Ut Southwestern Medical Center

NIH Research Projects · FY 2025 · 2021-09

Project Summary/Abstract Viruses have evolved sophisticated mechanisms to hijack host cellular machinery. For their part, hosts have developed their own intricate defense systems. Defining how these opposing strategies have co-evolved increases our understanding of infectious diseases and provides new opportunities to discover unexplored areas of biology. Our understanding of host cellular defense systems has been limited due to genetic redundancy in host genomes. A further complication is that successful pathogens are able to inactivate host antiviral networks during infection. We have recently established a new genetic screening platform that overcomes these roadblocks. First, we bypass genetic redundancy in host genomes through a gain of function screen that identifies antiviral genes. Second, we use both virulent and attenuated strains of viruses in our screening pipeline. Importantly, these attenuated viruses have deletions or mutations in critical, yet poorly understood, immune evasion proteins that antagonize host antiviral restriction factors. We predict that the avirulent strains of these viruses will become sensitive to restriction factors that have otherwise defied molecular identification. Using this strategy, one project will be to identify ancient antiviral genes that reveal unappreciated areas of host- pathogen conflict. For example, we are unmasking how the influenza immune evasion protein, NS1 orchestrates a complex, multifaceted rewiring of host antiviral networks using comparative screens with wild-type and NS1-deficient viruses. We are extending these studies to other virulent-attenuated virus pairings as well. Another project takes newly identified antiviral molecules that inhibit disparate virus families and define the molecular mechanism underlying viral inhibition. Here, we focus on the JADE family of proteins that regulate histone acetylation. We aim to uncover functional redundancy in the JADE family and to determine if it assembles a concerted antiviral epigenetic program. Another project is to leverage newly identified host-pathogen conflicts to discover the physiological functions of host- proteins that are poorly understood. In this regard, we are focusing on the CD300 family of receptor proteins. CD300 genes are undergoing rapid positive selection, yet their physiological function remains largely unexplored. We previously demonstrated that CD300lf is a protein receptor for murine norovirus. We will employ the biochemical, genetic, and cellular tools we developed for studying norovirus-CD300 interactions to define the physiological ligands of CD300 receptors. Determining the ligands for these orphan receptors will provide functional insight into these rapidly evolving proteins. Taken together, we plot a road map for discovering new host-pathogen interfaces, defining their molecular interactions during host defense, and how this relates to the physiological functions of these proteins and pathways.

NIH Research Projects · FY 2025 · 2021-09

Project Abstract Tumors frequently re-activate genes whose expression is otherwise restricted to gametogenic tissues including the ovary, placenta and testes. Tumorigenic expression of these genes, known collectively as cancer-testes antigens (CTAs), has been documented for over 25 years, however functional knowledge of the contribution of these gene products to tumorigenesis remains scant. We have recently discovered that expression of one of these CTAs, Cytochrome C Oxidase subunit 6B2 (COX6B2), is activated in lung adenocarcinoma. COX6B2 is essential for tumor survival in vitro and in vivo and its expression correlates with shortened patient survival time. We have found that expression of COX6B2 in cancer cells leads to an increase in activity of Complex IV (cytochrome c-oxidase) and ATP production. Based on these findings, we assert that tumor cells adopt metabolic mechanisms from one of the most ATP-intensive processes in the animal kingdom: sperm motility. To test this hypothesis, we propose to dissect the tumor-specific function of COX6B2 at multiple biological length scales incorporating structural, cell biological and whole animal approaches. Our specific aims are to 1) dissect the molecular mechanism by which COX6B2 enhances Cytochrome C oxidase activity, 2) determine how COX6B2 is activated and its consequences on survival 3) elaborate the contribution of COX6B2 to tumorigenesis and tumor survival in vivo. In Aim 1, we use a structure-guided approach to elucidate the mechanism by which COX6B2 modulates Cytochrome c oxidase activity. In Aim 2, we will investigate how the low oxygen tumor microenvironment activates COX6B2 and how COX6B2 promotes survival in hypoxia both in cancer and in sperm. In Aim 3, we will use an orthotopic xenograft to determine how COX6B2 expression influences tumorigenesis, tumor growth, and oxidative phosphorylation in vivo. The significance of the proposed work lies in the identification of novel mechanisms that tumor cells adopt to promote oxidative phosphorylation. Current electron transport chain inhibitors are hampered by a lack of therapeutic index due to the broad necessity of this process in healthy tissues. This study proposes an innovative new solution to this lack of specificity by presenting a target that is selectively expressed in cancer cells. The outcome of this work will be a novel therapeutic entry point for targeting oxidative phosphorylation exclusively in tumor tissues.

NIH Research Projects · FY 2025 · 2021-09

PROJECT SUMMARY Social wellbeing and cognitive health are two pillars of successful aging, and they are interrelated. Three decades of research have consistently shown that social isolation is associated with declines in cognitive functioning. Although existing literature has explored the individual-level risk and protective factors, researchers' attention has been less paid to the extent to which physical and social characteristics of the living environment affect social isolation and cognitive health. The Ecological Theory of Aging (ETA) posits environmental factors have significant influences on health and social wellbeing. Negative physical characteristics of the environment were hypothesized to be related to increased social isolation and cognitive decline longitudinally. An increasing amount of research investigated the association between ICT use and social isolation, yet whether ICT use would interact with the effect of environmental factors on the risk of social isolation has not yet been empirically tested. Besides, the influence of ICT use on cognitive health has not been well understood. Promoting social interaction using technology-based approaches have the potential to alleviate social isolation among older people and prevent cognitive decline. The proposed dissertation work (F99) will examine the effect of physical and social environments of living on social isolation and cognitive health. The effects of ICT use will be investigated within living contexts. Multi-level modeling and structural equation modeling methods will be employed to conduct secondary data analysis using data from the National Health & Aging Trend Study (NHATS) wave 5 to 9. The postdoctoral stage (K00) is guided by the overarching aim of building knowledge on the behavioral and contextual pathways that could explain the association between social isolation and cognitive decline and developing a community-based behavioral health intervention. At the K00 phase, the PI will increase understanding of the social isolation and cognitive health research landscape by conducting a systematic review. Longitudinal data from the ongoing Internet-based Conversational Engagement Clinical Trial (I-CONECT) will be analyzed to examine the association between social isolation and change in neuropsychological outcomes. The PI will utilize the longitudinal data collected with the Collaborative Aging Research Using Technology (CART) platform to investigate the relationships between daily activities, social engagement, and the cognitive health of older participants. Knowledge to be built at F99 and K00 stages will be synthesized to inform the participatory co-design of a technology-facilitated intervention program for social isolation and cognitive decline. The proposed research will produce knowledge on the influence of environmental factors on social isolation and cognitive health, and achieve the PI’s long-term professional goal of developing behavioral health intervention to address social isolation and prevent the onset of Alzheimer's Disease and Related Dementias.

NIH Research Projects · FY 2025 · 2021-09

SUMMARY: To grow and proliferate, cells need to fulfill three key metabolic demands: increased biosynthesis, sufficient energy supply, and maintenance of redox homeostasis. The latter demand is particularly important for sustained growth, because increased rate of cellular metabolic activity during cell proliferation results in elevated levels of reactive oxygen species (ROS), which can have detrimental effects on cell growth. Nicotinamide adenine dinucleotide phosphate (NADPH) is a principal supplier of reducing power for biosynthesis of macromolecules and protection against oxidative stress. The total cellular pool of NADPH is regulated by the activity of NAD kinases (NADK), enzymes that catalyze the phosphorylation of NAD+ to NADP+, the rate-limiting substrate for NADPH production. Mammalian cells express two NAD kinases, cytosolic NADK, and mitochondrial NADK2, which generate compartment-specific reducing power. Recently, we discovered that the activity of NADK is stimulated by the phosphoinositide 3-kinase (PI3K) - Akt pathway to boost the NADP(H) production for cell growth, but the importance of NADK2 and the overall role of mitochondrial NADPH in cell growth has yet to be established. Mutations in NADK2 have been observed in patients with various neurological and developmental disorders. Therefore, defining the key functions of NADK2 and mitochondrial NADP(H) is critical and relevant to human health. This proposal builds on our finding that decreasing mitochondrial NADP(H) levels through depletion of NADK2 renders cells uniquely proline auxotroph. Cells with NADK2 deletion fail to synthesize proline and rely on exogenous proline for their growth. Proline is critical for protein synthesis, and, unexpectedly, for nucleotide synthesis, in NADK2-deficient cells. We propose three Specific Aims to establish the functions of NADK2 and mitochondrial NADP(H) that are essential for cell growth and proliferation and relevant for proliferative diseases and NADK2 deficiency in humans. In Aim 1, we propose to define the molecular mechanisms by which NADK2 and mitochondrial NADPH support proline biosynthesis and evaluate the effects of NADK2 patient mutations on proline synthesis. In Aim 2, we will determine the mechanisms of how NADK2 deficiency and reduced proline abundance affect flux through the de novo and salvage nucleotide synthesis pathways. In Aim 3, we will assess the requirement of NADK2 and mitochondrial NADPH for tumor growth and evaluate the therapeutic potential of targeting NADK2 in combination with dietary restriction of proline. This proposal will establish the primary functions of mitochondrial NADP(H) and NADK2 that are essential for cell growth and proliferation, thereby informing us on new therapeutic strategies to combat proliferative diseases and NADK2 deficiency in humans.

NIH Research Projects · FY 2025 · 2021-09

Project Summary Many cardiovascular and neurological disorders, and oncogenesis result from changes in cell mechanics. Assessment of human pathophysiology in this context reveals that these diseases share a common root cause: abnormal mechanotransduction – the process by which cells respond to physical stress and forces. Mechanosensitive ion channels, the molecular machines by which cells convert external forces into electrical response, are therefore emerging targets of interest, for understanding biological processes and for therapeutic development. Piezo family (Piezo1 and Piezo2) was discovered in 2010 as the first excitatory mechanosensitive ion channels in vertebrates. Piezo channels are now known to be critical sensors of touch and pain (somatosensation), volume regulation (osmosensation), shear stress (cardiovascular tone), baroreception, proprioception and respiratory physiology, and may have other important functions yet to be discovered. Substantial efforts are made in the last decade to identify Piezo related diseases and incidents within the United State population. So far, Piezo dysfunction is linked to diverse pathologies including hypertension, lymphatic disease and anemias, somatosensory and neurological disorders, cancer and metastasis, amongst others. Despite their biological and medical relevance, the mechanism behind Piezo-dependent mechanotransduction remains elusive. Therefore, our lab’s goal is to understand how physical forces such as pressure and membrane tension control Piezo1 function in health and diseased state. This research proposal focuses on ion permeation and force-dependent gating mechanisms of Piezo1 channels, in cells, as well as in reconstituted lipid bilayer systems. We will employ biochemical and biophysical techniques in efforts to understand how lipid bilayer control the gating of Piezo1 and subsequent ion conduction across the membrane. Moreover, we have identified robust expression and protein purification protocols to examine the function of Piezo1 channels. Droplet lipid bilayers will be used to study the single channel conductance and open probability of the purified protein in biologically relevant lipid compositions. Structurally identified pore domain of Piezo1 will be used as a template to understand the pressure sensitivity and voltage-dependent inactivation - hallmark of Piezo channels - by constructing various deletion mutants- heterologous expression in HEK cells. The preliminary data is striking, and shows that the droplet bilayer approach coupled with traditional cellular patch clamp assays are ideally suited to study mammalian Piezo1 channel function. We are convinced that a comprehensive understanding of Piezo’s function is a timely contribution to the field of mammalian mechanotransduction. Our unique proposal represents the application of single molecule investigation of Piezos. Completion of this proposal will provide a path to dissect and kick-start the development of effective therapeutics targeted towards neuropathic pain, brain ischemia and gliomas, amongst others.

NIH Research Projects · FY 2025 · 2021-09

PROJECT SUMMARY Mitochondria are powerhouses of the cell that are often viewed as bacteria living in the cytosol. Stressed or damaged mitochondria induce inflammation through activation of multiple innate immune signaling pathways including the DNA sensing cGAS-STING pathway. We recently showed that deficiency of a cytoplasmic deglycosylase NGLY1 results in mitochondrial damage and potent activation of the STING pathway. Mutations in the NGLY1 gene are associated with an early childhood onset neurodegenetive disease with high mortality and no therapy. We recently established and characterized a Ngly1-deficient mouse model that develop early onset and progressive deteriorating coordination and motor functions. We also present exciting molecular and genetic evidence that implicate the mitochondrion-STING axis in neuropathology. The overall goal of this project is to define the neuro-pathological mechanism of NGLY1-deficiency, with a strong focus on the mechanism of the mitochondrion-STING axis. Aim 1 will define cell type specificity through genetic and single-cell approaches. Aim 2 will dissect intracellular signaling pathways in neuronal and glial cells. Aim 3 will evaluate therapies targeting the mitochondrion-STING axis. Studies proposed here should reveal the central mechanism of the mitochondrion-STING axis in neuropathology and neurodegeneration, which will have broad implications in many complex neurogenerative diseases that are affecting millions of people.

NIH Research Projects · FY 2025 · 2021-09

PROJECT SUMMARY The ability to vicariously experience the other's aversive feelings in a situation, a process called observational fear (OF), is critical to live in society. Malfunctions of OF represent well-described findings in individuals with autism spectrum disorders. In OF, an observer witnesses a demonstrator in an aversive situation and responds with fear behaviors. If the demonstrator's reaction is robust, the observer easily expresses OF without prior similar experience and social familiarity with demonstrator (we refer to as innate OF). However, in nature and our lives, the demonstrator's reaction is often ambiguous and thus difficult for the observer to understand. To fully understand the other's situation, the observer utilizes both prior similar own experience and social familiarity that facilitate OF (we refer to as experience-dependent OF; Exp OF). While innate OF primarily depends on the anterior cingulate cortex (ACC) and basolateral amygdala (BLA), the neural mechanisms of Exp OF remain unexplored. My goal is to elucidate the neural mechanisms of how both prior similar own experience and social familiarity enable the observer to fully understand the other's aversive situation from their ambiguous reaction, by examining hippocampal (HPC)-BLA neural circuits in a mouse OF model that delivers strong electrical shocks to the demonstrator eliciting a robust reaction or weak electrical shocks eliciting an ambiguous reaction. Recently, our preliminary studies showed that ACC is dispensable for Exp OF, while ACC is essential for innate OF. On the other hand, both dorsal HPC (dHPC) and ventral HPC (vHPC) are crucial for Exp OF, but not for innate OF. BLA is required for both Exp and innate OF. These findings lead us to propose distinct neural circuits in Exp and innate OF. The central hypothesis of this model is that the dorsoventral HPC to BLA pathways generate and reactivate a neural ensemble of BLA neurons encoding prior similar own fear Guided by strong preliminary data, first, we propose that the dorsoventral HPC to BLA pathways are crucial for Exp OF, while the ACC to BLA pathway is essential for innate OF. Second, we propose that the reactivation of a neural ensemble of BLA neurons encoding prior similar own fear experience (a fear memory ensemble) mediates Exp OF as the perception-action mechanisms. Third, we propose that the pathway from dHPC to BLA generates the fear memory ensemble in BLA during own fear experience and then vHPC neurons respond to the familiar demonstrator's fearful situation to reactivate the fear memory ensemble in BLA to elicit Exp OF. experience, which facilitates Exp OF, while ACC induces robust BLA activity for innate OF. Collectively, our proposed research will broadly impact the field of learning and memory by characterizing neural circuits and their neural process about how we understand the other's aversive situational experience. Potentially, the neural mechanisms can be generalized for other types of empathy to vicariously experience the other's feeling/situation.

NIH Research Projects · FY 2025 · 2021-09

Project Summary A balanced supply of deoxynucleoside triphosphates (dNTPs), the building blocks for DNA, is vital for the synthesis or repair of both nuclear and mitochondrial genomes, whereas its imbalance results in genome instability that precipitates cellular damage and breach of homeostasis. Research on dNTP metabolism has been traditionally conducted in highly proliferative (e.g. tumor cells), metabolically active (e.g. muscle cells) or virus-infected cells due to the key roles of dNTPs in fulfilling demands for cell growth, energy production and viral replication. However, little is known regarding the role of dNTP metabolism in innate immunity, especially in the context of nonpathogen-induced immune activation. A hallmark of innate immune activation is the assembly of the Nod-like receptor pyrin domain containing 3 (NLRP3) inflammasome—a dominant innate immune sensor for tissue damage. The NLRP3 inflammasome is composed of the sensor NLRP3, the adaptor ASC (apoptosis associated spike-like protein) and the effector pro-caspase-1. Assembly of the NLRP3 inflammasome proceeds with two distinct steps: ‘priming’ and ‘activation’. Priming entails rapid NF-kB activation for initiating de novo synthesis of pro-IL-1β as well as increasing the amount of NLRP3. In contrast, activation involves the assembly of the NLRP3 inflammasome machinery, resulting in autocleavage and activation of caspase-1 which then converts immature pro-IL-1β into bioactive IL-1β—a powerful proinflammatory cytokine that ignites inflammation. Although properly controlled NLRP3 inflammasome activity allows for restoration of homeostasis after traumatic tissue injury by stimulating damage clearance and tissue repair, its aberrant and prolonged activation also promotes the rapid progression of many devastating disorders, including gouty arthritis, Alzheimer’s disease, atherosclerosis, macular degeneration and cancer. It is therefore crucial to understand how NLRP3 inflammasome activity is regulated in innate immune cells. Recently, we discovered that genetic deletion of CMPK2 or SAMHD1, two key enzymes within the dNTP metabolic pathways responsible for synthesizing or degrading dNTPs respectively, orchestrates NLRP3 inflammasome activation. Therefore, the ultimate goal of this MIRA R35 project is to establish dNTP metabolism as a new layer for innate immune regulation and further delineate its underlying mechanism of action. To achieve this goal, three major scientific questions will be pursued: (1) how does inflammasome priming regulate the function of dNTP metabolic enzymes? (2) how does dNTP metabolism control NLRP3 inflammasome activation? Lastly, since NLRP3 inflammasome overactivation is a shared pathogenic hallmark of many diseases, we further asked: (3) do common disease risk factors, such as aging and obesity, dysregulate macrophage dNTP metabolism, thereby permitting NLRP3 inflammasome overactivation? Completion of this project will not only fill an important knowledge gap in the innate immunity field, but may also guide new therapy development to prevent NLRP3 inflammasome hyperactivation.

NIH Research Projects · FY 2025 · 2021-09

Abstract Heat waves are lethal and cause a disproportionate number of deaths in the elderly relative to any other age group. It is important to note that such deaths are primarily cardiovascular, not hyperthermia itself, in origin. Nevertheless, we know relatively little about the effects of aging on cardiovascular function during actual heat wave-like conditions. The central hypothesis of this work is that the elderly exhibit greater cardiovascular stress during heat wave conditions, which can be mitigated by employing low-energy demand cooling strategies. Aim 1 will test the hypothesis that recognized impairments in thermoregulatory capacity in the elderly will culminate in heightened cardiovascular stress during prolonged exposure to heat wave conditions. Comprehensive cardiovascular and thermal responses in the elderly, relative to younger adults, will be evaluated during exposure to two prolonged heat wave conditions: hot and humid (replicating the 1995 Chicago heat wave), very hot and dry (replicating the 2018 Los Angeles heat wave). Aim 2 will test the hypothesis that skin wetting is an effective cooling modality to attenuate elevations in core body temperature and accompanying cardiovascular stress during heat waves in the elderly, while the use of a fan may be detrimental depending on air temperature and whether skin wetting is employed. Though air conditioning is the most effective strategy to prevent heat- related morbidity and mortality, 1 in 8 (~12%) Americans do not have access to air conditioners, and this percentage is likely higher in the Midwest and Northeast United States where injury and deaths during heat waves are particularly high. Moreover, factors such as socio-economic status, power outages, government- imposed rolling blackouts, and COVID-19 related closures of public spaces (e.g., malls, libraries, senior centers, etc.) threaten region-wide access to air conditioning often at times when it is most needed. Therefore, it is essential to identify non-air conditioning dependent modalities that will attenuate excessive elevations in core body temperature and associated cardiovascular stress in the elderly during heat wave conditions. This aim will assess the efficacy of skin wetting only, fan use only, and a combination of skin wetting and fan use in mitigating excessive elevations in core body temperature and associated cardiovascular stress in the elderly during both types of heat waves outlined in Aim 1. The expected outcomes from this body of work will re-shape our understanding of the consequences of aging on cardiovascular function during heat waves, as well as identify the efficacy of low-energy cooling modalities directed towards saving the lives of this vulnerable population during heat wave exposure.

NIH Research Projects · FY 2025 · 2021-09

Project Summary/Abstract Large-scale studies have identified thousands of genetic variants linked to developmental defects, together with the regulatory elements harboring these variants and the cell types in which these variants likely function. This diversity of variants, regulatory elements, and cell types indicates that multiple mechanisms contribute to developmental defects. One key challenge to our understanding of these mechanisms is that the molecular, cellular, and functional phenotypes of each variant remain largely uncharacterized. Until these critical gaps in knowledge are addressed, the underlying molecular and cellular determinants of developmental disease susceptibility will remain incomplete. To bridge these gaps, we propose to establish the “UT Southwestern Center for Regulatory Element Variation and Function”. The primary goal of this Center is to systematically catalog molecular and cellular phenotypes for disease-associated enhancers in human development, with a focus on gaining insights into mechanisms of non-canonical human genetics and gene regulation. To build a generalizable framework to understanding the impact of human genetic variation on function, we propose a high throughput perturbation platform with three primary goals: (1) Contribute to a variant/element/phenotype catalog with relevance to diseases of human development, focusing on elements genetically associated with congenital heart disease (cardiomyocytes), autism (neurons), and placental defects (trophoblasts); (2) Contribute to a variant/element/phenotype catalog for non-canonical human genetics, focusing on two understudied topics in human genetics: pleiotropic effects and non-cell autonomous effects; and (3) Contribute to a variant/element/phenotype catalog with relevance to mechanisms of gene regulation, focusing on enhancer RNAs. The Center will take advantage of recent technological innovations in genome engineering, single-cell genomics, and high content screening to enable the multiscale functional characterization of genomic variation in human developmental disorders. Several of these techniques have been pioneered by investigators contributing to this project, including: the development of novel tools for enhancer perturbation and the coupling of endogenous enhancer perturbations with a single-cell RNA-Seq readout (Mosaic-Seq). Impact and Significance: The efforts on this project will lead to a number of key outcomes and deliverables, including (1) greater understanding of the relationships between sequence variation and genome function, (2) an extensive variant/element/phenotype catalog for the community, (3) tools for generating predictive models for the community, and (4) resources to enable future functional genomics studies. Together, our multifaceted and combinatorial approaches will open new horizons to understanding the impact of regulatory variants on developmental disease phenotypes.

NIH Research Projects · FY 2025 · 2021-09

The proposal details a comprehensive five years training program to expand my skills as a physician-scientist. The research plan is focused on JAK-STAT pathway in Celiac Disease (CeD), but the training plan includes extensive didactics in human subject research, bioinformatics and laboratory-based training on T cell immunology. CeD is an immune mediated gastrointestinal disease that arises in patients with a permissive HLA-DQ2/HLA-DQ8 genetic background. The only available treatment for CeD is to follow a gluten-free diet. Patients with germline gain-of-function (GOF) STAT3 mutations and Down Syndrome have an estimated 40- fold and 5-fold higher risk of CeD, respectively, than the general population, along with a predisposition for other autoimmune diseases, including thyroiditis and Type I Diabetes. Patients with Down Syndrome have three copies of the gene for interferon (IFN) receptors on chromosome 21 and thus constitutive JAK-STAT activation. The central hypothesis is that interferon and cytokines mediated JAK-STAT overactivation may cause loss of T cell tolerance to gluten and an increased risk for CeD. The overall goal of this research is to investigate the mechanism of JAK-STAT activation in the pathogenesis of CeD. To accomplish this, first, we will conduct genetic investigation in 100 index cases with familial CeD and search for genetic variants in the JAK-STAT pathway. We will also expand our cohort of CeD patients with known genetic diseases affecting JAK-STAT activation, include patients with GOF-STAT3 and DS. Second, we will assess the key molecules in the JAK-STAT pathway, including agonists, receptors, STAT and their phosphorylated forms, ISGs in monocyte and CD4+ T cells to identify a molecular signature of active CeD. Third, we will investigate the mechanisms of JAK-STAT overactivation on gluten-specific CD4+ T cells through both single cell RNA-Seq and amplified T cell libraries, followed by testing the functional impact of JAK inhibitors on gluten-specific T cells. The results of this study will delineate JAK-STAT activation and its potential as a therapeutic target for Celiac Disease. Furthermore, through the proposed complementary career development plan, I will gain additional training in clinical investigation on the genetics and immunology of CeD; advanced bioinformatic analysis with RNA-seq; and laboratory-based training in gluten specific CD4+ T cells biology. Throughout this research and career development activities, I will be mentored by a team lead by Dr. Timothy Wang, an internationally recognized physician-scientist and an expert in inflammatory cytokines and their role in human gastrointestinal diseases. I am committed to a career as an independent investigator in patient-oriented translational research; and have designed my training plan to acquire the knowledge and skills needed to make a meaningful and substantial contribution to the field by using a functional genetics approach to discover the therapeutic targets in gastrointestinal diseases.

NIH Research Projects · FY 2026 · 2021-09

PROJECT SUMMARY/ ABSTRACT The perturbation of phospho-tyrosine mediated signaling networks is an essential occurrence during the multistep process of tumor development and progression. As a result, the components of these phospho-tyrosine signaling networks, especially tyrosine kinases, have been shown to be a key reservoir of actionable molecular targets for the treatment of cancer. In recent years, it has been revealed that the tumor microenvironment plays a critical role in modulating the signaling pathways that govern tumor progression and metastasis. The features of a tumor's microenvironment have been shown to produce unique sensitivities and resistances to different treatment modalities. One major aspect of the tumor microenvironment which is often overlooked in preclinical studies is oxygen tension. This proposal seeks to understand the impact that oxygen tension has on phosphotyrosine-dependent signaling networks in solid tumors, and how the resultant vulnerabilities can be targeted to improve patient outcome. In Aim 1.1 (prior studies), we sought to identify alterations in signaling networks that occur when lung cancer cells colonize the brain, a hypoxic environment. We showed that brain- metastatic lung cancer cells elevate and have an increased dependence on a non-canonical HSF1-E2F transcriptional program for survival. Importantly, we identified that this transcriptional program is targetable through treatment with allosteric ABL2 tyrosine kinase inhibitors. In Aim 1.2 (proposed studies), using a small molecule screen, I have identified previously unrecognized modulators of the cellular response to hypoxia, a tumor microenvironment feature associated with increased metastasis and lower overall survival in patients with solid tumors. The top uncharacterized hit was the FDA-approved ABL1/2 tyrosine kinase inhibitor Dasatinib and my preliminary investigation has shown that the ABL kinases are critical regulators of HIF-1α protein stability. I will continue mechanistic investigation of the ABL- HIF-1α axis in vitro and in vivo. Finally, in Aim 2 (post-doctoral studies), I will focus on understanding the impact that tumor representative- oxygen tension has on protein tyrosine phosphatase activity. Extensive investigation has demonstrated that tumor hypoxia induces activation of phospho-tyrosine signaling networks, but current work has almost exclusively focused on the role of tyrosine kinases. I show that hypoxia induces inhibitory oxidation of protein tyrosine phosphatases (PTPs). Using mass- spectrometry based approaches, I will identify the oxidized- PTP landscape (ox-PTPome) of tumor samples and cancer cells at oxygen levels observed in tumors. Further, since PTPs restrain cellular signaling, I will employ high-throughput drug screening technologies in vitro to identify emergent sensitivities due to the loss of PTP activity that would not have been captured in the numerous normoxically (tumor-unrepresentative oxygen level) performed screens. Overall, the focus of my career is to understand how the different characteristics of the tumor microenvironment, such as hypoxia, modulates the signaling networks co-opted by cancer cells and translate this to the identification of biological mechanisms that may be amenable to therapeutic exploitation.

NIH Research Projects · FY 2024 · 2021-09

Project Summary/Abstract Brain metastasis is the development of secondary tumors within brain tissue which are typically derived from melanoma, lung cancer, and breast cancer. The prognosis for patients with brain metastasis is devastatingly poor with a median survival of less than six months. To reduce brain metastasis incidence and cancer mortality, rationally-designed therapeutic approaches targeting the mechanistic underpinnings of brain metastasis progression is imperative. It is increasingly appreciated that the immune cells within the brain metastatic niche have indisputable and ubiquitous roles in regulating brain tumor progression. Yet, the regulation of CNS immunity by peripheral and systemic factors during brain metastatic colonization and outgrowth are not completely understood. The gut microbiota composition plays a crucial role in regulating the host’s peripheral immune system, correlates with anti-cancer immunotherapy efficacy and has a profound influence on brain behavior and function by reshaping the brain immune niche. In this study, we aim to identify how gut microbiota modulation reshapes the brain’s immune landscape and subsequently influences the metastatic niche and progression of brain metastasis. Antibiotic-induced gut microbiota dysbiosis led to a significant increase in brain metastasis burden in contrast to a vehicle-treated control group, suggesting that gut microbiota dysbiosis remodeled the brain metastatic niche to a tumor-promoting environment. Using single-cell analysis, we revealed compositional and transcriptional differences of immune cells within the brain metastatic niche of mice with and without gut dysbiosis. These findings suggest that gut dysbiosis affects specific immune cell types to promote brain metastasis outgrowth. Here, we propose to dissect the roles of these immune cell types in promoting brain metastasis outgrowth under gut dysbiosis conditions. Furthermore, we will elucidate the spatial distribution of effector immune cells within the brain metastatic niche and functional impact of gut-derived signaling molecules in brain metastasis outgrowth. Understanding the constituents and host-intrinsic regulators of the brain metastatic niche shaped through gut-brain communication will guide the development of novel and feasible brain metastasis prevention strategies through gut microbiota modulation.

NIH Research Projects · FY 2024 · 2021-09

PROJECT SUMMARY/ABSTRACT The recent wave of cryo-electron microscopy (cryo-EM) studies on amyloid fibrils has highlighted the complexity of protein deposition amongst patients of amyloid diseases. A convincing example is found in tau, a microtubule binding protein whose pathological aggregation causes tauopathies. Each of these tauopathies seems to be associated with a particular structural assembly, suggesting causality. Similar to tauopathies, other amyloid diseases manifest differential prognosis, onset, and symptomatology. This is the case of TDP-43, α-synuclein, β-amyloid, or transthyretin (TTR), to name a few. We hypothesize that these phenotypical differences are driven by the formation of various structural assemblies, similar to what is found in tau. As a consequence, the detection of these disease-specific assemblies in a timely manner could ensure proper diagnosis and treatment. We aim to map the structural spectrum of amyloid fibrils in an amyloid disease model using cryo-EM and a co- culture system to be developed in our laboratory. Using the obtained structural information, our laboratory will design structure-specific peptides for the detection and inhibition of amyloid fibrils in cells, mice, and patient- derived samples. If successful, our study will serve as a launching platform for the development of personalized structure-based diagnostics and therapeutics for amyloid diseases.

NIH Research Projects · FY 2025 · 2021-09

ABSTRACT The hippocampus has been implicated in numerous functions related to normal memory and its dysfunction in several diseases. Our lab has investigated the hippocampus in relation to schizophrenia (SZ) pathophysiology using cognitive and behavioral outcomes(1-3) and brain image analyses(4-6). These consistently show that hippocampal (Hipp) activity is elevated in schizophrenic psychosis (SZ), especially in early illness. To test the molecular basis of this hyperactivity, we examined human postmortem hippocampal tissue by subfield, contrasting healthy and schizophrenia cases, using excitatory and inhibitory synaptic markers and Golgi. We found a reduction in GluN1 limited to dentate gyrus (DG) and an increase in markers of synaptic strength in CA3 in the SZ tissue; these changes are consistent with the observations that Lee et al(7) reported in hippocampal cell cultures (see A.1). Lee showed that CA3 pyramidal cell sensitivity is inversely and powerfully controlled by afferent input from DG, with decreased afferent input associated with increased pyramidal cell activity. We have been able to recapitulate this human-specific SZ pathology in a mouse using a DG-selective GluN1 knock out (KO)(8). This back-translation mouse KO demonstrated Hipp hyperactivity and alterations in Hipp-mediated behaviors (8). We are piloting an inhibitory DREADD technique in DG to mimic the DG-selective GluN1 KO mouse and saw evidence of a sensitive period during ‘adolescence’, when reduced DG activity could stimulate hyperactivity in CA3/CA1. This time phase cannot be resolved in the KO animal, so we had not seen it before and can only study it using DREADDs. The goal of these experiments are to causally define the mechanisms underlying the neurobiological outcomes of temporary DG hypofunction in mouse using DREADD constructs, and to show the extent, development, and critical periods of vulnerability of brain-wide changes. Having found a discrete circuit of Hipp projection regions hyperactive in the KO mouse, we will test human SZ vs HC tissue in these regions for evidence of hyperactivity and coherence with Hipp. The goal is to build a model of how hippocampal hyperactivity affects behavior and brain pathology, and specifically how this tissue pathology could support aberrant memories with psychotic content.

NIH Research Projects · FY 2024 · 2021-09

Project Summary Normal nervous system function depends on proper communications between neurons and non-neuronal cells. In the human brain, the majority of non-neuronal cells are called glia that consist of nearly half of total brain cells. Despite decades of work focusing on neurons and glia alone, how they communicate with each other remains poorly understood. This is partly due to a dearth of methods to comprehensively profile molecules that are enriched at the neuron-glial interface during dynamic cell-cell interactions. This proposal aims to develop a genetically-guided proteomic toolbox to spatiotemporally profile critical molecules enriched at neuron-glial interface in vivo, allowing for molecular and genetic dissection of neuron-glial interactions. We will first design and generate glial-specific cell surface proximity-labeling probes with the spatiotemporal precision. We will apply this platform in the oligodendrocytes, the sole myelin-producing cells in the central nervous system (CNS), to determine the molecular mechanisms governing the initiation of oligodendrocyte-axon ensheathment. We will leverage this new method, along with single-cell RNA sequencing, genome-wide CRISPR screens, and novel transgenic mouse strains, to interrogate a mysterious cell stage (the pre-myelinating oligodendrocytes) during developmental and adaptive myelination. To extend the glial surface proximity labeling toolkit, we will develop a neuron-glial complementary proximity labeling system allowing for visualization of transient neuron-glial interactions and capture of molecules only enriched at neuron-glial interface in vivo. Using this system we will address the molecular codes governing myelination selectivity between subsets of oligodendrocytes and functionally distinct neuronal subpopulations. This proposed work will fulfill the knowledge gap in myelin biology, and will have broader implications in understanding neuron-glial and glia-glial interaction mechanisms in general. The methods and reagents established by the work will also greatly enrich the methods in studying diverse cell- cell communications, including neuro-immune and cancer-immune interactions.

NIH Research Projects · FY 2025 · 2021-09

PROJECT SUMMARY Although genomics has led to an expansive set of predicted genes, functional annotation of gene products remains rate-limiting. To drive discovery of gene functions, we exploit host-virus interfaces and signatures of conflict. In addition to revealing host defense mechanisms, studies of infected cells and immune responses have led to the definition of fundamental cellular processes and key master regulators (e.g. SRC, P53). Here, we leverage our integrative framework – termed VIROLOG - for the discovery and characterization of novel host- virus interfaces. Specifically, we use genomic scars of conflict unique to factors linked to infection outcomes to identify uncharacterized genes combined with cell-based and viral infection assays. The merit of our strategy is illustrated by the identification of a vertebrate specific MItochondrial STress Response (MISTR) circuit. MISTR is executed by related electron transport chain factors and regulated by ultraconserved miRNAs induced by stress signals such as infection and hypoxia. Using the VIROLOG framework, this research program is defining new battlefronts in mitochondria highlighted by hundreds of viral-encoded factors that may target this organelle during infection to drive viral replication. As our multidimensional bioinformatic screens serve as fertile ground to identify host defenses and uncover new dimensions to textbook functions, we are developing VIROLOG as an interactive user database and interface. Using “classic” viruses such as vaccinia, the prototypical poxvirus, and virus vesicular stomatitis virus (VSV), a model RNA virus, along with the extensive molecular toolkit for key host defenses, we will narrow the gap of genes lacking function. Collectively, our innovative framework continues the rich history of using viral systems to drive biological discovery by exploiting a combination of classic evolutionary and molecular signatures paired with experimental analysis to characterize mechanisms. 1

NIH Research Projects · FY 2025 · 2021-09

PROJECT SUMMARY/ABSTRACT With this Mentored Patient-Oriented Research Career Development Award, the candidate – a psychiatrist with strong background in clinical research – aspires to achieve the long-term goal of becoming an independent, federally funded investigator who (i) uses a neuroscience-informed experimental medicine approach to identify neurocircuit mechanisms and (ii) conducts clinical trials to develop the next generation of circuit-specific antide- pressant treatments in order to ameliorate the public health burden of irritability, an important yet understud- ied feature of depression that is associated with poorer quality of life and elevated levels of suicidal ideation. Candidate proposes structured research experience and formal training in (1) ultra-high-field [7 Testa (7T)] func- tional magnetic resonance imaging (fMRI), (2) the affective neuroscience of irritability [including the use of task- based fMRI to model frustrative nonreward (FNR), an evolutionarily conserved response to omission of an ex- pected reward], and (3) experimental therapeutics approach to conduct clinical trials with changes in neurocircuit function as the outcome measure of interest, that will be supervised by a team of prominent experts in neuroim- aging, psychiatric neuroscience and experimental therapeutics. To fill the knowledge gaps regarding the neu- rocircuit mechanisms of irritability, candidate’s proposed research strategy aims to identify dysfunctions in neu- rocircuitry that engender irritability (Aim 1) and determine how changes in neurocircuit function relate to changes in irritability (Aim 2). The proposed study will enroll male and female adults aged 18-55 years [n=30 healthy controls (HC) and n=60 adults with major depressive disorder (MDD)]. All subjects will undergo resting-state and task-based 7T fMRI to characterize the (i) patterns of functional connectivity (Aim 1.1) and (ii) neural responses to a behavioral task of FNR (Aim 1.2) that are associated with irritability (quantified with the 5-item irritability domain of Concise Associated Symptom Tracking scale). The MDD cohort (n=60) will then be randomized to 2 weeks of twice-weekly intravenous infusions of either ketamine (0.5 mg/kg) or midazolam (0.02 mg/kg) in a double-blind parallel-arm fashion. Clinical assessments and MRI scans will be repeated after the last infusion to evaluate pre-to-post treatment changes in neurocircuit function using resting-state (Aim 2.1) and FNR (Aim 2.2) task-based fMRI with ketamine versus midazolam and to explore the association between changes in neurocir- cuit function and irritability. Candidate’s didactic and mentored training in 7T fMRI, affective neuroscience, and experimental therapeutic approach will enable successful execution of the research strategy. Research activities, such as collecting, storing, analyzing, and interpreting data from MRI scans and from clinical assessments, will provide valuable hands-on training. Successful completion of the proposed study will allow candidate to (1) ac- quire expertise in the conduct of neuroscience-informed studies that use experimental therapeutics approach and (2) generate pilot data in order to pursue NIH funding and build this highly innovative program of research.

NIH Research Projects · FY 2024 · 2021-08

PROJECT SUMMARY/ABSTRACT Seventy% of older adults (60+ yr) are overweight or obese and many are unable or unwilling to exercise due to exercise intolerance and/or dyspnea on exertion (DOE). We have identified numerous obesity- related effects that could influence exercise tolerance and DOE in obese adults. We have also identified many age-related ventilatory constraints in nonobese older adults. However, it is unclear whether obesity- related and aging-related effects combine to reduce exercise tolerance, provoke DOE, or contribute to respiratory symptoms in older obese adults. We propose that many of the obesity-related effects in older obese adults are the result of low lung volume breathing, i.e., a reduction in functional residual capacity (FRC) at rest and end-expiratory lung volume (EELV) during exercise. Increased fat on the chest wall produces low FRC and EELV levels, where breathing limitations like expiratory flow limitation (EFL) and enhanced perception of dyspnea are more likely to occur due to the age-related decline in maximal expiratory flow at low lung volumes. Our overall hypothesis is that respiratory limitations, exercise intolerance, DOE, and respiratory symptoms in older obese adults are due to mechanical loading of the thorax and low lung volume breathing. We propose to test this hypothesis with the use an external cuirass (i.e., a plastic shell over the thorax) to mechanically unload the chest wall. This will decrease the load on the thorax thereby increasing FRC at rest and EELV during exercise, and potentially decrease the work of breathing during exercise. The overall objective of this application is to investigate the effects of obesity on lung function, exercise tolerance, and DOE in older obese adults as compared with older adults without obesity, using a novel probe for mechanically unloading the thorax at rest and during exercise. We will use 1) continuous negative cuirass pressure, and 2) assisted biphasic cuirass ventilation to decrease obesity-related effects in older obese adults. Our approach will be to examine respiratory function, exercise tolerance, and DOE with and without mechanical unloading in older obese men and women (65-75 yr), including those with respiratory symptoms, as compared with older adults without obesity. Specific Aims: We will test the following hypotheses: Aim 1) Obesity will decrease respiratory function but to a greater extent in older obese adults with respiratory symptoms; Aim 2) Obesity will decrease exercise tolerance but not cardiorespiratory fitness, except in older obese adults with respiratory symptoms where both may be reduced; Aim 3) Obesity will increase DOE but to a greater extent in older obese adults with respiratory symptoms; and Aim 4) Mechanical unloading of the thorax will improve respiratory function, exercise tolerance, and DOE in older obese adults, but to a greater extent in older obese adults with respiratory symptoms. These results will have broad and immediate clinical impact on the care of older obese adults with DOE.

NIH Research Projects · FY 2023 · 2021-08

PROJECT SUMMARY Peripheral artery disease (PAD) is highly prevalent in patients with chronic kidney disease (CKD). In addition, the adverse consequences of PAD are more severe in patients with CKD compared to those without. The pathophysiology and clinical manifestations of PAD might be unique among CKD patients. Vascular calcification and arterial stiffness are common in CKD patients and cause non-compressible arteries and an artificial elevation of ankle brachial index (ABI). Our previous study reported that both reduced ABI (<1.0) and elevated ABI (≥1.4) were associated with a higher risk of PAD-related revascularization and amputation, as well as a higher risk of myocardial infarction, compared to those with an ABI between 1.0 to <1.4 in CKD patients. In addition, our pilot study suggested that the current diagnostic criteria of ABI ≤0.9 and toe-brachial index (TBI) ≤0.7 had poor sensitivity in detecting PAD defined as ≥50% artery stenosis in the lower extremities by Doppler ultrasound among CKD patients. PAD risk classification models including multiple cardiovascular disease (CVD) risk factors in addition to ABI or TBI significantly improved discrimination of those with and without PAD. Furthermore, the diagnostic value of TBI, a measurement of perfusion of the foot, especially in patients with incompressible vessels, has not been well evaluated in CKD patients. The overall objective of the proposed study is to identify more accurate cut-points of ABI and TBI for PAD screening and diagnosis and to develop a novel approach for PAD screening and diagnosis among CKD patients. The specific aims are: (1) to assess the accuracy of current ABI and TBI diagnostic criteria and to identify more accurate cut-points of ABI and TBI for PAD defined as ≥50% artery stenosis in the lower extremities by Doppler ultrasound; (2) to assess whether the inclusion of multiple CVD risk factors in addition to ABI or TBI will improve discrimination and to develop novel risk classification models for PAD screening and diagnosis; and (3) to compare the new cut- points of ABI for PAD vs. the conventional cut-point (ABI ≤0.9), as well as risk classification models vs. ABI alone, in predicting risk of clinical PAD, CVD, and all-cause mortality in CKD patients. We will recruit 420 pre- dialysis CKD patients from three Chronic Renal Insufficiency Cohort (CRIC) study centers and conduct ABI, TBI, and Doppler ultrasound tests. Color Doppler ultrasound, which will employ multiple features including reduction in luminal diameter, monophasic waveform, peak systolic velocity ratio >2.0, and presence of special broadcasting, will be used as the reference standard. The CRIC study recruited about 5,500 CKD patients with ABI measurements and followed clinical outcomes for up to 17 years. The proposed study has 99% statistical power to detect an area under the curve (AUC) of 0.70, with the null hypothesis AUC value of 0.50 and a 2- sided significance level of 0.05 (Aim 1), and over 90% power to detect the difference of comparing AUC of 0.85 vs. AUC of 0.75 (Aim 2). The proposed study may change clinical practice regarding the screening and diagnosis of PAD in CKD patients, with the aim of reducing PAD-related morbidity and mortality.

NIH Research Projects · FY 2025 · 2021-08

ABSTRACT The excitement about nanomedicine stems from the potential application of nanoscience to solve challenging medical problems. Inorganic nanoparticles (iNPs) exhibit unique properties that favor their diverse application in medicine, engineering, science, and technology. The large surface-to-volume ratio of these iNPs provides sites for the attachment of multiple drugs or imaging agents for therapy and imaging of diverse human diseases. Further conjugation of biological entities, such as proteins, nucleic acids, and lipids, confers specific targeting of these iNPs to desired tissues in vivo. Recent studies have shown that the intrinsic properties of some iNPs can be harnessed for therapeutic outcomes. Still, spontaneous stimulation of intrinsic therapeutic effects through interactions of the NPs with intracellular organelles, proteins, or molecular processes is difficult to control, leading to significant off-target toxicity. An alternative therapeutic approach is to transform some iNPs into nanoscale energy transducers. Quantum dots, upconversion NPs, carbon nanomaterials, and photocatalytic NPs are some nanoscale energy transducers that have shown promise in the treatment of human diseases. The excellent redox properties of these nanophotosensitizers offer high spatiotemporal control and precision phototherapy upon absorption of light. Two major limitations of current phototherapeutic interventions are the limited penetration of light used to activate the photosensitizers, which confines therapy to shallow lesions, and the frequent reliance on molecular oxygen to generate cytotoxic reactive oxygen species, a condition that precludes the effective treatment under the hypoxic conditions found in many solid and hematologic tumors. Recently, we developed radionuclide stimulated therapy that leverages the interaction of Cerenkov radiation emitting radionuclides to stimulate the production of reactive oxygen species from photosensitizers. The spatiotemporal therapeutic effects of these interactions allow the treatment of diverse diseases without tissue depth limitation that affects light-based therapies. Supported by new concepts grounded in robust preliminary data, we propose to (1) explore new nanostrategies to overcome the impediment to delivering NPs to tumors, (2) disrupt the protective interactions of cancer with stromal cells to enhance treatment response, and (3) exert sustainable therapeutic effect via multidimensional combination therapy to achieve disease-free survival. At the completion of this study, we would develop new nanoplatforms for the treatment and imaging of cancer and bone lesions.

NIH Research Projects · FY 2025 · 2021-08

Project Summary Centrosomes nucleate microtubule arrays and act as force-coordinating centers to position nuclei and segregate chromosomes, which are essential activities during early embryogenesis and neural development. While much is understood about the regulation of centrosome number, much less is known about molecular mechanisms determining centrosome size, microtubule nucleation capacity, and resistance to forces. The goal of this proposal is to reveal how molecular-level interactions between centrosome proteins determine the activity, emergent material properties, and ultrastructure of PCM, the most substantial layer of a centrosome. I hypothesize that PCM is an amorphous hydrogel whose material state (e.g., strength, elasticity) is regulated by phospho-tunable connections between coiled-coil scaffolding proteins. I further hypothesize that fine-tuning of scaffold structure and material properties regulates PCM size, activity, and resistance to microtubule-dependent pulling forces. I propose to test these hypotheses using two innovative techniques that I recently developed: a minimal PCM reconstitution system and an optical method to perform nano- rheology of PCM in living embryos. In addition, I propose to develop in-cell cryo-electron tomography to visualize PCM ultrastructure with sub-10 nm resolution. These experiments are designed to 1) identify the minimal components needed to generate consistently sized, fully active PCM, 2) discover key regulators and material design principles that allow PCM to resist microtubule-pulling forces, and 3) generate the highest- resolution structural atlas of native centrosomes to date. This proposal is significant because it will illuminate how centrosome function is determined and regulated at the molecular level, which will provide mechanistic insight into human disorders caused by centrosome dysfunction, such as microcephaly, primordial dwarfism, and various cancers.

NIH Research Projects · FY 2025 · 2021-08

Niemann-Pick disease type C (NPC) is a fatal neurodegenerative disease caused by genetic mutations in the NPC1 (95%) and NPC2 (5%) genes. Neurological features of NPC bear striking resemblances with Alzheimer’s disease (AD), leading to some experts considering NPC as “Childhood Alzheimer’s”. No FDA-approved therapy is available. Lipids and cholesterol defects associated with the NPC disease are well understood. However, immune pathways underlying neuroinflammation and Purkinje cell death in the NPC disease remain unknown. We recently found that the innate immune STING pathway is activated by NPC-deficiency, and genetic deletions of STING pathway components in mice are able to remarkably rescue NPC neuropathology. The overall goal of this project is to understand how STING drives neuropathology associated with the NPC disease. Aim 1 will focus on intracellular mechanisms, where we will dissect how STING signaling is activated by NPC-deficiency. Aim 2 will focus on brain tissue pathology, where we will comprehensively determine STING expression cell types, activities and their functional contribution to the NPC disease. Aim 3 will focus on therapeutic assessment, where we will treat Npc1-/- mice and NPC1 patient iPSC-derived cells with existing and new STING pathway inhibitors. STING has been implicated in several neurodegenerative diseases including Parkinson’s disease, ALS/FTD and now NPC1. Studies proposed here will provide a much-needed comprehensive understanding of STING pathway function in the brain as well as a step-by-step mechanism from STING activation to neuropathology.

NIH Research Projects · FY 2025 · 2021-08

PROJECT SUMMARY Alcoholic liver disease (ALD) is exacerbated by impaired liver regeneration. The cellular basis of liver regeneration is unclear and whether hepatocytes in different zones differ in regenerative activity is unclear, in part because fate-mapping has only been performed on a few hepatocyte subsets. The liver is organized into zones in which hepatocytes express different metabolic enzymes. To systematically compare the regenerative activities of these distinct subsets of hepatocytes, we developed twelve new CreER strains. Lineage tracing during normal homeostasis showed that cells from periportal zone 1 and pericentral zone 3 contracted in number, while cells from mid-lobular zone 2 expanded in number. Hepatocytes in different regions of the liver thus exhibit differences in turnover and zone 2 is an important source of new hepatocytes during homeostasis. Because zone 2 may represent a reserve population sheltered from pericentral and periportal liver injuries, we hypothesize that these cells also preferentially repopulate livers exposed to modest chronic injuries such as alcohol. Our preliminary scRNA-seq and in vivo CRISPR screens identified two critical zone 2 specific genes that regulate zone 2 hepatocyte proliferation and survival: Igfbp2 and Hamp2. Both of these secreted factors are suppressed in NAFLD and ALD in humans, which suggests functional importance in disease. Igfbp2 operates through mTOR and Ccnd1 to promote zone 2 hepatocyte proliferation. Hamp1 and Hamp2 encode for hepcidins, which negatively regulate iron uptake by inhibition of iron transporters and thus protects the body from iron overload. Patients with ALD accumulate hepatic iron through suppression of hepcidins. Free iron enhances reactive oxygen species (ROS) production in the liver, leading to alcohol-induced liver injury. We hypothesize that ALD pathogenesis is accelerated through the suppression of Igfbp2 and Hamp1/2, and that this involves changes in the number or function of zone 2 hepatocytes. In Aim 1, we will first use our CreER models to systematically determine the extent to which zone 2 cells repopulate the liver in the setting of ETOH. To understand how zone 2 cell might be involved in regeneration after ETOH, we will perturb two critical zone 2 genes using loss and gain of function approaches. In Aim 2, we will ask if Igfbp2 is necessary and sufficient to regulate the frequency or repopulating activities of zone 2 cells in the context of ETOH. In Aim 3, we will ask if Hamp1 and Hamp2 are necessary and sufficient to regulate the frequency or repopulating activities of zone 2 cells. Success in this project will for the first time define the cellular basis of regeneration in response to ETOH, allow us to focus on critical subpopulations, and determine the importance of two critical zone 2 specific genes in ALD.

NIH Research Projects · FY 2025 · 2021-07

PROJECT SUMMARY/ABSTRACT Derivation of pluripotent stem cells (PSCs) has revolutionized developmental biology and regenerative medicine. To stably maintain PSCs in culture and guide them to differentiate with high efficiency and fidelity into a variety of cell types, it is important to understand the molecular mechanisms governing pluripotency (the ability of a cell to generate any tissues in the body). Two phases of pluripotency, naïve and primed, have been defined and studied in detail thanks to the successful derivation of mouse embryonic stem cells (ESCs) and epiblast stem cells (EpiSCs), respectively. Mouse ESCs most closely resemble epiblast from a 4-day-old mouse blastocyst (~embryonic day 4, or E4), while “primed” EpiSCs display a global gene expression signature similar to the E7 epiblast of a post-implantation mouse embryo. Despite these advances, however, however, there is lack of a well-established PSC model that resembles E5-6 early post-implantation epiblast, which corresponds to the formative phase of pluripotency. Formative pluripotency exists within a time window during which naïve pluripotency is reconfigured to prepare for multilineage competency, including germ cells. Functionally, formative pluripotency is characterized by both chimera competency and permissiveness for direct primordial germ cell (PGC) induction. Several recent studies have attempted to define this state by transient epiblast-like cells (EpiLCs) differentiated from ESCs. To date, however, stable formative PSCs have not yet been generated. By modulating the FGF, TGF-β and WNT pathways, we recently derived PSCs from both mice and humans (referred to as FTW-PSCs) that are permissive for direct PGC-like cell induction in vitro and are capable of contributing to intra- or inter-species chimeras in vivo. FTW-PSCs harbor molecular, cellular and phenotypic features characteristic of formative pluripotency. The overall objective of this proposal is to use these newly established cell lines to comprehensively dissect the formative state across species. The proposed studies will elucidate the roles of several transcription factors in regulating mouse and human formative pluripotency, as well as demonstrate that FTW-PSCs are a robust platform for dissecting the molecular mechanisms underlying human and mouse PGC specification. In addition, we will establish an in vitro platform for the generation of functional mouse oocytes and human oogonia based on formative FTW-PSCs, thereby providing an invaluable resource for studying germ cell development and human infertility. Our proposal has tremendous potential to revolutionize regenerative medicine and reproductive biology.