Browsing by Author "Meyer, Joel N"

Mitochondria are essential organelles required for all eukaryotic life on earth. Each organelle contains multiple copies of the mitochondrial genome (mtDNA) that encodes genes essential for energy production. Mutations in the mitochondrial genome are associated with mitochondrial diseases and diseases of aging, particularly neurodegenerative diseases, such as Parkinson’s Disease, and cancer. mtDNA mutation rates are often higher than nuclear DNA mutation rates. However, the origin of mtDNA mutations is poorly understood. Mitochondria lack many of the basic DNA repair mechanisms that are in the nucleus, potentially rendering mtDNA vulnerable to DNA damage-induced mtDNA mutations. Very few studies have investigated the impact of chemical exposures, in particular pollutants, on mtDNA mutagenesis, as reviewed in Chapter 4 of this dissertation (published as Leuthner and Meyer, 2021). Therefore, the two research aims of this thesis were designed to investigate the role of chemical exposures on mtDNA damage and mutagenesis in two species, chosen based on particular research strengths that each offered. The overarching hypothesis of this dissertation was that exposure to known nuclear genotoxicants and mutagens would result in the accumulation of mtDNA damage, which would ultimately lead to mtDNA mutations. The first aim of this thesis was to investigate the impact of the ubiquitous pollutant, cadmium (Cd), on mtDNA mutagenesis in the aquatic keystone species, Daphnia pulex. Cd is a known nuclear mutagen and carcinogen, yet the effects of Cd exposure on mtDNA mutations remain unknown. D. pulex offers a number of major advantages for this investigation, as discussed in Chapter 2. A unique aspect for this thesis was the use of a wild, Cd-tolerant population of D. pulex. Remarkably, this population of D. pulex sampled from Simon Lake in Sudbury, Ontario, Canada has adapted to high levels of Cd due to over a century of exposure to pollution from mining and smelting processes. Thousands of generations of experimental evolution were performed under laboratory conditions (an approach termed Mutation Accumulation, or MA, lines) in a Simon Lake isolate and an isolate collected from pristine Buck Lake (Dorset, Ontario, Canada) in both the presence and absence of Cd. This allowed investigation of the effects of Cd on mtDNA mutagenesis in D. pulex populations with very different evolutionary histories. Whole genome sequencing was conducted and mtDNA reads were extracted for analysis of mtDNA mutation frequencies, rates, and signatures. Hundreds of single nucleotide mutates were detected after >2,000 and >12,000 total generations of mutation accumulation, or about 40 to 250 fold more mutations than previous Daphnia mtDNA MA line studies. This afforded the resolution to determine the mechanism of endogenous mtDNA mutagenesis in Daphnia for the first time; these results indicate that endogenous mtDNA mutagenesis is likely mostly driven by polymerase γ error at sites of oxidized and deaminated cytosines (G  A/C T). At the earlier timepoint, Cd exposure further increased the rate of this mutation in the Cd-sensitive Buke Lake Daphnia compared to the Cd-tolerant Simon Lake Daphnia by about 3.6-fold. The results of this research aim suggest that Cd has a small effect on mtDNA mutagenesis, and that the adapted population is resistant to Cd-induced mtDNA mutations. However, after an additional >10,000 generations of mutation accumulation, there was no effect of Cd on this mutation spectrum, and the number of mtDNA mutations that were present at very low frequency increased significantly compared to the earlier timepoint. A small number of mutations did reach fixation or near-fixation however, and these mutations are discussed individually in Chapter 2. We propose that this result is consistent with natural selection acting on germline mtDNA mutation rates and heteroplasmy. Mitochondria harbor various quality control mechanisms that act in response to stress and mitochondrial dysfunction, such as mitophagy, fission, and fusion. Previous studies indicate that mitophagy may be involved in purifying selection against deleterious mtDNA mutations, in addition to targeted degradation of organelles that contain damaged mtDNA. Therefore, the next research aim of this thesis was to investigate the role of mitophagy on the accumulation of mtDNA damage and mutagenesis after exposure to Cd and another genotoxin, Aflatoxin B1 (AfB1), in the organism Caenorhabditis elegans. C. elegans are often used for MA studies to investigate mutational processes in both the nuclear and mitochondrial genomes, and offer a variety of strengths for such studies, as discussed in Chapter 3. A particular strength for the purposes of this thesis was the ability to work with strains carrying mutations in mitophagy genes. A MA experiment was conducted in wild-type C. elegans and two mitophagy-deficient strains, dct-1 and pink-1, in control, 50µM Cd, and 10µM AfB1 conditions. AfB1 was selected because it causes mtDNA damage that is not expected to be efficiently repaired in mtDNA. After an average of 50 generations of MA, about 10 MA lines were selected for each strain/treatment combination for Duplex Sequencing. Duplex Sequencing is an ultra-sensitive, error-corrected sequencing approach that allows for detection of mutations as low as 1 in 10,000 base pairs. Until the preparation of these samples, no study had yet conducted targeted mtDNA Duplex Sequencing in C. elegans. Wild-type and mitophagy-deficient strains all had mutation spectra indicative of oxidative damage driving mtDNA mutagenesis (GT/CA), contrary to what was observed in Daphnia in Aim 1, and contrary to what has been reported in other organisms. However, this confirmed results from a very recent study that also used mtDNA targeted Duplex Sequencing of wild-type C. elegans. Surprisingly, even though more mtDNA mutations were detected and at a lower frequency than ever previously reported, there was no clear effect of either Cd of AfB1 exposure on mtDNA mutations in any strain, despite a marginally significant increase in G:C  A:T mutations in pink-1 AfB1 MA lines compared to wild-type AfB1 MA lines. Overall, this suggests that mitochondria are resistant to exogenous damage-induced point mutations in C. elegans. Further investigations into what mechanisms are responsible for maintaining mtDNA homeostasis that are independent of mitophagy are an exciting future next step. Understanding the impact of chemicals on mtDNA mutations is critical for human and environmental health, as addressed in the Chapter 4 “Mitochondrial DNA mutagenesis: A feature of and biomarker for environmental health,” which has been published as a review. The primary research chapters of this dissertation (Chapters 2 and 3) further contribute to understanding how chemicals impact mitochondrial genome quality and integrity. Collectively, it will be critical to continue to use improved sequencing technologies to continue to investigate the origin and mechanisms of mtDNA mutagenesis resulting from both endogenous and exogenous factors.

In order to develop a better understanding of the role environmental toxicants may play in the onset and progression of neurodegenerative diseases, it has become increasingly important to optimize sensitive methods for quickly screening toxicants to determine their ability to disrupt neuronal function. The model organism Caenorhabditis elegans can help with this effort. This species has an integrated nervous system producing behavioral function, allows for easy access for molecular studies, has a rapid lifespan, and is an inexpensive model. This project focuses on the development and optimization of a low-cost efficient behavioral tool for the identification of environmental toxicants with actions that disrupt dopamine function in the nematode C. elegans. Several dopamine-mediated locomotory behaviors, Area Exploration, Body Bends, and Reversals, as well as Swimming-Induced Paralysis and Learned 2-Nonanone Avoidance, were compared to determine the best behavioral method for screening purposes. These behavioral endpoints were also compared to morphological scoring of neurodegeneration in the dopamine neurons. We found that in adult worms, Area Exploration is more advantageous than the other behavioral methods for identifying DA-deficient locomotion and is comparable to neuromorphological scoring outputs. For larval stage worms, locomotion was an unreliable endpoint, and neuronal scoring appeared to be the best method. We compared the commonly used dat-1p::GFP reporter strains BY200 and BZ555, and we further characterized the dopamine-deficient strains, cat-2 e1112 and cat-2 n4547. We found that BY200 and BZ555 have differential responses in DA-targeted neurodegeneration using the model toxicant 6-hydroxydopamine, with BY200 appearing to have somewhat increased sensitivity to treatment. In contrast to published results, we found that the cat-2 strains slowed on food almost as much as N2s. Rather than being null mutants, both showed decreased levels of cat-2 mRNA and DA content, with cat-2 e1112 having the greatest reduction in DA content in comparison to N2. Ultimately, we concluded that while behavioral analysis is a promising endpoint, it is probably best used as a quick screening tool, with follow-up studies assessing affects to neuron morphology. A more informative readout of toxicant effects to the DA system would be best accomplished using a combination of the assays outlined in this project. We offer a table at the conclusion of this study comparing strengths, limitations, costs, and equipment needs for the methods of analysis of the dopaminergic system in C. elegans undertaken in this project.

Mitochondria are the main cellular producers of ATP, and play key roles in cellular signaling and apoptosis. Mitochondria also contain their own genomes (mtDNA), which encode 13 subunits of the electron transport chain (ETC), 22 tRNAs, and 2 rRNAs, making mtDNA integrity critical to both mitochondrial and organismal health. Mitochondria are dynamic organelles that fuse and divide to maintain mitochondrial shape, number, and size. However, mitochondrial fission and fusion also play a major role in the mitochondrial stress response. For example, mildly damaged mitochondria can fuse with healthy mitochondria allowing contents to mix, resulting in the generation of healthy mitochondria, which is known as functional complementation. Alternatively, when mitochondria become damaged beyond repair, they are targeted for autophagosomal degradation, or mitophagy. The overall importance of fission, fusion, mitophagy, and mtDNA is demonstrated by the fact that deficiencies in these processes and mtDNA content cause human disease. Interestingly, the age of onset, and severity of clinical manifestations of mitochondrial disease vary from patient to patient, even in individuals harboring identical mutations. These observations suggest a role for the environment in the development and progression of certain mitochondrial diseases; however, the relationship remains poorly understood.

To investigate the role of environmental toxicants in the development, progression, and exacerbation of mitochondrial disease I have taken two approaches using the in vivo model organism Caenorhabditis elegans. First, ten known and suspected mitochondrial toxicants (2,4-dinitrophenol (DNP), acetaldehyde, acrolein, aflatoxin B1 (AfB1), arsenite, cadmium, cisplatin, doxycycline, paraquat, rotenone) were screened for exacerbation of larval growth delay in wild-type, fission-, fusion-, and mitophagy-deficient nematodes using the COPAS Biosort. Second, a C. elegans model of mtDNA depletion was developed using chronic low-dose ethidium bromide exposure. Five toxicants (AfB1, arsenite, paraquat, rotenone, ultraviolet C radiation (UVC)) were tested for exacerbation of mitochondrial function (assessed via changes in steady-state ATP levels) in nematodes with reduced mtDNA content. Mitochondrial health was then further assessed for some of the identified gene-environment interactions. Mitochondrial respiration was measured using the Seahorse XFe24 Extracellular Flux Analyzer, while steady-state ATP levels were assessed using transgenic luciferase expression nematodes and traditional extraction protocols. Gene expression, mtDNA, and nuclear DNA copy number were assessed using real-time PCR, while enzyme activity was assessed using microplate reader-based assays.

Results from the fission, fusion, and mitophagy toxicant screen revealed that fusion-deficient nematodes were sensitive to a variety of toxicants (DNP, AfB1, arsenite, cisplatin, paraquat, rotenone), while pink-1 mitophagy-deficient nematodes were sensitive to rotenone, and fission- and pdr-1 mitophagy-deficient nematodes were only mildly sensitive to paraquat, and rotenone, respectively. As mitochondrial disease is rare, but chronic arsenite exposure is widespread, we further investigated the mechanisms underlying arsenite sensitivity in fission- and fusion-deficient nematodes. Although not sensitive in the larval growth assay, fission-deficient nematodes were sensitive to arsenite later in life in both reproduction and lethality assays. Seahorse and ATP analysis revealed that arsenite disrupts mitochondrial function in fusion-deficient nematodes at multiple life stages (L4, 8- and 12-days of age), while enhancing mitochondrial function in 8-day old wild-type nematodes, and has minimal effect on mitochondrial function in fission-deficient nematodes. Lastly, arsenite inhibited both pyruvate and isocitrate dehydrogenase activity in fusion-deficient nematodes, suggesting a disruption of pyruvate metabolism and Krebs cycle activity underlie the observed mitochondrial dysfunction. These results suggest that deficiencies in mitochondrial fusion may sensitive individuals to arsenite toxicity.

Lastly, I have found that reducing mtDNA content 35-55% only mildly sensitized nematodes to certain secondary toxicant exposures, including UVC and arsenite. Alternatively, reduced mtDNA content did not sensitize nematodes to acute or chronic paraquat or AfB1 exposure, and provided resistance to rotenone. However, we also found that EtBr can induce cytochrome P450s (CYPs), which play a major role in rotenone metabolism; thus, it is likely that induction of CYPs and not reduced mtDNA content is responsible for rotenone resistance. These results suggest that individuals with reduced mtDNA content may be sensitive to certain toxicant exposures, but also highlight the robust mechanism that exist to maintain the integrity of mitochondria and mtDNA.

Collectively, these results suggest individuals suffering from mitochondrial disease caused by mutations in mitochondrial fission, fusion, or mitophagy genes, or by depletion of mtDNA, may be especially sensitive to certain environmental toxicant exposures, including arsenic. Arsenic’s pervasive contamination of drinking water results in chronic exposure for over 100 million people worldwide; thus, dramatically increasing the probability of exposure for individuals suffering from mitochondrial disease, and warrants further investigation in the human populous.