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The development of the nervous system is regulated by numerous intracellular molecules and cellular signals that interact temporally and spatially with the extracellular microenvironment. The three major cell types in the brain, i.e., neurons and two types of glial cells (astrocytes and oligodendrocytes), are generated from common multipotent neural stem cells (NSCs) throughout life. However, NSCs do not have this multipotentiality from the beginning. During cortical development, NSCs sequentially obtain abilities to differentiate into neurons and glial cells in response to combinations of spatiotemporally modulated cell-intrinsic epigenetic alterations and extrinsic factors. After the completion of brain development, a limited population of NSCs remains in the adult brain and continues to produce neurons (adult neurogenesis), thus contributing to learning and memory. Many biological aspects of brain development and adult neurogenesis are regulated by epigenetic changes via behavioral control of NSCs. Epigenetic dysregulation has also been implicated in the pathogenesis of various brain diseases. Here, we present recent advances in the epigenetic regulation of NSC behavior and its dysregulation in brain disorders.

Although lineage reprogramming from one cell type to another is becoming a breakthrough technology for cell-based therapy, several limitations remain to be overcome, including the low conversion efficiency and subtype specificity. To address these, many studies have been conducted using genetics, chemistry, physics, and cell biology to control transcriptional networks, signaling cascades, and epigenetic modifications during reprogramming. Here, we summarize recent advances in cellular reprogramming and discuss future directions.

Abstract Several transcription factors, including NeuroD1, have been shown to act as neuronal reprogramming factors (RFs) that induce neuronal conversion from somatic cells. However, it remains unexplored whether expression levels of RFs in the original cells affect reprogramming efficiency. Here, we show that the neuronal reprogramming efficiency from two distinct glial cell types, microglia and astrocytes, is substantially dependent on the expression level of NeuroD1: low expression failed to induce neuronal reprogramming, whereas elevated NeuroD1 expression dramatically improved reprogramming efficiency in both cell types. Moreover, even under conditions where NeuroD1 expression was too low to induce effective conversion by itself, combined expression of three RFs (Ascl1, Brn2, and NeuroD1) facilitated the breaking down of cellular barriers, inducing neuronal reprogramming. Thus, our results suggest that a sufficiently high expression level of RFs, or alternatively their combinatorial expression, is the key to achieving efficient neuronal reprogramming from different cells.

Epilepsy is a chronic neurological disorder characterized by hypersynchronous spontaneous recurrent seizures, and affects approximately 50 million people worldwide. Cumulative evidence has revealed that epileptogenic insult temporarily increases neurogenesis in the hippocampus; however, a fraction of the newly generated neurons are integrated abnormally into the existing neural circuits. The abnormal neurogenesis, including ectopic localization of newborn neurons in the hilus, formation of abnormal basal dendrites, and disorganization of the apical dendrites, rewires hippocampal neural networks and leads to the development of spontaneous seizures. The central roles of hilar ectopic granule cells in regulating hippocampal excitability have been suggested. In this review, we introduce recent findings about the migration of newborn granule cells to the dentate hilus after seizures and the roles of seizure-induced ectopic granule cells in the epileptic brain. In addition, we delineate possible intrinsic and extrinsic mechanisms underlying this abnormality. Finally, we suggest that the regulation of seizure-induced ectopic cells can be a promising target for epilepsy therapy and provide perspectives on future research directions.

Post-mitotic neurons do exhibit DNA methylation changes, contrary to the longstanding belief that the epigenetic pattern in terminally differentiated cells is essentially unchanged. While the mechanism and physiological significance of DNA demethylation in neurons have been extensively elucidated, the occurrence of de novo DNA methylation and its impacts have been much less investigated. In the present study, we showed that neuronal activation induces de novo DNA methylation at enhancer regions, which can repress target genes in primary cultured hippocampal neurons. The functional significance of this de novo DNA methylation was underpinned by the demonstration that inhibition of DNA methyltransferase (DNMT) activity decreased neuronal activity-induced excitatory synaptogenesis. Overexpression of WW and C2 domain-containing 1 (Wwc1), a representative target gene of de novo DNA methylation, could phenocopy this DNMT inhibition-induced decrease in synaptogenesis. We found that both DNMT1 and DNMT3a were required for neuronal activity-induced de novo DNA methylation of the Wwc1 enhancer. Taken together, we concluded that neuronal activity-induced de novo DNA methylation that affects gene expression has an impact on neuronal physiology that is comparable to that of DNA demethylation. Since the different requirements of DNMTs for germ cell and embryonic development are known, our findings also have considerable implications for future studies on epigenomics in the field of reproductive biology.

Abstract The brain consists of three major cell types: neurons and two glial cell types (astrocytes and oligodendrocytes). Although they are generated from common multipotent neural stem/precursor cells (NS/PCs), embryonic NS/PCs cannot generate all of the cell types at the beginning of brain development. NS/PCs first undergo extensive self‐renewal to expand their pools, and then acquire the potential to produce neurons, followed by glial cells. Astrocytes are the most frequently found cell type in the central nervous system (CNS), and play important roles in brain development and functions. Although it has been shown that nuclear factor IA (Nfia) is a pivotal transcription factor for conferring gliogenic potential on neurogenic NS/PCs by sequestering DNA methyltransferase 1 (Dnmt1) from astrocyte‐specific genes, direct targets of Nfia that participate in astrocytic differentiation have yet to be completely identified. Here we show that SRY‐box transcription factor 8 (Sox8) is a direct target gene of Nfia at the initiation of the gliogenic phase. We found that expression of Sox8 augmented leukemia inhibitory factor (LIF)‐induced astrocytic differentiation, while Sox8 knockdown inhibited Nfia‐enhanced astrocytic differentiation of NS/PCs. In contrast to Nfia, Sox8 did not induce DNA demethylation of an astrocyte‐specific marker gene, glial fibrillary acidic protein (Gfap), but instead associated with LIF downstream transcription factor STAT3 through transcriptional coactivator p300, explaining how Sox8 expression further facilitated LIF‐induced Gfap expression. Taken together, these results suggest that Sox8 is a crucial Nfia downstream transcription factor for the astrocytic differentiation of NS/PCs in the developing brain.

Spiral ganglion neurons (SGNs) are primary auditory neurons in the spiral ganglion that transmit sound information from the inner ear to the brain and play an important role in hearing. Impairment of SGNs causes sensorineural hearing loss (SNHL), and it has been thought until now that SGNs cannot be regenerated once lost. Furthermore, no fundamental therapeutic strategy for SNHL has been established other than inserting devices such as hearing aids and cochlear implants. Here we show that the mouse spiral ganglion contains cells that are able to proliferate and indeed differentiate into neurons in response to injury. We suggest that SRY-box transcription factor 2/SRY-box transcription factor 10–double-positive (Sox2/Sox10–double-positive) Schwann cells sequentially started to proliferate, lost Sox10 expression, and became neurons, although the number of new neurons generated spontaneously was very small. To increase the abundance of new neurons, we treated mice with 2 growth factors in combination with valproic acid, which is known to promote neuronal differentiation and survival. This treatment resulted in a dramatic increase in the number of SGNs, accompanied by a partial recovery of the hearing loss induced by injury. Taken together, our findings offer a step toward developing strategies for treatment of SNHL.

Summary: Rett syndrome (RTT) is a severe neurological disorder, with impaired brain development caused by mutations in MECP2; however, the underlying mechanism remains elusive. We know from previous work that MeCP2 facilitates the processing of a specific microRNA, miR-199a, by associating with the Drosha complex to regulate neuronal functions. Here, we show that the MeCP2/miR-199a axis regulates neural stem/precursor cell (NS/PC) differentiation. A shift occurs from neuronal to astrocytic differentiation of MeCP2- and miR-199a-deficient NS/PCs due to the upregulation of a miR-199a target, Smad1, a downstream transcription factor of bone morphogenetic protein (BMP) signaling. Moreover, miR-199a expression and treatment with BMP inhibitors rectify the differentiation of RTT patient-derived NS/PCs and development of brain organoids, respectively, suggesting that facilitation of BMP signaling accounts for the impaired RTT brain development. Our study illuminates the molecular pathology of RTT and reveals the MeCP2/miR-199a/Smad1 axis as a potential therapeutic target for RTT.

Regulation of the epigenome during in vivo specification of brain stem cells is still poorly understood. Here, we report DNA methylome analyses of directly sampled cortical neural stem and progenitor cells (NS/PCs) at different development stages, as well as those of terminally differentiated cortical neurons, astrocytes, and oligodendrocytes. We found that sequential specification of cortical NS/PCs is regulated by two successive waves of demethylation at early and late development stages, which are responsible for the establishment of neuron- and glia-specific low-methylated regions (LMRs), respectively. The regulatory role of demethylation of the gliogenic genes was substantiated by the enrichment of nuclear factor I (NFI)-binding sites. We provide evidence that de novo DNA methylation of neuron-specific LMRs establishes glia-specific epigenotypes, essentially by silencing neuronal genes. Our data highlight the in vivo implications of DNA methylation dynamics in shaping epigenomic features that confer the differentiation potential of NS/PCs sequentially during development.

Currently, all methods for converting non-neuronal cells into neurons involve injury to the brain; however, whether neuronal transdifferentiation can occur long after the period of insult remains largely unknown. Here, we use the transcription factor NEUROD1, previously shown to convert reactive glial cells to neurons in the cortex, to determine whether astrocyte-to-neuron transdifferentiation can occur under physiological conditions. We utilized adeno-associated virus 9 (AAV9), which crosses the blood-brain barrier without injury, to deliver NEUROD1 to astrocytes through an intravascular route. Interestingly, we found that a small, but significant number of non-reactive astrocytes converted to neurons in the striatum, but not the cortex. Moreover, astrocytes cultured to minimize their proliferative potential also exhibited limited neuronal transdifferentiation with NEUROD1 expression. Our results show that a single transcription factor can induce astrocyte-to-neuron conversion under physiological conditions, potentially facilitating future clinical approaches long after the acute injury phase.

Suberoylanilide hydroxamic acid (SAHA) has been reported preclinically to diffuse across the placenta and to be found in fetal plasma, suggesting that it can influence the fetus if taken by a pregnant cancer patient. In utero exposure of SAHA to mouse embryos during mid-gestation was found to perturb corticogenesis. However, the influence of in utero administration of SAHA to mouse embryos during mid-gestation on astrocyte, glial cell, and inhibitory neurons (interneurons) is yet to be reported. Pregnant dams were divided into control and SAHA groups and given methyl cellulose (as control) and SAHA orally once a day for 3 days during mid-gestation, starting from embryonic day (E)12 until E14. Astrocyte, interneuron, and behavior analyses were performed on the pups from postnatal day 7 until adulthood (3 months old). Brains were harvested and immunohistochemistry, Western Blot, and RT-PCR were performed on their cortex area. Transient exposure of SAHA to mouse embryos resulted in a decrease and increase in cortical astrocyte and interneuron, respectively. Meanwhile, adult SAHA mice displayed significantly increased anxiety, decreased memory, altered long-term cognitive functions, and reduced social interactions. Our study suggests that exposure to SAHA during prominent neurogenic periods might imbalance the normal excitatory:inhibitory neuron ratio required for the precise regulation of physiological functions in the brain.

Human neural precursor cells (hNPCs) derived from pluripotent stem cells display a high propensity for neuronal differentiation, but they require long-term culturing to differentiate efficiently into astrocytes. The mechanisms underlying this biased fate specification of hNPCs remain elusive. Here, we show that hypoxia confers astrocytic differentiation potential on hNPCs through epigenetic gene regulation, and that this was achieved by cooperation between hypoxia-inducible factor 1α and Notch signaling, accompanied by a reduction of DNA methylation level in the promoter region of a typical astrocyte-specific gene, Glial fibrillary acidic protein. Furthermore, we found that this hypoxic culture condition could be applied to rapid generation of astrocytes from Rett syndrome patient-derived hNPCs, and that these astrocytes impaired neuronal development. Thus, our findings shed further light on the molecular mechanisms regulating hNPC differentiation and provide attractive tools for the development of therapeutic strategies for treating astrocyte-mediated neurological disorders.

Abstract Background Recent transcriptome analyses have shown that long non-coding RNAs (ncRNAs) play extensive roles in transcriptional regulation. In particular, we have reported that promoter-associated ncRNAs (pancRNAs) activate the partner gene expression via local epigenetic changes. Results Here, we identify thousands of genes under pancRNA-mediated transcriptional activation in five mammalian species in common. In the mouse, 1) pancRNA-partnered genes confined their expression pattern to certain tissues compared to pancRNA-lacking genes, 2) expression of pancRNAs was significantly correlated with the enrichment of active chromatin marks, H3K4 trimethylation and H3K27 acetylation, at the promoter regions of the partner genes, 3) H3K4me1 marked the pancRNA-partnered genes regardless of their expression level, and 4) C- or G-skewed motifs were exclusively overrepresented between−200 and−1 bp relative to the transcription start sites of the pancRNA-partnered genes. More importantly, the comparative transcriptome analysis among five different mammalian species using a total of 25 counterpart tissues showed that the overall pancRNA expression profile exhibited extremely high species-specificity compared to that of total mRNA, suggesting that interspecies difference in pancRNA repertoires might lead to the diversification of mRNA expression profiles. Conclusions The present study raises the interesting possibility that the gain and/or loss of gene-activation-associated pancRNA repertoires, caused by formation or disruption of the genomic GC-skewed structure in the course of evolution, finely shape the tissue-specific pattern of gene expression according to a given species.

Adult hippocampal neurogenesis requires the quiescent neural stem cell (NSC) pool to persist lifelong. However, establishment and maintenance of quiescent NSC pools during development is not understood. Here, we show that Suppressor of Fused (Sufu) controls establishment of the quiescent NSC pool during mouse dentate gyrus (DG) development by regulating Sonic Hedgehog (Shh) signaling activity. Deletion of Sufu in NSCs early in DG development decreases Shh signaling activity leading to reduced proliferation of NSCs, resulting in a small quiescent NSC pool in adult mice. We found that putative adult NSCs proliferate and increase their numbers in the first postnatal week and subsequently enter a quiescent state towards the end of the first postnatal week. In the absence of Sufu, postnatal expansion of NSCs is compromised, and NSCs prematurely become quiescent. Thus, Sufu is required for Shh signaling activity ensuring expansion and proper transition of NSC pools to quiescent states during DG development.

Tissue-specific stem cells give rise to new functional cells to maintain tissue homeostasis and restore damaged tissue after injury. To ensure proper brain functions in the adult brain, neural stem cells (NSCs) continuously generate newborn neurons that integrate into pre-existing neuronal networks. Proliferation, as well as neurogenesis of NSCs, are exquisitely controlled by extrinsic and intrinsic factors, and their underlying mechanisms have been extensively studied with the goal of enhancing the neurogenic capacity of NSCs for regenerative medicine. However, neurogenesis of endogenous NSCs alone is insufficient to completely repair brains damaged by neurodegenerative diseases and/or injury because neurogenic areas are limited and few neurons are produced in the adult brain. An innovative approach towards replacing damaged neurons is to induce conversion of non-neuronal cells residing in injured sites into neurons by a process referred to as direct reprogramming. This review describes extrinsic and intrinsic factors controlling NSCs and neurogenesis in the adult brain and discusses prospects for their applications. It also describes direct neuronal reprogramming technology holding promise for future clinical applications.

Prenatal exposure to valproic acid (VPA), an established antiepileptic drug, has been reported to impair postnatal cognitive function in children born to VPA-treated epileptic mothers. However, how these defects arise and how they can be overcome remain unknown. Using mice, we found that comparable postnatal cognitive functional impairment is very likely correlated to the untimely enhancement of embryonic neurogenesis, which led to depletion of the neural precursor cell pool and consequently a decreased level of adult neurogenesis in the hippocampus. Moreover, hippocampal neurons in the offspring of VPA-treated mice showed abnormal morphology and activity. Surprisingly, these impairments could be ameliorated by voluntary running. Our study suggests that although prenatal exposure to antiepileptic drugs such as VPA may have detrimental effects that persist until adulthood, these effects may be offset by a simple physical activity such as running.

Transcription factors (TFs) and epigenetic modifications function cooperatively to regulate various biological processes such as cell proliferation, differentiation, maturation, and metabolism. TF binding to regulatory regions of target genes controls their transcriptional activity through alteration of the epigenetic status around the binding regions, leading to transcription network formation regulating cell fates. Although nuclear factor I/A (Nfia) is a well-known TF that induces demethylation of astrocytic genes to confer astrocytic differentiation potential on neural stem/precursor cells (NS/PCs), the epigenetic role of NFIA in oligodendrocytic lineage progression remains unclear. Here, we show that oligodendrocyte differentiation/maturation is delayed in the brains of Nfia-knockout (KO) mice, and that NFIA-regulated DNA demethylation in NS/PCs plays an important role in determining the timing of their differentiation. We further demonstrate that the promoter activity of the oligodendrocyte transcription factor 1 (Olig1) gene, involved in oligodendrocyte differentiation/maturation, is suppressed by DNA methylation, which is in turn regulated by Nfia expression. Our results suggest that NFIA controls the timing of oligodendrocytic differentiation/maturation via demethylation of cell-type-specific gene promoters.

Rett syndrome (RTT) is a neurodevelopmental disorder caused by MECP2 mutations. Although emerging evidence suggests that MeCP2 deficiency is associated with dysregulation of mechanistic target of rapamycin (mTOR), which functions as a hub for various signaling pathways, the mechanism underlying this association and the molecular pathophysiology of RTT remain elusive. We show here that MeCP2 promotes the posttranscriptional processing of particular microRNAs (miRNAs) as a component of the microprocessor Drosha complex. Among the MeCP2-regulated miRNAs, we found that miR-199a positively controls mTOR signaling by targeting inhibitors for mTOR signaling. miR-199a and its targets have opposite effects on mTOR activity, ameliorating and inducing RTT neuronal phenotypes, respectively. Furthermore, genetic deletion of miR-199a-2 led to a reduction of mTOR activity in the brain and recapitulated numerous RTT phenotypes in mice. Together, these findings establish miR-199a as a critical downstream target of MeCP2 in RTT pathogenesis by linking MeCP2 with mTOR signaling.

Rett syndrome (RTT) is a devastating neurodevelopmental disorder that occurs once in every 10,000-15,000 live female births. Despite intensive research, no effective cure is yet available. Valproic acid (VPA) has been used widely to treat mood disorder, epilepsy, and a growing number of other disorders. In limited clinical studies, VPA has also been used to control seizure in RTT patients with promising albeit somewhat unclear efficacy. In this study we tested the effect of VPA on the neurological symptoms of RTT and discovered that short-term VPA treatment during the symptomatic period could reduce neurological symptoms in RTT mice. We found that VPA restores the expression of a subset of genes in RTT mouse brains, and these genes clustered in neurological disease and developmental disorder networks. Our data suggest that VPA could be used as a drug to alleviate RTT symptoms.

Epigenetic changes are thought to lead to alterations in the property of cells, such as differentiation potential. Neural precursor cells (NPCs) differentiate only into neurons in the midgestational brain, yet they become able to generate astrocytes in the late stage of development. This differentiation-potential switch could be explained by epigenetic changes, since the promoters of astrocyte-specific marker genes, glial fibrillary acidic protein (Gfap) and S100beta, have been shown to become demethylated in late-stage NPCs prior to the onset of astrocyte differentiation; however, whether demethylation occurs generally in other astrocyctic genes remains unknown. Here we analyzed DNA methylation changes in mouse NPCs between the mid-(E11.5) and late (E14.5) stage of development by a genome-wide DNA methylation profiling method using microarrays and found that many astrocytic genes are demethylated in late-stage NPCs, enabling the cell to become competent to express these genes. Although these genes are already demethylated in late-stage NPCs, they are not expressed until cells differentiate into astrocytes. Thus, late-stage NPCs have epigenetic potential which can be realized in their expression after astrocyte differentiation.

Although generating new neurons in the ischemic injured brain would be an ideal approach to replenish the lost neurons for repairing the damage, the adult mammalian brain retains only limited neurogenic capability. Here, we show that direct conversion of microglia/macrophages into neurons in the brain has great potential as a therapeutic strategy for ischemic brain injury. After transient middle cerebral artery occlusion in adult mice, microglia/macrophages converge at the lesion core of the striatum, where neuronal loss is prominent. Targeted expression of a neurogenic transcription factor, NeuroD1, in microglia/macrophages in the injured striatum enables their conversion into induced neuronal cells that functionally integrate into the existing neuronal circuits. Furthermore, NeuroD1-mediated induced neuronal cell generation significantly improves neurological function in the mouse stroke model, and ablation of these cells abolishes the gained functional recovery. Our findings thus demonstrate that neuronal conversion contributes directly to functional recovery after stroke. [ABSTRACT FROM AUTHOR]