Résumé :
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An autosomal form of centronuclear myopathy (CNM) spontaneously segregates in Labrador retrievers and constitutes a large animal model suitable for comparative functional analyses. In young affected dogs, muscle fibers exhibit size heterogeneity and Ttubule defects, followed by a late progressive centralization of nuclei. We previously identified a causative lossoffunction mutation in the PTPLA canine gene, which encodes an endoplasmic reticulum (ER) resident enzyme required for the elongation of Very Long Chain Fatty Acids (VLCFA). VLCFA are major components of sphingolipids and phosphoinositides which are complex lipids involved in membrane structure and trafficking, as well as in regulation of signaling pathways.Our goal is to decipher which roles PTPLA and VLCFA play in muscle physiology and to provide novel insights regarding CNM pathogenesis. To this purpose, we established murine C2C12 and canine PTPLA-deficient muscle cell lines as well as Ptpla- KO/KI mouse lines.First, we show that two PTPLA isoforms are expressed in wildtype muscle cells, mice or dogs. A PTPLA isoform lacking exon 5 is largely expressed and is predominant in smooth muscles; a full-length isoform is specifically expressed in differentiating myoblasts and mature striated muscles. In CNM affected dogs, these two isoforms are nearly absent whereas a third isoform, devoid of exons 2 to 5, is generated. Precise expression patterns of these three isoforms, revealed by RT-PCR and immunofluorescence, are shown. Consequences of their expression on ER and nuclear envelope organization are reported. In addition, we provide data revealing the endogenous Ptpla expression pattern through the analysis of mice carrying the KI LacZ reporter gene.Second, we describe VLCFA contents abnormalities in PTPLA-deficient conditions and provide evidence that lipid rafts organization is dependent upon PTPLA activity. Lipid rafts are membrane microdomains enriched in cholesterol and sphingolipids. Neuromuscular junctions (NMJ) constitute large and observable lipid rafts. Preliminary results indicate that in NMJ of muscle fibers from CNM-affected dogs, AchR are not wellorganized and cholesterol not enriched. Our current characterization of functional consequences of lipid rafts disorganization on the PI3-K hypertrophic pathway activation is presented.Finally, we hope to be able to provide an early report of the phenotype resulting from a complete loss-of-function of Ptpla in mice
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