University Of California, San Diego

NIH Research Projects · FY 2025 · 2017-07

Abstract Anesthesiology is an interdisciplinary medical specialty requiring its practitioners to have a broad fund of knowledge, including pharmacology, physiology, and engineering and the ability to apply this knowledge in both controlled and emergency situations. In addition to providing trainees with exceptional clinical knowledge and skills, the Anesthesiology Department as well as the larger academic community at the University of California, San Diego (UCSD), provides a world-class environment for high quality training in research. Two particular basic science research strengths at UCSD include the Departments of Pharmacology and Engineering. Anesthesiologists including both basic scientists and clinicians have productive interactions and collaborations with faculty in these two disciplines. Thus, the primary goal of this renewal T32 grant application remains to train the next generation of clinician scientist in the Department of Anesthesiology to address important questions in the discipline by leveraging the potential interdisciplinary training available in collaboration with faculty in pharmacology and engineering. These interactions serve as guidance for our fellows; however, fellows with their mentoring team have the ability to work beyond, within, and in larger collaboratives to undertake research at the broadest level to involve as many disciplines necessary to adequately address the questions asked. This goal will continue to be facilitated by approaching primary questions in anesthesia with an interdisciplinary training focus; however, the program will remain flexible to expand research scope as the interests of fellows develop. This training grant will provide post-graduate trainees (MDs or MD/PhDs) with a 2-year research experience consisting of a broad focus on the basic molecular mechanisms of drug action and/or exploration of a variety of engineering disciplines. Emphasis will be on defining novel mechanisms of drug action for therapeutic interventions, applying engineering principles to create devices, model physiology and pathophysiology using mathematical and theoretical approaches, investigate biomaterials to facilitate non-invasive monitoring, and generation of biometric and blood derived biological data, among other projects to enhance human health and the patient experience. To achieve this overall goal, the training experience has two specific aims: i) expose fellows to a culture of science and investigation present in the Department specifically and the campus broadly and ii) achieve competency in the allied elements of a research career, e.g. experimental design, data analyses, presentations, manuscript preparation and publication and how best to obtain research funding as well as lab management and job interview skills. We strongly emphasize collegiality and ethics in the research environment. In achieving our strong commitment to the development of the clinician scientist, our overriding mission is the training of the next generation of clinician scientists who have the foundation of knowledge and fundamental tools coupled with the passion and commitment to affect translational research.

NIH Research Projects · FY 2025 · 2017-07

PROJECT SUMMARY/ABSTRACT This proposal aims to determine how interoceptive nutrient detection modulates chemosensory responses and innate behavior. Protein-rich foods are widely considered beneficial to postpartum mothers by supplying energy and cellular building materials, or improving moods by acting as precursors for neurotransmitters. However, it remains unclear whether the nutrient’s restorative effect can also arise from other means: specifically, whether and how dietary amino acids can trigger signaling events to modulate neuronal function and behavior. To address these complex questions, it is necessary to dissociate the multifaceted functions of dietary proteins in neuromodulation or nutrition. Gut enteroendocrine cells, found in both flies and mammals, present excellent opportunities to address these questions, because these cells release peptide hormones in a nutrient-specific manner and, with modern genetic tools, can be specifically manipulated to release their hormones without ingestion of the nutrients. Using fruit flies as a model, this project leverages the powerful genetic toolkit and tractable nervous & gut enteroendocrine systems of D. melanogaster. Preliminary data showed that high-protein diet can accelerate receptivity recovery in mated females. At the molecular level, a subset of enteroendocrine cells exhibits heightened sensitivity to amino acids, thereby increasing the likelihood of releasing a specific peptide hormone from those cells upon protein ingestion. At the neuronal level, high-protein diet heightens the sensitivity of two types of olfactory receptor neurons (ORNs) to external social cues. Importantly, the specific gut-released peptide hormone is necessary for the ORN sensitization as well as the restorative effect of high protein on postmating receptivity. This proposal will test the hypothesis that mated females employ a “gut-to-nose” signaling pathway whereby dietary amino acids trigger the release of a peptide hormone from the gut to regulate sensitivity to social cues and restore critical innate behavior. The proposed experiments will characterize the regulation and function of the gut-released peptide hormone (Aim 1), identify the molecular mechanism by which the specific enteroendocrine cell subset responds to amino acids (Aim 2), and determine the role of peripheral chemosensory neuromodulation on receptivity recovery (Aim 3). The mechanistic insights expected from this research will shed light on how macronutrients impinge on particular internal physiological states to influence neuronal function and behavior—in a manner beyond nutrient assimilation or energy supply. Importantly, the proposed research reveals that nutrient responses of gut enteroendocrine cells can be modulated by mating status, and that the outcome of this modulated interoception (elevated release of a peptide hormone by dietary proteins) can in turn impact exteroception of social cues (ORNs’ sensitization to pheromones).

NIH Research Projects · FY 2026 · 2017-07

PROJECT SUMMARY New strategies to combat pneumonia caused by different pathogens are urgently needed. Inflammatory macrophages play an essential role in clearing bacteria, fungi, and viruses during infections; however, hyperinflammatory responses mediated by these cells can cause severe side effects, including death. Immunotherapy that modulates macrophage polarization has shown promise in suppressing hyperinflammatory responses while retaining the capability of macrophages to clear pathogens. To achieve successful immunotherapy with RNA therapeutics, the following obstacles must be overcome: (1) rapid clearance of RNA therapeutics by RNase in tissues; (2) poor cellular uptake of free RNA therapeutics; and (3) loss of RNA therapeutics to non-infected tissues and potential off-target side effects. We previously discovered and successfully transitioned to industry two nanotherapeutic systems that addressed these limitations for bacterial infections. The elements of the nanosystems relevant to the present proposal are: high loading capacity for RNA therapeutics; an ability to protect the RNA payload from degradation in vivo; and highly selective targeting of the macrophages via pendant peptides. The proposed project hypothesizes that this approach may be generally applicable across a spectrum of pathogens, and it aims to investigate treatment of viral and fungal pulmonary infections. To address these goals, a deeper understanding of macrophage function and nanoparticle interactions is needed, particularly in the context of pathogenic infections. Through three Specific Aims, we propose to optimize and then evaluate three major nanoplatform-based systems that have shown promise for nucleic acid delivery, and investigate the in vivo biological interactions of the targeted nanoplatforms to obtain deeper understanding of macrophage polarization in combating pulmonary infections: (1) Develop a targeting strategy for macrophage homing in infected lungs. We hypothesize designs that will allow the nanoparticle to reach the infected regions of the lungs while preserving the potency of the RNAi therapeutic, either by i.v. or by direct pulmonary delivery of nanoplatforms. This Aim will focus on screening for new peptides that target macrophages in lung infection models, using the existing macrophage-targeting peptide CRV as a benchmark. We will focus on well-established mouse models of pneumonia induced by carbapenem-resistant K. pneumoniae, A. fumigatus, and influenza A. (2) Develop, evaluate, and then downselect from three broad classes of nanoplatforms (i.e., lipid nanoparticles, tandem peptide nanoparticles, and fusogenic porous silicon nanoparticles) to load RNA therapeutics. These nanoplatforms will be targeted to macrophages in the infected lungs using peptides from Aim 1. Cytotoxicity, gene knockdown efficiency, and macrophage polarization will be evaluated in vitro. Pharmacokinetics including macrophage targeting and tissue distribution will be studied in vivo. (3) Evaluate leading candidate(s) from Aim 2 for therapeutic performance in vivo through intranasal or nebulizing administration. The goal of this Aim is to evaluate biosafety and therapeutic efficacy (i.e., pathogen burden clearance, tissue recovery, and improved survival). The significance of this project is that it will yield tools to actively target macrophages at infected lungs and it will identify the essential design rules for nanoplatforms that can provide immunotherapy to combat a wide range of pulmonary infections.

NIH Research Projects · FY 2025 · 2017-06

Project Summary: The effort to identify functional non-coding sequences has largely focused on the regulatory elements such as enhancers and non-coding RNAs. However, while ~70% of the human genome is epigenetically quiescent, i.e. not marked by any epigenetic modification indicating functional inactivity, the majority of somatic mutations associated with diseases occur in the non-coding regions. An emerging question is why and how somatic mutations in epigenetically quiescent loci would affect cellular functions and cause disease formation. A possible mechanism is that the mutations in these loci may alter the neighbor chromatin organization and such a change is propagated through the 3D genome to generate a profound impact on phenotype. Although this hypothesis is tempting, such non-coding loci have not been widely identified, characterized and analyzed in the human genome. In the proposed work, we aim to integrate computational prediction and CRISPR library screen to systematically uncover non-coding loci that do not host any functional element and are not even marked by histone modification, protein binding or open chromatin but are important for cell survival (Specific Aim 1 and 2). We will investigate the mechanisms of how the deletion of these loci lead to cell dysfunction using Hi-C, single cell RNA-seq and single ATAC-seq analyses (Specific Aim 3). Once completed, the proposed work will provide a new aspect of understanding the functions of epigenetically quiescent non-coding loci and facilitate developing genome-editing based therapeutics targeting these loci.

NIH Research Projects · FY 2026 · 2017-06

Summary/Abstract The overall vision of our research is to gain a comprehensive understanding of the molecular mechanisms driving the function of a major brake to cell survival signaling, protein kinase C (PKC), and its negative regulator, the PH domain Leucine-rich repeat Protein Phosphatase (PHLPP). The PKC family has been intensely investigated in the context of cancer since the discovery in the early 1980s that it is a receptor for the tumor-promoting phorbol esters. This led to the dogma that activation of PKC by phorbol esters promotes carcinogen-induced tumorigenesis. Nonetheless, PKC has been an elusive chemotherapeutic target despite decades of research. In 2015 we reversed a major paradigm by showing that PKC generally suppresses, rather than enhances, oncogenic signaling. This proposal aims to 1] understand the downstream substrates and molecular mechanisms by which PKC isozymes brake oncogenic signaling and 2] establish ways to restore PKC in cancer. Furthermore, we aim to understand the regulatory mechanisms of its negative regulator, PHLPP, which we discovered in a targeted search for a phosphatase that would dephosphorylate a conserved site on PKC and related kinases such as Akt. PHLPP functions both as a tumor suppressor and as an oncogene and whereas much is known about its substrates, downstream signaling pathways, and function, comparatively little is known about its own regulatory mechanisms. This proposal aims to understand the structure and regulatory mechanisms of PHLPP in order to inhibit target-specific roles of PHLPP, especially as a way to restore PKC. The overarching challenge of the proposed research is to fill gaps in our knowledge of the molecular mechanisms governing the regulation of, and signaling by, PKC and PHLPP in order to leverage this understanding to develop effective therapies when these mechanisms are disrupted in disease.

NIH Research Projects · FY 2026 · 2017-06

Project Summary Alcohol associated health problems are a major medical burden in industrialized countries. Patients with alcohol-associated liver disease show intestinal bacterial dysbiosis and increased intestinal permeability. Although there is considerable progress in understanding the interaction between the host and intestinal bacteria, the role of the intestinal fungal microbiome (also called mycobiome) in alcohol-associated liver disease is not very well understood. Results from our laboratories indicate a proportional increase of Candida albicans (C. albicans) and Malassezia restricta (M. restricta) in patients with alcohol use disorder. Results from chronic ethanol administration in mice or chronic alcohol abuse in patients show that C. albicans-specific T cell responses occur in the intestine. CD4+ T cells re-circulate to the liver, where they re-activate by translocated C. albicans antigens, produce interleukin 17 (IL17) and contribute to progression of ethanol-induced steatohepatitis. In addition, products from M. restricta translocate from the gut lumen to the systemic circulation and liver. M. restricta induces liver inflammation via ligation with the Dectin-2 (Clec4n) receptor on Kupffer cells and augments ethanol-induced liver disease in mice. The testable central hypothesis of this proposed collaborative and multidisciplinary research application implicates disturbances in the gut fungal mycobiota as an important etiological factor in the modulation of adaptive and innate immunity in the liver. Through the proposed study, we will characterize the host gut mycobiome and immune response in a human cohort. We will mechanistically test our hypothesis in a mouse model of ethanol-induced liver disease. Towards this goal, we will use pharmacological interventions, supplementation of fungi and genetically modified mice. We predict that two pathogenic factors contribute to dysfunction of the gut-liver axis in alcohol-associated liver disease: C. albicans overgrowth drives Th17 cell expansion contributing to liver inflammation and damage (Aim 1). Binding of M. restricta to Dectin-2 induces hepatic inflammation and exacerbates alcohol-associated liver disease (Aim 2). We believe these studies will provide important insights into alcohol-mediated changes of the intestinal mycobiome that result in an immune response contributing to alcohol-associated liver disease. Eventually this approach might lead to new therapeutic targets for patients with alcohol-associated liver disease.

NIH Research Projects · FY 2025 · 2017-05

PROJECT SUMMARY Chromosome missegregation or errors in cytokinesis produce aneuploidy, a chromosome content other than a multiple of the haploid number. The linkage of aneuploidy to tumorigenesis has long been recognized. A striking chromosomal abnormality linked to chromosome missegregation is chromothripsis (also known as chromoanagenesis), an event in which one (or two) chromosomes appear to have been shattered into tens to hundreds of small genomic fragments and religated back together in random order. Chromotriptic chromosomes are now recognized to be present in a broad range of cancers. With support from an NIGMS R35 grant, we have identified mechanisms of normal chromosome segregation that act to prevent aneuploidy in the normal situation and have determined that single chromosome missegregation or transient spindle pole amplification is a driver of tumorigenesis. We have identified the epigenetic mark of centromere identity and determined that DNA replication acts as an error correction mechanism to maintain that identity. We have identified key molecular mechanisms underlying the mitotic checkpoint (also known as the spindle assembly checkpoint), the primary guard against chromosome missegregation in mammals. We have identified how both mitotic checkpoint activation and silencing involve the catalytic action of a conformation altering AAA+ ATPase TRIP13. We have also determined that mitotic exit has an absolute requirement for TRIP13-mediated disassembly of the checkpoint inhibitor or the non-essential APC15 subunit of the E3 ubiquitin ligase that targets mitotic cyclin destruction. By exploiting a unique feature of the human Y centromere, we have produced cells in which we can induce selective, transient inactivation of the Y centromere, with the Y chromosome missegregated into micronuclei at high frequency. With these and whole genome sequencing, we determined that simple missegregation into a micronucleus can initiate chromothripsis and drive the complex genome rearrangements frequently found in human cancer. In the upcoming 5 years, we propose to determine mechanisms of fragmentation of a chromosome during chromothripsis, identify and validate nucleases that fragment micronuclear chromosomes, determine how shattered chromosomes are reassembled and produce extrachromosomal DNA (ecDNA), determine mechanisms of inheritance of ecDNA, and determine the role of spatial proximity in the inheritance of centromere identity, including neocentromere formation and other genomic abnormalities. We will also exploit our development over the last 15 years of antisense oligonucleotide (ASO) therapy for nervous system disease to undertake proof of principle therapy development targeting inactivation of the mitotic checkpoint by testing suppression of TRIP13/APC15 for the major brain cancer glioblastoma.

NIH Research Projects · FY 2025 · 2017-04

Abstract This research project focuses on the fibrinolysis system and its activity in regulating innate immunity. We have shown that tissue-type plasminogen activator (tPA) functions as an antagonist of pro-inflammatory responses triggered by Toll-like Receptors (TLRs) in macrophages and in vivo in mice. Mechanistically, the activity of tPA is mediated by a receptor system that includes the N-methyl-D-aspartate receptor (NMDA-R) and LDL Receptor- related Protein-1 (LRP1). Although the function of enzymatically-active tPA in innate immunity is regulated by the Serpin, PAI1, and by plasmin generation, enzymatically-inactive tPA (EI-tPA) is resistant to these regulatory pathways. EI-tPA fails to inhibit pro-inflammatory responses mediated by Pattern Recognition Receptors (PRRs) other than TLRs; however, in mouse models of disease in which multiple PRRs function in concert, including the Dextran Sulfate Sodium (DSS) colitis model and the K/BxN serum-transfer arthritis model, EI-tPA is efficacious as a candidate therapeutic. In this application for continued support, we propose studies to elucidate the activity of tPA and its receptors in regulating inflammation and determine the potential to generate novel anti-inflam- matory drugs based on the structure of tPA. Four specific aims are proposed. In Specific Aim 1, the structural elements in tPA required for regulation of innate immunity will be determined by genetic engineering. Novel recombinant derivatives of EI-tPA will be developed and tested with the goal of optimizing tPA for use as a candidate anti-inflammatory therapeutic in vivo. Specific Aim 2 is focused on understanding the anti-inflam- matory cell-signaling pathway activated by tPA downstream of the NMDA-R. New preliminary results implicate a novel system, involving Trk receptor transactivation by Src family kinases, which is previously undescribed in macrophages and other inflammatory cells. The proposed studies in Specific Aim 2 will not only contribute to our understanding of tPA signaling in general but also may identify novel intracellular targets for anti-inflam- matory drug development based on our analysis of tPA-activated cell-signaling. In Specific Aim 3, we will breed mice available in our laboratory to generate animals in which macrophages and other cells in which the LysM promoter is active do not express the NMDA-R. This will allow us to definitively test the hypothesis that the anti- inflammatory activity of EI-tPA in vivo, for example in neutralizing LPS toxicity, requires the NMDA-R and that macrophages and/or neutrophils are EI-tPA target cells. In Specific Aim 4, we will study the activity of tPA and its anti-inflammatory receptors, the NMDA-R and LRP1, in the DSS colitis model. Single-cell transcriptome profiling studies are proposed to identify, in an unbiased manner, colon cells targeted by EI-tPA in vivo and identify novel pathways by which EI-tPA elicits a favorable response in this model system. Collectively, the studies proposed herein should further our understanding of the interface between hemostasis and immunity and further efforts to mine the fibrinolysis system for novel anti-inflammatory drug development.

NIH Research Projects · FY 2025 · 2017-04

Abstract The purpose of the NIDA Animal Genetics Program is to identify genetic, genomic, epigenetic variants, physiology and brain functions that contribute to addiction-like behaviors, related behavioral endophenotypes, and behavioral comorbidities to substance use disorder. During the past four years, our multidisciplinary and highly collaborative consortium has been identifying gene variants that are associated with increased vulnerability to compulsive-like cocaine use by performing the first GWAS using an advanced model of chronic intravenous cocaine self-administration in N/NIH heterogeneous stock (HS). We have also created the first preclinical cocaine biobank which enables researchers who do not have the resources to perform chronic intravenous self-administration or next-generation genome sequencing to perform advanced genetic, molecular, and cellular studies to further our understanding of the biological changes underlying addiction-like behaviors. While these efforts have been very successful in achieving the planned milestones, it has become clear that our project would benefit from an even larger sample size. In particular, increasing sample sizes lead to exponential rather than linear increase in the number of loci identified. Moreover, in the past four years there has been tremendous technological advances in behavioral and genetic analysis that can be leveraged to provide unprecedented access to identify the single nucleotide and structural variants that contribute to complex behavioral endophenotypes of high relevance to cocaine use-disorders. The first goal of this competing renewal is to double the sample size of the current GWAS to increase the number of gene variants identified and meet the demands of the Biobank. The second goal is to use high-throughput behavioral phenotyping using markerless pose estimation based on transfer learning with deep neural network to identify behavioral endophenotypes that can help predict and identify individuals with a resistant, mild, moderate, or severe phenotype of cocaine addiction-like behaviors. The third goal is to use methodological improvements of the genetic analysis, including the analysis of structural variants and tandem repeats, as well as enhanced integration with gene expression data. The fourth goal is to strengthen the cocaine biobank infrastructure. This project is likely to continue having a sustained and powerful impact on the field because it will provide an exponential increase in the number of genetic loci identified, eQTLs and PheWAS analysis related to addiction- like behavior; establish the first high-throughput behavioral motifs analysis of addiction-like behaviors using parallel video-recording and automated machine learning analysis; identify novel behavioral endophenotypes of vulnerability/resistance to addiction-like behaviors; and expand and improve the Cocaine Biobank offering and infrastructure.

NIH Research Projects · FY 2025 · 2017-03

Project Summary The objective of this study, “Diagnostic Innovations in Glaucoma Study (DIGS): Glaucoma and High Myopia”, is to overcome barriers to the detection of open angle glaucoma (OAG) in individuals with high myopia (mypOAG). In 2010, there was an estimated 1.4 billion people worldwide with myopia and the prevalence is rapidly rising to an estimated 4.75 billion by 2050. Moreover, persons with high myopia are 2.5 times more likely to have OAG than those without high myopia. It is unclear why myopia increases the risk of OAG, but it is likely related at least in part to biomechanical factors; longer axial lengths in myopic eyes may result in deformation of the lamina cribrosa, temporal displacement of Bruch's membrane, parapapillary changes and vascular factors; these all lead to increased susceptibility of the optic nerve to OAG damage. Given the higher prevalence of tilted discs and peripapillary atrophy in myopic eyes, the structural and functional tests that usually guide treatment decisions are of diminished value. This proposal will provide essential follow-up to establish best practices for patient-centered detection of OAG progression in the challenging high myopia population. Specifically, this proposal will 1) identify optic nerve head (ONH) 3D morphologic parameters from optical coherence tomography (OCT) scans (segmented and unsegmented) to differentiate between myopia eyes with and without progressive OAG; 2) optimize change detection using novel OCT features (e.g. texture and microvasculature) from wide field of view (WFOV) maps merged from individual ONH and macula scans; and 3) develop novel longitudinal and multimodal deep learning (DL) models to predict OAG progression. Most importantly, we will improve our understanding of the complex temporal relationship between structural, functional and microvascular age- and OAG related changes in a diverse cohort across the range of myopia. Specifically, in Specific Aim 1 (To improve our understanding of the complex relationship between ONH morphology and structural, functional, and microvascular change in the aging and OAG eye), we address several hypotheses related to the characterization of myopic ONH morphology in healthy eyes with and without high myopia. We hypothesize that ONH morphology is predictive of age – and OAG related structural, functional and microvascular changes and that it is predictive of fast progression. In Specific Aim 2 (To improve detection of OAG progression in myopic eyes using WFOV maps, unsegmented 3D volumes, ONH morphology), we address several hypotheses designed to detect and predict OAG progression using novel DL approaches. In Specific Aim 3, we will establish a cloud-based pipeline for data curation and computation that will facilitate secure DL model development and extensive data sharing with the vision research community.

NIH Research Projects · FY 2026 · 2017-03

The NLRP3 inflammasome is a multi-protein cytoplasmic complex functioning as a pattern recognition receptor that has emerged as a key regulator of sterile inflammation and cell death. NLRP3 activation leads to secretion of mature IL-1β and IL-18. Caspase 1 activation can also initiate a distinct form of programmed cell death (pyroptosis) mediated via processing of Gasdermin D and formation of discrete pores in the plasma membrane. Using single cell RNA transcriptomics (scRNA-seq) we demonstrated that time-controlled, inducible global NLRP3 inflammasome activation results in 1) shifts in liver macrophages characterized by depletion of KCs and infiltration of monocyte-derived macrophages (MdMs); 2) liver infiltration with proinflammatory neutrophils; 3) increased activated myofibroblastic HSCs; 4) an enhanced role for NLRP3 inflammasome driven by IL-18; 5) These changes are associated with chronic inflammation, increased collagen deposition and development of liver fibrosis. Based on our published and novel preliminary data we propose the CENTRAL HYPOTHESIS that NLRP3 inflammasome regulated changes in the innate immune cell niche in the liver is a central mechanism that triggers myofibroblastic HSC activation, and liver fibrosis driven by IL-18. To investigate this hypothesis our proposal has following SPECIFIC AIMS. FIRST, we will determine the mechanisms and consequences of KC depletion induced by NLRP3 activation. We will test the hypothesis that NLRP3 inflammasome activation in KCs leads to pyroptotic cell death and the recruitment of MdMs to the liver. We will use a number of unique, genetically engineered mice. To study the effects of KC-specific loss of function of NLRP3 during NASH we will use our new floxed Nlrp3-/- mice on a fat-fructose-cholesterol diet that mimics human fibrotic-NASH. In vivo experiments will be complemented with ex vivo assays using purified KCs, iPSCs and sc- RNA transcriptomics. SECOND, we will dissect the role of cell-specific IL-18 signaling pathways in NLRP3 mediated HSC activation and liver fibrosis. We will test the hypothesis that NLRP3 inflammasome activation in neutrophils and MdMs, followed by IL-18 signaling pathway activation is a central driver of HSC activation and liver fibrosis. We will characterize the role of IL-18 signaling and identify novel downstream mediators responsible for liver fibrosis. We will generate floxed IL-18, or IL-18R1, or IL-18BP murine lines to study IL-18 signaling in a cell- specific manner using neutrophil or macrophage-specific, NLRP3-driven models. We will also assess cell-specific modulation of NLRP3/IL-18 signaling in murine NASH-associated fibrosis in vivo. Finally, we will investigate translational mechanisms targeting the IL-18 pathway in fibrotic liver disease.

NIH Research Projects · FY 2025 · 2017-02

Project Summary: This project develops cell and animal models to understand the role of a multivalent transcription factor, ZNF423, in integrating information from extracellular signaling and intracellular lineage pathways during hindbrain development. ZNF423 encodes a constitutively nuclear transcriptional regulatory protein that binds lineage differentiation factors of the EBF family and transcriptional effectors for canonical signling pathways, including SMAD, retinoic acid, and NOTCH intracellular domains. ZNF423 mutations are reported in rare Joubert syndrome (JBTS19) and nephronophthisis (NPHP14) ciliopathy patients. The ciliopathies comprise a broad family of individually rare disorders unified by signaling defects in primary cilia. Clinical presentations range mild to lethal and from primary involvement of a single organ to more pleiotropic presentations. The overwhelming majority of genes identified for ciliopathy disorders encode physical components of primary cilia. Regulatory genes that control cilium-dependent signaling and genetic modifiers that change the outcome of ciliary defects remain understudied with respect to pathogenic mechanisms and potential points for intervention in more typical cases. ZNF423 is thought to comprise an integrative node among several transcriptional complexes that respond to classical intercellular signals during brain development and to regulate SHH signaling through the primary cilium. Both reported patients and mouse models show hindbrain malformations that include hypoplasia or agenesis of the vermis. Aim 1 will test hypotheses for ZNF423 activity in canalizing information from complex signaling environments into predictable cell responses. Aim 2 will comprehensively test for modifier genes that alter cellular outcomes ex vivo in response to loss of ZNF423. Aim 3 will test hypotheses for ZNF423 participation in oligogenic brain malformations in a well-validated animal model.

NIH Research Projects · FY 2025 · 2017-01

OAT1 IN UREMIA PROJECT SUMMARY/ABSTRACT Organic anion transporter 1 (OAT1/SLC22A6), discovered by us (NKT), is the prototypical kidney organic anion (PAH) transporter responsible for the transport of many drugs (e.g., diuretics, antivirals, NSAIDs). Based on our in vivo studies of the Oat1 knockout mouse during the last project period and in vitro studies by us and others, OAT1 is now believed to be a central component of a proximal tubule sensing and elimination mechanism for gut microbe products and uremic toxins. Furthermore, recent data from our lab in rodents, as well as human studies by others, indicates that OAT1-dependent function is critical for residual kidney function in CKD. However, what is truly remarkable from our metabolomics and transcriptomics studies is the degree to which OAT1, which is almost exclusively expressed in the kidney, regulates systemic metabolism--beyond gut microbe products and uremic toxins. For example, it regulates many signaling lipids, citric acid cycle intermediates, bile acids, and vitamins/cofactors. Indeed, OAT1 may be the renal gene with the broadest effects on systemic metabolism. Although CKD is a multi-factorial disease, one of these factors is the metabolic consequence of the gradual loss of OAT1-dependent sensing and elimination as proximal tubule function declines. Thus, we hypothesize that, in CKD, the normal functioning of OAT1-mediated protein-bound metabolite sensing and signaling in the proximal tubule is severely disrupted--leading to major disruptions in small molecule metabolism and signaling. This is because of the endogenous role of OAT1 as a central component of a larger metabolic network involving gut microbe-derived metabolites, some of which participate in uremic toxicity in severe kidney disease but which also impact tryptophan and lipid metabolism as well as other metabolic processes. Using the latest approaches to integration of large omics datasets and a particularly novel multi-scale metabolic reconstruction approach (combining Recon3D with a genome-scale microbiome reconstruction), we will define the pathways in Oat1 KO mice under conditions in which: a) the gut microbiome is present or depleted; and b) kidney function is compromised. At the end, we will have fully analyzed combinations of Oat1 KO vs WT, healthy vs depleted gut microbiome, and sham operation vs 5/6 nephrectomy, as sampled in the serum, kidney, liver and feces. This will settle (in mice) the relative importance of each altered state on levels of uremic toxins, on biochemical pathways, and on overall multi-scale metabolic impact as determined by genome-scale metabolic reconstruction for each of the conditions. A portion of the omics data has already been obtained (KO effect, partial gut microbe effect). This project will thus produce a validated detailed map of OAT1-centered metabolism in normal physiology and in diseased states, possibly the first of its kind for any multi-specific “drug” transporter (Nigam, Nature Reviews Drug Discovery, 2015). The studies could lead to design of strategies for improving the metabolic abnormalities in CKD by affecting OAT1 function or expression.

NIH Research Projects · FY 2026 · 2017-01

Abstract There is an urgent need to develop prevention strategies to halt progression of oral premalignant lesions (OPL) into head and neck squamous cell carcinoma (HNSCC), a disease that results in over 300,000 deaths each year worldwide. Our team discovered that activation of the PI3K/mTOR signaling network is the most frequently dysregulated cancer-driving signaling mechanism in HNSCC and OPLs and that, in turn, PI3K/mTOR inhibition exerts potent antitumor activity in experimental OPL and HNSCC models. These findings provided the foundation for our Phase II clinical trial (NCT01195922) exploring the antitumor activity mTOR blockade in HNSCC patients. More recently, we showed that repurposing metformin, a drug safely used by millions of type 2 diabetes (T2DM) patients, decreases mTOR signaling and displays potent chemopreventive activity in experimental OPL models. Based on these findings, and epidemiological data showing a significantly lower HNSCC incidence in T2DM patients on metformin, we conducted a Phase IIa trial in individuals with OPL to explore the potential of metformin to prevent oral cancer (M4OC-Prevent trial; NCT02581137). This represented the first study evaluating the chemopreventive potential of metformin in OPL. The histologic response rate was 60%, and we found a significant correlation between histological response and mTOR inhibition in OPL. These results provided the foundation for launching a multi-institutional NCI-funded Cancer Prevention Clinical Trial (M4OC-Prevent 2.0), a Phase IIb randomized, double-blind, placebo-controlled trial in current and former smokers with OPL to explore the potential of metformin for oral cancer prevention. However, we still have an incomplete understanding of the molecular determinants of the therapeutic response to metformin in OPL or in any other precancerous lesions. The overall objective of this project is to elucidate the molecular mechanisms by which metformin acts on OPL and HNSCC to (a) identify biomarkers for predicting and monitoring clinical response, (b) provide a rationale for mechanism-based multimodal precision chemoprevention strategies, and (c) prevent and overcome drug resistance. Leveraging the wealth of data from clinical, genetic, and tissue specimens from our M4OC-Prevent trial with our team’s expertise in decoding cancer promoting pathways, the long-term goal of our project is to define (1) mechanistic biomarkers predicting a response to metformin and (2) suitable multimodal therapeutic options to overcome drug resistance. Our planned studies will uncover new mechanistic and genetic determinants of metformin sensitivity to inform patient selection in future precision prevention trials, unveil novel mechanisms by which metformin induces histological response in OPL by the concomitant inhibition of mTOR and YAP1 signaling, and provide insights into novel a precision immune prevention strategy for metformin- resistant OPL lesions. By focusing on a cancer with a well-recognized premalignant state and readily accessible lesions for histological and molecular evaluation, our findings will have a broad impact, as they will enable the development of metformin as an effective, safe, and low-cost preventive agent for multiple malignancies.

NIH Research Projects · FY 2024 · 2017-01

Project Summary/Abstract: Hematopoietic stem cells (HSCs) give rise to all terminally differentiated cells in the blood. The ability of HSCs to reconstitute these blood cell lineages for life underlies the efficacy of bone marrow transplantation therapy for treatment of various blood disorders, including leukemias, anemia, and autoimmunity. Although this is an established and effective treatment, two-thirds of patients in need of a transplant lack a matched donor. Therefore, alternative sources of therapeutic HSCs would be a boon to the field. Human pluripotent stem cells (hPSCs) represent a potential source for cell-based therapies, including the derivation of patient-specific transplantable HSCs, which would additionally circumvent immune rejection and alloreactivity, both major issues in the clinic. The proposed collaborative research leverages the expertise of two Principal Investigators with complementary research interests and skills in stem cell biology, zebrafish genetics and development, murine HSC biology, hematopoietic development, and Wnt biology and biochemistry. Using zebrafish and hPSCs as model systems, they seek to identify and characterize the molecular cues that direct hematopoietic development during early embryonic stages, with an emphasis on the role of Wnt signaling. The proposed studies will build on their finding that a signaling axis regulated by Wnt9a/Frizzled9/EGFR is specifically required for HSC emergence and expansion across vertebrate phyla. This proposal will leverage lineage tracing methods in zebrafish and in vitro differentiation protocols of hPSCs combined with single-cell sequencing approaches to determine the molecular mechanisms of this requirement, and to provide a new level of understanding of how posterior lateral mesoderm is instructed to generate HSCs. The long-term goal of these studies is to gain a better understanding of how HSCs develop in the embryo in order to translate this information to hPSCs. Successful completion of this research will have a profound impact on HSC derivation and expansion, and thereby will be instrumental in overcoming current obstacles to the effective treatment of diseases requiring bone marrow transplant therapy.

NIH Research Projects · FY 2026 · 2016-09

Project Summary/Abstract Aging-related diseases are associated with disruptions in protein homeostasis, or proteostasis. A major disruptor of proteostasis is infection with intracellular pathogens, but it is poorly understood how responses to these infections may promote proteostasis. Our long-term goal is to dissect how responses to infection and other proteotoxic stressors can protect overall organismal health and lifespan through characterizing a novel proteostasis response we discovered in the nematode C. elegans called the Intracellular Pathogen Response or IPR. Our previous work demonstrated how upregulation of IPR genes that encode components of a cullin ring ubiquitin ligase promote improved proteostasis, including increased thermotolerance. The objective of this proposal is to determine how this multi-subunit ubiquitin ligase is assembled, to identify its target(s), and to elucidate the fate of those targets and how they impact thermotolerance. The central hypothesis is that intracellular infection and other specific proteotoxic stressors induce mRNA expression of ubiquitin ligase subunits including: 1) the Cullin CUL-6, 2) the RING domain protein RCS-1, 3) a Skp-Related Protein SKR-3, 4 or 5, and 4) F-Box Proteins FBXA-75 or FBXA-158; and that redox-dependent dimerization of this CUL-6- containing ubiquitin ligase tetramer (to create an enzyme complex of eight subunits total) leads to its activation, and that this enzyme complex ubiquitylates a yet-to-be identified target that is then degraded by the lysosome to regulate proteostasis. The rationale is based on our published genetic and biochemical data about the assembly and function of RCS-1/CUL-6/SKR-3,4,5/FBXA-75/158, and our unpublished in vitro and in vivo data about redox-dependent dimerization of SKR-3, together with our unpublished genetic and pharmacological data indicating that the increased thermotolerance mediated by the CUL-6 ubiquitin ligase is dependent on the lysosome. Our work is innovative because we are pursuing the IPR, which is a recently described proteostasis response acting independently of canonical proteostasis pathways like the heat shock response and unfolded protein responses. We will test our hypothesis with three specific aims including Aim 1) Determine the dimerization, interactions and function of SKR-3, SKR-4, SKR-5, FBXA-75 and FBXA-158, both in vitro and in vivo; Aim 2) Identify the target(s) of the CUL-6 ubiquitin ligase; and Aim 3) Characterize the downstream fate of these targets, including possible degradation by the lysosome. The expected outcome is to determine which SKR proteins heterodimerize, which SKR protein interacts with which F-box protein, which proteins are targeted by this ubiquitin ligase complex, and which autophagy factors and other cellular components are involved in directing targets to the lysosome. The proposed research is significant, because it could lead to new treatments for aging-related diseases associated with disruptions in proteostasis.

NIH Research Projects · FY 2025 · 2016-09

The abuse of psychomotor stimulants, including cocaine and methamphetamine, as well as the synthetic cathinone drugs (“bathsalts”), continues to wreak havoc worldwide and in the United States of America, with most individuals that suffer with stimulant use disorders going untreated. The more established stimulants such as cocaine and methamphetamine are highly addictive, can be acutely lethal and can result in long-term brain alterations with many implications for health and well-being. Recent studies show that synthetic cathinone psychoactive drugs 3,4-methylenedioxypyrovalerone (MDPV), α-pyrrolidinopentiophenone (α-PVP) and associated close analogs are a highly potent and efficacious reinforcers in animal models. These compounds tend to be used as cheaper or more available substitutes in lower socioeconomic populations, by incarcerated individuals, etc. Efforts to develop small molecule or vaccine therapies for psychomotor stimulant dependence have not, as yet, succeeded. It continues to be critical to better understand how stimulant dependence is established and maintained so as to better prevent stimulant addiction before it is established. This project will investigate the situational and behavioral contributors to escalating stimulant drug taking in rat models of intravenous self-administration, consistent with the goals of NOSI NOT-DA-21-028. Studies proposed under Aim I will determine if binge-like acquisition of psychostimulant self-administration alters the propensity for escalated intake under extended access conditions. The goal is to understand the consequences of initial uncontrolled consumption and whether that has lasting consequences for dependence. Studies under Aim II will determine if the intra-cranial self-stimulation (ICSS) reward threshold indexes the escalation in self-administration under extended access conditions, in a novel test of a longstanding negative-reinforcement framework for understanding escalating stimulant use. Finally, investigations under Aim III will determine if alternating cathinone and methamphetamine use alters the escalation of self-administration. These novel poly-drug models will address an understudied phenomenon whereby some human stimulant users substitute drugs depending on cost and availability. In total, these proposed studies will advance our understanding of the development of dependence on psychomotor stimulant drugs.

NIH Research Projects · FY 2026 · 2016-08

Project Summary Psychiatric Genomics Consortium for PTSD Posttraumatic stress disorder (PTSD) occurs only in vulnerable individuals after exposure to severe traumatic events. This risk is due, in part, to 40-50% heritability of differential vulnerability. Due to increasing collaborations across the field of PTSD genomics and the advent of new analytical tools, it is a very exciting time for PTSD genetic risk discovery. The purpose of this application is to facilitate meta-analyses of genome- wide association study (GWAS) data for symptoms and diagnosis of PTSD. We propose to conduct large-scale meta-analyses through the PTSD group of the Psychiatric Genomics Consortium (PGC). The PGC was created in 2007 to conduct field-wide mega-analyses of individual data for 5 major psychiatric disorders. With its current 11 working groups, it is the largest consortium (>800 scientists from 40 countries) in the history of psychiatry. The PGC has produced major findings with regard to the genetic architecture of psychiatric disorders. The PGC-PTSD group was launched in 2013 and has been enormously successful. Currently our multi-ethnic data collection includes genotypes from 90 studies with a total N of over 1.25 million combined cases and trauma-exposed controls. We recently identified 95 genome-wide significant loci and generated a polygenic risk score to identify individuals at highest risk for PTSD after trauma exposure. We hypothesize that with an increased sample size and deeper phenotype characterization, the PGC- PTSD will accelerate our current understanding of the genetic architecture of PTSD. Our progress thus far demonstrates feasibility and successes of the proposed work. Aim 1 proposes to increase sample size to 450,000 PTSD cases, including 100,000 cases of non-European ancestry, conduct GWAS meta-analyses to detect novel common variants, and identify rare copy-number variants (CNVs) and single nucleotide variants (SNV) hypothesized to contribute to PTSD heritability. This aim will be supplemented by the contribution of diverse ancestry groups to ensure that advances in our genetic understanding of PTSD extend across ancestral backgrounds in Aim 2. Aim 3 is centered around the characterization of functional consequences of identified variants. Lastly, we will address the heterogeneity of PTSD and its overlap with internalizing disorders in Aim 4. Identifying the genetic pathways underlying PTSD will lead to improved neurobiological understanding, enhanced prevention, and improved treatment of this debilitating and prevalent syndrome.

NIH Research Projects · FY 2026 · 2016-08

PROJECT SUMMARY/ABSTRACT Pontocerebellar hypoplasia (PCH) is a heterogeneous group of mostly recessive pediatric brain disorders that show features of both impaired neurodevelopment and presence of neurodegeneration. PCH is characterized by severe age-dependent neurological impairment, and notable radiographic volume loss of the pons and cerebellum with loss of brainstem and cerebellar neurons. Currently there are 32 genes known mutated in PCH, but still more genes await to be discovered, and molecular mechanisms are poorly understood. Some of the genes implicate key steps in protein synthesis and genomic integrity including tRNA and mRNA splicing, suggesting disruption to homeostatic cellular functions, but many questions remain: 1] How many genetic subtypes remain to be discovered? 2] Why do loss of broadly expressed genes predispose specifically to neurons? 3] Are there convergent molecular pathways for PCH? Over the past 5 years, we have: 1] Grown our unique cohort of PCH patients, containing 248 families including 132 still without a molecular cause. 2] Applied a range of genomics and transcriptomics methods to uncovered mutations in several novel genes including TOE1, TBC1D23, PRP17 and PPIL1 leading to specific PCH subtypes. 3] Revealed defects in RNA splicing and genome integrity as underlying causes. 4] Uncovered the first spliceosome protein mutations. 5] Revealed new genotype phenotype correlations. In our preliminary data we have: 1] Secured resources to advance whole genome sequencing to evaluate our remaining unsolved cases. 2] Identified a further ten new genes as causes for PCH. 3] Remarkably, found that six of the novel causes encode spliceosome proteins. 4] Uncovered R-loop accumulation as a cause of DNA damage by which mutations lead to genotoxic stress. The goal of this application is to: 1] Identify the remaining ‘discoverable’ genes for PCH. 2] Functionally validate mutations within a pathogenic framework. 3] Test the hypothesis that PCH gene loss leads to neurons cell death through R-loop accumulation, DNA damage and genotoxic stress. This work will lead to insight into causes and mechanisms of an important cause of infantile encephalopathy, and uncover mechanisms of selective neuronal vulnerability and pediatric neurodegeneration underlying developmental brain disease.

NIH Research Projects · FY 2025 · 2016-07

ABSTRACT The mammalian secretory pathway regulates a cell’s extracellular interactions by making most signals and receptors that moderate communication. Furthermore, it makes cell adhesion molecules and the extracellular matrix components. Indeed it makes most proteins needed for a cell to interact with its extracellular environment, which includes ~⅓ of their protein-coding genes in the human genome. The pathway has hundreds of machinery proteins used for synthesizing and trafficking secreted proteins, but each of these have their unique role in the process. However, for the ~8000 protein products of the secretory pathway, it remains unclear what machinery is needed for each one. To further complicate this, each tissue expresses its own subset of genes for the secretory pathway. Understanding the organization of the pathway and the interactions between the secretory pathway machinery and secreted protein products is of considerable importance since many diseases involve changes in the abundance of secreted and/or membrane proteins, and these are not always accompanied by changes in mRNA. That is, the secretory pathway itself is regulating changes in hormone secretion, managing ER stress, or amyloid formation. Thus, there is a fundamental need to understand (1) what the secretory pathway machinery does, (2) which secreted and membrane proteins rely upon it, (3) how the hundreds of machinery proteins work together, and (4) what regulates their functions. All of these items require systems-level experiments, tools, and analyses to fully understand. Here we are developing such resources for the community to answer fundamental questions. In this work, we are mapping out all of the secretory pathway machinery and detailing their functions. We describe their functions mathematically, and build computational models to account for their concerted functions, even for individual tissues and cell types. Furthermore we are developing software and algorithms to help analyze the models and use them to diagnose the molecular bases underlying secretory disorders. We are further developing and deploying experimental techniques to more fully identify all of the secretory machinery proteins needed to facilitate the production of each specific secreted or membrane protein, and will use those techniques to identify these interactions for liver-secreted proteins. Finally, we are using genomics and systems biology techniques to unravel the regulatory mechanisms controlling the tissue-specific expression of the secretory pathway. Through this, valuable resources will be developed and shared with the community to make it easier to study the secretory pathway as a biomolecular system.

NIH Research Projects · FY 2025 · 2016-06

Project Summary/Abstract RNA quality control pathways play essential roles in ridding cells of defective RNAs that arise from RNA damage, misprocessing, or transcription of pseudogenes. Much has been learned over the past decades about the quality control pathways that monitor the integrity of protein-coding messenger (m)RNAs, such as the nonsense-mediated mRNA decay pathway. Much less is known about those quality control pathways that monitor non-coding (nc)RNAs, which make up ≈95% of the cell's RNA and are susceptible to the same types of damage as mRNAs. The primary goal of this research is to uncover the mechanisms whereby ncRNA quality control pathways distinguish normal from defective RNAs, how the defective RNAs are targeted for degradation, and what are the consequence of failures in these pathways to cell function and human health. To address these questions we will over the next five years focus on the quality control of abundant human stable small ncRNAs that are critical to cell function, including small nuclear (sn)RNAs of the spliceosome and 7SL RNA of the signal recognition particle. We will take advantage of the fact that 1,000s of pseudogenes of these RNAs exist in the human genome, many of which produce defective ncRNA variants that must be detected and degraded by quality control pathways. The features of these defective ncRNAs that are identified by quality control pathways and the factors involved in their degradation will be uncovered through targeted and global assays monitoring effects of degradation factor depletion and ncRNA mutagenesis on the stability of the ncRNA variants. We recently uncovered a central role for 3' end-processing machineries in one such quality control pathway that targets defective snRNAs, and will therefore additionally over the next five years pursue RNA targets and potential roles in RNA quality control of 3' end processing factors, including factors that when defective cause human neurodegenerative disorders. These efforts should uncover principles by which defective human small ncRNAs are detected and degraded by quality control pathways, a mostly unexplored yet critical aspect of gene expression. These efforts also have the potential to provide insights into defects in RNA processing that lead to human disease such as neurodegenerative disorders.

NIH Research Projects · FY 2026 · 2016-05

Project Summary/Abstract The major long-term objective of this research program is to understand the mechanisms of regulation of RNA polymerase II transcription in animals, particularly in humans. The basis of numerous biological phenomena, including many human diseases, can be traced to the proper or improper expression of a gene or set of genes. Why is it essential to study the mechanisms of gene expression? It is necessary to do more than to observe and to model the expression of genes, but also to understand how and why genes are expressed under different conditions. In this manner, insights into the mechanisms of gene expression will have a far-reaching impact upon the biological and biomedical sciences. Specific emphasis will be placed upon the study of the RNA polymerase II core promoter, which is the short stretch of DNA that directs the initiation of transcription. Why is it important to study the core promoter? First, the core promoter is the strategic site of convergence of the signals that lead to transcription initiation, and it is where the yes/no decision is made to initiate transcription. Second, the core promoter is varied in terms of its composition and function. Different core promoters have different properties, and thus, the core promoter is a transcriptional regulatory element. Two RNA polymerase II transcription systems in humans will be studied. The first system is based on the TATA box, and the second system is based on the DPR (downstream core promoter region). DPR-dependent transcription occurs by a different mechanism than TATA-dependent transcription. Furthermore, this project will investigate not only the DNA sequences of the DPR and TATA, but also the protein factors that differentially act upon those DNA motifs. The proposed research should provide researchers with the critical knowledge of the factors that transcribe and regulate human genes, most of which are TATA-less, as well as clarify the mechanisms by which sequence-specific DNA-binding proteins regulate gene activity. In addition, machine learning models would be generated that should enable the prediction of the hormone responsiveness of individual human genes. A second objective of this research program is to understand the biological functions of the HMGN (high mobility group N) proteins, which are abundant nucleosome-specific binding proteins in vertebrates. In the past, a key barrier to the analysis of the HMGN proteins has been the presence of multiple closely-related proteins. For instance, there are five HMGN proteins in humans. To address this problem, human HMGN null cell lines, which lack all five HMGN proteins, were created. These HMGN null cell lines now enable the genetic analysis of the HMGN nucleosome-binding proteins in humans. The ensuing experiments should add a new dimension to the understanding of the functions of non-histone chromosomal proteins in gene expression and chromatin structure and reveal new mechanisms by which genes are regulated.

NIH Research Projects · FY 2025 · 2016-05

Abstract Microglia are tissue macrophages that reside in the central nervous system (CNS) and perform unique and critical auxiliary functions important to CNS development, homeostasis, immunity and repair. These roles, along with the progressive appreciation that microglia can contribute to neurological disease processes, provides a compelling case to more clearly understand the mechanisms that regulate their development and functions. Major unanswered questions include determining the combination of signals within the brain that trigger the differentiation of erythromyeloid progenitor (EMP) cells to become mature microglia and how alterations in these signals specify distinct microglia phenotypes in health and disease. Studies performed under the support of this grant for the past four years provide the foundations for addressing these questions. Four Specific Aims are proposed. Specific Aim 1 is to define expression and chromatin Quantitative Trait Loci and collaborative transcription factors in human microglia. These studies will generate a valuable resource for the neuroscience community and inform studies in Aims 2, 3 and 4. Specific Aim 2 is to define cis regulatory elements that mediate brain environment-dependent regulation of microglia gene expression, focusing on the microglia-specific lineage determining factor SALL1. Importantly, our experimental plan will exploit the recent ability to achieve an in vivo human microglia phenotype within the mouse brain as the context for analysis of the function of environment- dependent enhancers. Specific Aim 3 is to test the hypothesis that brain environment-dependent genes can be activated in iPSC-derived microglia in vitro by conditional expression of environment-dependent transcription factors. Forced expression of these factors in human iPSC-derived microglia in vitro will provide insights into their molecular functions and may enable development of improved in vitro microglia model systems. Specific Aim 4 is to perform in vivo ASO-mediated loss of function experiments to identify transcriptional mediators of brain environmental factors. This aim is based on advances in anti-sense oligonucleotide (ASO) chemistry that now make it possible to use ASOs to significantly alter gene expression in microglia and other cell types of the brain in vivo. In concert, the proposed studies are intended to qualitatively advance understanding of mechanisms that establish the brain environment dependent program of human microglia gene expression.

NIH Research Projects · FY 2025 · 2016-05

SUMMARY Non-typhoidal Salmonella (NTS) causes 100 million infections per year worldwide. The pathogen infects the gastrointestinal tract, where it triggers intestinal inflammation. Essential to NTS pathogenesis is the ability to evade host responses and compete with the gut microbiota. Research in my laboratory has contributed to elucidating the duality of the mucosal response to Salmonella enterica serovar Typhimurium (STm), a highly prevalent NTS serovar. Particularly, we have shown that STm evades sequestration of essential metal nutrients, a process known as “nutritional immunity”, to thrive in the inflamed gut and compete with the microbiota. We discovered: (i) key host factors that modulate nutritional immunity in the inflamed gut, including the cytokine interleukin-22 (IL-22) and the antimicrobial proteins lipocalin-2 and calprotectin; (ii) several mechanisms and virulence factors that enable STm to overcome metal nutrient sequestration in the inflamed gut. We also discovered that, during colitis, the probiotic bacterium Escherichia coli Nissle 1917 (EcN) competes with STm for iron and zinc via multiple mechanisms, including expression of high affinity iron and zinc acquisition systems, and secretion of small antimicrobial molecules termed microcins. Building on this prior work, the primary objective of this application is to continue to investigate host-microbe and microbe-microbe interaction in the inflamed gut, a complex environment with unique nutritional challenges for both pathogens and the microbiota. We will continue to investigate the role of microcins and zinc acquisition systems in the inflamed gut, and we will expand to elucidating the role of secondary bile acids, including newly discovered microbial conjugated bile acids, in modulating nutritional immunity. Our central hypothesis is that the host inflammatory response changes the nutritional and metabolic landscape of the gut, triggering an environment that favors the bloom of Enterobacteriaceae and promotes microbial competition. In Aim 1, we will elucidate mechanisms of competition for metal nutrients in the inflamed gut between Salmonella, E. coli Nissle, and the gut microbiota. In Aim 2, we will evaluate the impact of Salmonella infection on bile acid pool composition, and the impact of these changes on nutritional immunity. Understanding the host and microbial factors that modulate host immunity, together with the mechanisms by which STm thrives in this environment, is essential for developing new approaches to limit STm replication in the inflamed gut.

NIH Research Projects · FY 2025 · 2016-05

Spermatogonial stem cells (SSCs) are self-renewing cells essential for adult spermatogenesis that hold promise for treating male infertility. In this proposal we focus on two topics revolving around SSCs. First, we will identify “transcription factor (TF) circuits” critical for SSC establishment. Identifying such TF circuits will allow us to ultimately define a functionally defined (not merely “omics” defined) TF network that drives immature germ cells to become stem cells. Second, we will follow-up on our surprising finding that knockout (KO) of a transcription factor important for spermatogenesis causes expansion of SSCs. Understanding the underlying mechanism for this potential compensatory response to genetic insult has the potential to reveal insights into how human SSCs respond to toxic insults and infertility. The molecular mechanisms by which SSCs are initial generated in vivo are poorly understood. One of the few proteins that have been shown to drive SSC establishment is the homeobox TF, RHOX10, which we showed promotes SSC precursor cell differentiation and the initial seeding of SSCs in the seminiferous epithelium. Given that TFs are regulatory factors that control batteries of downstream genes, our discovery that RHOX10 drives SSC establishment provided an opportunity to define key genes critical for this process. Indeed, we recently identified scores of direct RHOX10-target genes, including a remarkably large number of TF genes with known roles in SSCs. This led us to hypothesize that a TF network is critical for SSC establishment, which we propose to test. This is critical, as even though other studies have identified hundreds of downstream genes regulated by TFs at various germ cell stages, it has not been ascertained which of these downstream genes are functionally important for the action of a given TF. In Specific Aim 1, we will perform functional experiments to determine the regulatory relationships of TFs, thereby allowing us to define functional TF circuits. As evidence of feasibility, we recently defined—through rescue experiments—3 TFs that function in a TF circuit to drive SSC precursor cell differentiation. The other focus of this proposal revolves around our unexpected recent discovery that adult Rhox10-null mice have expanded numbers of SSCs. Understanding the mechanisms that drive SSC expansion is critical for devising approaches to treat infertility. Our preliminary data support 3 testable models to explain the SSC expansion in these KO mice, including a compensatory mechanism model in which SSCs undergo proliferative expansion when they sense downstream spermatogenic defects. In support of such a feedback mechanism operating in humans, it was recently reported that cryptozoospermia infertility patients have a selective and robust increase in the number of SSCs, as defined by single-cell RNAseq analysis. In Specific Aim 2, we will determine the cellular and molecular mechanisms responsible for the expansion of SSCs in Rhox10-null mice. Elucidating mechanisms controlling SSC expansion in vivo has the potential to translate into approaches into expanding SSCs in vitro for clinical applications.