Browsing by Subject "Caenorhabditis elegans"

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.

Early life exposure to mitochondrial toxicants, including paraquat, rotenone, and manganese, has been hypothesized to promote early onset of genetic mitochondrial disorders as well as common degenerative diseases such as Parkinson's Disease and Alzheimer's Disease. This dissertation aimed to investigate the biochemical and physiological effects of early life exposure to mitochondrial genotoxicants during development in the whole organism modelC. elegans. In the first experiment, a panel of model mammalian neurotoxicants and heavy metal ions was screened for mitochondrial genotoxicity by measuring mitochondrial DNA (mtDNA) copy number and damage in C. elegans. Exposures to paraquat, cumene hydroperoxide, rotenone, maneb, cadmium (II) chloride, and manganese (II) chloride have no significant effect on the mtDNA : nuclear DNA (nuDNA) ratio; only exposure to paraquat resulted in higher mtDNA than nuDNA damage level.In the second experiment, a laboratory method was developed to generate persistent mtDNA damage in larval C. elegans using serial ultraviolent C (UVC) exposures. While the mitochondrial DNA damage persisted from L1 to L4 stage, there was no difference between mitochondrial copy number of the control and UVC treated worms. The UVC treatment significantly inhibited both ATP level and oxygen consumption 24 and 48 hr after the exposure, while the mitochondrial mRNA expression was inhibited 3 hr after the exposure. The pink-1 mutation, a mitochondrial serine/threonine-protein kinase involved in the mitophagy process, appeared to limit the growth inhibitory effect of UVC treatment and increase the mitochondrial DNA content of the organism. In the third experiment,larval C. elegans was exposed to UVC and paraquat and examined using differential interference contrast and fluorescence confocal microscopy. Both resulted in detectable, dose-dependent lesions in dopaminergic CEP neurons in adult C. elegans. Neither significant lesions in the GABAergic dorsal nerve cord nor any sign of pharyngeal necrosis were detected. This work demonstrated a mechanism in which early life exposure to mitochondrial genotoxicants could result in both biochemical and physiological changes in later stages of life, thereby highlighting the potential health hazard of time-delayed effects of these chemicals in the environment.

The advent of high coverage and low cost sequencing technologies has allowed for

newer and more powerful approaches in molecular and population genetics. Transposon

sequencing, where genome-saturated mutant populations allele frequencies are

measured before and after selection, functionally characterizes each and every gene

in the genome in a single experiment. The approach has been successfully applied

to a variety of phenotypes in a variety of unicellular systems: growth and motility

in E. coli, synthetic genetic interactions in yeast, and in vitro pathogen-resistance in

mammalian cell lines. However, transposon insertion typically produces null alleles,

which can be valuable to identify gene function, but evolutionary insight relies on

identifcation of naturally occurring polymorphisms affecting the trait of interest.

Genome-wide association studies (GWAS) can be used to study the effect of natural

genetic variation on a trait, but they grow prohibitively expensive if the number of

individuals to genotype and phenotype becomes large.

Here I describe the application of transposon sequencing and pooled sequencing

GWAS in the whole metazoan model, Caenorhabditis elegans. Transposon sequencing

has not been previously implemented in an animal model. I have sequenced a control

library using our method, C. elegans transposon sequencing (CeTnSeq). We have

constructed a new Mos1 transposon mutator strain that is more convenient to use

than the existing strain and allows for extra-chromosomal insertions to be degraded

by restriction digest. My preliminary results show that our method is qualitatively

effective at identifying transposon insertion sites, but suffers from PCR duplication

error. I propose to optimize the number of PCR cycles in the library and to include

unique molecular identifiers (UMI) in the library adaptor. I also show that the

restriction digest is effective at removing extra-chromosomal array insertions from

the library.

I constructed simulation models to help design optimal Ce-TnSeq experiments

with respect to statistical power for a proposed starvation survival assay. I considered

many parameters affecting the design, including: culture size, number of generations,

expected effect size, sequencing coverage, and sample size. I show that the number

of homozygous mutant animals in the screen is a critical factor in the design of

experiments. I also saw diminishing returns with respect to increasing sample size

and sequencing depth. These simulations will be invaluable in designing future Ce-

TnSeq experiments and identifying critical aspects of the protocol to optimize.

We performed pooled sequencing (using restriction-site associated DNA sequencing)

on a population of 95 wild isolates subjected to starvation. I identified strains

that were resistant and sensitive to starvation, and we verified these results using

traditional methods. We used our population sequencing data to perform an association

study of starvation survival across the 95 strains, and identified two statistically

significant quantitative trait loci.

Mitochondria are the primary energy producers of the cell and play key roles in cellular signaling, apoptosis and reactive oxygen species (ROS) production. Mitochondria are the only organelles that contain their own genome which encodes for a small subset of electron transport chain (ETC) proteins as well as the necessary tRNAs and ribosomal subunits to translate these proteins. Over 300 pathogenic mitochondrial DNA (mtDNA) mutations have been shown to cause a number of mitochondrial diseases emphasizing the importance of mtDNA maintenance and integrity to human health. Additionally, mitochondrial dysfunction and mtDNA instability are linked to many wide-spread diseases associated with aging including cancer and neurodegeneration. Mitochondria lack the ability to repair certain helix-distorting lesions that are induced at high levels in mtDNA by important environmental genotoxins including polycyclic aromatic hydrocarbons, ultraviolet C radiation (UVC) and mycotoxins. These lesions are irreparable and persistent in the short term, but their long-term fate is unknown. Degradation of mitochondria and mtDNA is carried out by autophagy. Autophagy is protective against cell stress and apoptosis resulting from exposure to mitochondrial toxicants suggesting that it plays an important role in removal of unstable mitochondria that can serve as a source of ROS or initiate apoptotic cell death. Furthermore, dysfunctional mitochondria can be specifically targeted for degradation by the more specific process of mitophagy influenced in part by the processes of mitochondrial dynamics (i.e., fusion and fission).

The goals of this dissertation were to investigate the long-term fate of helix-distorting mtDNA damage and determine the significance of autophagy and mitochondrial dynamics in removal of and recovery from persistent mtDNA damage. Removal of irreparable mtDNA damage and the necessity of autophagy, mitophagy, fusion and fission genes in removal of this damage were examined using genetic approaches in adult Caenorhabditis elegans. In order to investigate the significance of autophagy, fusion and fission genes in recovery from mtDNA damage-induced mitochondrial dysfunction in vivo, an experimental method was developed to specifically induce persistent mtDNA damage and mitochondrial dysfunction without persistent nDNA damage in developing C. elegans. Additionally, the effect of persistent helix-distorting DNA damage on mitochondrial morphology, mitochondrial function and autophagy was investigated in C. elegans and in mammalian cell culture. The rate and specificity of mitochondrial degradation was further examined in cell culture using live-cell fluorescence microscopy and transmission electron microscopy.

Removal of UVC-induced mtDNA damage was detectable by 72 hours in C. elegans and mammalian cell culture, and required mitochondrial fusion, fission and autophagy, providing genetic evidence for a novel mtDNA damage removal pathway. UVC exposure induced autophagy with no detectable effect on mitochondrial morphology in both systems; mitochondrial function was inhibited in the C. elegans system but not in the cell culture system in which the degree of mtDNA damage induced was less. Furthermore, mutations in genes involved in these processes as well as pharmacological inhibition of autophagy exacerbated mtDNA damage-mediated larval arrest, illustrating the in vivo relevance of removal of persistent mtDNA damage. Mutations in genes in these pathways exist in the human population, demonstrating the potential for important gene-environment interactions affecting mitochondrial health after genotoxin exposure.