Perez, L. et al. (2019) International Worm Meeting "Caenorhabdidtis elegans mitochondrial bionergetics during development: advances using the Seahorse Extracellular Flux Analyzer."

Meyer, J.N., Perez, L., Mello, D.F.

[International Worm Meeting, 2019]

Mitochondrial biology has become an area of intense research owing to its unique role in several physiological processes and pathologies. The model organism Caenorhabditis elegans has been extensively used to study ageing and stress responses, which are often tightly associated with mitochondrial metabolism, highlighting the importance of improving our basic understanding about this organelle throughout worm lifespan. The Seahorse Extracellular Flux (XF) Analyzer measures oxygen consumption rates (OCR) of different biological samples in real-time, and has been a great asset to study mitochondrial function with relatively high throughput. The aim of this study was to characterize different mitochondrial parameters in all larval stages, as well as in young and older adult worms using the 24-well XF analyzer. Besides measuring whole worm basal OCR, with the use of different mitochondrial inhibitors we can also assess mitochondrial bioenergetics such as mitochondrial OCR, spare capacity, maximal OCR, ATP-linked OCR, and proton-leak, as well as non-mitochondrial OCR. In order to perform these measurements, we are first optimizing worm numbers and mitochondrial inhibitors concentrations for each life stage. During the optimization process we are also testing different worm media and possible drug interactions. Our preliminary results revealed that (i) the complex IV inhibitor sodium azide response seems to be affected if injected subsequently to the mitochondrial uncoupler FCCP and the ATPase inhibitor DCCD. We have also found that (ii) although L1 worms have very low OCR, their relative spare capacity can be even greater than in adult worms, which are known to have a higher metabolic rate. Moreover, (iii) we found significant differences between basal OCR in worms within a same larval stage. Late L3 worms had approximately a 2-fold increase in basal OCR when compared to early L3s that were only 3h younger. And finally, (iv) OCR measurements in adult worms appear to be much more variable than in larval stages, therefore slight changes in mitochondrial responses of adult worms might not be easily detectable. The careful characterization of the worm mitochondrial metabolism throughout different life stages using the XF analyzer should be a useful asset to the C. elegans research field, and so far has revealed that care must be taken with possible drug interactions, when choosing which life stage to analyze, and when treatments might affect the worm development.

Mello, D.F. et al. (2019) International Worm Meeting "Linking mitochondrial dysfunction during development with pathogen resistance later in life."

Baugh, R.L., Mello, D.F., Meyer, J.N., Bergemann, C.M.

[International Worm Meeting, 2019]

Growing evidence has shown that mitochondrial dysfunction not only compromises the energetic metabolism of cells, but also plays key roles in other physiological processes such as immunomodulation. We hypothesize that mitochondrial toxicity can be a common link between increased prevalence in immune-related disorders and toxic environmental exposures. To test this hypothesis, we are using the pesticide rotenone -a widely known complex I inhibitor- and the model organism Caenorhabditis elegans. Synchronized N2 eggs were exposed to rotenone (0-0.5 M) or 0.25% DMSO (control) in liquid with food (HB101) and harvested 52h later (L4 stage). After a further 48h depuration period, worm survival was followed in the presence of the pathogens Pseudomonas aeruginosa strain PA14, and Salmonella enterica serovar Typhimurium strain SL1344. Our first finding was that rotenone caused a dose-dependent decrease in worm size, which was associated to developmental delay. Worm vulval development was assessed to precisely determine the hours of developmental delay. Stage-synchronized worms exposed to 0.5 M rotenone had a longer median survival in SL1344 than control animals (~40%); but were more susceptible to PA14 (~15%). To validate whether these altered pathogen responses were due to rotenone-induced mitochondrial dysfunction, we analyzed different mitochondrial parameters. No significant differences were observed in preliminary measurements of worm basal oxygen consumption rate (OCR), spare capacity and ATP-linked OCR, or the ratio of mitochondrial to nuclear DNA copy number. This apparent lack of mitochondrial dysfunction after a developmental rotenone exposure may be due to a metabolic compensation in the worms, most likely through upregulation of complex II activity and the glyoxylate cycle, according to previous work. Thus, this appears to be a great model to study signaling between mitochondria and the immune system caused by metabolism shifts, without the detrimental effects of overt mitochondrial dysfunction. Now we are investigating the mechanisms by which mitochondrial signaling might be regulating the observed shifts in resistance to pathogens.

Hershberger, K.A. et al. (2019) International Worm Meeting "Transgenerational effects of early-life arsenic exposure on mitochondrial function in C. elegans."

Hershberger, K.A., Dinyari, A., Reed, C.B., Meyer, J.N.

[International Worm Meeting, 2019]

Arsenic is a well-established environmental toxicant that contributes to the pathogenesis of a number of diseases including cardiomyopathy, neuropathy, and cancers. Carcinogenic effects may be regulated via epigenetic mechanisms, suggesting that effects of arsenic exposure may persist through multiple generations. Arsenic inhibits a number of enzymes in energy production that results in metabolic shifts that support disease pathogenesis, making mitochondria an important target of arsenic toxicity to further study. Here, we use the model organism, C. elegans to study the effect of early-life arsenic exposure on mitochondrial function in the parental generation, and three subsequent generations of unexposed progeny. Age-synchronous worms were exposed to sodium arsenite at concentrations ranging from zero to 100% of the 48-hour LC10. In the exposed parental generation, there was a significant decrease in larval growth after 48 hours of exposure at all concentrations compared to controls. However, in each of the three subsequent generations, 48-hour larval growth was indistinguishable in the progeny of exposed worms compared to control progeny. No changes were observed in mitochondrial copy number or ATP content after exposure in the parental generation; oxygen consumption and reproduction analyses are ongoing. Thus, these levels of sodium arsenite exposure do not appear to have a dramatic effect on mitochondrial function in the parental generation. Further, in contrast to a recent publication on arsenite effects in worms, we have not yet detected transgenerational effects. Future studies include characterizing the epigenome in the parental generation and three subsequent generations to determine if epigenetic modifications are inherited even though we do not currently observe phenotypic inheritance of mitochondrial defects.

Hartman, J.H. et al. (2017) International Worm Meeting "Exercise in Caenorhabditis elegans promotes mitochondrial maintenance and protects against chemical-induced mitotoxicity."

Gordon, K.L., Driscoll, M., Sherwood, D.R., Meyer, J.N., Laranjeiro, R., Hartman, J.H.

[International Worm Meeting, 2017]

Health, disease, and aging are determined by genetic factors, environment, and lifestyle. In humans, environmental contributions to long-term health arise from ambient factors such as environmental chemical exposures and sun exposure, while lifestyle impacts health through mental health and stress, diet, drug usage, and exercise. In laboratory animals, the impact of environment and lifestyle are minimized through carefully controlled experimental conditions, and can therefore be modulated to study the effects of these factors. The effect of exercise on general health has been reported: positive impacts on cognitive function, maintenance of skeletal muscle, and protection from age-related diseases are increasingly recognized. However, molecular mechanisms underlying those protections are not well understood. Furthermore, it is unknown what impacts regular exercise training may have on other health-modifying factors such as toxic exposures from the environment. In this study, we used C. elegans to study the impact of regular exercise training on mitochondrial health and chemical toxicity. For exercise experiments, beginning at L4 stage, animals were transferred to unseeded agar plates without (control) or with liquid (causing worms to swim/thrash) for 90 minutes twice daily. This regimen was carried out for six days, and mitochondrial and toxicity outcomes were tested following exercise on adult day 6 and adult day 10. Preliminary results show that mitochondrial morphology is not significantly different between control and exercise groups at adult day 6 (p=0.64); however, on day 10, control animals have highly fragmented and disorganized mitochondria, while exercised animals exhibit significantly healthier mitochondrial morphology (p=0.0065). Furthermore, mitochondrial respiration significantly differed in spare capacity (p<0.001) on adult day 6, with exercised animals showing increased spare capacity compared to controls. Respiration experiments with day 10 adults are underway; total ATP, mitochondrial DNA copy number, and mitochondrial DNA lesions are also being investigated. Preliminary toxicity experiments showed that exercise-induced changes in mitochondrial health were accompanied by a 30-50% reduction in lethality induced by the mitochondrial toxicants arsenite and rotenone. Together, these data demonstrate that changes in physical activity result in altered mitochondrial health, which extends to protection against chemical toxicants known to damage mitochondria. Ongoing and future experiments will further explore the biochemical and metabolic changes underlying this phenomenon.

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.

Kristin R Armstrong et al. (2004) East Coast Worm Meeting "Alterations of the C. elegans Excretory Duct in Dauer Larvae"

Helen M Chamberlin, Kristin R Armstrong

[East Coast Worm Meeting, 2004]

We are interested in studying organ development and function in order to understand both the normal processes and how improper organ development and function can lead to birth defects or cancer. The research in this project focuses on how an organ's structure, function, and response can be modified by external stimuli, such as environmental conditions. The C. elegans excretory system is a good model for organ development and function because of its simplicity. The excretory system is composed of only four cell types, yet it performs all of the necessary functions of osmotic regulation and waste removal, similar to mammalian kidneys. The simple structure of the excretory system makes it possible to study development and function at the cellular level. Dauer formation is an example of a change induced by environmental conditions in C. elegans . During dauer formation, the morphology of numerous cells and organs is altered, including the pharynx, hypodermis, and excretory system (Riddle, Blumenthal, Meyer, and Priess, 1997). It was previously shown that the excretory system might play a role in the C. elegans ' morphological transition into dauer state (Nelson and Riddle, 1984). We are following up on this research using GFP markers to help visualize each component of the excretory system. We currently have markers for the excretory cell and excretory duct and are working on markers for the excretory pore and gland cells. Using the markers, we are able to observe the morphology of the excretory system during its development. As an initial experiment, we compared the morphology of the excretory duct in normal L3 larvae and L3 larvae that had entered dauer. Our studies showed there is a difference in the excretory duct morphology between dauer and non-dauer larvae. In dauer larvae, the duct's position had shifted, the duct cell body was elongated, and the appendages were hyper-extended as compared to normal L3 larvae. We plan to expand these studies to other excretory cell types, and to investigate how these changes alter the excretory system function. Riddle, D.L., Blumenthal, T., Meyer, B.J., and Priess, J.R., C. elegans II (1997): 739-768 Nelson, F.K and Riddle, D.L., J. Exp. Zool. (1984) Jul; 231 (1): 45-56

DeLong L et al. (1991) International C. elegans Meeting "Effects of dosage compensation disruption on the regulation of sex determination."

DeLong L, Meyer BJ

[International C. elegans Meeting, 1991]

Mutations that disrupt dosage compensation have a paradoxical feminizing effect in some masculinized XX animals. The sdc-3 gene ( formerly dpy-29), one of several genes that controls the hermaphrodite modes of sex determination and dosage compensation, provides a striking example of this phenomenon operating in a single gene. This phenomenon was revealed by the fact that even though sdc-3(null) mutations disrupt both processes, the sdc-3(null) XX animals are not masculinized. In contrast, sdc-3 mutations that disrupt only the sex- determination function but not the dosage compensation function result in fully masculinized XX animals. Analysis of sdc-3 has provided insights into the effects of dosagecompensation disruptions on sex determination. sdc-3 resembles other sdc genes in that it acts at the same point of the regulatory hierarchy. It is negatively regulated by xol-l and in turn negatively regulates her-l . sdc-3 differs from the other sdc genes in that its sex-deterrnination and dosage-compensation functions are independently mutable, resulting in three classes of alleles: those that affect only the sexdetermination function (sdc-3(Tra)), only the dosage-compensation function (sdc-3(Dpy)), or both functions ( sdc-3(null)). The sdc-3(Dpy) and sdc3(null) XX mutants are phenotypically indistinguishable (i.e. hermaphrodites). (DeLong, Plenefisch and Meyer, unpublished) Mutations that eliminate the sdc-3 dosage-compensation function are not the only alterations that suppresses the masculinizing effect of sdc-3(Tra) alleles. Mutations in the dosage compensation genes dpy-26, dpy-27 and dpy-28, as well as increases in X-chromosome dose also suppress this masculinization. Our results suggest that suppression acts via regulation of her-l or the sdc genes. Suppression in dpy; sdc- 3(Tra) mutant animals is correlated with reduction in the elevated her- l expression caused by the sdc-3(Tra) mutations.(see abstract by Klein and Meyer). Suppression is independent of xol-l function, suggesting that it cannot be attributed entirely to changes in the primary sex- determining signal. Additional data are most consistent with suppression resulting from elevated X-linked gene expression, suggesting that suppression results from increased expression of either X-linked negative regulators of her-l or Xlinked positive regulators of the sdc genes. These results suggest that assessing the level of X-linked gene expression might be part of the normal sex-determining process. Altemately, they might indicate that overexpression of genes not normally involved in sex determination have an effect on the process. Several models will be discussed.

Chin Yee Loh et al. (2001) International C. elegans Meeting "The nuclear receptor SEX-1 in C. eleganssex determination"

Barbara J Meyer, Chin Yee Loh

[International C. elegans Meeting, 2001]

In C. elegans , the primary sex determination signal that specifies the two sexes, XO males and XX hermaphrodites, is the number of X chromosomes relative to the sets of autosomes. The X-signal elements located on the X chromosome function cumulatively to repress the sex determination and dosage compensation switch gene xol-1 , whose level of expression sets the sexual fates. The X-signal element, sex-1 , encodes a nuclear receptor that transcriptionally regulates xol-1. Previously, we have identified a 2.8 kb region of the xol-1 promoter that binds SEX-1 in vivo (Carmi, 1998). A GFP reporter fused to this 2.8 kb promoter region is derepressed in a sex-1(gm41) mutant background, further suggesting that SEX-1 regulatory regions might be contained within this fragment of the xol-1 promoter. To determine whether SEX-1 binds directly to the xol-1 promoter, we are undertaking in vitro binding experiments with a partially-purified DNA-binding domain of SEX-1 and fragments of the xol-1 promoter. In these experiments, we are also interested in investigating whether SEX-1 functions as a monomer or dimer. Carmi I., Kopczynski J.B., and Meyer B.J. (1998). The nuclear hormone receptor SEX-1 is an X-chromosome signal that determines nematode sex. Nature 396 , 168-73.

PW Reddien et al. (1999) International C. elegans Meeting "The engulfment of dying cells contributes to the killing process of programmed cell death"

HR Horvitz, JS Cameron, PW Reddien

[International C. elegans Meeting, 1999]

The engulfment of a cell corpse by a neighboring cell typically occurs rapidly after the generation of a cell programmed to die. This engulfment can initiate before the completion of cell division, as shown by electron microscopy (1). Because cell corpses are generated in mutants defective in engulfment, the proposed function of engulfment has long been solely the removal of unwanted apoptotic cell bodies. We have discovered, however, that in addition to functioning in cell-corpse removal, engulfment assists in the killing of dying cells. Animals with strong loss-of-function mutations in the killer gene ced-3 lack most if not all programmed cell deaths. However, weaker ced-3 mutants exist that lack only a small percentage of programmed cell deaths. We found that such a partial block in the execution of programmed cell death is enhanced by mutations in genes that are involved in the engulfment process ( ced-1 , ced-2 , ced-5 , ced-6 , ced-7 , and ced-10 ). Furthermore, mutations in these engulfment genes alone are sufficient to result in a low-penetrance survival of some cells that normally die in the ventral cord. Lineage analysis shows that cells that fail to die initially show some morphological characteristics of programmed cell deaths but ultimately appear morphologically indistinguishable, using Nomarski optics, from living cells. Surviving Pn.aap cells in these ventral cord lineages are capable of expressing the cell-type specific reporter lin-11::gfp (see Cameron, Tsung, and Horvitz, this meeting), suggesting that they are capable of differentiating. We are analyzing the relative contributions of the different engulfment genes to cell killing, how the engulfment role in killing interacts genetically with ced-8 and ced-9 , and the contribution of the killing role of engulfment to the death of different cell types. 1. Sulston, J.E., Albertson, D.G., and Thomson, J.N. (1980). Dev. Biol. 78 , 542-576.