Tissue-specific roles for muscle-matrix adhesion in neuromuscular development and homeostasis - Musculoskeletal function requires integration of skeletal muscle with two different junctions: neuromuscular junctions (NMJs) and myotendinous junctions (MTJs). Action potentials are transmitted through NMJs to signal muscle contraction. Muscle contraction then transmits force through MTJs to move the skeleton. Disruption of either of these junctions can lead to incurable neuromuscular degenerative diseases that shorten lifespan and deleteriously impact quality of life. Despite their importance, the mechanisms underlying coordinated development and homeostasis of NMJs, muscle, and MTJs are not entirely understood. NMJs and MTJs connect to muscle through cell-extracellular matrix (ECM) adhesion complexes such as the Dystrophin Glycoprotein Complex. Dystroglycan (DG) is a critical transmembrane receptor in the Dystrophin Glycoprotein Complex that requires glycosylation to bind to the ECM. A critical building block for DG glycosylation is Dolichylphosphate mannose (Dol-P-Man), which is generated by the Dol-P-Man synthase complex. Humans with mutations in any of the three components of this complex (Dpm1,2,3) can show failure to thrive and they have progressive muscle degeneration, resulting in a life expectancy of 20 years or less. Thus, glycosylation plays critical roles in NMJ and MTJ integrity. We propose to use the zebrafish model to provide insight into the basic cell biology underlying neuromusculoskeletal diseases where glycosylation is disrupted as a necessary step towards identifying future therapeutic targets. To understand how loss of Dol-P-Man leads to muscle degeneration, we generated a zebrafish model with truncated Dpm3, the stabilizing subunit in Dol-P-Man synthesis. In dpm3 mutants, NMJ and MTJ development are disrupted, muscle growth is reduced, there is neuromuscular degeneration, and death occurs by 3 weeks. We next created dpm3;dg double mutants to determine whether glycosylation of DG by Dpm3 is responsible for this phenotype. The enhanced phenotype of dpm3;dg homozygous double mutants, with severe muscle degeneration and death by just 1 week, suggests that DG-independent actions of Dol-P-Man have been overlooked. We will use our unique genetic models to elucidate cell-type specific noncanonical functions for Dpm3 and Dg to test the overall hypothesis that DPM3 and DG play interrelated and independent roles that are critical for cell-matrix adhesion in NMJs, muscle, and MTJs formation and maintenance. We will do this by completing the following two aims. Aim 1: Elucidate motor neuron- and muscle-specific roles for Dol-P-Man and DG at the NMJ. Aim 2: Determine how Dol-P-Man and DG play unique and synergistic roles to promote cell-matrix adhesion during MTJ development and muscle growth. Taken together, experiments in this proposal will integrate our unique genetic models to elucidate cell-type specific canonical and noncanonical roles for Dpm3 and Dg with powerful imaging approaches to quantitatively understand the coordination between the NMJ and MTJ during development and homeostasis in normal and diseased states.
