Ortiz Cordero, Carolina2022-11-142022-11-142021-06https://hdl.handle.net/11299/243130University of Minnesota Ph.D. dissertation. June 2021. Major: Integrative Biology and Physiology. Advisor: Rita Perlingeiro. 1 computer file (PDF); x, 121 pages.Fukutin-related protein (FKRP) is a ribitol-5-phosphate transferase involved in the functional glycosylation of α-dystroglycan (DG). Mutations in FKRP lead to limb-girdle (LGMDR9) and congenital muscular dystrophies (CDM), for which there are no cure. While the main hallmark of these diseases is hypoglycosylation of α-DG, a complete understanding of the pathophysiology remains unknown. To address this, we established a pluripotent stem (PS) cell-derived myogenic model to investigate the molecular implications of FKRP mutations and uncover potential therapeutics. Using patient-specific Walker-Warburg syndrome (WWS), a severe type of CMD, and LGMDR9 myotubes, we investigated the autophagy-lysosome pathway. Myotubes from both disease phenotypes displayed a significant reduction in autophagy. Moreover, WWS myotubes exhibited decreased ERK1/2 activity and increased apoptosis, parameters that are restored in WWS gene-corrected cells. Lack of function studies for FKRP recapitulated findings from patient-derived myotubes, providing further evidence of the importance of FKRP for cell homeostasis. In parallel, we sought to investigate the therapeutic effect of metabolites of the pentose phosphate pathway to rescue α-DG functional glycosylation in WWS myotubes. We show that ribitol and ribose supplementation leads to a rescue in functional glycosylation of α-DG. Additionally, we found NAD+ combined with these metabolites results in a synergistic effect, as demonstrated by increased rescue of α-DG glycosylation. Moreover, we demonstrate that FKRP residual enzymatic capacity is required for the metabolite-mediated rescue using functional and structural mutational analysis. Our results establish a human in vitro system for disease modeling and drug discovery for FKRP-associated MD. Using this platform, we uncovered that the autophagy-lysosome pathway and apoptosis might contribute to the FKRP-associated muscular dystrophy (MD) pathogenesis. Furthermore, we discovered that patients with FKRP-associated MDs could potentially benefit from the combined treatment of NAD+ and ribitol or ribose to restore α-DG functional glycosylation.enin vitro modelingmuscular dystrophyThesis or Dissertation