Joel Meyer et al. (2007) International Worm Meeting "Nucleotide excision repair is required for normal lifespan and growth in genotoxin-stressed adult Caenorhabditis elegans."

Wade Lehmann, Astrid Haugen, Jonathan Freedman, Bennett Van Houten, Joel Meyer, Windy Boyd

[International Worm Meeting, 2007]

The importance of DNA repair to adult Caenorhabditis elegans is poorly understood, despite the importance of this organism in aging, neurodegeneration and carcinogenesis research. Using a quantitative PCR assay for DNA damage, we found that the kinetics of repair of ultraviolet C (UVC; 254 nm) radiation-induced DNA damage (t_1/2 ~16 hours) are similar to those observed in mammals, but slower than those observed in bacteria and yeast. We also found that repair of UVC-induced damage was reduced 30-50% in 10 of 10 examined nuclear DNA regions in 6-day adults when compared to 1-day adults, in keeping with in vitro experiments indicating reduced DNA repair with age in mammals. The age-related decrease in repair could not be explained by a reduction in expression of nucleotide excision repair (NER; the DNA repair pathway responsible in C elegans for repair of UVC-induced damage) genes, however, and we used gene expression profiling to explore possible mechanisms for this decline in repair. We identified an age-related decrease in many processes fundamental to homeostasis. In particular, genes coding for proteins required for ATP synthesis were expressed at lower levels in 6-day adults. This was true of both nuclear- and mitochondrial-encoded genes, and was also coincident with a decrease in mitochondrial DNA copy number. These data suggest the hypothesis that the age-related decline in DNA repair is due to a decline in mitochondrial function. To begin to test the importance of DNA repair to adult C elegans, we measured lifespan and adult growth in N2 and repair-deficient (RB864, carrying a loss-of-function mutation in the NER gene xpa-1) nematodes chronically exposed to UVC. Young adults were exposed to daily doses of 0, 6, 12, 25, 50, or 100 J/m2 UVC. Lifespan and adult growth in non-exposed xpa-1 nematodes were not detectably different than in N2 nematodes. Chronic UVC decreased lifespan in both strains in a dose-dependent fashion, but xpa-1 nematodes were much more affected. Adult growth was inhibited by UVC in N2 nematodes at high doses, but completely blocked by the lowest dose in xpa-1 nematodes. Finally, UVC exposure greatly inhibited eating in xpa-1 adults, but only mildly affected eating in N2 adults. Thus, NER is required for normal adult growth, feeding and lifespan in C elegans under conditions of genotoxic stress. We are now characterizing the gene expression response to UVC in C elegans.

Joel N Meyer et al. (2005) International Worm Meeting "DNA damage formation and removal in aging, repair-deficient, or frataxin-deficient Caenorhabditis elegans."

Windy A Boyd, Bennett Van Houten, Joel N Meyer, Jonathan H Freedman

[International Worm Meeting, 2005]

The ability to study DNA damage and repair in a whole organism is currently limited. To address this problem, we adapted a PCR-based assay previously developed for use in other organisms (Van Houten et al., 2000 Mutat Res 460: 81-94.) to C elegans. This assay permits detection of approximately 1 lesion per 105 bases in less than 1 g total genomic DNA, and does not require separation of mitochondrial from nuclear DNA, thus allowing direct comparison of mitochondrial and nuclear DNA damage. Using this assay, we detected a dose-dependent increase in UV (254 nm) radiation-induced lesion frequency, with a slope of 0.4-0.5 lesions/10kb/100J/m2 in young adults, in both nuclear and mitochondrial targets. L1 and dauer larvae were > 5-fold more sensitive than young adults. After 24 h, ~70% of UV-induced damage was repaired in the transcribed F33H2.5 (DNA polymerase epsilon; pol E) gene, but less than 50% was repaired in the nontranscribed nob-1 gene. These data suggest that transcription-coupled repair occurs in C elegans, as in mammals. Little or no repair was detected in the mitochondrial genome in the same time period. Approximately 30% of the initial damage was repaired after 24 h in the pol E gene in strain RB864, which carries a deletion in the xpa-1 gene (ortholog of human xeroderma pigmentosum complementation group A gene, involved in nucleotide excision repair). Senescent adults repair UV-induced damage more slowly than do 1-day-old adults (< 50% repair in the pol E gene in 24 h). Thus, we are currently further investigating the hypothesis that DNA repair capacity decreases with age. Finally, we are investigating the hypothesis that frataxin deficiency sensitizes mitochondrial DNA to exposure to iron and other prooxidant chemicals. Frataxin is a mitochondrial protein that, when present in reduced amounts (15-30%) in humans, causes Friedreichs ataxia. Among the symptoms of this disease are high levels of mitochondrial iron and oxidative damage to mitochondrial protein and DNA. However, the precise mechanism by which reduced frataxin causes illness is not clear. We are characterizing the effects of frataxin (frh-1) deficiency in the C elegans mutant strain VC389, as well as an RNAi knockdown population. Preliminary data indicate that VC389 nematodes, which are maintained as a population heterozygous for deletion of frataxin (frh-1 +/-), are more sensitive than wildtype C elegans to iron toxicity, as measured by reduced growth and fecundity.

Bodhicharla, Rakesh et al. (2013) International Worm Meeting "The tobacco-specific nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) induces mitochondrial and nuclear DNA damage in Caenorhabditis elegans."

G.L, Prasad, Bodhicharla, Rakesh, Meyer, Joel

[International Worm Meeting, 2013]

The metabolites of the tobacco-specific carcinogen NNK form DNA adducts in animal models. One report indicates that NNK could cause damage to the mitochondrial as well as nuclear genome in rats (Stepanov and Hecht, 2009 Chem. Res. Toxicol. 22: 406-414). Using a different DNA damage detection technology, we tested whether this could be repeated in the nematode Caenorhabditis elegans; we also evaluated whether mitochondrial function would be affected. We treated N2 strain (wild-type) nematodes with NNK in liquid culture. Quantitative PCR was applied to analyze NNK-induced nuclear and mitochondria DNA damage. This assay has the advantage of measuring all DNA lesions that inhibit the DNA polymerase, and normalizes results to mitochondrial DNA copy number (Hunter et al., 2010 Methods 51:444-451). Our results confirm that NNK causes both nuclear and mitochondrial DNA damage, but surprisingly nuclear DNA damage was greater than mitochondrial DNA damage in C. elegans. To test whether the mitochondrial DNA damage was associated with mitochondrial dysfunction, we used a transgenic nematode strain that permits in vivo measurement of ATP levels and found lower levels of ATP in NNK-exposed animals when compared to the unexposed controls. To test whether the lower levels of ATP were due to the inhibition of respiratory chain components we investigated oxygen consumption in whole C. elegans and found reduced oxygen consumption in exposed animals when compared to the unexposed controls. Our data suggest a model in which NNK causes damage to both nuclear and mitochondrial genomes, and support the hypothesis that the mitochondrial damage is functionally important. These results also represent a first step in developing this genetically tractable organism as a model for assessing NNK toxicity.

Morton, Katherine et al. (2021) International Worm Meeting "Refining Sugar: A neuroprotective role for high sugar diets despite lifespan and reproductive losses"

Heffernan, Nathan, Hartman, Jessica, Morton, Katherine, Meyer, Joel

[International Worm Meeting, 2021]

High sugar diets are cited as a causal factor in the ongoing increase in obesity rates as Americans consume over 75 g of refined sugar and corn syrup per day on average. Further, obesity is associated with increased risk for multiple negative health outcomes including the neurodegenerative disease Parkinson's Disease. Utilizing C. elegans to investigate the effects of two common dietary sugars, glucose and fructose, we investigated the systemic and dopaminergic neuron-specific effects of high sugar diets on bioenergetics and susceptibility to mitochondrial dysfunction. Through the use of in vivo fluorescent reporters and Seahorse XF whole worm respiratory analysis, we found that glucose-fed worms exhibited a 14.9% increase in basal oxygen consumption and alterations to dopaminergic neuronal mitochondrial dynamics and morphology. Our study also indicated that unlike exposures beginning during development, chronic adult high glucose and high fructose diets are protective from 6-hydroxydopamine-induced dopaminergic neurodegeneration, despite deleterious impacts on lifespan and reproduction. These findings support the idea that a shift to glycolytic metabolism protects from acute electron transport chain inhibition, while also supporting previously observed adverse impacts of high sugar diets.

Smith, Amanda M et al. (2009) International Worm Meeting "Mitochondrial fusion and autophagy aid in removal of bulky mitochondrial DNA adducts."

Meyer, Joel, Crocker, Tracey, Bernal, Autumn, Leung, Maxwell, Smith, Amanda M

[International Worm Meeting, 2009]

Mitochondrial DNA (mtDNA) integrity is critical for human health; however, it is unclear how bulky mtDNA adducts formed after exposure to environmentally important genotoxins such as ultraviolet radiation and PAHs are handled. mtDNA may be particularly susceptible to these genotoxins due to the absence of nucleotide excision repair, the primary repair mechanism for bulky DNA adducts in nuclear DNA. We investigated the removal of bulky mtDNA adducts via mitochondrial fusion and autophagy in Caenorhabditis elegans. Larval fusion and autophagy mutant C. elegans were exposed to serial UVC doses over 48 hours. This exposure protocol allows for accumulation of mtDNA damage without lasting damage to nuclear DNA and results in measurable larval growth arrest. Strains carrying mutations in the autophagy gene atgr-18 and fusion genes fzo-1 and eat-3 exhibited exacerbated larval growth arrest with little to no growth recovery after 72 hours. We concluded that these proteins are required for normal recovery from mtDNA damage-induced larval growth arrest. In order to test directly the contribution of autophagy and fusion proteins to removal of UVC-induced DNA damage, we performed RNAi knockdown of autophagy and fusion genes in UVC treated adult glp-1 C. elegans. Knockdown of autophagy and fusion genes inhibited removal of UVC-induced DNA damage, as measured by a quantitative PCR-based assay. These data suggest that autophagy and fusion processes are involved in the removal of bulky DNA damage in mitochondria. Mitochondrial dysfunction as a result of bulky mtDNA damage may trigger mitochondrial remodeling. Preliminary results indicate the relative ATP levels are reduced in UVC treated animals. Therefore, we hypothesize that UVC-induced mtDNA damage is removed via fusion-mediated mitochondrial remodeling and subsequent autophagy, possibly triggered by mitochondrial dysfunction. This research was supported by funding from the National Institutes of Environmental Health Sciences, 1 P30 ES-011961-01A1.

Rooney, John P et al. (2013) International Worm Meeting "Developmental Exposure to Ultraviolet C Radiation Results in Altered Energy Production Later in Life in Caenorhabditis elegans."

Ji, Alex, Leung, Maxwell, Rooney, John P, Bodhicharla, Rakesh, Ryde, Ian, Bess, Amanda, Meyer, Joel

[International Worm Meeting, 2013]

Mitochondrial genomes encode for 13 proteins that are essential components of the electron transport chain (ETC) and are normally present at between 1000 and 100,000 copies per cell. However, this number is greatly reduced during specific developmental stages, representing a potential critical window for mitochondrial genotoxicant exposure. Mitochondrial DNA (mtDNA) is more susceptible than nuclear DNA (nucDNA) to damage by many environmental pollutants, for reasons including absence of Nucleotide Excision Repair (NER). NER is a highly functionally conserved DNA repair pathway that removes bulky, helix distorting lesions such as those caused by ultraviolet C (UVC) radiation and many environmental toxicants. In the current experiment we tested the hypothesis that UVC induced mtDNA damage during larval development would alter energy production throughout the life of the nematode. ATP levels were measured as luminescence using the firefly luciferase expressing PE255 glp-4(bn2) strain (generously provided by Cristina Lagido, University of Aberdeen), and both mtDNA and nuclear DNA copy numbers were measured via quantitative real time PCR. We exposed first larval stage PE255 nematodes to a serial UVC dose that results in the accumulation of mtDNA damage while allowing for repair of nuclear DNA damage, and measured ATP levels, mtDNA and nucDNA copy numbers every 48 hours for 10 days. Immediately after treatment, ATP levels were nearly equal, however at post treatment days 2 through 10 ATP levels were 50-70% lower in UVC treated nematodes. Interestingly, there was no detectable change in either mtDNA or nucDNA copy numbers throughout the time course. These results indicate that mtDNA damage during larval development can alter energy production throughout the life of the nematode, and highlights the potential for a critical window of exposure to mtDNA damage. Furthermore, while it is well established that genetic manipulation of expression of ETC components results in reduced ATP levels and lifespan extension, our data suggest the same phenotypes can be induced via environmental exposures.

Leung, Maxwell C.K. et al. (2009) International Worm Meeting "Mitochondrial genotoxicity during development leads to dopaminergic neurodegeneration in adult Caenorhabditis elegans."

Meyer, Joel N., Arrant, Andrew E., McKeever, Madeline G., Crocker, Tracey L., Smith, Amanda M., Leung, Maxwell C.K.

[International Worm Meeting, 2009]

Mitochondrial DNA damage and mutation have been correlated with neurodegeneration, but causation is unclear. Epidemiological studies have identified an association between neurological disorders and exposure to environmental chemicals such as pesticides and heavy metals. The current study investigates the effect of environmental exposures on mitochondrial DNA damage as well as dopaminergic neurons in Caenorhabditis elegans. In the first experiment, adult germ-line deficient C. elegans (glp-1 strain) was exposed to aflatoxin B1, paraquat, cumene hydroperoxide, and manganese. Mitochondrial and nuclear DNA damage was quantified based on the amount of PCR product amplified from a specific nuclear or mitochondrial DNA segment. Exposure to aflatoxin B1 (100 uM), manganese (2.5 mM), and paraquat (20 mM) resulted in significant dose-dependent mitochondrial DNA damage (0.27, 0.13, and 0.82 lesion per 105 base pairs, respectively, p < 0.05 in all cases). Nuclear DNA damage, on the other hand, was detected only with aflatoxin B1 (0.08 lesion per 105 base pairs), but not with paraquat and manganese (p > 0.05). Exposure to cumene hydroperoxide resulted in no significant nuclear or mitochondrial DNA damage (p > 0.05) but a 30% decrease in the mitochondrial:nuclear DNA copy number ratio as compared to controls (p < 0.05). In the second experiment, the effect of larval exposure to aflatoxin B1, paraquat, and cumene hydroperoxide on the development of dopaminergic neurons was examined. Transgenic C. elegans expressing GFP in dopaminergic neurons (dat-1 strain) were treated with the three chemicals at the L1 stage. The initial observation revealed that exposure to any of the three chemicals can result in dose-dependent dendritic and cell body degeneration in later life stages. Furthermore, similar dopaminergic neurodegeneration could be induced using repeated low-dose ultraviolet C treatment, which resulted in persistent DNA damage in the mitochondria but not in the nucleus. The current findings suggest that (1) mitochondrial DNA is a potential target of environmental exposure; and (2) mitochondrial DNA damage leads to dopaminergic neurodegeneration in adult Caenorhabditis elegans.

Crowder CM et al. (2000) West Coast Worm Meeting "The Effect of Nonimmobilizers on C. elegans"

Crowder CM, Metz LB

[West Coast Worm Meeting, 2000]

The mechanism of action of volatile anesthetics (VAs) is still uncertain. The Meyer - Overton hypothesis tries to correlate anesthetic potency directly to lipid solubility. Yet this theory does not explain why some lipophilic compounds in many homologous series of anesthetics are devoid of any anesthetic potency. F6 and F8, so called nonimmobilizers, are two such compounds that should have anesthetic action but don't. Here, we test whether C. elegans is also unaffected by nonimmobilizers and whether mutants hypersensitive to VAs are sensitized to nonimmobilizers. Behavioral assays including locomotion and mating assays were all performed with concentrations of F6 and F8 at 3-4 times the EC50 (concentration when effect should be half-maximal) predicted by the Meyer-Overton hypothesis. The nonimmobilizers did not significantly effect N2 in any behavioral assay, even after 24-hr exposure. Strains extremely hypersensitive to bona fide VAs were tested with F6 and F8. At 4 times the EC50, F8 had a slight effect on ric-4(js20) while F6 showed none. Both F6 and F8 affected unc-64(js21) at those same high concentrations. These data suggest that reduced neurotransmission somehow sensitize animals to these drugs. To test whether F6 and F8 alter cholinergic neurotransmission as has been shown for true VAs, we measured the effect of F6 and F8 on aldicarb sensitivity (which correlates with the level of cholinergic neurotransmission). Neither F6 nor F8 had an effect on aldicarb sensitivity, and neither antagonized VA-induced aldicarb resistance. Thus, as in vertebrate models, nonimmobilizers do not affect the behavior of the wildtype C. elegans; however, the drugs do have some effect when synaptic transmitter release is reduced.

Katlin B Massirer et al. (2004) West Coast Worm Meeting "The role of PAZ/PIWI domain proteins in the sex-determination/dosage compensation pathway."

Katlin B Massirer, Amy Pasquinelli

[West Coast Worm Meeting, 2004]

In C. elegans sex is determined by the number of X chromosomes and dosage compensation is achieved by regulation of X-signal elements. In hermaphrodites, the nuclear receptor sex-1 transcriptionally represses xol-1 , which is important for the activation of dosage compensation and the hermaphrodite mode of sex determination (Carmi & Meyer, 1999). We found that a member of the PAZ/PIWI domain (PPD) family of proteins genetically interacts with the sex-1 gene. PPD proteins have been implicated in various biological pathways including embryonic patterning and stem cell maintenance. PPD proteins function in the RNAi and microRNA pathways and may elicit their biological effects by controlling the expression and function of the ~ 22 nucleotide RNAs that direct these processes. We are currently exploring the role of specific PPD proteins and miRNA in the C. elegans sex-determination/dosage compensation pathway.

Mello, Danielle et al. (2021) International Worm Meeting "Rotenone modulates the Caenorhabditis elegans immunometabolism and pathogen susceptibility"

Bijwadia, Shefali, Bergemann, Christina, Caldwell, Alexis, Meyer, Joel, Baugh, Ryan, Chitrakar, Rojin, Fisher, Kinsey, Mello, Danielle, Wang, Yang

[International Worm Meeting, 2021]

Mitochondria are central players in host immunometabolism as they function not only as metabolic hubs but also as signaling platforms regulating innate immunity. Environmental exposures to mitochondrial toxicants occur widely and are increasingly frequent. Many pesticides target mitochondrial function, including the well-characterized complex I inhibitor, rotenone (Rot). Exposures to these mitotoxicants can pose a serious threat to organismal health and the onset of diseases by disrupting immunometabolic pathways. Our hypothesis is that Rot can disrupt C. elegans immunometabolism and consequently alter pathogen survival. C. elegans eggs were exposed to Rot (0.5 microM) or vehicle (Ctrl - 0.125 uM DMSO) in liquid and harvested once they reached the L4 larval stage (which was ~24h later for the Rot treatment, as it caused growth delay). Inhibition of mitochondrial respiration by Rot was confirmed by measuring the worm oxygen consumption rate. To explore pathways that were modulated by Rot, we performed a transcriptomic analysis and found 179 differentially expressed genes. WormCat analysis revealed that the two major broad enriched categories were stress response -which was mostly represented by pathogen response and detoxification genes- and metabolism -which was mostly represented by lipid and mitochondrial metabolism genes. Next, Ctrl and Rot-exposed worms were depurated for 48h, and further exposed to Pseudomonas aeruginosa (PA14), and Salmonella enterica (SL1344). Rot-exposed worms were more resistant to SL1344 but more susceptible to PA14. The mitochondrial unfolded protein response (mitoUPR) is a well-known immunometabolic pathway in C. elegans which links mitochondria and immunity and provides resistance to pathogen infection. Rot activated the mitoUPR pathway, which was evidenced by increased hsp-6:GFP expression. Activation was also observed after 24h of exposure to PA14 and SL1344, however, Ctrl PA14-infected worms displayed lower hsp-6:GFP expression, and Rot rescued its expression only to the level of Ctrl OP50-raised worms. Thus, mitoUPR activation could be involved in the increased resistance to SL1344 but the level of activation in PA14 worms might not have been sufficient to promote resistance. By further exploring our transcriptomic dataset using WormExp and "module-weighted annotations" analysis tools, we identified genes that are known to confer resistance to PA14 that were downregulated by the Rot exposure, including HIF-dependent genes, which may underlie the increased susceptibility to PA14. Together, these results demonstrate that the mitotoxicant rotenone can modulate important pathways associated to the C. elegans immunometabolism and alter pathogen resistance.