The vitamin B3 precursor nicotinamide riboside (NR) and the ketone DL-body beta-hydroxybutyrate (BHB) are two of the most promising natural compounds yet identified for the treatment of aging and aging-related diseases. NR increases nicotinamide adenine dinucleotide (NAD) levels to activate sirtuin protein deacetylases and BHB is an anti-aging calorie restriction (CR) mimetic. Caenorhabditis elegans is a 1 mm long nematode worm used as a model system to study aging with a mean lifespan of roughly 2-3 weeks depending upon the exact temperature of culture. NR and BHB have previously been shown to increase lifespan when administered to C. elegans by roughly 20%. However, the effect on lifespan when both compounds are added together has not yet been studied. It is hypothesized that when added together the effect on lifespan will be slightly larger than when either compound is given alone, due to the activation of complementary signaling pathways. The results will help determine if humans could benefit from taking both compounds simultaneously as most signaling pathways that regulate lifespan are conserved from nematodes to humans. For these experiments cultures of mixed age C. elegans nematodes were first treated with alkaline-bleach to kill adult worms leaving only eggs that are protected by their thick eggshell. Using this protocol isolated eggs are age-synchronized to within 9 hours of each other. The eggs were then transferred into E. coli-permeable, but nematode impermeable 8 micron cell culture inserts placed in twelve well microplates with roughly 25-40 eggs per insert. 1.35 mL of liquid S-media containing 9 x109 E. coli cells/mL as food was added to each well. Microplates were shaken to provide culture aeration. After three days, the worms reached adulthood and 0.4 mM fluorodeoxyuridine (FUdR), a DNA synthesis inhibitor, was added to prevent C. elegans egg-laying to maintain age-synchrony. Every Monday, Wednesday, and Friday for the approximate 4 weeks of the lifespan experiments the worms were counted under a microscope and the culture media and bacteria replaced. Results were analyzed using Kaplan-Meier survival curves and Log-rank analysis. Results indicated that individual treatment with BHB or NR or both combined increased lifespan in the two trials performed thus far. Another trial is currently underway, and results will be analyzed after it is completed to determine if the combined treatment has a greater benefit on lifespan then either individual treatment. Future studies could also be performed to determine if either NR or BHB can further extend the lifespan of animals given rapamycin, a TOR kinase inhibitor, another promising anti-aging therapeutic.

Neuropsychiatric disorders severely impact quality of life in millions of patients, contributing more Disease Affected Life Years (DALYs) than cancer or cardiovascular disease. The human brain is a complex system of 100 billion neurons connected by 100 trillion synapses, and human studies of neural disease focus on network-level circuit activity changes, rather than on cellular mechanisms. To probe for neural dynamics on the cellular level, animal models such as the nematode C. elegans have been used to investigate the biochemical and genetic factors contributing to neurological disease. C. elegans are ideal for neurophysiological studies due to their small nervous system, neurochemical homology to humans, and compatibility with non-invasive neural imaging. To better study the cellular mechanisms contributing to neurological disease, we developed automated analysis methods for characterizing the behaviors and associated neural activity during sleep and learning in C. elegans: two neural functions that involve a high degree of behavioral and neural plasticity. We developed two methods to study previously uncharacterized spontaneous adult sleep in C. elegans. A large microfluidic device facilitates population-wide assessment of long-term sleep behavior over 12 hours including effects of fluid flow, oxygen, feeding, odors, and genetic perturbations. Smaller devices allow simultaneous recording of sleep behavior and neuronal activity. Since the onset of adult sleep is stochastically timed, we developed a closed-loop sleep detection system that delivers chemical stimuli to individual animals during sleep and awake states to assess state-dependent changes to neural responses. Sleep increased the arousal threshold to aversive chemical stimulation, yet sensory neuron (ASH) and first-layer interneuron (AIB) responses were unchanged. This localizes adult sleep-dependent neuromodulation within interneurons presynaptic to the AVA premotor interneurons, rather than afferent sensory circuits. Traditionally, the study of learning in C. elegans observes taxis on agar plates which present variable environmental conditions that can lead to a reduction in test-to-test reproducibility. We also translated the butanone enhancement learning assay such that animals can be trained and tested all within the controlled environment of a microfluidic device. Using this system, we demonstrated that C. elegans are capable of associative learning by observing stimulus evoked behavioral responses, rather than taxis. This system allows for more reproducible results and can be used to seamlessly study stimulus-evoked neural plasticity associated with learning. Together, these systems provide platforms for studying the connections between behavioral plasticity and neural circuit modulation in sleep and learning. We can use these systems to further our understanding of the mechanisms underlying neural regulation, function, and disorder using human disease models in C. elegans.

Mitochondria are organelles that contain their own genomes (mtDNA) however, the majority of the mitochondrial proteome is encoded by nuclear genes and imported into the mitochondria for assembly into various components. Mitochondria adapt metabolism and biomass to changes in cellular protein synthesis rates accompanying growth. The signaling mechanisms that precede or initiate a mitochondrial expansion program to coordinate mitochondria-to-nuclear communication during development is not well-understood. C. elegans undergo long bouts of starvation in their natural environment upon hatching and remain developmentally arrested as L1s (also known as “L1 diapause”) until they encounter food sources. Prolonged L1 diapause leads to manifestation of age-related phenotypes and mitochondrial remodeling. The mitochondrial unfolded protein response (UPRmt) is a transcriptional response mediated by the bZip protein ATFS-1. ATFS-1 scales mitochondrial expansion with protein synthesis during normal development by regulating genes involved in mitochondrial biogenesis. Here, we demonstrate that ATFS-1 is required for growth and establishment of mature germline upon exiting from starvation-induced L1 arrest. Starvation survival as well as mtDNA depletion during L1 arrest is independent of ATFS-1. Interestingly, we found that the mitochondrial-localized function of ATFS-1 is required for the recovery and expansion of mtDNA following feeding. Lastly, we demonstrate that ATFS-1 functions downstream of the insulin-IGF signaling pathway to regulate mtDNA quantity. The insulin receptor DAF-2 senses nutrient fluctuations and hypomorphic mutation in DAF-2 causes an increase in mtDNA level partly regulated by mitochondrial-localized ATFS-1. Together, our data indicate the physiological relevance and significance of UPRmt in recovering mitochondrial mass when growth and development resumes following starvation.

This thesis describes my work to develop methods and assays to study transgenerational inheritance in the widely used genetic model organism Caenorhabditis elegans (C. elegans). In the first chapter, I describe a novel method that uses an exogenous histamine-selective chloride channel (HisCl1) for negative selection in transgenesis. C. elegans transgenesis is a core technique used by most laboratories and often requires distinguishing between rare animals with a single-copy transgene inserted into the genome from more frequent animals that carry multiple copies of the transgene in extra-chromosomal arrays. I demonstrate that histamine-selection induces rapid and irreversible paralysis in only array animals thus allowing quick identification of the desired transgenic animals. In the second chapter, I develop a high-throughput assay for quantifying transgenerational epigenetic inheritance of endogenous gene silencing. Small RNA -mediated gene silencing leads to an increased incidence of males in the population which can be inherited for four to six generations. I identify a fluorescent marker that specifically fluoresces in males and show that I can use a large-particle particle sorter to quantify the frequency of males in a population. This automated system will allow me to follow inheritance patterns over at least ten generations in various mutant backgrounds in parallel to determine the genetic basis and the rules of epigenetic inheritance.

C. elegans is a commonly used genetic model organism due to the ease of genetic screens, transgenesis, and microscopy. Here, I describe methods that improve transgenesis in C. elegans and the development of a genetic screen to identify genes involved in the piRNA pathway. Transgenesis is commonly used for most laboratories that utilize C. elegans and improvements are therefore likely to facilitate research across many research areas. In the first chapter, I characterized a pan-muscular promoter that drives fluorophore expression to help identify C. elegans transgenesis. This promoter is an improved co-injection marker as it drives bright fluorescence with low toxicity and high efficiency. In the second chapter, I study piRNAs which are a large class of non-coding RNA that play important roles in protecting the genome from transposable elements in most animals. The study of piRNAs has mostly focused on their function in the germline, but recent evidence suggests functions in somatic cells such as neurons. To identify genes involved in the piRNA pathway in C. elegans, I performed a chemical genetic screen. I identified one mutant with a somatic phenotype and six mutants with a germline phenotype. I have focused on the germline and sequenced two strains and identified candidate genes involved in the piRNA pathway. Future work will focus on validating and identifying the remaining mutants.

Amino acids are the building blocks of protein, but also play important cellular signaling roles. The mechanisms through which altered levels of many amino acids are sensed and the signals transmitted are still largely unknown. Increasing evidence is showing that these signals may influence the aging process. In this regard, methionine restriction appears to be an evolutionary conserved mechanism to delay aging and supplementation with glycine can mimic methionine restriction to extend lifespan in rodents. Tryptophan restriction may also activate specific anti-aging pathways, but it could interfere with cognitive function. With exercise the consumption of dietary branched chain amino acids (BCAAs) may be beneficial in building muscle mass, but high levels of BCAAs as well as tyrosine and phenylalanine in the bloodstream are associated with metabolic disease such as insulin resistance. Individual supplementation or restriction of several different amino acids has shown promise in the treatment of insulin resistance in rodents. Much progress regarding the effects of amino acids on longevity has been made using yeast, nematodes, and fruit flies. Clearly, much more research is needed to understand the signaling pathways activated by amino acid imbalance before efficacious and well-tolerated dietary interventions can be developed for human aging and aging-related disorders. In this review the mechanisms through which altered dietary and cellular levels of the twenty proteogenic amino acids affect aging or aging-related disorders are discussed.

lin-28 was first characterized as a developmental timing regulator in Caenorhabditis elegans. Loss of lin-28 function (lin-28(lf)) mutants skip the hypodermal cell fates specific to the 2nd larval stage. Here, we studied two aspects of lin-28 which had not yet been investigated. First, we show that lin-28(lf) mutants exhibit reduced fertility associated with abnormal somatic gonadal morphology. In particular, the abnormal spermatheca-uterine valve morphology of lin-28(lf) hermaphrodites traps embryos in the spermatheca, which disrupts ovulation and causes embryonic lethality. The same genes downstream of lin-28 in the regulation of hypodermal developmental timing also act downstream of lin-28 in somatic gonadal morphogenesis and fertility. Importantly, we find that hypodermal expression, but not somatic gonadal expression, of lin-28 is sufficient for restoring normal somatic gonadal morphology in lin-28(lf) mutants. We propose that the abnormal somatic gonadal morphogenesis of lin-28(lf) hermaphrodites results from temporal discoordination between the accelerated hypodermal development and normally timed somatic gonadal development. Thus, our findings exemplify how a cell-intrinsic developmental timing program can also control proper development of other interacting tissues, cell non-autonomously. We also investigated the expression patterns and functions of two lin-28 isoforms in C. elegans. Our analysis of spatial expression patterns suggests that lin-28a and lin-28b are co-expressed in diverse tissues. Consistently, neither of isoform specific knock-out mutant, lin-28a(lf) or lin-28b(lf), exhibits defects in hypodermal development, somatic gonad, or fertility, indicating functional redundancy of two isoforms. Our study will contribute to further investigation of lin-28 isoforms by providing the mutants of each isoform as well as the primary analysis of their phenotypes.

Obesity and obesity related diseases represent a leading cause of mortality in the United States and worldwide. Our research is oriented towards the role of lipid metabolism in longevity. Adipose triglyceride lipase, or ATGL, is a rate limiting enzyme in the lipolytic pathway. The nematode, C. elegans has many conserved biologic pathways to mammals, and the lipolytic pathway is one of them. The homologues include the insulin receptor (DAF-2), FoxO1 (DAF-16), and ATGL (ATGL-1). In this study, we use C. elegans as a model to study the role of lipolysis in longevity. It has been previously shown in our lab that overexpression of ATGL-1::GFP increases lifespan. To confirm that the increase in longevity was due to the overexpression of ATGL-1, we have used RNA interference to downregulate expression of ATGL-1::GFP. We have corroborated that ATGL-1::GFP worms have longer lifespans, than wildtype N2 worms. We have also found that RNAi control diet does not affect lifespan of ATGL-1::GFP strains. However, ATGL-1::GFP strains on an RNAi GFP diet demonstrate reduced levels of ATGL-1::GFP and have shorter lifespans compared to their control counterparts. Our findings confirm that overexpression of ATGL-1 increases lifespan of C. elegans probably due to its role in reducing fat content.

No description available.

Parkinson’s disease (PD) is the second most common neurodegenerative disorder primarily affecting the aging population over the age of sixty. Characterized by the significant degeneration of dopaminergic (DAergic) neurons of the substantia nigra causing severe motor dysfunction. Although the exact pathogenesis of this disease is still unknown endoplasmic reticulum stress and mitochondrial dysfunction are believed to play a role. PD is diagnosed after severe DAergic neuron degeneration, and yet is still often misdiagnosed. There is a need for a definitive diagnostic test for the early detection of PD. Current therapies only relieve symptoms and do not stop disease progression. Neurotrophic factors (NTF) are naturally occurring proteins that promote the survival, differentiation and maintenance of neurons and present a promising candidate for the treatment of PD. Cerebral dopamine neurotrophic factor (CDNF) is a novel NTF that protects and rescues DAergic neurons. The present study investigated the role of DAergic activity and CDNF mRNA expression in C. elegans, as well as understanding how does PD affect the endogenous levels of CDNF protein and mRNA expression. We demonstrated that of the various dopamine (DA) synthesis and transport mutants tested, the impaired synthesis of DA from levodopa is linked to the up regulation of CDNF. Also, following unilateral 6-hydroxydopamine (6-OHDA) lesioning protein and mRNA expression of CDNF was not affected implicating ER stress as inducing a possible compensatory up regulation of CDNF, thus returning levels to normal. CDNF mRNA expression was determined to decline with age and possibly increase ones vulnerability to developing a neurodegenerative disorder. An increase mRNA expression of CDNF in the PD patient population was found to be specific to platelets. Stroke patients showed an increase in CDNF expression in whole blood. In conclusion, these findings highlight the importance of the relationship between CDNF and ER stress and warrants further investigation. / Thesis / Master of Science (MSc)