BCL7A-containing SWI/SNF/BAF complexes modulate mitochondrial bioenergetics during neural progenitor differentiation.

Mammalian SWI/SNF/BAF chromatin remodeling complexes influence cell lineage determination. While the contribution of these complexes to neural progenitor cell (NPC) proliferation and differentiation has been reported, little is known about the transcriptional profiles that determine neurogenesis or gliogenesis. Here, we report that BCL7A is a modulator of the SWI/SNF/BAF complex that stimulates the genome-wide occupancy of the ATPase subunit BRG1. We demonstrate that BCL7A is dispensable for SWI/SNF/BAF complex integrity, whereas it is essential to regulate Notch/Wnt pathway signaling and mitochondrial bioenergetics in differentiating NPCs. Pharmacological stimulation of Wnt signaling restores mitochondrial respiration and attenuates the defective neurogenic patterns observed in NPCs lacking BCL7A. Consistently, treatment with an enhancer of mitochondrial biogenesis, pioglitazone, partially restores mitochondrial respiration and stimulates neuronal differentiation of BCL7A-deficient NPCs. Using conditional BCL7A knockout mice, we reveal that BCL7A expression in NPCs and postmitotic neurons is required for neuronal plasticity and supports behavioral and cognitive performance. Together, our findings define the specific contribution of BCL7A-containing SWI/SNF/BAF complexes to mitochondria-driven NPC commitment, thereby providing a better understanding of the cell-intrinsic transcriptional processes that connect metabolism, neuronal morphogenesis, and cognitive flexibility.

Mathematical modeling of therapeutic neural stem cell migration in mouse brain with and without brain tumors.

Neural stem cells (NSCs) offer a potential solution to treating brain tumors. This is because NSCs can circumvent the blood-brain barrier and migrate to areas of damage in the central nervous system, including tumors, stroke, and wound injuries. However, for successful clinical application of NSC treatment, a sufficient number of viable cells must reach the diseased or damaged area(s) in the brain, and evidence suggests that it may be affected by the paths the NSCs take through the brain, as well as the locations of tumors. To study the NSC migration in brain, we develop a mathematical model of therapeutic NSC migration towards brain tumor, that provides a low cost platform to investigate NSC treatment efficacy. Our model is an extension of the model developed in Rockne et al. (PLoS ONE 13, e0199967, 2018) that considers NSC migration in non-tumor bearing naive mouse brain. Here we modify the model in Rockne et al. in three ways: (i) we consider three-dimensional mouse brain geometry, (ii) we add chemotaxis to model the tumor-tropic nature of NSCs into tumor sites, and (iii) we model stochasticity of migration speed and chemosensitivity. The proposed model is used to study migration patterns of NSCs to sites of tumors for different injection strategies, in particular, intranasal and intracerebral delivery. We observe that intracerebral injection results in more NSCs arriving at the tumor site(s), but the relative fraction of NSCs depends on the location of injection relative to the target site(s). On the other hand, intranasal injection results in fewer NSCs at the tumor site, but yields a more even distribution of NSCs within and around the target tumor site(s).

Single-cell analysis of early chick hypothalamic development reveals that hypothalamic cells are induced from prethalamic-like progenitors.

The hypothalamus regulates many innate behaviors, but its development remains poorly understood. Here, we used single-cell RNA sequencing (RNA-seq) and hybridization chain reaction (HCR) to profile multiple stages of early hypothalamic development in the chick. Hypothalamic neuroepithelial cells are initially induced from prethalamic-like cells. Two distinct hypothalamic progenitor populations then emerge and give rise to tuberal and mammillary/paraventricular hypothalamic cells. At later stages, the regional organization of the chick and mouse hypothalamus is highly similar. We identify selective markers for major subdivisions of the developing chick hypothalamus and many previously uncharacterized candidate regulators of hypothalamic induction, regionalization, and neurogenesis. As proof of concept for the power of the dataset, we demonstrate that prethalamus-derived follistatin inhibits hypothalamic induction. This study clarifies the organization of the nascent hypothalamus and identifies molecular mechanisms that may control its induction and subsequent development.

mTOR pathway: A potential therapeutic target for spinal cord injury.

Spinal cord injury (SCI) is the most common disabling spinal injury, and the complex pathological process can eventually lead to severe neurological dysfunction. Many studies have reported that the mammalian target of rapamycin (mTOR) signaling pathway plays an important role in synaptogenesis, neuron growth, differentiation, and survival after central nervous system injury. It is also involved in various traumatic and central nervous system diseases, including traumatic brain injury, neonatal hypoxic-ischemic brain injury, Alzheimer's disease, Parkinson's disease, and cerebral apoplexy. mTOR has also been reported to play an important regulatory role in various pathophysiological processes following SCI. Activation of mTOR signals after SCI can regulate physiological and pathological processes, such as proliferation and differentiation of neural stem cells, regeneration of nerve axons, neuroinflammation, and glial scar formation, through various pathways. Inhibition of mTOR activity has been confirmed to promote repair in SCI. At present, many studies have reported that Chinese herbal medicine can inhibit the SCI-activated mTOR pathway to improve the microenvironment and promote nerve repair after SCI. Due to the role of the mTOR pathway in SCI, it may be a potential therapeutic target for SCI. This review is focused on the pathophysiological process of SCI, characteristics of the mTOR pathway, role of the mTOR pathway in SCI, role of inhibition of mTOR on SCI, and role and significance of inhibition of mTOR by related Chinese herbal medicine inhibitors in SCI. In addition, the review discusses the deficiencies and solutions to mTOR and SCI research shortcomings. This study hopes to provide reference for mTOR and SCI research and a theoretical basis for SCI biotherapy.

Study on Active Components of Cuscuta chinensis Promoting Neural Stem Cells Proliferation: Bioassay-Guided Fractionation.

Neural stem cells (NSCs) exist in the central nervous system of adult animals and capable of self-replication. NSCs have two basic functions, namely the proliferation ability and the potential for multi-directional differentiation. In this study, based on the bioassay-guided fractionation, we aim to screen active components in Cuscuta chinensis to promote the proliferation of NSCs. CCK-8 assays were used as an active detection method to track the active components. On the basis of isolating active fraction and monomer compounds, the structures of these were identified by LC-MS and (1H, 13C) NMR. Moreover, active components were verified by pharmacodynamics and network pharmacology. The system solvent extraction method combined with the traditional isolation method were used to ensure that the fraction TSZE-EA-G6 of Cuscuta chinensis exhibited the highest activity. Seven chemical components were identified from the TSZE-EA-G6 fraction by UPLC-QE-Orbitrap-MS technology, which were 4-O-p-coumarinic acid, chlorogenic acid, 5-O-p-coumarinic acid, hyperoside, astragalin, isochlorogenic acid C, and quercetin-3-O-galactose-7-O-glucoside. Using different chromatographic techniques, five compounds were isolated in TSZE-EA-G6 and identified as kaempferol, kaempferol-3-O-glucoside (astragalin), quercetin-3-O-galactoside (hyperoside), chlorogenic acid, and sucrose. The activity study of these five compounds showed that the proliferation rate of kaempferol had the highest effects; at a certain concentration (25 µg/mL, 3.12 µg/mL), the proliferation rate could reach 87.44% and 59.59%, respectively. Furthermore, research results using network pharmacology techniques verified that kaempferol had an activity of promoting NSCs proliferation and the activity of flavonoid aglycones might be greater than that of flavonoid glycosides. In conclusion, this research shows that kaempferol is the active component in Cuscuta chinensis to promote the proliferation of NSCs.

In Vitro Studies on Therapeutic Effects of Cannabidiol in Neural Cells: Neurons, Glia, and Neural Stem Cells.

(‒)-Cannabidiol (CBD) is one of the major phytocannabinoids extracted from the Cannabis genus. Its non-psychoactiveness and therapeutic potential, partly along with some anecdotal-if not scientific or clinical-evidence on the prevention and treatment of neurological diseases, have led researchers to investigate the biochemical actions of CBD on neural cells. This review summarizes the previously reported mechanistic studies of the CBD actions on primary neural cells at the in vitro cell-culture level. The neural cells are classified into neurons, microglia, astrocytes, oligodendrocytes, and neural stem cells, and the CBD effects on each cell type are described. After brief introduction on CBD and in vitro studies of CBD actions on neural cells, the neuroprotective capability of CBD on primary neurons with the suggested operating actions is discussed, followed by the reported CBD actions on glia and the CBD-induced regeneration from neural stem cells. A summary section gives a general overview of the biochemical actions of CBD on neural cells, with a future perspective. This review will provide a basic and fundamental, but crucial, insight on the mechanistic understanding of CBD actions on neural cells in the brain, at the molecular level, and the therapeutic potential of CBD in the prevention and treatment of neurological diseases, although to date, there seem to have been relatively limited research activities and reports on the cell culture-level, in vitro studies of CBD effects on primary neural cells.

Astragalus saponins improves stroke by promoting the proliferation of neural stem cells through phosphorylation of Akt.

ETHNOPHARMACOLOGICAL RELEVANCE: As one of major components of Buyang Huanwu decoction, Astragali Radix is broadly used for stroke treatment. Astragalus saponins (AST), the main active compound from Astragali Radix has the potentials for neuroprotection and improving spatial memory without clear pharmacological mechanism. AIM OF THE STUDY: The aim of this study was to investigate that pretreatment of AST is beneficial to protect against focal ischemic stroke in mouse model and its related underlying mechanism. MATERIALS AND METHODS: The neurological and motor function of MCAO mice were assessed by TTC staining and CatWalk gait analysis. The effect of AST on proliferation of NSCs was showed by the expression of Ki67 of MCAO mice and the number and size of primary neurospheres cultured from adult SVZ. The intersection of stroke-related targets, neurogenesis targets and drug-related targets were identified by the online website (https://www.omicstudio.cn/index). Then GO functional annotation and KEGG pathway enrichment analysis were performed. Candidate target Akt was confirmed to increase proliferation of cultured NSCs from adult SVZ by CCK8 assay and Western blot. RESULTS: We found that with the prolongation of administration time, AST improved neurological and motor function of MCAO mice, by promoting the proliferation of NSCs both in vivo and in vitro. Then, the primary network among drug, genes and biological pathway was established by using compound-target-disease & function-pathway analysis of astragalus membranaceus. PI3K/Akt which plays a key role in cell proliferation was among the top 10 most significant GO terms from above three aspects. Further analysis using cultured NSCs from adult SVZ confirmed that AST, astragaloside I (A1) and astragaloside III (A3) increased the proliferation of NSCs through targeting Akt. CONCLUSION: The present study elucidated that Astragalus saponins pretreatment could provide a protective effect on experimental stroke mainly by enhancing proliferation of NSCs through targeting Akt. The findings provided a basis for the development of novel strategies for the treatment of stroke.

A subpopulation of embryonic microglia respond to maternal stress and influence nearby neural progenitors.

The interplay between hypothalamic neurons and microglia as they integrate stressors to regulate homeostasis is of growing interest. We asked if microglia in the embryonic hypothalamus were likewise stress responsive and, if so, whether their precocious activation perturbs nearby neural stem cell (NSC) programs. We performed single-cell transcriptomics to define embryonic hypothalamic microglia heterogeneity and identified four microglial subsets, including a subpopulation adjacent to NSCs that was responsive to gestational cold stress. Stress exposure elevated CCL3 and CCL4 secretion, but only in male brains, and ex vivo CCL4 treatment of hypothalamic NSCs altered proliferation and differentiation. Concomitantly, gestational stress decreased PVN oxytocin neurons only in male embryos, which was reversed by microglia depletion. Adult offspring exposed to gestational stress displayed altered social behaviors, which was likewise microglia dependent, but only in males. Collectively, immature hypothalamic microglia play an unappreciated role in translating maternal stressors to sexually dimorphic perturbation of neurodevelopmental programs.

Remyelination is enhanced by Astragalus polysaccharides through inducing the differentiation of oligodendrocytes from neural stem cells in cuprizone model of demyelination.

Demyelination is the hallmark of multiple sclerosis (MS). Promoting remyelination is an important strategy to treat MS. Our previous study showed that Astragalus polysaccharides (APS), the main bioactive component of Astragalus membranaceus, could prevent demyelination in experimental autoimmune encephalomyelitis mice. To investigate the effects of APS on remyelination and the underlying mechanisms, in this study we set up a cuprizone-induced demyelination model in mice and treated them with APS. It was found that APS relieved the neurobehavioral dysfunctions caused by demyelination, and efficaciously facilitated remyelination in vivo. In order to determine whether the mechanism of enhancing remyelination was associated with the differentiation of neural stem cells (NSCs), biomarkers of NSCs, astrocytes, oligodendrocytes and neurons were measured in the corpus callosum tissues of mice through Real-time PCR, Western blot and immunohistochemistry assays. Data revealed that APS suppressed the stemness of NSCs, reduced the differentiation of NSCs into astrocytes, and promoted the differentiation into oligodendrocytes and neurons. This phenomenon was confirmed in the differentiation model of C17.2 NSCs cultured in vitro. Since Sonic hedgehog signaling pathway has been proven to be crucial to the differentiation of NSCs into oligodendrocytes, we examined expression levels of the key molecules in this pathway in vivo and in vitro, and eventually found APS activated this signaling pathway. Together, our results demonstrated that APS probably activated Sonic hedgehog signaling pathway first, then induced NSCs to differentiate into oligodendrocytes and promoted remyelination, which suggested that APS might be a potential candidate in treating MS.

Printed Graphene Layer as a Base for Cell Electrostimulation-Preliminary Results.

Nerve regeneration through cell electrostimulation will become a key finding in regenerative medicine. The procedure will provide a wide range of applications, especially in body reconstruction, artificial organs or nerve prostheses. Other than in the case of the conventional polystyrene substrates, the application of the current flow in the cell substrate stimulates the cell growth and mobility, supports the synaptogenesis, and increases the average length of neuron nerve fibres. The indirect electrical cell stimulation requires a non-toxic, highly electrically conductive substrate material enabling a precise and effective cell electrostimulation. The process can be successfully performed with the use of the graphene nanoplatelets (GNPs)-the structures of high conductivity and biocompatible with mammalian NE-4C neural stem cells used in the study. One of the complications with the production of inks using GNPs is their agglomeration, which significantly hampers the quality of the produced coatings. Therefore, the selection of the proper amount of the surfactant is paramount to achieve a high-quality substrate. The article presents the results of the research into the material manufacturing used in the cell electrostimulation. The outcomes allow for the establishment of the proper amount of the surfactant to achieve both high conductivity and quality of the coating, which could be used not only in electronics, but also-due to its biocompatibility-fruitfully applied to the cell electrostimulation.

Characterization hiPSC-derived neural progenitor cells and neurons to investigate the role of NOS1AP isoforms in human neuron dendritogenesis.

Abnormal dendritic arbor development has been implicated in a number of neurodevelopmental disorders, such as autism and Rett syndrome, and the neuropsychiatric disorder schizophrenia. Postmortem brain samples from subjects with schizophrenia show elevated levels of NOS1AP in the dorsolateral prefrontal cortex, a region of the brain associated with cognitive function. We previously reported that the long isoform of NOS1AP (NOS1AP-L), but not the short isoform (NOS1AP-S), negatively regulates dendrite branching in rat hippocampal neurons. To investigate the role that NOS1AP isoforms play in human dendritic arbor development, we adapted methods to generate human neural progenitor cells and neurons using induced pluripotent stem cell (iPSC) technology. We found that increased protein levels of either NOS1AP-L or NOS1AP-S decrease dendrite branching in human neurons at the developmental time point when primary and secondary branching actively occurs. Next, we tested whether pharmacological agents can decrease the expression of NOS1AP isoforms. Treatment of human iPSC-derived neurons with d-serine, but not clozapine, haloperidol, fluphenazine, or GLYX-13, results in a reduction in endogenous NOS1AP-L, but not NOS1AP-S, protein expression; however, d-serine treatment does not reverse decreases in dendrite number mediated by overexpression of NOS1AP isoforms. In summary, we demonstrate how an in vitro model of human neuronal development can help in understanding the etiology of schizophrenia and can also be used as a platform to screen drugs for patients.

Cytotoxicity comparison of 35 developmental neurotoxicants in human induced pluripotent stem cells (iPSC), iPSC-derived neural progenitor cells, and transformed cell lines.

The Organization for Economic Co-operation and Development (OECD) test guideline 426 for developmental neurotoxicity (DNT) of industrial/environmental chemicals depends primarily on animal experimentation. This requirement raises various critical issues, such as high cost, long duration, the sacrifice of large numbers of animals, and interspecies differences. This study demonstrates an alternative protocol that is simple, quick, less expensive, and standardized to evaluate DNT of many chemicals using human induced pluripotent stem cells (iPSC) and their differentiation to neural progenitor cells (NPC). Initially, concentration-dependent cytotoxicity of 35 DNT chemicals, including industrial materials, insecticides, and clinical drugs, were compared among iPSC, NPC, and two transformed cells, Cos-7 and HepG2, using tetrazolium dye (MTS)-reducing colorimetric and ATP luciferase assays, and IC50 values were calculated. Next, inhibitory effects of the 14 representative chemicals (mainly insecticides) on iPSC differentiation to NPC were evaluated by measuring altered expression of neural differentiation and undifferentiation marker genes. Results show that both iPSC and NPC were much more sensitive to most DNT chemicals than the transformed cells, and 14 chemicals induced differential patterns of marker gene expression, highlighting the validity and utility of the protocol for evaluation and classification of DNT chemicals and preclinical DNT tests for safety assessment.

Cellular and molecular properties of neural progenitors in the developing mammalian hypothalamus.

The neuroendocrine hypothalamus is the central regulator of vital physiological homeostasis and behavior. However, the cellular and molecular properties of hypothalamic neural progenitors remain unexplored. Here, hypothalamic radial glial (hRG) and hypothalamic mantle zone radial glial (hmRG) cells are found to be neural progenitors in the developing mammalian hypothalamus. The hmRG cells originate from hRG cells and produce neurons. During the early development of hypothalamus, neurogenesis occurs in radial columns and is initiated from hRG cells. The radial glial fibers are oriented toward the locations of hypothalamic subregions which act as a scaffold for neuronal migration. Furthermore, we use single-cell RNA sequencing to reveal progenitor subtypes in human developing hypothalamus and characterize specific progenitor genes, such as TTYH1, HMGA2, and FAM107A. We also demonstrate that HMGA2 is involved in E2F1 pathway, regulating the proliferation of progenitor cells by targeting on the downstream MYBL2. Different neuronal subtypes start to differentiate and express specific genes of hypothalamic nucleus at gestational week 10. Finally, we reveal the developmental conservation of nuclear structures and marker genes in mouse and human hypothalamus. Our identification of cellular and molecular properties of neural progenitors provides a basic understanding of neurogenesis and regional formation of the non-laminated hypothalamus.

Symmetric neural progenitor divisions require chromatin-mediated homologous recombination DNA repair by Ino80.

Chromatin regulates spatiotemporal gene expression during neurodevelopment, but it also mediates DNA damage repair essential to proliferating neural progenitor cells (NPCs). Here, we uncover molecularly dissociable roles for nucleosome remodeler Ino80 in chromatin-mediated transcriptional regulation and genome maintenance in corticogenesis. We find that conditional Ino80 deletion from cortical NPCs impairs DNA double-strand break (DSB) repair, triggering p53-dependent apoptosis and microcephaly. Using an in vivo DSB repair pathway assay, we find that Ino80 is selectively required for homologous recombination (HR) DNA repair, which is mechanistically distinct from Ino80 function in YY1-associated transcription. Unexpectedly, sensitivity to loss of Ino80-mediated HR is dependent on NPC division mode: Ino80 deletion leads to unrepaired DNA breaks and apoptosis in symmetric NPC-NPC divisions, but not in asymmetric neurogenic divisions. This division mode dependence is phenocopied following conditional deletion of HR gene Brca2. Thus, distinct modes of NPC division have divergent requirements for Ino80-dependent HR DNA repair.

Dendrosomal nanocurcumin promotes remyelination through induction of oligodendrogenesis in experimental demyelination animal model.

Multiple sclerosis (MS) is an autoimmune disease, associated with central nervous system (CNS) inflammation, demyelination, and axonal loss. Myelin, a multilayer membranous that covers nerve fibers, is essential for rapid impulse conduction. Oligodendrocytes that are generated either from CNS-resident oligodendrocyte progenitor cells (OPCs) or subventricular zone-derived neural stem cells (NSCs) are the myelinating cells of the CNS. The adult CNS maintains a certain endogenous potential to repair myelin damage. However, this process often fails as MS progresses. The origin of this failure is not fully understood, but it is likely to relate to progenitors/stem cells' arrestment in a quiescent state, incapable of generating new oligodendrocyte. Current treatments for MS are immunomodulatory or immunosuppressive medications, with little to no effect on myelin restoration. Recent studies have provided proof-of-principle that CNS remyelination can be promoted either via enhancing endogenous remyelination or by transplanting myelinating cells. Curcumin, a natural polyphenolic compound, has been shown to have therapeutic properties in several neurodegenerative diseases. Here, we investigated the effect of a curcumin nanoformulation, dendrosomal nanoparticles (DNC) on oligodendrogenesis and remyelination, both in vitro and in animal model of demyelination. We indicated that DNC enhanced oligodendrogenesis from NSCs and OPCs, in vitro in dose dependent manner. DNC also induced in vivo remyelination via promotion of oligodendrogenesis. Furthermore, DNC enhanced remyelination capacity of transplanted NSCs through promoting their survival and oligodendrogenesis capacity. Our findings suggest that DNC has significant beneficial effects in demyelinating conditions, either as mono-therapy or as being paired with transplantation approaches.

Fibroblast growth factor 10 is a negative regulator of postnatal neurogenesis in the mouse hypothalamus.

New neurons are generated in the postnatal rodent hypothalamus, with a subset of tanycytes in the third ventricular (3V) wall serving as neural stem/progenitor cells. However, the precise stem cell niche organization, the intermediate steps and the endogenous regulators of postnatal hypothalamic neurogenesis remain elusive. Quantitative lineage-tracing in vivo revealed that conditional deletion of fibroblast growth factor 10 (Fgf10) from Fgf10-expressing ß-tanycytes at postnatal days (P)4-5 results in the generation of significantly more parenchymal cells by P28, composed mostly of ventromedial and dorsomedial neurons and some glial cells, which persist into adulthood. A closer scrutiny in vivo and ex vivo revealed that the 3V wall is not static and is amenable to cell movements. Furthermore, normally ß-tanycytes give rise to parenchymal cells via an intermediate population of α-tanycytes with transient amplifying cell characteristics. Loss of Fgf10 temporarily attenuates the amplification of ß-tanycytes but also appears to delay the exit of their α-tanycyte descendants from the germinal 3V wall. Our findings suggest that transience of cells through the α-tanycyte domain is a key feature, and Fgf10 is a negative regulator of postnatal hypothalamic neurogenesis.

Neuronal Differentiation from Mouse Embryonic Stem Cells In vitro.

The neural differentiation of mouse embryonic stem cells (mESCs) is a potential tool for elucidating the key mechanisms involved in neurogenesis and potentially aid in regenerative medicine. Here, we established an efficient and low cost method for neuronal differentiation from mESCs in vitro, using the strategy of combinatorial screening. Under the conditions defined here, the 2-day embryoid body formation + 6-day retinoic acid induction protocol permits fast and efficient differentiation from mESCs into neural precursor cells (NPCs), as seen by the formation of well-stacked and neurite-like A2lox and 129 derivatives that are Nestin positive. The healthy state of embryoid bodies and the timepoint at which retinoic acid (RA) is applied, as well as the RA concentrations, are critical in the process. In the subsequent differentiation from NPCs into neurons, N2B27 medium II (supplemented by Neurobasal medium) could better support the long term maintenance and maturation of neuronal cells. The presented method is highly efficiency, low cost and easy to operate, and can be a powerful tool for neurobiology and developmental biology research.

Quadrella incana (Capparaceae) Leaf Extract Enhances Proliferation and Maintenance of Neural Stem/Progenitor Cells through Upregulating Glycolytic Flux and Redox Potential.

Neural stem/progenitor cells (NSPCs) are self-renewing, multipotent cells located in the embryonic and adult central nervous system (CNS). Extensive preclinical and clinical studies have shed light on the potential of stem cell replacement therapy for various neurodegenerative diseases. The key prerequisite for the success of these clinical applications is the procurement of a sufficient number of high-quality NSPCs. In this study, we explored the biological activity of Quadrella incana leaf in NSPC homeostasis. We showed that the leaf extract of Quadrella incana upregulated NSPC marker and proliferative potential. On the other hand, Quadrella incana leaf suppressed spontaneous unintended NSPC differentiation. Mechanistically, Quadrella incana leaf contributed to the maintenance of NSPCs by upregulating glycolytic flux and redox potential.

Embryonic microglia influence developing hypothalamic glial populations.

BACKGROUND: Although historically microglia were thought to be immature in the fetal brain, evidence of purposeful interactions between these immune cells and nearby neural progenitors is becoming established. Here, we examined the influence of embryonic microglia on gliogenesis within the developing tuberal hypothalamus, a region later important for energy balance, reproduction, and thermoregulation. METHODS: We used immunohistochemistry to quantify the location and numbers of glial cells in the embryonic brain (E13.5-E17.5), as well as a pharmacological approach (i.e., PLX5622) to knock down fetal microglia. We also conducted cytokine and chemokine analyses on embryonic brains in the presence or absence of microglia, and a neurosphere assay to test the effects of the altered cytokines on hypothalamic progenitor behaviors. RESULTS: We identified a subpopulation of activated microglia that congregated adjacent to the third ventricle alongside embryonic Olig2+ neural progenitor cells (NPCs) that are destined to give rise to oligodendrocyte and astrocyte populations. In the absence of microglia, we observed an increase in Olig2+ glial progenitor cells that remained at the ventricle by E17.5 and a concomitant decrease of these Olig2+ cells in the mantle zone, indicative of a delay in migration of these precursor cells. A further examination of maturing oligodendrocytes in the hypothalamic grey and white matter area in the absence of microglia revealed migrating oligodendrocyte progenitor cells (OPCs) within the grey matter at E17.5, a time point when OPCs begin to slow their migration. Finally, quantification of cytokine and chemokine signaling in ex vivo E15.5 hypothalamic cultures +/- microglia revealed decreases in the protein levels of several cytokines in the absence of microglia. We assayed the influence of two downregulated cytokines (CCL2 and CXCL10) on neurosphere-forming capacity and lineage commitment of hypothalamic NPCs in culture and showed an increase in NPC proliferation as well as neuronal and oligodendrocyte differentiation. CONCLUSION: These data demonstrate that microglia influence gliogenesis in the developing tuberal hypothalamus.

Ginsenoside metabolite 20(S)­protopanaxadiol promotes neural stem cell transition from a state of proliferation to differentiation by inducing autophagy and cell cycle arrest.

20(S)­Protopanaxadiol (PPD) is an active ginseng metabolite and is the final form of protopanaxadiol saponins metabolized by human intestinal microflora. The neuroprotective effects and mechanisms underlying PPD on neural stem cells (NSCs) are not completely understood. The aim of the present study was to assess the effects of PPD on the proliferation and differentiation of neural stem cells. In the present study, following treatment with different concentrations of PPD for 24 h, the percentage of BrdU­positive cells decreased significantly with increasing concentrations of PPD. Moreover, flow cytometric analysis results indicated that PPD treatment increased the proportion of cells in the G0/G1 and G2/M phase and decreased the proportion of cells in the S phase. The activation of autophagy, determined by an increased number of autophagic vacuoles and light chain 3 lipidation, was associated with an increase in the expression of the neuronal marker tubulin­ß3 following PPD treatment. PPD also partially rescued NSCs from the inhibitory effects of the autophagic inhibitor wortmannin, suggesting that the effect of PPD on NSC differentiation was associated with autophagy. Collectively, the results indicated that PPD promoted the transition of NSCs from a state of proliferation to differentiation through the induction of autophagy and cell cycle arrest. Therefore, the present study may provide a basis for the development of regenerative therapies based on ginsenoside, an approved and safe drug.