Résumé :
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Communication n° 574. Myotubularin (MTM1) is a phosphoinositide phosphatase that is mutated in X-linked myotubular myopathy (XLMTM), a congenital myopathy characterized by hypotonia, generalized muscle weakness and increased centronucleation of myofibers. To better understand the function of myotubularin and its role in XLMTM, murine Mtm1 was manipulated in vitro using a selectable retroviral system in C2C12 cells. siRNA methods were used to knock-down (KD) myotubularin and expression profiling was performed on triplicate Mtm1 KD cells and controls at 3 time points of muscle differentiation: day -2 (myoblasts), day +2 (myogenic fusion) and day +6 (mature myotubes). The cells fused as expected on day 2 and showed well-differentiated myotubes on day 6 confirming no gross phenotypic changes. At day 6, T-tests and geometric fold analyses revealed 27 genes that were consistently up- or down-regulated in Mtm1 KD, including 5 membrane, 4 mitochondrial and 2 sarcomeric proteins. Of particular interest is doublecortin (Dcx) which was +3.3 fold upregulated in Mtm1 KD cells and absent in controls. Although doublecortin is normally not expressed in adult skeletal muscle, it was also +2.1 fold up-regulated in expression profiling data of XLMTM patients vs. controls. Quantitative real-time PCR revealed +3.8 and +5.1 fold increases respectively in cell lines derived from two different Mtm1 KD primers. Interestingly, there was also +2.3 fold increase in the levels of Dcx in cells over-expressing a catalytically inactive Mtm1. At the protein level, doublecortin was up-regulated in Mtm1 KD cells. Since we have observed changes of Dcx in multiple assays, we are intrigued by the possibility that an interaction between doublecortin and myotubularin is necessary for proper muscle maintenance. Doublecortin regulates microtubules which may play a role in the characteristic abnormalities of myofiber organization in XLMTM including the misplacement of organelles such as nuclei or mitochondria. The potential role of doublecortin in the pathogenesis of XLMTM is being studied by immunohistochemistry and functional experiments.
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