The mechanism of CELF1 upregulation and its role in the pathogenesis of Myotonic Dystrophy Type 1

Baylor College Of Medicine, Houston TX

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Abstract

Project Summary Myotonic Dystrophy Type 1 (DM1) is a multisystemic disorder characterized by progressive skeletal muscle weakness, muscle wasting, and myotonia. With a prevalence of 1 in 8500, DM1 is the most common cause of adult-onset muscular dystrophy. DM1 is caused by the expansion of CTG repeats in the 3' untranslated region of the Dystrophia Myotonica Protein Kinase (DMPK) gene. The RNA transcribed from the expanded DMPK allele contains expanded CUG repeats (CUGexp RNA) that sequester the paralogs of the Muscleblind Like (MBNL) family of RNA binding proteins, MBNL1 and MBNL2, resulting in their loss of function. In addition, a second RNA-binding protein, CUGBP Elav-like family member 1 (CELF1), is upregulated to a level shown to be toxic in DM1 skeletal muscle. While the mechanism of MBNL loss of function through sequestration is well established, the mechanism leading to CELF1 upregulation in skeletal muscle and its contribution to the muscle deficits in DM1 remain unknown. To model adult-onset DM1 in mice, we generated mice with floxed Mbnl1 and Mbnl2 alleles and induced conditional double knockout (Mbnl dcKO) in adult skeletal muscle. The Mbnl dcKO mice resemble adult-onset DM1 with striking splicing changes, significant muscle wasting, and expected mild histopathology. Interestingly, we also found an increase in CELF1 protein levels, revealing a previously unknown regulatory link between MBNL and CELF1, independent of CUGexp RNA. This discovery suggests that CELF1 upregulation and toxicity are a direct consequence of MBNL loss of function in DM1. The goal of this proposal is two-fold. In Aim 1, I will determine how MBNL loss of function leads to the upregulation of CELF1 protein and uncover the cis-regulatory motifs and trans-acting factors involved in the process. We previously demonstrated that CELF1 can be stabilized by Protein Kinase C (PKC)-mediated phosphorylation. As such, one particular interest will be to test if MBNL loss of function leads to an activation of PKC and increases the phosphorylation of CELF1 or if other mechanisms are involved in the upregulation of CELF1. As adult knockout of MBNL1 and MBNL2 in skeletal muscle has not been previously characterized, in Aim 2, I will first perform an in-depth characterization of the physiological, histopathological and molecular muscle phenotypes of Mbnl dcKO mice to establish robust and quantitative assays that will then be used to determine the extent CELF1 upregulation contributes to Mbnl dcKO muscle phenotypes. Upon completion of this study, I will have resolved the long-standing question in the DM1 field of how CELF1 is upregulated and determined the extent to which this upregulation contributes to the Mbnl dcKO muscle deficits. Our finding that CELF1 upregulation is caused by MBNL loss of function provides new insight into the complex combinatorial mechanisms of DM1 pathogenesis and will help future studies on DM1 disease causation and progression and will inform the development of future therapeutic approaches.