With age, proteins have an increased tendency to misfold, and the cellular processes that are meant to repair these aggregates begin to lose function. Alzheimer’s disease (AD) is partially characterized by the presence of neurofibrillary tangles and beta-amyloid senile plaques. These aggregates have been linked to neurotoxicity and the successive degeneration of neurons. We used a model organism, C. elegans, a nematode that has functional counterparts in humans, a fast life cycle, and transparent body plan. The GMC101 strain of C.elegans expresses the full-length human amyloid-beta protein, and this expression leads to quick and progressive paralysis in animals when they are temperature upshifted to 25°C at the fourth larval stage (pre-adulthood). The goal of this project is to test whether we can extend this model by inducing the paralysis phenotype, and by proxy, beta-amyloid accumulation, when animals are upshifted to a restrictive temperature in mid and late adulthood rather than pre-adulthood. Since AD is primarily a disease of old age in humans, recapitulating the beginning of symptoms in older age animals would make this model more relevant to late-onset AD. Based on preliminary results, we hypothesized that when they are upshifted to a restrictive temperature in mid or late adulthood, animals would paralyze as quickly as they do at pre-adulthood. If true, this would suggest that we can use the GMC101 model as a screening tool for late-onset AD. To test our hypothesis, I shifted animals of the GMC101 and control strain to the restrictive temperature at various adult ages and screened for paralysis using the WormBot system developed in our laboratory. I will describe results from this set of experiments and discuss preclinical screening strategies we are currently pursuing using this model.

Late Onset Alzheimer’s Disease (LOAD) is a common neurodegenerative disorder that affects ~5 million Americans. A primary risk factor in sporadic LOAD is the apolipoprotein E (APOE) gene, with carriers of the ɛ4 allele (ɛ3/ɛ4 heterozygotes or ɛ4/ɛ4 homozygotes) being at highest risk compared to ɛ3/ɛ3 homozygotes. LOAD is strongly sex-biased, with increased frequency in women (XX), but also increased severity in men (XY). The impact of genetic sex differences combined with APOE genotypes has not been studied. Our goal is to build models with various sex chromosome complements and APOE genotypes to understand interactions of these genetic factors in sex differences in LOAD pathology. The Disteche lab has derived isogenic pairs of human induced pluripotent stem cell (iPSC) lines with different numbers of sex chromosomes, e.g. XY/XXY, X0/XX, or X0/XXX. These pairs are genetically identical, save for their sex chromosomes. My project is to use CRISPR/Cas9 editing to generate different APOE genotypes in these paired lines. Starting from an XXY/XY isogenic pair from a heterozygote ɛ4/ɛ3, I am generating ɛ3/ɛ3 clones with either the XXY or XY genotype. To accomplish this, I transfect iPSCs with Cas9 and APOE-specific guide RNAs in the presence of a single-stranded DNA donor that contains the ɛ3 allele. After DNA cleavage and replacement, edited ɛ3/ɛ3 single-cell clones and control ɛ4/ɛ3 clones are identified using PCR and DNA sequencing, followed by karyotyping and verifying the absence of off-target effects and epigenetic instability. The new isogenic iPSC lines with different APOE genotypes and sex chromosome complements will be differentiated to LOAD-relevant cell types, including neurons, microglia, and cortical organoids, for transcriptomic and functional analyses to better understand how APOE genotypes and genetic sex interact to modulate risk in LOAD pathology.

The mechanistic target of rapamycin (mTOR) pathway is a central nutrient signaling pathway involved in regulating cell proliferation and metabolism. Targeting this pathway has promising implications for treating a variety of diseases, especially cancer and age-related diseases. Rapamycin is an allosteric inhibitor of mTOR that has been shown to extend longevity in the fruit fly Drosophila melanogaster. Despite this, rapamycin can interfere with healthy mTOR pathways necessary for survival, which is why further research on rapamycin’s mechanism is necessary to improve clinical usage. Previous research has shown that larval size is significantly decreased in fly larvae following inhibition of the mTOR pathway. However, this effect has not been studied across fly strains with differing levels of rapamycin sensitivity. In the Promislow Lab, we have found that fly strains vary in sensitivity to the effect of rapamycin on developmental timing. We are now comparing variation in sensitivity of development time with variation in rapamycin’s effect on larval size. Using D. melanogaster, we are able to selectively expose larvae to rapamycin from embryogenesis to model the effects of rapamycin. We are collecting larvae at various time points throughout their development, comparing individuals exposed to rapamycin or control conditions. We use ImageJ software to quantify larval size. Because of mTOR’s involvement in growth pathways, we hypothesized that larvae with greater rapamycin sensitivity would have significantly smaller sizes at the same time points compared with larvae with lesser rapamycin sensitivity following rapamycin exposure. These experimental results will provide insight into how rapamycin sensitivity is linked to the phenotypic effects of rapamycin on larval size. They will also help us investigate what contributes to the sensitivity differences seen between genotypes. The spectrum of rapamycin resistance in humans is unknown and so our work could help us identify targets for treatments and therapies of age-related diseases.

The human brain is highly sophisticated and its functions are influenced by a multitude of factors, many of which play a role in the complex aging process. Certain individuals appear to possess more resilience to environmental and biological stressors as they age compared to others. However, why they are more resilient is not understood. Resilience refers to an individual's capacity to respond to stress (physically, psychologically, emotionally) by resisting damage and bouncing back. In my research in the Promislow lab, I use the fruit fly, Drosophila melanogaster, to explore the intricate process of aging. In this experiment, I applied a biological stressor on the flies halfway through their lives and examined mortality and motor function as measures of health to study resilience throughout the fly’s lifespan. I stressed the flies with a sublethal dose of paraquat, a neurotoxin that causes oxidative stress and mitochondrial dysfunction upon acute exposure. If the stressed flies return to the mortality and motor function levels of the control flies, this tells us the flies are resilient. I hypothesize that all of the flies that receive the paraquat dosage will experience an increase in mortality and a decrease in motor function when compared to the control flies. While I think the majority of the flies will fail to recover from these stressed levels, I hypothesize a small number of flies will return to the mortality and motor function of the control flies, demonstrating resilience. In my project, I aim to understand how flies can recover from biological stressors, and how their ability to recover changes throughout their lives. My long-term goal is to understand how exposure to biological stressors affects the aging process, and in particular, how and why resilience varies with age.

Throughout our lives, we generally base our idea of age upon someone’s ‘chronological age’, or how many years they’ve been alive. This, however, is not always the best indicator of aging, as people reach social and biological milestones at different ages. As an alternative, someone’s ‘biological age’ can be more representative of their progression through life. As such, research has focused on identifying biomarkers of biological age to help us better understand aging. Recent work in our lab has sought to determine the impact of several metabolites - biomolecules used for metabolism - on the biological age of the fruit fly, Drosophila melanogaster. Among the metabolites studied, histamine - a neurotransmitter involved in wakefulness and visual processing - had one of the strongest correlations with lifespan, suggesting that it plays a role in aging. In this study, we attempted to manipulate the biological age of female D. melanogaster by altering either their metabolic levels of histamine, or their perception of histamine. To do this, flies were given food supplemented with histamine or with the antihistamine hydroxyzine, a competitive inhibitor of histamine receptors. These experimental conditions were compared to control food that lacked additives. Treatment was administered continuously starting at 4 weeks and the lifespans of flies in each condition were measured. Based on our previous results, we expected to see a negative effect of added histamine on lifespan and an increase in lifespan in response to antihistamine. Our study could highlight histamine’s role in aging and lay the foundation for demonstrating that biological age can be influenced by a single metabolite.

Why do identical twins have different lifespans? Beyond genes, what else might influence the aging process? Variation in any phenotype is due to the combined effects of genetic variation and environmental variation. In fact, there are two types of environmental variation—one is extrinsic environmental variation, such as food, temperature, etc., and the other is intrinsic environmental variation, which can lead to subtle differences in behavior, such as how much an individual eats, how long it sleeps, etc. We hypothesize that these differences can be predicted by an individual’s underlying metabolism. There are thousands of molecules that make up the structural and functional building blocks of all organisms, a domain known as the metabolome. Previously, many studies have shown that genotypes vary in lifespan, but even within a single genotype there is enormous variation in lifespan. Here we address how intrinsic environmental variation influences aging by controlling the genetic and extrinsic environmental variation under lab conditions. We designed an experiment using Drosophila melanogaster, and since Drosophila has a natural tendency to climb upwards against gravity, and climbing ability of flies decreases with age, we hypothesized that we might use climbing ability as a biomarker of future mortality risk. Using mid-life climbing ability, we separated genetically-identical flies and then analyzed each group’s lifespan. We found that within a genotype, strong climbers had a longer lifespan than non-climbers. Finding strong support for this hypothesis led us to propose that the metabolome between climbers and non-climbers might be different. Our goal is to understand the role of intrinsic variation in aging. If we can find metabolites that associate with climbing ability, and as we have shown, climbing ability is associated with aging, we might be a step closer to explaining how intrinsic environmental variation influences aging.

The mechanistic target of rapamycin (mTOR) is a protein kinase that is closely linked to growth and nutrient control in a multitude of organisms. Inhibiting TOR with the drug rapamycin has been shown to increase lifespan in many species. In the fruit fly, Drosophila melanogaster, rapamycin slows development, an outcome that is perhaps closely related to its effect on lifespan. Recent work in the Promislow lab on larval development has shown that the effect of rapamycin varies greatly across different genotypes, from no impact in the time of development to a nearly doubling of development time. However, it has not yet been determined which of the three larval stages is most sensitive to rapamycin. My project attempts to answer this question. I tested the delay in larval development of larvae treated with rapamycin across six different fly genotypes, four that are known to be sensitive to rapamycin treatment, and two that are resistant. After transferring eggs to food containing rapamycin or control food, I collected larvae over three days and staged them based on specific characteristics of each stage. The data were compared between treatments and genotypes to see if there was a delay in specific larval stage development that resulted in the overall delay seen in previous experiments. These data were analyzed using R, and the results indicate that there is a significant delay in development of the first instar larvae of the sensitive strains, and no delay in the resistant strains. Based on these results, I will next use single cell sequencing of first instar larvae raised on rapamycin-treated or normal food, with the goal of better understanding the specific mechanisms by which rapamycin leads to a decrease in larval development time, and the genetic basis of variation in the response to this treatment.

 In humans, gait changes with age; changes that have been associated with the onset of disease. We also see age-related changes in the fruit fly, Drosophila melanogaster, which shows a decrease in the ability to climb vertically. However, the effects of age on walking patterns of flies on a flat surface, which more closely mimics human walking, have not been fully characterized. In my research, I use D. melanogaster as a model to investigate such effects. During the past year, I followed cohorts of D. melanogaster over their lifespans and recorded videos of them walking in an enclosed arena. A wide-field camera captured the entire arena while a higher resolution camera captured the leg movements of individual flies. I analyzed the trajectories of each fly from the wide-field videos to evaluate walking velocity and duration. Based on my preliminary analysis, I have discovered that flies walk less frequently and at slower average speeds with increasing age. As a next step, I am analyzing the high resolution videos to investigate the possibility that changes in gait might explain the slower walking velocities at older age. To do this, I trained a neural network using multi-pose animal estimation software to track the movement of individual legs in relation to the fly’s thorax. This will allow me to look at gait (step length, swing duration, stance duration) as well as coordination. I expect to see age-related changes in gait and a loss of limb coordination over fly lifespan, which might explain why flies walk slower with increasing age. With the findings from my study, I hope to establish a foundation for how gait changes with age in D. melanogaster.

Recently, the Promislow lab found that levels of metabolites in the carnitine pathway can be used to estimate age in the fruit fly, Drosophila melanogaster. This ‘metabolite clock’ not only predicts an individual’s age, but also shows that when an individual’s predicted age is older than its chronological age, it has a higher mortality rate than other flies its age, and vice-versa. The carnitine pathway is required for energy production via fatty acid oxidation, for which carnitine also removes cellular waste products, and which may influence aging. I hypothesized that higher levels of carnitine would be associated with a longer lifespan, sustained by ongoing energy production and reduced cellular toxin accumulation. To test the effect of the carnitine pathway on fly aging, I measured the lifespan of flies while either supplying additional carnitine, or treating with the carnitine biosynthesis inhibitor etomoxir. I expect flies treated with supplemental carnitine to live longer than control flies, and that etomoxir-treated flies will live shorter than control flies. Approximately 125 female Drosophila melanogaster were assigned to food vials in each condition, plus a control condition that lacked added carnitine or etomoxir. I recorded deaths every two days, transferring remaining flies to fresh vials. Once all flies are dead, I use survival analysis to determine if either treatment affects lifespan, thus testing for a role of the carnitine pathway in fly mortality. Should the results support my hypothesis, I may explore the role that fatty acid oxidation has in aging, or to what degree the metabolome clock is affected by manipulation of the carnitine pathway.

In mammals, males have sex chromosomes XY while females have XX. To balance out the extra X sex chromosome, females undergo X-chromosome inactivation (XCI) which silences most genes on one of the two X chromosomes. However, some genes escape XCI and continue to be expressed on the inactive X chromosome causing a high expression level of these genes in females compared to XY males, leading to potential sex differences in health and disease. Shroom4, an X-linked gene that encodes an essential protein for cytoskeletal architecture, is an example of a gene that escapes XCI in mice. How Shroom4 escapes XCI is unclear. It has been proposed that CTCF, a master chromatin regulator that controls gene transcription through histone or chromatin modifications, could play a role in insulating escape genes from the silencing environment on the inactive X chromosome. Indeed, we found there is a strong CTCF peak between Shroom4 and the neighbor silenced gene Bmp15. To functionally test this insulation model, I am using CRISPR/Cas9 to edit the CTCF binding site and examining the effects of deletion and inversion of the site on Shroom4 allelic expression levels. This analysis will show whether the CTCF binding site and its correct orientation are necessary for Shroom4 escape from X inactivation. Through this project we are able to improve our understanding of the complex nature of XCI.

 Tuberculosis (TB) is the second leading cause of death due to infectious disease worldwide, accounting for 10 million infections and 1.5 million deaths in 2020. The ubiquitin-binding protein, Toll Interacting Protein (TOLLIP), is associated with TB susceptibility in genetic studies and small animal models. In mice lacking TOLLIP (Tollip-/-), lungs develop profuse lipid-laden foamy macrophages, which induces the integrated stress response (ISR), a cellular adaptation to harsh environmental conditions. However, how Mycobacterium Tuberculosis (Mtb) induces foam cells and the relationship between foam cells and the ISR is uncertain. We treated mice with the Mtb cell wall lipid mycolic acid (MA), a model of lipid accumulation, to understand how TOLLIP influences lipid accumulation and ISR activation in macrophages. MA was administered to Tollip-/- mice intraperitoneally and Mtb replication, lipid accumulation, and the ISR were evaluated. MA treatment was associated with increased mycobacterial replication. ISRIB treatment, a drug inhibitor of the ISR, restored immune control in Tollip-/- macrophages. Three and five days after MA administration, Tollip-/-  peritoneal macrophages (PEM) developed increased intracellular lipid accumulation measured by flow cytometry, demonstrating that TOLLIP is essential to prevent MA-induced foam cell formation and intracellular Mtb replication. Future studies will characterize the impact of MA treatment on host cytokine responses and the impact of the ISR on lipid accumulation feedback loops. These findings can contribute to novel therapeutics for the treatment of TB infection.

Alzheimer’s Disease (AD) is a neurodegenerative disease that is the most common cause of dementia. One hallmark of AD pathology is hyperphosphorylation of Tau protein. Tau is a neuronal-specific protein that stabilizes microtubules. Hyperphosphorylation of Tau leads to loss of its normal function and promotes aggregation into neurotoxic fibrillary tangles. While Tau aggregation is well-documented, the exact role of Tau loss-of-function in AD pathogenesis remains uncharacterized. The goal of our project is to determine the mechanism by which Tau loss-of-function contributes to AD pathogenesis. We hypothesize that Tau loss-of-function contributes to AD pathogenesis by activating the cellular stress response in neurons, characterized by DNA damage, stress granule formation, and immune activation. To test this hypothesis, we produced mixed cultures of neurons and astrocytes derived from human induced pluripotent stem cells. We generated three cell lines: one in which Tau expression was knocked out (Tau KO), another in which Tau expression was knocked down (shTau), and control lines. To determine whether genes implicated in the cellular stress response are upregulated in Tau KO neurons, we used RNAseq and real-time polymerase chain reactions (RT-PCR). Then, we used immunocytochemistry to detect protein markers of cellular stress in neural progenitor cells and neurons from all three lines. Preliminary results indicate upregulation of genes and proteins associated with neuroinflammation and stress granule formation. Regarding neuroinflammation, Tau-depleted neurons exhibit increased secretion of chemoattractant cytokines, and tau-depleted astrocytes demonstrate increased glial fibrillary acidic protein expression suggestive of pro-inflammatory cytokine induction. In terms of cellular stress, Tau-depleted neurons show increased levels of proteins involved in stress granule formation and cytoplasmic double-stranded RNA known to induce stress granules. By elucidating the role of Tau loss-of-function in AD pathogenesis, this research could inform therapeutic targets for AD.

Following a heart attack, clinically known as a myocardial infarction (MI), the heart undergoes changes that replace healthy heart muscle with rigid scar. Fibrotic scarring can lead to long term heart dysfunction, arrhythmias, and potentially heart failure. Currently, there are no anti-fibrotic therapies available to patients that can help to stop, slow, or reverse the heart's fibrotic response to injury. It is thought that the transition of fibroblasts to activated matrix secreting myofibroblasts underlie the hearts fibrotic response, and harnessing these transitions holds therapeutic promise. Although the mechanisms behind these transitions remain poorly understood, recent genetic mouse models removing the mitogen-activated protein kinase p38α in resident cardiac fibroblasts have shown that fibroblasts void of p38α fail to form activated myofibroblasts in response to MI. This resulted in over a 50% scar area reduction after MI, but these mice were prone to cardiac rupture since they failed to form an early protective scar crucial to maintaining the heart’s structural stability. Thus, when considering the therapeutic window for anti-fibrotic therapies, timing is essential. This led us to investigate the ideal therapeutic window for small molecule p38 inhibition to reduce scarring and preserve cardiac function in a mouse model of MI. A p38 inhibitor was administered to mice either immediately following MI, to reduce the formation of myofibroblasts, or 3 days following MI to try to initiate myofibroblast deactivation to reduce overall scarring but also give time for initial protective scar to form. Following 10 days post MI, mice receiving a p38 inhibitor showed preserved cardiac function and reduced pathological remodeling. Yet, fibrosis was only significantly reduced in mice receiving the inhibitor 3 days following MI. This data suggests that targeting myofibroblast deactivation holds therapeutic promise in reducing pathological fibrotic remodeling, although further studies are needed to fully validate these findings.