Ut Southwestern Medical Center

NIH Research Projects · FY 2025 · 2021-05

Summary Transmembrane proteins must adopt proper membrane topology to perform their function. In mammalian cells, the topology of transmembrane proteins is determined by the direction through which transmembrane helices are inserted into ER during their translation on ER-associated ribosomes. It has been assumed that protein translocation across the ER membranes is a constitutive process so that transmembrane proteins must adopt a fixed topology. This assumption has been challenged by our recent observation that the direction through which transmembrane helices are inserted into the ER can be reversed under certain physiological conditions. We reported that ceramide inverted the topology of two polytopic transmembrane proteins, namely TM4SF20 and CCR5. Since this regulatory mechanism does not flip transmembrane proteins that have already been synthesized but inverts the topology of newly synthesized proteins by changing the direction through which transmembrane helices are translocated across membranes, we designated this process as Regulated Alternative Translocation (RAT). This project is initiated to further characterize RAT by delineating the mechanism of this topological regulation. We will begin by testing the hypothesis that TRAM2 is the ceramide sensor that interacts with the nascent transmembrane helices subjected to RAT. The approaches developed for this study can be generalized to identify ceramide interactome, a finding that might reveal more signaling reactions mediated by the sphingolipid. These approaches may also be applied unbiasedly to identify proteins interacting with the nascent transmembrane helices subjected to RAT, thereby providing more mechanistic insights into this novel translocation regulation. This project will also identify proteins subject to RAT by a novel proteome-wide approach capable of measuring topology of transmembrane proteins globally. Achieving this part of the project will not only reveal the breadth of RAT but also provide essential experimental data for proteome- wide assembly of topology of transmembrane proteins. Considering that only 10% of mammalian transmembrane proteins have their topology defined by experimental evidence, accomplishing this project should greatly improve our understanding of transmembrane proteins.

NIH Research Projects · FY 2025 · 2021-05

Exploring mechanisms of cardiac pacemaker cell fate determination PROJECT SUMMARY Pacemaker (PM) cells reside within the sinoatrial node (SAN), which faithfully initiates over 3 billion heartbeats during the human lifespan. PM dysfunction often necessitates device implantation to prevent circulatory collapse from bradycardia. Despite the critical importance of cardiac PM function, the mechanisms by which PM cells undergo lineage commitment remain obscure. The long-term goal of our research program is to understand the mechanistic basis for cell fate determination within the cardiac conduction system. The overall objective for this proposal is to explore molecular strategies for PM cell lineage commitment. There is an urgent need to elucidate the molecular underpinnings of PM lineage commitment to understand the fundamental biology of PM fate de- termination and to inform future development of new therapeutic strategies. My lab recently reported on key mechanisms by which Hand2 regulates PM formation using conversion of fibroblasts into induced PM myocytes (iPMs) as a model system. Building upon this preliminary data, our central hypothesis is that Hand2 interacts with AP-1 to promote subtype diversity and cooperatively binds genomic targets to orchestrate PM specification. To test our central hypothesis, we propose the following Specific Aims: 1) Define the mechanisms by which Hand2 ensures cardiac subtype diversity, 2) Explore the basis for cardiac PM lineage commitment and alterna- tive fate restriction, and 3) Boost iPM reprogramming by component annotation and combinatorial perturbation. In Aim 1, we will use our iPM reprogramming system in conjunction with genomic occupancy analysis, co-im- munoprecipitation, immunocytochemistry (ICC), single-cell RNA sequencing (scRNA-seq), and confocal micros- copy to define biochemical interactions, perturb cardiac reprogramming, and characterize the resulting cell fates. In Aim 2, we will use scRNA-seq, cell fate trajectory mapping, ICC, genomic occupancy analysis, and protein- binding microarrays (PBMs) to analyze lineage regulators, alternative fate repressors, and combinatorial inter- actions during iPM reprogramming. In Aim 3, we will systematically annotate candidate factors curated from our preliminary data and the literature. In parallel, we will build PM regulatory networks from the ground-up using novel combinatorial genomic approaches that we have recently developed. Successful completion of the pro- posed project will provide critical details regarding the establishment and maintenance of PM cell identity. This contribution will be significant because it will provide detailed insight into how cell fate is accomplished and identify potential regulators and mechanisms of PM specification. Furthermore, the proposed research is inno- vative because our unique experimental approaches and multi-disciplinary research team promise to uncover new principles in cell fate determination. Taken together, we anticipate that the results of the proposed project will provide detailed knowledge of the gene regulatory networks that drive PM cell specification and guide future development of novel strategies to engineer therapeutic replacement cells for sinus node dysfunction.

NIH Research Projects · FY 2025 · 2021-04

Project Summary: Lipogenesis is essential for normal physiology and its dysregulation is a notable feature of obesity, diabetes, cardiovascular disease, cancer, neurodegeneration and infection. Classical regulation of de novo lipogenesis involves transcriptional regulation of lipogenic gene expression via hormone mediated SREBP activation, and/or carbohydrate sensing via ChREBP activation. However, neither program facilitates substrate handling nor set the cellular energy status amenable to lipogenic conditions. The mitochondria, specifically the TCA cycle, is the putative source of acetyl-CoA used for lipid synthesis but before transport to the cytosol, it is converted to citrate, a step that consumes TCA cycle intermediates. The balance between TCA cycle cataplerosis (loss of TCA cycle intermediates) and anaplerosis (replenishment of TCA cycle intermediates) and may help to determine the rate at which citrate can be used for lipid synthesis. These pathways are known to be disrupted in many diseases, that also have pathological lipid metabolism. Thus, we will examine how anaplerotic and cataplerotic pathways of the TCA cycle help to mediate the appropriate lipogenic response to nutrition, by promoting substrate (e.g. citrate) availability and/or cellular energy status necessary for lipogenic reactions. We will use state of the art stable isotope tracers, analytical chemistry platforms and mouse genetics to evaluate the role of these pathways in controlling rates of lipid synthesis. Completion of this project will identify new metabolic mechanisms for the regulation of lipid synthesis that complement transcriptional mechanisms and may have particular relevance to the growing list of diseases known to disrupt TCA cycle metabolism.

NIH Research Projects · FY 2026 · 2021-04

Project Abstract Individuals with neurofibromatosis type 1 (NF1) have an approximately 160-fold increased risk of developing malignant peripheral nerve sheath tumor (MPNST). As a leading cause of death for NF1 patients. MPNST has no effective therapy and thus there is an urgent need for new therapies. The dramatically increased risk of developing MPNSTs is caused by the presence of plexiform neurofibromas (PNFs), the major benign precursor lesion for NF1-MPNST. It has been proposed that PNFs are congenital lesions, arising from the early stages of nerve development when neural-crest stem cells differentiate into Schwann cell (SC) lineages, which give rise to either myelinating or nonmyelinating SCs (mSCs or nmSCs). In the normal nerve, unmyelinated axons are sorted and ensheathed by nmSCs into individual pockets, forming Remak bundles. Whereas no defect in SC precursors or mSCs was observed, Nf1 loss (Nf1-/-) during early nerve development induced a pocket defect in Remak bundles, characterized by abnormally sorted unmyelinated axons. These abnormal Remak pockets progress to a stage with axonal degeneration and abnormal proliferation of dissociated SCs, leading to the formation of PNFs. Axonal degeneration may contribute to PNF formation by inducing a nerve injury environment, a concept supported by the observation that Nf1 loss in mature SCs is not sufficient to induce PNFs unless an injury to the nerve occurs. Malignant transformation of PNFs to MPNSTs requires at least two additional genetic alterations: sequential inactivation of CDKN2A, and then either SUZ12 or EED - two essential components of Polycomb Repressive Complex 2 (PRC2). PRC2 catalyzes histone modification H3K27me3 to repress gene expression throughout the genome. Loss of PRC2 specifically observed in MPNSTs, but not in benign tumors, suggests that PRC2-mediated H3K27me3 normally represses expression of the oncogenic drivers responsible for malignant transformation of PNFs to MPNSTs. However, Eed/PRC2 is dispensable during normal mouse SC development and myelination. Further, loss of the Eed/PRC2 tumor suppressor unexpectedly inhibits proliferation of injury-induced reprogrammed SCs, accompanied by derepression of Cdkn2a expression. Here, we propose to test two related hypotheses: (1) the developmental Nf1-/- Remak pocket defect and its associated axonal degeneration (nerve injury) drive Nf1-/- SCs to form PNFs and (2) nerve injury response induces an epigenomic switch, rendering reprogrammed PNFs or SCs susceptible to malignant transformation by sequential loss of CDKN2A and PRC2. We will determine the role of the developmental Remak pocket defect in NF1-MPNST formation (Aim 1), investigate tumor suppressive mechanisms in injury-induced reprogramed SCs (Aim 2), and develop therapeutic strategies based on the injury-induced epigenomic switch in reprogrammed SCs (Aim 3). We will identify injury-induced and PRC2- repressed oncogenic drivers for MPNST formation via epigenomic approaches, develop synergistic therapies using a high-throughput drug repurposing screen, and test them GEM- and patient-derived preclinical models.

NIH Research Projects · FY 2025 · 2021-04

Project Summary Triple-Negative breast cancer (TNBC), which accounts for 15-20% of all breast cancer, represents an aggressive clinical history, development of distant metastasis, shorter survival and high mortaility rate compared with other subtypes of breast cancer. It is imperative to identity new therapeutic targets that are actionale in TNBC. Our lab has been focusing on studying a family of enzymes that uses oxygen, Fe2+ and 2-oxoglutarate (2-OG) for their enzymatic reactions. This enzyme family has been reported to be involved in the pathogenesis of cancers. We generated the custom siRNA library for all of 2-OG dependent enzymes and developed a stringent screening strategy by combining the functional readouts from both 2-D cell proliferation and 3-D soft agar growth assay with TNBC breast cancer cell lines. Our preliminary data show that gamma-butyrobetaine hydroxylase 1 (BBOX1) involved in carnitine biosynthesis pathway is essential for TNBC cell proliferation on 2-D and 3-D. Mechanistically, we show that BBOX1 binds with the calcium channel inositol-1,4,5-trisphosphate receptor type 3 (IP3R3), therefore promoting calcium release, mitochondrial function and glycolysis in TNBC. We hypothesize that BBOX1-IP3R3 signaling axis promotes TNBC by inducing calcium release and tumor metabolism. This is the first study directed at a pro-oncogenic function for BBOX1 in cancer, with our focus in TNBC. In Specific Aim 1, we will characterize the functional significance of BBOX1-IP3R3 signaling in TNBC. In Specific Aim 2, we will elucidate the molecular mechanism by which BBOX1-IP3R3 signaling promotes oncogenic phenotypes in TNBC. In Specific Aim 3, we will assess the therapeutic implications of targeting BBOX1 in TNBC xenografts and patient derived xenografts (PDXs). Successful completion of this proposal would establish the role of BBOX1 as a new oncogenic driver in TNBC and explore its therapeutic potential in this lethal disease.

NIH Research Projects · FY 2025 · 2021-04

Project Summary This is a Bioengineering Research Grant (BRG) proposal in response to PAR-19-158 to further develop and validate a non-invasive panel of the most critical glioma molecular markers (IDH, 1p/19q, MGMT) using standard clinical MRI T2-weighted images and deep learning, and extend the performance to tissue-level accuracies. Currently, the only reliable way of obtaining molecular marker status is through direct tissue sampling of the tumor, requiring either a craniotomy and stereotactic biopsy or a large open surgical resection. Noninvasive determination of molecular markers with tissue-level accuracy would be transformational in the management of gliomas, reducing or eliminating the risks and costs associated with a neurosurgical procedure, accelerating the time to definitive treatment, improving patient experience and ultimately patient outcomes and survival time. Artificial intelligence such as deep learning has emerged as a powerful method for classification of imaging data that can exceed human performance. Preliminary work using our novel voxel-wise classification-segmentation approach with the NIH/NCI TCIA glioma database has outperformed any prior noninvasive methods for determination of IDH, 1p/19q, and MGMT methylation, achieving accuracies of 97%, 93%, and 95%, respectively. The approach however, needs to be validated beyond the TCIA and accuracies need to be extended in order to achieve tissue level performance. This will be accomplished by using our top-performing voxel-wise classification framework, leveraging marker-specific targeted sample sizes, and gaining a final boost from deep-learning artifact correction networks. In Aim 1 we will curate a database of over 2000 gliomas including 500 subjects from our institution, 1200 subjects from our external collaborators, and over 300 subjects from the TCIA. We will train our voxel-wise deep learning classifiers to determine molecular status based on clinical T2-weighted MR images with target accuracies of 97%. In Aim 2 we will rigorously evaluate the motion and noise sensitivity of the networks and create an artifact correction network with the goals of 1) recovering accuracies in the setting of large amounts of motion/noise and 2) further boosting accuracy to tissue-level performance even in the absence of visible artifact. In Aim 3 we will deploy a complete end-to-end clinical workflow and evaluate real-world live performance of the AI tool on 300 prospectively acquired brain tumor cases and 300 subjects from our external collaborators. The AI tool will be made available for deployment at other medical centers. The developed framework can also be extended to additional markers in a straightforward fashion. In summary, this BRG proposal will further develop, refine and validate a non-invasive MRI-based method for determining the most critical glioma molecular markers rivaling tissue-level accuracies to significantly reduce and in many cases eliminate the need for stereotactic biopsy.

NIH Research Projects · FY 2025 · 2021-04

ABSTRACT Ion transfer across biological membranes is central to nerve excitation, muscle cell contraction, signal transduction, and hormone secretion. Ion channels play a vital role by providing a passageway within membranes to allow specific ions to traverse down their electrochemical gradient. The immense physiological importance of ion channels is reflected in the fact that their dysfunction underlies a variety of disabling human diseases including seizures, deafness, ataxia, long QT syndrome, and cardiac arrhythmias. There is a long history of physiological work and a large body of functional and structural data on tetrameric cation channels that are localized to the plasma membrane, including the K+, Ca2+, Na+, TRP and cyclic nucleotide-gated channels; however, relatively little is known about organellar cation channels, partly because of the difficulty in directly measuring their activities in organellar membranes. Currently, there is an emerging research interest in the recently identified organellar cation channels due to their importance in organelle physiology and cell signaling. This Maximizing Investigators' Research Award proposal will be focused on our ongoing efforts to dissect the structural and functional properties of two specific groups of organellar cation channels: the endolysosomal cation channels and the mitochondrial calcium uniporters. The insights gained from the proposed studies will facilitate our understanding of how these organellar channels regulate some basic biological functions of lysosome and mitochondria. Endosomes and lysosomes play crucial roles in many biological processes such as protein and lipid degradation, catabolite export, membrane trafficking, and metabolism-sensing, and defects to these processes can result in lysosomal storage diseases. These acidic organelles contain various ion channels that control endolysosomal pH and ionic homeostasis. One major research direction in my lab is designed to reveal the structural basis of gating and selectivity in endolysosomal cation channels, including two-pore channels (TPCs), transient receptor potential mucolipin channels (TRPMLs), and the non-canonical TMEM175 K+ channels. Mitochondria can take up large amounts of Ca2+ from cytosol, a process that can modulate ATP production, alter cytoplasmic Ca2+ dynamics, and trigger cell death. Mitochondrial calcium uptake is mediated by the mitochondria calcium uniporter (MCU), a highly selective Ca2+ channel that is localized to the inner mitochondrial membrane. In humans, the uniporter functions as a protein complex consisting of at least four components: the pore-forming MCU, the essential membrane-spanning subunit EMRE, and the Ca2+-sensing gate-keeping proteins MICU1 and MICU2. Another major project in the lab aims to reveal the structural basis of the human MCU complex assembly and the channel regulation. Our experimental approach utilizes single particle cryo-electron microscopy (cryo-EM) and protein crystallography to determine the three-dimensional structures of these channels, and electrophysiology to elucidate their biophysical properties.

NIH Research Projects · FY 2025 · 2021-04

Project Summary. Malaria remains one of the most serious infectious diseases, globally threatening nearly 50% of the world population, and leading to >400,000 deaths annually, mostly among young African children. There are no effective vaccines and the disease is managed through a combination of insecticides and drugs for both treatment and chemoprevention. The relentless ability of the parasite to acquire drug resistance necessitates that a continual pipeline of new drug candidates is maintained. We sought to identify novel chemical starting points for the discovery of new anti-malarial drugs by phenotypic screening against erythrocytic stage P. falciparum. We undertook a high-throughput screen of a newly acquired (in 2017) 100K chemical library reasoning that since it was recently purchased it might contain new chemical space that had not been previously screened. As part of our hit validation process we prioritized hits from the screen based on the following experimental measures: 1) potency versus the parasite against two cell lines, 2) selectivity versus a human cell line, 3) novelty of the chemical matter, 4) parasite kill rate (medium and fast kill being desirable) and 5) in vitro ADME properties including metabolic stability and solubility. We identified 16 chemical series that met our objectives of novelty and from these have selected 3 series for hit to lead chemistry. These include a piperidine carboxamide series (Alchm18) that has a moderate rate of kill, good starting potency (P. falciparum 3D7 EC50 <100 nM), and strong starting in vitro and in vivo ADME properties; a a tetrazole-based series (Alchm3) that shows fast kill kinetics, and a an azetidine amide (Alchm17), with good potency and solubility. We have validated synthetic strategies for all three series through synthesis of both the parent compound and analogs. The goal of this proposal is to conduct hit-to-lead chemistry on these three series, to evaluate their biological profiles, and to perform studies to identify their targets. The strongest series will then be prioritized for full scale lead optimization. Our project team of Phillips (parasite biology), Ready (medicinal chemistry) and Charman (ADME/PK) is highly experienced and has a long track record of working together. The project will also be a collaborative effort with the Medicines for Malaria Venture (MMV) who will provide in kind support and access to their in vitro and in vivo parasite efficacy models and project oversight. Upon completion of this proposal we will have substantial new insight into the developability of three new chemical series, we will have validated up to three additional new anti-malarial targets, and we will have progressed the strongest of our three chemical series through lead optimization to identify a potential preclinical development candidate.

NIH Research Projects · FY 2025 · 2021-04

Project Summary Radiotherapy (RT) aims to deliver tumoricidal dose to clinical target volume (CTV) while sparing organs at risk (OAR), for which proton and photon beams are naturally complementary to each other: protons are generally better for OAR sparing, while photons are more robust to delivery uncertainties for CTV coverage. The hybrid proton-photon RT has a long history. However, as it generates proton and photon plans separately without fully utilizing joint proton-photon optimization during the planning stage, current hybrid RT is pseudo- hybrid and very limited in plan quality, treatment sites, and broad applicability. The key to leapfrog from pseudo-hybrid to truly-hybrid RT is new joint proton-photon optimization method that synergizes complementary proton and photon beams. The hypothesis is that truly-hybrid RT via appropriate joint proton- photon optimization will be more favorable than proton or photon-only RT, in terms of CTV coverage robustness and OAR sparing optimality. Broad applicability of truly-hybrid RT to patients: (A) Clinical applicability: unlike pseudo-hybrid RT that is limited in plan quality and treatment sites, truly-hybrid RT may become a new paradigm for general cancer RT, owing to its superior plan quality and thus potentially clinical outcomes to proton-only or photon-only RT. (B) Clinical workflow: our truly-hybrid plans can be individually and safely delivered on existing proton and photon machines, and this effort envisions patients being treated in an integrated cancer center like ours with both proton and photon equipment, under the direction of a single physician, using shared immobilization devices, simulation procedure and structure set, and integrated treatment planning and delivery system. (C) Patient coverage: truly-hybrid RT can be made broadly available to many cancer patients through existing infrastructures in US, since (1) most hospitals with proton centers also have photon centers; (2) 76% of cancer patients live in the states with operational proton centers, while 85% are within 100-mile (2-hour-driving) distances to these proton centers; (3) cancer patients are more willing to travel for advanced treatment options. Proposed effort: Inspired by unprecedented plan quality and broad applicability of truly-hybrid RT via our joint proton-photon optimization method, the next step is to test the hypothesis prospectively via clinical trials. However, a missing prerequisite to advance truly-hybrid RT from research to clinic is a treatment planning system (TPS) that can generate clinically-deliverable hybrid plans. To meet this urgent need, this effort will develop novel optimization methods and TPS for clinically-deliverable truly-hybrid RT, which is a radical step towards prospective clinical trials for testing the hypothesis. Aim 1: Optimization methods and TPS for clinically-deliverable truly-hybrid RT. Aim 2: Optimization methods for accurate and efficient MCO truly-hybrid planning. Aim 3: Deep learning based optimization methods for efficient truly-hybrid planning.

NIH Research Projects · FY 2025 · 2021-04

PROJECT SUMMARY: We have developed methods to profile the metabolome of hematopoietic stem cells (HSCs) and other rare cell types purified from tissues. Each hematopoietic cell type had a distinct metabolite identity. Most metabolites were enriched or depleted in specific cell types, suggesting they may have novel cell-type specific roles. HSCs and multipotent progenitors (MPPs) in mouse and human bone marrow had high levels of ascorbate (Vitamin C), which promoted the activity of the enzyme TET2, a suppressor of HSC function. Hematopoietic-specific ascorbate deficiency promoted HSC function, myelopoiesis and the generation of inflammatory myeloid cells, and caused early lethality. Ascorbate deficiency is common in the human population because in early primate evolution we lost the ability to synthesize ascorbate. Ascorbate deficiency in healthy people is associated with increased risk of mortality for unknown reasons. Hematopoietic TET2 loss of function mutations are also common in humans, and drive a clonal expansion of mutant blood cells termed clonal hematopoiesis. TET2- deficient blood cells may contribute to an increased risk of mortality. This application’s objective is to understand the role of ascorbate in the regulation of myelopoiesis. Our central hypothesis is that ascorbate suppresses myelopoiesis, and that ascorbate deficiency increases myelopoiesis and inflammation after plasmodium infection. To test this hypothesis, we will use genetically engineered ascorbate deficient mice, to mimic the human condition, and Tet2-deficient mice. In Aim 1 we will test if ascorbate suppresses the generation of inflammatory myeloid cells by acting on HSCs or restricted myeloid progenitors, and if this is mediated by Tet2. In Aim 2 we will determine the effects of ascorbate deficiency or Tet2 deficiency on the myelopoietic response to Plasmodium infection in a mouse model of malaria. In Aim 3 we will investigate the mechanisms by which ascorbate deficiency and Tet2 deficiency promote morbidity and mortality in Plasmodium infection. These experiments may have significant public health implications. They could identify physiological situations, such as infection, in which the presence of ascorbate deficiency and Tet2-deficient clonal hematopoiesis are deleterious to the organism. They may also identify mechanisms by which aberrant myelopoiesis contributes to the pathogenesis of malaria which afflicts more than 200 million people worldwide.

NIH Research Projects · FY 2025 · 2021-04

PROJECT SUMMARY (See instructions): Mammalian cells tightly control the membrane lipid composition of their organelles, and the key homeostatic machinery for many lipids resides within the endoplasmic reticulum (ER). Proteins in the ER must respond to changes in lipid levels, but how proteins respond to changes in lipid composition remains largely unknown. Protein degradation is a key part of feedback loops that regulate cholesterol and sphingolipid biosynthetic pathways, yet how protein homeostasis is influence by the membrane itself is unknown. Resolving this basic knowledge gap will provide insights into membrane protein biology and potentially uncover new directions for treating metabolic diseases. To better understand how the ER membrane influences protein function and how this feeds back into lipid metabolism, we focus on the activity ER resident ubiquitin E3 ligases and their substrates. E3 ligases label substrate proteins with ubiquitin, which causes the substrate proteins to be degraded by the proteasome system. We will study how these enzymes and their substrate proteins are regulated in the ER membrane and how their activity, in turn, regulates lipid levels. In the first aim, we will study how membrane cholesterol levels influence the activity and substrate recognition of two ER-localized E3 ligases called MARCH6 and TRC8. We will use cellular and biochemical assays to determine the lipid binding specificity and activity relationships for these two enzymes. We will use cryo-electron microscopy to elucidate how these enzymes recognize sterols and how membrane lipids influence the ability of these enzymes to form active complexes with substrate proteins. Through these efforts we will discover biophysical principles that may apply to a broad range of membrane bound mammalian E3 ligases. In the second aim, we will begin to uncover how targeted protein degradation regulates sphingolipid metabolism. We will use molecular dissection techniques along with candidate and discovery-based approaches to elucidate the molecular determinants of the regulated degradation of regulatory proteins called ORMDLs that control sphingolipid biosynthesis. Together, these aims will provide the first insights into substrate recognition of mammalian membrane E3 ligases and a new understanding of how these membrane sensors function in cells.

NIH Research Projects · FY 2025 · 2021-04

Fragile X Syndrome (FXS) is the most common heritable autism spectrum disorder and is associated with auditory features such as hypersensitivity to sound (hyperacusis). FXS is caused by the absence of Fragile X mental retardation protein (FMRP), which is known to bind specific mRNAs and repress their translation. Little is known about how FMRP impacts the central auditory pathway. We will study FMRP effects on gene expression in the auditory brainstem, using the well-established fmr1-knockout (KO) mouse model, which exhibits auditory hypersensitivity and seizures in response to loud noise. Although the well-established role of FMRP is translational repression, it has been shown recently in neurons that FMRP can also change the level of many mRNAs. Whether this occurs in the auditory brainstem is unknown. We have novel transcriptome data (unpublished) showing that the levels of many mRNAs that are known to be bound by FMRP, and to function in synaptic pathways, are decreased in the fmr1-KO cochlear nucleus. How this occurs is not known. Aim 1 will test the hypothesis that direct FMRP binding stabilizes the bound mRNA, but in the absence of FMRP, these mRNAs have decreased stability (and, therefore, decreased level). We will also determine if decreased stabilization of mRNA leads to decreased protein level or if it is offset by the loss of FMRP-mediated translational repression so as to manifest as increased protein level. Another possibility is that FMRP acts indirectly through translational repression of factors, such nonsense-mediated mRNA decay (NMD) factor, UPF1. Unpublished data from my research mentor’s lab has shown that induced pluripotent stem cells derived from FXS-patient fibroblasts manifest an abnormally high level of UPF1 (whose mRNA is bound by FMRP), resulting in hyperactivated NMD and, as a consequence, reduced levels of cellular NMD target mRNAs. Based on these data, Aim 2 will test the hypothesis that NMD is hyperactivated in fmr1-KO cochlear nucleus, leading to gene downregulation. Lastly, it is known that FMRP is involved in activity dependent processes. For example, dendritic localization of FMRP is increased with glutamatergic signaling and loss of afferent activity can blunt translational repression by FMRP. Aim 3 will test the hypothesis that the FMRP effects on gene expression are dependent on afferent activity. We will examine an inducible deafness mouse model to determine if it can phenocopy the fmr1-KO, indicating that afferent activity is required for FMRP function. The PI has extensive molecular biology experience, and with the guidance of a primary mentor who is a respected RNA biologist. The PI will master current RNA techniques and work towards becoming an independent investigator. Results will reveal how FMRP regulates genes important for auditory development and plasticity. The ultimate goal is to reveal potential therapeutic targets to treat auditory hypersensitivity and processing disorders. The mechanism leading to hyperacusis is poorly understood, and because it is not limited to FXS and affects up to 15% of the population, the study is relevant to the general population.

NIH Research Projects · FY 2026 · 2021-04

Project Summary/Abstract The vast expanse of chemical space offers limitless possibilities for medicinal chemists, particularly in discovering novel scaffolds and pioneering chemical functional groups with drug-like properties. In this respect, various chemical functional groups, absent in natural metabolites, have been developed as useful building blocks and (bio)isosteres in medicinal chemistry during the past several decades. These structural and electronic unique functional groups exhibit the ability to modulate the pharmacokinetic and physicochemical properties of drug candidates, because of their unusual physical and chemical properties. However, these sterically hindered and uncommon functional groups also pose synthetic challenges in terms of their accessibility and subsequent functionalization. Among these functional groups, the persulfuranyl (with a six-coordinate hypervalent sulfur atom) group is long considered a possible (bio)isostere for tert-butyl and trifluoromethyl groups, but is synthetically challenging to access due to limited chemical reagents and methodologies. The scarce availability of persulfuranyl transfer agents has greatly restricted their broad application in discovery chemistry. In this regard, the development of novel and bench-stable general persulfuranyl and other sulfur(VI) group transfer reagents is still highly desirable. Building upon these guidelines, this proposal aims to general a series of user-friendly, shelf- stable sulfur(VI) transfer reagents. These reagents will be accessible, and the following methodologies will be optimized from the vantage points of operational simplicity and generating products with novel chemical space. These described bench-stable reagents and chemical transformations will enable facile access to sulfur(VI)- containing scaffolds of interest for medicinal chemistry and biological evaluation. The biological and pharmacokinetic properties of several families of compounds will be further investigated through our already established collaborations. Taken together, generating an armamentarium of these stable reagents and practitioner-friendly chemical methodologies will address the synthesis of challenging and medicinally relevant sulfur(VI) chemical scaffolds, accelerate discovery and allow us to further explore novel chemical space in medicinal chemistry, agrochemistry and other chemical and scientific disciplines.

NIH Research Projects · FY 2025 · 2021-04

Project Summary The proposed project focuses on our recent discovery that immunological production of the oxysterol 25- Hydroxycholesterol (25HC) potently inhibits the cellular dissemination of two globally important bacterial pathogens, Listeria monocytogenes and Shigella flexneri. The anti-bacterial activity of 25HC is mediated through mobilization of the accessible cholesterol pool from the plasma membrane (PM). Accessible cholesterol is one of three pools into which PM cholesterol is sub-divided and this pool regulates cellular signaling pathways that control lipid homeostasis and cell growth. By first characterizing the molecular mechanism by which 25HC induces internalization of accessible cholesterol (Aim 1), these studies will reveal how cholesterol can be rapidly transported in response to cytokine stimulation. Second, we will determine how remodeling of PM cholesterol suppresses Listeria and Shigella from penetrating the cell-to-cell contact junctions of the mucosal epithelium (Aim 2). This work will reveal how mammals enhance the barrier function of mucosal surfaces through cholesterol metabolic pathways and will identify points of weakness in the mucosal immune system that may be exploited by numerous microbial pathogens. Third, we will develop new technologies for monitoring cholesterol dynamics in the living organism and use these technologies to determine the tissues and cell types that mobilize accessible cholesterol in response to bacterial infection (Aim 3). Finally, the physiological significance of oxysterol-mediated immune pathways will be investigated in mammalian model organisms using three complementary mouse models that disrupt 25HC activation, production, and downstream activity (Aim 4). Insights gleaned from these studies, which range from basic biochemistry to mouse models of infection, will explain how the human immune system has adapted fundamental aspects of cholesterol metabolism to protect barrier cells from intracellular bacterial infection. Developing new drugs that mimic the molecular activity of 25HC as determined in this proposal would be an innovative approach to combat human infectious disease associated with pathogens that exploit host cholesterol metabolism. These studies will also provide new insights into the pathogenic mechanisms of an important infectious disease-causing agent and also into the biology of the human inflammatory response.

NIH Research Projects · FY 2025 · 2021-04

Project Summary/Abstract The molecular heterogeneity of cancers poses a major hurdle for treatment and drug discovery efforts. Previous studies have addressed this challenge by characterizing distinct molecular subtypes of specific cancers (e.g., breast cancers), based on cell type-specific patterns of gene expression. To interrogate the molecular underpinnings of cancer subtypes, the Kraus Lab has developed a robust and multi-faceted computational pipeline that integrates data from various genomic assays to define a Total Functional Score of Enhancer Elements (TFSEE) for each subtype. One outcome of this method is the identification of cancer subtype- enriched transcription factors (TFs) that promote subtype-specific enhancer formation and drive downstream transcriptional outcomes. In breast cancers, several TFSEE-identified subtype-specific TFs are uniquely required for the growth of the cognate breast cancer subtype, but do not affect the proliferation or viability of other subtypes. Recent studies have also shown that ADP-ribosylation (ADPRylation), a post-translational modification of proteins, varies dramatically across the different subtypes of breast cancers. ADPRylation is mediated by the Poly(ADP-ribose) polymerase (PARP) family of enzymes, including PARP-1, a nuclear enzyme which is the target of FDA-approved PARP inhibitor drugs. PARPs are well known for the roles in DNA repair, but recent studies suggest an important BRCA1/2-independent role in transcriptional regulation as well. In preliminary analyses, we have identified a cohort of cancer-related TFs that are ADPRylated in breast cancers. The long-term objective of these studies is to achieve a better understanding of the molecular and biochemical mechanisms underlying the regulation of breast cancer subtype-specific TFs by ADPRylation, as well as the responses of distinct breast cancer subtypes to PARP inhibitors. Our hypothesis is that ADPRylation of subtype-specific TFs dictates their function and may influence the response of breast cancer cells to PARP inhibitors. We have proposed a project that will use an integrated set of biochemical, molecular, cell-based, mouse-based, genomic, and proteomic assays to test our overarching and specific mechanistic hypotheses. Specifically, we will: (1) Identify TFs that are ADPRylated in breast cancers (Aim 1), (2) Determine how ADPRylation of TFs affects their molecular and biochemical functions (Aim 2), and (3) Determine the effects of TF ADPRylation on the responses of breast cancer cells to clinically used PARP inhibitors (Aim 3). These studies will take advantage of the expertise of the PI’s lab in PARPs, ADPRylation, enhancer function, and gene regulation in cancer. Although focused initially on breast cancers, our results should be broadly applicable across a variety of cancer types. Our integrative approach using ‘omics’ and functional assays will provide new insights into the regulation of TF ADP-ribosylation in breast cancers that will serve as a model for how to explore PARP function and ADP-ribosylation in cancer cells. The use of mouse-based models and patient samples will allow us to explore the clinical relevance of our mechanistic results. We anticipate that our studies will suggest new avenues for the therapeutic potential of PARP inhibitors in cancers beyond DNA repair pathways.

NIH Research Projects · FY 2025 · 2021-04

Project Summary The worldwide obesity epidemic presents a significant public health crisis that is progressively worsening. This has led to intensive efforts to identify the host and environmental factors that regulate human metabolism and energy homeostasis. The gut microbiota has been identified as an environmental factor that regulates lipid metabolism and absorption in the intestine, and thus promotes high fat diet-induced obesity. However, a major knowledge gap remains about the specific bacterial and host factors that regulate intestinal lipid absorption and metabolism. The overall goal of this proposal is therefore to identify the intestinal bacteria and host immune recognition pathways that regulate intestinal lipid metabolism. Prior work by our group identified the circadian transcription factor NFIL3 as essential for the gut microbiota’s role in driving obesity in mice fed a high-fat, high sugar Western-style diet. NFIL3 expression is regulated by the microbiota, and promotes the transcription of intestinal epithelial genes that regulate lipid absorption and metabolism. Preliminary studies of monocolonized mice revealed that flagellated Gram-negative bacteria induced NFIL3 expression. Therefore, my central hypothesis is that Gram-negative flagellated bacterial species, such as Escherichia coli, selectively promote lipid absorption in intestinal epithelium. I will test this hypothesis by using gnotobiotic mice and genetic manipulation of both mice and bacteria. My first aim is to identify intestinal bacteria that regulate intestinal lipid uptake and metabolism through NFIL3. Monocolonized mice will be fed a Western style diet and analyzed for lipid content, gene expression changes in the small intestine, and metabolic syndrome. I will then use genetically-altered bacteria to identify specific bacterial factors that are required to promote intestinal lipid absorption. My second aim is to identify the host immune pathways that are required for bacterial activation of NFIL3-regulated metabolic pathways. Genetically altered mice with deletions of specific pattern recognition receptors will be used to identify host factors required for lipid uptake and metabolism. These studies will provide new insight into how the microbiota regulates lipid metabolism of the host and should identify new avenues for therapeutic interventions into obesity.

NIH Research Projects · FY 2025 · 2021-03

ABSTRACT CAR-T cell therapy is an emerging option for cancer treatment, but its efficacy is limited, especially in solid tumors because the effector CD8+T cells become dysfunctional and exhausted in the tumor microenvironment (TME). However, the key pathways that define the delicate balance between the effector vs exhausted state of CD8+T cells remain unclear. Our preliminary studies demonstrate that sumoylation of the T-box transcription factor, Eomesodermin (Eomes), facilitates its association with Zbtb44, a member of the ThPOK family of transcription factors. The Zbtb44-Eomes complex promotes the effector function and anti-tumor activity of CD8+ tumor infiltrated lymphocytes (TILs). In exhausted CD8+ TILs, the ubiquitin ligase Trim47 targets Zbtb44 for degradation and disrupts the Zbtb44-Eomes complex. Furthermore, CRISPR-Cas9-mediated inhibition of Trim47 rescues exhausted CD8+ TILs and restores their effector function. These preliminary findings led us to hypothesize that ubiquitination and sumoylation of the Zbtb44/Eomes complex are critical molecular events that dictate the effector vs exhaustion of CD8+ TILs which can be therapeutically targeted. In Aim1, we will determine the mechanism by which the Zbtb44-Eomes complex promotes effector CD8+T cell function and anti-tumor immunity. We will use newly generated Zbtb44-/- mice to investigate how sumoylation of Eomes at Lys(K)-446 facilitates the formation of the Zbtb44-Eomes complex via the SUMO interacting motif (SIM) within Zbtb44. Further, we will delineate the mechanism by which the Zbtb44-Eomes complex cooperatively binds to and transactivates the IFN- promoter. In Aim 2, we will determine the mechanism by which Trim47-mediated ubiquitination of Zbtb44 leads to dysfunction of CD8+T cells. We will investigate how Trim47, which is upregulated in exhausted (PD1+Tim3+) CD8+ TILs, targets Zbtb44 for ubiquitination at K139 and promotes its degradation. Using newly generated Trim47-/- mice, we will determine how disruption of the Zbtb44- Eomes complex leads to the inhibitory transcriptional profile of exhausted CD8+T cells. In Aim 3, we will target the Zbtb44-Trim47 pathway to promote anti-tumor immunity. We will test the therapeutic potential of blocking Zbtb44 ubiquitination in CAR-T cells against carcinoembryonic antigen (CEA) in the MC38 and in a patient- derived xenograft (PDX) colon cancer model. Completion of these studies will lead to: 1) dissection of the novel Zbtb44-Eomes complex that is critical for effector CD8+ T cell function, 2) determination of how Trim47-mediated ubiquitination disrupts this complex leading to alternate transcription profile in exhausted CD8+ TILs, and 3) evaluate the means to target the Zbtb44- Trim47 pathway to overcome the current limitations of CAR-T cell therapy for solid tumors.

NIH Research Projects · FY 2025 · 2021-03

Project Abstract The success of any bacterial pathogen ultimately depends on its ability to multiply and transmit to new hosts. Mycobacterium tuberculosis (Mtb), the causative agent of the human disease tuberculosis and one of the most successful pathogens in human history, likely also employs sophisticated means to spread from one person to the next, including mediating caseation, tissue destruction, and airborne transmission. Yet, despite the toll Mtb has taken on world health, the molecular mechanisms responsible for Mtb transmission remain elusive. A major symptom of active tuberculosis is cough, and cough is a major mechanism of transmission. Although cough is a major route of aerosolization and transmission of Mtb, very little is known about the factors that produce cough during infection. Furthermore, epidemiologic studies have demonstrated that Mtb strains representing specific lineages are more prevalent in humans but whether differences in prevalence are due to differences in bacterial transmissibility and associated factors such as cough induction and aerosolization of bacteria is unknown. Thus, there is an urgent need to better characterize the transmission dynamics of Mtb and the relationship of cough to transmission. Because nociceptive neurons mediate cough, and some bacteria including mycobacteria secrete complex molecules targeting neurons, we hypothesized that Mtb produces molecules to trigger nociceptive neurons to activate the cough response, thereby facilitating transmission. We discovered and characterized the activity of one such molecule, sulfolipid-1, and recently identified a second molecule produced by virulent mycobacteria. In the proposed research we will (1) Identify and study the sulfolid-1 receptor in neurons and experimental animals, (2) Characterize the activity of the second nociceptive molecule in neurons and experimental animals, and determine how its activity combines with that of sulfolipid-1 (3) Develop and use a sophisticated Mtb transmission system to measure transmission, cough and aerosolized particles safely and quantitatively and use the system to compare the transmissibility of a variety of Mtb mutants lacking cough-inducing molecules. The proposed work is expected to identify novel factors associated with nociceptive neuron activation, cough and mycobacterial transmission.

NIH Research Projects · FY 2025 · 2021-03

Project Summary/Abstract Circadian rhythms are 24h oscillations in a variety of processes that are entrained by environmental cues including light. Molecularly, this “clock” is driven by key transcription factors and feedback loops that generate rhythmic expression of thousands of mammalian genes in a variety of tissues. Past work has revealed the impact of circadian rhythms on metabolism and immunity. However, the impact of circadian rhythms on infection, particularly enteric virus infection, is understudied. Preliminary experiments using the enteric virus coxsackievirus B3 (CVB3) revealed a profound circadian effect on infection: Mice orally inoculated with CVB3 in the morning had viral titers 10-100 fold lower than mice inoculated in the evening. Inhibition of viral replication in the morning correlated with increased expression of antiviral proteins at this time. Circadian effects on CVB3 infection were lost in mice lacking certain proteins involved in interferon-mediated antiviral responses, suggesting a possible link between circadian transcriptional control and innate immune responses in the intestine. Indeed, expression of an antiviral protein was lost in mice that lack activity of an important clock transcription factor. Thus, CVB3 infection is under circadian control and rhythmic host interferon responses contribute to these effects. However, several questions remain. In this work we will 1) examine mechanisms by which clock transcription factors control expression of innate immune genes, 2) examine the effect of clock transcription factors on infection with CVB3 and other enteric viruses, and 3) identify and evaluate cell types in the intestine that contribute to circadian control of enteric virus infection. Answering these questions will illuminate key, but unanticipated, aspects of intestinal biology that influence enteric virus infection.

NIH Research Projects · FY 2024 · 2021-03

PROJECT SUMMARY/ABSTRACT The overarching goal of this award is to prepare the applicant for an independent, sustained program of research that incorporates psychosocial, behavioral, and clinical concepts and methods to understand and design interventions to guide clinical translation of uncertain and reclassified genomic variants. Variants of uncertain significance (VUS) introduce uncertainty and can confuse clinical decision making for patients and providers. VUS are also frequently reclassified, especially in racial/ethnic minority populations, and can inform clinical decision making. However, insufficient evidence around the influences and outcomes of uncertain and reclassified variants presents a challenge for more diffuse clinical translation of these genetic variants. Such understanding is particularly important in clinical oncology, as identification of mutation carriers can significantly alter cancer prevention, screening, surgery recommendations, and treatment. The K99 phase is designed to augment the candidate's prior research experience though coursework, apprenticeships and directed readings with specific training in: 1) clinical health informatics, 2) psychometrics and survey methodology, and, 3) advanced qualitative methods. The proposed research will collect patient reported and electronic medical record data from six healthcare systems that provide clinical genetic services to a racially/ethnically diverse patient population. Aim 1 (K99 phase) surveys a national sample of oncology providers to understand their practices related to variant reclassification and recontact. Aim 2 (K99 phase) interviews patients to identify dimensions of reclassification associated psychosocial well-being. Aim 3 (R00 phase) uses data from aim 2 and existing literature to develop and pilot an instrument to measure genomic uncertainty in patients. Aim 4 (R00 phase) evaluates the clinical utility of variant reclassification. This work will generate evidence to inform institutional and professional practice around variant reclassification. Taken together, the findings from this study will contextualize, and provide tools for a future longitudinal study to determine the behavioral, psychosocial, and clinical consequences of receiving uncertain genetic test results. This project is a critical building block for the applicant's long-term research goal to develop and test interventions (at the levels of provider, patient and healthcare system) to facilitate the clinical translation of genomics into diverse health systems and into underserved populations. The proposed award will provide training, mentorship and research experience that will serve as the foundation for the applicant's career as an independently funded clinical investigator dedicating to improving health outcomes in translational genomics for underrepresented minority populations.

NIH Research Projects · FY 2025 · 2021-03

Loss of function mutations in SMARCA4/BRG1, a tumor suppressor and core component of SWI/SNF chromatin remodeling complexes, occur frequently in lung adenocarcinoma (LAD) and harbor a poor prognosis. As a tumor suppressor, aberrations of BRG1 have no actionable therapy. Modulation of GLI1, a transcription factor and target gene of the Hedgehog (Hh) signaling pathway, by alternative pathways has been reported and high expression of GLI1 is correlated with significantly poor survival of non-small cell lung cancer patients. BRG1-loss has been shown to up-regulate GLI1 independently of the Hh pathway in mouse embryonic fibroblasts. However, no such studies have been reported in lung cancers. We show that high expression of GLI1 in LAD cell lines depend upon BRG1-loss. Genetic and pharmacologic inhibition of GLI1 expression inhibit the growth and induce cell death in BRG1-deficient lung cancer cell lines. Therefore, we HYPOTHESIZE that loss of BRG1 upregulates GLI1 expression to drive LAD growth and that GLI1 is a candidate therapeutic target for BRG1-deficient LAD. The rationale for the proposed research is that elucidation of the mechanisms by which loss of BRG1 upregulates GLI1 expression will identify novel therapeutic candidates whose modulation will inhibit expression of the GLI1 transcription factor in BRG1-deficient lung cancers – a cancer type that has no readily actionable target for treatment. We propose to identify mechanisms for GLI1 suppression by BRG1 and for upregulation of GLI1 expression with BRG1 loss. We will also test three therapeutic regimens that inhibit GLI1 expression with drugs that are FDA-approved or in active clinical testing. We utilize a novel autochthonous mouse model and patient derived xenografts of BRG1- deficient LAD to test the regimens. We will also identify missense and nonsense mutations that upregulates GLI1 expression and thus, may serve as predictive biomarkers for the therapies tested here. If successful, our results will establish a firm scientific rationale for targeting BRG1- deficient lung cancers with compounds that inhibit GLI1 expression and that are readily available for clinical testing.

NIH Research Projects · FY 2025 · 2021-03

Friedreich’s ataxia (FRDA) is an autosomal recessive neurodegenerative disease caused by reduced expression of the mitochondrial protein frataxin (FXN). Frataxin is translated as a 210 amino acid (aa) precursor (FXN-P) that is imported into the mitochondrial matrix where it undergoes sequential cleavage steps, producing a 168 aa intermediate (FXN-I) and the mature isoform of 129 aa (FXN-M). Frataxin participates in iron-sulfur cluster (ISC) biosynthesis in the mitochondria, and many of the overt FRDA phenotypes result from deficient activity of ISC- containing enzymes. Currently, there is no cure for this debilitating disease. Most FRDA patients are homozygous for large expansions of GAA triplet repeat sequences in intron 1 of the FXN gene, while a subset of patients are compound heterozygotes with an expanded GAA repeat tract in one FXN allele and a missense or nonsense mutation in the other. Homozygous and compound heterozygous mutant genotypes both result in reduced levels of FXN-M protein when compared with healthy controls. The most prevalent missense mutation changes a glycine to valine at position 130 (G130V). FRDA G130V patients exhibit different clinical features than patients harboring homozygous GAA expansions, including lower limb spasticity rather than ataxia, preserved sensory responses, spared speech and upper limb functions, and slower disease progression. Paradoxically, substantially less FXN-M protein is detectable in G130V patient samples than in patient samples harboring two expanded alleles. Our preliminary data revealed that normal mitochondrial maturation processing of the FXN protein is perturbed by the G130V mutation, suggesting functional importance of an intermediate isoform (G130V-I). We hypothesize that the G130V mutation impairs FXN mitochondrial maturation processing and/or destabilizes the mature isoform. The unprocessed FXN-G130V-I isoform is functional and partially compensates for the substantial reduction of FXN-M, thus slowing disease progression and contributing to the distinct symptoms of FRDA G130V patients. To address these hypotheses, we will use novel cellular and mouse models of FRDA G130V. First, we will define the structural and functional properties of the FXN-G130V-I isoform to test whether this mutation confers a change of function that contributes to the atypical clinical presentation of FRDA G130V patients. Subsequently, we will determine mechanisms governing steady state levels and maturation processing of FXN-G130V in iPSC-derived cortical and sensory neurons. Finally, using FRDA patient-derived neuronal models as well as our novel Fxn G127V mouse model, we will define molecular mechanisms underlying the unique clinical presentation of FRDA G130V patients. Results of the proposed studies will have a broad impact on therapy development for all FRDA patients.

NIH Research Projects · FY 2025 · 2021-02

Project Summary/Abstract Cholinergic neurons (ChIs) are a central but poorly understood element of striatal circuitry. A considerable literature strongly implicates ChI dysfunction in the pathogenesis of abnormal movements, especially in dystonia and levodopa-induced dyskinesias in Parkinson disease. A common theme of these studies is that maladaptive plastic changes cause aberrant ChI output and connectivity, promoting motor dysfunction. The central goal of this proposal is to advance understanding of the cellular and synaptic mechanisms through which ChIs cause motor dysfunction by employing novel selective genetic and chemical strategies in a recently validated model of DYT1 dystonia. Conditional Knock Out of torsinA from all striatal neurons (using Dlx5/6-Cre; “Dlx-CKO”) causes selective neurodegeneration of dorsolateral striatal ChI. ChI degeneration occurs roughly coincident with the juvenile onset of abnormal twisting movements in these mice, and selective ChI abnormalities are also present in postmortem tissue from DYT1 subjects. These movements are suppressed by the same anti-muscarinic compounds used to treat patients with DYT1 dystonia, establishing model therapeutic validity and suggesting shared pathophysiology with human dystonia. Surviving striatal ChIs are enlarged and hyperexcitable, and receive aberrant synaptic inputs. Selective ablation of these surviving ChI suppresses abnormal twisting, implicating these cells as key contributors to abnormal movements. Based on these data, we hypothesize that maladaptations in surviving ChIs drive motor dysfunction. Successful completion of the proposed studies will fundamentally advance understanding of maladaptive mechanisms whereby ChI function and connectivity drive abnormal movements, information highly significant for multiple striatal diseases. We will first address our hypothesis by testing the necessity of striatal ChI dysfunction in abnormal movement generation by selectively restoring torsinA to these cells (Aim 1), decisively moving beyond the current association between these factors. We will determine if cholinergic dysfunction arises primarily from intrinsic ChI abnormalities or defects in how they respond to afferents (Aim 2), and, informed by Aims 1 and 2, will pursue translational studies (Aim 3) testing whether directly modulating the activity of surviving ChIs can suppress dystonic-like movements. This proposal is therefore highly signifiant because it will define a circuit-based model of motor dysfunction that will inform the design of targeted therapeutics.

NIH Research Projects · FY 2025 · 2021-01

PROJECT SUMMARY/ABSTRACT Alzheimer’s disease (AD) is the most common cause of dementia in the elderly with currently no cure or effective disease-modifying therapy. The pathogenesis of AD is unclear; however, a leading hypothesis is that accumulation of amyloid-beta (Aβ) peptides derived from the amyloid precursor protein is one of the earliest pathological events resulting in neuronal dysfunction, at least in part by dysregulating intracellular Ca2+ homeostasis, and disruption of neural networks culminating in dementia. While cognitive impairment is the major manifestation of AD, non-cognitive manifestations such as unintentional body weight loss often occurs prior to the cognitive decline. Furthermore, weight loss in AD correlates with worsening disease progression and increased mortality, while weight gain is protective. Collectively, this suggests that brain regions such as the hypothalamus that regulate body weight and systemic metabolism may be selectively vulnerable to Aβ early in the pathogenesis of AD during the presymptomatic or preclinical stages. However, the cellular and molecular mechanisms underlying the early systemic metabolic dysfunction in AD have remain largely unexplored. Therefore, the goal of this application is to test the hypothesis that hypothalamic networks regulating systemic metabolism are selectively vulnerable to Aβ pathology and contribute to the early pathogenesis of AD. We will use a “bench-to-bedside” strategy using state-of-the-art molecular, neurophysiological, imaging, and genomic approaches in genetic mouse models, and verify key findings in clinically relevant human studies. We will test the following working hypotheses: (a) disruption of intracellular Ca2+ homeostasis by Aβ is an early pathological event leading to dysfunction of leptin-responsive hypothalamic NPY/AgRP neurons; (b) Aβ causes disruption of hypothalamic networks regulating systemic metabolism; and (c) central leptin signaling dysfunction is an early manifestation of human subjects with Alzheimer’s disease. The findings from this project will shed light on the mechanisms underlying early selective vulnerability in the hypothalamic network regulating systemic metabolism and identify the cell types affected, thereby filling a knowledge gap in our understanding of one of the earliest clinical manifestations of AD.

NIH Research Projects · FY 2026 · 2020-12

PROJECT SUMMARY/ABSTRACT Tissues store excess lipids in cytoplasmic organelles termed lipid droplets (LDs) that are critical to cellular and organismal homeostasis. Whereas long-term lipid storage occurs in adipose tissues, the liver and kidney store lipids for shorter time-periods and must actively sense, compartmentalize, or secrete lipids or lipid signaling molecules to maintain human health. Defects in this lipid balance promote diseases including fatty liver disease and type 2 diabetes (T2D), and can aggravate acute kidney injury (AKI) that affects millions of Americans and is highly prevalent in hospital patients, especially diabetics1-5. To maintain lipid homeostasis, the liver and kidney must balance the storage of absorbed lipids from blood circulation (often called “old fat” since it is made elsewhere in the body) with locally produced lipids via de novo lipogenesis (termed “new fat”). Studies reveal “old” and “new” lipid pools play distinct roles in tissue physiology and disease6-11, but a pervasive question is how these lipid pools are sensed, functionally compartmentalized, and facilitate inter-organ signaling in human health and diseases. Here, we lead a multi-disciplinary team to dissect how liver-like and kidney tissues organize their lipid pools, and how these pools are sensed and signal to execute metabolic homeostasis and tissue repair. Capitalizing on a uniquely enabling genetic screening platform we developed in Drosophila during our previous funding period, we identify genes and pathways that compartmentalize “old” and “new” lipid pools into functionally distinct LD populations in the Drosophila fat body. Through this genetic platform we discover a novel cytokine termed cDIP. Human cDIP homologs are linked to diabetes through poorly defined mechanisms22-24. We find cDIP signaling is triggered by depletion of de novo lipogenesis lipids, and influences energy signaling and inter- organ crosstalk necessary for survival. In Aims 1 and 2, we capitalize on this cDIP-dependent pathway to mechanistically dissect how lipid pools are sensed and drive inter-organ signaling and metabolic adaptation. Capitalizing on our Drosophila findings, in Aim 3 we apply these principles to the mouse kidney to address a key knowledge gap in kidney injury and repair. Following ischemia reperfusion injury (IRI) induced AKI, kidney cells accumulate LDs, but whether these are a manifestation of pathology or pro-survival is unclear. We isolate IRI-induced LDs and discover specialized LDs decorated with proteins that drive arachidonic acid metabolism and the synthesis of eicosanoids, key lipid signaling molecules mediating tissue inflammation and repair. Injury-induced LDs are also loaded with oxidation-prone linoleic acid, and we provide evidence that LDs store these lipids to suppress lipid peroxidation-associated stress. We test the innovative hypothesis that IRI- induced LDs are thus functionally specialized to coordinate eicosanoid lipid signaling and storage of oxidation- prone lipids, promoting kidney health. Collectively, our work provides new mechanistic insights into how lipid pools are sensed and organized, and how lipids signal, highly relevant to liver and kidney diseases.