University Of California Los Angeles

NIH Research Projects · FY 2025 · 2021-07

Project Summary/Abstract Epilepsy is a severe and debilitating disease and a significant public health concern. Epilepsy is also a disease without a medical cure, and a disease where about 1 in 3 patients fails to respond to anti-seizure medications. In the most severe epilepsy syndromes of childhood, medical control of seizures can be even more challenging. Novel experimental platforms have the potential to play a critical role in advancing our understanding and treatment of epilepsy. Brain organoids derived from human embryonic or induced pluripotent stem cells are one such novel technology that has enormous potential. This is particularly true for severe childhood epilepsies, as organoids are ideally suited to model early neural development. Organoids are 3D structures that recapitulate complex elements of human brain such as its laminar organization and cell types seen in all six layers of human cortex. Since they can be human induced-pluripotent stem cell (hiPSC) derived, an organoid can be produced directly from patient tissue. Recent advances in organoid technology have resulted in the ability to generate distinct brain region-like organoids such as forebrain cortex and hippocampus and to make “fusion” structures with integration of inhibitory and excitatory cell types. In the following proposal I will leverage these advances and build on an organoid platform that I have recently developed to model brain circuit formation and dysfunction in epilepsy. Previously, l was able to recapitulate hyperexcitable electrographic features in organoids derived from a patient with Rett syndrome, a neurological disorder highly associated with seizures and epilepsy. I have now generated cortical and hippocampal organoids from hiPSCs harboring mutations in the SCN8A gene. This mutation results in a severe childhood epilepsy. I have found that the SCN8A mutant cortex organoids have a highly hyperexcitable pattern of physiological activity compared to controls, whereas the SCN8A mutant hippocampus lacks a particular type of neural oscillation that is important for memory consolidation called a sharp wave ripple. This finding suggests that the SCN8A mutation results in different physiological activity patterns in distinct brain regions. Based on published studies, I hypothesize that this difference is primarily due to dysfunction of excitatory neurons in the cortex versus inhibitory interneurons in the hippocampus. I will now use an array of techniques such as calcium indicator imaging, extracellular recordings, immunohistochemistry, and manipulation of the genetic background of excitatory and inhibitory neurons within the organoid to test this hypothesis. To increase the rigor and generalizability of my data, I will use hiPSC from three different patients with pathogenic SCN8A mutations. Finally, I will perform drug testing to further isolate the role of specific cell types to the observed phenotypes and for consideration as therapeutic agents in patients. I expect that this will both provide a blueprint for a novel methodology for epilepsy research and enhance our treatment and understanding of epilepsy and neural circuit dysfunction resulting from SCN8A mutations.

NIH Research Projects · FY 2025 · 2021-06

Project Summary The long-term objectives are to develop and validate rat models of disease that allow investigators to measure the differential effects of XX and XY sex chromosomes that protect from or exacerbate disease. Most human diseases occur differently in males and females, indicating that one sex is protected or vulnerable because of factors that are inherently different in the two sexes. Understanding the mechanisms of protection or vulnerability involves isolating different molecular pathways causing greater or less protection. Sex chromosomes (XX vs. XY) are one major source of sex bias within any type of cell, but this category has been difficult to discriminate from gonadal hormone effects that often co-vary with sex chromosome complement. To isolate and study sex chromosome effects, it is necessary to make experimental models comparing XX and XY animals with the same type of gonad. Such models have not been available to investigators who study rats, but have just become available. The modified rats have two genetic mutations, to introduce the testis-determining gene Sry onto a non-sex-chromosome, and to knock Sry out on the Y chromosome. These modifications produce XY and XX rats with ovaries, and XX and XY rats with testes. The proposal is to study the newly developed genetically modified rat lines, to establish the nature of genetic sequence in and near the two genetic modifications, and to determine how the modifications change the development of ovaries and testes. Rats bearing these modifications will be compared to normal rats, to measure: physiology of reproduction, sexual development of the brain, cardiac function, systemic and pulmonary hypertension, and hypertension- related cognitive function. Rats offer significant advantages as models of human physiology and disease, because of their large size, the large literature concerning basic physiology and sex differences in rats, their superior cognitive ability, and suitability of rats to research on specific diseases. The successful rat models will be deposited in the Rat Resources & Research Center and made widely available to other investigators.

NIH Research Projects · FY 2025 · 2021-06

PROJECT SUMMARY Retinopathy of prematurity (ROP) is a leading cause of childhood blindness, affecting approximately 1 in 3-4 extremely low birth weight premature infants. Preterm delivery requires exposing these neonates to relative hyperoxia because of their lung immaturity. Hyperoxia leads to vessel attenuation in the retina, which results in local hypoxia, which then fuels abnormal compensatory neovascularization (NV). This process is mediated by altered expression of growth factors such as vascular endothelial growth factor (VEGF). Though the neuroretina has been described to play a role in pro-angiogenic signaling in ROP, the mechanisms remain poorly understood. Current therapies for ROP treat late retinal neovascularization and do not address neuroretinal dysfunction in ROP. In this grant, we propose to understand the role of an upstream regulator of VEGF, epithelial membrane protein-2 (EMP2), in an oxygen-induced murine model of retinopathy. EMP2, a tetraspanin membrane protein important for cell-to-cell signaling, regulates angiogenesis via VEGF and hypoxia inducible factor (HIF)1α modulation in select cancers and placental diseases. We hypothesize that EMP2 serves as a regulator of hypoxia-mediated pathological neoangiogenesis in the eye as well. Our preliminary data from a mouse model of oxygen-induced retinopathy (OIR) demonstrates that genetic knock out of EMP2 attenuates NV and suppresses HIF1α and VEGFA expression in the neuroretina. Moreover, OIR induces EMP2 expression in the neuroretina, which in physiologic states, has low expression in the neuroretina and high expression in the RPE and cornea. However, the role for EMP2 expression and its function in the developing neuroretina is unknown. The goals of this proposal are to determine the temporal and spatial expression and function of EMP2 in normal retinal development as well as in pathologic conditions of OIR. Thus, we seek to understand the mechanisms by which EMP2 regulates neuroretinal angiogenic signaling. We hypothesize that EMP2 expression, normally isolated to the retinal pigment epithelium (RPE) in the adult mouse retina, is increased in neuroretinal cells in OIR in the developing eye (Aim 1), where it directly regulates HIF- mediated VEGF production from these cells (Aim 2). We further hypothesize that antibody-mediated targeting of EMP2 will safely and effectively attenuate pathologic NV (Aim 3). Our approach is multidisciplinary, with experts in neonatology/vascular disease in neonates, EMP2 biology, retinal diseases, genomics, and advanced imaging. We will utilize biochemical, physiological, genomic, and optical imaging methods in vivo to assess EMP2 expression, function, and the downstream angiogenic effect. The central innovations of this study are to: (1) further our understanding of the neuroretina’s role in oxygen- induced retinopathy via EMP2-mediated angiogenic growth factor production, and (2) apply the knowledge of EMP2’s effects on angiogenesis via VEGF expression in cancer, placentation, and adult eye disease to ROP.

NIH Research Projects · FY 2025 · 2021-06

PROJECT SUMMARY AND ABSTRACT Opioid use disorder (OUD) causes significant morbidity and mortality in the United States. Efficacious pharmacotherapies that can be delivered in a primary care or specialty clinic setting have been developed but most patients with OUD do not receive them. In recent years, three kinds of long-acting medication for OUD (MOUD) have been approved by the Food and Drug Administration (FDA): monthly injectable naltrexone (2010), six-monthly implantable buprenorphine (2016) and monthly injectable buprenorphine (2017). These modalities have several theoretical advantages over daily buprenorphine or methadone – e.g., stabilizes dosing, reduces frequency of follow-up visits, prevents diversion – but their uptake and dissemination in real- world settings have not been characterized. The proposed research will investigate provider- and systems- level barriers and facilitators to adoption of long-acting MOUD. In this mentored career development award (K01), Dr. Shover will characterize clinicians who are early adopters of long-acting MOUD, identify context- specific barriers and facilitators to prescribing and administering long-acting MOUD, and longitudinally compare dissemination of long-acting MOUD to that of daily MOUD. In Aim 1, Dr. Shover will use insurance claims data to characterize individual and organizational attributes of providers who prescribe long-acting buprenorphine or naltrexone to treat OUD in the first year of their FDA approval. In Aim 2, Dr. Shover will conduct qualitative interviews with primary care physicians, pain medicine physicians, and psychiatrists to identify subjective factors that influence willingness or capacity to prescribe long-acting MOUD. In Aim 3, Dr. Shover will conduct a longitudinal analysis of dissemination of long-acting MOUD in their first five years, comparing geographic and systems-level patterns diffusion to those of daily oral MOUD. Through the award, Dr. Shover will build on her doctoral training as an epidemiologist and experience in substance use policy and community-based HIV prevention to develop new skills needed to conduct impactful addiction health services research. These skills will include working with claims data, qualitative interviewing, implementation science, grant-writing, and scientific communication. Through coursework, clinical observation in psychiatry and pain medicine clinics, mentorship, and external conferences and workshops, Dr. Shover will gain the skills needed to apply for her first R01 and pursue a career as a tenure-track principal investigator.

NIH Research Projects · FY 2025 · 2021-05

Project Summary This proposal aims to enable precision nutrition by creating a wearable technology that can be scaled across the general population to non-invasively track the diurnal profiles of a panel of putative circulating nutrients and biomarkers. Accordingly, we will address fundamental and intermeshed engineering bottlenecks and scientific questions at sensor, device, and data analytics levels to realize a sweat-based wearable bioanalytical technology, equipped with autonomous sweat secretion modulation, biofluid management, and analysis capabilities. To illustrate our technology’s transformative potential, we will particularly position it to monitor a panel of nutrients and indicators of the metabolic and disease state that are relevant in cystic fibrosis (CF, the most common inherited multisystemic disorder), in order to enable individualized nutritional support, which is central to the CF treatment. Accordingly, in the first phase (R21), we will develop microsensor arrays targeting glucose, triglyceride, and β- hydroxybutyrate. We will incorporate our readily developed auxiliary sensing modalities (sweat sodium, chloride, pH, and sweat secretion rate sensing interfaces) to enable the in-situ characterization of the secretion profile (which is useful for the normalization of sweat readings and tracking of the CF progression). In parallel to these engineering efforts, we will conduct sweat characterization experiments to study the effect of the secretion rate on analyte partitioning from blood into sweat. These datasets will be augmented with state-of-art machine learning algorithms to formulate a dedicated analytical framework that accounts for sweat secretion variabilities and determines optimal sweat secretion condition(s) to provide undistorted and physiologically meaningful sweat readings. In the second phase (R33), we will establish the clinical utility of our technology by demonstrating the ability to non-invasively track the target nutrients’ temporal profiles in relation to their circulating levels in blood (in both healthy subjects and CF patients and through simple/mixed meal-modulated studies). Accordingly, we will first measure the sweat and blood analytes’ excursion profiles after controlled single/binary combinations of nutrients intake and develop a machine-learning based algorithm to correlate the sweat analyte readouts to their circulating concentrations. Then we will assess and characterize the predictive utility of our solution in the context of complex nutritional supplement studies. Upon its validation, we will recruit a cohort of CF patients and perform a longitudinal randomized nutritional support study to demonstrate the enabling remote patient monitoring capabilities rendered by our solution. The success of this work will represent a groundbreaking contribution towards the development of strategies to enable precision nutrition and personalized medicine.

NIH Research Projects · FY 2025 · 2021-05

PROJECT SUMMARY Large-scale genome-wide association studies (GWAS) have identified between a handful to hundreds of risk loci for each major type of neuropsychiatric disorders. One of the main challenges for the post-GWAS era is to determine the causal variants and dissect the regulatory mechanism in each of the risk loci. The analysis of causal genetic mechanisms for psychiatric diseases is confounded by the highly heterogeneous brain structures and cell types. We hypothesize that brain regions and cell types are selectively vulnerable to mental disorders and cell-type-specific gene regulation underlies such selectivity. In this proposed project, we aim to determine the causal probability of individual genetic variants with high spatial resolution with respect to brain regions and cell types. To this end, we will generate a unique dataset of single-nucleus joint profiling of chromatin conformation and DNA methylation (sn-m3C-seq) for 10 adult brain regions, allowing the cell-type-specific identification of regulatory elements, enhancer-gene looping and linking non-coding variants to their regulatory target. To further identify the genetic mechanisms for cell-type-specific regulation of gene expression, we will develop and apply cutting-edge statistical methods to existing and newly generated population single-nucleus RNA-seq datasets for the human brain cortex and hippocampus. We will develop CONtexT spEcific geNeTics (CONTENT) to distinguish tissue- or cell-type-specific from the tissue-shared genetic component of gene expression regulation. We will also apply the recently developed PopuLation Allele-Specific MApping (PLASMA) that integrates QTL and allele-specific QTL for regulatory variant fine-mapping. To validate our findings, we will experimentally determine the function of non-coding variants using both high-throughput CRISPR interference and precise variant replacement experiments, as well as apply orthogonal statistical approaches to link the functional properties of variants to disease causality. Our proposed project integrates diverse approaches including single-cell multi-omics, statistical fine-mapping, and genetic engineering and will likely provide new insights into the genetic mechanism of mental disorders.

NIH Research Projects · FY 2025 · 2021-05

Project Summary/Abstract Helicobacter pylori is a highly prevalent pathogen, with 50% of the world’s population infected. All H. pylori infections at minimum cause gastric inflammation. A fraction of those infected will eventually develop gastric or duodenal ulcer disease, atrophy, or gastric adenocarcinoma or MALT lymphoma. Gastric cancer is one of the leading causes of cancer death worldwide, and eradication of the infection leads to prevention or even regression of gastric cancer. Treatment is becoming more difficult because of widespread antibiotic resistance. It is not definitively known who will go on to develop advanced disease, although many different bacterial and host factors have been implicated. The focus of this research proposal is to study mechanisms related to novel host/bacterial connections that potentially lead to gastric injury. H. pylori is known to cause epithelial injury, and preliminary data suggest that the bacteria induce downregulation of the Na,K-ATPase, which is involved with critical transport functions via establishment of an inward sodium gradient and with cell adhsion. Decreased Na,K-ATPase activity in gastric epithelial cells leads to reduced barrier function and gastric injury. Downregulation of the transporter by H. pylori targets newly formed pumps and trafficking from the ER. The mechanism will be further investigated by studying post-translational modifications potentially induced by the bacteria, by looking at the physiologic consequences of decreased pump expression on gastric cells, and by further characterizing the mechanism of pump degradation. H. pylori bacterial factors also play an important role in induction of gastric injury. From the bacterial standpoint, the role of direct H. pylori adhesion in Na,K-ATPase downregulation will be delineated. Dependence on the virulance factor CagA and the CagPAI type 4 secretion system (T4SS) will be determined. The role of gastric injury via Na,K-ATPase downregulation in induction of signaling pathways from stomal cells will be studied in an enteroid-stromal co-culture model. A NanoString platform will be used to examine gene changes in bacteria and host simultaneously in order to expand the targets studied in barrier dysruption and ultimately initiation of oncogenesis. Coordinated signaling systems induced by bacteria and host that impact decrease in Na,K-ATPase will be delineated, specifically as related to the CagPAI T4SS; known pathways will be explored and novel pathways will be identified via innovative mass spectometry techniques. Completion of this work will help determine why and how H. pylori specifically targets the Na,K-ATPase, identify effector molecules aside from CagA that enter cells via CagPAI to affect Na,K-ATPase levels, and delineate how bacterial factors modified by host proteins induce signaling cascades, leading to the changes in transporter levels. The goal of this work is to gain new insight into the mechanism of gastric injury by H. pylori, which will lead to novel therapeutic protective and treatment options.

NIH Research Projects · FY 2025 · 2021-05

PROJECT SUMMARY/ABSTRACT Cumulative epidemiological and experimental evidence have shown that exposure to air pollutants leads to increased cardiovascular morbidity and mortality. These associations have been mostly ascribed to the particulate matter (PM) components. We have found that exposures to ambient ultrafine particles (UFP), with an aerodynamic diameter less than 0.18 µm, and/or diesel exhaust, rich in ultrafine PM, lead to enhanced lipid peroxidation, metabolic derangements, liver steatosis and atherosclerosis. Inhalation of PM exerts prooxidant actions in the lungs but the mechanisms as to how pulmonary effects are translated into systemic toxicity are still unknown. PM exposure also triggers antioxidant responses in pulmonary and systemic tissues, including activation of transcription factor Nrf2 and upregulation of its target gene heme oxygenase 1 (HO-1), which attempt to counteract the ensuing harmful effects. The observations that particle uptake by alveolar macrophages significantly correlates with the development of atherosclerotic plaques strongly suggest that these cells are likely mediators in translating effects from the lungs to the systemic tissues. Our overarching hypothesis is that PM exposure promotes cardiometabolic toxicity starting with oxidative actions in the lungs that lead to prooxidant and proinflammatory effects in the circulating blood and systemic tissues via activation of alveolar macrophages, all modulated by the degree of myeloid anti-oxidant protection. We will test this hypothesis via the following three specific aims: 1) Assess the kinetics and mechanisms of lipid peroxidation in the lungs after ultrafine particle exposure, and their relation to prooxidant effects in the circulating blood and the development of atherosclerosis. We will use lipid peroxidation byproducts as tracking signals of PM-induced biological effects, and assess the kinetics of their appearance in various tissues such as the lungs, blood, liver, adipose tissue and aorta of ApoE KO mice exposed to ultrafine particles vs. filtered air for various times. 2) Determine if the myeloid antioxidant defense protects against UFP-induced lipid peroxidation, pulmonary and cardiometabolic toxicity. Myeloid-specific Nrf2 and HO-1 KO mice as well as myeloid-specific HO-1 Transgenic overexpresser mice in the ApoE null background, recently developed by us, will be used to test the effects of decreased or increased antioxidant defense, respectively, in the toxicity induced by UFP. 3) Evaluate whether alveolar macrophages carry UFP-induced oxidative effects from the lungs to the circulating blood. We will develop alveolar and lung macrophage chimeras with ablated HO-1 in their alveolar/interstitial macrophages to dissect their contribution in translating effects from the lungs into the systemic vessels. The proposed studies will aid in identifying mechanisms involved in PM-induced cardiovascular toxicity, and characterizing promising novel biomarkers of health effects, with the potential to aid in the design of therapeutic and/or prophylactic interventions against the toxicity induced by air pollution.

NIH Research Projects · FY 2025 · 2021-05

PROJECT ABSTRACT. Kimberly Paul, PhD, MPH, is a neurologic and aging disease epidemiologist whose research career goals focus around using modern, “omic” technologies to define the neurodegenerative disease process and elucidate risk at a biologic level, integrating a systems biology approach, in population-based studies. The research she proposes, entitled “Immune System Aging in Parkinson’s and Alzheimer’s disease-related dementia: Epigenetics, biologic aging, and heightened immune states”, combines advanced statistical methods and a systems biology modeling approach with high-throughput omics markers to study immune system aging in neurodegenerative disease patients relative to community-based controls; followed by relating epigenetic markers of inflamm-aging to symptom development and multiple metabolomics measurements over time. Candidate & Mentoring Team: Dr. Paul is a Postdoctoral Scholar in the Department of Epidemiology at the Fielding School of Public Health (UCLA) and will transition to an Assistant Professor in the Department of Neurology, David Geffen School of Medicine. Dr. Paul was previously awarded an NIEHS F32 to investigate how metabolic dysfunction mediates the association between ambient environmental exposures and primarily Alzheimer’s disease-related dementia (ADRD). For this work, she was awarded the prestigious Chancellor’s Award for Postdoctoral Research at UCLA. Her research priorities have developed into bringing biology and mechanism into population and data science. The proposed career development plan will build upon her previous training with goals to enhance her trajectory toward becoming an independent investigator, including experiential and didactic learning in design, methods and data interpretation, while developing the leadership and professional skills required to lead an independent research lab and execute an R01. Dr. Paul has assembled a strong mentoring team with renowned expertise, commitment, and available resources to support her training. Research Summary: The research aims of this K01 proposal focus on using a systems biology pipeline to investigate immune system aging in Parkinson’s disease (PD) and ADRD patients, using blood-based epigenetics to define immune states. The purpose of this proposal is to investigate how these immune markers and measures of accelerated immune system aging relate to onset, symptom development, and trajectories of metabolomic changes using two population-based studies. The hypothesis being that immune profiles which reflect “inflamm-aging” (chronic, low-level inflammation states) and immunosenescence will be related to faster symptom development. Furthermore, longitudinal metabolomics and targeted gene sequencing will give insight into endogenous risk and the systemic response related to different immune states as symptoms develop. This award will provide Dr. Paul the research opportunity and career development resources to continue to excel and establish herself as an independent investigator and leader in her field, conducting innovative research, combining systems biology, omic technologies, and population-based research.

NIH Research Projects · FY 2025 · 2021-05

PROJECT SUMMARY For millions with movement disorders including paralysis and ALS, intracortical brain-machine interfaces (BMIs) are an emerging technology that aims to restore lost motor function and communication. The main component of a BMI is a decoder algorithm that translates neural activity from motor areas of the brain into the kinematics of a prosthetic device. Due to the complexity of these systems, which includes the BMI user interacting with the decoded kinematics in a closed-feedback loop, current technology requires expensive and invasive experiments to design, optimize, and validate decoder algorithms. The need for such experiments (1) results in slow develop- ment and evaluation of decoder algorithms, and (2) limits the scope of people who can work on these problems to a small group of nonhuman primate and clinical trial labs. As a consequence, BMIs have remained in pilot clinical trials since their first reported demonstration in 2004. We propose a new open-source simulator for multiple degree-of-freedom (DOF) BMI systems. The goals of this simulator are to (1) reduce the time it takes to evaluate and optimize BMI algorithms from months to minutes, and (2) significantly expand the community of researchers who develop testable algorithms for BMIs. To build the simulator, we propose neural encoding models that generate synthetic motor cortical activity for multiple DOF tasks. This is possible because neural population activity is relatively low-dimensional and has dynamics, which can be learned via recurrent neural networks (RNNs). We build our neural simulators using data collected from human clinical trials during point-to-point multi-DOF reaches. We also propose to develop new models of human controllers. This solves an important problem in BMIs: users learn new control strategies when controlling a particular BMI decoder algorithm. Our simulator uses deep imitation and reinforcement learning to solve this problem. It is constrained through imitation learning to perform actions like a human. It is optimized through reinforcement learning to explore new strategies – under the constraint of being human-like – to optimally control the BMI. Together, we expect these innovations will result in a purely software simulator that accurately predicts BMI performance and enables design and optimization. This tool will be open-sourced and available to all, enabling widespread development of BMIs.

NIH Research Projects · FY 2025 · 2021-05

PROJECT SUMMARY Current ideas that systemic factors regulate the aging of cells and tissues, in terms of both promoting and reversing the aging process, have emerged from studies of heterochronic parabiosis (HP) and heterochronic blood exchange (HBE) protocols. Our lab performed the first HP studies in order to assess cellular aging and rejuvenation as it relates to tissue homeostasis and repair and in terms of molecular determinants of cellular age. Initially, we focused changes in muscle stem cells (MuSCs), but we and others later explored these phenotypic changes in many different cell populations. Since then, we have explored the molecular mechanisms by which systemic factors might influence the aging process. Based on numerous lines of evidence, we have hypothesized that the alteration in cellular aging features is due to a form of epigenetic reprogramming that is akin to that which occurs during induced pluripotent stem cell generation but without the loss of cellular differentiation. A general feature of cellular aging is the loss of heterochromatin and subsequent dysregulation of transcriptional stability. Heterochromatin is prominently associated with specific histone modifications, most notably by di- and tri-methylation of lysine 9 on histone 3 (H3K9me2 and H3K9me3, respectively). In Preliminary Studies, we have shown the H3K9me3 and heterochromatin can be regulated in MuSCs, with loss of both leading to features seen in aging cells. Based on these data and related published findings, the primary hypothesis of this proposal is that a primary mediator of HP and HBE transposition of aging phenotypes to young and old cells is the regulation of the cellular epigenome, and with a specific focus on H3K9 methylation and heterochromatin formation. To test this hypothesis, this proposal is divided into three Specific Aims. Aim 1: To assess the transcriptional and epigenetic signature of MuSCs in response to HP. We will establish HP and control pairs, and we will assess the MuSC molecular signatures compared to control mice in assays of the transcriptome and the epigenome. Aim 2: To examine the epigenetic mechanisms underlying the transposition of aging phenotypes in MuSCs. Using genetic and pharmacologic tools, we will modulate H3K9 methylation in young and old MuSCs exposed to young or old serum in vitro. We will test for the effect of changes in H3K9 methylation on the phenotypic changes of MuSCs previously described in response to heterochronic serum exposure. Aim 3: To test in vivo for the essential roles of H3K9 methyltransferase and demethylase activities in mediating the effects of HP on MuSCs. We will use genetic and pharmacologic tools to modulate H3K9 methylation in MuSCs in vivo. We will assess epigenetic and heterochromatin status, as well as the functional changes in MuSCs in response to HP. Together, these studies will elucidate molecular mechanisms of epigenetic programming of age in response to HP and HBE.

NIH Research Projects · FY 2026 · 2021-05

Project Summary The proposed research addresses critical issues of high translational importance concerning the mechanisms and outcomes of partial dysfunction of the vestibular sensory epithelia, referred to as peripheral vestibular hypofunction. The research plan utilizes chemotoxin- induced hypofunction, the foundation for which was identified through recent work from the PI’s laboratory in an animal model enabling precise intraperilymphatic dosing resulting in the production of highly reproducible lesions. This provides the basis for producing lesions of graded magnitudes within the sensory neuroepithelia, documented through histopathologic analyses. The physiologic outcome of these lesions will be evaluated through recordings of single afferent neuron electrophysiology and the vestibulo-ocular reflex, providing the bases for establishing histologic and physiologic correlates to a direct behavioral test of vestibular function. Previous work has demonstrated that the afferent neuron calyx is highly labile to pathologic compromise, and owing to its important contribution to shaping neural dynamics in untreated epithelia it is a focus for assessing pathologic damage. The present research plan will enable the direct correlate of afferent discharge dynamics to critical cellular components of the calyx, including its morphology and expression of KCNQ4 and sodium-potassium ATPase. In addition, we will examine the distribution of synaptic ribbons within hair cells of lesioned epithelia, testing whether a systematic synaptopathy also contributes to the compromised vestibular function. In summary, the present investigation provides critical insight into the histopathologic substrates of vestibular hypofunction and the alterations in sensory coding that underlies the functional compromise. At the same time, however, this investigation will reveal important cellular and physiologic metrics that are required for normal vestibular function, addressing longstanding question in vestibular neurobiology.

NIH Research Projects · FY 2026 · 2021-04

PROJECT SUMMARY/ABSTRACT This proposal targets the design, development and distribution of Bayesian statistical methods and software to study the historical and real-time emergence of rapidly evolving pathogens, such as Dengue, Ebola, hepatitis B, HIV, influenza, mpox, SARS-CoV-2, and yellow fever viruses. The proposal exploits novel versatile and scalable modeling and inference techniques to equip us for large-scale epidemics and pandemics and address some of the pressing and long-standing questions in viral epidemiology. Our multidisciplinary team carries expertise across statistical thinking, data science, evolutionary biology and infectious diseases to leverage advancing se- quencing technology and high-throughput biological experimentation that can characterize 10,000s of pathogen genomes, phenotype measurements or covariates, including sampled geographic, epidemiologic, ecologic and clinical information, from a single outbreak, to help inform actionable public health policy. Our chief innovations are three-fold. First, we will invent versatile phylogenetic and phylodynamic models to better understand com- plex biological heterogeneity in the emergence and spread of rapidly evolving pathogens within and across host reservoirs. Second, we will foster scalable phylodynamic techniques to more accurately learn the dynamics of pathogen transmission between infected individuals and the effects of population structure. Third, we will de- sign markedly more time- and energy-efficient Bayesian phylogenetic software that exploits recent advances in artificial intelligence (AI) techniques and massively parallel computing. Although no alternative implementations exist for the phylogenetic, phylogeographic and phylodynamic models we are developing at this scale, we will compare restricted cases of our models with reduced datasets to current state-of-the-art approaches to evaluate computational performance improvement and statistical bias that these limitations inject using real-world exam- ples. We will demonstrate the value of our developments through high-impact applications across a network of on-going collaborations. These include testing for seasonal persistence of yellow fever virus and improving deep history reconstruction of hepatitis B through novel time-inhomogeneous evolutionary models, and measur- ing the impact of control strategies on Dengue virus and identifying the ecological drivers of highly-pathogenic avian influenza spread while mitigating persistent sampling bias. This proposal will deliver user-friendly software through a leap-forward version of the popular BEAST platform for deployment across a rapidly expanding range of large-scale problems in the molecular epidemiology of infectious disease.

NIH Research Projects · FY 2025 · 2021-04

PROJECT SUMMARY/ABSTRACT Sensory over-responsivity (SOR) is an impairing condition manifested as extreme sensitivity to stimuli such as loud noises or being touched. SOR is present across neurodevelopmental disorders, but is particularly prevalent in youth with autism spectrum disorders (ASD) with rates of at least 56-70%. SOR is a fundamental limitation to individuals’ ability to participate in the community, succeed in school, complete daily living tasks, and interact socially. Despite this, there are almost no empirically-based treatments for SOR, in part due to the lack of understanding of its underlying biological mechanisms. Furthermore, while SOR tends to decline across adolescence into adulthood, there is little understanding as to why and for whom it improves. Thus, the primary goals of this study are to identify the developmental course of SOR as well as neurobiological mechanisms through which it can be attenuated, both essential to developing interventions. Our team has conducted some of the first studies identifying key neural mechanisms of SOR across youth with ASD, including 1) over-reactive brain responses/reduced habituation in primary sensory cortices and amygdala, 2) reduced thalamic GABA, and 3) reduced prefrontal cortex (PFC)-amygdala functional connectivity during aversive sensory stimulation. Our prior studies also indicate that the subset of youth with ASD but low SOR show heightened amygdala-prefrontal connectivity during sensory stimulation, suggesting a mechanism for resilience against SOR. Our preliminary data also suggest that amygdala reactivity to aversive sensory stimulation declines with age while PFC activation increases. This proposal seeks to build on this foundation by examining biological mechanisms through which SOR may be attenuated either through natural development or through direct intervention, with the goal of proximate translation to treatment. Using a combined cross-sectional and longitudinal design, we will examine: 1) developmental changes in sensory reactivity in ASD compared to typically developing (TD) children; 2) developmental changes in two candidate neural mechanisms of sensory regulation (thalamic GABA and PFC- amygdala connectivity), and 3) the relative ability of two different emotion regulation strategies (attention cuing vs. reappraisal) in engaging sensory regulation. Based on our prior studies and preliminary data, we expect to see that behavioral and neural markers of SOR decrease with age, but that this decline happens later than is typical for youth with ASD, indicating a developmental delay in sensory regulation. We further expect that of our two candidate top-down mechanisms of sensory regulation that prefrontal-amygdala connectivity but not thalamic GABA will improve with development, which will inform our understanding of why SOR decreases with age and how best to treat it at different stages of development. Finally, we will compare the relative ability of attention cuing vs. reappraisal to engage PFC for youth of different ages and SOR severity. Results will directly inform both behavioral and psychopharmacological personalized interventions for SOR.

NIH Research Projects · FY 2025 · 2021-04

ABSTRACT Obesity is a major public health problem that is related to a variety of illnesses, such as heart disease and diabetes. Prior research indicates that social stressors contribute to risk for obesity, possibly through alterations in diet and physical activity. However, it is not fully clear how these alterations contribute to obesity. In this study, we examine how the stressors of social isolation and discrimination are related to eating behaviors and dietary patterns, and further, how these behaviors affect the brain-gut-microbiome (BGM) axis. Our prior research has suggested that BGM alterations play an important role in the development of obesity. Our study focuses on Mexican and Filipina women because research shows that they encounter a high burden of obesity as well as exposure to social stressors. We will screen and enroll approximately 300 Mexican and Filipina women who will then provide information about social stressors via a survey, dietician-administered 24-hour food recall, measured anthropometrics (e.g. waist circumference), questionnaire data regarding diet and eating behaviors, and accelerometer data on physical activity. Stool and serum to determine microbial-related measures (16sRNA sequencing, shotgun metagenomics, and metabolomics), and MRI to assess brain alterations in the extended reward network will be collected. Advanced multivariate analytic techniques will be used to integrate data from multiple data sources (neuroimaging, microbiome and metabolite profiles, survey data, diet, and clinical/behavioral data). This analysis will determine the unique variance associated with ethnicity and social stressors in moderating eating behaviors and dietary patterns, and the BGM axis related to obesity. An integrated systems investigative approach such as the one proposed is a critical step to understanding the mechanisms contributing to obesity. Further the impact of how environmental stressors “get under the skin” and impact eating behaviors and diet patterns and the BGM axis is important in addressing health disparities in ethnic groups and in women. This premise guides the specific aims of this proposal: Aim A: We will phenotype the influence of stressors on brain signatures and eating patterns in obesity. Aim B: We will phenotype the influence of stressors on gut microbiome and metabolites in obesity. Aim C: We will apply advanced analytical techniques in order to determine ethnic differences in the influence of stressors on the BGM axis in obesity. The results of this study will provide novel information about a possible pathway whereby social stressors affect behavioral, neurological and microbiome mechanisms related to obesity risk. It will also provide new information in BGM patterns in two understudied ethnic groups. In the long term, this research may suggest possible approaches for intervention that may help reduce inequalities in obesity and related health problems.

NIH Research Projects · FY 2025 · 2021-04

Project summary A significant percentage of people in the US suffer from disabilities resulting from traumatic injury, stroke, or degenerative disease which result in enigmatic deficits in cognitive function. Therefore, an understanding of the fundamental properties of neuronal circuits for complex brain function, and how these circuits are modulated by biogenic amines, will sharpen our understanding of the normal brain, thereby highlighting pathology and facilitating treatments. A major function of the brain is to integrate information across sensory modalities to enable sharp and robust perception. The cell-circuit mechanism for how different sensory modalities interact is not well understood. This project will capitalize on the significant experimental advantages of the fruit fly Drosophila to explore the molecular logic and neural connections that produce an elementary form of multisensory integration perception. The fly displays robust multisensory perception, integrating olfactory signals with visual processing to enhance perceptual abilities. Furthermore, sensory circuits have been shown to be under robust neuromodulatory control by biogenic amines. Similar processes have been localized to sub- cortical and cortical pathways in humans and non-human primates. The fly has a numerically compact nervous system, with which highly advanced genetic techniques can be used to identify, manipulate, and record activity from individual neurons and neurosecretory cells, as well as their upstream and downstream synaptic partners and molecular components. The PI hypothesizes that the fly brain couples olfactory sensory detection to visual processing through neuromodulatory cells that carry the chemical equivalent of norepinephrine. The PI will perform two-photon Ca2+ imaging to ‘read’ activity from live flies in response to stimuli the PI has discovered elicit robust multi-modal integration behavior within a virtual reality system. The PI will use optogenetics imaging to ‘write’ signals into these circuit pathways to assess input-output functions from an intact behaving fly.

NIH Research Projects · FY 2025 · 2021-04

ABSTRACT Glioblastoma (GBM) is the most frequent and deadly primary brain tumor in adults; the median survival of patients with GBM remains a dismal 14-16 months with no improvement over standard of care since its introduction 15 years ago. Thus, identifying new therapeutic strategies for GBM is an urgent unmet medical need. Significant evidence indicates that, similar to other cancers, GBM have reprogrammed metabolism to support the requisite demands to fuel malignant growth and survival. Notably, work from our group and others has demonstrated a link between specific genetic alterations (e.g., EGFR) in GBM and rewired metabolism, consequently revealing nodes of therapeutic intervention to exploit GBM metabolism. However, comprehensive molecular profiling has shown that there is considerable molecular heterogeneity among GBM patients, and an unbiased investigation into how this molecular diversity in GBM shapes the metabolome has yet to be conducted. In preliminary studies, we have used integrated next-generation sequencing (RNA and Exome Seq) together with large-scale “shotgun” lipidomics from over 50 GBM patient and patient-derived samples to determine if molecular heterogeneity influences the lipidome of GBM. Using this cutting edge, systems-level approach we have identified a unique lipid signature enriched in GBM tumors with deletion of the tumor suppressor, CDKN2A: the most frequently altered driver gene in GBM. Importantly, as an apparent consequence of this specific lipid enrichment, CDKN2A null GBM demonstrate selective susceptibility to ferroptosis – a recently described form of lipid-peroxidation dependent cell death. These exciting preliminary results have led to the following aims both to determine the underlying mechanistic basis for these observations and to evaluate the therapeutic potential of inducing ferroptosis in CDKN2A-deleted GBM mouse models. In Aim 1, stable isotope-labeled metabolic tracing and metabolic flux analysis will be conducted to evaluate how the loss of CDKN2A elicits a shift in fatty acid composition relative to CDKN2A WT GBM. Aim 2 investigates the molecular pathways underlying enhanced sensitivity to ferroptosis in CDNK2A null GBM. Finally, Aim 3 will assess whether the exploitation of ferroptosis can selectively inhibit growth of CDKN2A null patient-derived orthotopic GBM xenografts. Together, the proposed studies will provide mechanistic insight into a previously unappreciated link between a common genetic alteration in GBM (CDKN2A deletion) and composition of the GBM lipidome, and evaluate the therapeutic potential of ferroptosis in controlling growth of this genetically-defined subset of GBM tumors.

NIH Research Projects · FY 2025 · 2021-04

ABSTRACT HIV continues to be a global health concern that has claimed the lives of millions. Although anti-retroviral therapy (ART) slows disease progression, ART is not curative due to certain reservoirs of replication-competent virus that persist during therapy. Therefore, if ART is stopped, then virus can emerge from these reservoirs and rapidly spread, causing renewed progression towards AIDS. In addition, life-long use of ART is associated with issues related to cost, medical compliance, and adverse drug events. One strategy for clearing the reservoir of latently infected cells is to use a kick and kill approach, in which latent cells are “kicked” or activated from latency, and then concurrently cleared or “killed”. Latency reversal agents (LRA) can “kick” or induce HIV expression from latent cells, but thus far only a subset of activated latent cells die. Natural killer (NK) cells hold great promise as killing agents for HIV-infected cells as they re-emerge from latency due to their innate anti-viral recognition and cytotoxic function. The goal of this research project is to develop new methods to enhance the intrinsic killing activity of NK cells and to develop NK cell-based kick and kill strategies to reduce the need for life-long ART by decreasing or eliminating latent viral reservoirs. We intend to approach this proposal by using cutting-edge technology to engineer the enhanced survival and anti-viral function of NK cells, sophisticated humanized mouse models of HIV latency, and innovative tools to measure and study the effect of our treatments on the HIV reservoir. We will test our overall hypothesis that a kick and kill approach will decrease or eliminate the latent reservoir in the following aims: 1) engineer NK cells to enhance their elimination of HIV-infected cells using an innovative non-viral mRNA transfection technology, and 2) investigate the effect of novel latency reversal agents (LRAs) in combination with modified NK cells on HIV reservoirs in a humanized mouse model of HIV latency. This proposal utilizes Dr. Jerome Zack's (lead PI, UCLA) extensive background in HIV latency and animal modeling, Dr. Catherine Blish's (dual-PI, Stanford) expertise in NK cell immunobiology and cellular manipulation, and includes a unique collaboration with Dr. Paul Wender (Stanford), an expert in chemical synthesis, who has developed a globally unique library of latency reactivating agents (LRAs) with unprecedented latency reversal capabilities and expanded tolerability that will be tested individually and in synergistic combinations with NK cells. Together we hope to fully harness the potential of NK cellular therapies, and develop LRA and NK cell combination therapeutic approaches to provide patients with sustained virologic remissions or complete viral eradication.

NIH Research Projects · FY 2025 · 2021-04

PROJECT ABSTRACT The K01 Mentored Research Scientist Development Award will provide Dr. Michael Li with invaluable research experience, mentored training from interdisciplinary faculty, and training activities in a combination of behavioral and basic sciences, which will prepare him well in his career as a biobehavioral researcher in addiction medicine and HIV treatment/prevention. This K01 mechanism will support Dr. Li’s research and training efforts to develop expertise in the following areas: (1) neurally regulated “stress” gene expression markers and links to addiction and HIV disease progression; (2) cultural competence and ethical conduct; (3) technical assay and substantive analytic methods in gene expression research; (4) clinical trial methods; and (5) professional development. Dr. Li has assembled an interdisciplinary mentorship team who will support key aspects of his training and research. Dr. Steven Shoptaw is a highly productive and influential researcher in addiction medicine, and he has an extensive track-record mentoring people who later became successful independent researchers. Co-mentor Dr. Steven Cole has pioneered the field of social genomics, and will direct Dr. Li’s training in transcriptomic methods. Dr. Jesse Clark will guide Dr. Li in clinical trial operations and safety procedures, and Dr. Thomas Belin will provide extensive mentoring in advanced statistical methods and inferential frameworks in clinical trials. Dr. Li proposes to investigate whether a neurally regulated “stress” gene expression pattern can serve as a clinically meaningful, non-abstinence-based endpoint for contingency management for methamphetamine (METH) use disorder (MUD) in MSM living with HIV. Abstinence determined by urine testing has been the only standard clinical outcome for MUD treatment, but provides an incomplete picture of patient recovery. The gene expression pattern called the conserved transcription response to adversity (CTRA) may provide insight into changes in both psychosocial health and pathogenesis over the course of MUD treatment. The CTRA is marked by upregulated expression of pro-inflammatory genes and downregulated expression of Type I interferon genes in response to negative psychosocial experiences such as depression, anxiety, and violence, problems also comorbid with METH use. The CTRA also involves some of same gene regulatory pathways that contribute to METH-related pathogenesis, such as those involving inflammation and innate antiviral responses (relevant to PLWH). My proposed research will use a two-arm clinical trial design (N=55) with 35 HIV-positive MSM with MUD receiving contingency management for METH reduction, and 20 HIV-positive MSM who qualify as a non-substance-using control to accomplish the following aims: 1) to investigate whether CTRA gene expression coincides with METH use and viral load; 2) to investigate whether psychosocial indicators of addiction are associated with CTRA; and 3) to conduct an exploratory pilot investigation to determine the degree to which CTRA mediates the association between METH use and viral load. Together, this K01 research project and training plan will play a fundamental role in my early success as an independent substance use and HIV researcher.

NIH Research Projects · FY 2025 · 2021-04

Stem cells are responsible for homeostasis and repair of many tissues in the body, and stem cell exhaustion is one of the hallmarks of aging. In recent years, work from our group and others has drawn attention to the mechanisms by which the resilience of muscle stem cells (MuSCs) declines with age at the population level and at the single cell level. As one example, we have shown that a signaling pathway involving Notch activation and increased p53 activity prevents MuSCs from undergoing a form of cell death, mitotic catastrophe, as they activated out of quiescence and enter the cell cycle. This Notch/p53 axis declines with age and leads to an increased propensity of aged MuSCs to undergo mitotic catastrophe, leading to a decline in MuSCs over time. Furthermore, in preliminary studies, we have found that quiescent MuSCs exhibit evidence of replicative stress and that an ATR response to that stress prevents cell cycle entry and preserves the MuSC population. We have also found that dietary interventions, in particular fasting and a ketogenic diet, enhance MuSC resilience, perhaps mediated by HDAC activity and p53 acetylation. Together, these observations highlight robust processes to maintain MuSC resilience and prevent stem cell depletion, processes that go awry during the aging process. The primary goals of this proposal are to explore these processes in more detail, to identify the molecular mediators of each, to use unbiased screens to identify as yet unknown mediators, and to pursue rejuvenating interventions that restore resiliency to aged MuSCs. To address these issues, this proposal is divided into three Specific Aims. Aim 1: To examine changes of the Notch/p53 axis as a cause of the age-related reduction of MuSC resilience. We will use novel genetic models to modulate Notch signaling in MuSCs and test for resilience signatures of cells protected against mitotic catastrophe. We will also assess resilient cells for evidence of mediators downstream of p53 using single cell RNA-seq. Aim 2: To examine replicative stress and the ATR response in young and old MuSCs. We will examine a potential downstream mediator of ATR, CDK12, identified in a phosphoproteomic screen, in preserving resilience of the population. We will also test whether this replicate stress response pathway changes with age and protect MuSCs from undergoing mitotic catastrophe when they activate out of quiescence. Aim 3: To elucidate the mechanisms by which ketosis promotes MuSC resilience. We will test for enhancement of resilience using three different ketosis-inducing interventions, and we will test for mechanisms of action based on the well- documented role of the major circulating ketone body, beta-hydroxybutyrate (βHB), as an inhibitor of histone deacetylases (HDACs). We will also test whether ketosis enhances MuSC resilience, at least in part, by promoting p53 activity and preventing mitotic catastrophe. Together, these studies will advance our understanding of the mechanisms of stem cell resiliency and how to enhance the resilience of aged stem cells to promote tissue homeostasis and repair across the lifespan.

NIH Research Projects · FY 2025 · 2021-04

PROJECT SUMMARY/ABSTRACT Our recent discovery of orally brain permeable small molecules that mimic the bioactivity of Humanin (HN) peptide to enhance and normalize neuronal phospho-Akt (p-Akt) levels provides a unique opportunity for evaluating this new approach in Alzheimer's disease (AD). In this proposal, we will direct our efforts to optimize this new class of agents, focusing on screening additional hits, enhancing their potency, drug-like properties, solubility, oral brain permeability and efficacy in an AD model towards development of this novel therapeutic approach for AD. We will also use modeling to identify HN based peptidomimetics for testing. Our data show that these small molecule HN mimetics, like HN, can suppress neuronal death through its activation of the gp130 receptor and signaling via the PI3/Akt pathway and provide neuroprotection for primary hippocampal neurons against N-methyl D-aspartate (NMDA) and Aβ-induced neurotoxicity. HN is a naturally occurring mitochondrial-derived brain peptide that decreases with age and may act as a neuroprotective factor against AD-relevant neurotoxicity. Treatment of hippocampal neurons with our HN mimetic compound 2 resulted in an increase in p-Akt, and this correlated to its observed neuroprotective effects. In AD patients, a significant decrease in p-Akt has been reported. Similarly, in aged apolipoprotein E4 (ApoE4) mice, there is a significant decrease in p-Akt in the brain relative to age-matched ApoE3 mice suggesting that PI3/Akt signaling is affected by ApoE4, a risk factor in AD. Activation of PI3/Akt signaling can transcriptionally modulate genes related to memory such as choline acetyltransferase (ChAT) and vesicular acetylcholine transporter (VAChT) and may also regulate postsynaptic proteins involved in neuroplasticity. AD is the most prevalent age-related dementia, currently afflicting more than 5.4 million people in the US. Given the urgent need for new therapeutic approaches for AD, these HN mimetics could provide promising lead candidates for therapeutic development. In Aim 1, we plan to evaluate small molecule HN mimetics and peptidomimetics for activation of gp130 and normalization of p-Akt along with their neuroprotection against Aβ and NMDA induced neurotoxicity. In Aim 2, we would conduct a design and synthesis campaign using current SAR and new docking/modeling data to identify small mimetics, peptides and peptidomimetics. We will optimize potency, drug-like properties, solubility and oral brain bioavailability for efficacy testing. The best analogs/peptidomimetics from Aims 1 and 2 will undergo in vitro ADMET profiling and pharmacokinetic (PK) studies, along with phosphoproteomics analyses, in Aim 3 to prioritize the optimal compounds for in vivo efficacy testing in the ApoE4(TR):5XFAD murine model of AD as part of Aim 4. The goal is to identify orally available HN-mimetics that enhance/normalize brain p-Akt levels and improve cognition. Like HN itself, they could also have broader therapeutic applications in traumatic brain injury (TBI), stroke, Aβ-induced cerebrovascular dementia, amyotrophic lateral sclerosis (ALS) and could lead to a new class of preclinical candidates for AD.

NIH Research Projects · FY 2025 · 2021-04

Abstract Mitochondrial dysfunction is an important causal factor in normal aging, and aging-related diseases such as neurodegenerative disorders, Alzheimer's disease, cardiac disease, cancer, diabetes and muscle sarcopenia. Age is the strongest risk factor of Alzheimer’s disease, and the prevalence of Alzheimer’s disease doubles every 5 years in populations over the age of 65. The goal of this K07 Academic Career Leadership Award is to develop a world-class Center in Aging and Mitochondrial Health at UCLA to foster research collaborations, and to educate and train the next generation of leaders in this important field. The proposed Center will be the first of its kind at UCLA and will be led by Dr. Ming Guo. Dr. Guo is an established physician-scientist. She is a clinical specialist on dementia and cognitive impairment, who also investigates roles for mitochondria in aging and neurodegenerative diseases including Alzheimer’s and Parkinson’s disease in research realm. The Center's goals will be addressed in three aims: 1) to develop core resources to attract interdisciplinary investigators to engage in aging-related research and to enhance collaborative research projects, including a major effort in Alzheimer’s disease research; 2) to create a comprehensive interdisciplinary training and mentorship program in aging and mitochondrial biology; and 3) to serve as a local and national resource for research on mitochondrial biology in aging. The long-term goals of the Center will be to obtain extramural funding for multi-investigator driven proposals, as well as career development faculty grants and an institutional training grant to sustain the training of postdoctoral scholars and geriatric research fellows. Thus the Center will be a new initiative for UCLA and will be a beacon of excellence for investigators pursuing research into mitochondrial biology, aging and diseases of aging, with an emphasis on Alzheimer’s disease.

NIH Research Projects · FY 2025 · 2021-04

PROJECT SUMMARY/ABSTRACT Emerging viruses such as severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) have devastated the world. We are desperately in need of broadly acting antivirals that can curb the spread of the next emerging virus. However, a major knowledge gap exists due to our incomplete understanding of the cellular pathways that control or facilitate viral replication. Type I interferon (IFN) is the principal host innate immune response to viral infection and regulates the expression of an array of IFN-stimulated genes (ISGs). Zinc finger antiviral protein (ZAP) is one of the most potent ISGs that blocks the replication of diverse RNA and DNA viruses. ZAP attenuates alphavirus production by up to 8 logs and is indispensable for IFN efficacy against alphaviruses. Moreover, increased virulence of human immunodeficiency virus 1 (HIV-1) and SARS-CoV-2 associates with viral evasion from ZAP recognition. Importantly, highly pathogenic alphaviruses such as chikungunya virus (CHIKV) have developed resistance to ZAP, highlighting ZAP as a critical driver of viral pathogenesis. ZAP is proposed to act through viral translational inhibition (alphavirus and flavivirus) and viral RNA degradation (other viruses), however, how ZAP blocks viral replication and how viruses evade from ZAP recognition are still poorly understood. We recently made two important advances towards this goal: First, we showed that ZAP viral translational inhibition requires the host factor TRIM25, an E3 ubiquitin ligase. TRIM25-mediated ZAP antiviral mechanism is innovative, and independent of RIG-I, IRF3, and IFN. Using a novel “substrate trapping” approach we have now identified numerous cellular proteins with exciting roles in translation and RNA processes to be TRIM25 interactors and potential substrates. These preliminary findings connect the ubiquitination process to viral translational suppression by ZAP for the first time and provide cellular targets for antiviral therapy. Second, we identified differences in alphavirus sensitivity to ZAP. Highly virulent alphaviruses such as CHIKV that have infected millions of people in recent epidemics can evade from or counteract ZAP recognition through their viral non-structural gene region. Discovery of viral strategies for evasion and antagonism will identify druggable viral targets and inform development of vaccine strains with weakened ability to counteract ZAP. Taken together, our results have led us to hypothesize that ZAP recruits TRIM25 to ubiquitinate and modulate cellular factors leading to viral translational inhibition, and highly pathogenic alphaviruses have evolved strategies to escape and/or antagonize ZAP antiviral activity. We propose to elucidate the mechanism of TRIM25-mediated ZAP translational inhibition (Aim 1) and determine the mechanisms of viral resistance to ZAP (Aim 2). Success of these Aims will advance our understanding of how the IFN pathway co-opts cellular processes to block viral replication and drive viral pathogenesis, and provide promising host and viral targets for therapeutic intervention of alphavirus and other virus infections.

NIH Research Projects · FY 2025 · 2021-03

ABSTRACT Tuberculosis (TB), caused by Mycobacterium tuberculosis (Mtb), is one of the world's leading causes of death. BCG, the only licensed vaccine against TB, is an attenuated bacterium highly homologous to Mtb, yet safe in immunocompetent individuals because it has lost several genes that confer virulence. BCG has good efficacy against TB in children, but poor efficacy against TB in adolescents and adults. Hence, a vaccine much more potent than BCG is clearly needed. However, any replacement vaccine will almost certainly need to be based on modified (e.g. recombinant) BCG or attenuated Mtb to preserve the substantial benefits of BCG. The goal of this project is to develop an attenuated Mtb mutant that is safer and more potent than BCG. Our novel strategy involves manipulating two key characteristics of live vaccines: (1) their initial period of growth in the host and (2) their rate of elimination. The inadequate protective efficacy induced by BCG and non-replicating Mtb mutants can be attributed, at least in part, to their lack of replication in the host. Prolonged persistence in the host is also a negative factor, resulting in the generation of primarily effector and effector memory T cells rather than central memory T cells, important for long-term immunity. We hypothesize that limited replication of an Mtb mutant for a brief period after immunization, mimicking the early stage of a natural Mtb infection, followed by rapid clearance will induce a potent immune response and yet avoid the negative inflammatory responses induced by prolonged Mtb infection. To achieve our goal, we first shall engineer an attenuated Mtb mutant defective in both of its iron acquisi- tion pathways - siderophore-mediated iron acquisition (SMIA) and heme-iron acquisition (HIA). Such a mutant will be unable to obtain iron from the host but can be pre-loaded in vitro with the precise amount of iron to allow optimal replication in the host. Thus, an Mtb ∆SMIA ∆HIA mutant will allow us to address the first important factor - controlling the extent of replication in the host. While growth of Mtb ∆SMIA ∆HIA in the host will cease once it exhausts its supply of iron, the organism may persist for a prolonged period. Thus, to address the second important factor, the rate of clearance from the host, we shall further modify Mtb ∆SMIA ∆HIA, via two approaches – 1) knocking out persistence genes and 2) conditional silencing of essential genes. While both should result in improved clearance, conditional silencing likely will result in faster clearance. We shall vaccin- ate mice with persistence and conditional silencing mutants and perform clearance and protective efficacy studies to determine the optimal replication and clearance. We expect a replication- and persistence-limited Mtb mutant with rapid clearance will be much more efficacious than BCG and, in contrast to BCG, safe even in an immunocompromised host. Once we have optimized the vaccine for protective immunity in mice, we shall examine its immunogenicity in mice to assess preliminary correlates of protection, assess its safety in immuno- compromised SCID mice, and examine its safety and efficacy in a second animal model of TB - guinea pigs.

NIH Research Projects · FY 2025 · 2021-03

PROJECT SUMMARY The proposed project quantitatively and qualitatively investigates and disseminates the mechanisms underlying potential racial/ethnic and sex differences in risk for substance use disorder (SUD) and disparities in SUD treatment services among justice-involved adolescents (JIA). JIA are a critically underserved population who are especially vulnerable to SUD. Certain ethnic groups and females in the juvenile justice system are predisposed to harsher consequences of substance misuse and are more prone to suffer from undiagnosed SUDs. The cascade of care is a novel framework for investigating unmet treatment needs. The stress process model hypothesizes that disparities occur due to unequal distribution of stressors and resources, offering a major pathway linking race/ethnicity and sex to disparities at the cascade checkpoints. The study will synergize transdisciplinary theory and methodology through integrating a) a sociological theory of health disparities—the stress process model, b) a novel framework for measuring unmet treatment needs–the cascade of care, c) advanced quantitative methods –mediation analysis in structural equation modeling, and d) innovatory qualitative approaches–hybrid thematic analysis with cutting-edge technologies. The specific research aims are to 1) investigate if stress and resources mediate racial/ethnic and sex differences in substance misuse (SM) patterns; 2) test if stress and resources mediate ethnic and sex disparities in referral to SUD screening, diagnosis with SUD, treatment initiation and treatment completion; and 3) qualitatively explore how stress and resources relate to ethnic and sex disparities in SUD services and post-release outcomes. The study employs mediation analysis in structural equation modeling to analyze a statewide longitudinal database of 100,000 JIA from the third largest juvenile justice population in the United States. To uncover deeper insights into the statistical findings, the project conducts and analyzes in-depth interviews and surveys among 60 individuals who experienced SM and incarceration as minors. Hybrid thematic analysis will be employed to fuse the quantitative and qualitative data. These aims serve to fulfil NIDA’s mission to advance science on the etiology of SM, improve SUD prevention and treatment, and advance research on minorities, females, and other populations facing disproportionate consequences of misuse. To complete the research aims, an extensive training plan directed by a network of expert mentors who are leaders in the field will be executed. The training aims are to develop expertise in SUD services delivery in criminal justice systems; mediation analysis in SEM, psychometrics, censored data, and longitudinal design; interviewing methods in health research and analysis with the ATLAS.ti software; stress instrumentation; and implementation and translational science. These training and subsequent research activities are a major step towards developing and testing culturally appropriate, sex-responsive, and trauma-informed SUD risk assessment protocols and disparity reduction interventions that innovate SUD screening and treatment delivery in juvenile justice systems.