Date of Award

Spring 5-11-2019

Level of Access Assigned by Author

Open-Access Thesis

Degree Name

Doctor of Philosophy (PhD)

Department

Biological Sciences

Advisor

Clarissa Henry

Second Committee Member

Robert Wheeler

Third Committee Member

Benjamin King

Additional Committee Members

Thomas Gridley

Roger Sher

Abstract

Muscle development and homeostasis are critical for normal muscle function. A key aspect of muscle physiology during development, growth and homeostasis is modulation of protein turnover. Protein turnover is the balance between synthesis and degradation of muscle proteins. The rate of protein degradation is the major factor underlying overall muscle growth during development. Protein degradation mainly takes place in the lysosome. The efficiency of lysosomal protein degradation depends upon lysosomal pH, which is generated and maintained by proton pumps. Despite the importance of protein degradation in muscle growth during development, the impact of dysregulated lysosomal pH on muscle development, growth, and homeostasis is unknown. Spns1, a highly conserved gene that encodes a putative late endosome/lysosome carbohydrate/H+ symporter, plays a pivotal role in maintaining optimal lysosomal pH. We used the zebrafish homozygous mutant spns1-/- to investigate the requirement for lysosomal pH during muscle development. Muscle development proceeds normally in spns1-/-embryos; however, spns1-/- larvae experience rapid loss of muscle integrity. We found that the developmental isoform of laminin (laminin-111) is re-expressed in spns1-/- muscle at a time when the mature isoform of laminin (laminin-211) is normally expressed. We showed that Sonic hedgehog (Shh) signaling can either promote or inhibit laminin-111 expression in muscle basement membranes in response to different genetic backgrounds and/or developmental times.

To identify mechanisms upstream of laminin-111 re-expression, we focused on identifying the relevant laminin receptor. We found that laminin-111 re-expression is mediated by the laminin receptor integrin α6β1 (itga6b1) in spns1-/- larvae. We detected increased activation of the intracellular protein FAK in response to itga6b1-mediated laminin-111 re-expression. We showed that muscle fiber detachments in spns1-/- larvae predominantly result from sarcolemmal instability. We detected increased expression of the cytokine TWEAK and matrix metalloproteinase MMP-9 in spns1-/- larvae. We showed that TWEAK induces expression of MMP-9 independent of NF-kB signaling. Finally, we provided evidence that inhibition of TWEAK signaling does not reduce muscle damage in spns1-/- larvae. This result contrasts with previous data showing that inhibition of TWEAK reduces muscle damage. Taken together, these data indicate that dysregulated lysosomal pH has pleiotropic effects on zebrafish skeletal muscle.

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