Sex Differences in Mitochondrial Function and Susceptibility: Mechanisms of Establishment and Evolutionary Origins

Abstract

Mitochondria are complex organelles best known for their role in cellular metabolism and energy production. In addition to their role in generating ATP, these organelles are central to a variety of diverse signaling pathways involved with maintaining ion balance, regulating immune responses, apoptotic pathways, and many others. Disruptions to mitochondrial health and function have been linked to numerous diseases, including diabetes, Parkinson's disease, Alzheimer's disease, and more. Many environmental chemicals cause mitochondrial dysfunction through a variety of different mechanisms.Sex-linked differences in mitochondrial ATP production, enzyme activities, and reactive oxygen species generation have been reported in multiple tissue and cell types. Additionally, sex-variable toxicologic responses to environmental pollutants and drugs that cause mitochondrial toxicity have been observed. Recent research highlighting sex differences in mitochondrial function has provided evidence for the influence of hormones as a key driver of these sex differences. Additional biologic factors that may contribute to the establishment and maintenance of sex differences in mitochondrial function include the double dosing of many genes present on the X-chromosome and epigenetic regulation. It has also been hypothesized that uniparental inheritance of the mitochondrial genome may influence these sex differences, as this inheritance pattern has led to evolution of mtDNA in a female nuclear background, potentially resulting in enhanced mitochondrial-nuclear compatibility in females. Understanding the mechanisms responsible for the establishment and maintenance of these sex differences is critical to understanding the factors that may contribute to sex differences in mitochondrial-related disease incidence rates and outcomes.The overarching goal of this dissertation is to understand how epigenetic regulation of the nuclear and mitochondrial genomes, as well as uniparental inheritance of mtDNA, contribute to the establishment and maintenance of sex differences in mitochondrial function and sex-variable toxicological responses. Understanding sex differences in mitochondrial function contributes to our fundamental understanding of biology. Understanding the mechanisms that contribute to sex-variable toxicological responses is crucial to ensuring that the field of toxicology is well equipped to conduct chemical safety testing in a way that ensures people of all sexes are considered. The first aim of this dissertation addresses sex differences in a nuclear epigenetic modification, DNA methylation, at nuclear-encoded mitochondrial genes. We leveraged the Newborn Epigenetics STudy (NEST) cohort to assess sex differences in DNA methylation data specifically focused on nuclear-encoded mitochondrial genes. We also tested for sex-specific differences in DNA methylation alterations associated with exposure to in utero tobacco smoke exposure. We identified 596 differentially methylated sites corresponding to 324 different genes. We identified 17 genes with both sex differences in DNA methylation and gene expression, with a strong enrichment of electron transport chain genes, particularly genes associated with ATP synthase, or complex V of the electron transport chain. We also found that alterations in DNA methylation associated with in utero tobacco smoke exposure were highly sex-specific in these nuclear-encoded mitochondrial genes. Overall, our findings suggest that sex-specific DNA methylation may help establish sex differences in expression and function of a subset of nuclear-encoded mitochondrial proteins, and that sex-specific alterations in DNA methylation in response to exposures could contribute to sex-variable toxicological responses.The second aim of this dissertation focused on understanding the role that one aspect of the mitochondrial epigenome, the mtDNA packaging protein Transcription Factor A Mitochondrial (TFAM), may have in mediating mtDNA damage recognition and removal. A second goal was to understand the role that TFAM, which is downstream of estrogen response elements, may also have in providing protection against mtDNA damage. We identified a role for TFAM in mtDNA damage recognition through observations that TFAM compacts DNA containing photodimers more than it compacts intact, undamaged DNA. Additionally, we observed a reduction in TFAM sequence specificity associated with DNA containing photodimers. Overall, these results indicate that TFAM is able to sense photolesions on DNA, differentially bind to them, and alter the physical structure and compaction of DNA following damage. Further, overexpression of TFAM to levels similar to those observed previously via stimulation with 17- estradiol does not result in protection of the mtDNA from UVC-induced lesions. Thus, TFAM protein levels do not represent a mechanism through which sex differences in hormone production and regulation could lead to sex differences in mtDNA damage accumulation in the context of UVC exposure. The final aim of this work sought to address the extent to which uniparental inheritance of mtDNA could drive elevated mito-nuclear crosstalk and enhanced mitochondrial function in females, as previously hypothesized. To test the relevance of this hypothesis, we utilized the model organism, Caenorhabditis elegans, which exhibits maternal inheritance of the mitochondrial genome but lacks canonical sex hormones. We assessed sex differences in mitochondrial function in the C. elegans system via whole worm respirometry and determined whole worm ATP levels and mtDNA copy number. To probe whether sex differences might manifest only after stress, and to inform the growing use of C. elegans as a mitochondrial health and toxicologic model, we also assessed susceptibility to a classic mitochondrial toxicant, rotenone. Our results suggest few to no detectable differences in mitochondrial function in C. elegans sexes, which is inconsistent with the hypothesis that uniparental inheritance of mtDNA enhances mito-nuclear crosstalk. Additionally, this work provides useful information on the limitations of C. elegans as a toxicological model in the context of capturing sex-variable toxicological responses that exist in mammalian systems. The research chapters of this dissertation (Chapters 2, 3, and 4) further contribute to understanding how nuclear epigenetics, mitochondrial epigenetics, and inheritance patterns of mtDNA influence sex differences in mitochondrial function and the extent to which these differences impact certain chemical exposures. Collectively, it will be critical to continue to investigate the biologic factors that help to establish these sex differences and identify the extent to which they may influence health span and disease outcomes, as well as investigate the strengths and limitations of toxicological model systems for their ability to reliably detect sex-specific toxicological outcomes.