Icahn School Of Medicine At Mount Sinai

NIH Research Projects · FY 2026 · 2024-08

Abstract Diet and nutrition influence our cognitive abilities, how our brains age, and vulnerability to neurodegeneration. However, the interaction of diet with the human brain is complex and influenced by every person’s unique genetic composition. Clinical studies found that ketogenic diets and supplements improve cognition and protect against Alzheimer's disease (AD) in most individuals but have no beneficial effect on individuals with the strongest genetic risk factor for AD, APOE4. How APOE4 interacts with diet at the cellular and molecular level to influence AD is unknown. The primary metabolite of ketogenic diets beta-hydroxybutyrate freely passes across the blood-brain barrier into the human brain where it is converted to acetyl-CoA. Our data demonstrate that cholesterol transport is impaired in APOE4 glia leading to intracellular cholesterol accumulation which triggers inflammation, AD pathogenesis, and cognitive decline. As a compensatory mechanism to impaired cholesterol trafficking, APOE4 glia upregulates cholesterol biosynthesis, which uses acetyl-CoA to generate cholesterol. We hypothesize that impaired cholesterol trafficking and upregulation of cholesterol biosynthesis in APOE4 glia adversely interact with high-fat/ketogenic diets to exacerbate and accelerate AD pathogenesis. We established methods to mimic ketogenic diets in vitro. This revealed in APOE4 glia ketones increase aberrant intracellular cholesterol deposits and promote neuroinflammation and hypomyelination. We developed an in vitro model of human brain tissue that contains all the major cell types and tissues including cerebrovasculature, neurocircuits, myelination, and neuro-immune cells. Aim 1 will employ this system (miBrain) to further investigate the interaction of APOE genotype with high-fat/ketogenic diets and its contribution to AD pathogenesis in human brain tissue. Using transcriptomic and biochemical approaches we will discover the underlying mechanisms that we will modulate via chemical and genetic approaches to identify therapeutic targets for promoting beneficial APOE4-diet interactions. We will complement this with studies in APOE3/3 and APOE4/4 humanized mice in Aim 2 to mechanistically dissect the interaction between APOE genotype and high-fat/ketogenic diets at the organismal level. Together aims 1 and 2 will provide holistic insight into the peripheral and central interactions of APOE4 with ketogenic diets. Several other AD risk variants also have functional roles in cholesterol and lipid homeostasis. We further hypothesize that cholesterol dysregulation and its interaction with diet is a central pathogenic mechanism of AD. In Aim 3, we will investigate using isogenic human brain tissue generated from iPSCs harboring genetic risk factors in SORL1, TREM2, ABCA7, and APOE. We will determine how each risk variant interacts with ketogenic diets to influence pathogenic outcomes in AD. Collectively, this study will pioneer approaches and technology that will deliver a detailed molecular understanding of the interactions between genetics, diet, and neurodegeneration opening avenues to genetically informed therapeutic and diagnostic opportunities.

NIH Research Projects · FY 2024 · 2024-08

PROJECT SUMMARY Advances in single particle cryo-EM have transformed structural biology of ever-expanding biological systems. We request funds to purchase a Glacios 2 cryo-TEM (Thermo-Fisher) at the Icahn School of Medicine at Mount Sinai. The goal is to enable users throughout the Institution, working on different biological and disease mechanisms, to measure cryo-EM data at home, as well as provide a seamless transition for atomic resolution data collection on Titan cryo-TEMs at nearby NYSBC-MEMC and NCCAT. The microscope is equipped with a new generation Falcon 4i direct electron detection camera, and an ancillary vitrification device for the preparation of cryogenic grids. Importantly, the microscope features 200 kV X-FEG optics, and an Autoloader for cryogenic manipulation and loading of the biological samples. A suitable site for installation has already been identified, and as a proof of the Institution’s commitment to establishing cryo- EM, the Dean, Dr. Dennis Charney has agreed to lease the Glacios 2 under Thermo-Fisher’s “bridge-to-grant” mechanism with no obligation to buy, and no lease payment until 12 months after installation. Besides the construction costs, the institution is also deeply committed to all other associated costs in establishing cryo-EM at Mount Sinai, including yearly service contract, IT infrastructure, consumables, and salary for a cryo-EM manager. The success of this S10 application will help to meet a significant portion of the cost in establishing an expensive cryo-EM facility at Mount Sinai that is absolutely crucial for the continued success of our NIH-funded investigators, allowing them to fully partake in this new “resolution revolution” in structural biology. The Glacios 2 cryo-TEM majorly enhances the NIH-funded research projects of numerous investigators throughout Mount Sinai studying a wide range of biological systems that underpin human diseases.

NIH Research Projects · FY 2024 · 2024-08

Project Summary Advanced age is a major risk factor for age-related diseases such as type 2 diabetes (T2D), Alzheimer’s disease (AD) and many others. It remains elusive what aging mechanisms, or more generally what biological mechanisms are responsible for promoting the development of multiple age-related diseases. To address this question, we believe it is essential to investigate the heterogeneity of human aging. We posit that human aging can be generally divided into two major subtypes, i.e., the healthy and pathological aging, and the pathological aging contains critical information on the link between aging and age-related diseases. Using tissues’ transcriptomes, we can divide a cohort into two major subgroups which roughly correspond to healthy and pathological conditions. Molecular mechanisms associated with pathological tissue aging can be learned by comparing the differential gene regulations between the two major subgroups. Using this general approach, we will focus on studying the common pathological aging mechanisms between brain (e.g., hippocampus, and frontal cortex) and adipose tissues (e.g., visceral adipose tissue VAT and subcutaneous adipose tissue SAT). We will also examine the potential molecular mechanisms underlying the adipose-brain cross-tissue talk. We will perform experimental validation for the findings using mouse models. This proposal is innovative as to our best knowledge, the systematic study of the heterogeneity of human tissue aging, the classification of healthy and pathological aging, and identification of the molecular mechanisms shared between adipose-brain tissues and their crosstalk have not been well-examined. The project will help to elucidate the common molecular mechanisms related to pathological aging in metabolic and neurological disorders using an unbiased data-driven approach. We will work on the following two Specific Aim. In Aim 1, we will Identify common gene regulations between brain and adipose tissues in pathological subgroups and the gene regulations putatively mediating adipose-brain cross-tissue talk using GTEx data and other existing human tissue transcriptomic data. In Aim 2, we will experimentally validate the transcriptomic changes involved the adipose- brain axis that may mediate the inter-organ communication and promote pathological brain conditions. In summary, our proposed work represents a novel comprehensive unbiased framework to study the human tissue aging heterogeneity and to identify the key molecular mechanisms shared by brain and adipose tissue. It will generate important pilot results and new insights into the pathological aging mechanism across human body and how the pathological aging in peripheral tissues could contribute to the disease development in the brain.

NIH Research Projects · FY 2025 · 2024-08

Early life social experiences, especially social deprivation, can have long-lasting consequences on risk for developing psychiatric disorders including Substance Use Disorders (SUD). Animal studies have also shown that juvenile social isolation (jSI) dysregulates adult social and cognitive behaviors relevant to SUD. However, how early isolation alters the developmental trajectory of circuits and behaviors implicated in SUD is poorly understood. Our long-term goal is to elucidate neural mechanisms mediating the impact of jSI on neurobehavioral development and the risk for SUD in adolescence and adulthood. Among many brain regions, prefrontal cortex (PFC), which provides top-down control to sub-cortical areas essential for reward processing, has been extensively implicated to be dysregulated in SUD. it is hypothesized that jSI dysregulates adolescent reciprocal social interaction, which leads to an imbalance between the two types of subcortically projecting mPFC neurons, ultimately contributing to SUD-relevant cognitive behavior deficits. This project will conduct the preparative activities at the behavioral (Aim1) and circuit level (Aim2) that are essential to establish feasibility and validity to test the aforementioned hypothesis. To this end we will form an interdisciplinary team with expertise in developmental psychobiology, circuit manipulation/measurement, behavioral electrophysiology, machine learning-based behavioral analysis, and SUD-related cognitive behavior in rodent models, as well as expertise in human developmental psychology and human SUD-related developmental imaging studies. These preparatory activities will set a stage for a future project to conduct multimodal longitudinal study using rat models to examine the impact of juvenile social isolation on PFC circuit maturation, social play trajectory, and adult SUD related behavior.

NIH Research Projects · FY 2025 · 2024-08

Project Summary Interactions among different organs are pivotal in developing pathological conditions like obesity and type 2 diabetes. Substantial research efforts have been directed towards comprehending how communication between cell types such as adipocytes, hepatocytes, and islets contribute to and respond to specific disruptions associated with metabolic diseases. There remains an essential yet relatively unexplored question: how does the secretome of the mammary gland's luminal epithelium influence inter-organ communication in the context of obesity? Breastfeeding holds a significant role in promoting the health of mothers and reducing the risk of diabetes, benefiting both maternal and neonatal health. It also protects against cardiovascular diseases, obesity, and other metabolic disorders for both the mother and child. However, obesity can disrupt mammary gland function and development, potentially affecting maternal well-being and the health of offspring. Recent technological advancements, such as single-cell transcriptomics and precision proteomics, have opened up opportunities for unbiased exploration and the discovery of signaling molecules involved in inter-organ communication. We recently employed a bioinformatics framework based on single-cell transcriptomic correlations, integrating data from multiple datasets with publicly available resources to identify secretory factors from mammary duct luminal cells that influence surrounding adipocyte metabolism. We refer to these factors as "mammokines." Our innovative research endeavor seeks to combine transcriptomics and proteomics to unveil the ambiguities surrounding the mammary gland's function as an endocrine organ and the impact of obesity on its action. The primary goal of this project is to discover new mammokines that may have vital roles in paracrine and endocrine signaling, influencing the function and homeostasis of the liver, pancreas, adipose tissue, and the mammary gland itself, potentially affecting the progression of obesity and Type 2 diabetes phenotypes. These mammokines could serve as valuable biomarkers for obesity and diabetes in women. We also aim to determine which pathways are conserved from mice to humans and investigate the physiological consequences of disrupted endocrine communication through functional experiments. The success of these objectives relies on integrating transcriptomics, proteomics, computational, and experimental approaches, which is supported by the extensive training and expertise of the Principal Investigator and collaborators. We believe this research has great potential to advance our understanding of women's metabolic health and inspire innovative strategies for obesity and diabetes therapy.

NIH Research Projects · FY 2025 · 2024-08

PROJECT SUMMARY/ABSTRACT Human dyadic social communication entails a rich repertoire of expression, including not only face expression (and gaze), but also acoustics (prosody and pauses) turn-taking, gestures and language. Communication has evolved in humans within a social context, beginning with the parent-infant dyad, with mirroring of facial expressions and sounds. Its natural ecology is face-to-face dyadic interactions, both in-person and increasingly via remote platforms for teleconferencing and telehealth. Social communication is a “complex orchestration” in real time: its signals are multiple and temporally offset. It is a continuous exchange that is highly coordinated between speakers, with norms for turn-taking and alignment of face expression, gesture, semantic content and speech rates. As yet, a critical gap exists in that we lack the tools to quantify and analyze temporal patterns of multimodal communication behavior between two individuals in face-to-face communication, in an ecologically valid setting, that have the same rigor and reproducibility as do hyperscanning approaches to record brain activity during dyadic conversation. This tool must be developed to realize the true potential of second-person neuroscience. This planning proposal for tool development entails several key activities, beginning with the convening of a diverse multidisciplinary team of experts from various fields, including ethics/regulatory, anthropology, cognitive neuroscience, computer science, engineering, physics, mathematics, psychiatry and neurology. This team will discuss ethics, diversity, paradigm development, and computational frameworks, and providing iterative feedback and convening also with advocacy groups. Also, we will build two testing rooms for multimodal recording of dyadic communication, to demonstrate feasibility of acquiring and synching high temporal resolution data. Pilot EEG hyperscanning will be done concurrently in a subcohort. Further, given increased use of teleconferencing, dyadic communication data will be collected via remote platform and compared with in-person data, to determine how information may be degraded by differences in resolution and streaming delays. We will also develop computational frameworks for analyses of multimodal data.

NIH Research Projects · FY 2025 · 2024-08

There is an urgent need to identify effective treatments for intrahepatic cholangiocarcinoma (CCA), a highly desmoplastic tumor of the bile ducts with a five-year survival rate of 5%. Over half of iCCA cases are diagnosed after metastases are present, when treatment choices are limited and minimally effective. The long-term goal of our work is to identify novel therapeutic interventions to improve outcomes for this devastating malignancy. This proposal leverages our exciting preliminary data suggesting that the immune checkpoint protein B7-H4 is over- expressed in more than 60% of iCCA cases where, in addition to its known T cell immunosuppressive role mediated by its membrane-bound form, it exerts a critical role in the control of tumor growth and stromal deposition through a previously unknown cooperation of its intracellular form with the TGFβ pathway. Genetic ablation of B7-H4 in murine models generated by the co-expression of Notch1 intracellular domain and activated Akt1 (Nicd1/Akt1) using sleeping beauty transposon/transposase and hydrodynamic tail vein injection (HTVI) significantly prolongs survival and profoundly alters the surrounding desmoplasia. Mechanistically, B7-H4 over- expression increases the levels of the docking receptor for latent-TGβ1, GARP, and thus promotes the release of active TGFβ1 and activation of the downstream SMAD2/3-dependent pathway. We herein propose three aims to validate our hypothesis that, through activation of the TGFβ pathway, B7-H4 shapes a unique desmoplastic response and promotes tumor growth of iCCA, so that pharmacologic inhibition of this cross-talk will contribute to tumor inhibition through several complementary ways. In Aim 1, we will dissect the molecular mechanisms through which B7-H4 promotes cancer cell growth and motility by performing in vitro functional studies in iCCA cell lines (murine and human) and patient-derived organoids upon genetic manipulation of the B7-H4/TGFβ axis. Furthermore, we will unveil the full interactome of intracellular B7-H4 and its exact subcellular localization. In Aim 2, we will clarify how the newly identified axis shapes the desmoplastic reaction, immune infiltration and composition, and instructs other cell types, particularly cancer-associated fibroblasts, to deposit and organize collagen, in the genetically versatile HTVI-based Nicd1/Akt1 mouse model, which - as supported by our preliminary data - highly resembles human iCCA with endogenous expression of B7-H4. In Aim 3, we will assess the efficacy of pharmacologically modulating the B7-H4 axis using clinically-relevant drugs blocking TGFβ1 and other identified vulnerabilities in combination with a monoclonal antibody directed against B7-H4 in genetic and orthotopic murine models . Here, by combining our large collection of cell lines/organoids, human samples, mouse models with single cell-based approaches and a single-fiber artificial intelligence-based platform, we will dissect a previously unknown B7-H4/TGFβ cross-talk and yield new insights into iCCA biology. The proposed research is highly significant since its successful completion would establish the B7-H4 axis as a novel target for iCCA therapy that could improve the clinical outcome of this deadly cancer.

NIH Research Projects · FY 2025 · 2024-08

PROJECT SUMMARY Compulsivity, persisting with set ways of thinking and behaving in the face of negative consequences, is a prominent component of multiple psychiatric disorders. Given the many millions of patients and family members impacted by harms stemming from compulsive behavior, there is an urgent need to better understand the circuit basis of this quintessentially transdiagnostic phenomenon. The objective of this K23 application is to equip the candidate, a psychiatrist with a strong quantitative research background, to carry out intracranial studies of circuit level computational deficits in human subjects with serious psychiatric illness. A new generation of Food and Drug Administration (FDA) approved deep brain stimulation (DBS) devices, precisely targeted and chronically implanted, can both stimulate and record neural activity. Our preliminary data shows that this new technology can be successfully incorporated into the treatment of patients receiving therapeutic DBS for severe obsessive- compulsive disorder (OCD), making it possible to directly record from clinically relevant circuitry as patients go from sick to well. We propose to pair repeat measures of important cognitive processes (inhibitory control, reversal learning) with serial neural recordings of key cortico-basal ganglia-thalamo-cortical (CBGTC) circuit nodes. This experimental design will allow us to ascertain how clinical improvement is accompanied by changes in cognitive processes, and to uncover the relevant circuit activity. In Aim 1, to identify the cognitive changes caused by therapeutic brain stimulation we will repeatedly administer validated assays of flexibility (a reversal learning task) and inhibition (a stop signal task). At each time point we will computationally model relevant cognitive parameters, so that we can track evolution of decision-making dynamics as treatment progresses. In Aim 2, we will identify neural underpinnings of the cognitive processes changed by therapeutic brain stimulation. We have implemented an experimental paradigm that allows for millisecond-precision alignment of behavior data and neural activity. Each time the participants perform a task, local field potentials (LFPs) will be recorded from key CBGTC circuit nodes, never previously accessible for study in patients with compulsive disorders. With the unique opportunity to longitudinally record directly from the basal ganglia in these patients, we will be able to establish if these circuits do indeed subserve clinically relevant decision-making processes. This K23 proposal is supported by a carefully assembled, collaborative, and diverse mentorship team with the requisite expertise in psychiatric DBS, disorders of compulsivity, computational modeling of behavior, and human intracranial recordings. The candidate will emerge from the mentored research experience equipped to lead independent studies of deep brain circuit function in real world patients.

NIH Research Projects · FY 2025 · 2024-08

Project Summary The number of physician-scientists in the United States has decreased from its peak of 5% in 1980 to 1.5% today. This decline particularly threatens Pediatrics and child health research where only 12.6% of all MD/PhD program graduates choose residency training in Pediatrics and in Internal Medicine, while 40% of U.S. MD/PhD graduates enter Internal Medicine residency programs, these trainees only account for 3.6% of all Internal Medicine trainees. Of those, smaller numbers train in infectious diseases, clinical immunology and rheumatology. Together, these data represent a critical need, and opportunity, for new and sustained efforts to reinvigorate the physician-scientist pipeline infectious diseases, allergy and immunologic diseases. The Icahn School of Medicine at Mount Sinai is well poised to answer this call for novel on-ramps into the PhysicianScientist Pathway during residency given its established infrastructure for Research Tracks in Residency which successfully track accomplished researchers to academic research-focused fellowships and are synergistic with this proposal. The opportunity to cultivate opportunities for research during residency is ideally suited to our robust translational research programs. Our excellence in biomedical research across the basic, clinical, and translational spectrum, along with the ability to individualize and adapt to each physician-scientist trainee, makes our environment an ideal place to train future physician-scientists. The Mount Sinai Stimulating Access to Research in Residency (StARR) is a new program to enhance physician-scientist research training opportunities related to the mission of the National Institute of Allergy and Infectious Disease (NIAID). Mount Sinai StARR will provide Internal Medicine and Pediatrics residents an entrez into the physician-scientist pipeline, especially focused on those with a passion for inquiry, but who may not have had extensive research experience--the “late bloomers”. The program will provide a stepwise introduction to investigative research to recruit resident trainees and will be an attractive recruiting tool across and outside the Mount Sinai Health System that will build an immersive individualized research experience with the goal of accelerating and retaining research independence for resident investigators.

NIH Research Projects · FY 2025 · 2024-08

PROJECT SUMMARY Leukemia represents the most prevalent form of cancer among children. Sequencing studies from identical twins indicate that the initial genetic alterations can manifest as early as during fetal development. Acute myeloid leukemia (AML) is a heterogeneous disease, which is characterized by various genetic alterations including fusion oncoproteins. NUP98-NSD1 fusion-positive AML is a poor prognostic subtype that is commonly diagnosed in pediatric patients with cytogenetically normal AML. There, the nuclear pore complex member NUP98 fuses to the histone methyltransferase NSD1. Recently, NUP98-NSD1 gene fusions, in combination with transcription factor WT1 (Wilms' tumor 1) mutations, were identified in a chemotherapy resistant pediatric AML cohort, with a devastating 4-year event-free survival of less than 10%. The underlying reason why NUP98-NSD1 with WT1 mutations are associated with therapy resistance and relapse is not known. In preliminary experiments, we created a humanized system of NUP98-NSD1 rearranged leukemia with endogenous expression of the fusion gene, with and without WT1 loss of function mutations. Strikingly, NUP98- NSD1 fusion-driven leukemogenesis displayed a developmental dependency, where its expression transforms pre-natal fetal liver-derived hematopoietic stem cells (HSCs), to a lesser extent post-natal cord blood-derived HSCs, but unable to transform adult bone marrow-derived HSCs. In vitro, endogenous NUP98-NSD1 oncoprotein conferred clonal selection and proliferation advantage in fetal HSCs, while in vivo, through xenotransplantation in mice, NUP98-NSD1 faithfully recapitulated AML. Therefore, we hypothesize that the epigenetic state of fetal-derived HSCs is permissive of NUP98-NSD1-mediated leukemic transformation, but is repressed in developmental states after birth. The overall objective of our project is to define mechanisms of action of NUP98-NSD1 in HSCs across development, understand how WT1 mutations drive therapy resistance and identify novel therapeutic targets for NUP98-rearranged leukemia. In Aim 1, we are proposing to investigate the cellular and developmental dependency of NUP98-NSD1-mediated leukemic initiation by xenotransplantations. We will assess the single- cell epigenetic and transcriptional landscape of NUP98-NSD1 fusion-positive HSCs across ontogeny. In Aim 2, we plan to characterize the downstream effects of WT1 mutations in NUP98-rearranged leukemia. We observed that concurrent WT1 loss led to a more primitive stem cell hierarchy and an enrichment of quiescent leukemic stem cells. Finally, in Aim 3, we are proposing to identify therapeutic approaches against NUP98-rearranged leukemia. We will functionally assess the role of candidate genes using our established model and primary PDX patient samples. Our goal is to characterize novel therapeutic targets for NUP98-rearranged leukemia. The proposed research has the potential to change our understanding of why children are susceptible to NUP98-NSD1 rearranged AML and provide a strong rational for therapeutic intervention in the future.

NIH Research Projects · FY 2025 · 2024-08

Complex diseases are heterogeneous in their etiology and clinical manifestation. This heterogeneity hampers understanding of their causes, and requires new precision medicine strategies to apply targeted interventions in homogeneous disease subgroups. While statistical genetics methods have provided key insights into disease etiology in recent years, they are not optimized to expose the mechanisms underlying the heterogeneity observed among patients. Therefore, novel strategies to identify and understand disease heterogeneity are needed. In this K99/R00 proposal, we introduce PATHFINDER: a comprehensive research program to reveal the mechanisms leading to heterogeneity in complex diseases. This research program will be prototyped for schizophrenia, inflammatory bowel disease and coronary artery disease, selected for their high heritability, availability of powerful GWASs, and the rich functional genomic and clinical individual- level data available. These diseases provide, thus, excellent templates for extending this approach to a range of complex diseases. To achieve the program objectives, the following analytical strategy is proposed: - In Aim 1 (K99 phase), the context in which putative causal genes act will be determined. This involves identification of tissues and cell types with the highest gene expression and specificity, and identification of other genes functionally related to the genes of interest (e.g. based on metrics such as gene co-expression). - In Aim 2 (K99 phase), our recently developed PRSet software and tool will be utilized to compute pathway- based polygenic risk scores (PRSs), identifying, for each individual, the biological pathways with the highest genetic risk to disease. This will enable the identification of shared and distinct genetic profiles across patients. - In Aim 3 (R00 phase), Dr. García-González will apply the techniques and knowledge gained from Aims 1 and 2 to develop a precision medicine framework for stratifying patients from large biobanks. In Aim 3.1, PRSs associated with clinical factors and disease symptoms will be identified. In Aim 3.2, statistical and machine learning methods will be used to stratify patients into more genetically and clinically homogeneous subgroups. This research program is expected to have a significant translational impact by identifying more homogeneous disease subgroups that will inform mechanistic hypotheses, ultimately leading to more etiology-specific interventions and treatments. Furthermore, the training during the K99 phase will be crucial for Dr. García- González’s scientific development and to set the foundations of her independent research program. The combination of: (i) Dr. García-González’s background in experimental research and statistical genetics, (ii) the expertise of the mentoring team in PRSs methods development, functional genomics and experimental validation approaches, and (iii) the world-leading research performed on medical genetics and genomics at the Icahn School of Medicine at Mount Sinai in New York City, provides an ideal lever for Dr. García-González to climb the academic career ladder and to become an independent investigator.

NIH Research Projects · FY 2025 · 2024-08

Reproductive success and health depend on precise coordination between germ cells and their surrounding support cells, yet the mechanisms governing these interactions remain poorly understood. Our research team explores genetic, molecular, and cellular interactions between germ cells and somatic (non-germline) cells of the gonad. We focus on the basic mechanisms regulating reproductive system formation and function. Key questions are: 1) what establishes the cells that produce eggs and sperm? and 2) what maintains distinct cell types found in ovaries versus testes, including both germ cells and their supporting somatic cells, all of which are required for reproductive success and health. Our goal in pursuing this research program is to define the developmental mechanisms that regulate gonad and gamete development to prevent infertility. Extending the function of reproductive organs is expected to mitigate or eliminate health risks that accompany premature ovarian failure, perimenopause, and menopause. From a developmental perspective, disorders of sexual development (DSDs) are common, and include gonadal dysgenesis, dysregulated puberty, and sterility. Although common, the basis of most DSDs is unknown or results from genetic or hormonal anomalies during reproductive development. We will leverage powerful genetic tools, cell imaging, and comprehensive genomic and molecular profiling to define the developmental mechanisms and identify new therapeutic targets for preventing infertility and reproductive disorders.

NIH Research Projects · FY 2025 · 2024-08

PROJECT SUMMARY Sarcoidosis is a systemic condition characterized by non-caseating granulomas affecting ~200,000 Americans. Granuloma infiltration in the myocardium leads to inflammation, fibrogenesis, ventricular arrhythmias, and increased morbidity and mortality. Imaging determinants of cardiac sarcoidosis (CS) include late gadolinium enhancement (LGE) on cardiac MR (CMR) indicating fibrosis, uptake of 18F-FDG on PET indicating inflamma- tion, and perfusion deficits on 82Rb myocardial PET perfusion imaging (MPI) indicating microvascular constric- tion secondary to either fibrosis or inflammation. Each have been identified as predictors of future clinical events. While early immune-suppressant therapy (IST) can mitigate cardiac dysfunction and improve longer- term outcomes, treatment guidelines for CS are not well developed. Given the deleterious effects of prolonged IST (with corticosteroids), and the availability of combination approaches using steroids and steroid-sparing medications, optimization of the therapeutic strategy is an important clinical need. An elevated FDG signal (SUVmax) and its subsequent reduction are widely regarded as a trigger for IST and evidence of response to therapy, respectively, however agreed-upon quantitative thresholds for such determinations are lacking. Our overarching goals are, 1) to determine the imaging response to therapy of multiple imaging parameters (Aims 1&2) and to establish thresholds that are associated with changes in cardiac function (LVEF and NYHA Class) after IST; 2) to correlate imaging parameters with changes in cardiac function and change in LGE (fibrosis) after IST and with the occurrence of adverse clinical events (Aim 3). We propose a prospective study, with serial imaging beyond completion of IST to assess sustained changes after IST, comprehensive imaging (LGE- CMR, FDG PET, Rb MPI, advanced CMR mapping, and quantitative perfusion measurements), and a standardized IST regimen to improve generalizability of our findings. We will enroll 174 patients with confirmed CS (biopsy-proven extra-CS with cardiac involvement according to WASOG and HRS criteria or biopsy-proven CS with clinical evidence of extra-CS), a perfusion deficit on MPI, and who are treatment naïve at baseline. 112 patients with elevated FDG will undergo IST and serial imaging at Time Point (TP) 1 prior to IST, TP2, after IST (12-16 weeks), and TP3, after steroid tapering (28-36 weeks). Clinical adverse events (cardiac arrest, VT, heart failure, new heart block) will be monitored for the duration of the study. The central innovation in the study is the development of a multi-parametric approach and the hypothesis that composite multi- parametric measures will be superior predictors of overall risk and subsequently response to therapy. In Aim 1, we focus on SUVmax and other standard imaging parameters, addressing an immediate need. In Aim 2, we evaluate advanced CMR mapping parameters (T2, T1, extracellular volume (ECV)), quantitative myocardial perfusion parameters, and develop novel parameters using radiomics analysis. In Aim 3, we develop a multi- parametric model to predict future adverse events and develop a novel Artificial Intelligence-based approach.

NIH Research Projects · FY 2026 · 2024-08

PROJECT SUMMARY Choline is a vitamin-like metabolite that is indispensable for cellular and organismal viability which must be obtained through diet. Once imported into the cell, choline has multiple metabolic fates influencing diverse cellular processes ranging from membrane biosynthesis to epigenetics. Choline it is a key constituent of phospholipids, acetylcholine, and betaine which in turn impacts S-adenosylmethionine (SAM) and DNA methylation. Despite the influence of choline on diverse cellular processes, the identity of a high affinity choline transporter ubiquitously expressed across mammalian tissues was unknown. In our recently published preliminary data, we utilized genome-wide association studies (GWAS) of serum metabolites to identify a poorly characterized plasma membrane protein, feline leukemia virus subgroup C cellular receptor 1 (FLVCR1), as the predominant choline transporter in mammals. In human cells and the developing mouse embryo, FLVCR1 loss severely impacts choline metabolism resulting in depletion of betaine and phosphatidylcholine (PC) – the predominant phospholipid species in cellular and organellar membranes. Mechanistically, FLVCR1 directly transports choline into cells and we have recently used CryoEM to identify the residue necessary for transport. In this effort, we also discovered that FLVCR1 can also transport ethanolamine, suggesting that it may also affect phosphatidylethanolamine (PE) synthesis, the second most abundant membrane phospholipid. Taken together, these data suggest that FLVCR1 is a crucial transporter for phospholipid metabolism. Broadly, this proposal seeks to investigate the influence of phospholipid metabolism on cellular and organismal physiology. Utilizing a conditional knockout mouse, in Aim 1, we will study the role of FLVCR1 in organismal physiology and metabolism and assess the efficacy of FLVCR1 as a target in metabolic disease. Our preliminary data suggest that mitochondrial stress and activation of the integrated stress response are defining features of cells and embryos lacking FLVCR1. In Aim 2 we will study how FLVCR1 loss and phospholipid metabolism impacts mitochondrial function and the subsequent cellular stress response. In Aim 3 we seek to understand how phospholipid homeostasis is maintained and regulated. Spanning basic biochemistry to mouse modeling, this application will address outstanding fundamental questions in cellular metabolism and seek to apply these findings to the possible treatment of human disease. The innovative studies proposed in this application in addition to the personalized training plan, will provide rigorous scientific training and professional development which will enable my transition to independence and start my own laboratory as a tenure-track professor.

NIH Research Projects · FY 2025 · 2024-08

PROJECT SUMMARY Proper skin restoration after the damage is vital for organismal survival. The lymphatic vascular system is spread throughout the human body and has a critical function in mammalian physiology. In physiological conditions, its main function is the regulation of tissue drainage, immunosurveillance, and regeneration. Lymphatic dysfunction causes lymphedema a medical condition that manifests as tissue swelling and fibrosis. This serious condition results in profound severe delays in wound repair and the formation of chronic non-healing wounds. The mechanism by which lymphatic vessels regulate skin repair is unexplored. Additionally, while the role of lymphatic vessels in immune cell egress from tissues is well-established, how lymphatic vessels may directly modulate immune function in damaged tissues is poorly-defined. Recently, I discovered that lymphatic vessels are actively remodeled during wound healing to form small capillaries which are present in close localization to the wound front and hair follicles. This remodeling is critical for optimal repair as skin-specific loss of lymphatic vessels results in a significant delay in wound closure accompanied by a massive infiltration of immune cells. This proposal aims to leverage these observations by 1) delineating the mechanisms and consequences of lymphatic vessel remodeling during skin repair, and 2) determining the role of lymphatic vessels and fluid pressure in macrophage behavior during skin repair. This research stands to have a significant clinical impact because it can serve as a basis for developing new therapeutic avenues for lymphedema ulcers and chronic wound management. In addition, career-oriented guidance from my mentor and advisors, along with career development activated during the K99 phase that includes formal coursework on grant writing and project management, will further facilitate my transition to the R00 phase and my long-term productivity as an independent academic investigator. Collectively, the proposed research and career development plans are expected to generate data with a significant impact on understanding the repair and immunomodulatory functions of lymphatic vessels in skin repair and setting the basis of my future research as an independent researcher.

NIH Research Projects · FY 2025 · 2024-08

Project Summary/Abstract Inflammation is a central pathologic mechanism across a variety of chronic diseases, including cardiovascular, metabolic, and pulmonary disorders. Current approaches to disease management center on non-specific strategies to attenuate local and systemic inflammation to improve health outcomes. However, emerging data supports the presence of endogenous mechanisms increasing an individual’s capacity to resolve inflammation, highlighting a yet-untapped novel therapeutic target. New insights into the resolution of inflammation reveal this process can be mediated by lipid-derived specialized pro-resolving mediators (SPMs), which trigger a cascade of events that attenuate the inflammatory response. SPMs have been implicated in the resolution of inflammation in a variety of diseases such as cardiovascular disease, COVID-19, and asthma; however, their role in resolving inflammation and counteracting lung injury in COPD patients is unknown. The objective of this proposal is to determine if greater plasma circulating SPM concentrations (reflecting individual capacity to resolve inflammation) are associated with improved respiratory outcomes in individuals with emphysema. To accomplish this, we will leverage data from the robustly-phenotyped Subpopulations and Intermediate Outcome Measures in COPD Study (SPIROMICS) cohort with available biospecimens in BIOLINCC. We will then validate our findings in The Losartan Effects on Emphysema Progression (LEEP) cohort. This proposal will accomplish the following specific aims: in SA 1: we will characterize circulating SPM concentrations as a biomarker for decreased potential to resolve chronic inflammation across two different populations of emphysema patients and identify risk factors (e.g., race/ethnicity, economic deprivation, etc.) for low SPM status. We hypothesize that there will be clear disparities in SPM across subpopulations reflecting differences in resilience to inflammatory insults. In SA 2: we will determine the association between circulating SPM concentrations and COPD health outcomes (i.e., lung function, respiratory symptoms) across two populations of emphysema patients. We hypothesize that greater SPM status will be associated with greater lung function and decreased respiratory morbidity. This work is directly related to the mission of NHLBI as it will provide foundational evidence for SPMs as an emerging therapeutic target for the prevention and treatment of COPD, stimulating basic discoveries about the causes of, and resilience factors for, lung disease and enabling the translation of these findings into clinical practice.

NIH Research Projects · FY 2025 · 2024-08

Project Summary Eight out of ten children with mitochondrial disorders die before three years of age. The most critical predicting factor of such a fatal outcome is the age of disease onset. Children who manifest the first symptoms during the neonatal or early infantile periods have a ten times higher risk of dying than those who present later. Despite recent advances in prenatal diagnosis, there are no approved treatments for mitochondrial diseases during pregnancy. As a result, most children are born with severe malformations, mainly of the nervous system, rendering most postnatal therapies ineffective. We have identified a new drug to support intermediary metabolism during pregnancy and postnatal periods in a mouse model of one of the most common human mitochondrial diseases: pyruvate dehydrogenase deficiency (PDHD). Approximately 80% of babies with PDHD are born with structural defects of the brain, including cortical dysplasia, hypoplasia of the corpus callosum, and cerebellar atrophy, resulting in severe intellectual disability, intractable seizures, and short lifespan. To identify this treatment, we tracked glucose metabolism in vivo in a mouse model of PDHD to ascertain the metabolic pathways that are amenable to treatment. We found that the Krebs cycle is a critical pathway. In contrast to the control brain, the activity of the Krebs cycle is partially sustained by the incorporation of non-glucose-derived substrates at several entry points throughout the cycle. A main entry point is succinyl-CoA. A singular characteristic of succinyl-CoA and its precursors is that they provide net carbons to replace Krebs cycle intermediates that engage in biosynthesis. This finding is important because current treatments for PDHD do not support biosynthesis and the developmental processes that depend on it, including cell proliferation, migration, and myelination. We hypothesize that a specific precursor of succinyl-CoA could represent a new therapeutic strategy to improve neurodevelopmental outcomes in this condition. To test this hypothesis, two independent aims are proposed: 1) To analyze how a specific succinyl-CoA precursor is metabolized in the PDHD brain; 2) To evaluate its therapeutic impact on neurodevelopmental outcomes in the PDHD mouse model. The research proposed is significant because it aims to prevent, for the first time, neurodevelopmental deficits in PDHD and, by virtue, to improve the quality of life and life expectancy in this condition. This proposal is innovative because it will test a new drug to treat PDHD as early as the prenatal stages. Our preliminary data indicate that the administration of this drug is safe during pregnancy in mice. Positive results from these studies may have important implications for patient care because the drug is already FDA-approved for human use and can be applied to other metabolic conditions in which brain biosynthesis is impaired.

NIH Research Projects · FY 2025 · 2024-08

Cardiovascular disease (CVD) is the leading cause of death in women in the United States. Mounting evidence suggests that pregnancy is a key window when cardiovascular (CV) health is eroded, increasing future CVD risk. Further, the link between CV and lung health is well-established, and the critical prenatal period may influence multiple future morbidities simultaneously. Neighborhood-level factors may result in a pro-inflammatory state during pregnancy and impair postpartum cardiopulmonary health (CV and lung health) setting the stage for future chronic disease risk. For instance, evidence from our pregnancy cohort (Generation C) in NYC found that neighborhood measures were associated with preterm birth which separately has been linked to a life course two-fold risk of CVD and respiratory mortality. To better understand the effect of neighborhood during pregnancy on future cardiopulmonary health, we propose Gen C Mamas, a mixed-method longitudinal study of 440 women from the Generation C cohort, initially recruited during pregnancy in 2020-2022 in New York City. We will use the life course model of multimorbidity, resilience, and weathering in our proposed Gen C Mamas study. First, we will assess the association between neighborhood and cardiopulmonary health (e.g., systolic and diastolic blood pressure, lung function, and hemoglobin A1c) at 3 and 5 years postpartum. Then we will leverage previously collected data to examine how mid-pregnancy inflammation is associated with neighborhood factors during pregnancy and cardiopulmonary health. Finally, we will select 30 participants from this subgroup and invite them to participate in Photovoice data collection. We will hold focus group sessions where participants will narrate the stories of their photo choices, and these stories will be analyzed for themes and then mapped to our theoretical framework. Because of the central role of inflammation in health, our findings may provide a model that can be extended to additional chronic conditions.

NIH Research Projects · FY 2025 · 2024-08

We propose an Undiagnosed Diseases Network (UDN) Diagnostic Center of Excellence (DCoE) at the Mount Sinai Health System (MSHS) and the Icahn School of Medicine at Mount Sinai (ISMMS). The MSHS, a large health system in New York City, serves highly diverse populations. Our investigators are leaders in community-engaged genomic research with equitably diverse enrollment. Mount Sinai's DCoE will build upon our Undiagnosed Disease Program (UDP), modeled on the UDN approach, that has a strong track record of applying state-of-the-art genomic approaches, including using artificial intelligence (AI)-based variant prioritization and long-read genome sequencing. We routinely pursue functional genomic studies for high- quality candidate variants, through our Drosophila-based modeling core and collaboratively. Adapting to and complying with the UDN standardized approach will be our deep commitment. We will be active contributors to UDN governance. For Aim 1, we will incorporate Mount Sinai's UDP as a DCoE within the collaborative UDN ecosystem. Enrolled Tier 2-4 pediatric and adult patients (35–50/year) with rare, undiagnosed disorders will be assessed in the ISMMS Clinical Research Unit by a team of physicians with broad expertise in Genetics, Pediatrics, Medicine, and Neurology, among others. Our DCoE central group includes the persons who have driven the success of our existing UDP, which routinely screens referrals and analyzes exome, genome, and RNAseq data for unsolved cases. To enhance diverse UDP referrals from the NYC area, we will expand our existing outreach to the > 7,000 physicians in the MSHS as well as those with our community partner, the Institute for Family Health, and draw from unsolved cases within our clinical genetics services. We will seek to advance sustainability through a multi-prong approach including establishing clinical utility and conversations with payers, and by continued philanthropic donor engagement. For Aim 2, we will deploy the clinical and research benefits of our UDN DCoE equitably for New York City's highly diverse population. We will leverage the fact that the MSHS resides in one of the most ancestrally diverse places on earth, providing the potential for equitable inclusion. We will rely on our site's expertise with community-engaged participatory research, specifically for genomic medicine, in order to fashion strategies to make UDN participation accessible and attractive. The Mount Sinai DCoE will apply this proven approach to improve the diversity of UDN referrals. For Aim 3, we will undertake in-depth, research-based characterization of UDN patients with immunological disorders. We will perform extensive immune profiling of participants prioritized as more likely to have single- gene disorders of the immune system in order to drive understanding of trait pathogenesis. For adult patients likely to have traits with somatic gene variants, we will undertake deep sequencing to detect mosaicism. In summary, we are proposing a Mount Sinai DCoE that will draw upon a diverse urban population, experience with our own UDP, and expertise in community-engaged research to contribute to the UDNs Phase III goals.

NIH Research Projects · FY 2025 · 2024-08

Skeletal muscle regenerates following injury, with muscle stem cells (MuSCs) the source of new myofibers. MuSCs are quiescent during homeostasis. Quiescence is an actively maintained state, supported by signals from the MuSC niche. Upon muscle injury, MuSCs activate and enter the cell cycle, proliferate as myoblasts, and differentiate and fuse to form new myofibers. How MuSCs maintain quiescence is not well understood, and there is little known about the earliest events in the transition from quiescence to activation (the Q-A transition). Quiescent MuSCs in vivo have long, heterogeneous cellular projections that rapidly retract in response to muscle injury. Projections may therefore act as direct sensors of the niche environment. Projection retraction is driven by a Rac-to-Rho GTPase activity switch that promotes downstream MuSC activation events. These observations lead to several hypotheses: 1) MuSC projections are morphologically dynamic at quiescence, providing a surveillance function for muscle damage; 2) MuSC dynamics during quiescence are regulated by the relative balance of Rac and Rho activities promoted by niche-derived cues; and 3) there exist factors in muscle tissue that signal to stimulate MuSC projection outgrowth and, consequently, promote MuSC quiescence. Such factors are anticipated to be critical regulators of MuSC quiescence and the Q-A transition, but their identities are unknown. Moreover, new approaches are required to screen for candidate factors. We have developed an ex vivo live imaging assay for MuSCs within muscle bundles and used it to identify the axonal chemoattractant, netrin-1, as a candidate niche-derived regulator of MuSC projection dynamics. Multiple cell types in adult muscle express netrin-1, and MuSCs express the netrin-1 receptors, Neogenin (Neo1) and Dcc. Furthermore, MuSCs extend projections in response to recombinant netrin-1, as visualized in the ex vivo live imaging assay. It is proposed here to determine the role of netrin-1 signaling in MuSC quiescence and the Q-A transition. We will use conditional genetic removal of netrin-1 receptors from MuSCs to test directly the role of netrin-1 signaling in MuSC morphology, quiescence, and the Q-A transition. A combination of in vivo and ex vivo techniques will be used, including tissue clearing, in vivo analyses of uninjured and injured muscles, analysis of MuSCs on single myofibers; and live imaging of MuSCs within muscle bundles. Successful completion of the proposed work will provide strong evidence that: 1) regulation of MuSC projection dynamics is a key process in maintenance of quiescence; and 2) netrin-1 is the first member of a new class of MuSC quiescence regulator. Such findings will validate the use of our new ex vivo live imaging assay as a means to identify secreted signaling cues involved in this process.

NIH Research Projects · FY 2025 · 2024-08

PROJECT SUMMARY/ABSTRACT Multiple Sclerosis (MS) is an immune-mediated, demyelinating disorder of the central nervous system (CNS) and a leading cause of disability in young adults. MS white matter lesions are easily visible on clinical magnetic resonance imaging (MRI) and are the target of current treatments. However, white matter lesions are poor predictors of disability, and prevention of white matter lesion formation does not stop gradual disability worsening in later stages of disease, when white matter lesion formation is rare. Cortical lesions are also common in MS, can be extensive, and are associated with disability and disability worsening over time. Cortical lesions are thought to form due to overlying meningeal inflammation and thus they may respond differently to treatment than white matter lesions, in which inflammatory mediators come from parenchymal veins. Here we propose to further our understanding of MS cortical lesion formation and repair in early MS, the immunological mechanisms underlying these processes, and the impact of these processes on the clinical course of disease. New MRI methods applied at ultra-high field strength (7 tesla, T), some of which we helped to develop, now allow us to sensitively visualize cortical lesions in vivo and track their formation and repair. With these methods, we and others have demonstrated that cortical lesions are common, even early in disease, and are associated with disability. We have also found that cortical lesion burden, but not white matter lesion burden, predicts subsequent worsening of motor disability. Our recent data demonstrate that cortical lesion formation is rare in longstanding disease, and so we hypothesize that cortical lesions form early in disease and then lead to subsequent gradual worsening of disability over time. Here, we propose to follow a cohort of adults with newly diagnosed MS for 3 years with 7T MRI (including 0.5mm3 T1 and motion and B0-corrected T2* weighted imaging), motor and cognitive evaluation, and blood collection at baseline, year 1, and year 3. A subset of participants will also undergo cerebrospinal fluid (CSF) collection at baseline. We will determine how cortical and white matter demyelination are related in early MS and their relative contributions to physical and cognitive disability and disability worsening over time (Aim 1). Using annual MRI data as well as data from short interval MRI follow-up (baseline, month 3, month 6) in a subset of participants, we will measure changes in cortical lesions over time and characterize cortical lesion growth, repair, and chronic inflammation (Aim 2). Finally, we will use single cell transcriptomics, proteomics, and flow cytometry in blood and CSF to determine how cortical lesion burden is related to immune activation in the periphery and the CNS (Aim 3). This work will lead to key advances in our understanding of the pathophysiology, natural history, and clinical implications of cortical lesions in early MS, which will be essential the future development and testing of treatments targeting cortical lesion formation and repair.

NIH Research Projects · FY 2025 · 2024-08

PROJECT SUMMARY/ABSTRACT Hepatitis C virus (HCV) is a hepacivirus that chronically infects an estimated 58 million people worldwide and can result in end-stage liver disease. It has been well established that ineffectual T cell responses contribute to the establishment and maintenance of chronic HCV infection. Namely, T cell exhaustion, wherein T cells lose effector and proliferative functions due to chronic antigen stimulation, is a common phenomenon observed in chronic HCV patients and is known to enable viral persistence. However, it has proven difficult to define mechanistically different aspects of immune dysfunction in chronic hepacivirus infection, in part due to a lack of animal models that can support chronic HCV infection and elicit a similar immune response. Norway rat hepacivirus (NrHV) is a close relative of HCV that can establish chronic infection in immunocompetent mice and recapitulate key features of chronic HCV, thus serving as a physiologically relevant and experimentally tractable model for studying the immune response to chronic hepacivirus infection. Despite its promise as a model for studying various aspects of the immune response to chronic hepacivirus infection, an in-depth characterization of the immune landscape over time in chronic NrHV infection in mice has yet to be performed. Here, I propose performing high-resolution immunological characterizations of this novel murine model of chronic hepacivirus infection to study the evolution of immune dysfunction over the course of infection and to better understand the molecular drivers of T cell exhaustion and viral persistence in chronic hepacivirus infection. First, I will model transcriptional regulatory networks in chronic hepacivirus infection and other contexts of T cell exhaustion to identify common and distinct transcriptional programs defining immune dysfunction (Aim 1). My preliminary findings from existing transcriptomic data on this model suggest that there are two distinct lineages of exhausted T cells in NrHV infection which may recognize viral antigen. Therefore, I will define the phenotypic features of the two exhausted lineages in this model to elucidate the relationship between TCR specificity and cellular phenotypes of virus-specific exhausted T cells (Aim 2). These studies are expected to ultimately contribute to our understanding of the immunological mechanisms that drive T cell exhaustion, hinder hepacivirus clearance, and contribute to the pathogenesis of viral liver disease in chronic HCV infection. The proposed project provides an excellent training opportunity and environment that encompasses mastering cutting-edge single cell and immunological techniques, professional development, and mentorship that support my goals and growth as an independent research scientist.

NSF Awards · FY 2024 · 2024-08

This research project aims to develop a groundbreaking instrument capable of measuring mass changes in live single cells with unparalleled precision and speed. The instrument uses small pipettes that capture the cells using gentle pressure and measure tiny weight changes in thousandths of a second. By enabling scientists to observe how cells regulate their mass during crucial processes such as migration and differentiation, this tool will fill a significant gap in biological research. Additionally, the project will benefit society by advancing scientific knowledge and fostering new technological innovations. The project will also include educational outreach programs through internships to allow underrepresented and economically disadvantaged students to experience scientific research and entrepreneurship, promoting diversity and inclusion in STEM fields. The goal of this project is to develop a transformative instrument for measuring cellular mass changes with picogram accuracy and millisecond temporal resolution. The instrument will utilize small pipettes that can precisely attach to individual cells using gentle pressure, eliminating the need for adhesion molecules that might alter cell behavior. This technology will enable mass measurements of mammalian cells and support high-throughput analysis using pipette arrays with embedded sensors. By integrating several technical innovations, the instrument will achieve accurate and frequency measurements in liquid environments. This innovative approach will allow continuous monitoring of cell mass changes, applicable to studies on cell division, metabolism, migration, and more. Collaboration with industry partners will facilitate the commercialization of the instrument, ensuring its broad application in biological research and potential therapeutic developments. The research outcomes, including device designs and data, will be disseminated through peer-reviewed publications and made available on a stable URL: https://labs.icahn.mssm.edu/gaitaslab/. This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.

NIH Research Projects · FY 2025 · 2024-08

Project Summary Ebola virus (EBOV) is a member of the filoviridae family of non-segmented, negative sense RNA viruses known to cause sporadic and deadly outbreaks. Among the eight major translation products encoded in the filovirus genome, the viral protein 24 (VP24) is crucial for the production of infectious viral particles, viral nucleocapsid assembly, and the suppression of IFN signaling. To suppress IFN signaling, VP24 interacts with host importin- alpha (IMPA) nuclear transport proteins to competitively inhibit STAT1 interaction with IMPA, preventing STAT1 nuclear import and the induction of STAT1 dependent gene expression. While the VP24-IMPA interaction is well characterized in the suppression of IFN signaling, less is known about whether this has additional functional significance. Further, less is known about the importance of VP24 interaction with other host proteins. In a preliminary affinity purification-mass spectrometry (AP-MS) proteomic screen, we demonstrated that several host proteins identified as wildtype VP24 interactors were not identified as interactors for VP24 IMPA-binding mutants. Host proteins that were identified as interactors for wildtype but not mutant VP24 may use IMPA to bridge interaction with VP24. In contrast, host proteins that interact with both wildtype and IMPA binding mutant VP24s are potential direct VP24 interactors. Therefore, I hypothesize that VP24 can interact with host proteins through IMPA-independent and dependent binding modes, with IMPA-binding acting as a mechanism to expand the range of host proteins targeted by VP24. I also hypothesize that both modes of interaction are functionally significant for EBOV replication. The goal of this project is to define the characteristics and mechanistic significance of these two VP24 binding modes by examining representative host proteins identified in our AP- MS studies, with the host protein ANP32A representing IMPA-dependent VP24 interactors and EMD and ATP1A1 representing IMPA-independent VP24 interactors. In Aim 1, we will use structural and mutational approaches to characterize crucial binding determinants that mediate IMPA-dependent and independent interactions. In Aim 2, we will examine the functional significance of these binding modes. Using our transcription and replication competent virus-like particle (trVLP) EBOV life cycle modeling system, we will examine the impact of these host proteins on different stages of the EBOV life cycle. We will also use the previously described Ebola∆VP30-eGFP virus to examine how these host proteins impact viral replication. The results from these studies will provide additional clarity about the importance of the VP24-IMPA interaction and provide novel insight into cellular processes targeted by VP24. Together, these findings will advance our understanding of the crucial role for VP24 in EBOV replication. The studies proposed here will also help further my personal career goals by providing new training in filoviruses, nuclear trafficking, structural biology and new means to examine virus-host interactions.

NIH Research Projects · FY 2026 · 2024-08

PROJECT SUMMARY. Under homeostatic conditions, there is a balance between pro- and anti-inflammatory responses in the intestinal mucosa to provide a rapid response to pathogen while preventing aberrant tissue damage. In inflammatory bowel disease (IBD), disruption of this balance can lead to harmful inflammation resulting in disease initiation or relapse. gd intraepithelial lymphocytes (IEL) provide the first line of defense against luminal microorganisms and exhibit immunoregulatory capacity through the expression of CD39, which contributes to the hydrolysis of extracellular ATP to adenosine. We and others have reported that the total number of gd IELs, and CD39 expression on these cells, is reduced both in IBD patients and in a mouse model of Crohn’s disease-like ileitis; however, the regulation of CD39 and its influence on gd IEL effector function remains unclear. We now show that the frequency of immunoregulatory CD39+ gd IELs is decreased a month prior to the onset of Crohn’s disease-like ileitis and the microbiota contributes to gd IEL CD39 expression. Further, we recently described a novel transmissible gd IEL hyperproliferative (gdHYP) phenotype, in which the presence of a unique microbiota increased gd IEL number and surveillance behavior. Our preliminary data show that WT mice exhibiting the gdHYP phenotype also exhibit increased CD39 expression that is accompanied by a shift toward mitochondrial metabolism and reduced cytokine production. Therefore, we propose to interrogate the mechanisms by which CD39 expression is regulated in gd IELs, elucidate the extent to which CD39 is coupled to gd IEL bioenergetic capacity, and investigate the intersection between antibiotic treatment and gd IEL functionality in pouchitis. To address these questions, we will use a combination of ex vivo IEL culture, IEL/enteroid co-culture, single cell metabolomics assays, and unique gd T cell-specific mouse strains to dissect the molecular mechanisms involved in the upregulation of CD39 and immunometabolism in response to the microbiota. Moreover, we will leverage access to retrospective and prospective longitudinal biobank samples to both translate findings from animal studies and investigate the potential link between antibiotic therapy and gd IEL CD39 expression in the context of pouch inflammation. These studies will further our understanding of the cellular and molecular mechanisms involved in amplifying gd IEL regulatory function to maintain mucosal homeostasis with the ultimate goal of preventing disease relapse in IBD patients.