Washington University

NIH Research Projects · FY 2026 · 2024-01

Type 1 diabetes (T1D) is a chronic autoimmune disease caused by the destruction of pancreatic beta cells in the islets of Langerhans. Genome-wide associated studies (GWAS) have implicated several dozen genes in T1D, but few are expressed by or unique to beta cells. We hypothesize that a combination of genetic and environmental factors contributes to immune and exocrine-endocrine crosstalk leading to the loss of beta cell mass. In this multidisciplinary study, we will investigate the relationship between pancreatic tissue types, inflammation, and genotypes to uncover new insights into the initiating events of T1D. We will generate immune cells and pancreatic exocrine and endocrine organoids with induced pluripotent stem (iPS) cells derived from patients with T1D to produce a reproducible platform for our proposed study. Specific Aim 1 will delineate transcriptional and chromatin accessibility changes of pancreatic exocrine and endocrine tissues under inflammation with T1D-risk variants to identify T1D cell type-specific regulatory programs. Specific Aim 2 will determine changes in pancreatic exocrine and endocrine function on immune responses with T1D-risk variants. Specific Aim 3 will establish a microcontact printing co-culture system of pancreatic exocrine and endocrine tissue to investigate tissue interactions in the context of stress with T1D-risk variants. The multi- tissue assemblies will be evaluated with spatial transcriptomics to identify the relationship between tissue cross-talk and transcriptional identity. Successful completion of this study will provide a better understanding of the relationship between pancreatic exocrine and endocrine cells in the context of defined genotypes and environmental factors in the development of T1D. The data and novel co-culture platform generated in this study will benefit the T1D community.

NIH Research Projects · FY 2025 · 2024-01

PROJECT SUMMARY Hematopoietic stem cells (HSC) maintain both a self-renewal capacity to persist throughout life and an ability to differentiate to downstream progenitors to generate blood cell lineages. Stem cells balance these fates through symmetric and asymmetric divisions, respectively. HSC self-renewal and symmetric division are necessary for rapid expansion of HSCs in fetal development as well as repopulation of bone marrow following stem cell transplantation. Recently, the transcriptional regulator Bclaf1 (Bcl2 associated transcription factor 1) was shown to support both fetal and adult HSC function. Bclaf1 is ubiquitously expressed in hematopoietic cells but its function in normal hematopoiesis has not been elucidated. We recently characterized BCLAF1 as a modulator of ETS-family transcription factor activity in developing B cells. Bclaf1 is highly expressed in HSCs, which also rely on ETS-family transcription factors for development and function, consistent with a potential role for BCLAF1 in HSC activities. To test whether BCLAF1 regulates HSC development and hematopoiesis, we used murine models with germline loss of function and with selective deletion of BCLAF1 in hematopoietic cells. We find that Vav-cre:Bclaf1fl/fl mice have significantly reduced numbers of HSCs at embryonic day 17.5 (E17.5) that persists into adulthood, but does not exaggerate over time. To further characterize the function of BCLAF1 in HSCs in adult mice, we treated 8-week old Mx-cre:Bclaf1fl/fl mice with polyinosinic:polycytidylic acid (pIpC) to delete Bclaf1 in established adult HSCs. Loss of BCLAF1 did not alter HSC numbers in the adult bone marrow as compared to pIpC-treated Bclaf1fl/fl control mice. These findings demonstrate that BCLAF1 supports HSC development beginning in fetal populations but is not required for homeostasis of adult bone marrow HSCs. We performed competitive reconstitution assays with limited numbers of HSCs to investigate if BCLAF1 is required for HSC repopulation function. HSCs were sorted from fetal livers of BCLAF1-sufficient or -deficient mice and transplanted along with wild-type competitor bone marrow cells into lethally-irradiated recipient mice. Mice transplanted with Vav-cre:Bclaf1fl/fl HSCs had significantly reduced hematopoietic repopulation with lower percentage of donor-derived leukocytes and reduced percentage of donor HSCs at 16 weeks post-transplant than mice with Bclaf1fl/fl donor HSCs. This phenotype is consistent between fetal and adult donor HSCs from Vav-Cre:Bclaf1fl/fl mice. Additionally, single cell CITEseq reveals that BCLAF1-deficient HSCs have significantly upregulated AP-1 factors JUN and FOS. Collectively, our findings reveal that BCLAF1 is a novel regulator of fetal HSC development and HSC repopulation activity. On-going studies are investigating the mechanism of BCLAF1 function in HSCs and the activity of BCLAF1 as a transcription factor to regulate AP-1.

NIH Research Projects · FY 2026 · 2024-01

PROJECT SUMMARY / ABSTRACT Small cell lung cancer (SCLC) is a recalcitrant cancer that causes 250,000 deaths worldwide each year. Patients with SCLC initially respond to cytotoxic therapies but almost invariably relapse with therapy resistant disease. Moreover, when diagnosed, many patients have metastases in distant organs including the liver, which represents a major impediment to successful therapy. The metastatic cascade involves cancer cell interactions with many normal cell types, which are thought to provide by pro- and anti-metastatic signals. Recent studies have shown that endothelial cells (ECs) play important and diverse roles in metastasis of different cancer types. However, our understanding of the molecular and cellular interactions between SCLC cells and ECs in liver metastasis remains incomplete and no therapies exist to prevent or stop these interactions, which marks great therapeutic possibilities for target treatment for SCLC patients in clinic. In this proposed study, I hypothesize that SCLC-EC interactions alter the cell state of both cell types and these changes are critical for SCLC liver metastasis. To systemically study the reciprocal interactions between SCLC cells and ECs, I have developed an innovative method to qualitatively record physical interactions between various cell types (GFP- based Touching Nexus, G-baToN). Meanwhile, my mentors' labs have generated somatic CRISPR genome editing mouse models and high-throughput tumor barcode sequencing platforms which I will employ to deconvolute metastatic burden, metastatic seeding, and clonal expansion in SCLC liver metastasis. By leveraging all these cutting-edge techniques, this project aims to characterize the role of SCLC-activated ECs in SCLC liver metastasis through studying the function of the CXCLs-CXCR2 axis (Aim 1) and other cell-cell interaction related EC genes (Aim 2). On the other hand, this project will also quantitatively assess the impact of EC-induced SCLC alternations in liver metastasis (Aim 3). Taken together, this project will uncover new mechanistic insights in SCLC metastasis, establish new strategies for investigating cancer- stromal interactions in vitro and in vivo, and identify new therapeutic possibilities to better treat SCLC patients.

NIH Research Projects · FY 2025 · 2024-01

PROJECT SUMMARY Over the past 70 years, research supported by the Pediatric Heart Network (PHN) and the National Heart, Lung, and Blood Institute (NHLBI), among other organizations, has led to significant advances in congenital heart disease (CHD) care and outcomes. The GATeway to tHe wEst consoRtium (GATHER) is a collaboration between Washington University School of Medicine/St. Louis Children’s Hospital and the University of Colorado/Children's Hospital Colorado, aimed at continuing this progress as a PHN Clinical Research Center in the next funding cycle. Recognizing the importance of new and emerging research tools as well as key priorities for future research in CHD, the consortium brings together diverse expertise and resources in basic, clinical, and translational research, including multi-omics research, health services and outcomes research, machine learning and big data approaches, digital health technology, and social determinants of health (SDoH). The GATHER consortium is rooted in a strong track record of past and ongoing collaborations between investigators and is committed to training junior investigators using a shared mentorship model. The GATHER consortium is committed to enhancing diverse perspectives in clinical research through the recruitment and retention of diverse study populations and the training of junior investigators from diverse backgrounds. Strategies such as community engagement, leveraging technology for data collection, and making study participation more accessible are outlined. GATHER represents a unique addition to PHN’s roster of core sites by drawing from a racially, ethnically, and socioeconomically diverse patient population in the Midwest and Mountain regions, areas that have historically been under-represented in PHN studies. The GATHER consortium plans to develop a multi-center prospective registry, PROTECT-HF: Pediatric Registry Of heart failure Therapies to Embed Clinical Trials, its design and content informed by clinical experts, patient and family advocates, and community partners. The goals of this registry will be to study real world practice variation in guideline-directed medical therapy for heart failure, to examine the role of SDoH on healthcare utilization and outcomes, to characterize responders and non-responders to GDMT, and to develop risk-prediction models for disease-specific outcomes. This work will be facilitated by novel analytic approaches and automated data extraction to optimize efficiencies and insights. The PROTECT-HF registry will then be used as a framework in which to embed clinical trials of novel heart failure therapies, starting with an exploratory phase Ib/II clinical trial of omecamtiv mecabril, a cardiac myosin activator. The GATHER consortium and the PROTECT-HF registry’s goals align with the PHN mission of studying and improving health outcomes in pediatric congenital and acquired heart disease, engaging and supporting families, and training and educating new investigators.

NIH Research Projects · FY 2026 · 2024-01

PROJECT SUMMARY This proposal aims to elucidate the molecular circuitry that limits the efficacy of chimeric antigen receptor (CAR)-engineered T cells. CARs are synthetic proteins that, when expressed by T cells, can mediate potent activity against cancer. The most successful clinical application of this technology targets CD19 on the surface of B cell leukemia and lymphoma. While curative for some, durable remissions are seen in less than half of patients treated. Emerging data point to CAR T cell failure, as opposed to cancer cell resistance, as the driver of poor outcomes and disease progression. These findings highlight that understanding how CARs direct T cell function and dysfunction is of critical importance. The form of T cell dysfunction that is best understood is exhaustion, a cell state that develops as a result of chronic stimulation of the endogenous T cell receptor (TCR) in the setting of chronic viral infection. All CARs contain the primary signaling domain of the TCR as well as an additional costimulatory domain to enhance T cell activation. Of the six CAR products approved for clinical use in the US, four contain the costimulatory domain from 41BB while two contain the domain from CD28. To identify the CAR-activated molecular pathways responsible for T cell failure we developed an in vitro system that induces T cell dysfunction through chronic CAR stimulation. We observed that while 41BB and CD28-based CAR T cells both lose function as a result of chronic stimulation, these receptors activate divergent molecular circuitry as they lose function. CD28-based CAR T cells are phenotypically, transcriptionally and epigenetically similar to exhausted T cells. In contrast, 41BB-based CAR T cells do not bear the hallmarks of classic exhaustion but are distinct at all levels of programming. We observed activation of this unique transcriptional signature of 41BB-driven dysfunction in CAR T cells that failed to control disease in patients. We further identified that the transcription factor FOXO3 is highly active in dysfunctional 41BB-based CAR T cells and that deletion of FOXO3 improves CAR T cell function. Based on these observations, we hypothesize that chronic stimulation of 41BB triggers signaling pathways that aberrantly activate FOXO3 and that FOXO3 activates several distinct programs that suppress CAR T cell function. In the proposed studies we will use biochemical techniques and high-resolution microscopy to identify the intermediate signaling proteins that link 41BB and FOXO3 (Aim 1), use conditional expression systems to determine how FOXO3 suppresses T cell function (Aim 2) and identify other engineered T cell therapies that are vulnerable to 41BB-driven dysfunctional programs (Aim 3). Collectively, these studies will elucidate how chronic activation of 41BB leads to a novel state of T cell dysfunction and evaluate translationally-relevant strategies for advanced engineering that overcome this dysfunction-inducing circuitry.

NIH Research Projects · FY 2026 · 2024-01

Abstract My lab studies the mechanistic basis, and functional consequences, of ion channels, particularly inward rectifier (Kir) and ATP-sensitive (KATP) potassium channels, found throughout the cardiovascular system. Our work integrates studies at multiple levels, from the fundamental molecular basis of channel activity to animal models of pathologies associated with human disease. We are interested in how channels are constructed and function, how they regulate individual smooth and cardiac muscles, and how altered channel function contributes to the pathological consequences of aberrant function in the cardiovascular system. We have developed the capability to purify and to analyze channel proteins structurally, biochemically and functionally. This allows us to develop and address exciting new questions and hypotheses regarding the fundamental basis of Kir and KATP channel activity. KATP channels link metabolism to electrical activity in cardiac and smooth muscle and we have discovered how mutations in these KATP channel genes cause distinct human diseases. Cantu syndrome is caused by gain-of-function in vascular KATP channels, and associated with multiple pathological consequences, including reduced systemic vascular resistance, increased cardiac size and output, persistent fetal circulation, pericardial effusion, lymphedema, decreased vascular compliance and decreased gut motility. Unique cellular and animal models, as well as a unique research clinic, provide the tools for us to explain such features, and to develop appropriate therapies. Our recent work leads us to new hypotheses which will be explored using multiple cell biological and physiological approaches in cells, animals, and humans to reach a full understanding of the nature and role of KATP dependent excitability in regulation of cardiovascular function. These studies form the background to the development of novel pharmacological approaches and of appropriate specific therapies for KATP-dependent pathologies.

NIH Research Projects · FY 2026 · 2024-01

Project Summary This proposal tests the fundamental hypothesis that daily rhythms in neuronal activity promote growth of brain cancers and provide a temporal therapeutic window during which the efficacy of treatments may be substantially optimized, and novel targets discovered. Timed delivery of chemotherapy, or chronotherapy, has an established role in colorectal cancer and leukemia but has never been evaluated in brain tumors. Here we build on exciting preliminary data in which we have demonstrated that the efficacy of Temozolomide (TMZ), an established chemotherapeutic for glioblastoma (GBM), is substantially modulated by the time of day when it is administered. These findings suggest that we may already have the means of significantly improving outcome from this dismal disease. In this proposal, we will utilize novel intracranial xenograft models of GBM in which tumor cells have been engineered to serve as reporters of tumor circadian time with and without intrinsic clock function, and in which recipient mice have been genetically engineered to allow control of diurnal cycles in neuronal activity, hormone signaling or rest-wake behavior. We will test in mice how human and mouse GBM cells integrate into the host’s circadian system to drive proliferation. We will evaluate treatments targeting the tumor or host circadian systems for their ability to slow tumor progression. Results from these experiments should reveal a role for sex differences in circadian rhythms that affect tumor growth. Success in these studies will advance our basic understanding of GBM biology and will provide critical information for the translational application of chronotherapy to GBM care.

NIH Research Projects · FY 2025 · 2024-01

Project Summary Black college students who attend Predominately White Institutions (PWIs) in the United States (US) are disproportionately exposed to both explicit and subtle racial discrimination, which is associated with substance use, misuse, and substance use-related consequences. However, much of the existing research on racial discrimination and substance use in Black college students has focused on alcohol, using retrospective self- report and cross-sectional surveys. The lack of in vivo prospective data prevents us from understanding how Black college students’ substance use behaviors fluctuate in near real-time subsequent to racial discrimination experienced in near real-time. Examining racial discrimination and substance use at the momentary level is especially important for Black college students at PWIs who report facing unique race-related stressors compounded by stressors generally associated with the college environment (e.g., increased responsibilities, academic pressures) that are also known risk factors for substance use. In addition, US colleges are witnessing national increases in nicotine, marijuana, and other drug use. Therefore, examining racial discrimination as a risk factor for the use of multiple substances warrants greater empirical attention. The proposed study aims to fill these knowledge gaps by leveraging ecological momentary assessment, a repeated collection of real-time data, and advanced statistical analyses (e.g., multilevel modeling) to investigate how racial discrimination “gets under the skin” to increase Black college students’ substance use risk. Aim 1 addresses concurrent and lagged effects at the between-person and within-person levels: (1) Do Black college students who report higher experiences of racial discrimination on average (i.e., across time) report higher levels of substance use (between-person effects)? (2) Do Black college students report substance use more on days when they report more racial discrimination than usual (i.e., higher than their personal mean) (within- person effects)? In response to national priorities that call for empirical investigations of mechanisms linking racism to substance use and misuse in marginalized groups, this study draws on the Psychological Mediation Framework to generate knowledge about the indirect effect of racial discrimination on substance use through affective, social, and cognitive processes (Aim 2). This study represents an unprecedented opportunity to collect repeated real-time data on racial discrimination, substance use (alcohol, nicotine, marijuana, other drugs), and substance use patterns (e.g., types of use, quantity of use, co-use) among a subgroup of Black Americans in a developmental period known to be associated with high-risk substance use. In line with NIDA’s Racial Equity Initiative and 2022-2026 Strategic Plan, findings from this study will help identify specific mechanisms as intervention targets to address substance use disparities related to racism in the US.

NIH Research Projects · FY 2026 · 2024-01

ABSTRACT A variety of gene therapy strategies have been developed to achieve HIV cure. These strategies include genetic methods to render immune cells resistant to infection or to enhance immune effector cell anti-HIV activity. In this latter instance, genetic engineering of B cells has provided a highly novel means to achieve vectored immunotherapy for production of broadly neutralizing anti-HIV antibodies (bnAbs). Of note, the exceptional utilities of gene editing have been successfully employed to achieve precision genome modification of B cells to accomplish this technical end. In this approach, primary mature B cells from the periphery are modified to express HIV bnAbs as functional antigen receptors spliced to cell endogenous heavy chain constant genes. These cells are then expanded and affinity-matured, using vaccines or viral antigen in vivo, resulting in the elicitation of durable, self-tolerant, and isotyped-switched broadly neutralizing antibodies and memory. Here-to-fore this promising B cell approach has mandated ex vivo genetic modification to practically accomplish effective cell engineering. This facet of the strategy thereby entrains technical and methodological complexities practically limiting this approach. Clearly, the ability to accomplish genetic modification of B cells in vivo would render this approach of greater applicability and accessibility. Further to this end, the ability to achieve such in vivo B cell transduction, in an efficient and selective manner, would be key to realizing the benefits of this technology with an acceptable margin of safety. To this end, we have explored the utilities of replication defective adenoviral vectors (Ad) to address the mandates for in vivo transduction of B cells. In this regard, we have developed a novel “triple targeting” approach that allows efficient and selective gene delivery to key target cells in vivo. In addition, adenovirus provides a unique framework for effective and economical in vivo delivery of CRISPR/Cas9 and donor DNA for targeted integration of bnAb genes into the B cell genome by homology directed repair in situ. Of additional note, the use of non-human primate Ads allows derivation of vectors that can traverse immune barriers to human adenovirus-based vectors. In the aggregate, these combined facets of adenovirus provide the critical functional capacities allowing us to address the mandates of an in vivo applied anti-HIV B cell vectored immunotherapy strategy.

NIH Research Projects · FY 2026 · 2023-12

PROJECT SUMMARY: The objective of this multi-principal investigator application is to leverage the complimentary expertise of the investigators to develop and credential multiple new mouse models of high-risk, pediatric acute myeloid leukemia (AML). These will include both human and mouse AML with genotypes and cells of origin that are informed by real-world patient data and represent understudied high-risk molecular subtypes of the disease. This proposal will address a pressing clinical and scientific need. Children with AML continue to have an overall poor survival relative to other pediatric cancer patients. Advances in targeted therapies have been slow to develop, and up- front treatment protocols have remained remarkably stagnant for the past 4 decades. Efforts to derive new therapies have lagged, in part due to the marked genetic heterogeneity of pediatric AML. Indeed, we have established that there are over 20 unique molecular subtypes of pediatric AML that reflect unique driver mutations and expression signatures. Pediatric AML also harbor secondary mutations that further exacerbate genetic heterogeneity. Thus, therapies that might benefit patients with one genetic subtype of pediatric AML could have limited value in a different patient population. Unfortunately, current models of pediatric AML do not reflect the marked genomic diversity observed in patients. Current approaches commonly focus on a limited subset of molecular subtypes, typically avoiding rare high-risk subtypes, and they either do not co-model secondary mutations or they implement mutational combinations that are not reflected in patients. Thus, there remains a clear unmet need to advance pediatric AML modeling as a means to expand genetically informed therapeutic options. We will address this need through the completion of three Aims that exploit the unique skills of each investigator’s independent research programs and the exceptional institutional infrastructures at both St. Jude Children’s Research Hospital and Washington University School of Medicine. Aim 1: Develop novel syngeneic mouse models of high-risk pediatric AML; Aim 2: Develop human models of rare pediatric AMLs with patient- informed cooperating mutations; Aim 3. Credential established and newly developed AML models against one another and primary human specimens. To create AML models, we will deploy several innovative genetic strategies, including a novel approach to generate genetically complex murine AML from induced pluripotent stem cells and direct editing of human cord blood progenitors. Credentialing will involve a direct comparison of primary patient material with genetically matched murine and human models based on transcriptomic, epigenomic and proteomic strategies. The high-risk pediatric AML models developed through this comprehensive, cross-species proposal will provide the scientific community with an unparalleled collection of extensively characterized specimens for future investigations into disease mechanisms and therapeutic vulnerabilities.

NIH Research Projects · FY 2025 · 2023-12

Project Summary Inborn errors of immunity (IEI) are genetic disorders of the immune system that can manifest with a wide variety of presentations, including susceptibility to infection, lymphoproliferation, autoimmunity, and malignancy. Investigating IEI is critical not only to develop targeted molecular therapies for patients, but also to enhance our understanding of the human immune system. Indeed, fundamental advances of the human immune response have stemmed from studying patients with IEI. The long-term goal of this research is to investigate genetic mechanisms of novel IEI to enhance our understanding of the human immune system and improve patient care through the application of targeted molecular therapies. Here we propose to investigate patients with an autosomal dominant antibody deficiency and defect in memory B cells. These patients suffer from recurrent and severe infections due to an inability to mount appropriate antibody responses. Our preliminary studies have identified a defect in B cell maturation associated with increased proliferation. In Aim 1 of this application we will test B cell signaling and in vitro maturation in an effort to pinpoint important pathways that are altered and disease-causing. In Aim 2 of this application we focus on a genomic finding of a region of duplicated DNA with overexpression of a gene that we hypothesize leads to the altered B cell phenotype. We will use genomic and genome engineering approaches to better define this genetic change and determine whether it is responsible for disease in these patients. Results from these studies have the potential to molecularly define a new IEI and provide insight into novel genetic mechanisms of human disease.

NIH Research Projects · FY 2026 · 2023-12

PROJECT SUMMARY Bacteroides is one of the most abundant genera of the human gut microbiota, representing nearly one-third of the total composition. Bacteroides spp. participate in gut homeostasis and the development of the immune system, being able to promote both health and disease states. Bacteroides spp. unlike other bacteria, produce large quantities of uniformly-sized outer membrane vesicles (OMVs) that present distinct protein composition when compared to the outer membrane (OM). These OMVs are composed mainly of lipoproteins with protease and glycosidase activity. Bacteroides OMVs have been proposed to play pivotal roles in immune modulation, nutrient degradation, and interbacterial mutualistic interactions. Despite their physiological relevance, no mechanism for OMVs biogenesis has been yet established. Recently, we have developed molecular tools that allow the differentiation between bona fide vesicles from lysis byproducts in live Bacteroides strains expressing differentially labeled OMVs- and OM-specific proteins. Moreover, based on specific OMVs markers fused to nano-luciferase, we developed the first high-throughput screen to identify genes involved in OMV biogenesis. The screening for mutants displaying hyper- or hypo- vesiculation resulted in the identification of genes involved in OMV biogenesis. Mutagenes of the gene encoding the protein BT_3341 completely abrogated OMV formation. Preliminary experiments suggest that this strain is not able to surface display the respective glycosylhydrolases required for degradation of glycans. Additionally, we identified two anti-sigma factors with unique structural features. These two proteins appear to span across the bacterial outer and inner membranes, connecting the extracellular space with the bacterial cytoplasm. Thus, we named these proteins Dual Membrane-spanning Anti-sigma factors (DMA) family. Mutations of DMA1 and DMA2 dramatically increase vesiculation, suggesting that they regulate OMV biogenesis. The goal of this proposal is to further understand the mechanism of OMV biogenesis in Bacteroides. In Aim 1, we propose to characterize the novel DMA family and their role in OMV biogenesis. In Aim 2 we will determine the role of BT_3341 and identify additional components of this machinery in OMV biogenesis. In Aim 3 will quantify and visualize OMV formation and evaluate the importance of OMV for fitness in vivo. Our work will generate novel basic knowledge on this poorly understood process in bacteria, and lay the foundation for in- depth investigations into the role of Bacteroides OMVs within the gut. Due to their suspected role in healthy and disease states, this research may lead in the future to the production of novel OMV-based therapies applicable to diseases involving gut dysbiosis, such as inflammatory bowel diseases (IBD).

NIH Research Projects · FY 2026 · 2023-12

PROJECT SUMMARY The deadliest form of advanced-stage prostate cancer is treatment-resistant metastatic castration- resistant prostate cancer (mCRPC), which occurs in ~35% of patients after exposure to androgen receptor (AR)-directed therapies such as Abiraterone and Enzalutamide, resulting in a dismal median survival of only ~5.5 months. Unfortunately, detection of treatment-resistant disease typically occurs too late, when patients are already progressing on AR-directed drugs, after which they succumb rapidly to their disease. There is therefore a critical need to detect AR resistance more sensitively and earlier in the disease course. To address this, our team designed a novel cell-free DNA assay called Enhancer and Neighboring Loci of Androgen Receptor Sequencing (EnhanceAR-Seq), built upon the Cancer Personalized Profiling by deep Sequencing (CAPP-Seq) platform, and demonstrated superb sensitivity for detecting AR gene body and enhancer locus alterations in plasma collected from mCRPC patients. Notably, we applied our EnhanceAR-Seq technology to mCRPC patient plasma and showed that the results correlate strongly with clinically verified resistance to AR-directed therapy, and significantly outperform circulating tumor cell (CTC) AR-V7 detection. Building upon this, the current proposal will evaluate whether our EnhanceAR-Seq technology can be used to predict both (Aim 1) pre-treatment and (Aim 2) on-treatment resistance to AR-directed therapy in mCRPC patients. Further, in Aim 3 we will investigate whether integrating EnhanceAR-Seq with genome-wide methylation sequencing can granularly refine our ability to predict treatment resistance and monitor both clonal and phenotypic evolution in mCRPC. We are uniquely positioned to accomplish this through our interdisciplinary team comprised of cell-free DNA genomicists, bioinformaticians, oncologists, and statisticians. Overall, this study has significant translational impact by establishing cell-free genomic and epigenomic biomarkers of tumor evolution and resistance to AR-directed therapy in men with lethal prostate cancer.

NIH Research Projects · FY 2025 · 2023-12

Abstract About 18.7 million adults have asthma in the United States. The airway microbiome has emerged as an essential regulatory factor in asthma immune responses. Patients with asthma harbor altered bacterial compositions in their airways compared to healthy individuals; the most important alteration is a decrease in beneficial commensals and an increase in pathogenic bacteria. Our study that compared sputum bacterial compositions between adult asthma patients and healthy individuals revealed that asthma was associated with microbial alterations at the community and taxa levels, including changes in the abundances of Streptococcus salivarius, Lactobacillus species, and Haemophilus species. These results are consistent with those of previous independent human studies and have plausible biological mechanisms. However, current data on the airway microbiome in adult patients with asthma are predominantly derived from case-control and cross-sectional studies, which cannot evaluate temporal relationships between airway microbiota alterations and longitudinal asthma morbidity. There is strong evidence from multiple longitudinal studies on chronic obstructive pulmonary disease (COPD) suggesting that the airway microbial composition is closely correlated with asthma exacerbations and immune responses. However, it remains to be established whether these states are correlated in asthma. New study approaches are needed to fill this important knowledge gap. Here, we will perform a longitudinal cohort study with adult patients with asthma and healthy controls. We will collect induced sputum samples at several time points over 6 months and additional sputum samples during asthma exacerbations. We will utilize an FDA-approved electronic inhaler sensor system that will be attached to a rescue inhaler to identify asthma exacerbations and collect induced sputum soon after these exacerbations (within 24 hours). We will perform 16S ribosome RNA gene sequencing and shotgun metagenomic sequencing to identify the sputum bacterial community and relative abundance of different species. We have two aims in this study. Aim 1 is to evaluate compositional fluctuations of the sputum microbiome over time. Aim 2 is to Characterize temporal relationships of the sputum microbiome with asthma exacerbations. This proposed study will enable us to longitudinally evaluate the relationship of airway bacteria with asthma morbidity and characterize the changing dynamic patterns of the airway microbial community during asthma exacerbations. Successful completion of this study could provide valuable insights and evidence for a scaled study to include immune biomarkers and clinical indicators for further investigation.

NIH Research Projects · FY 2026 · 2023-12

PROJECT SUMMARY The overarching goal of this candidate’s program of research is to overcome social and environmental barriers to promote behavior change and cardiovascular health. The focus on this application is rural patients, since cardiovascular disease (CVD) prevalence is 40% higher among rural than urban residents. Health behavior counseling and follow-up care are required for patients with an elevated body mass index who have increased risk for CVD. Counseling is most effective when developed with, and tailored to, the patient and offered with resources that support healthy food intake and physical activity. Resource referral and follow-up is particularly important in rural residents who often have more severe social needs that impede healthy behaviors. The proposed research will leverage the candidate’s digital health tool (PREVENT) for healthcare teams to use within the clinic visit. PREVENT visually displays patient-reported and electronic health record (EHR) data to facilitate counseling and deliver tailored physical activity and healthy food intake goals and resources. PREVENT may improve the quality of required care and promote cardiovascular health. A K12 award enabled the candidate to show that PREVENT is feasible in urban clinics. This research will build upon her K12 work to: 1) collaborate with rural and clinic partners to modify and integrate the PREVENT tool for rural patients with obesity (Aim 1); and 2) conduct a pilot pragmatic clinical trial of PREVENT to optimize feasibility, acceptability, appropriateness, and potential health impact. To achieve the goals of this research and transition to independence, the candidate requires further training in: 1) rural health; 2) advanced dissemination and implementation science; 3) design and conduct of pragmatic clinical trials; and 4) team management and leadership. Together, the exceptional mentors, internal and external advisors, and institution are committed to assisting the candidate to reach her career development goals and to conduct the proposed research. In summary, this award will build the foundation for the candidate’s career as an independent investigator by providing: 1) the skillset to use dissemination and implementation science and rural health research methods to tailor and appropriately implement technology interventions; 2) methodologic and leadership skills to conduct pragmatic clinical trials; 3) a multilevel, scalable program for improving the quality of health behavior counseling; and 4) the pilot data to support the feasibility of a subsequent R01 trial of PREVENT in multiple clinic settings..

NIH Research Projects · FY 2026 · 2023-12

Abstract In response to viral infection or vaccination, antigen(Ag)-specific CD8 T cells that are present at low frequencies undergo rapid clonal expansion. While the majority of activated CD8 T cells become terminally differentiated effector T (T ECF) cells following expansion and die after Ag clearance, a small fraction of them persists as memory cells (Tr,1er,:) that contribute to long-term protection. However, it remains incompletely understood how cell-extrinsic stimuli through TCR and cytokine receptors establish the gene regulatory networks that determine the fates of activated CD8 T cells. The overall goal of this grant is to dissect the molecular and cellular mechanisms by which TCR and IL-2R signaling cooperatively regulates CD8 T cell differentiation through the control of the critical transcription factor TCF-1 encoded by Tcf7_ We will test the hypothesize that cooperative action between signals induced by antigen and IL-2, but not IL-15, specifically during priming induces expression of a set of transcription factors and epigenetic changes at a transcriptional silencer element in the Tcf7 locus. The establishment of stable Tcf7 repression requires the stimulationresponsive enhancement of IL-2R signaling, which establishes steady the TEFF- or T MEM-specific gene regulatory circuitry that can be stably maintained after the inducing cell extrinsic stimuli decays as immune responses resolve or become equilibrated. These studies will provide insights into the long standing question of the molecular mechanisms of CD8 T cell differentiation and potential application to programming improved immunotherapies.

NIH Research Projects · FY 2026 · 2023-12

Abstract. Oxidative imbalance mediates pathogenesis of neurodegenerative diseases including amyotrophic lateral sclerosis (ALS), Parkinson’s disease (PD), and Alzheimer’s disease (AD) and is shown to induce mitochondrial and synaptic dysfunction in neurons. The brains of patients with MCI and AD also have increased oxidative alterations, such as protein nitration and nucleic acid modifications. Combined factors provide compelling evidence for role of oxidative imbalance in conjunction with misfolded proteins (Aβ and p-Tau) and inflammation at the front and center of AD pathogenesis resulting in functional impairment of neurons. However, noninvasive imaging tools to investigate role of oxidative imbalance in vivo have been lacking and continues to be an unmet need. Compared with other standard of care techniques, molecular imaging with radiotracers offers advantage of enabling non-invasive, quantitative, and longitudinal analysis of the biochemical status of tissues and organs. To address this need, standard clinical 18F-FDG PET/CT lacks sensitivity and diagnostic robustness. Furthermore, the mechanism of its retention and trapping poorly correlates with oxidative imbalance. To address this critical gap in armamentarium of PET tracers, we have discovered a 2ndgeneration redox-sensitive molecular PET imaging probe (identified as 18F- SLN-128) through a rational design, wherein the probe penetrates neuronal cells, gets oxidized upon encountering oxidants, and trapped within cells to report on mitochondrial function. Using live-cell fluorescence imaging analysis, we demonstrate ability of molecular imaging probe (noncarrier added SLN 128) to detect LPS- and 3-nitropropionic acid (3-NP)-induced oxidative imbalance within mitochondria of the human glioblastoma U87 cells. Moreover, in a model of LPS induced systemic inflammation of mouse brain, dynamic PET/CT scans revealed a 2-fold higher 18F- SLN128 uptake and retention in LPS-treated brains relative to uninjured saline-treated cohorts. Furthermore, studies using a stereotaxic injection of 3-NP, a mitochondrial toxin into striatum demonstrates 2-fold higher retention of the radiotracer in brains of 3-NP treated mice compared with their saline treated counterparts. These data correlate with post-imaging quantitative biodistribution studies and immunohistochemical correlations thus providing evidence for microglial cell activation and neurodegeneration. Finally, dynamic PET/MR scan indicate ability of 18F- SLN-128 to penetrate brain (SUV= 3.5) in a nontargeted rhesus monkey following intravenous injection of the radiotracer. Armed with this provocative supporting data, aims of this preclinical imaging and translational MPI RO1 project are: Aim 1. Evaluate potential of 18F-SLN-128 to serve as a noninvasive imaging agent of 3-Nitropropionic acid (3 NP) induced mitochondrial dysfunction and neurodegeneration in presence or absence of N-acetyl cysteine (NAC) in mice; Aim 1 Sub Aims. Evaluate potential of 18F-SLN-128 to serve as a noninvasive imaging agent of ROS-mediated inflammation: 1.1. APP/PS1 (Aβ); mice and their age-matched control counterparts as a function of aging (pre-plaques (3 months), mild-moderate plaques, and severe plaques (12 months) through PET/CT imaging using 3-tracer paradigm imaging (11C-PiB for Aβ; 18F-SLN-128 for ROS; and 18F-FDG for glucose metabolism); Aim 2. Evaluate pharmacokinetics of 18F-SLN128, perform metabolite analysis from both venous and arterial outputs, and kinetic modeling in non-human primates; Aim 3. Perform radiation dosimetry to determine human effective dose equivalent (HED) and toxicology studies for 18F-SLN128 to prepare for GMP production of the PET tracer; and Aim 4. Perform three qualifying runs under GMP conditions to ascertain chemistry manufacturing controls (CMCs) to produce clinical doses of the PET radiopharmaceutical for compiling data for eIND filing; Aim 5: Perform first- in-human studies using 18F-SLN128: evaluate dosimetry, biodistribution, safety, and imaging characteristics of inflammation in AD participants compared to healthy controls. Successful accomplishment of proposed aims could deliver redox-sensitive PET molecular imaging agent for management of ROS-mediated pathogenesis in Parkinson’s disease, muscular dystrophy, multiple sclerosis, amyotrophic lateral sclerosis, stroke, traumatic brain injury, and chronic inflammation, thus extending benefits for functional imaging of mitochondrial function in vivo well-beyond its immediate utility in Alzheimer’s disease and ADRDs.

NIH Research Projects · FY 2026 · 2023-12

PROJECT SUMMARY/ABSTRACT Drug-resistant epilepsy affects approximately 1 million people in the United States. Responsive neural stimulation (RNS) and deep brain stimulation (DBS) offer novel treatment options for refractory seizures. These neurostimulation therapies have significant advantages over surgical resection, though they are generally less effective. Treatment optimization has been challenging due to inter-patient variability, electrode placement, and the vast range of available stimulation parameters. This project aims to provide a more comprehensive description of the spatial and temporal dynamics that underlie neurostimulation and evaluate novel therapeutic effects that could prevent and modify the disease course in epilepsy. This proposal also outlines a five-year career development program focused on acquiring the skills necessary to perform an in-depth mechanistic analysis of neurostimulation in a rodent model of temporal lobe epilepsy. Washington University provides an excellent environment of collaborators and resources to develop skills necessary for investigating neurostimulation as a treatment modality for epilepsy. The outlined proposal builds on Dr. Foutz's prior research in deep brain stimulation and his clinical training in the field of epilepsy. Under the mentorship of Michael Wong, M.D., Ph.D. (basic scientist, clinical epileptologist), and Dominique Durand, Ph.D. (basic scientist, biomedical engineer), he will investigate the spatiotemporal dynamics underlying electrical neurostimulation and evaluate anti-epileptogenic effects. Dr. Foutz has a career goal to become a translational scientist focused on developing and optimizing device-based neuromodulatory treatments for patients with epilepsy. The career development plan outlines formal and informal training in animal models of epilepsy and neurostimulation. This work's potential impact is to optimize existing devices, guide the development of next- generation neurostimulation technology and discover novel therapeutic effects of neurostimulation in epilepsy.

NIH Research Projects · FY 2026 · 2023-12

Project Summary/Abstract (30 lines) Immunotherapy has revolutionized cancer treatment and is a cornerstone of standard clinical practice and ongoing clinical trials. However, some immunotherapies including immune checkpoint inhibitors (ICIs) impart adverse cardiac events ranging from arrhythmias to fulminant myocarditis. While the mechanistic basis for adverse cardiac events from cancer therapies targeting immune checkpoints is not known, recent evidence suggests that macrophage infiltration and aberrant T-cell activation may be contributing factors. Ligand-receptor interactions between antigen presenting cells (APCs) and T-cells comprise current targets for immunotherapy. When used in combination with ICIs, CD40 agonists increase efficacy against previously untreatable tumors. CD40 signaling mediates activation of macrophages to heighten downstream T-cell responses. CD40 is primarily expressed on APCs such as macrophages, and its ligand is expressed on T- cells. Little is known regarding how CD40 agonists impact the heart including the potential for adverse cardiac events. The principal investigator (PI) has established that CD40 agonist treatment activates an inflammatory response in the heart that may lead to injury. In this proposal, the PI will test the hypothesis that CD40 signaling alters the immune landscape of the heart, priming the inflammatory response to myocardial injury. The scientific goals of this proposal are focused on identifying mechanisms of cardiac injury and remodeling as a result of cancer immunotherapies that regulate immune checkpoints, which may be targeted to improve clinical outcomes. The PI plans to investigate the immune cell target(s) of CD40 signaling (Aim 1), the key regulators and mechanism of signaling (Aim 2), and the effect the inflammatory response has on cardiac remodeling (Aim 3). Completion of these aims will allow the PI to gain technical expertise in flow cytometry, single cell sequencing, immune functional assays, and evaluation of cardiac injury and cancer models. The career development goals of this proposal are focused on developing the PI into an independent physician scientist in the field of cardiovascular research. The PI is an MD/PhD with advanced training in basic and translational cardiovascular research during his post-doctoral fellowship and has completed clinical training in Internal Medicine, Cardiovascular Diseases, and Cardio-Oncology. Within the next 5 years, the PI plans to: 1) develop collaborative relationships with his mentoring committee; 2) develop core and advanced competencies in bioinformatics with a focus on single cell sequencing analysis, and in immunology with a focus on cardiac immunology and immunotherapies; 3) strengthen grant writing, manuscript preparation, data presentation, and mentoring skills. At the conclusion of this mentored clinical scientist training proposal, the PI will have acquired mentorship, technical, didactic, and professional development training to become an independent and successful physician scientist.

NIH Research Projects · FY 2025 · 2023-11

ABSTRACT Viruses are obligate intracellular parasites that have evolved to co-exist with their hosts. As such, they have the ability to both hijack the necessary host machinery to promote viral proliferation as well as to antagonize host immune defenses. Despite extensive research in these areas there are still significant gaps in our knowledge of the repertoire of host genes that function in either pro-viral or anti-viral manner. A novel strategy to identify host genes that are important for mammalian virus infection takes advantage of the model organism C. elegans. Due to its simplicity and the fact that ~40% of its genes (~7500) are conserved with humans, C. elegans has played a key role in many fundamental discoveries in biology. However, its use in the study of virology had been limited by the lack of known viruses capable of naturally infecting C. elegans until the discovery in 2011 of Orsay Virus. Orsay virus is a non-enveloped positive sense RNA virus related to viruses in the families Picorna-, Calici- and Astroviridae, and thus it can serve as a model for many pathogenic mammalian viruses. Several published studies have demonstrated clear proof of concept that the Orsay virus-C. elegans model can be used to identify novel genes important for infection by mammalian viruses. For some genes that are absolutely required for Orsay virus infection in C. elegans, knockouts of the orthologous gene in human cell culture result in up to ~1,000-fold reductions in virus titers for viruses like encephalomyocarditis virus (EMCV) and Coxsackie Virus B3 (CVB3). To systematically identify genes that promote or antagonize Orsay virus infection in C. elegans, a large-scale RNAi screen will be performed. RNAi in C. elegans is both more facile and robust than corresponding siRNA screening in mammalian cells, and it is complementary to CRISPR screens in cell culture. The goals of this project are: (1) To systematically identify host factors that impact Orsay virus infection in C. elegans by performing RNAi against the ~ 7500 C. elegans genes with human orthologs. (2) Determine whether the human orthologs of genes identified in Aim 1 play roles in infection by a panel of mammalian viruses. This study will identify, in an unbiased fashion, novel genes with pro-viral and anti-viral functions.

NIH Research Projects · FY 2026 · 2023-11

PROJECT SUMMARY/ABSTRACT Cryptosporidium is a wide-spread enteric pathogen that causes severe diarrheal disease in immunocompromised patients and infants, especially in the developing world. There are more than 20 described species of Cryptosporidium some of which show narrow host ranges while others are quite broad. C. parvum has a relatively broad host range infecting many agricultural animals and causing zoonotic infections in humans. C. parvum is further divided into subtypes that undergo recombination in the wild often resulting in in differences in host range, infectivity, and virulence. The factors that control host range of C. parvum as well as their pathogenicity in different hosts are largely unknown, presenting a major gap in our understanding of this important group of parasites. In preliminary studies, we have defined major virulence differences between common laboratory variants of C. parvum during infection in immunocompromised mice. High-virulence isolates are associated with elevated oocyst shedding and lethality, while low-virulence isolates cause only mild, non-lethal infections. Whole genome sequencing identified numerous single nucleotide polymorphisms in candidate genes that likely control these phenotypic differences. We recently developed a stem-cell derived system for culturing differentiated intestinal enterocytes that allows complete development of C. parvum in vitro, including production of viable oocysts. In preliminary studies, we tagged C. parvum isolates with fluorescent reporters using CRISPR/Cas9 gene editing and used these lines to conduct genetic crosses in vitro. Analysis of the recombinant progeny from crossed lines demonstrates that meiosis results in Mendelian chromosome reassortment. In the proposed studies, we will cross fluorescently tagged virulent isolates with avirulent isolates in vitro, sort recombinant progeny using FACS, and expand them in mice to select for genes associated with increased fitness. The contributing loci will be mapped using Bulk Segregant Analysis to define candidate genes that will be confirmed by CRISPR/Cas editing. These studies should lead to a molecular understanding of pathogenicity of C. pavrum and may identify genes responsible for mediating infectivity and host-range.

NIH Research Projects · FY 2026 · 2023-11

ABSTRACT The intracellular parasite Toxoplasma gondii provides a model for studying structure-function relationships of conserved features of the phylum Apicomplexa. Several apicomplexan parasites are responsible for severe human diseases that persist despite efforts to control them. One limitation is that we lack fundamental understanding of essential parasite processes that could be used to develop new and/or improved therapies. Our work is motivated by the goal of understanding the unique complexity of structural features that form the cytoskeleton of apicomplexan parasites. Among the conserved features is the apical complex consisting of specialized tubulin rich cytoskeletal structure called the conoid, which provides a platform for other motor proteins and conduit for protein secretion. Toxoplasma has one of the most elaborate conoid structures, and while it is reduced in complexity in other apicomplexans, many of the core proteins are conserved. In T. gondii, the apical complex consists of several preconoidal rings, a prominent conoid made up of specialized microtubular conoidal fibers, two long intraconoidal microtubules, and the base formed by the apical polar ring. To date, only a few proteins have been studied functionally, although it is anticipated that conoid is comprised of ~100 proteins. Progress has been limited by the laborious process of identifying and functionally characterizing proteins in conoid, most of which have no orthologs in model systems. The proposed project aims to elevate the state of knowledge of the composition and function of the conoid by identifying all of the major proteins and localizing them in a composite 3-D structure of the organelle. This goal will be achieved through cell fractionation, quantitative mass spectrometry, and high-resolution single-particle cryo-electron microscopy (cryo-EM). We will also functionally characterize a number of new conoid proteins to define their roles in conoid assembly and function. These findings are expected to provide a near-complete atomic model of all the major components of the conoid, define the role of essential components in its assembly and function, and provide a framework for comparative studies of other apicomplexans.

NIH Research Projects · FY 2025 · 2023-11

Project summary. To maximize chances of survival, animals must not only be able to effectively predict the values of future outcomes, but also to learn from erroneous predictions. The basal ganglia (BG) are crucial for both processes. Value prediction is thought to rely on striatum, where cells store value representations in their synaptic weights. Value updating is thought to rely on modulation of these striatal synapses by dopamine, since dopamine cells encode a key updating signal in the form of reward prediction errors (RPE; the difference between expected and unexpected reward outcomes). However, anatomical studies suggest that another strong yet understudied source of striatal modulation arises from the GABAergic pallidum. Pallidum is known to have a crucial role in reward-guided behaviors. A subpopulation known as `arkypallidal' cells (in contrast to `prototypical' cells comprising the rest of pallidum) project exclusively to striatum and form massive, extremely dense axonal arborizations, making this population the largest known exogenous source of striatal inhibition. Despite anatomical evidence suggesting that arkypallidal cells are well positioned to modulate striatal value representations, no study to date has directly tested if these cells carry the signals necessary for value updating. Whether and how this important input to striatum participates in value updating to support flexible behavior remains unclear. Importantly, little is known about how pallidal cells in general signal basic motivational variables such as value or prediction error, let alone their function in complex value-guided tasks. We hypothesize that within pallidum, arkypallidal cells uniquely signal prediction errors and integrate across multiple dimensions of motivational variables to support value updating and flexible behavior. In aim 1, I will explicitly test in mice whether and how arkypallidal and prototypical cells encode motivational variables including reward and punishment value, uncertainty, and prediction error in support of value updating. Preliminary data suggest that putative arkypallidal cells preferentially signal the positive component of RPE more strongly and quickly compared to putative prototypical cells. In aim 2, I will test how pallidal cells in the non-human primate (NHP) encode and integrate across reward attributes in support of multi-attribute decision-making. Pallidum is crucial for reward-seeking behaviors, but it is not clear how it signals information when subjects must choose between reward options with multiple varying attributes. Preliminary data suggest that pallidal cells encode a wide range of attributes that subjects use to guide their choices. Further preliminary analyses suggest that putative arkypallidal cells preferentially encode reward value PE, and integrate across more decision offer attributes than putative prototypical cells. Subsequent analyses will test the hypothesis that putative arkypallidal cells compute PE signals that reflect subjects' overall value estimates of options, integrated over multiple option attributes. Subsequent experiments aim to verify these results using cell type-targeted approaches to identify arkypallidal cells in monkeys performing this multi-attribute decision-making task. These aims will elucidate the role of a poorly understood yet anatomically significant pallido-striatal projection in value-guided behavior. They will also bridge functional studies of BG circuitry across species, allowing for a more generalized understanding of BG to help guide human health.

NIH Research Projects · FY 2025 · 2023-11

PROJECT SUMMARY Rett syndrome is a devastating neurodevelopmental disorder with a significant burden on patients and their families. Patients present with an initial period of healthy neurological development, immediately followed by severe motor and cognitive regression during their first few years of life. Patients typically suffer from gait impairments, loss of speech, seizures, and sleep disturbances. Owing to its primarily neurological presentation, this X-linked disorder caused by mutations in the methyl-CpG binding protein 2 (MeCP2) has been mostly studied as a disease of neurons. While MeCP2 loss results in cell-intrinsic neuronal dysfunction, non-neuronal mechanisms also impact Rett syndrome progression, thus opening therapeutic avenues that bypass manipulation of neurons. Research over the past decade has shed light on neuroimmune interactions at the borders of the brain that are critical for healthy brain development and function, suggesting the possibility to uncover new mechanisms contributing to Rett syndrome. One major advance in the field of neuroimmunology has been the discovery of the meningeal lymphatic network. Meningeal lymphatic vessels reside in the brain’s outer membranous layer, the dura mater, where they regulate central nervous system (CNS) fluid volume and the continuous clearance of cerebrospinal fluid (CSF) out of the CNS. Ablation of these vessels impairs CSF clearance, resulting in extracellular waste accumulation and cognitive impairment. The clinical presentation of seizures, increased CSF volume, and inflammatory changes to the composition of CSF in Rett patients, suggests a potential impairment in the perfusion and clearance of the CSF that normally bathes the brain. Supporting this hypothesis, our preliminary data shows that CSF perfusion throughout the brain, as well as a major regulator of normal CSF dynamics, the meningeal lymphatics, is largely absent in an MeCP2-deficient mouse model of Rett syndrome. Here, we propose to test the hypothesis that the loss of meningeal lymphatics, resulting from a loss of meningeal macrophages, is an important contributor to Rett syndrome. Our preliminary data suggest that this decreased meningeal lymphatic coverage may result indirectly from the loss of meningeal macrophages, which typically provide trophic support to lymphatic vessels. Our data also points to an upstream, cell-intrinsic impairment in bone marrow hematopoiesis, or immune cell production, as the mechanism responsible for the observed loss in meningeal macrophages. The first aim will employ state-of-the-art transgenic mouse lines to study the cell type specific effects of MeCP2 loss on skull bone marrow hematopoiesis and macrophage differentiation. The second aim will employ advanced cell replacement strategies and viral overexpression tools to assess the functional relationship, as well as therapeutic potential, of meningeal macrophages and lymphatics in Rett syndrome. This work has the potential to both uncover new biology that is critical to Rett syndrome progression and identify new and accessible therapeutic targets to help ameliorate disease.

NIH Research Projects · FY 2025 · 2023-09

Modified Project Summary/Abstract Section Understanding the pathogenesis of glaucoma, a leading cause of blindness worldwide, is an important goal of vision research. As indicated in numerous population-based studies, individuals of various genetic profiles develop glaucoma at an earlier age with increased severity and risk of blindness. Many studies have identified oxidative damage to the trabecular meshwork (TM) cells, leading to decreased outflow facility and increased intraocular pressure. Although the evidence linking oxidative damage to glaucoma is strong, the causes of oxidative damage in glaucoma are not known. In the proposed studies, we will test the hypothesis that oxidative damage to the TM is associated with basic genetic and physiologic differences in oxygen metabolism leading to disparities in glaucoma. Our hypothesis is based on intraocular measurements of oxygen partial pressure (pO2) made in the human eye during surgery. We identified strong correlations between pO2 and important risk factors for glaucoma, including central corneal thickness. In addition, measurements of a cell marker for DNA oxidative damage were elevated in the aqueous humor of patients with severe glaucoma. Cultured TM cells from healthy donors compared to glaucomatous cells had altered energy (ATP) and reactive oxygen species production, implicating the energy processing organelles, the mitochondria. Our specific aims will test these hypotheses: (Aim 1) will evaluate differences in TM gene expression between donor controls and patients at various glaucoma stages in order to identify the genetic basis of disparities in tissue function (controlling eye pressure) and susceptibility to damage. Genes of interest will be further studied (RT-qPCR, in situ hybridization) and delineate protein expression (Western blot) in the tissue and potential biological pathways involved, focusing on mitochondrial function, oxidative stress and antioxidant protection. (Aim 2A) will study mitochondrial functions in primary human TM cell cultures from specimens of healthy donor controls and glaucoma patients at various severity stages. We will determine the effects of exposure to varying levels of pO2 on detailed analyses of mitochondrial quantities and function, reactive oxygen species levels and antioxidant protection. (Aim 2B) will also assess the cellular effects of potential antioxidative therapies. The proposed research is innovative because understanding these physiological mechanisms potentially contributing to disease based on genetic ancestry, confirmed by blood and tissue ancestral genotyping and mitochondrial DNA haplogroup stratification, will provide important information about the pathophysiology of open angle glaucoma by identifying factors responsible for the TM dysfunction and loss of aqueous outflow facility. The knowledge gained in these studies may lead to new therapies and strategies to prevent this blinding disease.