Michelle LeBlanc et al. (2008) C.elegans Aging, Stress, Pathogenesis, and Heterochrony Meeting "Oocyte Maturation and Ovulation Modulate Survival in Long-term Oxygen Deprivation in Caenorhabditis elegans in a daf-16 Independent Manner"

Alexander Mendenhall, Pamela Padilla, Desh Mohan, Michelle LeBlanc

[C.elegans Aging, Stress, Pathogenesis, and Heterochrony Meeting, 2008]

Identifying the genotypes and phenotypes that enhance an organism''s ability to survive stress is of interest. We used Caenorhabditis elegans mutants, RNA interference (RNAi), and the chemical 5-fluorodeoxyuridine (FUDR) to test the hypothesis that a reduction in progeny would enhance survival in oxygen deprivation. In the hermaphrodite gonad, germline processes can be simultaneously as well as independently disrupted by genetic mutations. We analyzed genetic mutants with reduced progeny production due to various germline or gonad function defects. Furthermore, we used RNAi to inhibit the function of gene products thought to function in the let-60/ras signaling pathway and play a role in gonad development and function. We determined that a decrease in progeny production enhanced anoxia survival; however, this enhanced survival was not present in sterile hermaphrodites undergoing meiotic maturation and ovulation, seen through the presence of laid unfertilized oocytes. Unlike daf-2(e1370) animals, previously shown to survive long-term anoxia in a daf-16 dependent manner, the mutants with reduced progeny survive in a daf-16 independent manner. This data suggest that the insulin signaling pathway is one mechanism but not the only mechanism to survive oxygen deprivation and that altering gonad function leads to a physiological state conducive for oxygen deprivation survival.

Alexander Mendenhall et al. (2006) Development & Evolution Meeting "Morphological, developmental and genetic analysis of post-embryonic Caenorhabditis elegans exposed to anoxia."

Bobby LaRue, Alexander Mendenhall, Pamela Padilla, Desh Mohan

[Development & Evolution Meeting, 2006]

Organisms have evolved the capacity to survive a variety of environmental stresses such as oxygen deprivation. Metazoan responses to oxygen deprivation include developmental arrest, reduction in developmental trajectory, physiological alterations, metabolic repression, and gene expression changes. Unlike many metazoans, C. elegans are tolerant to severe oxygen deprivation conditions in that all life-cycle stages are able to survive 24 hours of anoxia (< .001kPa O2) by arresting energy requiring processes such as developmental progression and motility. We have found that sex, life cycle stage, and genetics influence the ability to survive long-term anoxia (3-5 days of anoxia at 20 C) or the double stress of high-temperature and anoxia (1 day of anoxia at 28 C). For example, wild-type adult males exposed to anoxia have a significantly higher survival rate in comparison to adult hermaphrodites and the dauer larvae survive several days of anoxia. Furthermore, unlike the wild-type hermaphrodite, the daf-2(e1370) hermaphrodite has the ability to survive long-term anoxia and high temperature anoxia. The goal of this project is to identify mechanisms involved with enhanced anoxia survival. First, we used DIC microscopy to examine and compare tissue structure of wild-type adult hermaphrodites, males and daf-2(e1370) hermaphrodites. We determined, that unlike wild-type hermaphrodites adult, males and daf-2(e1370) hermaphrodites do not accumulate significant tissue damage in long-term anoxia, suggesting that long-term anoxia tolerant animals are better able to maintain tissue structure and function. Second, we used RNA interference to screen DAF-16 regulated genes that suppress the daf-2(e1370) enhanced anoxia-survival phenotype. We found that the enhanced anoxia survival phenotype of males and daf-2(e1370) hermaphrodites is dependent upon the small heat shock protein hsp-12.6. These data suggests that mechanisms have evolved to serve a protective role for animals exposed to severe oxygen deprivation. Identifying the mechanisms regulating oxygen deprivation tolerance in eukaryotic systems may be of interest to further understanding the pathology of environmental stress and genes regulating aging.

Alexander Mendenhall et al. (2007) International Worm Meeting "Sex-Dependent Responses to Oxygen Deprivation in C. elegans."

Pamela Padilla, Michelle LeBlanc, Alexander Mendenhall, Desh Mohan

[International Worm Meeting, 2007]

Oxygen deprivation is a medical, ecological, and economic issue for humans. Identification of the molecular mechanisms animals use to survive oxygen deprivation (anoxia or hypoxia) could result in new medical treatments or environmental policies. We have determined that adult male C. elegans, in comparison to adult hermaphrodites, are able to survive longer periods of anoxic exposure. Briefly, after three days of anoxic exposure males (wild-type or him-8 animals) display a high survival rate (~80%) whereas hermaphrodites have a very low survival rate (~10%). This data lead to the hypothesis that the sex of an organism could influence the response to oxygen deprivation. To test this hypothesis further we are examining genetic and environmental factors that may influence anoxia survival. First, since the daf-2/daf-16 signaling pathway is known to have a role in a variety of stresses including oxygen deprivation we are testing if daf-16 is required for male anoxia survival. Second, we are using RNA interference and genetic mutants to determine if egg production, male genotype or male phenotype influences the ability of an animal to survive anoxia. Third, we are altering animal growth conditions (population density) to analyze the influence pretreatments have on anoxia survival rate. These studies will lead to a greater understanding of the mechanisms nematodes use to survive the stress of anoxia. Furthermore, this work will address the question of whether intrinsic differences that exist between the sexes affect their ability to respond to and survive the stress of oxygen deprivation.

Bobby LaRue et al. (2007) International Worm Meeting "Identification of genes involved with prolonged anoxia survival in Caenorhabditis elegans."

Neil Stewart, Desh Mohan, Alexander Mendenhall, Pamela Padilla, Bobby LaRue

[International Worm Meeting, 2007]

Oxygen deprivation has an enormous economic and human health impact on our society. Understanding mechanisms that cells use to survive prolonged periods of oxygen deprivation have relevancy in human health and welfare. The nematode Caenorhabditis elegans has a remarkable ability to survive oxygen deprivation. Briefly, wild-type embryos and adults have a 90 percent survival rate after three days or one day of anoxia exposure (20 C), respectively. We are using a bifurcated approach to identify and understand the mechanisms used to survive anoxia. First, an RNA interference (RNAi) genomic screen was used to identify genes involved with embryonic anoxia survival. As determined by homology analysis, various classes of genes were identified with this preliminary screening approach. In this report we further examine a gene, that when RNAi reduces the gene product, the embryos display a reduced ability to survive prolonged anoxia (3 days of anoxia). In a second approach we are studying long-term anoxia survival in the adult animal. We conducted a genetic screen to identify mutants that could survive long-term anoxia. A long-term anoxia survival mutant (lans-1) that has a high survival rate when exposed to 4.5 days of anoxia (20 C) was isolated. Currently, phenotype analysis and complementation analysis with daf-2(e1370) is being conducted with lans-1. These studies could lead to a greater understanding of how cells, tissues and organisms at different developmental states, cope with and survive the stress of prolonged oxygen deprivation.

Guruprasada R. Sure et al. (2006) East Asia C. elegans Meeting "Identifying the players involved in the transport of mitochondria in Caenorhabditis elegans neurons"

Swetha Mohan, Guruprasada R. Sure, Shaili N Johri, Sandhya P Koushika

[East Asia C. elegans Meeting, 2006]

The growth, maintenance, and survival of neurons rely critically on organelle transport between the cell body and the synapse. Mitochondria are involved in the ATP production, calcium homeostasis and synaptic vesicles recycling. Mitochondria, unlike other organelles are transported bi-directionally and remain stationary 75% of the time. In neurodegenerative diseases such as Alzheimer""s and Parkinson""s, abnormal mitochondrial distribution has been observed. We are trying to identify the molecular players involved in the transport of mitochondria using classical genetic approaches. To address this question we take a classical genetic approach using a transgenic strain jsIs609 in which GFP is expressed in the mechanosensory neurons and targeted to the mitochondrial matrix. We have performed EMS mutagenesis and screened around 3000 genomes and isolated 17 mutants. These fall into different classes, such as 100% increase in number of mitochondria in axons, 50-75% fewer mitochondria in the axons, abnormal accumulation of mitochondria in minor neurites and large spherical mitochondria in the cell bodies of the neurons. We are currently performing complementation analyses of these mutants. We have also analyzed the distribution of mitochondria in mutants with known roles for the transport of organelles and in the dynamics of mitochondria, such as anc-1, unc-16, unc-116, rab-7 and unc-73. In unc-116 Kinesin-I mutants fewer mitochondria are observed in the axons whereas unc-16 ortholog of Sunday driver have more number of mitochondria in the axons. To analyze the mitochondrial behavior/dynamics with respect to metabolism we have performed starvation analyses. We find that the cell bodies of the neurons are fragmented during starvation but the axons are spared and are normal. We are currently imaging mitochondrial motility and dynamics across the developmental stages. # Guruprasada Reddy Sure and Swetha Mohan contributed equally to this work.