Project description:Duchenne muscular dystrophy (DMD) is a fatal X-linked disease caused by mutations in the dystrophin (DMD) gene, leading to the complete absence of DMD and progressive degeneration of skeletal and heart muscles. Expression of an internally shortened dystrophin in DMD subjects (DMDΔ52) can be achieved by skipping DMD exon 51 to reframe the transcript. To predict the best possible outcome of this therapeutic strategy, we generated transgenic pigs lacking DMD exon 51 and 52, additionally representing a new model for Becker muscular dystrophy (BMD). To inspect the proteome alterations caused by the different dystrophin mutations in an unbiased and comprehensive manner, we performed a label-free liquid chromatography-tandem mass spectrometry analysis (LC-MS/MS) of myocardial and skeletal muscle samples from wild-type (WT), DMDΔ52 and DMDΔ51-52 pigs.
Project description:Duchenne muscular dystrophy (DMD) is a fatal muscle-wasting disorder caused by mutations in the Dystrophin gene with no therapeutic option. To bridge the gap between preclinical and therapeutic evaluation studies, we have generated a rat model for DMD that carries an exon 52 deletion (R DMDdel52) causing a complete lack of dystrophin protein. Here we show that R DMDdel52 animals recapitulated human DMD pathophysiological trajectory more faithfully than the mdx mouse model. We report that R DMDdel52 rats displayed progressive and severe skeletal muscle loss associated with fibrotic deposition, fat infiltration and fibre type switch. Early fibrosis was also apparent in the cardiac muscle. These histological modifications led to severe muscle, respiratory and cardiac functional impairments leading to premature death around one year. Moreover, DMD muscle exhibited systemic inflammation with a mixed M1/M2 phenotype. A comparative single cell RNAseq analysis of the diaphragm muscle was performed, revealing cellular populations alteration and molecular modifications in all muscle cell types. We show that DMD fibroadipogenic progenitors produced elevated levels of cartilage oligomeric matrix protein (COMP), a glycoprotein responsible for modulating homeostasis of extracellular matrix, and whose increased concentration correlated with muscle fibrosis both in R DMDdel52 rats and human patients. Fibrosis is a component of tissue remodelling impacting the whole musculature of DMD patients, at the tissue level but most importantly at the functional level. We therefore propose that this specific biomarker can optimize the prognostic monitoring of functional improvement of patients included in clinical trials.
Project description:Dystrophin proteomic regulation in Muscular Dystrophies (MD) remains unclear. We report that a long noncoding RNA (lncRNA) H19 associates with dystrophin. To investigate the biological roles of this interaction in vivo, we performed mass spectrometry analysis of dystrophin and its associated proteins in H19-proficient and -deficient C2C12 myotubes. Mass spectrometry data indicated that in H19-proficient myotubes, dystrophin associates with components of dystrophin-associated protein complex (DPC); however, in H19-deficient myotubes, dystrophin associated with UBA1, UB2G1, TRIM63 ubiquitin E3 ligase and ubiquitin. In H19-deficient myotubes, dystrophin was post-translationally modified with K48-linked poly-ubiquitination at Lys3577 (referred to as Ub-DMD). This mass spectrometry study demonstrated that lncRNA H19, associates with dystrophin and inhibits E3 ligase-dependent Ub-DMD formation and its subsequent proteasomal degradation. Based on this study, H19 RNA oligonucleotides conjugated with a muscle homing ligand Agrin (referred to as AGR-H19) and Nifenazone, a TRIM63-specific small molecule inhibitor, reverses the dystrophin degradation in iPSC-derived skeletal muscle cells from Becker Muscular Dystrophy patients. Furthermore,treatment of mdx mice with exon-skipping reagent, in combination with either AGR-H19 or Nifenazone, dramatically stablized dystrophin, preserved skeletal/cardiac muscle histology, and improved strength/heart function. In summary, this mass spectrometry study paves the way to meaningful targeted therapeutics for BMD and certain DMD patients.
Project description:Postmenopausal osteoporosis (PMOP) is a major global public health concern and older women are more susceptible to experiencing fragility fractures. Our study investigated the associations between circulating proteins with bone mineral density (BMD) in postmenopausal women with or without low BMD (osteoporosis and osteopenia) to explore the pathogenesis of PMOP and discover novel biomarkers for this disease..
Project description:Duchenne muscular dystrophy (DMD) is a fatal muscle disorder characterized by cycles of degeneration and regeneration of multinucleated myofibers and pathological activation of a variety of other associated cell types. Here, we describe the creation of a new mouse model of DMD caused by deletion of exon 51 of the dystrophin gene, which represents a prevalent mutation in humans. To understand the transcriptional abnormalities and heterogeneity associated with the nuclei of myofibers, as well as other mononucleated cell types that contribute to DMD disease pathogenesis, we performed single nucleus transcriptomics of skeletal muscle of mice with exon 51 deletion. Our results reveal distinctive and previously unrecognized myonuclear subtypes within dystrophic myofibers and uncover degenerative and regenerative transcriptional pathways underlying DMD pathogenesis. Our findings provide new insights into the molecular underpinnings of DMD, controlled by the transcriptional activity of different types of muscle and nonmuscle nuclei.
Project description:We constructed the trans/mdx mice as a novel DMD disease model by crossing transgenic mice and mdx mice. The transgenic and trans/mdx mice have UAA incorporation system that can readthrough the nonsense mutation in the dmd gene with additional UAA. The whole transcriptomics of heart and muscle tissues of 5 mouse groups (n=2) were used to determine whether the introduced UAA incorporation system affect the normal gene expression of mice, or the safety of the system. Groups include: a) wild-type C57BL/6N mice; b) transgenic mice; c) mdx mice; d) trans/mdx mice without UAA; e) trans/mdx mice with UAA (50 mg NAEK every 2-3 day, treated for 4 weeks). The total numbers of expressed genes were around 11,000 in all 5 mouse groups. The expression levels of major genes (logFPKM>0) in 5 mouse groups were also close to each other. These data indicated that the UAA incorporation system was safe and could serve as a potential therapeutic strategy for DMD disease. The transgenic mice with UAA incorporation system we constructed could also cross with other nonsense mutation mice to build more functional animal models.