Ut Southwestern Medical Center

NIH Research Projects · FY 2025 · 2024-07

Project Summary The pathways regulating the development of early-onset glaucoma are not well understood. Despite evidence suggesting that a large proportion of early-onset glaucoma cases may have a genetic basis, known genes only account for about 20% of cases. In fact, only 12 genes have been described so far, for primary forms of this disease, compared to hundreds for retinal dystrophies. In this complex biological system, a Forward Genetics approach is an ideal strategy to ask, without preconceptions, which genes/molecules are important in regulating early-onset glaucoma. Our short-term goal is to identify and characterize gene/protein defects and molecular pathways that lead to early-onset glaucoma. The long-term goal is to leverage our research discoveries to understand this blinding disease, improve screening strategies, and identify novel therapeutic opportunities. We propose that a high-throughput and unbiased strategy provides an ideal approach to discovery of gene/phenotype associations for early-onset glaucoma. In collaboration with Nobel laureate Bruce Beutler, we have been employing a robust state-of-the-science and unbiased Forward Genetics pipeline in which random mutations are generated and mice can be screened for signs of glaucoma. We have already collected retinal images from 6000 mutagenized mice and proposing to use this extensive database to screen for genes that lead to inner retinal thinning. Our approach has significant advantages compared to other existing protocols. Most importantly, ours is the first and only protocol in which all mice have been pre-genotyped at all mutant loci. In addition, the large scale of our system and the large pedigree size will also add to the discovery power. Together, these advantages will allow us to identify and pursue novel gene/phenotype associations related to glaucoma. In our retinal studies we have identified over 45 gene-phenotype associations after covering just 5%-8% of the mouse genome. Of these, 20 genes have not been reported to be associated with the retina. These results attest to the strength of our pipeline. Having an excellent continuous variable parameter to monitor for early-onset glaucoma (ganglion cell complex OCT measurements) also supports the feasibility of our proposal. We will harness the power of CRISPR/Cas9 gene editing, single cell RNA sequencing, and co- immunoprecipitation experiments with highly sensitive mass spectrometry and proteomics analysis, and other techniques to explore the mechanisms of these associations. This proposed research will advance our knowledge of the genetic basis in early-onset glaucoma. We also anticipate that our results will lead to the identification of novel diagnostic and therapeutic avenues.

NIH Research Projects · FY 2025 · 2024-07

Project Summary Protein-protein interactions (PPIs) are essential in cellular processes and human diseases. It is estimated that 80% of proteins rely on PPIs to perform their primary functions. Thus, modulating PPIs should be a powerful way to interfere with pathological pathways and treat human diseases. However, PPIs are challenging targets for small-molecule drugs due to the lack of druggable pockets to achieve sufficient drug affinity. Recent advances in covalent inhibition of kinases have shown that targeting hyperreactive cysteines with small electrophilic molecules can achieve increased potency, prolonged target engagement, and improved selectivity. We hypothesize that electrophilic molecules forming covalent bonds with hyperreactive cysteines on PPI interfaces may represent a new avenue for developing PPI-modulating drugs. Building on the recent development in Artificial Intelligence (AI) methods for protein structure modeling and analysis, the ongoing efforts to predict and model 3D structures of human PPIs in my sponsor’s lab, and my co-sponsor’s expertise in developing covalent inhibitors, this project aims to predict and validate druggable hyperreactive cysteines located within PPI interfaces. Using a Convolutional Neural Network trained on a large dataset of reactive cysteines to integrate the physiochemical environment around a cysteine in the 3D space, I will develop a predictor for cysteine reactivity. I will identify reactive cysteines on human PPI interfaces by integrating experimental results and predictions on PPI structures and cysteine reactivity. Based on my preliminary data, I expect to find thousands of PPI interfaces with hyperreactive cysteines. Next, I will analyze the protein surface pockets around hyperreactive cysteines on PPI interfaces using both established tools to evaluate pocket druggability and new AI methods to characterize the geometric and chemical fingerprints of a pocket. Comparing the fingerprint of a potential drug pocket against the surface pockets of the entire human proteome will allow me to identify pockets with unique features for specific drug targeting. Results from these analyses will be incorporated into a comprehensive online database of reactive cysteines on PPI interfaces and their druggability. Combining the above analyses with other structural (such as interface size and other components in a complex) and functional considerations, I will choose several dozen target PPIs to perform virtual screens using multiple established methods to identify covalent drug candidates. Several promising drug candidates supported by multiple methods will be tested experimentally through pull-down assays. Experimentally tested candidates will be further studied by affinity chromatography and mass spectrometry to evaluate the off-target binding partners. Overall, this project will provide valuable insights into the identification and targeting of hyperreactive cysteines within PPI interfaces, offering new opportunities for the development of covalent inhibitors to modulate PPIs and treat various diseases and providing valuable resources for further research in the field.

NIH Research Projects · FY 2025 · 2024-07

PROJECT SUMMARY/ABSTRACT Cancer-associated chromosomal abnormalities frequently arise through punctuated episodes of genomic instability. This is exemplified by chromothripsis, the shattering and re-stitching of individual chromosomes, which generates a distinct rearrangement signature in ~30% of pan-cancer genomes. Chromothripsis is driven by mitotic errors and the formation of aberrant nuclear bodies termed micronuclei that entrap mis-segregated chromosomes outside of the nucleus. Due to defects in nuclear envelope assembly, micronuclei undergo frequent and irreversible rupture that inactivates normal nuclear processes, including DNA replication, DNA repair, and transcription. During mitotic entry, chromosomes in micronuclei undergo extensive breakage into tens to hundreds of fragments through incompletely defined mechanism(s). Our laboratory recently identified that fragmented chromosomes remain bound throughout mitosis by protein-mediated tethers, which facilitates the re-incorporation of the fragments into the nucleus and its subsequent reassembly. This cascade gives rise to the highly complex yet localized rearrangements that are often observed in cancer genomes. In addition to the deletion of tumor suppressor genes and/or formation of oncogenic fusion genes, chromothripsis can also result in the circularization and amplification of genes on extrachromosomal DNAs (ecDNAs). While several key mechanistic steps have been well characterized, the source(s) of mitotic chromosome fragmentation remain unclear. To identify genetic drivers of chromothripsis, I recently conducted pooled CRISPR-Cas9 screens using a chromosome-specific micronucleus system. I unexpectedly identified that a genome maintenance mechanism known as the Fanconi anemia (FA) pathway functions as a critical driver of chromothripsis and complex genomic rearrangements. My preliminary data suggest that the FA pathway promotes mitotic chromosome shattering through the recruitment of structure-specific DNA endonucleases to under-replicated DNA intermediates from micronuclei, which is then followed by mitotic DNA synthesis, a process that may be analogous to the processing of late-replicating fragile sites in the genome. Here I propose to further define the role of the FA pathway and mitotic DNA synthesis in chromothripsis. First, I will comprehensively identify regions of the micronucleated chromosome undergoing active processing during mitosis by the FA pathway. I will also determine whether mitotic DNA synthesis is required for priming fragments for reassembly in the next cell cycle. Second, I will leverage pre-clinical cell models to investigate whether inhibition of the FA pathway represents a feasible therapeutic strategy to suppress chromothripsis-induced emergence of drug-resistant cancer cells harboring ecDNAs. These studies will shed light on how a genome-protective DNA repair mechanism can be co-opted as a pathological driver of cancer genome instability. In addition to this research plan and with support from my sponsor, co-sponsor, and dissertation committee, I have also established a well-defined training and career development plan that will further bolster my abilities as an independent researcher focused on cancer biology.

NIH Research Projects · FY 2025 · 2024-07

PROJECT ABSTRACT Site-one protease (S1P) is a membrane anchored protease in the secretory system and a critical component of cellular signaling pathways including cholesterol biogenesis, the ER stress response, and lysosome biogenesis. S1P begins as an inactive pro-enzyme in the ER and becomes active through autoproteolysis of its inhibitory pro-domains as it folds in the ER and traffics to the Golgi, where it functions as an active enzyme. S1P is best studied in cholesterol metabolism. The spatial control of S1P activity, with the inactive form in the ER and the active form in the Golgi, underpins cholesterol metabolism in human cells. Lipogenic transcription programs that cause the uptake and synthesis of cholesterol are controlled by a family of transcription factor proteins known as Sterol response element binding proteins (SREBP). The SREBP precursors are folded in the ER, where they must be protected from proteolysis by S1P. When cholesterol levels are low, SREBP precursors are transported from the ER to the Golgi, where it they cleaved by S1P to initiate a cascade that results in the liberation of the SREBP transcription factor domain and the upregulation of lipogenesis. Ensuring S1P is active in the Golgi but inactive in the ER is critically important to cells and animals. SPRING (also C12ORF49) is a newly identified co-factor that is critical for the controlled maturation of S1P and understanding their relationship will provide new insights into cholesterol metabolism specifically and protease maturation in the secretory pathway more broadly. In preliminary work, we obtained a high-resolution structure of the soluble S1P-SPRING complex using cryo-electron microscopy (cryo-EM). Structural and biochemical data develop the hypothesis for a proposed mechanism where SPRING matures S1P by competing with and displacing an inhibitory pro-domain. Removing this pro-domain is necessary for S1P to proteolyze external substrates. Experiments with S1P trapped in different maturation stages suggest SPRING binds S1P at an intermediate stage of maturation as S1P traffics from the ER to the Golgi. In this proposal, we will use structural biology, biochemistry, and cellular biology to elucidate how S1P matures in the presence or absence of SPRING and how SPRING controls the enzymatic activity of S1P. In Aim 1, we will obtain cryo-EM reconstructions of S1P in distinct stages of maturation. We will use competition assays to test whether SPRING and the S1P pro-domains compete for binding at the same site of the S1P enzyme. In Aim 2, will use a peptide cleavage assay to determine the substrate specificity of the S1P-SPRING complex and which S1P substrate motifs require SPRING for proteolysis by S1P. In Aim 3, we will determine the functional consequences of disrupting the S1P-SPRING interaction in biochemical and cell-based signaling assays that measure SREBP activity and the ER stress response.

NIH Research Projects · FY 2026 · 2024-07

PROJECT SUMMARY / ABSTRACT Societies are organized into hierarchies, which fundamentally involve social conflict. As a result, there are winners and losers, and social defeat can profoundly impact animals, with the potential to induce abnormal behavior and disease. In this research interest statement, I propose the social ant Harpegnathos saltator as a novel model system to study how epigenetic factors and gene regulation modulate neuronal circuitry resulting in specific social behaviors. The social ant Harpegnathos saltator is an emerging model to study epigenetics of the social behavior because the distinct caste phenotypes of workers and gamergates are specified by the same genome. Unlike most ant species, Harpegnathos individuals can change caste through adulthood: when the queen dies or is removed from a colony, workers enter a dominance tournament until a few become reproductive individuals, called gamergates. Along with changes to behavior and physiology, gamergate status is accompanied by a six-fold lifespan extension and changes in neuronal gene expression and chromatin organization. This transition to gamergate can also be completely reversed by inducing social defeat in gamergates. Our research will address two major topics of epigenetic regulation in the social brain. First, how do social interactions like social bonding, aggression and social defeat induce epigenetic changes responsible for long-lasting behavioral phenotypes? Second, we plan to develop ant models of autism spectrum disorders by generating mutants of ant homologs to human ASD risk genes. We will study the impact of these mutations on chromatin organization, gene expression and behavior in both castes. Harpegnathos ants can provide mechanistic insights into connection between epigenetic pathways, behavior and neurodevelopmental disorders.

NIH Research Projects · FY 2025 · 2024-07

PROJECT SUMMARY Our proposed research employs innovative, unbiased approaches and novel preclinical models to elucidate how disseminated HER2+ breast cancer cells cells survive in the brain microenvironment and initiate metachronous metastasis. Through functional characterization of phenotypically stable preclinical models of HER2+ breast cancer brain metastasis, we discovered latent/dormant (Lat) HER2+ cells display stem cell-like characteristics, downregulate immune activating sensors and survive in equilibrium with innate immune surveillance, while brain metastatic cells escape and metastasize. Moreover, metabolically distinct HER2+ brain-tropic Lat cells and metachronous brain metastatic (M-BM) cells are resistant to radiation and systemic HER2 targeted therapies. AXL, a member of the TAM (TYRO3, AXL, MERTK) receptor tyrosine kinase family is enriched in Lat and M-BMs. AXL expression is enriched in metachronous brain metastatic lesions compared to matched primary tumors from HER2+ breast cancer patients. Of note, AXL is predominantly nuclear in these brain metastatic lesions and in our preclinical brain metastatic model systems. CRISPR affinity purification of in situ regulatory elements revealed enrichment of TEAD transcription factor at the AXL promoter region in Lat and M-BMs. AXL immunoprecipitation-mass spectrometry analysis identified WRN Helicase Interacting Protein 1 (WRNIP1) among other nuclear proteins that interact with AXL. WRNIP1 aids maintenance of genomic stability under replicative stress and promotes survival of Lat and M-BM cells. Depletion of AXL in Lat and M-BM cells or administration of small molecule AXL inhibitor (BGB324) results in attenuated metastasis initiating capacity. Noticeably, increased AXL expression and reduced cytotoxicity was also observed in tumor trained/exposed NK cells, while administration of BGB324 to augmented cytotoxicity. Thus, our central hypothesis is therapy resistant brain-tropic HER2+ breast cancer cells are dependent on nuclear AXL signaling response for survival and membranous AXL expression in NK cell results in dysfunction. The proposed aims will delineate how TEAD signaling response promotes brain metastasis and assess the impact of AXL inhibitors in limiting tumor cell survival and reactivating NK mediated innate immune surveillance.

NIH Research Projects · FY 2025 · 2024-06

Project Summary The inositol trisphosphate receptors (ITPR1, ITPR2, ITPR3) are present in the endoplasmic reticulum, forming homo- or hetero- tetrameric complexes to control calcium release. Upon binding to the second messenger, inositol 1,4,5 trisphosphate (IP3), the ITPR controlled release of calcium controls differentiation, proliferation, and death processes for every cell type in the body. We identified the same single allelic mutation in the Inositol Tris Phosphate Receptor Type 3 (ITPR3) in two unrelated children. The missense mutation creates a p.Arg2524Cys amino acid substitution within the calcium channel. These children have immunodeficiency, Charcot-Marie Tooth disease and partial to complete anhidrosis. While autosomal recessive ITPR3 mutations cause one form of CMT syndrome, immune system problems and sweating defects are not normally seen. In fact, most single allelic mutations in ITPR3 are benign due to compensatory functions by ITPR1 and ITPR2. To understand the basis for the more widespread clinical presentation our two patients with the single allelic p.R2524C variant, as well as an expanding number being reported elsewhere, we genocopied the mutation in mice. Our preliminary findings suggest this Itpr3 p.R2524C variant forms a cell-type specific dominant negative. In the current proposal, we will determine the impact of this Itpr3 mutation on cell signaling in the immune, neuronal, and eccrine systems. A characterization of the T and B cells of the adaptative immune system will be done with a combination of functional assays and signaling studies. Additionally, a characterization of the Schwann cells of the peripheral nervous system and eccrine cells will be undertaken. The studies will be complemented with knockdown approaches using shRNA technologies to determine if functionality can be restored by selectively targeting Itpr3.

NIH Research Projects · FY 2026 · 2024-06

PROJECT SUMMARY/ABSTRACT Dystrophinopathy is a group of X-linked neuromuscular disorders, resulting from mutations in the dystrophin genes. Duchenne muscular dystrophy (DMD) and a milder form, Becker muscular dystrophy (BMD), are the most common forms of dystrophinopathy. Both types of muscular dystrophy (MD) patients develop progressive wasting of skeletal muscle and heart failure, and currently there is no absolute cure. Despite extensive investigation into the management of MD, tools for monitoring the disease progression and the treatment response are yet to be established. Mitochondrial dysfunction and inflammation are indicative metabolic phenotypes of the severity of dystrophinopathy and precede muscle damage, playing causative roles in the pathogenesis of MD. Patients with MD have decreased level of glucose in the skeletal muscle and the myocardium, contributing to the low concentration of downstream metabolites and the subsequent energy deficiency in the muscle. However, how the myocytes utilize the fuel is underexplored. Pyruvate, the end-product of glycolysis, is positioned at a unique metabolic junction that can witness both mitochondrial dysfunction and inflammation via two enzymatic reactions: PDH and LDH. Pyruvate dehydrogenase (PDH) links glycolysis and the tricarboxylic acid (TCA) cycle in mitochondria. Lactate dehydrogenase (LDH) activity is often considered as a measure for glycolysis or anaerobic respiration and is positively correlated with tissue inflammation. Carbon- 13 (13C) MRI with an intravenous bolus injection of hyperpolarized (HP) [1-13C]pyruvate is a unique imaging method for estimating LDH activity and PDH flux by the in-vivo products, [1-13C]lactate and [13C]bicarbonate, respectively. Since hyperpolarization technology is using stable isotope (no ionizing radiation) and pyruvate is a natural metabolite, it is safe to inject HP [1-13C]pyruvate into both adult and pediatric patients. In this proposal, we will investigate mitochondrial dysfunction and inflammation in DMD and BMD patients using HP [1- 13C]pyruvate MRI. Thus, the overall goal of the study is to develop non-invasive biomarkers that detect early metabolic changes associated with myopathies in patients with MD. The underlying hypothesis is that metabolic alterations in myocardium and skeletal muscle precede myopathies associated with dystrophinopathy. The specific aims include to develop elevated lactate production as a biomarker for myocarditis and skeletal muscle inflammation (Aim 1), to develop limited bicarbonate production as a biomarker of mitochondrial dysfunction (Aim 2), and to assess early changes in lactate-to-bicarbonate ratio in predicting MD-associated myopathy (Aim 3). The imaging biomarkers will be compared to clinical cardiac parameters (e.g., ventricular ejection fraction) and other inflammatory markers or coronary risk indicators from blood samples (e.g., high-sensitivity troponin T). The research outcome of the proposed study will develop in-vivo imaging biomarkers that assess metabolic characteristics of the heart and skeletal muscle in patients with muscular dystrophy, and set a groundwork for clinical translation of this technique to muscular dystrophy patients.

NIH Research Projects · FY 2026 · 2024-06

Project Summary / Abstract Due to advances in biomedical engineering and medicine, joint replacement has restored function and relieved pain for millions of people. However, subsequent infection of implanted joints (known as periprosthetic joint infection or PJI) is a debilitating condition that can lead to multiple surgeries, long courses of intravenous antibiotics, and/or loss of limb or even life. Staphylococcus aureus is one of the most common and virulent organisms that causes PJI. S. aureus is also associated with significant morbidity and frequent relapse following treatment. Vaccination against S. aureus is a promising strategy to prevent periprosthetic joint infection. However, traditional vaccine systems designed to prevent S. aureus infection have failed in clinical trials. The goal of this proposal is to evaluate a novel approach to immunotherapy for S. aureus PJI by engineering a biomaterials-based vaccine platform (BiVAX). BiVAX is an injectable biodegradable scaffold that elutes cytokines to recruit dendritic cells, contains adjuvant for activation, and is loaded with antigen at high concentration for uptake by recruited cells. Our group has previously demonstrated that by using pathogen- associated molecular patterns as antigen in this biomaterials-based vaccine system, BiVAX is effective against preventing S. aureus abscesses and lethality from sepsis due to different gram-negative species in mouse models. PJI is a challenging biofilm-associated device infection, and it is unknown if vaccination can prevent disease. In this proposal, I will evaluate the effects of infection and vaccination on the host immune response and determine the efficacy of BiVAX for preventing PJI in murine and rabbit models of disease. I will determine host immune responses to PJI as a function of immunity and pathogen virulence factors in Aim 1 and evaluate the effect of BiVAX against PJI and compare its efficacy to conventional vaccination strategies in Aim 2. I am supported in these efforts by world experts in immunotherapy, vaccine science, and PJI including: primary mentor David Mooney (immunoengineering); co-mentor Ruanne Barnabas (vaccine science), collaborator David Scadden (stem cell biology/immunology); collaborator Jean Lee (murine S. aureus vaccination); collaborator Michael Super (immunology and glycobiology); advisor Antonia Chen (arthroplasty and human clinical trials for S. aureus vaccines); advisor Sandra Nelson (clinical periprosthetic joint infection); and advisor Hang Lee (biostatistics). The resource-rich ecosystem of Massachusetts General Hospital, Harvard University, and the Wyss Institute for Biologically Inspired Engineering have afforded me the necessary tools to conduct this multidisciplinary work at the interface of bioengineering, microbiology, and immunology. This proposal includes a robust training plan designed with my mentors to acquire additional skills in immunoengineering and vaccine science necessary for launching an independent career investigating immunity at the host/device/pathogen interface in device-related infection.

NIH Research Projects · FY 2026 · 2024-06

Project Summary The nasopharyngeal and respiratory mucosa represents a primary barrier to infection by inhaled organisms. Indeed, the earliest contact point between the airway pathogen Mycobacterium tuberculosis and the human body is the airway mucosa. As a result, M. tuberculosis has evolved to disseminate beyond the oral and respiratory mucosa to cause systemic disease, thus accounting for its global impact on human morbidity and mortality. Lining the airway mucosa are specialized epithelial cells that function to transcytose mucosal antigens from the mucosal surface to the basolateral space. These cells, known as microfold or M cells, overlie mucosal associated lymphatic tissue where macrophages and dendritic cells await to ingest and present antigens to B-cells and T-cells. We previously demonstrated that M. tuberculosis penetrates the mucosa via M cells by using a virulence factor called EsxA and a host receptor, scavenger receptor B1 (SR-B1). However, a major gap in our understanding of the impact of this very early event on the interaction of M. tuberculosis with the airway is that we do not fully understand M cell biology. Here, we will apply genetic, biochemical, immunologic, transcriptomic and animal approaches to determine the functional role of M-cells in mucosal and systemic immunity against M. tuberculosis. Thus, in the proposed research we will: (1) Determine the transcriptional profiles and functions of novel genes of primary human and mouse airway M cells at a single cell level under both homeostatic conditions and after experimental M. tuberculosis infection, (2) Identify and study unique cell-cell interactions of M cells with their adjacent epithelial and immune cells, (3) Characterize the mechanisms of binding and transcytosis of M. tuberculosis by airway M cells, (4) Determine the immunologic impact of M. tuberculosis transcytosis across airway M cells. The proposed work is expected to identify how M cells bind and transcytose M. tuberculosis and how this very early event in the life cycle of M. tuberculosis dictates the immunologic outcome of infection.

NIH Research Projects · FY 2025 · 2024-06

PROJECT SUMMARY Lysosomal storage disorder (LSD) is a collection of over 70 monogenic diseases associated with genetic mutations in lysosomal enzymes or proteins. The clinical manifestations of LSD are heterogenous but often include early-onset neurodegeneration with varying degrees of inflammation. The mechanism of inflammation associated with LSD is unknown. We recently showed in Niemann-Pick disease type C1 that an intracellular innate immune signaling pathway, STING, is activated due to impaired lysosomal degradation, leading to inflammation and neuropathology. In this R21 application, we aim to directly examine whether STING is required for pathogenesis of several lysosomal storage disorders using mouse models. We hypothesize that STING is an “immunosensor” of lysosome activity and that lysosomal dysfunction activates the STING- mediated immune signaling and lysosomal biogenesis. In Aim 1, we will breed mouse models of lysosomal storage disorder with Sting-/- mice or treat with a STING antagonist to determine whether STING is required for inflammation and disease pathogenesis. In Aim 2, we will elucidate the mechanisms of STING activation and STING-mediated lysosomal biogenesis. Together, we hope to define STING as a new drug target in lysosomal diseases. This study will also establish a new clinical paradigm for considering STING antagonists as first-line therapy for many lysosomal storage disorders.

NIH Research Projects · FY 2026 · 2024-06

Project Summary: The inability to distribute gene products broadly throughout the CNS is a major limitation of current gene delivery methods for brain disease. Employing white matter connectivity to map brain networks is a powerful, well- established method in laboratory studies, but has not been exploited clinically for therapeutic delivery. The central goal of this proposal is to advance a focused ultrasound (FUS)-based gene therapy delivery strategy – “circuit focused ultrasound” (CIFUS) – that utilizes brain connectivity to widely target interconnected brain regions. Our approach overcomes several longstanding obstacles that have thwarted the tremendous promise of gene therapy for central nervous system (CNS) diseases. This proposal will advance the development of CIFUS and test the hypothesis that this approach can functionally rescue key phenotypes in models of a degenerative and a circuit-based brain disease. We will use a mouse model of Alzheimer disease with tau pathology that spreads across multiple interconnected brain regions during maturation and aging.6,7 We will use mouse models of dystonia that recapitulates motor circuit dysfunction8,9 and neurodevelopmental neurodegeneration8,10. Successful completion of this proposal will advance development of a novel method of widespread, targeted gene delivery that will empower basic neuroscience research and lay the groundwork for human trials.

NIH Research Projects · FY 2026 · 2024-06

Sensory hyper- and hyposensitivity is a feature of neurodevelopmental disorders (NDDs), including autism spectrum disorder (ASD). It can cause significant quality of life deficits through maladaptive sensory seeking and avoiding behaviors. Foxp2 is a transcription factor expressed in the developing and mature brain. Mutations in human FOXP2 have been linked to NDDs, ASD, and most notably speech and language disorders. Foxp2 has selective expression in the brain including several areas involved in the auditory processing pathway including layer VI corticothalamic projection neurons (CThPNs), in the cells in auditory cortex (AC) and cells in the inferior colliculi (IC), a midbrain region crucial in auditory processing. Preliminary studies of the loss of Foxp2 in the mouse IC show sensory gating deficits and cell population changes. However, the role for Foxp2 in the developing and mature auditory sensory areas of the neocortex and brainstem, remains to be fully elucidated. This proposal will address the role of Foxp2 in the developing AC and IC at single nuclei resolution to identify downstream targets of and chromatin accessibility changes by Foxp2. With two regions, single nuclei transcriptomic (snRNA-seq) and chromatin accessibility data (snATAC-seq) can identify unique and overlapping genes and regulatory pathways which will help elucidate the role of Foxp2 in each region and region-specific cell types in sensory processing. Ex vivo recordings will also identify changes in cell and circuit function with electrophysiological recordings of single and paired cells to find circuit and synapse dysregulation and cellular excitability alterations caused by the loss of Foxp2. I hypothesize that Foxp2 has a critical role in sensory processing mediated through proper cortical and IC neural development. I predict Foxp2 regulates cell type-specific transcriptional networks with both cell autonomous and non-cell autonomous effects in gene regulation, chromatin accessibility, and influences cell and circuit dysfunction underlying the sensory processing deficits seen in ASD and other NDDs as a risk gene. Aim 1 will utilize paired snRNA-seq and snATAC-seq to determine how conditional deletion of Foxp2 affects cellular expression profiles of the AC and IC across development. I hypothesize that Foxp2-cKO will have ASD-related gene dysregulation, chromatin accessibility patterns, and cell type imbalances in cortical and IC neurons. Aim 2 will assess the cortical and IC cell and circuit electrophysiology regulated by Foxp2. I hypothesize that the loss of Foxp2 expression in the cortex and IC will have circuit and cellular level changes that will result in changes in intrinsic excitability, interfering with thalamic signaling. These data will be generated through whole cell recordings to determine the intrinsic electrophysiologic properties of both AC Layer VI CThPNs and IC excitatory neurons as well as paired stimulation and recording of IC and AC projections and synaptic connections to the thalamus. Together, these Aims will uncover the developmental contribution of Foxp2 to cell type specification and circuit function in brain regions important for auditory processing.

NIH Research Projects · FY 2026 · 2024-06

Abstract The inflammatory response is critical in tissue healing. While extensively explored at the site of injury, relatively little is known about how injury at a remote site communicates back to the bone marrow to regulate the systemic immune response. Previous studies have demonstrated that hematopoietic stem cells (HSCs) can directly respond to secreted cytokines derived from the injury site. However, this model is limited by the need to reach a critical threshold of cytokines within the blood prior to the bone marrow HSCs responding, limiting overall responsiveness. Using our established model of polytrauma-induced heterotopic ossification (HO), we have identified the immunomodulatory metabolite itaconate as being highly and specifically enriched within the injury site. Using single cell RNA sequencing and metabolomics, combined with molecular and histological validation, we found that itaconate is made exclusively within highly mature neutrophils. Our previous work has demonstrated a direct role of myeloid-lineage cell-derived itaconate in mitigating HO formation. However, these studies identified a secondary effect of itaconate as a novel modulator of hematopoiesis. Our preliminary data show that itaconate is made exclusively in injury site neutrophils, stimulated by TLR9 activation. Itaconate production in these mature, injury site neutrophils is facilitated through changes in neutrophil metabolism, including shifts in glucose and glutamine usage through the glycolytic and oxidative phosphorylation pathways. These itaconate-laden neutrophils are able to extravasate from the injured tissue and home back to the bone marrow. Once in the bone marrow, neutrophils are phagocytosed by macrophages, inhibiting macrophage Il1b expression, a known regulator of HSC differentiation, in an itaconate-dependent manner. These preliminary findings support our central hypothesis that neutrophils serve as an itaconate-dependent systemic sensor, coupling local extremity injury to systemic inflammation by regulating BM hematopoiesis. This conceptual model will be explored in studies divided into two Specific Aims. Specific Aim 1 will identify the transcriptional and metabolic regulation of neutrophils which induces expression of Acod1 and production of itaconate specifically within the injury site. Specific Aim 2 will map the cycling of itaconate-laden neutrophils from the injury site back to the bone marrow and determine how clearance of these neutrophils by macrophages results in hematopoietic skewing. Our results should provide a new mechanism through which the body coordinates activation of the local and systemic immune response following injury.

NIH Research Projects · FY 2026 · 2024-06

PROJECT SUMMARY The recent advancement of stereotactic body radiation therapy (SBRT) enables highly focused dose delivery to tumors while sparing surrounding normal tissues. Both past and emerging clinical evidence has strengthened the role of radiotherapy in lung cancer management, and SBRT is considered the standard of care for many diagnoses. However, radiation-induced toxicity, especially for cases with centrally located lung tumors, poses a lingering challenge to lung SBRT. The respiration-induced motion of lung tumors and surrounding organs-at-risk (OARs) introduces substantial uncertainties to the delivery accuracy of lung SBRT, causing under-dosing to tumors and over-irradiation to surrounding OARs. The current motion management techniques, including internal-target-volume (ITV)-based treatment, respiratory phase gating, breath hold, and motion tracking, all suffer from various sources of uncertainty and inaccuracy, potentially resulting in large dose deviations that miss the tumor and damage normal tissues. Obtaining intra-treatment tumor and OAR motion/deformation, and using such information to derive the actually delivered doses to optimize remaining radiotherapy treatments, will systematically address the intra-treatment motion challenge. However, reconstructing intra-treatment dynamic and volumetric images for motion/deformation tracking remains an unmet clinical need, mostly due to the challenging spatiotemporal inverse problem of reconstructing volumetric images from extremely under-sampled signals. In addition, currently, there are no existing techniques and workflows that use the actually delivered dose to optimize future SBRT plans and deliveries. The overarching goal of this project is to develop an intra-treatment dynamic imaging and plan adaptation (IDIPA) system, which is composed of two sub-systems, spatial and temporal implicit neural representation (STINR) and dosimetry-guided plan adaptation (DGPA). STINR solves dynamic cone-beam CT (CBCT) images and intra- treatment deformation vector fields (DVFs) from x-ray projections with each x-ray projection corresponding to a dynamic CBCT volume and a DVF. The solved dynamic CBCTs and DVFs will then be used by DGPA for treatment dose calculation, dose accumulation, and dosimetry-guided plan adaptation. The STINR sub-system uses an Artificial Intelligence-driven method to address the substantial challenge of dynamic CBCT reconstruction. The DGPA sub-system features the first closed-loop, dosimetry-guided optimization framework that uses delivered doses to adapt the following plans to ensure the treatment doses will be delivered to where it is intended. We have three Specific Aims for this project: 1) Develop and optimize the dynamic CBCT imaging sub-system (STINR), 2) Develop and optimize the dosimetry-guided plan adaptation sub-system (DGPA), and 3). Evaluate the overall IDIPA system via a clinical study. The success of the project will result in the first end-to-end system to improve the dose delivery accuracy of lung radiotherapy under intra-treatment motion, and to unleash the full potential of SBRT in advancing lung cancer care.

NIH Research Projects · FY 2026 · 2024-05

Abstract Multiple sclerosis (MS) is a heterogeneous neurological disorder characterized by autoimmune inflammation coupled to demyelination and eventual neurodegeneration, affecting more than 2 million people worldwide. Relaxation-based magnetic resonance imaging (MRI) is sensitive in revealing macroscopic tissue abnormalities in the brain, they are not specific to the pathological substrate of the MS lesion and have a limited prognostic role. These methods are sensitive to the MS lesions in white matter (WM), characterization of MS lesions in the cerebral cortex has been proven to be difficult by clinical MRI. Advanced diffusion MRI (dMRI) techniques offer the potential to improve the understanding of axon and dendrites damage in MS. Quantitative susceptibility mapping (QSM), as a novel MRI technique, has been demonstrated to show high correlations with myelin and iron content. Our long-term goal is to develop specific and reliable whole brain imaging biomarkers for early diagnosis of MS and monitoring the disease progression. We have developed the whole mouse brain dMRI and QSM methods at 25 µm isotropic resolution using 3D under sampling acquisition and nonlinear reconstruction. Our recent results have showed that QSM of corpus callosum decreases significantly (more diamagnetic) after 2 weeks cuprizone administration. Our hypothesis is that combining novel dMRI and QSM technologies at high spatial resolution affords robust and quantitative imaging-based biomarkers of MS by detecting the progression of iron dysregulation, demyelination, and axon damage through the whole brain. In this proposal, we will perform both in vivo and ex vivo MRI to quantify the whole brain demyelination, iron dysregulation, and axon damage using Thy-1 YFP-16 transgenic mice with cuprizone administration. The QSM values and dMRI outcomes from basic diffusion tensor imaging (DTI) model to the advanced neurite orientation dispersion and density imaging (NODDI) model and diffusion kurtosis imaging (DKI) model will be measured at different timing points (Aim 1). Currently, directly correlating MRI findings to histology is still challenging due to the limited spatial resolution and various image contrasts derived from water diffusion, relaxation, and magnetic susceptibility characteristics. The 3D MRI quantitative mappings will be validated against with the whole brain light sheet microscopy (LSM) at each timing point. The imaging-based biomarkers will be observed by the voxel- based comparison between MRI and LSM. The 3D co-registration comparison will also help us to fundamentally understand the origin of MR image contrasts and properties (Aim 2). The high-resolution multidimensional brain atlas at each timing point will be generated and shared at both Waxholm space and Allen Brain Mouse Atlas space at different spatial resolution, from 25 µm to 200 µm isotropic resolution (Aim 3). This project is expected to provide novel insights to improve the specificity of MRI for the diagnosis of MS and understand the complex mechanism of the disease.

NIH Research Projects · FY 2025 · 2024-05

Bordetella pertussis is the causative agent of pertussis, also known as whooping cough. Among the virulence factors produced by B. pertussis, the ABs toxin called pertussis toxin (PT) is strongly linked to disease symptoms and severity. Like other AB5 toxins, the PT holotoxin has five B subunits that recognize cell surface molecules and one A subunit that harbors an ADP-ribosylation activity. The nonulosonic acid N-acetylneuraminic acid (NeusAc) is commonly described to be the receptor for PT but it is unknown how other glycan features affect PT binding. Additionally, PT has been reported to bind to non-sialylated glycoconjugates. This proposal aims to define the glycoconjugate features that mediate PT recognition and intoxication of lymphocyte and respiratory epithelial cell surfaces, two cell types that are proposed to be targets of PT action in vivo. In Aim 1, CRISPR/Casg genome-wide knockout (KO) screening will be used to identify genes that impact PT binding to cell surfaces. Based on the results of these screens, individual KO cell lines will be constructed and assayed for PT binding, PT internalization, and PT intoxication. KO cell lines will also be evaluated for changes in glycosylation, to reveal which glycan structures are associated with susceptibility to PT. In Aim 2, photocrosslinking sugar technology will be used to identify protein component of glycoproteins that interact directly with PT. Based on the results of this analysis, KO cell lines will be constructed and assayed for PT binding, PT internalization, and PT intoxication. Together, the two aims will reveal the glycan and protein components of PT receptors. This information will explain how PT targets specific cell types and may suggest strategies to interfere with PT action therapeutically. Project Summary/Abstract

NIH Research Projects · FY 2026 · 2024-05

Summary Tumor formation and cancer cell proliferation depend on enhanced ribosome biogenesis and protein synthesis. Ribosomes are the molecular machines responsible for protein production within cells. Emerging data indicates that inhibition of ribosome formation and function can prevent cancers from forming in certain contexts. For example, liver disease predisposes patients for liver cancer, including hepatocellular carcinoma, and this transition is marked by enhanced ribosome biogenesis. Inhibition of ribosome function prevents liver tumor formation in mouse models. Thus, additional compounds that attenuate ribosome production and activity may be able to prevent the formation of tumors. Unfortunately, previously available methods used to study ribosome biogenesis are laborious and not easily scalable. This has significantly hampered the development of new ribosome biogenesis inhibitors. To overcome this critical barrier, we have developed an innovative platform that we call ribo-SNAP, which allows us to assay the dynamics of ribosome biogenesis at single cell resolution in living cells. Using this platform, we have successfully completed pilot compound screens and identified new genes and small molecules that inhibit the protein synthesis capacity of cancer cells. In parallel, we have also developed a second novel assay called SidBait, which allows us to robustly and rapidly identify compound targets within cells. Using these approaches, we are in a unique position to identify and develop ribosome biogenesis inhibitors as effective anti-cancer agents. We now seek to expand our efforts to the larger NCI natural product library. The proposal is divided into UG3 and UH3 sections. Under Aim 1, we will further optimize the ribo-SNAP platform and conduct a small pilot screen of a subset of fractions from the SOOK natural product library. In Aim 2, we will use Sid Bait to identify the targets of compounds identified in the pilot screen or our ongoing screen of an independent 3SOK compound library. Experiments described under Aim 3 will directly test the extent to which ribosome biogenesis inhibitors can prevent liver tumor formation in mouse models. These three aims have clear milestones and we anticipate that the successful completion of the UG3 phase will allow the project to advance. During the UH3 phase, the large of screening will expand to the entire SOOK library under Aim 4. Sid Bait will be used in Aim 5 to begin to determine the mechanism of action of specific compounds. Finally, under Aim 6, mouse models will be used to test whether lead molecule prevent cancer progression. The successful completion of the entire project described in this proposal will provide an unparalleled collection of ribosome biogenesis inhibitors that can be further developed into novel therapeutics for the prevention of liver cancer.

NIH Research Projects · FY 2026 · 2024-05

Project Summary Chronic kidney disease (CKD) is a common condition with limited treatment options. Because late-stage CKD is progressive and irreversible, molecular research to better understand the etiology of CKD progression and identify novel therapeutic targets is critically needed to improve clinical outcomes. Mitochondria play an important role in regulating kidney function, supporting the high energy demands of this end-organ. Mitochondrial DNA copy number (mtDNA-CN), a quantitative indicator of mitochondrial function, has been associated with the incidence of CKD. Our recently published data (CJASN 2022; 17: 966-975) further showed that lower baseline mtDNA-CN is associated with risk of CKD progression. However, longitudinal studies, with repeated measures of mtDNA-CN, have never been conducted in a CKD setting. Such research is needed to 1) evaluate whether mtDNA-CN decline predicts kidney function decline and 2) delineate its upstream determinants and downstream mechanisms. Kidney function may also be influenced by mtDNA quality, which can be assessed as mtDNA heteroplasmy (mtDNA-Het). mtDNA-Het reflects the presence of both mutant and wild-type mtDNA copies. In the only study to examine the association of mtDNA-Het with CKD progression, our pilot work targeting mutations at 25 random mtDNA markers showed that CKD patients with mtDNA-Het had a significant 2.4-fold higher hazard of CKD progression than those without mtDNA-Het. Sequencing of the entire mtDNA genome (16,569 markers) is needed to fully characterize the role of mtDNA-Het in CKD progression. Overall, our preliminary findings provide strong premise for our main hypothesis: mtDNA quality and quantity associate with CKD progression through known and novel CKD related risk factors. To characterize the role of mtDNA quality and quantity in CKD progression, we propose to measure mtDNA-Het at baseline and mtDNA-CN at baseline and 3- and 6-years of follow-up among 5,499 participants of the Chronic Renal Insufficiency Cohort (CRIC). The stringently ascertained CKD progression events and annually assessed data on clinical and molecular biomarkers in CRIC will allow us to assess the contribution of baseline mtDNA-Het (Aim 1) and mtDNA-CN changes (Aim 2) to CKD progression; delineate downstream mechanisms of mtDNA- Het and mtDNA-CN (Aim 3); and discover unique upstream determinants of mtDNA-CN decline (Aim 4) in a CKD setting. We will replicate CRIC findings among 3,498 CKD patients of the Trans-Omics for Precision Medicine program. The proposed work represents the first study to examine the role of mtDNA-Het in CKD progression and the first longitudinal study of mtDNA-CN decline in a CKD setting. Innovative methods, including state-of-the-art mtDNA variant calling pipeline to account for the circular nature of mtDNA and cross- lagged panel modeling to delineate causal directions, will be employed. Since mtDNA-CN and mtDNA-Het are modifiable, our findings have potential to not only reveal novel mechanisms of CKD progression but provide evidence-based targets for the development of new therapeutics to prevent and reverse CKD progression.

NIH Research Projects · FY 2024 · 2024-05

PROJECT SUMMARY The ability of the brain to utilize information from past experiences to guide future decisions, termed adaptive behavior, is critical for survival. To effectively adapt behaviors, the brain applies stored memory to new but similar situations (generalization), while also maintaining the capacity to distinguish unique stimuli (discrimination). When these critical processes (memory generalization or discrimination) go awry, it can lead to maladaptive disorders such as post-traumatic stress disorder (PTSD) and panic disorder. Despite their importance, mechanisms underlying memory discrimination and generalization remain largely unknown. This proposal will investigate the dynamic processes that underlie the utilization of an encoded memory to guide future behaviors, in particular the molecular, synaptic, and circuit mechanisms that govern the balance between discrimination and generalization. We have collected very exciting preliminary data showing that individual contextual fear memories are represented in the dentate gyrus (DG) by multiple functionally distinct neuronal ensembles defined by different activity-dependent transcriptional pathways, and that these ensembles bi-directionally regulate the discrimination-generalization balance. Based on these exciting findings, we hypothesize that the activity- dependent pathways target specific synaptic inputs on DG granule cells to differentially control memory discrimination and generalization. We aim to (1) uncover novel forms of learning-induced synaptic plasticity; (2) reveal underlying circuit mechanisms for memory discrimination and generalization; and (3) identify the molecular players important for this experience-dependent behavioral adaptation. The proposed research is both conceptually and technically innovative. It will experimentally demonstrate for the first time functionally distinct active neuronal ensembles coexisting within the memory engram, shed light on the synaptic and circuit mechanisms by which encoded memories directly drive experience-dependent behavioral outputs, and may lead to new treatment strategies for neuropsychiatric disorders, such as PTSD and panic disorder, which are caused by the imbalance between memory discrimination and generalization.

NIH Research Projects · FY 2026 · 2024-05

PROJECT SUMMARY Membrane tension is thought to be a long-range integrator of cell physiology. During migration, membrane tension has been proposed to enable cell polarity through front-back coordination and long-range protrusion competition. These roles necessitate effective tension transmission across the cell. However, it remains a source of significant debate as to whether cell membranes support or resist long-range membrane tension propagation. I speculated that this this discrepancy likely originates from the use of exogenous forces that may not accurately mimic endogenous forces. To overcome this complication, I used optogenetics to directly control localized actin-based protrusions or actomyosin contractions while simultaneously monitoring the propagation of membrane tension using dual-trap optical tweezers. My results led me to propose a unifying model of tension propagation in which actin-driven protrusions and actomyosin contractions both elicit rapid global membrane tension propagation, while forces applied to cell membranes alone do not. This work laid the foundations of my ongoing studies and raises many important questions. For some processes, membrane tension needs to act locally and in others, it needs to act at the range of the entire cell or multiple cells. What controls the range and efficiency of tension propagation (Aim 1)? The cell has many processes that can locally initiate changes in membrane tension (protrusions, contractions) and many cellular processes that are regulated by tension changes. Are there important functional differences for how these cellular programs transmit or receive membrane tension changes that may be important for cell polarity (Aim 2)? Migrating cells often need to coordinate their movement with other cells in order to achieve collective or cooperative motion. What is the impact of the membrane tension-polarity program in multicellular contexts such as encountered during collective or cooperative cell migration (Aim 3)? I will answer these questions by using a combination of optogenetics, force measurements, and advanced microscopy, and mathematical modeling. My research will elucidate the interplay between membrane mechanics and polarity during cell migration and will provide the foundation for my independent scientific niche of studying membrane mechanics during transendothelial migration. Towards this goal, I am supplementing the input of my postdoc supervisor, Dr Weiner (expert on cell polarity during migration) with a group of internationally recognized leaders at the interface of cell biology and biophysics: Carlos Bustamante (optical traps), Herve Turlier (mechanical modelling), Janis Burkhardt (cell migration). Japp van Buul and Ronen Alon, both experts on transendothelial migration. These scientists will act as both advisors and collaborators, helping me establish my own scientific niche in understanding the role of membrane mechanics in regulating transendothelial migration. I have worked with my mentor and network of collaborators and advisors to develop my research plan and construct a tailored career development program to help launch my independent career as a tenure-track principal investigator.

NIH Research Projects · FY 2026 · 2024-05

Healthy aging is a critical goal for the human population, and one solution to this problem is to extend healthspan and lifespan. In model organisms including rodents and non-human primates, caloric restriction (CR) is the most effective intervention for improving aging-related deterioration of biological functions and for extending lifespan. However, despite more than 80 years since its discovery, the underlying mechanisms for how caloric restriction extends lifespan are still largely unknown. A number of pathways have been associated with longevity including those involved with nutrient signaling, metabolism, growth, genome stability and oxidative stress. Our laboratories have been studying the behavioral effects of caloric restriction in mice and have found that CR leads to dramatic changes in the pattern of food intake. In contrast to normally fed mice, which distribute their food intake over the 24-hour day, mice on caloric restriction adopt a severe feeding and fasting pattern in which they consume all of their food within a few hours each day. In order to disentangle the contribution of calories, fasting and circadian alignment of eating on longevity, we recently showed that CR is sufficient to extend lifespan but that the pattern and circadian-alignment of feeding under CR acts synergistically to extend lifespan in male C57BL/6J mice. Calorie reduction alone increases lifespan only by ~10%, while time-restricted CR during the active phase extends lifespan more than 3 times longer (35%). Circadian alignment of feeding enhances CR-mediated benefits on survival independently of fasting duration and body weight. In Aim 1, because we have found that CR with time-restricted feeding (TRF) extends lifespan from 10% to 35%, we will test whether TRF without CR can extend lifespan. In Aim 2, we will test whether enhanced Clock gene expression can rescue the age-related decline in circadian gene expression and can improve health and extend lifespan. Aging promotes widespread increases in inflammation and decreases in metabolism in the livers from ad lib (AL) fed mice; whereas CR at night ameliorates these aging-related changes. In our previous work, we found that the gene expression of the cytokine, Interleukin-1 beta (IL-1b), is directly correlated with lifespan across 6 feeding conditions (AL vs. 5 CR groups). In Aim 3, we will test the hypothesis that IL-1b is necessary for the effects of CR on lifespan and whether calories, fasting and circadian alignment of feeding differentially effect lifespan in Il-1b null mice. In summary, we will test: whether circadian interventions such as TRF or genetic enhancement of CLOCK transcription can extend healthspan and lifespan and whether IL-1b is critical for these effects.

NIH Research Projects · FY 2026 · 2024-05

Project Summary/Abstract The molecular evolution underlying cell type diversity and function has facilitated the increased cognitive capacity of humans. While significant progress has been made to uncover cell type specific gene expression programs in the brains of rodents, only a modest amount of progress has been made in human brains. Moreover, the majority of the existing cell type genomic datasets focus on gray matter. Our recent work has identified important human-specific gene expression patterns relevant to non-neurons, especially oligodendrocytes. Human brain imaging studies in disorders such as autism spectrum disorders and schizophrenia have identified alterations in white matter tracts that are primarily comprised of oligodendrocytes. We therefore propose to profile white matter tracts that are anatomically comparable across primates: pyramidal tract, optic chiasm, cerebellar white matter, and corpus callosum. Because oligodendrocyte maturation varies between gray and white matter, we will profile tissue across development. This comparative cell type profiling between human and non-human primate brain will provide spatially defined epigenomic and transcriptomic data through the following four Aims: 1) Profile single cell level transcriptional maps and chromatin states of white matter tracts and functionally associated gray matter in human brain across development; 2) Profile single cell level transcriptional maps and chromatin states of white matter tracts and functionally associated gray matter across development in two non-human primates: rhesus macaque and the common marmoset; 3) Profile single cell level transcriptional maps and chromatin states of white matter tracts and functionally associated gray matter in additional tissue from adult great ape and monkey brains; and 4) Validate observed differences in white matter composition at the cell type level in human and non- human primates. Together, these aims will molecularly define understudied cell types from human and non- human primate brains.

NIH Research Projects · FY 2026 · 2024-04

PROJECT SUMMARY The brain serotonin (5-HT) system has been a target for multiple weight loss therapies. Compounds that elevate 5-HT content reduce food intake and body weight. Our previous and current findings show that 5-HT acts on two postsynaptic 5-HT receptors, Htr2c and Htr1b in the arcuate nucleus of the hypothalamus (ARH) to suppress food intake. Within the ARH, 5-HT reciprocally inhibits the orexigenic AgRP neurons (via Htr1b) while activating the anorectic POMC neurons (via Htr2c). Moreover, we have demonstrated that Htr2c and Htr1b in these neurons are necessary for the anorectic effects of 5-HT agents including the once-popular diet pill Fen/Phen. However, despite these significant findings, the precise source of the presynaptic 5-HT inputs to ARH neurons remains largely elusive. This has posed a considerable challenge due to the heterogeneity of 5-HT neurons in the midbrain dorsal raphe nucleus (DRN), comprising multiple subpopulations with distinct projection patterns and physiological functions. Consequently, our current proposal aims to unravel the neural circuit mechanism by which 5-HT regulates satiety. To accomplish this, we propose a multidisciplinary approach to isolate the 5-HT neurons that innervate the ARH (5-HTARH neurons). Our central hypothesis posits that these neurons provide direct synaptic inputs to the ARH and play a critical role in feeding regulation. To investigate this, we will use in vivo calcium imaging to determine the activity patterns of 5-HTARH neurons during hunger and satiety in behaving mice [Aim 1a]. Furthermore, we will manipulate the activity of these neurons in live mice by either stimulating or inhibiting them, aiming to uncover their specific contributions to food intake [Aim 1b]. Finally, we have developed a new intersectional genetic approach that enables us to selectively deplete 5-HT inputs to the ARH in adult mice and evaluate their physiological impact on energy homeostasis [Aim 2].

NIH Research Projects · FY 2025 · 2024-04

Targeting the prostate-specific membrane antigen (PSMA) with small molecules for imaging and therapy of prostate cancer (PC) has revitalized the field of nuclear medicine. Few targets have its combination of salutary attributes, namely, high concentration in malignant with restricted expression in normal tissues, easy access with recycling to and from the plasma membrane, an enzymatic active site toward which small molecules of high affinity and specificity can be designed, and biological relevance – an inverse relationship with androgen signaling while being directly related to degree of malignancy. The ureas that we initially described for imaging PSMA in 2002 have inspired a wide variety of cancer targeting species from radiotherapeutics to synthetic antibody mimics. Our goal is to use what we have learned from PSMA-targeted detection, imaging and treatment of PC to focus on highly aggressive disease, including that which does not express PSMA. We will deploy this prolific cancer target here by beginning with a project that leverages the considerable clinical data obtained during the last funding period to refine and simplify PSMA-targeted imaging with positron emission tomography (PET) – in a way agnostic to imaging agent employed. Complementing PET we will explore sensitive new PSMA- targeted agents and methods for photoacoustic (PA) imaging, which can characterize primary disease in new ways in an effort to uncover signatures that could separate aggressive from indolent cancer to prevent unnecessary surgery and its attendant morbidity. Because PC is a heterogeneous disease, in the second half of the project we will move from detection and characterization of PSMA-expressing PC to address highly aggressive, PSMA-negative adenocarcinoma and especially neuroendocrine PC (NEPC), a lethal and increasingly prevalent subtype with the proliferation of modern anti-androgen therapies. First, we will use a PSMA reporter gene strategy to track NEPC-targeted chimeric antigen receptor (CAR) T cells in order to gauge their spatial relationship to tumor, measure their expansion in vivo, and sense their microenvironment, with a view to improving this case of solid tumor CAR T cell therapy. Finally, we will use cancer cell specific promoter (CCSP) technology, which we developed for imaging and treating metastases, to enhance PSMA expression specifically within NEPC tissue so that it may become susceptible to the detection and treatment of its PSMA- expressing adenocarcinoma counterpart. In addition to using existing PSMA-targeted radiotherapy we will show how a new urea-drug conjugate we have developed can kill NEPC once it is re-programmed to express PSMA. To achieve these goals, we take the approach of beginning with a more sophisticated analysis of our clinical PSMA PET data then work toward more laboratory-based imaging and therapeutic studies also designed for translation. The team we have assembled is comprised of clinicians and scientists with a track record of high productivity and impact working together.