Date of Award

Summer 8-4-2025

Level of Access Assigned by Author

Open-Access Thesis

Degree Name

Doctor of Philosophy (Biomedical Science)

Department

Biological Sciences

First Committee Advisor

Clarissa Henry

Second Committee Member

Benjamin King

Third Committee Member

Robert Burgess

Additional Committee Members

Gregory Carter

Gregory Cox

Abstract

The function of the neuromuscular system is foundational to the overall health of the induvial. Coordinating the development and maintenance of this system involves a multitude of proteins – some of which need to be highly modified. One of these modified proteins is dystroglycan, a transmembrane protein that resides on the muscle membrane and is heavily glycosylated by several proteins. The terminal end of the glycan consists of tandem sugar repeats, which are responsible binding factors in the glycan. Dystroglycan’s ability to bind to ECM proteins provides structure and stability for the developing neuromuscular system. If Dystroglycan lacks the tandem sugar repeats, binding is lost and defects in muscle and neural tissue lead to pathological outcomes. We created the first CRISPR model of b4gat1-associated dystroglycanopathy utilizing the zebrafish. The b4gat1 mutant model possess a 7-bp deletion, resulting in the introduction of a premature stop codon. b4gat1-/- zebrafish exhibit hypoglycosylation of alpha-dystroglycan, confirming the loss of b4gat1 function in the line. Late defects in growth and reduced survival are preceded by mild muscle damage. Given the mild defects of the muscle, we investigated if a compensatory

mechanism was present. Our preliminary results indicate that Integrin alpha 7 may be providing support in the absence of Dystrolgycan’s matriglycan. Interestingly, the model exhibits increased susceptibility to muscle damage under stress, which seems to be rescued with the treatment of NAD+. However, the first phenotypes observed in the b4gat1 mutants are neural. Disruptions in motor neuron axon development are present by 24 hpf, with later defects in NMJ morphology observed at later timepoints. These results are the first of their kind, as no previously published papers implicate dystroglycan or its glycosylation proteins in motor neuron development. Treating the mutants with methylene blue, a neuroprotective dye, rescues the axonal defects in mutants. Taken together, these data offer an expansion of knowledge into the role of dystroglycan/laminin binding and b4gat1 function. The b4gat1 mutant line offers researchers a new tool in investigating dystroglycanopathies and potential therapeutic mechanisms.

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