Alzheimer's disease (AD) is the most common cause of dementia, a general term for memory loss and other cognitive abilities. Although this disease has been a major research focus since the 1980s, the pathological mechanisms are still not well understood, and therapeutic interventions have been ineffective. The most definitive method for classifying AD is through identifying accumulations of toxic amyloid-beta (Aβ) and tau proteins in post-mortem brain tissue. Dr. Su-in Lee’s lab has developed a machine learning method that integrates the pathological protein phenotypes with gene expression levels in the same brain tissue. They have highlighted 25 genes with expression level changes that correlate with the tau and Aβ protein aggregation phenotypes. For this proposal, I have integrated these human neuropathology-based phenotypes with the genetic power of Caenorhabditis elegans (C. elegans) to directly test the impact of these candidate genes on the cellular pathology. Previously, all C. elegans tau models had neuronal specific expression. However, neurons are resistant to RNAi. Therefore, I generated a novel transgenic C. elegans tau AD model that has been codon-optimized to express tau in body wall muscles instead of neurons. I measured the animal’s health with age in a series of phenotypic assays: egg-laying, growth, movement, paralysis, and lifespan analysis. This line exhibits premature paralysis and decreased crawling speeds, providing an easy to score phenotype. This new model allows for high-throughput RNAi screening to test the identified 25 genes’ effects on worm health by utilizing the automated worm-movement technology developed in the Matt Kaeberlein lab that can simultaneously determine the rate of paralysis of thousands of worms. The results of my genetic screening will lead to a better understanding of the human genes that are dysregulated in human AD brains, provide a basis for genetically-dissecting the pathways influencing tau toxicity, and suggest new therapeutic targets.

Alzheimer’s Disease (AD) is a neurodegenerative disorder characterized by the formation of senile plaques and neurofibrillary tangles through the accumulation of toxic amyloid-beta and Tau protein. There is growing recognition that extracellular vesicles (EVs) can package and transport toxic peptides associated with neurodegenerative disorders – such as AD – to other cells in the brain. Researchers in the Kaeberlein lab have designed methods to isolate these type of vesicles from C. elegans nematodes, a popular invertebrate genetic model. However, current nematode EV purification methods do not permit the following of EV signals from specific tissues when they are under AD proteotoxic-stress. I have generated a transgenic C. elegans AD model that has muscle specific expression of the pathogenic human Tau protein. The protein coding sequence was designed to use optimized codons to ensure high expression of the transgene. I have also generated transgenic nematode lines that express versions of known transmembrane proteins with small affinity tags in a tissue specific manner. The small affinity tags on the proteins make it possible to specifically pull down the EVs from designated tissues through standard immunohistochemistry techniques. The abundance of tissue-specific EV protein and RNA cargos from transgenic lines with or without human Tau have then been quantified using LC-MS-MS and RNAseq analyses, and parsed and condensed into a MySQL database via a C# program. The database allows for simple searching through large amounts of MS data, making data analysis more efficient and effective. Thus the methodology and tools I develop in this project could become a promising new approach for identifying novel therapeutic gene targets and biomarkers of AD stress.

Heart disease is a major health pandemic, and the myocardial infarction (MI), also known as a heart attack, is the leading cause of death. This is because adult cardiomyocytes (CMs) present in the heart cannot divide and proliferate, preventing the heart from regenerating itself and repairing tissue damage. From developmental studies in rodents, we know the Notch signaling pathway is crucial in mediating expansion and proliferation of CMs during development, as impairments in Notch lead to cardiac defects. Notch is inactive in adult CMs, which suggests it plays a role in CM renewal; however, this mechanism is still unknown. Thus, our goal is to investigate the role of Notch in CM proliferation and cell cycle regulation. For the purpose of this study, we are using CMs differentiated from a human embryonic stem cell line, RUES2. Differentiation was completed over a 17-day period by culturing the cells as a monolayer. On Day 0, Chiron 99021 is added to activate Wnt signaling, promoting mesoderm formation. Wnt-C59 is added at Day 2 to inhibit Wnt signaling and differentiate the cells into progenitors, and B27 supplement at Day 6 promotes full differentiation into CMs. This adherent protocol recapitulates every step of natural heart development in vitro. Purity of the cell populations, as assessed by flow cytometry staining for cardiac troponin T (cTnT), was 97.1 ± 0.9% cTnT+. We then determined the proliferative capabilities of CMs in the presence of a Notch inhibitor, DAPT. DAPT inhibits gamma-secretase, a transmembrane protein that normally proteolytically cleaves Notch during signaling, thus inactivating the Notch pathway. Treatment with DAPT significantly decreased cell proliferation by about 50%, confirming that Notch directly affects CM proliferation. The results of this study increase our knowledge of CM physiology and the mechanisms behind cell cycle withdrawal, and provide new insights into improving CM renewal.

X chromosome aneuploidy refers to an atypical number of X chromosomes, differing from two X chromosomes in females, or one X and one Y chromosome in males. Unusual numbers of chromosomes arise from errors in cell division that result in too many or too few chromosomes in a cell, with X aneuploidy reported to occur in around 1 in 1000 births depending on the disorder. X chromosome aneuploidy disorders, such as Klinefelter (47, XXY), Triple X (47, XXX) and Turner (45, XO) syndromes are associated with developmental abnormalities, including cognitive and cardiovascular defects. Our goal is to generate isogenic induced Pluripotency Stem Cells (iPSCs) with different numbers of X chromosomes from patients with X aneuploidy, and subsequently differentiate them into relevant cell types to identify genes affected by aneuploidy. Our lab has previously generated isogenic XXY and XY iPSCs from patients with Klinefelter’s Syndrome by removing the extra inactive X chromosome (Xi). My project is to establish and characterize isogenic lines from mosaic XXY/XY patients, who have a mixture of cell karyotypes. Specifically, I am screening iPSC clones derived from mosaic patients for expression of XIST, a gene expressed only from the Xi, by RT-PCR to determine presence or absence of the Xi. Isogenic control XY iPSC lines derived from the same patient serve as a control to XXY cells, lessening potential for confounding variables due to differences between individuals. Next, I will screen clones with different genotypes for integration of reprogramming vectors by genomic DNA-PCR. Integration-free isogenic XXY and XY clones will be differentiated into cardiomyocytes, neural progenitor cells, and cortical organoids, cell types that are associated with the adverse effects of X aneuploidy. By doing so, we hope to gain insight into gene expression and epigenetic changes associated with X aneuploidy phenotypes in a controlled genetic environment.
 

Mutations in Complex I (NADH:Ubiquinone Oxidoreductase) of the mitochondrial electron transport chain have been reported in up to 30% of pediatric mitochondrial diseases (MDs) and affect 1 in 5000 live births. One of such pathologies, Leigh Syndrome, is often fatal in the first three years of life and has no known cure. Knock-out (KO) of Ndufs4 in mice recapitulates several aspects of the disease, including lethargy, encephalopathy and retarded growth. Acarbose delays the onset of neurological symptoms and prolongs the lifespan of both Ndufs4−/− mice and heterogeneous wild type mice. Acarbose is a type 2 diabetes drug that inhibits alpha glucosidases, resulting in delayed absorption of complex carbohydrates and increased activity of the intestinal flora, including increased levels of short-chain fatty acids (SCFAs) and other fermentation products. No formal study has been conducted to determine whether increased SCFA concentration mediates the effects of acarbose on longevity and MD suppression. Our goal is to elucidate the mechanistic basis of acarbose. We hypothesize that acarbose delays MD progression in Nduf4-/- mice by increasing circulating levels of SCFAs. Thus, SCFAs supplementation should recapitulate the effects of acarbose. We will feed chow containing tributyrin to control and mutant mice from weaning until humane endpoint. We will measure body weight, observe the incidence of forelimb clasping behavior (a widely tested neurological symptom), and record percent survival daily. We expect to observe a delay in clasping behavior and prolonged survival in acarbose-treated KO mice compared to that of untreated KO mice, and that these effects are recapitulated more pronouncedly with higher tributyrin doses. SCFAs are promising therapeutics because they are natural metabolites that are inexpensive and can be manufactured into easy consumable pills. A successful outcome of this study will help progress therapies for patients with MDs and for anti-aging purposes.

A wide range of diseases and disabilities are associated with mitochondrial dysfunction including diabetes, cancer, Alzheimer’s disease, cardiovascular disease, and Parkinson’s disease. Mitochondrial dysfunction triggers activation of a novel mitochondrial retrograde response pathway in Caenorhabditis elegans termed the PMK-3 Retrograde Response, which upon activation leads to a reduction in mitochondrial stress and extension of lifespan. A mitogen-activated protein kinase (MAPK) cascade comprised of DLK-1, SEK-3, and PMK-3 forms the signaling core of the PMK-3 Retrograde Response. In this presentation, I will outline my attempts to develop photo-regulatable PMK-3 and DLK-1 kinases which are tools that can aid in the identification of other proteins involved in this pathway. These photo-regulatable kinases were made by engineering a recombinant form of the fluorescent protein Dronpa into two discrete sites of DLK-1 and PMK-3 to confer photosensitivity of the kinases. Photoregulation of the kinase occurs through dimerization of Dronpa in violet light (inactive kinase) and dissociation in cyan light (active kinase). After determining the identities of phosphorylated proteins purified from nematodes exposed to cyan and violet light using mass spectroscopy, a comparative analysis between the two datasets will suggest which proteins are involved in the PMK-3 Retrograde Response. Further investigation into these proteins could elucidate the role of these proteins in mitigating mitochondrial stress.

Rhabdomyosarcoma (RMS) is a devastating pediatric soft tissue cancer. Currently, the standard treatment regimen for RMS patients remains relatively unchanged. Conventional treatment of RMS includes a combination of chemotherapy, radiation, and surgical tumor resection. Unfortunately, with the heterogeneity of cancer between patients, these therapies are not always effective and can cause undesired health issues for the patient due to their non-specific effects. Targeted drug therapy can help patients live more normal lives. The angiotensin II receptor type 1 (AGTR1) and 2 (AGTR2) are potential targeted therapy targets that can inhibit RMS cancer growth and cause less side effects compared to conventional therapies. AGTR1/2 are the main effectors in the renin angiotensin system regulating cardiovascular health. While there are Federal Drug Administration (FDA) drugs blocking these receptors to treat high blood pressure (Irbesartan), AGTR1 and AGTR2 have yet to be investigated for their role in RMS. Tumor propagating cells (TPC), which function as tumor stem cells in RMS, are proposed to drive tumor metastasis and relapse through a process called self-renewal. Preliminary disruption of AGTR1 and AGTR2 in the two major subtypes of RMS (embryonal and alveolar) with the CRISPR/Cas9 gene editing system resulted in decreased tumor cell growth and self-renewal capabilities. RMS cell lines treated with Irbesartan also decreased in viability compared to untreated cells. Based on our preliminary results, I propose that AGTR1/2 plays a role in regulating RMS cell growth and self-renewal. Further functional characterization of AGTR1/2 and investigation of the cellular mechanism by which AGTR1/2 regulates RMS tumor cell growth and self-renewal can provide a strong rationale for prioritizing AGTR1/2 as targets for drug therapies to slow the progression of RMS without greatly compromising more of the patient's health.

Fanconi Anemia (FA) is a recessively inherited genetic disorder resulting from a loss-of-function mutation in any of the 23 genes that comprise the Fanconi (FANC) gene family. FA is characterized by congenital abnormalities, bone marrow failure, and a predisposition to hematologic and solid cancers. The elevated risk of cancer in FA is largely limited to the squamous mucosa lining of the oropharynx, esophagus and vulva, and is not ameliorated by successful prior bone marrow transplantation. Apart from surgery, effective treatment of these cancers is limited by patient hypersensitivity to standard-of-care therapies that include ionizing radiation and DNA cross-linking drugs. The pathogenesis of FA-derived cancers is still not well understood. As a result, we are developing a “Fanconi Anemia Cancer Cell Line Resource” to provide preclinical models for research on FA-derived head and neck squamous cell carcinoma. The resource will include genomically well-defined, isogenic HNSCC cell line pairs (FA-patient/complemented cells) from patients with FA and isogenic cell line trios (wild-type/FANCA-deficient/FANCA-complemented cells) from control sporadic HNSCC cells. FANCA-deficient control cells were generated by targeted Cas9-mediated deletions in both FANCA alleles. Complementation of the resulting FANCA-deficient cells and patient cells was accomplished by targeted FANC- transgene insertion at a chromosome 4 ‘safe harbor site’. The molecular and cellular phenotype of the clonally-derived knockout and knockout/complemented cell lines was verified by PCR analyses; by Western blot and Multiple Reaction Monitoring (MRM) mass spectrometry analysis of FANCA and FANCD₂ protein expression/modification ± DNA damage; and by cell survival as a function of mitomycin-C (MMC) dose. The FA-HNSCC and sporadic HNSCC cell lines will be available for investigators via the FARF- sponsored Fanconi Anemia Cancer Cell Line Repository. We anticipate this versatile resource will be valuable in the development of novel treatment and faciliate in improving our understanding of FA cancers.
 

Embryonal rhabdomyosarcoma (ERMS) is a common pediatric cancer that has poor prognosis for patients with relapsed disease. ERMS typically harbors mutations in one of 3 RAS proteins. Mutations in NRAS, a member of the RAS family, have been shown to be a driver for many different cancers, including ERMS. The Chen lab previously demonstrated that genetic disruption of the NRAS gene by the CRISPR/Cas9 technique successfully reduced ERMS tumor growth in a human xenograft mouse model. However, these mice also experienced disease relapse. I was able to confirm successful targeting of at least one copy of NRAS in ERMS cells. I have subsequently isolated clones of ERMS cells that continued to grow despite the presence of NRAS gene disruption. In my investigation of candidate genes and pathways that might be responsible for driving continued ERMS tumor cell growth, I saw an increase in the level of YAP1 being produced in NRAS-targeted ERMS cells when compared to the control cells. Based on my preliminary findings, once NRAS is successfully disrupted in ERMS cells, tumor relapse is then driven instead by YAP1. This study could provide novel insight into the mechanisms underlying cancer relapse in response to NRAS targeting and promise alternative treatment plans for ERMS patients.

Sarcopenia, the age-related of loss of muscle mass and function, is associated with a decline in quality of life in the elderly and has few effective treatment options. Sarcopenia is linked to mitochondrial dysfunction and elevated mitochondrial oxidant production. We are investigating the role of elevated mitochondrial oxidative stress in sarcopenia using a mitochondrial targeted therapeutic and a mouse model of accelerated sarcopenia. SS-31 is a mitochondrial targeted peptide that associates with cardiolipin, decreases oxidant production, and increases ATP production in vivo. Superoxide dismutase 1 knockout (Sod1KO) mice lack superoxide dismutase 1 (an enzyme that converts the oxidant superoxide into hydrogen peroxide and molecular oxygen) resulting in an accelerated sarcopenia phenotype. We hypothesize that improving mitochondrial function with SS-31 treatment will delay the decline in muscle function in the Sod1KO mice. To test this, we administered SS-31 to SOD1KO mice through surgically-inserted osmotic pumps for 8 weeks between 3 and 4 months of age, the published timeframe for the onset of skeletal muscle decline in SOD1KO mice. Muscle force generation and fatigue resistance was tested in vivo in the gastrocnemius before pump insertion and monthly after pump insertion for 4 months. At the end of the treatment we used histological and biochemical analyses of mouse tissue samples to determine skeletal muscle fiber type, metabolite and protein concentrations, and muscle fiber respiration and oxidant production. We expected SOD1KO mice with SS-31 to have a lower rate of decline in muscle force production and increased fatigue resistance over time, higher max ATP production, and decreased oxidative stress. The effect of SS-31 on muscle function, mitochondrial quality, and redox homeostasis has exciting potential as a translational therapeutic treatment for human sarcopenia.

Flavin-containing monooxygenases (FMOs) have historically been studied as xenobiotic metabolizing enzymes, but emerging data are consistent with a role for FMOs affecting longevity and metabolism through action on one or more endogenous substrates. Caenorhabditis elegans flavin-containing monooxygenase-2 (fmo-2) is necessary for the effects of multiple major longevity interventions including dietary restriction, and its overexpression is sufficient to extend lifespan. Despite this longevity-promoting role, the mechanisms by which FMO-2 extends lifespan remain undefined. We sought to test the hypothesis that fmo-2 interacts with the sulfur amino acid pathway to extend lifespan by screening RNAi clones of sulfur amino acid pathway genes for lifespan effects on wild type, FMO-2 overexpressor, and fmo-2(ok2147) loss-of-function mutant worms. We found that the increase in longevity induced by the overexpression of FMO-2 requires glutathione reductase, and that fmo-2(ok2147) worms are sensitive to the healthspan-shortening effects of glutathione synthesis RNAi. Additionally, HPLC analysis revealed that fmo-2(ok2147) worms synthesize significantly less glutathione. Our results are consistent with a model in which FMO-2 oxidizes glutathione to stimulate normal glutathione synthesis. This research serves to extend our understanding of the function of FMOs and provides insight into a mechanism by which cells maintain the reducing environment necessary for many metabolic pathways. 

In humans, aging is a major risk factor for high-mortality diseases such as cancer, heart disease, and Alzheimer’s, all of which do not currently have cures. One hypothesis is that by treating the aging process underlying these maladies, the progression of the diseases will also be alleviated. It has been found that many of the aging processes in the single celled eukaryote, Saccharomyces cerevisiae are also conserved in multi-cellular eukaryotes such as humans. One characteristic of aging yeast is the accumulation of extra-chromosomal rDNA circles (ERCs). rDNA is a repetitive region of the genome that encodes for ribosomes: cellular machinery that produces proteins. ERCs are small pieces of rDNA that become excised from the chromosome during homologous recombination. It is commonly thought that ERCs are a consequence of aging and that they build up over time and lead to cell death. My project investigates if ERCs have an evolutionary function that has been selected for. It is known that older yeast cells survive better on non- optimal carbon sources, such as galactose, compared to young yeast cells. I hypothesize that ERCs aid aging yeast cells in surviving on non-optimal carbon sources as an evolutionary adaptation. Sir2 suppresses the creation of ERCs and I have controled the amount of ERCs that accumulate in the cells by using a strain of yeast with a Sir2 deletion alongside a strain with Sir2 overexpression. I grew these strains on either dextrose or galactose to see if varying Sir2 activity affects old cells ability to grow on non-optimal carbon sources. Then I passaged these strains over many generations on glucose or galactose to see if ERC accumulation is favored in non-optimal carbon environments. We will also be quantifying ERC copy number through gel electrophoresis and quantitative Southern blotting.

Numerous interventions and genetic modifications have been shown to extend lifespan across a diversity of species. However, these studies often assume that extended lifespan is synonymous with extended healthspan. Recent research in the nematode worm, Caenorhabditis elegans, has questioned this assumption, and suggests that increasing lifespan can prolong the frailty associated with old age. This is particularly important for humans, as increasing lifespan without a corresponding increase in healthspan could spell disaster. The majority of healthcare costs are associated with aging-related pathologies, and prolonging life without prolonging health could radically inflate these costs. To parse out the genetic relationship between healthspan and lifespan, we have turned to Drosophila melanogaster, a well characterized model organism for studies on the genetics of aging. We have collected lifespan data as well as multiple measures of healthspan, such as negative geotaxis (climbing), intestinal permeability, Cold stress resistance, and metabolomics data across 16 inbred genotypes. We found a strong positive correlation between lifespan and climbing, and no correlation between cold stress resistance and lifespan. This confirms the importance of lifespan as a primary parameter in aging studies, but suggests additional measures of health are needed to accurately assess health. 

Aging is the single largest risk factor for many diseases, including Alzheimer’s disease (AD). Therefore, many of the molecular mechanisms that cause aging may also contribute to the onset and progression of aging-related diseases. Our approach to tackling the progression of AD takes a biology of aging standpoint, where screening compounds (FDA approved or naturopathic) that impact lifespan may help identify therapies for AD. This is a novel approach with the potential to accelerate clinical translation. This study uses a human Amyloid-beta (Aß) protein-expressing C. elegans strain that becomes progressively paralyzed with age. Utilizing a novel robotic imaging system (the WormBot), we show the impact these compounds have on AD progression through quantification of a delay in paralysis, then behavioral data and health metrics are collected by tracking worm motility over their entire lifespan. Three compounds have been identified to delay paralysis: Thioflavin T, alpha-lipoic acid and resveratrol, all of which we have shown to increase lifespan. We expect to see Thioflavin-T to have a strong influence on paralysis due to its disruption in Aß aggregation. Resveratrol and alpha-lipoic acid's inflence is expected to be associated with its impact on increasing lifespan. Both behavior and binary paralysis results under these three compounds exposure provide holistic insight into mechanisms of Aß toxicity and could lead to promising treatments that have the potential to increase the quality of life for Alzheimer’s disease patients.

Leigh syndrome is a severe mitochondrial disease that affects one in 40,000 newborn infants and typically results in death during early childhood. The disease is caused by genetic mutations that disrupt electron transport chain (ETC) function, which leads to serious impairment of energy production. Impaired energy production pushes cells with heavy energetic demands into a state of stress, which leads to neuronal dysfunction, progressive encephalopathy, loss of motor function, and respiratory failure. Therapeutics to treat Leigh syndrome are not available in the clinic. Recent studies in fly and mouse models of Leigh syndrome show that rapamycin improves survival and decreases neurological impairment. Rapamycin is a potent and specific inhibitor of the mechanistic Target Of Rapamycin (mTOR) signaling pathway, a crucial cellular nutrient-sensing regulatory pathway. We are interested in identifying other interventions to treat Leigh syndrome. In collaboration with researchers that use the mouse model to study Leigh syndrome, we are using the ND2 Drosophila melanogaster (fruit fly) Leigh syndrome model as a rapid screening tool to identify new interventions that prolong survival. The ND2 model contains a mutation in the mitochondrially-encoded ND2 gene that impairs ETC complex I function. To assess drug efficacy, we use a strategy where flies are treated during development, and post-developmental survival is measured. This strategy recapitulates treating Leigh syndrome in children, who are still developing. We have validated this approach by confirming that rapamycin extends adult lifespan when given only during development in our ND2 model. We are testing other interventions that regulate upstream or downstream elements of mTOR signaling or alter metabolism in a way that could improve mitochondrial function. Finding new interventions to treat Leigh syndrome could help improve thousands of children’s health outcomes and their survival.

Much of aging and longevity research is linked to the mitochondria; specifically mitochondrial dysfunction are an important factor in the etiology of age-related diseases. An important metabolic pathway for mitochondrial function is the Nicotinamide Adenine Dinucleotide (NAD+) biosynthesis pathway. NAD+ is a coenzyme found in hundreds of metabolic pathways within the body, and many are specific to high energy metabolic transformations within the mitochondria. This research introduces a multistep procedure which aims to quantify and identify specific metabolites within the NAD+ biosynthesis pathway. The procedure uses Liquid Chromatography-coupled Mass Spectrometry to detect and quantify fourteen metabolites in the NAD+ metabolome. This method can be used to analyze how specific mitochondrial dysfunction, for example deletion of the NADH Ubiquinone Oxidoreductase subunit 4 (Ndufs4), affects the NAD+ biosynthetic pathway, and to explore potential drug treatments for the improvement of such disorders.

Alzheimer’s disease is a neurodegenerative disease that results in deterioration of memory and cognitive function. One of the hallmarks of Alzheimer’s disease (AD) is the formation of tangled fibrils of the Tau protein. Tau is a microtubule-associated protein found in healthy neurons; in the disease state, it can aggregate and impair normal neuronal functions. Caenorhabditis elegans is a powerful genetic model that has been used to elucidate the cellular and genetic pathways that are impacted by AD-associated proteotoxic stress. All previous C. elegans Tau models have neuronal specific expression. However, neurons are resistant to RNAi. Therefore, we generated a novel transgenic C. elegans model of Tau hyperphosphorylation that has been codon-optimized to express Tau in body wall muscles instead of neurons. Two models were developed: an overexpression (OE) line and a single copy insert (SCI) line. We measured the animal’s health with age in a series of phenotypic assays: egg-laying, growth, movement, paralysis, and lifespan analysis. The OE Tau line displayed a significantly lower egg laying rate, developmental delay by approximately 1 day, and significantly reduced speed in comparison to synchronized N2 populations. The SCI Tau line displayed a significantly lower egg laying rate, smaller adults, and no significant reductions in speed in comparison to synchronized N2 populations. These phenotypic characteristics provide a quick, robust metric by which to measure Tau toxicity with age. The muscle expression opens up the possibility of genome wide RNAi screening to identify the genetic pathways underlying cellular responses to Tau toxicity. We will be screening candidate genetic suppressors of Tau toxicity using feeding RNAi. These experiments could point to genetic targets for future genetic therapies for AD.

Protein homeostasis is an essential cellular process that directs cellular pathways involved in maintaining the integrity of the proteome. An important part of maintaining protein homeostasis is the degradation of misfolded and damaged proteins. This degradation primarily occurs by two major pathways: autophagy and proteasome. The proteasome is a protein complex that degrades unneeded and damaged proteins by proteolysis. The proteasome system consists of the Ubiquitin-Dependent Proteasome System (UPS) and the Ubiquitin Independent Proteasome System (UIPS). Previous studies suggest that the UIPS, which consists of the 20S Core Particle (CP) preferentially degrades proteins that accumulate with age and in age-related neurodegenerative diseases such as Alzheimer’s (AD) and Huntington’s (HD). Proteasome Activator (PA) drugs stimulate the 20S CP, and recent evidence suggests that these therapies can lead to the preferential degradation of misfolded proteins in vitro. The goal of our project was to characterize the effects of PA drugs in vivo using C. elegans animal models of AD and HD. These transgenic models express human amyloid-beta and huntingtin proteins, which we quantified using Western Blotting after treatment with the drugs. We hypothesized that PA drugs would reduce the amount of amyloid-beta and huntingtin proteins and lead to an overall decrease in insoluble proteins that accumulate with age in both the wildtype and disease backgrounds. Our preliminary data suggest that some PA drugs improve survival in a wildtype background, as well as in a neurodegenerative model of AD. If these drugs are also effective in reducing toxic protein aggregates, they would represent an exciting avenue for determining the therapeutic potential of small molecule stimulators for the treatment of protein aggregation diseases.

The hypoxia response pathway, induced by genetic activation or by decreasing oxygen available, has been shown to extend the lifespan of C. elegans. A previous experiment conducted in our lab compared the transcriptomes of worms treated with normoxia, continuous hypoxia, and intermittent hypoxia therapy (IHT). This study showed that IHT doubles lifespan in C. elegans and was partially controlled by the enzyme inositol polyphosphate multikinase (IPMK-1), which suppresses some of the lifespan extension benefits of IHT. To further explore the genetic basis for the effect of IPMK-1 on IHT, we performed a forward genetic suppressor screen on the IPMK-1 animals. IPMK-1 animals die at elevated temperature so we mutagenized IPMK-1(sea9) worms and selected animals from the F2 generation that reached adulthood at 26.5^oC. Identifying the genetic changes in these suppressors will tell us more about the control of IHT and how it promotes longevity.

Mitochondrial disease refers to a group of disorders that affects the mitochondria and therefore influences energy production and metabolism. The main purpose of this study is to determine the impact of pharmacological interventions with known age-delaying activity on neurological mitochondrial disease. In order to achieve this, a mouse model of mitochondrial disease lacking a subunit of the NADH-Ubiquinone Oxidoreductase Complex (Ndufs4-/-) was used to conduct experiments. This model recapitulates Leigh Syndrome, a childhood mitochondrial disease characterized by progressive loss of psychomotor activity, retarded growth, and death within the first three years of life. Inhibition of mTOR (mechanistic Target of Rapamycin) with rapamycin increases lifespan across multiple model organisms. Rapamycin also increases lifespan in Ndufs4-/- mice. In this study, we tested whether acarbose, another drug that extends lifespan in mice, could also extend lifespan in Ndufs4-/- mice. Mice treated with acarbose had longer lifespan compared to untreated animals, and a significant delay in the onset of neurological symptoms. We also obtained brain tissue from these mice to determine whether rapamycin and acarbose are acting on the same biochemical pathways to rescue disease in these animals. Western blot analysis of brain protein extract from rapamycin treated mice showed no phosphorylation of S6 ribosomal protein, a marker of mTOR activity. Conversely, mice treated with acarbose showed phosphorylation of S6 ribosomal protein in the brain, suggesting that acarbose does not inhibit mTOR. Although both drugs prolonged lifespan in this model, these results suggest that they do not act on the same biochemical mechanisms. However, both rapamycin and acarbose appear to restore the NAD+/NADH ratio, reduce accumulation of glycolytic intermediates, and reduce acetylation of mitochondrial proteins in the brains of Ndufs4-/- mice, suggesting that the two drugs may have convergent effects on disease suppression.

One fundamental difference between sexes is that females have two X chromosomes, and males have one. This leads to an X chromosome gene dosage imbalance between sexes. X chromosome inactivation (XCI) in females is the process where one X chromosome is inactivated to balance gene dosage. However, some genes remain expressed from the inactive X (Xi) resulting in higher gene expression in females, suggesting these genes may play a female-specific role. My project focuses on Kdm6a, an X-linked escape gene that encodes a histone demethylase that removes trimethylation on lysine 27 of histone 3 (H3K27me3), a histone modification associated with gene repression and highly enriched on the Xi. Using hybrid embryonic stem cells (ES) with a Kdm6a knock out (KO), I contributed to a study demonstrating that KDM6A enhances gene expression in a maternally biased manner, suggesting it is capable of distinguishing parental alleles of genes. I then explored whether KDM6A also regulates allelic expression from the Xi. We hypothesized that Kdm6a KO will lead to decreased escape gene expression from the Xi via increased H3K27me3 at the promoters of escape genes. We have established an F1 hybrid ES cell model to ablate KDM6A protein levels by CRISPR/Cas9. Importantly, these cells have skewed XCI, which facilitates measurements of gene regulation by KDM6A on the Xi. So far, I have shown that Kdm6a KO leads to reduced expression and complete loss of the protein. I confirmed that KO cells retain both X chromosomes in culture and that KO results in reduced capability for differentiation. Next, we initiated studies to measure allele-specific expression of X-linked genes and to determine whether gain of H3K27me3 due to loss of KDM6A may explain expression changes on the Xi. Results from this study will help identify potential therapeutic targets for individuals with super numery X chromosomes.

Ageing is intrinsic to life, and its progression is a major risk factor for many high-morbidity diseases. Through examination of the cellular processes that govern aging, we hope to gain insight into how to reduce not only the rate of aging, but the incidence of associated diseases as well. Genomic instability is one of the key hallmarks of ageing and occurs in both the mitochondrial and nuclear genomes of both humans as well as less complex invertebrate models. Furthermore, loss of mitochondrial DNA stability is also associated with a loss of nuclear genome stability. In addition to producing essential electron transport chain proteins, mitochondria also produce essential iron-sulfur cluster proteins that are necessary for repair functions within the nuclear genome. Our goal is to disentangle the connections between nuclear and mitochondrial genome degradation using fluorescent reporters in Saccharomyces cerevisiae, in conjunction with a novel microfluidic system. Nuclear DNA degradation will be measured using RAD52::GFP, a component of the DNA-damage repair pathway, while the mitochondrial response will be observed with RTG1::mCh, which signals mitochondrial dysfunction. Since RTG1 and RAD52 both localize to the nucleus during DNA damage events, the relative concentrations of these proteins and their temporal patterning will reveal which system tends to fail first. We have engineering a novel strain of Saccharomyces cerevisiae that satisfies these flourescent properties, and will use a microfluidic chip to explore the interaction between both the nuclear and mitochondrial DNA, and determine the timing and causality of the genomic feedback loop that has been previously described.

More than 1 in 5,000 individuals are born with genetic mutations leading to severe mitochondrial diseases. A better understanding of the pathophysiology of disease progression could potentially lead to the discovery of novel interventions to treat these disheartening diseases. We are using a mouse strain that is deficient in the complex I subunit of the Electron Transport Chain (NDUFS4) as a model of mitochondrial disease. The neurometabolic disease known as Leigh syndrome is most often caused by mutations of proteins in the Electron Transport Chain and leads to severe mitochondrial dysfunction. Similar to patients with this disease, these mice exhibit symptoms including retarded growth, neuroinflammation, and loss of motor activity eventually leading to premature death. Our lab recently discovered that rapamycin, an FDA-approved inhibitor of the Mechanistic Target of Rapamycin (mTOR), delays disease progression and drastically increases the survival of these mice. By inhibiting both mTOR complex I and 2, rapamycin deactivates the Protein Kinase C (PKC) pathway. By doing so, inflammation is reduced due to the deactivation of the innate immune response in these mice. Thus, these mechanistic advances suggest targeting the PKC pathway may be beneficial in the discovery of new disease interventions.

Alzheimer’s Disease (AD) is a neurodegenerative disorder and is the major root cause of dementia. The most common AD cases are classified as sporadic AD, which means there are no known genetic or other underlying causes. Therefore, improving our understanding of the etiological factors of AD is highly important. Recent studies have illustrated that infection with Herpes Simplex Virus 1 (HSV1) is associated with AD, however this link has only recently been demonstrated and not yet fully understood. It has been shown that HSV-associated transcripts are enriched in patients with AD: Transcription Factor EB (TFEB), Integral Membrane Protein 2B (ITM2B) and ATRX chromatin remodeler (ATRX). Our goal is to further validate the link between TFEB, ITM2B, and ATRX expression and AD in clinical samples as well as experimental animals. We aim to develop a highly sensitive and quantitative assay utilizing quantitative real-time Polymerase Chain Reaction (qPCR) to investigate expression of TFEB, ITM2B and ATRX transcripts. We are currently in the preliminary stages of verifying our qPCR assay’s precision in quantifying target RNA expression by producing standard curves from recombinant DNA fragments as well as quantifying cDNA transcript samples extracted from human and mouse kidney cells. Through our future work with experimental animals, we seek to improve our mechanistic understanding of the association between HSV infection, HSV-associated transcripts, and AD.

A 2019 study by Lobas, et al. demonstrated that a circularly permuted form of Green Fluorescent Protein (GFP) can be created such that it only fluoresces when bound to adenosine 5’ triphosphate (ATP), thereby acting as an observable ATP sensor. The primary source of ATP production in cells is in the mitochondria, and loss of mitochondrial function is considered a hallmark of aging. Because ATP additionally reflects the energetic availability of tissue in multicellular animals, it is of interest to study how ATP levels change in an organism throughout aging and in response to environmental stressors. This study uses a novel plasmid construct that has been optimized to express the fluorescent ATP sensor in the nematode C. elegans. These nematodes are visualized using our fluorescent imaging robot to measure ATP levels throughout the whole lifetime of the worms in order to determine if cellular ATP levels serve as an aging biomarker. The first construct uses whole body expression of the ATP sensor, which is expected to show varying levels of ATP-reporting fluorescence throughout the life of each animal before darkening in response to age-induced paralysis and death. Subsequent studies employ different promoters in the plasmid to create tissue-specific fluorescence. This allows for a wide combination of experiments that test the effect of environmental, temporal, and genetic factors on specific tissue ATP levels and longevity in C. elegans. For example, expressing the ATP sensor in hepatocytes in organisms under cyanide conditions indicates the energetic response of these cells to toxin. Results from these follow-up studies indicate how cellular energy affects organisms’ lifespans and the ability to respond to stressors, as well as the role that varying biochemical pathways play in maintaining energetic homeostasis during aging.

Alzheimer’s disease (AD) is the most common cause of dementia. However, despite its wide prevalence and gravitas, the cause, molecular mechanism, and pathogenesis of AD are still not well understood. Prior studies indicate the formation and accumulation of amyloid-beta proteins may play a crucial role in the pathology of the disease. Furthermore, for a long time, investigators suspected that microbes may either directly or indirectly induce AD, characterizing AD’s pathology with an antimicrobial response. Some investigators found links between AD and Herpesviridae, specifically HSV-1 that’s highly prevalent in population, but have yet to find the exact relationship between AD and Herpes Simplex Virus (HSV-1). HSV-1 encodes an alkaline nuclease (UL12.5) known to cause degradation of the mitochondrial genome. We hypothesize that UL12.5 activity in the brain may inhibit amyloid-beta protein aggregation and predispose an individual to AD neuropathology. Here, we aim to control the amyloid-beta protein aggregation using a degron attached UL12.5, which will be induced by the plant hormone auxin through a molecular signaling pathway known as auxin-inducible degron. We have engineered an auxin UL12.5-degron construct in order to precisely control the temporal and cell type expression of UL12.5 in Caenorhabditis elegans (C.elegans). This construct was microinjected into the worms and by using auxin, and we controlled the expression of UL12.5 and tested its effects on amyloid-beta and Huntington protein aggregation. Ultimately, we aimed to elucidate the relationship between HSV-1 infection, UL12.5 expression, and neurodegenerative disease which may form the basis of novel treatments.

Knock out of Ndufs4, a gene that encodes a nuclear-encoded subunit of complex 1, models neurological mitochondrial disease in mice. Ndufs4^-/- mice are shorter lived, show fur loss around 21 days of age, and begin to show neurological symptoms around day 35. Acarbose is a FDA-approved anti-diabetic drug used to manage post-prandial glucose spikes. Administration of acarbose increases lifespan in Ndufs4^-/- mice and delays the onset of neurological symptoms. Importantly, acarbose does not appear to restore mitochondrial respiration; rather it decreases protein acetylation in the mitochondria of Ndufs4^-/- mice and restores the NAD+ /NADH ratio in the brain. Due to this observation, we wanted to look into how Ndufs4^-/-Sirt3^-/- mice responded to acarbose treatment because Sirt3 is a NAD-dependent deacetylase responsible for the deacetylation of proteins in the mitochondria. To test this, we crossed Ndufs4^+/- mice into a Sirt3^-/- strain of mice to determine whether Sirt3 is necessary to extend lifespan in response to acarbose. We set up 4 experimental groups consisting of Sirt3^+/+Ndufs4^-/- controls, Sirt3^-/-Ndufs4^-/- controls, Sirt3^+/+Ndufs4^-/- on continual 0.1% acarbose chow from day 21, and Sirt3^-/-Ndufs4^-/- on continual 0.1% acarbose chow from day 21. All mice were housed with companion mice that were heterozygous for the Ndufs4 gene to help with thermal regulation and prevent premature death. Mice were monitored and weighed daily and fed bi-weekly with 0.1% acarbose chow. Knock out of Sirt3 did not affect lifespan in Ndufs4^-/- mice. Furthermore, we measured extended lifespan in the mice treated with acarbose in both the Sirt3^+/+ and Sirt3^-/- genotypes indicating that Sirt3 is not required for lifespan extension from acarbose in this disease model. We are planning to collect brains from Ndufs4^-/- Sirt3^-/- animals to determine whether acarbose reduces acetylation in the mitochondria through a different mechanism, not the upregulation of Sirt3 deacetylase.