Impaired Neural Stem Cell Research Articles

Autocrine VEGF drives neural stem cell proximity to the adult hippocampus vascular niche.

The vasculature is a key component of adult brain neural stem cell (NSC) niches. In the adult mammalian hippocampus, NSCs reside in close contact with a dense capillary network. How this niche is maintained is unclear. We recently found that adult hippocampal NSCs express VEGF, a soluble factor with chemoattractive properties for vascular endothelia. Here, we show that global and NSC-specific VEGF loss led to dissociation of NSCs and their intermediate progenitor daughter cells from local vasculature. Surprisingly, though, we found no changes in local vascular density. Instead, we found that NSC-derived VEGF supports maintenance of gene expression programs in NSCs and their progeny related to cell migration and adhesion. In vitro assays revealed that blockade of VEGF receptor 2 impaired NSC motility and adhesion. Our findings suggest that NSCs maintain their own proximity to vasculature via self-stimulated VEGF signaling that supports their motility towards and/or adhesion to local blood vessels.

Autism-associated ANK2 regulates embryonic neurodevelopment

Autism spectrum disorder (ASD) is a neurodevelopmental condition characterized by altered social communication, restricted interests, and stereotypic behaviors. Although the molecular and cellular pathogeneses of ASD remain elusive, impaired neural stem cell differentiation and neuronal migration during cortical development are suggested to be critically involved in ASD. ANK2, which encodes for a cytoskeletal scaffolding protein involved in recruiting membrane proteins into specialized membrane domains, has been identified as a high-confidence ASD risk gene. However, the role of ANK2 in early neural development remains unclear. In this study, we analyzed the role of ANK2 in the cerebral cortex of developing mouse using in utero electroporation. We provide evidence suggesting that ANK2 regulates neural stem cell differentiation and neuronal migration in the embryonic cerebral cortex, where Ank2 is highly expressed. We also demonstrated that Ank2 knockdown alters the expression of genes involved in neural development. Taken together, these results support the view that ANK2 haploinsufficiency in patients may impair neural development, resulting in an increased risk of ASD. Our study findings provide new insights into the molecular and cellular pathogenesis of ASD, given that among high-confidence ASD genes, ANK2 is rare in that it encodes for a scaffolding protein for the membrane protein complex required for neuronal functions.

Absence of Both Thyroid Hormone Transporters MCT8 and OATP1C1 Impairs Neural Stem Cell Fate in the Adult Mouse Subventricular Zone.

SummaryAdult neural stem cell (NSC) generation in vertebrate brains requires thyroid hormones (THs). How THs enter the NSC population is unknown, although TH availability determines proliferation and neuronal versus glial progenitor determination in murine subventricular zone (SVZ) NSCs. Mice display neurological signs of the severely disabling human disease, Allan-Herndon-Dudley syndrome, if they lack both MCT8 and OATP1C1 transporters, or MCT8 and deiodinase type 2. We analyzed the distribution of MCT8 and OATP1C1 in adult mouse SVZ. Both are strongly expressed in NSCs and at a lower level in neuronal cell precursors but not in oligodendrocyte progenitors. Next, we analyzed Mct8/Oatp1c1 double-knockout mice, where brain uptake of THs is strongly reduced. NSC proliferation and determination to neuronal fates were severely affected, but not SVZ-oligodendroglial progenitor generation. This work highlights how tight control of TH availability determines NSC function and glial-neuron cell-fate choice in adult brains.

Neuroinflammation Induction and Alteration of Hippocampal Neurogenesis in Mice Following Developmental Exposure to Gossypol.

BackgroundNeurogenesis in the neonatal period involves the proliferation and differentiation of neuronal stem/progenitor cells and the establishment of synaptic connections. This process plays a critical role in determining the normal development and maturation of the brain throughout life. Exposure to certain physical or chemical factors during the perinatal period can lead to many neuropathological defects that cause high cognitive dysfunction and are accompanied by abnormal hippocampal neurogenesis and plasticity. As an endocrine disruptor, gossypol is generally known to exert detrimental effects in animals exposed under experimental conditions. However, it is unclear whether gossypol affects neurogenesis in the hippocampal dentate gyrus during early developmental stages.MethodsPregnant Institute of Cancer Research mice were treated with gossypol at a daily dose of 0, 20, and 50 mg/kg body weight from embryonic day 6.5 to postnatal day (P) 21. The changes of hippocampal neurogenesis as well as potential mechanisms were investigated by 5-bromo-2-deoxyuridine labeling, behavioral tests, immunofluorescence, quantitative reverse transcription-polymerase chain reaction, and western-blot analyses.ResultsAt P8, maternal gossypol exposure impaired neural stem cell proliferation in the dentate gyrus and decreased the number of newborn cells as a result of reduced proliferation of BLBP+ radial glial cells and Tbr2+ intermediate progenitor cells. At P21, the numbers of NeuN+ neurons and parvalbumin+ γ-aminobutyric acid-ergic interneurons were increased following 50 mg/kg gossypol exposure. In addition, gossypol induced hippocampal neuroinflammation, which may contribute to behavioral abnormalities and cognitive deficits and decrease synaptic plasticity.ConclusionsOur findings suggest that developmental gossypol exposure affects hippocampal neurogenesis by targeting the proliferation and differentiation of neuronal stem/progenitor cells, cognitive functions, and neuroinflammation. The present data provide novel insights into the neurotoxic effects of gossypol on offspring.

AdipoRon improves cognitive dysfunction of Alzheimer’s disease and rescues impaired neural stem cell proliferation through AdipoR1/AMPK pathway

Adult neurogenesis in hippocampus dentate gyrus (DG) is associated with the etiology on the early stage of Alzheimer's disease (AD). Factors that affect adult hippocampal neurogenesis have been shown to contribute to the neuropathology of AD. Adiponectin, a peptide hormone secreted by adipocytes, plays a critical role in insulin sensitizing, anti-inflammatory, and anti-diabetic effects in peripheral tissues. We previously showed that AdipoRon, as an agonist of adiponectin, promotes neurite outgrowth under ischemia. However, the role of AdipoRon on neural stem cells (NSCs) proliferation and cognitive dysfunction in the early stage of AD remains unknown. In this study, we investigated the role of AdipoRon on cognitive dysfunction and deficits of NSCs proliferation in AD. The in vivo study showed that AdipoRon improved either cognitive dysfunction or impaired NSCs proliferation in hippocampus DG region in APP/PS1 transgenic (Tg) mice. In addition, AdipoRon treatment also suppressed the β-amyloid (Aβ) deposition and inhibited β-secretase 1(BACE1) expression in both cortex and hippocampus of APP/PS1 Tg mice. The in vitro study further suggested that AdipoRon significantly alleviated Aβ-induced cell viability and neuronal morphology in primary neurons. Both AdipoR1 silencing and compound C, inhibitor of AMPK, completely abolished the effect of AdipoRon. Interestingly, AdipoRon also protected the dissipation of the ΔΨm caused by Aβ toxicity in primary neurons, which was reversed by compound C. In NE-4C NSCs, AdipoRon significantly promoted the Aβ-induced impaired cell proliferation through AdipoR1/AMPK/CREB pathway. Furthermore, inhibition of AMPK by compound C also reversed the promotive effects of AdipoRon on cognition and proliferation of NSCs of APP/PS1 Tg mice, suggesting a AMPK-dependent mechanism by AdipoRon in AD in vivo. Taken together, these results suggested that AdipoRon alleviated the cognitive dysfunction of AD mice, inhibited the Aβ deposition by inhibiting BACE1 expression and promoted the impaired hippocampal NSCs proliferation on the early stage in vivo. The mechanisms involved activation of AdipoR1/AMPK pathway. Therefore, AdipoRon might be a potential candidate for the treatment of AD on the early stage.

Decreased viability and neurite length in neural cells treated with chitosan-dextran sulfate nanocomplexes

CXCL12 is a chemokine known to regulate migration, proliferation, and differentiation of neural stem cells (NSCs) and to play a neuroprotective role in ischemic stroke. Chitosan-dextran sulfate nanocomplexes (Ch/DS NC) are known nanoparticulated systems used to efficiently deliver heparin-binding factors. Here we evaluate Ch/DS NC as carriers for CXCL12 in a mouse model of stroke. Free CXCL12 reduced the size of the ischemic brain lesion. However, when Ch/DS NC were administrated, the stroke volume increased. Neurotoxic screening revealed that Ch/DS NC reduced neuronal viability, decreased the extension of neurites and impaired NSC migration in vitro. To the best of our knowledge, neurotoxicity of Ch/DS NC has not been reported and further screenings will be needed in order to evaluate the biological safety of these nanocomposites. Our results add new data on nanoparticle neurotoxicity and may help us to better understand the complex interactions of the nanostructures with biological components.

Neural Stem Cell Fatty Acid Beta‐Oxidation and Autism Spectrum Disease

Inborn errors of metabolism occur with high incidence in human populations, and inborn deficiencies in fatty acid b‐oxidation (FAO) are clinically associated with neurological deficits such as speech weakness, motor delay, and autism. Using in vivo assays for analyzing neural stem cells (NSCs) in the developing mammalian forebrain, we find that neural stem cell (NSC)‐autonomous deficiencies in long‐chain FAO deplete neural stem cell (NSC) pools from mouse embryonic neocortex. Acute interference with activity of TMLHE (catalyzes the first step in carnitine biosynthesis), or with CPT1A (the rate‐limiting enzyme for carnitine‐dependent transport of long‐chain fatty acids into mitochondria), results in impaired NSC self‐renewal. Moreover, dorsal forebrain‐specific eviction of the Cpt1a gene resulted in reduced thickness of the murine neocortex at birth. Importantly, NSC deficits induced by TMLHE insufficiencies are rescued by exogenous carnitine in both cultured explants and by supplementation of the maternal diet. Taken together, the data argue for a direct role for FAO in controlling NSC self‐renewal vs differentiation in mammalian embryonic brain. The data further argue that deranged stem cell homeostasis is a significant mechanism for linking IEMs with human cognitive disorders such as autism. The implications of these findings for new strategies for management of autism risk will be discussed.Support or Funding InformationThis work was supported by the Robert A. Welch Foundation Award BE0017 and grant RO1GM112591 from the National Institute of HealthThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Methionine-Choline Deprivation Impairs Adult Hippocampal Neurogenesis in C57BL/6 Mice.

Methionine and choline, which are essential nutrients for mammalian animals, are important for cell composition, as metabolic factors, and for the synthesis of other biochemical compounds for cell metabolism. Methionine and choline, which are methyl group donors, play key roles in the homocysteine cycle and neuronal development and maintenance. In this study, we investigated the effects of methionine and choline deficiency on adult hippocampal neurogenesis and neural stem cell (NSC) lineage in the adult stage. For this study, we divided C57BL/6 mice into three groups as follows: normal chow (NC)-fed, methionine choline sufficient (MCS) diet-fed, and methionine choline deficient (MCD) diet-fed mice. The mice were fed the NC, MCS, and MCD diets for 4 weeks from the age of 8 weeks. MCD diet-fed mice showed significantly decreased proliferation and differentiation of NSCs when compared with the NC diet-fed or MCS diet-fed mice. In addition, the survival of newly generated neurons was critically impaired in the MCD diet-fed mice. We confirmed a decrease in the proliferation and differentiation of NSCs after 4 weeks of MCD diet administration, compared with that in NC- and MCS diet-fed mice. MCD diet critically impaired NSCs survival and survival of neurons during the 4 weeks. The number of phosphorylated cyclic AMP response element binding (pCREB) protein immunoreactive nuclei was decreased in the MCD diet-fed mice compared with that in the NC- or MCS diet-fed group. These results suggest that suitable levels of methionine and choline are essential for the maintenance of hippocampal neurogenesis in mice and affect NSC proliferation and differentiation through phosphorylation of CREB.

Amyloid β Peptide Compromises Neural Stem Cell Fate by Irreversibly Disturbing Mitochondrial Oxidative State and Blocking Mitochondrial Biogenesis and Dynamics.

Alzheimer's disease (AD) is the most common neurodegenerative disease and is characterized by the accumulation of amyloid β peptide (Aβ). Although most AD mouse models present a decline in neurogenesis, they express mutated genes which regulate neurogenesis per se and are not present in most AD patients, thus masking the real impact of Aβ on adult neurogenesis. Mitochondrion, a well-known target of Aβ in neurons, is a main regulator of neural stem cell (NSC) fate. Here, we aimed to investigate the impact of Aβ on NSC mitochondria and cell fate decisions, namely whether and how Aβ affects neurogenesis. NSC fate and mitochondrial parameters, including biogenesis, dynamics, and oxidative stress, were evaluated. Our results showed that Aβ impaired NSC viability and proliferation and indirectly blocked neurogenic differentiation, by disrupting mitochondrial signaling of self-renewing NSCs. Importantly, Aβ decreased ATP levels, generated oxidative stress, and affected the radical scavenger system through SOD2 and SIRT3. Aβ also reduced mtDNA and mitochondrial biogenesis proteins, such as Tfam, PGC-1α, and NRF1, and inhibited activation of PGC-1α-positive regulator CREB. Moreover, Aβ triggered mitochondrial fragmentation in self-renewing NSCs and reduced mitochondrial fusion proteins, such as Mfn2 and ERRα. Notably, Aβ compromised NSC commitment and survival by irreversibly impairing mitochondria and thwarting any neurogenic rescue through mitochondrial biogenesis, dynamics, or radical scavenger system. Altogether, this study brings new perspective to rethink the molecular targets relevant for endogenous NSC-based strategies in AD.

Impaired neural stem cell expansion and hypersensitivity to epileptic seizures in mice lacking the EGFR in the brain

Mice lacking the epidermal growth factor receptor (EGFR) develop an early postnatal degeneration of the frontal cortex and olfactory bulbs and show increased cortical astrocyte apoptosis. The poor health and early lethality of EGFR−/− mice prevented the analysis of mechanisms responsible for the neurodegeneration and function of the EGFR in the adult brain. Here, we show that postnatal EGFR‐deficient neural stem cells are impaired in their self‐renewal potential and lack clonal expansion capacity in vitro. Mice lacking the EGFR in the brain (EGFRΔbrain) show low penetrance of cortical degeneration compared to EGFR−/− mice despite genetic recombination of the conditional allele. Adult EGFRΔ mice establish a proper blood–brain barrier and perform reactive astrogliosis in response to mechanical and infectious brain injury, but are more sensitive to Kainic acid‐induced epileptic seizures. EGFR‐deficient cortical astrocytes, but not midbrain astrocytes, have reduced expression of glutamate transporters Glt1 and Glast, and show reduced glutamate uptake in vitro, illustrating an excitotoxic mechanism to explain the hypersensitivity to Kainic acid and region‐specific neurodegeneration observed in EGFR‐deficient brains.

Regulation of neural stem cell fate decisions by mitochondrial dynamics.

Regulation of neural stem cell fate decisions by mitochondrial dynamics.

Low-Grade Astrocytoma Mutations in IDH1, P53, and ATRX Cooperate to Block Differentiation of Human Neural Stem Cells via Repression of SOX2

Low-grade astrocytomas (LGAs) carry neomorphic mutations in isocitrate dehydrogenase (IDH) concurrently with P53 and ATRX loss. To model LGA formation, we introduced R132H IDH1, P53 shRNA, and ATRX shRNA into human neural stem cells (NSCs). These oncogenic hits blocked NSC differentiation, increased invasiveness invivo, and led to a DNA methylation and transcriptional profile resembling IDH1 mutant human LGAs. The differentiation block was caused by transcriptional silencing of the transcription factor SOX2 secondary to disassociation of its promoter from a putative enhancer. This occurred because of reduced binding of the chromatin organizer CTCF to its DNA motifs and disrupted chromatin looping. Our human model of IDH mutant LGA formation implicates impaired NSC differentiation because of repression of SOX2 as an early driver of gliomagenesis.

Pituitary Adenlylate Cyclase Activating Peptide Protects Adult Neural Stem Cells from a Hypoglycaemic milieu.

Hypoglycaemia is a common side-effect of glucose-lowering therapies for type-2 diabetic patients, which may cause cognitive/neurological impairment. Although the effects of hypoglycaemia in the brain have been extensively studied in neurons, how hypoglycaemia impacts the viability of adult neural stem cells (NSCs) has been poorly investigated. In addition, the cellular and molecular mechanisms of how hypoglycaemia regulates NSCs survival have not been characterized. Recent work others and us have shown that the pituitary adenylate cyclase-activating polypeptide (PACAP) and the glucagon-like peptide-1 receptor (GLP-1R) agonist Exendin-4 stimulate NSCs survival against glucolipoapoptosis. The aim of this study was to establish an in vitro system where to study the effects of hypoglycaemia on NSC survival. Furthermore, we determine the potential role of PACAP and Exendin-4 in counteracting the effect of hypoglycaemia. A hypoglycaemic in vitro milieu was mimicked by exposing subventricular zone-derived NSC to low levels of glucose. Moreover, we studied the potential involvement of apoptosis and endoplasmic reticulum stress by quantifying protein levels of Bcl-2, cleaved caspase-3 and mRNA levels of CHOP. We show that PACAP via PAC-1 receptor and PKA activation counteracts impaired NSC viability induced by hypoglycaemia. The protective effect induced by PACAP correlated with endoplasmic reticulum stress, Exendin-4 was ineffective. The results show that hypoglycaemia decreases NSC viability and that this effect can be substantially counteracted by PACAP via PAC-1 receptor activation. The data supports a potential therapeutic role of PAC-1 receptor agonists for the treatment of neurological complications, based on neurogenesis impairment by hypoglycaemia.

Mitochondrial Dynamics Impacts Stem Cell Identity and Fate Decisions by Regulating a Nuclear Transcriptional Program

Regulated mechanisms of stem cell maintenance are key to preventing stem cell depletion and aging. While mitochondrial morphology plays a fundamental role in tissue development and homeostasis, its role in stem cells remains unknown. Here, we uncover that mitochondrial dynamics regulates stem cell identity, self-renewal, and fate decisions by orchestrating a transcriptional program. Manipulation of mitochondrial structure, through OPA1 or MFN1/2 deletion, impaired neural stem cell (NSC) self-renewal, with consequent age-dependent depletion, neurogenesis defects, and cognitive impairments. Gene expression profiling revealed ectopic expression of the Notch self-renewal inhibitor Botch and premature induction of transcription factors that promote differentiation. Changes in mitochondrial dynamics regulate stem cell fate decisions by driving a physiological reactiveoxygen species (ROS)-mediated process, which triggers a dual program to suppress self-renewal and promote differentiation via NRF2-mediated retrograde signaling. These findings reveal mitochondrial dynamics as an upstream regulator of essential mechanisms governing stem cell self-renewal and fate decisions through transcriptional programming.

The RNA Binding Protein IMP2 Preserves Glioblastoma Stem Cells by Preventing let-7 Target Gene Silencing.

Cancer stem cells (CSCs) can drive tumor growth, and their maintenance may rely on post-transcriptional regulation of gene expression, including that mediated by microRNAs (miRNAs). The let-7 miRNA family has been shown to induce differentiation by silencing stem cell programs. Let-7-mediated target gene suppression is prevented by LIN28A/B, which reduce let-7 biogenesis in normal embryonic and some cancer stem cells and ensure maintenance ofstemness. Here, we find that glioblastoma stem cells (GSCs) lack LIN28 and express both let-7 and their target genes, suggesting LIN28-independent protection from let-7 silencing. Using photoactivatable-ribonucleoside-enhanced crosslinking and immunoprecipitation (PAR-CLIP), we show that insulin-like growth factor 2 mRNA-binding protein 2 (IMP2) binds to let-7 miRNA recognition elements (MREs) and prevents let-7 target gene silencing. Our observations define the RNA-binding repertoire of IMP2 and identify a mechanism whereby it supports GSC and neural stem cell specification.

Polo Kinase Phosphorylates Miro to Control ER-Mitochondria Contact Sites and Mitochondrial Ca2+ Homeostasis in Neural Stem Cell Development

Mitochondria play central roles in buffering intracellular Ca²⁺ transients. While basal mitochondrial Ca²⁺ (Ca²⁺ mito) is needed to maintain organellar physiology, Ca²⁺ mito overload can lead to cell death. How Ca²⁺ mito homeostasis is regulated is not well understood. Here we show that Miro, a known component of the mitochondrial transport machinery, regulates Drosophila neural stem cell (NSC) development through Ca²⁺ mito homeostasis control, independent of its role in mitochondrial transport. Miro interacts with Ca²⁺ transporters at the ER-mitochondria contact site (ERMCS). Its inactivation causes Ca²⁺ mito depletion and metabolic impairment, whereas its overexpression results in Ca²⁺ mito overload, mitochondrial morphology change, and apoptotic response. Both conditions impaired NSC lineage progression. Ca²⁺ mito homeostasis is influenced by Polo-mediated phosphorylation of a conserved residue in Miro, which positively regulates Miro localization to, and the integrity of, ERMCS. Our results elucidate a regulatory mechanism underlying Ca²⁺ mito homeostasis and how its dysregulation may affect NSC metabolism/development and contribute to disease.

Protein Palmitoylation Regulates Neural Stem Cell Differentiation by Modulation of EID1 Activity.

The functional significance of palmitoylation in the switch between self-renewal and differentiation of neural stem cells (NSCs) is not well defined, and the underlying mechanisms of protein palmitoylation are not well understood. Here, mouse NSCs were used as a model system and cell behavior was monitored in the presence of the protein palmitoylation inhibitor 2-bromopalmitate (2BRO). Our data show that 2BRO impaired the differentiation of NSCs into both neurons and glia and impaired NSC cell cycle exit. Moreover, the results show that palmitoylation modified E1A-like inhibitor of differentiation one (EID1) and this modification regulated EID1 degradation and CREB-binding protein (CBP)/p300 histone acetyltransferase activity at the switch between self-renewal and differentiation of NSCs. Our results extended the cellular role of palmitoylation, suggesting that it acts as a regulator in the acetylation-dependent gene expression network, and established the epigenetic regulatory function of palmitoylation in the switch between maintenance of multipotency and differentiation in NSCs.

Inhibitory Effects of Bisphenol-A on Neural Stem Cells Proliferation and Differentiation in the Rat Brain Are Dependent on Wnt/β-Catenin Pathway.

Neurogenesis, a process of generation of new neurons, occurs throughout the life in the hippocampus and sub-ventricular zone (SVZ). Bisphenol-A (BPA), an endocrine disrupter used as surface coating for packaged food cans, injures the developing and adult brain. However, the effects of BPA on neurogenesis and underlying cellular and molecular mechanism(s) are still unknown. Herein, we studied the effect(s) of prenatal and early postnatal exposure of low dose BPA on Wnt/β-catenin signaling pathway that controls different steps of neurogenesis such as neural stem cell (NSC) proliferation and neuronal differentiation. Pregnant rats were treated with 4, 40, and 400 μg BPA/kg body weight orally daily from gestational day 6 to postnatal day 21. Both in vivo and in vitro studies showed that BPA alters NSC proliferation and differentiation. BPA impaired NSC proliferation (5'-bromo-2'-deoxyuridine (BrdU(+)) and nestin(+) cells) and neuronal differentiation (BrdU/doublecortin(+) and BrdU/neuronal nuclei (NeuN(+)) cells) in the hippocampus and SVZ as compared to control. It significantly altered expression/protein levels of neurogenic genes and the Wnt pathway genes in the hippocampus. BPA reduced cellular β-catenin and p-GSK-3β levels and decreased β-catenin nuclear translocation, and cyclin-D1 and TCF/LEF promoter luciferase activity. Specific activation and blockage of the Wnt pathway suggested involvement of this pathway in BPA-mediated inhibition of neurogenesis. Further, blockage of GSK-3β activity by SB415286 and GSK-3β small interfering RNA (siRNA) attenuated BPA-induced downregulation of neurogenesis. Overall, these results suggest significant inhibitory effects of BPA on NSC proliferation and differentiation in the rat via the Wnt/β-catenin signaling pathway.

Retinoic Acid-Dependent Signaling Pathways and Lineage Events in the Developing Mouse Spinal Cord

Studies in avian models have demonstrated an involvement of retinoid signaling in early neural tube patterning. The roles of this signaling pathway at later stages of spinal cord development are only partly characterized. Here we use Raldh2-null mouse mutants rescued from early embryonic lethality to study the consequences of lack of endogenous retinoic acid (RA) in the differentiating spinal cord. Mid-gestation RA deficiency produces prominent structural and molecular deficiencies in dorsal regions of the spinal cord. While targets of Wnt signaling in the dorsal neuronal lineage are unaltered, reductions in Fibroblast Growth Factor (FGF) and Notch signaling are clearly observed. We further provide evidence that endogenous RA is capable of driving stem cell differentiation. Raldh2 deficiency results in a decreased number of spinal cord derived neurospheres, which exhibit a reduced differentiation potential. Raldh2-null neurospheres have a decreased number of cells expressing the neuronal marker β-III-tubulin, while the nestin-positive cell population is increased. Hence, in vivo retinoid deficiency impaired neural stem cell growth. We propose that RA has separable functions in the developing spinal cord to (i) maintain high levels of FGF and Notch signaling and (ii) drive stem cell differentiation, thus restricting both the numbers and the pluripotent character of neural stem cells.

The Effect of Ethanol on Cell Fate Determination of Neural Stem Cells

Recent studies have described the possible relevance of impaired neural stem cell (NSC) functions to the pathophysiology of psychiatric disorders, including alcoholism. However, relatively little is known about ethanol's effects on the determination of cell fate in NSCs. In this study, we investigated the effect of ethanol on neuronal and glial differentiation of NSCs. Under neuron-inductive culture conditions, NSCs were induced to differentiate and exposed to ethanol for 96 hr. Immunocytochemistry with cell-type-specific markers was performed (microtubule-associated protein 2 (MAP2) for neurons, glial fibrillary acidic protein (GFAP) for astrocytes and O4 for oligodendrocytes). The cells positive to MAP2, GFAP or O4 were counted, and the number of MAP2-positive cells was quantified by enzyme-linked immunosorbent assay (ELISA) following immunostaining with anti-MAP2 (MAP2-ELISA). The alteration of MAP2, GFAP or myelin basic protein (MBP, a marker for oligodendrocytes) expression was evaluated by Western blot analysis. Ethanol exposure increased astrocytic and oligodendrocytic differentiation with a statistically significant difference at 100 mM, while 25 to 100 mM ethanol reduced neuronal differentiation without affecting the viability of NSCs. The enhanced expression of glial markers was revealed by Western blot analysis for GFAP or MBP. Glial cells are known to increase in response to various kinds of insults to the central nervous system. It is possible that the increase of astrocytes and oligodendrocytes after ethanol exposure is a compensatory mechanism to repair the impaired neural network by promoting neurite outgrowth and increasing newly generated neurons.