記述言語：英語   掲載種別：研究論文（学術雑誌）  

DOI： 10.1093/hmg/ddac306

記述言語：英語   掲載種別：研究論文（学術雑誌）  

DOI： 10.3390/ijms24087420

記述言語：英語   掲載種別：研究論文（学術雑誌）  

DOI： 10.1007/s11102-023-01311-w

記述言語：英語   掲載種別：研究論文（学術雑誌）  

DOI： 10.1186/s12891-023-06318-9

記述言語：英語   掲載種別：研究論文（学術雑誌）  

DOI： 10.3390/biomedicines11030814

記述言語：英語   掲載種別：研究論文（学術雑誌）  

DOI： 10.3390/ijms24043730

記述言語：英語   掲載種別：研究論文（学術雑誌）  

DOI： 10.3892/etm.2022.11738

記述言語：英語   掲載種別：研究論文（学術雑誌）  

DOI： 10.3390/ijerph20010213

記述言語：英語   掲載種別：研究論文（学術雑誌）  

DOI： 10.1038/s41531-022-00428-2

記述言語：英語   掲載種別：研究論文（学術雑誌）  

DOI： 10.1038/s41598-022-25184-4

記述言語：英語   掲載種別：研究論文（学術雑誌）  

DOI： 10.3390/ijms232314750

記述言語：英語   掲載種別：研究論文（学術雑誌）  

DOI： 10.3390/ijms232314508

記述言語：英語   掲載種別：研究論文（学術雑誌）  

DOI： 10.15403/jgld-4313

記述言語：英語   掲載種別：研究論文（学術雑誌）  

DOI： 10.1038/s41598-022-15756-9

記述言語：英語   掲載種別：研究論文（学術雑誌）  

DOI： 10.1038/s41531-022-00328-5

記述言語：英語   掲載種別：研究論文（学術雑誌）  

DOI： 10.1038/s42003-022-03389-7

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  

KIAA1199 has a strong hyaluronidase activity in inflammatory arthritis. This study aimed to identify a drug that could reduce KIAA1199 activity and clarify its effects on inflammatory arthritis. Rat chondrosarcoma (RCS) cells were strongly stained with Alcian blue (AB). Its stainability was reduced in RCS cells, which were over-expressed with the KIAA1199 gene (RCS-KIAA). We screened the drugs that restore the AB stainability in RCS-KIAA. The effects of the drug were evaluated by particle exclusion assay, HA ELISA, RT-PCR, and Western blotting. We further evaluated the HA accumulation and the MMP1 and three expressions in fibroblast-like synoviocytes (FLS). In vivo, the effects of the drug on symptoms and serum concentration of HA in a collagen-induced arthritis mouse were evaluated. Ipriflavone was identified to restore AB stainability at 23%. Extracellular matrix formation was significantly increased in a dose-dependent manner (p = 0.006). Ipriflavone increased the HA accumulation and suppressed the MMP1 and MMP3 expression on TNF-α stimulated FLS. In vivo, Ipriflavone significantly improved the symptoms and reduced the serum concentrations of HA. Conclusions: We identified Ipriflavone, which has inhibitory effects on KIAA1199 activity. Ipriflavone may be a therapeutic candidate based on its reduction of KIAA1199 activity in inflammatory arthritis.

DOI： 10.3390/ijms23084089

記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：American Journal of Sports Medicine  

Background: Wnt/β-catenin signaling suppresses the differentiation of cultured tenocytes, but its roles in tendon repair remain mostly elusive. No chemical compounds are currently available to treat tendon injury. Hypothesis: We hypothesized that the inhibition of Wnt/β-catenin signaling would accelerate tendon healing. Study Design: Controlled laboratory study. Methods: Tendon-derived cells (TDCs) were isolated from rat Achilles tendons. The right Achilles tendon was injured via a dermal punch, while the left tendon was sham operated. A Wnt/β-catenin inhibitor, IWR-1, and an antihistamine agent, promethazine (PH), were locally and intramuscularly injected, respectively, for 2 weeks after surgery. The healing tendons were histologically and biomechanically evaluated. Results: The amount of β-catenin protein was increased in the injured tendons from postoperative weeks 0.5 to 2. Inhibition of Wnt/β-catenin signaling by IWR-1 in healing tendons improved the histological abnormalities and decreased β-catenin, but it compromised the biomechanical properties. As we previously reported that antihistamine agents suppressed Wnt/β-catenin signaling in human chondrosarcoma cells, we examined the effects of antihistamines on TDCs. We found that a first-generation antihistamine agent, PH, increased the expression of the tendon marker genes Mkx and Tnmd in TDCs. Intramuscular injection of PH did not improve histological abnormalities, but it decreased β-catenin in healing tendons and increased the peak force and stiffness of the healing tendons on postoperative week 2. On postoperative week 8, however, the biomechanical properties of vehicle-treated tendons became similar to those of PH-treated tendons. Conclusion: IWR-1 and PH suppressed Wnt/β-catenin signaling and improved the histological abnormalities of healing tendons. IWR-1, however, compromised the biomechanical properties of healing tendons, whereas PH improved them. Clinical Relevance: PH is a candidate repositioned drug that potentially accelerates tendon repair.

DOI： 10.1177/03635465221077116

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

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

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

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

記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：Annals of Nutrition and Metabolism  

DOI： 10.1159/000521993

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

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：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

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

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 Orthopaedic Research  

Heterotopic ossification (HO) is a pathological condition in which ectopic bone forms within soft tissues such as skeletal muscle. Human platelet-derived growth factor receptor α positive (PDGFRα+) cells, which were proved to be the original cells of HO were incubated in osteogenic differentiation medium with Food and Drug Administration-approved compounds. Alkaline phosphatase activity was measured as a screening to inhibit osteogenic differentiation. For the compounds which inhibited osteogenic differentiation of PDGFRα+ cells, we examined dose dependency of its effect using alizarin red S staining and its cell toxicity using WST-8. In addition, regulation of bone morphogenetic proteins (BMP)-Smad signaling which is the major signal of osteogenic differentiation was investigated by Western blotting to elucidate the mechanism of osteogenesis inhibitory effect by the compound. In vivo experiment, complete transverse incision of Achilles tendons in mice was made and mice were fed the compound by mixing with drinking water after operation. Ten weeks after operation, we assessed and quantified HO by micro-computed tomography scan. Intriguingly, we discovered desloratadine inhibited osteogenic differentiation of PDGFRα+ cells using the drug repositioning method. Desloratadine inhibited osteogenic differentiation of the cells dose dependently without cell toxicity. Desloratadine suppressed phosphorylation of Smad1/5/8 induced by BMP2 in PDGFRα+ cells. In Achilles tenotomy mice model, desloratadine treatment significantly inhibited ectopic bone formation compared with control. In conclusion, we discovered desloratadine inhibited osteogenic differentiation using human PDGFRα+ cells and proved its efficacy using Achilles tenotomy ectopic bone formation model in vivo. Our study paved the way to inhibit HO in early clinical use because of its guaranteed safety.

DOI： 10.1002/jor.24625

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  

During mRNA transcription, diverse RNA-binding proteins (RBPs) are recruited to RNA polymerase II (RNAP II) transcription machinery. These RBPs bind to distinct sites of nascent RNA to co-transcriptionally operate mRNA processing. Recent studies have revealed a close relationship between transcription and co-transcriptional RNA processing, where one affects the other’s activity, indicating an essential role of protein–RNA interactions for the fine-tuning of mRNA production. Owing to their limited amount in cells, the detection of protein–RNA interactions specifically assembled on the transcribing RNAP II machinery still remains challenging. Currently, cross-linking and immunoprecipitation (CLIP) has become a standard method to detect in vivo protein–RNA interactions, although it requires a large amount of input materials. Several improved methods, such as infrared-CLIP (irCLIP), enhanced CLIP (eCLIP), and target RNA immunoprecipitation (tRIP), have shown remarkable enhancements in the detection efficiency. Furthermore, the utilization of an RNA editing mechanism or proximity labeling strategy has achieved the detection of faint protein–RNA interactions in cells without depending on crosslinking. This review aims to explore various methods being developed to detect endogenous protein–RNA interaction sites and discusses how they may be applied to the analysis of co-transcriptional RNA processing.

DOI： 10.3390/ijms22105312

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

DOI： 10.1002/mus.27166

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

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

Background: The human microbiome forms very complex communities that consist of hundreds to thousands of different microorganisms that not only affect the host, but also participate in disease processes. Several state-of-the-art methods have been proposed for learning the structure of microbial communities and to investigate the relationship between microorganisms and host environmental factors. However, these methods were mainly designed to model and analyze single microbial communities that do not interact with or depend on other communities. Such methods therefore cannot comprehend the properties between interdependent systems in communities that affect host behavior and disease processes. Results: We introduce a novel hierarchical Bayesian framework, called BALSAMICO (BAyesian Latent Semantic Analysis of MIcrobial COmmunities), which uses microbial metagenome data to discover the underlying microbial community structures and the associations between microbiota and their environmental factors. BALSAMICO models mixtures of communities in the framework of nonnegative matrix factorization, taking into account environmental factors. We proposes an efficient procedure for estimating parameters. A simulation then evaluates the accuracy of the estimated parameters. Finally, the method is used to analyze clinical data. In this analysis, we successfully detected bacteria related to colorectal cancer. Conclusions: These results show that the method not only accurately estimates the parameters needed to analyze the connections between communities of microbiota and their environments, but also allows for the effective detection of these communities in real-world circumstances.

DOI： 10.1186/s12864-021-07401-y

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 Cell and Developmental Biology  

Meclozine has been developed as an inhibitor of fibroblast growth factor receptor 3 (FGFR3) to treat achondroplasia (ACH). Extracellular signal regulated kinase (ERK) phosphorylation was attenuated by meclozine in FGF2-treated chondrocyte cell line, but the site of its action has not been elucidated. Although orally administered meclozine promoted longitudinal bone growth in a mouse model of ACH, its effect on craniofacial bone development during the early stage remains unknown. Herein, RNA-sequencing analysis was performed using murine chondrocytes from FGF2-treated cultured tibiae, which was significantly elongated by meclozine treatment. Gene set enrichment analysis demonstrated that FGF2 significantly increased the enrichment score of mitogen-activated protein kinase (MAPK) family signaling cascades in chondrocytes; however, meclozine reduced this enrichment. Next, we administered meclozine to FGF2-treated larval zebrafish from 8 h post-fertilization (hpf). We observed that FGF2 significantly increased the number of ossified vertebrae in larval zebrafish at 7 days post-fertilization (dpf), while meclozine delayed vertebral ossification in FGF2-induced zebrafish. Meclozine also reversed the FGF2-induced upregulation of ossified craniofacial bone area, including ceratohyal, hyomandibular, and quadrate. The current study provided additional evidence regarding the inhibitory effect of meclozine on the FGF2-induced upregulation of MAPK signaling in chondrocytes and FGF2-induced development of craniofacial and vertebral bones.

DOI： 10.3389/fcell.2021.694018

記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：Annals of Nutrition and Metabolism  

Background: Parkinson's disease (PD) is caused by abnormal aggregation of α-synuclein fibrils, called the Lewy bodies, in the central nervous system. Accumulating knowledge points to the notion that α-synuclein fibrils start from the dorsal vagal nucleus and ascend to the locus ceruleus and the substantia nigra (SN). Even in healthy elderly subjects without motor or cognitive impairment, α-synuclein fibrils are frequently observed in the brain and sometimes in the intestinal neural plexus. Enteroendocrine cells have a direct synapse to the vagal afferents, and the vagal nucleus has synaptic pathways to the SN and the striatum. Intestinal bacteria are likely to be involved in the formation of intestinal α-synuclein fibrils. Summary: A nonparametric meta-analysis of intestinal microbiota in PD in 5 countries, as well as scrutinization of the other reports from the other countries, indicates that mucin-degrading Akkermansia is increased in PD and that short-chain fatty acid (SCFA)-producing bacteria are decreased in PD. Both dysbiosis should increase the intestinal permeability, which subsequently facilitates exposure of the intestinal neural plexus to toxins like lipopolysaccharide and pesticide, which should lead to abnormal aggregation of α-synuclein fibrils. Decreased SCFA also downregulates regulatory T cells and fails to suppress neuronal inflammation. Key Messages: Therapeutic intervention may be able to be established against these mechanisms. Additional biochemical, cellular, and animal studies are required to further dissect the direct association between intestinal microbiota and PD.

DOI： 10.1159/000518147

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

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 Genetics  

Prediction of the effect of a single-nucleotide variant (SNV) in an intronic region on aberrant pre-mRNA splicing is challenging except for an SNV affecting the canonical GU/AG splice sites (ss). To predict pathogenicity of SNVs at intronic positions −50 (Int-50) to −3 (Int-3) close to the 3’ ss, we developed light gradient boosting machine (LightGBM)-based IntSplice2 models using pathogenic SNVs in the human gene mutation database (HGMD) and ClinVar and common SNVs in dbSNP with 0.01 ≤ minor allelic frequency (MAF) < 0.50. The LightGBM models were generated using features representing splicing cis-elements. The average recall/sensitivity and specificity of IntSplice2 by fivefold cross-validation (CV) of the training dataset were 0.764 and 0.884, respectively. The recall/sensitivity of IntSplice2 was lower than the average recall/sensitivity of 0.800 of IntSplice that we previously made with support vector machine (SVM) modeling for the same intronic positions. In contrast, the specificity of IntSplice2 was higher than the average specificity of 0.849 of IntSplice. For benchmarking (BM) of IntSplice2 with IntSplice, we made a test dataset that was not used to train IntSplice. After excluding the test dataset from the training dataset, we generated IntSplice2-BM and compared it with IntSplice using the test dataset. IntSplice2-BM was superior to IntSplice in all of the seven statistical measures of accuracy, precision, recall/sensitivity, specificity, F1 score, negative predictive value (NPV), and matthews correlation coefficient (MCC). We made the IntSplice2 web service at https://www.med.nagoya-u.ac.jp/neurogenetics/IntSplice2.

DOI： 10.3389/fgene.2021.701076

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

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

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

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

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

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

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

掲載種別：研究論文（学術雑誌）  

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

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

Congenital myasthenic syndromes (CMS) are caused by mutations in molecules expressed at the neuromuscular junction. We report clinical, structural, ultrastructural, and electrophysiologic features of 4 CMS patients with 6 heteroallelic variants in AGRN, encoding agrin. One was a 7.9-kb deletion involving the N-terminal laminin-binding domain. Another, c.4744G>A - at the last nucleotide of exon 26 - caused skipping of exon 26. Four missense mutations (p.S1180L, p.R1509W, p.G1675S, and p.Y1877D) expressed in conditioned media decreased AChR clusters in C2C12 myotubes. The agrin-enhanced phosphorylation of MuSK was markedly attenuated by p.Y1877D in the LG3 domain and moderately attenuated by p.R1509W in the LG1 domain but not by the other 2 mutations. The p.S1180L mutation in the SEA domain facilitated degradation of secreted agrin. The p.G1675S mutation in the LG2 domain attenuated anchoring of agrin to the sarcolemma by compromising its binding to heparin. Anchoring of agrin with p.R1509W in the LG1 domain was similarly attenuated. Mutations of agrin affect AChR clustering by enhancing agrin degradation or by suppressing MuSK phosphorylation and/or by compromising anchoring of agrin to the sarcolemma of the neuromuscular junction.

DOI： 10.1172/jci.insight.132023

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

Transcription-coupled nucleotide excision repair (TC-NER) is initiated by the stalling of elongating RNA polymerase II (RNAPIIo) at DNA lesions. The ubiquitination of RNAPIIo in response to DNA damage is an evolutionarily conserved event, but its function in mammals is unknown. Here, we identified a single DNA damage-induced ubiquitination site in RNAPII at RPB1-K1268, which regulates transcription recovery and DNA damage resistance. Mechanistically, RPB1-K1268 ubiquitination stimulates the association of the core-TFIIH complex with stalled RNAPIIo through a transfer mechanism that also involves UVSSA-K414 ubiquitination. We developed a strand-specific ChIP-seq method, which revealed RPB1-K1268 ubiquitination is important for repair and the resolution of transcriptional bottlenecks at DNA lesions. Finally, RPB1-K1268R knockin mice displayed a short life-span, premature aging, and neurodegeneration. Our results reveal RNAPII ubiquitination provides a two-tier protection mechanism by activating TC-NER and, in parallel, the processing of DNA damage-stalled RNAPIIo, which together prevent prolonged transcription arrest and protect against neurodegeneration.

DOI： 10.1016/j.cell.2020.02.010

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

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

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

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

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

Background: One of the major challenges in microbial studies is detecting associations between microbial communities and a specific disease. A specialized feature of microbiome count data is that intestinal bacterial communities form clusters called as "enterotype", which are characterized by differences in specific bacterial taxa, making it difficult to analyze these data under health and disease conditions. Traditional probabilistic modeling cannot distinguish between the bacterial differences derived from enterotype and those related to a specific disease. Results: We propose a new probabilistic model, named as ENIGMA (Enterotype-like uNIGram mixture model for Microbial Association analysis), which can be used to address these problems. ENIGMA enabled simultaneous estimation of enterotype-like clusters characterized by the abundances of signature bacterial genera and the parameters of environmental effects associated with the disease. Conclusion: In the simulation study, we evaluated the accuracy of parameter estimation. Furthermore, by analyzing the real-world data, we detected the bacteria related to Parkinson's disease. ENIGMA is implemented in R and is available from GitHub (https://github.com/abikoushi/enigma).

DOI： 10.1186/s12864-019-5476-9

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

記述言語：英語   掲載種別：研究論文（学術雑誌）  

DOI： 10.15583/jpchrom.2019.014

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 Biosciences  

DOI： 10.3389/fmolb.2019.00053

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

Background: Establishing the relationship between microbiota and specific diseases is important but requires appropriate statistical methodology. A specialized feature of microbiome count data is the presence of a large number of zeros, which makes it difficult to analyze in case-control studies. Most existing approaches either add a small number called a pseudo-count or use probability models such as the multinomial and Dirichlet-multinomial distributions to explain the excess zero counts, which may produce unnecessary biases and impose a correlation structure taht is unsuitable for microbiome data. Results: The purpose of this article is to develop a new probabilistic model, called BERnoulli and MUltinomial Distribution-based latent Allocation (BERMUDA), to address these problems. BERMUDA enables us to describe the differences in bacteria composition and a certain disease among samples. We also provide a simple and efficient learning procedure for the proposed model using an annealing EM algorithm. Conclusion: We illustrate the performance of the proposed method both through both the simulation and real data analysis. BERMUDA is implemented with R and is available from GitHub ( https://github.com/abikoushi/Bermuda ).

DOI： 10.1186/s12859-018-2530-6

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

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

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  

DOI： 10.1002/mds.27472

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

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

Genes specifically expressed in neurons contain members with extended long introns. Longer genes present a problem with respect to fulfilment of gene length transcription, and evidence suggests that dysregulation of long genes is a mechanism underlying neurodegenerative and psychiatric disorders. Here, we report the discovery that RNA-binding protein Sfpq is a critical factor for maintaining transcriptional elongation of long genes. We demonstrate that Sfpq co-transcriptionally binds to long introns and is required for sustaining long-gene transcription by RNA polymerase II through mediating the interaction of cyclin-dependent kinase 9 with the elongation complex. Phenotypically, Sfpq disruption caused neuronal apoptosis in developing mouse brains. Expression analysis of Sfpq-regulated genes revealed specific downregulation of developmentally essential neuronal genes longer than 100 kb in Sfpq-disrupted brains; those genes are enriched in associations with neurodegenerative and psychiatric diseases. The identified molecular machinery yields directions for targeted investigations of the association between long-gene transcriptopathy and neuronal diseases. It has been a long-standing question how mammalian neuronal cells achieve full gene length transcription of extra-long genes. Takeuchi et al. show that RNA-binding protein Sfpq sustains long-gene transcription through Pol II-CTD activation. Loss of Sfpq caused long-gene transcriptopathy, which could be the cause of neurodegenerative and psychiatric disorders.

DOI： 10.1016/j.celrep.2018.03.141

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

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  

FGF receptor 2 is involved in the formation of the neuromuscular junction (NMJ), but its in vivo ligand remains to be determined. Laser capture microdissection of the mouse spinal motor neurons (SMNs) revealed that Fgf18 mRNA is highly expressed in SMNs in adults. Expression of Fgf18 mRNA was the highest in the spinal cord at embryonic day (E) 15.5, which gradually decreased to postnatal day 7. FGF18 protein was localized at the NMJs of the tibialis anterior muscle at E18.5 and in adults. Fgf18-/- mice at E18.5 showed decreased expressions of the NMJ-specific Chrne and Colq genes in the diaphragm. In Fgf18-/- diaphragms, the synaptophysin-positive areas at the nerve terminals and the acetylcholine receptor (AChR)-positive areas at the motor endplates were both approximately one-third of those in wild-type embryos. Fgf18-/- diaphragms ultrastructurally showed abnormal aggregation of multiple nerve terminals making a gigantic presynapse with sparse synaptic vesicles, and simplified motor endplates. In Fgf18-/- diaphragms, miniature endplate potentials were low in amplitude with markedly reduced frequency. In C2C12 myotubes, FGF18 enhanced AChR clustering, which was blocked by inhibiting FGFRs or MEK1. We propose that FGF18 plays a pivotal role in AChR clustering and NMJ formation in mouse embryogenesis.

DOI： 10.1038/s41598-017-18753-5

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

In schizophrenia (SCZ) and autism spectrum disorder (ASD), the dysregulation of glutamate transmission through N-methyl-d-aspartate receptors (NMDARs) has been implicated as a potential etiological mechanism. Previous studies have accumulated evidence supporting NMDAR-encoding genes' role in etiology of SCZ and ASD. We performed a screening study for exonic regions of GRIN1, GRIN2A, GRIN2C, GRIN2D, GRIN3A, and GRIN3B, which encode NMDAR subunits, in 562 participates (370 SCZ and 192 ASD). Forty rare variants were identified including 38 missense, 1 frameshift mutation in GRIN2C and 1 splice site mutation in GRIN2D. We conducted in silico analysis for all variants and detected seven missense variants with deleterious prediction. De novo analysis was conducted if pedigree samples were available. The splice site mutation in GRIN2D is predicted to result in intron retention by minigene assay. Furthermore, the frameshift mutation in GRIN2C and splice site mutation in GRIN2D were genotyped in an independent sample set comprising 1877 SCZ cases, 382 ASD cases, and 2040 controls. Both of them were revealed to be singleton. Our study gives evidence in support of the view that ultra-rare variants with loss of function (frameshift, nonsense or splice site) in NMDARs genes may contribute to possible risk of SCZ.

DOI： 10.1038/s41398-017-0061-y

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

Objective: Reversible myelin vacuolization is associated with variable conditions including mild encephalitis/encephalopathy with a reversible splenial lesion (MERS), which is characterized by mildly impaired consciousness and transient splenial lesion. Familial and/or recurrent cases with a clinical diagnosis of MERS suggest the presence of genetic factors. Methods: We examined a family in which the proband presented with a history of recurrent encephalopathy with extensive but reversible cerebral myelin vacuolization and neurological symptoms similar to those of MERS spanning 3 generations. Whole-exome sequencing was performed in family members. Results: Eight rare nonsynonymous single-nucleotide variants shared by all patients were identified. By filtering genes expressed in the corpus callosum, we identified a heterozygous c.1208A>G predicting p.Gln403Arg in the highly conserved DNA-binding domain in the myelin regulatory factor (MYRF) gene. We subsequently screened the coding regions of MYRF by Sanger sequencing in our cohort comprised of 33 sporadic cases with MERS and 3 cases in another family with extensive myelin vacuolization, and identified the same heterozygous c.1208A>G in all affected members in the second family. Luciferase assay revealed that transcriptional activity of the N-terminal region of MYRF was significantly diminished by introducing the c.1208A>G variant. Interpretation: MYRF is a transcriptional regulator that is necessary for oligodendrocyte differentiation and myelin maintenance. Functional defects of MYRF are likely to be causally associated with encephalopathy with extensive myelin vacuolization. We propose the term “MYRF-related mild encephalopathy with reversible myelin vacuolization.” Our findings provide a new perspective on the pathogenesis of myelin vacuolization. Ann Neurol 2018;83:98–106.

DOI： 10.1002/ana.25125

記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：Wiley Interdisciplinary Reviews: RNA  

Development of next generation sequencing technologies has enabled detection of extensive arrays of germline and somatic single nucleotide variations (SNVs) in human diseases. SNVs affecting intronic GT-AG dinucleotides invariably compromise pre-mRNA splicing. Most exonic SNVs introduce missense/nonsense codons, but some affect auxiliary splicing cis-elements or generate cryptic GT-AG dinucleotides. Similarly, most intronic SNVs are silent, but some affect canonical and auxiliary splicing cis-elements or generate cryptic GT-AG dinucleotides. However, prediction of the splicing effects of SNVs is challenging. The splicing effects of SNVs generating cryptic AG or disrupting canonical AG can be inferred from the AG-scanning model. Similarly, the splicing effects of SNVs affecting the first nucleotide G of an exon can be inferred from AG-dependence of the 3′ splice site (ss). A variety of tools have been developed for predicting the splicing effects of SNVs affecting the 5′ ss, as well as exonic and intronic splicing enhancers/silencers. In contrast, only two tools, the Human Splicing Finder and the SVM-BP finder, are available for predicting the position of the branch point sequence. Similarly, IntSplice and Splicing based Analysis of Variants (SPANR) are the only tools to predict the splicing effects of intronic SNVs. The rules and tools introduced in this review are mostly based on observations of a limited number of genes, and no rule or tool can ensure 100% accuracy. Experimental validation is always required before any clinically relevant conclusions are drawn. Development of efficient tools to predict aberrant splicing, however, will facilitate our understanding of splicing pathomechanisms in human diseases. WIREs RNA 2018, 9:e1451. doi: 10.1002/wrna.1451. This article is categorized under: RNA Processing > Splicing Regulation/Alternative Splicing RNA in Disease and Development > RNA in Disease RNA Methods > RNA Analyses In Vitro and In Silico.

DOI： 10.1002/wrna.1451

記述言語：英語   掲載種別：研究論文（学術雑誌）  

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

記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：Applied Physiology, Nutrition and Metabolism  

The impact of gene-environment interaction on diabetes remains largely unknown. We aimed to investigate if interaction between glucose metabolizing genes and lifestyle factors is associated with type 2 diabetes mellitus (T2DM). Interactions between genotypes of 4 glucose metabolizing genes (MTNR1B, KCNQ1, KLF14, and GCKR) and lifestyle factors were estimated in 722 T2DM patients and 759 controls, using multiple logistic regression. No significant associations with T2DM were detected for the single nucleotide polymorphisms of MTNR1B, KLF14 and GCKR. However, rs151290 (KCNQ1) polymorphisms were found to be associated with risk of T2DM. Compared with AA, the odds ratios (ORs) of AC or CC genotypes for developing T2DM were 1.545 (P = 0.0489) and 1.603 (P = 0.0383), respectively. In stratified analyses, the associations were stronger in smokers with CC than smokers with AA (OR = 3.668, P = 0.013); drinkers with AC (OR = 5.518, P = 0.036), CC (OR = 8.691, P = 0.0095), and AC+CC (OR = 6.764, P = 0.016) than drinkers with AA. Compared with nondrinkers with AA, drinkers who carry AC and CC had 12.072-fold (P = 0.0007) and 8.147-fold (P = 0.0052) higher risk of developing T2DM. In conclusions, rs151290 (KCNQ1) polymorphisms are associated with increased risk of T2DM, alone and especially in interaction with smoking and alcohol.

DOI： 10.1139/apnm-2017-0232

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

Achondroplasia (ACH) is one of the most common short-limbed skeletal dysplasias caused by gain-of-function mutations in the fibroblast growth factor receptors 3 (FGFR3) gene. Distraction osteogenesis (DO) is a treatment option for short stature in ACH in some countries. Although the patients with ACH usually show faster healing in DO, details of the newly formed bone have not been examined. We have developed a mouse model of DO and analyzed new bone regenerates of the transgenic mice with ACH (Fgfr3ach mice) histologically and morphologically. We established two kinds of DO protocols, the short-DO consisted of 5 days of latency period followed by 5 days of distraction with a rate of 0.4 mm per 24 h, and the long-DO consisted of the same latency period followed by 7 days of distraction with a rate of 0.3 mm per 12 h. The callus formation was evaluated radiologically by bone fill score and quantified by micro-CT scan in both protocols. The histomorphometric analysis was performed in the short-DO protocol by various stainings, including Villanueva Goldner, Safranin-O/Fast green, tartrate-resistant acid phosphatase, and type X collagen. Bone fill scores were significantly higher in Fgfr3ach mice than in wild-type mice in both protocols. The individual bone parameters, including bone volume and bone volume/tissue volume, were also significantly higher in Fgfr3ach mice than in wild-type mice in both protocols. The numbers of osteoblasts, as well as osteoclasts, around the trabecular bone were increased in Fgfr3ach mice. Cartilaginous tissues of the distraction region rapidly disappeared in Fgfr3ach mice compared to wild-type mice during the consolidation phase. Similarly, type X collagen-positive cells were markedly decreased in Fgfr3ach mice during the same period. Fgfr3ach mice exhibited accelerated bone regeneration after DO. Accelerated endochondral ossification could contribute to faster healing in Fgfr3ach mice.

DOI： 10.1016/j.bone.2017.05.016

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

Fatty degeneration of skeletal muscle leads to muscle weakness and loss of function. Preventing fatty degeneration in skeletal muscle is important, but no drug has been used clinically. In this study, we performed drug repositioning using human platelet-derived growth factor receptor α (PDGFRα)-positive mesenchymal progenitors that have been proved to be an origin of ectopic adipocytes in skeletal muscle. We found that promethazine hydrochloride (PH) inhibits adipogenesis in a dose-dependent manner without cell toxicity. PH inhibited expression of adipogenic markers and also suppressed phosphorylation of cAMP response-element binding protein, which was reported to be a primary regulator of adipogenesis. We established a mouse model of tendon rupture with intramuscular fat deposition and confirmed that emerged ectopic adipocytes are derived from PDGFRα+ cells using lineage tracing mice. When these injured mice were treated with PH, formation of ectopic adipocytes was suppressed significantly. Our results show that PH inhibits PDGFRα+ mesenchymal progenitor-dependent ectopic adipogenesis in skeletal muscle and suggest that treatment with PH can be a promising approach to prevent fatty degeneration of skeletal muscle.

DOI： 10.1016/j.ajpath.2017.08.008

記述言語：英語   掲載種別：研究論文（学術雑誌）  

DOI： 10.1016/j.jns.2017.08.2574

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

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  

GU-AG consensus sequences are used for intron recognition in the majority of cases of pre-mRNA splicing in eukaryotes. Mutations at splice junctions often cause exon skipping, short deletions, or insertions in the mature mRNA, underlying one common molecular mechanism of genetic diseases. Using N-ethyl-N-nitrosourea, a novel recessive mutation named seal was produced, associated with fragile bones and susceptibility to fractures (spine and limbs). A single nucleotide transversion (T → A) at the second position of intron 36 of the Col1a1 gene, encoding the type I collagen, α1 chain, was responsible for the phenotype. Col1a1 seal mRNA expression occurred at greatly reduced levels compared to the wild-type transcript, resulting in reduced and aberrant collagen fibers in tibiae of seal homozygous mice. Unexpectedly, splicing of Col1a1 seal mRNA followed the normal pattern despite the presence of the donor splice site mutation, likely due to the action of a putative intronic splicing enhancer present in intron 25, which appeared to function redundantly with the splice donor site of intron 36. Seal mice represent a model of human osteogenesis imperfecta, and reveal a previously unknown mechanism for splicing "rescue.".

DOI： 10.1038/s41598-017-10343-9

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

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  

Achondroplasia (ACH) is the most common short-limbed skeletal dysplasia caused by gain-of-function mutations in the fibroblast growth factor receptor 3 (FGFR3). No effective FGFR3-targeted therapies for ACH are currently available. By drug repositioning strategies, we identified that meclozine, which has been used as an anti-motion-sickness, suppressed FGFR3 signaling in chondrocytes and rescued short-limbed phenotype in ACH mouse model. Here, we conducted various pharmacological tests for future clinical application in ACH. Pharmacokinetic analyses demonstrated that peak drug concentration (Cmax) and area under the concentration-time curve (AUC) of 2 mg/kg of meclozine to mice was lower than that of 25 mg/body to human, which is a clinical usage for anti-motion-sickness. Pharmacokinetic simulation studies showed that repeated dose of 2 mg/kg of meclozine showed no accumulation effects. Short stature phenotype in the transgenic mice was significantly rescued by twice-daily oral administration of 2 mg/kg/day of meclozine. In addition to stimulation of longitudinal bone growth, bone volume and metaphyseal trabecular bone quality were improved by meclozine treatment. We confirmed a preclinical proof of concept for applying meclozine for the treatment of short stature in ACH, although toxicity and adverse events associated with long-term administration of this drug should be examined.

DOI： 10.1038/s41598-017-07044-8

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 Neurochemistry  

We humans have evolved by acquiring diversity of alternative RNA metabolisms including alternative means of splicing and transcribing non-coding genes, and not by acquiring new coding genes. Tissue-specific and developmental stage-specific alternative RNA splicing is achieved by tightly regulated spatiotemporal regulation of expressions and activations of RNA-binding proteins that recognize their cognate splicing cis-elements on nascent RNA transcripts. Genes expressed at the neuromuscular junction are also alternatively spliced. In addition, germline mutations provoke aberrant splicing by compromising binding of RNA-binding proteins, and cause congenital myasthenic syndromes (CMS). We present physiological splicing mechanisms of genes for agrin (AGRN), acetylcholinesterase (ACHE), MuSK (MUSK), acetylcholine receptor (AChR) α1 subunit (CHRNA1), and collagen Q (COLQ) in human, and their aberration in diseases. Splicing isoforms of AChET, AChEH, and AChER are generated by hnRNP H/F. Skipping of MUSK exon 10 makes a Wnt-insensitive MuSK isoform, which is unique to human. Skipping of exon 10 is achieved by coordinated binding of hnRNP C, YB-1, and hnRNP L to exon 10. Exon P3A of CHRNA1 is alternatively included to generate a non-functional AChR α1 subunit in human. Molecular dissection of splicing mutations in patients with CMS reveals that exon P3A is alternatively skipped by hnRNP H, polypyrimidine tract-binding protein 1, and hnRNP L. Similarly, analysis of an exonic mutation in COLQ exon 16 in a CMS patient discloses that constitutive splicing of exon 16 requires binding of serine arginine-rich splicing factor 1. Intronic and exonic splicing mutations in CMS enable us to dissect molecular mechanisms underlying alternative and constitutive splicing of genes expressed at the neuromuscular junction. This is an article for the special issue XVth International Symposium on Cholinergic Mechanisms. (Figure presented.).

DOI： 10.1111/jnc.13954

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

FUS, an RNA-binding protein (RBP), is mutated or abnormally regulated in neurodegenerative disorders. FUS regulates various aspects of RNA metabolisms. FUS-binding sites are rich in GU contents and are highly degenerative. FUS-binding motifs of GGU, GGUG, GUGGU and CGCGC have been previously reported. These motifs, however, are applicable to a small fraction of FUS-binding sites. As CLIP-seq tags are enriched in genes that are highly expressed, we normalized CLIP-seq tags by Nascent-seq tags or RNA-seq tags of mouse N2a cells. Nascent-seq identifies nascent transcripts before being processed for splicing and polyadenylation. We extracted frequently observed 4-nt motifs from Nascent-seq-normalized CLIP regions, RNA-seq-normalized CLIP regions, and native CLIP regions. Specific GU-rich motifs were best detected in Nascent-seq-normalized CLIP regions. Analysis of structural motifs using Nascent-seq-normalized CLIP regions also predicted GU-rich sequence forming a stem structure. Sensitivity and specificity were calculated by examining whether the extracted motifs were present at the cross-linking-induced mutation sites (CIMS), where FUS was directly bound. We found that a combination of six motifs (UGUG, CUGG, UGGU, GCUG, GUGG, and UUGG), which were extracted from Nascent-seq-normalized CLIP-regions, had a better discriminative power than (i) motifs extracted from RNA-seq-normalized CLIP regions, (ii) motifs extracted from native CLIP regions, (iii) previously reported individual motifs, or (iv) 15 motifs in SpliceAid 2. Validation of the 6 GU-rich (6GUR) motifs using CLIP-seq of the cerebrum and the whole brain showed that the 6GUR motifs were specifically enriched in CIMS. The number of the 6GUR motifs in an uninterrupted region was counted and multiplied by four to calculate the area, which was defined as the 6GUR-Score. The 6GUR-Score of 8 or more best discriminated CIMS from CIMS-flanking regions. We propose that the 6GUR motifs predict FUS-binding sites more efficiently than previously reported individual motifs or 15 motifs in SpliceAid 2.

DOI： 10.1016/j.gene.2017.04.008

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

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 Neurosurgery: Pediatrics  

OBJECTIVE: Achondroplasia (ACH) is the most common short-limbed skeletal dysplasia caused by gain-of-function mutations in the fibroblast growth factor receptor 3 (FGFR3) gene. Foramen magnum stenosis (FMS) is one of the serious neurological complications in ACH. Through comprehensive drug screening, the authors identified that meclozine, an over-the-counter drug for motion sickness, inhibited activation of FGFR3 signaling. Oral administration of meclozine to the growing ACH mice promoted longitudinal bone growth, but it did not prevent FMS. In the current study, the authors evaluated the effects of maternal administration of meclozine on FMS in ACH mice. METHODS: The area of the foramen magnum was measured in 17-day-old Fgfr3ach mice and wild-type mice using micro-CT scanning. Meclozine was administered to the pregnant mice carrying Fgfr3ach offspring from embryonic Day (ED) 14.5 to postnatal Day (PD) 4.5. Spheno-occipital and anterior intraoccipital synchondroses were histologically examined, and the bony bridges were scored on PD 4.5. In wild-type mice, tissue concentrations of meclozine in ED 17.5 fetuses and PD 6.5 pups were investigated. RESULTS: The area of the foramen magnum was significantly smaller in 17-day-old Fgfr3ach mice than in wild-type mice (p < 0.005). There were no bony bridges in the spheno-occipital and anterior intraoccipital synchondroses in wild-type mice, while some of the synchondroses prematurely closed in untreated Fgfr3ach mice at PD 4.5. The average bony bridge score in the cranial base was 7.053 ± 1.393 in untreated Fgfr3ach mice and 6.125 ± 2.029 in meclozine-treated Fgfr3ach mice. The scores were not statistically significant between mice with and those without meclozine treatment (p = 0.12). The average tissue concentration of meclozine was significantly higher (508.88 ± 205.16 ng/g) in PD 6.5 mice than in ED 17.5 mice (56.91 ± 20.05 ng/g) (p < 0.005). CONCLUSIONS: Maternal administration of meclozine postponed premature closure of synchondroses in some Fgfr3ach mice, but the effect on preventing bony bridge formation was not significant, probably due to low placental transmission of the drug. Meclozine is likely to exhibit a marginal effect on premature closure of synchondroses at the cranial base in ACH.

DOI： 10.3171/2016.7.PEDS16199

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

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

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

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  

Background: We previously reported gut dysbiosis in patients with Parkinson’s disease (PD). Objective: The aim of this study is to examine whether gut dysbiosis correlates with the progression of PD. Methods: We examined changes in gut microbiota and demographic features in 2 years in 36 PD patients. Results: A change of total UPDRS scores in 2 years was predicted by the counts of Bifidobacterium and Atopobium cluster at year 0 with a correlation coefficient of 0.52. Correlation analysis additionally revealed that low counts of Bifidobacterium and Bacteroides fragilis at year 0 were associated with worsening of UPDRS I scores in 2 years. In addition, low counts of Bifidobacterium at year 0 were associated with worsening of hallucinations/delusions in 2 years. Similarly, low counts of B. fragilis at year 0 were associated with worsening of motivation/initiative in 2 years. The patients were evenly divided into the deteriorated and stable groups based on the degree of worsening of total UPDRS scores. The deteriorated group had lower counts of Bifidobacterium, B. fragilis, and Clostridium leptium than the stable group at year 0 but not at year 2, suggesting that the deteriorated group may demonstrate accelerated lowering of these bacteria at year 0. Conclusions: The total counts of intestinal bacterial decrease in the course of PD progression. Temporal profiles of lowering of bacterial counts are likely to be different from bacteria to bacteria, and also between the deteriorating and stable groups, which may be able to be exploited to differentiate patients with rapidly and slowly progressive PD pathology.

DOI： 10.1371/journal.pone.0187307

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 Biology and Medicine  

Oxidative stress plays an important role in the pathogenesis of preeclampsia. Recently, molecular hydrogen (H2) has been shown to have therapeutic potential in various oxidative stress-related diseases. The aim of this study is to investigate the effect of H2 on preeclampsia. We used the reduced utero-placental perfusion pressure (RUPP) rat model, which has been widely used as a model of preeclampsia. H2 water (HW) was administered orally ad libitum in RUPP rats from gestational day (GD) 12–19, starting 2 days before RUPP procedure. On GD19, mean arterial pressure (MAP) was measured, and samples were collected. Maternal administration of HW significantly decreased MAP, and increased fetal and placental weight in RUPP rats. The increased levels of soluble fms-like tyrosine kinase-1 (sFlt-1) and diacron reactive oxygen metabolites as a biomarker of reactive oxygen species in maternal blood were decreased by HW administration. However, vascular endothelial growth factor level in maternal blood was increased by HW administration. Proteinuria, and histological findings in kidney were improved by HW administration. In addition, the effects of H2 on placental villi were examined by using a trophoblast cell line (BeWo) and villous explants from the placental tissue of women with or without preeclampsia. H2 significantly attenuated hydrogen peroxide-induced sFlt-1 expression, but could not reduce the expression induced by hypoxia in BeWo cells. H2 significantly attenuated sFlt-1 expression in villous explants from women with preeclampsia, but not affected them from normotensive pregnancy. The prophylactic administration of H2 attenuated placental ischemia-induced hypertension, angiogenic imbalance, and oxidative stress. These results support the theory that H2 has a potential benefit in the prevention of preeclampsia.

DOI： 10.1016/j.freeradbiomed.2016.10.491

記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：Chemico-Biological Interactions  

The low-density lipoprotein receptor-related protein 4 (LRP4) and the muscle-specific receptor tyrosine kinase (MuSK) form a tetrameric protein complex on the postsynaptic membrane at the neuromuscular junction (NMJ). Binding of agrin to LRP4 triggers phosphorylation of MuSK. Activated MuSK drives clustering of acetylcholine receptor (AChR). Wnt ligands also directly bind to MuSK to induce AChR clustering. MuSK anchors the acetylcholinesterase (AChE)/collagen Q (ColQ) complex to the synaptic basal lamina. In addition, an extracellular proteoglycan, biglycan, binds to MuSK. Anti-MuSK autoantibodies (MuSK-IgG) are observed in 5–15% of autoimmune myasthenia gravis (MG) patients. MuSK-IgG blocks both ColQ-MuSK and LRP4-MuSK interactions. MuSK-IgG, LRP4, ColQ, and biglycan bind to the immunoglobulin-like domains 1 and 4 of MuSK. Lack of the effects of cholinesterase inhibitors in MuSK-MG patients is likely due to hindrance of ColQ-MuSK interaction by MuSK-IgG and subsequent deficiency of AChE observed in model mice, which, however, has not been proven in MuSK-MG patients. As ColQ enhances expression of membrane-bound MuSK, inhibition of ColQ-MuSK interaction by MuSK-IgG may account for lack of AChR clusters in MuSK-MG. We thus made passive transfer models using Colq+/+ and Colq−/− mice to dissect the effect of ColQ on AChR clustering in MuSK-MG. We found that MuSK-IgG-mediated suppression of LRP4-MuSK interaction, not of ColQ-MuSK interaction, caused defective AChR clustering. We also unexpectedly observed that both MuSK-IgG and ColQ suppressed agrin/LRP4/MuSK signaling in dose-dependent manners. Quantitative comparison revealed that MuSK-IgG blocked agrin-LRP4-MuSK signaling more than ColQ. We propose that attenuation of AChR clustering in MuSK-MG is due to hindrance of LRP4-MuSK interaction in the presence of agrin by MuSK-IgG.

DOI： 10.1016/j.cbi.2016.04.019

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 Mutation  

We identify two novel mutations in acetylcholine receptor (AChR) causing a slow-channel congenital myasthenia syndrome (CMS) in three unrelated patients (Pts). Pt 1 harbors a heterozygous βV266A mutation (p.Val289Ala) in the second transmembrane domain (M2) of the AChR β subunit (CHRNB1). Pts 2 and 3 carry the same mutation at an equivalent site in the ε subunit (CHRNE), εV265A (p.Val285Ala). The mutant residues are conserved across all AChR subunits of all species and are components of a valine ring in the channel pore, which is positioned four residues above the leucine ring. Both βV266A and εV265A reduce the amino acid size and lengthen the channel opening bursts by fourfold by enhancing gating efficiency by approximately 30-fold. Substitution of alanine for valine at the corresponding position in the δ and α subunit prolongs the burst duration four- and eightfold, respectively. Replacing valine at ε codon 265 either by a still smaller glycine or by a larger leucine also lengthens the burst duration. Our analysis reveals that each valine in the valine ring contributes to channel kinetics equally, and the valine ring has been optimized in the course of evolution to govern channel gating.

DOI： 10.1002/humu.23043

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  

Molecular hydrogen (H2) is effective for many diseases. However, molecular bases of H2 have not been fully elucidated. Cumulative evidence indicates that H2 acts as a gaseous signal modulator. We found that H2 suppresses activated Wnt/β-catenin signaling by promoting phosphorylation and degradation of β-catenin. Either complete inhibition of GSK3 or mutations at CK1- and GSK3-phosphorylation sites of β-catenin abolished the suppressive effect of H2. H2 did not increase GSK3-mediated phosphorylation of glycogen synthase, indicating that H2 has no direct effect on GSK3 itself. Knock-down of adenomatous polyposis coli (APC) or Axin1, which form the β-catenin degradation complex, minimized the suppressive effect of H2 on β-catenin accumulation. Accordingly, the effect of H2 requires CK1/GSK3-phosphorylation sites of β-catenin, as well as the β-catenin degradation complex comprised of CK1, GSK3, APC, and Axin1. We additionally found that H2 reduces the activation of Wnt/β-catenin signaling in human osteoarthritis chondrocytes. Oral intake of H2 water tended to ameliorate cartilage degradation in a surgery-induced rat osteoarthritis model through attenuating β-catenin accumulation. We first demonstrate that H2 suppresses abnormally activated Wnt/β-catenin signaling, which accounts for the protective roles of H2 in a fraction of diseases.

DOI： 10.1038/srep31986

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：Clinical and Experimental Neuroimmunology  

Congenital myasthenic syndromes (CMS) are heterogeneous disorders caused by germline mutations in genes expressed at the neuromuscular junction. Mutations have been identified in 24 genes encoding acetylcholine receptor subunits (CHRNA1, CHRNB1, CHRND, CHRNE and CHRNG), skeletal muscle sodium channel (SCN4A), signaling molecules driving acetylcholine receptor subunits clustering (AGRN, LRP4, MUSK and DOK7), synaptic structural proteins (COLQ, LAMB2 and COL13A1), postsynaptic structural proteins (RAPSN and PLEC), presynaptic molecules (CHAT, SYT2), glycosylation enzymes (GFPT1, DPAGT1, ALG2, ALG14 and GMPPB), and other less characterized molecules (PREPL and SCL25A1). CMS are recessive disorders, except for slow channel CMS and synaptotagmin 2 (SYT2)-CMS. Onsets are largely less than 2 years, but adult-onset is not rare, especially in slow-channel CMS and limb-girdle type CMS caused by glycosylation defects and by DOK7 mutations. Clinical features include fatigable muscle weakness, amyotrophy and minor facial anomalies. Eye, facial and bulbar muscles are frequently affected, but sparing of these muscles is observed, especially in limb-girdle type CMS. Serum creatine kinase levels are frequently elevated in slow-channel CMS, glutamine-fructose-6-phosphate aminotransferase 1 (GFPT1)-CMS, and guanosine diphosphate mannose pyrophosphorylase B (GMPPB)-CMS. Electrophysiological findings supporting compromised neuromuscular signal transmission are a prerequisite for diagnosing CMS. Most CMS patients are likely to be underdiagnosed, and recognition of CMS in undiagnosed muscle weakness and/or amyotrophy is critical for diagnosing CMS.

DOI： 10.1111/cen3.12316

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 Neurology, Neurosurgery and Psychiatry  

DOI： 10.1136/jnnp-2016-313416

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

Background: Many pathogenic genetic variants have been shown to disrupt mRNA splicing. Besides splice mutations in the well-conserved splice sites, mutations in splicing regulatory elements (SREs) may deregulate splicing and cause disease. A promising therapeutic approach is to compensate for this deregulation by blocking other SREs with splice-switching oligonucleotides (SSOs). However, the location and sequence of most SREs are not well known. Results: Here, we used individual-nucleotide resolution crosslinking immunoprecipitation (iCLIP) to establish an in vivo binding map for the key splicing regulatory factor hnRNP A1 and to generate an hnRNP A1 consensus binding motif. We find that hnRNP A1 binding in proximal introns may be important for repressing exons. We show that inclusion of the alternative cassette exon 3 in SKA2 can be significantly increased by SSO-based treatment which blocks an iCLIP-identified hnRNP A1 binding site immediately downstream of the 5' splice site. Because pseudoexons are well suited as models for constitutive exons which have been inactivated by pathogenic mutations in SREs, we used a pseudoexon in MTRR as a model and showed that an iCLIP-identified hnRNP A1 binding site downstream of the 5' splice site can be blocked by SSOs to activate the exon. Conclusions: The hnRNP A1 binding map can be used to identify potential targets for SSO-based therapy. Moreover, together with the hnRNP A1 consensus binding motif, the binding map may be used to predict whether disease-associated mutations and SNPs affect hnRNP A1 binding and eventually mRNA splicing.

DOI： 10.1186/s12915-016-0279-9

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 Human Genetics  

Precise spatiotemporal regulation of splicing is mediated by splicing cis-elements on pre-mRNA. Single-nucleotide variations (SNVs) affecting intronic cis-elements possibly compromise splicing, but no efficient tool has been available to identify them. Following an effect-size analysis of each intronic nucleotide on annotated alternative splicing, we extracted 105 parameters that could affect the strength of the splicing signals. However, we could not generate reliable support vector regression models to predict the percent-splice-in (PSI) scores for normal human tissues. Next, we generated support vector machine (SVM) models using 110 parameters to directly differentiate pathogenic SNVs in the Human Gene Mutation Database and normal SNVs in the dbSNP database, and we obtained models with a sensitivity of 0.800±0.041 (mean and s.d.) and a specificity of 0.849±0.021. Our IntSplice models were more discriminating than SVM models that we generated with Shapiro-Senapathy score and MaxEntScan::score3ss. We applied IntSplice to a naturally occurring and nine artificial intronic mutations in RAPSN causing congenital myasthenic syndrome. IntSplice correctly predicted the splicing consequences for nine of the ten mutants. We created a web service program, IntSplice (http://www.med.Nagoya-u.ac.jp/neurogenetics/IntSplice) to predict splicing-affecting SNVs at intronic positions from -50 to -3.

DOI： 10.1038/jhg.2016.23

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  

At the neuromuscular junction (NMJ), acetylcholine receptor (AChR) clustering is mediated by spinal motor neuron (SMN)-derived agrin and its receptors on the muscle, the low-density lipoprotein receptor-related protein 4 (LRP4) and muscle-specific receptor tyrosine kinase (MuSK). Additionally, AChR clustering is mediated by the components of the Wnt pathway. Laser capture microdissection of SMNs revealed that a secreted activator of Wnt signaling, R-spondin 2 (Rspo2), is highly expressed in SMNs. We found that Rspo2 is enriched at the NMJ, and that Rspo2 induces MuSK phosphorylation and AChR clustering. Rspo2 requires Wnt ligands, but not agrin, for promoting AChR clustering in cultured myotubes. Leucine-rich repeat-containing G-protein coupled receptor 5 (Lgr5), an Rspo2 receptor, is also accumulated at the NMJ, and is associated with MuSK via LRP4. Lgr5 is required for Rspo2-mediated AChR clustering in myotubes. In Rspo2-knockout mice, the number and density of AChRs at the NMJ are reduced. The Rspo2-knockout diaphragm has an altered ultrastructure with widened synaptic clefts and sparse synaptic vesicles. Frequency of miniature endplate currents is markedly reduced in Rspo2-knockout mice. To conclude, we demonstrate that Rspo2 and its receptor Lgr5 are Wnt-dependent and agrin-independent regulators of AChR clustering at the NMJ.

DOI： 10.1038/srep28512

記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：Wiley Interdisciplinary Reviews: RNA  

Fused in sarcoma (FUS) is an RNA-binding protein that is causally associated with oncogenesis and neurodegeneration. Recently, the role of FUS in neurodegeneration has been extensively studied, because mutations in FUS are associated with amyotrophic lateral sclerosis (ALS), and the FUS protein has been identified as a major component of intracellular inclusions in neurodegenerative disorders including ALS and frontotemporal lobar degeneration. FUS is a key molecule in transcriptional regulation and RNA processing including processes such as pre-messenger RNA (mRNA) splicing and polyadenylation. Interaction of FUS with various components of the transcription machinery, spliceosome, and the 3′-end processing machinery has been identified. Furthermore, recent advances in high-throughput transcriptomic profiling approaches have enabled us to determine the mechanisms of FUS-dependent RNA processing networks at a cellular level. These analyses have revealed that depletion of FUS in neuronal cells affects alternative splicing and alternative polyadenylation of thousands of mRNAs. Gene ontology analysis has suggested that FUS-modulated genes are implicated in neuronal functions and development. CLIP-seq of FUS has shown that FUS is frequently clustered around these alternative sites of nascent RNA. ChIP-seq of RNA polymerase II (RNAP II) has demonstrated that an interaction between FUS and nascent RNA downregulates local transcriptional activity of RNAP II, which is critically involved in RNA processing. Both alternative splicing and alternative polyadenylation are fundamental processes by which cells expand their transcriptomic diversity, and are particularly essential in the nervous system. Dependence of transcriptomic diversity on FUS makes the nervous system vulnerable to neurodegeneration, when FUS is functionally compromised.

DOI： 10.1002/wrna.1338

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  

Myotonic dystrophy type 1 (DM1) is caused by abnormal expansion of CTG repeats in the 3′ untranslated region of the DMPK gene. Expanded CTG repeats are transcribed into RNA and make an aggregate with a splicing regulator, MBNL1, in the nucleus, which is called the nuclear foci. The nuclear foci sequestrates and downregulates availability of MBNL1. Symptomatic treatments are available for DM1, but no rational therapy is available. In this study, we found that a nonsteroidal anti-inflammatory drug (NSAID), phenylbutazone (PBZ), upregulated the expression of MBNL1 in C2C12 myoblasts as well as in the HSA LR mouse model for DM1. In the DM1 mice model, PBZ ameliorated aberrant splicing of Clcn1, Nfix, and Rpn2. PBZ increased expression of skeletal muscle chloride channel, decreased abnormal central nuclei of muscle fibers, and improved wheel-running activity in HSA LR mice. We found that the effect of PBZ was conferred by two distinct mechanisms. First, PBZ suppressed methylation of an enhancer region in Mbnl1 intron 1, and enhanced transcription of Mbnl1 mRNA. Second, PBZ attenuated binding of MBNL1 to abnormally expanded CUG repeats in cellulo and in vitro. Our studies suggest that PBZ is a potent therapeutic agent for DM1 that upregulates availability of MBNL1.

DOI： 10.1038/srep25317

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  

Endochondral ossification is a crucial process for longitudinal growth of bones. Differentiating chondrocytes in growth cartilage form four sequential zones of proliferation, alignment into column, hypertrophy, and substitution of chondrocytes with osteoblasts. Wnt/β-catenin signaling is essential for differentiation of proliferating chondrocytes into hypertrophic chondrocytes in growth cartilage. R-spondin 2 (Rspo2), a member of R-spondin family, is an agonist for Wnt signaling, but its role in chondrocyte differentiation remains unknown. Here we report that growth cartilage of Rspo2-knockout mice shows a decreased amount of β-catenin and increased amounts collagen type II (CII) and Sox9 in the abnormally extended proliferating zone. In contrast, expression of collagen type X (CX) in the hypertrophic zone remains unchanged. Differentiating chondrogenic ATDC5 cells, mimicking proliferating chondrocytes, upregulate Rspo2 and its putative receptor, Lgr5, in parallel. Addition of recombinant human Rspo2 to differentiating ATDC5 cells decreases expressions of Col2a1, Sox9, and Acan, as well as production of proteoglycans. In contrast, lentivirus-mediated knockdown of Rspo2 has the opposite effect. The effect of Rspo2 on chondrogenic differentiation is mediated by Wnt/β-catenin signaling, and not by Wnt/PCP or Wnt/Ca2+ signaling. We propose that Rspo2 activates Wnt/β-catenin signaling to reduce Col2a1 and Sox9 and to facilitate differentiation of proliferating chondrocytes into hypertrophic chondrocytes in growth cartilage.

DOI： 10.1016/j.bbrc.2016.03.089

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 Parkinson's Disease  

Background: Noninvasive biomarkers for Parkinson's disease (PD) are currently unavailable. Objective: To search for a biomarker unique to PD in sweat and serum. Methods: Sweat samples in 42 PD patients and 16 controls were analyzed using liquid chromatography/mass spectrometry (LC/MS). The principal component analysis (PCA) and the orthogonal projections to latent structures (OPLS) analysis were employed. Serum Phe and Tyr levels were determined using the HPLC-fluorescence detection system in 28 de novo PD patients, 52 L-Dopa-treated PD patients, and 27 controls. Results: PCA and OPLS analyses of LC/MS of sweat samples revealed that Tyr, Phe, Leu (Ile), and Asp have high effect sizes to differentiate PD and controls. As Phe and Tyr are precursors of dopamine, we quantified the serum Phe and Tyr levels in de novo and treated PD patients, as well as in controls. Phe was high in de novo patients, but not in treated patients. In contrast, Tyr tended to be low in treated patients, but not in de novo patients. Tyr/Phe ratios were lower in both de novo and treated patients than in controls. The Tyr/Phe ratios were all higher than 0.82 in controls, whereas 49% of the de novo and treated patients had Tyr/Phe ratios less than 0.82. The low Tyr/Phe ratios were associated with male patients and low doses of entacapone. However, Tyr/Phe ratios were not different between male and female patients, and between patients with and without entacapone. Conclusions: The low serum Tyr/Phe ratio differentiates PD from controls with sensitivity = 0.49, specificity = 1.00, positive predictive value = 1.00, and negative predictive value = 0.40.

DOI： 10.3233/JPD-150736

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

DOI： 10.4103/1673-5374.180727

記述言語：英語   掲載種別：研究論文（学術雑誌）  

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  

Genetic variants at KCNQ1 rs151290, KLF14 rs972283, GCKR rs780094 and MTNR1B rs10830963 have been associated with type 2 diabetes mellitus (T2DM), but the results are contradictory in Chinese populations. The aim of the present study was to investigate the association of these four SNPs with T2DM in a large population of Han Chinese at Henan province, China. Seven-hundred-thirty-six patients with T2DM (cases) and Seven-hundred-sixty-eight healthy glucose-tolerant controls were genotyped for KCNQ1 rs151290, KLF14 rs972283, GCKR rs780094 and MTNR1B rs10830963. The association of genetic variants in these four genes with T2DM was analyzed using multivariate logistic regression. Genotypes and allele distributions of KCNQ1 rs151290 were significantly different between the cases and controls (p < 0.05). The AC and CC genotypes and the combined AC + CC genotype of rs151290 in KCNQ1 were associated with increases risk of T2DM before (OR = 1.482, 95% CI = 1.062–2.069; p = 0.021; OR = 1.544, 95% CI = 1.097–2.172, p = 0.013; and OR = 1.509, 95% CI = 1.097–2.077, p = 0.011, respectively) and after (OR = 1.539, 95% CI = 1.015–2.332, p = 0.042; OR = 1.641, 95% CI = 1.070–2.516, p = 0.023; and OR = 1.582, 95% CI = 1.061–2.358, p = 0.024; respectively) adjustment for sex, age, anthropometric measurements, biochemical indexes, smoking and alcohol consumption. Consistent with results of genotype analysis, the C allele of rs151290 in KCNQ1 was also associated with increased risk of T2DM (OR = 1.166, 95% CI = 1.004–1.355, p = 0.045). No associations between genetic variants of KLF14 rs972283, GCKR rs780094 or MTNR1B rs10830963 and T2DM were detected. The AC and CC genotypes and the C allele of rs151290 in KCNQ1 may be risk factors for T2DM in Han Chinese in Henan province.

DOI： 10.3390/ijerph13030260

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  

Endochondral ossification is an essential process for reparative phase of fracture healing, which starts with the differentiation of mesenchymal cells into chondrocytes followed by substitution of bone tissue. It is strictly controlled by the expression of crucial transcriptional factors: SOX9 in the early phase and RUNX2 in the late phase. Screening of FDA-approved compounds revealed that an anti-allergic drug, tranilast, that has been used for more than 30 years in clinical practice, enhanced the SOX9 promoter in chondrogenic cells and the RUNX2 promoter in osteoblastic cells. We observed that tranilast increased mRNA expression of both Sox9 and Runx2 in differentiating ATDC5 chondrogenic progenitor cells. Tranilast upregulated mRNA expression of chondrogenic marker genes (Col2a1, Acan, Col10a1, and Mmp13) in differentiating ATDC5 cells. Moreover, tranilast upregulated mRNA expression of essential signaling molecules involved in endochondral ossification (Pthrp, Ihh, and Axin2). In the later phase of differentiation of ATDC5 cells, tranilast increased synthesis of matrix proteoglycans, induced the alkaline phosphatase activity, and tended to accelerate mineralization. Tranilast is a potential agent that accelerates fracture repair by promoting the regulatory steps of endochondral ossification.

DOI： 10.1016/j.bbrc.2016.01.044

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 Biology and Medicine  

Exposure to inflammation in utero is related to perinatal brain injury, which is itself associated with high rates of long-term morbidity and mortality in children. Novel therapeutic interventions during the perinatal period are required to prevent inflammation, but its pathogenesis is incompletely understood. Activated microglia are known to play a central role in brain injury by producing a variety of pro-inflammatory cytokines and releasing oxidative products. The study is aimed to investigate the preventative potential of molecular hydrogen (H2), which is an antioxidant and anti-inflammatory agent without mutagenicity. Pregnant ICR mice were injected with lipopolysaccharide (LPS) intraperitoneally on embryonic day 17 to create a model of perinatal brain injury caused by prenatal inflammation. In this model, the effect of maternal administration of hydrogen water (HW) on pups was also evaluated. The levels of pro-inflammatory cytokines, oxidative damage and activation of microglia were determined in the fetal brains. H2 reduced the LPS-induced expression of pro-inflammatory cytokines, oxidative damage and microglial activation in the fetal brains. Next, we investigated how H2 contributes to neuroprotection, focusing on microglia, using primary cultured microglia and neurons. H2 prevented LPS- or cytokine-induced generation of reactive oxidative species by microglia and reduced LPS-induced microglial neurotoxicity. Finally, we identified several molecules influenced by H2, involved in the process of activating microglia. These results suggested that H2 holds promise for the prevention of inflammation related to perinatal brain injury.

DOI： 10.1016/j.freeradbiomed.2015.12.015

記述言語：英語   掲載種別：研究論文（学術雑誌）  

DOI： 10.1371/journal.pone.0148470

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

The transcription factor, runt-related transcription factor 2 (Runx2), plays a pivotal role in the differentiation of the mesenchymal stem cells to the osteochondroblast lineages. We found by the drug repositioning strategy that a proton pump inhibitor, lansoprazole, enhances nuclear accumulation of Runx2 and induces osteoblastogenesis of human mesenchymal stromal cells. Systemic administration of lansoprazole to a rat femoral fracture model increased osteoblastogenesis. Dissection of signaling pathways revealed that lansoprazole activates a noncanonical bone morphogenic protein (BMP)-transforming growth factor-beta (TGF-β) activated kinase-1 (TAK1)-p38 mitogen-activated protein kinase (MAPK) pathway. We found by in cellulo ubiquitination studies that lansoprazole enhances polyubiquitination of the TNF receptor-associated factor 6 (TRAF6) and by in vitro ubiquitination studies that the enhanced polyubiquitination of TRAF6 is attributed to the blocking of a deubiquitination enzyme, cylindromatosis (CYLD). Structural modeling and site-directed mutagenesis of CYLD demonstrated that lansoprazole tightly fits in a pocket of CYLD where the C-terminal tail of ubiquitin lies. Lansoprazole is a potential therapeutic agent for enhancing osteoblastic differentiation.

DOI： 10.1016/j.ebiom.2015.11.024

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  

MuSK antibody-positive myasthenia gravis (MuSK-MG) accounts for 5 to 15% of autoimmune MG. MuSK and LRP4 are coreceptors for agrin in the signaling pathway that causes clustering of acetylcholine receptor (AChR). MuSK also anchors the acetylcholinesterase (AChE)/collagen Q (ColQ) complex to the synaptic basal lamina. We previously reported that anti-MuSK antibodies (MuSK-IgG) block binding of ColQ to MuSK and cause partial endplate AChE deficiency in mice. We here analyzed the physiological significance of binding of ColQ to MuSK and block of this binding by MuSK-IgG. In vitro plate-binding assay showed that MuSK-IgG blocked MuSK-LRP4 interaction in the presence of agrin. Passive transfer of MuSK-IgG to Colq-knockout mice attenuated AChR clustering, indicating that lack of ColQ is not the key event causing defective clustering of AChR in MuSK-MG. In three MuSK-MG patients, the MuSK antibodies recognized the first and fourth immunoglobulin-like domains (Ig1 and Ig4) of MuSK. In two other MuSK-MG patients, they recognized only the Ig4 domain. LRP4 and ColQ also bound to the Ig1 and Ig4 domains of MuSK. Unexpectedly, the AChE/ColQ complex blocked MuSK-LRP4 interaction and suppressed agrin/LRP4/MuSK signaling. Quantitative analysis showed that MuSK-IgG suppressed agrin/LRP4/MuSK signaling to a greater extent than ColQ.

DOI： 10.1038/srep13928

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 Thoracic and Cardiovascular Surgery  

Objective The pathogenesis of pulmonary arterial hypertension (PAH) involves reactive oxygen species and inflammation. Beneficial effects of molecular hydrogen, which exerts both anti-inflammatory and antioxidative effects, have been reported for various pathologic conditions. We therefore hypothesized that molecular hydrogen would improve monocrotaline (MCT)-induced PAH in rats. Methods Nineteen male Sprague-Dawley rats (body weight: 200-300 g) were divided into groups, receiving: (1) MCT + hydrogen-saturated water (group H); (2) MCT + dehydrogenized water (group M); or (3) saline + dehydrogenized water (group C). Sixteen days after substance administration, we evaluated hemodynamics, harvested the lungs and heart, and performed morphometric analysis of the pulmonary vasculature. Macrophage infiltration, antiproliferating cell nuclear antigen-positive cells, 8-hydroxy-deoxyguanosine (8-OHdG)-positive cells, and expressions of phosphorylated signal transducers and activators of transcription-3 (STAT3) and nuclear factor of activated T-cells (NFAT) were evaluated immunohistochemically. Stromal cell-derived factor-1 and monocyte chemoattractant protein-1 expressions were evaluated by quantitative reverse-transcription polymerase chain reaction. Results Pulmonary arterial hypertension was significantly exacerbated in group M compared to group C, but was significantly improved in group H. Vascular density was significantly reduced in group M, but not in group H. Adventitial macrophages, antiproliferating cell nuclear antigen - and 8-OHdG-positive cells, and stromal cell-derived factor-1 and monocyte chemoattractant protein-1 expressions were significantly increased in group M, but improved in group H. Expressions of phosphorylated STAT3 and NFAT were up-regulated in group M, but improved in group H. Conclusions Molecular hydrogen ameliorates MCT-induced PAH in rats by suppressing macrophage accumulation, reducing oxidative stress and modulating the STAT3/NFAT axis.

DOI： 10.1016/j.jtcvs.2015.05.052

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  

The catalytic subunits of acetylcholinesterase (AChE) are anchored in the basal lamina of the neuromuscular junction using a collagen-like tail subunit (ColQ) encoded by COLQ. Mutations in COLQ cause endplate AChE deficiency. An A-to-G mutation predicting p.E415G in COLQ exon 16 identified in a patient with endplate AChE deficiency causes exclusive skipping of exon 16. RNA affinity purification, mass spectrometry, and siRNA-mediated gene knocking down disclosed that the mutation disrupts binding of a splicing-enhancing RNA-binding protein, SRSF1, and de novo gains binding of a splicing-suppressing RNA-binding protein, hnRNP H. MS2-mediated artificial tethering of each factor demonstrated that SRSF1 and hnRNP H antagonistically modulate splicing by binding exclusively to the target in exon 16. Further analyses with artificial mutants revealed that SRSF1 is able to bind to degenerative binding motifs, whereas hnRNP H strictly requires an uninterrupted stretch of poly(G). The mutation compromised splicing of the downstream intron. Isolation of early spliceosome complex revealed that the mutation impairs binding of U1-70K (snRNP70) to the downstream 5′ splice site. Global splicing analysis with RNA-seq revealed that exons carrying the hnRNP H-binding GGGGG motif are predisposed to be skipped compared to those carrying the SRSF1-binding GGAGG motif in both human and mouse brains.

DOI： 10.1038/srep13208

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

Schwartz-Jampel syndrome (SJS) type 1 is characterized by short stature, myotonia, and chondrodysplasia, and is caused by partial loss-of-function mutations in HSPG2 encoding perlecan. Six missense mutations have been reported in SJS to date and only one has been characterized using a recombinant protein. We report an 11-year-old Japanese boy with SJS, who shows "rigid" walking with less flexion of knees/ankles and protruded mouth. His intelligence is normal. We identified by whole genome resequencing a heterozygous missense p.Leu1088Pro in domain III-2 and a heterozygous nonsense p.Gln3061Ter in domain IV of perlecan. Expression studies revealed that p.Leu1088Pro markedly reduces the cellular expression of domain III-2 and almost nullifies its secretion into the culture medium. As five of the seven missense mutations in SJS affect domain III of perlecan, domain III is likely to be essential for secretion of perlecan into the extracellular space.

DOI： 10.1016/j.nmd.2015.05.002

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

DOI： 10.1001/jamaneurol.2015.0853

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

Parkinson's disease (PD) is a progressive neurodegenerative disorder caused by the loss of nigrostriatal dopaminergic (DAergic) neurons and the depletion of striatal dopamine. Here we show that DAergic-neuron-like cells could be efficiently induced from stem cells derived from human exfoliated deciduous teeth (SHEDs), and that these induced cells had therapeutic benefits in a 6-OHDA-induced Parkinsonian rat model. In our protocol, EGF and bFGF signaling activated the SHED's expression of proneural genes, Ngn2 and Mash1, and subsequent treatment with brain-derived neurotrophic factor (BDNF) promoted their maturation into DAergic neuron-like SHEDs (dSHEDs). A hypoxic DAergic differentiation protocol improved cell viability and enhanced the expression of multiple neurotrophic factors, including BDNF, GDNF, NT-3, and HGF. Engrafted dSHEDs survived in the striatum of Parkinsonian rats, improved the DA level more efficiently than engrafted undifferentiated SHEDs, and promoted the recovery from neurological deficits. Our findings further suggested that paracrine effects of dSHEDs contributed to neuroprotection against 6-OHDA-induced neurodegeneration and to nigrostriatal tract restoration. In addition, we found that the conditioned medium derived from dSHEDs protected primary neurons against 6-OHDA toxicity and accelerated neurite outgrowth in vitro. Thus, our data suggest that stem cells derived from dental pulp may have therapeutic benefits for PD.

DOI： 10.1016/j.brainres.2015.04.001

掲載種別：研究論文（学術雑誌）  

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

More than half of all human genes produce prematurely terminated polyadenylated short mRNAs. However, the underlying mechanisms remain largely elusive. CLIP-seq (cross-linking immunoprecipitation [CLIP] combined with deep sequencing) of FUS (fused in sarcoma) in neuronal cells showed that FUS is frequently clustered around an alternative polyadenylation (APA) site of nascent RNA. ChIP-seq (chromatin immunoprecipitation [ChIP] combined with deep sequencing) of RNA polymerase II (RNAP II) demonstrated that FUS stalls RNAP II and prematurely terminates transcription. When an APA site is located upstream of an FUS cluster, FUS enhances polyadenylation by recruiting CPSF160 and up-regulates the alternative short transcript. In contrast, when an APA site is located downstream from an FUS cluster, polyadenylation is not activated, and the RNAP II-suppressing effect of FUS leads to down-regulation of the alternative short transcript. CAGE-seq (cap analysis of gene expression [CAGE] combined with deep sequencing) and PolyA-seq (a strand-specific and quantitative method for high-throughput sequencing of 3’ ends of polyadenylated transcripts) revealed that position-specific regulation of mRNA lengths by FUS is operational in two-thirds of transcripts in neuronal cells, with enrichment in genes involved in synaptic activities.

DOI： 10.1101/gad.255737.114

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

FUS is an RNA/DNA-binding protein involved in multiple steps of gene expression and is associated with amyotrophic lateral sclerosis (ALS) and fronto-temporal lobar degeneration (FTLD). However, the specific disease-causing and/or modifying mechanism mediated by FUS is largely unknown. Here we evaluate intrinsic roles of FUS on synaptic functions and animal behaviours. We find that FUS depletion downregulates GluA1, a subunit of AMPA receptor. FUS binds GluA1 mRNA in the vicinity of the 3′ terminus and controls poly (A) tail maintenance, thus regulating stability. GluA1 reduction upon FUS knockdown reduces miniature EPSC amplitude both in cultured neurons and in vivo. FUS knockdown in hippocampus attenuates dendritic spine maturation and causes behavioural aberrations including hyperactivity, disinhibition and social interaction defects, which are partly ameliorated by GluA1 reintroduction. These results highlight the pivotal role of FUS in regulating GluA1 mRNA stability, post-synaptic function and FTLD-like animal behaviours.

DOI： 10.1038/ncomms8098

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

Molecular hydrogen (H2) is an agent with potential applications in oxidative stress-related and/or inflammatory disorders. H2 is usually administered by inhaling H2-containing air (HCA) or by oral intake of H2-rich water (HRW). Despite mounting evidence, the molecular mechanism underlying the therapeutic effects and the optimal method of H2 administration remain unclear. Here, we investigated whether H2 affects signaling pathways and gene expression in a dosage- or dose regimen-dependent manner. We first examined the H2 concentrations in blood and organs after its administration and found that oral intake of HRW rapidly but transiently increased H2 concentrations in the liver and atrial blood, while H2 concentrations in arterial blood and the kidney were one-tenth of those in the liver and atrial blood. In contrast, inhalation of HCA increased H2 equally in both atrial and arterial blood. We next examined whether H2 alters gene expression in normal mouse livers using DNA microarray analysis after administration of HCA and HRW. Ingenuity Pathway Analysis revealed that H2 suppressed the expression of nuclear factor-kappa B (NF-κB)-regulated genes. Western blot analysis showed that H2 attenuated ERK, p38 MAPK, and NF-κB signaling in mouse livers. Finally, we evaluated whether the changes in gene expression were influenced by the route of H2 administration and found that the combination of both HRW and HCA had the most potent effects on signaling pathways and gene expression in systemic organs, suggesting that H2 may act not only through a dose-dependent mechanism but also through a complex molecular network.

DOI： 10.1007/s11010-015-2353-y

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

Background: l-dopa (l-3,4-dihydroxyphenylalanine) is commonly used for treating Parkinson's disease (PD). However, regardless of its prominent effect, therapeutic range of l-dopa narrows down with disease progression, which leads to development of motor complications including wearing off and dyskinesias. In addition, intestinal absorption of l-dopa is inversely correlated with the amount of oral protein intake, and shows intra- and inter-day variability. Hence, frequent monitoring of plasma l-dopa concentrations is beneficial, but frequent venipuncture imposes physical and psychological burdens on patients with PD. Methods: We investigated the usefulness of sweat samples instead of plasma samples for monitoring l-dopa concentrations. With a monolithic silica disk-packed spin column and the high-performance liquid chromatography-electrochemical detection system, l-dopa in sweat samples was successfully quantified and analyzed in 23 PD patients. Results: We found that the Pearson's correlation coefficient of the plasma and sweat l-dopa concentrations was 0.678. Although the disease durations and severities were not correlated with the deviation of the actual sweat l-dopa concentrations from the fitted line, acquisition of the sweat samples under a stable condition was technically difficult in severely affected patients. The deviations may also be partly accounted for by skin permeability of l-dopa. Conclusions: Measuring l-dopa concentrations in sweat is suitable to get further insights into the l-dopa metabolism.

DOI： 10.1016/j.cca.2014.12.032

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

Background: Identification of causative genes in mendelian forms of Parkinson's disease is valuable for understanding the cause of the disease. We did genetic studies in a Japanese family with autosomal dominant Parkinson's disease to identify novel causative genes. Methods: We did a genome-wide linkage analysis on eight affected and five unaffected individuals from a family with autosomal dominant Parkinson's disease (family A). Subsequently, we did exome sequencing on three patients and whole-genome sequencing on one patient in family A. Variants were validated by Sanger sequencing in samples from patients with autosomal dominant Parkinson's disease, patients with sporadic Parkinson's disease, and controls. Participants were identified from the DNA bank of the Comprehensive Genetic Study on Parkinson's Disease and Related Disorders (Juntendo University School of Medicine, Tokyo, Japan) and were classified according to clinical information obtained by neurologists. Splicing abnormalities of CHCHD2 mutants were analysed in SH-SY5Y cells. We used the Fisher's exact test to calculate the significance of allele frequencies between patients with sporadic Parkinson's disease and unaffected controls, and we calculated odds ratios and 95% CIs of minor alleles. Findings: We identified a missense mutation (. CHCHD2, 182C>T, Thr61Ile) in family A by next-generation sequencing. We obtained samples from a further 340 index patients with autosomal dominant Parkinson's disease, 517 patients with sporadic Parkinson's disease, and 559 controls. Three CHCHD2 mutations in four of 341 index cases from independent families with autosomal dominant Parkinson's disease were detected by CHCHD2 mutation screening: 182C>T (Thr61Ile), 434G>A (Arg145Gln), and 300+5G>A. Two single nucleotide variants (-9T>G and 5C>T) in CHCHD2 were confirmed to have different frequencies between sporadic Parkinson's disease and controls, with odds ratios of 2·51 (95% CI 1·48-4·24; p=0·0004) and 4·69 (1·59-13·83, p=0·0025), respectively. One single nucleotide polymorphism (rs816411) was found in CHCHD2 from a previously reported genome-wide association study; however, there was no significant difference in its frequency between patients with Parkinson's disease and controls in a previously reported genome-wide association study (odds ratio 1·17, 95% CI 0·96-1·19; p=0·22). In SH-SY5Y cells, the 300+5G>A mutation but not the other two mutations caused exon 2 skipping. Interpretation: CHCHD2 mutations are associated with, and might be a cause of, autosomal dominant Parkinson's disease. Further genetic studies in other populations are needed to confirm the pathogenicity of CHCHD2 mutations in autosomal dominant Parkinson's disease and susceptibility for sporadic Parkinson's disease, and further functional studies are needed to understand how mutant CHCHD2 might play a part in the pathophysiology of Parkinson's disease.

DOI： 10.1016/S1474-4422(14)70266-2

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

Achondroplasia (ACH) is one of the most common skeletal dysplasias causing short stature owing to a gain-of-function mutation in the FGFR3 gene, which encodes the fibroblast growth factor receptor 3. We found that meclozine, an over-The-counter drug for motion sickness, inhibited elevated FGFR3 signaling in chondrocytic cells. To examine the feasibility of meclozine administration in clinical settings, we investigated the effects of meclozine on ACH model mice carrying the heterozygous Fgfr3ach transgene. We quantified the effect of meclozine in bone explant cultures employing limb rudiments isolated from developing embryonic tibiae from Fgfr3ach mice. We found that meclozine significantly increased the full-length and cartilaginous primordia of embryonic tibiae isolated from Fgfr3ach mice. We next analyzed the skeletal phenotypes of growing Fgfr3ach mice and wild-type mice with or without meclozine treatment. In Fgfr3ach mice, meclozine significantly increased the body length after 2 weeks of administration. At skeletal maturity, the bone lengths including the cranium, radius, ulna, femur, tibia, and vertebrae were significantly longer in meclozine-treated Fgfr3ach mice than in untreated Fgfr3ach mice. Interestingly, meclozine also increased bone growth in wild-type mice. The plasma concentration of meclozine during treatment was within the range that has been used in clinical settings for motion sickness. Increased longitudinal bone growth in Fgfr3ach mice by oral administration of meclozine in a growth period suggests potential clinical feasibility of meclozine for the improvement of short stature in ACH..

DOI： 10.1210/en.2014-1914

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  

No clinically applicable drug is currently available to enhance neurite elongation after nerve injury. To identify a clinically applicable drug, we screened pre-approved drugs for neurite elongation in the motor neuron-like NSC34 cells. We found that zonisamide, an anti-epileptic and anti-Parkinson's disease drug, promoted neurite elongation in cultured primary motor neurons and NSC34 cells in a concentration-dependent manner. The neurite-scratch assay revealed that zonisamide enhanced neurite regeneration. Zonisamide was also protective against oxidative stress-induced cell death of primary motor neurons. Zonisamide induced mRNA expression of nerve growth factors (BDNF, NGF, and neurotrophin-4/5), and their receptors (tropomyosin receptor kinase A and B). In a mouse model of sciatic nerve autograft, intragastric administration of zonisamide for 1 week increased the size of axons distal to the transected site 3.9-fold. Zonisamide also improved the sciatic function index, a marker for motor function of hindlimbs after sciatic nerve autograft, from 6 weeks after surgery. At 8 weeks after surgery, zonisamide was protective against denervationinduced muscle degeneration in tibialis anterior, and increased gene expression of Chrne, Colq, and Rapsn, which are specifically expressed at the neuromuscular junction. We propose that zonisamide is a potential therapeutic agent for peripheral nerve injuries as well as for neuropathies due to other etiologies.

DOI： 10.1371/journal.pone.0142786

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  

Background The intestine is one of the first affected organs in Parkinson’s disease (PD). PD subjects show abnormal staining for Escherichia coli and α-synuclein in the colon. Methods We recruited 52 PD patients and 36 healthy cohabitants. We measured serum markers and quantified the numbers of 19 fecal bacterial groups/genera/species by quantitative RT-PCR of 16S or 23S rRNA. Although the six most predominant bacterial groups/genera/species covered on average 71.3% of total intestinal bacteria, our analysis was not comprehensive compared to metagenome analysis or 16S rRNA amplicon sequencing. Results In PD, the number of Lactobacillus was higher, while the sum of analyzed bacteria, Clostridium coccoides group, and Bacteroides fragilis group were lower than controls. Additionally, the sum of putative hydrogen-producing bacteria was lower in PD. A linear regression model to predict disease durations demonstrated that C. coccoides group and Lactobacillus gasseri subgroup had the largest negative and positive coefficients, respectively. As a linear regression model to predict stool frequencies showed that these bacteria were not associated with constipation, changes in these bacteria were unlikely to represent worsening of constipation in the course of progression of PD. In PD, the serum lipopolysaccharide (LPS)- binding protein levels were lower than controls, while the levels of serum diamine oxidase, a marker for intestinal mucosal integrity, remained unchanged in PD. Conclusions The permeability to LPS is likely to be increased without compromising the integrity of intestinal mucosa in PD. The increased intestinal permeability in PD may make the patients susceptible to intestinal dysbiosis. Conversely, intestinal dysbiosis may lead to the increased intestinal permeability. One or both of the two mechanisms may be operational in development and progression of PD.

DOI： 10.1371/journal.pone.0142164

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  

Therapeutic effects of molecular hydrogen for a wide range of disease models and human diseases have been investigated since 2007. A total of 321 original articles have been published from 2007 to June 2015. Most studies have been conducted in Japan, China, and the USA. About three-quarters of the articles show the effects in mice and rats. The number of clinical trials is increasing every year. In most diseases, the effect of hydrogen has been reported with hydrogen water or hydrogen gas, which was followed by confirmation of the effect with hydrogen-rich saline. Hydrogen water is mostly given ad libitum. Hydrogen gas of less than 4 % is given by inhalation. The effects have been reported in essentially all organs covering 31 disease categories that can be subdivided into 166 disease models, human diseases, treatment-associated pathologies, and pathophysiological conditions of plants with a predominance of oxidative stress-mediated diseases and inflammatory diseases. Specific extinctions of hydroxyl radical and peroxynitrite were initially presented, but the radical-scavenging effect of hydrogen cannot be held solely accountable for its drastic effects. We and others have shown that the effects can be mediated by modulating activities and expressions of various molecules such as Lyn, ERK, p38, JNK, ASK1, Akt, GTP-Rac1, iNOS, Nox1, NF-κB p65, IκBα, STAT3, NFATc1, c-Fos, and ghrelin. Master regulator(s) that drive these modifications, however, remain to be elucidated and are currently being extensively investigated.

DOI： 10.1186/s13618-015-0035-1

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

Myotonic dystrophy type 1 (DM1) is caused by transcription of CUG repeat RNA, which causes sequestration of muscleblind-like 1 (MBNL1) and upregulation of CUG triplet repeat RNA-binding protein (CUG-BP1). In DM1, dysregulation of these proteins contributes to many aberrant splicing events, causing various symptoms of the disorder. Here, we demonstrate the occurrence of aberrant splicing of LIM domain binding 3 (LDB3) exon 11 in DM1 skeletal muscle. Exon array surveys, RT-PCR, and western blotting studies demonstrated that exon 11 inclusion was DM1 specific and could be reproduced by transfection of a minigene containing the CTG repeat expansion. Moreover, we found that the LDB3 exon 11-positive isoform had reduced affinity for PKC compared to the exon 11-negative isoform. Since PKC exhibits hyperactivation in DM1 and stabilizes CUG-BP1 by phosphorylation, aberrant splicing of LDB3 may contribute to CUG-BP1 upregulation through changes in its affinity for PKC. © 2014 Elsevier Inc.

DOI： 10.1016/j.nbd.2014.05.026

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  

Endochondral ossification is an essential step for skeletal development, which requires chondrocyte differentiation in growth cartilage. The low-density lipoprotein receptor-related protein 4 (LRP4), a member of LDLR family, is an inhibitor for Wnt signaling, but its roles in chondrocyte differentiation remain to be investigated. Here we found by laser capture microdissection that LRP4 expression was induced during chondrocyte differentiation in growth plate. In order to address the roles, we overexpressed recombinant human LRP4 or knocked down endogenous LRP4 by lentivirus in mouse ATDC5 chondrocyte cells. We found that LRP4 induced gene expressions of extracellular matrix proteins of type II collagen (Col2a1), aggrecan (Acan), and type X collagen (Col10a1), as well as production of total proteoglycans in ATDC5 cells, whereas LRP4 knockdown had opposite effects. Interestingly, LRP4-knockdown reduced mRNA expression of Sox9, a master regulator for chondrogenesis, as well as Dkk1, an extracellular Wnt inhibitor. Analysis of Wnt signaling revealed that LRP4 blocked the Wnt/β-catenin signaling activity in ATDC5 cells. Finally, the reduction of these extracellular matrix productions by LRP4-knockdown was rescued by a β-catenin/TCF inhibitor, suggesting that LRP4 is an important regulator for extracellular matrix productions and chondrocyte differentiation by suppressing Wnt/β-catenin signaling. © 2014 Elsevier Inc. All rights reserved.

DOI： 10.1016/j.bbrc.2014.07.125

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 Neurology, Neurosurgery and Psychiatry  

DOI： 10.1136/jnnp-2013-306414

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 Molecular Neuroscience  

Acetylcholinesterase (AChE) at the neuromuscular junction (NMJ) is anchored to the synaptic basal lamina via a triple helical collagen Q (ColQ) in the form of asymmetric AChE (AChE/ColQ). We exploited the proprietary NMJ-targeting signals of ColQ to treat congenital myasthenia and to explore the mechanisms of autoimmune myasthenia gravis (MG). Mutations in COLQ cause congenital endplate AChE deficiency (CEAD). First, a single intravenous administration of adeno-associated virus serotype 8 (AAV8)-COLQ to Colq-/- mice normalized motor functions, synaptic transmission, and partly the NMJ ultrastructure. Additionally, injection of purified recombinant AChE/ColQ protein complex into gluteus maximus accumulated AChE in non-injected forelimbs. Second, MuSK antibody-positive MG accounts for 5-15 % of MG. In vitro overlay of AChE/ColQ to muscle sections of Colq-/- mice, as well as in vitro plate-binding of MuSK to ColQ, revealed that MuSK-IgG blocks binding of ColQ to MuSK in a dose-dependent manner. Passive transfer of MuSK-IgG to wild-type mice markedly reduced the size and intensity of ColQ signals at NMJs. MuSK-IgG thus interferes with binding of ColQ to MuSK. Elucidation of molecular mechanisms of specific binding of ColQ to NMJ enabled us to ameliorate devastating myasthenic symptoms of Colq-/- mice and also to reveal underlying mechanisms of anti-MuSK-MG. © 2013 Springer Science+Business Media.

DOI： 10.1007/s12031-013-0170-x

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  

Congenital myasthenic syndromes (CMS) are heterogeneous disorders in which the safety margin of neuromuscular transmission is compromised by one or more specific mechanisms. Using Sanger and exome sequencing in a CMS patient, we identified two heteroallelic mutations, p.Glu1233Lys and p.Arg1277His, in LRP4 coding for the postsynaptic low-density lipoprotein receptor-related protein 4. LRP4, expressed on the surface of the postsynaptic membrane of the neuromuscular junction, is a receptor for neurally secreted agrin, and LRP4 bound by agrin activates MuSK. Activated MuSK in concert with Dok-7 stimulates rapsyn to concentrate and anchor AChR on the postsynaptic membrane and interacts with other proteins implicated in the assembly and maintenance of the neuromuscular junction. LRP4 also functions as an inhibitor of Wnt/beta-catenin signaling. The identified mutations in LRP4 are located at the edge of its 3rd beta-propeller domain and decrease binding affinity of LRP4 for both MuSK and agrin. Mutations in the LRP4 3rd beta-propeller domain were previously reported to impair Wnt signaling and cause bone diseases including Cenani-Lenz syndactyly syndrome and sclerosteosis-2. By analyzing naturally occurring and artificially introduced mutations in the LRP4 3rd beta-propeller domain, we show that the edge of the domain regulates the MuSK signaling whereas its central cavity governs Wnt signaling. We conclude that LRP4 is a new CMS disease gene and that the 3rd beta propeller domain of LRP4 mediates the two signaling pathways in a position-specific manner.

DOI： 10.1093/hmg/ddt578

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 Biology and Medicine  

Molecular hydrogen (H2) scavenges hydroxyl radicals. Recently, H2 has been reported to prevent a variety of diseases associated with oxidative stress in model systems and in humans. Here, we studied the effects of H2 on rat fetal hippocampal damage caused by ischemia and reperfusion (IR) on day 16 of pregnancy with the transient occlusion of the bilateral utero-ovarian arteries. Starting 2 days before the operation, we provided the mothers with hydrogen-saturated water ad libitum until vaginal delivery. We observed a significant increase in the concentration of H 2 in the placenta after the oral administration of hydrogen-saturated water to the mothers, with less placental oxidative damage after IR in the presence of H2. Neonatal growth retardation was observed in the IR group, which was alleviated by the H2 administration. We analyzed the neuronal cell damage in the CA1 and CA3 areas of the hippocampus at day 7 after birth by immunohistochemical analysis of the 8-oxo-7,8-dihydro-2.©2014 ElsevierInc.Allrightsreserved.

DOI： 10.1016/j.freeradbiomed.2014.01.037

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

Objective: To identify other causative genes for Andersen-Tawil syndrome, which is characterized by a triad of periodic paralysis, cardiac arrhythmia, and dysmorphic features. Andersen-Tawil syndrome is caused in amajority of cases bymutations in KCNJ2,which encodes the Kir2.1 subunit of the inwardly rectifying potassium channel. Methods: The proband exhibited episodic flaccid weakness and a characteristic TU-wave pattern, both suggestive of Andersen-Tawil syndrome, but did not harbor KCNJ2 mutations. We performed exome capture resequencing by restricting the analysis to genes that encode ion channels/associated proteins. The expression of gene products in heart and skeletalmuscle tissues was examined by immunoblotting. The functional consequences of the mutation were investigated using a heterologous expression system in Xenopus oocytes, focusing on the interaction with the Kir2.1 subunit. Results: We identified a mutation in the KCNJ5 gene, which encodes the G-protein-activated inwardly rectifying potassium channel 4 (Kir3.4). Immunoblotting demonstrated significant expression of the Kir3.4 protein in human heart and skeletal muscles. The coexpression of Kir2.1 and mutant Kir3.4 in Xenopus oocytes reduced the inwardly rectifying current significantly compared with that observed in the presence of wild-type Kir3.4. Conclusions: We propose that KCNJ5 is a second gene causing Andersen-Tawil syndrome. The inhibitory effects of mutant Kir3.4 on inwardly rectifying potassium channels may account for the clinical presentation in both skeletal and heart muscles. © 2014 American Academy of Neurology.

DOI： 10.1212/WNL.0000000000000239

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 Human Genetics  

Myotonic dystrophy type 2 (DM2) is more common than DM1 in Europe and is considered a rare cause of myotonic dystrophies in Asia. Its clinical course is also milder with more phenotypic variability than DM1. We herein describe the first known Asian family (three affected siblings) with DM2 based on clinical and genetic analyses. Notably, two of the affected siblings were previously diagnosed with limb-girdle muscular dystrophy. Myotonia (the inability of the muscle to relax) was absent or only faintly present in these individuals. The third sibling had grip myotonia and is the first known Asian DM2 patient. The three DM2 siblings share several systemic characteristics, including late-onset, proximal-dominant muscle weakness, diabetes, cataracts and asthma. Repeat-primed PCR across the DM2 repeat revealed a characteristic ladder pattern of a CCTG expansion in all siblings. Southern blotting analysis identified the presence of 3400 repeats. Further DM2 studies in Asian populations are needed to define the clinical presentation of Asian DM2 and as yet unidentified phenotypic differences from Caucasian patients. © 2014 The Japan Society of Human Genetics All rights reserved 1434-5161/14.

DOI： 10.1038/jhg.2013.133

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 Molecular Medicine  

Marinesco-Sjögren syndrome (MSS) is a rare autosomal recessively inherited disorder with mental retardation (MR). Recently, mutations in the SIL1 gene, encoding a co-chaperone which regulates the chaperone HSPA5, were identified as a major cause of MSS. We here examined the pathophysiological significance of SIL1 mutations in abnormal corticogenesis of MSS. SIL1-silencing caused neuronal migration delay during corticogenesis ex vivo. While RNAi-resistant SIL1 rescued the defects, three MSS-causing SIL1 mutants tested did not. These mutants had lower affinities to HSPA5 in vitro, and SIL1-HSPA5 interaction as well as HSPA5 function was found to be crucial for neuronal migration ex vivo. Furthermore time-lapse imaging revealed morphological disorganization associated with abnormal migration of SIL1-deficient neurons. These results suggest that the mutations prevent SIL1 from interacting with and regulating HSPA5, leading to abnormal neuronal morphology and migration. Consistent with this, when SIL1 was silenced in cortical neurons in one hemisphere, axonal growth in the contralateral hemisphere was delayed. Taken together, abnormal neuronal migration and interhemispheric axon development may contribute to MR in MSS. © 2014 The Authors.

DOI： 10.1002/emmm.201303069

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  

In past years, the canonical Wnt/β-catenin signaling pathway has emerged as a critical regulator of cartilage development and homeostasis. FRZB, a soluble antagonist of Wnt signaling, has been studied in osteoarthritis (OA) animal models and OA patients as a modulator of Wnt signaling. We screened for FDA-approved drugs that induce FRZB expression and suppress Wnt/β-catenin signaling. We found that verapamil, a widely prescribed L-type calcium channel blocker, elevated FRZB expression and suppressed Wnt/β-catenin signaling in human OA chondrocytes. Expression and nuclear translocation of β-catenin was attenuated by verapamil in OA chondrocytes. Lack of the verapamil effects in LiCl-treated and FRZB-downregulated OA chondrocytes also suggested that verpamil suppressed Wnt signaling by inducing FRZB. Verapamil enhanced gene expressions of chondrogenic markers of ACAN encoding aggrecan, COL2A1 encoding collagen type II α1, and SOX9, and suppressed Wnt-responsive AXIN2 and MMP3 in human OA chondrocytes. Verapamil ameliorated Wnt3A-induced proteoglycan loss in chondrogenically differentiated ATDC5 cells. Verapamil inhibited hypertrophic differentiation of chondrocytes in the explant culture of mouse tibiae. Intraarticular injection of verapamil inhibited OA progression as well as nuclear localizations of β-catenin in a rat OA model. We propose that verapamil holds promise as a potent therapeutic agent for OA by upregulating FRZB and subsequently downregulating Wnt/β-catenin signaling. © 2014 Takamatsu et al.

DOI： 10.1371/journal.pone.0092699

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  

SMXA-5 mice are a high-fat diet-induced type 2 diabetes animal model established from non-diabetic SM/J and A/J mice. By using F2 intercross mice between SMXA-5 and SM/J mice under feeding with a high-fat diet, we previously mapped a major diabetogenic QTL (T2dm2sa) on chromosome 2. We then produced the congenic strain (SM.A-T2dm2sa (R0), 20.8-163.0 Mb) and demonstrated that the A/J allele of T2dm2sa impaired glucose tolerance and increased body weight and body mass index in the congenic strain compared to SM/J mice. We also showed that the combination of T2dm2sa and other diabetogenic loci was needed to develop the high-fat diet-induced type 2 diabetes. In this study, to narrow the potential genomic region containing the gene(s) responsible for T2dm2sa, we constructed R1 and R2 congenic strains. Both R1 (69.6-163.0 Mb) and R2 (20.8-128.2 Mb) congenic mice exhibited increases in body weight and abdominal fat weight and impaired glucose tolerance compared to SM/J mice. The R1 and R2 congenic analyses strongly suggested that the responsible genes existed in the overlapping genomic interval (69.6-128.2 Mb) between R1 and R2. In addition, studies using the newly established R1A congenic strain showed that the narrowed genomic region (69.6-75.4 Mb) affected not only obesity but also glucose tolerance. To search for candidate genes within the R1A genomic region, we performed exome sequencing analysis between SM/J and A/J mice and extracted 4 genes (Itga6, Zak, Gpr155, and Mtx2) with nonsynonymous coding SNPs. These four genes might be candidate genes for type 2 diabetes caused by gene-gene interactions. This study indicated that one of the genes responsible for high-fat diet-induced diabetes exists in the 5.8 Mb genomic interval on mouse chromosome 2. © 2014 Kobayashi et al.

DOI： 10.1371/journal.pone.0096271

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

TDP-43 and FUS are linked to amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD), and loss of function of either protein contributes to these neurodegenerative conditions. To elucidate the TDP-43- and FUS-regulated pathophysiological RNA metabolism cascades, we assessed the differential gene expression and alternative splicing profiles related to regulation by either TDP-43 or FUS in primary cortical neurons. These profiles overlapped by >25% with respect to gene expression and >9% with respect to alternative splicing. The shared downstream RNA targets of TDP-43 and FUS may form a common pathway in the neurodegenerative processes of ALS/FTLD. © 2014 The Authors.

DOI： 10.1016/j.fob.2013.11.001

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  

DOI： 10.1038/srep03301

掲載種別：研究論文（学術雑誌）   出版者・発行元：Scientific Reports  

DOI： 10.1038/srep03300

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

Background: Currently, there are no effective medical treatment options to prevent the formation of heterotopic bones in fibrodysplasia ossificans progressiva (FOP). By the drug repositioning strategy, we confirmed that perhexiline maleate (Pex) potentially ameliorates heterotopic ossification in model cells and mice. Here, we conducted a prospective study to assess the efficacy and safety of Pex in the treatment of FOP patients. Methods. FOP patients in this open-label single-center study were treated with Pex for a total of 12 months, and followed up for 12 consecutive months after medication discontinuation. The safety of the treatment was assessed regularly by physical and blood examinations. The efficacy of Pex for preventing heterotopic ossifications was evaluated by the presence of flare-ups, measurements of serum bone markers, and changes in the total bone volume calculated by the three-dimensional computed tomography (3D-CT) images. Results: Five patients with an average age of 23.4 years were enrolled. Within safe doses of Pex administration in each individual, there were no drug-induced adverse effects during the medication phase. Three patients showed no intense inflammatory reactions during the study period, while two patients had acute flare-ups around the hip joint without evidence of trauma during the medication phase. In addition, one of them became progressively incapable of opening her mouth over the discontinuation phase. Serum levels of alkaline phosphatase (ALP) and bone specific ALP (BAP) were significantly and synchronously increased with the occurrence of flare-ups. Volumetric 3D-CT analysis demonstrated a significant increase in the total bone volume of Case 2 (378 cm§ssup§3§ esup§) and Case 3 (833 cm§ssup§3§esup§) during the two-year study period. Conclusions: We could not prove the efficacy of oral Pex administration in the prevention of heterotopic ossifications in FOP. Serum levels of ALP and BAP appear to be promising biomarkers for monitoring the development of ectopic ossifications and efficacy of the therapy. Quantification of change in the total bone volume by whole body CT scanning could be a reliable evaluation tool for disease progression in forthcoming clinical trials of FOP. © 2013 Kitoh et al.; licensee BioMed Central Ltd.

DOI： 10.1186/1750-1172-8-163

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 Neurology, Neurosurgery and Psychiatry  

DOI： 10.1136/jnnp-2013-304931

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  

FUS is genetically and pathologically linked to amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD). To clarify the RNA metabolism cascade regulated by FUS in ALS/FTLD, we compared the FUS-regulated transcriptome profiles in different lineages of primary cells from the central nervous system. The profiles of FUS-mediated gene expression and alternative splicing in motor neurons were similar to those of cortical neurons, but not to those in cerebellar neurons despite the similarity of innate transcriptome signature. The gene expression profiles in glial cells were similar to those in motor and cortical neurons. We identified certain neurological diseases-associated genes, including Mapt, Stx1a, and Scn8a, among the profiles of gene expression and alternative splicing events regulated by FUS. Thus, FUS-regulated transcriptome profiles in each cell-type may determine cellular fate in association with FUS-mediated ALS/FTLD, and identified RNA targets for FUS could be therapeutic targets for ALS/FTLD.

DOI： 10.1038/srep02388

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 Mutation  

Acetylcholinesterase (AChE) at the neuromuscular junction (NMJ) is mostly composed of an asymmetric form in which three tetramers of catalytic AChE subunits are linked to a triple helical collagen Q (ColQ). Mutations in COLQ cause endplate AChE deficiency. We report three patients with endplate AChE deficiency with five recessive COLQ mutations. Sedimentation profiles showed that p.Val322Asp and p.Arg227X, but not p.Cys444Tyr, p.Asp447His, or p.Arg452Cys, inhibit formation of triple helical ColQ. In vitro overlay of mutant ColQ-tailed AChE on muscle sections of Colq-/- mice revealed that p.Cys444Tyr, p.Asp447His, and p.Arg452Cys in the C-terminal domain (CTD) abrogate anchoring ColQ-tailed AChE to the NMJ. In vitro plate-binding assay similarly demonstrated that the three mutants inhibit binding of ColQ-tailed AChE to MuSK. We also confirmed the pathogenicity of p.Asp447His by treating Colq-/- mice with adeno-associated virus serotype 8 carrying mutant COLQ-p.Asp447His. The treated mice showed no improvement in motor functions and no anchoring of ColQ-tailed AChE at the NMJ. Electroporation of mutant COLQ harboring p.Cys444Tyr, p.Asp447His, and p.Arg452Cys into anterior tibial muscles of Colq-/- mice similarly failed to anchor ColQ-tailed AChE at the NMJ. We proved that the missense mutations in ColQ-CTD cause endplate AChE deficiency by compromising ColQ-MuSK interaction at the NMJ. Missense mutations in COLQ encoding collagen Q (ColQ) compromise binding of ColQ to MuSK and lead to endplate acetylcholinesterase (AChE) deficiency. Defective binding has been proven by an in vitro overlay assay of mutant ColQ on skeletal muscle of Colq-/- mice and by an in vitro plate-binding assay on purified MuSK. We also confirmed that AAV8-mediated gene transfer of mutant ColQ into Colq-/- mice failed to express ColQ at the neuromuscular junction and to improve the motor deficits. © 2013 WILEY PERIODICALS, INC.

DOI： 10.1002/humu.22325

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

Background: Senescence-accelerated mice (SAM) are a series of mouse strains originally derived from unexpected crosses between AKR/J and unknown mice, from which phenotypically distinct senescence-prone (SAMP) and -resistant (SAMR) inbred strains were subsequently established. Although SAMP strains have been widely used for aging research focusing on their short life spans and various age-related phenotypes, such as immune dysfunction, osteoporosis, and brain atrophy, the responsible gene mutations have not yet been fully elucidated.Results: To identify mutations specific to SAMP strains, we performed whole exome sequencing of 6 SAMP and 3 SAMR strains. This analysis revealed 32,019 to 38,925 single-nucleotide variants in the coding region of each SAM strain. We detected Ogg1 p.R304W and Mbd4 p.D129N deleterious mutations in all 6 of the SAMP strains but not in the SAMR or AKR/J strains. Moreover, we extracted 31 SAMP-specific novel deleterious mutations. In all SAMP strains except SAMP8, we detected a p.R473W missense mutation in the Ldb3 gene, which has been associated with myofibrillar myopathy. In 3 SAMP strains (SAMP3, SAMP10, and SAMP11), we identified a p.R167C missense mutation in the Prx gene, in which mutations causing hereditary motor and sensory neuropathy (Dejerine-Sottas syndrome) have been identified. In SAMP6 we detected a p.S540fs frame-shift mutation in the Il4ra gene, a mutation potentially causative of ulcerative colitis and osteoporosis.Conclusions: Our data indicate that different combinations of mutations in disease-causing genes may be responsible for the various phenotypes of SAMP strains. © 2013 Tanisawa et al.; licensee BioMed Central Ltd.

DOI： 10.1186/1471-2164-14-248

記述言語：英語   掲載種別：研究論文（国際会議プロシーディングス）   出版者・発行元：Chemico-Biological Interactions  

Acetylcholinesterase (AChE) at the neuromuscular junction (NMJ) is anchored to the synaptic basal lamina via a triple helical collagen Q (ColQ) in the form of asymmetric AChE (AChE/ColQ). The C-terminal domain of ColQ binds to MuSK, the muscle-specific receptor tyrosine kinase, that mediates a signal for acetylcholine receptor (AChR) clustering at the NMJ. ColQ also binds to heparan sulfate proteoglycans including perlecan. Congenital defects of ColQ cause endplate AChE deficiency. A single intravenous administration of adeno-associated virus serotype 8 (AAV8)-COLQ to Colq-/- mice rescued motor functions, synaptic transmission, and the ultrastructure of NMJ. We also injected AAV1-COLQ-IRES-EGFP to the left tibialis anterior and observed colocalization of AChE/ColQ at all the examined NMJs of the non-injected limbs. Additionally, injection of purified recombinant AChE/ColQ protein complex into gluteus maximus accumulated AChE in non-injected forelimbs. These observations suggest that the tissue-targeting signal of ColQ can be exploited to specifically deliver the transgene product to the target tissue. MuSK antibody-positive myasthenia gravis (MG) accounts for 5-15% of autoimmune MG. As AChR deficiency is typically mild and as cholinesterase inhibitors are generally ineffective or worsen myasthenic symptoms, we asked if the patient's MuSK-IgG interferes with binding of ColQ to MuSK. In vitro overlay of AChE/ColQ to muscle sections of Colq-/- mice revealed that MuSK-IgG blocks binding of ColQ to the NMJ. In vitro plate-binding of MuSK to ColQ disclosed that MuSK-IgG exerts a dose-dependent block of MuSK-ColQ interaction. In addition, passive transfer of MuSK-IgG to mice reduced the size and density of ColQ to ∼10% of controls and had a lesser effect on the sizes and densities of AChR and MuSK. Elucidation of molecular mechanisms of specific binding of ColQ to the NMJ enabled us to ameliorate devastating myasthenic symptoms of Colq-/- mice and to reveal bases of anti-MuSK MG. © 2012 Elsevier Ireland Ltd. All rights reserved.

DOI： 10.1016/j.cbi.2012.08.020

記述言語：英語   掲載種別：研究論文（学術雑誌）  

DOI： 10.1186/2045-9912-3-6

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

Dynamic remodeling of the actin cytoskeleton is crucial for biological processes such as cell migration and cell spreading. S100A10 is a member of the S100 protein family and is involved in intracellular trafficking and cell migration. In this study, we examined the role of S100A10 in actin cytoskeletal organization and cell spreading. Depletion of S100A10 induced disruption of stress fiber formation and delay in cell spreading. Rac1 activation during spreading was suppressed by S100A10 knockdown, and exogenous expression of active Rac1 restored the ability of cells to spread in the absence of S100A10. Our results demonstrate the crucial role of S100A10 in actin dynamics promoting cell spreading via Rac1 activation. © 2012 Springer Science+Business Media New York.

DOI： 10.1007/s11010-012-1509-2

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 Bone and Mineral Metabolism  

Fibrodysplasia ossificans progressiva (FOP) is a rare autosomal dominant disorder characterized by progressive heterotopic ossification. FOP is caused by a gain-of-function mutation in ACVR1 encoding the bone morphogenetic protein type II receptor, ACVR1/ALK2. The mutant receptor causes upregulation of a transcriptional factor, Id1. No therapy is available to prevent the progressive heterotopic ossification in FOP. In an effort to search for clinically applicable drugs for FOP, we screened 1,040 FDA-approved drugs for suppression of the Id1 promoter activated by the mutant ACVR1/ALK2 in C2C12 cells. We found that that two antianginal agents, fendiline hydrochloride and perhexiline maleate, suppressed the Id1 promoter in a dose-dependent manner. The drugs also suppressed the expression of native Id1 mRNA and alkaline phosphatase in a dose-dependent manner. Perhexiline but not fendiline downregulated phosphorylation of Smad 1/5/8 driven by bone morphogenetic protein (BMP)-2. We implanted crude BMPs in muscles of ddY mice and fed them fendiline or perhexiline for 30 days. Mice taking perhexiline showed a 38.0 % reduction in the volume of heterotopic ossification compared to controls, whereas mice taking fendiline showed a slight reduction of heterotopic ossification. Fendiline, perhexiline, and their possible derivatives are potentially applicable to clinical practice to prevent devastating heterotopic ossification in FOP. © 2012 The Japanese Society for Bone and Mineral Research and Springer.

DOI： 10.1007/s00774-012-0380-2

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 Methods  

l-DOPA (l-3,4-dihydroxyphenylalanine) is commonly used in the treatment of Parkinson's disease. Monitoring the concentration of l-DOPA in human plasma will enable dose optimization, but is rarely performed because current quantification methods are tedious and time-consuming. In this study, a simple method for the determination of l-DOPA in the plasma of patients with Parkinson's disease was developed. A monolithic silica disk-packed spin column with a phenylboronate moiety, which forms stable anionic complexes with the cis-hydroxyl groups of l-DOPA, was used to extract l-DOPA from plasma with extraction recoveries exceeding 90%. The extracted l-DOPA was then separated on a reversed-phase column and detected electrochemically with a boron-doped diamond electrode. The method, which has a limit of detection of 10 fmol, was then successfully applied for the determination of l-DOPA in the plasma of healthy volunteers and patients with Parkinson's disease. © 2013 The Royal Society of Chemistry.

DOI： 10.1039/c3ay40934a

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  

Achondroplasia (ACH) is one of the most common skeletal dysplasias with short stature caused by gain-of-function mutations in FGFR3 encoding the fibroblast growth factor receptor 3. We used the drug repositioning strategy to identify an FDA-approved drug that suppresses abnormally activated FGFR3 signaling in ACH. We found that meclozine, an antihistamine drug that has long been used for motion sickness, facilitates chondrocyte proliferation and mitigates loss of extracellular matrix in FGF2-treated rat chondrosarcoma (RCS) cells. Meclozine also ameliorated abnormally suppressed proliferation of human chondrosarcoma (HCS-2/8) cells that were infected with lentivirus expressing constitutively active mutants of FGFR3-K650E causing thanatophoric dysplasia, FGFR3-K650M causing SADDAN, and FGFR3-G380R causing ACH. Similarly, meclozine alleviated abnormally suppressed differentiation of ATDC5 chondrogenic cells expressing FGFR3-K650E and -G380R in micromass culture. We also confirmed that meclozine alleviates FGF2-mediated longitudinal growth inhibition of embryonic tibia in bone explant culture. Interestingly, meclozine enhanced growth of embryonic tibia in explant culture even in the absence of FGF2 treatment. Analyses of intracellular FGFR3 signaling disclosed that meclozine downregulates phosphorylation of ERK but not of MEK in FGF2-treated RCS cells. Similarly, meclozine enhanced proliferation of RCS cells expressing constitutively active mutants of MEK and RAF but not of ERK, which suggests that meclozine downregulates the FGFR3 signaling by possibly attenuating ERK phosphorylation. We used the C-natriuretic peptide (CNP) as a potent inhibitor of the FGFR3 signaling throughout our experiments, and found that meclozine was as efficient as CNP in attenuating the abnormal FGFR3 signaling. We propose that meclozine is a potential therapeutic agent for treating ACH and other FGFR3-related skeletal dysplasias. © 2013 Matsushita et al.

DOI： 10.1371/journal.pone.0081569

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

Acetylcholinesterase (AChE) at the neuromuscular junction (NMJ) is anchored to the synaptic basal lamina via a triple helical collagen Q (ColQ). Congenital defects of ColQ cause endplate AChE deficiency and myasthenic syndrome. A single intravenous administration of adeno-associated virus serotype 8 (AAV8)-COLQ to Colq-/- mice recovered motor functions, synaptic transmission, as well as the morphology of the NMJ. ColQ-tailed AChE was specifically anchored to NMJ and its amount was restored to 89% of the wild type. We next characterized the molecular basis of this efficient recovery. We first confirmed that ColQ-tailed AChE can be specifically targeted to NMJ by an in vitro overlay assay in Colq-/- mice muscle sections. We then injected AAV1-COLQ-IRES-EGFP into the left tibialis anterior and detected AChE in noninjected limbs. Furthermore, the in vivo injection of recombinant ColQ-tailed AChE protein complex into the gluteus maximus muscle of Colq -/- mice led to accumulation of AChE in noninjected forelimbs. We demonstrated for the first time in vivo that the ColQ protein contains a tissue-targeting signal that is sufficient for anchoring itself to the NMJ. We propose that the protein-anchoring strategy is potentially applicable to a broad spectrum of diseases affecting extracellular matrix molecules. © The American Society of Gene & Cell Therapy.

DOI： 10.1038/mt.2012.34

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 Human Genetics  

Myotonic dystrophy type 1 (DM1) is an RNA gain-of-function disorder in which abnormally expanded CTG repeats of DMPK sequestrate a splicing trans-factor MBNL1 and upregulate another splicing trans-factor CUGBP1. To identify a diverse array of aberrantly spliced genes, we performed the exon array analysis of DM1 muscles. We analyzed 72 exons by RT-PCR and found that 27 were aberrantly spliced, whereas 45 were not. Among these, 25 were novel and especially splicing aberrations of LDB3 exon 4 and TTN exon 45 were unique to DM1. Retrospective analysis revealed that four parameters efficiently detect aberrantly spliced exons: (i) the signal intensity is high; (ii) the ratio of probe sets with reliable signal intensities (that is, detection above background P-value0.000) is high within a gene; (iii) the splice index (SI) is high; and (iv) SI is deviated from SIs of the other exons that can be estimated by calculating the deviation value (DV). Application of the four parameters gave rise to a sensitivity of 77.8% and a specificity of 95.6% in our data set. We propose that calculation of DV, which is unique to our analysis, is of particular importance in analyzing the exon array data. © 2012 The Japan Society of Human Genetics All rights reserved.

DOI： 10.1038/jhg.2012.37

記述言語：英語   掲載種別：研究論文（学術雑誌）  

DOI： 10.1186/2045-9912-2-15

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 the Neurological Sciences  

Mutations in the pore-forming subunit of the skeletal muscle sodium channel (SCN4A) are responsible for hyperkalemic periodic paralysis, paramyotonia congenita and sodium channel myotonia. These disorders are classified based on their cardinal symptoms, myotonia and/or paralysis. We report the case of a Japanese boy with a novel mutation of SCN4A, p.I693L, who exhibited severe episodic myotonia from infancy and later onset mild paralytic attack. He started to have apneic episodes with generalized hypertonia at age of 11 months, then developed severe episodic myotonia since 2 years of age. He presented characteristic generalized features which resembled Schwarz-Jampel syndrome. After 7 years old, paralytic episodes occurred several times a year. The compound muscle action potential did not change during short and long exercise tests. Functional analysis of the mutant channel expressed in cultured cell revealed enhancement of the activation and disruption of the slow inactivation, which were consistent with myotonia and paralytic attack. The severe clinical features in his infancy may correspond to myotonia permanence, however, he subsequently experienced paralytic attacks. This case provides an example of the complexity and overlap of the clinical features of sodium channel myotonic disorders. © 2011 Elsevier B.V. All rights reserved.

DOI： 10.1016/j.jns.2011.12.015

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 Human Genetics  

DOI： 10.1038/jhg.2011.152

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  

CUGBP1 and MBNL1 are developmentally regulated RNA-binding proteins that are causally associated with myotonic dystrophy type 1. We globally determined the in vivo RNA-binding sites of CUGBP1 and MBNL1. Interestingly, CUGBP1 and MBNL1 are both preferentially bound to 3′ UTRs. Analysis of CUGBP1-and MBNL1-bound 3′ UTRs demonstrated that both factors mediate accelerated mRNA decay and temporal profiles of expression arrays supported this. Role of CUGBP1 on accelerated mRNA decay has been previously reported, but the similar function of MBNL1 has not been reported to date. It is well established that CUGBP1 and MBNL1 regulate alternative splicing. Screening by exon array and validation by RT-PCR revealed position dependence of CUGBP1-and MBNL1-binding sites on the resulting alternative splicing pattern. This study suggests that regulation of CUGBP1 and MBNL1 is essential for accurate control of destabilization of a broad spectrum of mRNAs as well as of alternative splicing events.

DOI： 10.1038/srep00209

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  

Myotonic dystrophy type 2 (DM2) is a subtype of the myotonic dystrophies, caused by expansion of a tetranucleotide CCTG repeat in intron 1 of the zinc finger protein 9 (ZNF9) gene. The expansions are extremely unstable and variable, ranging from 75-11,000 CCTG repeats. This unprecedented repeat size and somatic heterogeneity make molecular diagnosis of DM2 difficult, and yield variable clinical phenotypes. To better understand the mutational origin and instability of the ZNF9 CCTG repeat, we analyzed the repeat configuration and flanking regions in 26 primate species. The 3′-end of an AluSx element, flanked by target site duplications (5′-ACTRCCAR-3′or 5′-ACTRCCARTTA-3′), followed the CCTG repeat, suggesting that the repeat was originally derived from the Alu element insertion. In addition, our results revealed lineage-specific repetitive motifs: pyrimidine (CT)-rich repeat motifs in New World monkeys, dinucleotide (TG) repeat motifs in Old World monkeys and gibbons, and dinucleotide (TG) and tetranucleotide (TCTG and/or CCTG) repeat motifs in great apes and humans. Moreover, these di- and tetra-nucleotide repeat motifs arose from the poly (A) tail of the AluSx element, and evolved into unstable CCTG repeats during primate evolution. Alu elements are known to be the source of microsatellite repeats responsible for two other repeat expansion disorders: Friedreich ataxia and spinocerebellar ataxia type 10. Taken together, these findings raise questions as to the mechanism(s) by which Alu-mediated repeats developed into the large, extremely unstable expansions common to these three disorders. © 2012 Kurosaki et al.

DOI： 10.1371/journal.pone.0038379

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  

FUS is an RNA-binding protein that regulates transcription, alternative splicing, and mRNA transport. Aberrations of FUS are causally associated with familial and sporadic ALS/FTLD. We analyzed FUS-mediated transcriptions and alternative splicing events in mouse primary cortical neurons using exon arrays. We also characterized FUS-binding RNA sites in the mouse cerebrum with HITS-CLIP. We found that FUS-binding sites tend to form stable secondary structures. Analysis of position-dependence of FUS-binding sites disclosed scattered binding of FUS to and around the alternatively spliced exons including those associated with neurodegeneration such as Mapt, Camk2a, and Fmr1. We also found that FUS is often bound to the antisense RNA strand at the promoter regions. Global analysis of these FUS-tags and the expression profiles disclosed that binding of FUS to the promoter antisense strand downregulates transcriptions of the coding strand. Our analysis revealed that FUS regulates alternative splicing events and transcriptions in a position-dependent manner.

DOI： 10.1038/srep00529

記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：Oxidative Medicine and Cellular Longevity  

Effects of molecular hydrogen on various diseases have been documented for 63 disease models and human diseases in the past four and a half years. Most studies have been performed on rodents including two models of Parkinson's disease and three models of Alzheimer's disease. Prominent effects are observed especially in oxidative stress-mediated diseases including neonatal cerebral hypoxia; Parkinson's disease; ischemia/reperfusion of spinal cord, heart, lung, liver, kidney, and intestine; transplantation of lung, heart, kidney, and intestine. Six human diseases have been studied to date: diabetes mellitus type 2, metabolic syndrome, hemodialysis, inflammatory and mitochondrial myopathies, brain stem infarction, and radiation-induced adverse effects. Two enigmas, however, remain to be solved. First, no dose-response effect is observed. Rodents and humans are able to take a small amount of hydrogen by drinking hydrogen-rich water, but marked effects are observed. Second, intestinal bacteria in humans and rodents produce a large amount of hydrogen, but an addition of a small amount of hydrogen exhibits marked effects. Further studies are required to elucidate molecular bases of prominent hydrogen effects and to determine the optimal frequency, amount, and method of hydrogen administration for each human disease. Copyright © 2012 Kinji Ohno et al.

DOI： 10.1155/2012/353152

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

Objective: Muscle-specific receptor tyrosine kinase (MuSK) antibody-positive myasthenia gravis (MG) accounts for 5%-15% of autoimmune MG. MuSK mediates the agrin-signaling pathway and also anchors the collagenic tail subunit (ColQ) of acetylcholinesterase (AChE). The exact molecular target of MuSK-immunoglobulin G (IgG), however, remains elusive. As acetylcholine receptor (AChR) deficiency is typically mild and as cholinesterase inhibitors are generally ineffective, we asked if MuSK-IgG interferes with binding of ColQ to MuSK. Methods: We used 3 assays: in vitro overlay of the human ColQ-tailed AChE to muscle sections of Colq-/- mice; in vitro plate-binding assay to quantitate binding of MuSK to ColQ and to LRP4; and passive transfer of MuSK-IgG to mice. Results: The in vitro overlay assay revealed that MuSK-IgG blocks binding of ColQ to the neuromuscular junction. The in vitro plate-binding assay showed that MuSK-IgG exerts a dosedependent block of MuSK binding to ColQ by but not to LRP4. Passive transfer of MuSK-IgG to mice reduced the size and density of ColQ tõ10% of controls and had a lesser effect on the size and density of AChR and MuSK. Conclusions: As lack of ColQ compromises agrin-mediated AChR clustering in Colq-/- mice, a similar mechanism may lead to AChR deficiency in MuSK-MG patients. Our experiments also predict partial AChE deficiency in MuSK-MG patients, but AChE is not reduced in biopsied NMJs. In humans, binding of ColQ to MuSK may be dispensable for clustering ColQ, but is required for facilitating AChR clustering. Further studies will be required to elucidate the basis of this paradox. Copyright © 2011 by AAN Enterprises, Inc.

DOI： 10.1212/WNL.0b013e318237f660

その他リンク： http://orcid.org/0000-0003-0092-7837

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 Genetics  

Autosomal dominant osteogenesis imperfecta (OI) is caused by mutations in COL1A1 or COL1A2. We identified a dominant missense mutation, c.3235G>A in COL1A1 exon 45 predicting p.G1079S, in a Japanese family with mild OI. As mutations in exon 45 exhibit mild to lethal phenotypes, we tested if disruption of an exonic splicing cis-element determines the clinical phenotype, but detected no such mutations. In the Japanese family, juvenile-onset hyperuricemia cosegregated with OI, but not in the previously reported Italian and Canadian families with c.3235G>A. After confirming lack of a founder haplotype in three families, we analyzed PRPSAP1 and PRPSAP2 as candidate genes for hyperuricemia on chr 17 where COL1A1 is located, but found no mutation. We next resequenced the whole exomes of two siblings in the Japanese family and identified variable numbers of previously reported hyperuricemia-associated SNPs in ABCG2 and SLC22A12. The same SNPs, however, were also detected in normouricemic individuals in three families. We then identified two missense SNVs in ZPBP2 and GPATCH8 on chromosome 17 that cosegregated with hyperuricemia in the Japanese family. ZPBP2 p.T69I was at the non-conserved region and was predicted to be benign by in silico analysis, whereas GPATCH8 p.A979P was at a highly conserved region and was predicted to be deleterious, which made p.A979P a conceivable candidate for juvenile-onset hyperuricemia. GPATCH8 is only 5.8 Mbp distant from COL1A1 and encodes a protein harboring an RNA-processing domain and a zinc finger domain, but the molecular functions have not been elucidated to date. © Springer-Verlag 2011.

DOI： 10.1007/s00439-011-1006-9

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  

Background: Molecular hydrogen has prominent effects on more than 30 animal models especially of oxidative stress-mediated diseases and inflammatory diseases. In addition, hydrogen effects on humans have been reported in diabetes mellitus type 2, hemodialysis, metabolic syndrome, radiotherapy for liver cancer, and brain stem infarction. Hydrogen effects are ascribed to specific radical-scavenging activities that eliminate hydroxyl radical and peroxynitrite, and also to signal-modulating activities, but the detailed molecular mechanisms still remain elusive. Hydrogen is a safe molecule that is largely produced by intestinal bacteria in rodents and humans, and no adverse effects have been documented. Methods. We performed open-label trial of drinking 1.0 liter per day of hydrogen-enriched water for 12 weeks in five patients with progressive muscular dystrophy (PMD), four patients with polymyositis/dermatomyositis (PM/DM), and five patients with mitochondrial myopathies (MM), and measured 18 serum parameters as well as urinary 8-isoprostane every 4 weeks. We next conducted randomized, double-blind, placebo-controlled, crossover trial of 0.5 liter per day of hydrogen-enriched water or placebo water for 8 weeks in 10 patients with DM and 12 patients with MM, and measured 18 serum parameters every 4 weeks. Results: In the open-label trial, no objective improvement or worsening of clinical symptoms was observed. We, however, observed significant effects in lactate-to-pyruvate ratios in PMD and MM, fasting blood glucose in PMD, serum matrix metalloproteinase-3 (MMP3) in PM/DM, and serum triglycerides in PM/DM. In the double-blind trial, no objective clinical effects were observed, but a significant improvement was detected in lactate in MM. Lactate-to-pyruvate ratios in MM and MMP3 in DM also exhibited favorable responses but without statistical significance. No adverse effect was observed in either trial except for hypoglycemic episodes in an insulin-treated MELAS patient, which subsided by reducing the insulin dose. Conclusions: Hydrogen-enriched water improves mitochondrial dysfunction in MM and inflammatory processes in PM/DM. Less prominent effects with the double-blind trial compared to the open-label trial were likely due to a lower amount of administered hydrogen and a shorter observation period, which implies a threshold effect or a dose-response effect of hydrogen. © 2011 Ito et al; licensee BioMed Central Ltd.

DOI： 10.1186/2045-9912-1-24

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  

Molecular hydrogen has been reported to be effective for a variety of disorders and its effects have been ascribed to the reduction of oxidative stress. However, we have recently demonstrated that hydrogen inhibits type I allergy through modulating intracellular signal transduction. In the present study, we examined the hydrogen effects on lipopolysaccharide/interferon γ LPS/IFNγ-induced nitric oxide (NO) production in murine macrophage RAW264 cells. Treatment with hydrogen reduced LPS/IFNγ-induced NO release, which was associated with a diminished induction of inducible isoform of nitric oxide synthase (iNOS). Hydrogen treatment inhibited LPS/IFNγ-induced phosphorylation of apoptosis signal-regulating kinase 1 (ASK1) and its downstream signaling molecules, p38 MAP kinase and JNK, as well as IκBα, but did not affect activation of NADPH oxidase and production of reactive oxygen species (ROS). As ROS is an upstream activator of ASK1, inhibition of ASK1 by hydrogen without suppressing ROS implies that a potential target molecule of hydrogen should be located at the receptor or immediately downstream of it. These results suggested a role for molecular hydrogen as a signal modulator. Finally, oral intake of hydrogen-rich water alleviated anti-type II collagen antibody-induced arthritis in mice, a model for human rheumatoid arthritis. Taken together, our studies indicate that hydrogen inhibits LPS/IFNγ-induced NO production through modulation of signal transduction in macrophages and ameliorates inflammatory arthritis in mice, providing the molecular basis for hydrogen effects on inflammation and a functional interaction between two gaseous signaling molecules, NO and molecular hydrogen. © 2011 Elsevier Inc.

DOI： 10.1016/j.bbrc.2011.06.116

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  

This report summarizes a brief description/history of the Hydrogen Research Meetings as well as key presentations/oral abstracts delivered in the most recent symposium. Additionally, we introduced 38 diseases and physiological states for which hydrogen exhibits beneficial effects. © 2011 Ohta et al; licensee BioMed Central Ltd.

DOI： 10.1186/2045-9912-1-10

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  

In pre-mRNA splicing, a conserved AG/G at the 3′-splice site is recognized by U2AF 35. A disease-causing mutation abrogating the G nucleotide at the first position of an exon (E +1) causes exon skipping in GH1, FECH and EYA1, but not in LPL or HEXA. Knockdown of U2AF 35 enhanced exon skipping in GH1 and FECH. RNA-EMSA revealed that wild-type FECH requires U2AF 35 but wild-type LPL does not. A series of artificial mutations in the polypyrimidine tracts of GH1, FECH, EYA1, LPL and HEXA disclosed that a stretch of at least 10-15 pyrimidines is required to ensure normal splicing in the presence of a mutation at E +1. Analysis of nine other disease-causing mutations at E +1 detected five splicing mutations. Our studies suggest that a mutation at the AG-dependent 3′-splice site that requires U2AF 35 for spliceosome assembly causes exon skipping, whereas one at the AG-independent 3′-splice site that does not require U2AF 35 gives rise to normal splicing. The AG-dependence of the 3′-splice site that we analyzed in disease-causing mutations at E +1 potentially helps identify yet unrecognized splicing mutations at E +1. © 2011 The Author(s).

DOI： 10.1093/nar/gkr026

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：Neurology  

Background: Plectin crosslinks intermediate filaments to their targets in different tissues. Defects in plectin cause epidermolysis bullosa simplex (EBS), muscular dystrophy (MD), and sometimes pyloric atresia. Association of EBS with a myasthenic syndrome (MyS) was documented in a single patient in 1999. OBJECTIVES:: To analyze the clinical, structural, and genetic aspects of a second and fatal case of EBS associated with a MyS and search for the genetic basis of the disease in a previously reported patient with EBS-MD-MyS. Methods: Clinical observations; histochemical, immunocytochemical, and electron microscopy studies of skeletal muscle and neuromuscular junction; and mutation analysis. Results: An African American man had EBS since early infancy, and progressive muscle weakness, hyperCKemia, and myasthenic symptoms refractory to therapy since age 3 years. Eventually he became motionless and died at age 42 years. At age 15 years, he had a marked EMG decrement, and a reduced miniature endplate potential amplitude. The myopathy was associated with dislocated muscle fiber organelles, structurally abnormal nuclei, focal plasmalemmal defects, and focal calcium ingress into muscle fibers. The neuromuscular junctions showed destruction of the junctional folds, and remodeling. Mutation analysis demonstrated a known p.Arg2319X and a novel c.12043dupG mutation in PLEC1. The EBS-MD-MyS patient reported in 1999 also carried c.12043dupG and a novel p.Gln2057X mutation. The novel mutations were absent in 200 Caucasian and 100 African American subjects. Conclusions: The MyS in plectinopathy is attributed to destruction of the junctional folds and the myopathy to defective anchoring of muscle fiber organelles and defects in sarcolemmal integrity. Copyright © 2011 by AAN Enterprises, Inc.

DOI： 10.1212/WNL.0b013e31820882bd

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  

Background: Oxidative stress is causally associated with the pathogenesis of Parkinson's disease (PD). Oxygen generates a large amount of reactive oxygen species (ROS). ROS including hydroxyl radicals and H2O2 react with guanine residues in DNA and produce 8-hydroxydeoxyguanosine (8-OHdG). 8-OHdG serves as a biomarker for oxidative stress in various diseases. Method: We investigated urinary 8-OHdG levels in 61 PD patients and 28 normal subjects to evaluate the correlation with various clinical features. We quantified disease severity using the Unified Parkinson's Disease Rating Scale for motor symptoms (UPDRS part 3), the Mini-Mental State Examination (MMSE) for mental function, and the Tottori University Hallucination Rating Scale (TUHARS) for quantifying hallucinations. Results: There were significant correlations between 8-OHdG and all the examined parameters, but the partial correlation coefficients excluding contributions of all the other parameters showed that only TUHARS and UPDRS part 3 are significantly related to 8-OHdG. In particular, TUHARS correlates best with urinary 8-OHdG levels. Conclusion: The significant correlation between urinary 8-OHdG levels and hallucinations but not with dementia suggests that hallucinations are likely to have unique but unidentified mechanisms that lead to excessive production of 8-OHdG. © 2010 Elsevier Ltd.

DOI： 10.1016/j.parkreldis.2010.11.004

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  

Molecular hydrogen ameliorates oxidative stress-associated diseases in animal models. We found that oral intake of hydrogen-rich water abolishes an immediate-type allergic reaction in mice. Using rat RBL-2H3 mast cells, we demonstrated that hydrogen attenuates phosphorylation of the FcεRI-associated Lyn and its downstream signal transduction, which subsequently inhibits the NADPH oxidase activity and reduces the generation of hydrogen peroxide. We also found that inhibition of NADPH oxidase attenuates phosphorylation of Lyn in mast cells, indicating the presence of a feed-forward loop that potentiates the allergic responses. Hydrogen accordingly inhibits all tested signaling molecule(s) in the loop. Hydrogen effects have been solely ascribed to exclusive removal of hydroxyl radical. In the immediate-type allergic reaction, hydrogen exerts its beneficial effect not by its radical scavenging activity but by modulating a specific signaling pathway. Effects of hydrogen in other diseases are possibly mediated by modulation of yet unidentified signaling pathways. Our studies also suggest that hydrogen is a gaseous signaling molecule like nitric oxide. © 2009 Elsevier Inc. All rights reserved.

DOI： 10.1016/j.bbrc.2009.09.047

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

Spinocerebellar ataxia type 10 is caused by ATTCT repeat expansion in the ATXN10 gene in humans. We studied the evolutionary history of the human genome to determine the time and mechanism of the acquisition of unstable ATTCT repeats in the genome. We found that long interspersed element-1 (LINE-1) was inserted into ATXN10 intron 9; Alu was then inserted in the middle of LINE-1; and endogenous retrovilcus K was lastly retrotransposed in the middle of Alu. The ATTCT repeat was located on the boundary between the 3′-end of the Alu element and the direct repeat arising from LINE-1. We determined nucleotide sequences of the orthologous region of 50 individuals representing 33 primate species and compared them with the human sequence. The analysis revealed that the ATTCT repeat is present only in human and apes. Old World monkeys also possess pentanucleotide repeats, but their motifs are TGTCT and GGTCT. New World monkeys and prosimians are not informative because they lack the corresponding region in ATXN10 intron 9. Our studies dictate two parsimonious scenarios of evolution. First, a TTTCT motif arose from a TTTTT motif at the junction of Alu and LINE-1, which was followed by introduction of A to make an ATTCT motif in hominoids. Second, an ATTCT motif was directly generated from an ancestral ATTTT motif in the common ancestor of catarrhines. We also demonstrate that orangutan uniquely introduced G to make a GTTCT motif and later C to make a GTTCC motif, where newly introduced nucleotides are underlined. Our studies reveal that nucleotide substitutions in a poly(A) tail of the Alu element and the following amplification of pentanucleotides occurred in the lineages of Old World monkeys and hominoids and that unstable ATTCT pentanucleotide repeats originated in the common ancestor of hominoids. These findings also highlight a new aspect of the role of retrotransposons in human disease and evolution, which might be useful in investigating the mystery of human uniqueness.

DOI： 10.1093/molbev/msp172

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

Background: Pathogenic mutations in rapsyn result in endplate acetylcholine receptor (AChR) deficiency and are a common cause of postsynaptic congenital myasthenic syndromes. Methods: Clinical, electrophysiologic, pathologic, and molecular studies were done in 39 patients. Results: In all but one patient, the disease presented in the first 2 years of life. In 9 patients, the myasthenic symptoms included constant or episodic ophthalmoparesis, and 1 patient had a pure limb-girdle phenotype. More than one-half of the patients experienced intermittent exacerbations. Long-term follow-up was available in 25 patients after start of cholinergic therapy: 21 became stable or were improved and 2 of these became asymptomatic; 3 had a progressive course; and 1 died in infancy. In 7 patients who had endplate studies, the average counts of AChR per endplate and the synaptic response to ACh were less reduced than in patients harboring low AChR expressor mutations. Eight patients were homozygous and 23 heterozygous for the common p.N88K mutation. Six mutations, comprising 3 missense mutations, an in-frame deletion, a splice-site mutation, and a nonsense mutation, are novel. Homozygosity for p.N88K was associated with varying grades of severity. No genotype-phenotype correlations were observed except in 8 Near-Eastern patients homozygous for the promoter mutation (c.-38A>G), who had a mild course. Conclusions: All but 1 patient presented early in life and most responded to cholinergic agonists. With early diagnosis and therapy, rapsyn deficiency has a benign course in most patients. There was no consistent phenotype- genotype correlation except for an E-box mutation associated with jaw deformities. Copyright © by AAN Enterprises, Inc.

DOI： 10.1212/WNL.0b013e3181ae7cbc

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

Molecular hydrogen serves as an antioxidant that reduces hydroxyl radicals, but not the other reactive oxygen and nitrogen species. In the past year, molecular hydrogen has been reported to prevent or ameliorate eight diseases in rodents and one in human associated with oxidative stress. In Parkinson's disease, mitochondrial dysfunction and the associated oxidative stress are major causes of dopaminergic cell loss in the substantia nigra. We examined effects of ∼50%-saturated molecular hydrogen in drinking water before or after the stereotactic surgery on 6-hydroxydopamine-induced nigrostrital degeneration in a rat model of Parkinson's disease. Methamphetamine-induced behavioral analysis showed that molecular hydrogen prevented both the development and progression of the nigrostrital degeneration. Tyrosine hydroxylase staining of the substantia nigra and striatum also demonstrated that pre- and post-treatment with hydrogen prevented the dopaminergic cell loss. Our studies suggest that hydrogen water is likely able to retard the development and progression of Parkinson's disease. © 2009 Elsevier Ireland Ltd. All rights reserved.

DOI： 10.1016/j.neulet.2009.02.016

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  

We recently reported that the intronic splice-site mutation IVS3-8G>A of CHRNA1 that encodes the muscle nicotinic acetylcholine receptor α subunit disrupts binding of a splicing repressor, hnRNP H. This, in turn, results in exclusive inclusion of the downstream exon P3A. The P3A(+) transcript encodes a non-functional α subunit that comprises 50% of the transcripts in normal human skeletal muscle, but its functional significance remains undetermined. In an effort to search for a potential therapy, we screened off-label effects of 960 bioactive chemical compounds and found that tannic acid ameliorates the aberrant splicing due to IVS3-8G>A but without altering the expression of hnRNP H. Therefore, we searched for another splicing trans -factor. We found that the polypyrimidine tract binding protein (PTB) binds close to the 3′ end of CHRNA1 intron 3, that PTB induces skipping of exon P3A and that tannic acid increases the expression of PTB in a dose-dependent manner. Deletion assays of the PTB promoter region revealed that the tannic acid-responsive element is between positions -232 and -74 from the translation initiation site. These observations open the door to the discovery of novel therapies based on PTB overexpression and to detecting possible untoward effects of the overexpression. © The Author 2009. Published by Oxford University Press. All rights reserved.

DOI： 10.1093/hmg/ddp023

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  

Spinocerebellar ataxia type 10 (SCA10) is an autosomal dominant neurodegenerative disease characterized by cerebellar ataxia and seizures. The disease is caused by a large ATTCT repeat expansion in the ATXN10 gene. The first families reported with SCA10 were of Mexican origin, but the disease was soon after described in Brazilian families of mixed Portuguese and Amerindian ancestry. The origin of the SCA10 expansion and a possible founder effect that would account for its geographical distribution have been the source of speculation over the last years. To unravel the mutational origin and spread of the SCA10 expansion, we performed an extensive haplotype study, using closely linked STR markers and intragenic SNPs, in families from Brazil and Mexico. Our results showed (1) a shared disease haplotype for all Brazilian and one of the Mexican families, and (2) closely-related haplotypes for the additional SCA10 Mexican families; (3) little or null genetic distance in small normal alleles of different repeat sizes, from the same SNP lineage, indicating that they are being originated by a single step mechanism; and (4) a shared haplotype for pure and interrupted expanded alleles, pointing to a gene conversion model for its generation. In conclusion, we show evidence for an ancestral common origin for SCA10 in Latin America, which might have arisen in an ancestral Amerindian population and later have been spread into the mixed populations of Mexico and Brazil. © 2009 Almeida et al.

DOI： 10.1371/journal.pone.0004553

掲載種別：研究論文（学術雑誌）  

DOI： 10.1016/j.cbi.2008.09.024

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

DOI： 10.1007/s10048-007-0117-x

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

Myotonic dystrophy type 2 (DM2) is caused by expansion of a tetranucleotide CCTG repeat in intron 1 of the ZNF9 gene on chromosome 3q21. All studied DM2 mutations have been reported in Caucasians and share an identical haplotype, suggesting a common founder. We identified a Japanese patient with DM2 and showed that the affected haplotype is distinct from the previously identified DM2 haplotype shared among Caucasians. These data strongly suggest that DM2 expansion mutations originate from separate founders in Europe and Japan and are more widely distributed than previously recognized. © 2007 Springer-Verlag.

DOI： 10.1007/s10048-007-0110-4

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  

We developed a simple algorithm, i-Score (inhibitory-Score), to predict active siRNAs by applying a linear regression model to 2431 siRNAs. Our algorithm is exclusively comprised of nucleotide (nt) preferences at each position, and no other parameters are taken into account. Using a validation dataset comprised of 419 siRNAs, we found that the prediction accuracy of i-Score is as good as those of s-Biopredsi, ThermoComposition21 and DSIR, which employ a neural network model or more parameters in a linear regression model. Reynolds and Katoh also predict active siRNAs efficiently, but the numbers of siRNAs predicted to be active are less than one-eighth of that of i-Score. We additionally found that exclusion of thermostable siRNAs, whose whole stacking energy (ΔG) is less than -34.6 kcal/mol, improves the prediction accuracy in i-Score, s-Biopredsi, ThermoComposition21 and DSIR. We also developed a universal target vector, pSELL, with which we can assay an siRNA activity of any sequence in either the sense or antisense direction. We assayed 86 siRNAs in HEK293 cells using pSELL, and validated applicability of i-Score and the whole ΔG value in designing siRNAs. © 2007 The Author(s).

DOI： 10.1093/nar/gkm699

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 Immunology  

Mast cells are pivotal effector cells in IgE-mediated allergic reactions. GATA transcriptional factors such as GATA-1 and GATA-2 are expressed in mast cells, and recent studies have revealed that both GATA-1 and GATA-2 are required for mast cell development. However, the role of GATA transcriptional factors in differentiated mast cells has remained largely unknown. In this study, we repressed the activity of GATA-1 and GATA-2 by using three different approaches (inducible overexpression of a dominant-negative form of GATA, pharmacological inactivation, or small interfering RNA technology), and analyzed the molecular mechanisms of GATA transcriptional factors in the activation of mast cells. Surprisingly, the repression of GATA activity in differentiated mast cells led to the impairment of cell survival, IgE-induced degranulation, and cytokine production. Signal transduction and histone modification in the chromatin related to protein kinase Cβ were defective in these cells. These results identify that GATA has a critical role in the activation of mast cell. Copyright © 2006 by The American Association of Immunologists, Inc.

DOI： 10.4049/jimmunol.178.1.360

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  

We have found that two previously reported exonic mutations in the PINK1 and PARK7 genes affect pre-mRNA splicing. To develop an algorithm to predict underestimated splicing consequences of exonic mutations at the 5′ splice site, we constructed and analyzed 31 minigenes carrying exonic splicing mutations and their derivatives. We also examined 189 249 U2-dependent 5′ splice sites of the entire human genome and found that a new variable, the SD-Score, which represents a common logarithm of the frequency of a specific 5′ splice site, efficiently predicts the splicing consequences of these minigenes. We also employed the information contents (Ri) to improve the prediction accuracy. We validated our algorithm by analyzing 32 additional minigenes as well as 179 previously reported splicing mutations. The SD-Score algorithm predicted aberrant splicings in 198 of 204 sites (sensitivity =97.1%) and normal splicings in 36 of 38 sites (specificity = 94.7%). Simulation of all possible exonic mutations at positions -3, -2 and -1 of the 189 249 sites predicts that 37.8, 88.8 and 96.8% of these mutations would affect pre-mRNA splicing, respectively. We propose that the SD-Score algorithm is a practical tool to predict splicing consequences of mutations affecting the 5′ splice site. © 2007 The Author(s).

DOI： 10.1093/nar/gkm647

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：Neuro-Ophthalmology Japan  

Congenital myasthenic syndromes (CMS) are caused by genetic defects of molecules expressed at the neuromuscular junction. Mutations identified to date include (1) MuSK, a receptor for neural agrin that plays a pivotal role in formation of the neuromuscular junction, (2) choline acetyltransferase that resynthesizes acetylcholine at the nerve terminal, (3) collagen Q that anchors acetylcholinesterase at the synaptic basal lamina, (4) rapsyn that clusters acetylcholine receptor (AChR) at the endplate, (5) AChR subunits, and (6) skeletal muscle voltage-gated sodium channel (Na v1.4). Mutations in the AChR subunit genes cause slow-channel syndrome in which channel opening events are prolonged, fast-channel syndrome that is characterized by short channel opening events, and/or endplate AChR deficiency. Slow channel syndrome is an autosomal dominant disorder, whereas the others are all caused by autosomal recessive mutations. Adult-onset cases of CMS are most commonly observed in slow channel syndrome, but are also observed in other types of CMS. CMS are frequently associated with muscle atrophy, and/or mild orofacial anomalies. CMS should be considered in diagnosing not only myasthenia gravis but also other types of myopathies. Low- and high-frequency repetitive nerve stimulations to any patient that exhibits muscle weakness are highly recommended for diagnosing CMS.

掲載種別：研究論文（学術雑誌）   出版者・発行元：Acta Myologica  

記述言語：英語   掲載種別：研究論文（学術雑誌）  

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 medical genetics  

DOI： 10.1136/jmg.2004.026682

掲載種別：研究論文（学術雑誌）   出版者・発行元：Human genetics  

記述言語：英語   掲載種別：研究論文（学術雑誌）  

記述言語：英語   掲載種別：研究論文（学術雑誌）  

記述言語：英語   掲載種別：研究論文（学術雑誌）  

記述言語：英語   掲載種別：研究論文（学術雑誌）  

掲載種別：研究論文（学術雑誌）   出版者・発行元：Human genetics  

掲載種別：研究論文（学術雑誌）   出版者・発行元：Human genetics  

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

We trace the cause of congenital myasthenic syndromes in two patients to mutations in the ε subunit of the muscle acetylcholine receptor (AChR). Both patients harbour deletion of an asparagine residue in the ε subunit (sN436del) at the C-terminus of the cytoplasmic loop linking the third (M3) and fourth (M4) transmembrane domains. The presence of a null mutation in the second allele of the ε subunit shows that εN346del determines the phenotype. Endplate studies show markedly reduced expression of the εN346del-AChR and compensatory accumulation of fetal γ-AChR. Expression studies in HEK cells reveal decreased expression of γN436del-AChR and abnormally brief channel openings. Thus, neuromuscular transmission is compromised by AChR deficiency, fast channel kinetics of the εN346del-AChR and incomplete phenotypic rescue by γ-AChR. Single-channel kinetic analysis shows that the εN436del shortens channel openings by reducing stability of the diliganded receptor: rates of channel closing and of ACh dissociation are increased and the rate of channel opening is decreased. In addition to shortening the M3-M4 loop, εN436del shifts a negatively charged aspartic acid residue adjacent to M4; the effects of εN436del are shown to result from shortening of the M3-M4 loop and not from juxtaposition of a negative charge to M4. To determine whether the consequences of εN346del are subunit-specific, we deleted residues that align with εN436 in β, δ and α subunits. Each deletion mutant reduces AChR expression, but whereas the β and δ mutants curtail channel open duration, the α mutant strikingly prolongs open duration. Kinetic analysis reveals that the α mutant increases the stability of the diliganded receptor: rates of channel closing and of ACh dissociation are decreased and the rate of channel opening is increased. The overall studies reveal subunit asymmetry in the contributions of the M3-M4 loops in optimizing AChR activation through allosteric links to the channel and the agonist binding site.

DOI： 10.1093/brain/awh364

掲載種別：研究論文（学術雑誌）   出版者・発行元：Advances in Molecular and Cell Biology  

DOI： 10.1016/S1569-2558(03)32005-3

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

DOI： 10.1212/01.WNL.0000125253.84724.B8

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  

DOI： 10.1038/sj.emboj.7600221

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 Biological Chemistry  

DOI： 10.1074/jbc.M305462200

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

DOI： 10.1016/s0960-8966(03)00241-4

記述言語：英語   掲載種別：研究論文（学術雑誌）  

DOI： 10.1136/jmg.2003.012245

記述言語：英語   掲載種別：研究論文（学術雑誌）  

DOI： 10.1093/hmg/ddg334

記述言語：英語   掲載種別：研究論文（学術雑誌）  

DOI： 10.1016/s0960-8966(03)00210-4

記述言語：英語   掲載種別：研究論文（学術雑誌）  

DOI： 10.1073/pnas.1230273100

記述言語：英語   掲載種別：研究論文（学術雑誌）  

DOI： 10.1023/B:NEUR.0000020639.22895.28

記述言語：英語   掲載種別：研究論文（学術雑誌）  

DOI： 10.1038/nrn1101

記述言語：英語   掲載種別：研究論文（学術雑誌）  

DOI： 10.1172/JCI16997

記述言語：英語   掲載種別：研究論文（学術雑誌）  

DOI： 10.1016/s1090-3798(03)00078-3

記述言語：英語   掲載種別：研究論文（学術雑誌）  

DOI： 10.1212/01.wnl.0000042422.87384.2f

記述言語：英語   掲載種別：研究論文（学術雑誌）  

DOI： 10.1385/MN:26:2-3:347

記述言語：英語   掲載種別：研究論文（学術雑誌）  

記述言語：英語   掲載種別：研究論文（学術雑誌）  

DOI： 10.1086/339465

記述言語：英語   掲載種別：研究論文（学術雑誌）  

DOI： 10.1212/wnl.58.4.603

記述言語：英語   掲載種別：研究論文（学術雑誌）  

DOI： 10.1007/s11910-002-0057-7

記述言語：英語   掲載種別：研究論文（学術雑誌）  

掲載種別：研究論文（学術雑誌）  

記述言語：英語   掲載種別：研究論文（学術雑誌）  

DOI： 10.1073/pnas.98.4.2017

記述言語：英語   掲載種別：研究論文（学術雑誌）  

DOI： 10.1085/jgp.116.3.449

記述言語：英語   掲載種別：研究論文（学術雑誌）  

DOI： 10.1002/1531-8249(200002)47:2<162::AID-ANA5>3.0.CO;2-Q

記述言語：英語   掲載種別：研究論文（学術雑誌）  

DOI： 10.1172/JCI8179

掲載種別：研究論文（学術雑誌）  

記述言語：英語   掲載種別：研究論文（学術雑誌）  

DOI： 10.1086/302551

記述言語：英語   掲載種別：研究論文（学術雑誌）  

DOI： 10.1016/s0960-8966(99)00007-3

記述言語：英語   掲載種別：研究論文（学術雑誌）  

DOI： 10.1038/6326

記述言語：英語   掲載種別：研究論文（学術雑誌）  

DOI： 10.1001/archneur.56.2.163

記述言語：英語   掲載種別：研究論文（学術雑誌）  

DOI： 10.1002/ana.410440214

記述言語：英語   掲載種別：研究論文（学術雑誌）  

DOI： 10.1073/pnas.95.16.9654

記述言語：英語   掲載種別：研究論文（学術雑誌）  

DOI： 10.1097/00001756-199806010-00044

記述言語：英語   掲載種別：研究論文（学術雑誌）  

DOI： 10.1111/j.1749-6632.1998.tb10921.x

記述言語：英語   掲載種別：研究論文（学術雑誌）  

DOI： 10.1111/j.1749-6632.1998.tb10926.x

記述言語：英語   掲載種別：研究論文（学術雑誌）  

DOI： 10.1111/j.1749-6632.1998.tb10925.x

記述言語：英語   掲載種別：研究論文（学術雑誌）  

DOI： 10.1111/j.1749-6632.1998.tb10928.x

記述言語：英語   掲載種別：研究論文（学術雑誌）  

DOI： 10.1016/S0928-4257(98)80147-2

記述言語：英語   掲載種別：研究論文（学術雑誌）  

DOI： 10.1016/s0896-6273(00)80996-4

記述言語：英語   掲載種別：研究論文（学術雑誌）  

DOI： 10.1523/JNEUROSCI.17-15-05651.1997

記述言語：英語   掲載種別：研究論文（学術雑誌）  

DOI： 10.1085/jgp.109.6.757

記述言語：英語   掲載種別：研究論文（学術雑誌）  

DOI： 10.1093/hmg/6.5.753

記述言語：英語   掲載種別：研究論文（学術雑誌）  

DOI： 10.1002/ana.410400521

記述言語：英語   掲載種別：研究論文（学術雑誌）  

DOI： 10.1093/hmg/5.9.1217

記述言語：英語   掲載種別：研究論文（学術雑誌）  

DOI： 10.1016/s0896-6273(00)80289-5

記述言語：英語   掲載種別：研究論文（学術雑誌）  

DOI： 10.1002/ana.410390612

掲載種別：研究論文（学術雑誌）  

記述言語：英語   掲載種別：研究論文（学術雑誌）  

DOI： 10.1016/0896-6273(95)90080-2

記述言語：英語   掲載種別：研究論文（学術雑誌）  

DOI： 10.1073/pnas.92.3.758

掲載種別：研究論文（学術雑誌）  

掲載種別：研究論文（学術雑誌）  

掲載種別：研究論文（学術雑誌）  

記述言語：英語   掲載種別：研究論文（学術雑誌）  

DOI： 10.1016/0006-291X(91)92014-B

掲載種別：研究論文（学術雑誌）  

記述言語：英語   掲載種別：研究論文（学術雑誌）  

DOI： 10.1016/S0006-291X(05)80276-1

掲載種別：研究論文（学術雑誌）  

掲載種別：研究論文（学術雑誌）  

掲載種別：研究論文（学術雑誌）  

掲載種別：研究論文（学術雑誌）  

掲載種別：研究論文（学術雑誌）  

掲載種別：研究論文（学術雑誌）  

掲載種別：研究論文（学術雑誌）  

掲載種別：研究論文（学術雑誌）  

記述言語：英語   掲載種別：研究論文（学術雑誌）  

DOI： 10.1016/0006-291X(90)92166-W

掲載種別：研究論文（学術雑誌）  

掲載種別：研究論文（学術雑誌）  

掲載種別：研究論文（学術雑誌）  

記述言語：英語   掲載種別：研究論文（学術雑誌）  

DOI： 10.1007/BF01799242

記述言語：英語   掲載種別：研究論文（学術雑誌）  

DOI： 10.1016/0006-291X(88)90272-0

記述言語：英語

記述言語：英語

記述言語：英語

記述言語：英語

記述言語：英語

記述言語：英語

記述言語：英語

記述言語：英語

記述言語：英語

記述言語：英語

記述言語：英語

記述言語：英語

記述言語：英語

記述言語：英語

記述言語：英語

記述言語：英語

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記述言語：英語

開催年月日： 2016年10月

記述言語：英語   会議種別：口頭発表（招待・特別）  

開催年月日： 2016年9月

記述言語：英語   会議種別：口頭発表（招待・特別）  

開催年月日： 2015年9月 - 2015年10月

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開催年月日： 2013年6月

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開催年月日： 2013年5月

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開催年月日： 2013年3月

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開催年月日： 2012年9月

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開催年月日： 2012年6月

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開催年月日： 2009年7月

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開催年月日： 2005年8月

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開催年月日： 2005年7月

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開催年月日： 2005年7月

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開催年月日： 1998年8月 - 1998年9月

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開催年月日： 1998年8月 - 1998年9月

記述言語：英語   会議種別：口頭発表（招待・特別）  

開催年月日： 1998年8月 - 1998年9月

記述言語：英語   会議種別：口頭発表（招待・特別）  

開催年月日： 1994年7月

記述言語：英語   会議種別：口頭発表（招待・特別）