University Of California, San Diego

NIH Research Projects · FY 2025 · 2021-09

Neurodevelopmental processes are shaped by dynamic interactions between genes and environments. Maladaptive experiences early in life can alter developmental trajectories, leading to harmful and enduring developmental sequelae. Pre- and postnatal hazards include maternal substance exposure, toxicant exposures in pregnancy and early life, maternal health conditions, parental psychopathology, maltreatment, structural racism, and excessive stress. To elucidate how various environmental hazards impact child development, it is imperative that a normative template of developmental trajectories over the first 10 years of life be established based on a sufficiently large and demographically diverse sample of the US population. To accomplish this, the Healthy Brain and Child Development National Consortium (HBCD-NC) has been formed to deploy a harmonized, optimized, and innovative set of neuroimaging (MRI, EEG) measures complemented by an extensive battery of behavioral, physiological, and psychological tools, and biospecimens to understand neurodevelopmental trajectories in a sample of 7,500 mothers and infants enrolled at 24 sites across the United States (US). The HBCD-NC will carry out a common research protocol under direction of the HBCD-NC Administrative Core (HCAC) and will assemble and distribute a comprehensive and well-curated research dataset to the scientific community at large under the direction of the HBCD-NC Data Coordinating Center (HDCC). The overarching goal of the HBCD-NC is to create a comprehensive, harmonized, and high- dimensional dataset that will characterize typical neurodevelopmental trajectories in US children and that will assess how biological and environmental exposures affect those trajectories. A special emphasis will be placed on understanding the impact of pre- and postnatal exposure to opioids, marijuana, alcohol, tobacco and/or other substances. To address these broad objectives, the sample of women enrolled will include: 1) a racially, ethnically, and socioeconomically diverse cohort that is representative of the US population; 2) pregnant women with use of targeted substances (opioids, marijuana, alcohol, tobacco); and 3) demographically and behaviorally similar women without substance use in pregnancy to enable valid causal inferences. In addition, the HBCD-NC will identify key developmental windows during which both harmful and protective environments have the most influence on later neurodevelopmental outcomes. The large, multi-modal, longitudinal, and generalizable dataset that will be produced for the first time by this study will provide novel insights into child development using state- of-the-art methods. The HBCD-NC study will inform public policy to improve the health and development of children across the nation.

NIH Research Projects · FY 2025 · 2021-09

Modified Project Summary/Abstract Section The SPARC Knowledge Management and Curation Core (K-Core) is engaged with the Common Fund Data Ecosystem (CFDE) through a number of activities: 1) Continue to actively participate in and engage with CFDE; 2) Continue to participate in working groups and carry out Working Groups’ recommendations; 3) Contribute SPARC (i) resource metadata to C2M2 [2], (ii) develop and maintain necessary services to support data within the CFDE Portal (e.g. DRS services), and (iii) ensure users are able to access SPARC data in the cloud; and 4) participate in other CFDE projects as needed. Metadata and asset ingestion into the data resource portal will include the production of C2M2 data submission manifests and the upload of these manifests to the CFDE Workbench including specification of ontology mappings. This will also entail maintenance of the SPARC data submission export pipeline to support updates in SPARC metadata and any updates to the CFDE submission process. As the SPARC DRC is now also supporting data publication through other initiatives (e.g. NIH HEAL) we will also work with CFDE to determine best ways to annotate these data assets that have begun to be published. Additional asset ingestion into the data resource portal will continue to include knowledge graph assertions from the SPARC Connectivity knowledge graph (SCKAN). In addition, we will pilot the contribution of assertions from the Functional Connectivity Map – which is focused on biological compartments, their connectivity, their protein and small molecule constituents, as well as related biophysical models that study related processes (e.g., fluid flow, drug transport). This FC Map currently contains knowledge for ~300 drugs and~900 related gene products, and explicitly represents knowledge about relevant tissues, cells, fluids, protein and small molecules. We will generate RDF models of FC Map metadata, leveraging semantic standards adopted by the Ontology Working Group for integration with CFDE.

NIH Research Projects · FY 2025 · 2021-09

Project Summary Trichomonas vaginalis is the causative agent of trichomoniasis, the most common, non-viral sexually- transmitted disease, with 5-7 million cases in the U.S. and >200 million in the world each year. In addition to infections of the urogenital tract, trichomoniasis increases the risk of adverse pregnancy outcomes, HIV transmission, and cervical and prostate cancer. Only two drugs of the same class are FDA-approved for treatment, the nitro drugs metronidazole and tinidazole. Although generally effective, treatment failures occur in a substantial fraction of patients and the drugs have significant liabilities, with moderate to severe adverse effects and poor compliance due to seemingly benign but common side effects such as metallic taste. Given its prevalence, its association with multiple disease outcomes, and an increase in nitro drug-resistant strains, new antimicrobials against T. vaginalis are urgently needed, particularly in women where infection can persist for months or even years compared to generally less than ten days in men. In extensive preliminary studies, we determined that inhibitors of the proteasome, an essential cellular machinery for the degradation and recycling of cell proteins, kill T. vaginalis at sub-micromolar levels. Importantly, the inhibitors overcome nitro drug resistance and are efficacious in a murine model of trichomonad infection. We have also isolated and biochemically characterized proteasomes from T. vaginalis and human HeLa cells and found that they display significant differences in their peptide substrate specificity, providing the rationale for designing new potent and parasite-selective proteasome inhibitors. Based on these promising findings, the project has the overall objective to develop novel proteasome inhibitors with greatly improved potency and selectivity for the treatment of trichomoniasis. Using a hit compound with 50-fold selectivity, we will systematically develop T. vaginalis- specific proteasome inhibitors using a comprehensive combination of medicinal chemistry efforts, functional testing with multiple clinical strains of T. vaginalis, biochemical and structural investigations of the parasite proteasomes, and efficacy and toxicity testing in murine infection models. We have assembled a superb team of investigators with complementary expertise in parasitology, protease biology, antimicrobial drug development, and medicinal and peptide chemistry. The team has the experience and track record to conduct the critical pre-clinical studies to establish proteasome inhibitors as a new class of agents in the therapeutic armamentarium against trichomoniasis.

NIH Research Projects · FY 2025 · 2021-09

PROJECT SUMMARY/ABSTRACT Despite its overall decreasing occurrence, colorectal cancer (CRC) remains the fourth most common cause of cancer deaths in the US. Unfortunately, epidemiological studies demonstrate an alarming increase in inci- dence in populations below the age of 50, who are not routinely screened. Furthermore, CRC detection is difficult in high-risk groups, including those with a genetic predisposition (e.g. familial adenomatous polyposis), disease traits (e.g. inflammatory bowel disease), or from certain demographics (e.g. Black-Americans). Thus, there is a significant need for the development of innovative solutions for the early detection of CRC and the prevention of the transition from adenoma to CRC. To address this need, our interdisciplinary research team will develop genetically engineered bacteria using synthetic biology approaches to identify early CRC development, monitor and report changes in the adenoma and CRC microenvironment, and prevent cancer progression. To achieve the above objectives, engineered bacteria have to engraft and colonize the hostile luminal environment, sense and distinguish an abnormal environmental signal, compute this signal, and express a reporter or a therapeutic agent. However, appropriate vectors with these features remain lacking, constraining synthetic biology applica- tions for cancer research. Importantly, CRC is highly associated with E. coli, for which we have many synthetic biology tools. Furthermore, our preliminary proof-of-concept studies have revealed that native E. coli can be engineered to perpetually colonize fully conventional (i.e. non-microbiome depleted) hosts and to execute func- tions of interest, e.g., deconjugation of luminal bile acids. Deconjugated bile acid and resultant farnesoid X re- ceptor (FXR) agonism can suppress CRC development, indicating a potential therapeutic use of engineered native bacteria. Building on our strong supportive preliminary results, we will identify native E. coli from healthy, adenoma, and CRC tissues of a genetic model of CRC and engineer them to detect and treat CRC in response to the cancer microenvironment. Furthermore, we will characterize the effects of different tumor environment factors on the colonization and performances of engineered native E. coli in the colon organoid model in an organ-on-chip with the support of mathematical modeling, thereby identifying specific CRC signals for program- ming the responses of engineered native E. coli as CRC reporters and therapeutics. Finally, we will engineer native bacteria to detect and attenuate the progression of CRC by quantitatively reporting the level of CRC- related cysteine proteases and selectively inhibiting their activity. The research described in this proposal will generate new, much-needed synthetic biology vectors that can be developed as biosensors and therapeutics of adenoma and CRC, as well as many other diseases. Furthermore, this project will enrich our fundamental knowledge about the CRC-microbiome relationship and elucidate the roles of cysteine proteases in CRC pro- gression and treatment.

NIH Research Projects · FY 2025 · 2021-09

ABSTRACT It is projected that there will be more than 500,000 childhood cancer survivors (CCS) living in the United States by 2020. Despite advances in treatment, the majority of CCS will experience a chronic or severe condition as a result of their treatment known as a “late effect. There is substantial evidence about the long-term effects of psychological distress, impaired health-related quality of life (HRQoL), and late mortality for CCS. Despite these serious and long-term health challenges, engagement with recommended survivorship care declines over time in this at-risk population. Throughout treatment and into long-term survivorship, parents of CCS are highly involved with medical care and play a role in helping CCS transition from pediatric to adult-focused survivorship care. Emerging studies suggest that parent’s psychological distress is related to poorer CCS HRQoL, and that Hispanic/Latino CCS may be at higher risk for such adverse outcomes. These studies suggest that there may be contextual and cultural factors that influence outcomes for Hispanic/Latino families, such as acculturation, immigration status, and linguistic aspects. Given the documented impact of treatment on adverse outcomes such as comorbid conditions and care engagement, and the key caregiving role that parents play for CCS, examining the relationships between parents, CCS, and medical providers is critical to identify ways to improve long-term care engagement for young adult CCS, particularly those at high risk for poorer outcomes such as ethnic minorities. The premise of the proposed research is that multi-level factors contribute to disparities in HRQoL and survivorship care among diverse populations of adult CCS. During my dissertation research (F99 phase), I focus on how survivor-caregiver dyads manage cancer, share appraisal, and how collaborative management of cancer optimizes both parent and child mental health among an ethnically diverse population with a focus on Hispanic/Latino families. During the K00 phase, I will extend this research and training by developing expertise in understanding healthcare system and policy-level factors that contribute to disparities in cancer survivorship. Results from this proposal will yield important information about how individual, interpersonal, cultural, and healthcare system factors impact medical decision-making and psychosocial health among an understudied and high-risk cancer population. The proposed F99/K00 proposal is also a priority area of scientific research under the Childhood Cancer Survivorship, Treatment, Access and Research (STAR) Act and is aligned with current NCI funding for intervention research to support pediatric and adolescent and young adult cancer survivor and caregivers. Ultimately, completing this integrated training will allow me to apply the skills learned at a research-intensive university, where I am at the forefront of research to build healthier communities, and develop new ways to address challenging cancer-related health problems, especially among historically marginalized and underserved populations.

NIH Research Projects · FY 2024 · 2021-09

This proposal will elucidate the function of the exocyst complex and actin dynamics in lipoprotein metabolism and its regulation by insulin. Our preliminary data reveal that insulin stimulates the recycling of the LDL Receptor in hepatocytes to increase the delivery of LPL into cells. We hypothesize that insulin controls the activity of the small GTPase RalA in hepatocytes by two pathways involving phosphorylation and inhibition of its GAP protein, and recruitment of its GEF protein. Once activated, RalA can interact with components of the targeting exocyst complex, resulting in the tethering of exocytotic vesicles containing the LDLR at discrete regions of the basolateral plasma membrane that are enriched in machinery required for fusion. We also hypothesize that insulin regulates the dynamics of cortical actin to propel LDLR endocytosis. We will evaluate: i) the role of the exocyst complex, and its regulators RalGAP, RalGEFs and RalA in the regulation of polarized LDLR exocytosis in hepatocytes; ii) the role of changes in the cortical actin cytoskeleton in governing LDLR endocytosis; iii) the physiological relevance of these hepatic signaling and trafficking events to overall lipoprotein metabolism. These new ideas and approaches will elucidate the key elements in control of these trafficking itineraries, and may ultimately generate valuable insights into the molecular mechanisms underlying dyslipidemia in obesity and Type 2 diabetes.

NIH Research Projects · FY 2025 · 2021-09

Project Summary/Abstract Little is known about structure-function relationships in the human placenta. As pathologists, we can view the placenta only after delivery, and correlate our findings to clinical and prenatal course, as well as neonatal outcome. At best, however, we have lesions which correlate with disease, but have yet to be validated, either directly, through molecular analysis, or indirectly, through manipulation and functional analysis of in vitro models. Placental dysfunction, manifested clinically as preeclampsia (PE) with or without fetal growth restriction (FGR), is associated with histopathologic lesions of maternal vascular malperfusion (MVM) and fetal vascular malperfusion (FVM). These lesions differentially affect the three trophoblast compartments: cytotrophoblast (CTB, the putative stem cell), syncytiotrophoblast (STB, the cell type responsible for gas/nutrient exchange), and extravillous trophoblast (EVT, the invasive cell type at the maternal-fetal interface). Nevertheless, our understanding of these lesions is limited to morphology and immunolocalization of a few markers. Over the past few years, multiple groups, including ours, have used scRNAseq to characterize cellular heterogeneity within both normal and diseased placentae; however, these studies remain mostly descriptive, lacking both spatial context of the molecular data and functional evaluation of the distinct cell types. We therefore propose to apply novel technologies, including digital spatial profiling (DSP) and tissue decellularization followed by extracellular matrix (ECM)-specific mass spectrometry, as discovery-based approaches to better characterize specific MVM and FVM lesions at the molecular level. We will then use primary term CTB, to reproducibly model these phenotypes and to perform functional validation as part of a targeted evaluation approach. We will test the hypothesis that late gestation CTB respond to specific cell- and ECM-derived signals to proliferate and/or differentiate into either STB or EVT, thus identifying potential regenerative capacity in this unique transient organ. We will also probe the cellular and ECM origins of PE-associated placental dysfunction, testing the hypothesis that this disease originates from alterations in ECM composition and paracrine signaling from both placental and decidual (maternal) cells. Successful completion of this proposal will establish a detailed cellular and matrix atlas of the human placenta, with validated structure-function relationships, laying the groundwork for probing placental regenerative capacity in the setting of placental dysfunction.

NIH Research Projects · FY 2025 · 2021-09

SUMMARY HIV assaults deep tissues including the gastrointestinal (GI) and genitourinary (GU) tract within days after transmission to a new person. It then quickly and irreversibly changes the local immune environment and establishes a reservoir in resident cells. Our current understanding of the different mechanisms that allow HIV persistence in GI, GU, and adipose tissues and of how the local immune environment impacts HIV reservoir persistence and dynamics remains superficial, however. The rationale for this project is that understanding these processes will be important for HIV cure efforts, which have until recently largely ignored non-blood reservoirs, and to improve the health of persons with HIV (PWH) as they age. In response to RFA DK-20-023, we built a team led by Drs. Smith and Rivera-Nieves (Co-PIs with complementary expertise in virology, gastroenterology and mucosal immunology) with the objective to precisely define the contributions of various viral and host mechanisms of HIV reservoir renewal and persistence across NIDDK targeted tissues and blood, using the novel Last Gift rapid autopsy cohort. Our overall hypothesis is that HIV reservoirs persist in NIDDK targeted tissues and are differentially renewed by various cellular and viral mechanisms. To address these open questions, our study will collect and analyze NIDDK targeted tissues throughout the human GI and GU tracts and intra-abdominal and subcutaneous adipose tissue of altruistic PWH enrolled in the Last Gift cohort, an ongoing rapid autopsy study. Immune cells collected from these individuals before death will also be analyzed, in parallel, in order to facilitate comparison with prior work. Some participants (n=15) will remain virally suppressed until the time of death, while others (n=5) will choose to stop their antiretroviral (ARV) treatment before death. The proposed research is innovative because we propose to map HIV burden and activity in tissues with different immune and ARV environments (Aim 1), to determine the role of clonal expansion as a driver of HIV persistence during treatment and viral rebound after treatment interruption (Aim 2) and to develop an integrative/innovative phylogeographic Bayesian approach to jointly analyze virological and immunological data to unravel viral dispersal and reseeding across the body in relation to local environments. By analyzing these connections, we expect to reveal pathways and interactions that may differentially impact HIV associated inflammation. We believe our proposed study to be significant because this is a unique opportunity to provide new insights into the mechanisms of HIV persistence. Such findings would be important for the development of strategies aimed to thwart local HIV-associated inflammation, which is associated with HIV pathogenesis in the gut, genital tract and adipose tissues.

NIH Research Projects · FY 2025 · 2021-09

ABSTRACT Multiple myeloma is a fatal plasma cell neoplasm that is characterized by the malignant expansion of abnormal antibody-producing cells. Despite recent therapeutic advances, most patients eventually relapse. Such high- risk patients typically have an overall survival rate of less than one year. These poor clinical outcomes occur as a result of uncontrolled regeneration of malignant stem-like cells in protective, inflammatory microenvironments. However, the precise transcriptional determinants of myeloma stemness in the bone marrow niche remain poorly understood. One intriguing candidate is a key B-cell transcription factor and myeloma cell survival factor, interferon response factor-4 (IRF4). IRF4 also governs normal plasma cell development and immune responses to inflammation, however the extent to which IRF4 promotes malignant progenitor generation in lymphoid or plasma cell neoplasms is unknown. A 50% reduction in IRF4 expression can disrupt myeloma cell survival, but IRF4 remains undeveloped as a therapeutic target because transcription factors are difficult to inhibit using traditional small molecule strategies. To overcome this challenge, proof-of- concept studies were performed to evaluate novel RNA-targeted therapeutic agents that directly inhibit human IRF4 RNA expression and protein translation. Based on extensive preliminary data that characterized IRF4 expression and activity in clinically-relevant in vitro and in vivo models, the central hypothesis of this project is that inhibition of IRF4 will disrupt stem cell pathway activation in multiple myeloma and synergize with standard-of-care drugs to reduce overall disease burden and prevent malignant stem cell generation. In the context of three independent yet conceptually interrelated aims, this hypothesis will be tested in comprehensive molecular and cellular assays through a rigorous scientific approach. Aim 1: Interrogating the role of IRF4 in functional myeloma stem cell generation. This aim will determine the cell-intrinsic mechanisms that link IRF4 to myeloma regeneration. Aim 2: IRF4 inhibitor monotherapy and combination drug treatment in pre-clinical models of multiple myeloma. This aim will develop combination drug treatment and biomarker detection strategies that leverage selective IRF4 inhibition. Aim 3: Elucidating the bone marrow microenvironment-derived signals that promote IRF4 pathway activation in myeloma regeneration. This aim will elucidate the paracrine mechanisms underlying myeloma regeneration and the activity of a novel cancer therapeutic. The proposed research will set the stage for rapid clinical translation of more selective combination therapies for myeloma. The research team unites cancer and stem cell biology experts, myeloma clinicians, and collaborators who are highly experienced in the translation of novel therapeutics for cancer. Together, this project is positioned to advance the rapid clinical translation of IRF4 inhibitor therapy for myeloma. These investigations will also open up new avenues for targeting malignant stem cell generation in other cancers and inflammation-associated diseases.

NIH Research Projects · FY 2025 · 2021-09

Project Summary/Abstract Understanding the mechanisms of gene and chromatin regulation and their roles within a multicellular organism has relevance across many disciples such as synthetic biology, medicine, developmental biology and neuroscience 1–3. Large-scale efforts of the genomics community have identified many of the functional genes and gene regulatory elements (GREs) including recent atlases with the specific expression of genes and their putative regulatory regions within different cell types of complex tissues 4,5. However, it remains unclear how the 3D organization of chromatin impacts gene regulation and vice versa. To build a mechanistic understanding of the interplay between chromatin organization and gene regulation, we would ideally simultaneously measure all the key elements - DNA sequences, regulatory proteins, and the transcribed RNA - at the genomic-scale, while maintaining information about cell type identity. To address this challenge, I will develop an imaging platform that can simultaneously measure the 3D structure of DNA together with the RNA expression of the regulated genes and their interaction with key structural proteins (Aim 1). While this method can be applied to many systems, a particularly suited example is the peripheral olfactory system. Olfaction, one of the main mammalian senses, is controlled by the largest family of genes comprising more than 1000 olfactory receptors 6,7. Large networks of regulatory sequences interact across the genome to establish more than 1000 neuronal types, each expressing one and only one receptor8. I will apply this imaging method to address the longstanding question: how do different olfactory sensory neurons establish their receptor expression? These integrated measurements relating chromatin organization and regulatory protein structures to transcriptional activity will provide a model of olfactory gene regulation. Aim 2, is to dissect this model and the roles of GRE-promoter interactions in achieving cell-type specific expression using a high-throughput synthetic biology approach. I will infect the olfactory epithelium with large pools of viral vectors that combine different regulatory elements and promoters, and determine the precise cell-type expression of these vectors using multiplexed imaging. There is an additional synergy between the two aims - the first aim provides measurements of the endogenous chromatin structure-transcription relationship which will be used to design transgenic control of specific subpopulation of cells. I will explore this capability to activate/inhibit specific sub-populations of olfactory receptor neurons and determine the behavior consequences of these manipulations.

NIH Research Projects · FY 2024 · 2021-09

Project Summary/Abstract Cells of the immune system, bacteria, and neurons are constantly interacting on every surface of the human body. Recent studies show that bacteria have evolved mechanisms to engage with the nervous system, often using the phagocytes that regulate inflammation. Neuroinflammation, or inflammation in neurological tissue, is thought to contribute to common neurodegenerative conditions, including Alzheimer's disease and Parkinson's disease, although the basic mechanisms of neuroinflammation remain incompletely understood. The long-term objective of this New Innovator Award proposal is to define the molecular mechanisms of neuroinflammation during infection. The innovation is our adaptation of the cutting-edge zebrafish molecular toolkit (optogenetics, biosensors, in vivo biotinylation) to host-pathogen interactions. With this system, we can genetically modify host and pathogen, then observe bacteria invading the transparent zebrafish brain in real-time. As pathogens, we use mycobacterial species that activate neuroinflammation in humans (TB meningitis, leprosy) and in zebrafish. We have also developed new zebrafish brain infection models, using human pathogens isolated from meningitis patients, in order to determine: Project 1: How do bacteria invade the brain from the blood? Approach: Define the endothelial mechanisms of brain invasion and vascular injury. Project 2: How do host versus pathogen factors drive catastrophic brain inflammation? Approach: Optogenetic (light-activated) control of phagocytes in vivo. Project 3: How do host versus pathogen factors injure neurons? Approach: Identifying infectious mechanisms of neuronal injury using biosensors. To answer these questions, this proposal combines immunobiology and neurobiology tools with brain infection models in zebrafish. Using live imaging of zebrafish brain infection, and genetic modification of fluorescent bacteria, neurons, and phagocytes, the earliest inflammatory and neurodegenerative events can be directly observed in unprecedented detail. Completion of this work fulfills two goals. First, it will provide needed mechanistic information on the host cells and molecules that mediate injury in understudied brain infections that disproportionately affect people living in poverty. This will support the goal of developing new therapies that limit brain inflammation. Beyond infection, the basic biological mechanisms of neuroinflammation, revealed by infection, will likely be relevant to the many non-infectious neurodegenerative conditions that are characterized by neuroinflammation.

NIH Research Projects · FY 2025 · 2021-09

Project Summary RNA-binding proteins (RBPs) play key roles in regulating gene expression and many cellular functions. A lot of RBPs are aggregation-prone due to their low complexity, prion-like domains. While naturally-occurring aggregation of RBPs is important for the compartmentalized control of RNA metabolism, aberrant aggregation is detrimental and is associated with many diseases, in particular, age-related diseases such as neurodegenerative diseases and cancers. However, a systematic analysis of RBP aggregation and its functional consequences during aging remains lacking. Here we propose to conduct a systems biology analysis of age- dependent RBP aggregation using the replicative aging of S.cerevisiae as a model system. Our initial screen has identified positive RBP candidates that aggregate upon aging-related perturbations. Building upon these findings, we will combine innovative microfluidics with single-cell imaging technologies to systematically characterize these RBP aggregates during aging and to evaluate how these aggregates influence gene expression, aging phenotypes and the lifespan of individual living cells. In Aim 1, we will systematically characterize each of the identified RBPs that aggregate during aging. We will determine the biophysical and biochemical properties, material state and phase transition of RBP aggregates at different stages of the lifespan, which will provide important clues about how these aggregates influence cell physiology during aging. In Aim 2, we will investigate the interplay between RBP aggregation and cellular aging, focusing on how aggregation of RBPs is regulated by conserved aging-related pathways or factors, and how these aggregates contribute to age- dependent cellular changes and the final lifespan. In Aim 3, we will evaluate how RBP aggregation contributes to the proteomic changes during aging. We will use a newly-developed high-throughput microfluidic platform to identify target genes that are regulated by RBP aggregation and will examine their influences on aging, establishing the functional links among RBP aggregation, proteomic changes and aging phenotypes. Finally, we will integrate all the data generated in Aims 1, 2 and 3, delineate a systems-level regulatory network of RBP aggregation during yeast replicative aging, and develop a dedicated website for sharing the data to the scientific community. The RBP-regulated network will provide mechanistic insights into the causes, control and consequences of pathological RBP aggregation in aging and will be used to guide the design of new hypotheses and experiments, laying the foundation for the development of therapeutic and preventive strategies towards age-associated diseases.

NIH Research Projects · FY 2025 · 2021-09

SUMMARY This project will leverage emerging knowledge about the role of immunometabolism in the pathophysiology of depression in people with HIV to generate hypotheses about potential future treatments. Depression in PWH is characterized by features that differentiate it from depression in people without HIV (PWoH). The first is the predominance of anhedonia - inability to gain enjoyment from activities. The second is a higher prevalence of depression that is resistant to treatment with standard antidepressant drugs in PWH versus PWoH. These clinical observations suggest not only that the underlying pathophysiology of depression is different between the two populations, but that different treatments are needed to successfully treat depression in PWH. Molecular studies of immunometabolism, defined as reciprocal interactions between immunity and metabolism that are dysregulated and linked to depression in PWH, support these distinctions. Based on our preliminary data and that in the literature, we propose to perform RNA-Seq transcriptomics, relating immunometabolic gene expression to protein and other biomarkers of immunometabolism, and to clinical depression as well as to cognitive processes impacted by depression. Our hypothesis-driven approach will focus on two interacting, co-regulated gene pathways central to depression and immunometabolism, the mammalian target of rapamycin (mTOR) and the NLRP3 inflammasome. We will study 80 newly recruited PWH and 40 age- matched PWoH. Given our early work suggesting that the relationship between immunometabolism and depression is sex-dependent, we aim for a 50/50 breakdown of men and premenopausal women in order to examine sex differences in these relationships. To optimize the spectrum of depressive symptom severity in our sample (e.g., avoid over-representation of individuals with minimal depressive symptoms), we will stratify with a 50/50 breakdown above and below the previously established Beck Depression Inventory-II cut-score for clinically-significant depressive symptoms (≥16). Since clinical studies cannot rigorously establish mechanistic relationships, we will study in parallel the roles of mTOR and NLRP3 inflammasome signaling in depression-like behaviors in a mouse model of HIV infection, EcoHIV, which expresses seven of nine key human HIV proteins. These pathways will be dissected using mTOR and NLRP3 inhibitors. Also, in these animals we will characterize immunometabolic markers in brain tissue, presumably the substrate of depression.

NIH Research Projects · FY 2025 · 2021-09

ABSTRACT The goal of this proposal is to understand how loss of protein homeostats (proteostasis) in aging blood-forming hematopoietic stem cells (HSCs) applies a selective pressure that promotes leukemia initiation. Loss of proteostasis is one of the least understood hallmarks of aging, particularly as it relates to malignant transformation. As an organism ages, misfolded proteins can accumulate in post-mitotic cells, or in cells that are largely quiescent. This stresses the cell and can drive adaptive changes that are required to rebalance proteostasis. HSCs are particularly susceptible to a loss of proteostasis. Adult HSCs have low rates of protein synthesis relative to more frequently dividing lineage-committed blood progenitors. This helps maintain proteostasis by preventing the biogenesis of misfolded proteins, and it is required to maintain adult HSC self- renewal capacity. However, we have discovered that aged HSCs experience significant protein stress in vivo, and proteostasis must be actively maintained through changes in gene expression and stress-response pathways to sustain HSC self-renewal activity and longevity. In this regard, we have generated preliminary data demonstrating that Hsf1, a critical proteostasis sensor that dynamically remodels the proteostasis network in response to stress, is activated within aging HSCs where it is required to attenuate protein synthesis and preserve HSC self-renewal capacity. These data indicate that aged HSCs must actively maintain proteostasis to remain functional, and loss of proteostasis may create a selective pressure that promotes clonal hematopoiesis and leukemia initiation. Based on these data, we hypothesize that a loss of proteostasis and pressure to maintain proteostasis in aging HSCs promotes clonal hematopoiesis and acute myeloid leukemia (AML) initiation in older adults. We propose two aims to test this hypothesis. In the first aim, we will use Aarssti/sti mice, which have a defect in tRNA editing activity, to disrupt proteostasis in young and old adult mice. We will test whether proteostasis disruption accelerates clonal hematopoiesis and AML initiation during aging in the setting of Dnmt3aR878H and Tet2D/D mutations. In the second aim, we will test if normal age-related activation of Hsf1 creates a permissive context for AML initiation in aging HSCs. We will conditionally delete Hsf1 in young and old adult HSCs in the setting of a Dnmt3aR878H mutation with and without a cooperating NrasG12D mutation to determine if it contributes to the emergence of clonal hematopoiesis and increased incidence of AML in older adults. These studies will open new lines of investigation into a previously unappreciated link between age-related loss of proteostasis (a hallmark of aging) and leukemia initiation. Therapies that mitigate proteostasis dysfunction could preserve HSC clonal diversity later in life while reducing susceptibility to AML.

NIH Research Projects · FY 2025 · 2021-09

PROJECT SUMMARY 1 The Mirarab laboratory designs leading computational methods for answering biological and biomedical ques- 2 tions, focusing on scalability and accuracy. These methods span several areas (e.g., microbiome profiling, 3 multiple sequence alignment, and phylogenomics), and a common thread among them is evolutionary mod- 4 eling. The lab has developed scalable and accurate methods for reconstructing evolutionary histories (i.e., 5 phylogenies) and using these histories in downstream biomedical applications. Reconstructing phylogenies is a 6 fundamental goal and a precursor to many biological analyses. Methods developed by this lab (e.g., ASTRAL) 7 are at the forefronts of modern genome-wide phylogenetics. Moreover, biomedical research increasingly uses 8 evolutionary histories in diverse areas like microbiome analyses, immunology, epidemiology, and comparative 9 genomics. While the lab has previously focused more on inferring species histories, it has recently started 10 to shift its focus to developing methods for microbiome analyses. The inference and the use of evolutionary 11 histories in analyzing environmental microbiome samples present a unique set of challenges. 12 In the next five years, the Mirarab lab will focus on designing, testing, and applying improved methods for 13 statistical analyses of microbiome data. These methods will target two questions. (i) Profiling: What organisms 14 constitute a given sample? (ii) Association: How are samples different in their organismal composition, and 15 how do these differences connect to measurable characteristics of their environment? While both questions 16 have been subject to considerable research, many computational challenges remain, providing an opportunity 17 for better methods to make a significant impact. Instead of focusing solely on new algorithms, the lab will 18 also work on building better reference datasets and combining data from multiple sources. Thus, the project 19 aims to harness the unprecedented computational power, large available datasets, and recent advances in 20 machine learning to improve state-of-the-art dramatically. The project will not use off-the-shelf machine learning 21 methods in a black-box fashion. Instead, it develops methods that incorporate biological knowledge (e.g., of the 22 evolutionary relationships) into machine learning methods in a principled biologically-motivated fashion. 23 The lab will pursue several ambitious goals for both profiling and association questions. The project will 24 (i) create methods to infer a continuously-updated reference alignment and tree encompassing all sequenced 25 prokaryotic genomes (half a million currently) to be used for profiling, (ii) build methods for ultra-sensitive sam- 26 ple profiling, (iii) use deep learning to connect data obtained using amplicon sequencing and metagenomics, 27 (iv) build discordance-aware phylogenetic measures of sample differentiation, and (v) develop machine learning 28 methods for associating a profiled microbiome to phenotypes of interest such as disease. These new methods 29 will draw on statistics, machine learning, discrete optimization, and high-performance computing. Consistent 30 with the goals of MIRA, the project may explore new unforeseen opportunities if they fit its general goals.

NIH Research Projects · FY 2025 · 2021-09

PROJECT SUMMARY The UC San Diego MPRINT CET, entitled “Optimization of Antibiotics in Mothers and Their Breastfed Infants Using Pharmacomicrobiomic and Metabolomic Analyses”, brings together a team of highly experienced and proven collaborative investigators with leadership roles in maternal and pediatric clinical pharmacology, fundamental research methods and technologies. Across its highly integrated and synergistic components, the UCSD MPRINT CET addresses critical barriers in maternal-infant pharmacology regarding (1) the pharmacokinetics of infant exposure to maternal antibiotic treatment via breastmilk or close contact, (2) the impact of maternal antibiotic therapy or prophylaxis on establishment of the normal infant microbiome and gut metabolome, (3) potential downstream effects of such antibiotic exposure on infant immune function and hepatic cytochrome P450 drug metabolizing enzymes, and (4) the pivotal role of breast milk both as a conduit for antibiotic transfer and source of beneficial human/mammalian milk oligosaccharides (HMOs/MMOs) that may support microbiome and immune integrity in face of antibiotic stress. The successful operation and outcome of our MPRINT CET is accomplished through 3 Projects (Clinical, Basic Science and Data Science) an Administrative Core and two Technology Cores, the Milk Analytics Core (MAC) and Pharmacometrics and Analytical Chemistry Core (PACC). In the Clinical Project “Antibiotic Treatment in Breastfeeding Mothers: Effects on Milk, Microbiome, and Infant Outcomes”, we have proven expertise and infrastructure and access to a high enrolling maternal-infant clinical cohort to study how maternal antibiotics alter breast milk composition and impact infant outcomes in clinical meaningful ways. In the Basic Science Project “The Impact of Ampicillin and Breast Milk Oligosaccharides on the Infant Microbiome and Immune Functions”, we leverage extensive experience in mouse models of neonatal host-pathogen interactions to probe functional effects of ampicillin and MMOs on infant immune function, including a novel cross-fostering strategy with wild-type and MMO-deficient mothers. In our Data Science Project “Impact of Maternal Antibiotics on the Breastfeeding Infant Microbiome and Metabolome”, we deploy advanced MS technology, non-invasive sampling and innovative molecular networking analytics in a cutting-edge study of the impact of breast milk antibiotic exposure on the infant microbiome, metabolome and hepatic Cyp enzymes. The MAC provides milk collection protocols and kits, near infrared spectroscopy and HPLC, HMO/MMO and nutritional composition analysis, and new assay validation expanding our MPRINT CET analytical capabilities, while the PAC develops and validates novel quantitative assays and physiologic and semi-physiologic models to describe and predict maternal and infant antibiotic PK during breastfeeding. Our Administrative Core oversees integration and performance of our research projects/cores and their milestones, connecting them to the national MPRINT CET HUB and unique training/pilot projects.

NIH Research Projects · FY 2025 · 2021-09

PROJECT SUMMARY This application is in response to RFA-DK-20-003, which is a limited competition opportunity with the objective of establishing the Liver Cirrhosis Network (LCN). Cirrhosis has doubled in prevalence in the last decade and is now the 11th leading cause of death in the United States. The number of cirrhosis-related deaths is projected to triple by 2030, with NASH projected to overtake HCV as the leading cause of liver transplantations. Severe limits on the number of liver transplantations performed each year and anticipated increases in the cirrhosis patient population create an urgent need to better understand predictors of mortality in cirrhosis and develop effective therapies to treat cirrhosis. Studies from our group and others suggest statins may reduce future risk of HCC and decompensation in patients with cirrhosis. Among statins, lipophilic statins such as atorvastatin have shown the greatest chemopreventive effects against HCC occurrence. Atorvastatin is associated with dose-dependent reduction in incident cirrhosis in HCV patients. Data supporting statin use for cirrhosis are promising, but larger, randomized controlled trials (RCT) are needed to determine whether they may be routinely recommended. To address the needs of the cirrhosis patient population, this research plan proposes the following aims: Aim 1: To conduct a prospective, longitudinal, multicenter, multi-ethnic cohort study of patients with cirrhosis. Among Hispanic men, cirrhosis is the 6th leading cause of mortality. To address this health disparity, we will test the hypothesis that Hispanic patients with cirrhosis are at higher risk for hepatic decompensation compared to non-Hispanics. We will establish and monitor a cohort of patients who have cirrhosis due to multiple etiologies. Primary endpoints include (a) a composite endpoint of hepatic decompensation, (b) liver transplantation, or (c) all–cause mortality. Ancillary studies will investigate associations between non-invasive imaging biomarkers, cirrhosis genetic risk score, and risk of decompensation. Following up on our previous work, we will also identify a microbiome signature that may predict decompensation risk. Aim 2: Phase 2, multi-center, double-blind, placebo-controlled, RCT evaluating efficacy and safety of atorvastatin 20 mg in subjects with compensated cirrhosis. The objective is to test the hypothesis that atorvastatin is more effective than placebo in reducing risk of decompensation, all-cause mortality and other liver-related clinical outcomes in cirrhosis patients. Primary outcome measure will be time to the first occurrence of any of the following adjudicated events: all-cause mortality, MELD score ≥ 15, liver transplant, ascites requiring medical intervention, hospitalization for onset of variceal bleeding, hepatic encephalopathy, spontaneous bacterial peritonitis, and development of hepatocellular carcinoma. Secondary outcomes include safety of atorvastatin, decrease in fibrosis, as measured by NITs and imaging biomarkers, and major adverse cardiovascular events. Exploratory analyses will investigate associations between treatment response and (a) delta MRE and VCTE, (b) genetic risk factors, and (c) changes in the gut microbiome.

NIH Research Projects · FY 2024 · 2021-09

ABSTRACT Myosteatosis is a measure of body composition, which measures the presence and degree of fatty infiltration into the muscle compartment to include deposition around the muscle fibers/bundles, as well as into the muscle tissue itself. Our work on this measure of ectopic fat has shown myosteatosis to be strongly and significantly associated with measures of glucose regulation, independent of visceral and hepatic adiposity. Given the strong links between the glucose dysregulation (i.e. metabolic syndrome and/or diabetes mellitus) and arterial stiffness, as well as left ventricular structure and function, we believe the study of myosteatosis may be particularly relevant as a determinant of heart failure. Compared to non-Hispanic Whites, Hispanic/Latino Americans have significantly higher rates of obesity, cardiometabolic risk factors and type 2 diabetes mellitus (T2DM), resulting in an increased risk for heart failure. To assess the risk factors for different chronic diseases in this burgeoning population, the NIH commissioned the Hispanic Communities Health Study – Study of Latinos (HCHS-SOL). In 2007, this study began recruitment at four different field centers and enrolled over 16,000 Hispanic/Latino Americans from diverse background heritage groups. The HCHS- SOL has now completed two clinic visits and is scheduled to begin enrollment for visit 3 in December 2019. At this visit, the study will update the medical history, obtain fasting blood samples that will be used to measure glucose, insulin and hemoglobin A1C, and will acquire a 2-hour glucose tolerance test. Additionally, the study will conduct the following assessments on the returning participants: 1) objectively measured physical activity and sedentary behavior; 2) peripheral arterial disease by the ankle and toe brachial indices, as well as Doppler waveforms and pulse volume recordings; 3) arterial stiffness by carotid femoral pulse wave velocity and 4) cardiac structure and function by magnetic resonance imaging. This ensemble of measurements can be utilized to assess the pathway from physical activity/sedentary behavior to body composition to cardiometabolic risk to arterial stiffness to cardiac structure and function. Given this, we propose to leverage the existing HCHS-SOL infrastructure and add the acquisition of new computed tomography scans of the abdomen and mid-thigh to obtain measurements of myosteatosis and skeletal muscle mass, as well as subcutaneous, visceral and hepatic adiposity. The goal of this project is to determine the applicability of myosteatosis to the aforementioned pathway among Hispanic/Latino Americans from diverse background heritages, independent of other measures of body composition.

NIH Research Projects · FY 2025 · 2021-09

Project Summary/Abstract Driven by new and emerging technologies for data acquisition, integrative genomic studies are revolutionizing the way we approach the study of cancer and charting the way to novel treatment regimens. They require the high-throughput generation and analysis of multiple and often complex types of genomic data. To address this challenge, and in active collaboration with end users, we developed, and released in 2008, a broadly applicable Integrative Genomics Viewer (IGV). IGV is an interactive, high-performance, user-friendly tool launched millions of times a year by investigators and clinicians worldwide. The goal of this project is to ensure that IGV continues to track and respond to advances in genomics technology and changing user needs, and maintains its high level of utility for the cancer research community. We will accomplish this through our three specific aims. Aim 1. Evolve IGV to keep pace with the needs of the cancer genomics community. We will continue to evolve IGV to leverage recent and future advances in genomics technologies and methods that are key to moving cancer research and treatment forward. We will add new visualizations and features to better enable interpretation and validation of complex variants from both short and long read data. Through collaborations with other projects we will add new capabilities to better assess and interpret single nucleotide and structural variants. Responding to IGV’s increased use for review of variants in patient tumors, we will enhance support for this task. Aim 2. Maintain IGV to ensure continued high levels of utility, usability, and reliability. We will continue to provide frequent software releases to address feature requests, bug reports, and other suggestions from IGV’s community of both users and developers. Based on community input, we will review the IGV user interface to improve usability. We will address the challenges and opportunities presented by the evolving computing and software technology environment. We will continue to maintain the code base and downloadable installers and update them for new releases of Java, JavaScript, external libraries, browsers, and operating systems. Aim 3. Support the IGV user and open-source developer communities. We will continue to provide a high level of support including rapid responses to help requests; in-depth documentation on software use; short videos to walk users through tasks with high utility; training materials and exercises. For our vibrant, open-source developer community that contributes to the IGV code base and uses IGV in their own applications or resources, support includes: documentation and code examples for integrating and extending IGV; guidance on new development through our GitHub forums; prompt review of contributions; and acknowledgement of their work. We have extensive software engineering experience, developing and distributing IGV and other software tools used by hundreds of thousands of biomedical researchers and clinicians worldwide. IGV’s success, user-driven development approach, collaborative tradition, and flexible architecture make us well poised to accomplish our aims to further transform data visualization and accelerate the pace of biomedical discovery.

NIH Research Projects · FY 2025 · 2021-09

PROJECT SUMMARY/ABSTRACT Pancreatic beta cells secrete insulin in order to maintain blood glucose homeostasis. Insulin secretion is tightly regulated by glucose and modulated by numerous environmental signals, including other nutrients, hormones, and inflammatory cytokines. Exposure of beta cells to environmental signals affects gene regulatory programs within hours, and these signal-dependent changes serve to adapt insulin secretion to changes in organismal states. Genetic variants associated with measures of insulin secretion are strongly enriched in genomic elements active in beta cells, and many of these variants are also associated with risk of diabetes. Beta cells therefore possess characteristics that make them an ideal cellular model for studying signal-dependent gene regulatory processes relevant to human health and disease. However, the specific genomic programs that drive signal- induced state changes in beta cells remain poorly characterized. Recent advances in the development of human pluripotent stem cell (hPSC)-derived multi-cellular islet organoid models by us and others provide a genetically tractable beta cell model for linking genomic activity to cellular phenotypes. Our group has further pioneered the development of numerous single cell assays, including chromatin accessibility, ultra-high-throughput paired chromatin accessibility and gene expression, and paired 3D chromatin interactions and DNA methylation; methods that we have successfully applied to both primary human islets and hPSC-islet organoids. We have further developed machine learning and network-based approaches for variant interpretation including from single cell RNA and epigenetic data. In this proposal we will develop novel gene regulatory network (GRN) models to predict network-level relationships among genomic elements, genes, and phenotypes derived from single cell multiomic maps charting signal- and time-dependent changes in hPSC-islet organoids. In Sections B and C we will measure genomic element activity, gene expression, and insulin secretion in hPSC-islet organoids exposed to ten different secretory signals each across four time points using paired single nucleus accessible chromatin and gene expression and paired single cell DNA methylation and 3D chromatin architecture assays. In Section D we will generate a GRN from these data, use machine learning to infer element-gene and element-phenotype relationships and use the trained models to refine the GRN. From the resulting GRN we will predict the effects of genetic variants in specific genomic elements on target gene expression, gene network activity, and cellular phenotype. In Section E we will validate and refine models by using medium-scale CRISPR interference of genomic elements individually and in combination as well as allele-specific gene editing of selected glucose-associated variants in hPSC-islet organoids and measuring gene expression changes in cis and trans. Together, the results, data, and methods from this project using a model of a cell type which both rapidly responds to environmental signals and has a quantifiable phenotypic output will be widely applicable to the community studying the dynamics of genomic regulation.

NIH Research Projects · FY 2025 · 2021-09

SUMMARY Age-related osteoporosis affects millions of American men and women, and is a major cause of fractures in those over age 50. Bone loss is due mainly to impaired bone formation, but FDA-approved agents with bone- anabolic effects (PTH analogs and the sclerostin-neutralizing antibody romosozumab) have serious limitations. We have found that pharmacologic or genetic stimulation of the NO/cGMP/protein kinase G (PKG) signaling pathway enhances bone formation and prevents bone loss in diabetic and aged mice; the mechanism is through increased Wnt/β-catenin signaling and reduced oxidative stress. Silent information regulator T1 (Sirtuin1, SIRT1) and its substrate NAD+ decrease with age, contributing to many age-related diseases, including osteoporosis. Osteoblast-specific Sirt1 knockout mice are osteoporotic while Sirt1 overexpressing mice have high bone mass. We found recently that PKG activation increases SIRT1 expression in osteoblasts, mesenchymal stem cells (MSCs), and bones of aged mice by stabilizing Sirt1 mRNA; the RNA binding protein hnRNPA1 is required. SIRT1 is known to increase NO synthesis, possibly leading to a positive feedback loop with PKG. Supporting positive feedback between PKG and SIRT1, we found they synergistically increase pro-survival genes in osteoblasts and prevent oxidative stress-induced apoptosis. As part of our interest in NO/cGMP/PKG signaling, we have developed nitrosyl-cobinamide (NO-Cbi), a NO donor that is also a strong anti-oxidant. Recent work shows that a 10 week course of NO-Cbi improved bone formation, architecture, and strength in 24 month-old mice. The overall goal of the proposed studies is to define the interactions between PKG and SIRT1 in bone, and devise a novel treatment strategy for age-related osteoporosis based on PKG and SIRT1 activators. In Aim 1 we will define how PKG increases Sirt1 mRNA in MSCs and osteoblasts, focusing on PKG regulation of hnRNPA1. In Aim 2, we will determine the consequences of PKG-SIRT1 crosstalk in bones of aged mice. We will determine whether PKG requires SIRT1 to improve bone formation and strength by examining the effects of PKG-activating agents on bone in conditional osteoblast-specific Sirt1 knockout mice. To determine if SIRT1- induced NO synthesis and PKG activation is necessary for the positive effects of SIRT1 activators in bone, we will examine the skeletal effects of these agents in mice with conditional deletion of Prkg2 in osteoblasts. In Aim 3, we will test whether combining PKG- and SIRT1-activating agents can synergistically increase bone formation in aged mice, compared to each treatment alone. We will use NO-Cbi and the guanylyl cyclase activator cinaciguat to activate PKG, and direct SIRT1 activators and nicotinamide riboside, a NAD+ precursor, to activate SIRT1. The proposed work will fill important knowledge gaps, defining the role of PKG-SIRT1 crosstalk in bone during aging and determining how PKG-SIRT1 reduce age-related oxidative stress in bone; importantly, the proposed work lays the foundation for developing novel bone-anabolic therapies for age-related osteoporosis.

NIH Research Projects · FY 2025 · 2021-09

SUMMARY The basal ganglia are a group of subcortical nuclei that regulates motor and cognitive functions. Recent identification of neuronal heterogeneity in the basal ganglia suggests that functionally distinct neural circuits defined by their molecular identity and efferent projections exist even within the same nuclei. This distinction may account for a multitude of symptoms associated with basal ganglia disorders such as Parkinson's disease (PD). However, our incomplete understanding of the basal ganglia functional organization has hindered further investigation of individual circuits that may underlie distinct behavioral symptoms in different disease states. The external globus pallidus (GPe) is a central basal ganglia nucleus that can influence numerous downstream regions. While the prevailing circuit model assumes that the GPe is a homogeneous population of neurons transferring the signal in the indirect pathway of the basal ganglia, accumulating evidence suggests that neurons in the GPe are more heterogeneous than previously appreciated. Although GPe is known to be a nucleus with GABAergic neurons, we have identified novel cell types expressing VGLUT2, glutamatergic neuronal marker, at the outer layer of GPe. In our careful anatomical and molecular examination showed that VGLUT2GPe neurons project mainly to inner part of GPe, making synaptic contacts onto other neuronal populations. Recent evidence showed that the distinct cell types in GPe may have different roles in modulating basal ganglia circuitry and associated behaviors. Thus, elucidating the anatomical and functional organization of VGLUT2GPe neurons will provide novel cellular and circuit information to understand basal ganglia function. The progressive nature of behavioral deficits associated with PD is very well documented in human patients. However, what neural adaptations associated with behavioral deficits at different stages of PD are not fully understood. In this application, we try to address this with two different animal models. First, as in our preliminary results and recent reports, we will administer different doses of neurotoxin administration to induce different degrees of DA neuronal loss, which elicit the different behavioral deficits. Second, we will confirm the neurotoxin- induced PD-related behaviors in MitoPark mice which show the progressive loss of DA neurons. Examining the circuit adaptation in two animal models will provide an important information on the neural mechanisms underlying the progressive nature of PD. Therefore, using cutting-edge techniques including optogenetic, genetic and viral-mediated manipulation, in vivo multi-unit recording, and so on, we will decipher roles of VGLUT2GPe neurons in behavioral deficits in these two animal models for PD.

NIH Research Projects · FY 2024 · 2021-09

PROJECT SUMMARY RNA-binding proteins (RBPs) interact with RNA molecules from synthesis to decay to control their metabolism, subcellular localization, stability and translation. Methods for transcriptome-wide detection of RBP-RNA interactions provide insights into how RBPs regulate gene expression programs and how RNA processing is disrupted in disease state. Despite their association with disease and although the importance of regulating gene expression is well appreciated, only a small fraction of the over 2,000 RBPs identified thus far have known RNA targets and molecular roles. Commonly, immunoprecipitation-based technologies coupled to high throughput (Illumina) sequencing, such as RNA immunoprecipitation (RIP) and Crosslinking Immunoprecipitation (CLIP), and ribosome profiling are used to identify RBP targets and binding sites across the transcriptome. However, these experimental protocols are labor-intensive, require large amounts of input material, are not adaptable to high-throughput workflows. To overcome these limitations, we develop a novel technology, reagent resource, experimental protocols and a computational framework, that we collectively term STAMP (Surveying Targets By APOBEC-Mediated Profiling), for detecting RBP-RNA targets and translation at the single-cell and single- molecule level. In preliminary data we demonstrate, for the first time in the field, discovery of RBP-RNA sites and translation states at single-cell resolution. We anticipate that STAMP can be used reliably to identify RNA targets, binding sites and even extract motifs from a few cells to a single cell, thus effectively increasing limits of detection over current methods by several orders of magnitude. Combined with simultaneous RNA-seq analyses, STAMP will enable the combined identification of RBP binding sites and global measurement of gene expression, a long- standing goal for the gene expression, genomics and RNA communities. As a corollary, even without single cell analyses, STAMP can accept ultra-low input material which enables rare cell-types to be collected and analyzed for RBP-interactomes. By applying STAMP to ribosomal proteins, we extend this approach for single-cell detection of ribosome association while simultaneously measuring gene expression. Our conceptual and technological innovations will, for the first time, enable translation efficiency and RBP-interactomes to be measured at single-cell level and at scale, opening up new paradigms of biological questions.

NIH Research Projects · FY 2025 · 2021-09

ABSTRACT Herpes Simplex Virus 1 (HSV1) can establish both lytic and latent infections in a cell type-specific fashion with known and emerging neuropathological ramifications, respectively. Provocative data now link reactivation of latent HSV1 infection to Alzheimer’s disease (AD), the etiological basis of which remains incompletely defined. Here we propose to employ powerful new genomic technologies to identify and characterize the actual cell types that harbor latent and reactivated HSV1, extending recent findings that have revealed an increased abundance of herpes virus transcripts in affected regions of human AD brains. Using a modified single-nucleus sequencing approach, which allows for DNA accessibility and global transcription to be assessed in the same nucleus, we will interrogate human control and AD brain samples as well a HSV1-infected brain organoids and mouse models of acute and progressive HSV1-induced neurotoxicity. These studies promise to reveal cell type-specific enhancer landscapes and transcriptional profiles consequent to lytic, latent, or reactivated HSV1 in the brain while also providing insights into the cell autonomous versus non-cell autonomous effects of its presence. In addition, we propose to elucidate a novel innate immune pathway by which HSV1 lytic transcripts trigger the sentinel kinase PKR to initiate a cascade of nuclear events that include the secondary activation of the transcriptional regulator PARP1 and culminate in a NF-kB-dependent inflammatory gene expression program, potentially providing a molecular mechanism by which occasional HSV1 reactivation in the brain could contribute to an inflammatory milieu that promotes the pathogenesis of AD. Furthermore, this molecular pathway may underlie diverse microbial and possibly non-microbial inflammatory triggers in the brain that have been implicated in AD. We also hypothesize that HSV1 latency-associated transcripts (LATs) have distinct and opposing genomic functions as well as non-genomic actions in host neurons and possibly non-neuronal brain cells, the balance of which preserves neuronal cell integrity but may facilitate low-grade, chronic inflammation in the context of latent infection irrespective of viral reactivation. Based on enticing preliminary evidence, we propose to investigate the idea that the sense (S) and antisense (AS) LATs impact transcription in a partially dichotomous fashion by associating with specific regulatory elements in the HSV1 and host genomes in collaboration with the KRAB zinc-finger protein (KZFP) co-regulator KAP1. We hypothesize that these genomic events influence the AD process by affecting neuronal function through modulation of KZFP-mediated regulation of human endogenous retrovirus (HERV) repeats. We further hypothesize that the LATs have a complementary non- genomic role that mitigates the innate immune response and suppresses cell death programs, at least in part, by inhibition of PKR. Finally, we propose to exploit these protective properties of the HSV1 LATs as a unique prophylactic strategy for AD.

NIH Research Projects · FY 2024 · 2021-09

Project Summary/Abstract This project addresses the role of astrocyte bone morphogenetic protein (BMP) signaling in the pathogenesis of Fragile X Syndrome (FXS). FXS is the most common inherited form of intellectual disability (ID) and autism spectrum disorder (ASD). FXS is caused by trinucleotide repeat expansion in the FMR1 gene promoter leading to transcriptional silencing, and FXS is most often modeled with the Fmr1 knockout (KO) mouse. Although research has implicated several pathways mediating the effects of the Fmr1 loss of function, most targeted treatments have failed in clinical trials, and FXS is predominantly treated by symptom only. The majority of research in FXS has focused on intrinsic changes within neurons. However, emerging research in FXS implicates astrocytes, specifically through astrocyte-secreted factors. Wild-type (WT) neurons cultured with astrocytes or astrocyte-conditioned media (ACM) from Fmr1 KO mice exhibit stunted neurite outgrowth and recapitulate the immature dendritic spine phenotype observed in vivo in Fmr1 KO mice and human FXS patients, providing direct evidence for a causal role of astrocyte-secreted factors in FXS. Preliminary data profiling FXS astrocyte transcription and protein secretion identified four proteins both overexpressed in mRNA and oversecreted, one of which is BMP6. Furthermore, activation of BMP signaling in WT astrocytes generates over a third of the protein secretion changes of FXS astrocytes, while abrogation of BMP signaling in FXS astrocytes abolishes neurite outgrowth deficits. The goal of this proposal is to test the hypothesis that BMP signaling in astrocytes is upstream of neurodevelopmental FXS deficits in vivo and to identify the astrocyte-secreted proteins that mediate this effect. A combined genetic and viral approach to selective knock out Bmpr2 or Smad4 in astrocytes will be used to assess whether downregulation of astrocyte BMP signaling can rescue in vivo FXS abnormalities in dendritic spines, plasticity, and behavior. An astrocyte-specific in vivo proteomic approach combined with characterization of specific proteins in vitro will identify proteins downstream of BMP signaling responsible for FXS deficits. These experiments will determine if BMP signaling in astrocytes mediates FXS deficits in vivo and elucidate mechanisms by which it occurs, thereby providing new insight into a previously underappreciated aspect of FXS pathophysiology. The proposed research will take place in the Allen Laboratory at the Salk Institute for Biological Studies, a collaborative research environment that provides access to all necessary equipment and training. Through theoretical and practical training in molecular neurobiology, collaboration with supporters of diverse research and clinical backgrounds, and a research team committed to mentorship, the proposed research training plan will enable rigorous instruction in research and lay the foundation for a future career as an independent physician-scientist.