Date of Graduation

7-2021

Document Type

Dissertation

Degree Name

Doctor of Philosophy in Cell & Molecular Biology (PhD)

Degree Level

Graduate

Department

Cell & Molecular Biology

Advisor/Mentor

Iyer, Shilpa

Committee Member

Rao, Raj R.

Second Committee Member

Pinto, Ines

Third Committee Member

Quinn, Kyle P.

Fourth Committee Member

Millett, Francis S.

Keywords

Cellular respiration; Leigh syndrome; Mitchondrial respiration; Mitochondria; Mitochondrial dynamics; Pathogenic mitochondrial DNA mutation

Abstract

Leigh syndrome (LS) is a rare fatal mitochondrial disorder of infants caused by pathogenic mutations in the nuclear (nDNA) or mitochondrial DNA (mtDNA) leading to mitochondrial dysfunction. The extent to which pathogenic mtDNA variants regulate disease severity in LS is not well understood. The heterogeneous nature of this disorder, based in part by complex mitochondrial genetics, and the nuclear and mitochondrial cross-talk has made it particularly challenging to investigate and develop therapies for treating LS . While the prognosis is poor, several studies are underway to understand the pathophysiology of LS. This dissertation provides a comprehensive structural and functional analysis of five patient fibroblast cells modeling LS, harboring pathogenic mtDNA variants; in order to gain a better understanding of disease severity and alleviate some of the mitochondrial dysfunction reported in LS.

The influence of pathogenic mtDNA on mitochondrial structure and function in LS is unknown. Therefore, in our first study, we conducted a comprehensive analysis and identified five mitochondrial morphologies individuals, networks, branches, length of branches and size of networks in single cells containing some of the most prevalent pathogenic mtDNA known to cause LS. Results indicated LS cells predominantly contained individual mitochondria with short branch lengths, characteristic of a fragmented state compared to a control line with long-branched mitochondrial networks.

To better understand disease severity in patient fibroblast cells modeling LS, (T8993G, T9185C, T10158C, T12706C) in MTATP6, MTND3, MTND5 genes affecting the function of complex V or complex I. In this study, we estimated a high percentage (> 90%) of pathogenic mtDNA (T8993G, T9185C ) in LS cells affecting complex V and a low percentage (< 39%) of pathogenic mtDNA (T10158C, T12706C ) in LS cells affecting complex I. Levels of defective enzyme activities of the electron transport chain correlated with the percentage of pathogenic mtDNA. Subsequent bioenergetics assays showed cell lines relied on both mitochondrial bioenergetics and glycolysis for meeting energy requirements. The CBHI ratio emerged as a sensitive biomarker to ascertain disease severity in LS lines specifically harboring pathogenic mtDNA variants. Higher CBHI values indicated milder forms of the disease and lower CBHI values indicated severe form of the disease. Our results suggest that whereas the precise mechanism of LS has not been elucidated, a multi-pronged approach taking into consideration the specific pathogenic mtDNA variant, and composite BHI ratio, can aid in better understanding the factors influencing disease severity in LS.

Finally, we explored the therapeutic effects of introducing citric acid cycle (TCA) intermediate substrate, succinate, as a cell permeable prodrug-NV118, to alleviate some of the mitochondrial dysfunction in LS. Results suggest that a 24-hour treatment with prodrug NV118 elicited an upregulation of glycolylysis and mitochondrial membrane potential (MMP) while inhibiting intracellular reactive oxygen species (ROS) in LS cells. The results from this study suggests an important role for TCA intermediates in the treatment of mitochondrial dysfunction in LS. With advances in available research tools leading to a better understanding of the mitochondria in health and disease, there is hope for novel treatment options in the future.

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