Washington University

NIH Research Projects · FY 2026 · 2024-06

PROJECT SUMMARY Brain aging and disease occur alongside the loss of essential proteins and protein functions. For example, thousands of inherited conditions are due to genetic haploinsufficiency, in which partial or complete loss of one normal allele is sufficient to cause disease. Likewise, advanced age involves the loss of protective signaling, such as through neurotrophic factors, that could prevent degenerative processes. In either case, strategies that restore these single gene targets may be therapeutic. Current protein upregulation strategies, such as direct protein delivery or viral gene therapies, are limited in the central nervous system as they lack regulatable dosing, may distribute poorly, and can elicit neuroimmune reactions. The goal of this proposal is to investigate mRNA regulation as an alternative approach to safely upregulate protein expression. Protein synthesis relies on mRNA stability and translation efficiency, which are both determined by regulatory sequences in the untranslated regions (UTRs) of mRNA. The 3’UTR encodes numerous regulatory elements that engage RNA-binding proteins and regulatory RNAs to control mRNA translation and stability. I predict that blocking repressors from engaging a 3’UTR could stimulate protein synthesis of a select gene of interest. Antisense oligonucleotides (ASOs) are short, single-stranded DNA sequences that bind complementarily to RNA with high affinity and once bound, can sterically block trans-acting elements from engaging the transcript. ASOs are a clinically proven technology that have entered clinical trials for many neurodegenerative conditions, but there are currently no ASOs in clinical trials that target 3’UTRs for gene-specific upregulation. Therefore, I hypothesize that ASOs that mask repressive regulatory sequences on a 3’UTR can be used to stimulate synthesis of proteins that prevent or protect against neurodegeneration or age-related diseases. I demonstrated the feasibility of this approach by increasing expression of TANK-binding kinase 1 (TBK1), a protein haploinsufficient in amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) caused by dominant TBK1 mutations. In Aim 1, I will test if our existing TBK1-upregulating ASOs can increase TBK1 expression and prevent neurodegenerative phenotypes in pre- clinical models of TBK1 haploinsufficiency. In Aim 2, I will evaluate if particular 3’UTR cis-elements may be universal targets for this ASO masking strategy. Aim 1 will establish the therapeutic relevance of 3’UTR-targeting ASOs and support advancement of TBK1-upregulating ASOs to human clinical testing. Aim 2 will greatly extend this approach to other gene targets, including neuroprotective protein factors. Completion of this proposal will establish 3’UTR-targeted ASOs as a generalizable strategy to stimulate protein expression to treat neurodegeneration and other age-related diseases. This training environment combines expertise in mRNA gene repression mechanisms with experience in ASO development for neurodegeneration and, therefore, is perfectly suited to support my endeavors to complete these aims and advance my development as a physician scientist.

NIH Research Projects · FY 2026 · 2024-06

ABSTRACT Recent studies in model organisms have demonstrated that the intertissue communications play a critical role in the regulation of aging and longevity. In mammals, we have demonstrated that the intertissue communication between the hypothalamus and adipose tissue, particularly mediated by extracellular vesicles-contained extracellular nicotinamide phosphoribosyltrasferase (eNAMPT), the rate-limiting NAD+ biosynthetic enzyme in mammals, functions to counteract age-associated physiological decline and promote longevity in mice. Most recently, we have demonstrated that Ppp1r17-positive neurons in the dorsomedial hypothalamus (DMHPpp1r17 neurons) regulate white adipose tissue (WAT) function, including lipolysis and eNAMPT secretion, through the sympathetic nervous system (SNS), and the feedback loop between DMHPpp1r17 neurons and WAT plays a critical role in the regulation of aging and longevity in mice. Our preliminary results suggest that this critical feedback loop between DMHPpp1r17 neurons and WAT wanes over age, which is one of the key triggers for aging. Indeed, chemogenetic stimulation of DMHPpp1r17 neurons in aged mice significantly ameliorates multiple aging phenotypes, decreases age-associated mortality rate, and extends longevity. However, why and how this critical feedback loop wanes over age remains unknown. In this research proposal, we hypothesize that adipose tissue starts decreasing adipose-resident immune cells, particularly type 2 innate lymphoid cells (ILC2s), and increases cellular senescence, causing WAT dysfunction and decreasing the content and the secretion of adipose EVs. Such WAT dysfunction then affects the regulation of Ppp1r17 function in DMHPpp1r17 neurons, affecting their function and accelerating WAT dysfunction through decreased SNS function. Maintaining this hypothalamus- WAT feedback loop is critical to counteract age-associated physiological decline and promote lifespan in mammals. To address this hypothesis, we propose the following three SPECIFIC AIMs: SPECIFIC AIM (1) will examine the effect of miR-20a, a microRNA species in adipose EVs, on the expression of Prkg1 in DMHPpp1r17 neurons during aging. SPECIFIC AIM (2) will elucidate how adipose immune cells are dysregulated during aging. We will particularly focus on ILC2s, which are dramatically reduced in adipose tissue during aging. SPECIFIC AIM (3) will address whether restoring ILC2 function by transplanting young ILC2s could delay aging and extend lifespan in mice. The anticipated outcome of the proposed research will advance our understanding of the importance of intertissue communications in mammalian aging and longevity control and open a new opportunity to develop an effective anti-aging intervention based on the intertissue communication between the hypothalamus and WAT.

NIH Research Projects · FY 2025 · 2024-06

PROJECT SUMMARY The identification of hundreds of genes associated with autism and related neurodevelopmental diseases (ASD/NDDs) in recent years has created and opportunity to leverage genetic discoveries into therapeutics for these disorders. Classically ASD/NDDs were assumed to be intractable due to disrupted development in utero, but studies in mice have demonstrated that restoring gene activity after birth can likely ameliorate phenotypes in some prominent ASD/NDDs. These findings create an imperative to systematically test the feasibility of genetically targeted therapies across ASD/NDDs with distinct genetic causes, but limited studies of this type have been carried out to date. A major class of disorders that has yet to be investigated in this paradigm is ASD/NDD caused by mutation of “epigenetic writer” enzymes. These enzymes chemically modify chromatin and DNA to control gene expression during development and throughout life. While the important potential role of these genes in early cell fate and patterning has suggested it will be it difficult to correct defects after altered development, recent studies have uncovered postnatal functions for these factors that are likely to be drivers of phenotypes in ASD/NDD. Therefore, in the proposed studies we will develop enabling mouse transgenic reagents to investigate the tractability of genetic rescue of ASD/NDDs caused by mutation of epigenetic writers. Our previous studies that have uncovered postnatal functions for the ASD/NDD-associated writer DNA methyltransferase 3A (DNMT3A) and defined robust phenotypes in heterozygous mutant mice the mimic human DNMT3A ASD/NDD mutations. We will therefore investigate the tractability of genetic reinstatement of the DNMT3A gene for rescue of molecular and organismal deficits in mice. We will implement a newly-established DNMT3A conditional genetic rescue strain to test the requirements for timing of DNMT3A reinstatement necessary to rescue molecular deficits in vivo (Aim 1). We will then perform proof-of-principle studies to investigate the degree to which behavioral and physiological function can be rescued by gene reactivation (Aim 2). Together these studies will establish enabling technologies and essential proof-of-principle findings to guide future large-scale development gene targeted therapeutics in DNMT3A disorders. Furthermore, by assessing the tractability of reinstatement for epigenetic writers in ASD/NDD, our studies can motivate therapeutic research efforts across a broad class of genetic causes for these disorders, advancing toward treatments for these devastating conditions.

NIH Research Projects · FY 2025 · 2024-06

PROJECT SUMMARY Within hospitals, infected patients are sources of bacterial transmission, often through the colonization of high- touch surfaces and equipment. Acinetobacter baumannii (Ab) is the Gram-negative bacterium with the highest rate of multidrug resistance, and is a common cause of hospital-acquired pneumonia, bloodstream, soft tissue, and urinary tract infections (UTIs). Although infected patients are sources of bacterial transmission within hospitals, no common reservoirs in the human body have been established for Ab. Hospitals often institute active surveillance strategies to mitigate the spread of bacteria between patients. However, outbreaks of Ab are still common. Moreover, there are increasing reports of community-acquired Ab infections across the globe, suggesting the existence of extra-hospital reservoirs. Two questions remain unanswered: how does the first patient get infected, and how are new Ab strains introduced into hospitals? We have recently demonstrated that Ab can hide undetected in murine bladder cells and then reactivate when stimulated by medical intervention. This lead to the hypothesis that Ab can be asymptomatically carried and introduced into hospitals by patients in intracellular reservoirs before hospitalization, and that subsequent medical interventions, such as the use of catheters, could trigger a resurgence of Ab infection from such reservoirs. Here we will employ murine models to explore this hypothesis. We recently developed catheter- and non-catheter-associated murine Ab UTI models by employing UPAB1, a recent MDR community-acquired urinary clinical isolate. In the non-catheter-associated UTI (ncUTI) model, although immunocompetent mice were not susceptible to long-term ncUTI, immunocompromised mice exhibited high bacterial burdens in urine, bladders, and kidneys, taking up to 8 weeks to completely resolve the infection. Notably, we found that introduction of a catheter into resolved mice led to the development of Ab CAUTI in ~50% of the mice in just 24 hours. Genetic characterization of the recovered bacteria confirmed that the same strain re-colonized the urinary tract. Moreover, intracellular bacteria were detected in the bladders of resolved mice up to two months after resolution of the initial infection. We named these small groups of intracellular bacteria ABIRs (Ab intracellular reservoirs). Furthermore, we also detected intracellular bacteria in alveolar macrophages during a respiratory infection. More than 300,000 patients receive mechanical ventilation in the US every year. Several complications often arise from the use of ventilators, such as ventilator-associated pneumonia (VAP). We have developed a murine model for VAP. Here we propose to investigate the relationship between ABIR formation and resurgent urinary and respiratory infections Understanding how Ab strains reside in the host and the events causing resurgence may lead, in the future, to modified surveillance strategies and/or preventative treatments to eradicate bacterial reservoirs previous to the use of ventilators, catheters, and other treatments that weaken the host immune system.

NIH Research Projects · FY 2026 · 2024-06

PROJECT SUMMARY/ABSTRACT There is an unmet critical need for techniques that achieve noninvasive molecular diagnosis of brain tumors. Our group is addressing this unmet need by introducing and developing the focused ultrasound (FUS)-enabled blood-based liquid biopsy technique, which we call sonobiopsy. NIH/NIBIB support (R01EB030102, funding period 8/1/2020–4/30/2024) allowed us to demonstrate the effectiveness and safety of sonobiopsy in mouse and pig models of glioblastoma, the most common primary brain tumor in adults. We also developed a clinical sonobiopsy device by integrating a single-element FUS transducer with a neuronavigation system. These breakthroughs led to our first-in-human clinical study, which demonstrated the initial feasibility and safety of sonobiopsy in patients with glioblastoma. In much the same way that MRI transformed the diagnostic capabilities of neurologic disease by providing anatomic and functional information, sonobiopsy has the potential to provide equally important and complementary molecular information about the brain that is not currently available. Sonobiopsy will be a platform technology that can be applied to the diagnosis and monitoring of various neurological diseases. The objective of this renewal application is to develop and validate a next-generation FUS device called sonocap, which will radically advance the clinical translation of sonobiopsy and enable its broad adoption. The sonocap device is patient-friendly, easily manufactured, accurate in tumor targeting, and safe. We will achieve this objective through two specific aims: Aim 1 will design and construct the wearable sonocap, and Aim 2 will validate the performance and safety of the sonocap in non-human primates (NHPs). The proposed sonocap is significant because it is a breakthrough FUS device that enhances our technical capability in interfacing with the brain using ultrasound, addresses a critical barrier to advancing the clinical translation of sonobiopsy, and improves the clinical practice in the diagnosis and monitoring of brain diseases through sonobiopsy. Our multidisciplinary team has expertise in ultrasound engineering, wearable device design, NHP research, and neurosurgery, and will advance sonocap through the development phase and into future clinical trials. This study has three main innovations: (1) sonobiopsy is a groundbreaking approach for interrogating the brain; (2) the wearable sonocap significantly departs from the status quo in the design of clinical FUS devices; (3) the proposed approach for developing the sonocap combines human head phantom testing and NHP validation. The project outcomes are expected to significantly impact the medical ultrasound field by driving the development of wearable FUS devices, collecting essential large animal data required for clinical translation, and ultimately achieving personalized patient care through noninvasive molecular diagnosis of brain tumors.

NIH Research Projects · FY 2026 · 2024-06

Project Summary/Abstract: Axonal degeneration is an early and likely initiating event in many of the most prevalent neurodegenerative diseases. DLK is a major neuronal stress kinase that we identified as the first gene required for pathological axon degeneration. Recently we defined the mechanism: DLK promotes the turnover of the axon maintenance factors NMNAT2 and stathmin2 (STMN2). DLK is activated in animal models of both Alzheimer's Disease and ALS and there are strong data that DLK is also activated in patients with these degenerative disorders. While other labs focus on DLK pro-apoptotic signaling, we demonstrated an independent function for DLK in stimulating SARM1-dependent axon loss. In these studies, we identified STMN2 as an axonal maintenance factor—loss of STMN2 promotes axon degeneration and increased levels of STMN2 inhibits axon degeneration. Recently, two prominent papers identified STMN2 as the major transcript misspliced and downregulated by TDP-43 dysfunction in human iPSCs as well as from spinal cords of ALS patients. TDP-43 dysregulation is an important cause of both frontotemporal dementia and ALS and has recently been implicated in more common dementias. Indeed, STMN2 is one of the most downregulated transcripts in neurons from Alzheimer's patients. The identification of STMN2 as a major target of both TDP-43 dysregulation and DLK activation, two mechanisms implicated in both dementias and ALS, identifies downregulation of STMN2 as a candidate mechanism promoting axon loss in these neurodegenerative diseases. These findings motivate our efforts to define the function of STMN2 for axonal maintenance. While it is known that STMN2 is a stathmin family member that regulates microtubule dynamics, it is not known how STMN2 promotes axon maintenance or the in vivo function of STMN2 for axon survival in health, injury, and disease. Here we will test the hypothesis that STMN2 promotes the activity of the axon maintenance factor NMNAT2 to inhibit the activity of SARM1, the executioner of pathological axon degeneration, and in vivo helps regulate the choice between axon survival and self-destruction in injury and disease. If successful, these studies will define the mechanism by which STMN2 promotes axonal survival and identify STMN2 as a therapeutic target in neurodegenerative disease.

NIH Research Projects · FY 2026 · 2024-06

PROJECT SUMMARY/ABSTRACT Fatty acid β-oxidation (FAO) is a central metabolic pathway of great physiological relevance to human health and disease. FAO provides fuel for energy production, generates building blocks for the biosynthesis of many cellular molecules, detoxifies damaging lipids, and produces key signaling molecules. Disruption of this pathway contributes to fatty liver disease, hypoketotic hypoglycemia, obesity, insulin resistance, type 2 diabetes, and chronic kidney disease. Importantly, many fatty acids that ultimately are catabolized by FAO are not compatible with FAO until they have first been modified by other enzymes. These lipids include the 4-hydroxy fatty acids (4- HAs)—fatty acids that possess an oxidized carbon adjacent to the third, or β, position. Recent analyses empowered by advances in mass spectrometry have revealed a wide range of 4-HAs and 4-HA precursors in human plasma, which can originate from dietary intake, lipid peroxidation, and certain drugs of abuse. Prior analyses in liver revealed that 4-HAs are indeed processed by FAO once they are converted to compatible substrates. However, the enzymes responsible for this conversion are unknown, and the physiological relevance of these lipids is largely unexplored. Recently, we identified two atypical, highly uncharacterized FAO-related enzymes, acyl-CoA dehydrogenase 10 and 11 (ACAD10 and ACAD11), that appear capable of processing 4- HAs into FAO-friendly substrates via a novel mechanism. The long-term goals of this proposal are to define the roles of ACAD10 and ACAD11 in the catabolism of 4-HAs and to establish the metabolic ramifications that result from the disruption of these enzymes. We first aim to establish the specific biochemical reactions catalyzed by ACAD10 and ACAD11 in vitro through enzymology and structural biology approaches. We hypothesize that these ACADs employ their unique kinase domain, which is not found in other ACADs, to phosphorylate the 4- hydroxy position as part of their enzymatic mechanism. We then aim to establish the cellular locations and activities of each enzyme via microscopy and metabolite tracing studies. We hypothesize that ACAD10 is a mitochondrial enzyme responsible for the conversion of shorter-chain 4-HAs and that ACAD11 is a peroxisomal enzyme that converts longer-chain 4-HAs. Last, we aim to reveal the physiological ramifications of ACAD10 and ACAD11 disruptions in worms, mice, and humans. Notably, ACAD10 and ACAD11 have putative links to T2DM/insulin resistance, kidney disease, and coronary artery disease via GWAS studies, including in the Akimel O’odham (Pima Indian) tribe, and mouse models have linked ACAD10 and ACAD11 collectively to insulin resistance, weight gain, ectopic lipid deposition, and rhabdomyolysis; however, none of these have been studied directly. We hypothesize that the loss of these enzymes in mice, or the expression of specific ACAD10 variants in Pima Indians, will result in the accumulation of plasma 4-HAs concomitant with the development of metabolic complications. Overall, this work aims to establish a fundamental new piece to our understanding of cellular FAO metabolism and to forge new links between a large, unexplored class of fatty acids and mammalian physiology.

NIH Research Projects · FY 2025 · 2024-06

PROJECT SUMMARY Existing delivery systems for CRISPR-Cas gene editing machinery have very little capacity for making multigenic insertions. As such, the scope of existing gene therapies has been limited primarily to monogenic diseases. Yet multigenic insertions could facilitate treatment of a much broader range of conditions. To address this challenge, I am developing a chimeric AdAAV gene editing delivery system by physically linking multiple adeno-associated viruses (AAVs) to the surface of adenovirus (Ad). I am using the SpyTag-SpyCatcher technology for site-specific covalent conjugation. In my design, the Ad will encode the Cas9 protein and the gRNAs while the AAVs will provide a single-stranded DNA template. To enable multigenic genetic insertions, I will conjugate a mixture of AAVs (with distinct genetic cargos) onto the Ads. Each AAV cargo sequence will undergo programmable insertion into a unique genomic target site via the Cas9 and gRNAs encoded in the Ad. I anticipate that the strong and targetable transduction of the Ad will efficiently drive the uptake of AdAAV particles into cells. I also anticipate that greater in vivo gene editing efficiencies may occur using this design because of the known benefits of single- stranded DNA templates for homology-directed repair (HDR). My fully novel AdAAV vector will act as an efficacious and versatile platform for multigenic gene therapy. AdAAVs will be particularly suited for treating aging and aging-related conditions, especially Alzheimer’s disease.

NIH Research Projects · FY 2025 · 2024-06

PROJECT SUMMARY/ABSTRACT Lumbar spine surgery for degenerative disease is one of the most common and most expensive surgeries performed in the United States. However, there is substantial variation in lumbar surgery rates, approaches, and patient-reported outcomes at the surgeon, hospital, and regional levels. One major cause of these inconsistencies is the lack of evidence-based tools to both predict outcome and personalize treatment recommendations. For example, while there has been growing recognition that pain symptoms reflect a complex interplay of biochemical, psychological, and social factors that influence surgical outcomes, these factors are not typically incorporated into surgical treatment plans. One important factor preventing the expansion of evidence-based treatments, particularly related to behavioral and cognitive interventions, is a lack of precision tools to measure dynamic symptom profiles for pain and related psychosocial comorbidities. In particular, traditional patient assessments use cross-sectional (i.e., one-time) questionnaires that are subject to recall bias and fail to capture longitudinal symptom dynamics. Mobile health (mHealth) technology has enabled a fundamentally new approach to collect intensive longitudinal patient-reported and biometric data to support individualized decision-making. In particular, ecological momentary assessment (EMA) is an emerging tool that leverages brief mobile surveys to obtain momentary, longitudinal assessments of core disease constructs. Complementing EMA, mobile fitness trackers, such as Fitbit, passively collect biometric data (e.g., activity, heart-rate, and sleep) that reflect the physiologic manifestations of lumbar spine-related disability and impaired psychosocial health. These innovative tools may provide a newfound ability to capture important biopsychosocial features that impact surgical outcome. Recognizing these evidence gaps and the value of emerging mHealth technology, this study’s overall objective is to establish the utility of using real-time patient-reported and biometric data to prognosticate and stratify lumbar spine patients, and to establish the value proposition for implementing these methods. This objective will be accomplished through the following Aims. In Aim 1 I will investigate the ability of mHealth assessments to identify novel disease features with prognostic importance for degenerative lumbar spine surgery patients. In Aim 2, I will use real-time mHealth assessments to identify novel phenotypes of lumbar disease. In Aim 3, I will define the value proposition and implementation context for using mHealth assessments to address functional and psychosocial influences on lumbar surgery outcomes. These research activities will be combined with a rigorous mentored training program incorporating chronic pain research, machine learning, behavioral intervention development, and implementation science. At the completion of this award, I will be prepared to submit an R01 application integrating data analytics, implementation science, and the biopsychosocial model of pain to test interventions to improve lumbar spine surgery outcomes.

NIH Research Projects · FY 2026 · 2024-05

ABSTRACT Clostridioides difficile (Cd) infection (CDI) is a leading cause of healthcare-associated infections in the U.S., affecting ~500,000 people and costing ~$5.4B annually. Despite substantial advances in investigating Cd pathogenesis and transmission over the past 20 years, CDI remains a significant public health burden. Conventional antimicrobial therapy often fails to clear Cd, can inflict extensive collateral damage to the commensal gut microbiome (GM), and frequently leads to recurrent CDI, for which treatment options are limited. A major impediment in mitigating Cd-associated disease is current unclear understanding of the biological basis for the broad spectrum of disease severity, from asymptomatic Cd colonization to recurrent fatal CDI. In this context, elucidating mechanisms of Cd colonization and disease necessitates explicit consideration of confounding GM interactions that increase a patient's CDI risk. Accordingly, we propose a quantitative investigation of pathogen-GM-host dynamics of clinically prominent Cd lineages and their contribution to CDI, leveraging a unique cohort of >30,000 Cd-associated patient stools with accompanying well-curated clinical metadata. We hypothesize that high-resolution genomic analyses of Cd strains and pathogen-GM interactions will enable sensitive identification of novel genomic elements in virulent lineages associated with variable CDI outcomes. The rationale for this proposal stems from i) the recognition that most Cd research relies on studying hypervirulent, previously epidemic lineages that are no longer prevalent in the clinic, ii) the need for stable, reproducible GM-humanized animal models of CDI to investigate pathogen and human GM variables, and iii) the observation that taxonomic and functional features of the GM, including bacteriophages, drive Cd's adaptation as a pathobiont. Our central motivation is to expand the representation of prominent Cd lineages and leverage our improved understanding of pathogen-GM dynamics, and our innovative technologies, to enable the design of novel diagnostic, therapeutic, and management avenues to improve patient outcomes. This will be achieved via three aims: 1) investigate pangenome determinants of CDI risk in clinically representative Cd strains, 2) model and predict how different patient GM taxonomic and functional architectures, integrated with clinical metadata, relate to CDI outcomes, and 3) characterize and predict Cd strain-specific dynamics by employing innovative, defined GM-humanized gnotobiotic mouse models of Cd colonization and CDI. Our analyses are significant as they pursue new avenues of precisely investigating pathobiont-commensal variables that influence CDI, a model bacterial-microbiome infectious disease with extensive morbidity and mortality. This proposal is innovative in improving understanding and prediction of Cd disease spectrum via novel complementary technologies including GM-humanized gnotobiotic mouse models to identify pathogen and GM drivers of clinical outcomes. The proposed work is impactful in its goal to address basic, translational, and clinically relevant questions in GM-mediated infectious diseases, to enable a new era of precision therapies.

NIH Research Projects · FY 2025 · 2024-05

Malaria remains a major global infectious disease that infects hundreds of millions of people and is responsible for hundreds of thousands of deaths annually. Two major species of parasite, Plasmodium falciparum and P. vivax are responsible for the majority of clinical cases. During the course of a malaria infection, parasites undergo repeated cycles of invasion and replication in red blood cells (RBC) which leads to all symptom of the disease. An essential step in this process is parasite invasion: a mature schizont will burst releasing 16 – 20 merozoites which will bind to and invade a new host RBC in a process that takes less than a minute. During this time, the merozoite deploys an array of invasion ligand proteins that facilitate the different steps of the invasion process. Over 70 candidate P. falciparum invasion ligand proteins have been identified and are thought to bind to cognate host receptor proteins on the surface of the RBC. However, to date less than a third of the candidate invasion ligand proteins have been demonstrated to bind to RBCs and even fewer host receptors have been identified. Comprehensive identification of a global P. falciparum invasion ligand bindome is a major gap in the field and would greatly facilitate prioritizing targets for a blood stage malaria vaccine. Here we propose a new approach: biotinylated supernatant erythrocyte binding assay proteomics (BSEP) in order to globally identify the P. falciparum bindome. In BSEP, purified schizont stage parasites are allowed to egress in protein free media to generate a supernatant enriched in invasion ligand proteins. These invasion ligand proteins are biotinylated, incubated with RBCs which are then spun through mineral oil. Bound proteins are eluted via high salt treatment, purified via streptavidin beads and subjected to quantitative tandem mass-tag based proteomics. In this proposal we aim to systematically develop the BSEP protocol by testing for the optimal way to generate and biotinylate parasite supernatants, and to determine binding specificity by using RBC binding saturation and supernatant depletion assays (with antibodies against known invasion ligands as controls). We will also use BSEP to identify invasion ligands binding to candidate host receptors by comparing the bindomes between WT and candidate host receptor knockouts generated in an immortalized erythroid cell line and already available in the lab. We will validate this approach using the well-known interaction between PfEBA175 invasion ligand and host glycophorin A (GypA) using a ∆GypA knockout. We will use a similar approach with our candidate host receptor glycophorin B (GypB). GypB is thought to interact with PfEBL-1 invasion ligand; however, GypB knockdown RBCs show a strong invasion defect with parasite strains that have either wild type or EBL-1 deletions, thus suggesting the presence of an alternative invasion. We will validate candidate invasion ligands by recombinant expression followed by flow cytometry binding assays with WT or ∆GypB cells. Once established, we believe that the BSEP approach will rapidly enhance our understanding of invasion ligand/host receptor interactions and would be highly generalizable to other malaria parasite species, including P. vivax, about which even less is known.

NIH Research Projects · FY 2026 · 2024-05

The “amyloid hypothesis” posits that Amyloid β (Aβ) misfolds, oligomerizes to soluble species, and thereby contributes to neurodegenerative Alzheimer’s disease (AD). Recent support for the hypothesis comes from a new antibody, Lecanemab, that targets Aβ and its aggregation and significantly reduces clinical decline. Still, the aggregation is not understood, leaving a weak foundation for building new therapeutics. In the transition from monomer to fibril, Aβ exists in soluble, putatively neurotoxic species in mixtures of varied sizes and morphologies. Complicating matters further is the unknown molecular role of apolipoprotein E (ApoE), a family of partially disordered proteins including an isoform, ApoE4, that is one of few biomarkers for AD. The aggregate mixture and the interactions of Aβ with apoE4 and with lipids constituting cell membranes are too complex for many structural characterization techniques. Protein footprinting coupled with mass spectrometry, however, can provide a measure of solvent accessibility, local dynamics and structure, hydrogen bonding, sites of ligand- binding, and oligomerization, with spatial resolution at the peptide and amino-acid residue levels. Considerable preliminary data establish that this technique can provide structural details not obtainable by other methods. This footprinting technique has not yet been systematically or rigorously applied to characterize the soluble intermediates formed in amyloidogenic protein aggregation, and this is our goal. We will chemically footprint Aβ and ApoE alone or together by using both pulsed hydrogen/deuterium exchange (HDX) and irreversible modification by highly reactive species (free radicals, carbenes, carbocations). For the latter, we will utilize the “Fast Photochemical Oxidation of Proteins” or “FPOP” platform, which we invented. Both HDX and FPOP are used in a differential mode where changes in structure are determined as a function of aggregation time or increasing media complexity. Utilization of optimized reagents will be coupled with separation and kinetic modeling to reduce heterogeneity and analyze the aggregation pathways and the varied structures or morphologies of the oligomers. We will then apply these optimized footprinting approaches in complex media containing lipids, small molecules, and other proteins including antibodies to reveal how aggregation changes upon perturbation with these substances. We will be guided by powerful Density Functional Theory that can probe small molecule/Aβ interactions Once the footprinting and data processing pass rigorous validation, we will apply them to Aβ and ApoE in a system of reprogrammed neurons that recapitulate the pathophysiology of AD patients. This system succeeds where pluripotent stem cells and mouse models do not because they don’t carry age information, and age correlates strongly with AD. The proposed research will bring new understanding of Aβ aggregation and of ApoE interactions with regional and amino acid resolution in relevant media. It will also define and characterize a novel and potentially informative approach for gaining insight on neurodegenerative diseases.

NIH Research Projects · FY 2026 · 2024-05

SUMMARY Alzheimer's disease (AD) was once considered a monolithic disorder of canonical symptoms and definitive pathology, but recent work has identified considerably heterogeneity in onset, progression, and histologic features. Despite this important shift in clinical perspective, we don't yet know if or how the range of brain pathologies explains the diversity in clinical trajectories. We also can't explain how neuropathological diversity arises in the first place, even for the canonical traits of amyloid, tangles, and neurodegeneration. It would be instructive to understand how such diverse structures arise and what if any role each plays in cognitive decline, but this insight has been stymied by human pathological heterogeneity combined with a paucity of appropriate animal models to replicate these extremes. We unexpectedly discovered a possible cellular explanation for the emergence of neuritic vs diffuse amyloid structures while characterizing two new transgenic lines that expressed the same APP construct selectively in glutamatergic or GABAergic neurons. Both models developed pronounced Ab deposits, but showed divergent patterns of pathology and Ab profiles. Glutamatergic APP mice formed cored, thioflavin-positive neuritic plaques composed of both Ab40 and 42, while GABAergic APP mice formed diffuse, thioflavin-negative plaques composed primarily of Ab42. These complementary APP transgenic models, with their respective diffuse and neuritic plaque pathology, now allow us to directly test the role of neuronal subtypes in plaque heterogeneity, identify the mechanistic basis for these differences in APP processing, and determine how distinct plaque structures may be pathogenic or protective through the divergent response of neighboring cells. Aim 1 will characterize the physical differences that distinguish plaques generated by GABAergic vs glutamatergic neurons using electron microscopy, in vivo seeding assays, and mass spectrometry. Aim 2 will determine how neuron-specific differences in APP processing arise using snRNAseq transcriptomic analysis of each neuronal subtype. This aim will also ascertain whether APP processing differences extend from overexpressed pathogenic APP to wild-type protein expressed at endogenous levels. Finally, Aim 3 will dissect the downstream consequences of each aggregate structure at the cell/molecular level using snRNAseq data and by testing cognitive function. Collectively we expect these studies may cast a new light on excitatory:inhibitory balance in AD and advance a new appreciation of neuronal heterogeneity as an important facet of disease pathogenesis.

NIH Research Projects · FY 2026 · 2024-05

The Midwest D-CFAR seeks to advance impactful HIV research and thereby catalyze greater effectiveness in the HIV response in our region and beyond. More than ever, the scientific progress required to make progress against the HIV pandemic requires a cross-disciplinary scientific workforce, novel trans-disciplinary insights and deep engagement with communities and stakeholders. The epidemic in the St. Louis and Missouri Region is in urgent need of such progress: the total annual number of new cases (approximately 500), deaths (approximately 200) and hospitalizations has remained steady over the last five years, even as these numbers have fallen in some areas in the US. At this moment, however, the region has unique opportunities: the End the HIV Epidemic Initiative has energized the response and mobilized resources. At the same time the NIH HIV funded research base at WU and SLU has grown from $8.2 to $14.8 million yearly, generating renewed scientific insights into cure, treatment and care delivery. In this environment, an investment from the NIH in a Developmental Center for AIDS Research (D-CFAR) can accelerate existing scientific investments and align scientific directions with the broader HIV response. After a formative process to identify strengths and gaps in our environment, we have assembled a stakeholder-engaged and data-driven proposal. We propose the D-CFAR to be jointly hosted by Washington University in St. Louis (WashU) and Saint Louis University (SLU) to synergize complementary strengths. We include an optional core in Dissemination and Implementation Science, which reflect our institutional strengths and regional needs. We are led by a distinguished multidisciplinary MPI team, Drs. Geng (WashU) and Iwelunmor (SLU), to draw a wide range of scientific expertise and institutional units together. Our specific aims are: Aim 1. Attract, advance, and retain HIV investigators to accelerate the scientific response to HIV regionally and globally. Aim 2. Develop and deploy institutional resources to promote multi-disciplinary, innovative research for an effective HIV response. Aim 3. Foster engagement with communities and regional public health authorities to ensure stakeholder-responsive research and rapid utilization of findings into practice. Aim 4. Undertake iterative evaluation of D-CFAR based on standard process evaluation as well as the Translational Sciences Benefits Model to prepare for a full CFAR application long-term impact. Our vision is to meaningfully improve the lives of people affected by the HIV epidemic. Our mission is to augment the scope, quality, and impact of people-centered science addressing HIV. Our goal is to transform our institutions to lead science aligned with the OAR priorities and contribute to turning the tide on this epidemic. In summary, the Midwest D-CFAR will provide scientific leadership, build infrastructure dedicated to HIV research to enable collaboration, and establish a rigorous framework to develop the next generation of scientific leaders in HIV research, and thereby contribute to turning the tide on the HIV epidemic in our region and beyond.

NIH Research Projects · FY 2025 · 2024-05

Abstract The spondyloarthropathies (SpAs) are human diseases, characterized by sacroiliitis and overlapping clinical features, of unknown pathogenesis. The prototypical SpA, ankylosing spondylitis (AS), is one of the best known and strongest examples of a disease associated with an HLA allele, specifically HLA-B27, an association known for 50 years. Since HLA-B27 is a major histocompatibility complex class I (MHC-I) molecule, it seems likely that it is involved in disease pathogenesis by presenting peptide antigens on antigen-presenting cells (APCs) to CD8+ T cells, the canonical function of MHC-I molecules. However, due to lack of direct support of this hypothesis, and conflicting data from previous HLA-B27 rodent models, other hypotheses became more favored, even though genome-wide association studies and lack of association of AS with certain HLA-B27 alleles that affect peptide display support a role for HLA-B27 in antigen presentation in disease. Recent studies from the applicant’s laboratory in collaboration with an international group showed clonal expansion of certain T cell receptors (TCRs) on CD8+ T cells in the joints of patients with AS and eye of patients with anterior uveitis, a frequent complication of the SpAs, that can occur in isolation and is also associated with HLA-B27. These TCRs were used to identify peptides from bacterial and human proteins that are presented by HLA-B27 to trigger TCR reporter cells. Importantly, several of these TCRs recognized both bacterial and self-peptides presented by HLA-B27. Crystallographic studies showed the basis for this TCR cross-reactivity. These studies strongly suggest the overall hypothesis to explain the pathogenesis of AS, the basis for this proposal: Antigen- specific CD8+ T cells in HLA-B27+ individuals clonally expand in response to bacterial challenge then attack normal tissues expressing the cross-reactive self-protein(s). To test this model, the applicant proposes here to create a mouse expressing a relevant TCR and HLA-B27 for such mechanistic immune response studies that are otherwise impossible to pursue in humans. To avoid other technical and knowledge limitations of prior HLA-B27 rodent models, they will produce other modifications. Therefore, the Specific Aims of this proposal are to: 1) Generate HLA-B27 mouse models; and 2) Perform functional analyses of T cells and APCs in HLA- B27 mice. Thus, this proposal will lead to the development of a mouse model for detailed mechanistic studies to further explore the pathogenesis of HLA-B27 in AS and related diseases, and indeed lay the foundation for mechanistic studies of other HLA-associated autoimmune disorders.

NIH Research Projects · FY 2025 · 2024-05

Project Summary/Abstract Nearly all US adolescents use social media (SM), and nearly half report almost constant use. Adolescence is marked by emotional intensity, difficulties regulating emotions, and increased risk for psychopathologies like depression. Despite this, due to mixed findings in the literature, little is known about the effects of SM use (SMU) on healthy adolescent emotional development. One reason for inconclusive results could be failure to account for different ways adolescents engage in SMU, which could lead to varied outcomes. Additionally, no studies have examined SMU effects on emotion in both controlled lab settings and real-life contexts, nor have they considered objective indicators like physiological reactivity. This lack of nuance hinders understanding of how specific SMU activities impact adolescent emotions generally (when effects are experimentally manipulated) and during daily life. The proposed investigation aims to untangle the positive and negative emotional effects of SMU in adolescents using a multimethod approach. Specifically, it seeks to examine how distinct SMU types identified in my prior research affect adolescents in the moment, how they are used for emotion regulation, and their association with depressive symptoms. With funds from a parent grant, participants aged 13-17 (N=145) will be recruited from the community. Participants will first undergo a lab-based SMU experiment, engaging in one of four SMU types at a time: Belief- (e.g., sharing negative opinions), comparison- (e.g., body comparison), image (e.g., monitoring "likes"), and consumption-based (e.g., watching videos) SMU. Physiological indicators of emotion will be recorded alongside self-report measures of emotion. These findings will provide insight into how each SMU type makes adolescents feel, both objectively (physiological indicators of stress or regulation) and subjectively. Ecological momentary assessment (EMA) will be used in the week following the experiment, where participants will report their emotions, extent of engagement in each SMU type, and to what extent they engaged in each type to feel better or worse. This approach will elucidate how SMU types predict adolescent emotions and are used to influence their emotions in daily life. Furthermore, self-report measures will be employed to examine the association between weekly engagement in each SMU type and depressive symptoms, revealing how habitual SMU affects adolescent emotional wellbeing. By taking a nuanced approach to measuring the emotional effects of specific SMU types, this study holds promise to inform novel interventions that encourage healthy adolescent SMU habits. The research aims to achieve several training goals, including expertise in physiological assessment, honing EMA methodological and analytic skills, applying digital emotion regulation theory in adolescents, and advancing knowledge in adolescent mental health. The training team assembled to assist in achieving these goals has substantial expertise in emotion and mental health, EMA assessment, adolescent emotional development, and physiological assessment. With their support, I will develop the skills needed to foster my research program and career ambitions to become a NIH-funded academic researcher.

NIH Research Projects · FY 2025 · 2024-05

PROJECT SUMMARY/ABSTRACT Muscle diseases caused by mutations in genes that encode for (co-)chaperones, called chaperonopathies, are characterized by muscle weakness, degeneration, and the accumulation of protein aggregates. These diseases currently lack a cure, highlighting an urgent need for a better understanding of the underlying mechanisms. Protein chaperones are needed to recognize misfolded proteins and facilitate their proper folding. The recognized misfolded proteins are termed “clients.” Classically, DNAJ proteins are thought to diversify the function of Hsp70 by determining client specificity, but little is known about how DNAJ proteins bind and select clients for Hsp70 and how dysfunction of DNJ proteins causes chaperonopathies in muscle. Recently, mutations in DNAJB4 were reported to cause a chaperonopathy with similar myopathology to the known chaperonopathy caused by DNAJB6 mutations although their phenotypes are distinctive. Our central hypothesis is that distinct client proteins of DNAJ proteins and chaperone-chaperone availability contribute to the phenotypic differences and selective muscle vulnerability in chaperonopathies. This study will experimentally evaluate client proteins of DNAJ proteins in muscle, the combinatory effect of DNAJ proteins, and skeletal muscle-specific tissue vulnerability in chaperonopathies, using recently developed innovations (e.g., proximity labeling approach, proteomics, RNAi, RNA-seq, and small animal imaging). In Aim 1, I will Aim 1 test the hypothesis that different DNAJ proteins have common and distinct client proteins in skeletal muscle. To identify client proteins, I will apply proteomics in TurboID proximity labeling method in cultured cells and model mice, DNAJ knockout myotubes, and laser microdissections of inclusions from patient muscle with DNAJB4, DNAJB5, and DNAJB6 mutations. In Aim 2, I will test the hypothesis that DNAJ-DNAJ interactions play a synergetic role in muscle maintenance. I will employ a knockdown approach to investigate how this network dysfunction impacts protein homeostasis, using myoblasts and proteostatic stress (e.g., drug, heat shock). Finally, in Aim 3, I will test the hypothesis that the skeletal muscle-specific tissue vulnerability is caused by a disruption of proteostasis, where key molecules are compromised. Using chaperonopathy mouse models, I will thoroughly analyze the myopathology and proteostatic capacity of different skeletal muscle groups, and perform single nucleus RNA sequencing on the most vulnerable muscle groups in model mice to identify define vulnerable myofiber subpopulations in chaperonopathies. The proposed study will address an unmet medical need, providing insight into disease mechanisms and thereby potentially may serve as a basis for the development of novel treatment for chaperonopathies and a broad range of diseases related to protein misfolding.

NIH Research Projects · FY 2026 · 2024-05

ABSTRACT Alzheimer disease (AD) is a neurodegenerative condition that causes progressive cognitive decline and death. The AD pathological hallmarks are extracellular amyloid β (Aβ) plaques and neurofibrillary tangles containing aggregated Tau protein. The US FDA recently approved aducanumab to promote Aβ plaque clearance, and antibody-mediated removal of Tau aggregates has also moved to phase II clinical trials. Despite the potential therapeutic impact of antibodies in AD, our understanding of natural B cell responses in AD remains limited. Our preliminary data demonstrate that meningeal B cells of young adult mice are not blood-borne as in aging mice, but rather derive from B cell progenitors that migrate from the skull bone marrow into the meninges and are selected by CNS antigens. Based on this premise, Aim1 will test the hypothesis that the BCR repertoire of ABand Tau-specific B cells, the affinity of the antibodies produced, and the T cell help in mouse models of Aβ plaques and tauopathy depend on the lymphopoietic niche in which they develop. Meningeal B cells derived from skull bone marrow and selected by Aβ and Tau should generate low affinity antibodies. Conversely, B cells that develop in the periphery, not selected in the CNS, should be strongly activated by Aβ and Tau, receive T cell help, and generate high affinity antibodies. Antibodies of different origins will be further tested for the ability to clear Aβ and Tau from the brain. Active Aβ immunization to induce antibody-mediated clearance of Aβ plaques has been tested in a human trial of active immunotherapy, but was discontinued because of aseptic meningoencephalitis in some cases. In this trial, Aβ peptide was injected subcutaneously together with the QS- 21 adjuvant that strongly augments T cell responses to vaccine antigens. Based on our preliminary data, Aim 2 will test the hypothesis that delivery of Aβ and Tau directly to the meninges and/or with the mild B cell selective adjuvant CpG-B will elicit anti-Aβ and Tau antibodies that clear plaques without inducing strong CNS autoimmunity. Several studies have reported that AD associates with changes in function and cytokine secretion of T cells. Our preliminary data delineates an antigen-experienced population of CD8 T cells with potent effector functions and the ability to secrete proinflammatory cytokines in the blood of AD patients, as well as clonal expansion in the CSF. Aim 3 will test the hypotheses that clonally expanding T cells are Aβ and/or Tau specific and have distinct transcriptional and phenotypic programs in AD patients versus controls; furthermore, these T cells correlate with clinical parameters. We will determine whether the BCR repertoires of Aβ- and Tau-specific B clonal expansions found in blood and CSF of AD patients and healthy controls are different and correlate with AD pathology and cognition defects. We will use innovative immunophenotyping combined with single cell TCR, BCR-, and RNA-seq methods. Results will lay the groundwork to understand anti-Aβ and anti-Tau T and B cell responses in mouse models and AD patients, which will help stratify patients before offering Aβ/Tau-based immunotherapy.

NIH Research Projects · FY 2026 · 2024-05

PROJECT SUMMARY Antimicrobial resistant organisms (ARO) have long been a concern in the acute healthcare setting, but community rates have increased exponentially over the past decade, with the Centers for Disease Control and Prevention (CDC) estimating that 47% of all extended-spectrum beta-lactamase producing Enterobacterales (ESBL-E) infections are now community-associated (CA). Despite this, very little is known about community- associated AROS (CA-ARO). The goals of this proposal are to characterize AROs in the community setting and to identify risk factors for CA-ARO infection and colonization. Our central hypotheses are that there are identifiable risk factors associated with CA-ARO infection and colonization, and that these data will lay the groundwork for interventions to reduce the transmission of AROs and to ultimately prevent ARO infections. In Aim 1, we will characterize clinical risk factors associated with CA-ARO infections utilizing a database of patients with urinary tract infections and bloodstream infections associated with both community and healthcare settings. In Aim 2 we will determine the prevalence and clinical risk factors associated with CA-ARO colonization in patients admitted to the acute care hospital setting. This will be achieved through recruiting patients admitted to the hospital from the community setting and interrogating clinical specimens with selective microbiologic culture to determine the prevalence of ARO colonization and identify risk factors for CA-ARO colonization in this population. In Aim 3 we will determine the prevalence and characteristics associated with ARO colonization in asymptomatic community volunteers and their home environment to identify risk factors and reservoirs of CA-ARO colonization. We will achieve this through a prospective cohort study of community volunteers and interrogation of human and environmental samples paired with detailed clinical data to document reservoirs of CA-ARO and potential risk factors for CA-ARO colonization. Together, this data will provide key insight to understand CA-AROs, a necessary first step towards creating interventions to combat AROs in community settings. Results of this study will directly impact clinical practice by providing key data to guide the use of infection prevention interventions for people at-risk for CA-ARO colonization or infection, environmental hygiene recommendations, and empiric antimicrobial use in the community setting. This study is directly responsive to the AHRQ FOA focused on the prevention of HAIs as it is aimed at reducing the transmission of AROs and preventing HAIs. This project represents a necessary and novel approach to targeting AROs within the community setting, and will lead to practice-changing paradigms to combatting AROs.

NIH Research Projects · FY 2026 · 2024-05

ABSTRACT Our vision is to unravel and ultimately reverse the intricate network of causal factors throughout the life course that disrupts biological homeostasis to promote colorectal cancer (CRC) among individuals younger than age 50 years. Uniting leading scientific minds in early-onset colorectal cancer (EOCRC) research and complementary fields, we have embraced disruptive, transdisciplinary approaches spanning cells to individuals to populations to address the core Grand Challenge to “Determine why the incidence of early-onset cancers is rising globally”. We will address specific questions of “the mechanisms linking lifetime exposures with cancer initiation and promotion” by focusing on EOCRC as an ideal model for early-onset cancer due to the availability of well- characterized animal models and a well-established and prevalent precursor lesion, the adenomatous polyp (adenoma), offering a unique opportunity for interception and prevention. Our work will transform the field by directly addressing our overarching goal to “identify and understand the processes through which different biological and environmental factors cause early-onset cancers”, and reverse the burden in a timely, effective, and feasible fashion. Our team, both working independently and in collaboration, has uncovered several risk factors that are likely to be drivers for the rising incidence for EOCRC. We are now uniquely positioned to translate etiologic understanding to actionable prevention by identifying novel factors, including environmental determinants, and deepening our understanding into overlooked dimensions of exposure throughout the life course. The unprecedented scope and scale of our proposal can only be supported through Cancer Grand Challenges since our “high-risk” disruptive approach requires deep interactions between work packages (WP)s led by leaders in distinct disciplines. This will enable incorporation of fresh perspectives to move beyond traditional risk-factor epidemiology toward an integrated, mechanistically-informed model with population scale and cellular resolution of the multiple and cumulative “hits” that promote EOCRC to inform the development of actionable prevention. Our innovations intersect epidemiology, small molecule discovery, genomics, stem cell biology, immunology, and computational biology with these key features: 1) harmonization of cohorts with data and biospecimens collected across the lifecourse; 2) innovative and reliable analysis of small molecules to detect novel exposures; 3) high-resolution technologies for analysis of target tissues; 4) model systems capable of interrogating accumulating exposures across the lifespan and their impact on the cellular ecosystem; 5) prevention through risk assessment and pharmacologic/lifestyle interventions. Collectively, our work will serve as an exemplar for transforming research into other early-onset cancers.

NIH Research Projects · FY 2025 · 2024-05

ABSTRACT Prescription opioids, such as oxycodone, remain a common treatment for pain disorders. Although effective in the short-term, long-term use can result in tolerance, dependence and fatal overdose. While oxycodone is not the primary driver of fatal overdoses from opioids, the exposure to and subsequent withdrawal from oxycodone can lead susceptible individuals to relapse, potentially substituting intake fentanyl or other more potent synthetic opioids. Thus, understanding the mechanisms of oxycodone-induced withdrawal will provide a better foundation for the treatment of opioid use disorder. The central nucleus of the amygdala (CeA) is an integrative brain region that contributes to the generation of negative affective states. Prior work has demonstrated increased neuronal excitability within the CeA in rodent models of morphine withdrawal, and inhibition or lesion of the CeA resulted in a reduction of the aversive, and, to a lesser extent, somatic withdrawal behaviors from morphine. Most studies investigating the CeA’s role in opioid withdrawal have done so in a global sense, ignoring potential contributions of specific cell types to the manifestation of opioid withdrawal behaviors. The central hypothesis in the present application is that activation of molecularly distinct neuronal subpopulations within the CeA are necessary and sufficient for the expression of somatic opioid withdrawal and contribute to future drug seeking behaviors. Uncovering the specific subtypes responsible for the expression of opioid withdrawal has the potential to lead to a more effective and specialized approach for the development of treatments of opioid use disorder. To this end, I will employ the use of targeted recombination in active populations mice, which express the tamoxifen-dependent CreERT2 recombinase from the Fos promoter to gain genetic access to oxycodone withdrawal activated (OWA) neurons. Using this strategy, I propose to evaluate the activity of OWA during oxycodone withdrawal and evaluate their contributions to the expression of behaviors related to somatic withdrawal and negative affect. In Aim 2, I will seek to use both known probes and single-cell RNA sequencing to identify CeA neuronal populations preferentially activated during oxycodone withdrawal. In the independent (R00) phase I will evaluate different subpopulations of CeA neurons based on hits from Aim 2. Highly enriched projections and genes will be evaluated for their ability to influence behaviors pertaining to oxycodone withdrawal and seeking. These experiments will provide me with an outstanding technical skillset and an enriched data set to build the foundation of my independent research program studying the framework of the CeA during opioid dependence and withdrawal. The results of these experiments will provide a clearer understanding of the CeA’s role during opioid withdrawal, potentially to leading to more targetable treatments for opioid use disorder.

NIH Research Projects · FY 2026 · 2024-05

PROJECT SUMMARY/ABSTRACT Epilepsy is a prevalent pediatric neurologic condition that remains treatment-resistant for 30% of patients. It is increasingly recognized as a disorder of brain networks, with network changes shown to underlie seizures, neurodevelopmental impairments, and treatment effects. Limited targeted therapies for epilepsy have been most successful in high-risk populations when administered before seizures start, suggesting that they may prevent pathologic brain network changes. Assessing the development of brain networks using functional connectivity (fc) is therefore a promising technique for early prediction of epilepsy and neurodevelopmental outcomes. Thus, to evaluate network changes as susceptibility markers, fc must be studied prospectively in populations at risk for epilepsy. Very preterm infants (VPT; ≤32 weeks’ gestation) with high-grade intraventricular hemorrhage (IVH) have increased rates of childhood epilepsy and may provide important insights into risk stratification if studied early. This innovative proposal capitalizes on a time-sensitive opportunity to capture a critical period before/during the development of epilepsy in an understudied, high-risk sample, enabling the identification of risk biomarkers translatable to future clinical screening and interventions. To identify prospective biomarkers of epilepsy susceptibility in VPT infants with IVH, the applicant will collect high-density (HD)-EEG data at the bedside in the Neonatal Intensive Care Unit (NICU) in parallel with a longitudinal R01 study collecting functional (f)MRI data and developmental assessments of VPT infants with and without high-grade IVH. This K23 also adds collection of early life epilepsy outcomes through a parent questionnaire and medical record review. The applicant will use HD-EEG to detect clinical EEG abnormalities among VPT infants with high-grade IVH (Aim 1), reductions in HD-EEG and fMRI measures of brain network connectivity, particularly in injured brain regions (Aims 1 & 2), relationships between HD-EEG and fMRI connectivity measures (Aim 2), and relationships between the strength of HD-EEG connectivity metrics, developmental outcomes, and a diagnosis of epilepsy in early life (Aim 3). This award provides the applicant, a uniquely qualified pediatric epileptologist, formal training in quantitative HD-EEG analyses and source localization, multimodal fc analyses, and developmental assessments at an institution deeply committed to early career physician scientists, featuring field leaders in epilepsy, neuroimaging, computational neuroscience, neurodevelopment, and one of the country’s largest NICUs. The applicant’s multidisciplinary mentoring team assembles senior investigators with expertise in early life epilepsy, neonatology, neurodevelopment, electrophysiology, functional neuroimaging, and advanced computational methods. The foundational data and training from this K23 award will play a pivotal role in launching the applicant’s independent career as a physician-scientist, preparing her to direct future studies to identify epilepsy risk markers and assess disease-modifying treatments for infants and children at highest risk.

NIH Research Projects · FY 2026 · 2024-05

PROJECT SUMMARY Idiopathic Pulmonary Fibrosis (IPF) constitutes a tremendous burden to public health. IPF is a rapidly progressive lung disease that results from the aberrant accumulation of extracellular matrix proteins (ECM) in fibroblasts with an estimated survival of 3-4 years. Amino acids are required to provide the critical biomass for proliferating fibroblasts. The varied mechanisms controlling amino acid transport and metabolism represent a key opportunity for drug development and precision medicine. SLC7A5 (Solute Carrier Family 7 Member 5) mediates the uptake of essential amino acids primarily leucine and efflux glutamine out of the cell. As leucine is critical for the activation of mTOR and aberrant mTOR activation is a hallmark of pulmonary fibrosis, collectively our preliminary findings motivate our novel hypothesis that SLC7A5 promotes myofibroblast differentiation, mTOR activation, apoptosis and mitophagy resistance and by targeting SLC7A5 which could capable of abrogating multiple facet of fibroblast activation, may represent a efficacious approach towards developing new therapeutic strategies to treat fibroproliferative diseases. These questions will be addressed by 3 highly interrelated Specific Aims. Aim 1. We will define the biological roles, metabolic and molecular mechanism(s) by which SLC7A5 regulate profibrotic TGF-β signaling and whether the induction of apoptosis by inhibiting SLC7A5 “chemosensitize” fibrotic foci. Aim 2. We will elucidate detailed role(s) of SLC7A5 mediated mitochondrial alteration in controlling fibroblast apoptosis and mitophagy. We will also investigate whether SLC7A5 inhibition induces mitophagy and inhibits lung fibrosis development in the setting of insufficient mitophagy as seen in IPF. Aim 3. We will determine the in vivo efficacy of targeting SLC7A5 in a therapeutic model of lung fibrosis and aging. The completion of these specific aims will provide important mechanistic as well as preclinical information on the role(s) of SLC7A5 in mediating the fibroproliferative actions of TGF-β and a new therapeutic approach for the treatment of pulmonary fibrosis.

NIH Research Projects · FY 2025 · 2024-05

PROJECT SUMMARY/ABSTRACT Spinal cord injury in mammals triggers a cascade of cellular events that lead to the loss of sensory and motor function caudal to the site of injury. Following spinal cord injury, immune cells, including microglia and macrophages, infiltrate into the lesion site and become activated. Depleting microglia and macrophages in mammalian systems has shown both beneficial and detrimental effects post-injury. Identifying the specific regenerative immune requirements in mammalian systems has proven difficult due to a complex combination of anti-regenerative barriers. In contrast, zebrafish spontaneously regenerate a fully severed spinal cord and provide a platform for identifying pathways necessary for spinal cord regeneration. The zebrafish immune system is conserved with mammals, and therefore provides a unique system to identify pro-regenerative immune pathways. In preliminary data, I found microglia and macrophages are necessary for functional and anatomical recovery post-injury, but the pathways directing microglia/macrophage-dependent spinal cord regeneration are not known. Microglia and macrophages are highly plastic cells, and their gene expression and behavior have direct implications on functional outcomes following neural injury. This proposal will identify microglia/ macrophage-specific cellular identities, gene expression, and pathways that are necessary for spinal cord regeneration in the adult zebrafish. First, two of the most important functions of microglia and macrophages following spinal cord injury are to direct the healing of injured tissue and clear the lesion site of cellular debris. Aim 1 (K99 Phase) will utilize loss-of-function mutants to define genes upstream of wound healing that are necessary for re-establishing immune privilege of the spinal cord after injury. Aim 2 (K99/R00 Phase) will move from the adult zebrafish spinal cord to a human cell culture system to visualize behavior in human iPSC-derived microglia and test the conservation of pro-regenerative gene function in human cells. Lastly, the origin of immune cells will dictate their cellular function and effect on regeneration, and the origins of pro-regenerative microglia and macrophages are unknown. In Aim 3 (R00 Phase), I will perform lineage tracing in the adult zebrafish regenerating spinal cord to characterize the origin of expanding immune cells post-injury. These Aims are designed to apply my strengths in zebrafish genetics and regeneration to the new field of neuroimmunology. To facilitate my ability to carry out these proposed experiments, I have assembled a team of advisors and collaborators, taking advantage of the vibrant neural injury and neuroimmunology communities at Washington University School of Medicine. This proposal will generate novel tools and protocols to measure the immune events during spinal cord regeneration and offers a foundational niche in the spinal cord injury field through which I can launch a future tenure-track research faculty position. Additionally, work proposed here will identify pro-regenerative pathways that have direct relevance to human health and provide potential therapies for human spinal cord injury patients.

NIH Research Projects · FY 2025 · 2024-05

Abstract Single cell profiling has revealed a striking cellular diversity and complexity in the mammalian nervous system. Neurons develop their unique features by intrinsic factors, but also must adapt to local environmental features to produce functional circuits. This raises the question: how do neurons maintain or return to stable cellular states in the context of variable inputs? Evidence suggests that this process requires transcriptional regulation by DNA methylation and the methyl-binding protein MeCP2. The levels of 5-methylcytosine (mC) and its oxidized form, 5-hydroxymethylcytosine (hmC), dramatically change in the developing mammalian brain in response to intrinsic cues and environmental input, and these marks show unique genomic patterns in different cell-types. MeCP2 modulates the effects of mC and hmC in neurons and is commonly mutated in Rett syndrome. Further, mutations in the TET enzymes responsible for converting mC to hmC have been recently associated with neurological disorders. Meanwhile, developmental studies indicate that neuron-specific non-CG methylation (mCH), as well as hmC and MeCP2 build up during the postnatal period, when neurons integrate into circuits and complete their final maturation into specific functional subtypes. This leads to the intriguing hypothesis that mC, hmC, and MeCP2 are critical for the establishment and maintenance of diverse, specialized neuronal subtypes within microcircuits. Recent results from us and others indicate that mC and hmC play distinct roles in neuron-specific gene regulation and function depending on sequence context (CG vs CH) and the genomic feature (promoter, enhancer, gene body). However, a critical barrier to testing our hypothesis is the lack of available methods to simultaneously analyze mC and hmC across different genomic contexts on an individual allele. The primary limitation is that the bisulfite chemistry that underlies all high-resolution mC profiling methods shears DNA to under 300 bp. Here we propose to take advantage of the long-read capabilities of nanopore sequencers to develop a method to accurately simultaneously profile mC and hmC in both CG and non-CG contexts across gene promoters and bodies (Aim 1). This will allow us to address how mC and hmC coordinate in different contexts to affect MeCP2 binding and transcriptional programs in Granule and Pukinje neuronal subtypes in the cerebellum (Aim 2). More specifically we will address how differences in mC and hmC profiles across cell types are read out by MeCP2 to maintain differential subtype-specific transcriptomes. We will further dissect how allele specific patterns of this methylation, which can only be decoded using long read sequencing, contribute to neuronal gene regulation. In the future, our approach, which can be easily implemented into standard nanopore workflows, will enable complete sequencing and methylation analysis of clinical or research samples in a single assay. This could lead to a profound increase in the number of available methylomes and our ability to interpret and exploit methylation information for clinical utility.