The goal of this project is to determine how the RNA binding protein CELF1 (also known as CUGBP1) contributes to skeletal muscle wasting in myotonic dystrophy type 1 (DM1). DM1 is an adult-onset neuromuscular disorder and more than half of DM1 mortality is due to muscle wasting. The mechanisms leading to skeletal muscle wasting remain unknown. DM1 is caused by expansion of a CTG repeat in the 3' UTR of the Dystrophia Myotonica-Protein Kinase (DMPK) gene. The CTG repeats are transcribed and DM1 pathogenesis is due to a gain of function of the expanded CUG repeat mRNA. The expanded repeat RNA disrupts the function of RNA binding proteins. The RNA sequesters the MBNL family of RNA binding proteins producing a loss of function which leads to RNA processing abnormalities resulting in disease features. A second RNA binding protein effected by the repeat RNA is CELF1. CELF1 protein is post-transcriptionally up- regulated 2-4 fold in DM1 skeletal muscle. The sponsor?s lab previously reported that inducible overexpression of CELF1 in adult skeletal muscle of transgenic mice reproduces DM1-like defects in muscle function and histology. CELF1 functions in the nucleus as a splicing regulator and in the cytoplasm as a regulator of mRNA stability and translation. I have recently generated and tested transgenic mice for skeletal muscle-specific and tetracycline-inducible expression of active CELF1 derivatives localized exclusively to either the nucleus or the cytoplasm. These transgenic animals will be used for muscle-specific expression to determine whether a gain of CELF1 nuclear or cytoplasmic functions contribute to DM1 pathogenesis and muscle wasting. I will achieve this through two specific aims. In the first specific aim transgenic mice inducibly expressing modified versions of mCherry-tagged human CELF1 that localize exclusively to the nucleus or cytoplasm will be used to determine whether the nuclear and/or cytoplasmic roles of CELF1 leads to pathogenesis. I will validate effects on CELF1 nuclear and cytoplasmic targets and systematically characterize muscle function and histology. In the second specific aim I will use RNA-seq to identify CELF1-associated transcriptome changes likely to contribute to skeletal muscle wasting in DM1. RNA-seq from skeletal muscle of either the nuclear or cytoplasmic line (or both) will be filtered against available RNA-seq data sets from skeletal muscle of individuals affected by DM1, a mouse model in the sponsor?s lab that express CUG repeat RNA and show muscle wasting, as well as models of CELF1 up-regulation. Additionally, I will determine the extent to which the muscle wasting phenotype of animals that express expanded repeat RNA is rescued by Celf1 loss of function by AAV-shRNA mediated Celf1 knockdown. RNA-seq from rescued animals will provide an additional data set to identify the molecular changes that correlate with phenotypic rescue.
CELF1 is a developmentally regulated RNA binding protein that plays a role in the severe myopathy associated with myotonic dystrophy. CELF1 protein levels are normally down-regulated during development to ensure normal skeletal muscle physiology, however in DM1 CELF1 levels are increased 2-4 fold contributing to muscle pathology. An enhanced understanding of how CELF1 contributes to muscle pathology in DM1 will significantly progress the DM1 field since the cause of muscle wasting remains unknown, and will also provide insight as to how CELF1 contributes to normal muscle physiology during development. !