Aging is a degenerative process characterized by a progressive deterioration of cellular components and organelles resulting in mortality. The nematode Caenorhabditis elegans has been used extensively to study the biology of aging, and several determinants of C. elegans longevity are conserved in higher order organisms. The specific mechanisms of longevity in C. elegans have not been completely identified and many pathways are still poorly understood. Dietary restriction is one promising pro-longevity intervention, though its mechanism is poorly defined. DR, a reduction in caloric intake in the absence of malnutrition, extends lifespan in C. elegans and in a wide range of other species, including yeast, flies, mice, and primates. However, this extension is highly dependent on genetic background. To investigate this, we are performing a genome-wide screen of the Vidal RNA interference (RNAi) Library, which has a catalog of 11,511 clones. By knocking down specific genes within the C. elegans genome through RNAi, we are able to observe an extension or reduction in longevity in response to DR. We have screened over 1500 genes, 61 of which are short-lived and 67 of which are long-lived. We are currently following up on our collected data by measuring the full lifespans using the RNAi clones that significantly shortened or extended lifespans in our screen. By screening such a large number of genes, the widespread effect, and hopefully mechanism, of DR will be elucidated. We hope to see genes significantly affected by DR cluster into groups with common functionality, or along the same pathway, and then follow up on those specific pathways to clarify the mechanisms underlying DR. If these pathways are conserved, research findings relating to C. elegans may be applicable to other organisms. We hope to confirm these results and further investigate the interaction between DR and other potential longevity pathways.

In an effort to study evolution and mechanisms of adaptation, seven populations of S. cerevisiae were cultivated in chemostat under nutrient limitation. Whole genome sequencing and Single Nucleotide Polymorphisms (SNPs) analysis were performed on clones isolated at 200 generations. I sought to test the effect of each mutation on the cellular fitness thus to characterize beneficial and passengers mutations. By combining PCR and Sanger sequencing, I confirmed five mutations (4 SNPs, one indel) detected by whole genome sequencing on one clone. Using backcross-mating techniques with the ancestral strains, I was able to isolate each mutation and test their individual fitness. I found that 4 of the mutations are passenger mutations that do not impact the fitness of the cells. One of the mutations, a non-synonymous change in SGF73 leading to the truncation of the protein, increased the competitive fitness by 24%. Mutations leading to the truncation of SGF73 were also found in three other independent clones. The presence of similar mutations in four independent populations suggests that the loss of the protein is an important process of adaptation to sulfate limitation. I am now studying the dynamic and the spread of this mutation in the population over 200 generations. SGF73 is the human ortholog of ataxin-7 of the SAGA complex associated with spinocerebellar ataxia diseases and our findings may be important to study the role of this protein in the development of human disease.

The hypoxia-inducible factor (HIF-1) is an oxygen-dependent transcription factor that plays a crucial role in an animal’s response to changing oxygen availability. When oxygen is plentiful, HIF-1 is ubiquitinated and targeted for degradation by the von Hippel Lindau protein (VHL-1). Recent studies have shown that HIF-1 stabilization through mutation of vhl-1 increases longevity in the model organism Caenorhabditis elegans. This is contrary to humans, where VHL-1 mutations cause a disease characterized by angiomas, renal carcinomas and various other tumors as a direct result of aberrant HIF-1 activation. Since nematodes are post-mitotic organisms in adulthood, constitutive activation of HIF-1 does not cause tumor formation and is able to increase lifespan. In order to better understand the how HIF-1 increases longevity and to relate these findings to mammals, our project aims to find the specific genes and tissues downstream of HIF-1 necessary for its effect on worm longevity. Our goal is to find genes downstream of HIF-1 that benefit longevity without the consequences of VHL disease. To identify these genes, we conducted an RNAi screen for genes that are necessary for HIF-1-mediated longevity. Using the results of this screen, we are creating transgenic worms that overexpress these genes under specific nematode promoters. To test for the tissue specificity of HIF-1-mediated longevity, we are stabilizing HIF-1 in different tissues using tissue specific promoters. Our initial data are promising; we have found two genes downstream of HIF-1 that when overexpressed, increase nematode longevity. At least one of these genes, flavin-monooxygenase-2, is a well-conserved protein known to be involved in xenobiotic metabolism. Additionally, we have found that expressing stabilized HIF-1 in neurons is sufficient to increase nematode lifespan. Our continuing studies will identify additional target genes and tissues important for HIF-1 mediated longevity and will attempt to elucidate the mechanism for their action.

Organisms have evolved specific mechanisms to respond to molecular and environmental stress. These stress-response pathways are evolutionarily conserved and can modify lifespan in model organisms, making them popular targets of aging research. Using the nematode Caenorhabditis elegans (C. elegans), we have found the loss of important genes in stress-response pathways can cause a phenomenon called age-related vulval integrity defects (Avid). Worms with Avid show a small protrusion near their vulva that can eventually lead to expulsion of the intestine and premature death. While many labs have noted this phenotype, it is unknown if Avid is a natural part of C. elegans aging or if genetic and environmental factors induce this phenotype. Because of this, there is confusion as to whether worms with Avid should be censored in aging experiments. The goal of our project is to determine the mechanism of Avid and whether it is part of normal worm aging. Our initial work has identified several conditions and genetic pathways important for Avid, including temperature, food availability, the hypoxic response pathway, the oxidative stress response pathway, and the protein assembly pathway. We have also identified that the reproductive period of the C. elegans lifecycle is influential on Avid frequency, despite the phenotype occurring post-reproductively. We are currently testing the hypothesis that Avid is caused by secretions from the bacteria worms eat that influence nematode physiology and lead to Avid. We hypothesize that the pathways that influence Avid frequency do so by altering the digestion of bacteria in the intestine or by modifying the worm’s immune response. To test our hypothesis we measured the quantity of bacteria ingested and digested by various worm strains at different temperatures and tested whether bacterial metabolites can cause Avid. We intend to combine our results and develop a more complete model of Avid.

Leigh syndrome is a clinically defined collection of diseases comprised of multiple genetic defects related to mitochondrial function. This syndrome results in early death in children typically due to respiratory failure as a consequence of progressive neuropathy, and currently no viable treatment options exist. Leigh syndrome can result from mutations in several different mitochondrial genes, one of which encodes the NDUFS4 subunit of complex I in the mitochondrial electron transport chain. As the NDUFS4 knockout mice phenocopy human patients with Leigh syndrome remarkably well, this model was utilized for the purpose of this study. Dietary restriction (DR) was identified as a possible treatment for mitochondrial deficiencies through a yeast genetic screen, which found that mitochondrial gene mutants generally received a robust lifespan benefit from DR. Thus, the primary focus of our research is to determine the efficacy of rapamycin (a DR mimetic) as an intervention in the NDUFS4 -/- mitochondrial disease mouse model. Rapamycin functions as a DR mimetic through its inhibition of the mechanistic target of rapamycin (mTOR) signaling pathway, which is responsible for modulating a variety of metabolic and cellular processes. Our model suggests that the increased NADH/NAD ratio resulting from a complex I deficiency in these knockout mice is sensed as energy abundance and results in systemic metabolic derailment. We propose that inhibiting mTOR signaling addresses this defect by altering intracellular energy sensing and modifying metabolic status. In order to test the efficacy of rapamycin treatment, NDUFS4 -/- mice were treated with daily injections of rapamycin or vehicle. Lifespan data for each treatment and additional assays for health related parameters were conducted to determine the response to the treatment regime. Investigating Leigh syndrome in this context will allow us to better understand the underlying mechanisms of pathogenesis, and directly test novel interventions aimed at attenuating this disease.

Ethosuximide is a succinimide anticonvulsant used in the clinical treatment of petit mal epileptic seizures in humans. Although its exact mechanism is not fully elucidated, it is believed to inhibit T-type calcium channels. In addition to its medical use, ethosuximide has been found to increase lifespan in the model organism, the nematode C. elegans. Nematodes exhibit lifespan extension when treated with ethosuximide at concentrations below a toxic threshold. While the published experimental results on ethosuximide-treated nematodes are promising, we have discovered that ethosuximide breaks down in the presence of 254 nm ultra-violet (UV) light, forming a toxic by-product. This change in ethosuximide that results in toxicity is specifically caused by irradiation, not heat. Acute exposure of C. elegans to ethosuximide treated with UV light results in death at lower dosages compared to nematodes treated with the control, native compound. UV-treated ethosuximide toxicity is independent of chemosensation (i.e. the sensing of chemical stimuli) as evidenced by the fact that chemosensory mutants do not show enhanced resistance to the UV-treated drug. Some human patients taking ethosuximide experience a butterfly rash under the eyes following exposure to sunlight. We speculate that this rash may be indicative of a similar mechanism of UV-induced cytotoxicity occurring in these patients. Taken together, these studies will help explicate the mechanism of toxicity resulting from exposure of ethosuximide to UV light, as well as the mechanism of action of native ethosuximide.

It is well known that mammals contain several types of phospholipase A2. The cytosolic phospholipases A2 (cPLA2s) are one type, and are composed of six enzymes: cPLA2alpha, cPLA2beta, cPLA2gamma, cPLA2delta, cPLA2epsilon, and cPLA2zeta. There has been significant interest in the cPLA2alpha isoform because of the enzyme's 10 fold preference for the hydrolysis at the sn-2 position of the glycerol backbone in phospholipids, resulting in the liberation of arachidonic acid. Arachidonic acid serves as a precursor for several highly regulated inflammatory mediators that play an important role in asthma, atherosclerosis, and arthritis. To address these problems, inhibitors that target cPLA2alpha have been developed to serve as anti-inflammatory therapeutics. Wyeth pharmaceuticals has released a class of indole inhibitors that inhibit cPLA2zeta at <10 nM IC50. However, a recent study has shown that in cPLA2alpha-/-stimulated lung fibroblasts arachidonic acid production is lessened but still present. cPLA2zeta has been identified as the other enzyme involved in the release of arachidonic acid. To further our limited understanding of cPLA2zeta's role in the inflammatory process, we are developing selective and potent inhibitors of the cPLA2zeta isoform. Our synthetic strategy is to modify the scaffold of Wyeth's cPLA2alpha inhibitor. Analysis of the structure-activity relationships and 3D molecular modeling has revealed 4 different places on the scaffold to introduce substitutions. To date, we have generated and assayed a panel of inhibitors. These inhibitors display low nanomolar potency, but have only minimal selectivity for cPLA2zeta. Thus, our main focus is to increase the selectivity of our inhibitors towards cPLA2zeta in order to further our understanding of cPLA2zeta's role in arachidonic acid production.