PROJECT SUMMARY / ABSTRACT The proposed research plan will offer critical insight into the cellular and molecular mechanisms that drive the pathogenesis of myotonic dystrophy type 1 (DM1). One prominent feature of DM1 is debilitating, progressive skeletal muscle weakness, for which there is no cure or effective treatment. Skeletal muscles from patients with DM1 exhibit abnormalities in alternative splicing, a post-transcriptional mechanism that enables one gene to produce two or more protein isoforms. As a result, many protein isoforms are inappropriately expressed in DM1 muscles. The alternative splicing of the clathrin heavy chain (Cltc) gene is mis-regulated in skeletal muscles from DM1 patients, resulting in aberrant protein isoform expression during postnatal development and adulthood. Cltc mis-splicing reduces muscle contractile function in mice. CLTC is the main player in clathrin mediated endocytosis, yet also serves as a scaffold for cytoskeletal and endosomal components. The objective of this proposal is to determine the mechanisms by which the mis- regulation of Cltc splicing during development causes muscle weakness. The overall hypothesis is that Cltc mis-splicing, which blocks the inclusion of a 21-nucleotide micro exon during postnatal development, prevents protein scaffolding events that are critical for neonatal muscles to mature into muscles capable of supporting adult contractile needs. Aim 1 will determine the mechanisms by which Cltc mis-splicing disrupts the formation of transverse tubules, structures that are required for contraction and are abnormal in DM1 muscles. Aim 2 will define how Cltc splicing regulates the endo-lysosomal trafficking of ubiquitinated proteins, which is responsible for the downregulation of ion channels and membrane receptors. Aim 3 will determine the functional impact of Cltc mis-splicing on skeletal muscles with different cell type compositions; the pathology of DM1 and other muscle diseases is often cell type specific. Together, these studies will expand our understanding of the role alternative splicing plays in muscle health and disease. One exciting possibility is that Cltc mis-splicing could be redirected by antisense oligonucleotides to treat DM1. These studies will provide outstanding training for the applicant, whose long-term goal is to lead an academic research team. Through this work, she will master skills that are critical for her scientific development including primary muscle cell culture, live-cell trafficking studies, in situ hybridization, muscle contractility studies, molecular cloning, and versatile computational skills. The applicant will undergo exceptional training from her sponsor, co-sponsor, and collaborators at the University of North Carolina at Chapel Hill. She will receive additional support from the institutional environment through research core facilities, topical group meetings, and her department. The proposed research and training plan will build a very strong ...