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

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

Blood flow in microcirculation is a significant physiological parameter that reflects the adaptive response of organs to disease, trauma, and cancer. Although ultrasound Doppler imaging was previously unable to assess blood flow in the microvasculature (< 0.5 cm/sec), the introduction of microbubble contrast agents has removed this limitation. However, blood flow of the entire vascular tree is mixed together during imaging. We present a method that segments and visualizes the entire vascular tree, including capillary blood flow, larger sub-spatially resolved vasculature and larger vasculature (>50 µm). In this work, we present an approach that decomposes nonlinear Doppler acquisitions into different groups of velocity projections. We demonstrated the ability to segment these different levels of vasculature in a rat spinal cord injury model where the varying rates of low velocity microbubble decorrelations captured by our high frame rate acquisitions enable us to quantify microvascular blood flow. This approach overcomes limitations encountered in conventional imaging methods by removing tissue signal before Doppler processing by combining high-frame rate plane wave imaging, microbubble nonlinear pulse sequences, and Doppler segmentation of blood flow. Singular value decomposition was used to segment the nonlinear Doppler signal. Our results successfully illustrate the segmentation of lower velocity sub-resolution microvascular flow and higher velocity flow in larger vessels in a rat spinal cord injury model. We isolated low and mid-velocity flow in sub-resolution vasculature (<20 µm). We observed different spatial distribution and bolus kinetics between low- mid- and higher velocity Doppler projections. We are assessing the utility of these different blood flow features for the management of spinal cord injury and other applications (e.g. oncological).

The link between protein sequence and structure is not always apparent. The dogma is that sequence determines structure, but it is not clear how very different sequences can give rise to the same structure. Here, we employ high temperature molecular dynamics unfolding simulations to probe the pathways and specific interactions that direct the folding and unfolding of the SH3 domain, a family of small proteins consisting of two β-sheets arranged to form a barrel. SH3 domain proteins are involved in various functions including protein binding, cell signaling, and nucleic acid modification. The SH3 metafold consists of 753 proteins with the same structure but varied sequence and function. To investigate the relationship between sequence and structure, we selected 17 SH3 proteins with an average pairwise sequence identity of only 27%. Six unfolding simulations were performed for each protein and unfolding transition states were determined, revealing two unfolding/folding pathways. Transition states were also expressed as mathematical graphs of contacts between chemical groups, and three positions in the transition state structure were consistently more connected to the rest of the graph than other nearby positions. These positions represent a folding hub connecting different portions of the structure in the transition state. Analysis of the multiple sequence alignment and covariation also highlighted positions with high conservation due to packing constraints and long-range contacts. This study demonstrates that the SH3 domain can fold through two distinct pathways, but certain folding/unfolding characteristics are conserved independent of sequence and unfolding pathway. By identifying similar interactions, we demonstrate how different sequences can have the same influence on folding pathway and final structure.

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

Native mass spectrometry (MS) experiments provide direct mass measurements of intact proteins and protein complexes. Protein samples for native MS are prepared in solutions that mimic physiological conditions, which maintain a protein’s native folded state before entering the gas phase of the mass spectrometer. Ammonium acetate solution is typically used due to its volatility and relevant ionic strength. However, protein purification protocols typically require inorganic salts and detergents to maintain protein stability. Native MS experiments can be hindered or made uninterpretable by those salts and detergents. Furthermore, the presence of protein modifications or multiple proteins can make native mass spectra difficult to interpret. Anion exchange chromatography (AEX) is well suited for the requirements of native MS, as it can simultaneously desalt, remove non-ionic detergents, and separate proteins or proteoforms directly into an ammonium acetate solution. This project seeks to develop a comprehensive method for desalting, removing non-ionic detergents, and separating proteins through an ammonium acetate-based anion exchange chromatography method. Preliminary experiments in egg whites, a complex matrix with a high sodium concentration, showed separation and four distinct proteins using an AEX pH gradient from pH 10 100 mM ammonium acetate to pH 4 100 mM ammonium acetate. Native MS analysis showed low interference from sodium or other contaminants and the various modified forms of those proteins were identified. Refinement of this preparation technique can result in the improvement and efficiency of native MS analysis of proteins.

This study investigates the ways in which parents consider and rate their child’s temperament in relation to the ways in which their child interacts with them during play. Participants were typically developing children (TD, n = 26) ages 2-5, children with autism spectrum disorder (ASD, n = 25) ages 2-12, and children with fetal alcohol spectrum disorder (FASD, n = 18) ages 4-9. Attentional focusing was assessed with a parent-report child temperament questionnaire; child’s age determined which Rothbart Childhood Temperament Questionnaire was given (for example, the Children’s Behavior Questionnaire for ages 3-7). Play sessions were unscripted, free play time between the parent and child with a consistent set of toys (M = 14.4 minutes, SD = 2.8). The number of child attention switches (to a different toy, person, or object) was coded from video. Preliminary results indicate that parents rated TD children as more attentive in comparison to children with ASD and FASD based on the attentional focusing score from the Child Temperament Questionnaire (p< .001). There were fewer attention switches during the play sessions by TD children in comparison to children with ASD and FASD (p<.05). There is no significant difference in either of these measures between children with ASD and FASD. There is also no correlation between parent-reported attentional focusing scores and the observed number of attention switches. These results suggest that TD children demonstrate more attentiveness in both parent-reported and examiner-rated measures. Children with ASD and FASD were not distinguishable from each other based on parent report or direct observation of attention. Overall, attention should be considered as a contributor in a child’s interactions with their environment, relationships to others, and further growth and development.

Executive function (EF), a broad term for an individual’s higher-order cognitive abilities, has been shown to be an important factor in proper development of play in childhood. Children with autism spectrum disorder (ASD) have been noted to score significantly lower on tasks requiring EF and are often noted to engage in more simplistic levels of play compared to typically developing peers. We investigated within-group associations of average play level for children with ASD, as observed during parent-child play sessions, in relation to parent-reported EF scores, as measured by the Behavior Rating Inventory of Executive Function (BRIEF). Participants with ASD (n = 28, age = 3-11 years) and participants with typical development (n = 27, age = 2-7 years) engaged in a video-recorded, 15-minute unscripted parent-child play session. Blind coders determined the child’s level of play, ranging from object manipulations to pretend play, on a numeric scale of 1-13. The highest level of play was coded at each one-minute epoch of engagement using Behavior Observation Research Interactive Software. Participant’s play scores were averaged and analyzed with their BRIEF scores using Pearson’s correlations. Results indicated no significant correlation between average play level and the BRIEF working memory, planning, and inhibition subscales, with Pearson’s correlations ranging from less than .01 to 0.03 (p > 0.8). Likewise, for participants with typical development, there was no correlation between average play level and BRIEF global composite scores, with Pearson’s correlations less than 0.01 (p > 0.9). Our current analysis did not account for parental support of the child’s play, which may contribute to why parent-reported EF scores did not relate to child play level in these unscripted parent-child play sessions. Future directions include examining the relationship between EF and play in children with other developmental disabilities.

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

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

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

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

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

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

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

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

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

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

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

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

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