Role of the Pvr signaling Pathway in a Fly Model for Myotonic Dystrophy Type 1

By Maya Takashima, Huda Al-Bana, Grace Holcomb

Faculty Mentor: Professor Ginny Morriss

Myotonic Dystrophy Type 1 (DM1) is a multi-systemic genetic disorder that causes severe muscle weakening and wasting. The phenotype is caused by a CTG repeat expansion in the 3’ untranslated region in the DMPK gene. Studies have shown several different signaling pathways implicated in the DM1 muscle phenotype; however, one pathway that has not been implicated in DM1 is the platelet-derived growth factor receptor beta (PDGFRβ) signaling pathway. PDGFRβ is involved in cell growth, survival, and skeletal muscle hypertrophy. With this involvement, the pathways deregulation could contribute to the muscle wasting seen in DM1. This project uses a fly model to understand the role of the pvr signaling pathway (PDGFRβ fly equivalent) in muscle wasting due to DM1. Through this fly model, it will be determined which downstream pathway is primarily affected during deregulation. The two downstream pathways targeted are PI3K/Akt and Ras/Mek/Erk. Flybase was utilized to determine the downstream gene targets and fly lines needed to start the fly model. After selecting the gene targets, fly lines were picked that were either overexpression of the gene (UAS – upstream activating sequence) or a knockout. Gal4 promoter lines and CTG repeat lines are also needed. Gal4 promoters drive the expression of the desired gene, and the CTG repeat represents the CTG repeat of DM1. With the fly lines chosen, mating schemes were planned to determine what crosses needed to occur to get the desired progeny. The goal was to end up with a progeny with the Gal4 promoter, CTG repeat, and stock line all in the same fly. In the future, using these progeny, different tests will be completed to look at how the overexpression or knockout affects the muscle wasting phenotype.

Differential Myokine Expression in a Model for Myotonic Dystrophy Type I

By Huda Al-Bana, Maya Takashima, Grace Holcomb

Faculty Mentor: Professor Ginny Morriss

Myotonic dystrophy type 1 (DM1) is a multi-systemic disease resulting in severe muscle weakening and wasting. Skeletal muscle wasting is the predominant cause of morbidity and mortality and is responsible for 60% of DM1-associated deaths. DM1 is caused by CTG repeat expansion in the 3’ untranslated region of the dystrophia myotonica protein kinase (DMPK) gene. Myokines are proteins that are produced and released by muscle cells in response to muscular contractions. Previous studies indicated that myokine signaling is deregulated in a mouse model expressing expanded CUG repeat (CUGexp) RNA. The goal of this work was to assess muscle-specific differential expression of myokines in mice expressing CUGexp RNA, relative to non-CUGexp RNA expressing control mice. Previously published RNA-seq datasets were compared to determine which putative myokines, identified in skeletal muscle samples of healthy subjects, were differentially expressed in CUGexp expressing mice. Myokines that display significant differential expression at 6, 12, and 20 weeks of repeat expression include the Cx3cl1, Cxcl10, and Gdf5. Primers were designed customary for these genes and specified for regions with constitutive exons to allow consideration of overall expression levels. Primers were then optimized using RT-PCR. The developed primers will be used to validate differential expression levels of these myokines using quantitative RT-PCR. Validated myokines will be assessed in unaffected and DM1 human myoblast cell lines to determine the contribution of these myokines to the skeletal muscle phenotype in DM1.