Myotonic Dystrophy type 1 (DM1) is a multisystemic, neuro-muscular disease without cure. DM1 pathology is mainly mediated by untranslated RNA CUG repeats which misregulate two families of RNA CUG-binding proteins, CUGBP1 and MBNL. In DM1, CUG repeats increase protein levels of CUGBP1. Consistent with the elevation of CUGBP1 in DM1 patients, the overexpression of CUGBP1 in mice causes muscular dystrophy, myotonia and a delay of skeletal muscle development and differentiation. It has been recently found that the silencing of CUG repeats in mouse DM1 model reverses myotonia and muscular dystrophy through normalization of CUGBP1. Despite this progress, mechanisms by which CUG repeats alter activities of CUGBP1 in DM1 cells are not well understood. We found that RNA binding activities of CUGBP1 and interactions of CUGBP1 with initiation translation factor 2, eIF2, are regulated by Akt and cyclinD3/cdk4 signal-transduction pathways. Akt controls cytoplasm/nucleus distribution of CUGBP1. CyclinD3/cdk4 increases interactions of CUGBP1 with eIF2. In DM1 muscle, these signal transduction pathways are misregulated. Cyclin D3 levels are reduced in DM1 myogenic cells, while Akt is up-regulated. These alterations change expression of CUGBP1 targets. Cdk4-mediated phosphorylation of CUGBP1 seems to be critical for the prevention of interaction of CUGBP1 with expanded CUG repeats since the mutant S302G molecule binds significantly stronger to RNA CUG expansion. In addition, recent data showed that CUG repeats also increase PKC-dependent phosphorylation of CUGBP1 leading to the stabilization of CUGBP1. Thus, the main hypothesis of this application is that in DM1 cells CUGn-mediated alterations of Akt, cyclinD3/cdk4, and PKC signal transduction pathways lead to (1) increase of CUGBP1 levels;(2) a delay of muscle differentiation;(3) increase of protein levels of a new CUGBP1 target, histone deacetylase 1 (HDAC1), which will inhibit transcription of certain genes in DM1. In this application, we will test the role of these upstream signal transduction pathways in the regulation of CUGBP1 stability, in the regulation of intracellular localization of CUGBP1 and in the epigenetic control of gene expression in DM1. We will examine the role of cyclin D3-cdk4 pathway in the delay of muscle differentiation by generation and examination of CUGBP1-S302G knock in mice. The role of Akt-CUGBP1 pathway in the increased proliferative rate of DM1 muscle will be examined in DM1 cultured cells and in CUGBP1-S28A knock in mice. Results of this study will help to develop approaches to normalize CUGBP1 activity in DM1 and to correct skeletal muscle differentiation in DM1 patients.
Myotonic Dystrophy type 1 (DM1) is a multisystem, neuro-muscular disease which is caused by expansion of un-translated CTG repeats increasing protein levels of CUG-binding protein, CUGBP1. My application proposes the elucidation of molecular mechanisms miss-regulating CUGBP1 activity in DM1 muscle. Results of this study will help to develop therapy to normalize CUGBP1 activity in DM1 patients.
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