Embryonic Neural Stem Cells Research Articles

Quercetin-3-O-glucuronide promotes the proliferation and migration of neural stem cells

Quercetin is a bioactive compound exerting therapeutic effects on in vivo animal models of neurodegeneration or neurotoxicity. However, the narrow therapeutic dose-range of quercetin has been a point of concern since previous studies have demonstrated that quercetin induces cytotoxicity in vitro. Quercetin is metabolized to quercetin glucuronates such as quercetin-3-O-glucuronide (Q3GA), primarily detected in the plasma and the brain. Here, we examined whether and how quercetin or Q3GA regulates neural stem cells (NSCs) in vivo and in vitro. Immunohistochemistry showed that oral administration of quercetin increased nestin-, DCX-, BrdU/DCX-, and BrdU/NeuN-positive cells in the dentate gyrus of mice. However, quercetin decreased the viability of human embryonic NSCs in culture, accompanied by decreased Akt phosphorylation and increased cleavage of caspase-3 and PARP. In contrast, Q3GA increased BrdU-positive cell proliferation, Akt phosphorylation, and cyclin D1 expression. PI3K/Akt inhibitor LY294002 reversed Q3GA-induced Akt phosphorylation and cyclin D1 expression, thereby reducing Q3GA-induced proliferation. Furthermore, Q3GA increased the protein secretion of BDNF and its blockade using anti-BDNF antibody reversed Q3GA-induced proliferation. Under differentiation state, Q3GA promotes NSC migration, along with increased mRNA expression of CXCR4. Moreover, Q3GA significantly reversed scopolamine-induced reduction of Akt phosphorylation in the mouse hippocampus and ameliorated scopolamine-induced memory impairments. Our results demonstrate that quercetin and its metabolite Q3GA control NSC viability in a converse manner through contrary regulation of Akt, accounting for the conflicting effects of quercetin in vivo and in vitro. This study provides a novel mechanism for the positive effects of Q3GA on neurogenesis and suggests its therapeutic potential in neurodegenerative diseases.

EphB4 Regulates Self-Renewal, Proliferation and Neuronal Differentiation of Human Embryonic Neural Stem Cells in Vitro

Background/Aims: EphB4 belongs to the largest family of Eph receptor tyrosine kinases. It contributes to a variety of pathological progresses of cancer malignancy. However, little is known about its role in neural stem cells (NSCs). This study examined whether EphB4 is required for proliferation and differentiation of human embryonic neural stem cells (hNSCs) in vitro. Methods: We up- and down-regulated EphB4 expression in hNSCs using lentiviral over-expression and shRNA knockdown constructs and then investigated the influence of EphB4 on the properties of hNSCs. Results: Our results show that shRNA-mediated EphB4 reduction profoundly impaired hNSCs self-renewal and proliferation. Furthermore, detection of differentiation revealed that knockdown of EphB4 inhibited hNSCs differentiation towards a neuronal lineage and promoted hNSCs differentiation to glial cells. In contrast, EphB4 overexpression promoted hNSCs self-renewal and proliferation, further induced hNSCs differentiation towards a neuronal lineage and inhibited hNSCs differentiation to glial cells. Moreover, we found that EphB4 regulates cell proliferation mediated by the Abl-CyclinD1 pathway. Conclusion: These studies provide strong evidence that fine tuning of EphB4 expression is crucial for the proliferation and neuronal differentiation of hNSCs, suggesting that EphB4 might be an interesting target for overcoming some of the therapeutic limitations of neuronal loss in brain diseases.

TRBP maintains mammalian embryonic neural stem cell properties by acting as a novel transcriptional coactivator of the Notch signaling pathway.

Transactivation response element RNA-binding protein (TRBP; TARBP2) is known to play important roles in human immunodeficiency virus (HIV) replication and microRNA biogenesis. However, recent studies implicate TRBP in a variety of biological processes as a mediator of cross-talk between signal transduction pathways. Here, we provide the first evidence that TRBP is required for efficient neurosphere formation and for the expression of neural stem cell markers and Notch target genes in primary neural progenitor cells in vitro Consistent with this, introduction of TRBP into the mouse embryonic brain in utero increased the fraction of cells expressing Sox2 in the ventricular zone. We also show that TRBP physically interacts with the Notch transcriptional coactivation complex through C promoter-binding factor 1 (CBF1; RBPJ) and strengthens the association between the Notch intracellular domain (NICD) and CBF1, resulting in increased NICD recruitment to the promoter region of a Notch target gene. Our data indicate that TRBP is a novel transcriptional coactivator of the Notch signaling pathway, playing an important role in neural stem cell regulation during mammalian brain development.

In vitro models reveal differences in the developmental neurotoxicity of an environmental polycylic aromatic hydrocarbon mixture compared to benzo[a]pyrene: Neuronotypic PC12 Cells and embryonic neural stem cells.

In addition to their carcinogenic activity, polycyclic aromatic hydrocarbons (PAHs) are suspected to be developmental neurotoxicants. We evaluated the effects of PAHs with two in vitro models that assess distinct "decision nodes" in neurodifferentiation: neuronotypic PC12 cells, which characterize the transition from cell replication to neurodifferentiation, neurite outgrowth and neurotransmitter specification; and embryonic neural stem cells (NSCs), which evaluate the origination of neurons and glia from precursors. We compared an environmentally-derived PAH mixture from a Superfund contamination site (Elizabeth River Sediment Extract, ERSE) to those of a single PAH, benzo[a]pyrene (BaP). In PC12 cells, BaP impaired the transition from cell replication to neurodifferentiation, resulting in higher numbers of cells, but with reduced cell size and deficits in all indices of neuronal features (neurite formation, development of dopamine and acetylcholine phenotypes). ERSE was far less effective, causing only modest changes in cell numbers and size and no impairment of neurite formation or neurotransmitter specification; in fact, ERSE evoked a slight increase in emergence of the acetylcholine phenotype. In the NSC model, this relationship was entirely reversed, with far greater sensitivity to ERSE than to BaP. Furthermore, ERSE, but not BaP, enhanced NSC differentiation into neurons, whereas both ERSE and BaP suppressed the glial phenotype. Our studies provide a cause-and-effect relationship for the observed association of developmental PAH exposure to behavioral deficits. Further, PAH sensitivity occurs over developmental stages corresponding to rudimentary brain formation through terminal neurodifferentiation, suggesting that vulnerability likely extends throughout fetal brain development and into early childhood.

Skin-derived precursors possess the ability of differentiation into the epidermal progeny and accelerate burn wound healing.

Skin-derived precursors (SKPs) are remnants of the embryonic neural crest stem cells that reside in the dermis until adulthood. Although they possess a wide range of differentiation potentials, their differentiation into keratinocyte-like cells and their roles in skin wound healing are obscure. The present study aimed to investigate the differentiation of SKPs into keratinocyte-like cells and evaluate their role in healing of third degree burn wounds. To this aim, SKPs were differentiated into keratinocyte-like cells on tissue culture plate and collagen-chitosan scaffold prepared by freeze-drying. Their differentiation capability was detected by real-time RT-PCR and immunofluorescence. Thereafter, they were cultured on scaffold and implanted in a rat model of burn wound. Histopathological and immunohistochemical analyses were employed to examine the reconstituted skin. The research findings revealed that SKPs were able to differentiate along the epidermal lineage and this ability can be enhanced on a suitable scaffold. Additionally, the results indicated that SKPs apparently promoted wound healing process and accelerate its transition from proliferating stage to maturational phase, especially if they were differentiated into keratinocyte-like cells. Regarding the results, it is concluded that SKPs are able to differentiate into keratinocyte-like cells, particularly when they are cultured on collagen-chitosan scaffold. Moreover, they can regenerate epidermal and dermal layers including thick collagen bundles, possibly through differentiation into keratinocyte-like cells.

Enhancement of Neural Stem Cell Survival, Proliferation, Migration, and Differentiation in a Novel Self-Assembly Peptide Nanofibber Scaffold.

Considerable efforts have been made to combine biologically active molecules into the self-assembling peptide in order to improve cells growth, survival, and differentiation. In this study, a novel three-dimensional scaffold (RADA4GGSIKVAV; R-GSIK) was designed by adding glycine and serine between RADA4 and IKVAV to promote the strength of the peptide. The cell adhesion, viability, proliferation, migration, and differentiation of rat embryonic neural stem cells (NSCs) in R-GSIK were investigated and compared to laminin-coated, two-dimensional, and Puramatrix cultures. The scanning electron microscopy studies of the R-GSIK showed an open porous structure and a suitable surface area available for cell interaction. R-GSIK promoted the cell adhesion, viability, proliferation, and migration compared to the other cultures. In addition, the R-GSIK enhanced NSCs differentiation into neuronal cells. The NSCs injected in R-GSIK had a lower glial differentiation rate than in the Puramatrix. The results suggest that R-GSIK holds great promise for cell therapies and neuronal tissue repair.

Sex-Specific Effects of Testosterone on the Sexually Dimorphic Transcriptome and Epigenome of Embryonic Neural Stem/Progenitor Cells

The mechanisms by which sex differences in the mammalian brain arise are poorly understood, but are influenced by a combination of underlying genetic differences and gonadal hormone exposure. Using a mouse embryonic neural stem cell (eNSC) model to understand early events contributing to sexually dimorphic brain development, we identified novel interactions between chromosomal sex and hormonal exposure that are instrumental to early brain sex differences. RNA-sequencing identified 103 transcripts that were differentially expressed between XX and XY eNSCs at baseline (FDR = 0.10). Treatment with testosterone-propionate (TP) reveals sex-specific gene expression changes, causing 2854 and 792 transcripts to become differentially expressed on XX and XY genetic backgrounds respectively. Within the TP responsive transcripts, there was enrichment for genes which function as epigenetic regulators that affect both histone modifications and DNA methylation patterning. We observed that TP caused a global decrease in 5-methylcytosine abundance in both sexes, a transmissible effect that was maintained in cellular progeny. Additionally, we determined that TP was associated with residue-specific alterations in acetylation of histone tails. These findings highlight an unknown component of androgen action on cells within the developmental CNS, and contribute to a novel mechanism of action by which early hormonal organization is initiated and maintained.

Diverse neurotoxicants target the differentiation of embryonic neural stem cells into neuronal and glial phenotypes.

The large number of compounds that needs to be tested for developmental neurotoxicity drives the need to establish in vitro models to evaluate specific neurotoxic endpoints. We used neural stem cells derived from rat neuroepithelium on embryonic day 14 to evaluate the impact of diverse toxicants on their ability to differentiate into glia and neurons: a glucocorticoid (dexamethasone), organophosphate insecticides (chlorpyrifos, diazinon, parathion), insecticides targeting the GABAA receptor (dieldrin, fipronil), heavy metals (Ni2+, Ag+), nicotine and tobacco smoke extract. We found three broad groupings of effects. One diverse set of compounds, dexamethasone, the organophosphate pesticides, Ni2+ and nicotine, suppressed expression of the glial phenotype while having little or no effect on the neuronal phenotype. The second pattern was restricted to the pesticides acting on GABAA receptors. These compounds promoted the glial phenotype and suppressed the neuronal phenotype. Notably, the actions of compounds eliciting either of these differentiation patterns were clearly unrelated to deficits in cell numbers: dexamethasone, dieldrin and fipronil all reduced cell numbers, whereas organophosphates and Ni2+ had no effect. The third pattern, shared by Ag+ and tobacco smoke extract, clearly delineated cytotoxicity, characterized by major cell loss with suppression of differentiation into both glial and neuronal phenotypes; but here again, there was some selectivity in that glia were suppressed more than neurons. Our results, from this survey with diverse compounds, point to convergence of neurotoxicant effects on a specific "decision node" that controls the emergence of neurons and glia from neural stem cells.

Neurotrophic Factor-α1: A Key Wnt-β-Catenin Dependent Anti-Proliferation Factor and ERK-Sox9 Activated Inducer of Embryonic Neural Stem Cell Differentiation to Astrocytes in Neurodevelopment.

Embryonic neurodevelopment involves inhibition of proliferation of multipotent neural stem cells (NSCs) followed by differentiation into neurons, astrocytes and oligodendrocytes to form the brain. We have identified a new neurotrophic factor, NF-α1, which inhibits proliferation and promotes differentiation of NSC/progenitors derived from E13.5 mouse cortex. Inhibition of proliferation of these cells was mediated through negatively regulating the Wnt pathway and decreasing β-catenin. NF-α1 induced differentiation of NSCs to astrocytes by enhancing Glial Fibrillary Acidic Protein (GFAP) expression through activating the ERK1/2-Sox9 signaling pathway. Cultured E13.5 cortical stem cells from NF-α1-knockout mice showed decreased astrocyte numbers compared to wild-type mice, which was rescued by treatment with NF-α1. In vivo, immunocytochemistry of brain sections and Western blot analysis of neocortex of mice showed a gradual increase of NF-α1 expression from E14.5 to P1 and a surge of GFAP expression at P1, the time of increase in astrogenesis. Importantly, NF-α1-Knockout mice showed ∼49% fewer GFAP positive astrocytes in the neocortex compared to WT mice at P1. Thus, NF-α1 is critical for regulating antiproliferation and cell fate determination, through differentiating embryonic stem cells to GFAP-positive astrocytes for normal neurodevelopment. Stem Cells 2017;35:557-571.

Accelerated generation of oligodendrocyte progenitor cells from human induced pluripotent stem cells by forced expression of Sox10 and Olig2.

Oligodendrocyte progenitor cells (OPCs) hold great promise for treatment of dysmyelinating disorders, such as multiple sclerosis and cerebral palsy. Recent studies on generation of human OPCs mainly use human embryonic stem cells (hESCs) or neural stem cells (NSCs) as starter cell sources for the differentiation process. However, NSCs are restricted in availability and the present method for generation of oligodendrocytes (OLs) from ESCs often requires a lengthy period of time. Here, we demonstrated a protocol to efficiently derive OPCs from human induced pluripotent stem cells (hiPSCs) by forced expression of two transcription factors (2TFs), Sox10 and Olig2. With this method, PDGFRα+ OPCs can be obtained in 14 days and O4+ OPCs in 56 days. Furthermore, OPCs may be able to differentiate to mature OLs that could ensheath axons when co-cultured with rat cortical neurons. The results have implications in the development of autologous cell therapies.

The Synergistic Effect of Cationic Moieties and GRGDSF-Peptides in Hydrogels on Neural Stem Cell Behavior.

This article reports the behavior of embryonic neural stem cells on a hydrogel that combines cationic, non-specific cell adhesion motifs with glycine-arginine-glycine-aspartic acid-serine-phenylalanine (GRGDSF)-peptides as specific cell adhesion moieties. Therefore, three hydrogels are prepared by free radical polymerization that contains either a GRGDSF-peptide residue (P1), amino ethylmethacrylate as a cationic residue (P2), or a combination of both motifs (P3). For each gel, cross linker concentrations of 8 mol% is used to have a comparable gel stiffness of 8-9 kPa. The cell experiments indicate a synergistic effect of the non-specific, cationic residues, and the specific GRGDSF-peptides on embryonic neural stem cell behavior that is especially pronounced in the cell adhesion experiments by more than doubling the number of cells after 72 h when comparing P3 with P2 and is less pronounced in the proliferation and differentiation experiments.

Donepezil promotes differentiation of neural stem cells into mature oligodendrocytes at the expense of astrogenesis.

Oligodendrocytes are the myelin-forming cells of the central nervous system. Oligodendrocyte loss and failure of myelin development result in serious human disorders, including multiple sclerosis. Previously, using oligodendrocyte progenitor cells, we have shown that donepezil, which is an acetylcholinesterase inhibitor developed for the treatment of Alzheimer's disease, stimulates myelin gene expression and oligodendrocyte differentiation. Here, we aimed to analyze the effects of donepezil on primary mouse embryonic neural stem cells (NSCs). Donepezil treatment led to impaired self-renewal ability and increased apoptosis. These effects appeared to be mediated through the Akt/Bad signaling pathway. Using neurosphere differentiation analysis, we observed that donepezil leads to reduced numbers of astrocytes and increased numbers of oligodendrocytes and neurons. Consistent with this finding, mRNA and protein levels for the oligodendrocyte markers myelin-associated glycoprotein, 2', 3'-cyclic-nucleotide 3'-phosphodiesterase (CNPase), and myelin basic protein, as well as the neuronal marker β-tubulin type III (Tuj1) were up-regulated. In contrast, the expression of the astrocyte marker glial fibrillary acidic protein (GFAP) was down-regulated by donepezil in a dose- and time-dependent manner. Moreover, donepezil increased oligodendrocyte differentiation, resulting in a reduction in the differentiation of NSCs into astrocytes, by suppressing the activation of signal transducer and activator of transcription 3 (STAT3), SMAD1/5/9, and the downstream target gene GFAP, even under astrocyte-inducing conditions. These results suggest that efficient differentiation of NSCs into oligodendrocytes by donepezil may indicate a novel therapeutic role for this drug in promoting repair in demyelinated lesions in addition to its role in preventing astrogenesis.

Prenatal arsenic exposure alters REST/NRSF and microRNA regulators of embryonic neural stem cell fate in a sex-dependent manner

Exposure to arsenic, a common environmental toxin found in drinking water, leads to a host of neurological pathologies. We have previously demonstrated that developmental exposure to a low level of arsenic (50ppb) alters epigenetic processes that underlie deficits in adult hippocampal neurogenesis leading to aberrant behavior. It is unclear if arsenic impacts the programming and regulation of embryonic neurogenesis during development when exposure occurs. The master negative regulator of neural-lineage, REST/NRSF, controls the precise timing of fate specification and differentiation of neural stem cells (NSCs). Early in development (embryonic day 14), we observed increased expression of Rest, its co-repressor, CoREST, and the inhibitory RNA binding/splicing protein, Ptbp1, and altered expression of mRNA spliced isoforms of Pbx1 that are directly regulated by these factors in the male brain in response to prenatal 50ppb arsenic exposure. These increases were concurrent with decreased expression of microRNA-9 (miR-9), miR-9*, and miR-124, all of which are REST/NRSF targets and inversely regulate Rest expression to allow for maturation of NSCs. Exposure to arsenic decreased the formation of neuroblasts in vitro from NSCs derived from male pup brains. The female response to arsenic was limited to increased expression of CoREST and Ptbp2, an RNA binding protein that allows for appropriate splicing of genes involved in the progression of neurogenesis. These changes were accompanied by increased neuroblast formation in vitro from NSCs derived from female pups. Unexposed male mice express transcriptomic factors to induce differentiation earlier in development compared to unexposed females. Thus, arsenic exposure likely delays differentiation of NSCs in males while potentially inducing precocious differentiation in females early in development. These effects are mitigated by embryonic day 18 of development. Arsenic-induced dysregulation of the regulatory loop formed by REST/NRSF, its target microRNAs, miR-9 and miR-124, and RNA splicing proteins, PTBP1 and 2, leads to aberrant programming of NSC function that is perhaps perpetuated into adulthood inducing deficits in differentiation we have previously observed.

Effect of propofol on microRNA expression in rat primary embryonic neural stem cells.

BackgroundPropofol is a widely used intravenous anesthetic that is well-known for its protective effect in various human and animal disease models. However, the effects of propofol on neurogenesis, especially on the development of neural stem cells (NSCs), remains unknown. Related microRNAs may act as important regulators in this process.MethodsPublished Gene Expression Omnibus (GEO) DataSets related to propofol were selected and re-analyzed to screen neural development-related genes and predict microRNA (miRNA) expression using bioinformatic methods. Screening of the genes and miRNAs was then validated by qRT-PCR analysis of propofol-treated primary embryonic NSCs.ResultsFour differentially expressed mRNAs were identified in the screen and 19 miRNAs were predicted based on a published GEO DataSet. Two of four mRNAs and four of 19 predicted miRNAs were validated by qRT-PCR analysis of propofol-treated NSCs. Rno-miR-19a (Rno, Rattus Norvegicus) and rno-miR-137, and their target gene EGR2, as well as rno-miR-19b-2 and rno-miR-214 and their target gene ARC were found to be closely related to neural developmental processes, including proliferation, differentiation, and maturation of NSCs.ConclusionPropofol influences miRNA expression; however, further studies are required to elucidate the mechanism underlying the effects of propofol on the four miRNAs and their target genes identified in this study. In particular, the influence of propofol on the entire development process of NSCs remains to be clarified.Electronic supplementary materialThe online version of this article (doi:10.1186/s12871-016-0259-1) contains supplementary material, which is available to authorized users.

Identification of Noncanonical Wnt Receptors Required for Wnt-3a-Induced Early Differentiation of Human Neural Stem Cells.

Wnt proteins preferentially activate either β-catenin-dependent or β-catenin-independent signals, but the activity of a particular Wnt also depends on cellular context and receptor availability. We previously reported that Wnt-3a induces neural differentiation of human embryonic stem cell-derived neural stem cells (NSCs) in a β-catenin-independent manner by activating a signal involving JNK and the AP-1 family member ATF-2. Here, we report the results of a gene silencing approach to identify the Wnt receptors that mediate this response to Wnt-3a. Silencing of ROR2 increased neuronal differentiation, as measured by expression of the genes DCX, NEUROD1, and NGN1, suggesting ROR2 signals normally prevent differentiation. Silencing of the other Wnt receptors singly did not affect Wnt-3a-induced neuronal differentiation. However, pairwise silencing of ROR1 and FZD4 or FZD5 and of LRP6 and FZD4 or FZD5 inhibited neuronal differentiation, as detected by reductions in the expression of neuronal genes and immunocytochemical detection of DCX,NEUROD1 and DCX. Ectopic expression of these receptors in HEK 293 cells increased ATF2-dependent transcription. In addition, ROR1 coimmunoprecipitated with FZD4 and LRP6 in transfected HEK 293 cells and colocalized with FZD4 and with LRP6 at the cell surface of transfected L cells. Wnt-3a did not appear to affect these interactions but did alter the interactions between LRP6 and FZD4/5. Together, these observations highlight roles for ROR1, LRP6, FZD4, and FZD5 in neural stem cell differentiation and provide support for a model in which dynamic interactions among these receptors mediate Wnt-3a activation of ATF2 signaling.

A Glo1-Methylglyoxal Pathway that Is Perturbed in Maternal Diabetes Regulates Embryonic and Adult Neural Stem Cell Pools in Murine Offspring

Maternal diabetes is known to adversely influence brain development in offspring. Here, we provide evidence that this involves the circulating metabolite methylglyoxal, which is increased in diabetes, and its detoxifying enzyme, glyoxalase 1 (Glo1), which when mutated is associated with neurodevelopmental disorders. Specifically, when Glo1 levels were decreased in embryonic mouse cortical neural precursor cells (NPCs), this led to premature neurogenesis and NPC depletion embryonically and long-term alterations in cortical neurons postnatally. Increased circulating maternal methylglyoxal caused similar changes in embryonic cortical precursors andneurons and long-lasting changes in cortical neurons and NPCs in adult offspring. Depletion of embryonic and adult NPCs was also observed in murine offspring exposed to a maternal diabetic environment. Thus, the Glo1-methylglyoxal pathway integrates maternal and NPC metabolism to regulateneural development, and perturbations in this pathway lead to long-lasting alterations in adult neurons and NPC pools.

Regulation of adult neural progenitor cell functions by purinergic signaling.

Extracellular purines are signaling molecules in the neurogenic niches of the brain and spinal cord, where they activate cell surface purinoceptors at embryonic neural stem cells (NSCs) and adult neural progenitor cells (NPCs). Although mRNA and protein are expressed at NSCs/NPCs for almost all subtypes of the nucleotide-sensitive P2X/P2Y, and the nucleoside-sensitive adenosine receptors, only a few of those have acquired functional significance. ATP is sequentially degraded by ecto-nucleotidases to ADP, AMP, and adenosine with agonistic properties for distinct receptor-classes. Nucleotides/nucleosides facilitate or inhibit NSC/NPC proliferation, migration and differentiation. The most ubiquitous effect of all agonists (especially of ATP and ADP) appears to be the facilitation of cell proliferation, usually through P2Y1Rs and sometimes through P2X7Rs. However, usually P2X7R activation causes necrosis/apoptosis of NPCs. Differentiation can be initiated by P2Y2R-activation or P2X7R-blockade. A key element in the transduction mechanism of either receptor is the increase of the intracellular free Ca2+ concentration, which may arise due to its release from intracellular storage sites (G protein-coupling; P2Y) or due to its passage through the receptor-channel itself from the extracellular space (ATP-gated ion channel; P2X). Further research is needed to clarify how purinergic signaling controls NSC/NPC fate and how the balance between the quiescent and activated states is established with fine and dynamic regulation. GLIA 2017;65:213-230.

The sphingosine-1-phosphate analogue, FTY-720, promotes the proliferation of embryonic neural stem cells, enhances hippocampal neurogenesis and learning and memory abilities in adult mice.

Fingolimod (FTY-720) is the first oral therapeutic drug approved for the relapsing-remitting forms of multiple sclerosis. Neural stem cells (NSCs) are capable of continuous self-renewal and differentiation. The dentate gyrus of the hippocampus in the adult mammalian brain contains a population of NSCs and is one of the regions where neurogenesis takes place. FTY-720 has been shown to have neuroprotective effects in several model systems, so we investigated the direct effects of FTY-720 on NSCs and adult neurogenesis. We assessed the effects of FTY-720 on the proliferation and differentiation of cultured embryonic hippocampal NSCs using the 5-bromo-2-deoxyuridine incorporation assay, the neurosphere formation assay and western blot analysis. Receptor selective agonists and antagonists were used to identify the mechanisms involved. Neurogenesis in the hippocampus of C57BL/6 mice was also assessed by immunohistochemistry. The Morris water maze and fear conditioning tests were used to detect the learning and memory abilities of mice. FTY-720 promoted the proliferation of embryonic hippocampal NSCs probably via the activation of ERK signalling, Gi/o proteins and S1P1 receptors. However, FTY-720 did not affect the differentiation of cultured hippocampal NSCs. In vivo, chronic treatment with FTY-720 promoted hippocampal neurogenesis in adult C57BL/6 mice and enhanced their learning and memory abilities. Our results suggest a new target for the activation of NSCs and provide an insight into the therapeutic effects of FTY-720 in neuropsychiatric disorders, neurodegenerative diseases and age-related cognitive decline where hippocampal neurogenesis is compromised.

627 - Oxidized products of α-linolenic acid negatively affect cell survival and motility of breast cancer cells

During neural development, embryonic neural stem cells (eNSCs) differentiate toward glial, oligodendrocytic, and neuronal cells. Dysregulation of polyunsaturated fatty acids (PUFAs) induce a wide range of neurological and developmental disorders. In this study, we investigated the effect of various concentrations and ratios of linoleic acid (LA) and alpha linolenic acid (ALA), which belong respectively to omega-6 and omega-3 PUFAs, on the proliferation and differentiation of eNSCs.Results showed that low (25 and 50 μM) or high (100 and 200 μM) concentrations of ALA, but not LA, and the ratio of 1:3 of LA/ALA significantly increased neurospheres size, frequency and cell numbers, in comparison to controls. Moreover, low or high concentrations of ALA, but not LA, and different ratios of LA/ALA resulted in a significant increase in mRNA expression levels of Notch1, Hes1 and Ki-67, and the differentiation of eNSCs toward astrocytes (GFAP) and oligodendrocytes (MBP), but not neurons (β-III Tubulin), with the highest increase being for LA/ALA ratio of 1:3, in comparison to controls. These results demonstrate the importance of higher concentrations of ALA in enhancing proliferation and differentiation of eNSCs, which could be used in diet to help preventing neurodevelopmental syndromes, cognitive decline during aging, and various psychiatric disorders.

Long noncoding RNA BDNF-AS regulates ketamine-induced neurotoxicity in neural stem cell derived neurons.

Ketamine is an anesthetic commonly used in both humans and animals. Emerging evidence has demonstrated that ketamine may induce neurotoxicity in neural stem cell-derived neurons. In this work, we investigated whether long noncoding RNA (lncRNA) Brain derived neurotrophic factor antisense (BDNF-AS) was involved in ketamine-induced neurotoxicity in differentiation of mouse embryonic neural stem cells. Mouse embryonic neural stem cells were differentiated in vitro, and treated with ketamine. The corresponding change in gene expression levels of BDNF and BDNF-AS were assessed by qRT-PCR. BDNF-AS was subsequently downregulated by siRNA. And its effect on protecting neuronal apoptosis, promoting neurite regrowth, and activating TrkB signaling pathways were assessed by TUNEL assay, neurite outgrowth assay, and western blot assay, respectively. In ketamine-injured mouse embryonic neural stem cell-derived neurons, BDNF was downregulated, whereas BDNF-AS was upregulated in dose-dependent manner. SiRNA-mediated BDNF-AS downregulation ameliorated neuronal apoptosis, induced neurite outgrowth, and phosphorylated TrkB signaling pathway after ketamine-induce neurotoxicity in mouse embryonic neural stem cell-derived neurons. Inhibition of BDNF-AS is a novel method to protect ketamine-induced neurotoxicity in mouse embryonic neural stem cell-derived neurons, very likely through the activation of TrkB signaling pathway.