University Of California Los Angeles

NIH Research Projects · FY 2024 · 2022-07

Project Summary/Abstract Panic disorder affects millions of adults in the U.S. every year. Panic attacks are characterized by overwhelming fear, difficulty breathing, accelerated heart rate, and an urge to escape. Panic therapies are often ineffective, emphasizing the need to develop a novel mechanistic understanding of panic attacks to advance treatment. The periaqueductal gray region has been strongly implicated in panic, as electrical stimulation of the periaqueductal grey (PAG) induces panic in humans and induces escape, freezing, and other defensive behaviors in rodents. However, the genetic identity of the specific cell population that selectively drives escape is unknown. Cholecystokinin (cck), a neuropeptide, is expressed in the lateral and ventrolateral PAG (l/vlPAG) columns. I propose to dissect a novel neural circuit involving cck+ neurons in the lateral ventrolateral PAG (l/vlPAG) underlying escape in mice during an ethological predator-exposure behavioral assay to elucidate the mechanism of escape. In Aim 1, I will test if cck+ l/vlPAG neural activity is sufficient and necessary for escape from a live predator using chemogenetic manipulations. In Aim 2, I will examine if cck+ l/vlPAG neural activity predicts escape using miniaturized microscope calcium imaging in freely-moving mice in the presence of a live rat. In Aim 3, I will test if cck+ l/vlPAG cells contribute to encoding of escape and threat in pan-neuronal PAG cells using chemogenetics to manipulate cck+ l/vlPAG neural activity and recording subsequent neural activity in cck- PAG cells using miniaturized microscopes. Together, these three Aims will serve as a comprehensive approach to elucidating the neural mechanism of escape, which will provide better insight into understanding panic mechanisms.

NIH Research Projects · FY 2026 · 2022-07

Accumulating evident support intercellular transmission and subsequent amplification of pathological tau as a key mechanism for the progression of Alzheimer’s disease (AD) and other tauopathies. Blocking this transmission process is a promising therapeutic strategy to slow down disease progression. However, the molecular mechanisms that regulate pathological tau transmission remains largely unknown. Previous studies of tau transmission have been focused on pathological tau or the ‘seed’ itself. But, the amplification of pathological tau requires both the ‘seed’ and soluble tau, the ‘substrate’. What has generally been ignored is the potential effect of soluble tau on the amplification of pathological tau. Our preliminary study demonstrated that soluble α-synuclein (a-syn) post-translational modifications (PTMs) would dramatically affect the amplification of pathological a-syn, which highlights for the first time that PTMs on soluble protein would affect amplification of the corresponding pathological protein. Since many PTMs were identified on soluble tau, we hypothesize that soluble tau PTMs will also affect pathological tau amplification. Indeed, our preliminary data demonstrated that soluble tau acetylation could dramatically modulate the amplification of pathological tau prepared from AD brains (AD-tau). More interestingly, this effect is highly pathological tau conformation dependent. Pathological tau from corticobasal degeneration brains (CBD-tau) shows very different responses to soluble tau PTMs compared with AD-tau. Here, we propose to systematically explore how soluble tau PTMs would modulate the amplification of pathological tau in AD and other tauopathies. Firstly, PTMs on soluble tau from tauopathy brains has not been systematically identified and quantified. Therefore, we propose to systematically identify and quantify soluble tau PTMs in AD and other tauopathies by LC-MS/MS. Secondly, we will explore how soluble tau PTMs and PTM combinations would affect the amplification of pathological tau in AD and other tauopathies. Thirdly, the effects of soluble tau PTMs are highly pathological tau conformation dependent. Therefore, we propose to evaluate whether pathological tau in different disease subtypes, brain regions, and cell types (neurons and glial cells), would have different responses to soluble tau PTMs. Fourthly, it has been shown that different intracellular environment in neurons and glial cells would affect the amplification of pathological proteins. We propose to test whether different intracellular environment would also change the effect of soluble tau PTMs. Finally, the effects of soluble tau PTMs on AD-tau amplification will be evaluated in mouse. In summary, the proposed study represents the first to systematically explore how soluble tau PTMs would affect the amplification of pathological tau in AD and other tauopathies, which is a novel mechanism that modulates tau transmission. Successful performance of the proposed study will not only provide critical new insights into tau transmission but also dramatically facilitate the development of novel therapies targeting soluble tau PTMs for AD and other tauopathies.

NIH Research Projects · FY 2026 · 2022-07

PROJECT SUMMARY / ABSTRACT Although many well-studied aspects of neural function involve activity driven by sensory inputs or occurring at the time of motor actions, the brain often links such fleeting sensory and motor signals with persistent activity. Somehow, neural circuits and individual neurons are capable of maintaining activity without additional input. This is a fundamental aspect of neural function and a critical building block of cognition. The mechanisms underlying persistent neural activity have long been considered in both experiment and theory, but there is little definitive mechanistic understanding of the circuit and cellular contributions to persistent activity. Indeed, theories of persistent activity are far more biologically nuanced than current empirical knowledge— especially in the nonhuman primate, from which our understanding should have greatest clinical relevance given the number of disorders that involve persistent activity. Here, we propose work that leverages advanced techniques for multiple scales (and specificities) of neural recordings with corresponding analyses of large- scale datasets to test detailed theories of how the brain generates and maintains persistent activity. Specific Aim 1. Establish the marmoset as a powerful complementary model system for dissecting persistent activity mechanisms in primate brains. We will demonstrate the viability of studying memory-guided saccades and persistent activity in the marmoset, using successful training approaches, electrophysiology, and calcium imaging to elicit the key behavior and to characterize the important brain areas in this exciting primate model system. Specific Aim 2. Characterize the large-scale circuitry underlying oculomotor persistent activity. Using large scale recordings of extracellular activity across multiple brain regions collecting during performance of a memory-guided saccade task, we will acquire a dataset of unprecedented scale to assess the large-scale circuitry underlying persistent activity. We will adapt, develop, and deploy advanced statistical models to capture the functional interactions between neurons and brain areas. Specific Aim 3. Test and refine theories of persistent activity with novel measurements at fine spatial and genetic resolution. We will perform both 2-photon imaging and high density electrophysiological measures of neural activity. The imaging will allow us to test the local circuit components of the theory, as well as to assess cell-type-specific contributions to persistent activity. High density electrophysiology will reveal the local circuit architecture and signal flow that are not accessible with coarser techniques. Integrated within our analysis framework, the resultant model of persistent activity will be supported and refined by multiple scales and forms of empirical evidence, all collected in the primate brain.

NIH Research Projects · FY 2026 · 2022-07

PROJECT SUMMARY/ABSTRACT Understanding cellular processes and pathways in depth requires methods that not only investigate the composition and arrangement of various protein assemblies, but that also elucidate their dynamics and functional regulation. Experimental strategies delivering structural information beyond primary sequence; i.e., higher-order structure and protein modifications, connect proteomics to structural biology in ways yielding potential insights into complex disease mechanisms. New techniques based on mass spectrometry (MS), native MS (i.e., measuring biomolecules in their native solution environment preserving ligand- and other molecular interactions), and top-down MS/proteomics will be advanced and applied to facilitate the characterization of proteins and protein assemblies. Coupling sensitive ionization, solution- and gas-phase separations, new electron-based dissociation methods (e.g., electron capture dissociation, ECD; electron ionization dissociation, EID) and other activation/dissociation techniques to ultra-high resolution mass spectrometry will provide an experimental platform for complete sequence and proteoform coverage. These advanced tools will be employed to characterize important biological complexes for which high-resolution structures have been difficult to obtain, including G-coupled protein receptors (GPCRs) and other membrane proteins. The exploration of links between post-translational modifications (PTMs) and proteoforms to the aggregation and toxicity of neurodegenerative diseases such as Alzheimer's disease (AD) will be furthered by our advanced MS methods. Our experimental strategies apply broadly and integrate with numerous biophysical techniques to enable the detailed structural study of large and complex molecular machines and to provide insights into their dynamics. Native top-down MS promises to advance structural biology and to hasten drug discovery and development. Improvements in MS-based technologies can advance our understanding of how proteins and protein machines drive biology.

NIH Research Projects · FY 2025 · 2022-07

PROJECT SUMMARY Cardiac fibrosis is a common adverse myocardial remodeling event that worsens heart failure caused by chronic ventricular pressure overload and other cardiac pathologies such as heart failure with preserved ejection fraction. Due to our incomplete understanding of the pathogenesis of cardiac fibrosis, no specific antifibrotic therapy exists, and affected patients are usually treated by heart transplantation, a therapy that has many limitations. Motivated by this clinical problem, our research has recently uncovered new insights into the regulation of cardiac collagen content and fibrosis, and, in this application, we propose to extend this work to gain further understanding into cellular and molecular mechanisms. In our recent work, we discovered that global deficiency of SLIT3, a secreted glycoprotein which binds to the ROBO receptors, leads to a marked decrease in cardiac collagen content, improved diastolic function in adult mice, and decreased cardiac fibrosis and improved survival after left ventricle (LV) pressure overload induced by transverse aortic constriction (TAC). We also found that SLIT3 stimulates cardiac fibroblast function, intracellular signaling, and collagen production in vitro. Collectively, these results support a developmental link between SLIT3 and collagen deposition during both normal cardiac homeostasis and pathologic stress. However, as these studies relied on SLIT3 global knockout mice where SLIT3 knockout potentially affected developmental programs, the more specific, postnatal functions of SLIT3 - as well as the details of the cellular and molecular signaling - remain undefined in the adult state. New preliminary data using SLIT3 floxed mice and inducible Cre promoters, we find that SLIT3, generated by either cardiac pericytes or fibroblasts, regulates collagen expression in vitro and in vivo in the postnatal state, most likely, by signaling through ROBO1. Based on these results, our overarching hypothesis is that cardiac pericyte and fibroblast-mediated SLIT3 regulate fibroblast activation and function via ROBO1 during conditions of homeostasis and pressure overload. As such, we propose the following aims: (1) Characterize the role of SLIT3 in regulating adult cardiac fibroblast function in vivo. (2) Determine the details of how SLIT3/ROBO1 regulates adult cardiac fibroblast activation and function in vivo and in vitro. (3) Characterize the role of cardiac pericyte and fibroblast-mediated SLIT3 on the ROBO1-dependent response of cardiac fibroblasts during LV pressure overload in vivo. This research will reveal new cellular and molecular concepts of cardiac fibroblast regulation and will further advance our understanding of the pathogenesis of cardiac fibrosis. The results of this research may provide the rationale for targeting SLIT3-ROBO1 to mitigate pressure overload-induced cardiac fibrosis, thereby benefiting patients with heart failure and other forms of cardiac disease.

NIH Research Projects · FY 2025 · 2022-07

PROJECT SUMMARY/ABSTRACT Alternative splicing is a key process in shaping the functional complexity of the brain. Accordingly, many neurologic and psychiatric disorders are caused by mutations in RNA binding proteins or their targets in alternative splicing. The Rbfox family of RNA Binding proteins regulate alternative splicing during neuronal development and their mutations have been implicated in autism spectrum disorder and various familial epileptic disorders. To understand the role of Rbfox in these diseases, many studies have focused on how individual Rbfox proteins bind to their targets and regulate their splicing. However, Rbfox proteins are also known to interact with cofactors that influence their function. We previously found that nuclear Rbfox proteins in the mouse brain are almost exclusively bound to a large assembly of splicing regulators (LASR). However, it remains unknown what the full subunits of the neuronal LASR complex are and how they interact with Rbfox to regulate alternative splicing in neurons. In this proposal, I will define the neuronal components of LASR, determine the transcriptome-wide targets of the LASR/Rbfox complex and its subunits in the mouse brain, and analyze how components of LASR affect Rbfox’s splicing regulatory activity in neurons. These studies will elucidate how combinatorial interactions between RNA binding proteins in a novel splicing regulatory protein complex shape the gene regulatory circuit of the brain and will further our understanding of the function of the Rbfox proteins which have been implicated in epilepsy and autism spectrum disorder.

NIH Research Projects · FY 2025 · 2022-06

ABSTRACT Chimeric Antigen Receptor (CAR) T-cells have emerged as a promising immunotherapy in controlling HIV-1 infection. However, CAR-T cells are also subject to immune dysfunction/exhaustion mediated by persistent inflammation during chronic HIV infection. Strategies to prevent exhaustion/restore functions of anti- HIV CAR-T cell are critical for ultimately achieving HIV functional cure. Our preliminary studies have showed that autophagy induction can improve mitochondria function and promote CAR-T cell cytotoxic T lymphocyte activity in vitro. Importantly, we found that induction of autophagy can prevent excessive IFN-I signaling and in vivo treatment with autophagy inducer rapamycin in chronically HIV infected humanized mice can decrease inflammation, restore exhausted anti-viral T cell function, and reduce viral loads. In addition, we found that autophagy inducers such as rapamycin allow efficient HIV-1 latency reversal by PKC activator bryostatin-1 while reducing T cell activation associated immune toxicity. Therefore, we hypothesize that autophagy induction can enhance ‘kick and kill’ HIV cure approaches by improving the survival, persistence and function of anti-HIV CAR-T cells and facilitating effective and safe latency reversal by PKC modulators. We will utilize our well-established humanized mouse model engineered with anti-HIV CD4CAR T cells to investigate the therapeutic potentials of autophagy induction for HIV ‘kick and kill’ cure approaches. Our study will also provide mechanistic insights into the development of immune exhaustion and autophagy’s regulation of CAR-T cell function and will thus have a wide impact beyond HIV cure research.

NIH Research Projects · FY 2026 · 2022-06

Project Summary/Abstract Affiliative social interactions play an essential role in the reproduction and survival of social species including humans. Its disruption in neuropsychiatric conditions or during times of social isolation such as the COVID-19 pandemic can take a heavy toll on mental and physical well-being. However, the neural circuit mechanisms governing affiliative social behaviors are not well understood. Allogrooming (grooming behavior directed toward another individual) is a major form of affiliative social contact through which animals may form, maintain, and strengthen social relationships and is conserved in a wide range of social species, such as birds, bats, rodents, canids, cats, equids, and primates. However, the neural circuitry underlying allogrooming has been sparsely explored and few brain areas that encode and promote affiliative allogrooming have been identified. Deciphering the neural circuit mechanisms of affiliative allogrooming will provide key insights into the neural basis underlying social affiliation and attachment. Given the prominent impairment in affiliative social behavior in several neuropsychiatric disorders, including autism and schizophrenia, this understanding can guide circuit-level investigation of disease mechanisms and development of interventions. In recent studies, we established an ethologically relevant and experimentally tractable paradigm for studying allogrooming behavior in laboratory mice and uncovered a key role of a medial amygdala (MeA)-to-medial preoptic area (MPOA) circuit in controlling this behavior. These findings open up valuable opportunities for in-depth dissection of the functional circuitry underlying allogrooming behavior. The central objective of this application is to elucidate the neural circuit mechanisms through which the MPOA controls allogrooming, which represents a critical next step toward defining the functional organization of the neural circuitry of affiliative social behavior. We propose a series of experiments to comprehensively probe whether and how the activity of select MPOA neuronal subpopulations and their downstream targets regulate allogrooming behavior. Specifically, we will address the following important questions: (Aim 1) Is allogrooming behavior controlled by select, molecularly defined MPOA subpopulations? (Aim 2) Whether and how neural activity dynamics in MPOA neurons encodes social sensory cues and allogrooming behavior? (Aim 3) What are the neural circuits downstream of the MPOA that mediate allogrooming behavior? Our proposed research will integrate state-of-the-art techniques for functional manipulation of specific neuronal subpopulations, in vivo imaging of neuronal activity dynamics in awake, freely behaving animals, and functional mapping of neural projections to reveal how specific MPOA neuronal subpopulations respond to conspecific cues and control the display of allogrooming through their downstream projections. This investigation will yield novel, critical insights into the neural circuitry underlying an evolutionarily conserved, major form of affiliative social behavior. Such insights will impact our understanding of social cohesion and disconnection, such as in individuals experiencing social isolation or neuropsychiatric disorders.

NIH Research Projects · FY 2026 · 2022-06

Project Summary This project aims to advance our understanding of major depressive disorder (MDD) through the analysis of electronic medical records, biobanks and associated genetic data. MDD is the commonest psychiatric disorder and recognized as the world’s leading cause of disability, yet current treatments are relatively ineffective: only about half of patients will show signs of improvement after three months of therapy. Genetic approaches are a proven path to identifying causal factors and hence finding novel treatments, but they are hard to apply to MDD without obtaining large samples of cases. We propose using the very large numbers of cases available through electronic medical records by applying statistical methods that accurately identify MDD. Our methods provide a “best-guess” diagnosis by a process known as imputation. We then identify features that are specific to MDD. Our insight is that since non-genetic and non-specific factors explain large components of variability in traditional MDD phenotypes, algorithmically removing them increases the signal from the core biological drivers. We assume that non-specificity can be attributed to latent factors capturing the relationship between MDD, comorbid disease, and pleiotropic factors. By identifying and removing these signals, we increase specificity, and thus identify features that reflect the episodic severe shifts of mood, associated with neurovegetative and cognitive changes, that are central to MDD. Our project has three aims: first, to impute phenotypes of a large sample of MDD cases and controls in biobank data and determine the best approximation to MDD; second, to identify and characterise specific and non-specific genetic effects on MDD, and finally to identify genes involved in MDD by associating the cases defined via our first two aims with rare coding variants.

NIH Research Projects · FY 2025 · 2022-06

PROJECT SUMMARY Quantitative dynamic contrast enhancement (DCE) MRI metrics such as tissue perfusion rates, kinetic parameters, extravascular volume, and plasma volume allow characterization of subtle differences in tissue states related to ischemia, vascularity, inflammation, and fibrosis in neurological and cardiovascular diseases, and in cancers such as pancreatic adenocardinoma (PDAC). The reproducible nature of quantitative imaging makes it more suitable for multi-center or longitudinal studies than conventional “qualitative” imaging. Quantitative DCE metrics have been shown to be important for risk assessment, early detection, staging, characterization, and treatment monitoring of PDAC and other diseases. DCE MRI performs imaging before, during, and after injection of a gadolinium (Gd)-based contrast agent. There are several major challenges, especially in moving organs: i) cardiac motion must be dealt with for heart scans, generally by syncing acquisition with an ECG signal, leading to difficulty in arrhythmia patients as well as low imaging efficiency and challenges for whole-heart 3D imaging; ii) respiratory motion must be dealt with, typically by patient breath-holding; iii) safety questions surrounding Gd contrast agents lower the benefit-to-risk ratio in many situations. The objective of this project is to develop low-dose, motion-resolved, quantitative dynamic contrast enhanced (DCE) MRI for PDAC. This will be accomplished by developing and validating the MR multitasking framework for multi-dynamic, highly time-resolved T1 mapping, and correlating MRI measurements to histology in patients undergoing surgical resection. Multitasking designs DCE MRI around the concept of images as functions of multiple time dimensions, each corresponding to a different dynamic process (e.g., motion, T1, DCE). It integrates machine learning, low-rank tensor modeling, compressed sensing, and deep learning to extract reproducible, quantitative measurements from 6D DCE images, even under free-breathing conditions. The resulting technology would be a powerful tool for quantitative MRI tissue characterization in moving organs, and would be promising as a tool for monitoring neoadjuvant therapies prior to pancreatic resection.

NIH Research Projects · FY 2025 · 2022-06

Project Summary/Abstract Poor sleep is very common in persons with Alzheimer’s disease and related dementias (ADRD) and their caregivers. It is significantly associated with adverse mental and physical health outcomes and well-being in both members of the group. Unmanaged poor sleep will further impact the quality of care that the caregivers provide for the patients. This suggests a critical need of sleep management in this vulnerable population. Unfortunately, an intervention addressing sleep problems of both members of the dyad simultaneously is lacking, particularly using the behavioral strategies that have been effective in other groups. Effects of sleep intervention programs in different delivery modalities are also unknown in this group. The current proposal aims to examine the efficacy of a dyadic sleep intervention program for ADRD patients and their caregivers, that is built upon PI’s prior work. We propose a 3-arm randomized controlled trial design (Stage II), including both in-person (n=70 dyads) and telehealth (n=70 dyads) delivery of the intervention, compared to in-person sleep education control (n=70 dyads). The dyadic intervention is a 5-week, manual-based program, which incorporates key components of cognitive behavioral therapy for insomnia, daily light exposure and walking, and a problem-solving approach for ADRD-related problematic nighttime behaviors and other caregiving challenges. All intervention sessions will be delivered by a sleep educator. Primary outcomes include subjective and objective sleep quality of the dyads. Secondary outcomes include the patients’ dementia-related behaviors and quality of life, and the caregivers’ burden, depression, and perceived health. We will also explore effects of the dyadic sleep program on inflammatory markers among caregivers. All outcomes will be measured at baseline, post-intervention (i.e., immediately after the last session of the intervention), and 6-month after the last session. Both superior (both in-person and telehealth interventions versus control) and non-inferior effects (in-person versus telehealth intervention) will be tested. A unique aspect of the proposed work is that the program is tailored to address sleep problems of both patients and caregivers, and includes inflammatory biomarkers to evaluate a key mechanism of intervention benefits that can be further explored in future research. The knowledge gained from this study has the potential to improve the lives of ADRD patients and their caregivers. Our dyadic sleep intervention can be disseminated to multiple communities serving ADRD patients and/or caregivers, including those that lack access to traditional in-person sleep treatment. The intervention manual can also be used to train health professionals and staff in various types of community programs.

NIH Research Projects · FY 2025 · 2022-06

ABSTRACT The incidence of oral squamous cell carcinoma (OSCC), the most common type of head and neck cancer, continues to rise among numerous demographic groups in the US, yet its 5-year survival rate has not improved for several decades. Despite advances in targeted therapy, immunotherapy and other novel adjuvant regimens, patients with locoregionally advanced and recurrent/metastatic disease continue to have extremely poor outcomes, underscoring the need for more effective treatments for OSCC, which often goes undiagnosed until it has reached late stages. Primary risk factors for OSCC include tobacco use, alcohol consumption, and for oropharyngeal cancers, HPV infection, but cancer incidence is influenced by additional elements such as anatomic site, patient demographics, and likely the individual’s oral microbiome. Given that cases of OSCC are increasing despite targeted head and neck cancer prevention efforts in the US such as smoking cessation and HPV vaccination, there is a strong rationale for exploring other modifiable risk factors that, together with new therapies, can improve outcomes for patients with OSCC as well as other aggressive head and neck cancers. The oral microbiome is a complex and dynamic community of commensal organisms that can become imbalanced (“dysbiotic”) in response to dietary intake, tobacco and alcohol use, and poor dental hygiene. Dysbiosis can pre-dispose an individual to oral disease, including cancer, by enriching for bacterial pathogens that promote carcinogenesis, secrete carcinogenic compounds, and promote chronic inflammation. Thus, reversing oral dysbiosis is a promising approach for protecting against tumorigenesis. The food additive nisin, which is bactericidal against a broad range of pathogens, has been shown to restore oral microbiome diversity, suppress inflammation, and stimulate anti-tumor cellular responses in vitro and in a polymicrobial mouse model of oral cancer, while maintaining its well-established safety profile. However, the potential clinical benefit of nisin for treating OSCC in humans has not been investigated. Here, we propose a Phase I/IIa trial to establish the tolerability and feasibility of administering nisin to OSCC patients who represent high-risk populations. In parallel, we will perform mechanistic studies of nisin and its effects on oral microbiome community structure, inflammasome expression, and anti-cancer cellular responses of the study participants. We will also analyze the emergence of nisin resistance among key oral bacteria, which could provide insight into circumventing nisin resistance in other clinical contexts. We hypothesize that nisin will be well-tolerated among OSCC patients and will counter dysbiosis by inhibiting bacterial pathogen growth and promoting an anti-tumorigenic environment via immunomodulation and anti-cancer cell activity. Our long-term goals are to validate nisin as a promising candidate for OSCC treatment and demonstrate that oral dysbiosis is a major driver of tumorigenesis in humans that can be manipulated, thus highlighting the important yet mostly unrecognized protective role that antimicrobials can exert against cancer in humans.

NIH Research Projects · FY 2025 · 2022-06

PROJECT SUMMARY/ABSTRACT The continuous discovery of new biological targets presents opportunities to dramatically improve our understanding of diseases and normal function and provides new avenues for treatment. In vivo imaging of these targets via positron-emission tomography (PET) is an especially powerful tool to understand the initiation and progression of disease and to aid in the development of novel therapeutics. The major benefits of PET are the very high sensitivity (enabling imaging of rare targets such as neuroreceptors without saturating them), and the ability to image deep tissues (which provides translatability from preclinical research to later clinical use). But the development of useful and validated tracers can take years or decades. A significant limiting factor is the complexity and cost of radiochemistry, and the difficulty in using current technologies to optimize synthesis conditions – a key step toward achieving reliable production with sufficient yield to support initial imaging studies. Slow throughput and high reagent and isotope consumption mean that optimization studies are very expensive and time-consuming, and thus such studies tend to be very limited and are unlikely to find globally optimal conditions. These limitations also create pressures in other aspects of new probe development, e.g., significant efforts are made to reduce the number of “hits” so only a very small number of compounds are labeled and studied via in vitro and ex vivo assays and in vivo imaging. However, this selection process is imperfect as it sometimes leads to the pursuit of dead-ends while it excludes promising candidates. To more rapidly leverage preclinical and clinical imaging of new biological targets, the radiochemistry field is in urgent need of new tools to improve the tracer discovery and development process. Our proposed solution is the development of high-throughput radiochemistry methods. Arrays of droplet reactions have recently been introduced as a way to rapidly perform dozens of reactions in parallel from a single batch of radioisotope, with total reagent consumption of those reactions similar to a single batch on a conventional system. Furthermore, the droplet reactions can readily be scaled to quantities for preclinical or even clinical imaging. These methods could be used to efficiently explore a vast reaction parameter space in a matter of days (instead of weeks to months), or they could be used to label dozens of candidate compounds in parallel to perform screening based on the most relevant metric: in vivo properties. While these reaction arrays, operated using manual pipetting, have revealed the benefits and potential of high-throughput radiochemistry, this new technology requires significant further development and automation to increase safety and speed, and reduce the chance for human error. We propose to (1) integrate in situ radiation detectors to quantify the radioactivity at various stages of each reaction, (2) integrate a method to automatically sample the reactions for analysis (radio-TLC or radio-UPLC), and (3) use high-throughput methods to optimize the synthesis of 5 neurotracers that currently have low yield, develop at least one novel tracer, and develop best practices for high-throughput optimization in radiochemistry.

NIH Research Projects · FY 2025 · 2022-06

Project Summary Generativity—defined as concern and care for the well-being of others, especially younger generations—is related to better physical and mental health in older adults. Despite the potential for generativity interventions to serve as an important method for improving health and well-being in this population, this is a highly understudied area of research. Furthermore, neurobiological mechanisms behind the effects of generativity on health and well-being have never been examined. As such, this proposal aims to fill these critical gaps in the literature. The goal of this NIA R01 is to investigate the effect of a writing-based generativity intervention on well-being and inflammation in older adults, as well as to examine underlying neurobiological mechanisms behind improvements. Participants (ntotal=200) will be randomly assigned to a 6- week intervention aimed at increasing perceptions of generativity (vs. a control condition). During pre- and post-intervention sessions, all participants will complete: 1) self-report measures of social, mental, and physical well-being, 2) a blood draw (in order to assess multiple markers of inflammation), and 3) a neuroimaging session (in order to assess the caregiving system as a potential neurobiological mechanism). It is hypothesized that participants in the generativity intervention, compared to those in the control condition, will show: 1) improvements in multiple domains of well-being, 2) improvements in biological markers of inflammation (e.g., decreases in pro-inflammatory gene expression), and 3) activation of the neural caregiving system (i.e., increases in neural activity in caregiving-related regions and decreases in threat-related neural activity). Furthermore, it is hypothesized that activation of the neural caregiving system will mediate observed self- reported improvements in social, mental, and physical well-being, as well as inflammatory activity. This study will fill a crucial gap in our understanding of the effect of generativity on well-being and inflammation, as well as the underlying neurobiology of these effects. Finally, these results may ultimately have large-scale public health implications, as they could inform a low-cost, low-effort method for improving health and well-being in older adults.

NIH Research Projects · FY 2026 · 2022-06

ABSTRACT Despite the prevalence and public health significance of depression, up to 40% of depressed adolescents do not respond to first-line antidepressants (i.e., serotonin selective reuptake inhibitors [SSRIs]). Adolescents with treatment non-response (TNR) are at high risk for physical and mental health difficulties associated with ineffectively treated depression, including cardiovascular disease and suicide. Thus, identifying the neurobiological mechanisms that underlie TNR in adolescents is a critical step toward optimizing treatment plans for those who do not respond to first-line treatments. In this context, sustained threat to social stressors, as measured by elevated inflammatory profiles to stressful stimuli, has been shown to drive the onset and maintenance of depression among adolescents and is associated with TNR. The mechanisms by which elevated inflammation impact the brain in depressed adolescents, however, are unclear. To address these gaps in our knowledge, we will test our central hypothesis that excessive glutamate (Glu) in depression-related corticolimbic circuits—including the anterior cingulate cortex, ventromedial prefrontal cortex, amygdala, and hippocampus—is a critical mediator between peripheral inflammation and TNR in depressed adolescents. Specifically, we will conduct a prospective 18-month study of 160 unmedicated treatment-seeking depressed adolescents (ages 14-18) using state-of-the-art multimodal neuroimaging data at 7 Tesla. At Time 1 (prior to SSRI treatment) and Time 2 (after an open-label 12-week SSRI trial), we will assess peripheral measures of pro-inflammatory cytokines and glutamate in corticolimbic circuits before and after a well-validated adolescent- version of the Trier Social Stress Test (TSST). We also will use a well-validated fMRI task designed to probe behavioral and neural responses to negative peer evaluation, a salient form of social threat for adolescents. At Time 1, we will test if TSST induces increases in inflammation and glutamate in corticolimbic circuits in unmedicated adolescents with depression. At Time 2, we will use machine learning methods to identify multi- level predictors of TNR based on behavioral, inflammatory, and neural indicators of sustained threat to social stress; we will also test whether glutamate in corticolimbic circuits mediates the association between baseline levels of inflammation and TNR. Finally, we will continue to clinically assess depression symptoms and collect information on social stressors (e.g., context, severity, duration) every 3 months for 15 months following Time 2 (i.e., from Time 3 to Time 7), which will enable us to use functional clustering analyses to identify subgroups of adolescents on the basis of depression trajectories (e.g., persistent depression, gradual remission, etc), and identify predictors of these subgroups and other related clinical outcomes (e.g., remission status), while accounting for the effects of TNR status and any changes in treatment (and other related factors, including stressful life events). Results from this work will motivate future studies testing alternative therapeutics for depressed adolescents at risk for treatment resistant depression.

NIH Research Projects · FY 2026 · 2022-05

Project Summary Sleep disruption during college presents a significant public health concern, with studies documenting clinically-significant sleep disruption in 40-60% of college students. Poor sleep contributes to rising anxiety, depression, and loneliness as well as declining positive affect, motivation, and sense of purpose faced by many students as they attempt to navigate a successful path through college. Disrupted sleep also negatively impacts physical health, in part through upregulating inflammatory processes that can have acute and more chronic effects on mental and physical health. In response, many colleges and universities have embarked on efforts to improve the sleep hygiene of their students. The challenge is to identify programs that can simultaneously improve sleep, be delivered at scale, and be easily completed by students. Mindfulness-based interventions (MBIs), including a six-week Mindful Awareness Practices (MAPs) intervention developed by our group, have been shown to improve sleep quality and associated psychosocial and biological outcomes among adults. MBIs are well-positioned between interventions targeting clinical insomnia (e.g., CBT-I) and mass-delivered sleep education programs, the latter of which have been rolled-out by many universities despite evidence of limited effectiveness. Only four published RCTs, however, have tested the effect of MBIs among college students and none targeted sleep as a primary outcome. To address this important public health problem, we propose to conduct a randomized controlled trial (RCT) of 240 first-year college students at a four-year university that serves an ethnically and economically diverse student population. Our two-arm, parallel group RCT will test the efficacy of the validated, group- based, six-week MAPs intervention vs. sleep education, an active time and attention matched control condition, for students who report poor sleep at this critical transition year. Effects will be assessed at post- intervention and at 3-, 9-, and 12-month follow-ups to assess persistence. Our project brings together a diverse team with expertise in sleep, mindfulness-based interventions, and youth development to pursue four aims: (1) determine effects of MAPs vs. sleep education on subjective and objective markers of sleep; (2) evaluate effects of MAPs vs. sleep education on negative and positive psychosocial symptoms associated with sleep disruption; (3) determine effects of MAPs vs. sleep education on inflammatory processes associated with sleep disruption and relevant for long-term health; and (4) explore potential sex and ethnic variations in intervention effects.

NIH Research Projects · FY 2026 · 2022-05

PROJECT SUMMARY/ABSTRACT The proposed project, “Mapping Cellular Resolution Connectopathies in Aging and Alzheimer's Disease,” will systematically and comprehensively characterize cell-type specific anatomical and molecular phenotypes across aging and Alzheimer’s disease (AD) in the entorhinal cortex (ENT): the ground zero of AD pathology. We will map cellular resolution, age-dependent morpho-molecular phenotypes of ENT projection neurons by performing single-nucleus RNA-sequencing and methylomic analysis in 2m, 9m, 18m APPSAA-(KI/KI) male and female mice. Novel genetic sparse labeling will be used to label and characterize morphology of ENT pyramidal neurons in different cortical layers in APPSAA-(KI/KI)/MORF3/Cux2-CreER and APPSAA-(KI/KI)/MORF3/Etv1-CreER mice. These studies will provide comprehensive data on how molecularly defined ENT neuronal cell types interact with age-, sex- and Aβ pathology to confer progressive transcriptomic/epigenomic, morphological, and synaptic deficits in vivo. In addition, we will map the age-dependent morpho-molecular phenotypes of ENT projection neurons in humanized Tau models, MAPT(H1)-GR*N279K and their MAPT(H1) controls. A combined single- nucleus transcriptomics and genome-wide chromatin accessibility assays will be applied to MAPT(H1)- GR*N279K and MAPT(H1) male and female mice at 2m, 9m, and 18m to define integrated transcriptomic/epigenomic ENT neuronal cell types, and to identify neuronal subsets undergoing age-dependent multi-modal molecular dysregulation in mutant “humanized” Tau mouse models. RNAscope multiplex in situ hybridization and GeoMX digital spatial profiling analyses will be performed to identify morpho-molecular types of neurons in ENT that are most affected in MAPT(H1)-GR*N279K knock-in mice compared to MAPT(H1) mice during aging. To identify age-related connectional vulnerabilities and to map connectivity disruptions in AD, we will systematically quantify changes of axonal outputs arising from genetically and connectionally defined ENT cell types using 2m, 9m, 18m male and female Cux2-CreER and Etv1-CreER mice. Cell-type specific connectivity disruptions also will be examined in two next generation AD mouse models, APP knock-in (APPSAA-KI/KI with wildtype controls) and MAPT(H1)-GR*N279K [with MAPT(H1) controls], across ages and in both sexes. Novel viral sparse labeling will be used to characterize age- and AD-related axonal dystrophy, while genetic sparse labeling in newly generated MORF3 mouse lines will help to identify local morphological changes in ENT cell types. Age- and AD-related morphological compromises to ENT input neurons will be studied along with their synaptic disruptions onto different ENT cell types. Finally, we will establish a cloud-based visualization platform to map the integrated molecular-anatomic circuit deficits of aging and AD to the Allen Common Coordinate Framework to facilitate dissemination and analysis of the data. Although the focus of the current project is the ENT, the pipelines established for data production, collection, and analysis can be scaled up to identify brainwide cell-type specific anatomic-molecular deficits in aging and other late-onset AD mouse models.

NIH Research Projects · FY 2026 · 2022-05

PROJECT SUMMARY/ABSTRACT Due to advances in geroscience, aging must be considered a modifiable risk factor for the major causes of death. Interventions targeting human aging are ongoing, but there is a lack of predictive biomarkers to measure the effectiveness of these interventions. With age, somatically-derived mitochondrial DNA (mtDNA) deletions clonally accumulate within individual cells across a variety of tissues. Attaining high intracellular abundance, mtDNA deletions disrupt oxidative phosphorylation, cellular function, and result in cell death. We hypothesize that human mtDNA deletion frequency predicts the risk of morbidity and mortality and responds to interventions that alter healthspan. We have developed a digital PCR assay that quantifies mtDNA deletion frequency using total DNA samples from any tissue in rodents and humans. This assay provides absolute quantitation, is amenable to a 96-well format, correlates strongly with the subsequent cellular phenotypes including cell death, and has a detection limit below one part per million. MtDNA deletion mutation frequency increases exponentially with age and this increase parallels the age-induced accumulation of dysfunctional cells, tissue degeneration, and mortality. This project will further develop and validate mtDNA deletion frequency as a measure of cell death in human aging. We are validating the test in accordance with FDA guidelines for bioanalytical assays. We are measuring mtDNA deletion frequency in a number of human tissues and biofluids across the human lifespan and will establish the relationship between mtDNA deletion frequency, chronological age, clinical and physiological outcomes, and interventions targeting human aging.

NIH Research Projects · FY 2026 · 2022-05

ABSTRACT Chronic pain is second only to bipolar disorder as the major cause of suicide among all medical illnesses, where prevalence of depression ranges between 30 to 80%. The importance of this negative affect is reflected in studies that show co-existing psychopathology increases pain intensity, pain-related disability and susceptibility for opioid use disorder. Allostatic adaptations caused by chronic opioid drug exposure, diminish reward, however, paradoxically, they reinforce drug-seeking behavior that contributes to the high rates of opioid misuse and development of opioid use disorder in chronic pain patients. One of the opponent processes to chronic drug administration is engagement of extra-hypothalamic stress systems, including increased activity of corticotropin-releasing factor and dynorphin within the extended amygdala (which includes the central nucleus of the amygdala, CeA). The extended amygdala integrates stress and reward systems to produce drug withdrawal-induced negative affective states. Additionally, the lateral CeA responds predominantly to painful stimuli being termed the ‘nociceptive amygdala’ and a circuit from the parabrachial nucleus to the CeA was recently shown to be involved in aversive learning of noxious (painful) stimuli. Dynorphin neurons are present in the lateral CeA, of which ~1/3 co-express corticotrophin releasing factor. This brain region has been implicated in both drug consumption and pain. For example, administration the kappa opioid receptor (KOR) antagonist nor-BNI into the CeA decreased excessive alcohol intake and chemo- genetic silencing CeA dynorphin neurons reduced alcohol drinking. KOR signaling in the CeA was also shown to contribute to chronic pain-induced aversion. Given the involvement of the CeA in aversive learning related to ongoing pain, and the involvement of these neurons in drug-seeking behavior, the primary aim of our application is to determine the extent amygdala KOR system contributes to increased drug-seeking behavior in chronic pain. We will use a mouse model of opioid intravenous self-administration focusing on a reinstatement paradigm that models relapse of drug-seeking behavior. This paradigm allows us to determine the extent KOR systems contribute to stress-induced reinstatement. Our central hypothesis is that chronic pain states lead to activation of KOR systems in the CeA that are involved in stress-induced reinstatement of opioid drug-seeking behavior. Aim 1 of the proposal will determine the necessity and sufficiency of CeA dyn-KOR system in negative reinforcement. Aim 2 will determine the sufficient and necessity of the CeA dynorphin/kappa opioid system in reinstatement of opioid place preference. Aim 3 will determine the extent stress-induced reinstatement of opioid self-administration is driven by the kappa opioid system in the CeA.

NIH Research Projects · FY 2025 · 2022-05

Project Summary The photoreceptor cilium is the most elaborate of all primary cilia. Its plasma membrane is extensively amplified to form a stack of disk membranes that contain a very high concentration of the visual receptor, opsin. This stack of disks forms the photoreceptor outer segment, and its organization is central to visual sensitivity and spatial resolution. The overall goal of the proposed research is to understand the cellular mechanisms involved in the morphogenesis of the disk membranes. The research plan is built on the application of molecular tools, with the use of advanced microscopy, including EM tomography and live-cell super-resolution microscopy, for high resolution, 3-D analysis. The long-term goal of the proposed research is to provide a molecular understanding of photoreceptor disk morphogenesis, including the mechanisms for delivering essential proteins to the site of membrane growth. The Specific Aims will address unknown aspects of the ciliary localization of opsin, and the roles of disk morphogenetic proteins in initiating lamellar growth, using both mouse rod photoreceptors in vivo and cultures of cells bearing unspecialized cilia. Our findings will provide understanding of a key area of photoreceptor cell biology. They will also be fundamental to our understanding of the pathogenesis of retinal disease that ensues from perturbations of disk membrane morphogenesis.

NIH Research Projects · FY 2026 · 2022-05

Project Summary Twenty million Americans suffer from peripheral nerve injury, which results in approximately $150 billion health-care expenses annually in the United States. Approximately half of patients treated with nerve grafts have an inadequate level of function. Twenty million Americans suffer from peripheral nerve injury, which results in approximately $150 billion health-care expenses annually in the United States. Among various factors, axon growth rate and the lack of reinnervation and neuromuscular regeneration are two major road barriers. In many cases, during peripheral regeneration, the muscle undergoes atrophy and becomes un-receptive to reinnervation, and even the sprouting axons that regenerate across the gap cannot form functional neuromuscular junctions (NMJs). While most of the previous studies focus on accelerating peripheral nerve growth, novel approaches to maintain the neuromuscular receptivity and delay the degeneration of muscle need to be developed and integrated. Therefore, an integrative approach that combines the acceleration of axon growth and the prevention of muscle degeneration is required to address this unmet medical need, and we propose to use multimodal electrical stimulation (ES) to achieve this goal. Our recent studies have shown that repetitive ES either at the proximal or distal stumps of transected nerve can be more effective than one-time ES to further improve the therapeutic outcome, but their relative contributions to and their combined effects on axon growth, the slow-down of muscle atrophy and reinnervation remain to be investigated. Therefore, to investigate the potential synergistic effects of proximal and distal ES, we hypothesize that programmable ES at the proximal and distal stumps of sciatic nerve following a transection injury can promote axon growth and maintain muscle receptivity respectively and synergize neuromuscular regeneration. We will test this hypothesis by developing a wireless, stretchable, bioresorbable and miniaturized system that allows repetitive ES with versatile protocols. To address the aforementioned challenges and test our hypothesis, we have assembled a multidisciplinary team, and performed pilot studies to demonstrate the feasibility. We propose three Specific Aims: (1) To develop and characterize a bioresorbable, stretchable and wireless bioelectronic device for repetitive electrical stimulation. (2) To determine how the time periods of repetitive proximal and distal ES regulate muscle functional recovery. (3) To investigate the combined effects of proximal and distal ES on neuromuscular regeneration. This proposed project is timely with the recent advancement in regenerative engineering, micro/nanotechnologies, miniaturized wireless point-of-care devices, and bioresorbable and stretchable electrodes. This innovative bioelectronic device provides a novel and minimally invasive approach for neuromuscular regeneration, and will have wide applications in regenerative medicine and therapy.

NIH Research Projects · FY 2026 · 2022-05

Project Summary In 1995, following the Institute of Medicine’s report, “Emerging Infections,” and in response to the CDC’s strategic plan to enhance surveillance, EMERGEncy ID NET was established. EMERGEncy ID NET's goal was to address the threat of emerging infectious diseases by assessing disease prevalence, risk factors, and management practices for acute presentations from the community among a diverse and underserved population of patients presenting to US emergency departments (EDs). A CDC cooperative grant has funded the network for the last 25 years. Due to the ability to prospectively collect clinical data and specimens for on-site laboratory analysis 24/7 from acutely ill patients from the community, EMERGEncy ID NET has been able to produce translational research that has impacted physician practices and informed public health policy. The research network demonstrated its capability to successfully address an urgent public health threat during the COVID-19 pandemic by rapidly implementing 20-site and 16-site public health surveillance projects of ED patient care-related infection risk and vaccine effectiveness among frontline health care personnel. Numerous peer-reviewed publications have resulted from EMERGEncy ID NET research, including in high-impact journals such as The New England Journal of Medicine, the Journal of the American Medical Association, Clinical Infectious Diseases, Emerging Infectious Diseases, and Annals of Emergency Medicine. Aims of EMERGEncy ID NET for the next 5 years are to: 1) identify emerging infections and risk factors for these conditions affecting US ED patients, including among underserved groups; 2) leverage EMERGEncy ID NET’s findings to create new collaborations to develop and improve diagnostic tests, treatments, and vaccines; and 3) disseminate results at national medical conferences, in high-impact journals, and on infectious diseases, public health, and emergency medicine social media outlets to inform treatment and public health policy, and educate the public. Support of EMERGEncy ID NET for the next 5 years will ensure that the network can continue to answer new questions about emerging infections and related areas of high public health priority.

NIH Research Projects · FY 2026 · 2022-05

Project Summary The somatosensory system permits us to perceive and react to the environment through modalities that include touch, nociception, thermosensation and proprioception. Somatosensory information is received peripherally and then relayed centrally by different populations of dorsal interneurons (dIs; dI1-dI6) in the spinal cord. Our research objectives are to understand the mechanisms that establish dIs during development and then apply these principles towards designing differentiation protocols to direct the formation of specific populations of dIs from pluripotent stem cells. These cells have the potential to repair damaged sensory circuits and act as substrates for drug screening platforms. We have focused on dissecting the role of the bone morphogenetic protein (BMP) family in dI fate specification. BMPs were widely assumed to act as morphogens, patterning the dorsal spinal cord in a concentration-dependent mechanism similar to the manner in which sonic hedgehog (Shh) patterns the ventral spinal cord. However, our recent studies using mouse, chicken, and mouse embryonic stem cell (mESC) models have found that no evidence that BMPs act as morphogens. Rather, BMPs have signal-specific activities, with differential abilities to direct dorsal progenitor (dP) patterning and/or differentiation through specific type I BMP receptors. Our recent in vivo and in vitro studies have also suggested that dI fates are established in a series of nested choice points. In our model, spinal progenitors are dorsalized by retinoic acid (RA), subdivided into multipotential dP subgroups by BMP signaling, and then resolve into specific dI fates. Since little is known about this patterning process, we will assess in Aim 1 how multipotential dP fates are first established by RA and BMP signaling and identify the mechanisms directing multipotential dPs into specific dI identities. In Aim 2, we will determine the nature of the intracellular response that permits specific BMPs to drive dPs towards different dI identities, addressing two unresolved questions: [1] are canonical receptor regulated (R)-Smads activated in a BMP-specific manner to result in distinct patterning activities? And [2] what factors do the R-Smads in turn regulate to promote dI fates? Together, these studies will investigate a long-standing problem in developmental biology, i.e., understanding how specific outcomes arise from a common signal, and shed light on the specification of dIs, cell types needed to permit paralyzed patients to again interpret their sensory environment. Our specific aims are as follows: Aim 1: Identify the mechanism(s) that establish multipotential dPs and assign them into individual dI fates. Hypothesis: RA±BMP4 direct the formation of multipotential dP subgroups, which resolve into specific dI fates by the asymmetric activation of additional endogenous signaling pathways, such as the Wnt pathway. Aim 2: Assess the role of the Smad and Id families in BMP-induced dI fate specification. Hypothesis: BMPs differentially activate Smad1 and/or Smad5, to regulate the activity of Id family members.

NIH Research Projects · FY 2026 · 2022-05

PROJECT SUMMARY Students in marginalized communities who `strive' to rise above adversity to achieve academic success are considered `resilient'. However, youths' resilience in one domain (i.e. academic) can come at a cost in other domains including physical and mental health morbidities that are under-identified and under-treated. Previous research suggests that Black, Indigenous and People of Color (BIPOC) who exhibit a “striving persistent behavioral style” in the face of adversity evince later health morbidities. Ironically, the same self- regulatory skills that promote academic achievement amid chronic stress can also result in physiological dysregulation that harms health and mental health1–3. Self-regulatory processes that involve emotion suppression, experiential avoidance, and unmodulated perseverance can culminate in allostatic load which fuels health disparities1,4 and internalizing symptoms of depression and anxiety5. The proposed mechanistic trial will utilize mindfulness training to permit examination of questions about the causal role of emotion regulation strategies linked to the striving persistent behavioral style in driving mental health and health morbidities among BIPOC78. The proposed Project STRIVE (STudents RIsing aboVE) will identify BIPOC students who are academically resilient in the face of disadvantage and will offer a tailored mindfulness intervention targeting self-regulation processes as a putative mechanism to interrupt the links between the striving persistent behavioral style and negative health outcomes. We propose a multisite randomized trial randomizing 504 high achieving, socioeconomically disadvantaged Black, Latinx and Asian American students in 18 schools to receive a mindfulness intervention or an attention control condition focused on study skills. The study will: (1) test the effects of the STRIVE intervention on putative self-regulation mechanisms (emotion suppression, experiential avoidance, and unmodulated perseverance) among. (2) test the effects of the STRIVE intervention on health and mental health outcomes at 12-month post-treatment, including biomarkers of allostatic load (cortisol, blood pressure, body-mass-index, waist/hip/neck circumference), health complaints, and internalizing symptoms, and (3) examine the mechanistic model linking striving persistent behavioral style and health outcomes within the STRIVE trial. If successful, this trial will build toward a scalable, secondary preventive intervention with potential for preventing both health and mental health disparities among underserved BIPOC youth at the population-level.

NIH Research Projects · FY 2025 · 2022-05

Abstract A global obesity epidemic is driving the concomitant rapid increase in the prevalence of cardiometabolic disorders (CMDs), including hypertriglyceridemia, type 2 diabetes (T2D), hypertension, and non-alcoholic fatty liver disease (NAFLD). Population- and sex-specific differences in CMD predisposition exist; however, the biological mechanisms underlying these differences are not well understood. Previous large-scale genome-wide association studies (GWAS) have reliably identified CMD-associated variants in multiple populations; however, functional understanding of the biological mechanisms of the GWAS variants remains challenging. One major obstacle is the limited knowledge of the relevant cell types in which GWAS variants affect gene expression. Bulk tissue gene expression data exist for CMD-relevant tissues, such as subcutaneous adipose, but these data exhibit considerable heterogeneity, including both cell type and cell state within each cell type. Subcutaneous adipose is an important human endocrine tissue for CMDs, and it is possible to collect high-quality adipose tissue samples from healthy individuals. However, the contributions of many adipose genes to CMDs and CMD traits are still poorly understood. The current lack of cell-type expression reference data sets limits fine-scale regional transcriptional assessment of GWAS variant effects. In addition, local expression quantitative trait locus (cis- eQTL) analyses are confounded by cell-type-specific expression differences, which hamper replication efforts across independent bulk RNA-sequenced (RNA-seq) cohorts. To address these knowledge gaps and identify genetic effects on adipose cell-type gene expression, we will perform single nucleus RNA-sequencing (snRNA- seq) in frozen subcutaneous adipose tissue biopsy samples from 300 well-characterized individuals, generate fine-scale estimates of cell-type proportions, identify study-wide and personalized cell-type-specific differences corresponding to cardiometabolic trait levels, and experimentally test GWAS variants for allelic effects on cell- type-specific expression. We hypothesize that by elucidating adipose tissue cell-type expression from 300 existing frozen adipose biopsies, we can leverage available GWAS and adipose bulk RNA-seq data (n=3,230; 45% female) from diverse populations to identify the relevant cell types for hundreds of CMD genes. In our preliminary studies, we have successfully performed snRNA-seq in frozen human subcutaneous adipose tissue biopsies and performed multi-omic studies integrating GWAS results with bulk adipose RNA-seq data, identifying hundreds of colocalized loci for CMD traits. Our approach will leverage the existing wealth of information available in GWAS and bulk adipose RNA-seq cohorts to elucidate the largely unknown cell types of biological mechanisms in the adipose tissue that drive CMDs. Success of the proposed study will substantially improve understanding of cell-type-specific transcriptional mechanisms of CMD diseases and traits in a key human metabolic tissue.