University Of Southern California

NIH Research Projects · FY 2025 · 2022-01

Project Description 1 Introduction More than any other phenomena in recent history, the COVID-19 pandemic has challenged how we approach patient-care due to the huge burden it has placed on hospitals, clinics, and health professionals. The health community has responded to this trend with research and technology leveraging data that goes beyond what is customarily thought of as “health data”, such as commu- nity and contextual data, social media, traffic, and mobility data. For example, Nsoesie et al.[84] analyzed hospital traffic and search engine data in Wuhan to infer early disease activity in Fall 2019. These new efforts, including our own work in utilizing mobility data to forecast COVID- 19’s transmission risk [94], uses what this NSF call-for-proposal refers to as “non-traditional health data”. In this proposal, we focus on one specific type of non-traditional health data, wearable data, which are also fast becoming an important source of health and disease data as they inform on a variety of personal, behavioral, social, contextual, and environmental health-relevant factors. Wearables have been primarily used for activity tracking [96, 15, 20, 80] and gained popularity with fitness applications; however, more recently, these devices have been used in an increasing number of health applications, including health monitoring, clinical-care, remote clinical-trials, drug delivery, and disease characterization to name a few. In fact, wearables have been found useful in a num- ber of applications and diseases (e.g., Parkinson’s disease, epilepsy and stroke [57], sleep disor- ders [12], cardiac disorders [90, 63] and cancer [75]). This trend is accelerating with the COVID-19 epidemic, e.g., smartphones have been proposed to track symptoms [64], monitor effectiveness of non-pharmaceutical interventions, assess potential spread, and support contact tracing [45]. Wearable measurements differ from traditional clinical measurements. When a patient visits a clinic, vitals and lab tests are collected in a “controlled” environment in a short duration of time using multiple devices. We define this monitoring in the controlled environment as Snapshot In-Clinic monitoring, abbreviated as SIC. Meanwhile, the recent growth and accessibility of the wearable devices such as smartphones and watches [97] with embedded activity and mobile sensors [114] enables the continuous monitoring of patients’ vital signs and other health indicators over a long duration of time. Patient monitoring using wearable devices typically happens in an “uncontrolled” setup at home or at work in a non-intrusive fashion with only a few sensors. This trend has also been encapsulated by the NIH mHealth’s initiatives, resulting in the evolution of new healthcare models such as “home healthcare” [9, 40] and “minute clinic” [125], which goes hand in hand with both ubiquitous sensors in smartphones and custom sensors like glucose monitors [62]. We define this monitoring in the uncontrolled environment as Longitudinal In-Field monitoring, abbreviated as LIFE. Clearly these are wordplay, i.e., SIC is for “sick” capturing patients’ state of mind when they visit a clinic/hospital vs. LIFE for when patients live their normal “life” at home and at work. LIFE monitoring makes up for greater than 99% of patients’ time, enabling outpatient monitoring of the effects of disease and its therapy on patient performance and quality of life. In fact, our preliminary data show that in some cases, such as assessment of performance status in cancer patients, LIFE data outperform in-office SIC assessments [82]. SIC monitoring is the current standard of care and is driven by improving outcomes in measurable Page 72

NIH Research Projects · FY 2025 · 2021-12

Abstract The long-term goal of this project is to develop better therapies for respiratory inflammation and allergic asthma. Group 2 innate lymphoid cells (ILC2) are a recently identified cell population producing type 2 cytokines in response to a growing number of environmental signals and epithelial cell-derived cytokines. Studies show increased ILC2 activity in asthma and many widespread diseases, as ILC2s are sufficient to induce airway inflammation independent of adaptive immunity in mice. The proposed research plan is motivated by recent new observations from our laboratory and others that the TNF/TNFR2 axis controls ILC2- dependent airway inflammation (Cell Report 2019, J Allergy Clin Immunol. 2020). High levels of TNF are found in the lungs of asthmatic patients, however anti-TNF therapy is generally associated with systemic toxicity due to the existence of two distinct functionally different receptors for TNF: TNFR1 and TNFR2. Our results suggest that TNF enhanced the secretion of ILC2 effector cytokines IL-5 and IL-13 and increased survival via TNFR2 signaling, leading to airway inflammation. However, how the TNF/TNFR2 axis mechanistically affects ILC2s and subsequent development of airway inflammation remains to be explored. Based on our data, we hypothesize that blocking TNFR2 on ILC2s induces an immunoregulatory phenotype fueling on a distinct metabolic source, together favoring the reduction of AHR. In Specific Aim 1 (SA1), we have designed several approaches to characterize the effects of TNF on ILC2 effector functions and lung inflammation. Our preliminary data suggest that TNFR2 is heterogeneously expressed on activated ILC2s. We will therefore characterize the transcription factors driving the effects of TNF in ILC2s using a combination of single cell genomic and transcriptomic analysis. Furthermore, data from our laboratory and others suggest that metabolic processes in ILC2s are dependent on the generation of energy from fatty acid oxidation (FAO) and oxidative phosphorylation. Interestingly our results clearly show a metabolic shift towards glycolysis in TNFR2-/- ILC2s. Based on these results we intend to assess glycolysis and FAO mechanisms in WT and TNFR2-/- ILC2s in SA2. Finally, we previously showed that human ILC2s express TNFR2 and humanized ILC2 mice developed TNFR2-dependent AHR in response to TNF. Therefore, we intend to assess in the SA3 the relevance of our findings in asthmatic patients. We will collect lung and blood ILC2s from carefully selected cohorts of mild/moderate, severe asthmatics and healthy donors and correlate the levels of TNF in the BAL to the numbers of ILC2s/expression of TNFR2, as well as monitor other cells that express TNFR2. These studies, based on strong preliminary data, will focus on developing novel therapy for allergic asthma. In order to achieve these results we have assembled a team including leading experts in lung biology and the chief of clinical pulmonology to complement our extensive experience in pre-clinical models.

NIH Research Projects · FY 2026 · 2021-12

Near-roadway and regional air pollution, industrial releases, goods movement and growing oil and gas production in urban areas vulnerable to wildfires all threaten to increase the burden of environmental disease. In California and worldwide, these threats disproportionately affect children. Air pollution has adverse effects on childhood respiratory health, obesity and metabolic outcomes, and neurodevelopment. New children’s environmental health science (CEHS) translation is needed to develop and implement effective, science-based interventions to address these unfavorable trends. The mission of the Southern California Center for Children’s Environmental Health Translational Research (SC-CCEHTR) is to leverage scientific knowledge to reduce the burden of environmentally related diseases by developing: (1) multidisciplinary CEHS translational teams building an innovative framework for multidirectional, action-oriented engagement with communities, academia and stakeholders, and (2) model collaborations supporting junior investigators and communities to use emerging CEHS, leading to better decision-making. Accordingly, the theme of the SC-CCEHTR is Urbanism, Air Pollution and Children’s Health. The SC-CCEHTR will build on a foundation of a large CEHS grant base across three NIEHS Centers and of innovative multidirectional engagement with communities and stakeholders. The proposed SC-CCEHTR framework includes novel approaches to youth engagement and community science, urban design and public health interventions, and communication and public knowledge. Investigators new to CEHS from communication, urban design, sociology, dramatic arts, education, network analysis and implementation science will bring fresh approaches to this framework, focused on identifying solutions to urban air pollution by “re-imagining” the design of the city to reduce air pollution exposure and improve children’s health. A Translation Core will bring the SC-CCEHTR tools to bear on the development of pilot projects to better translate CEHS into community knowledge and action. A Developmental Core responds to career development needs of junior investigators and to emerging CEHS challenges. Innovative translational and career development collaborations will be promoted with the Moving Forward Network, the International Society for Children’s Health and the Environment, other Children's Environmental Health Research Translation Centers and NIEHS P30 Core Centers, and stakeholders across the country.

NIH Research Projects · FY 2025 · 2021-12

Estrogen-related receptor (ERR) plays critical roles in the transcriptional regulation of genes involved in mitochondrial bioenergetics, TCA cycle, mitochondrial oxidative phosphorylation, and fatty acid -oxidation. This project intends to discover the roles of ERR as novel transcriptional factor for lipid metabolism and its involvement in pathology development in the liver. To address the function of ERR in dyslipidemia, we developed a novel small molecule inhibitor (ERR-PA) that can block the binding of ERRs to the promoters of their target genes. Using this compound in diet and genetic models of liver steatosis (NAFLD/ALD) and steatohepatitis (NASH/ASH), our preliminary studies showed that inhibiting ERR robustly blocks the development of steatosis and reverses the lipid accumulation in models where liver steatosis is induced by diet and ethanol feeding as well as genetic alterations. ERR-PA also significantly reduced the fibrosis and inflammation occurring in established steatohepatitis. Using these in vivo as well as in vitro systems, we will explore the molecular mechanisms by which ERR inhibition suppresses the progression of liver disease. The hypothesis to be tested is that ERRs positively regulate transcription of genes encoding enzymes for anabolic lipid metabolism and inhibiting this action blocks steatosis and associated inflammation and fibrosis in NASH/ASH. We will address this hypothesis with the following three aims. Aim1 will investigate the regulation of liver lipid metabolism via the transcriptional activity of ERRs. This aim will explore the transcriptional complex by which ERR regulates de novo lipogenesis, glycerolipid biosynthesis and fatty acid -oxidation. Aim2 will determine the effect of ERR inhibition on oxidative lipid damage during ASH/NASH development. This aim will investigate lipid, oxidized lipid and other derivatives, ROS production and their contribution to liver damage. The proposed project explores ERR as a potential target for inhibiting and reversing fatty liver diseases. The mechanistic and translational approaches will uncover novel biology for lipid metabolism as well as test the therapeutic effect of a small molecule polyamide.

NIH Research Projects · FY 2025 · 2021-09

PROJECT SUMMARY Uveitis is an important cause of visual morbidity, responsible for an estimated 10% of blindness in the United States with an annual associated cost of $242.6 million. Prior studies have sought to elucidate the incidence and prevalence of uveitis; however, these studies have been limited in scope by age subset, localized geography, referral practice setting, or specialized health systems environment. The primary objectives of this K23 career development proposal are: 1) to demonstrate the benefit of using multiple databases to characterize the epidemiology of uveitis across the U.S., elucidating risk factors for its development, persistence, and progression; and 2) to provide an academic uveitis and vitreoretinal surgeon with the training and mentored research experience necessary to conduct independent clinical research. Achieving these objectives will provide critical skills and experiences necessary to establish an independent research program focused on applying clinical informatics to improve the clinical care of patients with uveitis. The proposed K23 application will provide additional training in four vital areas: 1) epidemiology and mechanisms of chronic disease; 2) quantitative characterization of treatment patterns and health economics; 3) biostatistical methods for health services research; 4) validation and analysis of very large clinical databases. The proposed research will capitalize on the strengths of two unique databases containing large cohorts of patients, including health claims-based data from the Optum Clinformatics Data Mart and electronic medical record-derived data from the Los Angeles County Department of Health Services safety net healthcare system, to generate real-world knowledge on the epidemiology and treatment of ocular inflammatory diseases. After validating longitudinal samples of patients with uveitis, the prevalence of uveitis will be estimated across distinct datasets. Risk factors for the development of uveitis and its sequelae will be incorporated in developing and testing models to investigate to what degree risk factors might affect health outcomes. Smoking status will be a particular factor of interest. Population-based treatment patterns and health care costs associated with non-infectious uveitis will be characterized. Treatment patterns in the utilization of steroid-sparing immunomodulatory therapy will be explored. The results of the proposed research will provide the foundation for a future longitudinal study examining the benefit of targeted interventions in patients at higher risk of blindness from uveitis. The ultimate goal of my research is to develop risk-stratification and treatment protocols that guide the standardized care of patients with uveitis and decrease the incidence of ocular inflammatory disease and its associated ocular morbidity.

NIH Research Projects · FY 2025 · 2021-09

PROJECT SUMMARY/ABSTRACT Down syndrome (DS) is the most common genetic cause of intellectual disability, with virtually all DS individuals having AD by age 60. Despite exciting advances over the past 20 years in our understanding of DS and how genetics confer susceptibility for AD, there have been few clinical trials to treat AD in the DS population (DS-AD) and currently no means to clinically impact disease progression. We hypothesize that the biological makeup of DS-AD is as heterogeneous as the sporadic AD population and that identifying specific subsets of DS-AD for particular treatments will yield meaningful therapeutic responses. We propose to test a validated biomarker for sporadic AD, our established proinflammatory endophenotype analysis. This analysis will allow for an understanding of differences in immune signaling between DS-AD individuals and how this may impact anti- inflammatory effects on AD biomarkers. For this study we will leverage biobanked samples from the previously completed Phase 3 clinical trial, “Vitamin E in Aged Persons with Down Syndrome (NCT00056329).” For our proposed work we will employ high throughput proteomics on native plasma and exosome subpopulations to validate new techniques and develop a framework for designing better therapeutic trials for DS-AD

NIH Research Projects · FY 2025 · 2021-09

PROJECT SUMMARY/ABSTRACT The MarkVCID consortium was established in 2016 to pursue the initial stages of multi-site validation of 7 imaging-based and 4 fluid-based candidate biomarkers for small vessel vascular contributions to cognitive impairment and dementia (VCID). As one of the 7 participating sites, our team at the University of Southern California (USC) has analyzed and optimized candidate VCID biomarkers during the UH2 phase (years 1-2), and participated in the consortium-wide program of biomarker scaling-up, multi-site protocol implementation, and initial multi-site validation during the UH3 phase (years 3-5). USC has led the consortium wide development and implementation of the optical coherence tomography angiography (OCTA) biomarker kit which is based on an FDA approved device that allows in vivo and completely noninvasive imaging of retinal capillaries with a spatial resolution of ~10 microns. We have also participated in the validation of the other 6 MRI based candidate biomarkers, while contributing blood samples for the validation of the 3 plasma-based candidate biomarkers. The primary objective of the next 5 years is to carry out comprehensive multi-site clinical validation of candidate VCID biomarkers in longitudinal studies of diverse populations that are typical in clinical settings in the US. Capitalizing on the high racial diversity in Los Angeles, we plan to enroll a multiethnic longitudinal cohort of at least 200 subjects that are enriched for small vessel VCID, including 100 Latinx subjects from the Los Angeles Latino Eye Study (LALES), 50 African Americans (AAs) enrolled in the African American Eye Disease Study (AFEDS), and 50 Caucasian participants from the Clinical Core of the USC ADRC. We have performed pilot studies in both Latinx and AA subjects (two largest minority groups in the US) using MarkVCID protocols to show the feasibility for conducting such studies in a multiethnic cohort. We have also built a multiethnic team of investigators, technical staff, research coordinators, and community outreach staff to streamline enrollment, achieve the recruitment milestones and perform longitudinal follow-up studies. Our team includes established expertise, equipment and infrastructure for OCTA, MRI, fluid biomarker and clinical/cognitive evaluation of VCID, as well as a proven track record in the sharing of de-identified clinical data through the Laboratory of Neuro Imaging (LONI) for the second phase of MarkVCID project.

NIH Research Projects · FY 2025 · 2021-09

Abstract Dementia is the progressive loss in memory and cognition of the brain. Alzheimer’s disease (AD) is the leading cause of dementia in those over the age of 65. Currently, there are ~5.6 million Americans age 65 and older have AD, and the projected AD patients will double by 2050. The social and economic burden of neurodegenerative diseases is enormous and no cure or prevention is available to date. Late onset AD accounts more than 98% of all AD cases and is a multifactorial disease, with aging being the most prominent risk factor. In addition to the genetic makeup of a patient, environmental factors, such as microbial infection, contribute significantly to the development and outcome of AD. Herpesviruses are ubiquitous in human and their infection is often asymptomatic in immune- competent individuals. Recent studies suggest a causal role of several herpesviruses, particularly herpes simplex virus 1 (HSV-1), in AD and other related dementia. How HSV-1 contributes to AD pathogenesis is not well understood. In studying host innate immunity against herpesvirus, we discovered that NAMPT, the rate-limiting enzyme of the salvage NAD synthesis pathway, potently restricts HSV-1 lytic replication. Loss of NAMPT greatly increases HSV-1 replication in mice. To counteract the NAMPT-mediated restriction, HSV-1 deploys deamidation to inactivate NAMPT and promote viral replication. Collateral to the HSV-1-induced immune evasion, deamidated NAMPT is severely impaired in synthesizing NAD+. Thus, HSV-1-induced NAMPT deamidation and subsequent impaired salvage synthesis of NAD+ likely contribute to the HSV-1-induced neurodegeneration. Interestingly, aging also induces NAMPT deamidation in the brain. In this study, we will delineate the role of deamidation in host defense and salvage NAD+ synthesis in neurons and in mice. We will also determine how aging and HSV-1 infection synergize to promote NAMPT deamidation and NAD+ depletion, thus fueling neurodegeneration in normal mouse strains. Finally, we will develop a modality to resist NAMPT deamidation that impedes or reverts neurodegeneration and AD development. This study will elucidate an innovative mechanism by which collateral damage of viral immune evasion and aging collaborate to induce neurodegeneration, offering new insight into possible avenues to thwart AD and other neurodegenerative diseases associated with aging and microbial infection.

NIH Research Projects · FY 2025 · 2021-09

Colorectal cancer (CRC) is the second leading cause of cancer-related deaths in the US. Populations exhibiting variability in colorectal cancer outcomes, especially those experiencing higher incidence rates and early-onset CRC, require more thorough genomic characterization. Current genomic research has extensively characterized CRC but lacks sufficient representation of patients with early onset and adverse clinical outcomes. Comprehensive molecular characterization is crucial for determining whether observed clinical variations are correlated with specific genomic factors or biological pathways. To achieve significant advances in genomic characterization research, improved strategies for participant recruitment, consent, and engagement are required. Effective participant involvement in research is crucial for advancing precision medicine initiatives and ensuring robust translational biomedical research outcomes. Developing optimized methods to engage and retain research participants throughout all stages of genomic research, from initial recruitment through results dissemination, will enhance scientific validity, strengthen participant-researcher relationships, and potentially improve clinical outcomes. To address these needs, we established the USC Center for Optimization of Participant Engagement in Cancer Characterization (COPECC). This Center specializes in optimizing recruitment and retention methods for genomic research related to colorectal cancer. As part of the NCI U2C Participant Engagement and Cancer Genome Sequencing (PE-CGS) Network, USC COPECC employs rigorous scientific methodologies for participant engagement, data collection, genomic analysis, and results dissemination. The overall objective of USC COPECC is to generate scientifically robust data and standardized engagement practices to enhance research participation and optimize outcomes in CRC genomic research across populations with different genomic profiles.

NIH Research Projects · FY 2025 · 2021-09

Cirrhosis is the 11th leading cause of death in the United States and its incidence is rising. Major causes of cirrhosis include chronic viral infections (hepatitis B and C), alcohol-associated cirrhosis (ALD) and non- alcoholic steatohepatitis (NASH). Stable compensated cirrhosis offers an opportunity to intervene to prevent progression to decompensation and thereby reduce liver-related deaths. Two stages of compensated cirrhosis are recognized; with stage 2 identified by the presence of clinically significant portal hypertension (CSPH, defined by hepatic venous pressure gradient of 10 mmHg or higher). Statins are a novel drug class for consideration in treatment of cirrhosis as preliminary data show reductions in both portal hypertension and improved survival and offer promise as a therapeutic intervention in stage 2 compensated cirrhosis. This multicenter study will establish a prospective cohort of 2000 patients with compensated cirrhosis and CSPH for longitudinal follow-up of changes in clinical, lifestyle, laboratory, and imaging characteristics combined with a rich biospecimen collection for future translational studies. Genetic variants, especially the role of patatin-like phospholipase domain-containing protein 3 (PNPLA-3), in cirrhosis progression and response to statin therapy. The primary goal of this longitudinal cohort is to characterize that natural history of compensated cirrhosis in a representative and contemporary population, and to refine the risk profiles for progression versus non- progression. Additionally, social determinants of health and their influence on healthcare engagement and promotion of healthy liver lifestyle will be examined, with further exploration of how these factors affects liver- related outcomes. Finally, a randomized, placebo-controlled phase 3 study of rosuvastatin will be conducted in 200 patients with compensated cirrhosis and CSPH due to treated hepatitis B or C, ALD or NASH. The treatment duration is 96 weeks with the primary endpoint being survival without decompensation. Secondary endpoints include liver-related mortality, cardiovascular events, diabetes and statin safety, including rates of hepatotoxicity and myotoxicity. The City of Angeles Clinical Center for Cirrhosis (CACC), located at the University of Southern California in Los Angeles, has a unique, ethnically diverse population, that well- represents the population experiencing rising rates of cirrhosis. Through this collaborative network, CACC will contribute to an: 1) enhanced patient-centered model of care; 2) build a rich biorepository to support ancillary and translational studies to improve our understanding of the pathobiology of cirrhosis progression and regression; and 3) conduct a rigorous clinical trial to define the role of statins, with particular attention to the role of PNPLA3 genetic variants on efficacy. Ultimately, the goal of these studies is to improve the clinical outcomes of patients with compensated cirrhosis, especially those from underrepresented minorities.

NIH Research Projects · FY 2024 · 2021-09

As a result of mitigation policy and behavioral changes, the COVID-19 pandemic significantly reduced access to primary care, which is likely to have lasting effects on chronic disease management and health outcomes, particularly for under-resourced populations with existing chronic conditions. Newly available longitudinal individual-level data allows us to rigorously examine these effects for the first time. Although there is a large and growing literature quantifying racial/ethnic disparities in outcomes related to COVID-19, relatively less is known about how the pandemic has affected access to primary and preventive care as well as associated health outcomes among under-resourced populations. Understanding how the pandemic affected the utilization of preventive health care and management of chronic conditions, such as diabetes mellitus (DM) and hypertension (HTN), as well as the downstream outcomes, is critical to addressing resultant health disparities and mitigating the impact of future disruptions to the healthcare system and advancing interventions and policies to reduce health disparities We will leverage this newly available and timely data to examine changes in receipt of healthcare during the pandemic. Our data will include commercial health insurance claims in addition to a unique dataset containing billing data for a large, urban safety-net county health system serving a low-income patient population with a large population of racial/ethnic minorities. Within those populations, we will include a special focus examining individuals with DM or HTN. Using a mixed-methods design, we will examine changes in health care utilization and the predictors of disruptions over time. To more comprehensively understand contextual influences related to disruptions in care, we will obtain complementary qualitative data through interviews with patients, providers and clinic staff, and health systems leaders from the second largest municipal health system in the United States that will explore the drivers of underutilization of primary and preventive care during and after the pandemic. Our specific aims are to: (1) quantify the impact of the pandemic on racial/ethnic disparities in receipt of appropriate primary care, chronic disease management, and downstream health outcomes; (2) compare the differential effect of pandemic mitigation efforts between a commercially insured population and a safety net population; and (3) contextualize the secondary health effects of the pandemic using qualitative interviews with health systems stakeholders. Using established and novel data sets, supplemented by qualitative interviews we will be able to shed light on the changes in patterns of care management both during and after the pandemic for under-resourced patients. Such information is crucial to determining how we should direct health care resources during national crises. However, these results are equally important in a post-pandemic landscape, in which the way patients seek care and health systems offer care is permanently altered.

NIH Research Projects · FY 2025 · 2021-09

Project Summary The goal of this research program is to provide tools for the discovery of transcriptional networks that control cell fate decisions. Cell fate decisions driven by cell state transitions underlie essential cell processes from development to cellular reprogramming. There is an opportunity to make use of publicly available genomic data to develop predictive computational models of cell state transition dynamics. The methods proposed will offer means to gain insight into cell fate decision-making and how it is transcriptionally regulated, given specific cell fate decision points and suitable data. Examples of such decision points include control of epidermal regeneration, or the maintenance of balance among myeloid cell fates during hematopoiesis. In order to bridge the gap between genomics and cell dynamics, statistical and computational modeling challenges must be overcome. Two key challenges form the basis of this research program: 1) developing statistical methods to infer regulatory networks while accounting for the levels of variability between single cells, and 2) developing computational models to couple gene regulatory dynamics within cells and cell-cell communication between cells. To address the first challenge, we will develop machine learning models to predict gene expression dynamics from time-series data. These models will be able to classify genes by their temporal patterns, and the results will inform gene network inference. We will then develop methods for network inference that integrate muti-modal data (single-cell RNA and ATAC sequencing) as well as cell-cell signaling information to learn networks that control specific cell state transitions. To address the second challenge, we will develop differential equation-based multiscale models of the gene regulatory network dynamics coupled with the cell- external signaling dynamics. This will allow us to capture both molecular and cellular dynamics in high resolution, and thus identify which parameters exert key control over the system. We will use Bayesian methods for parameter inference to fit models to data and perform model selection, adapting methods where needed for multiscale model inference. Models will be rigorously evaluated through their application to specific systems, including cell differentiation (e.g. myeloid fate decisions during hematopoiesis) and development (e.g. nephron progenitor cell fate decisions). In each of these organ systems, models predictions will be tested experimentally via collaborations. Following iterative testing, open-source, validated methods will be made widely available for the study of the dynamic processes of cell fate decision-making.

NIH Research Projects · FY 2025 · 2021-09

Abstract Huntington disease (HD) is one of many neurodegenerative diseases wherein accumulation of misfolded, aggregated protein is a pathogenic mechanism. HD is caused by polyglutamine expansions in the huntingtin protein which make it and its naturally occurring exon 1 fragment (Httex1), more aggregation prone. We have shown that Httex1 aggregation is a stepwise process, wherein the monomer gives rise to different aggregation intermediates prior to formation of fibrils. Although there is good consensus that Httex1 aggregation plays a key role in disease pathogenesis, less is known about the 3D structures of Httex1 aggregation intermediates and how each conformer contributes to toxicity. A major obstacle in the field has been the difficulty in obtaining homogeneous population of these conformers for their biochemical characterization. We have recently identified, stably prepared, and characterized different different intermediates during Httex1 aggregation. We propose to extend this work by determining the structure of key conformers (α-helical oligomer and unbundled fibril) and by investigating different mechanism by which misfolding and toxicity can be inhibited. Using our array of different conformers, we also expect to obtain detailed insight into the how chaperones recognize Httex1 conformers. By combining EPR, solid-state and solution NMR, cryo-EM, and cell toxicity assays, our team is in a unique position to successfully accomplish these goals. In Aim 1, we will combine EPR, NMR, cryo-EM and computational refinement to determine the structure of unbundled fibrils from Httex1 proteins with different Q-lengths. By learning about the structures of these toxic conformers, we enable future efforts aimed at finding biomarkers and aggregation inhibitors. The structure of the earliest misfolding intermediate, the α-helical oligomer, will be determined in Aim 2A. This will be done using EPR, solution NMR, and cryo-EM. We also obtained a fibril binder from small, multimerized N17Q7 peptides which potently inhibits Httex1 aggregation. Specific aim 2B tests the hypothesis that this binder inhibits aggregation by interfering with primary and/or secondary seeding. Moreover, we will optimize the inhibitor and test its ability to protect from toxicity in a cellular setting. Specific aim 3 determines how chaperones recognize Httex1 misfolding. Using a combination of biochemical methods, EPR, NMR and cryo-EM, we will identify the molecular mechanism by which chaperones (DNAJB1 and DNAJB6) bind to Httex1 by determining which Httex1 conformers the they bind to and which epitope they are recognizing.

NIH Research Projects · FY 2025 · 2021-09

Project Summary/Abstract Glioblastoma (GBM), the most common primary brain tumor, has a 15 month patient survival due to inevitable recurrence of tumor despite standard treatment – temozolomide chemotherapy (TMZ), radiation, and surgery. It is critical to characterize and target genes modulating GBM chemoresistance. Long noncoding RNAs (lncRNAs) are a novel class of genetic transcripts comprising 80% of the genome. These lncRNAs induce profound alterations in transcriptional regulation and phenotype, including chemotherapy response. Because few GBM lncRNAs have been studied, characterizing new candidates among this abundant and novel class of genes may significantly improve chemotherapeutic efficacy. To assess lncRNAs involved in GBM chemotherapy resistance, we identified linc02454 among the most highly upregulated lncRNAs following TMZ treatment. Knockdown (KD) of linc02454 decreased in vitro GBM cell viability in response to TMZ, while RNA- seq identified CXC-chemokine receptor type 4 (CXCR4) among the most heavily downregulated genes after lncRNA KD. CXCR4 is a well-characterized modulator of GBM chemoresistance, currently targeted in multiple clinical trials. We hypothesize TMZ regulates linc02454 expression and that linc02454 exerts GBM resistance to TMZ through modulation of CXCR4. In this project, we will evaluate the role of linc02454 on GBM response to TMZ treatment. In aim 1, we identify if linc02454 relies upon increased CXCR4 expression, assessing functional interaction via epistasis and CXCR4 rescue, as well as evaluating whether linc02454 directly regulates CXCR4 via Fluorescence in Situ Hybridization. In aim 2, we will assess how TMZ increases linc02454. SA2.1 will perform integrative analysis of lncRNA methylation at DNA loci (including linc02454) and methylation-dependent lncRNA transcription at initial resection/recurrence. SA2.2 will assess chromatin state of linc02454 before and after TMZ. In aim 3, we will assess if linc02454 repression, when combined with TMZ + radiotherapy, improves treatment response, in vitro and in vivo. These aims will examine whether lin02454 meditates GBM response to TMZ via CXCR4 and how linc02454 is regulated by TMZ exposure. We will also test whether linc02454 is a potential therapeutic target in GBM therapy. If successful, this five-year study will provide a more thorough understanding of lncRNA biology in the context of TMZ-induced treatment resistance. With my mentorship group, we have developed a career development plan for tailored training in transcriptional regulation of GBM lncRNAs. The project will be closely supervised through regular one-on-one interaction with primary mentor Dr. Yali Dou, an international expert in chromatin and transcriptional regulation in cancer. NIH-funded co-mentors, Dr. Behnam Badie, a neurosurgeon focusing on GBM biology, and Dr. William Mack also have extensive experience mentoring neurosurgeon-scientists to independence, and have functioned as close mentors. Importantly, by completing this work, I will develop expertise in transcriptional regulation, a critical component in the function of lncRNAs and treatment-induced changes in GBM.

NIH Research Projects · FY 2025 · 2021-09

The past decade of genome-wide association studies (GWASs) has seen thousands of complex traits and diseases studied and identified thousands of reproducibly associated genetic variants. GWAS has helped characterize the complexity of common genetic architectures and shed light on the role of genetics in disease risk. A large body of works have demonstrated that risks of complex traits are highly enriched in functional regions of the genome, which indicates that risk is mediated through perturbed regulatory action on relevant susceptibility genes. Similarly, multiple recent works have found that disease risks are shaped by forces of natural selection, which kept the frequencies of deleterious alleles low in the population. Together, the functional mechanisms and their interplay with natural selection can be coupled under a general mechanism we refer to as the evolutionary architecture. Current frameworks to infer the evolutionary architecture for common complex diseases are only applicable to relatively homogenous populations. Several recent works have demonstrated that integrating multi-population GWAS data substantially improves statistical power to identify causal factors underlying complex traits and diseases due to the increased heterogeneity in allele frequencies. Current approaches evolutionary architecture are unable to appropriately model the heterogeneity across populations with respect to allele frequencies and linkage disequilibrium. Similarly, the resolution of these methods is currently limited to complex diseases and phenotypes, whose inferred architectures, while informative, fail to describe regulatory network mechanisms that mediate risk. Methods capable of analyzing many molecular phenotypes simultaneously have the potential to identify shared architectures, and pinpoint core genes relevant for disease risk. Lastly, several works have shown that integrating functional information with GWAS substantially improves polygenic risk prediction. Together, these issues and opportunities highlight the need for new computational approaches that can scale to multiple populations and large-scale molecular phenotype catalogues while accounting for underlying heterogeneity and shared signals. Here, we propose novel approaches to integrate GWAS data from multiple populations and phenotypes to characterize the population-specific and shared evolutionary architectures. Importantly, our approaches run directly on summary data, which enables immediate large-scale analysis. We propose to apply our novel approaches to large-scale multi-population GWAS data. Together, our work will systematically characterize evolutionary architectures for complex diseases and molecular phenotypes and populations in a robust, open, and reproducible approach.

NIH Research Projects · FY 2025 · 2021-09

With treatment advances for childhood cancer, over 80% of patients achieve long-term survival. However, cancer treatments often lead to other serious issues, including chronic health problems and early mortality. These health problems are referred to as “late effects,” defined as any adverse medical or psychosocial outcome that develops or persists after treatment. By 25 years following treatment, over two-thirds of childhood cancer survivors (CSS) have developed at least one clinically significant late effect, with over one-third developing one or more severe or life-threatening late effect. As a result, life-long cancer-focused long-term follow-up care (LTFU) is essential for CCS to screen for, prevent, and treat late effects. Our current understanding of among CCS comes primarily from a cohort of adult CCS diagnosed between 1970 and 1986 (The Childhood Cancer Survivor Study-CCSS) which is comprised of predominantly white survivors. However, studies have shown that CCS from other groups experience significant health challenges, and thus more research is required to understand their issues to address and reverse them. Rates of LTFU and factors influencing cancer-focused care among Asian American (AA) CCS are unknown. AA populations represent the fastest growing group in the United States, with 59% of the US Asian population born in another country. This group is socioeconomically heterogenous and has grown 72% between 2000 and 2015 is projected to grow to more than 10% of the US population by 2050. Thus, the number of AA CCS is expected to increase, and a greater focus on facilitators and barriers to LTFU is needed in this population. Further, many Asians live in neighborhoods with a high concentration of residents who share similar backgrounds (“enclaves”) which may influence engagement in LTFU for AA CCS. We propose to recruit a population-based cohort of roughly 330 young adult AA CCS and their parents (N=100) in Los Angeles and Orange Counties to examine factors related to survivorship care utilization. Our aims are: Aim 1: We will characterize on a population basis the utilization of cancer-focused survivorship care among Asian CCS. Aim 2: We will examine through qualitative interviews and survey methods individual- and family-level factors associated with the utilization of cancer-focused survivorship care among AA CCS and their parents. Aim 3: We will investigate the effect of residing in an enclave on the utilization of LTFU. The proposed research will provide guidance to identify and address needs among AA CCS for survivorship care utilization to increase outreach and intervention strategies to engage and retain these CCS in care.

NIH Research Projects · FY 2025 · 2021-09

PROJECT SUMMARY Perivascular spaces (PVS) are the area around arterioles, venules and capillaries which accommodate the clearance of the metabolic waste via Aquaporin-4 channels expressed on astrocytic endfeet and are involved in blood-brain barrier transport. PVS have come to prominence recently through potential roles in brain interstitial fluid drainage and waste clearance, and in the pathogenesis of Alzheimer’s disease (AD) and other neurodegenerative disorders. While animal studies have precisely clarified this mechanism, data from humans are relatively crude and limited to visual counting of visible PVS on clinical magnetic resonance images, and this limits our understands of the morphology, biophysical properties and distribution of the PVS in human aging and AD. The long-term goal is to understand the role of PVS in brain health and the degree to which it alters in AD. The objective of this project is to map morphologic and diffusion characteristics of the PVS fluid in healthy aging, and how it is altered in the early stages of AD. The central hypothesis is that imaging-derived PVS fluid characteristics, such as volume occupied and diffusion properties, are differentially and selectively altered in cognitive decline and AD in comparison with normal aging. The rationale underlying this proposal is that in vivo noninvasive mapping of the PVS provides mechanistic insight about AD pathophysiology. The proposed work will also develop widely applicable open resources to map PVS morphologic and diffusion properties that can be applied to a wide range of neurological disorders. The central hypothesis will be tested by pursuing three specific aims: 1) Characterize changes in PVS fluid in normal aging individuals with no cognitive decline nor evidence of AD pathology and evaluate the effect of image acquisition strategy on outcome measures; 2) Identify PVS alteration in cognitive decline and in individuals with AD pathology; and 3) Determine the extent to which PVS is associated with cardiovascular risk factors versus AD pathology. We will pursue these aims by applying innovative MRI-based computational techniques on recently available data of normal aging and patients with cognitive decline from multiple large NIH-funded studies. Computational techniques include both recently developed neuroimaging techniques sensitized to the PVS and more established structural image analysis techniques. The proposed research is significant, because it will aid in the understanding of AD pathophysiological mechanisms and consequently assist in the early diagnosis and disease monitoring of AD. It is also significant because it will make public resources available that can be used to study other neurological disorders characterized by impaired PVS in the brain. The results will have an important positive impact immediately because they will identify and map, for the first time, PVS alteration in human brain across aging, cognitively impaired and AD individuals. Our shared PVS imaging tools can also be more broadly applied to a wide range of clinical and research applications by other groups as well.

NIH Research Projects · FY 2025 · 2021-09

With nearly 6 million cases of Alzheimer’s disease and related dementias (ADRD) diagnosed in the United States in 2019, the health and well-being of ADRD caregivers is of critical importance. Promoting the health and well-being of ADRD caregivers is an effective way to support these unpaid staples of ADRD care. Although interventions targeting ADRD caregivers continues to accelerate, little attention has been paid to caregivers’ use of supportive services, which remains strikingly low. The objective of this K99/R00 application is to enable Dr. Falzarano to develop the necessary expertise to design, implement, and evaluate studies focused on leveraging technology to support and address ADRD caregivers’ multifarious needs. In the K99 phase, Dr. Falzarano will work toward achieving five training objectives that will prepare her to transition to become an independent investigator who designs and evaluates evidence-based technologies to enhance tailored caregiver support. First, she will extend her knowledge in substantive areas including dementia caregiving and front-end and back-end software development. Second, she will learn how to develop, implement, and test behavioral interventions with an emphasis on collaboration with community partners. Third, she will receive training in statistical methods to examine feasibility, acceptability, and preliminary effectiveness in randomized controlled trials (RCTs). Fourth, she will learn about measurement development and psychometric validation of assessments for ADRD caregivers. Fifth, Dr. Falzarano will pursue professional development activities, particularly in the areas of grant writing, leadership, and integration of research and web development teams. The four proposed research aims were chosen to complement the areas identified for career development. Aim 1a will provide the requisite training and experiences to provide Dr. Falzarano with the critical skills needed to conduct the proposed study. Aim 1b will include interviews with ADRD caregivers to identify needs, awareness of, barriers to, and willingness to use services. Aim 1c will leverage key stakeholder (dementia-care experts) input to identify existing services corresponding to areas of need identified in Aim 1b. Aim 2 will leverage data collected from caregivers and stakeholders to inform the iterative development and design of the Caregiver Resource Room (CRR) website, which will include a digital self-assessment tool using machine learning models to identify and categorize needs and provide an output of targeted service recommendations. Aim 3 will include an analysis of the tool’s content, design, features, and services output, which will be iteratively refined based on ADRD caregiver feedback. A randomized controlled trial will be conducted in Aim 4 to determine the feasibility, acceptability, and preliminary efficacy of the CRR as a tool to enhance awareness of and access to relevant services to address unmet needs and improve mental health. The proposed research is well-aligned with the NIA’s strategic initiative to foster the development of research scientists in aging and to develop and test promising interventions to improve ADRD caregiver outcomes.

NIH Research Projects · FY 2024 · 2021-09

Abstract Exosomes are extracellular vesicles secreted by many types of cells and play important roles in mediating intercellular communications. Endogenous exosomes have been emerging as a new and attractive class of therapeutics, owing to their unique and important characteristics. However, effective approaches remain limited for targeting exosomes to desired cells and tissues. And few methods are available for active loading of protein cargos into exosomes for cellular delivery. To address these challenges for broadening therapeutic utility of exosomes and unleashing their full potential, we propose to develop an innovative platform technology, termed synthetic multivalent antibodies retargeted exosomes (SMART-Exos). This will be achieved through genetically and chemically engineering exosomes with functional antibodies, ligands, and enzymes, combined with encapsulation of different types of therapeutic payloads. By integrating knowledge and technologies in exosome biology, protein engineering, synthetic chemistry, and enzyme inhibitor design, we aim to design and generate a series of SMART-Exos with new and/or enhanced functions and properties through distinct but complementary strategies. The resulting SMART-Exos are expected to redirect exosomes toward target cells in high specificity for modulating cellular functions and processes through receptor engagement and/or cargo delivery, leading to the development of a general and versatile platform technology for next-generation investigational and therapeutic exosomes.

NIH Research Projects · FY 2024 · 2021-09

Project Summary: Shifting Suicide Prevention Paradigms: Looking Beyond Clinical Settings Every 10 minutes one person dies by suicide in the US, a result of a public health epidemic that has increased over the last 15 years and incurred over $70 billion in medical costs and lost productivity. A common lamentation in the wake of suicide is, “If we had only known how bad things were.” A crucial scientific gap in suicide prevention stems from the majority of suicide research relying on individual psychopathology, largely overlooking meaningful constellations of risk in a broader context of social environmental life disruptions that commonly precede suicide. Job loss, financial strain, divorce, legal problems, housing instability – these life disruptions can be semaphores of despair that are misinterpreted as coincidental rather than causal; thus overlooked as points for timely detection and intervention. I propose a paradigm shift in suicide research by prioritizing social determinants to develop public health research and prevention through broad, but strategic, partnerships with industries outside of mental health and health care, including the specific industries of family law, mortgage foreclosure, and unemployment services. This proposal uses a two- pronged approach that ventures beyond traditional funding mechanisms by challenging what we typically consider to be “health” vs. “non-health”-related suicide prevention research and intervention. First, by taking the tested methodology of psychological autopsy and reinventing it as a social autopsy, we will dive deeply both into the nature of life disruptions that often precede suicide and the contacts with non-medical services that a suicide decedent may have made prior to their death. Second, by surveying and interviewing employees of industries that commonly deal with life disruptions (i.e., divorce, mortgage foreclosure, and job loss), we will explore their experiences with suicidal clients, such as occupational wisdom and intuition around warning signs among clients, training around suicide prevention, knowledge about suicide, and approaches they may have used when working with clients in distress. Seeking unconventional upstream strategies to identify and reach people at risk for suicide is all the more urgent against the backdrop of the COVID-19 pandemic, which created historic job loss, relationship strain, and increased potential of mortgage foreclosures. As a researcher trained in public health sciences, I investigate the social production of health problems, exemplified in my focus of how social determinants contribute to poor mental health, suicidal ideation, and suicide attempt. My perspective, coupled with extensive experience in survey data research and efforts in training medicolegal death investigators, uniquely qualify me to successfully implement these novel approaches to change suicide risk detection and prevention. True integration of social determinants into suicide prevention research requires rethinking the problem of suicide as solely a clinical mental health problem requiring clinical solutions, to suicide as a problem at a social and clinical nexus, thus necessitating both social and clinical solutions.

NIH Research Projects · FY 2025 · 2021-09

PROJECT SUMMARY Stroke is a leading cause of long-term disability around the world and disproportionately affects older adults. After a stroke, older adults commonly exhibit gait dysfunction (e.g., slow, unstable, asymmetric walking), which is the top patient-reported rehabilitation target. In addition to motor deficits, approximately 70% of people living with chronic stroke also have post-stroke cognitive impairment (PSCI). While post-stroke gait rehabilitation interventions effectively mitigate gait dysfunction in aging adults, there remains a significant degree of inter- individual variability in walking outcomes. We posit this is because current gait rehabilitation interventions are predominantly structured to leverage explicit versus implicit locomotor learning. Importantly, explicit motor learning is cognitively demanding. It occurs through intentional changes to a movement based on external feedback and relies primarily on the dorsolateral prefrontal cortex. Whereas, implicit motor learning is cerebellum-dependent and occurs more automatically in response to proprioceptive feedback. Despite evidence of shared neural resources between cognitive and motor processing, the impact of PSCI on the capacity for these forms of motor learning during walking remains largely unexplored. The overall goal of this proposal is to demonstrate that PSCI moderates explicit, but not implicit, locomotor learning in older adults with chronic stroke and identify the role of the dorsolateral prefrontal cortex in this relationship. Under this career development award, the PI (Leech) will develop skills in neuropsychology, structural neuroimaging, and advanced statistics. This training will promote the achievement of her long-term career goal: as an independent investigator, to develop effective, comprehensive, individualized interventions to reduce disability in older adults. This project has three research aims. Aim 1 will determine the impact of PSCI severity on explicit locomotor learning in older adults post-stroke. Aim 2 will test the impact of the PSCI severity the capacity for implicit locomotor learning in the same group of participants. Finally, Aim 3 will examine the role of dorsolateral prefrontal cortex structural integrity in explicit locomotor learning post-stroke. Older adults with chronic stroke who have a range of PSCI severity will complete the procedures necessary for all three aims over a total of 2 experimental sessions. We expect to show that PSCI severity limits an individual’s capacity to learn a more symmetric walking pattern through explicit, but not implicit, locomotor learning and that this relationship will be mediated by the structural integrity of the dorsolateral prefrontal cortex. This work will provide necessary scientific foundation for a program of research to develop an individualized gait rehabilitation intervention anchored to cognitive impairment and a comprehensive predictive model of locomotor learning in older adults post-stroke.

NIH Research Projects · FY 2025 · 2021-09

ABSTRACT Parkinson’s disease (PD) is a devastating, progressive neurodegenerative brain disease, with no known cure. The disease afflicts 10 million people worldwide - ~1.5 million in the U.S. alone, and ~50k-60k new cases are diagnosed annually. Risk factors or interventions are extremely hard to evaluate as we lack objective metrics of how PD affects the brain. The vast global availability of brain imaging has led to several promising metrics to gauge PD progression in the brain - structural changes in the basal ganglia and motor cortex, abnormalities in neural connectivity seen with diffusion MRI (dMRI), and disruptions of the brain’s functional synchrony across regions, seen with resting state functional MRI (fMRI). Despite these findings, factors that affect disease severity are difficult to discover, as most imaging studies of PD scan <100 patients. Most PD research is conducted in isolated cohorts from the US and Europe, limiting worldwide generalizability. Factors that affect PD progression are hard to verify, leading to a crisis of reproducibility. Responding to NIH’s call for more reproducible studies, here we launch ENIGMA’s Worldwide Parkinson’s Initiative. ENIGMA recently published the largest neuroimaging studies of schizophrenia, bipolar disorder, major depression, epilepsy, and autism spectrum disorder. With ENIGMA’s globally coordinated, highly powered consortium approach, we plan to overcome the crisis of small studies with poor power and reproducibility. Pooling anatomic, diffusion and resting state functional MRI metrics from 21 deeply assessed international cohorts - from the US, Brazil, Taiwan, New Zealand, the Netherlands, Italy, Switzerland, South Africa, China, and Russia - we ask: How does the illness affect the brain's structure, neural connectivity, and functional synchrony? What imaging biomarkers track disease progression and consistently predict clinical outcomes? Do genetic risk loci for PD help predict brain decline? What PD subtypes, or clusters, can imaging identify? Combining multimodal data from 2,307 patients and 1,264 controls, we will thoroughly evaluate predictors and brain biomarkers in PD. Our aims are to: (1) Evaluate and rank structural, diffusion, and resting state functional MRI biomarkers of PD worldwide; (2) Evaluate the added value of polygenic risk scores (PRS) in predicting PD brain biomarkers; (3) Predict future functional decline in PD with machine learning, multi-modal imaging and genomics. We will use genetic data and baseline clinical variables from PD patients and healthy controls across our cohorts to construct an ensemble of models to predict the annual rate of change in combined scores from the Movement Disorder Society—Unified Parkinson's Disease Rating Scale parts II and III. We will rank the best predictors of decline, and assess how robust they are internationally. By better modeling variance in patient outcomes, our multimodal predictive model will empower PD clinical trials by ranking biomarkers of disease burden and determining the contexts where they are reliable, accurate, and feasible to use.

NIH Research Projects · FY 2025 · 2021-08

Project summary The overarching goal of my lab is to understand understudied mechanisms of genomic regulation, and how they influence lifelong Vertebrate health and disease. In multi-cellular organisms, diverse cell types are characterized by specific genomic regulation patterns, and the precise control of these patterns is key not only for development, but also for cell/tissue homeostasis in adults. Indeed, loss of fine control in genomic regulation has been linked to disease (e.g. cancer, neurodegeneration) and age-related functional decline. An interesting and understudied family of genomic elements lies in dormant genetic parasites (e.g. transposons, also called “jumping genes”). Although transposons can represent up to 80% of some eukaryotic genomes, they remain critically understudied, since they were historically dismissed as unimportant (i.e. “junk DNA”), and their high copy numbers and repetitive nature pose unique technical challenges. Consistent with their potential impact in health and disease, the ability of cells to suppress transposon activity is disrupted with disease and with aging. In addition, accumulating evidence suggests that many aspects of biology and genomic regulation differ between males and females, including emerging data suggesting potential sex-dimorphism in transposon activity. However, how transposable elements are regulated throughout life in healthy somatic tissues and across biological sexes, and how they influence vertebrate health, remains largely unknown. Thus, we propose to decipher how transposons are controlled in healthy somatic cells (including in male vs. female cells), and how loss of that control could influence Vertebrate health and disease. To explore this question, my group will use a unique combination of ‘omics’ approaches, machine-learning, and experimental validation in animal models. We use two vertebrate models for their respective strengths: the laboratory mouse (e.g. powerful genetics, validated antibodies, etc.) and the African turquoise killifish, a naturally short-lived model organism I have helped develop (e.g. short generation time/lifespan, strain diversity, cost-effectiveness, etc.). First, we will decipher sex-dimorphic regulation of transposon activity, determining the impact of gonadal hormones vs. sex- chromosomes on such regulation. Second, we will use functional genomics to identify new regulators of transposon activity in somatic cells. Finally, we will evaluate the impact of transposon control in key somatic tissues and across sexes on lifelong vertebrate health using the naturally short-lived African turquoise killifish as a model. Ultimately, understanding the fine control of transposon in healthy cells will help devise strategies to prevent their misregulation in disease, by allowing us to maintain youthful and healthy genomic regulation landscapes. 1

NIH Research Projects · FY 2025 · 2021-08

In addition to coding proteins, RNA plays fundamental roles in virtually every aspect of biology. The extreme functional diversity of RNA stems from its ability to fold into complex structures and, like machines, dynamically take input, transmit signal and force, and execute genetic instructions. RNA structures regulate every step of gene expression in cells and control the life cycle of RNA viruses. As a result, physiological and abnormal activities underlie a variety of human diseases. In recent years, targeting RNA has transitioned from an interesting academic idea to a reality in the clinic, with the development of oligonucleotides and small molecules that bind specific RNA sequences and structures, ushering in a new era in RNA medicine. Despite decades of technology development, RNA structure analysis remains a major challenge, especially compared to proteins. Traditional physical methods such as crystallography, NMR and cryo-EM has only been applied to purified “well-behaving” samples in vitro, leaving the vast majority of cellular and viral RNAs beyond reach. Recent chemical probing methods provided experimental constraints that improved de novo modeling but has so far been limited to small and simple RNAs. This RNA structure analysis bottleneck has significantly limited functional studies and therapeutic development. In this MIRA application, I outline a research program to tackle the ultimate challenge in RNA structure biology: in vivo determination of structures and dynamics for any RNA in any biological sample at high resolution. This proposal is based on the simple mathematical theory that the 3D structure of any object is equivalent to the spatial distances among its components. Therefore, RNA 3D structure determination can be transformed into a problem of measuring spatial distances among the nucleotides. To achieve this goal, we will develop ic3D (in vivo crosslinking of 3D structures, or “I see 3D”), a technology that uses 3 new classes of “molecular rulers” - reversible chemical crosslinkers with defined lengths - to precisely measure inter-nucleotide distances at the atomic level. Coupled with proximity ligation, high throughput sequencing and Rosetta-based 3D modeling, ic3D enables in vivo global analysis of RNA structures and ensembles of conformations. We will perform rigorous benchmarking against a wide selection of simple and complex models that represent the full diversity of possible RNA structures in vivo. We will use ic3D to discover and model 3D structures across the transcriptome. The completion of this project will have broad impact in understanding the structural basis of RNA functions, mechanisms of RNA-mediated diseases, and revealing new structure targets for therapeutic interventions.

NIH Research Projects · FY 2025 · 2021-08

Neurodevelopmental disorders (NDDs) comprise a group of genetically and phenotypically heterogeneous pathologies commonly characterized by psychiatric impairment. The molecular basis of these neuropathologies remains poorly understood. Recent whole-genome-sequencing studies revealed that mutations in genes encoding heterochromatin modifiers are significantly associated with NDDs. This class of transcriptional regulators is thought to stabilize neural cell identity and function by enforcing heritable silencing of lineage non-specific genes through epigenetic chromatin modifications. However, since most heterochromatin modifiers are ubiquitously expressed and lack sequence-specificity, (1) how precise targeting of repressive chromatin is controlled and (2) how mutations in general heterochromatin modifiers contribute to NDD- associated neuronal defects remains unclear. To gain experimental traction on these questions, we will examine the mechanism by which a high-confidence NDD risk gene, ZNF462, recruits the heterochromatin modifiers EHMT1/2. We will test whether and how ZNF462 restricts lineage non-specific gene expression and maintains neural cell identity. ZNF462 haploinsufficiency causes Weiss-Kruszka syndrome, a complex NDD characterized by neurodevelopmental defects including developmental delay and autism. However, the neurodevelopmental role of the C2H2 zinc finger protein is unknown. We previously discovered that mouse Zfp462, is required for endodermal gene repression, directing Ehmt1/2-dependent heterochromatin to transposable element (TE)-derived enhancers in neural progenitor cells. We hypothesize that human ZNF462 controls facultative heterochromatin formation, by specifically restricting non-neural gene expression during neurogenesis. However, we predict that due to rapid species-specific evolution of TEs, ZNF462 will have novel human targets and control a distinct gene regulatory network. W e will therefore: (Aim 1) employ neural differentiation of human embryonic stem cells (hESCs) coupled to epigenome and transcriptome profiling to investigate the impact of ZNF462 heterozygosity on maintenance of neural gene expression, (Aim 2) perform structure-function analysis and functional complementation in mESCs to identify ZNF462 protein domains responsible for homodimerization, DNA binding and transcriptional repression and (Aim 3) profile CTCF binding and three-dimensional chromosome conformation in neuroepithelial stem cells (NESCs) to investigate the impact of ZNF462 heterozygosity on neuro-specific genome architecture. Our proposal provides a path to novel insight into the molecular mechanism of ZNF462-dependent gene silencing, and enhance our understanding of the etiology of Weiss-Kruszka syndrome. The following strategy will reveal new concepts in gene regulation and neurobiology and elucidate the link between mutations in heterochromatin modifiers and NDDs. Overall, our work will inform novel strategies to prevent and treat NDDs arising from epigenetic dysregulation.