Language：English   Publisher：Hearing Research  

The regenerative capacity of inner ear hair cells in mammals varies between the cochlea and the vestibular system. Hair cells in the cochlea lack regenerative ability, whereas those in the vestibular system exhibit limited regenerative potential. However, supporting cells in the cochlea retain proliferative capacity, making them a key focus in auditory regeneration research. Similarly, spiral ganglion neurons actively proliferate until birth but lose this ability within a week postnatally, sharing the regenerative limitations of hair cells. This study investigated the role of the canonical Wnt signaling pathway as a potential regulator of these cells. Wnt signaling plays a crucial role in otic development and inner ear morphogenesis. Using reporter mice, we analyzed the activity of the Wnt canonical pathway in the inner ear at the cellular stages from embryonic to adult stages, assessing fluorescence intensities as an indicator of signaling activity. Our findings demonstrate that Wnt signaling remains active in the vestibular hair cells and in the supporting cells of both the cochlea and vestibule throughout development and into adulthood. In addition, Wnt activity was observed in spiral ganglion neurons up to 7 days after birth, coinciding with their period of proliferative potential. These findings suggest that Wnt signaling is integral to cell proliferation in the inner ear both before and after birth.

DOI： 10.1016/j.heares.2025.109282

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Language：English   Publisher：FEBS Letters  

During cortical development, neural stem/precursor cells (NS/PCs) sequentially produce neurons, astrocytes, and oligodendrocytes. Before producing these cells, human (h) NS/PCs undergo prolonged self-renewal to form a larger cortex than other mammals, although the mechanisms are mostly unknown. Here, we performed a gene knockout screen using the CRISPR/Cas9 system to search for genes involved in hNS/PC self-renewal. We identified RMND5A, encoding an E3 ubiquitin ligase, among the candidate genes. We further demonstrated that knockdown of RMND5A decreased proliferation and promoted neuronal differentiation of hNS/PCs through the activation and suppression of the Wnt and mTOR signaling pathways, respectively. Taken together, our findings suggest that RMND5A participates in the maintenance of hNS/PC self-renewal by modulating the Wnt and mTOR signaling pathways. Impact statement During cortical development, human neural stem/precursor cells (hNS/PCs) undergo prolonged self-renewal to form a larger cortex than other mammals, although the mechanisms are mostly unknown. We identified RMND5A, an E3 ubiquitin ligase, as essential for maintaining self-renewal of hNS/PCs, providing valuable insights into the evolutionary expansion of the human brain.

DOI： 10.1002/1873-3468.70067

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Language：English   Publisher：(公社)日本農芸化学会  

Language：English   Publisher：Bioscience Biotechnology and Biochemistry  

Amyloid fibril formation is associated with various amyloidoses, including neurodegenerative diseases such as Alzheimer's and Parkinson's diseases. Despite the numerous studies on the inhibition of amyloid formation, the prevention and treatment of a majority of amyloid-related disorders are still challenging. In this study, we investigated the effects of various plant extracts on amyloid formation of α-synuclein. We found that the extracts from Eucalyptus gunnii are able to inhibit amyloid formation, and to disaggregate preformed fibrils, in vitro. The extract itself did not lead to cell damage. In the extract, miquelianin, which is a glycosylated form of quercetin and has been detected in the plasma and the brain, was identified and assessed to have a moderate inhibitory activity, compared to the effects of ellagic acid and quercetin, which are strong inhibitors for amyloid formation. The properties of miquelianin provide insights into the mechanisms controlling the assembly of α-synuclein in the brain.

DOI： 10.1093/bbb/zbae114

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Language：English   Publisher：Stem Cell Reports  

Although microglia are macrophages of the central nervous system, their involvement is not limited to immune functions. The roles of microglia during development in humans remain poorly understood due to limited access to fetal tissue. To understand how microglia can impact human neurodevelopment, the methyl-CpG binding protein 2 (MECP2) gene was knocked out in human microglia-like cells (MGLs). Disruption of the MECP2 in MGLs led to transcriptional and functional perturbations, including impaired phagocytosis. The co-culture of healthy MGLs with MECP2-knockout (KO) neurons rescued synaptogenesis defects, suggesting a microglial role in synapse formation. A targeted drug screening identified ADH-503, a CD11b agonist, restored phagocytosis and synapse formation in spheroid-MGL co-cultures, significantly improved disease progression, and increased survival in MeCP2-null mice. These results unveil a MECP2-specific regulation of human microglial phagocytosis and identify a novel therapeutic treatment for MECP2-related conditions.

DOI： 10.1016/j.stemcr.2024.06.013

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：EMBO Reports  

Adult neural stem cells (NSCs) in the hippocampal dentate gyrus continuously proliferate and generate new neurons throughout life. Although various functions of organelles are closely related to the regulation of adult neurogenesis, the role of endoplasmic reticulum (ER)-related molecules in this process remains largely unexplored. Here we show that Derlin-1, an ER-associated degradation component, spatiotemporally maintains adult hippocampal neurogenesis through a mechanism distinct from its established role as an ER quality controller. Derlin-1 deficiency in the mouse central nervous system leads to the ectopic localization of newborn neurons and impairs NSC transition from active to quiescent states, resulting in early depletion of hippocampal NSCs. As a result, Derlin-1-deficient mice exhibit phenotypes of increased seizure susceptibility and cognitive dysfunction. Reduced Stat5b expression is responsible for adult neurogenesis defects in Derlin-1-deficient NSCs. Inhibition of histone deacetylase activity effectively induces Stat5b expression and restores abnormal adult neurogenesis, resulting in improved seizure susceptibility and cognitive dysfunction in Derlin-1-deficient mice. Our findings indicate that the Derlin-1-Stat5b axis is indispensable for the homeostasis of adult hippocampal neurogenesis.

DOI： 10.1038/s44319-024-00205-7

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Language：English   Publisher：Epigenomes  

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.

DOI： 10.3390/epigenomes8020022

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：Cells  

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.

DOI： 10.3390/cells13080707

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Publisher：株式会社医学書院  

DOI： 10.11477/mf.2425201815

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Language：Japanese   Publisher：The Japanese Society of Toxicology  

<p>Autism spectrum disorder (ASD) is a developmental disorder characterized by impaired social communication, repetitive behaviors, and obsessiveness. Its prevalence has been increasing year by year and is currently very high: about 1 in 50 people. Although several causative genes for ASD have been identified, in most cases the causative gene has not even been identified, and effective treatment strategies have yet to be established.</p><p>ASD is known to be caused by abnormalities in brain development beginning in the fetal period. Previous studies have reported abnormal expression of the retrotransposon LINE1 (L1) in the postmortem brains of ASD patients, but the impact of L1 expression on normal brain development is unknown. It has been reported that L1 expression is basically maintained at a low level, but exceptionally, its expression is upregulated in neural progenitor cells in the brain and germ cells. Recent studies have also revealed that L1-cDNA, which is reverse-transcribed from L1-mRNA, regulates intracellular signaling by acting as a functional molecule. Therefore, we analyzed the role of L1 in normal brain development focusing on L1-cDNA.</p><p>First, we examined L1 expression during brain development and found that L1 expression increased as brain development progressed. Next, the reverse transcription of L1 mRNA to cDNA was inhibited by administering a reverse transcriptase inhibitor (3TC) to the mother mice from day 9 of gestation, and the proliferative ability of neural stem cells in the fetal brain at day 15 of gestation was evaluated. The results showed that treatment with 3TC increased the number of Sox2-positive (stem cell marker) and Ki67-positive (proliferation marker) neural stem cells. To elucidate the mechanism by which 3TC treatment increases neural stem cells, we administered 3TC to mice expressing EGFP downstream of the Sox2 promoter, collected EGFP-positive neural stem cells, and performed RNA-sequencing. GO analysis of the genes upregulated by 3TC treatment revealed that the expression of genes related to cell proliferation downstream of Yap signaling was increased. Furthermore, since the cGAS-STING pathway is known to be a major pathway for recognizing intracellular DNA, and activation of this pathway has been reported to inhibit Yap activation, we investigated whether the phenotype observed by 3TC treatment is also observed in STING-Knockout (KO) mice. We found that the number of Sox2-positive and Ki67-positive neural stem cells was increased in STING-KO mice as well as in 3TC-treated mice. Furthermore, when we waited until the embryonic 3TC-exposed mice reached adulthood to analyze their behavior, we observed abnormalities in their social behavior. The above results suggest that the presence of a certain amount of endogenous DNA is essential for normal brain development and that when this endogenous DNA is reduced by drugs or environmental changes, the activity of the cGAS-STING pathway is suppressed and YAP is activated, causing abnormal brain development and resulting in the observation of abnormal social behavior. </p>

DOI： 10.14869/toxpt.51.1.0_s23-2

CiNii Research

Publishing type：Research paper (scientific journal)   Publisher：Knowledge Enterprise Journals  

Stem cells are the source of diverse cell types, and therefore their fate decisions are tightly regulated by multiple layers of controls in each tissue. Without a doubt, the brain is one of the most complex and highly functional tissues, as we now know more than 70 million neurons and even more non-neuronal cells are distributed across dozens of cortical areas in the mouse cerebral cortex, and a single region of cortex contains more than 40 cell types. These diverse neuronal cells emerge from initially homogeneous neural stem cells during embryonic development. In the course of differentiation, neural stem cells undergo cellular division to produce daughter cells with new cellular identities, during which epigenetic and transcriptional regulations determine their fate. Recent advances in the field of neural stem cell biology have revealed that not only epigenetic regulators and transcription factors but also specialized intracellular organelles regulate many aspects of stem cell functions and fate choices, and therefore it is timely to review the mechanisms of sophisticated changes of the properties of neural stem cells during development and how they impact the function of the daughter cells.

DOI： 10.18103/mra.v12i3.5084

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Authorship：Last author,　Corresponding author   Language：English   Publisher：Proceedings of the National Academy of Sciences of the United States of America  

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.

DOI： 10.1073/pnas.2307972120

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Language：Japanese   Publisher：(公社)日本生化学会  

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：Journal of Neurotrauma  

Neonatal spinal cord injury (SCI) shows better functional outcomes than adult SCI. Although the regenerative capability in the neonatal spinal cord may have cues in the treatment of adult SCI, the mechanism underlying neonatal spinal cord regeneration after SCI is unclear. We previously reported age-dependent variation in the pathogenesis of inflammation after SCI. Therefore, we explored differences in the pathogenesis of inflammation after SCI between neonatal and adult mice and their effect on axon regeneration and functional outcome. We established two-day-old spinal cord crush mice as a model of neonatal SCI. Immunohistochemistry of the spinal cord revealed that the nuclear translocation of NF-jB, which promotes the expression of chemokines, was significantly lower in the astrocytes of neonates than in those of adults. Flow cytometry revealed that neonatal astrocytes secrete low levels of chemokines to recruit circulating neutrophils (e.g., Cxcl1 and Cxcl2) after SCI in comparison with adults. We also found that the expression of a chemokine receptor (CXCR2) and an adhesion molecule (b2 integrin) quantified by flow cytometry was lower in neonatal circulating neutrophils than in adult neutrophils. Strikingly, these neonate-specific cellular properties seemed to be associated with no neutrophil infiltration into the injured spinal cord, followed by significantly lower expression of inflammatory cytokines (Il-1b, Il-6 and TNF-a) after SCI in the spinal cords of neonates than in those of adults. At the same time, significantly fewer apoptotic neurons and greater axonal regeneration were observed in neonates in comparison with adults, which led to a marked recovery of locomotor function. This neonate-specific mechanism of inflammation regulation may have potential therapeutic applications in controlling inflammation after adult SCI.

DOI： 10.1089/neu.2023.0044

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Language：English   Publisher：Pediatric Blood and Cancer  

DOI： 10.1002/pbc.30379

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：Genes to Cells  

Neuronal regeneration to replenish lost neurons after injury is critical for brain repair. Microglia, brain-resident macrophages that have the propensity to accumulate at the site of injury, can be a potential source for replenishing lost neurons through fate conversion into neurons, induced by forced expression of neuronal lineage-specific transcription factors. However, it has not been strictly demonstrated that microglia, rather than central nervous system-associated macrophages, such as meningeal macrophages, convert into neurons. Here, we show that NeuroD1-transduced microglia can be successfully converted into neurons in vitro using lineage-mapping strategies. We also found that a chemical cocktail treatment further promoted NeuroD1-induced microglia-to-neuron conversion. NeuroD1 with loss-of-function mutation, on the other hand, failed to induce the neuronal conversion. Our results indicate that microglia are indeed reprogrammed into neurons by NeuroD1 with neurogenic transcriptional activity.

DOI： 10.1111/gtc.13033

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Language：Japanese   Publishing type：Research paper (scientific journal)  

Publisher：医歯薬出版  

DOI： 10.32118/ayu28411869

CiNii Research

Language：English   Publishing type：Research paper (scientific journal)   Publisher：Frontiers in Neuroscience  

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.

DOI： 10.3389/fnins.2023.1150283

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Language：Japanese   Publishing type：Research paper (scientific journal)  

Language：Japanese   Publisher：The Japanese Society of Toxicology  

<p>Linkage between early-life exposure to anesthesia and subsequent learning disabilities is of great concern to children and their families. Here we show that early-life exposure to midazolam (MDZ), a widely used drug in pediatric anesthesia, persistently alters chromatin accessibility and the expression of quiescence-associated genes in neural stem cells (NSCs) in the mouse hippocampus. The alterations led to a sustained restriction of NSC proliferation toward adulthood, resulting in a reduction of neurogenesis that was associated with the impairment of hippocampal-dependent memory functions. Moreover, we found that voluntary exercise restored hippocampal neurogenesis, normalized the MDZ-perturbed transcriptome, and ameliorated cognitive ability in MDZ-exposed mice. Our findings thus explain how pediatric anesthesia provokes long-term adverse effects on brain function and provide a possible therapeutic strategy for countering them.</p>

DOI： 10.14869/toxpt.50.1.0_s31-1

CiNii Research

Language：English   Publishing type：Research paper (scientific journal)   Publisher：Journal of Cancer  

In our comprehensive analysis of pancreatic cancer pathology, we found that the C4orf47 molecule was upregulated in hypoxic environments. C4orf47 is reported to be a centrosome-associated protein, but its biological significance in cancer is completely unknown; therefore, we assessed its role in pancreatic cancer. We found that C4orf47 was a direct target of HIF-1α and is upregulated in hypoxic conditions, in which it suppressed the cell cycle and inhibits cell proliferation through up-regulation of the cell cycle repressors Fbxw-7, P27, and p57; and the down-regulation of the cell cycle promoters c-myc, cyclinD1, and cyclinC. Furthermore, C4orf47 induced epithelial-mesenchymal transition and enhanced their cell plasticity and invasiveness. In addition, the p-Erk/p-p38 ratio was significantly enhanced and down-regulated CD44 expression by C4orf47 suppression, suggesting that C4orf47 is involved in pancreatic cancer dormancy under hypoxic conditions. Furthermore, the potential of C4orf47 expression was a good prognostic biomarker for pancreatic cancer. These results contribute to the elucidation of the pathology of refractory pancreatic cancer and the development of novel therapeutic strategies.

DOI： 10.7150/jca.78993

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Language：Japanese   Publishing type：Research paper (scientific journal)   Publisher：Scientific Reports  

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.

DOI： 10.1038/s41598-022-22802-z

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Publisher：Tetsu-To-Hagane/Journal of the Iron and Steel Institute of Japan  

The contact angle between molten Fe-Al alloy with 0.03, 0.3, and 3 mass% Al composition, and Y2O3 matrix oxide substrate with 0.002, 0.32, and 1 SiO2 activity was measured using sessile drop method in Ar atmosphere at 1873 K, and the interfacial tension was evaluated. The contact angle and interfacial tension between the molten Fe-0.3 Al alloy and the Y2Si2O7 + SiO2 (aSiO2 = 1) substrate decreased over time during 60 s after the molten alloy was dropped onto the substrate. The decrease of the contact angle was 20°, and that of the interfacial tension was 628 mN・m−1 Conversely, the other contact angles and the other interfacial energies were almost stable during the same period. The decrease of the contact angles ranged between 0° and 7°, and that of the interfacial tensions ranged 4 and 195 mN・m−1. By observing the wetting behavior for 60 min, it was recognized that the interfacial reaction between the Fe-Al alloy and the oxide substrate was the redox reaction between Al composition in the alloy and SiO2 composition in the substrate, composed of SiO2 decomposition reaction and Al2O3 formation reaction between oxygen absorbed at the interface and Al composition in the alloy. In addition, it was indicated from the interfacial tension dependence on SiO2 activity that the medium SiO2 volume slag for the molten low-Al steel and the low SiO2 volume slag for the molten high-Al steel were effective in preventing the small droplets of molten slag into the molten steel

DOI： 10.2355/tetsutohagane.TETSU-2021-110

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Language：Japanese   Publisher：(公財)鈴木謙三記念医科学応用研究財団  

Publisher：ISIJ International  

The contact angle between molten Fe–Al alloy with 0.03, 0.3, and 3 mass% Al composition, and Y2O3 matrix oxide substrate with 0.002, 0.32, and 1 SiO2 activity was measured using sessile drop method in Ar atmosphere at 1 873 K, and the interfacial tension was evaluated. The contact angle and interfacial tension between the molten Fe-0.3 Al alloy and the Y2Si2O7 + SiO2 (aSiO2 = 1) substrate decreased over time during 60 s after the molten alloy was dropped onto the substrate. The decrease of the contact angle was 20°, and that of the interfacial tension was 628 mN·m-1 Conversely, the other contact angles and the other interfacial energies were almost stable during the same period. The decrease of the contact angles ranged between 0° and 7°, and that of the interfacial tensions ranged 4 and 195 mN·m-1. By observing the wetting behavior for 60 min, it was recognized that the interfacial reaction between the Fe–Al alloy and the oxide substrate was the redox reaction between Al composition in the alloy and SiO2 composition in the substrate, composed of SiO2 decomposition reaction and Al2O3 formation reaction between oxygen absorbed at the interface and Al composition in the alloy. In addition, it was indicated from the interfacial tension dependence on SiO2 activity that the medium SiO2 volume slag for the molten low-Al steel and the low SiO2 volume slag for the molten high-Al steel were effective in preventing the small droplets of molten slag into the molten steel.

DOI： 10.2355/isijinternational.ISIJINT-2022-093

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Publisher：Conference Proceedings - IEEE International Conference on Systems, Man and Cybernetics  

Assisting patients with dementia is a significant social issue. Currently, to assist patients with dementia, a multimodal care technique called Humanitude is gaining popularity. In Humanitude, the patients are assisted through various techniques to stand up independently by utilizing their motor functions as much as possible. Humanitude care techniques encourage caregivers to increase the area of contact with patients during the sit-to-stand motion. However, Humanitude care techniques are not accurately performed by novice caregivers. Therefore, in this study, a smock-type wearable sensor was developed to measure the proximity between caregivers and care recipients during sit-to-stand motion assistance. A measurement experiment was conducted to evaluate the proximity differences between Humanitude care and simulated novice care. In addition, the effects of different care techniques on the center of mass (CoM) trajectory and muscle activity of the care recipients were investigated. The results showed that the caregivers tend to bring their top and middle trunk closer in Humanitude care compared with novice simulated care. Furthermore, it was observed that the CoM trajectory and muscle activity under Humanitude care were similar to those observed when the care recipient stands up independently. These results validate the effectiveness of Humanitude care and provide useful information for teaching techniques in Humanitude.

DOI： 10.1109/SMC53654.2022.9945156

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Language：English   Publishing type：Research paper (scientific journal)  

Gene expression programs and concomitant chromatin regulation change dramatically during the maturation of postmitotic neurons. Subnuclear positioning of gene loci has been shown to be relevant to transcriptional regulation. However, little is known about subnuclear genome positioning in neuronal maturation. Using cultured murine hippocampal neurons, we found genomic locus 14qD2 to be enriched with genes that are upregulated during neuronal maturation. Reportedly, the locus is homologous to human 8p21.3, which has been extensively studied in neuropsychiatry and neurodegenerative diseases. Mapping of the 14qD2 locus in the nucleus revealed that it was relocated from the nuclear periphery to the interior. Moreover, we found a concomitant decrease in lamin B1 expression. Overexpression of lamin B1 in neurons using a lentiviral vector prevented the relocation of the 14qD2 locus and repressed the transcription of the Egr3 gene on this locus. Taken together, our results suggest that reduced lamin B1 expression during the maturation of neurons is important for appropriate subnuclear positioning of the genomic and transcriptional programs.

DOI： 10.1016/j.neures.2021.05.011.

Repository Public URL： http://hdl.handle.net/2324/4798375

Language：English   Publishing type：Research paper (scientific journal)  

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.

DOI： 10.1262/jrd.2021-106

Language：English   Publishing type：Research paper (scientific journal)  

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.

DOI： 0.1002/prp2.749

Language：English   Publishing type：Research paper (scientific journal)  

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 Sox2/Sox10-double-positive Schwann cells sequentially start to proliferate, lose Sox10 expression, and become neurons, although the number of new neurons generated spontaneously was very small. To increase the abundance of newborn neurons, we treated mice with two 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 moreover 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.

Language：English   Publishing type：Research paper (scientific journal)  

Microglia are the resident immune cells of the brain, and play essential roles in neuronal development, homeostatic function, and neurodegenerative disease. Human microglia are relatively different from mouse microglia. However, most research on human microglia is performed in vitro, which does not accurately represent microglia characteristics under in vivo conditions. To elucidate the in vivo characteristics of human microglia, methods have been developed to generate and transplant induced pluripotent or embryonic stem cell-derived human microglia into neonatal or adult mouse brains. However, its widespread use remains limited by the technical difficulties of generating human microglia, as well as the need to use immune-deficient mice and conduct invasive surgeries. To address these issues, we developed a simplified method to generate induced pluripotent stem cell-derived human microglia and transplant them into the brain via a transnasal route in immunocompetent mice, in combination with a colony stimulating factor 1 receptor antagonist. We found that human microglia were able to migrate through the cribriform plate to different regions of the brain, proliferate, and become the dominant microglia in a region-specific manner by occupying the vacant niche when exogenous human cytokine is administered, for at least 60 days.

DOI： 10.1002/glia.23985

Language：English   Publishing type：Research paper (scientific journal)  

Transcription factor 4 (TCF4) is a crucial regulator of neurodevelopment and has been linked to the pathogenesis of autism, intellectual disability and schizophrenia. As a class I bHLH transcription factor (TF), it is assumed that TCF4 exerts its neurodevelopmental functions through dimerization with proneural class II bHLH TFs. Here, we aim to identify TF partners of TCF4 in the control of interhemispheric connectivity formation. Using a new bioinformatic strategy integrating TF expression levels and regulon activities from single cell RNA-sequencing data, we find evidence that TCF4 interacts with non-bHLH TFs and modulates their transcriptional activity in Satb2+ intercortical projection neurons. Notably, this network comprises regulators linked to the pathogenesis of neurodevelopmental disorders, e.g. FOXG1, SOX11 and BRG1. In support of the functional interaction of TCF4 with non-bHLH TFs, we find that TCF4 and SOX11 biochemically interact and cooperatively control commissure formation in vivo, and regulate the transcription of genes implicated in this process. In addition to identifying new candidate interactors of TCF4 in neurodevelopment, this study illustrates how scRNA-Seq data can be leveraged to predict TF networks in neurodevelopmental processes.

DOI： 10.1242/dev.196022

Language：English   Publishing type：Research paper (scientific journal)  

Oocytes mature in a specialized fluid-filled sac, the ovarian follicle, which provides signals needed for meiosis and germ cell growth. Methods have been developed to generate functional oocytes from pluripotent stem cell-derived primordial germ cell-like cells (PGCLCs) when placed in culture with embryonic ovarian somatic cells. In this study, we developed culture conditions to recreate the stepwise differentiation process from pluripotent cells to fetal ovarian somatic cell-like cells (FOSLCs). When FOSLCs were aggregated with PGCLCs derived from mouse embryonic stem cells, the PGCLCs entered meiosis to generate functional oocytes capable of fertilization and development to live offspring. Generating functional mouse oocytes in a reconstituted ovarian environment provides a method for in vitro oocyte production and follicle generation for a better understanding of mammalian reproduction.

DOI： 10.1126/science.abe0237

Language：English   Publishing type：Research paper (scientific journal)  

Measles virus (MeV), an enveloped RNA virus in the family Paramyxoviridae, is still an important cause of childhood morbidity and mortality worldwide. MeV usually causes acute febrile illness with skin rash, but in rare cases persists in the brain, causing a progressive neurological disorder, subacute sclerosing panencephalitis (SSPE). The disease is fatal, and no effective therapy is currently available. Although trans-synaptic cell-to-cell transmission is thought to account for MeV propagation in the brain, neurons do not express the known receptors for MeV. Recent studies have shown that hyperfusogenic changes in the MeV fusion (F) protein play a key role in MeV propagation in the brain. However, how such mutant viruses spread in neurons remains unexplained. Here we show that cell adhesion molecule 1 (CADM1, also known as IGSF4A, Necl-2, SynCAM1) and CADM2 (also known as IGSF4D, Necl-3, SynCAM2) are host factors which enable MeV to cause membrane fusion in cells lacking the known receptors and to spread between neurons. During enveloped virus entry, a cellular receptor generally interacts in trans with the attachment protein on the envelope. However, CADM1 and CADM2 interact in cis with the MeV attachment protein on the same cell membrane, causing the fusion protein triggering and membrane fusion. Knockdown of CADM1 and CADM2 inhibits syncytium formation and virus transmission between neurons that are both mediated by hyperfusogenic F proteins. Thus, our results unravel the molecular mechanism (receptor-mimicking cis-acting fusion triggering) by which MeV spreads trans-synaptically between neurons, thereby causing SSPE.ImportanceMeasles virus (MeV), an enveloped RNA virus, is the causative agent of measles, which is still an important cause of childhood morbidity and mortality worldwide. Persistent MeV infection in the brain causes a fatal progressive neurological disorder, subacute sclerosing panencephalitis (SSPE), several years after acute infection. However, how MeV spreads in neurons, which are mainly affected in SSPE, remains largely unknown. In this study, we demonstrate that cell adhesion molecule 1 (CADM1) and CADM2 are host factors enabling MeV spread between neurons. During enveloped virus entry, a cellular receptor generally interacts in trans with the attachment protein on the viral membrane (envelope). Remarkably, CADM1 and CADM2 interact in cis with the MeV attachment protein on the same membrane, triggering the fusion protein and causing membrane fusion, as viral receptors usually do in trans Careful screening may lead to more examples of such "receptor mimicking cis-acting fusion triggering" in other viruses.

DOI： 10.1128/JVI.00528-21

Language：English   Publishing type：Research paper (scientific journal)  

Spinal cord injury (SCI) causes motor and sensory deficits and is currently considered an incurable disease. We have previously reported that administration of anti-High Mobility Group Box-1 monoclonal antibody (anti-HMGB1 mAb) preserved lesion area and improved locomotion recovery in mouse model of SCI. In order to further enhance the recovery, we here examined combinatorial treatment of anti-HMGB1 mAb and epothilone B (Epo B), which has been reported to promote axon regeneration. This combinatorial treatment significantly increased hindlimb movement compared with anti-HMGB1 mAb alone, although Epo B alone failed to increase functional recovery. These results are in agreement with that anti-HMGB1 mAb alone was able to decrease the lesion area spreading and increase the surviving neuron numbers around the lesion, whereas Epo B facilitated axon outgrowth only in combination with anti-HMGB1 mAb, suggesting that anti-HMGB1 mAb-dependent tissue preservation is necessary for Epo B to exhibit its therapeutic effect. Taken together, the combinatorial treatment can be considered as a novel and clinically applicable strategy for SCI.

DOI： 10.1016/j.neures.2021.04.002

Language：English   Publishing type：Research paper (scientific journal)  

During female germline development, oocytes become a highly specialized cell type and form a maternal cytoplasmic store of crucial factors. Oocyte growth is triggered at the transition from primordial to primary follicle and is accompanied by dynamic changes in gene expression1, but the gene regulatory network that controls oocyte growth remains unknown. Here we identify a set of transcription factors that are sufficient to trigger oocyte growth. By investigation of the changes in gene expression and functional screening using an in vitro mouse oocyte development system, we identified eight transcription factors, each of which was essential for the transition from primordial to primary follicle. Notably, enforced expression of these transcription factors swiftly converted pluripotent stem cells into oocyte-like cells that were competent for fertilization and subsequent cleavage. These transcription-factor-induced oocyte-like cells were formed without specification of primordial germ cells, epigenetic reprogramming or meiosis, and demonstrate that oocyte growth and lineage-specific de novo DNA methylation are separable from the preceding epigenetic reprogramming in primordial germ cells. This study identifies a core set of transcription factors for orchestrating oocyte growth, and provides an alternative source of ooplasm, which is a unique material for reproductive biology and medicine.

DOI： 10.1038/s41586-020-3027-9

Language：English  

DOI： 10.1016/j.stemcr.2020.08.010

Language：English   Publishing type：Research paper (scientific journal)  

We previously reported that protein tyrosine phosphatase non-receptor type 3 (PTPN3), which is upregulated in activated lymphocytes, acts as an immune checkpoint. However, the mechanism by which PTPN3 expression is enhanced in activated lymphocytes is unknown. In this study, we analyzed the mechanism of PTPN3 expression in activated lymphocytes with a view for developing a novel immune checkpoint inhibitor that suppresses PTPN3. Through the activation process, lymphocytes showed enhanced NFκB activation as well as increased PTPN3 expression. NFκB enhanced proliferation, migration, and cytotoxicity of lymphocytes. Furthermore, NFκB enhanced PTPN3 expression and tyrosine kinase activation. TGFβ reduced PTPN3 expression and NFκB activation in the cancer microenvironment, and suppressed the biological activity of lymphocytes. The results of this study are expected to provide significant implications for improving existing immunotherapy and developing novel immunotherapy.

DOI： 10.1016/j.cellimm.2020.104237

Language：English  

DNA methylation controls gene expression, and once established, DNA methylation patterns are faithfully copied during DNA replication by the maintenance DNA methyltransferase Dnmt1. In vivo, Dnmt1 interacts with Uhrf1, which recognizes hemimethylated CpGs. Recently, we reported that Uhrf1-catalyzed K18- and K23-ubiquitinated histone H3 binds to the N-terminal region (the replication focus targeting sequence, RFTS) of Dnmt1 to stimulate its methyltransferase activity. However, it is not yet fully understood how ubiquitinated histone H3 stimulates Dnmt1 activity. Here, we show that monoubiquitinated histone H3 stimulates Dnmt1 activity toward DNA with multiple hemimethylated CpGs but not toward DNA with only a single hemimethylated CpG, suggesting an influence of ubiquitination on the processivity of Dnmt1. The Dnmt1 activity stimulated by monoubiquitinated histone H3 was additively enhanced by the Uhrf1 SRA domain, which also binds to RFTS. Thus, Dnmt1 activity is regulated by catalysis (ubiquitination)-dependent and -independent functions of Uhrf1.

DOI： 10.1111/gtc.12732

Language：English   Publishing type：Research paper (scientific journal)  

Permethrin, a pyrethroid chemical, is widely used as a pesticide because of its rapid insecticidal activity. Although permethrin is considered to exert very low toxicity in mammals, the effects of early, low-level, chronic exposure on the adult central nervous system are unclear. In this study, we investigated the effects of low-level, chronic permethrin exposure in early life on the brain functions of adult mice, using environmentally relevant concentrations. We exposed mice to the acceptable daily intake level of permethrin (0.3 ppm) in drinking water during the prenatal and postnatal periods. We then examined the effects on the central nervous system in adult male offspring. In the permethrin group, we detected behavior that displayed incomplete adaptation to a novel environment, as well as an impairment in learning and memory. In addition, immunohistochemical analysis revealed an increase in doublecortin- (an immature neuron marker) positive cells in the hippocampal dentate gyrus in the permethrin exposure group compared with the control group. Additionally, in the permethrin exposure group there was a decrease in astrocyte number in the hilus of the dentate gyrus, and remaining astrocytes were often irregularly shaped. These results suggest that exposure to permethrin at low levels in early life affects the formation of the neural circuit base and behavior after maturation. Therefore, in the central nervous system of male mice, low-level, chronic permethrin exposure during the prenatal and postnatal periods has effects that were not expected based on the known effects of permethrin exposure in mature animals.

DOI： 10.1002/jat.3882

Language：Others  

Suppressor of fused controls perinatal expansion and quiescence of future dentate adult neural stem cells.

DOI： 10.7554/eLife.42918

Language：English   Publishing type：Research paper (scientific journal)  

Minimal sets of transcription factors can directly reprogram somatic cells into neurons. However, epigenetic remodeling during neuronal reprogramming has not been well reconciled with transcriptional regulation. Here we show that NeuroD1 achieves direct neuronal conversion from mouse microglia both in vitro and in vivo. Exogenous NeuroD1 initially occupies closed chromatin regions associated with bivalent trimethylation of histone H3 at lysine 4 (H3K4me3) and H3K27me3 marks in microglia to induce neuronal gene expression. These regions are resolved to a monovalent H3K4me3 mark at later stages of reprogramming to establish the neuronal identity. Furthermore, the transcriptional repressors Scrt1 and Meis2 are induced as NeuroD1 target genes, resulting in a decrease in the expression of microglial genes. In parallel, the microglial epigenetic signature in promoter and enhancer regions is erased. These findings reveal NeuroD1 pioneering activity accompanied by global epigenetic remodeling for two sequential events: onset of neuronal property acquisition and loss of the microglial identity during reprogramming.

DOI： 10.1016/j.neuron.2018.12.010

Language：Others  

Nox4 Promotes Neural Stem/Precursor Cell Proliferation and Neurogenesis in the Hippocampus and Restores Memory Function Following Trimethyltin-Induced Injury.

DOI： 10.1016/j.neuroscience.2018.11.046

Language：English  

Rett syndrome (RTT) is a devastating neurodevelopmental disorder resulting from mutations in the X-linked gene encoding methyl-CpG-binding pro- tein 2 (MECP2). MECP2 mutations are also associated with other neurodevelop- mental diseases, including autism and schizophrenia. Therefore, elucidating the mechanism of RTT can contribute to understanding the pathogenesis of a wide range of neurodevelopmental diseases. Despite its importance, however, the RTT pathogenesis remains unclear, and effective therapeutic treatment has not been developed. Offering an opportunity to move toward this goal, however, is the recent advance in the stem cell research field of the development of induced pluripotent stem cell (iPSC) technology. This technology can yield important insights into dis- ease pathophysiology and has the potential to provide disease models for screening new drugs. Here, we discuss applications of recent stem cell technology to the field of research on RTT and describe the stem cell biology of RTT pathogenesis.

DOI： 10.1007/978-3-319-90695-9_3

Language：English   Publishing type：Research paper (scientific journal)  

Together with residual host neurons, transplanted neural stem cell (NSC)-derived neurons play a critical role in reconstructing disrupted neural circuits after spinal cord injury (SCI). Since a large number of tracts are disrupted and the majority of host neurons die around the lesion site as the damage spreads, minimizing this spreading and preserving the lesion site are important for attaining further improvements in reconstruction. High mobility group box-1 (HMGB1) is a damage-associated molecular pattern protein that triggers sterile inflammation after tissue injury. In the ischemic and injured brain, neutralization of HMGB1 with a specific antibody reportedly stabilizes the blood-brain barrier, suppresses inflammatory cytokine expression, and improves functional recovery. Using a SCI model mouse, we here developed a combinatorial treatment for SCI: administering anti-HMGB1 antibody prior to transplantation of NSCs derived from human induced pluripotent stem cells (hiPSC-NSCs) yielded a dramatic improvement in locomotion recovery after SCI. Even anti-HMGB1 antibody treatment alone alleviated blood-spinal cord barrier disruption and edema formation, and increased the number of neurites from spared axons and the survival of host neurons, resulting in functional recovery. However, this recovery was greatly enhanced by the subsequent hiPSC-NSC transplantation, reaching an extent that has never before been reported. We also found that this improved recovery was directly associated with connections established between surviving host neurons and transplant-derived neurons. Taken together, our results highlight combinatorial treatment with anti-HMGB1 antibody and hiPSC-NSC transplantation as a promising novel therapy for SCI. Stem Cells 2018
36:737–750.

DOI： 10.1002/stem.2802

Language：Others  

Canonical TGF-β Signaling Negatively Regulates Neuronal Morphogenesis through TGIF/Smad Complex-Mediated CRMP2 Suppression.

DOI： 10.1523/JNEUROSCI.2423-17.2018

Language：English  

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.

DOI： 10.4308/hjb.25.2.70

Language：English   Publishing type：Research paper (scientific journal)  

Epilepsy is a neurological disorder often associated with seizure that affects ∼0.7&#37; of pregnant women. During pregnancy, most epileptic patients are prescribed antiepileptic drugs (AEDs) such as valproic acid (VPA) to control seizure activity. Here, we show that prenatal exposure to VPA in mice increases seizure susceptibility in adult offspring through mislocalization of newborn neurons in the hippocampus. We confirmed that neurons newly generated from neural stem/progenitor cells (NS/PCs) are integrated into the granular cell layer in the adult hippocampus
however, prenatal VPA treatment altered the expression in NS/PCs of genes associated with cell migration, including CXC motif chemokine receptor 4 (Cxcr4), consequently increasing the ectopic localization of newborn neurons in the hilus. We also found that voluntary exercise in a running wheel suppressed this ectopic neurogenesis and countered the enhanced seizure susceptibility caused by prenatal VPA exposure, probably by normalizing the VPA-disrupted expression of multiple genes including Cxcr4 in adult NS/PCs. Replenishing Cxcr4 expression alone in NS/PCs was sufficient to overcome the aberrant migration of newborn neurons and increased seizure susceptibility in VPA-exposed mice. Thus, prenatal exposure to an AED, VPA, has a long-term effect on the behavior of NS/PCs in offspring, but this effect can be counteracted by a simple physical activity. Our findings offer a step to developing strategies for managing detrimental effects in offspring exposed to VPA in utero.

DOI： 10.1073/pnas.1716479115

Language：English   Publishing type：Research paper (scientific journal)  

Background: Although quiescent neural stem cells (NSCs) in the adult hippocampus proliferate in response to neurogenic stimuli and subsequently give rise to new neurons continuously throughout life, misregulation of NSCs in pathological conditions, including aging, leads to the impairment of learning and memory. High mobility group B family 1 (HMGB1) and HMGB2, HMG family proteins that function as transcriptional activators through the modulation of chromatin structure, have been assumed to play some role in the regulation of adult NSCs
however, their precise functions and even expression patterns in the adult hippocampus remain elusive. Results: Here we show that expression of HMGB2 but not HMGB1 is restricted to the subset of NSCs and their progenitors. Furthermore, running, a well-known positive neurogenic stimulus, increased the proliferation of HMGB2-expressing cells, whereas aging was accompanied by a marked decrease in these cells. Intriguingly, HMGB2-expressing quiescent NSCs, which were shifted toward the proliferative state, were decreased as aging progressed. Conclusions: HMGB2 expression is strongly associated with transition from the quiescent to the proliferative state of NSCs, supporting the possibility that HMGB2 is involved in the regulation of adult neurogenesis and can be used as a novel marker to identify NSCs primed for activation in the adult hippocampus. Developmental Dynamics 247:229–238, 2018. © 2017 Wiley Periodicals, Inc.

DOI： 10.1002/dvdy.24559

Language：English   Publishing type：Research paper (scientific journal)  

Neural stem cells (NSCs) undergo self-renewal and generate neurons and glial cells under the influence of specific signals from surrounding environments. Glioblastoma multiforme (GBM) is a highly lethal brain tumor arising from NSCs or glial precursor cells owing to dysregulation of transcriptional and epigenetic networks that control self-renewal and differentiation of NSCs. Highly tumorigenic glioblastoma stem cells (GSCs) constitute a small subpopulation of GBM cells, which share several characteristic similarities with NSCs. GSCs exist atop a stem cell hierarchy and generate heterogeneous populations that participate in tumor propagation, drug resistance, and relapse. During multimodal treatment, GSCs de-differentiate and convert into cells with malignant characteristics, and thus play critical roles in tumor propagation. In contrast, differentiation therapy that induces GBM cells or GSCs to differentiate into a neuronal or glial lineage is expected to inhibit their proliferation. Since stem cell differentiation is specified by the cells’ epigenetic status, understanding their stemness and the epigenomic situation in the ancestor, NSCs, is important and expected to be helpful for developing treatment modalities for GBM. Here, we review the current findings regarding the epigenetic regulatory mechanisms of NSC fate in the developing brain, as well as those of GBM and GSCs. Furthermore, considering the similarities between NSCs and GSCs, we also discuss potential new strategies for GBM treatment.

DOI： 10.1007/s10565-017-9420-y

Language：English  

Injury to the spinal cord causes transection of axon fibers and neural cell death, resulting in disruption of the neural network and severe functional loss. Reconstruction of the damaged neural circuits was once considered to be hopeless as the adult mammalian central nervous system has very poor ability to regenerate. For this reason, there is currently no effective therapeutic treatment for spinal cord injury (SCI). However, with recent developments in stem cell research and cell culture technology, regenerative therapy using neural stem cell (NSC) transplantation has rapidly been developed, and this therapeutic strategy makes it possible to rebuild the destroyed neural circuits. In this review, we discuss the recent breakthroughs in NSC transplantation therapy for SCI. Developmental Dynamics 247:75–84, 2018. © 2017 Wiley Periodicals, Inc.

DOI： 10.1002/dvdy.24558

Language：English  

Severe spinal cord injury (SCI) leads to almost complete neural cell loss at the injured site, causing the irreversible disruption of neuronal circuits. The transplantation of neural stem or precursor cells (NS/PCs) has been regarded as potentially effective for SCI treatment because NS/PCs can compensate for the injured sites by differentiating into neurons and glial cells (astrocytes and oligodendrocytes). An understanding of the molecular mechanisms that regulate the proliferation, fate specification and maturation of NS/PCs and their progeny would facilitate the establishment of better therapeutic strategies for regeneration after SCI. In recent years, several studies of SCI animal models have demonstrated that the modulation of specific epigenetic marks by histone modifiers and non-coding RNAs directs the setting of favorable cellular environments that promote the neuronal differentiation of NS/PCs and/or the elongation of the axons of the surviving neurons at the injured sites. In this review, we provide an overview of recent progress in the epigenetic regulation/manipulation of neural cells for the treatment of SCI.

DOI： 10.1007/s00441-017-2656-2

Language：English   Publishing type：Research paper (scientific journal)  

Adult neurogenesis is a process of generating new neurons from neural stem/precursor cells (NS/PCs) in restricted adult brain regions throughout life. It is now generally known that adult neurogenesis in the hippocampal dentate gyrus (DG) and subventricular zone participates in various higher brain functions, such as learning and memory formation, olfactory discrimination and repair after brain injury. However, the mechanisms underlying adult neurogenesis remain to be fully understood. Here, we show that Nuclear protein 95 KDa (Np95, also known as UHRF1 or ICBP90), which is an essential protein for maintaining DNA methylation during cell division, is involved in multiple processes of adult neurogenesis. Specific ablation of Np95 in adult NS/PCs (aNS/PCs) led to a decrease in their proliferation and an impairment of neuronal differentiation and to suppression of neuronal maturation associated with the impairment of dendritic formation in the hippocampal DG. We also found that deficiency of Np95 in NS/PCs increased the expression of tumor suppressor genes p16 and p53, and confirmed that expression of these genes in NS/PCs recapitulates the phenotype of Np95-deficient NS/PCs. Taken together, our findings suggest that Np95 plays an essential role in proliferation and differentiation of aNS/PCs through the regulation of tumor suppressor gene expression in adult neurogenesis.

DOI： 10.1016/j.neures.2018.05.007

Language：English  

Emerging evidence has demonstrated that epigenetic programs influence many aspects of neural stem cell (NSC) behavior, including proliferation and differentiation. It is becoming apparent that epigenetic mechanisms, such as DNA methylation, histone modifications, and noncoding RNA expression, are spatiotemporally regulated and that these intracellular programs, in concert with extracellular signals, ensure appropriate gene activation. Here we summarize recent advances in understanding of the epigenetic regulation of human NSCs directly isolated from the brain or produced from pluripotent stem cells (embryonic and induced pluripotent stem cells, respectively).

DOI： 10.1007/978-3-319-93485-3_5

Language：English   Publishing type：Research paper (scientific journal)  

Spinal cord injury (SCI) is a devastating neurologic disorder that often leads to permanent disability, and there is no effective treatment for it. High mobility group box-1 (HMGB1) is a damage-associated molecular protein that triggers sterile inflammation upon injuries. We have previously shown that two administrations of neutralizing monoclonal antibody (mAb) against HMGB1 (immediately after (0 h) and 6 h after) SCI dramatically improves functional recovery after SCI in mice. However, when considering clinical application, 0 h after SCI is not practical. Therefore, in this study, we examined the therapeutic time window of the mAb administration. Injection at 3 h after SCI significantly improved the functional recovery comparably to injection immediately after SCI, while injection at 6 h was less effective, and injection at 9 or 12 h had no therapeutic effect. We also found beneficial effects of injection at 3 h after injury on blood-spinal cord barrier maintenance, inflammatory-related gene expression and preservation of the damaged spinal cord tissue. Taken together, our results suggest that a single administration of anti-HMGB1 mAb within a proper time window could be a novel and potential therapeutic strategy for SCI.

DOI： 10.1016/j.neures.2018.03.004

Language：English  

Astrocytes, which support diverse neuronal functions, are generated from multipotent neural stem/precursor cells (NS/PCs) during brain development. Although many astrocyte-inducing factors have been identified and studied in vitro, the regions and/or cells that produce these factors in the developing brain remain elusive. Here, we show that meninges-produced factors induce astrocytic differentiation of NS/PCs. Consistent with the timing when astrocytic differentiation of NS/PCs increases, expression of astrocyte-inducing factors is upregulated. Meningeal secretion-mimicking combinatorial treatment of NS/PCs with bone morphogenetic protein 4, retinoic acid and leukemia inhibitory factor synergistically activate the promoter of a typical astrocytic marker, glial fibrillary acidic protein. Taken together, our data suggest that meninges play an important role in astrocytic differentiation of NS/PCs in the developing brain.

DOI： 10.1002/1873-3468.12881

Language：English   Publishing type：Research paper (scientific journal)  

Astrocytes, which support diverse neuronal functions, are generated from multipotent neural stem/precursor cells (NS/PCs) during brain development. Although many astrocyte-inducing factors have been identified and studied in vitro, the regions and/or cells that produce these factors in the developing brain remain elusive. Here, we show that meninges-produced factors induce astrocytic differentiation of NS/PCs. Consistent with the timing when astrocytic differentiation of NS/PCs increases, expression of astrocyte-inducing factors is upregulated. Meningeal secretion-mimicking combinatorial treatment of NS/PCs with bone morphogenetic protein 4, retinoic acid and leukemia inhibitory factor synergistically activate the promoter of a typical astrocytic marker, glial fibrillary acidic protein. Taken together, our data suggest that meninges play an important role in astrocytic differentiation of NS/PCs in the developing brain.

DOI： 10.1002/1873-3468.12881

Language：English   Publishing type：Research paper (scientific journal)  

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.

DOI： 10.1016/j.celrep.2017.08.086

Language：English   Publishing type：Research paper (scientific journal)  

Arginine methylation is a post-translational modification which is catalyzed by protein arginine methyltransferases (PRMTs). Here, we report that PRMT1 is highly expressed in neural stem/precursor cells (NS/PCs) of mouse embryos, and knockdown of PRMT1 in NS/PCs suppresses the generation of astrocytes. The luciferase assay demonstrated that knockdown of PRMT1 inhibits activation of the promoter of a typical astrocytic marker gene, glial fibrillary acidic protein (Gfap), in NS/PCs. The transcription factor signal transducer and activator of transcription 3 (STAT3) is known to generally be critical for astrocytic differentiation of NS/PCs. We found that PRMT1 methylates arginine residue(s) of STAT3 to regulate its activity positively, resulting in the promotion of astrocytic differentiation of NS/PCs.

DOI： 10.1111/jnc.14123

Language：English  

In the developing brain, the three major cell types, i.e., neurons, astrocytes and oligodendrocytes, are generated from common multipotent neural stem cells (NSCs). In particular, astrocytes eventually occupy a great fraction of the brain and play pivotal roles in the brain development and functions. However, NSCs cannot produce the three major cell types simultaneously from the beginning; e.g., it is known that neurogenesis precedes astrogenesis during brain development. How is this fate switching achieved? Many studies have revealed that extracellular cues and intracellular programs are involved in the transition of NSC fate specification. The former include growth factor- and cytokine-signaling, and the latter involve epigenetic machinery, including DNA methylation, histone modifications, and non-coding RNAs. Accumulating evidence has identified a complex array of epigenetic modifications that control the timing of astrocytic differentiation of NSCs. In this review, we introduce recent progress in identifying the molecular mechanisms of astrogenesis underlying the tight regulation of neuronal-astrocytic fate switching of NSCs.

DOI： 10.2183/pjab.93.024

Language：English   Publishing type：Research paper (scientific journal)  

Currently, all methods for converting non-neuronal cells into neurons involve injury to the brain; however, whether neuronal trans-differentiation 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.

DOI： 10.1016/j.stemcr.2017.04.013

Language：English   Publishing type：Research paper (scientific journal)  

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 1 alpha 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.

DOI： 10.1016/j.stemcr.2017.05.001

Language：English   Publishing type：Research paper (scientific journal)  

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.

DOI： 10.1186/s12864-017-3662-1

Language：English  

In mammals, transcription in the zygote begins after fertilization. This transcriptional wave is called zygotic gene activation (ZGA). During ZGA, epigenetic modifications, such as DNA methylation and histone modifications, are dynamically and drastically reconstructed in a sequence-specific manner. However, how such orchestrated gene upregulation is regulated remains unknown. Recently, using microinjection techniques, we have revealed that a class of long noncoding RNAs, named promoter-associated noncoding RNAs (pancRNAs), mediates specific gene upregulation through promoter DNA demethylation during ZGA. Here, we describe the experimental methods available to control the expression levels of pancRNAs and to evaluate epigenetic status after pancRNA manipulation.

DOI： 10.1007/978-1-4939-6716-2_16

Language：English  

Development of high-throughput sequencing technologies has uncovered the immensity of the long noncoding RNA (lncRNA) world. Divergently transcribed lncRNAs from bidirectional gene promoters, called promoter-associated noncoding RNAs (pancRNAs), account for ~20% of the total number of lncRNAs, and this major fraction is involved in many biological processes, such as development and cancer formation. Recently, we have found that the pancRNAs activate their partner genes, as represented by the fact that pancIl17d, a pancRNA that is transcribed from the antisense strand of the promoter region of Interleukin 17d (Il17d) at the onset of zygotic gene activation (ZGA), is essential for mouse preimplantation development through Il17d upregulation. The discovery of the expression of a specific set of pancRNAs during ZGA was achieved by using a method that generates directional RNA-seq libraries from small-scale samples. Although there are several methods available for small-scale samples, most of them require a pre-amplification procedure that frequently generates some amplification biases toward a subset of transcripts. We provide here a highly sensitive and reproducible method based on the preparation of directional RNA-seq libraries from as little as 100 mouse oocytes or embryos without pre-amplification for the quantification of lncRNAs as well as mRNAs.

DOI： 10.1007/978-1-4939-6988-3_6

Language：English   Publishing type：Research paper (scientific journal)  

The female germ line undergoes a unique sequence of differentiation processes that confers totipotency to the egg(1,2). The reconstitution of these events in vitro using pluripotent stem cells is a key achievement in reproductive biology and regenerative medicine. Here we report successful reconstitution in vitro of the entire process of oogenesis from mouse pluripotent stem cells. Fully potent mature oocytes were generated in culture from embryonic stem cells and from induced pluripotent stem cells derived from both embryonic fibroblasts and adult tail tip fibroblasts. Moreover, pluripotent stem cell lines were re-derived from the eggs that were generated in vitro, thereby reconstituting the full female germline cycle in a dish. This culture system will provide a platform for elucidating the molecular mechanisms underlying totipotency and the production of oocytes of other mammalian species in culture.

DOI： 10.1038/nature20104

Language：English   Publishing type：Research paper (scientific journal)  

Despite the importance of DNA methylation in health and disease, technologies to readily manipulate methylation of specific sequences for functional analysis and therapeutic purposes are lacking. Here we adapt the previously described dCas9-SunTag for efficient, targeted demethylation of specific DNA loci. The original SunTag consists of ten copies of the GCN4 peptide separated by 5-amino-acid linkers. To achieve efficient recruitment of an anti-GCN4 scFv fused to the ten eleven (TET) 1 hydroxylase, which induces demethylation, we changed the linker length to 22 amino acids. The system attains demethylation efficiencies >50&#37; in seven out of nine loci tested. Four of these seven loci showed demethylation of >90&#37;. We demonstrate targeted demethylation of CpGs in regulatory regions and demethylation-dependent 1.7- to 50-fold upregulation of associated genes both in cell culture (embryonic stem cells, cancer cell lines, primary neural precursor cells) and in vivo in mouse fetuses.

DOI： 10.1038/nbt.3658

Language：English   Publishing type：Research paper (scientific journal)  

Bidirectional promoters are the major source of gene activation-associated noncoding RNA (ncRNA). PC12 cells offer an interesting model for understanding the mechanism underlying bidirectional promoter-mediated cell cycle control. Nerve growth factor (NGF)-stimulated PC12 cells elongate neurites, and are in a reversible cell-cycle-arrested state. In contrast, these cells irreversibly differentiate and cannot re-enter the normal cell cycle after NGF plus cAMP treatment. In this study, using directional RNA-seq, we found that bidirectional promoters for protein-coding genes with promoter-associated ncRNA (pancRNA) were enriched for cAMP response element consensus sequences, and were preferred targets for transcriptional regulation by the transcription factors in the cAMP-dependent pathway. A spindle-formation-associated gene, Nusap1 and pancNusap1 were among the most strictly co-transcribed pancRNA-mRNA pairs. This pancRNA-mRNA pair was specifically repressed in irreversibly differentiated PC12 cells. Knockdown (KD) and overexpression experiments showed that pancNusap1 positively regulated the Nusap1 expression in a sequence-specific manner, which was accompanied by histone acetylation at the Nusap1 promoter. Furthermore, pancNusap1 KD recapitulated the effects of cAMP on cell cycle arrest. Thus, we conclude that pancRNA-mediated histone acetylation contributes to the establishment of the cAMP-induced transcription state of the Nusap1 locus and contributes to the irreversible cell cycle exit for terminal differentiation of PC12 cells.

DOI： 10.1093/nar/gkw113

Language：English   Publishing type：Research paper (scientific journal)  

Proper dendritic elaboration of neurons is critical for the formation of functional circuits during brain development. Defects in dendrite morphogenesis are associated with neuropsychiatric disorders, and microRNAs are emerging as regulators of aspects of neuronal maturation such as axonal and dendritic growth, spine formation, and synaptogenesis. Here, we show that miR-214 plays a pivotal role in the regulation of dendritic development. Overexpression of miR-214 increased dendrite size and complexity, whereas blocking of endogenous miR-214-3p, a mature form of miR-214, inhibited dendritic morphogenesis. We also found that miR-214-3p targets quaking (Qki), which is implicated in psychiatric diseases such as schizophrenia, through conserved target sites located in the 3-untranslated region of Qki mRNA, thereby down-regulating Qki protein levels. Overexpression and knockdown of Qki impaired and enhanced dendritic formation, respectively. Moreover, overexpression of Qki abolished the dendritic growth induced by miR-214 overexpression. Taken together, our findings reveal a crucial role for the miR-214-Qki pathway in the regulation of neuronal dendritic development.

DOI： 10.1074/jbc.M115.705749

Language：English   Publishing type：Research paper (scientific journal)  

Development of the hippocampal dentate gyrus (DG) in the mammalian brain is achieved through multiple processes during late embryonic and postnatal stages, with each developmental step being strictly governed by extracellular cues and intracellular mechanisms. Here, we show that the maintenance DNA methyltransferase 1 (Dnmt1) is critical for development of the DG in the mouse. Deletion of Dnmt1 in neural stem cells (NSCs) at the beginning of DG development led to a smaller size of the granule cell layer in the DG. NSCs lacking Dnmt1 failed to establish proper radial processes or to migrate into the subgranular zone, resulting in aberrant neuronal production in the molecular layer of the DG and a reduction of integrated neurons in the granule cell layer. Interestingly, prenatal deletion of Dnmt1 in NSCs affected not only the developmental progression of the DG but also the properties of NSCs maintained into adulthood: Dnmt1-deficient NSCs displayed impaired neurogenic ability and proliferation. We also found that Dnmt1 deficiency in NSCs decreased the expression of Reelin signaling components in the developing DG and increased that of the cell cycle inhibitors p21 and p57 in the adult DG. Together, these findings led us to propose that Dnmt1 functions as a key regulator to ensure the proper development of the DG, as well as the proper status of NSCs maintained into adulthood, by modulating extracellular signaling and intracellular mechanisms.

DOI： 10.1523/JNEUROSCI.0512-16.2016

Language：English   Publishing type：Research paper (scientific journal)  

Chromosomes and genes are non-randomly arranged within the mammalian cell nucleus, and gene clustering is of great significance in transcriptional regulation. However, the relevance of gene clustering and their expression during the differentiation of neural precursor cells (NPCs) into astrocytes remains unclear. We performed a genome-wide enhanced circular chromosomal conformation capture (e4C) to screen for genes associated with the astrocyte-specific gene glial fibrillary acidic protein (Gfap) during astrocyte differentiation. We identified 18 genes that were specifically associated with Gfap and expressed in NPC-derived astrocytes. Our results provide additional evidence for the functional significance of gene clustering in transcriptional regulation during NPC differentiation.

DOI： 10.1038/srep23903

Language：English   Publishing type：Research paper (scientific journal)  

Neural stem cells (NSCs) have the ability to self-renew and give rise to neurons and glial cells (astrocytes and oligodendrocytes) in the mammalian central nervous system. This multipotency is acquired by NSCs during development and is maintained throughout life. Proliferation, fate specification, and maturation of NSCs are regulated by both cell intrinsic and extrinsic factors. Epigenetic modification is a representative intrinsic factor, being involved in many biological aspects of central nervous system development and adult neurogenesis through the regulation of NSC dynamics. In this review, we summarize recent progress in the epigenetic regulation of NSC behavior in the embryonic and adult brain, with particular reference to DNA methylation, histone modification, and noncoding RNAs.

DOI： 10.1016/j.nepig.2016.01.001

Language：English   Publishing type：Research paper (scientific journal)  

Despite recent advances in our understanding of epigenetic regulation of central nervous system development, little is known regarding the effects of epigenetic dysregulation on neurogenesis and brain function in adulthood. In the present study, we show that prenatal deletion of DNA methyltransferase 1 (Dnmt1) in neural stem cells results in impaired neurogenesis as well as increases in inflammatory features (e.g., elevated glial fibrillary acidic protein [GFAP] expression in astrocytes and increased numbers of microglia) in the adult mouse brain. Moreover, these mice exhibited anxiety-like behavior during an open-field test. These findings suggest that Dnmt1 plays a critical role in regulating neurogenesis and behavior in the developing brain and into adulthood.

DOI： 10.1080/23262133.2016.1232679

Language：English   Publishing type：Research paper (scientific journal)  

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.

DOI： 10.1016/j.stemcr.2015.10.012

Language：English   Publishing type：Research paper (scientific journal)  

Three-dimensional (3D) open-field gait analysis of mice is an essential procedure in genetic and nerve regeneration research. Existing gait analysis systems are generally expensive and may interfere with the natural behaviors of mice because of optical markers and transparent floors. In contrast, the proposed system captures the subjects shape from beneath using a low-cost infrared depth sensor (Microsoft Kinect) and an opaque infrared pass filter. This means that we can track footprints and 3D paw tip positions without optical markers or a transparent floor, thereby preventing any behavioral changes. Our experimental results suggest with healthy mice that they are more active on opaque floors and spend more time in the center of the open-field, when compared with transparent floors. The proposed system detected footprints with a comparable performance to existing systems, and precisely tracked the 3D paw-tip positions in the depth image coordinates. (C) 2015 The Authors. Published by Elsevier Ireland Ltd.

DOI： 10.1016/j.neures.2015.06.006

Language：English   Publishing type：Research paper (scientific journal)  

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.

DOI： 10.1016/j.celrep.2015.08.028

Language：English   Publishing type：Research paper (scientific journal)  

DOI： 10.2174/1574888X1005150820103608

Language：English   Publishing type：Research paper (scientific journal)  

Adult neurogenesis persists throughout life in the dentate gyrus (DG) of the hippocampus, and its importance has been highlighted in hippocampus-dependent learning and memory. Adult neurogenesis consists of multiple processes: maintenance and neuronal differentiation of neural stem/precursor cells (NS/PCs), followed by survival and maturation of newborn neurons and their integration into existing neuronal circuitry. However, the mechanisms that govern these processes remain largely unclear. Here we show that DNA methyltransferase 1 (DNMT1), an enzyme responsible for the maintenance of DNA methylation, is highly expressed in proliferative cells in the adult DG and plays an important role in the survival of newly generated neurons. Deletion of Dnmtl in adult NS/PCs (aNS/PCs) did not affect the proliferation and differentiation of aNS/PCs per se. However, it resulted in a decrease of newly generated mature neurons, probably due to gradual cell death after aNS/PCs differentiated into neurons in the hippocampus. Interestingly, loss of DNMT1 in post-mitotic neurons did not influence their survival. Taken together, these findings suggest that the presence of DNMT1 in aNS/PCs is crucial for the survival of newly generated neurons, but is dispensable once they accomplish neuronal differentiation in the adult hippocampus. (C) 2015 Elsevier Ireland Ltd and the Japan Neuroscience Society. All rights reserved.

DOI： 10.1016/j.neures.2015.01.014

Language：English   Publishing type：Research paper (scientific journal)  

Although the central nervous system is considered a comparatively static tissue with limited cell turnover, cells with stem cell properties have been isolated from most neural tissues. The spinal cord ependymal cells show neural stem cell potential in vitro and in vivo in injured spinal cord. However, very little is known regarding the ependymal niche in the mouse spinal cord. We previously reported that a secreted factor, chick Akhirin, is expressed in the ciliary marginal zone of the eye, where it works as a heterophilic cell-adhesion molecule. Here, we describe a new crucial function for mouse Akhirin (M-AKH) in regulating the proliferation and differentiation of progenitors in the mouse spinal cord. During embryonic spinal cord development, M-AKH is transiently expressed in the central canal ependymal cells, which possess latent neural stem cell properties. Targeted inactivation of the AKH gene in mice causes a reduction in the size of the spinal cord and decreases BrdU incorporation in the spinal cord. Remarkably, the expression patterns of ependymal niche molecules in AKH knockout (AKH-/-) mice are different from those of AKH+/+, both in vitro and in vivo. Furthermore, we provide evidence that AKH expression in the central canal is rapidly upregulated in the injured spinal cord. Taken together, these results indicate that M-AKH plays a crucial role in mouse spinal cord formation by regulating the ependymal niche in the central canal. (c) 2014 Wiley Periodicals, Inc. Develop Neurobiol 75: 494-504, 2015

DOI： 10.1002/dneu.22238

Language：English   Publishing type：Research paper (scientific journal)  

Background: Rett syndrome (RTT) is one of the most prevalent neurodevelopmental disorders in females, caused by de novo mutations in the X-linked methyl CpG-binding protein 2 gene, MECP2. Although abnormal regulation of neuronal genes due to mutant MeCP2 is thought to induce autistic behavior and impaired development in RTT patients, precise cellular mechanisms underlying the aberrant neural progression remain unclear.
Results: Two sets of isogenic pairs of either wild-type or mutant MECP2-expressing human induced pluripotent stem cell (hiPSC) lines were generated from a single pair of 10-year-old RTT-monozygotic (MZ) female twins. Mutant MeCP2-expressing hiPSC lines did not express detectable MeCP2 protein during any stage of differentiation. The lack of MeCP2 reflected altered gene expression patterns in differentiated neural cells rather than in undifferentiated hiPSCs, as assessed by microarray analysis. Furthermore, MeCP2 deficiency in the neural cell lineage increased astrocyte-specific differentiation from multipotent neural stem cells. Additionally, chromatin immunoprecipitation (ChIP) and bisulfite sequencing assays indicated that anomalous glial fibrillary acidic protein gene (GFAP) expression in the MeCP2-negative, differentiated neural cells resulted from the absence of MeCP2 binding to the GFAP gene.
Conclusions: An isogenic RTT-hiPSC model demonstrated that MeCP2 participates in the differentiation of neural cells. Moreover, MeCP2 deficiency triggers perturbation of astrocytic gene expression, yielding accelerated astrocyte formation from RTT-hiPSC-derived neural stem cells. These findings are likely to shed new light on astrocytic abnormalities in RTT, and suggest that astrocytes, which are required for neuronal homeostasis and function, might be a new target of RTT therapy.

DOI： 10.1186/s13041-015-0121-2

Language：English   Publishing type：Research paper (scientific journal)  

Pathological conditions such as epilepsy cause misregulation of adult neural stem/progenitor populations in the adult hippocampus in mice, and the resulting abnormal neurogenesis leads to impairment in learning and memory. However, how animals cope with abnormal neurogenesis remains unknown. Here we show that microglia in the mouse hippocampus attenuate convulsive seizure-mediated aberrant neurogenesis through the activation of Toll-like receptor 9 (TLR9), an innate immune sensor known to recognize microbial DNA and trigger inflammatory responses. We found that microglia sense self-DNA from degenerating neurons following seizure, and secrete tumour necrosis factor-a, resulting in attenuation of aberrant neurogenesis. Furthermore, TLR9 deficiency exacerbated seizure-induced cognitive decline and recurrent seizure severity. Our findings thus suggest the existence of bidirectional communication between the innate immune and nervous systems for the maintenance of adult brain integrity.

DOI： 10.1038/ncomms7514

Language：English   Publishing type：Research paper (scientific journal)  

In mice, zygotic activation occurs for a wide variety of genes, mainly at the 2-cell stage. Long noncoding RNAs (lncRNAs) are increasingly being recognized as modulators of gene expression. In this study, directional RNA-seq of MII oocytes and 2-cell embryos identified more than 1000 divergently transcribed lncRNA/mRNA gene pairs. Expression of these bidirectional promoter-associated noncoding RNAs (pancRNAs) was strongly associated with the upregulation of their cognate genes. Conversely, knockdown of three abundant pancRNAs led to reduced mRNA expression, accompanied by sustained DNA methylation even in the presence of enzymes responsible for DNA demethylation. In particular, microinjection of siRNA against the abundant pancRNA partner of interleukin 17d (II17d) mRNA at the 1-cell stage caused embryonic lethality, which was rescued by supplying IL17D protein in vitro at the 4-cell stage. Thus, this novel class of lncRNAs can modulate the transcription machinery in cis to activate zygotic genes and is important for preimplantation development.

DOI： 10.1242/dev.116996

Language：English  

Nuclear protein 95 KDa (Np95, also known as UHRF1 or ICBP90) plays an important role in maintaining DNA methylation of newly synthesized DNA strands by recruiting DNA methyltransferase 1 (DNMT1) during cell division. In addition, Np95 participates in chromatin remodeling by interacting with histone modification enzymes such as histone deacetylases. However, its expression pattern and function in the brain have not been analyzed extensively. We here investigated the expression pattern of Np95 in the mouse brain, from developmental to adult stages. In the fetal brain, Np95 is abundantly expressed at the midgestational stage, when a large number of neural stem/precursor cells (NS/PCs) exist. Interestingly, Np95 is expressed specifically in NS/PCs but not in differentiated cells such as neurons or glial cells. Furthermore, we demonstrate that Np95 is preferentially expressed in type 2a cells, which are highly proliferative NS/PCs in the dentate gyrus of the adult hippocampus. Moreover, the number of Np95-expressing cells increases in response to kainic acid administration or to voluntary running, which are known to enhance the proliferation of adult NS/PCs. These results suggest that Np95 participates in the process of proliferation and differentiation of NS/PCs, and that it should be a useful novel marker for proliferating NS/PCs, facilitating the analysis of the complex behavior of NS/PCs in the brain.

DOI： 10.4161/23262133.2014.976026

Language：English  

The regenerative capacity of an injured spinal cord is limited, and once paralysis occurs, it is difficult to improve the situation. We have developed a treatment method called the HINT method (HDAC Inhibitor and Neural stem cell Transplantation) that improves hind limb motor functionality in mice with severe spinal cord injuries by using a combination of antiepileptic drugs and neural stem cells. This chapter will introduce you to the new neural damage reconstruction mechanism employed in the HINT method.

DOI： 10.1007/978-4-431-54502-6_20

Language：English  

The regenerative capacity of an injured spinal cord is limited, and once paralysis occurs, it is difficult to improve the situation. We have developed a treatment method called the HINT method (HDAC Inhibitor and Neural stem cell Transplantation) that improves hind limb motor functionality in mice with severe spinal cord injuries by using a combination of antiepileptic drugs and neural stem cells. This chapter will introduce you to the new neural damage reconstruction mechanism employed in the HINT method.

DOI： 10.1007/978-4-431-54502-6_20

Language：English  

In the mammalian brain, epigenetic mechanisms are clearly involved in the regulation of self-renewal of neural stem cells and the derivation of their descendants, i.e. neurons, astrocytes and oligodendrocytes, according to the developmental timing and the microenvironment, the 'niche'. Interestingly, local epigenetic changes occur, concomitantly with genome-wide level changes, at a set of gene promoter regions for either down- or upregulation of the gene. In addition, intergenic regions also sensitize the availability of epigenetic modifiers, which affects gene expression through a relatively long-range chromatinic interaction with the transcription regulatory machineries including non-coding RNA (ncRNA) such as promoter-associated ncRNA and enhancer ncRNA. We show that such an epigenetic landscape in a neural cell is statically but flexibly formed together with a variable combination of generally and locally acting nuclear molecules including master transcription factors and cell-cycle regulators. We also discuss the possibility that revealing the epigenetic regulation by the local DNA RNA protein assemblies would promote methodological innovations, e.g. neural cell reprogramming, engineering and transplantation, to manipulate neuronal and glial cell fates for the purpose of medical use of these cells.

DOI： 10.1098/rstb.2013.0511

Language：English   Publishing type：Research paper (scientific journal)  

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.

DOI： 10.1371/journal.pone.0100215

Language：English  

For higher-order functions of the mammalian brain such as the regulation of motor behavior, consciousness, emotion, learning, and memory, neurons have to establish complicated and elaborate networks. In addition, the functions of neurons are critically supported by glial cells (astrocytes and oligodendrocytes). All of these neural cell types (i.e., neurons, astrocytes, and oligodendrocytes) are generated from common neural stem cells (NSCs), which also have self-renewal activity. Accumulating evidence suggests that the behavior of NSCs is influenced spatiotemporally by both cell-extrinsic factors, including cytokine signaling, and cell-intrinsic epigenetic changes, which together regulate the proliferation and fate decisions of NSCs to produce glial cells or neurons, including different neuronal subtypes, in a spatiotemporal manner. In the first half of this chapter, we summarize recent advances in elucidating the role of epigenetic control in the differentiation of NSCs. In postmitotic neurons, as well as NSCs, several orchestrated epigenetic mechanisms underlie neuronal functioning critical for memory formation. Recent studies have revealed the presence and physiological significance of changes of the epigenetic modifications within a neuron of an already defined cell fate. A dynamic change of epigenetic status induced by neuronal activity can alter synaptic plasticity, which constitutes part of the mechanisms of learning and memory. In the latter half of the chapter, we describe the role of epigenetic plasticity in non-dividing neurons. We also discuss the robust identity of the neuronal cell fate, as exemplified by the extremely poor ability of neurons to be reprogrammed to pluripotent stem cells.

DOI： 10.1016/B978-0-12-417114-5.00004-8

Language：English  

For higher-order functions of the mammalian brain such as the regulation of motor behavior, consciousness, emotion, learning, and memory, neurons have to establish complicated and elaborate networks. In addition, the functions of neurons are critically supported by glial cells (astrocytes and oligodendrocytes). All of these neural cell types (i.e., neurons, astrocytes, and oligodendrocytes) are generated from common neural stem cells (NSCs), which also have self-renewal activity. Accumulating evidence suggests that the behavior of NSCs is influenced spatiotemporally by both cell-extrinsic factors, including cytokine signaling, and cell-intrinsic epigenetic changes, which together regulate the proliferation and fate decisions of NSCs to produce glial cells or neurons, including different neuronal subtypes, in a spatiotemporal manner. In the first half of this chapter, we summarize recent advances in elucidating the role of epigenetic control in the differentiation of NSCs. In postmitotic neurons, as well as NSCs, several orchestrated epigenetic mechanisms underlie neuronal functioning critical for memory formation. Recent studies have revealed the presence and physiological significance of changes of the epigenetic modifications within a neuron of an already defined cell fate. A dynamic change of epigenetic status induced by neuronal activity can alter synaptic plasticity, which constitutes part of the mechanisms of learning and memory. In the latter half of the chapter, we describe the role of epigenetic plasticity in non-dividing neurons. We also discuss the robust identity of the neuronal cell fate, as exemplified by the extremely poor ability of neurons to be reprogrammed to pluripotent stem cells.

DOI： 10.1016/B978-0-12-417114-5.00004-8

Language：English   Publishing type：Research paper (scientific journal)  

Understanding how dopamine (DA) phenotypes are acquired in midbrain DA (mDA) neuron development is important for bioassays and cell replacement therapy for mDA neuron-associated disorders. Here, we demonstrate a feed-forward mechanism of mDA neuron development involving Nurr1 and Foxa2. Nurr1 acts as a transcription factor for DA phenotype gene expression. However, Nurr1-mediated DA gene expression was inactivated by forming a protein complex with CoREST, and then recruiting histone deacetylase 1 (Hdac1), an enzyme catalyzing histone deacetylation, to DA gene promoters. Coexpression of Nurr1 and Foxa2 was established in mDA neuron precursor cells by a positive cross-regulatory loop. In the presence of Foxa2, the Nurr1-CoREST interaction was diminished (by competitive formation of the Nurr1-Foxa2 activator complex), and CoRESTHdac1 proteins were less enriched in DA gene promoters. Consequently, histone 3 acetylation (H3Ac), which is responsible for open chromatin structures, was strikingly increased at DA phenotype gene promoters. These data establish the interplay of Nurr1 and Foxa2 as the crucial determinant for DA phenotype acquisition during mDA neuron development.

DOI： 10.1242/dev.095802

Language：English   Publishing type：Research paper (scientific journal)  

Background: The majority of non-coding RNAs (ncRNAs) involved in mRNA metabolism in mammals have been believed to downregulate the corresponding mRNA expression level in a pre- or post-transcriptional manner by forming short or long ncRNA-mRNA duplex structures. Information on non-duplex-forming long ncRNAs is now also rapidly accumulating. To examine the directional properties of transcription at the whole-genome level, we performed directional RNA-seq analysis of mouse and chimpanzee tissue samples.
Results: We found that there is only about 1&#37; of the genome where both the top and bottom strands are utilized for transcription, suggesting that RNA-RNA duplexes are not abundantly formed. Focusing on transcription start sites (TSSs) of protein-coding genes revealed that a significant fraction of them contain switching-points that separate antisense- and sense-biased transcription, suggesting that head-to-head transcription is more prevalent than previously thought. More than 90&#37; of head-to-head type promoters contain CpG islands. Moreover, CCG and CGG repeats are significantly enriched in the upstream regions and downstream regions, respectively, of TSSs located in head-to-head type promoters. Genes with tissue-specific promoter-associated ncRNAs (pancRNAs) show a positive correlation between the expression of their pancRNA and mRNA, which is in accord with the proposed role of pancRNA in facultative gene activation, whereas genes with constitutive expression generally lack pancRNAs.
Conclusions: We propose that single-stranded ncRNA resulting from head-to-head transcription at GC-rich sequences regulates tissue-specific gene expression.

DOI： 10.1186/1471-2164-15-35

Language：English   Publishing type：Research paper (scientific journal)  

In the mammalian central nervous system, astrocytes are the most abundant cell type and play crucial roles in brain development and function. Astrocytes are known to be produced from multipotent neural stem cells (NSCs) at the late gestational stage during brain development, and accumulating evidence indicates that this stage-dependent generation of astrocytes from NSCs is achieved by systematic cooperation between environmental cues and cell-intrinsic programs. Exemplifying the former is cytokine signaling through the gp130-Janus kinase/signal transducer and activator of transcription 3 pathway, and exemplifying the latter is epigenetic modification of astrocyte-specific genes. Here, we introduce recent advances in our understanding of the mechanisms that coordinate astrocytogenesis from NSCs by modulating signaling pathways and epigenetic programs, with a particular focus on the developing mammalian forebrain.

DOI： 10.1016/j.conb.2013.06.002

Language：English   Publishing type：Research paper (scientific journal)  

The cerebral cortex comprises over three quarters of the brain, and serves as structural basis for the sophisticated perceptual and cognitive functions. It develops from common multipotent neural stem cells (NSCs) that line the neural tube. Development of the NSCs encompasses sequential phases of progenitor expansion, neurogenesis, and gliogenesis along with the progression of developmental stages. Interestingly, NSCs steadfastly march through all of these phases and give rise to specific neural cell types in a temporally defined and highly predictable manner. Herein, we delineate the intrinsic and extrinsic factors that dictate the progression and tempo of NSC differentiation during cerebral cortex development, and how epigenetic modifications contribute to the dynamic properties of NSCs. (C) 2013 ISDN. Published by Elsevier Ltd. All rights reserved.

DOI： 10.1016/j.ijdevneu.2013.02.006

Language：English   Publishing type：Research paper (scientific journal)  

Suberoylanilide hydroxamic acid (SAHA) is one of the epidrugs developed for cancer treatment that works epigenetically by inhibiting histone deacetylases (HDACs). SAHA has been reported to diffuse across the placenta and found in fetal plasma in preclinical study, implying that it can influence fetus if taken by pregnant cancer patients. However, report regarding this aspect and the study of in utero HDAC inhibition by SAHA especially on fate specification of neural stem/progenitor cells within the developing mammalian cortex, is yet unavailable. Here we show that transient exposure of SAHA to mouse embryos during prominent neurogenic period resulted in an enhancement of cortical neurogenesis, which is accompanied by an increased expression of proneuronal transcription factor Neurog1 Neurogenesis was enhanced due to the increase number of proliferating Tbr2+ intermediate progenitor cells following SAHA exposure. In this relation, we observed that SAHA perturbed neonatal cortical lamination because of the increased production of Cux1+ and Satb2+ upper-layer neurons, and decreased that of Ctip2+ deep-layer neurons. Furthermore, an upper-layer neuronal lineage determinant Satb2 was also up-regulated, whereas those of deep-layer ones Fezf2 and Ctip2 were down-regulated by SAHA treatment. Taken together, our study suggests that proper regulation of HDACs is important for precise embryonic corticogenesis. (C) 2013 Elsevier Ireland Ltd and the Japan Neuroscience Society. All rights reserved.

DOI： 10.1016/j.neures.2013.06.004

Language：English   Publishing type：Research paper (scientific journal)  

DNA demethylation of astrocyte-specific gene promoters and STAT3 activation in neural precursor cells (NPCs) are essential for astrogliogenesis in the developing brain. To date, it remains unclear whether DNA methylation is the sole epigenetic determinant responsible for suppressing astrocyte-specific genes. Here, we used mouse embryonic stem cells (TKO ESCs) that lacked all 3 DNA methyltransferase genes, Dnmt1, Dnmt3a, and Dnmt3b, and thereby exhibit complete demethylation of the astrocyte-specific glial fibrillary acidic protein (Gfap) gene promoter. We found that although the Gfap promoter was demethylated, STAT3 failed to bind to its cognate element to induce Gfap transcription, whereas it induced transcription of a different target gene, Socs3. Moreover, although the Gfap promoter region containing the STAT3-binding site (GSBS) is enriched with transcription-repressive histone modifications, such as methylation of H3 at lysine 9 (H3K9me3) and H3K27me3, the reduction of these modifications in TKO ESCs was not sufficient for binding of STAT3 at GSBS. Furthermore, GSBS was digested by micrococcal nuclease in late-gestational NPCs that express GFAP upon LIF stimulation, but not in cells that show no expression of GFAP even in the presence of LIF, indicating that STAT3 can access GSBS in the former cells. We further showed that expression of NF-1A, which is known to potentiate differentiation of mid-gestational NPCs into astrocytes, increased its accessibility. Taken together, our results suggest that chromatin accessibility of GSBS plays a critical role in the regulation of Gfap expression.

DOI： 10.1247/csf.12034

Language：English   Publishing type：Research paper (scientific journal)  

ASIS is a member of the CREB/ATF family of transcription factors and modulates cell-or tissue-specific unfolded protein response signalling. Here we show that this modulation has a critical role in the differentiation of neural precursor cells into astrocytes. Cerebral cortices of mice specifically deficient in OASIS (Oasis(-/-)) contain fewer astrocytes and more neural precursor cells than those of wild-type mice during embryonic development. Furthermore, astrocyte differentiation is delayed in primary cultured Oasis(-/-) neural precursor cells. The transcription factor Gcm1, which is necessary for astrocyte differentiation in Drosophila, is revealed to be a target of OASIS. Introduction of Gcm1 into Oasis(-/-) neural precursor cells improves the delayed differentiation of neural precursor cells into astrocytes by accelerating demethylation of the Gfap promoter. Gcm1 expression is temporally controlled by the unfolded protein response through interactions between OASIS family members during astrocyte differentiation. Taken together, our findings demonstrate a novel mechanism by which OASIS and its associated family members are modulated by the unfolded protein response to finely control astrocyte differentiation.

DOI： 10.1038/ncomms1971

Language：English   Publishing type：Research paper (scientific journal)  

Because of their ability to self-renew, to differentiate into multiple lineages, and to migrate toward a damaged site, neural stem cells (NSCs), which can be derived from various sources such as fetal tissues and embryonic stem cells, are currently considered to be promising components of cell replacement strategies aimed at treating injuries of the central nervous system, including the spinal cord. Despite their efficiency in promoting functional recovery, these NSCs are not homogeneous and possess variable characteristics depending on their derivation protocols. The advent of induced pluripotent stem (iPS) cells has provided new prospects for regenerative medicine. We used a recently developed robust and stable protocol for the generation of long-term, self-renewing, neuroepithelial-like stem cells from human iPS cells (hiPS-lt-NES cells), which can provide a homogeneous and well-defined population of NSCs for standardized analysis. Here, we show that transplanted hiPS-lt-NES cells differentiate into neural lineages in the mouse model of spinal cord injury (SCI) and promote functional recovery of hind limb motor function. Furthermore, using two different neuronal tracers and ablation of the transplanted cells, we revealed that transplanted hiPS-lt-NES cell-derived neurons, together with the surviving endogenous neurons, contributed to restored motor function. Both types of neurons reconstructed the corticospinal tract by forming synaptic connections and integrating neuronal circuits. Our findings indicate that hiPS-lt-NES transplantation represents a promising avenue for effective cell-based treatment of SCI. STEM CELLS2012;30:11631173

DOI： 10.1002/stem.1083

Language：English   Publishing type：Research paper (scientific journal)  

Oxygen Levels Epigenetically Regulate Fate Switching of Neural Precursor Cells via Hypoxia-Inducible Factor 1 alpha-Notch Signal Interaction in the Developing Brain
Oxygen levels in tissues including the embryonic brain are lower than those in the atmosphere. We reported previously that Notch signal activation induces demethylation of astrocytic genes, conferring astrocyte differentiation ability on midgestational neural precursor cells (mgNPCs). Here, we show that the oxygen sensor hypoxia-inducible factor 1 alpha (HIF1 alpha) plays a critical role in astrocytic gene demethylation in mgNPCs by cooperating with the Notch signaling pathway. Expression of constitutively active HIF1 alpha and a hyperoxic environment, respectively, promoted and impeded astrocyte differentiation in the developing brain. Our findings suggest that hypoxia contributes to the appropriate scheduling of mgNPC fate determination. STEM CELLS 2012;30:561-569

DOI： 10.1002/stem.1019

Language：English   Publishing type：Research paper (scientific journal)  

Within the developing mammalian cortex, neural progenitors first generate deep-layer neurons and subsequently more superficial-layer neurons, in an inside-out manner. It has been reported recently that mouse embryonic stem cells (mESCs) can, to some extent, recapitulate cortical development in vitro, with the sequential appearance of neurogenesis markers resembling that in the developing cortex. However, mESCs can only recapitulate early corticogenesis; superficial-layer neurons, which are normally produced in later developmental periods in vivo, are under-represented. This failure of mESCs to reproduce later corticogenesis in vitro implies the existence of crucial factor(s) that are absent or uninduced in existing culture systems. Here we show that mESCs can give rise to superficial-layer neurons efficiently when treated with valproic acid (VPA), a histone deacetylase inhibitor. VPA treatment increased the production of Cux1-positive superficial-layer neurons, and decreased that of Ctip2-positive deep-layer neurons. These results shed new light on the mechanisms of later corticogenesis. (C) 2011 Elsevier Ireland Ltd and the Japan Neuroscience Society. All rights reserved.

DOI： 10.1016/j.neures.2011.09.012

Language：English   Publishing type：Research paper (scientific journal)  

In the present study, we demonstrated that insulin is produced not only in the mammalian pancreas but also in adult neuronal cells derived from the hippocampus and olfactory bulb (OB). Paracrine Wnt3 plays an essential role in promoting the active expression of insulin in both hippocampal and OB-derived neural stem cells. Our analysis indicated that the balance between Wnt3, which triggers the expression of insulin via NeuroD1, and IGFBP-4, which inhibits the original Wnt3 action, is regulated depending on diabetic (DB) status. We also show that adult neural progenitors derived from DB animals retain the ability to give rise to insulin-producing cells and that grafting neuronal progenitors into the pancreas of DB animals reduces glucose levels. This study provides an example of a simple and direct use of adult stem cells from one organ to another, without introducing additional inductive genes.

DOI： 10.1002/emmm.201100177

Language：English   Publishing type：Research paper (scientific journal)  

The Hedgehog pathway functions as an organizer in embryonic development. Aberrant activation of the Hedgehog pathway has been reported in various types of malignant tumours. The GLI2 transcription factor is a key mediator of Hedgehog pathway but its contribution to neoplasia is poorly understood. To establish the role of GLI2 in osteosarcoma, we examined its expression by real-time PCR using biopsy tissues. To examine the function of GLI2, we evaluated the growth of osteosarcoma cells and their cell cycle after GLI2 knockdown. To study the effect of GLI2 activation, we examined mesenchymal stem cell growth and the cell cycle after forced expression of GLI2. We found that GLI2 was aberrantly over-expressed in human osteosarcoma biopsy specimens. GLI2 knockdown by RNA interferences prevented osteosarcoma growth and anchorage-independent growth. Knockdown of GLI2 promoted the arrest of osteosarcoma cells in G(1) phase and was accompanied by reduced protein expression of the cell cycle accelerators cyclin D1, SKP2 and phosphorylated Rb. On the other hand, knockdown of GLI2 increased the expression of p21(cip1). In addition, over-expression of GLI2 promoted mesenchymal stem cell proliferation and accelerated their cell cycle progression. Finally, evaluation of mouse xenograft models showed that GLI2 knockdown inhibited the growth of osteosarcoma in nude mice. Our findings suggest that inhibition of GLI2 may represent an effective therapeutic approach for patients with osteosarcoma. Copyright (C) 2011 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

DOI： 10.1002/path.2880

Language：English   Publishing type：Research paper (scientific journal)  

Long interspersed nuclear elements-1 (LINE-1 or L1s) are abundant retrotransposons that comprise approximately 20% of mammalian genomes(1-3). Active L1 retrotransposons can impact the genome in a variety of ways, creating insertions, deletions, new splice sites or gene expression fine-tuning(4-6). We have shown previously that L1 retrotransposons are capable of mobilization in neuronal progenitor cells from rodents and humans and evidence of massive L1 insertions was observed in adult brain tissues but not in other somatic tissues(7,8). In addition, L1 mobility in the adult hippocampus can be influenced by the environment(9). The neuronal specificity of somatic L1 retrotransposition in neural progenitors is partially due to the transition of a Sox2/HDAC1 repressor complex to a Wnt-mediated T-cell factor/lymphoid enhancer factor (TCF/LEF) transcriptional activator(7,10). The transcriptional switch accompanies chromatin remodelling during neuronal differentiation, allowing a transient stimulation of L1 transcription(7). The activity of L1 retrotransposons during brain development can have an impact on gene expression and neuronal function, thereby increasing brain-specific genetic mosaicism(11,12). Further understanding of the molecular mechanisms that regulate L1 expression should provide new insights into the role of L1 retrotransposition during brain development. Here we show that L1 neuronal transcription and retrotransposition in rodents are increased in the absence of methyl-CpG-binding protein 2 (MeCP2), a protein involved in global DNA methylation and human neurodevelopmental diseases. Using neuronal progenitor cells derived from human induced pluripotent stem cells and human tissues, we revealed that patients with Rett syndrome (RTT), carrying MeCP2 mutations, have increased susceptibility for L1 retrotransposition. Our data demonstrate that L1 retrotransposition can be controlled in a tissue-specific manner and that disease-related genetic mutations can influence the frequency of neuronal L1 retrotransposition. Our findings add a new level of complexity to the molecular events that can lead to neurological disorders.

DOI： 10.1038/nature09544

Language：English   Publishing type：Research paper (scientific journal)  

The body's capacity to restore damaged neural networks in the injured CNS is severely limited. Although various treatment regimens can partially alleviate spinal cord injury (SCI), the mechanisms responsible for symptomatic improvement remain elusive. Here, using a mouse model of SCI, we have shown that transplantation of neural stem cells (NSCs) together with administration of valproic acid (VPA), a known antiepileptic and histone deacetylase inhibitor, dramatically enhanced the restoration of hind limb function. VPA treatment promoted the differentiation of transplanted NSCs into neurons rather than glial cells. Transsynaptic anterograde corticospinal tract tracing revealed that transplant-derived neurons reconstructed broken neuronal circuits, and electron microscopic analysis revealed that the transplant-derived neurons both received and sent synaptic connections to endogenous neurons. Ablation of the transplanted cells abolished the recovery of hind limb motor function, confirming that NSC transplantation directly contributed to restored motor function. These findings raise the possibility that epigenetic status in transplanted NSCs can be manipulated to provide effective treatment for SCI.

DOI： 10.1172/JCI42957

Language：English   Publishing type：Research paper (scientific journal)  

Chromatin remodeling is a dynamic alteration of chromatin structure that regulates several important biological processes. It is brought about by enzymatic activities that catalyze covalent modifications of histone tail or movement of nucleosomes along the DNA, and these activities often require multisubunit protein complexes for its proper functions. In concert with DNA methylation and noncoding RNA-mediated processes, histone modification such as acetylation and methylation regulates gene expression epigenetically, without affecting DNA sequence. Recent advances have revealed that this intrinsic regulation, together with protein complexes such as RE1 silencer of transcription/neuron-restrictive silencer factor (REST/NRSF) and switch/sucrose nonfermentable (SWI/SNF), play critical roles in neural stem cell fate determination.

DOI： 10.1016/j.conb.2010.04.001

Language：English  

The central nervous system (CNS) is composed of three major cell types - neurons, astrocytes, and oligodendrocytes - which differentiate from common multipotent neural stem cells (NSCs). This differentiation process is regulated spatiotemporally during the course of mammalian development. It is becoming apparent that epigenetic regulation is an important cell-intrinsic program, which can interact with transcription factors and environmental cues to modulate the differentiation of NSCs. This knowledge is important given the potential of NSCs to produce specific CNS cell types that will be beneficial for clinical applications. Here we review recent findings that address molecular mechanisms of epigenetic and transcription factor-mediated regulation that specify NSC fate during CNS development, with a particular focus on the developing mammalian forebrain.

DOI： 10.1111/j.1440-169X.2010.01175.x

Language：English   Publishing type：Research paper (scientific journal)  

Neural stem cells (NSCs) in the adult hippocampus divide infrequently, and the molecules that modulate their quiescence are largely unknown. Here, we show that bone morphogenetic protein (BMP) signaling is active in hippocampal NSCs, downstream of BMPR-IA. BMPs reversibly diminish proliferation of cultured NSCs while maintaining their undifferentiated state. In vivo, acute blockade of BMP signaling in the hippocampus by intracerebral infusion of Noggin first recruits quiescent NSCs into the cycle and increases neurogenesis; subsequently, it leads to decreased stem cell division and depletion of precursors and newborn neurons. Consistently, selective ablation of Bmpr1a in hippocampal NSCs, or inactivation of BMP canonical signaling in conditional Smad4 knockout mice, transiently enhances proliferation but later leads to a reduced number of precursors, thereby limiting neuronal birth. BMPs are therefore required to balance NSC quiescence/proliferation and to prevent loss of the stem cell activity that supports continuous neurogenesis in the mature hippocampus.

DOI： 10.1016/j.stem.2010.04.016

Language：English   Publishing type：Research paper (scientific journal)  

Once they have differentiated, cells retain their individual character and repress genes that are specifically expressed in other cell lineages, but how alternative fate choice is restricted during and/or after differentiation remains unclear. In the mammalian central nervous system, neurons, astrocytes, and oligodendrocytes are generated throughout life from common tripotent neural progenitor cells (NPCs). Bone morphogenetic proteins (BMPs) are well-known astrocyte-inducing cytokines. We show here that the expression of a transcriptional repressor, RE1 silencer of transcription (REST)/neuron-restrictive silencer factor (NRSF), is up-regulated and sustained by BMP signal activation in the course of astrocytic differentiation of NPCs, and restricts neuronal differentiation. We further show that, in differentiated astrocytes, endogenous REST/NRSF associates with various neuronal genes and that disruption of its function resulted in their derepression, thereby explaining how ectopic neuronal gene expression is prevented in cells with astrocytic traits. Collectively, our results suggest that REST/NRSF functions as a molecular regulator of the nonneuronal phenotype in astrocytes.

DOI： 10.1083/jcb.200908048

Language：English   Publishing type：Research paper (scientific journal)  

Neurons, astrocytes, and oligodendrocytes-the three major cell types that comprise the central nervous system-are generated from common multipotent neural precursor cells (NPCs). Members of the interleukin-6 family of cytokines, including leukemia inhibitory factor (LIF), induce astrocyte differentiation of NPCs by activating the transcription factor signal transducer and activator of transcription 3 (STAT3). We show here that retinoic acid (RA) facilitates LIF-induced astrocyte differentiation of NPCs. RA and LIF synergistically activate the promoter of gfap, which encodes the astrocytic marker glial fibrillary acidic protein, and a putative RA response element in the promoter was found to be critical for this activation. Histone H3 acetylation around the STAT-binding site in the gfap promoter was increased in NPCs treated with RA, allowing STAT3 to gain access to the promoter more efficiently. These results suggest that RA acts in concert with LIF to induce astrocyte differentiation of NPCs through an epigenetic mechanism that involves cross-talk between distinct signaling pathways. Stem Cells 2009; 27: 2744-2752

DOI： 10.1002/stem.176

Language：English   Publishing type：Research paper (scientific journal)  

Methyl-CpG-binding protein 2 (MeCP2), a methyl-CpG-binding domain protein family member which is expressed predominantly in neurons in the nervous system, acts as a transcriptional repressor by binding to methylated genes, and mutations in mecp2 cause the neurological disorder known as Rett syndrome (RTT). Although MeCP2 has been reported to regulate neuronal maturation rather than fate specification of neural precursor cells (NPCs), we have previously shown that it inhibits astrocyte differentiation of NPCs when ectopically expressed. Here, we show that expression of MeCP2 in NPCs not only suppresses astrocytic differentiation but actually promotes neuronal differentiation, even in the presence of well-known astrocyte-inducing cytokines. This dual function of MeCP2 was abolished by the MEK inhibitor U0126. Moreover, we observed that a truncated form of MeCP2 found in RTT patients fails to promote neuronal differentiation. We further demonstrate that transplanted MeCP2-expressing NPCs differentiate in vivo into neurons in two non-neurogenic regions, striatum and spinal cord. These results suggest a possible therapeutic application for MeCP2 in neurodegenerative diseases and injuries to the central nervous system. (C) 2009 Elsevier Inc. All rights reserved.

DOI： 10.1016/j.expneurol.2009.05.001

Language：English   Publishing type：Research paper (scientific journal)  

In adult hippocampus, new neurons are continuously generated from neural stem cells (NSCs), but the molecular mechanisms regulating adult neurogenesis remain elusive. We found that Wnt signaling, together with the removal of Sox2, triggered the expression of NeuroD1 in mice. This transcriptional regulatory mechanism was dependent on a DNA element containing overlapping Sox2 and T-cell factor/lymphoid enhancer factor (TCF/LEF)-binding sites (Sox/LEF) in the promoter. Notably, Sox/LEF sites were also found in long interspersed nuclear element 1 (LINE-1) elements, consistent with their critical roles in the transition of NSCs to proliferating neuronal progenitors. Our results describe a previously unknown Wnt-mediated regulatory mechanism that simultaneously coordinates activation of NeuroD1 and LINE-1, which is important for adult neurogenesis and survival of neuronal progenitors. Moreover, the discovery that LINE-1 retro-elements embedded in the mammalian genome can function as bi-directional promoters suggests that Sox/LEF regulatory sites may represent a general mechanism, at least in part, for relaying environmental signals to other nearby loci to promote adult hippocampal neurogenesis.

DOI： 10.1038/nn.2360

Language：English   Publishing type：Research paper (scientific journal)  

During midgestation, mammalian neural precursor cells (NPCs) differentiate only into neurons. Generation of astrocytes is prevented at this stage, because astrocyte-specific gene promoters are methylated. How the subsequent switch from suppression to expression of astrocytic genes occurs is unknown. We show in this study that Notch ligands are expressed on committed neuronal precursors and young neurons in mid-gestational telencephalon, and that neighboring Notch-activated NPCs acquire the potential to become astrocytes. Activation of the Notch signaling pathway in midgestational NPCs induces expression of the transcription factor nuclear factor 1, which binds to astrocytic gene promoters, resulting in demethylation of astrocyte-specific genes. These findings provide a mechanistic explanation for why neurons come first: committed neuronal precursors and young neurons potentiate remaining NPCs to differentiate into the next cell lineage, astrocytes.

DOI： 10.1016/j.devcel.2008.12.014

Language：English   Publishing type：Research paper (scientific journal)  

To understand the cellular and molecular mechanisms regulating cytogenesis within the neocortical ventricular zone, we examined at high resolution the spatiotemporal expression patterns of Ngn2 and Tbr2. Individually Dil-labeled daughter cells were tracked from their birth in slice cultures and immunostained for Ngn2 and Tbr2. Both proteins were initially absent from daughter cells during the first 2 h. Ngn2 expression was then initiated asymmetrically in one of the daughter cells, with a bias towards the apical cell, followed by a similar pattern of expression for Tbr2, which we found to be a direct target of Ngn2. How this asymmetric Ngn2 expression is achieved is unclear, but gamma-secretase inhibition at the birth of daughter cells resulted in premature Ngn2 expression, Suggesting that Notch signaling in nascent daughter cells suppresses a Ngn2-Tbi-2 cascade. Many of the nascent cells exhibited pin-like morphology with a short ventricular process, Ksuggesting periventricular presentation of Notch ligands to these cells. (C) 2008 Elsevier Inc. All rights reserved.

DOI： 10.1016/j.mcn.2008.10.007

Language：English   Publishing type：Research paper (scientific journal)  

The major cell types in the central nervous system, neurons and glial cells (astrocytes and oligodendrocytes), are generated from common neural stem cells (NSCs). However, neurogenesis precedes gliogenesis, which normally commences at later stages of fetal brain development. Recent studies have shown that epigenetic gene regulation, in concert with cues from outside the cells, plays important roles in regulating the differentiation potential and fate specification of NSCs. In this review, we discuss emerging molecular explanations for the transitions that occur during the stepwise process of NSC fate specification.

DOI： 10.4161/epi.4.2.8233

Language：English  

Committed neuronal precursors confer astrocytic potential on residual neural precursor cells.
During midgestation, mammalian neural precursor cells (NPCs) differentiate only into neurons. Generation of astrocytes is prevented at this stage, because astrocyte-specific gene promoters are methylated. How the subsequent switch from suppression to expression of astrocytic genes occurs is unknown. We show in this study that Notch ligands are expressed on committed neuronal precursors and young neurons in mid-gestational telencephalon, and that neighboring Notch-activated NPCs acquire the potential to become astrocytes. Activation of the Notch signaling pathway in midgestational NPCs induces expression of the transcription factor nuclear factor I, which binds to astrocytic gene promoters, resulting in demethylation of astrocyte-specific genes. These findings provide a mechanistic explanation for why neurons come first: committed neuronal precursors and young neurons potentiate remaining NPCs to differentiate into the next cell lineage, astrocytes.

DOI： 10.1016/j.devcel.2008.12.014

Language：English   Publishing type：Research paper (scientific journal)  

Neural stem/progenitor cells (NSCs/NPCs) give rise to neurons, astrocytes, and oligodendrocytes. It has become apparent that intracellular epigenetic modification including DNA methylation, in concert with extracellular cues such as cytokine signaling, is deeply involved in fate specification of NSCs/NPCs by defining cell-type specific gene expression. However, it is still unclear how differentiated neural cells retain their specific attributes by repressing cellular properties characteristic of other lineages. In previous work we have shown that methyl-CpG binding protein transcriptional repressors (MBDs), which are expressed predominantly in neurons in the central nervous system, inhibit astrocyte-specific gene expression by binding to highly methylated regions of their target genes. Here we report that oligodendrocytes, which do not express MBDs, can transdifferentiate into astrocytes both in vitro (cytokine stimulation) and in vivo (ischemic injury) through the activation of the JAK/STAT signaling pathway. These findings suggest that differentiation plasticity in neural cells is regulated by cell-intrinsic epigenetic mechanisms in collaboration with ambient cell-extrinsic cues.

DOI： 10.1073/pnas.0808417105

Language：English   Publishing type：Research paper (scientific journal)  

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 S100b, 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.

DOI： 10.1371/journal.pone.0003189

Language：English   Publishing type：Research paper (scientific journal)  

Neural stem cells (NSCs) possess the ability to self-renew and to differentiate along neuronal and glial lineages. These processes are defined by the dynamic interplay between extracellular cues including cytokine signalling and intracellular programmes such as epigenetic modification. There is increasing evidence that epigenetic mechanisms involving, for example, changes in DNA methylation, histone modification and non-coding RNA expression are closely associated with fate specification of NSCs. These epigenetic alterations could provide coordinated systems for regulating gene expression at each step of neural cell differentiation. Here we review the roles of epigenetics in neural fate specification in the mammalian central nervous system.

DOI： 10.1098/rstb.2008.2262

Language：Others  

[Epigenetic regulation involved in fate specification of neural cells].

Language：English   Publishing type：Research paper (scientific journal)  

TLX is an orphan nuclear receptor that is expressed exclusively in vertebrate forebrains. Although TLX is known to be expressed in embryonic brains, the mechanism by which it influences neural development remains largely unknown. We show here that TLX is expressed specifically in periventricular neural stem cells in embryonic brains. Significant thinning of neocortex was observed in embryonic d 14.5 TLX-null brains with reduced nestin labeling and decreased cell proliferation in the germinal zone. Cell cycle analysis revealed both prolonged cell cycles and increased cell cycle exit in TLX-null embryonic brains. Increased expression of a cyclin-dependent kinase inhibitor p21 and decreased expression of cyclin D1 provide a molecular basis for the deficiency of cell cycle progression in embryonic brains of TLX-null mice. Furthermore, transient knockdown of TLX by in utero electroporation led to precocious cell cycle exit and differentiation of neural stem cells followed by outward migration. Together these results indicate that TLX plays an important role in neural development by regulating cell cycle progression and exit of neural stem cells in the developing brain.

DOI： 10.1210/me.2007-0290

Language：English   Publishing type：Research paper (scientific journal)  

Astrocytes play important roles in brain development and injury response. Transcription factors STAT3 and Smad1, activated by leukemia inhibitory factor (LIF) and bone morphogenetic protein 2 (BMP2), respectively, form a complex with the coactivator p300 to synergistically induce astrocytes from neuroepithelial cells (NECs) (K. Nakashima, M. Yanagisawa, H. Arakawa, N. Kimura, T. Hisatsune, M. Kawabata, K. Miyazono, and T. Taga, Science 284:479482, 1999). However, the mechanisms that govern astrogliogenesis during the determination of the fate of neural stem cells remain elusive. Here we found that LIF induces expression of BMP2 via STAT3 activation and leads to the consequent activation of Smad1 to efficiently promote astrogliogenic differentiation of NECs. The BMP antagonist Noggin abrogated LIF-induced Smadl activation and astrogliogenesis by inhibiting BMPs produced by NECs. NECs deficient in suppressor of cytokine signaling 3 (SOCS3), a negative regulator of STAT3, readily differentiated into astrocytes upon activation by LIF not only due to sustained activation of STAT3 but also because of the consequent activation of Smadl. Our study suggests a novel LIF-triggered positive regulatory loop that enhances astrogliogenesis.

DOI： 10.1128/MCB.02435-06

Language：English   Publishing type：Research paper (scientific journal)  

In the developing mouse brain, the highest Bcl-x(L) expression is seen at the peak of neurogenesis, whereas the peak of Bax expression coincides with the astrogenic period. While such observations suggest an active role of the Bcl-2 family proteins in the generation of neurons and astrocytes, no definitive demonstration has been provided to date. Using combinations of gain- and loss-of-function assays in vivo and in vitro, we provide evidence for instructive roles of these proteins in neuronal and astrocytic fate specification. Specifically, in Bax knockout mice, astrocyte formation was decreased in the developing cortices. Overexpression of Bcl-X-L and Bax in embryonic cortical precursors induced neural and astrocytic differentiation, respectively, while inhibitory RNAs led to the opposite results. Importantly, inhibition of caspase activity, dimerization, or mitochondrial localization of Bcl-X-L/Bax proteins indicated that the differentiation effects of Bcl-X-L/Bax are separable from their roles in cell survival and apoptosis. Lastly, we describe activation of intracellular signaling pathways and expression of basic helix-loop-helix transcriptional factors specific for the Bcl-2 protein-mediated differentiation.

DOI： 10.1128/MCB.00031-07

Language：English   Publishing type：Research paper (scientific journal)  

The conceptual understanding of hippocampal function has been challenged recently by the finding that new granule cells are born throughout life in the mammalian dentate gyrus (DG). The number of newborn neurons is dynamically regulated by a variety of factors. Kainic acid-induced seizures, a rodent model of human temporal lobe epilepsy, strongly induce the proliferation of DG neurogenic progenitor cells and are also associated with long-term cognitive impairment. We show here that the antiepileptic drug valproic acid (VPA) potently blocked seizure-induced neurogenesis, an effect that appeared to be mainly mediated by inhibiting histone deacetylases (HDAC) and normalizing HDAC-dependent gene expression within the epileptic dentate area. Strikingly, the inhibition of aberrant neurogenesis protected the animals from seizure-induced cognitive impairment in a hippocampus-dependent learning task. We propose that seizure-generated granule cells have the potential to interfere with hippocampal function and contribute to cognitive impairment caused by epileptic activity within the hippocampal circuitry. Furthermore, our data indicate that the effectiveness of VPA as an antiepileptic drug may be partially explained by the HDAC-dependent inhibition of aberrant neurogenesis induced by seizure activity within the adult hippocampus.

DOI： 10.1523/JNEUROSCI.0110-07.2007

Language：English   Publishing type：Research paper (scientific journal)  

Conversion of mammalian Muller glial cells into a neuronal lineage by in vitro aggregate-culture
Mammalian Muller glial cells are major glial cells in the retina. Here we report that these glial cells can be redirected towards a neuronal lineage by an aggregate-culture in vitro. Rat and macaque Muller glial cells did not express neuronal markers except after transfer to adhesive conditions. Furthermore, this expression could only take place in the presence of platelet-derived growth factor and valproic acid. We compared a normal monolayer-culture and an aggregate-culture, and rat Muller glial cells could only differentiate into neurons under non-adhesive conditions. However, Muller glial cells did not express the photoreceptor markers in vitro. After transplantation into the subretinal space, a retina-specific niche, rat Muller glial cells expressed the photoreceptor-specific marker, opsin (RET-P1). We demonstrate the potential of mammalian Muller glial cells as a source of photoreceptors, which may possibly contribute to the treatment of degenerative retinal diseases such as retinitis pigmentosa. (c) 2006 Elsevier Inc. All rights reserved.

DOI： 10.1016/j.bbrc.2006.10.072

Language：English   Publishing type：Research paper (scientific journal)  

Neurons and astrocytes are generated from common neural precursors, yet neurogenesis precedes astrocytogenesis, which normally commences at later stages of development. We have previously reported that a particular cytosine residue within a STAT3-binding site in the astrocyte-specific marker glial fibrillary acidic protein (GFAP) gene promoter becomes demethylated in neuroepithelial cells as gestation proceeds. This demethylation correlates tightly with the onset of astrocyte differentiation, suggesting that a change in DNA methylation at cell-type-specific gene promoters controls the switch from neurogenesis to astrocytogenesis in the developing brain. Here, we show that late-gestation neuroepithelial cells, which have already lost the methylation in the STAT3-binding site within the GFAP promoter, can still give rise to neurons and that these neurons do not respond to a STAT3-activating cytokine to express GFAP. Members of a transcriptional repressor family, the methylated-CpG binding proteins (MBDs), including MeCP2, are predominantly expressed in neurons, and ectopic MeCP2 expression inhibited astrocyte differentiation of neuroepithelial cells. Moreover, we found that exon 1 of the GFAP gene remains hypermethylated even in neuroepithelial cells at a late developmental stage and in neurons differentiated from such neuroepithelial cells. We further demonstrate that MeCP2 actually binds to the highly methylated exon 1 of the GFAP gene in neurons. These results suggest that region-specific DNA methylation and MBDs play an important role in the regulation of differentiation plasticity in neurons. (c) 2006 Wiley-Liss, Inc.

DOI： 10.1002/jnr.21001

Language：English   Publishing type：Research paper (scientific journal)  

Neuropoietin (NP) is a recently identified member of the interleukin (IL)-6 family of cytokines, which share glycoprotein 130 (gp130) as a signal-transducing receptor component, and is highly expressed in embryonic brain. In this study, we show that NP has the potential to induce neuroepithelial cells to differentiate into astrocytes. NP stimulation leads to promoter activation of the gene for an astrocyte marker, glial fibrillary acidic protein (GFAP), which is clearly inhibited by either expression of a dominant-negative form of a transcription factor, signal transducer and activator of transcription 3 (STAT3) or by a nucleotide-substitution in the STAT3-binding element within the gene promoter. We further show that NP induces binding of endogenous STAT3 to its cognate sequence within the gfap gene promoter in neuroepithelial cells. Moreover, like the other IL-6 cytokine family members, NP promotes astrocyte differentiation in a synergistic manner with bone-morphogenetic protein (BMP)-2. Taken together, our data indicate that NP can be considered as a new astrocyte-inducing cytokine in the developing brain. (c) 2006 Elsevier Ltd. All rights reserved.

DOI： 10.1016/j.cyto.2006.10.007

Language：English   Publishing type：Research paper (scientific journal)  

ES cells represent a valuable model for investigating early embryo development and hold promise for future regenerative medicine strategies. The self-renewal of pluripotent mouse ES cells has been shown to require extrinsic stimulation by the bone morphogenetic protein (BMP) and leukemia inhibitory factor signaling pathways and the expression of the transcription factors Oct4 and Nanog. However, the network of interactions among extrinsic and intrinsic determinants of ES cell pluripotency is currently poorly understood. Here, we show that Nanog expression is up-regulated in mouse ES cells by the binding of T (Brachyury) and STAT3 to an enhancer element in the mouse Nanog gene. We further show that Nanog blocks BMP-induced mesoderm differentiation of ES cells by physically interacting with Smad1 and interfering with the recruitment of coactivators to the active Smad transcriptional complexes. Taken together, our findings illustrate the existence of ES cell-specific regulatory networks that underlie the maintenance of ES cell pluripotency and provide mechanistic insights into the role of Nanog in this process.

DOI： 10.1073/pnas.0506945103

Language：English   Publishing type：Research paper (scientific journal)  

Multipotent neural stem/progenitor cells (NSPCs) can be isolated from many regions of the adult central nervous system (CNS), yet neurogenesis is restricted to the hippocampus and subventricular zone in vivo. Identification of the molecular cues that modulate NSPC fate choice is a prerequisite for their therapeutic applications. Previously, we demonstrated that primary astrocytes isolated from regions with higher neuroplasticity, such as newborn and adult hippocampus and newborn spinal cord, promoted neuronal differentiation of adult NSPCs, whereas astrocytes isolated from the nonneurogenic region of the adult spinal cord inhibited neural differentiation. To identify the factors expressed by these astrocytes that could modulate NSPC differentiation, we performed gene expression profiling analysis using Affymetrix rat genome arrays. Our results demonstrated that these astrocytes had distinct gene expression profiles. We further tested the functional effects of candidate factors that were differentially expressed in neurogenesis-promoting and -inhibiting astrocytes using in vitro NSPC differentiation assays. Our results indicated that two interleukins, IL-1 beta and IL-6, and a combination of factors that included these two interleukins could promote NSPC neuronal differentiation, whereas insulin-like growth factor binding protein 6 (IGFBP6) and decorin inhibited neuronal differentiation of adult NSPCs. Our results have provided further evidence to support the ongoing hypothesis that, in adult mammalian brains, astrocytes play critical roles in modulating NSPC differentiation. The finding that cytokines and chemokines expressed by astrocytes could promote NSPC neuronal differentiation may help us to understand how injuries induce neurogenesis in adult brains.

DOI： 10.1089/scd.2006.15.407

Language：Others  

Mechanisms of neural stem cell fate determination: extracellular cues and intracellular programs.

DOI： 10.2174/157488806776956887

Language：English   Publishing type：Research paper (scientific journal)  

Embryonic stem cells (ESCs) can be propagated indefinitely in culture, while retaining the ability to differentiate into any cell type in the organism. The molecular and cellular mechanisms underlying ESC pluripotency are, however, poorly understood. We characterize a population of early mesoderm-specified (EM) progenitors that is generated from mouse ESCs by bone morphogenetic protein stimulation. We further show that pluripotent ESCs are actively regenerated from EM progenitors by the action of the divergent homeodomain-containing protein Nanog, which, in turn, is upregulated in EM progenitors by the combined action of leukemia inhibitory factor and the early mesoderm transcription factor T/Brachyury. These findings uncover specific roles of leukemia inhibitory factor, Nanog, and bone morphogenetic protein in the self-renewal of ESCs and provide novel insights into the cellular bases of ESC pluripotency. © 2006 Nature Publishing Group.

DOI： 10.1038/ncpcardio0442

Language：English   Publishing type：Research paper (scientific journal)  

Human embryonic stem cells are pluripotent entities, theoretically capable of generating a whole-body spectrum of distinct cell types. However, differentiation of these cells has been observed only in culture or during teratoma formation. Our results show that human embryonic stem cells implanted in the brain ventricles of embryonic mice can differentiate into functional neural lineages and generate mature, active human neurons that successfully integrate into the adult mouse forebrain. Moreover, this study reveals the conservation and recognition of common signals for neural differentiation throughout mammalian evolution. The chimeric model will permit the study of human neural development in a live environment, paving the way for the generation of new models of human neurodegenerative and psychiatric diseases. The model also has the potential to speed up the screening process for therapeutic drugs.

DOI： 10.1073/pnas.0509315102

Language：English   Publishing type：Research paper (scientific journal)  

Mammalian cells that have been committed to a certain cell lineage cannot be directed to other lineages. However, some astrocytes in the mammalian brains have been reported to represent plasticity to redirect to other cell lineages. We found that mouse hippocampal astrocytes cultured in aggregate forms of "astrosphere", redirected to MAP2-positive immature neurons. In astrospheres, basic HLH factors positively regulating neuronal differentiation were up-regulated and Id3 inhibiting basic HLH factors was down-regulated. Ectopic Id3 induction repressed redirection of astrocytes to a neuronal lineage, suggesting that astrosphere formation induced plasticity of astrocytes by changing the gene expression patterns. (c) 2005 Elsevier Ireland Ltd and the Japan Neuroscience Society. All rights reserved.

DOI： 10.1016/j.neures.2005.06.015

Language：English   Publishing type：Research paper (scientific journal)  

Activation of the transcription factor STAT3 is important for astrocyte differentiation during neural development. Demethylation of the methyl-CpG dinucleotide in the STAT3 binding site in the promoter of the glial fibrillary acidic protein (GFAP) gene, a marker for astrocytes, was previously shown to be a crucial cue for neural progenitors to express this gene in response to astrogenic signals during brain development. In this study, we analyzed the methylation status of the STAT3 binding site in the GFAP gene promoter during neural cell development from mouse embryonic stem (ES) cells in vitro. The CpG dinucleotide in the STAT3 binding site in the GFAP gene promoter exhibited a high incidence of cytidine-methylation in undifferentiated pluripotent ES cells. The high incidence of methylation of this particular cytidine was maintained in ES cell-derived neuroectoderm-like cells, but it underwent demethylation when the neural lineage cells became competent to express GFAP in response to a STAT3 activation signal. In contrast, hypermethylation of the CpG site was maintained in non-neural cells generated from the same ES cells. Progressive demethylation of the STAT3 binding site in the GFAP gene promoter was also observed in primary embryonic neuroepithelial cells during in vitro culture, whereas non-neural cells maintained hypermethylation of this site even after culture. Taken together, these results demonstrate that the astrocyte gene-specific cytidine-demethylation is programmed when neural progenitors from pluripotent cells are committed to a neural lineage that is capable of producing astrocytes.

DOI： 10.1111/j.1471-4159.2005.03031.x

Language：English   Publishing type：Research paper (scientific journal)  

It has become apparent that chromatin modification plays a critical role in the regulation of cell-type-specific gene expression. Here, we show that an inhibitor of histone deacetylase, valproic acid (VPA), induced neuronal differentiation of adult hippocampal neural progenitors. In addition, VPA inhibited astrocyte and oligodendrocyte differentiation, even in conditions that favored lineage-specific differentiation. Among the VPA-up-regulated, neuron-specific genes, a neurogenic basic helix-loop-helix transcription factor, NeuroD, was identified. Overexpression of NeuroD resulted in the induction and suppression of neuronal and glial differentiation, respectively. These results suggest that VPA promotes neuronal fate and inhibits glial fate simultaneously through the induction of neurogenic transcription factors including NeuroD.

DOI： 10.1073/pnas.0407643101

Language：English   Publishing type：Research paper (scientific journal)  

Spontaneous recovery after spinal cord injury is limited. Transplantation of neural precursor cells (NPCs) into lesioned adult rat spinal cord results in only partial functional recovery, and most transplanted cells tend to differentiate predominantly into astrocytes. In order to improve functional recovery after transplantation, it is important that transplanted neural precursor cells appropriately differentiate into cell lineages required for spinal cord regeneration. In order to modulate the fate of transplanted cells, we advocate transplanting gene-modified neural precursor cells. We demonstrate that gene modification to inhibit bone morphogenetic protein (BMP) signaling by noggin expression promoted differentiation of neural precursor cells into neurons and oligodendrocytes, in addition to astrocytes after transplantation. Furthermore, functional recovery of the recipient mice with spinal cord injury was observed when noggin-expressing neural precursor cells were transplanted. These observations suggest that gene-modified neural precursor cells that express molecules involved in cell fate modulation could improve central nervous system (CNS) regeneration. (C) 2004 Elsevier Inc. All rights reserved.

DOI： 10.1016/j.expneurol.2003.12.007

Language：English   Publishing type：Research paper (scientific journal)  

Astrocytes are generated from neuroepithelial cells after neurons during brain development. However, the mechanism of this sequential generation is not fully understood. Here, we show that a particular cytosine residue in the promoter of the gene encoding the immature astrocyte marker, S100beta, becomes demethylated, correlating with the time when the S100beta expression commences at embryonic day (E)14. In addition, astrocyte-inducing cytokine, BMP2, increased histone acetylation around the CpG site in neuroepithelial cells at E14 but not Ell when S100beta expressing astrocytes are absent. Furthermore, binding of a methyl DNA binding protein, MeCP2, to the S1000 gene promoter in neuroepithelial cells was reduced at E14 compared to E11. Thus, demethylation of specific CpG site is suggested to be a critical determinant in regulating astrocyte differentiation in the developing brain. (C) 2004 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

DOI： 10.1016/j.febslet.2004.07.029

Language：English   Publishing type：Research paper (scientific journal)  

Somatic stem cells have been claimed to possess an unexpectedly broad differentiation potential ( referred to here as plasticity) that could be induced by exposing stem cells to the extracellular developmental signals of other lineages in mixed-cell cultures(1-6). Recently, this and other experimental evidence supporting the existence of stem-cell plasticity have been refuted because stem cells have been shown to adopt the functional features of other lineages by means of cell-fusion-mediated acquisition of lineage-specific determinants (chromosomal DNA) rather than by signal-mediated differentiation(1,2,5,7,8). In this study we co-cultured mouse neural stem cells (NSCs), which are committed to become neurons and glial cells(9,10), with human endothelial cells, which form the lining of blood vessels(11). We show that in the presence of endothelial cells six per cent of the NSC population converted to cells that did not express neuronal or glial markers, but instead showed the stable expression of multiple endothelial markers and the capacity to form capillary networks. This was surprising because NSCs and endothelial cells are believed to develop from the ectoderm and mesoderm, respectively. Experiments in which endothelial cells were killed by fixation before co-culture with live NSCs (to prevent cell fusion) and karyotyping analyses, revealed that NSCs had differentiated into endothelial-like cells independently of cell fusion. We conclude that stem-cell plasticity is a true characteristic of NSCs and that the conversion of NSCs to unanticipated cell types can be accomplished without cell fusion.

DOI： 10.1038/nature02604

Language：Others  

[Mechanism of neural stem cell fate specification]

Language：English   Publishing type：Research paper (scientific journal)  

Regional evolution of venom-gland phospholipase A2 isoenzymes of Trimeresurus flavoviridis snakes in the southwestern islands of Japan.

Language：English   Publishing type：Research paper (scientific journal)  

Discovering the molecular mechanisms that regulate neuron-specific gene expression remains a central challenge for CNS research. Here, we report that small, noncoding double-stranded [ds) RNAs play a critical role in mediating neuronal differentiation. The sequence defined by this dsRNA is NRSE/RE1, which is recognized by NRSF/REST, known primarily as a negative transcriptional regulator that restricts neuronal gene expression to neurons. The NRSE dsRNA can trigger gene expression of neuron-specific genes through interaction with NRSF/REST transcriptional machinery, resulting in the transition from neural stem cells with neuron-specific genes silenced by NRSF/REST into cells with neuronal identity that can express neuronal genes. The mechanism of action appears to be mediated through a dsRNA/protein interaction, rather than through siRNA or miRNA. The discovery of small modulatory dsRNAs (smRNAs) extends the important contribution of noncoding RNAs as key regulators of cell behavior at both transcriptional and posttranscriptional levels.

DOI： 10.1016/S0092-8674(04)00248-X

Language：English  

A new drug that activates the Wnt pathway maintains the undifferentiated state of pluripotent human and mouse embryonic stem cells. This finding opens the door to defining the precise molecular mechanism of embryonic stem cell self-renewal, which is crucial for providing a steady supply of embryonic stem cells for regenerative medicine (pages 55-63).

DOI： 10.1038/nm0104-23

Language：English   Publishing type：Research paper (scientific journal)  

Adult multipotent neural progenitor cells can differentiate into neurons, astrocytes, and oligodendrocytes in the mammalian central nervous system, but the molecular mechanisms that control their differentiation are not yet well understood. Insulin-like growth factor I (IGF-I) can promote the differentiation of cells already committed to an oligodendroglial lineage during development. However, it is unclear whether IGF-I affects multipotent neural progenitor cells. Here, we show that IGF-I stimulates the differentiation of multipotent adult rat hippocampus-derived neural progenitor cells into oligodendrocytes. Modeling analysis indicates that the actions of IGF-I are instructive. Oligodendrocyte differentiation by IGF-I appears to be mediated through an inhibition of bone morphogenetic protein signaling. Furthermore, overexpression of IGF-I in the hippocampus leads to an increase in oligodendrocyte markers. These data demonstrate the existence of a single molecule, IGF-I, that can influence the fate choice of multipotent adult neural progenitor cells to an oligodendroglial lineage.

DOI： 10.1083/jcb.200308101

Language：English   Publishing type：Research paper (scientific journal)  

The finding of neurogenesis in the adult brain led to the discovery of adult neural stem cells(1). TLX was initially identified as an orphan nuclear receptor expressed in vertebrate forebrains(2) and is highly expressed in the adult brain(3). The brains of TLX-null mice have been reported to have no obvious defects during embryogenesis(4); however, mature mice suffer from retinopathies(5), severe limbic defects, aggressiveness, reduced copulation and progressively violent behaviour(4,6). Here we show that TLX maintains adult neural stem cells in an undifferentiated, proliferative state. We show that TLX-expressing cells isolated by fluorescence-activated cell sorting (FACS) from adult brains can proliferate, self-renew and differentiate into all neural cell types in vitro. By contrast, TLX-null cells isolated from adult mutant brains fail to proliferate. Reintroducing TLX into FACS-sorted TLX-null cells rescues their ability to proliferate and to self-renew. In vivo, TLX mutant mice show a loss of cell proliferation and reduced labelling of nestin in neurogenic areas in the adult brain. TLX can silence glia-specific expression of the astrocyte marker GFAP in neural stem cells, suggesting that transcriptional repression may be crucial in maintaining the undifferentiated state of these cells.

DOI： 10.1038/nature02211

Language：English   Publishing type：Research paper (scientific journal)  

The signaling systems of Notch and bone morphogenetic protein (BMP) are highly conserved from flies to mammals and have been shown to be important in the development of multiple organs. For instance, in the fate determination of mouse neuroepithelial cells, Notch signaling plays a role in keeping the progenitors from differentiating into neurons. BMP is also known to inhibit neuronal differentiation. In this paper, we show that BMP2 enhances Notch-induced transcriptional activation of Hes-5 and Hesr-1 in mouse neuroepithelial cells. BMP2 stimulation, in addition to the introduction of the intracellular domain of Notch (NIC), resulted in enhanced activation of the Hes-5 gene promoter. RBP-Jkappa binding to its target sequence is important not only for Notch signaling, but also for BMP2 signaling, to activate the Hes-5 gene promoter. Smad1, a Smad species that is activated by BMP2, barely interacted with NIC, but did form a complex with NIC in the simultaneous presence of the coactivators P/CAF and p300. Recruitment of p300 to the NIC-containing complex was facilitated by activated Smad1, which is suggested to contribute to BMP2-mediated enhancement of Notch-induced Hes-5 expression. These data suggest a novel functional cooperation between Notch signaling and BMP signaling.

DOI： 10.1093/nar/gkg778

Language：English  

Enhanced gene activation by Notch and BMP signaling cross-talk.
The signaling systems of Notch and bone morphogenetic protein (BMP) are highly conserved from flies to mammals and have been shown to be important in the development of multiple organs. For instance, in the fate determination of mouse neuroepithelial cells, Notch signaling plays a role in keeping the progenitors from differentiating into neurons. BMP is also known to inhibit neuronal differentiation. In this paper, we show that BMP2 enhances Notch-induced transcriptional activation of Hes-5 and Hesr-1 in mouse neuroepithelial cells. BMP2 stimulation, in addition to the introduction of the intracellular domain of Notch (NIC), resulted in enhanced activation of the Hes-5 gene promoter. RBP-Jkappa binding to its target sequence is important not only for Notch signaling, but also for BMP2 signaling, to activate the Hes-5 gene promoter. Smad1, a Smad species that is activated by BMP2, barely interacted with NIC, but did form a complex with NIC in the simultaneous presence of the coactivators P/CAF and p300. Recruitment of p300 to the NIC-containing complex was facilitated by activated Smad1, which is suggested to contribute to BMP2-mediated enhancement of Notch-induced Hes-5 expressi

Language：English   Publishing type：Research paper (scientific journal)  

Oct3/4 plays a critical role in maintaining embryonic stem cell pluripotency. Regulatable transgene-mediated sustained Oct3/4 expression in ES cells cultured in serum-free LIF-deficient medium caused accelerated differentiation to neuroectoderm-like cells that expressed Sox2, Otx1 and Emx2 and subsequently differentiated into neurons. Neurogenesis of ES cells is promoted by SDIA (stromal cell-derived inducing activity), which accumulates on the PA6 stromal cell surface. Oct3/4 expression in ES cells was maintained by SDIA whereas without it expression was promptly downregulated. Suppression of Oct3/4 abolished neuronal differentiation even after stimulation by SDIA. In contrast, sustained upregulated Oct3/4 expression enhanced SDIA-mediated neurogenesis of ES cells. Therefore, Oct3/4 appears to promote neuroectoderm formation and subsequent neuronal differentiation from ES cells.

DOI： 10.1242/dev.00476

Language：English   Publishing type：Research paper (scientific journal)  

DNA methylation-mediated epigenetic regulation plays critical roles in regulating mammalian gene expression, but its role in normal brain function is not clear. Methyl-CpG binding protein 1 (MBD1), a member of the methylated DNA-binding protein family, has been shown to bind methylated gene promoters and facilitate transcriptional repression in vitro. Here we report the generation and analysis of MBD1(-/-) mice. MBD1(-/-) mice had no detectable developmental defects and appeared healthy throughout life. However, we found that MBD1(-/-) neural stem cells exhibited reduced neuronal differentiation and increased genomic instability. Furthermore, adult MBD1(-/-) mice had decreased neurogenesis, impaired spatial learning, and a significant reduction in long-term potentiation in the dentate gyrus of the hippocampus. Our findings indicate that DNA methylation is important in maintaining cellular genomic stability and is crucial for normal neural stem cell and brain functions.

DOI： 10.1073/pnas.1131928100

Language：English   Publishing type：Research paper (scientific journal)  

Objective. Definitive hematopoiesis starts in the aorta-gonad-mesonephros (AGM) region during mouse development and remarkably expands in the liver at a later stage of ontogeny. gp130 is a signal transducing receptor component shared by all the IL-6 family cytokines, whose gene ablation in mouse results in the significant reduction in the fetal liver hematopoiesis. The present study aims to evaluate the role of gp130 signaling in the fetal mouse AGM hematopoiesis. Materials and Methods. Mouse AGM regions from the wild-type and gp130-deficient mice on embryonic day 11.5 were dissociated and cultured with a mixture of cytokines, including one which activates gp130. Wild-type human gp130 and its mutant constructs were introduced into cultured gpl30-deficient AGM cells using retrovirus system. To further analyze gp130 downstream signaling, a dominant-negative mutant of STAT3 was also introduced. Results. The gp130 deficiency in the culture of fetal mouse AGM cells resulted in the failure of the expansion of the c-kit(+), Sca-1(+), and lineage markers(-) population. Such failure was rescued by introduction of a wild-type gp130 expression construct but not its mutant constructs having no ability to activate STAT3. In the normal AGM cell culture, introduction of a dominant-negative form of STAT3 in which Y-705 was changed to phenylatanine suppressed the expansion of hematopoietic cell colonies. Conclusion. gp130 plays an indispensable role in the expansion of hematopoietic precursor cells in the fetal mouse AGM. In particular, the activation of STAT3 by gp130 is found to be important in this process. (C) 2003 International Society for Experimental Hematology. Published by Elsevier Science Inc.

DOI： 10.1016/S0301-472X(03)00025-0

Language：English   Publishing type：Research paper (scientific journal)  

Trimeresurus flavoviridis snakes inhabit the southwestern islands of Japan. A phospholipase A(2) (PLA(2)) named PL-Y, was isolated from Okinawa T.flavoviridis venom and its amino acid sequence was determined from both protein and cDNA. PL-Y was unable to induce edema. In contrast, PLA-B, a PLA(2) from Tokunoshima T. flavoviridis venom, which is different at only three positions from PL-Y, is known to induce edema. A new PLA(2), named PLA-B', which is similar to PLA-B, was cloned from Amami-Oshima T. flavoviridis venom gland. Three T. flavoviridis venom basic [Asp(49)]PLA(2) isozymes, PL-Y (Okinawa), PLA-B (Tokunoshima), and PLA-B' (Amami-Oshima), are identical in the N-terminal half but have one to four amino acid substitutions in the PI-sheet and its vicinity. Such interisland sequence diversities among them are due to isolation in the different environments over 1 to 2 million years and appear to have been brought about by natural selection for point mutation in their genes. Otherwise, a major PLA(2), named PLA(2), ubiquitously exists in the venoms of T. flavoviridis snakes from the three islands with one to three synonymous substitutions in their cDNAs. It is assumed that the PLA(2) gene is a prototype among T. flavoviridis venom PLA(2) isozyme genes and has hardly undergone nonsynonymous mutation as a principal toxic component. Phylogenetic analysis based on the amino acid sequences revealed that T. flavoviridis PLA(2) isozymes are clearly separated into three groups, PLA(2) type, basic [ASP(49)]PLA(2) type, and [LyS(49)]PLA(2) type. Basic [ASP(49)]PLA(2)-type isozymes may manifest their own particular toxic functions different from those of the isozymes of the PLA(2) type and [LyS(49)]PLA(2) type.

DOI： 10.1007/s00239-002-2400-7

Language：English   Publishing type：Research paper (scientific journal)  

The role of gp130 in cerebral cortical histogenesis remains unknown. Mice lacking gp130 showed a hypoplastic cortical plate and decreased incorporation of 5-bromo-2'-deoxyuridine (BrdU) in progenitor cells of the developing cerebrum. In contrast, injection of leukemia inhibitory factor (LIF), a gp130 ligand, into the lateral cerebral ventricle of wild-type embryos exo utero induced hyperplasia of the cerebral cortex and increased the incorporation of BrdU in progenitor cells. Furthermore, chronologically controlled injection of LIF followed or preceded by BrdU revealed that gp130-mediated signals promote the progenitor cells to reenter the stem cell cycle without affecting the duration of cell cycle and enhance the migration of postmitotic neurons in the developing cerebrum.

Language：English   Publishing type：Research paper (scientific journal)  

Neurons, astrocytes, and oligodendrocytes, the three major cell types in the nervous system, are generated from common neural stem cells during development. Recent studies have provided evidence that neural stem cells are preserved in the adult brain, where, until recently, neurogenesis had not been considered to take place. The mechanisms that govern the fate of neural stem-cell determination have yet to be clarified. It is becoming apparent that soluble protein mediators referred to as cytokines play critical roles in cell-fate determination. For instance, bone morphogenetic proteins (BMPs) alter the fate of developing brain cells from a neurogenic differentiation to an astrocytic one. Different types of cytokines sometimes cooperate to modulate differentiation. For example, the interleukin-6 (IL-6) family cytokines and the BMP family cytokines act in synergy to elaborate astrocyte differentiation. In this review, we focus on recent progress that addresses the molecular mechanisms whereby cytokines regulate the fate of cells in neural lineages. We also discuss possible clinical applications of these findings to minimize the undesirable gliogenesis that occurs after neural stem-cell implantation and nerve injury.

DOI： 10.1385/MN:25:3:233

Language：English   Publishing type：Research paper (scientific journal)  

Cardiotrophin-like cytokine (CLC), also known as novel neurotrophin-1/B cell stimulating factor-3 (NNT-1/BSF-3), is a recently identified member of the interleukin (IL)-6 family of cytokines that share gp130 as a signal-transducing receptor component. In this study, we demonstrate that CLC is expressed in fetal mouse neuroepithelial cells and has a potential to induce their astrocyte differentiation in a synergistic manner with bone-morphogenetic protein (BMP)-2, which is also expressed in the fetal mouse brain. CLC-stimulation led to promoter activation of the gene for an astrocyte marker, glial fibrillary acidic protein (GFAP), which was clearly inhibited by expression of a dominant negative form of a transcription factor, STAT3, or by introduction of a mutation in a single STAT3-binding site in the promoter, suggesting a critical role of STAT3 in the CLC-induced GFAP transcription.
These results suggest that CLC plays a role in astrocyte differentiation via STAT3 activation within the developing brain. (C) 2002 Elsevier Science Ltd. All rights reserved.

DOI： 10.1006/cyto.2002.1006

Language：English   Publishing type：Research paper (scientific journal)  

Background: The precise mechanism governing the generation of haematopoietic stem cells still remains to be understood, partly because the molecules required for early haematopoiesis have not fully been identified.
Results: We have identified a novel gene expressed in embryonic haematopoietic tissues, designated ELYS (for embryonic large molecule derived from yolk sac), which has no significant homology with any other known molecules. Based on the cDNA sequence, mouse ELYS protein is composed of 2243 amino acid residues and contains an AT-hook DNA-binding domain, eight nuclear localization signals (NLSs) at the C-terminal region, three nuclear export signals (NESs) and two WD repeats at the N-terminal region. ELYS has a potential to shuttle between the cytoplasm and nucleus. When in the nucleus, ELYS is present in the nuclear matrix. Fusions of the yeast GAL4 DNA-binding domain and various ELYS mutants reveal the presence of transcriptional activation and inhibitory domains. The ELYS gene is predominantly expressed in embryonic haematopoietic tissues, i.e. foetal liver, spleen, and thymus, whereas the expression is down-regulated in the adult. In the aorta-gonad-mesonephros (AGM) region of an 11.5 dpc mouse embryo, ELYS is expressed in the endothelium lining the dorsal aorta. In the adult bone marrow, ELYS is notably expressed in the Lin(-)/c-kit(+)/Sca-1(+) population.
Conclusions: We have reported the isolation and characterization of a novel molecule, ELYS. ELYS seems to be a nuclear transcription factor associated with both early and mature haematopoietic events.

DOI： 10.1046/j.1365-2443.2002.00529.x

Language：English   Publishing type：Research paper (scientific journal)  

Astrocyte differentiation, which occurs late in brain development, is largely dependent on the activation of a transcription factor, STAT3. We show that astrocytes, as judged by glial fibrillary acidic protein (GFAP) expression, never emerge from neuroepithelial cells on embryonic day (E) 11.5 even when STAT3 is activated, in contrast to E14.5 neuroepithelial cells. A CpG dinucleotide within a STAT3 binding element in the GFAP promoter is highly methylated in E11.5 neuroepithelial cells, but is demethylated in cells responsive to the STAT3 activation signal to express GFAP. This CpG methylation leads to inaccessibility of STAT3 to the binding element. We suggest that methylation of a cell type-specific gene promoter is a pivotal event in regulating lineage specification in the developing brain.

DOI： 10.1016/S1534-5807(01)00101-0

Language：English   Publishing type：Research paper (scientific journal)  

It has been reported that bone morphogenetic proteins (BMPs) are involved in the generation of the central nervous system during development. However, the roles of BMPs in mature spinal cord have not been clarified. We examined the expression of BMP7 mRNA before and after traumatic injury of the adult rat spinal cord. BMP7 mRNA was already detectable at a relatively low level in uninjured spinal cord, but was dramatically increased after injury. Semiquantitative RT-PCR study further confirmed upregulation of BMP7 mRNA in injured spinal cord. In situ hybridization indicated that expression of BMP7 mRNA was present only in glial cells in uninjured spinal cord. After injury, the number of BMP7-expressing glial cells was increased, BMP7 expression also became apparent in motor neurons. It has been suggested that BMPs promote survival of subventricular zone cells in adult rats. Thus, our results suggest that increase in the expression of BMP7 promotes survival of neurons and glial cells after acute traumatic injury. In contrast, there is increasing evidence that BMPs inhibit neurogenesis and alternatively promote gliogenesis of neural progenitors, which are also present in adult spinal cord, suggesting that injury-upregulated BMP7 may regulate differentiation of glial cells from neural progenitors and may induce gliosis after central nervous system injury. (C) 2001 Elsevier Science B.V. All rights reserved.

DOI： 10.1016/S0006-8993(01)03123-7

Language：English   Publishing type：Research paper (scientific journal)  

Background: BMP2 is known to play a wide variety of roles, including some in the development of the nervous system. This cytokine has been reported to induce neurite outgrowth in rat pheochromocytoma PC12 cells via the activation of a p38 MAP kinase, although its regulatory mechanism remains largely to be elucidated.
Results: BMP2-induced neurite outgrowth in PC12 cells was inhibited by the introduction of a kinase-negative form of a MAP kinase kinase kinase, TAK1, an upstream regulatory kinase for p38 kinase. Following BMP2 stimulation, the expression of Smad6 and Smad7, inhibitory Smad species that are known to inhibit the BMP2-restricted Smad species, Smad1, Smad5 and Smad8, was up-regulated. Unexpectedly, over-expression of either Smad6 or Smad7 in PC12 cells repressed the BMP2-induced neurite outgrowth and severely impeded the p38 kinase pathway. Both of these inhibitory Smads were found to interact physically with TAK1-binding protein, a molecule required for TAK1 activation.
Conclusions: This study demonstrates that BMP2-induced neurite outgrowth in PC12 cells involves activation of the TAK1-p38 kinase pathway which is inhibited by Smad6 and Smad7.

DOI： 10.1046/j.1365-2443.2001.00483.x

Language：English   Publishing type：Research paper (scientific journal)  

Bone morphogenetic proteins (BMPs), a class of cytokines belonging to the transforming growth factor-beta superfamily, have been shown to play a wide variety of roles during development including those in the central nervous system. We here report that BMP2, BMP4 and BMP7 have an equivalent potential to inhibit neurogenesis and concomitantly induce astrocytogenesis of mouse fetal neuroepithelial cells. We further show that these BMPs activate a promoter of the gene for negative helix-loop-helix (HLH) factor, Id1, which is known to inhibit the function of such neurogenic transcription factors as Mash1 and neurogenin. Those results suggest that BMP2, BMP4 and BMP7 alternate the fate of neuroepithelial cells from neuronal type to astrocytic one via a common mechanism involving negative HLH factor. (C) 2001 Elsevier Science Ireland Ltd and the Japan Neuroscience Society. All rights reserved.

DOI： 10.1016/S0168-0102(01)00297-8

Language：English   Publishing type：Research paper (scientific journal)  

Bone marrow stromal cells are able to differentiate into adipogenic, chondrogenic, myogenic, osteogenic, and cardiomyogenic lineages, all of which are limited to a mesoderm-derived origin. In this study, we showed that neurons, which are of an ectoderm-origin, could be generated from marrow-derived stromal cells by specific inducers, fibronectin/ornithine coating, and neurosphere formation. The neurons generated from marrow stroma formed neurites, expressed neuron-specific markers and genes, and started to respond to depolarizing stimuli as functional mature neurons, Among stromal cells, isolated mature osteoblasts which had strong in vivo osteogenic activity could be efficiently converted into functional neurons. This transdifferentiation or meta-differentiation was enhanced by Noggin, an inhibitor of bone morphogenetic proteins, in comparison with 5-azacytidine, a demethylating agent capable of altering the gene expression pattern. Marrow stroma is therefore a potential source of cells for neural cell transplantation.

Language：English   Publishing type：Research paper (scientific journal)  

Recent studies have suggested the existence of progenitors common to hematopoietic and endothelial cells, called hemangioblasts, in, for instance, embryonic dorsal aorta. To identify a membrane-bound or secretory molecule regulating early hematopoiesis, we screened a cDNA library from dorsal aortas of embryonic day (E) 10.5 mice by a signal sequence trap method and obtained a clone encoding a sialoprotein, endomucin-1. Immunohistochemistry revealed that the endomucin-1 transcript was specifically expressed in the endothelial cells of dorsal aorta of E10.5 mouse embryo. Overexpression of endomucin-1 strongly inhibited adhesion and aggregation of cells, including cultured endothelial cells from E10.5 dorsal aorta. These data suggest that endomucin-1 may play a role in detachment of hematopoietic cells from endothelium during early hematopoiesis. (C) 2001 Academic Press.

DOI： 10.1006/bbrc.2001.5587

Language：English   Publishing type：Research paper (scientific journal)  

Three-dimensional structure of the ligand binding domain (LBD) of the vitamin D receptor (VDR) docked with the natural ligand 1 alpha ,25-dihydroxyvitamin D-3 [1,25-(OH)(2)D-3] has been mostly solved by the X-ray crystallographic analysis of the deletion mutant (VDR-LBD Delta 165-215). The important focus, from now on. is how the VDR recognizes and interacts with potent synthetic ligands. We now report the docking models of the VDR with three functionally and structurally interesting ligands, 22-oxa-1.25-(OH)(2)D-3 (OCT), 20-epi-1,25-(OH)(2)D-3 and 20-epi-22-oxa-24,26,27-trihomo-1.25-(OH)(2)D-3. In parallel with the computational docking studies, we prepared twelve one-point mutants of amino acid residues lining the ligand binding pocket of the VDR and examined their transactivation potency induced by 1125-(OH)(2)D-3 and these synthetic ligands. The results indicate that L233, R274, W286, H397 and Y401 are essential for holding the all ligands tested, S278 and Q400 are not important at all, and the importance of S237, V234, S275, C288 and H305 is variable depending on the side-chain structure of the ligands. Based on these studies, we suggested key structural factors to bestow the selective action on OCT and the augmented activities on 20-epi-ligands. Furthermore, the docking models coincided well with our proposed active space-region theory of vitamin D based on the conformational analyses of ligands. (C) 2001 Elsevier Science Ltd. All rights reserved.

DOI： 10.1016/S0968-0896(01)00060-8

Language：English   Publishing type：Research paper (scientific journal)  

Cardiotrophin-1 (CT-1) belongs to the interleukin (IL-)6 family of cytokines that share membrane glycoprotein 130 (gp130) as a receptor component critical for signal transduction, We here observed that CT-1 was expressed in mouse fetal neuroepithelial cells, and was capable of inducing astrocyte differentiation from these cells in a synergistic manner with bone morphogenetic protein (BMP)-2, whose expression was also found in the fetal brain. CT-1-induced astrocyte differentiation was solely gp130-dependent. CT-1-stimulation led to promoter activation of the gene for an astrocyte marker, glial fibrillary acidic protein (GFAP), which was clearly inhibited by expression of a dominant negative form of a gp130-downstream transcription factor, signal transducer and activator of transcription 3(STAT3), or by introduction of a mutation in a single STAT3-binding site in the promoter, suggesting a critical role of STAT3 in the CT-1-induced GFAP transcription. These results suggest that astrocyte differentiation in the developing brain involves CT-1-signaling which cooperates with BMP2. (C) 2001 Academic Press.

DOI： 10.1006/cyto.2001.0883

Language：English   Publishing type：Research paper (scientific journal)  

We show that when telencephalic: neural progenitors are briefly exposed to bone morphogenetic protein 2 (BMP2) in culture, their developmental fate is changed from neuronal cells to astrocytic cells. BMP2 significantly reduced the number of cells expressing microtubule-associated protein 2, a neuronal marker, and cells expressing nestin, a marker for undifferentiated neural precursors, but BMP2 increased the number of cells expressing S100-beta, an astrocytic marker. In telencephalic neuroepithelial cells, BMP2 up-regulated the expression of negative helix-loop-helix (HLH) factors Id1, Id3, and Hes-5 (where Hes is homologue of hairy and Enhancer of Split) that inhibited the transcriptional activity of neurogenic HLH transcription factors Mash1 and neurogenin. Ectopic expression of either Id1 or Id3 (where Id is inhibitor of differentiation) inhibited neurogenesis of neuroepithelial cells, suggesting an important role for these HLH proteins in the BMP2-mediated changes in the neurogenic fate of these cells. Because gliogenesis in the brain and spinal cord, derived from implanted neural stem cells or induced by injury, is responsible for much of the failure of neuronal regeneration, this work may lead to a therapeutic strategy to minimize this problem.

DOI： 10.1073/pnas.101109698

Language：English   Publishing type：Research paper (scientific journal)  

Signals of interleukin 6 (IL-6) are transduced by binding of IL-6 to its cell surface receptor (IL-6R) and subsequent association of the resultant IL-6/IL-6R complex with gp130, the signal transducing receptor component utilized in common by all the IL-6 family of cytokines, A soluble form of IL-6R (sIL-6R), which lacks transmembrane and cytoplasmic regions, retains the ability to bind IL-6 and signal through gp130, We show here that a fusion protein of sIL-6R and IL-6 without a polypeptide linker, termed FP6, induces differentiation of astrocytes from fetal mouse neuroepithelial cells as potently as a representative IL-6 family cytokine, leukaemia inhibitory factor (LIF). FP6 has a potential to activate a transcription factor, signal transducer and activator of transcription 3 (STAT3), and mitogen-activated protein kinases, ERK1 and ERK2, in these cells as does LIF, FP6 activates a promoter of the gene for an astrocytic marker, glial fibrillary acidic protein (GFAP), in neuroepithelial cells. This activation is virtually abolished by ectopic expression of a dominant-negative form of STAT3, or by introducing a point mutation into the STAT3 response element located in the GFAP promoter. These results suggest that FP6 induces astrocyte differentiation from neuroepithelial cells through STAT3 activation and that FP6 could be of use as a substitute for natural IL-6 family cytokines, (C) 2001 Academic Press.

DOI： 10.1006/cyto.2000.0831

Language：English   Publishing type：Research paper (scientific journal)  

We here show that hone morphogenetic protein (BMP) 7 acted in synergy with the distinct type of cytokines, leukemia inhibitory factor (LIF) and interleukin (IL) 6 that are in the IL-6 family, to induce astrocyte differentiation from neuroepithelial cells as assessed by expression of glial fibrillary acidic protein (GFAP), In this synergistic action, transcription factors, Smads and STAT3 (for signal transducer and activator of transcription 3) activated by respective group of cytokines, as well as a transcriptional coactivator p300 were essential. Taken together with our previous finding that the synergistic astrocyte induction by BMP2 and LIF is attributed to the complex formation of Smads and STAT3 bridged by p300, it is conceivable that this complex formation is a mechanism utilized in common by two different types of cytokines belonging to the BMP and IL-6 families in order to synergistically induce astrocyte differentiation. (C) 2001 Federation of European Biochemical Societies, Published by Elsevier Science B.V. All rights reserved.

DOI： 10.1016/S0014-5793(01)02095-6

Language：English   Publishing type：Research paper (scientific journal)  

Signal transduction from cell surface receptors to the nucleus is regulated in most part by protein phosphorylation. For the purpose of identification of kinases which play an important role at a particular phosphorylation step in a series of signal transduction pathways, we have developed a new expression-screening method using a phosphorylation site specific antibody and a vector encoding substrate polypeptide. We have applied this method for screening kinases which phosphorylate STAT3 at serine(727). In this screening, antibody (PS727 antibody) specifically recognizing STAT3 in which serine(727) is phosphorylated was first prepared. Escherichia coli, bacteria expressing a serine(727)-containing fragment of STAT3 which was fused to glutathione-S-transferase (GST) (GST-STAT3-WT) were infected by lambda phage cDNA expression libraries. Phosphorylation of GST-STAT3-WT was effectively performed in E. coli as expected, and clones positive for PS727 antibody immunoreactivity were selected. Isolated 53 clones encode four serine/threonine kinases; extracellular signal regulated kinase 1 (ERK1/p44-MAPK), dual specificity Yak1 related kinase (DYRK), dual specificity Yak1 related kinase 2 (DYRK2) and homeodomain interacting protein kinase 2 (HIPK2), These kinases have a potential to phosphorylate serine(727) in STAT3 protein also in mammalian cells. The present method is considered to be applicable in general to isolate kinases. (C) 2001 Elsevier Science B.V. All rights reserved.

DOI： 10.1016/S0022-1759(00)00313-6

Language：English   Publishing type：Research paper (scientific journal)  

In the present study, we have identified a novel gene, NDRP (for neuronal differentiation-related protein), which is predominantly expressed in developing and regenerating neurons. The predicted NDRP comprises 1,019 amino acid residues and has 6 WD repeats in the N-terminal half and multiple potential nuclear localization signals (NLSs) at the C-terminal part. This molecule shows no significant structural similarity with any other molecules in available databases. In situ hybridization and immunohistochemistry revealed the highest expression of NRDP in sensory neurons, for instance, olfactory epithelia and neural layer of retina during embryonic development, as well as in perinatal dorsal root ganglions. The expression of this gene in intact motor neurons such as in the hypoglossal nerve was undetectable but became obvious after axotomy, These results suggest that the product of this gene might be involved in the development of sensory neurons as well as the regeneration of motor neurons.

Language：English   Publishing type：Research paper (scientific journal)  

We previously demonstrated that oncostatin M (OSM) promotes hepatic development in concert with glucocorticoid. The livers from mice deficient for gp130, a signaling subunit of the OSM receptor, displayed reduced expression of hepatic differentiation marker and defective glycogenic function. However, these phenotypes were not completely abolished in gp130(-/-) mice, suggesting that there is an alternative pathway regulating hepatic development in vivo. To test this possibility, we cultured gp130(-/-) fetal hepatic cells and investigated a signal that induces hepatic differentiation. When hepatocytes were forced to interact with each other by inoculating cells at high densities, hepatic differentiation was induced even in the absence of gp130. Moreover, cells stimulated with OSM and/or cultured at a high density possess many other metabolic functions. These observations suggest that fetal hepatic cells acquire multiple characteristics of differentiated hepatocytes in response to the signals generated by cell-cell contacts as well as by OSM. (C) 2000 Academic Press.

DOI： 10.1006/bbrc.2000.3635

Language：English   Publishing type：Research paper (scientific journal)  

Members of the T-box family are known to play critical roles in the embryonic development of most animal species. Recently, we have isolated its new mammalian member, Tbr2, from mouse embryonic brain. We have also shown that the expression patterns of Tbr2 and the closely related Tbr1 appear to be reciprocal in the developing brain; Tbr2 is expressed in mesencephalon and rhombencephalon, but expression of Tbr1 is restricted to telencephalon. To investigate possible structural and functional relationships of Tbr2 and other T-box containing genes, we analyzed genomic organization of the murine Tbr2 gene. The Tbr2 gene is composed of six exons (1353, 155, 122, 159, 62 and 1035 bp), and five introns (920, 643, 602, 85 and 2036 bp). This exon/intron organization is very similar to that of Tbr1. We also analyzed the 3.9 kb sequence of the 5' promoter region flanking the Tbr2 gene and the corresponding region of the Tbr1 gene. The sites for Brn-2 and Tst-1 were found in the promoter of Tbr2 but not Tbr1. On the contrary, there were eight HNF-3 beta binding sites in the Tbr1 gene promoter but only three in the Tbr2 promoter. The differential presence of putative binding sites for these brain-specific transcription factors may explain the reciprocal expression of Thr1 and Tbr2. Furthermore, a single chromosomal locus for mouse Tbr2 was assigned to 9F3 by fluorescence in-situ hybridization. (C) 2000 Elsevier Science B.V. All rights reserved.

DOI： 10.1016/S0378-1119(00)00290-0

Language：English   Publishing type：Research paper (scientific journal)  

Bone morphogenetic protein 2 (BMPS), a member of the transforming growth factor-beta (TGF-beta) superfamily, regulates a variety of cell fates and functions. At present, the molecular mechanism by which BMPS induces apoptosis has not been fully elucidated. Here we propose a BMPS signaling pathway that mediates apoptosis in mouse hybridoma MH60 cells whose growth is interleukin-6 (IL-6)-dependent. BMP2 dose-dependently induces apoptosis in MH60 cells even in the presence of IL-6. BMPS has no inhibitory effect on the IL-g-induced tyrosine phosphorylation of STAT3, and the bcl-2 gene expression which is known to be regulated by STAT3, suggesting that BMP2-induced apoptosis is not attributed to alteration of the IL-6-mediated bcl-2 pathway. We demonstrate that BMPS induces activation of TGF-beta-activated kinase (TAK1) and subsequent phosphorylation of p38 stress-activated protein kinase. In addition, forced expression of kinase-negative TAXI in MH60 cells blocks BMP2-induced apoptosis. These results indicate that BMP2-induced apoptosis is mediated through the TAK1-p38 pathway in MH60 cells. We also show that MH60-derived transfectants expressing Smad6 are resistant to the apoptotic signal of BMPB. Interestingly, this ectopic expression of Smad6 blocks BMP2-induced TAXI activation and p38 phosphorylation. Moreover, Smad6 can directly bind to TAK1. These findings suggest that Smad6 is likely to function as a negative regulator of the TAK1 pathway in the BMP2 signaling, in addition to the previously reported Smad pathway.

DOI： 10.1074/jbc.M908622199

Language：English   Publishing type：Research paper (scientific journal)  

The interleukin (IL)-6 family cytokines utilize membrane glycoprotein gp130 in common as a critical signal-transducing receptor component. IL-11, a cytokine initially identified as a plasmacytoma growth factor, belongs to this family. We show here that IL-11 and its cognate receptor components are expressed in fetal mouse neuroepithelial cells. We also show that after 4 days of culture with it-if, cells with typical astrocytic morphologies expressing glial fibrillary acidic protein (GFAP; a marker for astrocytes) come out. This differentiation process is totally dependent on the gp130-mediated signal-transduction pathway involving activation of a latent cytoplasmic transcription factor, STAT3 (for signal transducer and activator of transcription 3), because (a) IL-11-induced astrocyte differentiation is not observed when neuroepithelial cells prepared from gp130-deficient mice were used, (b) stimulation of neuroepithelial cells by IL-11 rapidly induces tyrosine-phosphorylation of STAT3, and (c) transfection of neuroepithelial cells with a dominant-negative form of STAT3 inhibits IL-11-induced activation of the GFAP gene promoter. We have further identified, in the GFAP promoter region, a STAT3 site at which nucleotide substitutions almost completely abolished the IL-11-induced GFAP promoter activation. Taken together, it is suggested that IL-11 contributes to astrocytogenesis in fetal brain via activation of gp130 and STAT3.

DOI： 10.1046/j.1471-4159.2000.0741498.x

Language：English   Publishing type：Research paper (scientific journal)  

Regional evolution of venom-gland phospholipase A2 isoenzymes of Trimeresurus flavoviridis snakes in the southwestern islands of Japan

DOI： 10.1042/0264-6021:3470491

Language：English   Publishing type：Research paper (scientific journal)  

The ligand binding domain of the human vitamin D receptor (VDR) was modeled based on the crystal structure of the retinoic acid receptor. The ligand binding pocket of our VDR model is spacious at the helix 11 site and confined at the beta-turn site. The ligand 1 alpha,25-dihydroxyvitamin D-3 was assumed to be anchored in the ligand binding pocket with its side chain heading to helix 11 (site 2) and the A-ring toward the beta-turn (site 1). Three residues forming hydrogen bonds with the functionally important 1 alpha- and 25-hydroxyl groups of 1 alpha,25-dihydroxyvitamin D-3 were identified and confirmed by mutational analysis: the 1 alpha-hydroxyl group is forming pincer-type hydrogen bonds with S237 and R274 and the 25-hydroxyl group is interacting with H397. Docking potential for various ligands to the VDR model was examined, and the results are in good agreement with our previous three-dimensional structure-function theory.

DOI： 10.1073/pnas.020522697

Language：English   Publishing type：Research paper (scientific journal)  

Leukemia inhibitory factor (LIF) and bone morphogenetic protein (BMP) 2 signal via different receptor systems. We have recently demonstrated that simultaneous stimulation of fetal mouse neuroepithelial cells with these distinct types of cytokines synergistically induces astrocyte differentiation in a 2-day culture. Here we show that astrocytes spontaneously emerge in vitro without exogenously added LIF and BMP2 in the culture of neuroepithelial cells for a much longer period. This spontaneous astrocyte differentiation is completely impaired when neuroepithelial cells deficient for gp130, a signal transducing receptor component for LIF, are used. We also show that LIF and BMP2 as well as related cytokines and respective receptor molecules are expressed in fetal mouse brain and cultured neuroepithelial cells. Taken together with our previous finding that prenatal mouse brain deficient for gp130 exhibits a severe reduction of astrocyte, it is suggested that LIF acts cooperatively with BMP2 in vivo to induce astrocyte differentiation in mouse developing brain, (C) 1999 Federation of European Biochemical Societies.

DOI： 10.1016/S0014-5793(99)00997-7

Language：English   Publishing type：Research paper (scientific journal)  

LIM-homeodomain (LHX) transcription factors play critical roles in cell fate determination during development, in particular, in CNS, The transcriptional activity of several LHX proteins is postulated to be regulated by interaction with an LIM-domain binding protein, Ldb1, We have now identified a novel LHX molecule, termed Lhx6.1, that is closely related to a recently reported Lhx6 molecule. The Lhx6.1 transcript is found in several restricted regions in the developing CNS, mostly within the embryonic forebrain, We further show that Lhx6.1 interacts with Ldb1 through tandem LIM-domains, implying transcriptional regulation of Lhx6.1 by Ldb1.

Language：English   Publishing type：Research paper (scientific journal)  

The interleukin (IL) -6 family of cytokines share gp130 as a signal-transducing receptor component. We here show that oncostatin M (OSM), a member of this family, and its receptor components were expressed in embryonic mouse brain and that OSM induced astrocytes in cultured fetal mouse neuroepithelial cells. OSM induced promoter activation of the gene for an astrocyte marker, glial fibrillary acidic protein. The activation was completely blocked by expression of a dominant negative form of STATE, a transcription factor activated downstream of gp130, or by introduction of a mutation in the STATE binding motif in the promoter. Taken together with its expression, we suggest that OSM contributes to the induction of astrocyte differentiation in a fetal developing brain via STATE activation. (C) 1999 Elsevier Science Ireland Ltd. All rights resented.

DOI： 10.1016/S0304-3940(99)00447-4

Language：English   Publishing type：Research paper (scientific journal)  

gp130 is a signal-transducing receptor component used in common by the interleukin-6 (IL-6) family of hematopoietic and neurotrophic cytokines, including IL-6, IL-11, leukemia-inhibitory factor, ciliary neurotrophic factor, oncostatin-M, and cardiotrophin-1. We have examined in this study a role of gp130 in the nervous system by analyzing developmental cell death of several neuronal populations and the differentiation of astrocytes in gp130-deficient mice. A significant reduction was observed in the number of sensory neurons in L5 dorsal root ganglia and motoneurons in the facial nucleus, the nucleus ambiguus, and the lumbar spinal cord in gp130 -/- mice on embryonic day 18.5. On the other hand, no significant neuronal loss was detectable on day 14.5, suggesting a physiological role of gp130 in supporting newly generated neurons during the late phase of development when naturally occurring cell death takes place. Moreover, expression of an astrocyte marker, GFAP, was severely reduced in the brain of gp130 -/- mice. Our data demonstrate that gp130 expression is essential for survival of subgroups of differentiated motor and sensory neurons and for the differentiation of major populations of astrocytes in vivo.

Language：English   Publishing type：Research paper (scientific journal)  

We have identified and characterized a new member of the mammalian brain-specific T-box gene family, Tbr2, which is closely related to mouse Tbr1, and to the Xenopus earliest mesodermal gene, Eomesodermin. As Tbr1, Tbr2 is predominantly expressed in some regions of the developing brain, but in a strikingly complementary manner. On embryonic day 14.5 (E14.5), Tbr2 mRNA expression was observed in the mesencephalon and rhombencephalon in contrast to Tbr1 which was expressed mostly in the telencephalon. At this stage, Tbr2 mRNA was readily detectable in the postmitotic and differentiating neurons located in various brain regions, i.e., oculomotor, red, trigeminal, vestibular, facial, and hypoglossal nuclei. However, expression of Tbr2 in these nuclei became undetectable on E18.5. In contrast, Tbr2 mRNA expression was detected in the hippocampus only from E18.5 onwards. Whereas Tbr2 expression disappeared in most parts of the mature adult brain, it remained detectable in the hippocampus and olfactory bulb, regions where some neuronal precursors retain their differentiation potential. These results suggest that Tbr2 may play a crucial role in differentiating neurons rather than in proliferating or already differentiated neurons. In addition, similarly to Xenopus Eomesodermin, mouse Tbr2 showed biphasic expression; a first peak around E6.5 and a second peak around E14.5, suggesting that Tbr2 may also be important at early stages of gastrulation. (C) 1999 Elsevier Science B.V. All rights reserved.

DOI： 10.1016/S0165-3806(99)00064-4

Language：English   Publishing type：Research paper (scientific journal)  

Fetal liver, the major site of hematopoiesis during embryonic development, acquires additional various metabolic functions near birth. Although liver development has been characterized biologically as consisting of several distinct steps, the molecular events accompanying this process are just beginning to be characterized. In this study, we have established a novel culture system of fetal murine hepatocytes and investigated factors required for development of hepatocytes, We found that oncostatin M (OSM), an interleukin-6 family cytokine, in combination with glucocorticoid, induced maturation of hepatocytes as evidenced by morphological changes that closely resemble more differentiated hepatocytes, expression of hepatic differentiation markers and intracellular glycogen accumulation, Consistent with these in vitro observations, livers from mice deficient for gp130, an OSM receptor subunit, display defects in maturation of hepatocytes, Interestingly, OSM is expressed in CD45(+) hematopoietic cells in the developing liver, whereas the OSM receptor is expressed predominantly in hepatocytes. These results suggest a paracrine mechanism of hepatogenesis; blood cells, transiently expanding in the fetal liver, produce OSM to promote development of hepatocytes in vivo.

DOI： 10.1093/emboj/18.8.2127

Language：English   Publishing type：Research paper (scientific journal)  

The cytokines LIF (leukemia inhibitor factor) and BMP2 (bone morphogenetic protein-2) signal through different receptors and transcription factors, namely STATs (signal transducers and activators of transcription) and Smads. LIF and BMP2 were found to act in synergy on primary fetal neural progenitor cells to induce astrocytes. The transcriptional coactivator p300 interacts physically with STAT3 at its amino terminus in a cytokine stimulation-independent manner, and with Smad1 at its carboxyl terminus in a cytokine stimulation-dependent manner. The formation of a complex between STAT3 and Smad1, bridged by p300, is involved in the cooperative signaling of LIF and BMP2 and the subsequent induction of astrocytes from neural progenitors.

DOI： 10.1126/science.284.5413.479

Language：English   Publishing type：Research paper (scientific journal)  

Complementary DNAs homologous to a rat 42-kDa choline/ethanolamine kinase [C. Aoyama et al., Biochim. Biophys. Acta 1390 (1998) 1-7] and to a 50-kDa choline kinase [T. Uchida and S. Yamashita, J. Biol. Chem. 267 (1992) 10156-10162] were isolated from a 17-day post coitum mouse embryo cDNA library and their sequences were compared with the two murine species, respectively. The nucleotide sequence homology (within the coding sequence) between mouse and rat 50-kDa choline kinases (96.0%) was considerably higher than that between their 42-kDa choline/ethanolamine kinases (92.4%). Northern blot and RT-PCR studies on several rat tissues demonstrated that both isozymes are expressed ubiquitously with the highest level in testis. Copyright (C) 1998 Elsevier Science B.V.

DOI： 10.1016/S0005-2760(98)00062-9

Language：English   Publishing type：Research paper (scientific journal)  

Language：English   Publishing type：Research paper (scientific journal)  

A part of the 3'-flanking region of BP-II gene, which is one of Trimeresurus flavoviridis venom gland phospholopase A(2) (PLA(2)) isozyme genes, has a region homologous to avian chicken repeat 1 (CR1)-element. In the present study, ten CR1-like elements were further identified in T. gramineus venom gland PLA(2) isozyme genes, T. flavoviridis PLA(2) inhibitor (PLI) genes, and T. flavoviridis and T. gramineus TATA-box binding protein (TBP) genes. Southern blot analysis using a probe for CR1 showed that Crotalinae snake genomes contain a number of CR1-like elements. (C) 1998 Elsevier Science Ltd. All rights reserved.

DOI： 10.1016/S0041-0101(97)00104-9

Language：English   Publishing type：Research paper (scientific journal)  

Inhibitors (PLIs) against snake venom gland phospholipases A(2) (PLA(2)s) have been found in their sera. A cDNA encoding a PLI from Trimeresurus flavovirdis (Tf, habu snake, Crotalinae) serum, cPLI-A, was isolated from the Tf liver cDNA library and sequenced. Northern blot analysis with cPLI-A showed that PLIs are expressed only in liver. Genes for PLIs, gPLI-A and gPLI-B, were isolated from the Tf genomic DNA library and their nucleotide (nt) sequences were determined. The genes consisted of four exons and three introns, and exon 4 encoded the carbohydrate recognition domain (CRD)-like motif. Comparison of the nt sequences between gPLI-A and gPLI-B showed that these genes are highly homologous, including introns, except that exon 3 is rich in nonsynonymous nt substitutions which are almost four times as frequent as synonymous nt substitutions. This evolutionary feature of PLI genes is different from that of venom gland PLA(2) isozyme genes in which nonsynonymous nt substitutions are spread over the entire mature protein-coding region. (C) 1997 Elsevier Science B.V.

DOI： 10.1016/S0378-1119(97)00024-3

Language：English   Publishing type：Research paper (scientific journal)  

Leptin (OB) exerts weight-reducing effects in mice. The structure of the receptor for this factor, OB-R, is considerably similar to those of gp130, the common signal transducing receptor component for the interleukin-6 (IL-6) family of cytokines, and leukemia inhibitory factor receptor (LIPR). Since the IL-6 family of cytokines signal through gp130 homodimer or gp130/LIFR heterodimer, we have examined in this study the possible involvement of gp130 and LIPR in leptin signaling through OB-R. Leptin stimulation induces tyrosine phosphorylation of neither gp130 nor LIFR, while LIF stimulation does both. As examined by using two differently epitope-tagged OB-R molecules, the spontaneous homo-oligomerization of OB-R has been elucidated. Ba/F3 cells, which do not express gp130, are non-responsive to leptin and exhibit increased DNA synthesis in response to leptin after transfection of OB-R cDNA alone. OB-R appears to transduce the signal via its homooligomerization without interaction with gp130 or LIFR. (C) 1997 Federation of European Biochemical Societies.

DOI： 10.1016/S0014-5793(97)00013-6

Language：English   Publishing type：Research paper (scientific journal)  

The structure of leptin receptor (OB-R) is highly homologous to that of gp130, the common signal transducing receptor component for the interleukin-6 family of cytokines, Based on this structural similarity, we examined signaling processes initiated by OB-R in comparison with those by gp130, Stimulation of either a long form of OB-R or gp130 led to tyrosine phosphorylation of STAT3, whereas stimulation of the truncated form of OB-R that is predominantly expressed in db/db mice failed to do so, Stimulation of the long form OB-R did not induce tyrosine phosphorylation of a Src homology domain 2 containing protein tyrosine phosphatase, SHP-2, while stimulation of gp130 did, In contrast, activation of p42(ERK2) is mediated by either the long form OB-R or gp130. Two closely related molecules, OB-R and gp130, thus appear to mediate overlapping but distinct signaling procedures.

DOI： 10.1016/S0014-5793(96)01430-5

Language：English   Publishing type：Research paper (scientific journal)  

The nucleotide sequences of two cDNAs and four genes encoding Trimeresurus gramineus venom gland phospholipase A(2) (PLA(2)) isozymes were determined and compared internally and externally with those encoding Trimeresurus flavoviridis venom gland PLA(2) isozymes. It was revealed that the protein-coding regions are much more diversified than the 5' and 3' untranslated regions (UTRs) and the introns except for the signal peptide domain. The numbers of nucleotide substitutions per site (K-N) for the UTRs and the introns were approximately one-quarter of the numbers of nucleotide substitutions per synonymous site (K-s) for the protein-coding regions and were at the same level as the K-N value of T. gramineus and T. flavoviridis TATA box-binding protein (TBP) genes, indicating that the protein-coding regions of PLA(2) isozyme genes are unusually variable and that the UTRs including the introns of venom gland PLA(2) isozyme genes have evolved at similar rate to those of non-venomous genes. The numbers of nucleotide substitutions per non-synonymous site (K-A) values were close to or larger than the K-s values for the protein-coding regions in venom gland PLA(2) isozyme genes, indicating that the protein-coding regions of snake venom gland PLA(2) isozyme genes have evolved via accelerated evolution. Furthermore, the evolutionary trees derived from the combined sequences of the 5' and 3' UTRs and the signal peptide domain of cDNAs were in accord with the consequences from taxonomy. In contrast, the evolutionary trees from the mature protein-coding region sequences of cDNAs and from the amino acid sequences showed random patterns. Estimations of nucleotide divergence of genes and the phylogenetic analysis reveal that snake venom group II PLA(2) isozyme genes have been evolving under adaptive pressure to acquire new physiological activities. Copyright (C) 1996 Elsevier Science Ltd

DOI： 10.1016/S0041-0101(96)00112-2

Language：English   Publishing type：Research paper (scientific journal)  

Eight cDNAs encoding serine proteases isolated from Trimeresuras flavoviridis (habu snake) and T. gramineus (green habu snake) venom gland cDNA libraries showed that nonsynonymous nucleotide substitutions have accumulated in the mature protein-coding regions to cause amino acid changes, Southern blot analysis of T. flavoviridis genomic DNAs using two proper probes indicated that venom gland serine protease genes form a multigene family in the genome. These observations suggest that venom gland serine proteases have diversified their amino acid sequences in an accelerating manner. Since a similar feature has been pre,iously discovered in crotalinae snake venom gland phospholipase A(2) (PLA(2)) isozyme genes, accelerated evolution appears to be universal in plural isozyme families of crotalinae snake venom gland.

DOI： 10.1016/S0014-5793(96)01144-1

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Three Trimeresurus okinavensis (To; himehabu snake, Crotalinae) venom gland phospholipase A(2) (PLA(2)) isozyme-encoding genes, gPLA(2)-o1, gPLA(2)-o(2) and gPLA(2)-o3, were isolated from its genomic DNA library. The nucleotide (nt) sequence analysis revealed that two of the three genes (gPLA(2)-o2 and gPLA(2)-o3) occasionally have been converted to inactivated genes by introduction of one base insertion or substitution. It was confirmed from Southern blot analysis that the To haploid genome contains only three venom gland PLA(2) isozyme genes herein isolated. Comparison of these genes showed that nonsynonymous nt substitutions have occurred more frequently than synonymous nt substitutions in the protein-coding regions, except for the signal-peptide coding domain, implying that To venom gland PLA(2) isozyme genes have evolved via accelerated evolution, Such an evolutionary feature of To venom gland PLA(2) isozyme genes proves the general universality of accelerated evolution previously drawn for venom gland PLA(2) isozyme genes of other crotalinae snakes. The variability in the mature protein-coding regions of three To venom gland PLA(2) isozyme genes appears to have been brought about by natural selection for point mutations.

DOI： 10.1016/0378-1119(96)00186-2

Language：English   Publishing type：Research paper (scientific journal)  

Trimeresurus flavoviridis (Habu snake) venom aspartate-49-phospholipase A(2) (Asp-49-PLA(2)) was reacted at pH 9.0 with a 2-fold molar excess of 2,4,6-trinitrobenzenesulfonate in the absence of Ca2+ and two trinitrophenylated derivatives were isolated by HPLC. One was a derivative modified at Lys-ll and its activity was mostly retained, The other was a derivative modified at both Lys-ll and Lys-72 and its activity was 40% that of unmodified enzyme, Trinitrophenylation of Lys-72 appeared to bring about a conformational disorder at the lipid-water interface recognition site and thus a reduction of activity, When the enzyme was modified in the presence of Ca2+, activity decreased at a rate much faster than that in the absence of Ca2+ and Lys-69 came to be modified. These results suggested that conformational displacement of Asp-49-PLA, of a local to global type occurs upon the binding of Ca2+, The derivative modified at Lys-69 had 28% activity and existed as a monomer, This supports a previous assumption that Lys-69 participates in dimerization of group II Asp-49-PLA(w)s [Brunie et al, (1985) J. Biol. Chem. 260, 9742-9749] and shows that dimerization is not necessarily essential for activity manifestation.

DOI： 10.1002/(SICI)1099-1352(199601)9:1<23::AID-JMR235>3.0.CO;2-P

Language：English   Publishing type：Research paper (scientific journal)  

The nucleotide sequences of 13 cDNAs encoding group II phospholipases A(2) (PLA(2)s), which are from viperidae snake venoms and from mammalian sources, were aligned and analyzed by phylogenetic trees constructed using various components of the sequences. The evolutionary trees derived from the combined sequences of the untranslated (5' and 3') region and the signal peptide region of cDNAs were in accord with the consequences from taxonomy. In contrast, the evolutionary trees from the mature protein-coding region sequences of cDNAs and from the amino acid sequences showed random patterns. These observations indicated that the mature protein-coding region has evolved through a process differently from the untranslated and signal peptide regions. The trees built from the nucleotide differences at each of three positions of codons in the mature protein-coding region suggested that snake-venom-gland PLA(2) genes have evolved via a process different from mammalian PLA(2) genes. The occurrence of accelerated evolution has been recently discovered in Trimeresurus flavoviridis venom-gland group II PLA(2) isozyme genes (Nakashima et al. 1993, Proc Natl Acad Sci USA 90:5964-5968), so the present phylogenetic analysis together with the estimation of nucleotide divergence of cDNAs provides further evidence that snake-venom-group II PLA(2) isozyme genes have evolved by accelerated evolution to gain diverse physiological activities.

Language：English   Publishing type：Research paper (scientific journal)  

Basic proteins I and II (BP-I and BP-II) isolated from the venom of Trimeresurus flavoviridis (Habu snake) are isozymes of highly active Asp-49-phospholipase A(2) (Asp-49-PLA(2)) and classified into the group Lys-49-PLA(2). BP-II was found to elicit a strong contraction of guinea pig ileum, and this activity was inhibited completely by 1 mu M indomethacin, an inhibitor of the arachidonate cascade. BP-II was inactive in the Ca2+-free medium, and p-bromophenacylated His-48-BP-II was also inactive, BP-II exhibited no binding affinity for the cells expressing PLA(2) receptors. These results indicated that the contraction elicited by BP-II is due to the hydrolytic action of BP-II, liberating arachidonic acid from the ileum phospholipid biomembranes, In spite of its limited lipolytic activities (av. 0.9% of Asp-49-PLA(2)) against monomers and micelles of synthetic phospholipids, BP-II hydrolyzed considerably strongly the phospholipids in the artificial bilayer vesicles. Arachidonic acid released from liposomes of beta-arachidonoyl-gamma-stearoyl-L-alpha-phosphatidylcholine was determined by HPLC, and the activity of BP-II was estimated to be about 75% as compared to Asp-49-PLA(2). Liposomes encapsulating carboxyfluorescein exhibited a strong dye-leakage induced by BP-II in a concentration-dependent manner, only in the Ca2+-containing buffer. The net result from all these observations was that BP-II, a Lys-49-PLA(2), is an enzyme that hydrolyzes the membrane phospholipids. In contrast to BP-II, BP-I was found to be considerably weak in hydrolyzing membrane phospholipids, although its activities were distinct, BP-I and BP-II share a common sequence with the sole exception of Asp-67 (BP-I) and Asn-67 (BP-II) in the aligned sequences, This implies that the amino acid at position 67 of Lys-49-PLA(2)s is the residue required for discriminatory recognition of beta-arachidonoyl-phospholipid membranes.

Language：English   Publishing type：Research paper (scientific journal)  

Four acidic phospholipase A(2) (PLA(2)) isozymes named PLA(2)-I, II, III and IV have previously been isolated from Trimeresurus gramineus (green habu snake) venom and sequenced [Oda et al. (1991) Toxicon 29, 157; Fukagawa et al, (1992) Toxicon 30, 1131; Fukagawa et al, (1993) Toxicon 31, 957]. They contain aspartate-49 which is known to bind Ca2+, essential for catalysis. In the present study, a basic PLA(2) named PLA(2)-V containing lysine-49 was newly isolated from the same snake venom. Its isoelectric point was 9.4 and considerably higher than those (c. 4.5) of PLA(2)-I-IV. PLA(2)-V was 1.1% as active as PLA(2)-I toward egg-yolk emulsion but exhibited strong myotoxicity. The amino acid sequence of PLA(2)-V was determined by sequencing the S-carboxamidomethylated derivative and its peptide fragments produced by enzymatic (clostripain, chymotrypsin, Achromobacter protease I and Staphylococcus aureus V8 protease) cleavages, PLA(2)-V consists of 122 amino acid residues and is highly homologous (72-78%) to Lys-49 PLA(2)s so far isolated from Viperidae snake venoms but less homologous (52%) to PLA(2)-I. The presence of Asn-28, which is characteristic of Lys-49 PLA(2)s, was confirmed.

DOI： 10.1016/0041-0101(95)00090-9

Language：English   Publishing type：Research paper (scientific journal)  

The nucleotide sequences of four genes encoding Trimeresurus gramineus (green habu snake, crotalinae) venom gland phospholipase A(2) (PLA2; phosphatidylcholine 2-acylhydrolase, EC 3.1.1.4) isozymes were compared internally and externally with those of sis genes encoding Trimeresurus flavoviridis (habu snake, crotalinae) venom gland PLA2 isozymes, The numbers of nucleotide substitutions per site (K-N) for the noncoding regions including introns were one-third to one-eighth of the numbers of nucleotide substitutions per synonymous site (K-S) for the protein-coding regions of exons, indicating that the noncoding regions are much more conserved than the protein-coding regions, The K-N values for the introns were found to be nearly equivalent to those of introns of T. gramineus and T. flavoviridis TATA box-binding protein genes, which are assumed to be a general (nonvenomous) gene, Thus, it is evident that the introns of venom gland PLA2 isozyme genes have evolved at a similar rate to those of nonvenomous genes, The numbers of nucleotide substitutions per nonsynonymous site (K-A) were close to or larger than the K-S values for the protein-coding regions in venom gland PLA2 isozyme genes, All of the data combined reveal that Darwinian-type accelerated evolution has universally occurred only in the protein-coding regions of crotalinae snake venom PLA2 isozyme genes.

DOI： 10.1073/pnas.92.12.5605

Language：English   Publishing type：Research paper (scientific journal)  

Structures of Genes Encoding TATA Box-binding Proteins from Trimeresurus gramineus and T. flavoviridis Snakes

DOI： 10.1016/0378-1119(94)00681-H

Language：English   Publishing type：Research paper (other academic)  

As a step towards understanding the structure and function of phospholipases A(2) (PLA(2)s), five cDNAs encoding Trimeresurus flavoviridis venom gland PLA(2) isozymes have been sequenced. They revealed that the 5' and 3' untranslated regions are much more homologous than the protein-coding regions and that base substitutions have occurred at similar rates for the three positions of codons in the protein-coding regions. Such novel findings are of great interest from the viewpoint of molecular evolution. To gain a further insight into this evolutional phenomenon, we have isolated and sequenced six T. flavouiridis PLA(2) isozyme genes. Each gene consisted of four exons and three introns and encoded protein of 138 amino-acid residues, including the signal sequence of 16 amino-acid residues. The introns were much more homologous than the protein-coding regions of exons except for the signal peptide-coding region of the first exon. The absence of apparent functional role in the introns suggested that the protein-coding regions, except for the signal peptide-coding domains, have evolved at greater substitution rates than introns. The fact that the numbers of nucleotide substitutions per non-synonymous site are close to or larger than the numbers of nucleotide substitutions per synonymous site for relevant pairs of genes revealed that Darwinian-type accelerated substitutions have occurred in the protein-coding regions of exons. This is compatible with the presence of PLA(2) species with diverse physiological activities in the venom.

DOI： 10.1002/jmr.300080107

Language：English  

Trimeresurvus flavoviridis venom gland phospholipase A(2) (PLA(2)) genes pgPLA 1a and pgPLA 2a encode Asp-49-PLA(2) and genes pgPLA 1b and pgPLA 2b encode an isozyme of Asp-49-PLA(2). Polymorphisms were found in pairs of pgPLA 1a and pgPLA 2a and of pgPLA 1b and pgPLA 2b for individuals of T. flavoviridis. The occurrence of both homozygotes and heterozygotes was demonstrated.

DOI： 10.1271/bbb.58.1510

Language：English   Publishing type：Research paper (scientific journal)  

Darwinian Evolution of Trimeresurus flavoviridis Gland Phospholipase A2 Isozymes

DOI： 10.1351/pac199466040715

Language：English   Publishing type：Research paper (scientific journal)  

Two phospholipases A2 named PLA2-III and IV were newly isolated from Trimeresurus gramineus (green habu snake) venom in addition to PLA2-I and II reported previously [ODA et al. (1991) Toxicon 29, 15 7; FUKAGAWA et al. (1992) Toxicon 30, 133]. Their isoelectric points were determined to be about 4.5. PLA2-III and IV exhibited almost unchanged lipolytic activity toward egg-yolk when compared with PLA2-I. The amino acid sequences were determined by sequencing the native proteins and the peptides produced by enzymatic (Achromobacter protease I and clostripain) and chemical (hydroxylamine) cleavages of the S-carboxamidomethylated derivative of the proteins. Both proteins consisted of 122 amino acid residues. When compared with PLA2-I, PLA2-III showed only a single amino acid substitution at the N-terminal position; namely from His to Asn. PLA2-IV also showed a single substitution from Ala to Asp at position 72. It was inferred that these amino acid substitutions between PLA2-I and PLA2-III or IV are due to the single base substitution at the corresponding codons of genes, which might be preserved independently. The unique presence of Phe at position 28, where Tyr is commonly located and assumed to be a part of the Ca2+-binding loop, was conserved in both PLA2-III and IV as in PLA2-I. There was no significant difference in the dissociation constants (4.3-5.2 x 10(-4) M) for Ca2+ between these PLA2s and Tyr-28-containing PLA2S. These results suggested that the p-hydroxy group of Try-28 does not play a crucial role in binding of PLA2s to Ca2+.

DOI： 10.1016/0041-0101(93)90255-H

Language：English   Publishing type：Research paper (scientific journal)  

Six Trimeresurus flavoviridis (Habu snake) venom gland phospholipase A2 (PLA2) isozyme genes were found to consist of four exons and three introns and to encode proteins of 138 amino acid residues, including the signal sequence of 16 amino acid residues. Comparison of the nucleotide sequences showed that the introns are much more homologous than the protein-coding regions of exons except for the signal peptide-coding region of the first exon. The numbers of nucleotide substitutions per site (K(N)) for introns are approximately one-fourth of the numbers of nucleotide substitutions per synonymous site (K(S)) for the protein-coding regions, indicating that the introns are unusually conserved. The absence of an apparent functional role for the introns suggests that the protein-coding regions, except for the signal peptide-coding domains, have evolved at greater substitution rates than introns. The fact that the numbers of nucleotide substitutions per nonsynonymous site (K(A)) are close to or larger than K(S) values for relevant pairs of genes revealed that Darwinian-type accelerated substitutions have occurred in the protein-coding regions or exons. This is compatible with the presence of PLA2 species with diverse physiological activities in the venom.

DOI： 10.1073/pnas.90.13.5964

Language：English   Publishing type：Research paper (scientific journal)  

Trimeresurus flavoviridis (Habu snake) venom contains phospholipase A2 (PLA2) isozymes which are rich in disulfide bond and show diverse activities in spite of their highly homologous structures. When reduced form of Tyr(NO2)-67-PLA2, which is a mimic of Asp-49-PLA2 mutant, was oxidized at pH 8.0 in the presence of 5 mM L-Cysteine and 5 mM Ca2+, native form of Tyr(NO2)-67-PLA2 was produced as being judged from both activity regeneration and adequate HPLC profile. Reduced forms of two Lys-49-PLA2 isozymes with extremely low lipolytic activity, which are called basic proteins I and II, also generated native forms of proteins when oxidized under the same conditions as above. Protein disulfide isomerase accelerated proper folding of reduced Tyr-(NO2)-67-PLA2 at an initial phase of oxidation and was effective for rearrangement of incorrectly paired disulfide bonds of Asp-49-PLA2 to native construction. However, this isomerase failed to convert reduced form of partially active L-fragment, Asp-49-PLA2 lacking N-terminal octapeptide, to the native form, indicating that the entire sequence of protein in a nascent state is primarily required for proper folding. The present data together with the fact that reduced Asp-49-PLA2 folded properly (S. Tanaka et al. J. Biochem., 96, 1443 (1984)) afforded a strong basis for the structure and function study of T. flavoviridis PLA2s by means of in vitro mutagenesis technique.

Language：English   Publishing type：Research paper (scientific journal)  

As a step toward understanding the structure and function of phospholipases A2 (PLA2s), we isolated and sequenced several cDNAs encoding Trimeresurus flavoviridis venom PLA2 isozymes including two [Lys49]PLA2s called basic proteins I and II, [Thr37]PLA2, and PLX'-PLA2. Comparison of the nucleotide sequences of these cDNAs with the previously isolated [Asp49]PLA2 cDNA revealed some interesting findings from the viewpoint of evolution. First, the homologies of the 5' and 3' untranslated regions (98% and 89%, respectively) were much higher than that of the protein-coding regions (67%). The predicted secondary structure showed the characteristic stem-loop structures for both the untranslated regions of the mRNAs, suggesting that these regions play some functional role(s) in translation or stability of mRNAs. Second, base substitutions appeared to have occurred at similar rates for the three positions of codons among these PLA2s. The results are discussed in terms of evolution of PLA2s. Northern blot analysis showed that these PLA2s are specific to venom gland.

DOI： 10.1073/pnas.89.18.8557

Language：English   Publishing type：Research paper (other academic)  

Cloning and sequence analysis of cDNA for Trimeresurus flavoviridis phospholipase A2 isozymes : Unusually high conservation of untranslated region sequences
P. Goparakrishnakone and C. K. Tan, eds

Event date： 2023.10 - 2023.11

Language：Japanese  

Venue：福岡市  

Event date： 2023.10 - 2023.11

Language：Japanese  

Venue：福岡市   Country：Japan  

Event date： 2023.10 - 2023.11

Venue：福岡市   Country：Japan  

Event date： 2023.3 - 2023.6

Language：Japanese  

Venue：新潟大学五十嵐キャンパス   Country：Japan  

Event date： 2023.3

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Venue：国立京都国際会館、京都   Country：Japan  

Event date： 2023.3

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Venue：Okinawa Japan   Country：Japan  

Event date： 2022.7 - 2023.6

Language：Japanese  

Country：Japan  

Event date： 2022.6 - 2022.7

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Venue：沖縄コンベンションセンター、宜野湾市   Country：Japan  

Event date： 2022.6

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Venue：金沢文化ホール   Country：Japan  

Event date： 2021.11

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Venue：web開催   Country：Japan  

Event date： 2021.10

Language：Japanese  

Venue：web開催   Country：Japan  

Event date： 2021.9 - 2021.10

Language：Japanese  

Venue：web開催   Country：Japan  

Event date： 2021.7

Language：Japanese  

Venue：web開催   Country：Japan  

Event date： 2021.5

Language：Japanese  

Venue：web開催   Country：Japan  

Event date： 2021.3

Language：Japanese   Presentation type：Symposium, workshop panel (public)  

Venue：福岡国際会議場   Country：Japan  

Event date： 2020.6 - 2020.7

Language：Japanese  

Venue：web開催   Country：Japan  

Event date： 2020.3

Language：Japanese   Presentation type：Symposium, workshop panel (public)  

Venue：Web開催   Country：Japan  

Event date： 2020.3

Language：Japanese   Presentation type：Symposium, workshop panel (public)  

Venue：Web開催   Country：Japan  

Event date： 2020.3

Language：English   Presentation type：Oral presentation (general)  

Venue：Melia Nassau Beach All Inclusive   Country：Bahamas  

Usefulness of neural stem cell (NSC) transplantation to replenish neurons has been extensively investigated. However, because of gliogenic environment in the lesion site, effective neuronal production has not been satisfactorily successful. We have previously found that antiepileptic drug valproic acid, also known as a histone deacetylase inhibitor, induced neuronal differentiation of NSCs, and reported that combinatorial treatment with VPA and NSC transplantation can supply newly generated neurons in injured spinal cord, leading to dramatic functional recovery after spinal cord injury (SCI). In the subsequent study, we have also shown that human iPSC-derived NSCs, which are epigenetically restricted not to differentiate into glial cells, efficiently give rise to neurons in the injured spinal cord, inducing recovery of locomotor functions of mice after SCI. Although it is now understood that supply of new neurons has beneficial effects on the functional recovery, there still remain critical problems, such as tumorigenicity and time-consuming preparation of cells if we utilize iPSC-derived cells. As an alternative strategy to replenish new neurons in vivo, direct conversion from endogenous cells in non-neuronal lineages has recently attracted much attention. In this regard, we have found that microglia, which converge at lesion site after injury, can be converted to functional neurons both in vitro and in vivo by the expression of a single proneural transcription factor NeuroD1. In this talk, I would like to introduce these our achievements and discuss future directions regarding treatments of CNS injuries.

Other Link： https://www.fusion-conferences.com/conference/107

Event date： 2020.2

Language：Japanese   Presentation type：Oral presentation (invited, special)  

Venue：琵琶湖ホテル   Country：Japan  

Event date： 2020.1

Language：Japanese   Presentation type：Oral presentation (invited, special)  

Venue：熊本大学医学部山崎記念館   Country：Japan  

Event date： 2019.11

Language：Japanese   Presentation type：Oral presentation (general)  

Venue：神戸市 sheraton Kobe Bay Hotel&Towers   Country：Japan  

Event date： 2019.11

Language：Japanese   Presentation type：Oral presentation (general)  

Venue：岡山県、岡山大学Junko Fukutake Hall   Country：Japan  

Event date： 2019.9

Language：Japanese   Presentation type：Symposium, workshop panel (public)  

Venue：福井県、福井大学文京キャンパス   Country：Japan  

Event date： 2019.9

Language：Japanese   Presentation type：Oral presentation (general)  

Venue：福岡県、国民宿舎マリンテラスあしや   Country：Japan  

Event date： 2019.6

Language：Japanese   Presentation type：Symposium, workshop panel (public)  

Venue：徳島県、アスティとくしま   Country：Japan  

Event date： 2019.6

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Venue：徳島県、アスティとくしま   Country：Japan  

Event date： 2019.2

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Venue：中之島センター   Country：Japan  

Event date： 2018.11

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Venue：パシフィコ横浜   Country：Japan  

Event date： 2018.10

Language：Japanese   Presentation type：Oral presentation (general)  

Venue：名古屋市立大学病院   Country：Japan  

Event date： 2018.10

Language：Japanese   Presentation type：Oral presentation (general)  

Venue：秋葉原コンベンションホール   Country：Japan  

Event date： 2018.9

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Venue：神戸国際会議場   Country：Japan  

Event date： 2018.7

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Venue：神戸コンベンションセンター   Country：Japan  

Event date： 2018.3

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Venue：香川県   Country：Japan  

Event date： 2018.3

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Venue：金沢市   Country：Japan  

Event date： 2017.9 - 2017.10

Language：Japanese   Presentation type：Oral presentation (invited, special)  

Venue：名古屋市   Country：Japan  

Event date： 2017.9

Language：Japanese   Presentation type：Oral presentation (general)  

Venue：北海道   Country：Japan  

Event date： 2017.7

Language：Japanese   Presentation type：Symposium, workshop panel (public)  

Venue：幕張メッセ   Country：Japan  

Event date： 2017.1

Language：English   Presentation type：Oral presentation (general)  

Venue：Olympic Valley   Country：United States  

Language：Japanese   Publishing type：Article, review, commentary, editorial, etc. (scientific journal)  

Language：Japanese   Publisher：(公財)金原一郎記念医学医療振興財団  

＜文献概要＞ヒトの個性はどのようにして決まるのだろうか。われわれヒトを含む生物は,遺伝子情報に基づいてその生命体を構成・維持している。しかし,ヒトの個性を決めるのは単に遺伝子情報だけではない。がん家系に生まれた人でも,お酒やたばこを控えるなど周囲の環境を改善することでがんにならずに済む人もいる。近年,われわれの体を構成する細胞には,DNAの配列変化を伴わずに,遺伝子発現の強弱あるいはオン/オフを制御するしくみが存在しており,その制御は環境要因によって動的に変化することが科学的に明らかになってきた。この制御を"エピジェネティクス"と呼び,遺伝子配列情報だけでは理解できない事象を理解するために非常に重要である。本稿ではエピジェネティクスの概要と,それが生み出すと考えられる個性について紹介する。

Language：Japanese   Publishing type：Article, review, commentary, editorial, etc. (scientific journal)  

Language：Japanese   Publisher：(株)ニュー・サイエンス社  

脳梗塞などの中枢神経損傷は,重度の神経障害につながり,現在の治療やリハビリテーションでは失われた機能の回復には限界がある。失われたニューロンを補填するための治療法として,最近注目され始めているのがダイレクトリプログラミング法である。ダイレクトリプログラミング法とは,終末分化細胞に,細胞系譜特異的な転写因子を強制発現させ,幹細胞状態を介さずに,別の系統の終末分化細胞に誘導する方法である。筆者らは最近,ダイレクトリプログラミング法を用いて,脳梗塞モデルマウスの脳損傷部位に自然集積するミクログリアにNeuroD1を強制発現させることによって,ミクログリアをニューロンに変換し,脳梗塞により失われた機能を回復させることができるという予備的実験結果を得た。本稿では,ダイレクトリプログラミングによる神経損傷に対する再生医療の現状を述べる。(著者抄録)

Language：Japanese   Publisher：東京 : ニューサイエンス社  

CiNii Books

CiNii Research

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Language：English   Publishing type：Article, review, commentary, editorial, etc. (scientific journal)  

DOI： 10.3389/fnins.2023.1150283

Language：Japanese  

Language：Japanese   Publisher：医歯薬出版(株)  

ヒトを含む成体哺乳類の中枢神経系では,神経幹細胞からニューロンが新生される.しかし,その量は虚血性・外傷性脳損傷や神経変性疾患などにより失われた神経機能を回復するには十分でない.近年,こうした損傷や疾患によって失われたニューロンを再生するために着目されているのが,生体内ダイレクトリプログラミング(以下,直接分化転換法)である.これは生体内で,体細胞を特徴付けるために鍵となる因子の発現を操作することで,幹細胞などの多能性を持つ細胞状態を経由せず,意図した細胞へ直接分化転換する方法である.幹細胞状態を経由しないため,腫瘍化のリスクがきわめて少なく,必要細胞種を治療が必要なときに,免疫拒絶反応を引き起こさず患者体内へ供給可能である.本稿では,アストロサイトやミクログリアを用いた直接分化転換法による神経再生の最新の成果について概説する.(著者抄録)

J-GLOBAL

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Language：Japanese   Publishing type：Article, review, commentary, editorial, etc. (scientific journal)  

Language：English   Publishing type：Article, review, commentary, editorial, etc. (scientific journal)  

Neural stem cells (NSCs) in the adult hippocampus generate new neurons via a process referred to as neurogenesis, supporting cognitive functions. Since altered neurogenesis has been reportedly associated with several diseases such as epilepsy, the molecular basis of NSC activity is an important focus in the study of neurogenesis. Furthermore, facilitation of neurogenesis in the injured brain would be an ideal approach to replenish lost neurons for damage recovery. However, natural neurogenesis by endogenous NSCs in the adult brain is insufficient for complete recovery after severe injury. Recent advances in understanding forced neurogenesis from brain-resident non-neuronal cells by direct reprogramming and clearing hurdles to achieve it have improved the ability to replace damaged neurons in the brain. In this review, we describe molecular mechanisms underlying natural and forced neurogenesis, and discuss future directions for treatments of diseases in the central nervous system.

DOI： 10.1016/j.neures.2020.05.011

Language：English   Publishing type：Article, review, commentary, editorial, etc. (scientific journal)  

https://hyoka-lab.ir.kyushu-u.ac.jp/search/FSIServ

DOI： 10.3390/cells10051145

Language：Japanese  

Language：Japanese  

Language：Japanese  

Language：Japanese  

Language：Japanese  

Language：English  

The central nervous system (CNS) is composed of three major cell types, neurons, astrocytes, and oligodendrocytes, which differentiate from common multipotent neural stem/precursor cells (NS/PCs). However, NS/PCs do not have this multipotentiality from the beginning: neurons are generated first and astrocytes are later during CNS development. This developmental progression is observed in vitro by using human (h) NS/PCs derived from pluripotent cells, such as embryonic- and induced pluripotent-stem cells (ES/ iPSCs), however, in contrast to rodent’s pluripotent cells, they require quite long time to obtain astrocytic differentiation potential. Here, we show that hypoxia confers astrocytic differentiation potential on hNS/PCs through epigenetic alteration for gene regulation. Furthermore, we found that these molecular mechanisms can be applied to functional analysis of patient’ iPSC-derived astrocytes. In this review, we summarize recent findings that address molecular mechanisms of epigenetic and transcription factor-mediated regulation that specify NS/PC fate and the development of potential therapeutic strategies for treating astrocyte-mediated neurological disorders.

DOI： 10.1254/fpj.153.54

Language：Japanese  

低用量ペルメトリン早期慢性暴露によるマウス次世代雄個体行動影響

Language：Japanese  

Language：Japanese  

子どもへの低用量化学物質暴露が誘発する情動認知行動影響とその評価系の開発 幼若期ネオニコチノイド投与が成体マウス脳トランスクリプトームに及ぼす影響

Language：Japanese  

子どもへの低用量化学物質暴露が誘発する情動認知行動影響とその評価系の開発 幼若期ネオニコチノイド投与が成体マウス脳トランスクリプトームに及ぼす影響

Language：English   Publishing type：Article, review, commentary, editorial, etc. (scientific journal)  

Severe spinal cord injury (SCI) leads to almost complete neural cell loss at the injured site, causing the irreversible disruption of neuronal circuits. The transplantation of neural stem or precursor cells (NS/PCs) has been regarded as potentially effective for SCI treatment because NS/PCs can compensate for the injured sites by differentiating into neurons and glial cells (astrocytes and oligodendrocytes). An understanding of the molecular mechanisms that regulate the proliferation, fate specification and maturation of NS/PCs and their progeny would facilitate the establishment of better therapeutic strategies for regeneration after SCI. In recent years, several studies of SCI animal models have demonstrated that the modulation of specific epigenetic marks by histone modifiers and non-coding RNAs directs the setting of favorable cellular environments that promote the neuronal differentiation of NS/PCs and/or the elongation of the axons of the surviving neurons at the injured sites. In this review, we provide an overview of recent progress in the epigenetic regulation/manipulation of neural cells for the treatment of SCI.

DOI： 10.1007/s00441-017-2656-2

Language：English   Publishing type：Article, review, commentary, editorial, etc. (scientific journal)  

Neural stem cells (NSCs) undergo self-renewal and generate neurons and glial cells under the influence of specific signals from surrounding environments. Glioblastoma multiforme (GBM) is a highly lethal brain tumor arising from NSCs or glial precursor cells owing to dysregulation of transcriptional and epigenetic networks that control self-renewal and differentiation of NSCs. Highly tumorigenic glioblastoma stem cells (GSCs) constitute a small subpopulation of GBM cells, which share several characteristic similarities with NSCs. GSCs exist atop a stem cell hierarchy and generate heterogeneous populations that participate in tumor propagation, drug resistance, and relapse. During multimodal treatment, GSCs de-differentiate and convert into cells with malignant characteristics, and thus play critical roles in tumor propagation. In contrast, differentiation therapy that induces GBM cells or GSCs to differentiate into a neuronal or glial lineage is expected to inhibit their proliferation. Since stem cell differentiation is specified by the cells’ epigenetic status, understanding their stemness and the epigenomic situation in the ancestor, NSCs, is important and expected to be helpful for developing treatment modalities for GBM. Here, we review the current findings regarding the epigenetic regulatory mechanisms of NSC fate in the developing brain, as well as those of GBM and GSCs. Furthermore, considering the similarities between NSCs and GSCs, we also discuss potential new strategies for GBM treatment.

DOI： 10.1007/s10565-017-9420-y

Language：English   Publishing type：Article, review, commentary, editorial, etc. (scientific journal)  

Injury to the spinal cord causes transection of axon fibers and neural cell death, resulting in disruption of the neural network and severe functional loss. Reconstruction of the damaged neural circuits was once considered to be hopeless as the adult mammalian central nervous system has very poor ability to regenerate. For this reason, there is currently no effective therapeutic treatment for spinal cord injury (SCI). However, with recent developments in stem cell research and cell culture technology, regenerative therapy using neural stem cell (NSC) transplantation has rapidly been developed, and this therapeutic strategy makes it possible to rebuild the destroyed neural circuits. In this review, we discuss the recent breakthroughs in NSC transplantation therapy for SCI. Developmental Dynamics 247:75–84, 2018.

DOI： 10.1002/dvdy.24558

Language：English   Publishing type：Article, review, commentary, editorial, etc. (scientific journal)  

In mammals, transcription in the zygote begins after fertilization. This transcriptional wave is called zygotic gene activation (ZGA). During ZGA, epigenetic modifications, such as DNA methylation and histone modifications, are dynamically and drastically reconstructed in a sequence-specific manner. However, how such orchestrated gene upregulation is regulated remains unknown. Recently, using microinjection techniques, we have revealed that a class of long noncoding RNAs, named promoter-associated noncoding RNAs (pancRNAs), mediates specific gene upregulation through promoter DNA demethylation during ZGA. Here, we describe the experimental methods available to control the expression levels of pancRNAs and to evaluate epigenetic status after pancRNA manipulation.

DOI： 10.1007/978-1-4939-6716-2_16

Language：Japanese  

Language：English   Publishing type：Article, review, commentary, editorial, etc. (scientific journal)  

Development of high-throughput sequencing technologies has uncovered the immensity of the long noncoding RNA (lncRNA) world. Divergently transcribed lncRNAs from bidirectional gene promoters, called promoter-associated noncoding RNAs (pancRNAs), account for ~20% of the total number of lncRNAs, and this major fraction is involved in many biological processes, such as development and cancer formation. Recently, we have found that the pancRNAs activate their partner genes, as represented by the fact that pancIl17d, a pancRNA that is transcribed from the antisense strand of the promoter region of Interleukin 17d (Il17d) at the onset of zygotic gene activation (ZGA), is essential for mouse preimplantation development through Il17d upregulation. The discovery of the expression of a specific set of pancRNAs during ZGA was achieved by using a method that generates directional RNA-seq libraries from small-scale samples. Although there are several methods available for small-scale samples, most of them require a pre-amplification procedure that frequently generates some amplification biases toward a subset of transcripts. We provide here a highly sensitive and reproducible method based on the preparation of directional RNA-seq libraries from as little as 100 mouse oocytes or embryos without pre-amplification for the quantification of lncRNAs as well as mRNAs.

DOI： 10.1007/978-1-4939-6988-3_6

Language：English   Publishing type：Article, review, commentary, editorial, etc. (scientific journal)  

In the developing brain, the three major cell types, i.e., neurons, astrocytes and oligodendrocytes, are generated from common multipotent neural stem cells (NSCs). In particular, astrocytes eventually occupy a great fraction of the brain and play pivotal roles in the brain development and functions. However, NSCs cannot produce the three major cell types simultaneously from the beginning; e.g., it is known that neurogenesis precedes astrogenesis during brain development. How is this fate switching achieved? Many studies have revealed that extracellular cues and intracellular programs are involved in the transition of NSC fate specification. The former include growth factor- and cytokine-signaling, and the latter involve epigenetic machinery, including DNA methylation, histone modifications, and non-coding RNAs. Accumulating evidence has identified a complex array of epigenetic modifications that control the timing of astrocytic differentiation of NSCs. In this review, we introduce recent progress in identifying the molecular mechanisms of astrogenesis underlying the tight regulation of neuronal-astrocytic fate switching of NSCs.

DOI： 10.2183/pjab.93.024

Language：Japanese   Publishing type：Article, review, commentary, editorial, etc. (scientific journal)  

DOI： 10.14952/SEIKAGAKU.2017.890051

Language：English  

Neural stem cells (NSCs) have the ability to self-renew and give rise to neurons and glial cells (astrocytes and oligodendrocytes) in the mammalian central nervous system. This multipotency is acquired by NSCs during development and is maintained throughout life. Proliferation, fate specification, and maturation of NSCs are regulated by both cell intrinsic and extrinsic factors. Epigenetic modification is a representative intrinsic factor, being involved in many biological aspects of central nervous system development and adult neurogenesis through the regulation of NSC dynamics. In this review, we summarize recent progress in the epigenetic regulation of NSC behavior in the embryonic and adult brain, with particular reference to DNA methylation, histone modification, and noncoding RNAs.

DOI： 10.1016/j.nepig.2016.01.001

Language：English   Publishing type：Article, review, commentary, editorial, etc. (scientific journal)  

A population of proliferating neural stem/progenitor cells located in the subgranular zone of the adult hippocampal dentate gyrus (DG) gives rise to new neurons continuously throughout life, and this process is referred to as adult hippocampal neurogenesis. To date, it has generally been accepted that impairments of adult hippocampal neurogenesis resulting from pathological conditions such as stress, ischemia and epilepsy lead to deficits in hippocampus-dependent learning and memory tasks. Recently, we have discovered that microglia, the major immune cells in the brain, attenuate seizure- induced aberrant hippocampal neurogenesis to withstand cognitive decline and recurrent seizure. In that study, we further showed that Toll-like receptor 9, known as a pathogen-sensing receptor for innate immune system activation, recognizes self-DNA derived from degenerating neurons to induce TNF-a production in the microglia after seizure, resulting in inhibition of seizure-induced aberrant neurogenesis. Our findings provide new evidence that interaction between the innate immune and nervous systems ensures homeostatic neurogenesis in the adult hippocampus and should pave the way for the development of new therapeutic strategies for neurological diseases including epilepsy.

DOI： 10.1080/23262133.2015.1081714

Language：English   Publishing type：Article, review, commentary, editorial, etc. (scientific journal)  

Differentiation of neural stem/precursor cells (NS/PCs) into neurons, astrocytes and oligodendrocytes during mammalian brain development is a carefully controlled and timed event. Increasing evidences suggest that epigenetic regulation is necessary to drive this. Here, we provide an overview of the epigenetic mechanisms involved in the developing mammalian embryonic forebrain. Histone methylation is a key factor but other epigenetic factors such as DNA methylation and noncoding RNAs also partake during fate determination. As numerous epigenetic modifications have been identified, future studies on timing and regional specificity of these modifications will further deepen our understanding of how intrinsic and extrinsic mechanisms participate together to precisely control brain development.

DOI： 10.2217/epi.14.53

Language：English   Publishing type：Article, review, commentary, editorial, etc. (scientific journal)  

In the mammalian brain, epigenetic mechanisms are clearly involved in the regulation of self-renewal of neural stem cells and the derivation of their descendants, i.e. neurons, astrocytes and oligodendrocytes, according to the developmental timing and the microenvironment, the 'niche'. Interestingly, local epigenetic changes occur, concomitantly with genome-wide level changes, at a set of gene promoter regions for either down- or upregulation of the gene. In addition, intergenic regions also sensitize the availability of epigenetic modifiers, which affects gene expression through a relatively long-range chromatinic interaction with the transcription regulatory machineries including non-coding RNA (ncRNA) such as promoter-associated ncRNA and enhancer ncRNA. We show that such an epigenetic landscape in a neural cell is statically but flexibly formed together with a variable combination of generally and locally acting nuclear molecules including master transcription factors and cell-cycle regulators. We also discuss the possibility that revealing the epigenetic regulation by the local DNA-RNA-protein assemblies would promote methodological innovations, e.g. neural cell reprogramming, engineering and transplantation, to manipulate neuronal and glial cell fates for the purpose of medical use of these cells.

DOI： 10.1098/rstb.2013.0511

Language：English   Publishing type：Article, review, commentary, editorial, etc. (scientific journal)  

In the mammalian central nervous system, astrocytes are the most abundant cell type and play crucial roles in brain development and function. Astrocytes are known to be produced from multipotent neural stem cells (NSCs) at the late gestational stage during brain development, and accumulating evidence indicates that this stage-dependent generation of astrocytes from NSCs is achieved by systematic cooperation between environmental cues and cell-intrinsic programs. Exemplifying the former is cytokine signaling through the gp130-Janus kinase/signal transducer and activator of transcription 3 pathway, and exemplifying the latter is epigenetic modification of astrocyte-specific genes. Here, we introduce recent advances in our understanding of the mechanisms that coordinate astrocytogenesis from NSCs by modulating signaling pathways and epigenetic programs, with a particular focus on the developing mammalian forebrain.

DOI： 10.1016/j.conb.2013.06.002

Language：English   Publishing type：Article, review, commentary, editorial, etc. (scientific journal)  

The cerebral cortex comprises over three quarters of the brain, and serves as structural basis for the sophisticated perceptual and cognitive functions. It develops from common multipotent neural stem cells (NSCs) that line the neural tube. Development of the NSCs encompasses sequential phases of progenitor expansion, neurogenesis, and gliogenesis along with the progression of developmental stages. Interestingly, NSCs steadfastly march through all of these phases and give rise to specific neural cell types in a temporally defined and highly predictable manner. Herein, we delineate the intrinsic and extrinsic factors that dictate the progression and tempo of NSC differentiation during cerebral cortex development, and how epigenetic modifications contribute to the dynamic properties of NSCs.

DOI： 10.1016/j.ijdevneu.2013.02.006

Language：Japanese  

妊娠期投与による胎生期バルプロ酸暴露マウスは学習記憶異常と海馬抑制系の減弱を示す

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アストロサイト分化における小胞体ストレス応答の役割(The role of endoplasmic reticulum stress response in astrocyte differentiation)

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DOI： 10.11251/ojjscn.41.411

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脊髄損傷に対するエピジェネティック治療

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神経新生・幹細胞の生物学 REST/NRSFを介した骨形成因子BMPによる新規ニューロン分化抑制機構(A inhibitory mechanism of neuronal differentiation mediated by BMP-induced REST/NRSF)

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エピジェネティクスにより制御される神経幹細胞運命決定機構

Language：Japanese  

ヒストン脱アセチル化酵素阻害剤バルプロ酸による神経幹細胞分化制御機構の解明と損傷脊髄再生治療への応用

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生命現象との関わり 2.幹細胞分化とエピジェネティクス―神経幹細胞をモデルとして

Language：Others  

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Notchシグナルによる発生段階依存的な神経幹細胞成熟機構の解析

Language：Japanese  

レチノイン酸とLIFによる神経幹細胞の相乗的アストロサイト分化誘導

Language：Japanese  

メチル化DNA結合蛋白質による神経系細胞分化可塑性制御機構の解析

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メチル化DNA結合タンパク質による神経系細胞分化可塑性制御

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神経幹細胞多分化能獲得機構の解析

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DOI： 10.11477/mf.1431100002

Language：Japanese  

ハブ毒ホスホリパーゼA₂アイソザイムの島内および島間多様性の比較

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Neurogenin1は神経幹細胞においてintegrinα5の発現を抑制する

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マウスAGM造血期におけるgp130シグナルの役割

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DOI： 10.11477/mf.2425902275

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胎生10.5日マウスdorsal aortaで発現する分泌蛋白と膜蛋白のcDNAクローニング

Language：Japanese  

日本南西諸島におけるハブ毒腺ホスホリパーゼA2アイソザイムの孤立による進化

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IL‐6ファミリーサイトカインによる胎生期マウス神経上皮細胞のアストロサイトへの分化誘導とその分子機構

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LIFとBMP2のシグナルクロストークによる未分化神経上皮細胞の分化制御

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インターロイキンIIにより誘導される未分化神経上皮細胞のアストロサイトへの分化

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Accelerated evolution of viper (Vipera ammodytes Serpentes) subfamily snake venom gland thrombin-like enzyme isoenzyme.

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Accelerated Evolution of Snake Venom Phospholipase A2 Isozymes to Acquire Multiple Functions.

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DOI： 10.1271/kagakutoseibutsu1962.32.702

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Multiple Functions of Snake Venom Gland phospholipase A2 Isozymes Acquired by Adaptive Evolution: Approach by Protein Engineering.

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グリーンハブ毒腺塩基性Lys‐49‐ホスホリパーゼA₂の精製と一次構造

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ヒメハブ毒ホスホリパーゼA₂アイソザイムcDNA及び遺伝子のクローニング

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ハブ属異種ヘビ間におけるホスホリパーゼA₂アイソザイム遺伝子の比較

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II型ホスホリパーゼA₂の分子進化

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ハブ毒腺ホスホリパーゼA₂アイソザイムの機能の多様性と適応進化

Language：Japanese  

ハブ毒ホスホリパーゼA₂アイソザイム遺伝子の多様性及び構造解析

Type：Academic society, research group, etc. 

Type：Competition, symposium, etc. 

Number of participants：100

Type：Competition, symposium, etc. 

Number of participants：350

Type：Academic society, research group, etc.