Student Award Details

As we age, cells accumulate damage, become less efficient at converting energy and produce more toxic byproducts leading to an overall decline of the human body. This decline is linked to mitochondria, the cells energy supplier. Normally mitochondria rely on oxygen to provide energy, which comes at the cost of producing toxic molecules in the process. However, it was recently found that a previously unknown mammalian molecule can reprogram the mitochondrial energy supply route to become independent of oxygen reducing toxic byproduct levels. This project will explore how this alternative energy pathway changes with age and whether activating it can restore mitochondrial health in older mice. By tracing metabolic changes in reprogrammed mitochondria, the goal is to find out if this “low-damage” energy mode can slow or even reverse some of the effects of aging. This could lead to new ways to support healthy aging at the cellular level.

Zoonotic transmission of avian Influenza A virus (IAV) to humans poses a pandemic threat. Recent transmission of avian IAV to dairy cattle, and to dairy farm employees emphasizes the importance of identifying molecular mechanisms that regulate zoonotic events. It is thought that the IAV envelope glycoprotein, hemagglutinin (HA), must adapt its receptor specificity to bind α2,6-linked SA that predominates the human upper airway to initiate infection. However, recognition of α2,6-linked SA by HA is insufficient for avian H5N1 IAVs to enter human cells, indicating that other adaptations are necessary for zoonotic transmission to occur. Although evidence suggests that the pH and temperature sensitivity of HA are important factors that govern host tropism of IAV, a molecular mechanism that explains the temperature and pH dependence is missing. To define the pH and temperature dependence of HAs pre- to post-fusion conformational change, we first focused on visualizing conformational dynamics of HA with single-molecule FRET (smFRET). Preliminary data reveal that under neutral pH and room temperature conditions, the head domains of A/Vietnam/1194/2004 (H5N1) (VN04) HA, HA1, undergo a breathing motion where the heads are either caged, or uncaged. At pH8.0 HA spends more time in the fully caged conformation and less time in the uncaged conformation. Decreasing the pH to 6.5 shifts the equilibrium to where HA spends more time in the caged conformation and 80% of the time in the uncaged conformation. These data allow us to establish a conformational phenotype for HA where can determine the pH and temperature dependence of a given conformational phenotype. Thus, the central hypotheses of this proposal are that (1) the regulation of the pre- to post-fusion conformational change of avian HA is differentially regulated by pH and temperature compared to human adapted HA and (2) the membrane fusion phenotypes of HA are different. Experiments performed in Aim 1 will characterize the pH and temperature dependence of HAs conformational phenotype by monitoring conformational dynamics of HA at pH 8.0, pH 6.5, and pH 5.5 as well as at room temperature, 32°C and 37°C. I will implement the smFRET for three HA serotypes: A/Vietnam/1194/2004 (H5N1) (VN04), A/California/07/2009 (H1N1) (CA09) and A/Hong Kong/1/1968 (H3N2) (HK68). Furthermore, mutations that alter pH and temperature stability will be characterized to evaluate a molecular mechanism that can explain the differences in the conformational phenotype between the different HA serotypes. Aim 2 will characterize the pH and temperature dependence of HA-mediated membrane fusion by monitoring the extent and rates of fusion of HA pseudo-typed virus with the three previously mentioned HA serotypes. Additionally, the same pH and temperature stability mutations will be used to assess their impact on membrane fusion. Collectively, these data will reveal a mechanism that explains the zoonotic transmission potential of IAV and help improve genetic surveillance efforts.

In the context of human immunodeficiency virus (HIV), both CD4+ T cells and macrophages contribute to the viral reservoir within people living with HIV on combination antiretroviral therapy. Natural killer (NK) cells are the major cytolytic cells of the innate immune system, and their failure to effectively eliminate infected cells allows propagation and persistence of infection. In in vitro models of infection, HIV-infected macrophages are more resistant to NK cell-mediated killing than their CD4+ T cell counterparts, implicating macrophage-specific mechanisms of resistance. However, these mechanisms have yet to be determined. The overall objective of this proposal is to define macrophage-specific mechanisms that facilitate increased resistance to NK cellmediated killing. The central hypothesis is that HIV-infected macrophages resist NK cell-mediated killing by reducing immediate NK cell lytic function, and antagonizing death receptor signaling. To address this hypothesis, the following aims will be pursued: Specific Aim #1 will characterize release of HIV-infected macrophage lysosomal granules towards NK cells. Published work shows that melanoma cells being targeted by CD8+ cytolytic T lymphocytes release their lysosomes at the immunological synapse to degrade perforin, protecting them from elimination. To measure lysosome release, the investigators used CD107a, which lines the membrane of lysosomes and lytic granules and is surface exposed following degranulation. My preliminary data shows that HIV-infected macrophages increase surface CD107a expression upon co-culture with autologous NK cells. Therefore, I will investigate whether this mechanism is also being used by HIV-infected macrophages to neutralize NK cell degranulation. Specific Aim #2 will define mechanisms of HIV-infected macrophage resistance to NK cell FasL-mediated killing. Preliminary data shows that HIV-infected macrophages, but not CD4+ T cells, are not susceptible to apoptosis induced by incubation with recombinant FasL, despite both cells expressing the Fas receptor. I will investigate whether this resistance is due to increased anti-apoptotic activity of the protein cFLIP, which regulates caspase-8 activity. To complement these in vitro experiments, I will also analyze published single-cell RNA-sequencing data sets to determine tissue resident macrophage expression of cFLIP and other anti-apoptotic proteins. Results from these studies will elucidate how HIV-infected macrophages escape NK cell-mediated killing and will ultimately inform clinicals strategies utilizing NK cells to control pathogenesis. The work outlined above will be conducted at the University of Massachusetts Chan Medical School, within the Immunology and Microbiology Ph.D. program. The training plan, which will help me achieve my goal of becoming an independent investigator, includes coursework on basic/advanced principles of immunology and ethics of research. Finally, I will attend national/international conferences where I will be able to receive feedback from external scientists on my research and engage with others performing cutting edge research.

Despite progress in pharmacological advances, the rate of obesity incidence rises every year with an increased risk of developing insulin resistance, cardiovascular disease, and other cardiovascular risk factors including type 2 diabetes (T2D). Current treatments are not curative and typically require compliance with life-long, daily treatments. Thus, there is a critical need for novel therapeutic advancements to combat obesity and its comorbidities. We plan to generate metabolically active, thermogenic adipocytes from human white adipose tissue (WAT) to advance a cell therapy strategy for alleviating insulin resistance and cardiovascular risk in T2D. Using a CRISPR-based system to target a thermogenic suppressor gene, Nrip1/RIP140, we were able to induce "browning" in mouse and human white adipocytes with high efficiency. The browning of WAT is favorable as the brown-like human adipocytes express a brown fat-specific marker, uncoupling protein 1 (UCP1), and other beneficial secreted factors that can improve metabolism in obese mice. Although our published method is effective in vivo, we only manage to achieve ~10% of the brown fat UCP1 expression, implying that there is room for more browning to generate a more potent thermogenic fat. PPAR and cAMP signaling are key in the activation of brown fat. Thus, our goal is to generate fully brown adipocytes from human white adipocytes by combining Nrip1 disruption with PPAR and cAMP activation. We predict that this combination will result in maximally induced brown human adipocytes that can improve systemic metabolism in our mouse model. The long-term goal in our lab is to genetically modify human adipocytes ex vivo to enhance therapeutic activity and implant the metabolically active and thermogenic adipocytes into obese patients.

Neurodegenerative diseases (NDDs) are devastating conditions that rob individuals of their cognitive function, mobility, and ability to function in the world. Ultimately, many of these diseases are fatal. Today, a combined 6.5 million Americans suffer from NDDs, encompassing Alzheimer’s disease (AD), Parkinson’s disease (PD), multiple sclerosis (MS), amyotrophic lateral sclerosis (ALS), and Huntington’s disease (HD). However, by 2030, 1 in 5 Americans will be over the age of 65, and because NDDs strike primarily in mid- to late-life, the incidence is expected to soar as our population ages. These circumstances highlight the increasing urgency for the development of effective treatments and cures for NDDs, which are currently lacking. While NDDs differ in their inciting mechanisms, research has now clearly demonstrated the presence of shared features of downstream pathophysiology, notably, the role of a dysregulated neuroinflammatory system. Recent studies in humans and animal models have uncovered a population of microglia (Disease-associated microglia, DAMs), that are defined by a distinct transcriptional signature, and are conserved across several different NDDs. Intriguingly, the DAM signature includes upregulation of a number of different members of the MS4A gene family, for which polymorphisms have been linked to AD by numerous genome-wide association studies (GWAS). The upregulation of AD-associated MS4A genes in DAMs begs the questions of whether MS4A genes might play a common role in NDDs, which share a DAM signature, and furthermore, whether MS4A genes impact the functional properties of microglia in the context of NDDs. Excitingly, our lab has found that across three animal NDD models (5XFAD, MAPT, SOD1G93A), mice deficient for either of two individual Ms4a family members exhibit improved disease phenotypes, extension of lifespan, and amelioration of histopathological disease hallmarks. Thus, this proposal will test the hypothesis that multiple MS4As act in concert to drive pathology in a mouse model of ALS and regulate microglial transcriptional as well as functional responses to the NDD milieu. To test this hypothesis, aim 1 will investigate the impact of simultaneous deletion of the entire MS4A gene family on ALS. To this end, we have generated a novel mouse genetic reagent in which the entire MS4A gene cluster is deleted. These mice will be crossed to the SOD1G93A ALS mouse model and pathological features of ALS, including motor defects, lifespan, microgliosis, neuronal loss, and synapse elimination will be assessed. Aim 2 will examine the impact of MS4A deficiency on the transcriptional and functional properties of spinal cord microglia isolated from end-stage SOD1G93A mice. Specifically, I will utilize single-cell RNA sequencing (scRNA-seq) in tandem with spatial transcriptomics (MERFISH) to evaluate the impact of MS4A deletion on the DAM population. In parallel, I will examine how MS4A deficiency in vivo affects the key microglial function of phagocytosis. Together, these aims will provide fundamental new insight into the role of MS4As in ALS and contribute to the development of therapeutic approaches targeting MS4A receptors.

The sliding clamp (PCNA) is an interaction hub for over one hundred protein partners during DNA replication, DNA repair, and DNA recombination, among other tasks. PCNA is a planar ring that slides along DNA and serves as a scaffold for enzymes that act on DNA. PCNA is loaded onto DNA by the "clamp loader" RFC protein complex. Despite the fact that clamp loading is essential to all life, we still do not fully understand its core molecular mechanisms. I use structural biology, biochemical and computational characterization to understand how RFC regulates its clamp loading process.

During obesity, white adipose tissue (WAT) expands to store excess calories from the diet. WAT expands either by increasing the size of preexisting adipocytes (hypertrophy) or by increasing the number of adipocytes through the differentiation (hyperplasia). Hypertrophic growth has been associated with hypoxia (low oxygen (O2)) in WAT due to ineffective vascularization during its expansion. Hypoxia in WAT has been linked to insulin resistance, ectopic lipid deposition, and mitochondrial dysfunction, although the mechanisms connecting hypoxia to these effects are not well understood. A major function of O2 is that it serves as the terminal electron acceptor (TEA) in the electron transport chain (ETC), which sustains mitochondrial functions including de novo pyrimidine synthesis, reactive oxygen species production, and ATP generation. However, it is unknown how obesityinduced hypoxia impacts the ETC, and if these changes mechanistically explain adipocyte dysfunction upon obesity. In preliminary work, we discovered rhodoquinone (RQ), a novel mammalian metabolite that functions as an electron carrier in the ETC. The RQ-directed ETC circuit employs fumarate, instead of O2, as the TEA, enabling the RQ circuit to support certain mitochondrial functions in hypoxia. Through lipidomic analysis of tissues from two distinct obese mouse models, we found that RQ levels profoundly and specifically rise in the WAT of obese mice. These data inspired the hypothesis that obese adipose tissue reprogram their ETC to the RQ/fumarate circuit to support mitochondrial functions in WAT during hypertrophic/hypoxic expansion. To address this hypothesis, Aim 1 will leverage primary human adipocytes to explore how the RQdirected ETC circuit impacts differentiation and lipid droplet formation. This will be achieved using stable isotope tracing to measure lipogenesis and western blotting to monitor signaling cascades associated with lipid droplet formation and turnover. In Aim 2 we will test which ETC circuit (UQ/O2 or RQ/fumarate) is preferentially used in the WAT of lean versus obese mice. To this end, we will perform stable isotope tracing assays and respirometry experiments that distinguish these two ETC pathways in vivo. Moreover, this aim will test the therapeutic potential of reprogramming the ETC to the RQ/fumarate circuit during diet-induced obesity using small molecule analogs of RQ. We will determine the impact of RQ on insulin sensitivity, lipid storage, and metabolic parameters via metabolic cages in lean and obese conditions to reveal if RQ can mitigate obesity-induced metabolic dysfunction. Taken together, this work will explore the role of a novel mammalian metabolite in adipocytes during differentiation and upon obesity induction. Beyond defining the fundamental metabolic changes induced by the RQ circuit in differentiating adipocytes, the proposed research is translational, as it will investigate, for the first time, the therapeutic potential of reprogramming the ETC in diet-induced obesity.

Opioid addiction is a major public health crisis, with many individuals experiencing both dependence and sleep disturbances. These disturbances, such as poor sleep quality and disrupted sleep rhythms, make recovery more difficult by increasing cravings and the risk of relapse. Our study examines how opioid addiction affects molecular rhythms in the human brain. In the first part of our research, we will study post-mortem brain tissue from individuals with opioid use disorder. We will use advanced techniques to analyze how molecular rhythms differ across various types of brain cells, focusing specifically on the striatum, a key region involved in reward and motivation. By comparing individuals with and without opioid use disorder, we aim to identify cell-type-specific changes in the brain associated with addiction. In the second part of the study, we will use a preclinical mouse model to observe how disruptions in molecular rhythms in the brain can influence addiction-like behaviors and sleep patterns. We will monitor sleep using specialized brain recordings called EEG and EMG (electroencephalography and electromyography) and measure the animals’ motivation to seek opioids. Our goal is to better understand the link between disrupted brain rhythms, opioid addiction, and sleep disturbances. This research may help identify new treatment strategies that improve recovery and reduce relapse by targeting sleep and circadian rhythms.

Familial dilated cardiomyopathy (DCM) is a heritable disorder characterized by progressive enlargement of the heart's ventricles and impaired contraction, often leading to early-onset heart failure. A significant subset of inherited DCM cases is attributed to mutations in an 18-bp segment of the RNA binding motif protein 20 (RBM20) gene, which causes aberrant splicing of critical cardiac genes. While current treatments offer symptomatic relief, there is an unmet need for therapies that correct the underlying genetic cause. This project seeks to develop a universal CRISPR gene editing strategy to replace the entire 18-bp RBM20 pathogenic cluster with a synonymous DNA sequence. The approach utilizes prime editing (PE), focusing on the optimization of the PE3b system, which has been identified as a promising strategy for achieving high precision and low indel rates. This optimization is being conducted via a high-throughput, self-targeting lentiviral screen to identify optimal guide RNA configurations. The optimized designs identified through this screen will then be validated in isogenic induced pluripotent stem cell (iPSC) lines carrying pathogenic RBM20 variants and differentiated cardiomyocytes (CMs) to assess restoration of normal cellular phenotype. This work will inform the development of an efficient, safe PE platform to treat genetically diverse cases of RBM20-DCM.

The germline contains the genetic information that will be passed to future generations. Therefore, maintaining the germline genome is essential for fertility and species survival. One mechanism of germline genome maintenance is that of the PIWI/piRNA pathway, in which small RNAs known as piRNAs interact with a PIWI Argonaute protein to form what is known as a piRNA-Induced Silencing Complex (piRISC). Through its endonuclease activity, piRISC silences repetitive elements (i.e., transposons) to protect the genome for future generations and regulates gene expression to ensure proper germ cell development and function. Loss of PIWI function leads to infertility in at least one sex in many animals, including human males. Recent work has revealed that the small zinc-finger protein, gametocyte-specific factor 1 (GTSF1), accelerates piRISC target cleavage. Loss of GTSF1 function in mice and human males leads to infertility. Preliminary kinetic evidence suggests that GTSF1 is not required for target binding or target release. However, the piRISC catalytic states associated with GTSF1 binding have not been explored. Seven GTSF1 residues have been identified as key for target cleavage, but most of these amino acids are not conserved in the other mouse GTSF paralogs, GTSF1L and GTSF2, even though they also accelerate piRISC target cleavage. This proposal seeks to test the hypothesis that mammalian GTSF proteins stabilize a catalytically active state of piRISC via key contacts with both the piRNA-target RNA duplex and PIWI protein. Aim 1 will use single-molecule FRET to probe piRISC conformational changes in the absence and presence of GTSF proteins to determine which, if any, catalytic state is stabilized in the presence of GTSF. Aim 2 will employ a high-throughput screening method to identify all amino acid positions across GTSF paralogs which are required to interact with piRISC. This study will provide insights into how GTSF1 accelerates piRISC target cleavage and determine which GTSF residues are key for its function, providing insight into why some human GTSF1 mutations lead to infertility. The proposed research will provide training in microscopy, in vitro biochemistry, and bioinformatics to prepare the fellow for a postdoc studying epigenetic inheritance and a future career as an independent investigator.