Alzheimer’s Disease (AD) is one of the leading causes of death in the United States. Studies suggest that the root cause of AD is due to two proteins, Aβ1-42 and tau, which accumulate and aggregate into amyloid plaques and neurofibrillary tangles, respectively. Current studies on the heritability and genetic foundation for AD are not well resolved. In order to understand which genes are best able to resist the effects of Aβ1-42 and tau, we utilized an AD fruit fly model. More specifically, we used a mutant line of Drosophila melanogaster, which we call R32, in which human transgenes of Aβ1-42 and tau are expressed in the fly eye. We used this reagent to express AD proteins in the Drosophila Genome Reference Panel (DGRP), a set of 200 fully sequenced, inbred lines derived from a wild population, in order to simulate a model of natural genetic variation. Aβ1-42 and tau proteins lead to degradation in the eyes causing fusions and irregular shaping of ommatidia. Accordingly, we imaged fly eyes at 28 days of age. We found significant genetic variation in ommatidial circularity and area, two measures of eye quality. We performed a genome-wide association study to identify specific single nucleotide polymorphisms (SNPs) that act as markers signaling degradation in the ommatidia. Significant SNPs were found linked to genes associated with neurotransmitter receptors, neural signalling, and photoreceptor differentiation. Further studies will try to validate the effect of the identified genes and quantify the effects of the SNPs on the degenerative eye phenotype. Additionally, metabolomic studies as well as a model that expresses the AD-related proteins in the brain would be utilized to study the effects on lifespan, memory, and locomotion.

The Kaeberlein laboratory utilizes the budding yeast Saccharomyces cerevisiae to study aging, a progression characterized by the gradual deterioration of cellular components and bodily functions. Aging is a progression every organism eventually undergoes, and by better understanding the molecular mechanisms behind it, we can determine how to delay its progression and increase our healthspan, the portion of our lifespan in which we are healthy and free from serious disease. S. cerevisiae functions as an effective model organism because it contains many conserved biological processes, making lifespan simple and fast to measure. The replicative lifespan assay (RLS) is used to quantify the lifespan of the yeast. Individual yeast cells undergo asymmetric mitotic division producing a daughter cell that is distinctively smaller than the original mother cell. Yeast are incubated on growth promoting plates, and the yeast produce daughter cells that are microdissected, counted, and moved away. The cycle repeats until the original mother cell can no longer produce a daughter. The total number of daughter cells produced by individual mother cells is then used to understand the effect of the gene on the lifespan of the yeast. This technique aids in developing models that can be used to understand the conserved biological pathways that influence longevity. Aging is an underlying risk factor for many non-communicable diseases such as Alzheimer’s Disease, Cancer, and heart disease. Our laboratory has performed thousands of gene knockouts on S. cerevisiae and has identified several genes that modulate replicative lifespan. This research is focused on identifying the genes that play a role in reducing the rate of aging to delay the onset of age associated diseases. By understanding how specific genes play a role in aging, we can apply this research to other organisms, with the ultimate intent of increasing the length of a healthier human life.

The incidence of many human diseases, such as cancer, Alzheimer's and heart disease, increases with age. Studying the aging process could provide insight into new treatments and therapies for these aging-related diseases. Our research uses the yeast species, Saccharomyces cerevisiae, as a model organism to explore the effects of specific genetic and environmental factors on aging. Aging in yeast can be measured in terms of replicative lifespan, which is the number of progeny (daughter) cells produced by a parental (mother) cell. In order to measure the replicative lifespan, we utilize a microdissection microscope outfitted with a fiber optic needle that is used to separate individual mother cells from their daughter cells in order to quantify the number of daughter cells produced. The total number of daughters produced by a mother cell is the replicative lifespan of that cell. To determine which genes play an instrumental role in yeast replicative lifespan, our lab has studied the lifespan of 4,698 yeast mutants containing single gene deletions. Many mutants were identified that increase the lifespan of S. cerevisiae. In this study, we have re-evaluated our data to identify potential false-negative results from our initial work, in order to identify additional genes that may influence lifespan. Identifying the false-negative results will be beneficial in finding genes that may play a role in the aging process and had been previously overlooked. This will allow future research to continue in developing treatments that target the prevention of age-associated disease in humans, potentially allowing people to live healthier lives and decreasing the prevalence of cardiovascular disease and cancers. 

Alzheimer’s Disease (AD) is a progressive brain disorder that results in middle to late life dementia. AD, the most common form of dementia, is the sixth leading cause of death in the United States, and the third leading cause of death for older people over 65 years of age. AD is pathologically characterized by the accumulation and aggregation of an alternatively spliced amyloid-beta (Aβ) and hyperphosphorylated tau, which lead to the formation of neuritic plaques and neurofibrillary tangles (NFT’s) in the brain and ultimately to decreased neural function and cell death. Previous studies indicate that the extent to which an individual is affected by the interaction of these proteins and the severity of AD is largely influenced by genetic variation. However, the specific genes involved, their regulation and expression remain largely unknown. Our ongoing project explores the genetic variation underlying Drosophila susceptibility to severe eye cell degeneration caused by human Aβ and tau. We express Aβ and tau in the fly eye through the UAS-GAL4 system to create our AD model Drosophila, and cross them with a panel of flies (the Drosophila Genome Reference Panel, DGRP) that allows us to map natural genetic variation for resistance to Aβ and tau. Our preliminary results show significant variation in neuronal eye cell degeneration among DGRP lines, implying that neurodegeneration would also differ between DGRP lines if Aβ and tau were expressed in the nervous system. Based on current progress, this new project will continue exploring variation in DGRP lines, but this time we will express them in the fly brain. I will measure how different genotypes shape the effect of brain-specific Aβ and tau on lifespan and protein aggregation, and will use Genome Wide Association Studies to identify single nucleotide polymorphisms associated with variation in expression of Aβ and tau.

Mutator phenotypes due to mutations in genes encoding DNA polymerases or mismatch repair proteins lead to increased error rates during DNA replication that accelerate the evolution of cancer cells. Work in the yeast Saccharomyces cerevisae indicates that excessive DNA replication errors lead to error-induced extinction (EEX), where every cell within the population dies due to the random loss of essential functions. One escape route from this lethality is the acquisition of antimutator alleles that lower the overall mutation rate. A possible direction for cancer therapy, then, may be to exploit these antimutators in order to modulate the mutation rate of mutator cells with drugs – either to increase mutation rates to a lethal level or suppress mutation rates to the baseline level of non-cancerous cells, thus slowing the rate of tumor evolution. Our lab has identified several different antimutator mutations in genes that regulate the production of dNTPs, the building blocks of DNA. Examination of cancer genomes from mutator tumors reveal numerous candidate antimutator mutations in the corresponding human genes. We reverse-engineered these candidates into mutator yeast strains to test whether they indeed confer an antimutator phenotype. Evidence that mutations exist in mutator driven tumors that confer an antimutator phenotype would suggest that the unrestrained mutator phenotypes may be lethal to cancer cells. These findings raise the possibility of new methods of human cancer therapy by which specific genes can be targeted to manipulate mutation rates.

Elevated mutation rates due to mutations in the POLE gene, encoding DNA polymerase (Pol) Epsilon, drive a subset of colorectal carcinoma and endometrial cancers, and may accelerate tumor malignancy and pharmacological resistance in other cancers. Work with Saccharomyces cerevisiae revealed that classic mutator alleles encoding defects to Pol Epsilon proofreading mutators rely on the S-phase checkpoint, which regulates dNTP (deoxyribonucleotide triphosphates) levels by controlling the expression and activity of ribonucleotide reductase (RNR). This raises the question, do human POLE cancer alleles share this dependency on the S-phase checkpoint? And if so, do they activate the S-Phase checkpoint by creating replication stress? To investigate these questions, we created mutator yeast strains that mimic human POLE cancer alleles and deleted the S-Phase checkpoint kinase gene, DUN1, which works by indirectly controlling the expression of RNR, and thus dNTP production. Here we show that there is a consistent suppression of mutation rates in diploid strains lacking DUN1 compared to those with one or two working copies. We further extended these studies by monitoring key indicators of checkpoint signaling and replication stress. Our work suggests that targeting dNTPs in order to modulate the mutator phenotype represents a potential therapy for POLE mutator driven tumors.

Cancer, the leading cause of death worldwide, results from a combination of mutagenesis and selection for malignant phenotypes. Mutations from DNA replication errors may initiate as many as two thirds of all human cancers. Accurate DNA replication requires proofreading domains found on the major DNA polymerases as well as mismatch repair (MMR) proteins that detect and repair replication errors after DNA synthesis. Cancer-causing defects in proofreading and/or MMR lead to “mutator phenotypes”, marked by elevated mutation rates and increased cellular mutation burden. Such mutator cells occur spontaneously and drive evolution by generating mutations that enhance population survival. However, mutator cells also accumulate detrimental mutations that compromise fitness. Combined defects in polymerase proofreading and MMR cause error-induced extinction (EEX), which imposes a strong selection for cells with “antimutator” mutations that suppress the mutator phenotype. Using yeast (Saccharomyces cerevisiae), we isolated mutants that survived EEX and mapped the underlying determinants to candidate mutations (APC1, MCM5, PMS1). We are engineering these mutations into yeast strains to test whether they confer an antimutator phenotype. Understanding how mutator cells suppress elevated mutation rates will give us insights into how cancer cells survive and thrive in the face of strong mutagenesis and may suggest novel therapeutic strategies to combat this disease.

Mutator phenotypes due to mutations in genes encoding DNA polymerases or mismatch repair proteins lead to increased error rates during DNA replication that accelerate the evolution of cancer cells and contribute to chemotherapy resistance. Work in the yeast Saccharomyces cerevisiae indicates that excessive DNA replication errors can lead to error-induced extinction (EEX), where every cell within the population dies due to a random lethal mutation. Thus, a possible direction for cancer therapy may be to target antimutators, which can modulate mutation rates of mutator cells and suppress mutation rates, in order to slow the rate of tumor evolution. In a screen isolating antimutators, we identified an EEX mutation in Chromosome Transmission Fidelity 18 (ctf18-K666fs). Ctf18 directly associates with the N-terminus of DNA Polε, including part of the proofreading domain, and thus may influence mutation rates by directly affecting Polε function — a novel finding that would expand our understanding of mutator polymerases. In the current study, I determined whether Ctf18 exerts its antimutator phenotype independent of the S phase checkpoint pathway, the pathway by which all other previously isolated antimutators, such as Dun1, are known to modulate mutation rates. To accomplish this, I compared the mutation rates of double mutant strains, dun1Δ ctf18-K666fs and dun1Δ ctf18Δ, to their respective single mutants in pol2-L439V cells, where lower mutation rates would indicate additive antimutator effects from independent pathways. I also observed the degree of activation of this pathway by fluorescently tagging Sml1, a protein downstream of Dun1 in the S phase checkpoint, expecting to see decreased expression of Sml1 in cells in which the S phase checkpoint was activated. Expanding our understanding of mechanisms by which antimutators can modulate mutation rates may contribute to novel approaches for cancer treatments by targeting the mutator phenotype.

Microglia are the innate immune cells of the central nervous system that exhibit a sustained pro-inflammatory response in the Alzheimer’s disease (AD) brain. Regulation of inflammatory gene expression in microglia by microRNA miR-155 modulates transition between distinct phases of the inflammatory response. Though altered expression profiles of miR-155 is seen in other neurodegenerative disorders, the precise role of this microRNA in modulating inflammation and downstream behavioral deficits in mouse models of AD remains unknown. We hypothesize that microglia specific deletion of miR-155 will alter neuroinflammation and behavioral phenotypes in transgenic mice expressing human mutant amyloid precursor protein and presenilin 1 (APP/PS1), an AD model that exhibits Aβ pathology and memory impairments. We generated trigenic (Cx3cr1-Cre^+/-/Floxed-miR155^+/+/APP/PS1^+/-) to acutely induce microglia specific Cx3cr1 driven Cre-mediated deletion of floxed miR-155 alleles in the APP/PS1 mouse AD model. Changes in inflammatory gene and microRNA expression in microglia 6 and 9 months post miR-155 deletion were assessed by qPCR. We expect that conditional deletion of miR-155 leads to anti-inflammatory gene expression and thus improve cognitive performance. To measure anxiety, spatial memory, and spatial learning, we employ open field chambers with and without novel object recognition and T-maze assessments. Preliminary results support the hypothesis that conditional miR-155 deletion specifically in microglia alters innate immune gene expression and behavioral phenotypes in the APP/PS1 mouse model of AD, further elucidating the impact of the molecular regulators in neuroinflammation in AD.

Rhabdomyosarcoma (RMS) is a rare and devastating pediatric soft tissue sarcoma, predominantly diagnosed in children and adolescents. Metastases and disease relapse rates continue to remain poor with a 5-year survival rate of less than 30%. Current therapeutic methods continue to remain inefficient in causing complete remission. Cancer stem cells (CSCs), a subpopulation of cells within tumors, are able to resist standard therapeutic treatments leading to disease relapse and metastases. Studies using human cells and a zebrafish model of RMS has shown that a population of CSCs exists within RMS. Thus, I am interested in characterizing potential genes that serve as a marker for CSCs in RMS. I am currently pursuing two candidate genes called PAX7 and CD82. Both PAX7 and CD82 have been demonstrated to play an essential role in regulating the function of skeletal muscle stems cells. The molecular signature of the CSCs in RMS is similar to that of skeletal muscle stem cells. Our preliminary data in the Chen lab also demonstrated increased expression of CD82 in a sphere assay, a surrogate in vitro assay to assess stem-like features in tumor cells. Based on these findings, my central hypothesis is that PAX7 and CD82 can potentially serve as specific markers of the CSCs in RMS. To test the hypothesis, I tagged PAX7 and CD82 with the aid of the CRISPR/Cas9 genome editing technology in order to isolate populations of RMS cells that either express PAX7 or CD82. I will perform cell-based assays in order to assess whether the stem-like qualities are enriched in isolated PAX7 and CD82-labeled RMS cell population. The identification of the CSCs in RMS will provide insight for a novel solution in overcoming drug-resistant RMS, tumor recurrence, and metastasis, through CSC-targeted drug therapy.

Rhabdomyosarcoma (RMS) is a rare pediatric cancer characterized by abnormal muscle development. In order to provide insight into muscle development and the pathogenesis of rhabdomyosarcoma, I developed a muscle cell lineage-specific CRISPR/Cas9 gene targeting system in zebrafish that will enable the study of gene function specifically in muscle cells. To create the zebrafish muscle tissue-specific CRISPR/Cas9 gene-editing model without disrupting endogenous gene function, I inserted the Cas9 gene at the end of the zebrafish myf5 gene locus. Myf5 is a gene expressed in developing skeletal muscle tissue and muscle stem cells. By inserting Cas9 downstream of the myf5 gene, Cas9 expression will be induced by the myf5 promoter resulting in gene targeting specifically in muscle tissue. I accomplished this by microinjecting zebrafish embryos with DNA containing the Cas9 gene flanked by short myf5 sequences matching the genome. Cas9 will be inserted at the myf5 locus through a homology independent CRISPR/Cas9-mediated gene knock-in approach which uses a cut and paste mechanism to insert genes into specific locations in the genome. All injected zebrafish embryos were screened for correct integration using polymerase chain reaction (PCR). Founder zebrafish (those with correct integration) were bred to create a stable line of myf5-Cas9 zebrafish. The myf5-Cas9 expressing zebrafish line will be used to target genes specifically in muscle cells and in our zebrafish model of rhabdomyosarcoma (RMS) to identify genes essential for growth of RMS cancer cells. Identification of genes essential for RMS growth will enable the development of new targeted therapies to improve survival of cancer patients.

Cas9 has long been used as an effective genome-editing tool for its ability to knock out genes with high efficiency; only in recent years has it been used to activate genes as well. Our current research aims to develop Cas9 as a gene activating tool in zebrafish, and to use that tool to target genes associated with rhabdomyosarcoma, a devastating pediatric cancer, in order to better understand the genetic factors and interactions leading to tumor growth and progression. When a dead form of Cas9 is used (dCas9), it will bind to the specified site without cutting/knocking out the gene. We have developed a gene-activating system using dead Cas9 fused with a transcriptional activator (dCas9-VPR), and are currently optimizing its use with Csy4 in order to activate multiple genes in a pathway or genetic network. We have seen low efficiency success so far by injecting zebrafish with DNA coding for Csy4, dCas9-VPR, and guide RNAs downstream of the ubiquitin promoter. Once the system is optimized, we hope to use it to test interactions between multiple genes simultaneously, which would allow us to determine how interactions between genes impact cell behavior.
This will allow us to gain a more wholistic understanding of how a genetic pathway works.

Multiple factors interact to determine the lifespan of an organism. The Promislow lab uses the fruit fly Drosophila melanogaster to study the interaction between the metabolome (the profile of all small molecules within an organism), and the lifespan of a fly under stress. In a study of metabolome and lifespan data for many genotypes of Drosophila, our lab found that fly strains with relatively long lifespans when exposed to oxidative stress (peroxide food), had high levels of maltose, a disaccharide of glucose. We hypothesized that maltose was beneficial to flies on peroxide food, and tested this by supplementing the diet with maltose to see if this would extend lifespan. Flies fed supplemental maltose prior to exposure to peroxide food lived longer than flies fed unsupplemented food, supporting our hypothesis. Maltose could extend lifespan by providing energy to the fly via metabolism into glucose, or through another function as a disaccharide. To distinguish between these possibilities, we tested lactose, a disaccharide, to determine if any disaccharide could extend lifespan. Lactose did not extend lifespan, suggesting that disaccharides in general do not extend lifespan under stress. We found that glucose extended lifespan, supporting the hypothesis that maltose extends lifespan via conversion to glucose. Maltose can be stored as glycogen, a polysaccharide, and glucose is derived from glycogen by glycogen phosphorylase, encoded by the gene GlyP. To test the role of glycogen metabolism on lifespan under stress, we manipulated the expression of GlyP. Several transgenes were used to reduce the expression of GlyP by RNA interference (RNAi). RNAi of GlyP decreased lifespan, which supports our hypothesis that glucose derived from glycogen promotes survival. Our work suggests that glucose derived from glycogen or maltose is an important determinant of lifespan under stress, furthering our understanding of links between metabolism and complex phenotypes, like lifespan.

Irreversible damage to organs due to various illnesses that impacts millions of people daily are derived from a common problem: a lack of oxygen. This includes myocardial infarctions that reduce oxygen to the heart and lead to extensive tissue damage. Stem cell-derived cardiomyocytes have been used for direct injection into the heart after an infarct to increase muscle regeneration, but the survival rate and engraftment of these cells are found to be only five to ten percent due to the ischemic conditions. This project aims to increase cardiac regeneration after myocardial infarction through the development of photosynthetic stromal cells that can be injected alongside the stem cell-derived cardiomyocytes. Our initial research has been focused on combining mammalian cells with chloroplasts. We isolated chloroplasts from spinach and measured their oxygen output to demonstrate that chloroplasts produce oxygen. We have then successfully inserted the chloroplasts into mouse fibroblast by incubating them together in the presence of Epidermal Growth Factor as a proof of concept. Once the chloroplasts are successfully uptaken by stromal cells, their ability to produce oxygen upon light exposure will allow surrounding stem cell-derived cardiomyocytes to survive in ischemic conditions. Application of these cells would not be limited to cardiac regeneration, as they could be injected with other cell types into damaged tissues from ischemic conditions to increase oxygen and facilitate regeneration.

Parkinson’s Disease (PD), a neurodegenerative disorder, features accumulation and transmission of toxic species of the protein a-synuclein in specific brain regions. Because examination of such proteins in the brain is invasive and expensive, a robust diagnostic or predictive blood biomarker for PD is strongly required. Previous studies have observed that oligomeric a-synuclein was present in the red blood cells (RBCs) of PD patients, but it did not work well as a biomarker. Because toxic forms of proteins are secreted from cells to the extracellular space in membrane-bounded vesicles called exosomes, therefore we believe that the RBC derived exosomes could be an potential biomarker for PD diagnosis. Also, isolation of specific type of exosome could be equivalent for testing the originated cells. However, there are all types of exosomes in blood released not only by the RBC but also by most cell types, particularly large fenestrated organs such as liver and kidney. To test these vesicles, we have developed the technique of immunoprecipitation to purify distinct kinds of exosomes by targeting its novel unique markers, such as the RBC-specific membrane protein CD235a, and quantifying the amount and size by nanoparticle tracking analysis. My preliminary data have shown that the antibody of CD235a can successfully target the membrane proteins on RBC membrane and its derived exosomes and can collect pure extract of exosomes by immunoprecipitation. In sum, we think the RBC derived exosomes is a promising candidate for PD diagnosis that is worth further investigation. However, the ratio of CD235a positive exosome in whole plasma is still unknown and further tests need to be done on the amount and size distribution of RBC derived exosome in a PD cohort.

C. elegans is a prolific model organism that is well established in the field of aging research and age-related diseases. C. elegans can be genetically manipulated to express human toxic proteins associated with neurodegenerative diseases. Its amenability to genetic screening and short lifespan make it an ideal animal model for studying the genetic basis for the neurological health-declines associated with Alzheimer’s disease (AD). Here we introduce new experimental approaches for quantifying the organism-wide impacts of nervous system specific expression of human AD-associated toxic proteins.. The pharynx in C. elegans is an oral pumping structure used in feeding. The pharyngeal nervous system, comprised of only twenty neurons, dictates the rate of pharynx pumping in the animal. Pharyngeal pumping rates have been shown to change and decay with the age of the animal, making it an ideal metric in aging research. The frequency of pharynx pumping can be measured via an electrophysiological recording of the pharynx’s contractions. We plan to use this electropharyngeogram (EPG) as a measure of neurodegeneration to compare wildtype animals with our AD-model mutants. Furthermore, the proper function of extracellular vesicles (ECVs) are thought to be important in the clearance of toxic peptides associated with AD. The only reported phenotype for ECV signaling is the differential development of the cuticle, the animal’s outer-most layer of epidermis. The cuticle’s formation can be assessed through the appearance of the worm’s alae, a set of three lateral stripes running across the animal from head to tail. The appearance of alae has been observed to be different in our AD model mutants, indicating that ECV signaling has been disrupted. Using these AD-model mutants, we aim to establish these novel phenotypes as a means to further investigate the physiological consequences of AD-associated toxic peptide expression.

Alzheimer’s disease (AD) is a progressive degenerative disorder that affects over 5.5 million Americans, resulting in memory loss and cognitive decline over time. AD is characterized by the accumulation of neurofibrillary tangles composed of pathogenic proteins, such as alpha-synuclein. Recent findings suggest that extracellular vesicles (EVs), which primarily function in intercellular communication, may play a critical role in the propagation of these pathogenic proteins in the brain. However, the cellular pathways that load AD-associated toxic proteins into EVs remain unknown. This research project aims to investigate the cellular pathways that influence the biogenesis, secretion, and uptake of EVs carrying AD-associated toxic proteinsusing the powerful genetic model system Saccharomyces cerevisiae. This simple brewer’s yeast has already been proven to be a useful model for understanding AD-related toxicity. When alpha synuclein is overexpressed in S. cerevisiae, it localizes to cell membranes and results in cytoplasmic aggregation that is observed in humans. We purified EVs from yeast expressing human AD-associated toxic proteins (Tau, alpha-synuclein) to establish whether they contain the human transgenes using Western blot analysis. We developed an immunohistochemistry protocol to rapidly quantify the levels of proteins secreted into the extracellular enviroment. We cultured yeast, optimized Western blotting conditions, purified extracellular vesicles, and used imaging techniques in our project.

Aging is a major risk factor for the most common causes of death in the developed world which include cancer, heart disease, and neurological disorders. Because of this, therapies that target the hallmarks of aging are a promising area of study. The mechanistic target of rapamycin (mTOR) kinase is a major regulator of growth, aging, and survival in cells. Inhibition of the mTOR signaling pathway is associated with increased longevity in organisms including yeast, nematode worms, fruit flies, and mice. Inhibition of mTOR can be achieved through pharmacologic intervention, which is a promising strategy to extend healthy lifespan. To better understand how mTOR inhibitor response varies between genetically-diverse populations, we utilized a set of 90 wild and domesticated yeast strains. We tested a set of mTOR inhibitory compounds on these strains to identify strains with increased sensitivity or resistance to mTOR inhibition. We have identified genetic variants that drive drug sensitivity or resistance in these strains. We then engineered mutations into laboratory strains to confirm that these mutations recapitulate the phenotype of interest. By understanding drug response across genetically-diverse populations, we will create the framework for a precision medicine approach to developing healthspan-enhancing compounds that can be translated to humans.

The development of neurodegenerative diseases, such as Parkinson’s, Huntington’s, and Alzheimer’s affect millions of people every year. Previous studies have shown that an enzyme found in the endemic Herpes Simplex Virus (HSV-1); UL12.5, degrades the mitochondrial genome, which we hypothesize may predispose patients to neurodegenerative conditions. A majority of the human population is infected with HSV-1 and other herpesviridae, which encode similar enzymes, and their reactivation is commonly seen throughout life. We have found that the degradation of mitochondrial DNA by UL12.5 activity is widely conserved and when UL12.5 is expressed in the model organism, Caenorhabditis elegans (C.elegans), mitochondrial DNA content drops nearly 10-fold. Interestingly, we find that activation of the HSV-1 UL12.5 enzyme alters cellular proteostasis; the cell’s mechanism for maintaining properly folded and functional proteins. This change in proteostasis alters the aggregation of misfolded proteins, especially those involved in the cause of neurodegenerative diseases, and leads to neurological phenotypes like paralysis, which we can quantify in the worm. To study the enzymatic activity of the UL12.5, we used modified worms expressing the protein using an inducible transgene that can be verified by fluorescent microscopy. The effects of the UL12.5 activity is determined by tracking lifespan of worms, performing paralysis assays, and counting protein aggregates with controls that use a catalytically inactive version of UL12.5. We have also found that Emodin, a compound found in traditional Chinese medicine, which has previously been shown to inhibit UL12.5, alters the activity of UL12.5 in a worm model of Huntington’s disease. We hypothesize that activation of this enzyme may cause similar pathologies in the human brain and may be a causative factor in human neurodegenerative disease. Further, inhibitors of UL12.5 like Emodin may prove useful therapeutics for treating neurodegenerative diseases.

As the average population lifespan increases in many countries, study into age-associated diseases and the basic biology of aging has become even more important. Studying age-associated changes and lifespan-altering genes in the budding yeast has revealed fundamental insights into the aging process. To measure replicative lifespan of budding yeast cells, we image hundreds of isolated yeast cells trapped in a microfluidic device over the aging process. Using fluorescently labeled strains allows the measurement of protein expression and localization during aging.We observe that Msn2, a general stress-response transcription factor, becomes increasingly activated with age in the budding yeast. Paradoxically, knockout of Msn2 and its homolog Msn4 results in increased lifespan, indicating that the Msn2-driven transcriptional stress response is detrimental to longevity. This effect is mediated by the inappropriate upregulation of a cohort of genes associated with the glucose starvation response despite replete glucose conditions. Deletion of these genes—glucokinase (Glk1), phosphoglucomutase (Pgm2), and glycogen synthase (Gsy1)—also results in increased lifespan. These genes are associated with the accumulation of glycogen during glucose starvation, and by staining old cells trapped in our microfluidic device, we find that glycogen content increases with age. We see that overexpression of the glycogen catabolism gene glycogen phosphorylase (Gph1) increases lifespan, indicating that the mechanisms underlying the detrimental effects of the Msn2-driven age-associated transcriptional program may be driven, at least in part, by the build-up of glycogen in aged cells. Accumulated glycogen is seen in the aged cells of a number of evolutionarily distant species including bacteria and human brain tissue and is implicated in multiple human diseases. Thus, our work may elucidate the details of a fundamental frailty of the metabolic network during aging.

Familial Platelet Disorder with Predisposition for Acute Myeloid Leukemia (FPD/AML) is an autosomal dominant human disorder caused by germline, heterozygous mutations in RUNX1. RUNX1 is a master regulator of hematopoiesis and has specific roles in megakaryocyte maturation and the production of platelets. Monoalellic mutations in RUNX1 result in haploinsufficiency of RUNX1 protein, leading to thrombocytopenia and impaired platelet function before leukemic transformation later in life. RUNX1 is controlled through direct positive auto-activation and its half-life is tightly regulated through ubiquitin-mediated proteasomal degradation. Tightly regulated auto-regulatory circuits are known to have capacity as centers for epigenetic cellular memory. Cellular memory effect offers the opportunity to transiently manipulate steady state product levels and observe maintenance that continues the newly attained levels.The goal of this work is to determine if the positive feedback loop controlling RUNX1 expression has capacity for epigenetic cellular memory that is responsive to transient RUNX1 overexpression. We hypothesize that increasing the presence of endogenous RUNX1 in RUNX1-deficient cells will correct megakaryocyte maturation and platelet formation. To initially determine if an increase in the half-life of RUNX1 will correct megakaryopoiesis, a series of ubiquitylnation and proteasome inhibitors were administered to HEK 293t cells for 6hr, 12hr, and 24hr periods to determine their effect of RUNX1 production and stability, showing increases in RUNX1 expression at RNA and protein levels. iPSC's derived from FPD/AML patients will be ultimately differentiated and analyzed via flow cytometry to observe the inhibitors' effects on platelet production. Anticipated results from this work hope to confirm a dose-dependant response in the epigenetic programming of RUNX1 that could illuminate novel therapies for individuals with FPD/AML.

Aging is associated with a decline in functionality and cellular organization. These changes are reflected in the morphology of cellular organelles, such as the cytoskeleton, which plays an integral role in cell movement and signaling. Age-related changes in the cytoskeletal system make it a great point of interest in age-related studies. However, its roles in aging and longevity are still largely unknown. We hypothesized that alteration of the actin cytoskeleton results in an accumulation of age-related cellular pathologies. To test this hypothesis, we examined cellular markers of aging in C. elegans worms with deactivated actin proteins. The genes for the cytoskeletal system are well conserved between humans and Caenorhabditis elegans, making it a great and popular model system for examining the role of the cytoskeleton in aging. We used RNA interference (RNAi) to knock down the expression of cytoskeletal actins in the worms. Afterwards, we used fluorescent microscopy to examine markers of cellular aging: pH, nucleoskeleton integrity morphology, and mitochondrial network morphology. We have found that perturbation of actin cytoskeleton partially mimics age-related cellular changes. We expect the results to be of help in the ongoing study of the role of the cellular cytoskeletal complex in the human aging process.

Aging is a phenomenon that brings about the onset of many diseases and increases mortality. Understanding aging mechanisms can help us increase longevity and delay the onset of multiple chronic diseases. It has been previously observed that dietary restriction is a method that increases lifespan and delays the loss of function caused by age-associated pathologies. We used the roundworm, Caenhorbabditis elegans, to dissect the mechanisms of dietary restriction-induced longevity. Specifically, we focused on the phenomenon of adult reproductive diapause (ARD). ARD is induced when pre-reproductive juvenile larvae are subjected to starvation. Upon reintroduction to food after prolonged starvation, ARD worms undergo a morphological rejuvenation and resume a normal lifespan. Mechanisms that control post-diapause recovery are still quite unknown. We have found that the TGF-β/BMP signaling pathway is required for post ARD rejuvenation. The TGF-beta signaling pathway is a critical factor for growth processes including cell growth, differentiation, embryonic development, and much more. We aimed to characterize the role of this signaling pathway in ARD maintenance and post-ARD recovery. We hypothesized that TGF-β/BMP mutants accumulate excessive amounts of cellular damage during ARD and are unable to recover from ARD. To address this hypothesis, we analyzed markers of cellular aging among diapaused animals using fluorescent microscopy, in both wild type and TGF-β/BMP mutants. Using transgenic fluorescent reporter strains we examined pH, nucleolar morphology, mitochondria, and cytoskeleton and nucleoskeleton integrity for aging-associated phenotypes. These insights can help us understand the role of the conserved TGF-β/BMP pathway in dietary restriction induced longevity.

Rhabdomyosarcoma (RMS) is a devastating pediatric soft tissue sarcoma. The major RMS subtype, embryonal RMS (ERMS), is often driven by abnormal RAS activity. Activating mutations in the NRAS oncogene, for example, drive cell growth in many types of cancer, including RMS. Unfortunately, there are currently no drugs that can block NRAS activity. Recently developed CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats)/Cas9 gene editing technology has become a powerful tool to disrupt gene function in a wide range of organisms and cell types. My research investigates the therapeutic potential of using CRISPR/Cas9 gene editing in RMS to target a cancer-causing mutation in the NRAS oncogene. To target the mutation in NRAS, I used a Gibson molecular cloning strategy to create a DNA construct expressing the SaCas9 coding sequence DNA and two CRISPR gRNAs. One of the gRNAs precisely matches the activating A183T NRAS mutation found in some ERMS tumors. Lentiviruses were produced from these constructs to deliver the gene editing NRAS A183T therapy to ERMS cancer cells. The efficiency for targeted disruption of the NRAS mutation by the CRISPR/Cas9 technology has been confirmed as comparable to general targeting of NRAS, and the mutations produced by this therapy have been profiled through next-generation sequencing to analyze targeting specificity in normal cells. If this therapy is effective in inhibiting RMS tumor growth, this mutation-specific CRISPR/Cas9 targeting strategy can be further developed into a treatment for cancer caused by this mutation. In contrast to conventional chemotherapy treatment, which kills both normal and cancer cells, this CRISPR/Cas9 gene editing therapy could be introduced with the aid of a suitable delivery system into the cancer cells of a patient carrying the NRAS A183T mutation. The treatment would then target and selectively kill NRAS mutant cancer cells, thereby achieving therapeutic results without causing significant side effects.

Cell divisions require proper replication and distribution of genetic materials between mother and daughter cells, and mistakes made during the process may result in aneuploidy (abnormal number of chromosomes in a cell) and possible carcinogenesis. Thus, checkpoints exist along the cell cycle to ensure that cellular errors are corrected. Likewise, during the mitotic processes of budding yeast, Saccaromyces cerevisiae, numerous checkpoint mechanisms evolved to prevent catastrophic genomic missegregations. By observing the replicative life span of aging yeasts fluorescently tagged with histone 2B through single cell imaging, we identified a new mechanism that is needed in aging cells for correcting nuclear missegregation. This Retrograde Transport Nuclear (RETRN) pathway fixes genomic missegregation by delaying the incorrect mitotic division and returning the genetic material from the daughter to the mother cell. Following the correction, mother cells could continue to divide and produce healthy daughter cells. In our research, we generated new strains of mutant yeasts that underwent incorrect mitotic divisions to further observe the activation of the RETRN mechanism. Each mutant strain had different non-essential genes relevant to the cell cycle deleted. We confirmed the genetic makeup of each mutant strain through replica plating and PCR. Then, we imaged mother cells from verified mutant strains and observed the budding events throughout their lifespans. This allowed us to see specific points along the lifespan where mitotic divisions occurred, and whether the deletions affected the RETRN pathway. We have speculated that this mechanism is a result of cellular damage due to increasing genomic instability in aging cells, and thus is rarely observed in young, healthy cells. The RETRN pathway could be part of many new pathways needed when cells age and become genomically unstable. Since mammalian cells also become genomically unstable with age, similar mechanisms may be necessary for age-associated genomic instability in multicellular eukaryotes.

Rhabdomyosarcoma (RMS) is an aggressive malignant pediatric cancer characterized by pathological skeletal muscle development. Due to a small accumulation of genetic mutations, RMS tumor progression is believed to be driven by epigenetic regulators, which modify chromatin structures to control gene expression. Through gene expression and knockout experiments, our lab identified components of the Switch/Sucrose non-fermentable (SWI/SNF) complex as being potentially involved in RMS growth. SWI/SNF is a family of chromatin remodeling complexes that may function in histone binding and chromatin organization to regulate gene expression. To identify any possible interactions and understand the role SWI/SNF genes in RMS, we developed a large-scale multiplex CRISPR/Cas9 genetic screening system that targets 50 epigenetic regulators, including all the SWI/SNF components. These genes are targeted individually and in every possible two-gene combination. CRISPR, a gene editing technology which uses guide RNA (gRNA) sequences to direct double-stranded cuts in DNA, allows us to knock out genes in cells to gain insight into their function. I have made the CRISPR targeting DNA constructs for the SWI/SNF genes to be included in the library of epigenetic regulators for the genetic screen. We are currently introducing the CRISPR library into human RMS cells to assess the effects of all possible combinations of single-gene and dual gene-knockouts on tumor cell growth. Using this large-scale multiplex genetic interaction screen, we hope to identify genes and gene combinations that are essential to the growth of RMS cells. If two genes have cooperative or redundant function in promoting RMS tumor growth, a dual-gene knockout is expected to result in a more significant reduction in cell growth compared to targeted disruption of either gene alone. The study will provide valuable insight into genetic interactions among key epigenetic regulators in RMS and potentially identify novel therapeutic targets for the treatment of RMS.

Human genetic variation is ubiquitous and its impacts are often not fully understood. The purpose of this study is to examine and catalog genetic variation in the specific context of the Fanconi Anemia disease family. Fanconi Anemia (FA) results from mutations in any of 22 FANC (Fanconi) genes. FA disease-associated pathogenic variants lead, in almost all instances, to loss of Fanconi protein expression and/or function: this is consistent with the recessive Mendelian inheritance of FA as a clinical disease phenotype. Recent work has identified aldehyde damage as an important driver of FA pathway function, and an important contributor to FA disease pathogenesis. Aldehydes are ubiquitous, potentially toxic molecules that are generated as part of normal cellular metabolism. Aldehyde damage is limited by the actions of aldehyde (ALDH) and alcohol (ADH) dehydrogenases, two gene families encoded by 26 different genes. To systematically characterize base pair-level pathogenic and human population-level genetic variation in the FANC, ADH and ALDH gene families, we built new databases of human genetic variation for clinically-ascertained FANC gene pathogenic variants (n = 1,015 variants), and for population genetic variants in the 22 FANC genes (n = 13,306 variants) and 26 ADH/ALDH genes (n = 8,388 variants). Population genetic variation was sourced from over 60,000 individuals included in the Exome Sequencing (ESP), the 1000 Genomes (1KGP) and the Exome Aggregation Consortium (ExAC) projects data. In order to interrogate potential pathogenicity we used CADD (Combined Annotation-Dependent Depletion) scoring, which provides a robust assessment of the predicted deleteriousness of base substitution and short indel variants by objectively integrating many diverse annotations into a single measure (C score) for each variant. This project tested the implementation of CADD scoring as a model and identified new potential pathogenic candidates in the ADH/ALDH gene families for future experimental confirmation.

Aging is characterized by the increasing loss of physiological and cellular functionality, however the mechanism that underlies this deterioration is still unclear. Emerging evidence indicates that aging is associated with increased cell-to-cell variation in gene expression within tissues: homologous cells within tissues express the same gene at varying levels. The causes of this age-related variation of gene expression are not known. Here we aim to investigate the mechanisms of increased cell-to-cell variation in gene expression with age using C. elegans as a model system. By characterizing aging in C. elegans, we hope to provide further insight into the molecular characteristics of aging in humans, and possible points of intervention. We hypothesize that stochastic noise of transcription can lead to increased gene expression variation with age. Previously we have found that stochastic noise of gene expression is incredibly restricted in young C. elegans animals. Using quantitative fluorescent microscopy we have analyzed expression of reporter genes in old animals and have dissected the potential contribution of stochastic transcription noise into age-related variation of gene expression.