A zebrafish model demonstrating that DPM3 functions in both dystroglycan dependent and independent roles in neuromuscular disease progression


Topic:

Pre-Clinical Research

Poster Number: T365

Author(s):

Amanda Ignacz, BS, University of Maine, Claire Schaffer, BS, University of Maine, Mary Astumian, University of Maine, Clarissa Henry, PhD, University of Maine

Skeletal muscle development, growth, and homeostasis relies on post-translational modifications, such as glycosylation. Healthy muscle function also requires the development of neuromuscular junctions (NMJs) and myotendinous junctions (MTJs), all interconnected via cell-matrix adhesion complexes that are replete with glycosylated proteins. Glycosylation is notably disrupted in dystroglycanopathies, which are identified by their role in glycosylation of dystroglycan (DG), a critical transmembrane receptor that anchors the intracellular cytoskeleton to the extracellular matrix. An important question that remains unanswered is if dystroglycanopathy genes contribute to the glycosylation of the myriad of other glycosylated proteins that promote adhesion to the extracellular matrix, and what aspects of neuromusculoskeletal degeneration are specifically due to the disruption of DG glycosylation versus disruption of glycosylation of other proteins. One such dystroglycanopathy gene is dolichyl phosphate mannosyltransferase 3 (dpm3), which contributes a foundational mannose residue as a building block for the glycosylation of DG. We developed a zebrafish model of DPM3-dystroglycanopathy through CRISPR/Cas9 technology that recapitulates the interesting phenotypic variation and disease progression observed in human DPM3 patients. Through use of dpm3 mutants, as well as dpm3;dg double mutants, we have developed a model to elucidate what aspects of DPM3 function are DG-dependent and DG-independent in disease progression. While dg -/- mutants do harbor dystrophy and muscle fiber detachments, the loss of even one copy of wild-type dpm3 dramatically exacerbates NMJ, MTJ, and skeletal muscle degeneration. Interestingly, we have also observed heterogeneity within the dpm3 +/-;dg -/- genotype, ranging from mild posterior muscle degeneration to severe multi-segment degeneration early in development. These findings indicate that DPM3 likely contributes to neuromuscular disease progression via both DG-dependent and DG-independent pathways, and possibly acts in a gene-dose dependent manner. Ultimately, we aim to elucidate DG-independent roles of dystroglycanopathy genes to gain fundamental knowledge for future therapeutic strategies in treating neuromuscular diseases.