Boston Children'S Hospital

NIH Research Projects · FY 2026 · 2026-06

Project Summary Botulinum neurotoxins (BoNTs) are a family of bacterial toxins including seven serotypes (BoNT/A-G). They cause botulism and are classified as one of the most dangerous potential bioterrorism agents. BoNTs enter neurons and remain active in neurons for a long period of time, causing persistent paralysis in humans and animals. There is currently no inhibitor available that can block BoNT activity once they enter neurons, and such a post-symptom inhibitor is urgently needed for treating botulism patients and for biodefense. We previously developed an intraneuronal delivery protein based on a BoNT-like toxin, BoNT/X, which was discovered in our lab, with its receptor-binding domain replaced by the corresponding region of a BoNT. Such a chimeric protein can send anti-toxin nanobodies into motor neurons in vivo and reverse BoNT intoxication inside neurons. Besides BoNT/X, our lab recently uncovered a series of novel BoNT- like toxins, many of which exhibit biochemical properties for recombinant expression superior to those of BoNT/X. In addition, a series of new nanobodies against BoNT/A and BoNT/B have been generated, with higher binding affinity or neutralization efficacy than the previously available nanobodies. Therefore, in the exploratory R61 Phase, we propose to build and evaluate a new generation of delivery proteins based on novel BoNT-like toxins (Aim 1). We will also screen newly available nanobodies to identify those with higher efficacy as a cargo for intraneuronal delivery than the previous nanobodies (Aim 2). We will then fuse them to create at least one improved therapeutic protein that can reverse BoNT intoxication (our milestone for transition from R61 to R33 Phase). Once such a lead protein is finalized, we will then collaborate with the experienced team at the Biological Process Development Facility (BPDF) at the University of Nebraska – Lincoln to develop and establish the scale-up production process, analytical assays, and pharmacokinetic parameters (Aim 3). BPDF will then finish the large-scale (150-L fermentation) engineering production of a toxicity batch (Aim 4). Success of our proposal will generate and produce a first-in-class therapeutic protein that can reverse BoNT intoxication.

NIH Research Projects · FY 2026 · 2026-06

THRIVE – Abstract Extracorporeal membrane oxygenation (ECMO) is a life-saving therapy for children with severe cardiopulmonary failure, yet mortality remains high, and survivors often experience long-term complications, including kidney injury and neurodevelopmental impairment. ECMO introduces mechanical and biochemical stressors—including red blood cell (RBC) hemolysis, nitric oxide (NO) scavenging, and endothelial injury—that are further exacerbated by transfusion of stored RBCs. Despite widespread use, transfusion practices remain largely empirical, and the mechanisms by which hemolysis and transfusion contribute to organ dysfunction are poorly understood. We hypothesize that subclinical hemolysis and NO depletion drive ECMO-associated organ injury and that specific transfusion practices amplify this risk. This R01 application, titled THRIVE, will leverage the multicenter Trial of Indication-Based Transfusion of Red Blood Cells in ECMO (TITRE), which enrolled over 220 children and collected time-resolved plasma samples, transfusion metadata, and detailed outcome data. Aim 1 will quantify biomarkers of hemolysis and NO scavenging (e.g., free hemoglobin, haptoglobin, hemopexin, nitrite/nitrate, von Willebrand factor) and link them to clinical outcomes including survival, long term kidney injury (LKI), and validated neurodevelopmental measures. Aim 2 will assess how transfusion variables—volume, frequency, donor source, and storage age—modulate hemolysis, endothelial dysfunction, and organ injury. Aim 3 will apply high-throughput proteomics (SomaScan and LC-MS/MS) to define biomarker fingerprints predictive of ECMO complications, integrating these data through machine learning for early risk stratification and future therapeutic targeting. This project represents the first integrated mechanistic, transfusion-focused, and proteomic analysis of pediatric ECMO-related injury. By reframing hemolysis as a modifiable pathophysiologic driver and leveraging a unique multicenter biorepository, this work will generate actionable insights to inform precision-based transfusion and therapeutic strategies— ultimately aiming to improve survival and long-term outcomes for critically ill children.

NIH Research Projects · FY 2026 · 2026-06

PROJECT SUMMARY Brief resolved unexplained events (BRUE) are frightening episodes in previously healthy infants characterized by appearance of life-threatening respiratory symptoms, pallor, cyanosis, and limpness. These common events are resource-intensive and current management approaches inadequately address persistent symptoms. Infants with BRUE commonly have oropharyngeal dysphagia with aspiration, which is a modifiable risk factor for persistent symptoms, but appropriate diagnosis and treatment are necessary to prevent severe symptoms and recurrent hospitalization along with lasting risks of severe pulmonary sequelae. Diagnosis of aspiration requires videofluoroscopic swallow study (VFSS) in infants due to the high prevalence of silent aspiration in this age group, but this test is limited by radiation exposure, the need for patient cooperation for reliable results, and lack of speech language pathologists in under resourced areas. In order to address these challenges, there is great need for a non-invasive test for aspiration, which may be used to screen, diagnose and monitor the progress of swallow dysfunction in infants with BRUE. Studies in adults with oropharyngeal dysphagia have identified salivary substance P (SP) as a neuropeptide signaling molecule involved in the pathogenesis of swallow dysfunction, with reduced levels in patients with neurologic causes of dysphagia. Despite significant overlap in pathophysiology, no studies of salivary biomarkers have been performed in any pediatric population. The objective of the proposed research is to perform the first-ever study of novel biomarkers of aspiration in infants. Systematic investigation of the relationship between saliva biomarker levels and aspiration risk is urgently needed, as this approach has vast potential to optimize diagnosis of aspiration and thereby improve clinical outcomes and quality of life and reduce healthcare utilization. The innovative studies outlined in this proposal will allow us to define the link between salivary SP and aspiration in infants and test the hypothesis that these non-invasive biomarkers can inform our understanding of both aspiration risk and persistent symptom risk following BRUE. This proposal has three aims: (1) to validate, using a cross-sectional study design, biomarker levels with the presence and severity of swallow dysfunction; (2) to determine, using a prospective longitudinal cohort study design, if biomarker levels change within patients over time with improvement in swallowing skills; and (3) to determine, using a prospective longitudinal cohort study design, the link between biomarker levels and persistent aspiration symptoms after BRUE. This paradigm-shifting research will address a common and costly problem by defining the relationship between salivary SP levels and aspiration in infants. Ultimately, we anticipate successful biomarker identification will transform care for children with BRUE by offering a non-invasive approach for diagnosis and management of aspiration.

NIH Research Projects · FY 2026 · 2026-06

PROJECT SUMMARY/ABSTRACT Pediatric Amplified Musculoskeletal Pain Syndrome (AMPS) is becoming increasingly common, affecting between 11 – 38% of youth. AMPS is associated with significant stress, psychological impairment, and functional disability, resulting in tremendous healthcare costs. Moreover, AMPS conditions often persist into adulthood, increasing lifetime risk for psychiatric and medical comorbidities and earlier mortality. There is a critical lack of understanding of the processes involved in the onset, maintenance, and exacerbation of chronic pain conditions in youth, hampering efforts to develop interventions, which are currently limited and ineffective for a significant subset of patients. Comprehensive understanding of the biopsychosocial factors underlying chronic pain disorders like AMPS are needed to inform the development of novel, effective prevention and treatment approaches. Data suggest that altered stress physiology (e.g., allostatic load) may play a role in chronic pain conditions, with possible intergenerational implications. Parallel lines of research indicate that adversity exposure (e.g., abuse, violence), neurobiological functioning, and psychological impairment across youth-parent dyads contribute to the onset and maintenance of pain. The overall goal of the proposed project is to test an intergenerational model of chronic amplified musculoskeletal pain syndrome (AMPS) in youth and their biological mother/birthing parent that incorporates these constructs to elucidate mechanisms responsible for AMPS, thereby identifying potential prevention and intervention targets. The project aims will be accomplished by following a cohort of pediatric AMPS patient-parent dyads (N = 170) and healthy control – parent dyads (N = 85) over a 2-year period, repeatedly assessing pain characteristics (e.g., intensity, frequency), stress exposures, neurobiological functioning (conceptualized as allostatic load), psychological impairment, and psychosocial- biobehavioral resilience. Analyses will (a) identify the combination of neurobiological stress measures in isolation and cumulatively (as allostatic load) most predictive of AMPS symptoms in youth; (b) define correspondence of these measures in parent-child dyads; (c) identify parental characteristics (e.g., adversity history, parent mental health, parent stress neurobiology, parent-child relationship quality) most predictive of AMPS symptoms in youth; (d) identify psychosocial and biobehavioral resilience characteristics (e.g., coping style, social support, physical activity) that buffer the effects of intergenerational adversity exposure on pain characteristics; and (e) compare neurobiological stress processes in chronic pain patients and sociodemographically-matched healthy controls. The findings may be translated into the development of mechanistically informed prevention and treatment methods that target the most influential modifiable biopsychosocial pathways involved in AMPS and stress physiology and that may be personalized based on individual and familial risk profiles for maximum benefit. Studying these phenomena in youth-parent dyads is critical given that the majority of research in this area has focused on adult populations or with individually focused (i.e., pediatric pain patients) cross-sectional designs.

NIH Research Projects · FY 2026 · 2026-06

PROJECT SUMMARY/ABSTRACT Induced pluripotent stem cells (iPSCs) offer a promising platform for regenerative medicine, with their ability to self-renew indefinitely and differentiate into vascular cells. However, their clinical translation in vascular diseases is limited by major challenges: the impracticality of autologous therapy, immune rejection in allogeneic transplantation, and inefficient differentiation. Allogeneic iPSC-derived cells are rapidly recognized and eliminated by the host immune system, triggering both innate and adaptive immune responses. Additionally, current differentiation protocols yield vascular cells with low and inconsistent efficiency (~20%), limiting scalability and therapeutic viability. To overcome these challenges, this study aims to engineer hypoimmunogenic iPSC-derived vascular cells that evade immune detection while efficiently integrating into host vasculature. Simultaneously, spatial transcriptomics will be utilized to map immune- vascular interactions and uncover pathways that drive immune tolerance and vascular remodeling. To overcome differentiation inefficiencies, we have developed a transcription factor-driven strategy using ETV2 and NKX3.1, enabling >95% efficiency in generating endothelial and mural progenitor cells, respectively. Their ability to integrate and enhance perfusion will be tested in ischemic hindlimb models. The project comprises three aims. First, we will engineer hypoimmunogenic vascular cells by knocking out B2M/CIITA to eliminate highly polymorphic MHC expression, preventing adaptive immune recognition and overexpressing CD47 to prevent innate immune clearance. Second, we will assess vascular integration and perfusion enhancement in ischemic models by transplanting the engineered vascular cells. Third, we will apply multiplexed spatial transcriptomics to map immune-vascular interactions, identifying pathways that regulate immune evasion and vascular remodeling. This study will provide critical insights into immune-vascular dynamics, establishing a foundation for hypoimmunogenic iPSC-based therapies that achieve long-term immune tolerance and functional vascular regeneration in ischemic diseases. The training will occur under the mentorship of Dr. Juan Melero-Martin at Boston Children's Hospital/Harvard Medical School. I will be co-mentored by an extraordinary team of scientists on my advisory committee, including Dr. Torsten Meissner and Dr. Kaifu Chen, for new training goals in immune biology, vascular biology, transcriptomics as well as career guidance. Through this training, I will acquire advanced conceptual, technical, and professional skills, preparing me for an independent research career in translational regenerative medicine.

NIH Research Projects · FY 2026 · 2026-05

PROJECT SUMMARY The overarching goal of this research is to elucidate the genetic underpinnings of B cell activation dysregulation in lupus, a leading cause of morbidity among youth in the United States. By decoding the complex interactions at the genetic and cellular levels, this project aims to unveil new targets for therapeutic intervention, ultimately reducing the disease burden. The genetic contribution to lupus is high and genome-wide association studies (GWAS) have found >150 risk variants for lupus. However, linking GWAS risk variants to mechanism has been achieved for only a small proportion of loci given most of the likely causal variants are non-coding. Our preliminary data identified a relevant B cell activation condition at which a great proportion of lupus risk variants are likely exerting their regulatory effects. We have further preliminary data focused on 2 specific lupus risk loci, representing two classes of mechanisms by non-coding risk variants: one class that affects gene expression levels of the target gene, and one that affects isoform usage of the target gene. For each locus we plan to establish the most likely causal risk variant, the mechanism by which the risk allele causes dysregulation, and the function of the target gene in B cell differentiation. We plan to study these aspects in both healthy subjects and lupus patients, and by utilizing advanced technologies including single cell profiling, CRISPR methods, tonsil organoids and long read RNA sequencing. Furthermore, we plan to uncover additional loci with differential isoform usage driven by lupus risk loci. Overall, this research is poised to provide critical insights into the molecular mechanisms of lupus, offering potential strategies for targeted treatment. By enhancing our understanding of B cell biology in the context of chronic autoimmune disease, our findings could lead to the prioritization of novel genetic targets, laying the groundwork for innovative therapeutic approaches in lupus and other immune-mediated diseases.

NIH Research Projects · FY 2026 · 2026-05

PROJECT SUMMARY Sterile inflammation underlies a broad range of human diseases, including atherosclerosis and monogenic autoinflammatory disorders like Cryopyrin-Associated Periodic Syndromes (CAPS). A central driver of sterile inflammation is the NLRP3 inflammasome, a cytosolic multiprotein complex that mediates the release of the proinflammatory cytokine IL-1β. While inflammasome activation is protective in infections, dysregulated activation contributes to diseases. The classical model of NLRP3 activation involves a two-step process: priming, typically provided by microbial ligands through NF-κB signaling and posttranslational modifications, and activation by danger signals. However, the mechanisms driving inflammasome activation in sterile inflammatory diseases remain poorly defined. In my K99 phase, I will investigate how oxidized phospholipids (oxPAPC), which accumulate in atherosclerotic plaques, function as “all-in-one” sterile signals to both prime and activate the NLRP3 inflammasome in macrophages. Our preliminary data show that oxPAPC drives NRF2-dependent metabolic rewiring, leading to glycosylation of NLRP3 – a novel regulatory mechanism required for inflammasome priming and activation. I will dissect these mechanisms using a combination of structural biology, glycoproteomics, and spatial transcriptomics. This work will identify key posttranslational modifications controlling inflammasome activity and map the spatial organization of IL-1β–producing macrophages within atherosclerotic lesions. In the R00 phase, I will apply the conceptual and technical framework developed during the K99 to study CAPS, a group of rare autoinflammatory diseases caused by NLRP3 gain-of-function mutations. I hypothesize that CAPS-associated variants mimic or bypass the priming step through loss of inhibitory phosphorylation and competition with glycosylation. I will generate a novel inducible knock-in mouse model to investigate how these mutations alter inflammasome activation and tissue inflammation in vivo, combining single-cell transcriptomics and immunofluorescence to uncover immune–stromal crosstalk in inflamed skin and joints. By integrating studies of metabolic inflammation and monogenic autoinflammation, this project will uncover shared checkpoints of NLRP3 regulation, with the ultimate goal of identifying new therapeutic targets that suppress pathological inflammation while preserving host defense.

NIH Research Projects · FY 2026 · 2026-05

Project Summary Mammalian neonatal hearts have the ability to regenerate after ischemic injury, a capacity lost in adulthood. Understanding the mechanisms underlying the regenerative ability of neonatal cardiomyocytes (CMs) could offer critical insights into strategies for repairing injured adult hearts. Our current understanding of neonatal heart regeneration focuses primarily on transcriptomic regulation, despite proteins are the primary effectors of tissue repair. In this project, we investigate the mechanism that drives proteome remodeling and protein homeostasis maintenance during neonatal heart regeneration, which will provide insights into how neonatal CMs rapidly adapt to injury stress and balance protein anabolic and catabolic processes for self-renewal. We observed an acute increase in newly synthesized proteins and enhanced proteasomal activity in regenerative neonatal hearts post-myocardial infarction (MI), which is a response absent in non-regenerative hearts. While protein synthesis and degradation are crucial for tissue growth, the mechanisms that coordinate these processes to maintain protein homeostasis during tissue regeneration and how they are activated by injury remain largely unknown. Our preliminary data indicate that mTORC1 activation in regenerative mouse CMs drives increased protein synthesis following MI and is essential for neonatal heart regeneration. While mTORC1 is known to suppress autophagy, our findings suggest it also promotes protein degradation by regulating Nrf1, an ER-bound transcription factor that controls proteasomal gene expression. Our previous studies showed that Nrf1 is required for neonatal heart regeneration by regulating proteasome activity to mitigate proteolytic stress during injury. We hypothesize that the mTORC1-Nrf1 regulatory axis is activated in neonatal regenerative CMs to enhance both protein synthesis and degradation, promoting critical proteostasis and proteome remodeling during heart regeneration. In Aim 1, we test the hypothesis that mTORC1 activation is a prerequisite for the regenerative response of neonatal CMs after MI by driving rapid translatome remodeling during heart regeneration. Aim 1 also studies how amino acid metabolism, particularly through the LAT1 transporter, which is highly activated in neonatal CMs post-MI, contributes to regenerative mTORC1 activation. Aim 2 investigates the role of the mTORC1-Nrf1 axis in protein homeostasis and proteome remodeling during heart regeneration and examines the function of the proteasome in maintaining proteostasis and supporting heart regeneration. Aim 2 also investigates whether activating the mTORC1-Nrf1 axis can benefit adult heart repair. This proposal will provide important insights into heart regeneration by revealing the role of the mTORC1–Nrf1 axis in regulating protein homeostasis and proteome remodeling during neonatal heart regeneration, which has been overlooked by previous transcriptomic-focused studies.

NIH Research Projects · FY 2026 · 2026-05

Project Summary The goal of this proposal is to identify how Toll-like Receptors (TLRs) stimulate diverse cellular responses in macrophages and dendritic cells (DCs), and to understand how these responses influence DC-based cancer immunotherapies. The ability of TLRs to induce inflammatory gene expression has been under investigation for twenty years, with distinct signaling pathways mediated by the MyD88 and TRIF adaptors explaining all transcriptional responses. It has only recently become appreciated that TLRs also drive metabolic changes in responding cells, such as the rapid induction of aerobic glycolysis. During the previous funding period, we discovered that the TLR-induced myddosome complex contains two classes of proteins. One class is necessary for myddosome assembly (e.g. MyD88) and represents the core of this signaling structure. The second class is not necessary for myddosome assembly (e.g. TRAF6), but rather operates to recruit enzymes that diversify the effector functions of the myddosome. Specifically, we identified the kinase TBK1 as a myddosome component that is recruited by TRAF6 and is dedicated specifically to induce glycolysis. The myddosome therefore serves as a subcellular site of signals that activate diverse cellular responses. Our understanding of how these activities are regulated in vitro and their impact on T cell mediated protective immunity remains limited. In addition to the myddosome, select TLRs (e.g. TLR4 and TLR3) engage the triffosome. The central triffosome regulator is TRIF, which stimulates interferon (IFN) responses, NF-kB and MAPK activation, necroptosis and glycolysis. While the importance of TRIF in immunity has long-been recognized, the means by which it activates these diverse responses is unclear. This gap in knowledge is not merely an academic curiosity, as TRIF is essential for the ability of the LPS receptor TLR4 to stimulate adaptive immunity. Understanding regulatory events that stimulate myddosome- and TRIF-dependent responses will enable discussions of how TLRs drive protective immunity against infection and cancer. In this application, we propose to explore myddosome activities in vitro and in the context of cancer immunotherapies (Aim 1). In Aim 2, we offer an innovative synthetic biology-based approach to define the mechanisms of TRIF signaling and how these mechanisms relate to those induced by complementary innate immune pathways. Our focus on the two major signaling pathways activated by TLRs should provide an operational view of this important family of receptors.

NIH Research Projects · FY 2026 · 2026-05

Project Summary/Abstract: Congenital heart disease (CHD) affects nearly 1% of live births, yet the molecular mechanisms underlying many forms remain poorly defined. Left ventricular noncompaction (LVNC), characterized by excessive trabeculation and myocardial thinning, is associated with defects in endocardial signaling. Recent data suggest that endocardial G protein-coupled receptor (GPCR) trafficking plays a crucial role in modulating cardiogenesis. My research identifies sphingosine-1-phosphate receptor 1 (S1PR1), a GPCR, as a key regulator of Notch1 signaling during late heart development. I discovered that excessive internalization of S1PR1 reroutes cleaved Notch1 from the nucleus to lysosomal degradation, disrupting Notch signaling and contributing to LVNC pathogenesis. The proposed research will elucidate the spatiotemporal regulation and functional consequences of endocardial S1PR1 internalization using advanced mouse genetics and biochemical tools. Specifically, I will (Aim 1) determine the impact of endocardial-specific S1PR1 gain-of-function on trabecular remodeling and Notch activity; and (Aim 2) evaluate whether targeted pharmacologic inhibition of S1PR1 rescues defective endocardial signaling and cardiac morphology. This K99/R00 project is embedded in a rich and collaborative environment at Boston Children’s Hospital and Harvard Medical School under the mentorship of Dr. Timothy Hla, a pioneer in S1P biology. My Advisory Committee includes leaders in developmental cardiology and vascular biology. During the mentored phase, I will expand my expertise in GPCR signaling, single-cell transcriptomics, and in vivo cardiac phenotyping. I will also receive structured training in grant writing, lab management, and scientific communication. My long-term goal is to become an independent investigator leading a research program on GPCR signaling and endocardial biology in heart development and disease. The K99/R00 award will facilitate my transition to independence and support a research trajectory with direct implications for understanding and treating CHDs.

NIH Research Projects · FY 2026 · 2026-05

PROJECT SUMMARY/ ABSTRACT Blood vessels are the earliest organs formed during embryonic development, and the embryonic cardiovas- cular system is required for nutrient exchange and organogenesis. As early as mouse embryonic day (E) 7.5 in the extraembryonic yolk sac blood islands and embryos, mesodermal precursors undergo the first cell fate de- cision to form the endothelial cell (EC) lineage. ECs further differentiate into arterial, venous, and lymphatic subtypes that are necessary to build the vascular system. However, the clonal relationships and spatial origins of EC subtypes that form the vasculature are poorly defined, and the mechanisms of EC specification and dif- ferentiation in vivo remain incompletely understood, posing a barrier to vascular regenerative medicine. Etv2, an ETS-family transcription factor, is a master regulator of EC specification. Etv2 is transiently ex- pressed in mesoderm progenitors in the yolk sac and developing mouse embryo from E7.5 - E9.5. Mice lacking Etv2 fail to develop blood or vasculature, and Etv2 is responsible for endothelial and hematopoietic lineage specification. Conversely, forced expression of Etv2 induces EC reprogramming. We have carefully interro- gated mechanisms by which ETV2 promotes EC specification in mesoderm progenitors derived from human induced pluripotent stem cells (iPSCs), using scRNA-seq, scATAC-seq, CUT&RUN, and functional CRISPR screens. Novel observations included: (1) the strength or timing of ETV2 expression influenced arteriovenous EC differentiation; (2) in addition to stimulating EC specification, ETV2 also suppressed specification of other mesodermal lineages; and (3) ETV2 suppression of alternative fates required its recruitment of the transcrip- tional repressor REST. These observations lead to our central hypothesis that ETV2 drives EC specification and arteriovenous differentiation, with REST cooperating with ETV2 to restrict alternative lineages during em- bryonic vasculogenesis. To track cell state transitions and fate of Etv2-expressing cells during vasculogenesis, we will integrate cut- ting edge single cell, spatial transcriptomics, and barcoded lineage tracing approaches to track the progeny of Etv2-expressing progenitors. To focus our efforts, in Aim 1 we will study the initial steps of vessel formation in the yolk sac. We will perform spatial and clonal analysis of Etv2-lineage cells during early yolk sac vasculogen- esis and late vascular plexus remodeling. To determine the role of Rest in Etv2-directed EC specification, in Aim 2 we will determine the effect of Rest inactivation on vasculogenesis and Etv2-lineage diversification, un- cover the Rest-Etv2 transcriptional regulatory network, and identify Rest-regulated genes and TFs in Etv2-line- age progenitors required for EC specification. The work will produce a high-resolution phylogenetic tree of vas- cular development originating from individual Etv2+ progenitors and bring new insights into the molecular mechanisms that direct EC subtype differentiation, informing future efforts in vascular regeneration.

NIH Research Projects · FY 2026 · 2026-05

PROJECT SUMMARY The earth's 24 hour light/dark cycle imposes predictable environmental variability to the nervous system. To synchronize internal cellular and organismal states with this daily geophysical oscillation, a multi-scaled circadian clock orchestrates global behaviors such as sleep/wake as well as circuit-specific behaviors like associative learning. Disruption of circadian rhythms and sleep loss are endemic in technological cultures and have been directly linked to sleep disorders, neurodegenerative and neuropsychiatric disorders. Since behavioral plasticity is rooted in the ability of synapses to adjust their computations, a crucial goal is to define how circadian rhythms regulate synaptic function to modulate behavior with the time of day. On the molecular level, circadian timing is rooted in a transcriptional-translational feedback loop present in all cells driven by the transcription factor BMAL1. In this collaborative proposal, we, a circadian biologist and synapse neuroscientist, capitalize on our recent discovery that BMAL1 is rhythmically localized to synapses in a manner dependent on its phosphorylation at Ser42 (pBMAL1S42). pBMAL1S42 regulates the timing of the key synaptic kinase CaMKIIα. We have engineered phosphorylation-incompetent Bmal1S42A mice, that strongly support roles for pBMAL1S42 in orchestrating synaptic vesicle (SV) dynamics, hippocampal memory, and sleep/wake. We have found that pBMAL1S42 is potentiated by serotonin, a crucial neuromodulator of SV dynamics, synaptic plasticity, and sleep/wake behavior. Based on these preliminary data, we hypothesize that synaptic BMAL1 – whose phosphorylation is regulated by serotonin – regulates CaMKIIα, SV dynamics, and sleep need in a phosphorylation-dependent manner. In Aim1, we will dissect the structure-function relationship between BMAL1 and CaMKIIα and define how BMAL1 modulates the biochemistry and biophysics of CaMKIIα to regulate presynaptic function. The goal of Aim 2 is to define the mechanisms by which serotonin potentiates pBMAL1S42 in brain to regulate SV dynamics. In Aim 3, we will define how pBMAL1S42 coordinates SV dynamics and serotonin signaling to consolidate wakefulness at the right time of day. Together, these experiments will expand our fundamental knowledge about the mechanisms by which the circadian clock facilitates behavioral plasticity by orchestrating foundational processes of neurotransmission and computation. Our proposal will provide new insights into the complex relationships between circadian rhythms and the nervous system and should have generalizable applicability to disorders like Alzheimer's disease and schizophrenia, which commonly share defects in serotonin signaling, circadian rhythms, and synaptic function.

NIH Research Projects · FY 2026 · 2026-05

Project Summary/Abstract Chronic glucocorticoids can suppress the hypothalamic-pituitary-adrenal (HPA) axis for up to a year after steroids are withdrawn, rendering patients vulnerable to life-threatening adrenal crises. In humans, this has been largely attributed to prolonged hypothalamic-pituitary suppression after glucocorticoid withdrawal. In a novel preclinical model of glucocorticoid-induced adrenal insufficiency (GIAI), we demonstrated that mice have delayed recovery of corticosterone (CORT) secretion after chronic dexamethasone (DEX) treatment despite early restoration of hypothalamic-pituitary function. Previously undescribed, giant macrophages infiltrated DEX- exposed adrenals, but CORT secretion was disproportionately low relative to the size of the adrenal cortex even after excluding these macrophages for up to 2 weeks after DEX withdrawal. Having established the adrenal as the major site of post-withdrawal GIAI, we then tested whether trophic support to the gland during the period of DEX exposure prevented GIAI. CORT secretion recovered more slowly in mice treated with dexamethasone and daily, intraperitoneal cosyntropin vs. DEX alone. However, maintenance of endogenous ACTH signaling during the period of DEX exposure using transgenic mice preserved adrenal mass and function on long-term steroids. Aim 1 of this grant tests two potential mechanisms of ACTH hypo- responsiveness after cessation of chronic glucocorticoids. First, we hypothesize that actively replicating zona fasciculata (zF) cells downregulate steroidogenic pathway genes as a trade-off between proliferative vs. biosynthetic function (Aim 1a). This will be assayed in a spatially informed, single-cell manner using fluorescent in situ hybridization for steroidogenic pathway genes multiplexed with immunofluorescence for proliferative markers. Aim 1b tests the hypothesis that activated, Trem2-positive adrenal macrophages contribute to post- withdrawal GIAI via paracrine suppression of StAR, the rate limiting enzyme in CORT biosynthesis, in zF cells. CORT secretion and StAR expression will be measured one week after discontinuing chronic DEX in mice following 1) global macrophage depletion with a CSF1R-blocking antibody, and 2) macrophage-specific Trem2 deletion vs. controls. In Aim 2b, we aim to develop a pharmacologic strategy that phenocopies our prior genetic model and prevents GIAI by providing constant, potentially CRH-driven, ACTH stimulation to the adrenal during the period of glucocorticoid exposure. Mice will be treated with long-term DEX and either constant a) CRH, b) cosyntropin, or c) vehicle via osmotic minipump. HPA axis function and histology will be assessed at the time of and 1 week after treatment completion. Pharmacokinetic studies will quantify systemic ACTH exposure on treatment. In Aim 3, we investigate whether early hypothalamic-pituitary recovery occurs in children withdrawn from chronic glucocorticoids, as observed in mice. Early, post-withdrawal ACTH levels will be compared to controls, and median time to axis recovery will be calculated. These studies may collectively inform the development of therapies that prevent or expedite recovery from GIAI.

NIH Research Projects · FY 2026 · 2026-05

Functions and mechanisms of a molecularly defined prefrontal cortex neuron subtype in drug addiction Abstract Drug addiction is a chronic, relapsing brain disorder characterized by compulsive drug seeking and use despite harmful consequences. It is an urgent social and health problem contributing to more than 90,000 deaths and incurs a yearly cost of over $700 billion in the United States (see NIDA website). Yet, there is no treatment due to the lack of molecular understanding of addiction pathophysiology. The prefrontal cortex (PFC), a main cortical center regulating reward, is one of the key brain regions that regulate voluntary drug taking. Dysfunction of PFC is well-known in addiction. However, the PFC is also a center for cognitive and executive function. Its tremendous molecular, cellular and anatomical heterogeneity makes it challenging to identify specific neuronal types and circuits modulating addiction that can be selectively targeted. To overcome this problem, we have used single cell RNA sequencing and spatial transcriptomic technique MERFISH and decoded the heterogeneity of the mouse PFC by identifying the molecular composition and anatomical location of all constituent neuronal subtypes. We have also identified a distinct L5 neuron subtype, uniquely marked by the expression of the Pou3f1 gene, that sends specific projections to the midbrain peri-aqueductal gray (PAG) to be responsive to cocaine taking. We further generated a Pou3f1-Cre mouse line, and demonstrated in an intravenous cocaine self-administration (IVSA) mouse model that chemogenetic activation of the Pou3f1+ neurons reduced the drug seeking behavior supporting that the Pou3f1+ neurons play an important role in addiction pathogenesis. To understand the functions and mechanisms of how Pou3f1+ neurons and the PFCàPAG circuit regulate addiction, we have established the following specific aims: 1) To dissect the role of the L5 PFC Pou3f1+ neurons in chronic drug abuse and relapse; 2) To map the downstream circuit of Pou3f1+ neurons that modulate addiction behavior; 3) To identify molecular changes in the Pou3f1+ neurons and the PFC at large, under chronic drug abuse. Completion of the proposed study will not only reveal how the PFC Pou3f1 neuron subtype regulates addiction, but also provide potential targets for therapeutic intervention.

NIH Research Projects · FY 2026 · 2026-05

Project Summary Mitochondria play a crucial role in maintaining neuronal health and function. Dysfunction of these organelles leads to various neurological diseases. Retinal ganglion cells (RGCs), the primary output neurons in the retina, are particularly vulnerable to mitochondrial damage. The two most common hereditary optic neuropathies characterized by RGC degeneration, Autosomal Dominant Optic Atrophy (ADOA) and Leber Hereditary Optic Neuropathy (LHON), both stem from mitochondrial dysfunctions. ADOA arises from mutations in OPA1, which regulates inner mitochondrial membrane fusion, while LHON is caused by mutations in the complex I subunit genes encoded by the mitochondrial genome. Currently, no effective treatments exist for either condition, underscoring a critical unmet need to unravel the disease mechanisms and develop therapies to safeguard RGCs from degeneration. The project’s significance lies in investigating the role of SARM1, a trigger of neurodegeneration, in mitochondria-induced RGC degeneration. Our lab has built a novel ADOA mouse model carrying the pathogenic Opa1R290Q/+ mutation. This model recapitulates key features of human ADOA, including mitochondrial fragmentation, aberrant glutathione redox, age-related RGC degeneration, and declines in RGC function. We found that knocking out Sarm1 in these ADOA mice nearly completely protects against all the degenerative phenotypes, suggesting that SARM1 activation drives RGC death in ADOA. Given the similarities between ADOA and LHON, we hypothesize that the same mitochondria-SARM1 pathway also contributes to LHON pathology. Therefore, the central hypothesis of the project posits that mitochondria-induced SARM1 activation leads to RGC death in both ADOA and LHON, and inhibiting SARM1 represents a promising therapeutic approach. Aim 1 of the proposal aims to identify specific mitochondrial defects triggering SARM1 activation in OPA1 mutant RGCs, and elucidate the underlying mechanisms. Aim 2 seeks to establish a dominant-negative SARM1-based therapeutic approach in ADOA mice. During the R00 phase in Aim 3, I will characterize a LHON mouse model and examine whether Sarm1 KO provides protective effects. I have assembled an advisory committee to provide conceptual and technical guidance as I pursue this study. Furthermore, I have also formulated a comprehensive training and career development plan to be executed during the grant period. This integrated proposal, encompassing the research plan and mentoring activities, will provide me with a solid foundation to embark on an independent academic career.

NIH Research Projects · FY 2026 · 2026-05

Summary/Abstract This R00 proposal describes a three-year research plan that will facilitate the transition of Dr. Patricia Davenport to an independent academic researcher in the field of neonatal platelet (PLT) biology and transfusion medicine. Dr. Davenport has shown herself to be a productive and dedicated early physician scientist who completed her post-graduate training in neonatal-perinatal medicine. PLTs are active participants in both hemostasis and inflammation, yet the clinical awareness of their immune properties has lagged behind research discoveries. Preterm infants are at high risk of spontaneous bleeding and receive PLT transfusions (Txs) at higher PLT counts than adults or children in hopes of preventing bleeding, but without consideration of their inflammatory activity. The PlaNet-2 trial, the largest randomized trial of PLT Tx in preterm neonates, found that liberal PLT Txs increased neonatal mortality and risk of bronchopulmonary dysplasia (BPD), or chronic lung disease of prematurity. The two-year outcomes from this trial were recently published and found an increased incidence of death or major neurodevelopmental disability, as well as increased need for supplement oxygen, again in the infants randomized to the liberal compared to the restrictive PLT Tx threshold. The mechanisms mediating the harmful effects of PLT Txs on lung and brain development are unknown and will be the focus of this proposal. When transfused, neonates receive adult PLTs, which are known to be hyperreactive and potentially more pro- inflammatory compared to neonatal PLTs. The overarching goal of this proposal is to improve the management of neonatal thrombocytopenia through a better understanding of the organ-specific consequences of PLT Txs given to neonates with different underlying pathologies. The overall hypothesis is that adult PLT Txs act as a “second hit” to an initial inflammatory stimulus to trigger or amplify neonatal immune and inflammatory responses, resulting in neonatal lung and brain injury. This hypothesis will be tested with the following specific aims: 1.To characterize the neonatal hepatic response to PLT Tx and how it contributes to the systemic inflammatory state, 2. To establish the mechanisms through which PLT Txs worsen neonatal lung injury and promote the development/severity of BPD, and 3. To determine the effects of PLT Tx in a mouse model of neonatal brain injury. These studies have high clinical and translational relevance, with the potential to lead to clinical practice change. The Division of Newborn Medicine at BCH is committed to Dr. Davenport’s success and has assured at least 75% protected time to devote to the activities described in this proposal. The expertise and knowledge gained from this R00 Award will enable Dr. Davenport to obtain future R01 funding and transition to an independent research career focused on understanding the mechanisms underlying the harm associated with neonatal PLT Tx.

NIH Research Projects · FY 2026 · 2026-04

Project Summary The transcription regulator Aire plays a pivotal role in immune tolerance by enforcing the expression of peripheral tissue antigens (PTAs) in medullary thymic epithelial cells (mTECs), selecting autoreactive T cells for clonal deletion or regulatory T cell differentiation. As such, defects in Aire result in manifestations of autoimmune polyglandular syndrome type 1 (APS-1), a form of primary immune deficiency diseases. The goal of this grant is to elucidate the molecular mechanism by which Aire engages with PTA loci and activates their transcription. Studies in mouse mTECs revealed that Aire preferentially binds to super-enhancers (to be referred as group- 1 targets), but predominantly induces genes located outside these regions (group-2 targets). However, Aire ChIP signals at group-2 loci are weak or undetectable. This raises fundamental questions about the relationship between group-1 and group-2 targets and how group-2 genes are activated by Aire. Our recent study suggest that group-1 and group-2 targets are mechanistically distinct. Group-1 loci are bound by Aire through direct interaction with pre-clustered p300/CBP and exhibit high level of basal transcription, which is modestly increased by Aire. These sites serve as a nucleation platform for Aire to form condensates, connecting multiple active loci into a transcription hub. In contrast, group-2 genes, including many PTA genes, are transcriptionally silent prior to Aire expression, and strongly induced by Aire with ordered stochasticity. Importantly, we now have multiple lines of evidence suggesting that these group-2 genes are not secondary to group-1 gene expression. Instead, Aire-p300/CBP condensates may directly contribute to group-2 gene activation. Based on our preliminary findings, we hypothesize that group-2 genes are also direct targets of Aire but are activated through transient interactions with Aire transcriptional condensates formed at p300/CBP-rich group-1 loci. We here propose two specific aims to test our hypothesis. First, we will determine the spatial relationship between Aire condensates and group-2 loci (Aim 1). Second, we will define the temporal relationship between group-1 and group-2 gene transcription (Aim 2). We expect that the proposed work will address key gaps in our understanding of how Aire regulates PTA gene expression. Furthermore, the proposed research will have a broad impact on the field of transcriptional regulation.

NIH Research Projects · FY 2026 · 2026-04

PROJECT SUMMARY Broadly neutralizing antibodies (bnAbs) and the antibody-like biologic eCD4-Ig (eCD4) can suppress established HIV-1 and SHIV infections when they are present at sufficient concentrations. BnAbs and eCD4 can be delivered passively or expressed from a recombinant adeno-associated virus (rAAV) vector. rAAV expression bypasses cost and compliance concerns associated with periodic antibody infusions, as well as side-effects associated with a life-time of use of conventional or long-acting antiretroviral therapy (ART). Moreover, unlike ART, long- term antibody-mediated control engages multiple effector arms of the immune system, potentially accelerating the rate of decay of the proviral reservoir. eCD4, a potency and half-life enhanced CD4-Ig fused to a coreceptor-mimetic sulfopeptide, has several advantages over bnAbs. It is exceptionally broad, neutralizing all 200-plus HIV-1 and SIV isolates assayed. rAAV-expressed eCD4 (rAAV-eCD4) can protect from multiple high-dose challenges of both SHIV and SIV. eCD4 can combine with non-neutralizing CD4-inducible antibodies common in the sera of infected persons to mediate very potent antibody-dependent cell-mediated cytotoxicity (ADCC). More recently, we have shown that more potent and bioavailable forms of eCD4, combined with the bnAb 10-1074, can fully suppress established SHIV infections in both infant and adult macaques. We are therefore close to robust functional cures in rhesus macaques. The current application seeks to bring us across the finish line and identify antibodies and eCD4-Ig variants that would best function in a human clinical trial. To do so, we will therefore in Aim 1 compare new eCD4 sulfopeptide variants, determine the Fc-domain that best limits anti-drug antibodies and enhances expression, and ensure in vivo that eCD4 poses no risk of immune interference. Aim 2 presumes that a bnAb partner of eCD4 will be useful in maintaining long term viral suppression and asks what class of antibodies works with eCD4 most effectively. Several properties are considered: their inherent bioavailablity, potency, and breadth, and how they complement eCD4 to limit potential viral escape. Aim 3 then seeks to improve the former properties by employing a novel system whereby human antibodies affinity mature in wild-type mice, starting with bnAbs modified with largely germline framework regions, and selecting in vivo those with greater potencies and longer half-lives. Aim 4 applies knowledge that we have accumulated from the previous decades and insights from the previous aims to establish and compare functional cures mediated by eCD4 paired with optimized forms of CD4-binding site bnAbs and V3-glycan antibodies. Collectively, these aims will directly inform human clinical trials designed to suppress established HIV-1 infections without ART.

NIH Research Projects · FY 2026 · 2026-04

ABSTRACT Our understanding of the genetic basis for inherited retinal disorders (IRDs) is in a fast forward mode. Yet the majority of the millions of individuals with IRDs remain untreated. Children with untreated IRDs often confront a lifetime of blindness with a host of concomitant burdens. Antisense oligonucleotides (ASOs) are a promising but incompletely realized option for critically needed IRD treatment. In this 2-year planning grant, a team with complementary expertise, including ASO drug development and pediatric retina and low vision, propose a step- wise plan to create a robust pathway to future treatment of pediatric IRDs due to ASO amenable genetic variants. With reference to a database of >3,000 well characterized children with IRDs, the team will streamline the analysis to determine genetic eligibility (Aim 1) namely identification of a suitable cryptic splicing defect. The records of the genetically eligible children will be reviewed to analyze clinical eligibility (Aim 2); evidence of rescuable photoreceptors will be the primary criterion. Children with rescuable photoreceptors are expected to have broad clinical spectra embracing a range of retinal diseases, syndromes, medical conditions, and developmental levels. Acknowledging that the realistic path to future ASO treatment is a patient-centric, N-of- 1/few approach, the team will develop procedures for considering not only scientific and medical criteria, but also feasibility and ethical criteria as they weigh clinical eligibility. Based on the lessons learned from Aim 1 and Aim 2, the team will build a versatile template for regulatory review, including the manual of procedures (Aim 3) in anticipation of future N-of-1 trials of ASO treatment of pediatric IRDs. The team’s work will clarify indications to ASO treatment of pediatric IRDs and the pathway to treatment. Early, effective ASO treatment of children with IRD has potential to accelerate the creation of the next generation management and treatment of IRDs.

NIH Research Projects · FY 2026 · 2026-03

PROJECT SUMMARY ABSTRACT Effective interventions to reduce the functional impact of core features of autism spectrum disorder (ASD) in school-aged children are critically needed. This R61/R33 application proposes to test whether in-person computer training delivered individually by a coach engages an electroencephalographic (EEG) biomarker of cognitive control (N2 event-related potential [ERP] amplitude) and whether changes in the target neural response mediate the reduction of restricted and repetitive behaviors and interests. An extensive clinical and cognitive neuroscience literature documents reduced cognitive control among autistic children compared to neurotypical children and a relation between cognitive control and repetitive features of autism––providing a solid rationale for our training program. Based on this work, we predict that developing more effective cognitive control, metacognition, and working memory will enhance neural responses to conflicting information (i.e., a neural marker of effective cognitive control) and changes will correspond with decreases in restricted and repetitive behaviors and interests. The R61 study will randomly assign 95 autistic children (ages 8-11yrs) to a novel computer-based Cognitive Control Training combined with Metacognition Coaching or to a waitlist control group. Before and after intervention, EEG will be used to examine engagement of the target neural responses. We expect the group assigned to Cognitive Control Training + Metacognition Coaching to exhibit significantly larger changes in N2 ERP amplitude in incongruent relative to congruent trials than the waitlist group. If this hypothesis is supported, the R33 will be implemented and 140 autistic children (8-11yrs) will be randomly assigned to either: 1) Cognitive Control Training + Metacognition Coaching; or 2) MentalUP Educational Games, an active control condition that provides computer-based cognitive training. Both Cognitive Control Training + Metacognition Coaching and MentalUP will be delivered individually during 15 in-person sessions. Before and after intervention, we will collect neural responses and behavioral measures of cognitive control and working memory. We expect target engagement (greater differentiation of N2 amplitude) to be associated with reduced restricted and repetitive behaviors and interests. This study is innovative in several ways and has the potential for large clinical and scientific impact. It is the first study to examine a cost-effective computer-based cognitive control intervention for ASD that provides in-person metacognition coaching. This could increase functioning for autistic children at a time when intensive intervention delivery is waning. The study will use biomarkers and validated behavioral measures of cognitive control and metacognition in the context of an ASD clinical trial. Finally, the study will provide critical information about the relation between cognitive control and clinically relevant ASD outcomes, thereby providing insight into the mechanisms underlying behavioral challenges for autistic children. Promising results from this study would provide the basis for a larger clinical trial to investigate the efficacy of cognitive control training and mediators and moderators of its effects.

NIH Research Projects · FY 2026 · 2026-03

SUMMARY The brain is bathed in cerebrospinal fluid (CSF), a medium rich in health- and growth-promoting factors, metabolites, nucleic acids, and more, whose composition changes profoundly throughout the lifespan and can be used to diagnose certain conditions. Most CSF is produced by the choroid plexus (ChP), a highly vascularized epithelium located in each ventricle of the brain. In addition, the sheet of tight junction-coupled ChP epithelial cells provides a critical blood-CSF barrier that protects the central nervous system from peripheral challenges such as bloodborne pathogens. The ChP detects and responds to changes in CSF composition. Abnormal levels of neurotransmitters in the CSF including glutamate have been reported in a wide range of neurologic and psychiatric disorders that are also characterized by ChP/barrier pathology and inflammation. We hypothesize a causal link between these observations. Specifically, we propose that excessive CSF-glutamate impairs ChP barrier integrity and that it does so through the metabotropic glutamate receptor 8 (mGluR8). Importantly, cAMP has been shown to enhance endothelial tight junctions at the blood-brain barrier (BBB), and experiments in cultured ChP cells suggest that cAMP may similarly regulate blood-CSF barrier permeability. Among the G-Protein Coupled Receptors (GPCRs) that we identified to be predominately and robustly expressed by mouse and human ChP epithelial cells, mGluR8 is coupled to the Gi/o-protein and therefore expected to inhibit cAMP/PKA signaling, consistent with negative regulation of barrier permeability. Our first set of experiments will test our hypothesis by studying cAMP regulation in ChP epithelial cells. Using methods we developed in collaboration with Mark Andermann (BIDMC/Harvard) for in vitro and in vivo two-photon imaging of ChP structure and function at subcellular resolution, we will visualize cAMP with a fluorescent indicator selectively expressed in ChP epithelial cells (cADDis). We will then use a newly generated mGluR8 knockout mouse (shared by Danny Winder, UMass), to test if mGluR8 is necessary and sufficient to mediate glutamate- evoked modulation of cAMP signaling (Aim 1). Next, we will test the hypothesis that CSF glutamate dose- dependently reduces barrier integrity in vivo and thereby causes ChP inflammation (Aim 2). To do so we will leverage technologies we recently developed based on GRAB-sensor technology for real-time tracking of neurotransmitters and neuromodulators in CSF. We will measure CSF glutamate levels with cell-based sensor iGluSnFR and correlate these levels with measurements of barrier permeability and inflammation.

NIH Research Projects · FY 2026 · 2026-02

Project Summary The goal of this proposal is to identify how the Gram-positive pathogen Staphylococcus aureus manipulates inflammasome activities that are important for host defense. With no vaccine in place and antibiotic resistance on the rise, understanding immunological aspects of S. aureus infections is of paramount importance. S. aureus strains of diverse genetic and epigenetic phenotypes cause infections, which can be difficult to treat and are subject to relapse. Acute infections are associated with so-called toxigenic strains of bacteria, which produce pore-forming toxins that stimulate the inflammasome regulatory protein NLRP3. Non- toxigenic bacterial strains are increasingly recognized for their association with difficult-to-clear infections including persistent bacteremia, endocarditis, and cystic fibrosis-associated pneumonia. Published work from us and others have demonstrated that non-toxigenic S. aureus retain the ability to stimulate inflammasomes, yet the mechanisms to explain these activities are unclear. This proposal is based on our recent discovery that Pattern Recognition Receptor AIM2 (but not NLRP3) is the primary mediator of inflammasome activities induced by diverse species and strains of Staphylococcus. Commonly used laboratory strains and those derived from S. aureus USA300, which accounts for >97% of skin infections in the United States, all induced AIM2-dependent inflammasome activities in macrophages. A genome-wide genetic screen in S. aureus identified the enzyme TarM as a mediator of AIM2 activation. TarM is an enzyme that glycosylates wall teichoic acid (WTA), a component of peptidoglycan (PGN). Interestingly, the WTA attachment site on PGN can be acetylated by a negative regulator of inflammasome activities, the S. aureus enzyme O- acetyltransferase A (OatA). TarM and OatA therefore display competing activities towards PGN modifications and towards AIM2 inflammasome activities. No current model of AIM2 functions can explain how TarM promotes or how OatA inhibits AIM2 during S. aureus infections. Based on our discoveries, we hypothesize that glycosylated WTA is a direct or indirect mediator of AIM2 activation during S. aureus infection, and that OatA prevents AIM2 activation by reducing the presence of glycosylated WTA on the bacterial cell wall. The relationship between these activities and how bacterial or host DNA activates AIM2 will be explored. We plan to define how TarM mediates AIM2 activation during infection in cultured cells, and determine the impact of these activities on acute inflammation and long term adaptive immunity in mice (specific aim 1). We further plan to determine the molecular mechanism of AIM2 activation in macrophages and how these events are coordinated with phagosome activities that may mediate DNA release into the cytosol (specific aim 2). Central to our mechanistic work is stoichiometric tandem-mass tagging (TMT) mass spectrometry, which will define kinetic interaction modules that regulate AIM2 activation events during S. aureus infection. Such analyses will be coupled with direct visualization of AIM2-inflammasomes within living cells, which will provide a comprehensive analysis of inflammasome activities during a bacterial infection.

NIH Research Projects · FY 2026 · 2026-02

Project Summary/Abstract The immune system can be categorized into innate and adaptive immunity. Innate immune cells detect and respond according to danger associated signals and initiate a coordinated antigen-specific response by the adaptive system to eliminate threats such as viruses, bacteria and tumor cells. Immunosuppressive drugs inhibit adaptive responses and are used following organ transplantation to prevent acute transplant rejection but do not generally target innate immunity. It was recently discovered that innate cells can be licensed to detect foreign allogenic tissue and initiate responses against the transplant in the absence of danger- associated signals. Additionally, innate cells show accelerated activation responses following re-exposure to the identical foreign tissue, called innate memory or trained immunity. These findings suggest that innate cells are chronically stimulated and activated following transplantation, and it is possible that persistent innate responses participate in or drive chronic transplant rejection, the main reason for graft loss. Little is known about the mechanisms that control and modulate innate allo-activity and no therapeutic is clinically available to resolve persistent innate alloimmunity following transplantation. In this proposal, we hypothesize that Semaphorin 3F (SEMA3F)-Neuropilin 2 (NRP2) interactions on monocytes can inhibit and/or resolve persistent innate alloimmune responses following transplantation. In preliminary experiments, we find that monocytes express NRP2 following initial alloimmune activation in recipients of fully MHC mismatched cardiac allografts. We also find that the administration of the NRP2-ligand SEMA3F prolongs allograft survival in models of cardiac transplantation. In Aim 1, we will assess SEMA3F- induced innate responses following transplantation and we will employ transcriptomics and confirmatory protein-based methods to define SEMA3F-dependent signaling networks within monocytic subsets. In Aim 2, we will use a monocyte-specific tamoxifen-inducible NRP2 KO mouse model and evaluate intrinsic effects of NRP2 signaling on monocyte responses following transplantation. We predict that the administration of SEMA3F prolongs allograft survival by inhibiting monocyte and macrophage alloimmune memory responses. We also predict that deletion of NRP2 from monocytes results in accelerated graft rejection and that SEMA3F- NRP2 interactions are required for long term graft survival. If successful, the impact and relevance of these studies are the identification of SEMA3F as a novel immunoregulatory ligand that suppresses innate immunity following transplantation, the identification of NRP2 as a co-inhibitory receptor on innate cells, and that they enable the development of immunomodulatory drugs that resolve persistent innate alloimmune responses post transplantation.

NIH Research Projects · FY 2026 · 2026-02

PROJECT SUMMARY This proposal responds to the Notice of Special Interest “Understanding Sudden Death in the Young” (SDY) (NOT-HL-22-040) by building on our previous body of research in Robert’s Program on SUDP (Sudden Unexpected Death in Pediatrics) at Boston Children’s Hospital with additional phenotypic and genetic data collected in the NIH/CDC SDY Registry, to better define the molecular underpinnings of sudden and unexpected death in individuals 0-20 years of age (definition of SUDP and SDY). Sudden infant death syndrome (SIDS) and sudden unexplained death in childhood (SUDC) (included in the definition of SUDP and SDY) are responsible for nearly one in ten deaths in US children. While public health efforts aim to minimize risk in the sleep environment for SIDS, the persistent mortality indicates that affected children may possess intrinsic vulnerabilities that increase their susceptibility to death. Robert’s Program on SUDP uses deep phenotyping, genetic analysis, and engagement of families to study SUDP as a constellation of undiagnosed diseases. Our premise is that intrinsic biological contributions to SUDP include neurodevelopmental, epilepsy- related, cardiac, metabolic, and respiratory factors that have a genetic basis. Our multidisciplinary approach mirrors undiagnosed disease programs, using deep phenotyping and comprehensive genomic analysis to identify unrecognized conditions. Our previous “proof of concept” study, funded in part as an R21, utilized exome sequencing and found evidence of genetically based susceptibilities in 11% of SUDP cases in genes related to neurological, cardiac, and systemic disease. For this proposal we hypothesize that we will identify contributions to SUDP through a multiomics approach, using GS and metabolomic data to identify potential underlying Mendelian disorders and to investigate genetic risk using polygenic scores (PGS) for phenotypes of interest in SUDP. In Aim 1 we will identify monogenic contributions to SUDP by phenotyping and performing genome sequencing (GS) in prospective cases from Robert’s Program trios (estimated 270 over the grant period) and analyzing these data together with GS and phenotype data from the SDY Registry (estimated 250 prospective cases over the grant period). When added to the existing cohort of nearly 1000 cases from the SDY Registry and Robert’s Program, total data will include over 1400 cases. We will also conduct long-read sequencing in Robert’s Program cases to identify monogenic contributions missed by GS alone. In Aim 2 we will explore risk for SUDP using validated PGS with phenotypic overlap to SUDP mechanisms (e.g., epilepsy and Brugada syndrome) in Robert’s Program and SDY Registry cases. In Aim 3 we will utilize metabolomics in Robert’s Program cases to uncover biochemical signatures of underlying metabolic disease. The impact of this research is the elucidation of genetic mechanisms in SUDP, advancing the forensic molecular autopsy in establishing a cause of mortality and assessing novel methods of identifying genetic and metabolomic underpinnings of SUDP.

NIH Research Projects · FY 2026 · 2026-02

Nephrotic syndrome (NS) is a rare form of chronic kidney disease resulting from glomerular filtration barrier failure and massive proteinuria. Morbidity and mortality from NS is related both the disease itself and the non- specific, immune-modulating medications used to try to treat it. NS has long been classified and treated according to either histologic appearance or ability of immunomodulating drugs to achieve remission (“steroid sensitive” [SSNS] or “steroid resistant” [SRNS]). These classifications are nonspecific and don't illuminate specific pathobiology. To achieve increasingly effective care for NS, a more precise understanding of its underlying molecular mechanisms is necessary. Human genetics studies in NS using family- and population- based methods have proven effective in empowering (1) patient classification by genetic subtype and (2) target identification for development of biomarkers and therapeutics. We are focusing on the genetic basis of pediatric SSNS, which is the most common pediatric subtype long known to have an immune component to its pathogenesis. Compared to children with SSNS, those with ISNS are less likely to reach end stage kidney disease (ESKD). However, because these children respond to steroids their lifetime burden of immunosuppression can be very high, with all the attendant infectious risks and side effects such as hypertension, osteoporosis, infertility, and cataracts. And for those children with initial SSNS who eventually reach ESKD, they have significantly increased odds of recurrent NS in their transplanted kidney. The genetic architecture of pSSNS is polygenic and published genome-wide association studies (GWAS) of small sample sizes (n=200-900 cases) had discovered four significant loci. Many of these signals are immune- related. We recently completed a global GWAS of pSSNS with 2440 cases, discovering seven more novel risk loci, establishing a pSSNS polygenic risk score (PRS), and colocalizing signals to specific genes across cell types. Here, we will dissect these pSSNS GWAS loci computationally and functionally, with a focus on integrative analysis using patient-derived molecular datasets and model systems. In Aim 1, we will discover the consequences of pSSNS risk alleles on the immune cell transcriptome of 310 with pSSNS through colocalization with immune-cell eQTLs and PRS-mRNA expression association studies. In Aim 2, we will discover the consequences of pSSNS risk alleles on the proteome of 364 children with NS through genetic associations with large-scale plasma proteomic datasets and targeted protein quantitation. In Aim 3, we will focus on furthering our mechanistic understanding and validation of top candidates through eQTL/pQTL integration, experimental systems, and targeted follow-up in independent human samples. Altogether, these studies will deepen our molecular knowledge and clinical impact of specific pediatric SSNS genetic subtypes.