Résumé :
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Communication n° 491 INTRODUCTION : Oculopharyngeal muscular dystrophy (OPMD) is an autosomal dominant, late-onset disease caused by the expansion of an alanine (ala) tract at the N terminus of the nuclear poly(A)-binding protein (PABPN1) from 10 to 12-17 ala. A hallmark of OPMD is the presence of intranuclear inclusions within a proportion of myofibre nuclei. These contain PABPN1, and co-stain for ubiquitin, proteasome subunits and heat shock proteins. OBJECTIVES : Others have modelled OPMD in cellular systems, but these study transient expression of PABPN1 in non-muscle cells. Our aim was to generate a stable model of OPMD in a physiologically relevant muscle cell type. METHODS : Wild-type (WT) and 7 alanine-expanded (+7ala) PABPN1, under the control of the muscle-specific human desmin control region, were used to generate stable cell clones of primary mouse myoblasts. Clones were analysed for transgene copy number, expression (by western blot and by quantitative RT-PCR) and inclusion presence (by immunofluorescence). In addition, microarray analysis was performed on RNA from untransfected and WT or +7ala expressing clones. Cells were treated with Congo Red and Doxycycline to try to reduce inclusion formation. RESULTS: We chose 4 cell lines for analysis: 2 expressing WT PABPN1 and 2 expressing +7ala PABPN1 at similar levels. On fusion into myotubes, these form intranuclear inclusions, which stain for PABPN1, ubiquitin and poly(A)polymerase (PAP). Congo Red and Doxycycline reduce the inclusion percentage. Microarray analysis reveals a number of genes that are either up- or down-regulated in the transfected clones, including several genes involved in the synthesis of extracellular matrix (ECM) components. CONCLUSIONS : We have made a stable model of OPMD in a muscle cell line that reproduces phenotypic hallmarks seen in patients. We have looked for potential pathways that could be involved in OPMD and are following up the possibility that OPMD pathogenesis may involve the ECM.
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