Boston Children'S Hospital

NIH Research Projects · FY 2025 · 2024-07

ABSTRACT Acute lymphoblastic leukemia (ALL) is the most common childhood malignancy and arises predominantly from developing B-cell precursors. B-cell ALL (B-ALL) remains a leading cause of cancer-related mortality in children, and B-ALL survivors face long-term therapy-related morbidities. Moreover, B-ALL disproportionately impacts children of Hispanic/Latino ethnicity, who have the highest risk of disease and inferior patient outcomes compared to non-Hispanic white children. Prevention of B-ALL remains an essential goal, to mitigate childhood mortality and suffering and to reduce disparities, and this necessitates an understanding of the disease’s root causes. Paramount to this will be elucidating the mechanisms of genetic predisposition to B-ALL. Previous genome-wide association studies (GWAS) have identified at least 15 risk loci associated with childhood B-ALL, but in most cases the causal variants have not been identified. Our proposal aims to integrate results from a large multi-ancestry GWAS of childhood ALL with single-cell epigenomic data followed by targeted base editing in hematopoietic stem and progenitor cells (HSPCs), to pinpoint causal variants and discern their impact on normal human B cell development. Through this innovative and collaborative approach, we hypothesize that we will uncover mechanistic insights into the root causes of B-ALL development that could inform future preventive strategies. In our first aim, we will conduct a multi-ancestry GWAS followed by statistical fine-mapping analysis of known B-ALL risk loci using existing genotype data. These results will be integrated with single-cell accessible chromatin data spanning human hematopoiesis and B cell development using the SCAVENGE method we have developed previously, to identify specific B cell developmental stages that underlie B-ALL predisposition and to identify causal variants at each B-ALL risk locus. In our second aim, we will generate new single-cell multi-omic data, including from joint scRNA- and scATAC-seq in single cells, from human HSPCs undergoing B cell differentiation in vitro. These data will enable regulatory mapping in cells across B cell development, including rare intermediate cells, and will be used to map relevant cell states and target genes for the fine-mapped B-ALL risk variants. Finally, in our third aim, we will investigate the functional impact of B-ALL risk variants using genome editing. We will prioritize six causal variants from our other mapping approaches, including variants in ARID5B and GATA3 that have previously been identified as causal and that contribute to the increased B-ALL risk in Hispanic/Latino children. We will recreate these six variants individually and in specific combinations via base editing in human HSPCs from multiple donors that will then be subject to B cell differentiation and investigate their effects on B cell development and on illegitimate V(D)J recombination, a known driver of B-cell leukemogenesis. Our innovative variant-to-function mapping approach will reveal the precise mechanisms through which B-ALL association loci may increase a child’s risk of developing leukemia and may highlight novel therapeutic targets and inform future approaches for childhood leukemia prevention.

NIH Research Projects · FY 2024 · 2024-07

PROJECT SUMMARY/ABSTRACT Endometriosis is a painful gynecological inflammatory disease that affects up to 15% of people born with a uterus. While effective for a fraction of patients, current therapies such as hormones and NSAIDs present several side effects. Therefore, new medical therapies and targets that provide long-term benefit are still needed. Here, we propose to validate drugs that block neuroimmune communication as well as macrophage-targeting drugs as novel, non-hormonal, and non-opioid approaches for the treatment of endometriosis-associated pain. The project also addresses a previous unknown mechanism by which CGRP contributes to a pro-endometriosis phenotype in macrophages and pinpoints a nociceptor-responsive macrophage population that drives omental colonization for subsequent pain and lesion formation in endometriosis. My preliminary data show that nociceptor to macrophage signaling via CGRP/RAMP1 contributes lesion formation and endometrial cell growth. However, the mechanisms by which CGRP programs (re)macrophages to a pro-endometriosis phenotype is not known. Therefore, we will (Aim 1) identify the inflammatory mediators released and the signaling pathways activated in macrophages upon CGRP stimulation using bulk RNAseq analysis. I will further use FDA approved drugs such as pexidartinib to the measure the effect of macrophage-targeting in my mouse model. I also have preliminary data showing that Maresin-1 (MaR1) a pro-resolving lipid mediator reduces pain and stimulates efferocytosis by macrophages. I will next determine the mechanisms by which MaR1 resolves pain and inflammation in endometriosis. For that, I will perform scRNAseq of MaR1-treated mice at different timepoints to understand the dynamics and mechanism of pain resolution during endometriosis. I also have preliminary scRNAseq data showing that endometriosis completely changes the immune cell landscape in the peritoneal cavity with a decrease in the macrophages that migrate to the omentum. Therefore, during my R00 phase (Aim 2) I will determine the extent to which neuroimmune communication drives omental colonization by macrophages as well as pinpoint the nociceptor-responsive population of macrophages in the omentum that is responsible for endometriosis pain and lesion formation. I will then use transgenic mice to deplete that macrophage population to determine the extent to which those cells contribute to pain and lesion formation. To reach these long-term goals, I have outlined a detailed career development plan, which will provide me with the technical and leadership skills to establish a successful research laboratory. The K99 phase of research will be conducted under the excellent co-mentorship of Drs. Michael Rogers and Clifford Woolf. My Research Advisory Committee and collaborator are leading experts in neuroimmune communication (Dr. Chiu), endometriosis (Dr. Missmer), and pharmacology of pain and neuroimmune communication (Dr. Cunha). This K99/R00 award will provide me key support for my successful transition to an independent investigator studying neuroimmune communication during endometriosis.

NIH Research Projects · FY 2025 · 2024-07

PROJECT SUMMARY This project proposes to develop and refine computational tools to advance the safety and efficacy of genome editing, addressing the critical need for precise quantification and prediction of on- and off-target effects. Genome editing technologies have the potential to revolutionize the treatment and prevention of disease by enabling precise modifications to DNA sequences. However, their clinical application is currently limited by significant challenges in detecting and avoiding off-target effects, as well as quantifying and understanding the heterogeneity of on-target edits. In Aim 1, we will enhance the capabilities of our recently developed tool, CRISPRme, which predicts off-target effects across diverse human genomes. We propose to develop a new tool, CRISPRus, which will leverage variation graphs, complete genomes, and pangenomes to provide a more comprehensive understanding of potential off-target effects. Aim 2 will focus on the characterization of complex on-target editing outcomes. We will develop and refine tools to enable accurate quantification from long-read and single-anchor sequencing methods. This will provide a more detailed and nuanced understanding of the outcomes of genome editing, including the potential for complex, multi-site edits. In Aim 3, we will tackle the statistical challenges inherent in quantifying genome editing events. We propose to develop DE-CRISPR, a comprehensive framework that will provide simulations and statistical models to assess the significance of editing events at the locus, repair-pathway, and individual allele levels. This will enable researchers to design experiments with adequate power to detect editing events and to interpret their results with greater confidence and precision. Accompanying these aims, we will produce comprehensive benchmark datasets, including of 6 editing approaches in 6 donors; comparing nuclease, base, and prime editing; comparing approaches across a range of anticipated specificity; targeting primary clinically relevant cells of unknown genotype (a real-world scenario) and cell lines with complete telomere-to-telomere genome assemblies described. The deliverables of this project, a comprehensive toolkit of computational tools including CRISPRus, CRISPRessoPE, CRISPRuni, CRISPRlungo and DE-CRISPR, will empower researchers to more safely and effectively harness the power of genome editing technologies. This work is essential to ensuring the robustness and reproducibility of genome editing, ultimately promoting the safety and efficacy of therapeutic development. By addressing current limitations in genome editing off-target prediction, complex edit characterization, and statistical analysis, we aim to keep pace with advances in genome editing technology and contribute to the realization of its full therapeutic potential.

NIH Research Projects · FY 2026 · 2024-07

Project Summary In pediatric epilepsy patients with drug-resistant seizures, surgical resection is the most effective treatment option. The goal of resective surgery is to maximize removal of epileptic foci to attain seizure-freedom while minimizing damage to surrounding brain regions to avoid permanent post-surgical functional loss. Functional MRI enables rapid and non-invasive pre-surgical mapping of language, motor skills and other critical functional brain regions with high spatial resolution. However, excessive head motion presents a major limitation for acquiring high-quality fMRI in pediatric patients with focal brain lesions, who usually have difficulty remaining still for long fMRI scan durations. Unfortunately, current retrospective and prospective approaches cannot adequately compensate for the complex effects of motion in fMRI. As echo planar imaging (EPI) is highly susceptible to local magnetic field variations, motion-induced geometric distortions and blood oxygenation level-dependent (BOLD) contrast changes can lead to potentially significant mislocalization of activation regions, even with accurate head motion tracking. The overarching goal of the research proposed under this application to the NIH is to dramatically improve the quality of fMRI for pre-surgical mapping in pediatric epilepsy patients. We are proposing a solution based on 3D EPI, which is more robust to spin history artifacts and has higher signal-to-noise ratio (SNR) compared to conventional 2D EPI, combined with real-time motion and field compensation. In particular, we will use ultra-fast free induction decay (FID) navigators, which can be embedded in each shot of the 3D EPI acquisition without affecting BOLD contrast or reducing acquisition efficiency. These navigator measurements will be used to produce accurate motion and field estimates that can be used to update the imaging volume and magnetic field in real time. We hypothesize that this improved functional MRI acquisition strategy will produce technically useful activation maps in pediatric epilepsy patients evaluated for a resection surgery at a higher rate than previously thought possible. To achieve these ambitious goals, we will undertake the following specific aims: 1) develop and evaluate a novel technology using FID navigators to measure head motion and magnetic field fluctuations in multi-shot 3D EPI; 2) develop and evaluate prospective motion correction and dynamic shimming utilizing real-time motion and field measurements; 3) apply and evaluate motion and distortion compensation in fMRI of pediatric epilepsy surgery candidates. If successful, our project will facilitate widespread clinical adaptation of fMRI for pre-surgical mapping in epilepsy, and enable high resolution fMRI for research studies in incompliant patient populations.

NIH Research Projects · FY 2025 · 2024-07

PROJECT SUMMARY/ABSTRACT Lack of oxygenation and nutrients that result from narrowed arteries and reduced blood flow debilitates the normal function of the heart, leading to severe morbidity and early mortality worldwide. This devastating acute medical episode is often seen in patients with obstructive plaques in their arteries. As a result, coronary artery disease is the root cause of ischemic cardiovascular diseases such as myocardial infarction. Despite that a healthy lifestyle and standard care may deter development of these diseases, risk factors such as obesity, dyslipidemia, diabetes, and high blood pressure elevate the odds of recurrent myocardial infarction, leading to heart failure, ischemic cardiomyopathy, or even death as well as a major financial burden to the society. Reperfusion after acute myocardial infarction introduced by coronary angioplasty and direct stenting can cause further damage to the injured heart. Unfortunately, the scarcity of novel molecular targets that stimulate natural new blood vessel growth to restore blood flow and augment heart repair and regeneration creates a daunting fight against this deleterious condition, for which there is currently no cure. The long-term goal of our research is to discern molecular insight into endothelial regulation and heart regeneration in health and disease. In this application, we aim to decipher molecular mechanisms behind the action of cardiac endothelial epsins in regulating in myocardial ischemia. Epsins are a family of prominent endocytic adaptor proteins that were originally discovered by the PI. We recently reported that targeting epsins in macrophages in atherosclerotic plaques by nanoparticle-mediated delivery of epsin siRNAs prevents atheroma progression and promotes atheroma regression, suggesting epsins as an emerging target for treating coronary artery disease in preclinical models. Harnessing newly developed snRNA sequencing technologies, we made a potentially groundbreaking discovery where the loss of cardiac endothelial epsins potentiates endothelial cell expansion and precursor cell transition to fuel vascularization in neonatal hearts. We see heightened neovascularization, bolstered cardiomyocyte proliferation in neonatal hearts of inducible endothelial-specific epsin 1 and 2 double knockout (EC-iDKO) mice. Consistently, elevated revascularization, enhanced cardiac function and improved heart repair and recovery from myocardial infarction in adult EC-iDKO mice. We posit that inhibition of cardiac endothelial epsins fortifies angiocrine signals such as CXCL12-CXCR4 signaling, enable creation of a vast endothelial cell reservoir to build vascular networks in the heart. Our central hypotheses are that epsins in the cardiac endothelium suppress endothelial expansion, in part, by repressing CXCR4 signaling and hindering heart regeneration and repair following myocardial ischemia. If successful, our proposed work will explore uncharted territory, uncover original regulatory mechanisms, identify novel modulators, and inaugurate new classes of restorative and regenerative therapies to treat deadly ischemic heart diseases and heart failure.

NIH Research Projects · FY 2026 · 2024-07

Project Summary/Abstract Asthma is the #1 chronic disease in childhood: prevention is an unmet need and pressing priority. We are conducting the first randomized, double-blind, placebo controlled trial in 200 high risk children 2-3 years of age to determine whether 2 years of treatment with omalizumab (anti-IgE) will prevent asthma and/or diminish asthma severity 2 years after treatment is stopped. Our hypothesis revolves around the role that IgE plays in the development of persistent asthma by catalyzing allergic type 2 driven recurrent wheezing and augmenting virally-induced exacerbations in susceptible young children. Recent studies have demonstrated the feasibility and power of using new and novel systems-scale network analysis of transcriptional pathways and epigenetic signatures to assess airway responses in readily obtained nasal samples. We hypothesize that patients who respond to anti-IgE may have upregulated expression of genes related to antiviral responses, epithelial cell structure and barrier integrity. This project will allow us to complete the trial and use state of the art epigenomic and transcriptomic techniques with integrative approaches to explore this hypothesis and examine the role of specific alterations in Treg cells and antigen-specific T cells in transducing clinical responses seen in the trial. Our first aim is to complete the trial to assess whether interfering with environmental allergen IgE-mediated immunological responses can prevent or moderate a progression to asthma in susceptible children. Our second aim is to examine the relationship between anti-viral and epithelial integrity pathways and the response to anti-IgE through DNA methylation and transcriptomic signatures and associated effects on Type 2 inflammation, wheezing episodes, asthma, and asthma severity. We will extend our findings to include single- cell transcriptional profiling in a subset to more fully inform the cellular sources of bulk transcriptome responses and provide important molecular details about individual cell heterogeneity and rare cell populations that relate to clinical responsiveness and clinical outcomes. We will integrate multi-omic and clinical data and perform analyses to define the combined transcriptomic and epigenetic changes underlying response to anti-IgE treatment and the persistence or resolution of benefit (including asthma outcome) following cessation of therapy. These signatures will provide comprehensive mechanistic insights on clinical treatment effects of responders vs. non-responders, and specific DNA methylation and gene expression molecular pathways that impact study outcomes and elucidate the disease modifying effects of anti-IgE. Differences in these profiles could provide potential biomarkers predictive of clinical and immunologic response to anti-IgE. This study is potentially paradigm shifting – regardless of the outcomes of the trial, we will gain considerable knowledge on the pathophysiology of the disease and the impact of interfering with IgE mediated processes at a critical time period where the immunologic and clinical phenotype is evolving but not yet fully established.

NIH Research Projects · FY 2026 · 2024-07

SUMMARY. From initially cataloging the microbiome, we can now causally link specific strains and metabolites of the microbiome with protective and pathogenic roles in a broad range of human disease. We are now faced with the challenge of shaping beneficial microbiomes, such as at the strain-level, and enhancing microbiome function, such as levels of microbiome-derived factors critical to the host, in order to harness the potential of the microbiome for human health. The Bacteroides are the most abundant Gram-negative bacteria of the human gut and play central roles in health and disease at the genus-, species- and strain- level, such as the role of pro- carcinogenic B. fragilis EBTF+ strains in colon cancer. This motivates the need of both a comprehensive understanding of the ecological factors impacting the Bacteroides and interventions to shape pro-health Bacteroides communities. The Bacteroides survive by utilizing glycans, the basis for using ‘prebiotic’ dietary glycans to target specific beneficial gut microbes such as members of the Bacteroides. Here, we reveal that butyrate, abundant in the gut and critical to host physiology, demonstrates strain-specific inhibition against the Bacteroides. Remarkably, butyrate inhibition is glycan dependent. Depending on the specific glycan used, a strain is rendered differentially vulnerable to butyrate. Within a Bacteroides species, the same glycan can render one strain vulnerable to butyrate and the other protected, suggesting that specific glycan-butyrate combinations may be exploited to enhance or suppress specific Bacteroides strains. Defense from butyrate is mediated by the activity of Acyl-CoA metabolic genes which differ among the Bacteroides. Unexpectedly, we find that extracellular butyrate is taken up and metabolized by members of the Bacteroides, demonstrating the Bacteroides as one of the first gut bacteria to take up butyrate and act as a potential microbial butyrate ‘sink’. Our findings revealing the impact of butyrate on the glycan-dependent and strain-specific survival of the Bacteroides and the ability of the Bacteroides to take-up and metabolize butyrate drive our central hypotheses: butyrate impacts the Bacteroides in vivo, we can leverage dietary glycan-butyrate interactions to shape strain-specific Bacteroides communities, and the Bacteroides modulate butyrate levels in the gut. To test these, we propose to: Aim 1. Define the impact of butyrate on the composition of the Bacteroides in vivo Aim 2. Determine the role of the Bacteroides in butyrate homeostasis in the gut Successful completion of this proposal will establish a new function for butyrate in gut microbial ecology, develop needed interventions to enhance/suppress Bacteroides strains by exploiting glycan-butyrate interactions, and establish both the role of microbial ‘sinks’ in gut butyrate homeostasis and the Bacteroides as a target to optimize butyrate levels in vivo. Our work is poised to have broad impact for our understanding of microbial ecology, diet- microbe-host interactions, microbial metabolism, and colonic health, with potential wide-ranging translational applications to shape Bacteroides communities and optimize butyrate levels in heath and disease.

NIH Research Projects · FY 2025 · 2024-07

PROJECT SUMMARY While immunotherapies have proved compelling efficacy against other leukemias, their application for acute myeloid leukemia (AML) is still hampered by the absence of tumor-restricted targets. The most suitable AML targets are shared with healthy hematopoietic stem/progenitor cells (HSPCs) or mature myeloid cells, leading to on-target/off-tumor toxicity and impairment of hematopoietic reconstitution. To address this issue, we hypothesized that epitope-engineering of donor HSPCs used for conventional bone marrow transplantation can endow hematopoietic lineages with selective resistance to CAR-T or monoclonal antibodies (mAb), without affecting protein function or regulation. This strategy allows targeting genes essential for leukemia survival regardless of shared expression on HSPCs, thus reducing the risk of tumor immune escape by antigen downregulation/loss. We have already identified single amino-acid (aa) changes that abrogate the binding of therapeutic mAb targeting FLT3, CD123, and KIT and optimized a base-editing approach to introduce them into CD34+ HSPCs, which retain long-term engraftment and multilineage differentiation capacity. We confirmed the in vivo resistance of epitope-edited hematopoiesis to CAR-T treatment and the concomitant eradication of patient-derived AML xenografts (Casirati et al., Nature 2023). Here, we will capitalize on these achievements and exploit state-of-the-art genetic engineering tools and in vivo modeling with the objectives to i) generate and functionally validate “stealth” FLT3, KIT, and IL3RA genes by multiplex base-editing; ii) identify the best- performing CAR configuration for multi-Ag targeting on AML samples and iii) validate resistance of edited HSPC to new immunotherapies directed against these targets. This project will provide fundamental advancement of new and more effective immunotherapy approaches for AML that should additionally have broad applicability to several other hematopoietic malignancies.

NIH Research Projects · FY 2025 · 2024-07

PROJECT SUMMARY Obesity is a serious health concern in the United States, affecting 41.9% of the population and resulting in an estimated annual medical cost of nearly $179 billion. Notably, obesity increases the risk of cardiovascular diseases due to inflammation and thrombotic events. Individuals with obesity often exhibit hyper-reactive platelets and reduced sensitivity to anti-platelet therapy, yet the mechanisms driving these altered platelet phenotypes remain poorly understood, representing a significant knowledge gap in platelet biology. Our preliminary data reveal that platelets from obese mice exhibit an altered lipidome that corresponds with enhanced reactivity. Further, we previously demonstrated that platelet-generating cells, megakaryocytes (MKs), can effectively incorporate fatty acids into their membrane, both in vivo and in vitro. However, it is unknown if and how this uptake directly translates into alterations in platelet membrane composition in disease. As such, our central hypothesis is that the hyper-reactive platelet phenotype observed in obesity arises from the increased incorporation of dietary saturated fatty acids (SFAs) into platelet membranes, leading to a higher membrane lipid saturation that in turn enhances receptor accumulation within lipid rafts, ultimately augmenting platelet reactivity. To test this hypothesis, Aim 1 will unravel how fatty acid uptake influences platelet membrane composition. Through in vitro experiments and in vivo studies utilizing high-fat diets with varying SFA concentrations, I aim to determine whether platelets acquire fatty acids from both MKs and the plasma. Aim 2 will test the influence of dietary SFAs on platelet reactivity and thrombus formation. By conducting in vivo studies with escalating SFA content in diets, I will establish a direct link between dietary factors and altered platelet phenotypes. Aim 3 will investigate whether dietary SFA-induced platelet hyper-reactivity is caused by changes in lipid raft content and subsequent receptor signaling. My investigations will include characterizing lipid raft density and receptor content, as well as analyzing the effects on downstream signaling both in vitro and in vivo. Overall, this proposal aims to uncover the precise mechanisms by which dietary SFAs are integrated into platelet membranes and how this integration influences platelet function. The main aims of this proposal are the identification of new avenues for pharmacological interventions targeting lipid receptors in platelets or of novel dietary modifications for obese individuals, ultimately contributing to a better management of obesity-related cardiovascular complications.

NIH Research Projects · FY 2025 · 2024-07

Project Summary The goal of this project is to investigate the structure and function of a poorly understood family of ion channels known as TMEM63s. There are three members of the family in animals, known as TMEM63A, B and C. TMEM63s form mechanosensitive ion channels and are thought to be the animal orthologues of the plant OSCA channels. TMEM63A mutations have been identified in young patients with hypo-myelinating leukodystrophies characterized by myelin deficits suggesting a functional role of TMEM63A in myelination and neuronal development. TMEM63B deficiency in mice leads to deafness and it is hypothesized to act as an osmosensor in auditory hair cells. TMEM63B mutations in humans lead to a range of disorders including severe neurodevelopmental disorders, epileptic encephalopathy, hematological abnormalities, and hearing loss. A genomic analysis in hypertensive rats implied a role for TMEM63C in kidney damage. When expressed in heterologous cells, TMEM63A and TMEM63B can be activated by mechanical stimulation, revealing small currents of 10s to 100s of pA. These data, while intriguing, raise fundamental questions regarding the structure and function TMEM63 mechanosensitive ion channels and whether TMEM63 channels share common molecular, structural, and gating mechanisms with other mechanosensitive ion channels. The project is organized around three specific aims. For aim one, we will determine the structures of human p.V44M variant of TMEM63B using single-particle cryo-EM. We hypothesize this variant biases the channel toward the open state. For aim two, we will investigate the function of TMEM63B in proteoliposomes and in heterologous cell lines. We will evoke mechanically activated currents using pressure steps and perfusion of hypoosmotic bath solutions. We will investigate structural and functional aspects of WT TMEM63B and will use site-directed mutagenesis to investigate the function of various structural features, including the putative pore region, the intracellular loop, IL2, and the hypothesized gating helices. We will also investigate the functional consequences of human TMEM63B mutations including the p.V44M variant. For the third aim, we will investigate the physiological contributions of TMEM63B expressed in inner ear hair cells. We hypothesize that intense auditory stimulation promotes massive influx of potassium and calcium in hair cells, raising intracellular osmolarity and causing ionic imbalance. With the inside of the cell being hypertonic relative to external hypotonic bath solution, cell swelling may ensue which may, in turn, activate TMEM63B channels. Calcium influx via TMEM63B is postulated to activate neighboring calcium-activated potassium channels promoting potassium efflux and ionic equilibration. Disruption of this pathway is thought to lead to hair cell death and hearing loss. The experiments proposed herein will shed light on the role of TMEM63B in hair cells and hearing and will illuminate structural and functional features of this newly discovered, poorly understood family of mechanosensitive ion channels.

NIH Research Projects · FY 2026 · 2024-07

PROJECT SUMMARY The proposed project will fill an important knowledge gap in the field by providing a mechanistic understanding of how FLT3 signaling regulates neuronal function and circuit excitability in healthy and epileptic brains, and this work will advance the field by identifying new avenues for drug intervention to rescue impaired GABAergic inhibition that leads to brain disorders. GABAergic inhibition interacts with glutamatergic excitation to determine the level of neural activities in the nervous system. The neuronal chloride transporter KCC2 plays a pivotal role in regulating the polarity and efficacy of GABAergic signaling. Dysregulation of KCC2 is associated with impaired GABAergic inhibition present in various brain disorders including epilepsy, indicating KCC2 as a promising –yet to be explored– drug target for suppressing pathological brain hyperexcitability. Our published work discovered that small molecule compounds inhibiting the FLT3 kinase signaling are capable of enhancing the level of KCC2 protein expression in neurons to strengthen GABAergic inhibition. Although FLT3 has been historically studied in the context of blood cancer, our preliminary results show for the first time that in the brain FLT3 is specifically expressed in neurons, indicating that this kinase has functions in neurons yet to be explored. Leveraging a newly-developed neuron-specific Flt3 conditional knockout mouse line, we demonstrated that knocking out Flt3 gene from neurons, or treating mice with an FLT3 pathway inhibitor drug, substantially reduces seizure susceptibility. Specifically, we propose to further investigate the functional role of FLT3 in regulating the development of the GABAergic inhibition system in the mouse brain (Aim 1), and to examine to what extent the FLT3 signaling cascade regulates the onset and recurrence of epileptic seizures in mice (Aim 2). We will also elucidate the molecular and cellular mechanisms underlying FLT3 signaling in the brain (Aim 3).

NIH Research Projects · FY 2024 · 2024-07

PROJECT SUMMARY The overarching goal of Dr. Wang’s proposal is to reduce the known risk of renal injury from febrile urinary tract infection (fUTI) in children by implementing a practical, validated clinical decision support algorithm to promptly identify unsafe anatomy before injury occurs. Dr. Wang’s proposal has identified significant care gaps in the care of fUTI children. The long-term goal is to contribute to optimal management for fUTI in children through implementation of novel, high-value, self-renewing machine learning (ML) models. The overall objective is to identify those critical elements necessary for the development and implementation of predictive modeling to identify children who would benefit most from early vs later voiding cystourethrogram (VCUG) in primary care (NOT-HS-22-011). The central hypothesis is that ML models can provide accurate prediction of risky fUTI, and thus assist clinicians/families to choose the best timing for VCUG. The rationale is to offer a scientific roadmap and pilot new strategies that incorporate and implement prediction models to provide true value-based care (NOT-HS-19-011) and equitable resource utilization for children (NOT-HS-21-015, NOT-HS-21-014). This hypothesis has been formulated based on Dr. Wang’s previous work that demonstrates 1) high variability in post-UTI VCUG practice patterns; and 2) ML models can serve as a promising basis to reliably identify children with high risk for damaging UTI. Leveraging the data from a large pediatric practice network within Boston Children’s Hospital, the following specific aims are proposed: 1) assess determinants for successful ML algorithm implementation for pediatric fUTI care, 2) prospectively collect data to optimize and validate novel ML algorithms in fUTI children, 3) pilot prediction of pediatric fUTI algorithm implementation to iteratively testing, implementing, and adapting the algorithm using the principles of implementation and behavioral science to maximize adoption and sustained implementations. In this proposal, Dr. Wang has assembled a multi- disciplinary mentorship team consisting of experts in, qualitative methods, informatics, infectious disease, machine learning, implementation, and behavioral science to help him achieve his goals and has designed a comprehensive training plan to acquire necessary expertise. Dr. Wang’s unique background combined with his career development plan, and the rich supporting environment (Boston Children’s Hospital, Harvard system, and MIT) position him well to attain the proposed training goals and specific aims, and eventually lead to his transition to independent surgeon-scientist. Combining machine-learning technology and real-life implementation to tackle the challenge and change the status quo by translating actionable ML results to the bedside is novel and innovative. The study is significant in that successful implementation of an algorithm for UTI will be proof-of-concept to catalyze a similar approach to optimal clinical decision support for other conditions. This work has broad impact and can be scaled to other institutions and conditions, facilitating interactive improvements that empower clinicians and caregivers to meet the diverse clinical needs of children.

NIH Research Projects · FY 2025 · 2024-07

Congenital heart disease (CHD) is the most common birth defect in the U.S. and affects ~1% of all live births. Over 85% live well into adulthood, and over 50% of those with moderate or complex CHD suffer from neurodevelopmental disabilities - most commonly impaired being executive function (EF). As EF is important for independent living and mental health, predicting who will have greater EF impairment and needs intervention is important as EF is particularly amenable to treatment. However, current routinely measured patient and medical factors do not reliably predict EF in CHD - better predictors are needed to appropriately allocate services and improve outcomes. In utero and postnatal factors may lead to impaired EF in CHD. Lack of substrate in utero or abnormal gene expression may impact early neurodevelopmental (ND) processes. This includes formation of the white matter “backbone”, the structural “rich club” of the brain that is critical for EF. Additional environmental factors, including surgery, parenting style and life experiences, further alter brain structure through secondary adaptive processes which include development of cortico-cortical connections, axonal pruning and myelination. Differentiating between early in utero factors from later adaptive changes is key to understanding the potential and optimal timing of interventions and the relative importance of developing novel in utero therapies. We propose to employ our advanced connectome measures of “information transport” to understand early and later environmental effects on white matter connectivity, by determining the impact of CHD on rich club versus secondary, non-rich club connections. We leverage two cohorts with dextro-transposition of the great arteries (d- TGA) CHD – infants with pre-operative MRI and 22-month ND outcomes, and adults aged 26-34 years old with MRI and cognitive testing. d-TGA is the more common severe CHD that is corrected soon after birth and additional surgery is rare. Thus d-TGA patients have the most uniform postnatal course of all CHDs but, like other CHDs, have significant yet variable impairment in EF. The overarching goal is to understand how brain structure in d-TGA patients differ to controls, and the impact of pre- and post-natal factors on outcome measures. This will help us to identify when to intervene, and therefore better manage the appropriate factors that can improve longer-term ND and EF outcomes. To address this, Aim 1 is to determine how the connectome is altered in pre-operative infants, and whether an altered prenatally established rich club is associated with outcomes at 22-months. Postnatal measures on surgery, socioeconomic status and upbringing will be modelled to study their influence on this association. In Aim 2, we investigate alterations in rich club/non-rich club connectivity in the d- TGA adults and their association with EF. We include similar clinical and environmental factors to this model to ascertain whether they play a role in the above relationship. Successful completion will determine the effect of pre- and post-natal factors on brain structure during two stages of life, and the relative roles of the rich club and secondary adaptive pathways on cognitive outcomes in d-TGA CHD.

NIH Research Projects · FY 2026 · 2024-06

Abstract Idiopathic hypersomnia (IH) is a chronic, disabling central nervous system disorder of hypersomnolence that typically begins in adolescence. Progress in understanding IH pathophysiology and identifying effective treatments is stymied by the commonly used diagnostic test, the multiple sleep latency testing (MSLT). This test is very reliable in classic narcolepsy but has poor validity and reliability in people with IH. In addition to this poor clinical performance, the current diagnostic use of the MSLT also hinders clinical trials by enrollment of clinically heterogeneous IH cohorts with more variable outcomes. Thus, there is a critical need to develop and validate an objective IH biomarker signature to improve diagnostic accuracy. The most striking IH features occur in the nocturnal sleep period. Most IH patients report non-restorative sleep and profound difficulty waking from sleep in the morning despite normal to long sleep durations. In our prior work, we showed IH is characterized by shorter (less stable) bouts of NREM 3 (N3) sleep (the most restorative sleep stage), longer bouts of less refreshing NREM 2 (N2) sleep, long sleep times, and high sleep efficiency. These findings may reflect proposed mechanisms of IH including heightened GABA receptor potentiation or prolonged circadian period. As a multi-disciplinary team with expertise in modeling neurophysiological processes, biomarker discovery, statistics, and hypersomnia disorders, we hypothesize that measures of nocturnal sleep stability will define the IH signature and more accurately identify IH than the current MSLT method. In the R61 grant, our objectives are to: (Aim 1) identify nocturnal sleep features of hypersomnia patients using polysomnograms retained in the National Sleep Research Resource; (Aim 2) develop and test a sleep biomarker signature from polysomnogram signals (IH signature) for IH diagnosis in a clinical population across 5 sites using current diagnostic criteria and cutoff scores on validated IH symptom scales as the “gold standard” IH classification. In meeting our robust “go criteria” showing high sensitivity and specificity of our IH signature for IH diagnosis, we will proceed with the R33 phase. In this next grant phase, we will validate the IH signature in a separate and new clinical cohort of patients (Aim 3) and test the reliability of the IH signature (Aim 4). Through this work, we will identify an accurate and stable biomarker signature that will provide more accurate diagnosis of IH using nocturnal PSG testing. This will improve diagnostic certainty in the clinic and reduce clinical heterogeneity for research, enabling study of underlying causal mechanisms and future drug development.

NIH Research Projects · FY 2025 · 2024-06

Project summary/abstract (30 lines) Succinic Semialdehyde Dehydrogenase Deficiency (SSADHD) is a rare genetic metabolic disorder caused by ALDH5A1 mutations. ALDH5A1 encodes SSADH essential for the catabolism of the inhibitory neurotransmitter γ-aminobutyric acid (GABA). In SSADHD, pathologic accumulation of GABA and metabolite γ-hydroxybutyrate (GHB) leads to broad spectrum encephalopathy. Paradoxically, despite heightened ambient GABA, patients with SSADHD are susceptible to seizures and sudden unexpected death in epilepsy (SUDEP), highlighting the significance of compensatory down-regulation of GABA receptors over pathologic GABA build-up. A major unmet medical need for SSADHD is treatment directly addressing the underlying enzyme deficiency such as gene restoration therapy. Proof-of-concept aldh5a1 restoration via adeno-associated virus (AAV) increased survival of SSADH-deficient aldh5a1lox-STOP mice and reversed SSADHD-relevant phenotypes. Furthermore, restoration of ~15% SSADH in relevant cell types is sufficient for enhanced survival. An AAV encompassing a human ALDH5A1 full-length native promoter (FLnP) driving a functional recombinant ALDH5A1 gene is a potential cure for patients with SSADHD. However, it is unclear whether AAV-FLnP-ALDH5A1 drives sufficient functional SSADH expressions in patient cells, and whether systemic AAV delivery (requiring high AAV dose) might lead to therapeutically meaningful brain target engagement. We propose to use patient induced pluripotent stem cells (iPSC)-derived neurons and the SSADH-deficient aldh5a1lox-STOP mouse model to study efficacy and safety of this novel AAV-FLnP-ALDH5A1 construct. R61 Aim 1: Measure SSADH restoration, cultured medium GHB content, and functional properties of patient iPSC-derived neurons. Go-no-go: At least 50% SSADH restored, functional phenotype returned to control. R61 Aim 2: Measure SSADH restoration in brain tissues, GABA receptors, GHB content in blood, electrographic seizures, and survival in SSADH-deficient aldh5a1lox-STOP mice. Go-no-go: GHB reduced to <15µM, reduced seizures and two-fold lifespan extension. R33 Aim 3: Utilize dose de-escalation to determine minimal AAV-FLnP-ALDH5A1 (in AAV9 serotype) threshold for therapeutic effects, and long-term toxicity in SSADH-deficient mice. The project goal is two-fold: 1) Provision of molecular insights into whether AAV-FLnP-ALDH5A1 is a viable clinical candidate for SSADH gene therapy. The proposed study provides necessary insights into whether this novel AAV construct is sufficient for phenotypic reversal in patient-derived iPSC, and whether this gene expression cassette FLnP-ALDH5A1 is effective in various cell types including excitatory and inhibitory neurons. 2) Establishment of practical AAV dosage for therapeutic SSADH gene restoration therapy. The proposed study provides necessary insights into minimal threshold for systemic delivery of translatable AAV serotype leading to brain-wide coverage and associated phenotypic reversal in SSADH-deficient mice, advancing future translational and clinical development of SSADH gene therapy.

NIH Research Projects · FY 2025 · 2024-06

PROJECT SUMMARY The neurocognitive side effects of cancer chemotherapy in pediatric patients are heart-breaking; not only must these children deal with the hardship of the disease and harsh treatment, but they often also face neurocognitive impairments that affect their performance in school and their daily life as cancer survivors. These brain toxicities, named “chemobrain”, or Chemotherapy-related Cognitive Impairment (CRCI), include immediate symptoms (confusion, memory impairment, ataxia), as well as prolonged neurocognitive malfunctions. THE GAP IN THE FIELD: Not enough is known about the molecular mechanisms of chemotherapy-induced toxicity. Specifically, metabolic aberrations in the brain that ensue following therapy with metabolic drugs such as the anti-folate MTX, a standard of care for childhood leukemia, is an unexplored field. To be able to offer solutions to chemotherapy-induced neurotoxicity it is critical to first understand the molecular mechanisms of the toxicity caused by common chemotherapies. Therefore, THE GOAL OF THIS PROJECT is to elucidate targetable mechanisms that mediate and modify chemotherapy-induced neurotoxicity with focus on oxidative distress. HYPOTHESIS: A good understanding of the molecular mechanisms of CRCI is required to be able to offer preventive care or treatment. PRELIMINARY DATA: We applied metabolite profiling of MTX-treated mouse and human cerebrospinal fluid (CSF) to study the metabolic impact of the drug and revealed oxidative distress in the CSF and the choroid plexus (ChP), the organ that produces the CSF. We found that in addition to damaging the ChP and CSF, MTX also caused toxicity to neurons in the hippocampus. However, these findings offer some hope: the CSF can serve as a conduit for therapy because it is relatively accessible for clinical intervention and reaches all parts of the brain. Indeed, our discovery that MTX treatment causes reduction in CSF levels of the secreted antioxidant enzyme SOD3, both in mice and in MTX-treated patients, led us to test CSF-based therapy; ChP-targeted gene therapy with exogenous SOD3 expression replenishes CSF's protective capacity, preventing metabolic damage in the hippocampus and even rescues MTX-induced behavioral deficits in mice. APPROACH: We will test our hypothesis that the ChP-CSF system can be harnessed to alter the brain redox environment to reduce adverse effects of chemotherapy on non- cancerous brain cells by assessing the mechanisms by which CSF-SOD3 protects the hippocampus from MTX-induced toxicity (Aim 1), by studying the role of nitric oxide (NO) and peroxynitrite in the MTX-induced oxidative damage in ChP cells and neurons (Aim 2), and by testing if resuming oxidative balance in the CSF can be achieved by peripheral and brain-specific injection of antioxidants, and whether these are sufficient to mitigate oxidative damage to ChP and hippocampus (Aim 3). IMPACT: Our study will reveal actionable tools to mitigate the neurological damage induced by MTX, and other chemotherapies, and the immediate potential of antioxidants co-administration with chemotherapy for amelioration of CRCI and improved survivorship.

NIH Research Projects · FY 2026 · 2024-05

Summary Virus entry begins with the first encounter between the virus and the cell surface and ends with delivery of the contents of the virus into the host cell. HIV-1 membrane fusion is the first key delivery step, mediated by the virus-encoded envelope glycoprotein [Env; trimeric (gp160)3 cleaved to (gp120/gp41)3], which belongs to the group of class I viral fusion proteins including influenza hemagglutinin, SARS-CoV-2 spike protein and Ebola glycoprotein. A mature Env spike has three copies each of noncovalently-associated receptor-binding subunit gp120 and fusion subunit gp41. Sequential binding of gp120 to the primary receptor CD4 and a coreceptor (chemokine receptor CCR5 or CXCR4) leads to large, irreversible structural rearrangements in gp41, which drive fusion. This picture, derived largely from structural studies of the soluble fragments and from cellular studies with inhibitors and antibodies, is still incomplete because it lacks extension to a high-resolution picture of the complete Env trimer in the context of a lipid-bilayer membrane, which is the substrate of the fusion reaction. We have determined by NMR the structures of the HIV-1 Env transmembrane domain (TMD), membrane proximal external region (MPER), and cytoplasmic tail (CT) in bicelles that mimic lipid bilayers. These regions all form well-ordered, trimeric clusters in a lipid bilayer. Disruption of any of them can reduce membrane fusion efficiency and alter the antigenic structure of the entire Env, suggesting that they have structural and functional roles in fusion and in trimer conformational stabilization. Very recently, we have completed a high-resolution structure of the intact SARS-CoV-2 postfusion spike in membrane, showing how the functionally critical membrane-interacting regions interact with membrane and with each other. These findings are the basis of our overall hypothesis that that structures of the HIV-1 fusion complex either alone or bound with fusion inhibitors in membrane will reveal new structural features of the membrane-interacting regions and substantially advance our mechanistic understanding of the viral fusion and its inhibition, thereby informing future development of intervention strategies. We will apply advanced technologies in cryogenic electron microscopy (cryo-EM) and tomography (cryo-ET) to study structural and functional properties of the HIV-1 fusion complex, as reconstituted in membranes and on the surface of a virus particle. We will also investigate molecular mechanisms of HIV-1 inhibition by two distinct types of fusion inhibitors. Our goal is a "molecular movie" of HIV-1 fusion, to inform development of new intervention strategies. We propose the following Specific Aims to address our hypothesis: 1) We will determine structure of the HIV-1 fusion complex containing intact Env, CD4 and CCR5 in the context of a lipid bilayer. 2) We will investigate molecular mechanism of HIV-1 fusion inhibition by anti-CD4 antibody ibalizumab. 3) We will dissect mechanism of action of small-molecule fusion inhibitors targeting the MPER of HIV-1 Env.

NIH Research Projects · FY 2025 · 2024-05

Project Summary/Abstract Celiac disease is a chronic immune disorder driven by gluten that typically develops in childhood. Over 1% of the world population is affected, many of whom are undiagnosed. Many more have an uncertain diagnosis because current diagnostic tests are not reliable in individuals following a gluten-free diet. Therefore, there is a strong need for better diagnostic methods that reflect the underlying causes of celiac disease and overcome the limitations of existing tests. Previous studies have shown that an increase in a cytokine called interleukin 2 (IL- 2) is the earliest and most sensitive marker of acute gluten ingestion in adults with celiac disease who are on a gluten-free diet. This method is not practical for diagnosis because patients who are on a gluten-free diet do not want to suffer symptoms of gluten ingestion and it is impractical for patients who are eating gluten to go on a gluten-free diet treatment just to confirm the diagnosis. An alternative to challenging patients with gluten is to challenge the immune cells in blood with gluten. In over 300 adults with celiac disease on a gluten-free diet, an ex vivo whole blood gluten challenge assay in which IL-2 is measured after blood has been incubated with gluten had near perfect specificity and high sensitivity for adults with celiac disease treated with a gluten-free diet. There is a need to conduct studies in children because celiac disease typically develops in this age group. As well, there may be age-related differences in immune responses. Therefore, the main goal of this research grant is to further investigate the immune response to gluten in children with celiac disease using the ex vivo gluten challenge whole blood assay. A cohort of 80 children with celiac disease (40 treated and 40 untreated) will be recruited. In Aim 1, the sensitivity of IL-2 rise in the ex vivo gluten challenge whole blood assay will be determined in the overall cohort. In a pre-planned secondary analysis, sensitivity in treated and untreated celiac disease will be compared. In Aim 2, an immune response signature of ex vivo gluten challenge will be determined using a panel of cytokines known to be elevated following oral gluten challenge (CCL20, CXCL9, IFN-γ, IL-2, IL-8, IL-10, IL-17A, IL-22, IP-10, and TNF-α). The sensitivity of each cytokine for detecting celiac disease will be determined as well as whether using a combination of cytokines improves the accuracy of the test. This novel study will determine whether IL-2 release in ex vivo gluten challenge whole blood assay is a sensitive marker of celiac disease in children. Critically, it will also be determined whether ex vivo gluten challenge evokes an immune response in children with untreated celiac disease. Results of this study will guide protocol development for a multi-center clinical trial to determine the diagnostic accuracy of cytokine release following gluten challenge for diagnosis of celiac disease. Results of this trial may redefine celiac disease and revolutionize diagnosis.

NIH Research Projects · FY 2026 · 2024-05

ABSTRACT During fetal skin development, over thirty distinct cell types emerge to form a solid barrier capable of immune surveillance and sensory detection. Due to its complexity, the skin has been challenging to reproduce in cultures. Maintenance of excised skin has only been possible for short durations. Yet, a longer-term skin culture system could greatly benefit efforts to model congenital diseases, investigate the mechanisms of cancer initiation, or mimic the site of infection, inflammation, and wounds. A critical obstacle to progress has been our inability to identify culture conditions that satisfy the metabolic needs of cells found in every skin subdomain, including skin appendages, such as hair follicles and sweat glands, and accessory structures, such as vasculature and nerves. Incorporating diverse immune cell populations has evaded previous engineering attempts. Recently, we invented a novel culture system that uses human pluripotent cells (hPSCs) to generate full-thickness skin with many of the cellular components of normal skin, including epidermis, dermis, hair follicles, and sensory nerves—collectively known as skin organoids. Although an improvement over previous skin models in terms of completeness, the resulting skin arises as a massive floating tissue that is challenging to monitor over time in culture and is devoid of immune cells. Here, we will build on preliminary data showing that skin organoids can reformat onto easily imaged microfluidic chips that we have maintained for over 100 days without apparent tissue degradation. For Goal 1, we will optimize a manufacturing process for creating hair-bearing skin organoids-on-a-chip by iteratively evaluating chip geometry, matrix composition, mechanical properties, and chemical treatments supplied to the developing tissue. We will use quantitative imaging and single-cell and spatial transcriptomics to assess the quality of our chip designs. For Goal 2, we will evaluate a novel chip design that better integrates neuro-vascular structures into the skin organoid chips. For Goal 3, we will build on exciting preliminary data to integrate myeloid and lymphoid lineage cells into developing skin organoid chips. We will define the proper timing and medium requirements for immune cell seeding and assess the fidelity of immuno-competent skin organoid chips through direct comparison to fetal histological specimens. We will test the system by simulating immune reactions due to hPSC donor incompatibility and bacterial infection. These experiments will yield chips with stereotyped neural inputs and vascular networks. Our collaborative team has strong bioengineering, material science, neuroscience, and immunology expertise and is uniquely suited to execute the project goals. Our research strategy considers how others could adopt our methodology, and we include plans for beta-testing tissue chip production in less equipped laboratories. We anticipate that our skin organoid tissue chips will provide researchers with a powerful new tool to watch and learn as human skin develops under normal and diseased conditions.

NIH Research Projects · FY 2026 · 2024-05

PROJECT SUMMARY/ABSTRACT Mural cells, including pericytes and smooth muscle cells, are critical for vascular development, function, and stability. Dysregulation of mural cells can lead to vascular abnormalities, emphasizing the need for generating functional mural cells to explore novel therapeutic approaches in vascular disorders, tissue repair, and regenerative medicine. Human induced pluripotent stem cells (iPSCs) offer a promising method for obtaining patient-specific mural cells; however, conventional chemical-based differentiation methods are limited in scope and precision. Inducible transcription factors (TFs) have gained traction as a differentiation strategy, offering precise temporal control and the potential for simultaneous differentiation of multiple cell types. However, identifying TFs for mural cell differentiation remains challenging. Our overarching goal is to develop TF-driven strategies for the effective differentiation of human iPSCs into competent vascular cells for regenerative medicine. Our group previously demonstrated the successful generation of vascular endothelial cells (iECs) from iPSCs using ETV2, a pioneer TF. More recently, we identified that another TF, NK3 Homeobox 1 (NKX3.1), facilitates the generation of mural progenitor cells (iMPCs) from iPSCs. These iMPCs display crucial mural cell characteristics and mature into fully differentiated mural cells upon interaction with ECs. Our group has also developed a novel vascular organoid (VO) model that allows concurrent co-differentiation of iPSCs into iECs and iMPCs and facilitates the maturation of iMPCs. Our central hypothesis is that NKX3.1 dictates mural cell lineage fate, and its activation can effectively generate iMPCs, introducing a novel method for creating an unlimited supply of functional mural cells for regenerative medicine. To test these hypotheses and elucidate the mechanisms underlying NKX3.1-driven mural cell differentiation and maturation, we propose three specific aims. In Aim-1, we seek to elucidate the transcriptional regulation of iMPC specification using NKX3.1. Aim-2 delves into the mechanisms of mural cell maturation in the VO model, examining how EC interactions affect iMPC maturation. In Aim-3, we seek to evaluate the therapeutic potential of iMPCs in ischemic tissues, determining whether iMPCs can act as a source of functional perivascular cells in vivo, supporting EC engraftment and contributing to the stability of newly generated blood vessels. We anticipate that these investigations will uncover the molecular underpinnings of mural cell lineage determination and functionality, ultimately advancing our understanding of mural cell development and therapeutic potential. This knowledge will pave the way for patient- specific strategies for treating vascular disorders and advancing vascular regenerative medicine.

NIH Research Projects · FY 2026 · 2024-05

Project Summary Nothing is more central to the human experience than memory, and no human capacity is more devastating when it is lost. Memory has been intensely studied in basic and disease research for many decades but to my knowledge we do not have any physical explanation of how memories alter complex behavior. This is in large part because we do not have a mechanistic explanation of how most behaviors are executed in the first place, but also because memory formation is best understood in brain regions like the hippocampus, which are far removed from action control centers, which themselves are not well understood in the mammalian brain. Drosophila courtship behavior stands out from other complex behaviors because it is exceptionally well understood at the circuit level. Here I describe a new and robust paradigm for courtship learning in which the memories appear to be formed, stored, modified, implemented, and erased within core courtship circuitry. In both insects and mammals, dopamine has long been known to translate memory-relevant information from the outside world into an internal teaching signal. In our new courtship learning paradigm we find that dopamine signaling is both necessary and, under the appropriate circumstances, sufficient for memory formation. I propose to test the hypothesis that dopamine-induced memory is written directly within courtship circuitry, likely within a set of command neurons called P1. We will identify the specific dopaminergic neurons required to write the memory, as well as localize the neurons that receive the dopamine signal. We will then use our established activity monitoring and behavioral assays, together with the courtship wiring diagram, to understand how memory alters the flow of information through behavioral circuitry. The results will provide new insights into how memories directly impact behavioral decision-making, a goal that has been unachievable in systems lacking detailed behavioral circuit maps. The conserved role of dopamine in memory writing suggests that our findings will generalize to mammals. Learning usually requires repetition or extreme events. This prevents the over-generalization that might occur with single-trial learning. By analyzing the contributions of various known components of the courtship circuitry to memory formation, we find a striking and novel role for a group of neurons, called mAL, in setting a threshold for memory formation. mAL stimulation can even erase old memories, but only while they are being recalled. I believe this is the first circuit-level manipulation that has been shown to disrupt memory reconsolidation. I outline experiments designed to leverage this new behavioral paradigm and its circumscribed neuronal populations to understand the nature of memory thresholding, maintenance, and removal in behavioral circuitry at the molecular, circuit, and physiological levels. We will work to establish the new and potentially transformative hypothesis that within populations of neurons that inhibit the execution of behaviors, there exist subsets of neurons that can limit or even erase memories associated with those behaviors.

NIH Research Projects · FY 2026 · 2024-05

Abstract/Project Summary Myelination facilitates rapid axonal conduction, enabling efficient communication across different parts of the nervous system. Demyelination associated with CNS trauma or diseases such as multiple sclerosis (MS) and glaucoma contributes significantly to behavioral deficits. Despite tremendous progress in understanding regulatory mechanisms of myelination, there are no pro-myelination treatments in the clinical setting. In our recent studies, using optic nerve/tract injury models, we discovered that injured axons could regenerate following intervention to elevate the regenerative ability of retinal ganglion cells (RGCs), yet these regenerated axons fail to be myelinated. Further, we showed that oligodendrocyte precursor cells (OPCs) do proliferate but fail to differentiate and mature into myelinating oligodendrocytes in response to ONC. With these unique models, we further demonstrated that blockade of muscarinic receptor 1 (M1R, or Chrm1) or GPR17 promoted OPC differentiation while depleting activated microglia facilitated the maturation or survival of newly formed oligodendrocytes. Thus, at least two overarching mechanisms contribute to the observed myelination failure: OPC intrinsic mechanisms preventing OPC differentiation, and microglia-relevant factors inhibiting the formation of mature oligodendrocytes by unknown mechanism(s). Importantly, treatments acting on these mechanisms promoted de novo myelination of regenerated axons and point to potential translatable pro- myelination strategies. In this application, following up with these initial findings, we will investigate the following questions: how do M1R and GPR17 regulate OPC differentiation? Do they work within the same signaling pathway(s)? How do activated microglia affect oligodendrocytes? Does treatment-induced de novo myelination improve behavioral outcome(s)? We expect that our studies will inform the mechanisms and therapeutic potential of myelination regeneration and visual restoration.

NIH Research Projects · FY 2026 · 2024-05

PROJECT SUMMARY Brain malformations comprise a group of genetic developmental brain disorders that present in childhood with epilepsy, intellectual, and other neurologic features, causing substantial morbidity, mortality, and health care costs. To date, mutations in hundreds of genes have been linked to brain malformations. The long-term goal of this project is to create a resource of well-categorized and expertly curated genes and variants responsible for causing brain malformations, to help clinicians navigate diagnosis and inform management. To this end, we have assembled an expert panel of clinician-investigators with broad, complementary expertise in brain malformations in the domains of gene discovery, neurobiology, clinical phenotype, radiographic presentation, and treatment. This panel will survey the literature to curate genes and variants associated with brain malformations, assess the strength of the evidence for these associations using ClinGen criteria, and organize them into biologically and clinically useful groups. They will work together with the ClinGen infrastructure and partner with biocurators from the Broad Institute to curate brain malformation genes through the Brain Malformations Gene Curation Expert Panel and variants in these genes through the Brain Malformations Variant Curation Expert Panel. A special focus of this group is the development of rigorous approaches to assess brain malformations caused by somatic mutations, a molecularly and clinically distinct subgroup of disorders (including those associated with focal cortical dysplasia (FCD), hemimegalencephaly (HME), and polymicrogyria (PMG) with megalencephaly, all recently associated with de novo germline or post-zygotic variants that result in activation of the mTOR-PI3K-AKT pathway) for which there are potentially important implications for treatment. Having created a blueprint for mosaic variant curation with recently published curation rules, this project will foster best practices in the clinical application of genomics to the care of patients with brain malformations: selecting appropriate diagnostic testing, interpreting variants, and ultimately choosing appropriate management.

NIH Research Projects · FY 2025 · 2024-05

PROJECT SUMMARY This proposal concerns the broad question of how relevant information is extracted from the environment and formatted according to animal needs. We focus on the process by which light synchronizes the circadian clock with the solar day and thus sets appropriate patterns of physiology and gene expression throughout the body. In mammals, this process requires neurons in the eye that sense light directly using a molecule called melanopsin and indirectly by receiving input from retinal circuitry. These melanopsin neurons wire into the principal circadian clock—the suprachiasmatic nucleus of the brain—via the retinohypothalamic pathway. We have developed new methods of investigating this pathway ex vivo under naturalistic conditions. First is a means of identifying melanopsin neurons that maintains their maximum photosensitivity (both intrinsic and extrinsic). Second is an experimental preparation in which the retina and suprachiasmatic nucleus retain functional connectivity. Using these methods, we will investigate how photic information arises in the retina and acutely drives neurons of the suprachiasmatic nucleus. We will focus on how this process produces a representation of the overall light intensity, which reflects the sun’s position in the sky and is used by the clock to maintain alignment with the day/night cycle. Specifically, we will ask how retinal and suprachiasmatic circuitry smooth away rapid fluctuations in light intensity, which tend to be uninformative for the clock (being caused, for example, by a passing cloud or flash of lightning). We expect that this work will provide knowledge of how neural processing is tailored to specific tasks and how environmental cues interact with internal states according to animal needs.

NIH Research Projects · FY 2025 · 2024-05

PROJECT SUMMARY Pediatric hydrocephalus is a life-threatening condition denoted by excessive cerebrospinal fluid (CSF) accumulation in the brain's ventricles. The most common causes of pediatric hydrocephalus are a previous infection or intraventricular hemorrhage from prematurity, which have been proposed to be driven by neuroinflammation. Recent in vivo experiments in adult mice have established that inflammation contributes to the development of hydrocephalus, and preliminary data that I have generated demonstrate that inflammation in utero also causes hydrocephalus. Human pathological studies and rodent models have highlighted that intraventricular macrophages are necessary to clear infection and blood but also may cause off-target damage. The most abundant macrophages in the ventricle are the epiplexus cells on the apical side of the choroid plexus (ChP), the essential brain barrier that synthesizes and regulates CSF composition. However, the core function of these macrophages remains elusive. Following innate immune activation, a diverse population of macrophages accumulates at the ChP, likely with distinct roles to upregulate or downregulate inflammation and prepare for tissue repair. Recent data from the Lehtinen lab has highlighted that the ChP epithelial cells express an essential macrophage survival factor, macrophage colony-stimulating factor (M-CSF), during homeostasis and is upregulated during inflammation. Additionally, differential expression of the M-CSF receptor, CSF-1R, is detected in macrophage populations at the ChP following innate immune activation. Despite the critical importance of brain resident macrophages for proper brain development and the deep understanding of microglia, the core functions of macrophage populations in the ventricle during homeostasis and neuroinflammation are still poorly understood. Is the ChP a source of M-CSF to maintain epiplexus macrophage survival required for core ChP function? Does the ChP M-CSF signaling regulate specific macrophage populations following in utero neuroinflammation? I will address these questions with innovative approaches to manipulate gene expression at the ChP in vivo and characterize macrophages at the ChP with single-cell RNA sequencing, histology, and advanced imaging techniques. I will uncover how the ChP regulates macrophages through M-CSF signaling during homeostatic and inflammatory conditions. Ultimately, I will test whether these populations can be therapeutically targeted with M-CSF gene therapy at the ChP to prevent inflammation-induced hydrocephalus. The training plan developed around this research proposal takes full advantage of the expertise in the Lehtinen lab and the unprecedented resources at Boston Children's Hospital to fulfill my goal of studying the core CSF and ChP biology. Ultimately this fellowship will enable me to establish the foundation for my future research program as a tenure-track professor in neurological infectious disease and neuroinflammation.