USING HUMAN CELL LINE MODELS TO UNDERSTAND THE MECHANISMS OF POLG MITOCHONDRIAL DISEASE AND MEHP-INDUCED MITOCHONDRIAL TOXICITY

Author

Md Mostafijur Rahman, Southern Illinois University CarbondaleFollow

Date of Award

12-1-2024

Degree Name

Doctor of Philosophy

Department

Molecular Biology, Microbiology and Biochemistry

First Advisor

Young, Matthew

Abstract

Mitochondrial DNA (mtDNA) is an essential component of mitochondria and consists of 13 protein coding genes which encode 13 subunits of the oxidative phosphorylation (OXPHOS) machinery. According to widely accepted theory, the mitochondrion was derived from an alpha-proteobacteria which was engulfed by an early eukaryotic cell and eventually evolved into the organelle through a process called endosymbiosis. Most of the proteins located in mitochondria are encoded by nuclear genes. Through the endosymbiotic process most of the bacterial genes were transferred to the nucleus leaving a small genome inside mitochondria (mtDNA). Looking at the DNA replication machinery of mtDNA, the components share structural similarity with viral replication machinery making it vulnerable to drugs targeted for antiviral therapy. Additionally, mitochondrion is one of the primary sources of cellular reactive oxygen species (ROS) production. Different chemical compounds and environmental toxicants can disrupt mitochondrial function resulting in increased ROS production leading to mitochondrial damage. Damaged mitochondria are detrimental to cellular health as the ROS produced by mitochondria can damage other cellular components. To protect the cell from damaged mitochondria, a specialized autophagy process is required called mitophagy which is responsible for clearing damaged mitochondria through the autophagic pathway. Moreover, damaged mitochondria can activate apoptotic signaling leading to cell death. To investigate the role of replication machinery on mitochondrial function, we utilized a cell line harboring a mutation in the POLG gene leading to a change in the amino acid position 955 from Tyr to Cys (Y955C) situated at the active site of the mitochondrial DNA polymerase, Polγ (chapter 2). We observed reduced growth rate, reduced mtDNA copy number, and increased sensitivity to the mtDNA toxicant dideoxycytidine (ddC) in the variant cell line relative to control. Additionally, there were reduced mtDNA nucleoids which support decreased mtDNA copy number. One and two-dimensional agarose gel electrophoresis analysis showed an increased abundance of Y955C mtDNA replication intermediates and linear molecules which likely result from double strand breaks due to replication stress. Moreover, the mtDNA encoded complex I subunit, NADH dehydrogenase subunit 3 and nuclear DNA encoded complex I subunit, NADH:ubiquinone oxidoreductase subunit B8 protein levels decreased in the variant cell line compared to control which indicates OXPHOS complex I activity and assembly is affected by the Y955C mutation. This indicates that dysfunction in mtDNA replication can affect mitochondrial function and cellular growth. Phthalates are a class of chemical compounds which are used as plasticizers. During the use of plastics, phthalates can leach out from the plastic and go inside the body. The detrimental effect of phthalate toxicity on mitochondrial function is reported in chapter 3 & 4. We explored the clearance of damaged mitochondria through PINK1-PARKIN pathway during MEHP exposure. MEHP exposure also affects mtDNA copy number in undifferentiated HepaRG cells. Also, one of the essential components of mtDNA replisome, MGME1 could be affected by MEHP exposure. This indicates that MEHP toxicity affects mitochondrial function and mtDNA copy number leading to mitochondrial damage which is likely cleared by the autophagy pathway. As mitochondria play an important role in apoptosis, the effect of MEHP exposure on the induction of cell death was assessed. During MEHP exposure, apoptotic cell death was observed, and apoptotic factors were induced. This indicate that acute MEHP toxicity disrupt mitochondrial function leading to autophagic clearance of mitochondria and induction of apoptotic cell death. Our observations explored the role of mitochondrial replication machinery in maintaining mitochondrial health and how disruption of mtDNA replication can affect mtDNA maintenance and cellular growth. Also, the toxic effect of MEHP exposure on mitochondrial function was observed which is evident by induction of mitophagy and apoptosis during exposure of MEHP. Insights from these studies help us to understand the mechanism of mitochondrial disease related to mutations in the mtDNA replication machinery and the consequence of environmental toxicant exposure on mitochondrial and cellular fate.