Orai1–STIM1 Regulates Increased Ca²⁺ Mobilization, Leading to Contractile Duchenne Muscular Dystrophy Phenotypes in Patient-Derived Induced Pluripotent Stem Cells

デュシェンヌ型筋ジストロフィーにおける筋収縮力低下のメカニズムの一端を解明. 京都大学プレスリリース. 2021-11-05.Ca²⁺ overload is one of the factors leading to Duchenne muscular dystrophy (DMD) pathogenesis. However, the molecular targets of dystrophin deficiency-dependent Ca²⁺ overload and the correlation between Ca²⁺ overload and contractile DMD phenotypes in in vitro human models remain largely elusive. In this study, we utilized DMD patient-derived induced pluripotent stem cells (iPSCs) to differentiate myotubes using doxycycline-inducible MyoD overexpression, and searched for a target molecule that mediates dystrophin deficiency-dependent Ca²⁺ overload using commercially available chemicals and siRNAs. We found that several store-operated Ca²⁺ channel (SOC) inhibitors effectively prevented Ca²⁺+ overload and identified that STIM1–Orai1 is a molecular target of SOCs. These findings were further confirmed by demonstrating that STIM1–Orai1 inhibitors, CM4620, AnCoA4, and GSK797A, prevented Ca²⁺+ overload in dystrophic myotubes. Finally, we evaluated CM4620, AnCoA4, and GSK7975A activities using a previously reported model recapitulating a muscle fatigue-like decline in contractile performance in DMD. All three chemicals ameliorated the decline in contractile performance, indicating that modulating STIM1–Orai1-mediated Ca²⁺+ overload is effective in rescuing contractile phenotypes. In conclusion, SOCs are major contributors to dystrophin deficiency-dependent Ca²⁺+ overload through STIM1–Orai1 as molecular mediators. Modulating STIM1–Orai1 activity was effective in ameliorating the decline in contractile performance in DMD

A muscle fatigue-like contractile decline was recapitulated using skeletal myotubes from Duchenne muscular dystrophy patient-derived iPSCs

デュシェンヌ型筋ジストロフィー患者由来iPS細胞を用いて、筋疲労に似た収縮力低下を培養細胞で再現する事に成功. 京都大学プレスリリース. 2021-06-07.Stopping muscles fatigue. 京都大学プレスリリース. 2021-06-21.Duchenne muscular dystrophy (DMD) is a muscle degenerating disease caused by dystrophin deficiency, for which therapeutic options are limited. To facilitate drug development, it is desirable to develop in vitro disease models that enable the evaluation of DMD declines in contractile performance. Here, we show MYOD1-induced differentiation of hiPSCs into functional skeletal myotubes in vitro with collagen gel and electrical field stimulation (EFS). Long-term EFS training (0.5 Hz, 20 V, 2 ms, continuous for 2 weeks) mimicking muscle overuse recapitulates declines in contractile performance in dystrophic myotubes. A screening of clinically relevant drugs using this model detects three compounds that ameliorate this decline. Furthermore, we validate the feasibility of adapting the model to a 96-well culture system using optogenetic technology for large-scale screening. Our results support a disease model using patient-derived iPSCs that allows for the recapitulation of the contractile pathogenesis of DMD and a screening strategy for drug development

The whole blood transcriptional regulation landscape in 465 COVID-19 infected samples from Japan COVID-19 Task Force

「コロナ制圧タスクフォース」COVID-19患者由来の血液細胞における遺伝子発現の網羅的解析 --重症度に応じた遺伝子発現の変化には、ヒトゲノム配列の個人差が影響する--. 京都大学プレスリリース. 2022-08-23.Coronavirus disease 2019 (COVID-19) is a recently-emerged infectious disease that has caused millions of deaths, where comprehensive understanding of disease mechanisms is still unestablished. In particular, studies of gene expression dynamics and regulation landscape in COVID-19 infected individuals are limited. Here, we report on a thorough analysis of whole blood RNA-seq data from 465 genotyped samples from the Japan COVID-19 Task Force, including 359 severe and 106 non-severe COVID-19 cases. We discover 1169 putative causal expression quantitative trait loci (eQTLs) including 34 possible colocalizations with biobank fine-mapping results of hematopoietic traits in a Japanese population, 1549 putative causal splice QTLs (sQTLs; e.g. two independent sQTLs at TOR1AIP1), as well as biologically interpretable trans-eQTL examples (e.g., REST and STING1), all fine-mapped at single variant resolution. We perform differential gene expression analysis to elucidate 198 genes with increased expression in severe COVID-19 cases and enriched for innate immune-related functions. Finally, we evaluate the limited but non-zero effect of COVID-19 phenotype on eQTL discovery, and highlight the presence of COVID-19 severity-interaction eQTLs (ieQTLs; e.g., CLEC4C and MYBL2). Our study provides a comprehensive catalog of whole blood regulatory variants in Japanese, as well as a reference for transcriptional landscapes in response to COVID-19 infection

DOCK2 is involved in the host genetics and biology of severe COVID-19

「コロナ制圧タスクフォース」COVID-19疾患感受性遺伝子DOCK2の重症化機序を解明 --アジア最大のバイオレポジトリーでCOVID-19の治療標的を発見--. 京都大学プレスリリース. 2022-08-10.Identifying the host genetic factors underlying severe COVID-19 is an emerging challenge. Here we conducted a genome-wide association study (GWAS) involving 2, 393 cases of COVID-19 in a cohort of Japanese individuals collected during the initial waves of the pandemic, with 3, 289 unaffected controls. We identified a variant on chromosome 5 at 5q35 (rs60200309-A), close to the dedicator of cytokinesis 2 gene (DOCK2), which was associated with severe COVID-19 in patients less than 65 years of age. This risk allele was prevalent in East Asian individuals but rare in Europeans, highlighting the value of genome-wide association studies in non-European populations. RNA-sequencing analysis of 473 bulk peripheral blood samples identified decreased expression of DOCK2 associated with the risk allele in these younger patients. DOCK2 expression was suppressed in patients with severe cases of COVID-19. Single-cell RNA-sequencing analysis (n = 61 individuals) identified cell-type-specific downregulation of DOCK2 and a COVID-19-specific decreasing effect of the risk allele on DOCK2 expression in non-classical monocytes. Immunohistochemistry of lung specimens from patients with severe COVID-19 pneumonia showed suppressed DOCK2 expression. Moreover, inhibition of DOCK2 function with CPYPP increased the severity of pneumonia in a Syrian hamster model of SARS-CoV-2 infection, characterized by weight loss, lung oedema, enhanced viral loads, impaired macrophage recruitment and dysregulated type I interferon responses. We conclude that DOCK2 has an important role in the host immune response to SARS-CoV-2 infection and the development of severe COVID-19, and could be further explored as a potential biomarker and/or therapeutic target

A human iPS cell myogenic differentiation system permitting high-throughput drug screening

Muscular dystrophy is a disease characterized by progressive muscle weakness and degeneration. There are currently no available treatments for most muscular diseases, such as muscular dystrophy. Moreover, current therapeutics are focused on improving the quality of life of patients by relieving the symptoms or stress caused by the disease. Although the causative genes for many muscular diseases have been identified, the mechanisms underlying their pathogenesis remain unclear. Patient-derived induced pluripotent stem cells (iPSCs) have become a powerful tool for understanding the pathogenesis of intractable diseases, as well as for phenotype screening, which can serve as the basis for developing new drugs. However, it is necessary to develop an efficient and reproducible myogenic differentiation system. Previously, we reported a tetracycline-inducible MyoD overexpression model of myogenic differentiation using human iPSCs (hiPSCs). However, this model has certain disadvantages that limit its use in various applications, such as a drug screening. In this study, we developed an efficient and reproducible myogenic differentiation system by further modifying our previous protocol. The new protocol achieves efficient differentiation of feeder-free hiPSCs to myogenic cells via small-scale culture in six-well microplates to large-scale culture in 384-well microplates for high-throughput applications

Beta3Gn-T7 Is a Keratan Sulfate β1,3 N-Acetylglucosaminyltransferase in the Adult Brain

International audienceKeratan sulfate (KS) glycan is covalently attached to a core protein of proteoglycans. KS is abundant in neuropils and presents densely in close proximity to the perineuronal region of the perineuronal net-positive neurons in the adult brain under physiological conditions. We previously showed that the synthesis of KS positive for the R-10G antibody in the adult brain is mediated by GlcNAc-6-sulfotransferase 3 (GlcNAc6ST3; encoded by Chst5). Deficiency in both GlcNAc6ST3 and GlcNAc6ST1, encoded by Chst2, completely abolished KS. Protein-tyrosine phosphatase receptor type z1 (Ptprz1)/phosphacan was identified as a KS scaffold. KS requires the extension of GlcNAc by β1,3 N-acetylglucosaminyltransferase (Beta3Gn-T). Members of the Beta3Gn-T family involved in the synthesis of adult brain KS have not been identified. In this study, we show by a method of gene targeting that Beta3Gn-T7, encoded by B3gnt7, is a major Beta3Gn-T for the synthesis of KS in neuropils and the perineuronal region in the adult brain. Intriguingly, the B3gnt7 gene is selectively expressed in oligodendrocyte precursor cells (OPCs) and oligodendrocytes similar to that of GlcNAc6ST3. These results indicate that Beta3Gn-T7 in oligodendrocyte lineage cells may play a role in the formation of neuropils and perineuronal nets in the adult brain through the synthesis of R-10G-positive KS-modified proteoglycan

GlcNAc6ST2/CHST4 Is Essential for the Synthesis of R-10G-Reactive Keratan Sulfate/Sulfated <i>N</i>-Acetyllactosamine Oligosaccharides in Mouse Pleural Mesothelium

We recently showed that 6-sulfo sialyl N-acetyllactosamine (LacNAc) in O-linked glycans recognized by the CL40 antibody is abundant in the pleural mesothelium under physiological conditions and that these glycans undergo complementary synthesis by GlcNAc6ST2 (encoded by Chst4) and GlcNAc6ST3 (encoded by Chst5) in mice. GlcNAc6ST3 is essential for the synthesis of R-10G-positive keratan sulfate (KS) in the brain. The predicted minimum epitope of the R-10G antibody is a dimeric asialo 6-sulfo LacNAc. Whether R-10G-reactive KS/sulfated LacNAc oligosaccharides are also present in the pleural mesothelium was unknown. The question of which GlcNAc6STs are responsible for R-10G-reactive glycans was an additional issue to be clarified. Here, we show that R-10G-reactive glycans are as abundant in the pulmonary pleura as CL40-reactive glycans and that GlcNAc6ST3 is only partially involved in the synthesis of these pleural R-10G glycans, unlike in the adult brain. Unexpectedly, GlcNAc6ST2 is essential for the synthesis of R-10G-positive KS/sulfated LacNAc oligosaccharides in the lung pleura. The type of GlcNAc6ST and the magnitude of its contribution to KS glycan synthesis varied among tissues in vivo. We show that GlcNAc6ST2 is required and sufficient for R-10G-reactive KS synthesis in the lung pleura. Interestingly, R-10G immunoreactivity in KSGal6ST (encoded by Chst1) and C6ST1 (encoded by Chst3) double-deficient mouse lungs was markedly increased. MUC16, a mucin molecule, was shown to be a candidate carrier protein for pleural R-10G-reactive glycans. These results suggest that R-10G-reactive KS/sulfated LacNAc oligosaccharides may play a role in mesothelial cell proliferation and differentiation. Further elucidation of the functions of sulfated glycans synthesized by GlcNAc6ST2 and GlcNAc6ST3, such as R-10G and CL40 glycans, in pathological conditions may lead to a better understanding of the underlying mechanisms of the physiopathology of the lung mesothelium

Complementary Role of GlcNAc6ST2 and GlcNAc6ST3 in Synthesis of CL40-Reactive Sialylated and Sulfated Glycans in the Mouse Pleural Mesothelium

International audienceSialyl 6-sulfo Lewis X (6-sulfo sLeX) and its derivative sialyl 6-sulfo N-acetyllactosamine (LacNAc) are sialylated and sulfated glycans of sialomucins found in the high endothelial venules (HEVs) of secondary lymphoid organs. A component of 6-sulfo sLeX present in the core 1-extended O-linked glycans detected by the MECA-79 antibody was previously shown to exist in the lymphoid aggregate vasculature and bronchial mucosa of allergic and asthmatic lungs. The components of 6-sulfo sLeX in pulmonary tissues under physiological conditions remain to be analyzed. The CL40 antibody recognizes 6-sulfo sLeX and sialyl 6-sulfo LacNAc in O-linked and N-linked glycans, with absolute requirements for both GlcNAc-6-sulfation and sialylation. Immunostaining of normal mouse lungs with CL40 was performed and analyzed. The contribution of GlcNAc-6-O-sulfotransferases (GlcNAc6STs) to the synthesis of the CL40 epitope in the lungs was also elucidated. Here, we show that the expression of the CL40 epitope was specifically detected in the mesothelin-positive mesothelium of the pulmonary pleura. Moreover, GlcNAc6ST2 (encoded by Chst4) and GlcNAc6ST3 (encoded by Chst5), but not GlcNAc6ST1 (encoded by Chst2) or GlcNAc6ST4 (encoded by Chst7), are required for the synthesis of CL40-positive glycans in the lung mesothelium. Furthermore, neither GlcNAc6ST2 nor GlcNAc6ST3 is sufficient for in vivo expression of the CL40 epitope in the lung mesothelium, as demonstrated by GlcNAc6ST1/3/4 triple-knock-out and GlcNAc6ST1/2/4 triple-knock-out mice. These results indicate that CL40-positive sialylated and sulfated glycans are abundant in the pleural mesothelium and are synthesized complementarily by GlcNAc6ST2 and GlcNAc6ST3, under physiological conditions in mice

GlcNAc6ST3 is a keratan sulfate sulfotransferase for the protein-tyrosine phosphatase PTPRZ in the adult brain

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Additional file 2: Figure S1. of Pigment Epithelium-Derived Factor (PEDF) mediates cartilage matrix loss in an age-dependent manner under inflammatory conditions

Immunofluorescence controls. The supplemental figure includes images of sections stained in parallel with each respective staining protocol which received no primary antibody. (PPTX 2875 kb