Aging in humans is often characterized by an accumulation of various multifocal degenerative pathologies whose distribution is relatively random within a particular anatomical site. The result is that no two people, not even identical twins, age in exactly the same way. Our hypothesis is that variegated gene expression may be an evolutionary adaptable mechanism which, via epigenetic drift, progresses to develop into age-related cellular degeneration. Our belief is that there exist genetic loci capable of diversifying the degree of gene expression over a range of different proteins. Using Ethyl Methane Sulfonate, we introduced a host of point mutations into cultured fibroblast clone cells. We followed this mutagenesis by a double-selection protocol with Cadmium and 6-Thioguanine. In order for the fibroblast cells to survive this selection, they must simultaneously down regulate the expression of a transferase enzyme while up regulating the expression of metallothionein proteins. We have successfully isolated an extreme class of mutant fibroblast cells which, compared to the parent cell line, show a high degree of variegation in protein expression. These preliminary studies suggest that individual cells can be induced to epigenetically regulate their gene expression. While this epigenetic gambling is adaptive under the selection conditions, once it is initiated, it can progress on its own towards deleterious consequences. It is possible that such epigenetic gambling can escape selection in aging humans and be responsible for a host of age-related diseases, including various neoplasms as well as Alzheimer's dementia.

Aging research seeks to determine the processes that cause degeneration and disease in aged organisms, and to discover ways to prevent and/or slow their progression. Previous studies have shown that aging can be slowed in several model organisms through various means; however much is still not understood. In the model organism Caenorhabditis elegans, previous work shows that decreasing oxygen levels (hypoxia) significantly increases lifespan. The hypoxia-induced lifespan increase is observed when worms are exposed to hypoxia either from a late larval stage for life or if they are deprived of oxygen for just one day at the same larval stage. The low oxygen environment activates the hypoxic response pathway, mediated by the hypoxia-inducible factor (HIF-1). Previous data show that an increase of the HIF-1 protein, via genetic mutation or alterations in atmospheric oxygen, is sufficient to extend lifespan. However, much about this pathway remains unknown, including the mechanism for increased longevity, the difference between genetic and environmental manipulations of HIF-1, and the downstream proteins involved. My project examines the genetic components responsible for extending lifespan in hypoxia. To test this, we measured whether hypoxia increases the lifespans of worm strains with deletions or transgenic overexpression of known aging genes. Our results suggest that disruption of most of the non-HIF-1-related pathways have no effect on hypoxia-induced lifespan extension. However, the data show that the forkhead transcription factor, DAF-16, is required for hypoxia to increase lifespan, and subsequent work has shown that hypoxia causes DAF-16 to enter the nucleus. Interestingly, results indicate that hypoxia decreases lifespan when the oxidative stress response factor, SKN-1, is overexpressed, suggesting that SKN-1 is somehow toxic to worms in low oxygen. Collectively, the results show that hypoxic treatment increases lifespan in worms, but that the hypoxic pathway is intertwined with other stress responsive pathways.

Spinocerebellar ataxia 7 (SCA7) is a heritable disease caused by a mutation in the gene for the protein ataxin-7. SCA7 results in progressive neurodegeneration in the cerebellum, brain stem, and retina, which causes patients to lose motor control and vision. The disease disrupts the intricate circuitry of the cerebellum, impacting cells such as Purkinje cell (PC) neurons and the Bergmann glia (BG) that support them. BG and PC function is interdependent such that dysfunction in one cell type causes damage in the other. I set out to characterize how BG function is altered in SCA7 by examining brain tissue from 40 week old mice with or without the ataxin-7 mutation. The tissue was analyzed for various markers of BG function and morphology. I addressed whether mutant ataxin-7 expression in BG leads to changes in their morphology and identity as radial glia using markers for radial glia such as Sox9. I also used the marker for GLAST, a glutamate transporter, to study changes in their ability to support proper PC neurotransmission through glutamate uptake from the synapses. BG distribution in the cerebellum was studied using the markers Calbindin and S100β to mark PC and BG respectively. The APC/β-catenin intracellular signaling pathway in BG was studied using markers for APC and β-catenin, as this pathway may impact BG’s ability to maintain its structure and thus, its function. The results of this study may suggest which BG functions are affected by SCA7. These functional changes may then provide insight into possible mechanisms for cerebellar neurodegeneration in SCA7.

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 a vulval integrity defect (Vid). Worms with Vid show a small protrusion near their vulva that can eventually lead to rupture of the gonad and premature death. While many labs have noted this phenotype, it is unknown if Vid is a natural part of C. elegans aging or if other genetic and environmental factors induce this phenotype, leading to confusion as to whether worms with Vid should be censored in aging experiments. The goal of our project is to determine the mechanism of Vid and whether it is part of normal worm aging. To further define the mechanism of Vid, we are using three experimental approaches: (1) using mutant worm strains, we are testing the genetic pathways important for causing and preventing Vid, (2) because Vid is temperature dependent, with frequency increasing inversely with temperature, we are testing at what point in their lifespan worms can still be rescued from Vid, (3) beginning with the gonad and vulval muscle, we are looking for cellular death and/or structural weaknesses that could be the direct mechanism behind Vid. Our initial work has identified several genes and genetic pathways important for Vid, including the hypoxic response pathway, the oxidative stress response pathway, and the protein assembly pathway. Our current work is close to identifying when worms are most susceptible to Vid and has begun to describe the physical mechanism of Vid. We intend to combine the results from these experiments to develop a model for Vid.

The regulation of organismal life cycles by genetic components is a research area that has shown great promise in recent years. The process of aging is becoming more important to understand, not only due to the increasing incidence of age-related diseases, but also in order to determine what factors contribute to prolonged health and longevity. A variety of physiological pathways can regulate lifespan in C. elegans nematodes. Among these, one pathway of great interest involves dietary restriction. It is known that “stresses” on the worm, such as reduced food intake, can extend its lifespan. A mutation in the eat-2 gene affects the pharyngeal pumping mechanism whereby the worms ingest bacteria, thus creating a genetic model for dietary restriction. This research investigates the role of steroid signaling in dietary restriction-induced lifespan extension and improved stress resistance, using eat-2 mutant worms as a background and using a heat shock assay to induce stress at various points during the worms’ life cycle. By studying genetic determinants of longevity in C. elegans, my research will help to uncover more information about what can make nematodes long-lived, thus contributing to our understanding of the genetic basis for longevity regulation. More importantly, the existence of human orthologs for many longevity genes in C. elegans suggests that our findings will be valuable in identifying factors that are crucial for human longevity and health.

Although aging has been studied extensively, the molecular mechanisms of longevity are unclear. Dietary restriction (DR), a reduction in calories without malnutrition, increases lifespan in most model organisms tested, including rats, flies, and the round worm, Caenorhabditis elegans. Although DR is the most proven longevity-promoting intervention, the mechanism by which it works has not been fully elucidated. A reduction of metabolic rate and reduction of insulin signaling have been implicated in longevity, though neither appear to be sufficient on their own. Another highly supported mechanism of longevity includes the link between TOR (Target of Rapamycin) signaling and DR. Recent data suggests that TOR and DR act along the same pathway in respect to longevity; however, it is widely accepted that DR influences other pathways in addition to TOR. There are many approaches to DR in C. elegans. Using a bacterium carrying an RNAi plasmid, we feed the worms from egg through the first four days of adulthood and are therefore able to knockout specific genes in temperature sterile nematodes. The worms are then transferred to a liquid medium in the absence of the bacterial food. This process allows us to test the effects of dietary restriction on specific genes and determine whether the mutants will respond synergistically or antagonistically to the intervention. These results will be followed up to determine common pathways relating to longevity. Caloric intake was believed to be the sole mechanism of lifespan extension in DR; however recent data supports the idea that different mechanisms appear to be involved. This screen will provide us with a basis of which knockouts extend or shorten lifespan allowing us to relate the biology of DR with the pathways recognized as determinants of aging. If these pathways are conserved, these research findings may be applicable to other organisms.

Oxidative stress and aggregation of misfolded proteins have been linked to aging in a range of model organisms, including the nematode Caenorhabditis elegans. Reduced expression of genes involved in mitochondrial respiration, such as those coding for electron transport chain (ETC) components, have been found to ameliorate these decreases in lifespan. This rescue is thought to act through upregulation of the mitochondrial unfolded protein response (UPRmt) in a cell-non-autonomous manner. Induction of the UPRmt is essential for degradation of misfolded mitochondrial proteins, and has been correlated with longevity in ETC mutant worms. While the mechanism underlying the UPRmt and its effect on gene expression knockdown is not well understood, there is evidence that knockdown induces the UPRmt. Previous studies demonstrate that knockdown of ETC components, as well as knockdown of a subset of mitochondrial proteins both induce the GFP reporters for the mitochondrial chaperone, hsp-6. To identify novel genes involved in the UPRmt, we are performing a forward genome-wide RNAi screen using the hsp-6::GFP reporter and measuring the UPRmt activity by fluorescent microscopy. We expect genes induced by the UPRmt to cluster into groups with common functionality, or along the same metabolic pathways. These hits may include genes coding for the ETC or other mitochondrial proteins, as well as genes outside of the mitochondrion. We will follow up on our screen data results by sequence verifying RNAi mutants and completing full lifespans to elucidate the interaction between the UPRmt and gene knockdown.