Publications by authors named "Kinichi Nakashima"

114 Publications

CADM1 and CADM2 triggers neuropathogenic measles virus-mediated membrane fusion by acting in .

J Virol 2021 Apr 28. Epub 2021 Apr 28.

Department of Virology, Faculty of Medicine, Kyushu University, Fukuoka, Japan

Measles virus (MeV), an enveloped RNA virus in the family , 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 with the attachment protein on the envelope. However, CADM1 and CADM2 interact in 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 -acting fusion triggering) by which MeV spreads trans-synaptically between neurons, thereby causing SSPE.Measles 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 with the attachment protein on the viral membrane (envelope). Remarkably, CADM1 and CADM2 interact in with the MeV attachment protein on the same membrane, triggering the fusion protein and causing membrane fusion, as viral receptors usually do in Careful screening may lead to more examples of such "receptor mimicking -acting fusion triggering" in other viruses.
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http://dx.doi.org/10.1128/JVI.00528-21DOI Listing
April 2021

Combinatrial treatment of anti-High Mobility Group Box-1 monoclonal antibody and epothilone B improves functional recovery after spinal cord contusion injury.

Neurosci Res 2021 Apr 17. Epub 2021 Apr 17.

Department of Stem Cell Biology and Medicine, Graduate School of Medical Sciences, Kyushu University, Fukuoka, Japan. Electronic address:

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.
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http://dx.doi.org/10.1016/j.neures.2021.04.002DOI Listing
April 2021

Reconstitution of the oocyte transcriptional network with transcription factors.

Nature 2021 01 16;589(7841):264-269. Epub 2020 Dec 16.

Department of Stem Cell Biology and Medicine, Graduate School of Medical Sciences, Kyushu University, Fukuoka, Japan.

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 expression, 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.
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http://dx.doi.org/10.1038/s41586-020-3027-9DOI Listing
January 2021

NFκB and TGFβ contribute to the expression of PTPN3 in activated human lymphocytes.

Cell Immunol 2020 12 17;358:104237. Epub 2020 Oct 17.

Department of Surgery and Oncology, Graduate School of Medical Sciences, Kyushu University, Fukuoka, Japan.

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.
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http://dx.doi.org/10.1016/j.cellimm.2020.104237DOI Listing
December 2020

Identification of Qk as a Glial Precursor Cell Marker that Governs the Fate Specification of Neural Stem Cells to a Glial Cell Lineage.

Stem Cell Reports 2020 10 24;15(4):883-897. Epub 2020 Sep 24.

Department of Anatomy and Developmental Biology, Graduate School of Medicine, Kyoto University, Sakyo-ku, Kyoto 606-8501, Japan.

During brain development, neural stem cells (NSCs) initially produce neurons and change their fate to generate glias. While the regulation of neurogenesis is well characterized, specific markers for glial precursor cells (GPCs) and the master regulators for gliogenesis remain unidentified. Accumulating evidence suggests that RNA-binding proteins (RBPs) have significant roles in neuronal development and function, as they comprehensively regulate the expression of target genes in a cell-type-specific manner. We systematically investigated the expression profiles of 1,436 murine RBPs in the developing mouse brain and identified quaking (Qk) as a marker of the putative GPC population. Functional analysis of the NSC-specific Qk-null mutant mouse revealed the key role of Qk in astrocyte and oligodendrocyte generation and differentiation from NSCs. Mechanistically, Qk upregulates gliogenic genes via quaking response elements in their 3' untranslated regions. These results provide crucial directions for identifying GPCs and deciphering the regulatory mechanisms of gliogenesis from NSCs.
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http://dx.doi.org/10.1016/j.stemcr.2020.08.010DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7562946PMC
October 2020

Natural and forced neurogenesis in the adult brain: Mechanisms and their possible application to treat neurological disorders.

Neurosci Res 2021 May 1;166:1-11. Epub 2020 Jun 1.

Department of Stem Cell Biology and Medicine, Graduate School of Medical Sciences, Kyushu University, Fukuoka, Japan.

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.
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http://dx.doi.org/10.1016/j.neures.2020.05.011DOI Listing
May 2021

Enhanced processivity of Dnmt1 by monoubiquitinated histone H3.

Genes Cells 2020 Jan 3;25(1):22-32. Epub 2019 Dec 3.

Laboratory of Epigenetics, Institute for Protein Research, Osaka University, Suita, Japan.

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.
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http://dx.doi.org/10.1111/gtc.12732DOI Listing
January 2020

Early-life exposure to low levels of permethrin exerts impairments in learning and memory with the effects on neuronal and glial population in adult male mice.

J Appl Toxicol 2019 12 15;39(12):1651-1662. Epub 2019 Aug 15.

Laboratory of Animal Reproduction and Development, Graduate School of Agricultural Science, Tohoku University, Sendai, Japan.

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.
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http://dx.doi.org/10.1002/jat.3882DOI Listing
December 2019

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

Elife 2019 04 11;8. Epub 2019 Apr 11.

Programs in Neuroscience and Developmental Biology, Eli and Edythe Broad Center of Regeneration Medicine and Stem Cell Research, University of California, San Francisco, San Francisco, United States.

Adult hippocampal neurogenesis requires the quiescent neural stem cell (NSC) pool to persist lifelong. However, establishment and maintenance of quiescent NSC pools during development is not understood. Here, we show that Suppressor of Fused (Sufu) controls establishment of the quiescent NSC pool during mouse dentate gyrus (DG) development by regulating Sonic Hedgehog (Shh) signaling activity. Deletion of in NSCs early in DG development decreases Shh signaling activity leading to reduced proliferation of NSCs, resulting in a small quiescent NSC pool in adult mice. We found that putative adult NSCs proliferate and increase their numbers in the first postnatal week and subsequently enter a quiescent state towards the end of the first postnatal week. In the absence of Sufu, postnatal expansion of NSCs is compromised, and NSCs prematurely become quiescent. Thus, Sufu is required for Shh signaling activity ensuring expansion and proper transition of NSC pools to quiescent states during DG development.
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http://dx.doi.org/10.7554/eLife.42918DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6459675PMC
April 2019

[Hypoxia epigenetically bestows astrocytic differentiation potential on human pluripotent cell-derived neural stem/precursor cells].

Nihon Yakurigaku Zasshi 2019;153(2):54-60

Department of Stem Cell Biology and Medicine, Graduate School of Medical Sciences, Kyushu University.

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.
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http://dx.doi.org/10.1254/fpj.153.54DOI Listing
August 2019

Pioneer Factor NeuroD1 Rearranges Transcriptional and Epigenetic Profiles to Execute Microglia-Neuron Conversion.

Neuron 2019 02 9;101(3):472-485.e7. Epub 2019 Jan 9.

Department of Stem Cell Biology and Medicine, Graduate School of Medical Sciences, Kyushu University, Fukuoka, Japan. Electronic address:

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.
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http://dx.doi.org/10.1016/j.neuron.2018.12.010DOI Listing
February 2019

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

Neuroscience 2019 02 5;398:193-205. Epub 2018 Dec 5.

Department of Medicine and Clinical Science, Graduate School of Medical Sciences, Kyushu University, 3-1-1 Maidashi, Higashi-ku, Fukuoka 812-8582, Japan.

Reactive oxygen species (ROS) modulate the growth of neural stem/precursor cells (NS/PCs) and participate in hippocampus-associated learning and memory. However, the origin of these regulatory ROS in NS/PCs is not fully understood. In the present study, we found that Nox4, a ROS-producing NADPH oxidase family protein, is expressed in primary cultured NS/PCs and in those of the adult mouse brain. Nox inhibitors VAS 2870 and GKT137831 or Nox4 deletion attenuated bFGF-induced proliferation of cultured NS/PCs, while lentivirus-mediated Nox4 overexpression increased the production of HO, the phosphorylation of Akt, and the proliferation of cultured NS/PCs. Nox4 did not significantly affect the potential of cultured NS/PCs to differentiate into neurons or astrocytes. The histological and functional development of the hippocampus appeared normal in Nox4 mice. Although pathological and functional damages in the hippocampus induced by the neurotoxin trimethyltin were not significantly different between wild-type and Nox4 mice, the post-injury reactive proliferation of NS/PCs and neurogenesis in the subgranular zone (SGZ) of the dentate gyrus were significantly impaired in Nox4 animals. Restoration from the trimethyltin-induced impairment in recognition and spatial working memory was also significantly attenuated in Nox4 mice. Collectively, our findings suggest that Nox4 participates in NS/PC proliferation and neurogenesis in the hippocampus following injury, thereby helping to restore memory function.
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http://dx.doi.org/10.1016/j.neuroscience.2018.11.046DOI Listing
February 2019

Epigenetic Regulation of Human Neural Stem Cell Differentiation.

Results Probl Cell Differ 2018;66:125-136

Department of Stem Cell Biology and Medicine, Graduate School of Medical Sciences, Kyushu University, Fukuoka, Japan.

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).
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http://dx.doi.org/10.1007/978-3-319-93485-3_5DOI Listing
July 2019

Np95/Uhrf1 regulates tumor suppressor gene expression of neural stem/precursor cells, contributing to neurogenesis in the adult mouse brain.

Neurosci Res 2019 Jun 31;143:31-43. Epub 2018 May 31.

Stem Cell Biology and Medicine, Department of Stem Cell Biology and Medicine, Graduate School of Medical Sciences, Kyushu University, Fukuoka, Japan. Electronic address:

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.
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http://dx.doi.org/10.1016/j.neures.2018.05.007DOI Listing
June 2019

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

J Neurosci 2018 05 25;38(20):4791-4810. Epub 2018 Apr 25.

Department of Stem Cell Biology and Medicine, Graduate School of Medical Sciences, Kyushu University, Higashi-ku, Fukuoka, Fukuoka 812-8582, Japan,

Functional neuronal connectivity requires proper neuronal morphogenesis and its dysregulation causes neurodevelopmental diseases. Transforming growth factor-β (TGF-β) family cytokines play pivotal roles in development, but little is known about their contribution to morphological development of neurons. Here we show that the Smad-dependent canonical signaling of TGF-β family cytokines negatively regulates neuronal morphogenesis during brain development. Mechanistically, activated Smads form a complex with transcriptional repressor TG-interacting factor (TGIF), and downregulate the expression of a neuronal polarity regulator, collapsin response mediator protein 2. We also demonstrate that TGF-β family signaling inhibits neurite elongation of human induced pluripotent stem cell-derived neurons. Furthermore, the expression of TGF-β receptor 1, Smad4, or TGIF, which have mutations found in patients with neurodevelopmental disorders, disrupted neuronal morphogenesis in both mouse (male and female) and human (female) neurons. Together, these findings suggest that the regulation of neuronal morphogenesis by an evolutionarily conserved function of TGF-β signaling is involved in the pathogenesis of neurodevelopmental diseases. Canonical transforming growth factor-β (TGF-β) signaling plays a crucial role in multiple organ development, including brain, and mutations in components of the signaling pathway associated with several human developmental disorders. In this study, we found that Smads/TG-interacting factor-dependent canonical TGF-β signaling regulates neuronal morphogenesis through the suppression of collapsin response mediator protein-2 (CRMP2) expression during brain development, and that function of this signaling is evolutionarily conserved in the mammalian brain. Mutations in canonical TGF-β signaling factors identified in patients with neurodevelopmental disorders disrupt the morphological development of neurons. Thus, our results suggest that proper control of TGF-β/Smads/CRMP2 signaling pathways is critical for the precise execution of neuronal morphogenesis, whose impairment eventually results in neurodevelopmental disorders.
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http://dx.doi.org/10.1523/JNEUROSCI.2423-17.2018DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6596019PMC
May 2018

Ectopic neurogenesis induced by prenatal antiepileptic drug exposure augments seizure susceptibility in adult mice.

Proc Natl Acad Sci U S A 2018 04 2;115(16):4270-4275. Epub 2018 Apr 2.

Department of Stem Cell Biology and Medicine, Graduate School of Medical Sciences, Kyushu University, 812-8582 Fukuoka, Japan;

Epilepsy is a neurological disorder often associated with seizure that affects ∼0.7% 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 (), 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 in adult NS/PCs. Replenishing 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.
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http://dx.doi.org/10.1073/pnas.1716479115DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5910824PMC
April 2018

Therapeutic time window of anti-high mobility group box-1 antibody administration in mouse model of spinal cord injury.

Neurosci Res 2019 Apr 28;141:63-70. Epub 2018 Mar 28.

Department of Stem Cell Biology and Medicine, Graduate School of Medical Sciences, Kyushu University, 3-1-1 Maidashi, Higashi-ku, Fukuoka, 812-8581, Japan. Electronic address:

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.
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http://dx.doi.org/10.1016/j.neures.2018.03.004DOI Listing
April 2019

Prior Treatment with Anti-High Mobility Group Box-1 Antibody Boosts Human Neural Stem Cell Transplantation-Mediated Functional Recovery After Spinal Cord Injury.

Stem Cells 2018 05 8;36(5):737-750. Epub 2018 Mar 8.

Department of Stem Cell Biology and Medicine, Graduate School of Medical Sciences, Kyushu University, Higashi-ku, Fukuoka, Japan.

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.
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http://dx.doi.org/10.1002/stem.2802DOI Listing
May 2018

New aspects of glioblastoma multiforme revealed by similarities between neural and glioblastoma stem cells.

Cell Biol Toxicol 2018 12 31;34(6):425-440. Epub 2018 Jan 31.

Division of Basic Stem Cell Biology, Department of Stem Cell Biology and Medicine, Graduate School of Medical Sciences, Kyushu University, 3-1-1 Maidashi, Higashi-Ku, Fukuoka, 812-8582, Japan.

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.
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http://dx.doi.org/10.1007/s10565-017-9420-yDOI Listing
December 2018

Synergistic induction of astrocytic differentiation by factors secreted from meninges in the mouse developing brain.

FEBS Lett 2017 11 31;591(22):3709-3720. Epub 2017 Oct 31.

Department of Stem Cell Biology and Medicine, Graduate School of Medical Sciences, Kyushu University, Fukuoka, Japan.

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.
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http://dx.doi.org/10.1002/1873-3468.12881DOI Listing
November 2017

DNA Methylome Analysis Identifies Transcription Factor-Based Epigenomic Signatures of Multilineage Competence in Neural Stem/Progenitor Cells.

Cell Rep 2017 Sep;20(12):2992-3003

Department of Stem Cell Biology and Medicine, Graduate School of Medical Sciences, Kyushu University, 3-1-1 Maidashi, Higashi-ku, Fukuoka 812-8582, Japan. Electronic address:

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.
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http://dx.doi.org/10.1016/j.celrep.2017.08.086DOI Listing
September 2017

HMGB2 expression is associated with transition from a quiescent to an activated state of adult neural stem cells.

Dev Dyn 2018 01 6;247(1):229-238. Epub 2017 Sep 6.

Stem Cell Biology and Medicine, Department of Stem Cell Biology and Medicine, Graduate School of Medical Sciences, Kyushu University, Fukuoka, Japan.

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.
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http://dx.doi.org/10.1002/dvdy.24559DOI Listing
January 2018

Neural stem cell therapy aiming at better functional recovery after spinal cord injury.

Dev Dyn 2018 01 6;247(1):75-84. Epub 2017 Sep 6.

Department of Stem Cell Biology and Medicine, Graduate School of Medical Sciences, Kyushu University, Fukuoka, Japan.

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.
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http://dx.doi.org/10.1002/dvdy.24558DOI Listing
January 2018

PRMT1 regulates astrocytic differentiation of embryonic neural stem/precursor cells.

J Neurochem 2017 Sep 2;142(6):901-907. Epub 2017 Aug 2.

Department of Stem Cell Biology and Medicine, Graduate School of Medical Sciences, Kyushu University, Higashi-ku, Fukuoka, Japan.

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.
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http://dx.doi.org/10.1111/jnc.14123DOI Listing
September 2017

Epigenetic regulation of neural stem cell differentiation towards spinal cord regeneration.

Cell Tissue Res 2018 01 10;371(1):189-199. Epub 2017 Jul 10.

Department of Stem Cell Biology and Medicine, Graduate School of Medical Sciences, Kyushu University, 3-1-1 Maidashi, Higashi-ku, Fukuoka, 812-8582, Japan.

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.
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http://dx.doi.org/10.1007/s00441-017-2656-2DOI Listing
January 2018

Emerging mechanisms underlying astrogenesis in the developing mammalian brain.

Proc Jpn Acad Ser B Phys Biol Sci 2017 ;93(6):386-398

Division of Basic Stem Cell Biology, Department of Stem Cell Biology and Medicine, Graduate School of Medical Sciences, Kyushu University.

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.
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http://dx.doi.org/10.2183/pjab.93.024DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5709539PMC
July 2017

Hypoxia Epigenetically Confers Astrocytic Differentiation Potential on Human Pluripotent Cell-Derived Neural Precursor Cells.

Stem Cell Reports 2017 06;8(6):1743-1756

Department of Stem Cell Biology and Medicine, Graduate School of Medical Sciences, Kyushu University, 3-1-1 Maidashi, Higashi-ku, Fukuoka, Fukuoka 812-8582, Japan; Laboratory of Molecular Neuroscience, Graduate School of Biological Sciences, Nara Institute of Science and Technology, Ikoma, Nara 630-0192, Japan. Electronic address:

Human neural precursor cells (hNPCs) derived from pluripotent stem cells display a high propensity for neuronal differentiation, but they require long-term culturing to differentiate efficiently into astrocytes. The mechanisms underlying this biased fate specification of hNPCs remain elusive. Here, we show that hypoxia confers astrocytic differentiation potential on hNPCs through epigenetic gene regulation, and that this was achieved by cooperation between hypoxia-inducible factor 1α and Notch signaling, accompanied by a reduction of DNA methylation level in the promoter region of a typical astrocyte-specific gene, Glial fibrillary acidic protein. Furthermore, we found that this hypoxic culture condition could be applied to rapid generation of astrocytes from Rett syndrome patient-derived hNPCs, and that these astrocytes impaired neuronal development. Thus, our findings shed further light on the molecular mechanisms regulating hNPC differentiation and provide attractive tools for the development of therapeutic strategies for treating astrocyte-mediated neurological disorders.
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http://dx.doi.org/10.1016/j.stemcr.2017.05.001DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5470174PMC
June 2017

NEUROD1 Instructs Neuronal Conversion in Non-Reactive Astrocytes.

Stem Cell Reports 2017 06 11;8(6):1506-1515. Epub 2017 May 11.

Departments of Molecular Biology, Neurology and Neurotherapeutics, and Hamon Center for Regenerative Science and Medicine, UT Southwestern Medical Center, Dallas, TX 75390, USA. Electronic address:

Currently, all methods for converting non-neuronal cells into neurons involve injury to the brain; however, whether neuronal transdifferentiation can occur long after the period of insult remains largely unknown. Here, we use the transcription factor NEUROD1, previously shown to convert reactive glial cells to neurons in the cortex, to determine whether astrocyte-to-neuron transdifferentiation can occur under physiological conditions. We utilized adeno-associated virus 9 (AAV9), which crosses the blood-brain barrier without injury, to deliver NEUROD1 to astrocytes through an intravascular route. Interestingly, we found that a small, but significant number of non-reactive astrocytes converted to neurons in the striatum, but not the cortex. Moreover, astrocytes cultured to minimize their proliferative potential also exhibited limited neuronal transdifferentiation with NEUROD1 expression. Our results show that a single transcription factor can induce astrocyte-to-neuron conversion under physiological conditions, potentially facilitating future clinical approaches long after the acute injury phase.
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http://dx.doi.org/10.1016/j.stemcr.2017.04.013DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5470076PMC
June 2017

Detection of Bidirectional Promoter-Derived lncRNAs from Small-Scale Samples Using Pre-Amplification-Free Directional RNA-seq Method.

Methods Mol Biol 2017 ;1605:83-103

Department of Stem Cell Biology and Medicine, Graduate School of Medical Sciences, Kyushu University, Maidashi 3-1-1, Fukuoka 812-8582, Japan.

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.
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http://dx.doi.org/10.1007/978-1-4939-6988-3_6DOI Listing
February 2018

Evolutionary acquisition of promoter-associated non-coding RNA (pancRNA) repertoires diversifies species-dependent gene activation mechanisms in mammals.

BMC Genomics 2017 04 7;18(1):285. Epub 2017 Apr 7.

Department of Stem Cell Biology and Medicine, Graduate School of Medical Sciences, Kyushu University, Fukuoka, 812-8582, Japan.

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.
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http://dx.doi.org/10.1186/s12864-017-3662-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5383967PMC
April 2017