallel, human endothelial progenitor cells (hEPC), which are part of the endogenous repair response to vascular injury, were collected from ischaemic stroke patients. hEPC treated with RA-NP had significantly higher proliferation, which further highlights the therapeutic potential of this formulation. To conclude, RA-NP protected endothelial cells from ischaemic death and stimulated the release of pro-survival, proliferation-stimulating factors and differentiation cues for NSC. RA-NP were shown to be up to 83-fold more efficient than free RA and to enhance hEPC proliferation. These data serve as a stepping stone to use RA-NP as vasculotrophic and neurogenic agents for vascular disorders and neurodegenerative diseases with compromised vasculature.

Neuroprotective Effects of Acetyl-L-Carnitine Against Oxygen-Glucose Deprivation-Induced Neural Stem Cell Death.

Deprivation of oxygen and glucose is the main cause of neuronal cell death during cerebral infarction and can result in severe morbidity and mortality. In general, the neuroprotective therapies that are applied after ischemic stroke have been unsuccessful, despite many investigations. Acetyl-L-carnitine (ALCAR) plays an important role in mitochondrial metabolism and in modulating the coenzyme A (CoA)/acyl-CoA ratio. We investigated the protective effects of ALCAR against oxygen-glucose deprivation (OGD) in neural stem cells (NSCs). We measured cell viability, proliferation, apoptosis, and intracellular signaling protein levels after treatment with varying concentrations of ALCAR under OGD for 8h. ALCAR protected NSCs against OGD by reducing apoptosis and restoring proliferation. Its protective effects are associated with increases in the expression of survival-related proteins, such as phosphorylated Akt (pAkt), phosphorylated glycogen synthase kinase 3b (pGSK3b), B cell lymphoma 2 (Bcl-2), and Ki-67 in NSCs that were injured by OGD. ALCAR also reduced the expression of death-related proteins, such as Bax, cytosolic cytochrome C, cleaved caspase-9, and cleaved caspase-3. We concluded that ALCAR exhibits neuroprotective effects against OGD-induced damage to NSCs by enhancing the expression of survival signals and decreasing that of death signals.

Activation of Type 4 Metabotropic Glutamate Receptor Attenuates Oxidative Stress-Induced Death of Neural Stem Cells with Inhibition of JNK and p38 MAPK Signaling.

Promoting both endogenous and exogenous neural stem cells' (NSCs) survival in the hostile host environments is essential to cell replacement therapy for central nervous system (CNS) disorders. Type 4 metabotropic glutamate receptor (mGluR4), one of the members of mGluRs, has been shown to protect neurons from acute and chronic excitotoxic insults in various brain damages. The present study investigated the preventive effects of mGluR4 on NSC injury induced by oxidative stress. Under challenge with H2O2, loss of cell viability was observed in cultured rat NSCs, and treatment with selective mGluR4 agonist VU0155041 conferred protective effects against the loss of cellular viability in a concentration-dependent manner, as shown by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay. Pretreatment of VU0155041 (30 μM) also inhibited the excessive NSC death induced by H2O2, and group III mGluRs antagonist (RS)-a-methylserine-O-phosphate (MSOP) or gene-targeted knockdown abolished the protective action of mGluR4, indicated by propidium iodide-Hoechst and terminal deoxynucleotidyl transferase-mediated UTP nick end labeling (TUNEL) staining. Western blot assay demonstrated that mGluR4 activation reversed the decreased procaspase-8/9/3and the destructed Bcl-2/Bax expressing balance, and likewise, MSOP and mGluR4 knockdown abrogated the action of mGluR4 activity. Furthermore, inhibition of JNK and p38 mitogen-activated protein kinases (MAPKs) were observed after mGluR4 activation, and as paralleling control, JNK-specific inhibitor SP600125 and p38-specific inhibitor SB203580 significantly rescued the H2O2-mediated NSC apoptosis and cleavage of procaspase-3. We suggest that activation of mGluR4 prevents oxidative stress-induced NSC death and apoptotic-associated protein activities with involvement of inhibiting the JNK and p38 pathways in cell culture. Our findings may help to develop strategies for enhancing the resided and transplanted NSC survival after oxidative stress insult of CNS.

Calpain Determines the Propensity of Adult Hippocampal Neural Stem Cells to Autophagic Cell Death Following Insulin Withdrawal.

Programmed cell death (PCD) has significant effects on the function of neural stem cells (NSCs) during brain development and degeneration. We have previously reported that adult rat hippocampal neural stem (HCN) cells underwent autophagic cell death (ACD) rather than apoptosis following insulin withdrawal despite their intact apoptotic capabilities. Here, we report a switch in the mode of cell death in HCN cells with calpain as a critical determinant. In HCN cells, calpain 1 expression was barely detectable while calpain 2 was predominant. Inhibition of calpain in insulin-deprived HCN cells further augmented ACD. In contrast, expression of calpain 1 switched ACD to apoptosis. The proteasome inhibitor lactacystin blocked calpain 2 degradation and elevated the intracellular Ca(2+) concentration. In combination, these effects potentiated calpain activity and converted the mode of cell death to apoptosis. Our results indicate that low calpain activity, due to absence of calpain 1 and degradation of calpain 2, results in a preference for ACD over apoptosis in insulin-deprived HCN cells. On the other hand, conditions leading to high calpain activity completely switch the mode of cell death to apoptosis. This is the first report on the PCD mode switching mechanism in NSCs. The dynamic change in calpain activity through the proteasome-mediated modulation of the calpain and intracellular Ca(2+) levels may be the critical contributor to the demise of NSCs. Our findings provide a novel insight into the complex mechanisms interconnecting autophagy and apoptosis and their roles in the regulation of NSC death.

Neural stem cell progeny regulate stem cell death in a Notch and Hox dependent manner.

Cell death is a prevalent, well-controlled and fundamental aspect of development, particularly in the nervous system. In Drosophila, specific neural stem cells are eliminated by apoptosis during embryogenesis. In the absence of apoptosis, these stem cells continue to divide, resulting in a dramatically hyperplastic central nervous system and adult lethality. Although core cell death pathways have been well described, the spatial, temporal and cell identity cues that activate the cell death machinery in specific cells are largely unknown. We identified a cis-regulatory region that controls the transcription of the cell death activators reaper, grim and sickle exclusively in neural stem cells. Using a reporter generated from this regulatory region, we found that Notch activity is required for neural stem cell death. Notch regulates the expression of the abdominalA homeobox protein, which provides important spatial cues for death. Importantly, we show that pro-apoptotic Notch signaling is activated by the Delta ligand expressed on the neighboring progeny of the stem cell. Thus we identify a previously undescribed role for progeny in regulating the proper developmental death of their parental stem cells.

A role for TRAIL/TRAIL-R2 in radiation-induced apoptosis and radiation-induced bystander response of human neural stem cells

Adult neurons, which are terminally differentiated cells, demonstrate substantial radioresistance. In contrast, human neural stem cells (NSC), which have a significant proliferative capacity, are highly sensitive to ionizing radiation. Cranial irradiation that is widely used for treatment of brain tumors may induce death of NSC and further cause substantial cognitive deficits such as impairing learning and memory. The main goal of our study was to determine a mechanism of NSC radiosensitivity. We observed a constitutive high-level expression of TRAIL-R2 in human NSC. On the other hand, ionizing radiation through generation of reactive oxygen species targeted cell signaling pathways and dramatically changed the pattern of gene expression, including upregulation of TRAIL. A significant increase of endogenous expression and secretion of TRAIL could induce autocrine/paracrine stimulation of the TRAIL-R2-mediated signaling cascade with activation of caspase-3-driven apoptosis. Furthermore, paracrine stimulation could initiate bystander response of non-targeted NSC that is driven by death ligands produced by directly irradiated NSC. Experiments with media transfer from directly irradiated NSC to non-targeted (bystander) NSC confirmed a role of secreted TRAIL for induction of a death signaling cascade in non-targeted NSC. Subsequently, TRAIL production through elimination of bystander TRAIL-R-positive NSC might substantially restrict a final yield of differentiating young neurons. Radiation-induced TRAIL-mediated apoptosis could be partially suppressed by anti-TRAIL antibody added to the cell media. Interestingly, direct gamma-irradiation of SK-N-SH human neuroblastoma cells using clinical doses (2-5Gy) resulted in low levels of apoptosis in cancer cells that was accompanied however by induction of a strong bystander response in non-targeted NSC. Numerous protective mechanisms were involved in the maintenance of radioresistance of neuroblastoma cells, including constitutive PI3K-AKT over-activation and endogenous synthesis of TGFβ1. Specific blockage of these survival pathways was accompanied by a dramatic increase in radiosensitivity of neuroblastoma cells. Intercellular communication between cancer cells and NSC could potentially be involved in amplification of cancer pathology in the brain.

Neural stem cell apoptosis after low‐methylmercury exposures in postnatal hippocampus produce persistent cell loss and adolescent memory deficits

The developing brain is particularly sensitive to exposures to environmental contaminants. In contrast to the adult, the developing brain contains large numbers of dividing neuronal precursors, suggesting that they may be vulnerable targets. The postnatal day 7 (P7) rat hippocampus has populations of both mature neurons in the CA1-3 region as well as neural stem cells (NSC) in the dentate gyrus (DG) hilus, which actively produce new neurons that migrate to the granule cell layer (GCL). Using this well-characterized NSC population, we examined the impact of low levels of methylmercury (MeHg) on proliferation, neurogenesis, and subsequent adolescent learning and memory behavior. Assessing a range of exposures, we found that a single subcutaneous injection of 0.6 µg/g MeHg in P7 rats induced caspase activation in proliferating NSC of the hilus and GCL. This acute NSC death had lasting impact on the DG at P21, reducing cell numbers in the hilus by 22% and the GCL by 27%, as well as reductions in neural precursor proliferation by 25%. In contrast, non-proliferative CA1-3 pyramidal neuron cell number was unchanged. Furthermore, animals exposed to P7 MeHg exhibited an adolescent spatial memory deficit as assessed by Morris water maze. These results suggest that environmentally relevant levels of MeHg exposure may decrease NSC populations and, despite ongoing neurogenesis, the brain may not restore the hippocampal cell deficits, which may contribute to hippocampal-dependent memory deficits during adolescence.

Interplay between autophagy and programmed cell death in mammalian neural stem cells

Mammalian neural stem cells (NSCs) are of particular interest because of their role in brain development and function. Recent findings suggest the intimate involvement of programmed cell death (PCD) in the turnover of NSCs. However, the underlying mechanisms of PCD are largely unknown. Although apoptosis is the best-defined form of PCD, accumulating evidence has revealed a wide spectrum of PCD encompassing apoptosis, autophagic cell death (ACD) and necrosis. This mini-review aims to illustrate a unique regulation of PCD in NSCs. The results of our recent studies on autophagic death of adult hippocampal neural stem (HCN) cells are also discussed. HCN cell death following insulin withdrawal clearly provides a reliable model that can be used to analyze the molecular mechanisms of ACD in the larger context of PCD. More research efforts are needed to increase our understanding of the molecular basis of NSC turnover under degenerating conditions, such as aging, stress and neurological diseases. Efforts aimed at protecting and harnessing endogenous NSCs will offer novel opportunities for the development of new therapeutic strategies for neuropathologies. [BMB Reports 2013; 46(8): 383-390]

Involvement of CD137 ligand signaling in neural stem cell death.

CD137 is a member of the tumor necrosis factor/nerve growth factor receptor superfamily. Interaction of CD137 with its ligand (CD137L) affects the apoptosis, proliferation and differentiation of immune cells. Interestingly, the CD137 receptor/ligand system involves the bi-directional transduction of signals. The expression of CD137 and its ligand is not restricted to immune organs, but can also be detected in a wide variety of tissues such as the brain, kidney, lung and heart. However, its role in brain is largely unknown. This study was performed to determine the role of CD137L reverse signaling in the apoptosis of neural stem cells. We identified the expression of CD137 and its ligand in C17.2 neural stem cells derived from mouse embryonic cerebellum. We found that the activation of CD137L reverse signaling by CD137 resulted in a decrease in cell adhesion to the fibronectin-coated culture basement, thus causing detachment-induced cell death. Furthermore, we showed that the cell death induced by CD137 was completely ameliorated by integrin activators and caspase inhibitors. Therefore we suggest that CD137L reverse signaling exerts a pro-apoptotic effect by suppressing integrin-mediated survival signals in neural stem cells.

The p21-activated kinase Mbt is a component of the apical protein complex in central brain neuroblasts and controls cell proliferation

The final size of the central nervous system is determined by precisely controlled generation, proliferation and death of neural stem cells. We show here that the Drosophila PAK protein Mushroom bodies tiny (Mbt) is expressed in central brain progenitor cells (neuroblasts) and becomes enriched to the apical cortex of neuroblasts in a cell cycle- and Cdc42-dependent manner. Using mushroom body neuroblasts as a model system, we demonstrate that in the absence of Mbt function, neuroblasts and their progeny are correctly specified and are able to generate different neuron subclasses as in the wild type, but are impaired in their proliferation activity throughout development. In general, loss of Mbt function does not interfere with establishment or maintenance of cell polarity, orientation of the mitotic spindle and organization of the actin or tubulin cytoskeleton in central brain neuroblasts. However, we show that mbt mutant neuroblasts are significantly reduced in cell size during different stages of development, which is most pronounced for mushroom body neuroblasts. This phenotype correlates with reduced mitotic activity throughout development. Additionally, postembryonic neuroblasts are lost prematurely owing to apoptosis. Yet, preventing apoptosis did not rescue the loss of neurons seen in the adult mushroom body of mbt mutants. From these results, we conclude that Mbt is part of a regulatory network that is required for neuroblast growth and thereby allows proper proliferation of neuroblasts throughout development.

A Global Transcriptome Analysis Reveals Molecular Hallmarks of Neural Stem Cell Death, Survival, and Differentiation in Response to Partial FGF-2 and EGF Deprivation

Neurosphere cell culture is a commonly used model to study the properties and potential applications of neural stem cells (NSCs). However, standard protocols to culture NSCs have yet to be established, and the mechanisms underlying NSC survival and maintenance of their undifferentiated state, in response to the growth factors FGF-2 and EGF are not fully understood. Using cultures of embryonic and adult olfactory bulb stem cells (eOBSCs and aOBSCs), we analyzed the consequences of FGF-2 and EGF addition at different intervals on proliferation, cell cycle progression, cell death and differentiation, as well as on global gene expression. As opposed to cultures supplemented daily, addition of FGF-2 and EGF every 4 days significantly reduced the neurosphere volume and the total number of cells in the spheres, mainly due to increased cell death. Moreover, partial FGF-2 and EGF deprivation produced an increase in OBSC differentiation during the proliferative phase. These changes were more evident in aOBSC than eOBSC cultures. Remarkably, these effects were accompanied by a significant upregulation in the expression of endogenous Fgf-2 and genes involved in cell death and survival (Cryab), lipid catabolic processes (Pla2g7), cell adhesion (Dscaml1), cell differentiation (Dscaml1, Gpr17, S100b, Ndrg2) and signal transduction (Gpr17, Ndrg2). These findings support that a daily supply of FGF-2 and EGF is critical to maintain the viability and the undifferentiated state of NSCs in culture, and they reveal novel molecular hallmarks of NSC death, survival and the initiation of differentiation.

Effects of Homocysteine on ERK Signaling and Cell Proliferation in Fetal Neural Stem Cells In Vitro

The aim of the present study was to determine if the excitatory amino acid homocysteine (Hcy) alters ERK signaling and cell proliferation in fetal neural stem cells (NSCs) in vitro. NSCs were isolated from fetal rats and grown in serum-free suspension medium. The cells were identified as NSCs by their expression of immunoreactive Sox2. NSCs were assigned to one of four treatment groups: vehicle control, low-dose Hcy group (Hcy-L, medium contained 30 μmol/L Hcy), middle-dose Hcy group (Hcy-M, 100 μmol/L Hcy) and high-dose Hcy group (Hcy-H, 300 μmol/L Hcy). Cell proliferation was evaluated by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. Protein expression levels of ERK1/2 and phosphorylated ERK1/2 were detected by Western blot. The effects of Hcy on NSC death, including apoptosis, were assessed by using flow cytometry and trypan blue exclusion. The results showed that NSCs grew as neurospheres in the serum-free medium. Hcy decreased ERK1/2 protein phosphorylation and NSC proliferation, but it did not induce cell death or apoptosis within the concentration from 30 to 300 μmol/L. The above results are consistent with the hypothesis that Hcy decreases fetal NSC proliferation by inhibiting ERK signaling.

Appearance of neural stem cells around the damaged area following traumatic brain injury in aged rats

We have previously reported free radical production after traumatic brain injury (TBI), which induces neural stem cell (NSC) degeneration and death. However, the effects of aging on NSC proliferation around the damaged area following TBI have not been investigated. Therefore, in this study, we used 10-week (young group) and 24-month-old (aged group) rat TBI models to investigate the effects of aging on NSC proliferation around damaged tissue using immunohistochemical and ex vivo techniques. Young and aged rats received TBI. At 1, 3 and 7days after TBI, immunohistochemical and lipid peroxidation studies were performed. Immunohistochemistry revealed that the number of nestin-positive cells around the damaged area after TBI in the aged group decreased significantly when compared with those in the young group (P<0.01). However, the number of 8-hydroxy-2'-deoxyguanosine-, 4-hydroxy-2-nonenal- and single-stranded DNA (ssDNA)-positive cells and the level of peroxidation around the damaged area after TBI significantly increased in the aged group, compared with those in the young group (P<0.01). Furthermore, almost all ssDNA-positive cells in young and aged groups co-localized with NeuN and nestin staining. Ex vivo studies revealed that neurospheres, which differentiated into neurons and glia in culture, could only be isolated from injured brain tissue in young and aged groups at 3days after TBI. These results indicate that, although there were fewer NSCs that have the potential to differentiate into neurons and glia, these NSCs escaped free radical-induced degeneration around the damaged area after TBI in the aged rat brain.