研究者詳細 - 伊藤　美佳子

Extremely Low-Frequency Electromagnetic Field (ELF-EMF) Increases Mitochondrial Electron Transport Chain Activities and Ameliorates Depressive Behaviors in Mice 査読有り

Soy protein β-conglycinin ameliorates pressure overload-induced heart failure by increasing short-chain fatty acid (SCFA)-producing gut microbiota and intestinal SCFAs 査読有り Open Access

Furukawa, N; Kobayashi, M; Ito, M; Matsui, H; Ohashi, K; Murohara, T; Takeda, JI; Ueyama, J; Hirayama, M; Ohno, K

CLINICAL NUTRITION 43 巻 ( 12 ) 頁： 124 - 137 2024年12月

記述言語：英語出版者・発行元：Clinical Nutrition

Background and aims: Soybeans and their ingredients have antioxidant and anti-inflammatory effects on cardiovascular diseases. β-Conglycinin (β-CG), a major constituent of soy proteins, is protective against obesity, hypertension, and chronic kidney disease, but its effects on heart failure remain to be elucidated. We tested the effects of β-CG on left ventricular (LV) remodeling in pressure overload-induced heart failure. Methods: A transverse aortic constriction (TAC)-induced pressure overload was applied to the heart in 7-week-old C57BL6 male mice that were treated with β-CG, GlcNAc, or sodium propionate. Gut microbiota was analyzed by 16S rRNA sequencing. Fecal short-chain fatty acids (SCFAs) were quantified by GC-MS. The effects of oral antibiotics were examined in β-CG-fed mice. Results: β-CG ameliorated impaired cardiac contractions, cardiac hypertrophy, and myocardial fibrosis in TAC-operated mice. As β-CG is a highly glycosylated protein, we examined the effects of GlcNAc. GlcNAc had similar but less efficient effects on LV remodeling compared to β-CG. β-CG increased three major SCFA-producing intestinal bacteria, as well as fecal concentrations of SCFAs, in sham- and TAC-operated mice. Oral administration of antibiotics nullified the effects of β-CG in TAC-operated mice by markedly reducing SCFA-producing intestinal bacteria and fecal SCFAs. In contrast, oral administration of sodium propionate, one of SCFAs, ameliorated LV remodeling in TAC-operated mice to a similar extent as β-CG. Conclusions: β-CG was protective against TAC-induced LV remodeling, which was likely to be mediated by increased SCFA-producing gut microbiota and increased intestinal SCFAs. Modified β-CG and/or derivatives arising from β-CG are expected to be developed as prophylactic and/or therapeutic agents to ameliorate devastating symptoms in heart failure.

DOI： 10.1016/j.clnu.2024.09.045

A class of chemical compounds enhances clustering of muscle nicotinic acetylcholine receptor in cultured myogenic cells 査読有り国際誌 Open Access

Miyairi, Y; Ohkawara, B; Sato, A; Sawada, R; Ishii, H; Tomita, H; Inoue, T; Nakashima, H; Ito, M; Masuda, A; Hosono, Y; Imagama, S; Ohno, K

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS 731 巻頁： 150400 - 150400 2024年10月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：Biochemical and Biophysical Research Communications

Neuromuscular signal transmission is affected in various diseases including myasthenia gravis, congenital myasthenic syndromes, and sarcopenia. We used an ATF2-luciferase system to monitor the phosphorylation of MuSK in HEK293 cells introduced with MUSK and LRP4 cDNAs to find novel chemical compounds that enhanced agrin-mediated acetylcholine receptor (AChR) clustering. Four compounds with similar chemical structures carrying benzene rings and heterocyclic rings increased the luciferase activities 8- to 30-folds, and two of them showed continuously graded dose dependence. The effects were higher than that of disulfiram, a clinically available aldehyde dehydrogenase inhibitor, which we identified to be the most competent preapproved drug to enhance ATF2-luciferase activity in the same assay system. In C2C12 myotubes, all the compounds increased the area, intensity, length, and number of AChR clusters. Three of the four compounds increased the phosphorylation of MuSK, but not of Dok7, JNK. ERK, or p38. Monitoring cell toxicity using the neurite elongation of NSC34 neuronal cells as a surrogate marker showed that all the compounds had no effects on the neurite elongation up to 1 μM. Extensive docking simulation and binding structure prediction of the four compounds with all available human proteins using AutoDock Vina and DiffDock showed that the four compounds were unlikely to directly bind to MuSK or Dok7, and the exact target remained unknown. The identified compounds are expected to serve as a seed to develop a novel therapeutic agent to treat defective NMJ signal transmission.

DOI： 10.1016/j.bbrc.2024.150400

Teranishi, M; Ito, M; Huang, ZZ; Nishiyama, Y; Masuda, A; Mino, H; Tachibana, M; Inada, T; Ohno, K

INTERNATIONAL JOURNAL OF MOLECULAR SCIENCES 25 巻 ( 20 ) 頁： 11315 - 11315 2024年10月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：International Journal of Molecular Sciences

Compromised mitochondrial electron transport chain (ETC) activities are associated with depression in humans and rodents. However, the effects of the enhancement of mitochondrial ETC activities on depression remain elusive. We recently reported that an extremely low-frequency electromagnetic field (ELF-EMF) of as low as 10 μT induced hormetic activation of mitochondrial ETC complexes in human/mouse cultured cells and mouse livers. Chronic social defeat stress (CSDS) for 10 consecutive days caused behavioral defects mimicking depression in mice, and using an ELF-EMF for two to six weeks ameliorated them. CSDS variably decreased the mitochondrial ETC proteins in the prefrontal cortex (PFC) in 10 days, which were increased by an ELF-EMF in six weeks. CSDS had no effect on the mitochondrial oxygen consumption rate in the PFC in 10 days, but using an ELF-EMF for six weeks enhanced it. CSDS inactivated SOD2 by enhancing its acetylation and increased lipid peroxidation in the PFC. In contrast, the ELF-EMF activated the Sirt3-FoxO3a-SOD2 pathway and suppressed lipid peroxidation. Furthermore, CSDS increased markers for mitophagy, which was suppressed by the ELF-EMF in six weeks. The ELF-EMF exerted beneficial hormetic effects on mitochondrial energy production, mitochondrial antioxidation, and mitochondrial dynamics in a mouse model of depression. We envisage that an ELF-EMF is a promising therapeutic option for depression.

DOI： 10.3390/ijms252011315

Muscle-specific lack of Gfpt1 triggers ER stress to alleviate misfolded protein accumulation 査読有り国際誌

Zhang, RC; Farshadyeganeh, P; Ohkawara, B; Nakajima, K; Takeda, JI; Ito, M; Zhang, SC; Miyasaka, Y; Ohno, T; Mori-Yoshimura, M; Masuda, A; Ohno, K

DISEASE MODELS & MECHANISMS 17 巻 ( 8 ) 2024年8月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：DMM Disease Models and Mechanisms

Pathogenic variants in GFPT1, encoding a key enzyme to synthesize UDP-N-acetylglucosamine (UDP-GlcNAc), cause congenital myasthenic syndrome (CMS). We made a knock-in (KI) mouse model carrying a frameshift variant in Gfpt1 exon 9, simulating that found in a patient with CMS. As Gfpt1 exon 9 is exclusively expressed in striated muscles, Gfpt1-KI mice were deficient for Gfpt1 only in skeletal muscles. In Gfpt1-KI mice, (1) UDP-HexNAc, CMP-NeuAc and protein O-GlcNAcylation were reduced in skeletal muscles; (2) aged Gfpt1-KI mice showed poor exercise performance and abnormal neuromuscular junction structures; and (3) markers of the unfolded protein response (UPR) were elevated in skeletal muscles. Denervation-mediated enhancement of endoplasmic reticulum (ER) stress in Gfpt1-KI mice facilitated protein folding, ubiquitin-proteasome degradation and apoptosis, whereas autophagy was not induced and protein aggregates were markedly increased. Lack of autophagy was accounted for by enhanced degradation of FoxO1 by increased Xbp1-s/u proteins. Similarly, in Gfpt1-silenced C2C12 myotubes, ER stress exacerbated protein aggregates and activated apoptosis, but autophagy was attenuated. In both skeletal muscles in Gfpt1-KI mice and Gfpt1-silenced C2C12 myotubes, maladaptive UPR failed to eliminate protein aggregates and provoked apoptosis.

DOI： 10.1242/dmm.050768

Calcitriol ameliorates motor deficits and prolongs survival of Chrne-deficient mouse, a model for congenital myasthenic syndrome, by inducing Rspo2. 査読有り国際誌

Ohkawara B, Tomita H, Inoue T, Zhang S, Kanbara S, Koshimizu H, Miyasaka Y, Takeda JI, Nishiwaki H, Nakashima H, Ito M, Masuda A, Ishiguro N, Ogi T, Ohno T, Imagama S, Ohno K

Neurotherapeutics : the journal of the American Society for Experimental NeuroTherapeutics 21 巻 ( 2 ) 頁： e00318 2024年1月

Splicing regulation of GFPT1 muscle-specific isoform and its roles in glucose metabolisms and neuromuscular junction 査読有り Open Access

Farshadyeganeh, P; Nazim, M; Zhang, RC; Ohkawara, B; Nakajima, K; Rahman, MA; Nasrin, F; Ito, M; Takeda, JI; Ohe, K; Miyasaka, Y; Ohno, T; Masuda, A; Ohno, K

ISCIENCE 26 巻 ( 10 ) 頁： 107746 2023年10月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：iScience

Glutamine:fructose-6-phosphate transaminase 1 (GFPT1) is the rate-limiting enzyme of the hexosamine biosynthetic pathway (HBP). A 54-bp exon 9 of GFPT1 is specifically included in skeletal and cardiac muscles to generate a long isoform of GFPT1 (GFPT1-L). We showed that SRSF1 and Rbfox1/2 cooperatively enhance, and hnRNP H/F suppresses, the inclusion of human GFPT1 exon 9 by modulating recruitment of U1 snRNP. Knockout (KO) of GFPT1-L in skeletal muscle markedly increased the amounts of GFPT1 and UDP-HexNAc, which subsequently suppressed the glycolytic pathway. Aged KO mice showed impaired insulin-mediated glucose uptake, as well as muscle weakness and fatigue likely due to abnormal formation and maintenance of the neuromuscular junction. Taken together, GFPT1-L is likely to be acquired in evolution in mammalian striated muscles to attenuate the HBP for efficient glycolytic energy production, insulin-mediated glucose uptake, and the formation and maintenance of the neuromuscular junction.

DOI： 10.1016/j.isci.2023.107746

Extremely low-frequency electromagnetic field induces acetylation of heat shock proteins and enhances protein folding 査読有り国際誌 Open Access

Huang, ZZ; Ito, M; Zhang, SC; Toda, T; Takeda, JI; Ogi, T; Ohno, K

ECOTOXICOLOGY AND ENVIRONMENTAL SAFETY 264 巻頁： 115482 - 115482 2023年10月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：Ecotoxicology and Environmental Safety

The pervasive weak electromagnetic fields (EMF) inundate the industrialized society, but the biological effects of EMF as weak as 10 µT have been scarcely analyzed. Heat shock proteins (HSPs) are molecular chaperones that mediate a sequential stress response. HSP70 and HSP90 provide cells under undesirable situations with either assisting covalent folding of proteins or degrading improperly folded proteins in an ATP-dependent manner. Here we examined the effect of extremely low-frequency (ELF)-EMF on AML12 and HEK293 cells. Although the protein expression levels of HSP70 and HSP90 were reduced after an exposure to ELF-EMF for 3 h, acetylations of HSP70 and HSP90 were increased, which was followed by an enhanced binding affinities of HSP70 and HSP90 for HSP70/HSP90-organizing protein (HOP/STIP1). After 3 h exposure to ELF-EMF, the amount of mitochondria was reduced but the ATP level and the maximal mitochondrial oxygen consumption were increased, which was followed by the reduced protein aggregates and the increased cell viability. Thus, ELF-EMF exposure for 3 h activated acetylation of HSPs to enhance protein folding, which was returned to the basal level at 12 h. The proteostatic effects of ELF-EMF will be able to be applied to treat pathological states in humans.

DOI： 10.1016/j.ecoenv.2023.115482

Fusobacterium infection facilitates the development of endometriosis through the phenotypic transition of endometrial fibroblasts 査読有り国際誌

Muraoka, A; Suzuki, M; Hamaguchi, T; Watanabe, S; Iijima, K; Murofushi, Y; Shinjo, K; Osuka, S; Hariyama, Y; Ito, M; Ohno, K; Kiyono, T; Kyo, S; Iwase, A; Kikkawa, F; Kajiyama, H; Kondo, Y

SCIENCE TRANSLATIONAL MEDICINE 15 巻 ( 700 ) 頁： eadd1531 2023年6月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：Science Translational Medicine

Retrograde menstruation is a widely accepted cause of endometriosis. However, not all women who experience retrograde menstruation develop endometriosis, and the mechanisms underlying these observations are not yet understood. Here, we demonstrated a pathogenic role of Fusobacterium in the formation of ovarian endometriosis. In a cohort of women, 64% of patients with endometriosis but <10% of controls were found to have Fusobacterium infiltration in the endometrium. Immunohistochemical and biochemical analyses revealed that activated transforming growth factor–β (TGF-β) signaling resulting from Fusobacterium infection of endometrial cells led to the transition from quiescent fibroblasts to transgelin (TAGLN)–positive myofibroblasts, which gained the ability to proliferate, adhere, and migrate in vitro. Fusobacterium inoculation in a syngeneic mouse model of endometriosis resulted in a marked increase in TAGLN-positive myofibroblasts and increased number and weight of endometriotic lesions. Furthermore, antibiotic treatment largely prevented establishment of endometriosis and reduced the number and weight of established endometriotic lesions in the mouse model. Our data support a mechanism for the pathogenesis of endometriosis via Fusobacterium infection and suggest that eradication of this bacterium could be an approach to treat endometriosis.

DOI： 10.1126/scitranslmed.add1531

Interactions between muscle glucose homeostasis and neuromuscular signal transduction - Lessons learned from lack of a muscle-specific long isoform of GFPT1 査読有り

Farshadyeganeh, P; Nazim, M; Ohkawara, B; Masuda, A; Ito, M; Takeda, J; Ohe, K; Ohno, K

EUROPEAN JOURNAL OF HUMAN GENETICS 31 巻頁： 31 - 31 2023年5月

Interactions between muscle glucose homeostasis and neuromuscular signal transduction - Lessons learned from lack of a muscle-specific long isoform of GFPT1 査読有り

Farshadyeganeh, P., Nazim, M., Ohkawara, B., Masuda, A., Ito, M., Takeda, J., Ohe, K., Ohno, K.

EUROPEAN JOURNAL OF HUMAN GENETICS 31 巻頁： 31 - 31 2023年5月

A mutation in DOK7 in congenital myasthenic syndrome forms aggresome in cultured cells, and reduces DOK7 expression and MuSK phosphorylation in patient-derived iPS cells 査読有り Open Access

Zhang, SC; Ohkawara, B; Ito, M; Huang, ZZ; Zhao, F; Nakata, T; Takeuchi, T; Sakurai, H; Komaki, H; Kamon, M; Araki, T; Ohno, K

HUMAN MOLECULAR GENETICS 32 巻 ( 9 ) 頁： 1511 - 1523 2023年4月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：Human Molecular Genetics

At the neuromuscular junction, the downstream of tyrosine kinase 7 (DOK7) enhances the phosphorylation of muscle-specific kinase (MuSK) and induces clustering of acetylcholine receptors (AChRs). We identified a patient with congenital myasthenic syndrome (CMS) with two heteroallelic mutations in DOK7, c.653-1G>C in intron 5 and c.190G>A predicting p.G64R in the pleckstrin homology domain. iPS cells established from the patient (CMS-iPSCs) showed that c.653-1G>C caused in-frame skipping of exon 6 (120 bp) and frame-shifting activation of a cryptic splice site deleting seven nucleotides in exon 6. p.G64R reduced the expression of DOK7 to 10% of wild-type DOK7, and markedly compromised AChR clustering in transfected C2C12 myotubes. p.G64R-DOK7 made insoluble aggresomes at the juxtanuclear region in transfected C2C12 myoblasts and COS7 cells, which were co-localized with molecules in the autophagosome system. A protease inhibitor MG132 reduced the soluble fraction of p.G64R-DOK7 and enhanced the aggresome formation of p.G64RDOK7. To match the differentiation levels between patient-derived and control induced pluripotent stem cells (iPSCs), we corrected c.190G>A (p.G64R) by CRISPR/Cas9 to make isogenic iPSCs while retaining c.653-1G>C (CMS-iPSCsCas9). Myogenically differentiated CMS-iPSCs showed juxtanuclear aggregates of DOK7, reduced expression of endogenous DOK7 and reduced phosphorylation of endogenous MuSK. Another mutation, p.T77M, also made aggresome to a less extent compared with p.G64R in transfected COS7 cells. These results suggest that p.G64R-DOK7 makes aggresomes in cultured cells and is likely to compromise MuSK phosphorylation for AChR clustering.

DOI： 10.1093/hmg/ddac306

Neural Isoforms of Agrin Are Generated by Reduced PTBP1-RNA Interaction Network Spanning the Neuron-Specific Splicing Regions in AGRN 査読有り国際誌 Open Access

Bushra, S; Lin, YN; Joudaki, A; Ito, M; Ohkawara, B; Ohno, K; Masuda, A

INTERNATIONAL JOURNAL OF MOLECULAR SCIENCES 24 巻 ( 8 ) 2023年4月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：International Journal of Molecular Sciences

Agrin is a heparan sulfate proteoglycan essential for the clustering of acetylcholine receptors at the neuromuscular junction. Neuron−specific isoforms of agrin are generated by alternative inclusion of three exons, called Y, Z8, and Z11 exons, although their processing mechanisms remain elusive. We found, by inspection of splicing cis−elements into the human AGRN gene, that binding sites for polypyrimidine tract binding protein 1 (PTBP1) were extensively enriched around Y and Z exons. PTBP1−silencing enhanced the coordinated inclusion of Y and Z exons in human SH−SY5Y neuronal cells, even though three constitutive exons are flanked by these alternative exons. Deletion analysis using minigenes identified five PTBP1−binding sites with remarkable splicing repression activities around Y and Z exons. Furthermore, artificial tethering experiments indicated that binding of a single PTBP1 molecule to any of these sites represses nearby Y or Z exons as well as the other distal exons. The RRM4 domain of PTBP1, which is required for looping out a target RNA segment, was likely to play a crucial role in the repression. Neuronal differentiation downregulates PTBP1 expression and promotes the coordinated inclusion of Y and Z exons. We propose that the reduction in the PTPB1−RNA network spanning these alternative exons is essential for the generation of the neuron−specific agrin isoforms.

DOI： 10.3390/ijms24087420

Effects of Pesticide Intake on Gut Microbiota and Metabolites in Healthy Adults 査読有り国際誌 Open Access

Ueyama, J; Hayashi, M; Hirayama, M; Nishiwaki, H; Ito, M; Saito, I; Tsuboi, Y; Isobe, T; Ohno, K

INTERNATIONAL JOURNAL OF ENVIRONMENTAL RESEARCH AND PUBLIC HEALTH 20 巻 ( 1 ) 2023年1月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：International Journal of Environmental Research and Public Health

Animal experiments have indicated that pesticides may affect gut microbiota, which is responsible for the production of short-chain fatty acids (SCFAs) and polyamines. Here, we present a preliminary observation of the relationship between pesticide exposure and fecal SCFAs and polyamines in Japanese adults. In total, 38 healthy adults aged 69 ± 10 years (mean ± SD) were recruited and subjected to stool and spot urine tests. Urinary dialkylphosphates (DAP), 3-phenoxybenzoic acid, and glyphosate were assayed as pesticide exposure markers of organophosphorus insecticide (OP), a pyrethroid insecticide, and glyphosate, respectively. Significant negative correlations (p < 0.05, Spearman’s rank correlation coefficient) were found between urinary DAP, fecal acetate (r = −0.345), and lactate (r = −0.391). Multiple regression analyses revealed that urinary DAP was a significant explanatory variable of fecal acetate concentration (p < 0.001, β = −24.0, SE = 4.9, t = −4.9) with some vegetable intake (adjusted R-square = 0.751). These findings suggest that OP exposure is independently associated with lower fecal acetate levels, which may contribute to human health in middle-aged and older adult groups. Given that the human gut environment has long-term effects on the host, studies on wide-range age groups, including children, are necessary.

DOI： 10.3390/ijerph20010213

Gut microbiota in dementia with Lewy bodies 査読有り国際誌 Open Access

Nishiwaki, H; Ueyama, J; Kashihara, K; Ito, M; Hamaguchi, T; Maeda, T; Tsuboi, Y; Katsuno, M; Hirayama, M; Ohno, K

NPJ PARKINSONS DISEASE 8 巻 ( 1 ) 頁： 169 - 169 2022年12月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：npj Parkinson's Disease

Gut microbiota and fecal bile acids were analyzed in 278 patients with α-synucleinopathies, which were comprised of 28 patients with dementia with Lewy bodies (DLB), 224 patients with Parkinson’s disease (PD), and 26 patients with idiopathic rapid eye movement sleep behavior disorder (iRBD). Similarly to PD, short-chain fatty acids-producing genera were decreased in DLB. Additionally, Ruminococcus torques and Collinsella were increased in DLB, which were not changed in PD. Random forest models to differentiate DLB and PD showed that high Ruminococcus torques and high Collinsella, which presumably increase intestinal permeability, as well as low Bifidobacterium, which are also observed in Alzheimer’s disease, were predictive of DLB. As Ruminococcus torques and Collinsella are also major secondary bile acids-producing bacteria, we quantified fecal bile acids and found that the production of ursodeoxycholic acid (UDCA) was high in DLB. Increased UDCA in DLB may mitigate neuroinflammation at the substantia nigra, whereas neuroinflammation may not be critical at the neocortex. Theraeutic intervention to increase Bifidobacteirum and its metabolites may retard the development and progression of DLB.

DOI： 10.1038/s41531-022-00428-2

Examination of Abnormal Alpha-synuclein Aggregates in the Enteric Neural Plexus in Patients with Ulcerative Colitis 査読有り国際誌 Open Access

Gibo, N; Hamaguchi, T; Miki, Y; Yamamura, T; Nakaguro, M; Ito, M; Nakamura, M; Kawashima, H; Hirayama, M; Hirooka, Y; Wakabayashi, K; Ohno, K

JOURNAL OF GASTROINTESTINAL AND LIVER DISEASES 31 巻 ( 3 ) 頁： 290 - 300 2022年9月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：Journal of Gastrointestinal and Liver Diseases

Background & Aims: Parkinson’s disease (PD) is the second most neurodegenerative disease after Alzheimer’s disease. Accumulating knowledge points to the notion that abnormal aggregation of alpha-synuclein (αSyn) starts in the gut and ascends to the substantia nigra via the vagus nerve in about a half of PD patients. Epidemiological studies revealed that ulcerative colitis (UC) increased the a risk for PD 1.3 to 1.8-folds. However, it remains unknown whether αSyn is abnormally aggregated in the enteric neurons in UC patients. Methods: We first inspected and optimized the immunostaining protocols with an anti-phosphorylated αSyn antibody, pSyn#64, using the brain and the gut of eight autopsied cases (five with PD and three without PD). Then, we examined abnormal αSyn aggregation in the enteric neurons in 23 and 18 colectomized patients with and without UC, respectively. Five or more sections were stained for αSyn in each of 87 and 25 paraffin-embedded blocks in patients with and without UC, respectively. Results: Ten different protocols of epitope exposure appropriately stained aggregated αSyn in the brain, but only a complete lack of epitope exposure stained aggregated αSyn in the colon with low background. Abnormal αSyn aggregates, which was confirmed by co-localization of p62, in the enteric neurons were detected in a single patient with UC but in no patient without UC. Conclusions: Omission of epitope exposure enabled us to immunostain aggregated αSyn in the colon by pSyn#64 with low nonspecific staining, but the number of 23 UC patients was not high enough to discern whether abnormal αSyn aggregation in the colonic neural plexus was increased in UC or not.

DOI： 10.15403/jgld-4313

Short chain fatty acids-producing and mucin-degrading intestinal bacteria predict the progression of early Parkinson's disease 査読有り国際誌 Open Access

Nishiwaki, H; Ito, M; Hamaguchi, T; Maeda, T; Kashihara, K; Tsuboi, Y; Ueyama, J; Yoshida, T; Hanada, H; Takeuchi, I; Katsuno, M; Hirayama, M; Ohno, K

NPJ PARKINSONS DISEASE 8 巻 ( 1 ) 頁： 65 - 65 2022年6月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：npj Parkinson's Disease

To elucidate the relevance of gut dysbiosis in Parkinson’s disease (PD) in disease progression, we made random forest models to predict the progression of PD in two years by gut microbiota in 165 PD patients. The area under the receiver operating characteristic curves (AUROCs) of gut microbiota-based models for Hoehn & Yahr (HY) stages 1 and 2 were 0.799 and 0.705, respectively. Similarly, gut microbiota predicted the progression of Movement Disorder Society-Unified Parkinson’s Disease Rating Scale (MDS-UPDRS) III scores in an early stage of PD with AUROC = 0.728. Decreases of short-chain fatty acid-producing genera, Fusicatenibacter, Faecalibacterium, and Blautia, as well as an increase of mucin-degrading genus Akkermansia, predicted accelerated disease progression. The four genera remained unchanged in two years in PD, indicating that the taxonomic changes were not the consequences of disease progression. PD patients with marked gut dysbiosis may thus be destined to progress faster than those without gut dysbiosis.

DOI： 10.1038/s41531-022-00328-5

Extremely low-frequency pulses of faint magnetic field induce mitophagy to rejuvenate mitochondria 査読有り国際誌 Open Access

Toda, T; Ito, M; Takeda, J; Masuda, A; Mino, H; Hattori, N; Mohri, K; Ohno, K

COMMUNICATIONS BIOLOGY 5 巻 ( 1 ) 頁： 453 - 453 2022年5月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：Communications Biology

Humans are frequently exposed to time-varying and static weak magnetic fields (WMF). However, the effects of faint magnetic fields, weaker than the geomagnetic field, have been scarcely reported. Here we show that extremely low-frequency (ELF)-WMF, comprised of serial pulses of 10 µT intensity at 1–8 Hz, which is three or more times weaker than the geomagnetic field, reduces mitochondrial mass to 70% and the mitochondrial electron transport chain (ETC) complex II activity to 88%. Chemical inhibition of electron flux through the mitochondrial ETC complex II nullifies the effect of ELF-WMF. Suppression of ETC complex II subsequently induces mitophagy by translocating parkin and PINK1 to the mitochondria and by recruiting LC3-II. Thereafter, mitophagy induces PGC-1α-mediated mitochondrial biogenesis to rejuvenate mitochondria. The lack of PINK1 negates the effect of ELF-WMF. Thus, ELF-WMF may be applicable for the treatment of human diseases that exhibit compromised mitochondrial homeostasis, such as Parkinson’s disease.

DOI： 10.1038/s42003-022-03389-7

Extremely low-frequency pulses of faint magnetic field induce mitophagy to rejuvenate mitochondria 査読有り

Takuro Toda; Mikako Ito; Jun-ichi Takeda; Akio Masuda; Hiroyuki Mino; Nobutaka Hattori; Kaneo Mohri; Kinji Ohno

Communications Biology 5 巻頁： 453 2022年5月

Molecular Hydrogen Enhances Proliferation of Cancer Cells That Exhibit Potent Mitochondrial Unfolded Protein Response 査読有り国際誌 Open Access

Hasegawa, T; Ito, M; Hasegawa, S; Teranishi, M; Takeda, K; Negishi, S; Nishiwaki, H; Takeda, JI; LeBaron, TW; Ohno, K

INTERNATIONAL JOURNAL OF MOLECULAR SCIENCES 23 巻 ( 5 ) 2022年3月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：International Journal of Molecular Sciences

Molecular hydrogen ameliorates pathological states in a variety of human diseases, animal models, and cell models, but the effects of hydrogen on cancer have been rarely reported. In addition, the molecular mechanisms underlying the effects of hydrogen remain mostly unelucidated. We found that hydrogen enhances proliferation of four out of seven human cancer cell lines (the responders). The proliferation-promoting effects were not correlated with basal levels of cellular reactive oxygen species. Expression profiling of the seven cells showed that the responders have higher gene expression of mitochondrial electron transport chain (ETC) molecules than the non-responders. In addition, the responders have higher mitochondrial mass, higher mitochondrial superoxide, higher mitochondrial membrane potential, and higher mitochondrial spare respiratory capacity than the non-responders. In the responders, hydrogen provoked mitochondrial unfolded protein response (mtUPR). Suppression of cell proliferation by rotenone, an inhibitor of mitochondrial ETC complex I, was rescued by hydrogen in the responders. Hydrogen triggers mtUPR and induces cell proliferation in cancer cells that have high basal and spare mitochondrial ETC activities.

DOI： 10.3390/ijms23052888

Meclozine ameliorates skeletal muscle pathology and increases muscle forces in mdx mice 査読有り国際誌 Open Access

Kawamura, Y; Hida, T; Ohkawara, B; Matsushita, M; Kobayashi, T; Ishizuka, S; Hiraiwa, H; Tanaka, S; Tsushima, M; Nakashima, H; Ito, K; Imagama, S; Ito, M; Masuda, A; Ishiguro, N; Ohno, K

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS 592 巻頁： 87 - 92 2022年2月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：Biochemical and Biophysical Research Communications

We screened pre-approved drugs for the survival of the Hu5/KD3 human myogenic progenitors. We found that meclozine, an anti-histamine drug that has long been used for motion sickness, promoted the proliferation and survival of Hu5/KD3 cells. Meclozine increased expression of MyoD, but reduced expression of myosin heavy chain and suppressed myotube formation. Withdrawal of meclozine, however, resumed the ability of Hu5/KD3 cells to differentiate into myotubes. We examined the effects of meclozine on mdx mouse carrying a nonsense mutation in the dystrophin gene and modeling for Duchenne muscular dystrophy. Intragastric administration of meclozine in mdx mouse increased the body weight, the muscle mass in the lower limbs, the cross-sectional area of the paravertebral muscle, and improved exercise performances. Previous reports show that inhibition of phosphorylation of ERK1/2 improves muscle functions in mouse models for Emery-Dreifuss muscular dystrophy and cancer cachexia, as well as in mdx mice. We and others previously showed that meclozine blocks the phosphorylation of ERK1/2 in cultured cells. We currently showed that meclozine decreased phosphorylation of ERK1/2 in muscles in mdx mice but not in wild-type mice. This was likely to be one of the underlying mechanisms of the effects of meclozine on mdx mice.

DOI： 10.1016/j.bbrc.2022.01.003

Altered gut microbiota in Parkinson's disease patients with motor complications 査読有り国際誌 Open Access

Takahashi, K; Nishiwaki, H; Ito, M; Iwaoka, K; Takahashi, K; Suzuki, Y; Taguchi, K; Yamahara, K; Tsuboi, Y; Kashihara, K; Hirayama, M; Ohno, K; Maeda, T

PARKINSONISM & RELATED DISORDERS 95 巻頁： 11 - 17 2022年2月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：Parkinsonism and Related Disorders

Introduction: Parkinson's disease (PD) is associated with gut dysbiosis. However, whether gut dysbiosis can cause motor complications is unclear. Methods: Subjects were enrolled from four independent movement disorder centers in Japan. We performed 16S ribosomal RNA gene sequence analysis of gut microbiota. Relative abundance of gut microbiota and relationships between them and clinical characteristics were statistically analyzed. Analysis of co-variance (ANCOVA) was used to assess altered gut microbiota associated with wearing-off or dyskinesia. Results: We enrolled 223 patients with PD. Wearing-off was noted in 47.5% of patients and dyskinesia in 21.9%. We detected 98 genera of bacteria. Some changes in the gut microbiota were observed in patients with PD and motor complications. After Bonferroni correction, patients with wearing-off showed decreased relative abundance of Lachnospiraceae Blautia (p < 0.0001) and increased relative abundance of Lactobacillaceae Lactobacillus (p < 0.0001), but patients with dyskinesia no longer showed significant changes in the gut microbiota. Adjustment with two models of confounding factors followed by ANCOVA revealed that age (p < 0.0001), disease duration (p = 0.01), and wearing-off (p = 0.0004) were independent risks for the decreased relative abundance of Lachnospiraceae Blautia, and wearing-off (p = 0.009) was the only independent risk factor for the increased relative abundance of Lachnospiraceae Lactobacillus. Conclusion: Relative abundance of Lachnospiraceae Blautia and Lactobacillaceae Lactobacillus was significantly decreased and increased, respectively, in the gut microbiota of PD patients with motor complications. This indicates that an altered gut microbiota is associated with the development of motor complications in patients with advanced PD.

DOI： 10.1016/j.parkreldis.2021.12.012

Enhancement of ethanol production and cell growth in budding yeast by direct irradiation of low-temperature plasma 査読有り Open Access

Tanaka, H; Matsumura, S; Ishikawa, K; Hashizume, H; Ito, M; Nakamura, K; Kajiyama, H; Kikkawa, F; Ito, M; Ohno, K; Okazaki, Y; Toyokuni, S; Mizuno, M; Hori, M

JAPANESE JOURNAL OF APPLIED PHYSICS 61 巻 ( SA ) 2022年1月

記述言語：日本語掲載種別：研究論文（学術雑誌）出版者・発行元：Japanese Journal of Applied Physics

Ethanol production by budding yeast was compared between direct and indirect plasma irradiation. We observed enhancement of ethanol production and cell growth not by indirect plasma irradiation but by direct plasma irradiation. Glucose consumption was increased in budding yeast by direct plasma irradiation. Extracellular flux analysis revealed that glycolytic activity in the budding yeast was elevated by direct plasma irradiation. These results suggest that direct plasma irradiation enhances ethanol production in budding yeast by elevating the glycolytic activity.

DOI： 10.35848/1347-4065/ac2037

Intestinal Collinsella may mitigate infection and exacerbation of COVID-19 by producing ursodeoxycholate 査読有り国際誌 Open Access

Hirayama, M; Nishiwaki, H; Hamaguchi, T; Ito, M; Ueyama, J; Maeda, T; Kashihara, K; Tsuboi, Y; Ohno, K

PLOS ONE 16 巻 ( 11 ) 頁： e0260451 2021年11月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：PLoS ONE

The mortality rates of COVID-19 vary widely across countries, but the underlying mechanisms remain unelucidated. We aimed at the elucidation of relationship between gut microbiota and the mortality rates of COVID-19 across countries. Raw sequencing data of 16S rRNA V3-V5 regions of gut microbiota in 953 healthy subjects in ten countries were obtained from the public database. We made a generalized linear model (GLM) to predict the COVID- 19 mortality rates using gut microbiota. GLM revealed that low genus Collinsella predicted high COVID-19 mortality rates with a markedly low p-value. Unsupervised clustering of gut microbiota in 953 subjects yielded five enterotypes. The mortality rates were increased from enterotypes 1 to 5, whereas the abundances of Collinsella were decreased from enterotypes 1 to 5 except for enterotype 2. Collinsella produces ursodeoxycholate. Ursodeoxycholate was previously reported to inhibit binding of SARS-CoV-2 to angiotensin-converting enzyme 2; suppress pro-inflammatory cytokines like TNF-α, IL-1β, IL-2, IL-4, and IL-6; have antioxidant and anti-apoptotic effects; and increase alveolar fluid clearance in acute respiratory distress syndrome. Ursodeoxycholate produced by Collinsella may prevent COVID-19 infection and ameliorate acute respiratory distress syndrome in COVID-19 by suppressing cytokine storm syndrome.

DOI： 10.1371/journal.pone.0260451

Regulated splicing of large exons is linked to phase-separation of vertebrate transcription factors 査読有り国際誌 Open Access

Kawachi, T; Masuda, A; Yamashita, Y; Takeda, J; Ohkawara, B; Ito, M; Ohno, K

EMBO JOURNAL 40 巻 ( 22 ) 頁： e107485 2021年11月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：EMBO Journal

Although large exons cannot be readily recognized by the spliceosome, many are evolutionarily conserved and constitutively spliced for inclusion in the processed transcript. Furthermore, whether large exons may be enriched in a certain subset of proteins, or mediate specific functions, has remained unclear. Here, we identify a set of nearly 3,000 SRSF3-dependent large constitutive exons (S3-LCEs) in human and mouse cells. These exons are enriched for cytidine-rich sequence motifs, which bind and recruit the splicing factors hnRNP K and SRSF3. We find that hnRNP K suppresses S3-LCE splicing, an effect that is mitigated by SRSF3 to thus achieve constitutive splicing of S3-LCEs. S3-LCEs are enriched in genes for components of transcription machineries, including mediator and BAF complexes, and frequently contain intrinsically disordered regions (IDRs). In a subset of analyzed S3-LCE-containing transcription factors, SRSF3 depletion leads to deletion of the IDRs due to S3-LCE exon skipping, thereby disrupting phase-separated assemblies of these factors. Cytidine enrichment in large exons introduces proline/serine codon bias in intrinsically disordered regions and appears to have been evolutionarily acquired in vertebrates. We propose that layered splicing regulation by hnRNP K and SRSF3 ensures proper phase-separation of these S3-LCE-containing transcription factors in vertebrates.

DOI： 10.15252/embj.2020107485

Plasma-activated Ringer's lactate solution inhibits the cellular respiratory system in HeLa cells 査読有り

Tanaka, H; Maeda, S; Nakamura, K; Hashizume, H; Ishikawa, K; Ito, M; Ohno, K; Mizuno, M; Motooka, Y; Okazaki, Y; Toyokuni, S; Kajiyama, H; Kikkawa, F; Hori, M

PLASMA PROCESSES AND POLYMERS 18 巻 ( 10 ) 2021年10月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：Plasma Processes and Polymers

Nonequilibrium atmospheric pressure plasma has enabled a variety of new applications in medicine, agriculture, and other industries. It is particularly noteworthy that plasma itself and/or plasma-activated culture medium have been shown to preferentially kill various cancer cells. We have previously developed a plasma-activated Ringer's lactate solution (PAL) for use as a new cancer treatment. In this study, behaviors of extracellular and intracellular reactive oxygen and nitrogen species in the cellular respiratory system of PAL-treated HeLa cells were investigated using an extracellular flux analyzer and a probe to measure mitochondrial membrane potential. In PAL-treated HeLa cells, extracellular hydrogen peroxide in PAL was found to be responsible for the induction of intracellular hydrogen peroxide and apoptosis, while other components in PAL are responsible for the induction of non-H2O2 intracellular ROS and non-apoptotic cell death, which should be clarified by further experiments. We believe that these are long-lived species derived from plasma-activated lactates. Furthermore, we found that the plasma-activated lactates inhibited glycolysis and the tricarboxylic acid (TCA) cycle, but not the electron transport chain in HeLa cells. These results suggest that PAL induces multiple modes of cell death, including apoptosis through hydrogen peroxide, and non-apoptotic cell death associated with the impairment of mitochondrial functions (glycolysis and TCA cycle). These findings shed light on the novel mechanism underlying plasma-activated lactate-induced cell death.

DOI： 10.1002/ppap.202100056

Zonisamide upregulates neuregulin-1 expression and enhances acetylcholine receptor clustering at the in vitro neuromuscular junction 査読有り国際誌

Inoue, T; Ohkawara, B; Bushra, S; Kanbara, S; Nakashima, H; Koshimizu, H; Tomita, H; Ito, M; Masuda, A; Ishiguro, N; Imagama, S; Ohno, K

NEUROPHARMACOLOGY 195 巻頁： 108637 - 108637 2021年9月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：Neuropharmacology

Decreased acetylcholine receptor (AChR) clustering compromises signal transmission at the neuromuscular junction (NMJ) in myasthenia gravis, congenital myasthenic syndromes, and motor neuron diseases. Although the enhancement of AChR clustering at the NMJ is a promising therapeutic strategy for these maladies, no drug is currently available for this enhancement. We previously reported that zonisamide (ZNS), an anti-epileptic and anti-Parkinson's disease drug, enhances neurite elongation of the primary spinal motor neurons (SMNs). As nerve sprouting occurs to compensate for the loss of AChR clusters in human diseases, we examined the effects of ZNS on AChR clustering at the NMJ. To this end, we established a simple and quick co-culture system to reproducibly make in vitro NMJs using C2C12 myotubes and NSC34 motor neurons. ZNS at 1–20 μM enhanced the formation of AChR clusters dose-dependently in co-cultured C2C12 myotubes but not in agrin-treated single cultured C2C12 myotubes. We observed that molecules that conferred responsiveness to ZNS were not secreted into the co-culture medium. We found that 10 μM ZNS upregulated the expression of neuregulin-1 (Nrg1) in co-cultured cells but not in single cultured C2C12 myotubes or single cultured NSC34 motor neurons. In accordance with this observation, inhibition of the Nrg1/ErbB signaling pathways nullified the effect of 10 μM ZNS on the enhancement of AChR clustering in in vitro NMJs. Although agrin was not induced by 10 μM ZNS in co-cultured cells, anti-agrin antibody attenuated ZNS-mediated enhancement of AChR clustering. We conclude that ZNS enhances agrin-dependent AChR-clustering by upregulating the Nrg1/ErbB signaling pathways in the presence of NMJs.

DOI： 10.1016/j.neuropharm.2021.108637

Zonisamide ameliorates neuropathic pain partly by suppressing microglial activation in the spinal cord in a mouse model 査読有り

Secreted Signaling Molecules at the Neuromuscular Junction in Physiology and Pathology 査読有り国際誌 Open Access

Ohkawara, B; Ito, M; Ohno, K

INTERNATIONAL JOURNAL OF MOLECULAR SCIENCES 22 巻 ( 5 ) 頁： 1 - 16 2021年3月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：International Journal of Molecular Sciences

Signal transduction at the neuromuscular junction (NMJ) is affected in many human dis-eases, including congenital myasthenic syndromes (CMS), myasthenia gravis, Lambert–Eaton my-asthenic syndrome, Isaacs’ syndrome, Schwartz–Jampel syndrome, Fukuyama-type congenital muscular dystrophy, amyotrophic lateral sclerosis, and sarcopenia. The NMJ is a prototypic cholinergic synapse between the motor neuron and the skeletal muscle. Synaptogenesis of the NMJ has been extensively studied, which has also been extrapolated to further understand synapse formation in the central nervous system. Studies of genetically engineered mice have disclosed crucial roles of secreted molecules in the development and maintenance of the NMJ. In this review, we focus on the secreted signaling molecules which regulate the clustering of acetylcholine receptors (AChRs) at the NMJ. We first discuss the signaling pathway comprised of neural agrin and its receptors, low-den-sity lipoprotein receptor-related protein 4 (Lrp4) and muscle-specific receptor tyrosine kinase (MuSK). This pathway drives the clustering of acetylcholine receptors (AChRs) to ensure efficient signal transduction at the NMJ. We also discuss three secreted molecules (Rspo2, Fgf18, and con-nective tissue growth factor (Ctgf)) that we recently identified in the Wnt/β-catenin and fibroblast growth factors (FGF) signaling pathways. The three secreted molecules facilitate the clustering of AChRs by enhancing the agrin-Lrp4-MuSK signaling pathway.

DOI： 10.3390/ijms22052455

Koshimizu, H; Ohkawara, B; Nakashima, H; Ota, K; Kanbara, S; Inoue, T; Tomita, H; Sayo, A; Kiryu-Seo, S; Konishi, H; Ito, M; Masuda, A; Ishiguro, N; Imagama, S; Kiyama, H; Ohno, K

LIFE SCIENCES 263 巻頁： 118577 - 118577 2020年12月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：Life Sciences

Neuropathic pain is caused by a lesion or a functional impairment of the sensory nervous system and allodynia is one of the frequently observed symptoms in neuropathic pain. Allodynia represents abnormal pain due to a non-noxious stimulus that does not normally provoke pain. Cellular mechanisms underlying neuropathic pain remain mostly elusive, and partial pain relief can be achieved in a limited number of patients by antidepressants, anticonvulsants topical anesthetics, and others. Zonisamide (ZNS) is widely used as an anti-epileptic and anti-Parkinson's disease drug. A recent report shows that ZNS suppresses neuropathic pain associated with diabetes mellitus in a mouse model. We made a mouse model of neuropathic pain in the hindlimb by cutting the nerve at the intervertebral canal at lumbar level 4 (L4). At 28 days after nerve injury, ZNS ameliorated allodynic pain, and reduced the expression of inflammatory cytokines and the nerve injury-induced increase of Iba1-positive microglia in the spinal dorsal horn at L4. In BV2 microglial cells, ZNS reduced the number of lipopolysaccharide-induced amoeboid-shaped cells, representing activated microglia. These results suggest that ZNS is a potential therapeutic agent for neuropathic pain partly by suppressing microglia-mediated neuroinflammation.

DOI： 10.1016/j.lfs.2020.118577

A set of random forest models for each amino acid substitution to predict pathogenicity of missense variants in the human genome

Takeda J., Nanatsue K., Yamagishi R., Ito M., Ohno K.

EUROPEAN JOURNAL OF HUMAN GENETICS 28 巻 ( SUPPL 1 ) 頁： 1003 - 1003 2020年12月

A set of random forest models for each amino acid substitution to predict pathogenicity of missense variants in the human genome 査読有り

Takeda J., Nanatsue K., Yamagishi R., Ito M., Ohno K.

EUROPEAN JOURNAL OF HUMAN GENETICS 28 巻 ( SUPPL 1 ) 頁： 1003 - 1003 2020年12月

Short-Chain Fatty Acid-Producing Gut Microbiota Is Decreased in Parkinson's Disease but Not in Rapid-Eye-Movement Sleep Behavior Disorder 査読有り国際誌 Open Access

Nishiwaki, H; Hamaguchi, T; Ito, M; Ishida, T; Maeda, T; Kashihara, K; Tsuboi, Y; Ueyama, J; Shimamura, T; Mori, H; Kurokawa, K; Katsuno, M; Hirayama, M; Ohnoa, K

MSYSTEMS 5 巻 ( 6 ) 頁： e00797-20 2020年11月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：mSystems

Gut dysbiosis has been repeatedly reported in Parkinson's disease (PD) but only once in idiopathic rapid-eye-movement sleep behavior disorder (iRBD) from Germany. Abnormal aggregation of a-synuclein fibrils causing PD possibly starts from the intestine, although this is still currently under debate. iRBD patients frequently develop PD. Early-stage gut dysbiosis that is causally associated with PD is thus expected to be observed in iRBD. We analyzed gut microbiota in 26 iRBD patients and 137 controls by 16S rRNA sequencing (16S rRNA-seq). Our iRBD data set was meta-analyzed with the German iRBD data set and was compared with gut microbiota in 223 PD patients. Unsupervised clustering of gut microbiota by LIGER, a topic model-based tool for single-cell RNA sequencing (RNA-seq) analysis, revealed four enterotypes in controls, iRBD, and PD. Short-chain fatty acid (SCFA)-producing bacteria were conserved in an enterotype observed in controls and iRBD, whereas they were less conserved in enterotypes observed in PD. Genus Akkermansia and family Akkermansiaceae were consistently increased in both iRBD in two countries and PD in five countries. Short-chain fatty acid (SCFA)-producing bacteria were not significantly decreased in iRBD in two countries. In contrast, we previously reported that recognized or putative SCFA-producing genera Faecalibacterium, Roseburia, and Lachnospiraceae ND3007 group were consistently decreased in PD in five countries. In a-synucleinopathy, increase of mucin-layer-degrading genus Akkermansia is observed at the stage of iRBD, whereas decrease of SCFA-producing genera becomes obvious with development of PD. IMPORTANCE Twenty studies on gut microbiota in PD have been reported, whereas only one study has been reported on iRBD from Germany. iRBD has the highest likelihood ratio to develop PD. Our meta-analysis of iRBD in Japan and Germany revealed increased mucin-layer-degrading genus Akkermansia in iRBD. Genus Akkermansia may increase the intestinal permeability, as we previously observed in PD patients, and may make the intestinal neural plexus exposed to oxidative stress, which can lead to abnormal aggregation of prion-like a-synuclein fibrils in the intestine. In contrast to PD, SCFA-producing bacteria were not decreased in iRBD. As SCFA induces regulatory T (Treg) cells, a decrease of SCFA-producing bacteria may be a prerequisite for the development of PD. We propose that prebiotic and/or probiotic therapeutic strategies to increase the intestinal mucin layer and to increase intestinal SCFA potentially retard the development of iRBD and PD.

DOI： 10.1128/mSystems.00797-20

Identification of Qk as a Glial Precursor Cell Marker that Governs the Fate Specification of Neural Stem Cells to a Glial Cell Lineage 国際誌 Open Access

Takeuchi, A; Takahashi, Y; Iida, K; Hosokawa, M; Irie, K; Ito, M; Brown, JB; Ohno, K; Nakashima, K; Hagiwara, M

STEM CELL REPORTS 15 巻 ( 4 ) 頁： 883 - 897 2020年10月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：Stem Cell Reports

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.

DOI： 10.1016/j.stemcr.2020.08.010

Gene Expression Profile at the Motor Endplate of the Neuromuscular Junction of Fast-Twitch Muscle 国際誌 Open Access

Huang, K; Li, J; Ito, M; Takeda, JI; Ohkawara, B; Ogi, T; Masuda, A; Ohno, K

FRONTIERS IN MOLECULAR NEUROSCIENCE 13 巻頁： 154 - 154 2020年9月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：Frontiers in Molecular Neuroscience

The neuromuscular junction (NMJ) is a prototypic chemical synapse between the spinal motor neuron and the motor endplate. Gene expression profiles of the motor endplate are not fully elucidated. Collagen Q (ColQ) is a collagenic tail subunit of asymmetric forms of acetylcholinesterase and is driven by two distinct promoters. pColQ1 is active throughout the slow-twitch muscle, whereas pColQ1a is active at the motor endplate of fast-twitch muscle. We made a transgenic mouse line that expresses nuclear localization signal (NLS)-attached Cre recombinase under the control of pColQ1a (pColQ1a-Cre mouse). RiboTag mouse expresses an HA-tagged ribosomal subunit, RPL22, in cells expressing Cre recombinase. We generated pColQ1a-Cre:RiboTag mouse, and confirmed that HA-tagged RPL22 was enriched at the NMJ of tibialis anterior (TA) muscle. Next, we confirmed that Chrne and Musk that are specifically expressed at the NMJ were indeed enriched in HA-immunoprecipitated (IP) RNA, whereas Sox10 and S100b, markers for Schwann cells, and Icam1, a marker for vascular endothelial cells, and Pax3, a marker for muscle satellite cells, were scarcely detected. Gene set enrichment analysis (GSEA) of RNA-seq data showed that “phosphatidylinositol signaling system” and “extracellular matrix receptor interaction” were enriched at the motor endplate. Subsequent analysis revealed that genes encoding diacylglycerol kinases, phosphatidylinositol kinases, phospholipases, integrins, and laminins were enriched at the motor endplate. We first characterized the gene expression profile under translation at the motor endplate of TA muscle using the RiboTag technique. We expect that our gene expression profiling will help elucidate molecular mechanisms of the development, maintenance, and pathology of the NMJ.

DOI： 10.3389/fnmol.2020.00154

Meta-Analysisof Gut Dysbiosis in Parkinson's Disease 国際誌 Open Access

Nishiwaki, H; Ito, M; Ishida, T; Hamaguchi, T; Maeda, T; Kashihara, K; Tsuboi, Y; Ueyama, J; Shimamura, T; Mori, H; Kurokawa, K; Katsuno, M; Hirayama, M; Ohno, K

MOVEMENT DISORDERS 35 巻 ( 9 ) 頁： 1626 - 1635 2020年9月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：Movement Disorders

Background: PD may begin with the intestinal accumulation of α-synuclein fibrils, which can be causally associated with gut dysbiosis. The variability of gut microbiota across countries prevented us from identifying shared gut dysbiosis in PD. Objectives: To identify gut dysbiosis in PD across countries. Methods: We performed 16S ribosomal RNA gene sequencing analysis of gut microbiota in 223 patients with PD and 137 controls, and meta-analyzed gut dysbiosis by combining our dataset with four previously reported data sets from the United States, Finland, Russia, and Germany. We excluded uncommon taxa from our analyses. For pathway analysis, we developed the Kyoto Encyclopedia of Genes and Genomes orthology set enrichment analysis method. Results: After adjusting for confounding factors (body mass index, constipation, sex, age, and catechol-O-methyl transferase inhibitor), genera Akkermansia and Catabacter, as well as families Akkermansiaceae, were increased, whereas genera Roseburia, Faecalibacterium, and Lachnospiraceae ND3007 group were decreased in PD. Catechol-O-methyl transferase inhibitor intake markedly increased family Lactobacillaceae. Inspection of these bacteria in 12 datasets that were not included in the meta-analysis revealed that increased genus Akkermansia and decreased genera Roseburia and Faecalibacterium were frequently observed across countries. Kyoto Encyclopedia of Genes and Genomes orthology set enrichment analysis revealed changes in short-chain fatty acid metabolisms in our dataset. Conclusions: We report that intestinal mucin layer-degrading Akkermansia is increased and that short-chain fatty acid–producing Roseburia and Faecalibacterium are decreased in PD across countries. © 2020 International Parkinson and Movement Disorder Society.

DOI： 10.1002/mds.28119

CTGF/CCN2 facilitates LRP4-mediated formation of the embryonic neuromuscular junction 査読有り国際誌 Open Access

Ohkawara, B; Kobayakawa, A; Kanbara, S; Hattori, T; Kubota, S; Ito, M; Masuda, A; Takigawa, M; Lyons, KM; Ishiguro, N; Ohno, K

EMBO REPORTS 21 巻 ( 8 ) 頁： e48462 2020年8月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：EMBO Reports

At the neuromuscular junction (NMJ), lipoprotein-related receptor 4 (LRP4) mediates agrin-induced MuSK phosphorylation that leads to clustering of acetylcholine receptors (AChRs) in the postsynaptic region of the skeletal muscle. Additionally, the ectodomain of LRP4 is necessary for differentiation of the presynaptic nerve terminal. However, the molecules regulating LRP4 have not been fully elucidated yet. Here, we show that the CT domain of connective tissue growth factor (CTGF/CCN2) directly binds to the third beta-propeller domain of LRP4. CTGF/CCN2 enhances the binding of LRP4 to MuSK and facilitates the localization of LRP4 on the plasma membrane. CTGF/CCN2 enhances agrin-induced MuSK phosphorylation and AChR clustering in cultured myotubes. Ctgf-deficient mouse embryos (Ctgf−/−) have small AChR clusters and abnormal dispersion of synaptic vesicles along the motor axon. Ultrastructurally, the presynaptic nerve terminals have reduced numbers of active zones and mitochondria. Functionally, Ctgf−/− embryos exhibit impaired NMJ signal transmission. These results indicate that CTGF/CCN2 interacts with LRP4 to facilitate clustering of AChRs at the motor endplate and the maturation of the nerve terminal.

DOI： 10.15252/embr.201948462

Zonisamide ameliorates progression of cervical spondylotic myelopathy in a rat model 国際誌 Open Access

Kanbara, S; Ohkawara, B; Nakashima, H; Ohta, K; Koshimizu, H; Inoue, T; Tomita, H; Ito, M; Masuda, A; Ishiguro, N; Imagama, S; Ohno, K

SCIENTIFIC REPORTS 10 巻 ( 1 ) 頁： 13138 - 13138 2020年8月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：Scientific Reports

Cervical spondylotic myelopathy (CSM) is caused by chronic compression of the spinal cord and is the most common cause of myelopathy in adults. No drug is currently available to mitigate CSM. Herein, we made a rat model of CSM by epidurally implanting an expanding water-absorbent polymer underneath the laminae compress the spinal cord. The CSM rats exhibited progressive motor impairments recapitulating human CSM. CSM rats had loss of spinal motor neurons, and increased lipid peroxidation in the spinal cord. Zonisamide (ZNS) is clinically used for epilepsy and Parkinson's disease. We previously reported that ZNS protected primary spinal motor neurons against oxidative stress. We thus examined the effects of ZNS on our rat CSM model. CSM rats with daily intragastric administration of 0.5% methylcellulose (n = 11) and ZNS (30 mg/kg/day) in 0.5% methylcellulose (n = 11). Oral administration of ZNS ameliorated the progression of motor impairments, spared the number of spinal motor neurons, and preserved myelination of the pyramidal tracts. In addition, ZNS increased gene expressions of cystine/glutamate exchange transporter (xCT) and metallothionein 2A in the spinal cord in CSM rats, and also in the primary astrocytes. ZNS increased the glutathione (GSH) level in the spinal motor neurons of CSM rats. ZNS potentially ameliorates loss of the spinal motor neurons and demyelination of the pyramidal tracts in patients with CSM.

DOI： 10.1038/s41598-020-70068-0

InMeRF: prediction of pathogenicity of missense variants by individual modeling for each amino acid substitution 査読有り Open Access

Takeda, J; Nanatsue, K; Yamagishi, R; Ito, M; Haga, N; Hirata, H; Ogi, T; Ohno, K

NAR GENOMICS AND BIOINFORMATICS 2 巻 ( 2 ) 頁： lqaa038 2020年6月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：NAR Genomics and Bioinformatics

In predicting the pathogenicity of a nonsynonymous single-nucleotide variant (nsSNV), a radical change in amino acid properties is prone to be classified as being pathogenic. However, not all such nsSNVs are associated with human diseases. We generated random forest (RF) models individually for each amino acid substitution to differentiate pathogenic nsSNVs in the Human Gene Mutation Database and common nsSNVs in dbSNP. We named a set of our models ‘Individual Meta RF’ (InMeRF). Ten-fold cross-validation of InMeRF showed that the areas under the curves (AUCs) of receiver operating characteristic (ROC) and precision–recall curves were on average 0.941 and 0.957, respectively. To compare InMeRF with seven other tools, the eight tools were generated using the same training dataset, and were compared using the same three testing datasets. ROC-AUCs of InMeRF were ranked first in the eight tools. We applied InMeRF to 155 pathogenic and 125 common nsSNVs in seven major genes causing congenital myasthenic syndromes, as well as in VANGL1 causing spina bifida, and found that the sensitivity and specificity of InMeRF were 0.942 and 0.848, respectively. We made the InMeRF web service, and also made genome-wide InMeRF scores available online (https://www.med.nagoya-u.ac.jp/neurogenetics/InMeRF/).

DOI： 10.1093/nargab/lqaa038

tRIP-seq reveals repression of premature polyadenylation by co-transcriptional FUS-U1 snRNP assembly 査読有り国際誌 Open Access

Masuda, A; Kawachi, T; Takeda, J; Ohkawara, B; Ito, M; Ohno, K

EMBO REPORTS 21 巻 ( 5 ) 頁： e49890 2020年5月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：EMBO Reports

RNA processing occurs co-transcriptionally through the dynamic recruitment of RNA processing factors to RNA polymerase II (RNAPII). However, transcriptome-wide identification of protein–RNA interactions specifically assembled on transcribing RNAPII is challenging. Here, we develop the targeted RNA immunoprecipitation sequencing (tRIP-seq) method that detects protein–RNA interaction sites in thousands of cells. The high sensitivity of tRIP-seq enables identification of protein–RNA interactions at functional subcellular levels. Application of tRIP-seq to the FUS-RNA complex in the RNAPII machinery reveals that FUS binds upstream of alternative polyadenylation (APA) sites of nascent RNA bound to RNAPII, which retards RNAPII and suppresses the recognition of the polyadenylation signal by CPSF. Further tRIP-seq analyses demonstrate that the repression of APA is achieved by a complex composed of FUS and U1 snRNP on RNAPII, but not by either one alone. Moreover, our analysis reveals that FUS mutations in familial amyotrophic lateral sclerosis (ALS) that impair the FUS-U1 snRNP interaction aberrantly activate the APA sites. tRIP-seq provides new insights into the regulatory mechanism of co-transcriptional RNA processing by RNA processing factors.

DOI： 10.15252/embr.201949890

SRSF3 and hnRNP K Antagonistically Regulate Splicing of Large Exons

tRIP-seq reveals repression of premature polyadenylation by co-transcriptional FUS-U1 snRNP assembly

Masuda Akio, Kawachi Toshihiko, Takeda Jun-ichi, Ohkawara Bisei, Ito Mikako, Ohno Kinji

EMBO REPORTS 21 巻 ( 5 ) 頁： . 2020年5月

Kawachi, T; Masuda, A; Takeda, J; Ito, M; Hamaguchi, T; Ohno, K

FASEB JOURNAL 34 巻 2020年4月

Fluctuating Weak Magnetic Fields Induce Marked Mitophagy

Toda, T; Ito, M; Mori, K; Ohno, K

FASEB JOURNAL 34 巻 2020年4月

Molecular Hydrogen Manages Mitochondrial Proteostatic Stress and Induces Cellular Response

Hasegawa, T; Ito, M; Hasegawa, S; Takeda, K; Nishiwaki, H; Negishi, S; Ohno, K

FASEB JOURNAL 34 巻 2020年4月

Mianserin suppresses R-spondin 2-induced activation of Wnt/β-catenin signaling in chondrocytes and prevents cartilage degradation in a rat model of osteoarthritis (vol 12, 4512, 2019) 査読有り国際誌 Open Access

Okura, T; Ohkawara, B; Takegami, Y; Ito, M; Masuda, A; Seki, T; Ishiguro, N; Ohno, K

SCIENTIFIC REPORTS 10 巻 ( 1 ) 頁： 2995 - 2995 2020年2月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：Scientific Reports

This Article contains errors in the Reference list. Reference 21 is incorrectly listed as: Masahiro N. et al. A genome-wide association study identifies susceptibility loci for ossification of the posterior longitudinal ligament of the spine. Nature Genetics 46(9), 1012–1016 (2014). The correct reference is listed below as ref. 1. Additionally, Reference 23 is incorrectly listed as: Rita D. Hypertrophic differentiation of chondrocytes in osteoarthritis: the developmental aspect of degenerative joint disorders. Arthritis Research & Therapy 12(5), 216 (2010). The correct reference is listed below as ref. 2. Furthermore, Reference 25 is incorrectly listed as: Marlies, B. et al. The Reason for Discontinuation of the First Tumor Necrosis Factor (TNF) Blocking Agent Does Not Influence the Effect of a Second TNF Blocking Agent in Patients with Rheumatoid Arthritis. The Journal of Rheumatology 36(10), 2171–2177 (2009). The correct reference is listed below as ref. 3. In addition, there is an error in the legend of Figure 4, where: “The number of β-catenin-positive cells is divided by the number of DAPI signals to calculate the ratio of nuclear β-catenin-positive cells. The number of β-catenin-positive cells is divided by the number of DAPI signals to calculate the ratio of nuclear β-catenin-positive cells. *P < 0.05 by one-way ANOVA followed by Tukey’s post-hoc test. Scale = bar 200 μm (A), 100 μm (C), and 25 μm (D). (G) Model of Rspo2-mediated OA development, and the effect of mianserin.” should read:“The number of β-catenin-positive cells is divided by the number of DAPI signals to calculate the ratio of nuclear β-catenin-positive cells. *P < 0.05 by one-way ANOVA followed by Tukey’s post-hoc test. Scale bar = 200 μm (A), 100 μm (C), and 25 μm (D). (G) Model of Rspo2-mediated OA development, and the effect of mianserin.”.

DOI： 10.1038/s41598-020-60112-4

Inhibition of cyclooxygenase-1 by nonsteroidal anti-inflammatory drugs demethylates MeR2 enhancer and promotes Mbnl1 transcription in myogenic cells 査読有り国際誌 Open Access

Huang, K; Masuda, A; Chen, GY; Bushra, S; Kamon, M; Araki, T; Kinoshita, M; Ohkawara, B; Ito, M; Ohno, K

SCIENTIFIC REPORTS 10 巻 ( 1 ) 頁： 2558 - 2558 2020年2月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：Scientific Reports

Muscleblind-like 1 (MBNL1) is a ubiquitously expressed RNA-binding protein, which is highly expressed in skeletal muscle. Abnormally expanded CUG-repeats in the DMPK gene cause myotonic dystrophy type 1 (DM1) by sequestration of MBNL1 to nuclear RNA foci and by upregulation of another RNA-binding protein, CUG-binding protein 1 (CUGBP1). We previously reported that a nonsteroidal anti-inflammatory drug (NSAID), phenylbutazone, upregulates MBNL1 expression in DM1 mouse model by demethylation of MeR2, an enhancer element in Mbnl1 intron 1. NSAIDs inhibit cyclooxygenase (COX), which is comprised of COX-1 and COX-2 isoforms. In this study, we screened 29 NSAIDs in C2C12 myoblasts, and found that 13 NSAIDs enhanced Mbnl1 expression, where COX-1-selective NSAIDs upregulated Mbnl1 more than COX-2-selective NSAIDs. Consistently, knockdown of COX-1, but not of COX-2, upregulated MBNL1 expression in C2C12 myoblasts and myotubes, as well as in myotubes differentiated from DM1 patient-derived induced pluripotent stem cells (iPSCs). Luciferase assay showed that COX-1-knockdown augmented the MeR2 enhancer activity. Furthermore, bisulfite sequencing analysis demonstrated that COX-1-knockdown suppressed methylation of MeR2. These results suggest that COX-1 inhibition upregulates Mbnl1 transcription through demethylation of the MeR2 enhancer. Taken together, our study provides new insights into the transcriptional regulation of Mbnl1 by the COX-1-mediated pathway.

DOI： 10.1038/s41598-020-59517-y

Hydrogen water alleviates obliterative airway disease in mice 査読有り

Ozeki, N; Yamawaki-Ogata, A; Narita, Y; Mii, S; Ushida, K; Ito, M; Hirano, S; Kurokawa, R; Ohno, K; Usui, A

GENERAL THORACIC AND CARDIOVASCULAR SURGERY 68 巻 ( 2 ) 頁： 158 - 163 2020年2月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：General Thoracic and Cardiovascular Surgery

Objective: Bronchiolitis obliterans syndrome arising from chronic airway inflammation is a leading cause of death following lung transplantation. Several studies have suggested that inhaled hydrogen can protect lung grafts from ischemia–reperfusion injury via anti-inflammatory and -oxidative mechanisms. We investigated whether molecular hydrogen-saturated water can preserve lung allograft function in a heterotopic tracheal allograft mouse model of obliterative airway disease Methods: Obliterative airway disease was induced by heterotopically transplanting tracheal allografts from BALB/c donor mice into C57BL/6 recipient mice, which were subsequently administered hydrogen water (10 ppm) or tap water (control group) (n = 6 each) daily without any immunosuppressive treatment. Histological and immunohistochemical analyses were performed on days 7, 14, and 21. Results: Hydrogen water decreased airway occlusion on day 14. No significant histological differences were observed on days 7 or 21. The cluster of differentiation 4/cluster of differentiation 3 ratio in tracheal allografts on day 14 was higher in the hydrogen water group than in control mice. Enzyme-linked immunosorbent assay performed on day 7 revealed that hydrogen water reduced the level of the pro-inflammatory cytokine interleukin-6 and increased that of forkhead box P3 transcription factor, suggesting an enhancement of regulatory T cell activity. Conclusions: Hydrogen water suppressed the development of mid-term obliterative airway disease in a mouse tracheal allograft model via anti-oxidant and -inflammatory mechanisms and through the activation of Tregs. Thus, hydrogen water is a potential treatment strategy for BOS that can improve the outcome of lung transplant patients.

DOI： 10.1007/s11748-019-01195-3

Freeze-drying enables homogeneous and stable sample preparation for determination of fecal short-chain fatty acids 査読有り国際誌

Ueyama, J; Oda, M; Hirayama, M; Sugitate, K; Sakui, N; Hamada, R; Ito, M; Saito, I; Ohno, K

ANALYTICAL BIOCHEMISTRY 589 巻頁： 113508 - 113508 2020年1月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：Analytical Biochemistry

Background: The analysis methods for fecal short-chain fatty acids (SCFAs) have evolved considerably. Recently, the role of SCFAs in gastrointestinal physiology and their association with intestinal microbiota and disease were reported. However, the intra-fecal variability and storage stability of SCFAs have not been extensively investigated. The aim of this study was to understand the limitations of the measurement of SCFAs in crude feces and develop a useful pre-examination procedure using the freeze-drying technique. Methods: SCFAs in crude feces, obtained from healthy volunteers, and freeze-dried feces were determined by derivatization with isobutyl chloroformate, followed by liquid–liquid extraction with hexane, and separation and analysis using gas chromatography–mass spectrometry. Results: Among the SCFAS, the maximum intra-fecal variability was observed for iso-butyrate (coefficient of variation of 37.7%), but the freeze-drying procedure reduced this variability (coefficient of variation of 7.9%). Similar improvements were also observed for other SCFAs. Furthermore, significant decreases in the SCFA amounts were observed with storage at 4 °C for 24 h. Conclusions: The freeze-drying procedure affords fecal SCFA stability, even with storage at room temperature for 3 d. The freeze-drying procedure allows reliable SCFA measurements without labour-intensive processes. Therefore, the freeze-drying procedure can be applied in basic, clinical, and epidemiological studies.

DOI： 10.1016/j.ab.2019.113508

Lewy小体病患者のメタメタ解析を用いた腸内細菌叢の関与の解明査読有り

平山正昭, 石田智大, 九鬼桃茄, 浜口知成, 伊藤美佳子, 上山純, 島村徹平, 阿部興, 勝野雅央, 大野欽司

臨床神経学 59 巻 ( Suppl. ) 頁： S301 - S301 2019年11月

パーキンソン病(PD)における腸内細菌査読有り

石田智大, 九鬼桃茄, 浜口知成, 上山純, 伊藤美佳子, 柏原健一, 前田哲也, 坪井義夫, 阿部興, 島村徹平, 森宙史, 黒川顕, 勝野雅央, 大野欽司, 平山正昭

パーキンソン病・運動障害疾患コングレスプログラム・抄録集 13回巻頁： 102 - 102 2019年7月

神経変性疾患における短鎖脂肪酸の役割査読有り

九鬼桃茄, 石田智大, 浜口知成, 伊藤美佳子, 木村郁夫, 長谷耕二, 橋本款, 大野欽司, 平山正昭

パーキンソン病・運動障害疾患コングレスプログラム・抄録集 13回巻頁： 102 - 102 2019年7月

Mianserin suppresses R-spondin 2-induced activation of Wnt/β-catenin signaling in chondrocytes and prevents cartilage degradation in a rat model of osteoarthritis 査読有り国際誌 Open Access

Okura, T; Ohkawara, B; Takegami, Y; Ito, M; Masuda, A; Seki, T; Ishiguro, N; Ohno, K

SCIENTIFIC REPORTS 9 巻 ( 1 ) 頁： 2808 - 2808 2019年2月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：Scientific Reports

Aberrant activation of the Wnt/β-catenin signaling pathway promotes the progression of osteoarthritis (OA). We previously reported that R-spondin 2 (Rspo2), an activator of the Wnt/β-catenin signaling, facilitates differentiation of proliferating chondrocytes into hypertrophic chondrocytes by enhancing Wnt/β-catenin signaling in endochondral ossification. However, the role of Rspo2 in OA remains elusive. Here, we showed that the amounts of Rspo2 protein in synovial fluid were increased in OA patients. We searched for a preapproved drug that suppresses Rspo2-induced Wnt/β-catenin signaling in chondrogenic cells and reduces joint pathology in a rat model of OA. In Rspo2-treated ATDC5 cells, mianserin, a tetracyclic antidepressant, inhibited Wnt/β-catenin signaling, increased proteoglycan production, and upregulated chondrogenic marker genes. Mianserin suppressed Rspo2-induced accumulation of β-catenin and phosphorylation of Lrp6. We identified that mianserin blocked binding of Rspo2 to its receptor Lgr5. We also observed that intraarticular administration of mianserin suppressed β-catenin accumulation and prevented OA progression in a rat model of OA. We conclude that mianserin suppresses abnormally activated Wnt/β-catenin signaling in OA by inhibiting binding of Rspo2 to Lgr5.

DOI： 10.1038/s41598-019-39393-x

Quantification of hydrogen production by intestinal bacteria that are specifically dysregulated in Parkinson's disease 査読有り国際誌 Open Access

Suzuki, A; Ito, M; Hamaguchi, T; Mori, H; Takeda, Y; Baba, R; Watanabe, T; Kurokawa, K; Asakawa, S; Hirayama, M; Ohno, K

PLOS ONE 13 巻 ( 12 ) 頁： e0208313 2018年12月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：PLoS ONE

Oral administration of hydrogen water ameliorates Parkinson’s disease (PD) in rats, mice, and humans. We previously reported that the number of putative hydrogen-producing bacteria in intestinal microbiota is low in PD compared to controls. We also reported that the amount of hydrogen produced by ingestion of lactulose is low in PD patients. The decreased hydrogen production by intestinal microbiota may be associated with the development and progression of PD. We measured the amount of hydrogen production using gas chromatography by seven bacterial strains, which represented seven major intestinal bacterial groups/ genera/species. Blautia coccoides and Clostridium leptum produced the largest amount of hydrogen. Escherichia coli and Bacteroides fragilis constituted the second group that produced hydrogen 34- to 93-fold lower than B. coccoides. Bifidobacterium pseudocatenulatum and Atopobium parvulum constituted the third group that produced hydrogen 559- to 2164-fold lower than B. coccoides. Lactobacillus casei produced no detectable hydrogen. Assuming that taxonomically neighboring strains have similar hydrogen production, we simulated hydrogen production using intestinal microbiota that we previously reported, and found that PD patients produce a 2.2-fold lower amount of intestinal hydrogen compared to controls. The lower amount of intestinal hydrogen production in PD was also simulated in cohorts of two other countries. The number of hydrogen-producing intestinal bacteria may be associated with the development and progression of PD. Further studies are required to prove its beneficial effect.

DOI： 10.1371/journal.pone.0208313

Inhalation of hydrogen gas elevates urinary 8-hydroxy-2'-deoxyguanine in Parkinson's disease. 査読有り国際誌 Open Access

Hirayama M, Ito M, Minato T, Yoritaka A, LeBaron TW, Ohno K

Medical gas research 8 巻 ( 4 ) 頁： 144 - 149 2018年10月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：Medical Gas Research

Hyposmia is one of the earliest and the most common symptoms in Parkinson's disease (PD). The benefits of hydrogen water on motor deficits have been reported in animal PD models and PD patients, but the effects of hydrogen gas on PD patients have not been studied. We evaluated the effect of inhalation of hydrogen gas on olfactory function, non-motor symptoms, activities of daily living, and urinary 8-hydroxy-2′-deoxyguanine (8-OHdG) levels by a randomized, double-blinded, placebo-controlled, crossover trial with an 8-week washout period in 20 patients with PD. Patients inhaled either 1.2-1.4% hydrogen-air mixture or placebo for 10 minutes twice a day for 4 weeks. Inhalation of low dose hydrogen did not significantly influence the PD clinical parameters, but it did increase urinary 8-OHdG levels by 16%. This increase in 8-OHdG is markedly less than the over 300% increase in diabetes, and is more comparable to the increase after a bout of strenuous exercise. Although increased reactive oxygen species is often associated with toxicity and disease, they also play essential roles in mediating cytoprotective cellular adaptations in a process known as hormesis. Increases of oxidative stress by hydrogen have been previously reported, along with its ability to activate the Nrf2, NF-κB pathways, and heat shock responses. Although we did not observe any beneficial effect of hydrogen in our short trial, we propose that the increased 8-OHdG and other reported stress responses from hydrogen may indicate that its beneficial effects are partly or largely mediated by hormetic mechanisms. The study was approved by the ethics review committee of Nagoya University Graduate School of Medicine (approval number 2015-0295). The clinical trial was registered at the University Hospital Medical Information Network (identifier UMIN000019082).

DOI： 10.4103/2045-9912.248264

Differential effects of spinal motor neuron-derived and skeletal muscle-derived Rspo2 on acetylcholine receptor clustering at the neuromuscular junction 査読有り Open Access

Li, J; Ito, M; Ohkawara, B; Masuda, A; Ohno, K

SCIENTIFIC REPORTS 8 巻 ( 1 ) 頁： 13577 - - 2018年9月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：Scientific Reports

We recently reported that R-spondin 2 (Rspo2), a secreted activator of Wnt/β-catenin signaling, promotes acetylcholine receptor (AChR) clustering and neuromuscular junction (NMJ) formation via its receptor, Lgr5. Rspo2 is expressed highly in spinal motor neurons (SMNs) and marginally in the skeletal muscle, but the origin of Rspo2 at the NMJ remains elusive. We rescued Rspo2-deficient (Rspo2−/−) mice by specifically expressing Rspo2 in the skeletal muscle and SMNs. SMN-specific Rspo2 mitigated or over-corrected abnormal features of the NMJs and AChR clusters observed in Rspo2−/− mice including (i) abnormal broadening of enlarged AChR clusters, (ii) three of six abnormal ultrastructural features, and (iii) abnormal expression of nine genes in SMNs and the diaphragm. In contrast, muscle-specific Rspo2 normalized all six abnormal ultrastructural features, but it had no effect on AChR clustering and NMJ formation at the light microscopy level or on abnormal gene expression in SMNs and the diaphragm. These results suggest that SMN-derived Rspo2 plays a major role in AChR clustering and NMJ formation in the postsynaptic region, and muscle-derived Rspo2 also plays a substantial role in juxtaposition of the active zones and synaptic folds.

DOI： 10.1038/s41598-018-31949-7

Randomized, Double-Blind, Multicenter Trial of Hydrogen Water for Parkinson's Disease 査読有り国際誌

Yoritaka, A; Ohtsuka, C; Maeda, T; Hirayama, M; Abe, T; Watanabe, H; Saiki, H; Oyama, G; Fukae, J; Shimo, Y; Hatano, T; Kawajiri, S; Okuma, Y; Machida, Y; Miwa, H; Suzuki, C; Kazama, A; Tomiyama, M; Kihara, T; Hirasawa, M; Shimura, H; Oda, E; Ito, M; Ohno, K; Hattori, N

MOVEMENT DISORDERS 33 巻 ( 9 ) 頁： 1505 - 1507 2018年9月

Protein-anchoring therapy to target extracellular matrix proteins to their physiological destinations 査読有り国際誌

Ito, M; Ohno, K

MATRIX BIOLOGY 68-69 巻 ( - ) 頁： 628 - 636 2018年8月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：Matrix Biology

Endplate acetylcholinesterase (AChE) deficiency is a form of congenital myasthenic syndrome (CMS) caused by mutations in COLQ, which encodes collagen Q (ColQ). ColQ is an extracellular matrix (ECM) protein that anchors AChE to the synaptic basal lamina. Biglycan, encoded by BGN, is another ECM protein that binds to the dystrophin-associated protein complex (DAPC) on skeletal muscle, which links the actin cytoskeleton and ECM proteins to stabilize the sarcolemma during repeated muscle contractions. Upregulation of biglycan stabilizes the DPAC. Gene therapy can potentially ameliorate any disease that can be recapitulated in cultured cells. However, the difficulty of tissue-specific and developmental stage-specific regulated expression of transgenes, as well as the difficulty of introducing a transgene into all cells in a specific tissue, prevents us from successfully applying gene therapy to many human diseases. In contrast to intracellular proteins, an ECM protein is anchored to the target tissue via its specific binding affinity for protein(s) expressed on the cell surface within the target tissue. Exploiting this unique feature of ECM proteins, we developed protein-anchoring therapy in which a transgene product expressed even in remote tissues can be delivered and anchored to a target tissue using specific binding signals. We demonstrate the application of protein-anchoring therapy to two disease models. First, intravenous administration of adeno-associated virus (AAV) serotype 8-COLQ to Colq-deficient mice, resulting in specific anchoring of ectopically expressed ColQ-AChE at the NMJ, markedly improved motor functions, synaptic transmission, and the ultrastructure of the neuromuscular junction (NMJ). In the second example, Mdx mice, a model for Duchenne muscular dystrophy, were intravenously injected with AAV8-BGN. The treatment ameliorated motor deficits, mitigated muscle histopathologies, decreased plasma creatine kinase activities, and upregulated expression of utrophin and DAPC component proteins. We propose that protein-anchoring therapy could be applied to hereditary/acquired defects in ECM and secreted proteins, as well as therapeutic overexpression of such factors.

DOI： 10.1016/j.matbio.2018.02.014

Molecular hydrogen upregulates heat shock response and collagen biosynthesis, and downregulates cell cycles: meta-analyses of gene expression profiles 国際誌

Loss of Sfpq Causes Long-Gene Transcriptopathy in the Brain

Takeuchi Akihide, Iida Kei, Tsubota Toshiaki, Hosokawa Motoyasu, Denawa Masatsugu, Brown J. B., Ninomiya Kensuke, Ito Mikako, Kimura Hiroshi, Abe Takaya, Kiyonari Hiroshi, Ohno Kinji, Hagiwara Masatoshi

CELL REPORTS 23 巻 ( 5 ) 頁： 1326-1341 2018年5月

Nishiwaki, H; Ito, M; Negishi, S; Sobue, S; Ichihara, M; Ohno, K

FREE RADICAL RESEARCH 52 巻 ( 4 ) 頁： 434 - 445 2018年

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：Free Radical Research

Molecular hydrogen exerts its effect on multiple pathologies, including oxidative stress, inflammation, and apoptosis. However, its molecular mechanisms have not been fully elucidated. In order to explore the effects of molecular hydrogen, we meta-analysed gene expression profiles modulated by molecular hydrogen. We performed microarray analysis of the mouse liver with or without drinking hydrogen water. We also integrated two previously reported microarray datasets of the rat liver into meta-analyses. We used two categories of meta-analysis methods: the cross-platform method and the conventional meta-analysis method (Fisher’s method). For each method, hydrogen-modulated pathways were analysed by (i) the hypergeometric test (HGT) in the class of over-representation analysis (ORA), (ii) the gene set enrichment analysis (GSEA) in the class of functional class scoring (FCS), and (iii) the signalling pathway impact analysis (SPIA), pathway regulation score (PRS), and others in the class of pathway topology-based approach (PTA). Pathways in the collagen biosynthesis and the heat-shock response were up-regulated according to (a) HGT with the cross-platform method, (b) GSEA with the cross-platform method, and (c) PRS with the cross-platform method. Pathways in cell cycles were down-regulated according to (a) HGT with the cross-platform method, (b) GSEA with the cross-platform method, and (d) GSEA with the conventional meta-analysis method. Because the heat-shock response leads to up-regulation of collagen biosynthesis and a transient arrest of cell cycles, induction of the heat-shock response is likely to be a primary event induced by molecular hydrogen in the liver of wild-type rodents.

DOI： 10.1080/10715762.2018.1439166

Fluoxetine ameliorates cartilage degradation in osteoarthritis by inhibiting Wnt/β-catenin signaling 査読有り Open Access

Miyamoto, K; Ohkawara, B; Ito, M; Masuda, A; Hirakawa, A; Sakai, T; Hiraiwa, H; Hamada, T; Ishiguro, N; Ohno, K

PLOS ONE 12 巻 ( 9 ) 頁： e0184388 - - 2017年9月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：PLoS ONE

Abnormal activation of the Wnt/β-catenin signaling is implicated in the osteoarthritis (OA) pathology. We searched for a pre-approved drug that suppresses abnormally activated Wnt/β-catenin signaling and has a potency to reduce joint pathology in OA. We introduced the TOPFlash reporter plasmid into HCS-2/8 human chondrosarcoma cells to estimate the Wnt/β-catenin activity in the presence of 10 μM each compound in a panel of pre-approved drugs. We found that fluoxetine, an antidepressant in the class of selective serotonin reuptake inhibitors (SSRI), down-regulated Wnt/β-catenin signaling in human chondrosarcoma cells. Fluoxetine inhibited both Wnt3A- and LiCl-induced loss of proteoglycans in chondrogenically differentiated ATDC5 cells. Fluoxetine increased expression of Sox9 (the chondrogenic master regulator), and decreased expressions of Axin2 (a marker for Wnt/β-catenin signaling) and Mmp13 (matrix metalloproteinase 13). Fluoxetine suppressed a LiCl-induced increase of total β-catenin and a LiCl-induced decrease of phosphorylated β-catenin in a dose-dependent manner. An in vitro protein-binding assay showed that fluoxetine enhanced binding of β-catenin with Axin1, which is a scaffold protein forming the degradation complex for β-catenin. Fluoxetine suppressed LiCl-induced β-catenin accumulation in human OA chondrocytes. Intraarticular injection of fluoxetine in a rat OA model ameliorated OA progression and suppressed β-catenin accumulation.

DOI： 10.1371/journal.pone.0184388

SRSF1 suppresses selection of intron-distal 5′ splice site of DOK7 intron 4 to generate functional full-length Dok-7 protein 査読有り Open Access

Bin Ahsan, K; Masuda, A; Rahman, MA; Takeda, J; Nazim, M; Ohkawara, B; Ito, M; Ohno, K

SCIENTIFIC REPORTS 7 巻 ( 1 ) 頁： 10446 - - 2017年9月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：Scientific Reports

Dok-7 is a non-catalytic adaptor protein that facilitates agrin-induced clustering of acetylcholine receptors (AChR) at the neuromuscular junction. Alternative selection of 5′ splice sites (SSs) of DOK7 intron 4 generates canonical and frame-shifted transcripts. We found that the canonical full-length Dok-7 enhanced AChR clustering, whereas the truncated Dok-7 did not. We identified a splicing cis-element close to the 3′ end of exon 4 by block-scanning mutagenesis. RNA affinity purification and mass spectrometry revealed that SRSF1 binds to the cis-element. Knocking down of SRSF1 enhanced selection of the intron-distal 5′ SS of DOK7 intron 4, whereas MS2-mediated artificial tethering of SRSF1 to the identified cis-element suppressed it. Isolation of an early spliceosomal complex revealed that SRSF1 inhibited association of U1 snRNP to the intron-distal 5′ SS, and rather enhanced association of U1 snRNP to the intron-proximal 5′ SS, which led to upregulation of the canonical DOK7 transcript. Integrated global analysis of CLIP-seq and RNA-seq also indicated that binding of SRSF1 immediately upstream to two competing 5′ SSs suppresses selection of the intron-distal 5′ SS in hundreds of human genes. We demonstrate that SRSF1 critically regulates alternative selection of adjacently placed 5′ SSs by modulating binding of U1 snRNP.

DOI： 10.1038/s41598-017-11036-z

Wnt/β-catenin signaling suppresses expressions of Scx, Mkx, and Tnmd in tendon-derived cells 査読有り Open Access

Kishimoto, Y; Ohkawara, B; Sakai, T; Ito, M; Masuda, A; Ishiguro, N; Shukunami, C; Docheva, D; Ohno, K

PLOS ONE 12 巻 ( 7 ) 頁： e0182051 2017年7月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：PLoS ONE

After tendon injuries, biomechanical properties of the injured tendon are not fully recovered in most cases. Modulation of signaling pathways, which are involved in tendon development and tendon repair, is one of attractive modalities to facilitate proper regeneration of the injured tendon. The roles of TGF-β signaling in tendon homeostasis and tendon development have been elucidated. In contrast, the roles of Wnt/β-catenin signaling in tendon remain mostly elusive. We found that the number of β-catenin-positive cells was increased at the injured site, suggesting involvement of Wnt/β-catenin signaling in tendon healing. Activation of Wnt/β-catenin signaling suppressed expressions of tenogenic genes of Scx, Mkx, and Tnmd in rat tendon-derived cells (TDCs) isolated from the Achilles tendons of 6-week old rats. Additionally, activation of Wnt/β-catenin reduced the amounts of Smad2 and Smad3, which are intracellular mediators for TGF-β signaling, and antagonized upregulation of Scx induced by TGF-β signaling in TDCs. We found that Wnt/β-catenin decreased Mkx and Tnmd expressions without suppressing Scx expression in Scx-programmed tendon progenitors. Our studies suggest that Wnt/β-catenin signaling is a repressor for tenogenic gene expressions.

DOI： 10.1371/journal.pone.0182051

Protein-Anchoring Therapy of Biglycan for Mdx Mouse Model of Duchenne Muscular Dystrophy 査読有り

Ito, M; Ehara, Y; Li, J; Inada, K; Ohno, K

HUMAN GENE THERAPY 28 巻 ( 5 ) 頁： 428 - 436 2017年5月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：Human Gene Therapy

Duchenne muscular dystrophy (DMD) is a devastating muscle disease caused by loss-of-function mutations in DMD encoding dystrophin. No rational therapy is currently available. Utrophin is a paralog of dystrophin and is highly expressed at the neuromuscular junction. In mdx mice, utrophin is naturally upregulated throughout the muscle fibers, which mitigates muscular dystrophy. Protein-anchoring therapy was previously reported, in which a recombinant extracellular matrix (ECM) protein is delivered to and anchored to a specific target using its proprietary binding domains. Being prompted by a report that intramuscular and intraperitoneal injection of an ECM protein, biglycan, upregulates expression of utrophin and ameliorates muscle pathology in mdx mice, protein-anchoring therapy was applied to mdx mice. Recombinant adeno-associated virus serotype 8 (rAAV8) carrying hBGN encoding human biglycan was intravenously injected into 5-week-old mdx mice. The rAAV8-hBGN treatment improved motor deficits and decreased plasma creatine kinase activities. In muscle sections of treated mice, the number of central myonuclei and the distribution of myofiber sizes were improved. The treated mice increased gene expressions of utrophin and β1-syntrophin, as well as protein expressions of biglycan, utrophin, γ-sarcoglycan, dystrobrevin, and α1-syntrophin. The expression of hBGN in the skeletal muscle of the treated mice was 1.34-fold higher than that of the native mouse Bgn (mBgn). The low transduction efficiency and improved motor functions suggest that biglycan expressed in a small number of muscle fibers was likely to have been secreted and anchored to the cell surface throughout the whole muscular fibers. It is proposed that the protein-anchoring strategy can be applied not only to deficiency of an ECM protein as previously reported, but also to augmentation of a naturally induced ECM protein.

DOI： 10.1089/hum.2015.088

Competitive regulation of alternative splicing and alternative polyadenylation by hnRNP H and CstF64 determines acetylcholinesterase isoforms 査読有り Open Access

Nazim, M; Masuda, A; Rahman, MA; Nasrin, F; Takeda, JI; Ohe, K; Ohkawara, B; Ito, M; Ohno, K

NUCLEIC ACIDS RESEARCH 45 巻 ( 3 ) 頁： 1455 - 1468 2017年2月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：Nucleic acids research

Acetylcholinesterase (AChE), encoded by the ACHE gene, hydrolyzes the neurotransmitter acetylcholine to terminate synaptic transmission. Alternative splicing close to the 3΄ end generates three distinct isoforms of AChET, AChEH and AChER. We found that hnRNP H binds to two specific G-runs in exon 5a of human ACHE and activates the distal alternative 3΄ splice site (ss) between exons 5a and 5b to generate AChET. Specific effect of hnRNP H was corroborated by siRNA-mediated knockdown and artificial tethering of hnRNP H. Furthermore, hnRNP H competes for binding of CstF64 to the overlapping binding sites in exon 5a, and suppresses the selection of a cryptic polyadenylation site (PAS), which additionally ensures transcription of the distal 3΄ ss required for the generation of AChET. Expression levels of hnRNP H were positively correlated with the proportions of the AChET isoform in three different cell lines. HnRNP H thus critically generates AChET by enhancing the distal 3΄ ss and by suppressing the cryptic PAS. Global analysis of CLIP-seq and RNA-seq also revealed that hnRNP H competitively regulates alternative 3΄ ss and alternative PAS in other genes. We propose that hnRNP H is an essential factor that competitively regulates alternative splicing and alternative polyadenylation.

DOI： 10.1093/nar/gkw823

Molecular hydrogen alleviates motor deficits and muscle degeneration in mdx mice 査読有り Open Access

Hasegawa, S; Ito, M; Fukami, M; Hashimoto, M; Hirayama, M; Ohno, K

REDOX REPORT 22 巻 ( 1 ) 頁： 26 - 34 2017年

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：Redox Report

Objective: Duchenne muscular dystrophy (DMD) is a devastating muscle disease caused by a mutation in DMD encoding dystrophin. Oxidative stress accounts for dystrophic muscle pathologies in DMD. We examined the effects of molecular hydrogen in mdx mice, a model animal for DMD. Methods: The pregnant mother started to take supersaturated hydrogen water (>5 ppm) ad libitum from E15.5 up to weaning of the offspring. The mdx mice took supersaturated hydrogen water from weaning until age 10 or 24 weeks when they were sacrificed. Results: Hydrogen water prevented abnormal body mass gain that is commonly observed in mdx mice. Hydrogen improved the spontaneous running distance that was estimated by a counter-equipped running-wheel, and extended the duration on the rota-rod. Plasma creatine kinase activities were decreased by hydrogen at ages 10 and 24 weeks. Hydrogen also decreased the number of central nuclei of muscle fibers at ages 10 and 24 weeks, and immunostaining for nitrotyrosine in gastrocnemius muscle at age 24 weeks. Additionally, hydrogen tended to increase protein expressions of antioxidant glutathione peroxidase 1, as well as anti-apoptotic Bcl-2, in skeletal muscle at age 10 weeks. Discussion: Although molecular mechanisms of the diverse effects of hydrogen remain to be elucidated, hydrogen potentially improves muscular dystrophy in DMD patients.

DOI： 10.1080/13510002.2015.1135580

Agrin-LRP4-MuSK signaling as a therapeutic target for myasthenia gravis and other neuromuscular disorders 査読有り

Ohno, K; Ohkawara, B; Ito, M

EXPERT OPINION ON THERAPEUTIC TARGETS 21 巻 ( 10 ) 頁： 949 - 958 2017年

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：Expert Opinion on Therapeutic Targets

Introduction: Signal transduction at the neuromuscular junction (NMJ) is compromised in a diverse array of diseases including myasthenia gravis, Lambert-Eaton myasthenic syndrome, Isaacs’ syndrome, congenital myasthenic syndromes, Fukuyama-type congenital muscular dystrophy, amyotrophic lateral sclerosis, and sarcopenia. Except for sarcopenia, all are orphan diseases. In addition, the NMJ signal transduction is impaired by tetanus, botulinum, curare, α-bungarotoxin, conotoxins, organophosphate, sarin, VX, and soman to name a few. Areas covered: This review covers the agrin-LRP4-MuSK signaling pathway, which drives clustering of acetylcholine receptors (AChRs) and ensures efficient signal transduction at the NMJ. We also address diseases caused by autoantibodies against the NMJ molecules and by germline mutations in genes encoding the NMJ molecules. Expert opinion: Representative small compounds to treat the defective NMJ signal transduction are cholinesterase inhibitors, which exert their effects by increasing the amount of acetylcholine at the synaptic space. Another possible therapeutic strategy to enhance the NMJ signal transduction is to increase the number of AChRs, but no currently available drug has this functionality.

DOI： 10.1080/14728222.2017.1369960