Date of Award

2019

Embargo Period

7-8-2024

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Drug Discovery and Biomedical Sciences

College

College of Graduate Studies

First Advisor

Sherine Chan

Second Advisor

John J. Lemasters

Third Advisor

Craig C. Beeson

Fourth Advisor

James Chou

Fifth Advisor

Seok-hyung Kim

Sixth Advisor

Yukiko Sugi

Abstract

Mitochondria are critical for energy production and cell signaling, possessing their own DNA (mtDNA) which encodes essential proteins for oxidative phosphorylation (OXPHOS). Lack of mtDNA stability, i.e. diminished mtDNA quality and quantity, is implicated in many diverse diseases and are encompassed under the term mtDNA depletion syndrome (MDS). Two major disease loci are OPA1 and POLG. OPA1 is needed for mitochondrial quality control and mtDNA stability, and POLG is the sole replicative DNA polymerase for mtDNA replication and repair. Current models for pathologies resulting from mutations at these loci do not capture the wholebody aspect of mutations nor the early onset of disease and death. The dearth of adequate animal models underscores the difficulty in developing therapeutics for MDS, for which there are currently no effective treatments. To address this, we have developed two models of MDS resulting from the opa1 and polg loci in zebrafish. The mutant zebrafish had massive mtDNA copy number depletion beginning at 7 dpf and showed decreased movement prior to a severe decline in survival. They were unable to persist beyond 3-4 weeks post fertilization, which corresponds to the early mortality reported in humans with MDS. The opa1 model was further investigated, with homozygotes having altered mitochondrial morphology while showing decreased oxygen consumption rate (OCR), reactive oxygen species (ROS), and increased AMP/ATP and AMP/ADP ratios. After establishing our new animal models of MDS, we tested novel Vitamin K (VK) analogs to potentially mitigate downstream pathologies. The Chan and Chou labs have shown these compounds can stabilize mitochondrial dysfunction in mammalian neurons and have efficacy in models of epilepsy and Parkinson’s disease, two pathologies linked with mutations in OPA1 and POLG. Literature shows that increasing the absolute level of mtDNA copy number in an afflicted tissue can mitigate pathology that results from phenotypic expression of mtDNA mutation load under depleted conditions, leading us to investigate whether VK analogs could induce an overall change in mtDNA copy number to improve survival in our mutant zebrafish. We assessed a chronic dosing of 10nM NT108 or 10nM NT117 in the treatment of MDS. While we did not observe an increase in survival of the homozygotes of our models with treatment of either compound, there was a significant increase in mtDNA copy number. Interestingly, the treatments increased movement in the opa1 model but not the polg model. This study is an exciting opportunity to mitigate diseases that result from the loss of mtDNA copy number while verifying that these unique vertebrate models can be leveraged to improve our understanding of rare genetic mitochondrial diseases and the development of therapeutics for their treatment.

Rights

All rights reserved. Copyright is held by the author.

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