Protein synthesis is a tightly regulated cellular process important for all of life. Specific diseases such as cancer can hijack the process of protein synthesis to drive specific disease states. My research focuses on determining if the protein making factories of a cancer cell can be secifically therapeutically targeted. I have focused my studies on prostate and bladder cancer. Prostate cancer is the second leading cause of cancer death in men. A well-studied signaling pathway that is altered in prostate cancer is the PI3K-AKT-mTOR pathway. This pathway converges on and activates the apparatus necessary to start protein synthesis through a protein called the eukaryotic initiation factor 4E (eIF4E). eIF4E is an mRNA cap-binding protein which recruits another translation factor to start protein synthesis in cells. Ribavirin is an mRNA cap-mimetic that can compete with eIF4E to inhibit mRNA translation. I will present my results from a preclinical trial using Ribavirin and a mouse model of prostate cancer. In addition to my work in prostate cancer I have also initiated preclinical studies in bladder cancer. Bladder cancer is the 5^th leading cause of cancer death in men and there have been no new theurapeutics for two decades. ARID1A is an ATP-dependent chromatin modifier that is one of the most mutated genes in bladder cancer. The Hsieh lab has discovered an intimate link between ARID1A levels and mRNA translation. I hypothesize that protein synthesis machinery represents a unique vulnerability, which can be therapeutically targeted in bladder cancer. I will present data on my findings using protein synthesis inhibitors such as puromycin and an ATP-site inhibitor of mTOR in ARID1A wild-type and mutant bladder cancer cell lines. Together, my work in the Hsieh lab points to a new treatment paradigm for targeting the protein synthesis machinery in human cancers.

Cancers are fueled by mutations which impart new capabilities to incipient cancer cells. Spontaneous mutations are, however, more likely to be deleterious than beneficial based on random chance alone. Therefore, inducing more mutations in an heavily mutated population could lead to an “error catastrophe” once the mutation level rises past a certain threshold. Accumulating too many of these debilitating mutations consequently leads to a reduction in the overall population fitness. Our hypothesis is that mutagenic versions of nucleoside analogs (modified versions of the building blocks of DNA) can induce more mutations to reach this ‘error’ threshold and beyond, and that this can selectively kill cancer cells. To evaluate the effectiveness of this strategy, termed therapeutic mutagenesis, we conducted cell culture experiments on various human colon cancer cell lines: HCT116 (mismatch repair deficient colorectal cancer) cells, SWS620 (non-MMR-deficient colorectal cancer), and a control normal human fibroblast line. We screened a library of candidate nucleoside analogues (e.g. O4-Ethyl-Thymidine) for mutagenicity and viability at physiologically relevant concentrations. I then quantified the short-term viability of the treated cell lines with a clonogenic survival assay. I used the highly accurate Duplex genetic sequencing methodology to monitor the changes in a cell line’s overall mutation load after treatment. After validating the concept of therapeutic mutagenesis in cancer, our next steps will be to determine whether possible animal and clinical trials would be possible in the future. Our approach may open up new therapeutic possibilities for patients in the most advanced stages of cancer, especially in the context of complementing increasingly common immunotherapies.

The transcription factor hypoxia inducible factor-1 (HIF-1) plays a central role in responding to changes in environmental oxygen levels in both mammals and nematodes. HIF-1 stabilization in Caenorhabditis elegans via knockout of the E3 ubiquitin ligase von Hippel-Lindau 1 gene (vhl-1) improves proteostasis and lifespan. In humans, however, stabilization of HIF-1 via loss of VHL results in von Hippel-Lindau syndrome, a disease characterized by angiomas and renal carcinomas. Because it is a transcription factor, HIF-1 activates multiple genes that are likely responsible for its effects – whether they are improved proteostasis and lifespan in worms or neoplastic disease in humans. Previously, my lab performed a screen to determine which genes downstream of hypoxic signaling are responsible for the beneficial effects of HIF-1 stabilization in worms. This screen yielded a list of genes which were subsequently tested for whether they were necessary and/or sufficient to promote health and longevity, leading to the isolation of flavin-containing monooxygenase-2 (FMO-2), as a protein that increases lifespan and improves multiple measures of healthspan in nematodes. My project involves first characterizing the sensitivity of the yeast FMO1 deletion strain (fmo1Δ) to specific forms of toxic stress. Having established these phenotypes, I am performing a screen to find genes that, when transgenically expressed, rescue the effects of the FMO1 deletion. My strategy is to overexpress a library of other yeast genes as well as orthologous FMO genes to isolate those that rescue the fmo1Δ phenotypes. Additionally, I will overexpress FMO1, and any genes of interest from the screen, for analysis in replicative lifespan assays, testing whether they also play a role in increasing yeast longevity. This screen and the associated replicative lifespan assays will help us characterize the role of yeast FMO1 and the conserved role of FMOs in general, furthering our understanding of the process of aging.

Regulation of metabolic pathways is often implicated in many normal as well as disease processes in organisms. Our previous work discovered changes in the metabolome associated with longevity in eleven different species of fruit flies. The metabolic pathways associated with longevity included gluconeogenesis, the Krebs Cycle, and amino acid degradation. However, it is still unknown how basic nutrient parameters influence metabolic pathways in flies. We used mass spectrometry coupled with liquid chromatography to quantify the small molecules present in whole fly tissue. After finding metabolites associated with age, we completed nutrition assays to determine if there were associations between nutrient parameters and metabolomic profiles. The measurement of basic nutrient parameters consisted of colorimetric assays for total glucose and protein levels. Data analysis revealed correlations between the levels of each nutrient found in whole fly tissue and corresponding metabolomic profiles. In our future work, we plan to manipulate metabolic pathways or nutrient parameters found to be significantly associated with age and observe whether there are changes in longevity.

Human genetic variants—genetic differences between people—are common. The functional significance of these variants is often not known, hence they are frequently referred to as variants of unknown significance (VUS). We have focused on developing experimental approaches to determine the functional significance of VUS, as in silico approaches have low predictive power. Our genes of choice are mutated in Fanconi anemia (FA), a rare recessive human disease that leads to early bone marrow failure and a risk of leukemia followed by a high lifetime risk of head and neck cancer. We have focused further on the two FANC genes that are mutated in 2/3 of FA patients, FANCA and FANCG. In order to functionally phenotype all small genetic variants in these two genes, we are first generating a plasmid library of 593 different mutant alleles, each linked to a unique barcode sequence. These plasmids are then used to generate a corresponding library of isogenic U-2 OS cells that each expresses a single FANCA or FANCG variant protein. Our functional assay is to determine how well each variant protein supports cell survival, after depletion of the corresponding endogenous FANC protein, upon treatment with increasing doses of the DNA cross-linking drug MMC. Mutant survival or fitness of specific alleles as a function of MMC dose is determined by barcode sequencing and counting to compare specific variant allele loss or persistence versus wildtype and dead mutant protein controls. My presentation will include assay development, validation results, and phenotyping data on a small number of FANCG genetic variants. This approach and methods are general, and should be applicable for determining the functional phenotype of VUS in any gene for which there is a good cell-based functional assay. 

As the primary producers of energy (ATP) in a cell, the mitochondria are key players in controlling cell aging and death. Interestingly, mild oxidative stress, derived from mitochondrial dysfunction or exogenous agents, evokes adaptive stress response pathways that can increase healthspan and lifespan in the nematode worm Caenorhabditis elegans. One of the pathways that responds to mitochondrial stress is the mitochondrial unfolded protein response (UPR^mt). The UPR^mt responds to stress from unfolded proteins or oxidative damage by upregulating expression of chaperones that stabilize and assist folding of unfolded proteins as well as proteases that can degrade proteins within the mitochondria. To search for genes that regulate the UPR^mt, we performed a genome-wide RNAi screen and found that knockdown of the tald-1 gene activates the UPR^mt. The tald-1 gene encodes transaldolase, an enzyme in the pentose phosphate pathway (PPP). One role of the PPP is to generate NADPH, which is required for cellular redox homeostasis. Although transaldolase deficiency has been linked to mitochondrial dysfunction in humans and mice, how the PPP controls mitochondrial function is not well understood. The focus of our study was to determine the relationship between PPP inhibition, mitochondrial function, and longevity by investigating the role of transaldolase in redox homeostasis and regulation of redox sensitive signaling pathways. Here we report that transaldolase deficient worms induced a starvation-like metabolic response that requires JNK MAPK signaling, a pathway that is directly activated by oxidative stress, to increase lifespan. Since the UPR^mt and PPP are correlated with several age-related diseases such as cancer and neurodegenerative disease, understanding the underlying genetics and molecular mechanisms of these pathways and their role in aging is likely to be relevant for human health.
 

The mitochondrial unfolded protein response (UPR^mt) is a key cellular mechanism for maintaining mitochondrial function in the face of oxidative and proteotoxic stress. This pathway upregulates mitochondrial chaperones and proteases that aid in folding and degradation of aberrant proteins. Induction of the UPR^mt was observed to correlate with increased longevity in the nematode worm Caenorhabditis elegans, a model whose genetic system is widely used to study the molecular mechanisms of aging. Through a genome-wide RNAi screen for regulators of the UPR^mt, we found that deficiency in the transaldolase (tald-1) resulted in activation of the UPR^mt. TALD-1 is responsible for the production of NADPH in the oxidative phase of the pentose phosphate pathway (PPP). In subsequent studies, we have found that knockdown of transaldolase reduces in vivo mitochondrial respiration, changes the overall metabolism of lipids, and affects the activity of transcription factors that regulate a starvation response and expression of metabolic genes. Among the genes transcriptionally altered by transaldolase knockdown is the flavin-containing monooxygenase-2 (fmo-2) gene, which has been previously found to be regulated by starvation. Like starvation, tald-1(RNAi) induced nuclear localization of HLH-30, a transcription factor responsible for the expression of autophagy genes. HLH-30 is also required for fmo-2 up-regulation. Epistasis lifespan analyses using tald-1 (RNAi) and deletion mutant strains of C. elegans, quantitative gene expression studies, and fluorescence reporter assays were used to determine the mechanistic relationship between tald-1, hlh-30, and fmo-2 as regulators of longevity in C. elegans.

Rapamycin, an FDA approved drug that inhibits the mTOR pathway, is known to prolong lifespan in yeast, invertebrates, and vertebrates. In aged mice, reports of lifespan extension had been demonstrated using continuous treatment. More recently, our lab showed that a shorter three-month transient treatment of rapamycin sufficiently extends lifespan. However, some questions remain. Is there an optimal rapamycin dosage and treatment length that results in a robust effect on healthspan and lifespan? We had noted a sex-specific increase in cancer incidence and no lifespan extension in females that received daily intraperitoneal (IP) injections of rapamycin, the same regimen that robustly extended lifespan in male mice. Currently, we are testing the effects of daily, once weekly, and twice weekly rapamycin injections in female mice. We have already uncovered an increased incidence of eye inflammation in the animals that received daily drug treatment compared to all the other cohorts. Interestingly, this increase in eye inflammation also occurred in mice that were fed high-dose rapamycin. These data suggest that high dosages present unwanted side effects in female mice and it is crucial to find an optimal treatment regimen to minimize the undesirable effects of rapamycin. Additional assays to measure physiological parameters such as body fat mass, body temperature, muscle function, cognitive function, and lifespan will be done in our study. We hope to reveal the proper drug dose and intervention frequency that provides the best outcome, enhancing longevity while minimizing or even abolishing the detrimental outcomes associated with rapamycin.

Leigh Syndrome (LS), one of the most common childhood mitochondrial diseases, typically arises during the first year of life and results in a wide array of metabolic and motor deficiencies leading to death within a couple of years. LS is associated with mutations in any of several mitochondrially encoded genes, including NADH Dehydrogenase Subunit 2 (ND2). The ND2 gene codes for a subunit of complex I within the respiratory chain of the mitochondria. Among patients with the same ND2 mutation, the progression and pathology of LS varies widely. The variation might be due to the effects of variation at other genes that interact with ND2. To test this hypothesis, we have turned to a Drosophila model of LS, in which flies carry an ND2 loss-of-function mutation. Drosophila with the ND2 mutation experience LS-like symptoms, including decreased lifespan, metabolic dysfunction, and paralysis, when exposed to mechanical stress, termed ‘bang sensitivity’. To identify other genes that affect the ND2 mutation in Drosophila, we crossed female ND2 flies with males from the Drosophila Genetic Reference Panel (DGRP); a panel of 200 inbred, fully sequenced lines. The resulting offspring are aged 21 days and assayed for changes in bang sensitivity— the time it takes for individual flies to recover from paralysis. By comparing the effects of ND2 on the bang sensitivity phenotype in each DGRP background, we hope to identify specific genes that either worsen or ameliorate the phenotype. If successful, this study could help us identify genetic modifiers of this mitochondrial disease, and thus help us identify novel therapeutic targets.

Exposure to manganese (Mn) causes a neurodegenerative syndrome with clinical manifestations of Parkinsonism (PS) similar to Parkinson’s disease (PD). Although both PD and Mn-induced PS feature movement dysfunction resulting from decreased striatal dopamine, pathologically, Mn-induced PS is thought to be related to striatal degeneration, while in PD, dopaminergic neuron bodies residing in the substantia nigra pars compacta are largely affected. Recent evidence has suggested that astrocytes, glial cells that play a vital role in maintaining homeostasis such as uptake and release of important molecules like precursors for neurotransmitters, may play a vital role in neuronal survival in PD and PS. In order to determine whether the regional differences in sensitivity to Mn are conferred by inherent differences in astrocytes from these brain regions I examined the role of such astrocytes in neuron survival. Contact co-cultures of two neuron-like immortalized cell lines, MES and SH-SY5Y cells, were made with astrocytes from the ventral mesencephalon (vm), striatum (str), and cortex (ctx). These were treated with 10μM and 100μM of Mn and stained for the markers MAP2, TH, and GFAP, imaged, and neurons were counted. Based on previous results, MES cells grown with str astrocytes are expected to have a lower survival rate when treated with Mn, versus those plated with vm and ctx astrocytes. If this trend is observed in the separate SH-SY5Y cell line, this would suggest other neuron-like cell lines and dopaminergic neurons respond similarly to Mn. By examining if Mn affects astrocytes, and in turn neuron survival, this could not only elucidate the pathogenesis of Mn-induced PS, but also elucidate the region specific astrocyte-neuron relationships in the brain.

In the context of the growing aging population and constantly improving medical advancement, studies aiming to delay or reverse age-related health issues and enhance longevity have become increasingly relevant, especially in pre-clinical trials with drug interventions. One pharmacological agent, rapamycin which inhibits mTOR (mechanistic target of rapamycin), has been shown to robustly promote longevity in multiple animal model systems. Previous studies in the Kaeberlein lab showed that transient rapamycin treatment in aged C57BL/6JNia mice dramatically extended life expectancy, improved measures of healthspan, and remodeled the gut microbiome. We were particularly interested in the last discovery—specifically whether the increased prevalence of segmented filamentous bacteria (SFB) or C. savagella, commensal microbes shown to modulate the gut immune response, plays a role in rapamycin’s robust effects on longevity and health. To test this hypothesis, we will transplant SFB into aged Balbc By mice, obtained from the National Institute of Aging (NIA), that do not harbor SFB in their gut. The effects of SFB inoculation, paired with transient rapamycin treatment in these mice, will be evaluated by measuring changes in their lifespan and healthspan. We will also assess the changes in their gut microbiome after rapamycin interventions. This set of experiments provides an avenue to evaluate two important aspects of rapamycin intervention. Firstly, since this drug has not been tested in Balbc By mice, we can assess the role of genetic background on rapamycin’s effect on longevity. Secondly, we can establish whether the remodeling of the gut microbiome is relevant to the drug’s role in longevity and health. Additionally, does SFB play a role in this process? Answers to these questions can have a significant impact on how we view the aging process and may help pave the way for possible clinical interventions that can attenuate the negative effects associated with human aging.

Formalin-fixed, paraffin-embedded (FFPE) tissue is the clinical standard for histopathological analysis. FFPE stores and protects for long-term clinical follow-up the tissues and biomarkers which may serve as detectors for cancers and other diseases. However, the traditional method of extracting RNA from FFPE tissue has been a challenge because formalin creates covalent bonds and often causes fragmentations during the extraction process. The goal of our project is to compare the traditional lysis buffer method alone and with the addition of the M220 focused-ultrasonicator. Measurable endpoints include four criteria: time, cost, quality and quantity of extracted RNA. I performed the RNA extraction processes for both protocols using 5 challenging FFPE samples (2 cell lines, 1 liver, 1 lung, and 1 kidney). To acquire the most accurate results, we used the Thermo Scientific UV-Vis micro volume spectrophotometer NanoDrop 1000, bioanalyzers, and a size ladder of polymerase chain reactions (PCR) to access the quality of the starting material. We measured and compared the concentration of RNA (ng/ml) and purity of RNA with the ratios 260/280 (the ratio of RNA over protein) and 260/230 (the ratio of RNA over contaminants) from two protocols. The results demonstrated that on average M220 (117.14 ng/ul) extracted less RNA than the traditional method (323.48 ng/ul). The 260/280 and 260/230 ratios of the RNA extract with M220 were lower than the traditional method. The traditional method kit (20 steps) costs $8.34 per sample, took 5 hours 30 minutes, while M220 (18 steps) costs $12 per sample, and took 4 hours 35 mins. In conclusion, in very preliminary studies M220 appears to decrease time process and complexity, but it doesn’t appear to improve extraction yield or quality of challenging specimens (over-fixed specimens, autopsy specimens).

Exposure to high levels of manganese (Mn) causes a form of Parkinsonism (PS) with clinical features similar to, but distinguishable from, Parkinson’s disease (PD). Though the clinical phenotype of Mn-induced PS is similar to PD, PD is characterized by neurodegeneration in the substantia nigra, whereas Mn-induced PS features striatal neurodegeneration. Mortalin, an important mitochondrial chaperone protein involved with the response to oxidative stress, has shown to be downregulated in PD. Moreover, in recent studies we found mortalin expression to be decreased in striatal astrocytes of Mn exposed miners and in astrocytes of the substania nigra of PD patients. Additionally, we found that in vitro, a reduction in mortalin expression impairs astrocytic protection of neurons. Importantly, Mn-induced astrocyte dysfunction is substantially reduced in cells lacking expression of α-Synuclein (α-Syn), suggesting that their interaction modulates astrocyte function, potentially affecting their neuro-protective role in PS. The interaction between α-Syn and mortalin was investigated in astrocytes isolated from brain regions associated with PD and of Mn-induced neurotoxicity. We employed an animal model of cultured astrocytes from the ventral mesencephalon, striatum, and cerebral cortex to examine this interaction. The cells were treated with 0, 10, or 100 μM Mn, then examined using western blotting and immunofluorescence microscopy. The relative levels of mortalin and α-Syn were then quantified and analyzed. We saw a decrease in the availability of functional astrocytic mortalin with increasing Mn in striatal cells compared to other regions and substantial aggregation of α-Syn, reflecting the astrocytic dysfunction. This study helps bring light to the potentially critical association between mortalin and α-Syn in motor-impaired neurodegenerative diseases and can have important implications for understanding the pathogenesis of α-Syn aggregation and ultimately, PS related neurodegeneration.
 

The Ndufs4 gene knockout (Ndufs4-/-) mice show the phenotype of severe neurodegenerative mitochondrial disease analogous to human Leigh syndrome. Phosphoproteomics is a large-scale study of phosphorylated proteins, which are post-translational modifications that can turn on and off biological signaling pathways. Phosphoproteomics and proteomics experiments using mass spectrometry can allow us to quantify protein levels in mouse tissues. The broad goal of my project is to define the underlying mechanism of mitochondrial dysfunction using a phosphoproteomics approach in the Ndufs4-/- mouse model and translate the findings to improve the health of patients with severe mitochondrial disorders. Our lab previously showed that rapamycin, a drug that inhibits mTOR kinase, partially rescued the disease phenotype of Ndufs4-/- mice. However, the downstream biological pathways affected by rapamycin in this setting are not understood yet. Those may be discovered by analyzing differences in phosphorylated proteins among brain tissues of wildtype, Ndufs4-/-, and rapamycin treated Ndufs4-/- mice. For the initial analysis, protein data sets with quantitative measurements of more than 4,000 proteins were analyzed. Proteins that may be important and most affected by mitochondrial disease and rescued by rapamycin were pinpointed. After the analysis of the data was complete, the proteins that passed the filtering thresholds were quantified with Western blots, a biochemical technique to quantify protein level individually. We have already identified two signaling pathways from the phosphoproteomics and will test the effect of inhibitors of these pathways in the mice. It is our hope that a better mechanistic understanding of disease progression in this mitochondrial disease mouse model will yield new therapies to treat mitochondrial disease in patients for whom there is not currently any effective treatment.
 

In mammals, fundamental differences evolved between the sex chromosomes of males (XY) and females (XX), resulting in specific regulatory mechanisms, which restore a balanced expression of X-linked genes between the sexes. To do this, one of the female’s X chromosomes is silenced (i.e. X inactivation). However, some genes escape this inactivation and remain bi-allelically expressed in females, resulting in higher expression levels potentially contributing to sex differences. The Car5b gene, which encodes a mitochondria carbonic anhydrase important for heart function, escapes X inactivation in both mouse and human. The CCCTC-binding factor (CTCF) often binds near the boundaries between escape and silenced genes. Car5b is inactivated in mouse brain, but it escapes silencing in embryonic kidney (Patski) cells derived from a F1 hybrid female mouse. Selective CTCF binding at a site near Car5b in Patski cells but not in brain suggests the possibility that a chromatin loop that includes the Car5b promoter may facilitate escape in the cell line. To test this looping escape model, we are deleting the CTCF binding site on the inactive X (BL6 allele) and will evaluate its effects on escape. To establish an allelic deletion at the Car5b locus, a CRISPR/cas9 approach (DNA modifier) is being used with two BL6-specific small guide RNAs spanning the CTCF binding site upstream of the Car5b promoter. We’ve completed single-cell cloning for Patski cells transfected with CRISPR constructs. These cell clones are currently being verified for correct deletion by PCR and Sanger sequencing. Results have shown a positive deletion in the cell cloning colony. Allelic expression of Car5b will then be examined in the clones to investigate whether deletion of the CTCF binding site affects its escape (chromatin architecture). If results are conclusive, such an approach will be utilized to examine other escape gene regions.

 Neurodegeneration from proteostatic stress spreads from cell to cell. However, the cellular pathways that contribute to this spreading are poorly understood because there is no simple genetic model established to study this. We can use Caenorhabditis Elegans as the model system because the nematode secretes the ECVs outside into the extracellular environment. I have found that transgenic nematodes (Caenorhabditis elegans) that express human versions of the toxic proteins characteristic of Alzheimer's disease (Amyloid beta) are transported by extracellular vesicles (ECVs). Therefore, I am now developing this novel model system for studying extracellular vesicles signaling and its role in spreading age-related neurodegeneration. The powerful genetics of C. elegans will allow me to study the cellular pathways that contribute to that spreading of these toxic molecules. By harvesting the nematodes from growth plates and centrifuging the worm pellet in buffer, we are able to separate the worms from the raw wash, which contains the ECVs. The raw wash is then run through a size exclusion column to separate the ECVs, and the different size samples are either run through a BCA Protein Assay to determine amount of protein in each sample based on an absorbance reading, or run through an SDS-PAGE Protein Gel and treated with antibodies to isolate the aβ protein. My preliminary results show that the aβ protein is in fact located within a specific range of samples from the size exclusion column. The ability to isolate the extracellular vesicles and locate the source of aβ protein and its proliferation should have a significant impact on the future of Alzheimer’s therapy. If we know more about the exact location and behavior of the deleterious protein, we can apply the knowledge to studies on human cerebral spinal fluid and develop drugs that target this specific pathway in humans.

Flavin containing monooxygenases (FMO) are a highly conserved and ancient protein family. They have been suggested to have evolved pre-Last Universal Common Ancestor (LUCA) and to be present in all kingdoms of life. FMOs are known as catalysts for detoxification of xenobiotic compounds, and their activity in the nematode Caenorhabditis elegans, animal model par excellence for aging research, has recently been found to modulate stress resistance and aging. However, the mechanisms by which FMOs affect these processes remain vastly undercharacterized. Our project seeks to define the subcellular localization and endogenous substrates of the C. elegans FMO, FMO-2, as well as investigate its role in redox balance and iron homeostasis. To compare fmo-2 knockout worms, FMO-2 overexpressor worms, and wild-type worms, we crossed the three strains into various established reporters, biochemically stained worms for changes in iron and fat, and performed stress epistasis experiments feeding them RNAi. We also assessed subcellular localization via several fluorescent translational fusion reporters including crossing them into organelle-marked worms. With FMOs’ newly uncovered role as longevity- and health-promoting enzymes, characterizing them more fully may lead in time to human therapies making use of these enzymes.

The drug rapamycin is a specific inhibitor of the mechanistic target of rapamycin (mTOR) and has been previously shown to increase lifespan in yeast, worms, flies, and mice. Rapamycin inhibits mTOR by binding the Fpr1 protein (FKBP12 in mammals); the rapamycin-Fpr1 complex then dissociates the mTOR complex I. In order to identify new, natural product inhibitors of mTOR that function similarly to rapamycin we have developed a high resolution assay for growth inhibition in yeast using a Bioscreen C MBR machine. We are currently testing twenty-two natural product compounds, and their mixes in this assay to determine if they inhibit the TOR pathway, change mitochondrial function, or have any effect on replicative lifespan in yeast. Each compound and mixture is being assessed for its ability to alter the growth kinetics of four yeast strains: wild-type, TOR1 deletion, FPR1 deletion, and SIT4 deletion. Tor1p is a kinase in the TOR complex, and deletion of TOR1 leads to rapamycin hypersensitivity. The fpr1Δ mutant is resistant to rapamycin. The sit4Δ deletion is sensitive to multiple drugs. Compounds were dissolved in the solvent DMSO, or in some cases other solvents, and diluted to 1 µg/ml, 10 µg/ml, and 100 µg/ml in culture medium. All tests were performed in yeast extract peptone dextrose (YEPD) solution. We used a Bioscreen C MBR analyzer to measure the optical density of growing yeast in the YEPD with the compounds. The bioscreen analyzer generates an optical density curve over time that can be further analyzed by using the web tool, Yeast Outgrowth Data Analyzer (YODA), to selectively examine and compute the doubling time of the cultured yeast cells. The doubling time data was used to evaluate the effect of supplements on growth rate compared to rapamycin, in order to identify compounds of interest for further study.

Cardiovascular disease is the leading cause of death worldwide due largely to the inability of the heart to respond effectively to injury through tissue regeneration. Coronary artery narrowing initiates cessation of arterial blood supply to the heart generating an ischemic injury, characterized by low oxygen and nutrient supply and the accumulation of metabolic waste. Currently, research approaches to study ischemia rely on high-cost and low-throughput in vivo models. As such, there is a need for an in vitro platform for mimicking ischemia/reperfusion injury in a three dimensional cellular environment. The research and design pursued here addresses this deficit in modeling capabilities by developing an in vitro ischemic injury and reperfusion model utilizing engineered cardiac micro tissues. Cardiomyocyte microtissue particles (MTPs) are formed through centrifugation of a single cell suspension of stem cell derived cardiomyocytes without scaffold materials into polydimethylsiloxane micro-wells. Ischemic injury is induced in MTPs by exposing tissue cultures to mineral oil layers on top of small volumes of spent culture media to restrict oxygen supply and metabolic waste exchange. This simulated injury results in an observable reduction in live cells as evidenced by a decreased live cell fluorescence signal associated with duration of injury from a calcein and EthD-1 live/dead stain. Optimization of this staining protocol for characterizing the extent of the induced injury in an adapted plate reader format is currently underway. Thus far, percent of live cells has been shown to positively correlate with normalized calcein fluorescence intensity and current efforts are directed towards increased efficacy of EthD-1 dead cell marking and sufficient tissue infiltration of the stain. Implementation of this protocol will create a baseline metric for monitoring the state of the engineered cardiac tissue during injury and reperfusion and provide a foundation for development of an accurate and cost effective cardiac ischemia injury model.

There is no known therapy for the loss of cardiac function associated with aging. Aging has been long associated with oxidative damage from reactive oxygen species (ROS), which are mostly produced at the mitochondria. In mammalian cells, catalase is an enzyme located in peroxisomes that breaks down damaging hydrogen peroxide. Methods that target catalase to the mitochondria, where most of the oxidative damage occurs, have been shown to increase life span and attenuate age-related pathologies in mice. Adeno-associated virus (AAV) has been used for gene transfer in mice and humans for decades with no adverse effects. We are investigating if the presence of AAV-mCAT leads to an improvement in cardiac function, the percentage of cells in each tissue that are expressing AAV-mCAT at the time of improvement in function, and if mCAT is expressed in other tissues aside from the heart. My role in particular is to determine the expression levels of catalase in various tissues, which could affect cardiac function. We present evidence of improvement of diastolic function by longitudinal echocardiography, and expression of the mitochondrial-targeted catalase gene (MCAT) in various tissues, after injection of AAV expressing MCAT (AAV9-MCAT) into aged female mice. This pilot project shows that the AAV9-MCAT has the potential to be an eventual therapy for aging-related diastolic dysfunction, for which there is presently no effective therapy. Thus, AAV9-MCAT is a novel and promising method of reversing the effects of aging on cardiac health.

In the aging community, our ultimate goal is to lifespan and healthspan, the period of time that an individual lives healthily. Previous studies have found that aging results in decline of heart functions and increases prevalence of cardiovascular disease. Characteristics of cardiac aging include enlargement of the left ventricle, impaired diastolic functions, and reduced myocardial performance. Oxidative stress has been implicated as one of the mechanisms underlying cardiac aging. SS-31 peptide, a tetrapeptide that targets the inner membrane of the mitochondria, has been shown to interact with cardiolipin and enhance the electron carrying function of cytochrome c, an essential component of the electron transport chain. SS-31 peptide also limits oxidative damage to cardiolipin, a phospholipid that is almost exclusively located in the inner mitochondrial membrane, by reducing peroxidase activity of cytochrome C. SS-31 treatment has been shown to protect mitochondria in pressure-overload induced heart failure and oxidative injury in other tissues. In this study, we performed echocardiography and treadmill running and showed that 8 weeks of the SS-31 treatment improved diastolic function, myocardial performance, and enhanced exercise endurance in old mice. Using quantitative real-time PCR, I am assessing the gene expression changes regulated by the SS-31 treatment. Understanding the mechanism of how SS-31 improves cardiac aging will provide insight on potential therapies to improve cardiac health in the elderly populations.

Aging is characterized by time-dependent deterioration of cellular and physiological functions. Due to the numerous challenges and limitations of studying aging in humans, model organisms are used to further extend our understanding of basic mechanisms of aging. The Kaeberlein laboratory uses the single celled budding yeast Saccharomyces cerevisiae as one such model. Advantages of this model organism include the ability to measure the lifespan of great numbers of organisms under various environmental conditions. One method of measuring a yeast cell’s longevity is by assaying its replicative lifespan (RLS). The RLS model refers to the number of daughter cells that can be produced by a mother cell before senescence. The number of budded daughters can be determined by micro-dissection with fiber optic needles from mother cells after each round of incubation. This process is repeated until mother cells no longer generate daughters. Approximately 600 mother cells are assessed weekly and incubation cycles last one month. Yeast lifespan can be manipulated through genetic changes, altering media content, addition of pharmacological agents, and change of temperature. Data collected is compared to control groups and to past and expected results. These results clarify how senescence is regulated. RLS is thought to be a better representation of senescence in mitotically active cells, like certain tissues in multicellular organisms. By analyzing thousands of cells, a variety of genes and environmental conditions that impact aging have been identified. These genes and interventions can be further studied and applied to other organisms. Studying budding yeast enhances our understanding of the molecular basis of aging.​

 Eukaryotic cells contain membrane-bound organelles that serve to compartmentalize certain cellular functions. Different organelles contain optimal pH levels necessary for the organelle function. The vacuolar ATPase (V-ATPase) is a highly evolutionarily conserved proton pump that is responsible for acidification of many organelles. This proton pump consists of various protein subunits and loss of any these subunits results in disruption of the entire protein complex, which in return is not compatible with life for most organisms. Within certain conditions, S.cerevisiae (yeast) is able to survive in the absence of a functional V-ATPase, which makes it an ideal model organism to study V-ATPase function. In yeast, the proteins that comprise the V-ATPase are encoded for by vacuolar membrane ATPase (VMA) genes. Loss of any individual VMA gene results in pleiotropic phenotypes including an inability to grow on nonfermentable carbon sources and shortened replicative lifespan. In order to understand more about how the V-ATPase functions, we have performed multiple screens to identify genes that when overexpressed or mutated can suppress the respiratory phenotype of VMA mutants. Our results implicate iron homeostasis as a process that is crucially disturbed when the V-ATPase is disrupted.

The budding yeast, Saccharomyces cerevisiae, has been an invaluable model organism in the study of aging. Yeast cells divide asymmetrically. During division, the “mother” cell retains any accumulated cellular damage and produces a rejuvenated “daughter” cell. Thus, each yeast cell has a finite replicative lifespan defined by the number of daughter cells it produces before senescence. The historical belief in the field of aging has been that the asymmetrical inheritance of damage is absolute; until the last few divisions of a mother cell’s lifespan, daughters are born with a full lifespan potential, completely independent of the mother’s remaining lifespan. In this project we measure the replicative lifespans of daughter cells that budded off from mothers at different ages. We found a loss of daughter lifespan potential and an increase in mother-daughter remaining lifespan correlation in middle-aged mother cells. By tracking the granddaughter lifespans of aged mother cells, we find that this effect is compounded across generations. The implications of this research are to improve our understanding of yeast replication so as to better interpret the data gained from experiments that, as an example, use yeast aging as a model for human cell aging.

Dopamine deficiency within the brain is a key characteristic observed in patients afflicted with Parkinson's disease. In the Darvas lab, we induce dopamine deficiency in mice by genetically targeting dopamine neurons to express the human diphtheria toxin (DT) receptor (DTR, Slc6a3^DTR mice), which allows us to selectively ablate targeted dopamine neurons in Slc6a3^DTR mice with intramuscular DT injections. After DT-mediated ablation of dopamine neurons in Slc6a3^DTR mice, we observe severe physiological changes including an inability to maintain motor balance on a rotating rod and, unfortunately, a precipitous drop in weight that results in mortality for a majority of mice. This loss of weight is attributed to a lack of appetite resulting from the dopamine depletion. In an attempt to increase the survival rate of dopamine-depleted adult Slc6a3^DTR mice, we have introduced a high-fat diet to both raise the body mass and stimulate the appetite of mice to prevent them from starving. Mice are weaned from their parents and put on the high-fat diet for 12 weeks after which they receive two DT injections, one 48 hours after the other. For 2 weeks post-injection mice are weighed daily and at 3 weeks are tested for motor balance to confirm successful dopamine depletion. There was no improvement in the survival rate of Slc6a3^DTR mice on the high-fat diet and severe dehydration was observed in a majority of Slc6a3^DTR mice past 10 days post-injection. Dopamine depletion in Slc6a3^DTR mice affects both appetite and thirst meaning the high-fat diet alone could not increase survival rate.

A key goal of aging research is to identify cellular factors that slow aging in hopes of one day developing treatments to combat age related diseases. Caenorhabditis elegans is a roundworm that has been used extensively to study the biology of aging. Low oxygen (hypoxic) environments have been shown to increase the longevity of C. elegans significantly, but the specific mechanisms of hypoxia induced longevity are still not completely understood. Temperature is one of the key determinants of lifespan in cold-blooded (poikilothermic) animals and also greatly influences the physiological response to decreased oxygen. This study aims to identify the relationship between temperature, oxygen levels, and development in C. elegans. Using a temperature gradient within a sealed gas environment we analyzed C. elegans development, growth and reproduction over a wide range of temperatures and oxygen concentrations. Our results show that under normal atmospheric oxygen concentrations (21%) growth is fastest at 26.7C. Interestingly, at extremely low oxygen concentrations (0.1%), development was extremely slow and was fastest at 16C. We hypothesize that increased oxygen demand at normally optimal growth temperatures prevents growth in hypoxia leading to the observed decrease in maximum growth temperature. We are continuing our work with various mutant strains to identify the genetic factors controlling the responses to low oxygen across a range of temperatures. These findings will provide insight into the fundamentals mechanisms of development and aging, and may facilitate development of therapies to delay the progression of age related diseases in humans. 

The efficient production of cellular energy via the mitochondrial electron transport chain is necessary for the growth, health, and lifespan of all organisms. However, the genetic pathways that impact the regulation of energy production are still largely undefined. This is largely because the electron transport chain is so essential for life that genetic mutations which disrupt its functioning are lethal, preventing their study. The small roundworm Caenorhabditis elegans has been a useful model because its powerful genetics have allowed researchers to perform forward screens to identify mutations to electron transport chain proteins that can significantly reduce ATP production but are not severe enough to kill the animal. Preceding studies in my lab (Dr. Kaeberlein, Pathology) have identified how mutations that on the ISP-1 molecule itself can restore more normal functioning. My research focuses on identifying genes other than ISP-1 that can also functionally compensate for the reduction in ATP production efficiency. I am currently identifying the genetic loci of three mutations that suppress the loss of ATP production through single nucleotide polymorphism (SNP) gene mapping, and whole genome sequencing. These process require genetic crosses and high-throughput PCR and restriction digest protocol. Results from this experiment would give the first insights into the cellular pathways that can impact energy production.

Aging and severe mitochondrial disease are disparate, complex processes whose causes have yet to be fully understood. One protein involved in both is the mechanistic target of rapamycin (mTOR), which regulates cell growth and metabolism. mTOR protein is a component of two complexes, the mTOR complex 1 (mTORC1) and mTOR complex 2 (mTORC2). Inhibiting mTORC1 with the drug rapamycin increases lifespan in model organisms and suppresses severe mitochondrial disease and shortened lifespan in the Ndufs4 knockout (KO) mouse model of Leigh Syndrome. In mouse models, KO of the S6K1 protein, a substrate of mTORC1, also increases lifespan. Therefore, we hypothesized that KO of S6K1 in the whole body and specific tissues (brain, liver, fat) would rescue the short lifespan of Ndufs4 KO mice. A breeding strategy utilizing Cre-recombinase mice, S6K1^Flox/Flox mice, and Ndufs4 +/- mice was employed to ultimately produce tissue-specific Cre +/- Ndufs4 -/- S6K1^Flox/Flox mice. PCR and Western blot analysis confirmed the whole body and tissue-specific deletion of S6K1 gene and depletion of the protein in each mouse. Our findings indicate that whole body and liver-specific S6K1 KO modestly increased lifespan and delayed the onset of neurological symptoms originating from the mitochondrial disease. However, S6K1 KO is still less effective in rescuing mitochondrial dysfunction than the optimal dose of rapamycin. We speculate that S6K1 partially contributes to the mitochondrial disease of Ndufs4 KO mice, and the liver may play an important role in regulating lifespan. In the long run, these findings may enable us to potentially produce drug treatments to extend the lifespan of healthy humans as well as Leigh Syndrome patients.