Washington University

NIH Research Projects · FY 2026 · 2024-05

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

NIH Research Projects · FY 2026 · 2024-04

PROJECT SUMMARY/ABSTRACT Type 2 diabetes is associated with derangements in a variety of pathways of intermediary metabolism. In addition to the widely-known effects on glucose metabolism, diabetes is also associated with abnormalities in lipid and amino acid metabolism including elevations in branched chain amino acids (BCAA). BCAAs are increased in people with obesity and insulin resistance and high plasma BCAA concentrations are predictive of future development of diabetes. Dietary restriction of BCAAs in rats improves insulin sensitivity and genome- wide association studies in humans have demonstrated that genetic variations in enzymes controlling BCAA metabolism are associated with increased BCAA concentrations and are also linked to the development of insulin resistance. These data suggest that high BCAA concentrations play a causative role in the development of insulin resistance and that strategies that correct accumulation of these bioactive amino acids may have value as therapeutic avenues for treating diabetes. Circulating BCAA concentrations are regulated by dietary intake, rates of protein synthesis and proteolysis, and BCAA catabolism. To be oxidized, BCAAs are first reversibly converted to branched chain keto acids (BCKA) by branched chain aminotransferases. Next, BCKA undergo oxidation that is catalyzed by the BCKA dehydrogenase (BCKDH) enzyme complex in the mitochondrial matrix. BCKDH is regulated by inhibitory phosphorylation and is activated by a protein phosphatase (PPM1K) that removes this covalent modification. Genetic or pharmacologic approaches to reduce BCKDH phosphorylation lead to enhanced BCKA catabolism and improved insulin sensitivity in rodent models of obesity. This application is designed to test the novel hypothesis that PPM1K phosphorylation at a conserved serine residue suppresses the activity of this phosphatase leading to increased BCKDH phosphorylation. Our studies are designed to identify mechanisms by which this phosphorylation is regulated, define the effects of this covalent modification on intrinsic PPM1K activity, and determine the effects of PPM1K phosphorylation on BCAA metabolism in liver of diabetic rodents.

NIH Research Projects · FY 2026 · 2024-04

PROJECT SUMMARY/ABSTRACT: Numerous biochemical and biophysical studies have demonstrated that amyloid-β (Aβ) aggregates into toxic oligomers and fibrils when the concentration of the peptide is elevated and the pH is low (Paredes-Rosan et al. 2019; Zhao et al. 2018). Aβ aggregates can be detected neuron throughout the endo-lysosomal pathway (Brewer et al. 2020) where pH ranges from 4.5-6.5 which are prime conditions for Aβ to aggregate. However, what physiological processes influence Aβ aggregation in vivo within this compartment remain unknown. Elevated synaptic activity increases the formation of Aβ within endosomes before it is secreted into the brain interstitial fluid (ISF) (Cirrito et al. 2005, 2008). We hypothesize that elevated synaptic activity not only causes formation of Aβ, but also directly drives formation and release of Aβ aggregates. We propose that low, physiological synaptic activity causes release of Aβ monomer, whereas high bursts of aberrant activity reach a threshold when the combination of low pH endosomes with sufficiently high Aβ levels induces aggregation. We have developed a novel electrochemical micro-immunoelectrode (MIE) technology to measure Aβ40, Aβ42, or Aβ oligomer levels every 60 seconds in an awake, moving mouse for up to 6 hours in order to tightly link the amount of synaptic activity with the level and conformation of Aβ. Furthermore, we will determine if glutamatergic or GABAergic neurons are primarily responsible for producing Aβ and if one type of neuron is more prone to generate aggregates. All studies will use the APPNLF/NLF knock-in mouse model of amyloidosis in order to preserve normal expression patterns of APP which is critical for these types of studies. Our preliminary data demonstrates the tight link between synaptic activity and Aβ generation, as well as the characterization and specificity of the MIE technology for real-time detection of Aβ levels and aggregates. Using the MIE, preliminary data suggest that only a slight increase in neuronal activity produces Aβ monomer but that higher levels of activity induce secretion of Aβ aggregates. Also, our data suggests that excitatory neurons produce more Aβ than inhibitory neurons. While we propose that synaptic activity is a potent regulator of Aβ oligomer and fibril formation, we acknowledge that there are several means that will induce and influence aggregation, such as chaperones like apoE, and that synaptic activity is just mechanism that can generate these toxic species. Aim 1 will determine the threshold of synaptic activity that causes Aβ to aggregate into soluble oligomers then get released into the brain ISF. Aim 2 will determine whether excitatory or inhibitory neurons release Aβ monomer or Aβ aggregates, and if that differs between wake and sleep states, since inhibitory tone is much greater during sleep. Aim 3 will determine if chronically modulating activity of excitatory or inhibitory neurons within the hippocampus alters development of Aβ plaques. How synaptic activity impacts Aβ species and aggregation state is unclear. The scientific premise of this proposal is to understand the mechanisms that contribute to Aβ generation and aggregation in vivo. The FDA recently approved a monoclonal antibody, Lecanemab, that targets Aβ oligomers and finally has clinical benefits to patients. Understanding the source of oligomers could inform on risk factors that influence aggregation and lead to new therapies targeting oligomers in the future or identifying novel biomarkers for disease development.

NIH Research Projects · FY 2026 · 2024-04

Project Summary/Abstract: Axon maintenance is regulated by components of the programmed axon degeneration (AxD) pathway, most importantly the axon survival factor, NMNAT2, and the chief axon executioner, SARM1, an NAD hydrolase that initiates local metabolic catastrophe when activated by axon damage. Chronic SARM1 activation that contributes to degeneration is called sarmopathy, and it is believed to play a role in diseases including amyotrophic lateral sclerosis (ALS), Charcot-Marie-Tooth disease type 2A, and peripheral neuropathy. SARM1 and NMNAT2 were previously believed to regulate axon integrity exclusively cell-autonomously, but we discovered that SARM1 plays a role in cell-extrinsic mechanisms of AxD, namely macrophage recruitment and activation contributing to the initial phase of axon dysfunction and degeneration. We developed a human disease model featuring pure sarmopathy based on patients with a rare early-onset, progressive polyneuropathy caused by NMNAT2 mutations. Macrophage depletion delays axon loss and significantly improves motor function even after the onset of symptom in this model, indicating that SARM1 initiates macrophage-mediated axon elimination months before stressed axons would otherwise succumb to cell-intrinsic metabolic failure. We also found that blocking axonal externalization of lyso-phosphatidylserine (lyso-PS), a phagocytic “eat-me” signal that promotes macrophage clearance of apoptotic cells, delays motor dysfunction in the model. Manipulating lyso-PS was achieved by virally overexpressing the phosphatidylserine lipase ABHD12 in mouse spinal cord, demonstrating a completely novel potential therapeutic strategy for peripheral neuropathy. In this proposal, we outline experiments to elucidate the SARM1-dependent pro-degenerative neuroinflammatory response. We will characterize immunocytes and other cells in mouse model nerves to determine their provenance and unique transcriptional profiles to identify key genes and pathways that shape sarmopathic neuroinflammation. We will test the roles of repair SCs and candidate chemoattractants/ receptors in SARM1-dependent macrophage recruitment and activation by testing whether sarmopathy is modified in combination with informative gene knockout models. We will also specifically address the role of phosphatidylserine (PS) exposure in sarmopathy by manipulating PS and PS receptors genetically in vivo, including via ABHD12 overexpression. Results of these studies will establish the through-line connecting SARM1 activation, inflammation, and the development of peripheral neurodegenerative diseases, and drive forward the development of anti-inflammatory axoprotective therapeutics for these devastating disorders.

NIH Research Projects · FY 2026 · 2024-04

DESCRIPTION Catheter-associated urinary tract infection (CAUTI) is a costly clinical problem that affects millions of patients worldwide. CAUTI are characterized by infection of the bladder and pathogen colonization of the catheter surface, making them especially difficult to treat. Catheter modifications have been employed to reduce pathogen colonization, including infusion of antibiotics. However, with a rise in antibiotic resistance among uropathogen strains, there is a great need to develop alternative strategies for effective CAUTI treatment and prevention. Lactobacillus probiotics offer promise for a “bacterial interference” approach to prevent CAUTI because they not only could compete for adhesion to the catheter surface but they also produce and secrete antimicrobial compounds that are effective against uropathogens. Three-dimensional (3D)-bioprinting has enabled fabrication of well-defined, cell-laden architectures with tailored release of active agents. We hypothesized that 3D- bioprinting could offer a novel means for sustained probiotic delivery. We have capitalized on this technology to design and fabricate a prototype 3D-bioprinted catheter tubing containing the widely used probiotic strain Lactobacillus rhamnosus GG. Our preliminary data already demonstrate that our initial bioprint prototype 1) maintains probiotic viability upon extended storage, 2) displays sustained release of live L. rhamnosus, lactic acid and hydrogen peroxide in vitro, 3) develops surface-associated L. rhamnosus biofilms, 4) inhibits uropathogenic E. coli in vitro and 4) maintains L. rhamnosus viability and release in vivo in a mouse model. Here, we will test the overarching hypothesis that the combination of probiotic bacterial interference and 3D- bioprinting technologies provides an optimal framework for effective CAUTI prevention The objective of this proposal is to optimize the design and fabrication of Lactobacillus 3D bioprints and to confirm their safety and efficacy in vitro and in a preclinical mouse model, while advancing our understanding of probiotic interactions with the host and uropathogenic bacteria. Successful completion of the aims will deliver validated 3D-bioprinted prototypes that accomplish long-acting delivery of probiotic species that ultimately improve outcomes in preclinical models of CAUTI. These studies will provide the foundation for translation to reduce risks associated with urinary catheterization, while providing new insights into the effects of lactic acid-based and probiotic therapeutics on host inflammatory response and CAUTI disease markers and progression. Moreover, outcomes of this research will have a significant impact on the development of future probiotic approaches in the context of other medical device-associated infections. The successful completion of this project will deliver validated 3D-printed prototypes that enable long-acting delivery of probiotic bacteria for the prevention and treatment of CAUTI.

NIH Research Projects · FY 2025 · 2024-04

SUMMARY Peripheral nerve injuries are common and debilitating conditions affecting more than one hundred thousand people annually in the United States. Prognosis for recovery of severe nerve injuries is poor, as high-grade axonotmetic and neurotmetic injuries usually do not spontaneously recover. These injuries require surgical intervention and if the nerve injury is located far from the target muscles, nerve repairs fail to provide useful function in half of patients- leading to permanent physical disabilities and enormous emotional stress. To improve clinical outcomes, a better understanding of the molecular mechanisms involved in nerve injury is critical. The traditional view that Wallerian degeneration (WD) is inevitable after nerve injury has recently been challenged with the discovery of the role of nicotinamide adenine dinucleotide (NAD) in supporting maintenance of viable axons. SARM1 with its NADase enzymatic activity, has been identified as a key gatekeeper of WD. After a severe nerve injury, SARM1 rapidly degrades NAD resulting in catastrophic structural changes to the distal axon. Blockade of this phenomenon, combined with the promise of fusogens can provide a potential mechanism for transected nerves to rapidly recover after injury. Optical imaging with its high spatial and temporal resolution is highly promising to visualize the entire process in the nerve tissue, however high scattering from the skin and other adjacent organs limit the application to only in vitro or ex vivo studies. To address these limiting issues, we developed a minimally invasive in vivo model that enables continuous imaging of a peripheral nerve injury with a high, single axon resolution. Our approach uses a flexible skin-embedded transparent optical window with the nerve surgically repositioned above the muscle layer. This modality allows daily or even hourly, longitudinal imaging of the nerve with virtually any optical reporter that can be used in living animals. When combined with fluorescent reporters and a high-resolution imaging system (i.e., two-photon imaging), this method generates a 3D view of the nerve with unprecedented resolution. In Aim 1 we will develop a double transgenic mouse reporter model (THY-1/CFP and S100/GFP) expressing different levels of SARM1 (SARM1-/-, SARM1-/+, SARM1+/+). We will then monitor differences in the degree and timing of axonal degeneration after a unilateral sciatic nerve injury, using our optical window. In Aim 2, we will synthesize a library of small activatable fluorogenic probes that mimic NAD+ to directly measure the activity of SARM1 in the distal stump. Overall, this imaging approach will provide direct visualization of the morphologic and metabolic characteristics of the distal stump degeneration in live animals. It will also establish a conceptual framework for future investigation of the fundamental processes during nerve regeneration. This approach will lead to new discoveries in the biology of these processes and spur new approaches toward developing novel therapeutics for nerve injury.

NIH Research Projects · FY 2026 · 2024-04

Project Summary Artificial intelligence (AI) systems are increasingly used in radiation oncology for tasks such as image reconstruction and registration, autosegmentation, synthetic CT generation, and treatment planning. However, AI design fundamentally challenges existing quality assurance (QA) paradigms which imperils the quality and safety of AI for clinical use. Addressing the unmet need of QA for clinical AI is critical as the potential for performance degradation of AI systems in the clinic is high. Domain shift - when the distribution of data used during training is different from the distribution of data encountered during deployment - is a critical problem that can lead to significant errors in AI performance. This is a common occurrence in clinical environments, where scanner performance varies over time due to changes in imaging protocols or sequences, equipment degradation, or replacement with a different make or model. Monitoring clinical AI system performance for signs of domain shift is of utmost importance to ensure safe and high-quality use. Development of robust QA tools and practices to verify and monitor the performance of AI systems is therefore critical as these systems enter the clinical arena. In this project, we will develop a new type of QA approach amenable for closed-source, clinical AI systems. Our approach supported by our preliminary data is to design a series of detectors which monitor the input imaging data and AI system output for changes and link these changes to an actionable tolerance through a prediction model, without the need to access the AI system internals. Our overall hypothesis is that the expected performance of clinical AI systems is predicted within 5% error by monitoring only the AI system inputs and outputs. In Specific Aim 1, we will develop a QA framework for AI systems which were trained with a ground truth set of labels, using autosegmentation as a model system. We will build compression algorithms to encode features from the distribution of inputs (images) and separately on the distribution of outputs (contours). A prediction model taking these distributions as input and predicting the contour accuracy will be built. We will develop the QA framework on a set of existing AI systems including two commercial AI and several in-house autosegmentation algorithms. In Specific Aim 2, we will focus on AI systems which do not use a ground truth during training, using synthetic CT generation as a model system. A similar approach as in SA1 using compression to build distributions of input and output latent features will be used. Instead of predicting accuracy (which requires a ground truth), we will develop a model to monitor the distribution of outputs. In Specific Aim 3, we will deploy our quality assurance frameworks in a prospective clinical study involving multiple institutions and evaluate effectiveness in ensuring the safe and high-quality deployment of clinical AI systems. We will also share our frameworks and data with the broader community to promote best practices in AI quality assurance. We expect that our QA framework will significantly improve the safety and effectiveness of clinical AI systems in radiation oncology, by ensuring that these systems are robust to domain shift and other sources of error.

NIH Research Projects · FY 2026 · 2024-04

Although significant advances in the treatment of opiate addiction have been made, relapse to opiate use after abstinence continues to impede successful treatment, highlighting the need for efforts to dissect the mechanism of opiate-dependent changes in brain function. Long-lasting associations between opiates and the context in which they are taken result in cues that lead to drug craving and ultimately relapse. The hippocampus represents a key structure in the integration of emotional processing, learning and memory, and reward-related behaviors. While the ventral subdivision of the hippocampus (vHPC) is involved in processing emotional values of salient stimuli and goal-directed behaviors, the dorsal hippocampus (dHPC) plays a critical role in episodic, spatial, and associative memory. In addition, it has been shown that the dHPC is necessary for context- and cue-associated reward behaviors, including the expression of reward seeking. Further, we found that chemogenetic inhibition of glutamatergic dHPC neurons reduces cue-induced morphine-seeking in an instrumental model of relapse. Together, these findings indicate that the dHPC is important for the development and maintenance of opioid-cue associations necessary for drug-seeking behavior. Integration of rewarding and aversive stimuli relies on the mesolimbic reward circuit, where the nucleus accumbens (NAc) plays a crucial role. The NAc facilitates reward seeking by integrating dopaminergic reinforcement signals with glutamate-encoded environmental and cue stimuli. Although glutamatergic inputs to the NAc from the ventral hippocampus have been reported, our preliminary data show that dense projections to the NAc also originate in the dHPC. Interestingly, we found that photo-stimulation of excitatory dHPC neurons is rewarding and reinforcing using a real-time place preference test and instrumental self-stimulation, respectively. Interestingly, this dHPC stimulation was accompanied by enhanced activity of accumbal neurons and evoked local field potentials within the NAc. No studies have examined whether the dHPCNAc is involved in opioid-seeking behavior. Characterization of the cellular mechanisms initiated by the alterations in glutamatergic transmission from the dHPC to the NAc observed during opioid self-administration is necessary to identify novel molecular targets that might prevent drug relapse triggered by the exposure to drug-associated cues. Therefore, the overall goal of this proposal is to investigate the role of excitatory transmission from the dHPC to the NAc in opioid-seeking behavior and identify novel molecular targets that may mitigate the influence of drug-cue associations on relapse. Accordingly, the aims here will 1) determine whether dHPC to NAc projecting neurons are necessary and sufficient for fentanyl-seeking; 2) examine the effects of fentanyl-seeking behavior on function and plasticity of NAc neurons downstream of dHPC projections; 3) determine the role of dHPCNAc dynorphin- or enkephalin-containing synapses in fentanyl-seeking behavior. Findings generated from this project will have significant translational potential for understanding the mechanisms of drug relapse.

NIH Research Projects · FY 2026 · 2024-04

PROJECT SUMMARY Aneurysms, which are defined as a 50% increase in the diameter of a blood vessel, are an important cause of death worldwide. Aortic aneurysms (AAs) are the most common type and they are divided into two groups: thoracic (TAAs) and abdominal (AAAs). All aneurysms share similar histopathologic features of elastic fiber fragmentation, disorganization and loss of smooth muscle cells (SMCs) and accumulation of collagen and proteoglycans, however, TAAs and AAAs differ in their pathogenesis. While TAAs have a strong genetic predilection, AAAs are considered of atherothrombotic origin. Regardless of type, there is currently no directed medical therapy for AAs, which progressively enlarge over time, increasing the risk of vessel rupture and fatal hemorrhage. Therefore, there is an urgent need to understand the pathogenesis of AAs to devise targeted therapeutic strategies. A single gene mutation is identified in nearly 20% of individuals with TAAs and the genes identified to date largely belong to three functional groups: 1) extracellular matrix (ECM) organization, 2) transforming growth factor b signaling and 3) SMC contractility, which underscores the importance of the interplay between the cell, signaling and the ECM in the pathogenesis of AAs. SMCs have been the focus of investigation in AA pathogenesis, however it is increasingly recognized that endothelial cells (ECs) play a role through endothelial dysfunction. We and others identified mutations in lysyl oxidase (LOX), a gene that encodes an enzyme important for collagen and elastin crosslinking, as a cause of inherited TAAs in humans. Mice homozygous for a Lox mutation identified in humans die perinatally of aneurysm rupture similar to mice lacking LOX, therefore it is challenging to study AA pathogenesis and progression using these models. Our collaborator, Dr. Philip Trackman at the Forsyth Institute, generated a conditional Lox mouse model, which when bred to mice carrying Cre recombinase in certain cell types will lead to deletion or loss of LOX from that cell type. Dr. Trackman generously shared this mouse model with us and we have bred to mice expressing Cre recombinase in ECs, SMCs as well as specific SMCs of the ascending aorta, which are derived from two developmental origins. Our preliminary data show increased proliferation markers in the ascending aorta where aneurysms are present. This observation led us to hypothesize that loss of LOX leads to a phenotypic change in the cell from a differentiated phenotype to a proliferative phenotype contributing to aneurysmal disease. This hypothesis will be explored in this application. Results generated from the proposed studies will not only further our understanding of the role LOX plays in arterial development and maintenance, but they will also help identify new pathways that may be leveraged to develop targeted therapeutic strategies for aneurysmal disease.

NIH Research Projects · FY 2026 · 2024-04

PROJECT SUMMARY High SERPINB3 is a biomarker of radioresistance, and a potential therapeutic target for cervical cancer. Our recent work demonstrated that CRISPR-Cas9 knock out of SERPINB3, an inhibitor of lysosomal proteases, sensitized cervical tumor cells to radiation therapy (RT)-induced cell death. In vivo targeting of SERPINB3 with siRNA not only altered the tumor microenvironment, but significantly sensitized SERPINB3-high tumor models to RT. Importantly, we established that tumor cells died via lysoptosis, a newly described and evolutionarily conserved mode of cell death that is dependent upon lysosomal membrane permeabilization (LMP) and leakage of potent proteases into the cytoplasm. SERPINB3-KO tumor cells underwent widespread lysoptosis when treated with a variety of cytotoxic agents and chemotherapies. This suggests that loss of SERPINB3 exposed lysoptosis as a default cell death mechanism in these cells. The molecular features of tumor cells that are susceptible to RT-induced LMP and lysoptosis are unknown. Validation that lysoptosis leads directly to tumor control is required. Additionally, the mechanism through which RT induces LMP is unknown. New unpublished data suggest that SERPINB3-high tumors have a distinct molecular signature characterized by enrichment of lysosome-related pathways. In these cells, LMP occurs soon after treatment with clinically relevant doses of RT, while lysosomal rupture occurs days later just prior to cell death. Finally, we have identified candidate small molecules that bind specifically to SERPINB3. The long-term goal of this work is to define new treatment approaches for radioresistant SERPINB3-high tumors. This proposal tests the hypothesis that targeting SERPINB3 in SERPINB3-high tumor cells renders them susceptible to RT-induced lysoptosis, and that LMP is the initiating event that results from unrepaired lysosomal damage. Three aims are proposed to directly address this hypothesis: Aim 1: Determine the predominant cell death mechanism induced by RT in SERPINB3-high tumors; Aim 2: Determine the mechanism of therapy-induced LMP leading to lysoptosis; Aim 3: Identify SERPINB3-targeting strategies that specifically sensitize tumor cells and not normal cells to RT. To accomplish these aims, we propose to use ex vivo organelle-based approaches, step-wise in vitro and in vivo analysis of cell death mechanisms leading to tumor control, and an innovative pre-clinical brachytherapy system that we have developed to mimic treatment delivered in the clinic. Success of this proposal will confirm that SERPINB3 is a predictive biomarker for lysoptosis-inducing CRT strategies, generate new knowledge of the critical events leading to RT-induced LMP and cell death, and provide innovative therapeutic strategies to target SERPINB3 and improve survival for patients with cervical cancer using a personalized treatment approach.

NIH Research Projects · FY 2025 · 2024-04

PROJECT SUMMARY Nigeria has the highest burden of maternal mortality globally with hypertensive disorders of pregnancy (HDP) contributing significantly to these deaths and posing long-term risks for adverse cardiovascular health. A large proportion of maternal deaths occur during the postpartum phase when care transitions between obstetricians and other providers. This phase has become crucial for monitoring and treating women, emphasizing the urgent need to address the knowledge-to-action gap in implementing evidence-based postpartum care practices. Dr. Mahmoud led the design and conduct of a feasibility study to contextualize and evaluate implementation of a postpartum blood pressure (BP) monitoring program in women with HDP in Abuja, Nigeria. The study achieved high adoption (100%) and 6-week retention rates (97%), demonstrating the feasibility of implementing a postpartum home BP monitoring program. Based on these findings, the program was adapted to address key barriers identified during the formative study resulting in the creation of the HDP Implementation Bundle. This career development award proposal includes a type 1 hybrid, stepped-wedge cluster randomized trial among 4 sites and 900 women with HDP in Nigeria, to evaluate the preliminary effectiveness and implementation of the HDP Implementation Bundle in improving postpartum BP control compared with usual care. The study will include a formative qualitative study to guide further adaptation of strategies included in the HDP Implementation Bundle using FRAME-IS, an evaluation of preliminary effectiveness that will be assessed by between-group difference in change in systolic BP from baseline to 6 weeks (primary outcome), and an assessment of implementation using the RE-AIM (Reach, Effectiveness, Adoption, Implementation and Maintenance) framework. This proposal aligns with research priorities identified by NHLBI and will leverage implementation science frameworks to guide local adaptation and enhance success. Dr. Mahmoud proposes training in qualitative research methods, proficiency in implementation, translation and dissemination science to build on her background in cardio-obstetrics and health policy. This career development plan will offer pragmatic training in a highly supportive environment with experienced mentors led by Dr. Mark Huffman at Washington University in St. Louis alongside Drs. Ojji (Abuja), Dávila- Román (WashU) and Lindley (Vanderbilt). This career development award will provide the necessary training and support for Dr. Mahmoud to achieve her overarching goal of becoming an independent physician-scientist in global cardio-obstetrics. Insights gained from this study will pave way for a larger trial to assess the implementation and effects of the HDP Implementation Bundle on cardiovascular outcomes in a wider population through an R01 or similar-level grant to support the candidate’s development as an independent physician-scientist with expertise in global cardio-obstetrics.

NIH Research Projects · FY 2026 · 2024-04

Program Director/Principal Investigator (Last, First, Middle): GUAN, JIANJUN Project Summary Following myocardial infarction (MI, or heart attack), the prolonged inflammation due to delayed transition of proinflammatory phase to anti-inflammatory phase, and uncontrolled cardiac fibrosis following myofibroblast differentiation, deteriorate cardiac function. As such, timely transitioning the proinflammatory phase to the anti- inflammatory phase, and inhibiting myofibroblast formation will improve cardiac function. However, the effective therapies to simultaneously achieve both goals remain to be established. Currently, systemic delivery of anti- inflammatory agents did not show consistent outcomes in clinical trials, mainly because the drugs either inhibit cardiac repair or are not delivered in a spatiotemporal manner to effectively target specific inflammatory signals. To attenuate cardiac fibrosis, systemic delivery of TGFβ inhibitors and antibodies represents a major strategy. Yet, the efficacy is not satisfactory because these therapies cannot essentially inhibit TGFβ pathway, and also cannot simultaneously prevent other major pathways-induced myofibroblast formation, e.g., IL-1 pathway. In this project, we propose a new approach to address the above limitations. We will directly deliver, at acute MI stage, secretome of M2 macrophages (M2Mφs), a dominated cell type in the anti-inflammatory phase, to timely decrease inflammation in the infarcted hearts. Besides anti-inflammation function, we found that M2Mφ-secretome increases cardiac cell survival and promotes angiogenesis in the infarcted hearts. Thus, it is multifunctional. Yet, M2Mφ-secretome has not been explored for cardiac therapy before. To prevent cardiac fibrosis, we propose to deliver a newly synthesized, peptide-based inhibitor RPE that simultaneously inhibits TGFβ and IL-1 pathways-induced myofibroblast differentiation. Our preliminary studies demonstrate that RPE significantly reduced myofibroblast density in the infarcted hearts. To the best of our knowledge, there is no known inhibitors that simultaneously inhibit these 2 pathways. To deliver the M2Mφ-secretome and RPE into infarcted hearts at the critical time window to attenuate inflammation and myofibroblast formation - acute MI stage, while considering that direct myocardial injection has safety concerns at this stage, we will deliver them by IV injection, after encapsulating them into infarcted heart-targeting nanoparticles. These nanoparticles will specifically accumulate in infarcted hearts following IV injection. They will then spatiotemporally release M2Mφ-secretome and RPE. We hypothesize that IV delivery of infarcted heart-targeting nanoparticles capable of releasing M2Mφ-secretome, and RPE at the acute MI stage will efficiently decrease tissue inflammation, increase cardiac cell survival, promote angiogenesis, and attenuate cardiac fibrosis, leading to a significant increase of cardiac function. AIM 1 will test the hypothesis that optimal M2Mφ-secretome composition will efficiently decrease cell proinflammatory cytokine secretion, improve cardiac cell survival under ischemia, and promote endothelial cell morphogenesis. AIM 2 will test the hypothesis that optimal RPE release profiles will simultaneously prevent TGFβ and IL-1 pathways-induced cardiac fibroblast differentiation into myofibroblast. AIM 3 will test efficacy of the M2Mφ- secretome and RPE releasing nanoparticles delivered at different times in the acute MI stage. OMB No. 0925-0001/0002 (Rev. 03/2020 Approved Through 02/28/2023) Page Continuation Format Page

NIH Research Projects · FY 2026 · 2024-04

Project Summary/Abstract Losing weight can be life saving for people with obesity. However, among patients that do lose significant weight, most have trouble keeping the weight off. People with obesity that lose weight experience physiological, neural, and behavioral changes that drive weight regain. These changes resemble adaptive mechanisms that defend body weight during periods of food scarcity, but for people trying to achieve a healthy body weight and stay there, these mechanisms are decidedly maladaptive. Intervening to counteract them has the potential to revolutionize the clinical approach to weight loss for people with obesity. The objective of this proposal is to understand how the function of a brain area known as the nucleus accumbens is altered across the weight “gain-loss-regain” cycle in mice. Our central hypothesis is that obesity is associated with adaptations in the brain’s reward circuitry that enhance the pursuit of palatable foods, promoting weight regain after obese animals lose weight, and thereby perpetuating this cycle. In Aim 1, we will use ex vivo electrophysiological approaches to monitor changes in intrinsic and synaptic properties of accumbal neurons as obese mice lose weight, critically determining adaptations that persist after weight loss. In Aim 2, we will employ in vivo calcium imaging to measure the activity of specific populations of accumbal neurons as mice gain, lose, and regain weight. Finally, in Aim 3 we will use viral genetic strategies to selectively silence specific populations of accumbal neurons to determine whether this: 1) facilitates weight loss in obese mice that remain on a high-fat diet; and/or 2) inhibits weight re-gain in formerly obese mice who have lost weight. Our long-term goal is to understand how obesity alters reward circuitry and discover methods for reversing these changes. This research will provide a critical foundation to advance efforts to improve weight loss outcomes in people with obesity.

NIH Research Projects · FY 2025 · 2024-04

PROJECT SUMMARY/ABSTRACT Tuberous sclerosis complex (TSC) is a relatively common genetic disorder, which features hamartoma or tumor growth in multiple organs, including the brain, and causes a variety of neurological and neuropsychiatric symptoms, including epilepsy, intellectual disability, and autism. Mutation of the TSC1 or TSC2 genes leads to hyperactivation of the mechanistic target of rapamycin (mTOR) pathway, which drives tumor growth and epileptogenesis in TSC. Epilepsy occurs in up to 90% of TSC patients and is intractable to treatment in the majority of cases, often leading to life-long disabling seizures. While significant advances in treatment of epilepsy in TSC have been made, including recent FDA approval of an mTOR inhibitor for epilepsy, most TSC patients continue to have intractable seizures and significant side effects, which strongly affect quality of life and may exacerbate co-morbidities of cognitive and behavioral disorders. So, novel therapeutic approaches to epilepsy in TSC are greatly needed. An intriguing, unexplored avenue to investigate epilepsy in TSC relates to the regulation of intracellular chloride homeostasis, which is critical for facilitating synaptic inhibition in the brain, particularly as driven by gamma-aminobutyric acid A (GABAA) receptors. The K+-Cl- cotransporter 2 (KCC2) extrudes chloride from neurons, leading to a relatively low intracellular concentration and negative reversal potential for chloride. Thus, activation of GABAA receptors results in a hyperpolarizing chloride influx and synaptic inhibition of neurons in the normal mature brain. However, during early brain development or under certain pathological conditions, KCC2 expression may be relatively low, leading to elevated intracellular chloride concentrations and a depolarizing response to GABA, increasing neuronal excitability and the propensity for seizures. Reduced KCC2 expression in pathological brain specimens from TSC patients with epilepsy have been reported, but the functional consequences of this abnormality and its potential role in causing epilepsy in TSC have not been previously investigated. In this grant, we test the novel hypothesis that decreased KCC2 expression may cause or contribute to epilepsy in mouse models of TSC and that pharmacological modulators that increase KCC2 expression may be effective treatments for seizures in TSC. This work is innovative in investigating a novel mechanism of epileptogenesis in the genetic epilepsy of TSC. The findings from these studies may have strong clinical significance and impact for developing novel therapies for epilepsy in TSC. In particular, some KCC2 modulators are already FDA-approved for other indications and could be repurposed and tested in clinical trials for epilepsy in TSC patients, potentially making the path to clinical translation and applications of this grant relatively immediate. In addition, as TSC is often viewed as a model disease, findings from this proposal may have relevance for the potential role of KCC2 in other epilepsies due to other causes.

NIH Research Projects · FY 2026 · 2024-04

PROJECT SUMMARY Type 1 diabetes (T1D) is caused by autoimmune destruction of β-cells that causes dependence on exogenous insulin. Despite remarkable advances in diabetes device technology, less than 25% of adults with T1D achieve the recommended HbA1c target of <7.0%. Moreover, subcutaneous delivery of insulin eliminates the normal portal-to-peripheral insulin gradient and causes “iatrogenic hyperinsulinemia” and whole-body insulin resistance, which increases the risk of cardiovascular complications. Novel therapies that improve glycemic control and reduce insulin requirements are needed to improve outcomes in people with T1D. A very-low-carbohydrate ketogenic diet (≤50 g carbohydrate/day) could reduce glycemic variability, total daily insulin requirement, and HbA1c in people with T1D. Indeed, several case series and observational studies of using a ketogenic diet (KD) in people with T1D have observed such benefits. However, we are not aware of any randomized controlled trials (RCTs) that evaluated the efficacy of KD for >7 days in people with T1D. In addition, there are serious concerns regarding the safety and tolerability of a KD in patients with T1D, including the potential for an increased risk of hypoglycemia, diabetic ketoacidosis, dyslipidemia, insulin resistance, decreased bone mineral density, and impaired quality of life. The purpose of this proposal is to conduct a two-site (Washington University School of Medicine in St. Louis, MO and Sansum Diabetes Research Institute in Santa Barbara, CA) 26-week RCT to evaluate the clinical efficacy, metabolic function, safety, socio-behavioral impact, acceptability and potential for dissemination of an isocaloric KD compared with an American Diabetes Association-recommended control diet in 80 adults with T1D. The following specific aims will be addressed: 1) determine the clinical efficacy and cardiometabolic effects of KD therapy in patients with T1D, including glycemic control [percent time-in-range 70-180 mg/dL (primary outcome), percent time in 70-140 mg/dL, mean glucose, glucose variability, and HbA1c], insulin sensitivity (determined by the hyperinsulinemic-euglycemic clamp procedure) (primary outcome), daily insulin requirement, 24-hour serial plasma glucose, FFA, triglyceride, insulin, glucagon, and ketone body concentrations; plasma lipid profile, hepatic de novo lipogenesis and cholesterol synthesis, body composition (fat-free mass, fat mass, appendicular lean mass, intra-abdominal adipose tissue, and intrahepatic triglyceride content), and selected plasma markers of inflammation; 2) assess the safety of KD therapy with respect to hypoglycemia, hyperketonemia, renal function and bone health; and 3) assess socio-behavioral factors and implementation outcomes, including sociodemographic factors (social determinants, unmet social needs), behavioral factors (eating behaviors, food cravings, diabetes distress and quality-of-life); and implementation outcomes (acceptability, feasibility, practicality, cost, complexity, and adherence).

NIH Research Projects · FY 2026 · 2024-04

ABSTRACT Maternal undernutrition affects 50% of the world’s female population. Maternal weight before pregnancy is a strong predictor of intrauterine growth restriction (IUGR), low birth weight, and future childhood stunting. While humanitarian efforts to improve nutritional status have focused primarily on children; there is little understanding of mechanisms underlying maternal undernutrition and its transmission to children. Our lab has identified perturbations in postnatal gut microbiota development that are evident in feces and the small intestine (SI). Our gnotobiotic mouse models have provided preclinical evidence that the SI microbiota of Bangladeshi children with stunting is causally related to the pathogenesis of a poorly understood enteropathy [environmental enteric dysfunction (EED)] characterized by loss of SI villi, reduction in intestinal absorptive area, epithelial barrier dysfunction and associated intestinal and systemic inflammation. The goal my project is to determine the impact of the SI microbiota of women with and without EED on arterial-remodeling at the maternal-fetal interface and maternal immune responses to inflammation at the maternal-fetal interface. Tissue-resident, non-cytotoxic uterine natural killer (uNK) cells produce IFN-𝛾 to remodel uterine spiral arteries into large luminal decidual arteries that maximize blood flow and nutrient delivery to the fetus. I hypothesize that maternal EED-associated SI microbiota leads to dysregulated vascular remodeling by impairing uNK cell function; this in turn reduces fetal growth by limiting nutrient availability. To test this hypothesis, Aim 1 will use germ-free (GF), conventionally-raised (CONV-R), and conventionalized mice (CONV-D = GF mice gavaged with CONV-R cecal contents) to first characterize the effects of the gut microbiota on placental/fetal development, including placental/decidual histological structures, NK cell composition at early (E11.5) and late (E17.5) stages of pregnancy. Bulk RNA-seq, single-nucleus RNA-seq, flow cytometry, multiplex assays of tissue/serum proteins, and histo/immunohistochemical methods will be used to define the cellular transcriptional/signaling/metabolic profiles of their placenta, decidua, and intestines. The resulting datasets will be analyzed using a suite of computational tools that I have applied to a substantial amount of preliminary results. Aim 2 will use groups of gnotobiotic mice colonized with i) a consortium of cultured SI bacteria from undernourished (low-BMI) Bangladeshi women with EED (based on histopathology of their duodenal mucosa obtained by endoscopy) and ii) a consortium of cultured SI bacteria from healthy (normal BMI) Bangladeshi women without histologic evidence of EED. I will apply methods from Aim 1 to E11.5 and E17.5 mice representing the 2 treatments groups to characterize placental/fetal development (transcriptional, signaling, metabolic and immunologic characteristics of placenta, decidua, and intestines including spiral artery development and NK cell biology). These experiments should advance knowledge about how the SI microbiota in EED impacts the maternal-fetal interface and could yield new therapeutic concepts/targets.

NIH Research Projects · FY 2025 · 2024-04

Economic choice is specifically disrupted in mental and neurological disorders such as major depression, frontotemporal dementia, and drug addiction. Established literature links this behavior to the orbitofrontal cortex (OFC). In recent years, my lab has examined neuronal activity in the OFC of monkeys and mice choosing between different juice types. Our studies revealed the presence of different cell groups: offer value cells encoding the value of individual offers; choice outcome cells encoding the identity of the chosen offer or the chosen action; and chosen value cells. In a computational sense, these variables capture both the input (offer value) and the output (choice outcome, chosen value) of the choice process, suggesting that the cell groups identified in OFC constitute the building blocks of a decision circuit. Indirect evidence supports this hypothesis. However, the anatomical organization of this circuit and the contributions of different cell groups are not known. Consequently, the decision mechanisms remain poorly understood. To shed light on these fundamental questions, we developed a preparation in which mice perform economic choices while we record from OFC using two-photon (2P) calcium (Ca2+) imaging through a GRIN lens. Longitudinal recordings showed that the encoding of specific decision variables by individual cells is very stable. Moreover, the representation of decision variables differs significantly across cortical layers: input variables populate mostly layers 2/3 (L2/3), while spatial and output variables populate mostly layer 5 (L5). These results support the notion of a stable decision circuit and provide a glimpse of its anatomical organization. However, to truly unravel the decision mechanisms, one needs to have a detailed understanding of how different cell groups connect with each other, how they influence each other’s activity, and how the activity of each cell group affects choices. Here we propose to address these questions using a combination of 2P imaging and single cell optogenetics. In Aim 1, we will assess the effective connectivity between cell groups. Mice will (a) be implanted with a GRIN lens accessing L2/3 and/or L5, (b) express a green Ca2+ indicator and a red shifted channelrhodopsin (ChR), and (c) perform choices under the microscope. In each mouse, we will image ~500 cells. First, we will assess the variable encoded by each cell. In separate sessions, while animals rest, we will holographically stimulate one specific cell group while imaging the entire population. Measuring the activity evoked by the stimulation, we will assess the effective connectivity between different cell groups. In Aim 2, we will use the same preparation, but we will deliver the stimulation while mice perform choices (in half of the trials). We will measure how optical stimulation affects performance, and thus assess the causal links between individual cell groups and choices. Relevance to R21. These experiments are innovative and technically challenging (high risk), but they can potentially provide unprecedented insights into the mechanisms underlying economic decisions (high reward). The proposal is exploratory, but we have the background and expertise to successfully conduct this research.

NIH Research Projects · FY 2026 · 2024-04

This program is an extension of our successful HIV basic science training program with the HIV Cure Research Infrastructure Studies (H-CRIS) based at the University of Ghana - an ongoing collaboration between Washington University in St. Louis (WU) and the University of Ghana (UG). Finding a permanent cure for people living with HIV, a key priority for the NIH, requires rigorous and basic science research. The main obstacle to an HIV cure is the persistence of transcriptionally silent and immunologically unrecognizable proviruses in quiescent memory CD4+ T cell reservoirs in people who are on ART. Although over 70% of HIV patients live in Africa, very little of the basic science of cure research involves African patients, scientists, or the subtypes in Africa. This is a clear deficiency in the current research efforts that needs immediate remedy. Most HIV basic science research on a cure has been conducted on one viral subtype (HIV subtype B). However, Africa has many viral subtypes, including A, C, G, and recombinant forms such as CRF2_AG, CRF2_AE, HIV-2, and others, which can all affect the latent viral reservoir. In addition, African patients also have co-infections like tuberculosis, hepatitis, and malaria, which can all determine CD4+ T cell responses. Therefore, training African scientists in HIV basic science will help account for these deficiencies in the cure research efforts, which will benefit people living with HIV in the United States. Since 2018, a partnership between WU and UG has set up the HIV Cure Research Infrastructure Studies (H-CRIS) at the Noguchi Memorial Institute for Medical Research, UG. H-CRIS is training graduate students and postdoctoral fellows in HIV basic science and cure research with outstanding success; many of the trainees have obtained independent grant funding. Over the five years of the proposed D43 program, we expect to enroll 3 PhDs (4-year program) and 4 MPhil students (2-year master's with a laboratory research thesis), and to provide a two-year intensive basic laboratory research training and mentorship for four postdoctoral fellows (11 trainees in all). In addition, our grants and manuscript writing workshops will be open to students and faculty at the UG and are expected to benefit over 80 additional scientists at the University of Ghana. The predominant training site will be the UG, with medium- and short-term experiences at WU. Our specific aims are: 1. To build capacity and experience at the University of Ghana for basic science researchers to design and conduct HIV cure-related research in Ghana. 2. To equip trainees with research skills through 2-year intensive and mentored postdoctoral research projects focused on HIV basic science and cure. 3. To establish and maintain a mentorship plan for trainees at UG who aspire to become world-class, independently funded investigators in the basic science of HIV research.

NIH Research Projects · FY 2026 · 2024-04

ABSTRACT Chloride channels contribute to airway health and disease by modulating airway secretion and mucociliary function. We discovered that the channel regulator CLCA1 is a potent and specific potentiator of the TMEM16A calcium-activated chloride channel, but there remains a major gap in understanding how this pair influences airway biology. In order to fill this gap and develop therapies for mucus-obstructive diseases that target CLCA1-mediated TMEM16A potentiation, the following are required: 1) a comprehensive understanding of TMEM16A isoform expression patterns and biophysical properties in diseased airway; 2) an understanding of how CLCA1-TMEM16A impact mucus properties in disease; and 3) the structural basis for CLCA1 potentiation of TMEM16A. These points are addressed by each aim of this project. Since it is challenging to fully investigate the potential mucus-modifying abilities of other channels in normal human airways due to the dominant activity of cystic fibrosis transmembrane regulator (CFTR), we will utilize human cystic fibrosis (CF) models to study CLCA1-TMEM16A biology in more depth. Investigating this system in CF also provides an opportunity to evaluate other channels that may compensate for CFTR as therapeutic targets for patients unresponsive to CFTR modulators. We have made a series of technical advances to facilitate our proposed studies investigating the role this pair plays in human airway biology. First, we discovered a minimal domain of CLCA1 (CLCA1 VWA) that potentiates TMEM16A in cells, is stable and can be purified in large quantities for functional studies. We have also devised ex vivo airway and human cellular models, using cystic fibrosis (CF) airway tissue, primary CF cells and cell lines for detailed studies of TMEM16A activation in regulating mucociliary function. Last, we have developed the tools required to determine the molecular structure of the CLCA1- TMEM16A complex. To address the function of the CLCA1-TMEM16A system in human airway health and disease, we propose a multidisciplinary investigation that incorporates banked human specimens, single cell and spatial ‘omics, multicellular human models and state-of-the-art structural approaches. In Aim 1, we will use single-nucleus RNAseq and merFISH to investigate expression dynamics of TMEM16A isoforms in CF disease. In Aim 2, we will potentiate TMEM16A with CLCA1 VWA in CF airway tissue and cellular models to characterize the impact of TMEM16A activation on beneficial mucus properties. In Aim 3, we will define the molecular mechanism for CLCA1 activation of TMEM16A using cryo-electron microscopy (cryo-EM). These studies will examine the impact of CLCA1 potentiation of TMEM16A in human airway disease, which will provide critical insight into broader regulation of TMEM16 family members relevant to lung biology and produce the molecular framework to develop much needed novel therapeutics for muco-obstructive diseases.

NIH Research Projects · FY 2026 · 2024-03

PROJECT SUMMARY / ABSTRACT While the promise of gene-based therapies for disabling neuromuscular diseases is finally becoming a reality, research efforts thus far have primarily focused on gene replacement strategies for recessive, loss-of-function disorders. Such strategies are not translatable to most dominant muscular dystrophies, hindering the development of new treatment strategies.1,2 Our group recently identified mutations in DNAJB6 that cause limb girdle muscular dystrophy D1 (LGMDD1), a dominantly inherited disabling myopathy with no current treatment options.3 The overarching goal of this proposal is to develop novel therapies for this devastating disease. Addressing this unmet need will advance the field’s understanding of how to treat LGMDD1 and establish the optimal approach to therapeutic development for other dominantly inherited disorders with complex, heterogeneous disease mechanisms.1,2 Several lines of preliminary data indicate that mutations in DNAJB6 exert a dominant effect via a toxic gain-of-function.4,5 The potential for deleterious effects preclude a global DNAJB6 knockdown strategy, as DNAJB6 knockout (KO) mice are embryonic lethal due to aggregation of client proteins.6 Haploinsufficiency appears to be tolerated, as heterozygous KO mice are viable, with no apparent skeletal muscle phenotype, and DNAJB6 frameshift and nonsense mutations are seen in healthy “control” patients in genetic databases. We propose to selectively knockdown mutant DNAJB6 using silencing RNA (siRNA). Our preliminary results indicate that allele specific knockdown (ASKD) is feasible and capable of correcting a proteomic signature of LGMDD1 disease in vitro. Thus, our central hypothesis is that ASKD of mutant DNAJB6 is a viable therapeutic approach to address the toxic gain-of-function mechanism of LGMDD1, while avoiding complete knockdown. In this project we will validate that ASKD of mutant DNAJB6 improves disease phenotypes in a LGMDD1 mouse model (Aim 1) and in vitro human LGMDD1 models (Aim 2). Finally, we will optimize ASKD targeting a common DNAJB6 single nucleotide polymorphism (SNP) to expand the applicability of this therapy to multiple DNAJB6 mutations (Aim 3). We have generated a knock in LGMDD1 mouse with a heterozygous p.F90I mutation, orthologous to human p.F89I, and a FLAG-tagged wild type (WT) allele, enabling size-based distinction of WT vs. mutant RNA and protein. We also generated induced pluripotent stem cells (iPSCs), gene-corrected isogenic controls, and primary myoblast cultures from LGMDD1 patients. Successful completion of the proposed aims will produce essential preclinical data supporting the therapeutic translation of ASKD for LGMDD1.

NIH Research Projects · FY 2024 · 2024-03

ABSTRACT (verbatim, original text) The overarching goal of this proposal is to lower the age of detection in autism to early infancy, making presymptomatic (i.e., before the emergence of ASD-specific behavioral features) intervention feasible. Infants with an older autistic sibling have up to a 20% risk of developing autism spectrum disorder (ASD). Prospective high familial risk (HR) infant sibling studies have shown that the defining behaviors of ASD do not emerge until the latter part of the first year and into the second year of life. Therefore, the vast majority of affected children are diagnosed after age 2. No behavioral markers in the first year of life have yet been identified that can predict later ASD diagnosis with sufficient accuracy (i.e., positive predictive value: PPV ≥ 80%) to justify presymptomatic intervention. We recently published two independent approaches that use brain imaging in the first year of life to predict which HR infants will be diagnosed with ASD at 2 years of age. Specifically, structural MRI (sMRI) at 6 and 12 months of age, and resting state functional connectivity MRI (fcMRI) at 6 months of age independently predicted later ASD diagnosis in HR infants with over 80% PPV. Our preliminary data show that a third MRI approach, using regions of CSF volume and cortical shape at 6 months of age can also accurately predict later ASD diagnosis. If we replicate and extend these findings, we will be able to identify individual infants at “ultra- high risk” (80% chance) of developing ASD, rather than being limited to group-level risk (20% chance), where we do not know who will later be affected. This R01 application aims to move our initial findings toward a clinical test for ASD in HR infants in the first year of life. Aim 1 will validate our previous findings in a new, independent sample of HR infants, extend our methods to a new MRI platform, and examine whether fcMRI and/or sMRI, with and without behavioral information, during the presymptomatic period in infancy, accurately predict ASD diagnosis at 24 months of age. Aim 2 will move beyond predicting categorical diagnosis to predicting dimensional, clinically-relevant characteristics for individual infants. Specific dimensional targets include expressive language level, social responsiveness, initiation of joint attention, and repetitive behavior. Validating and extending our findings on presymptomatic prediction of ASD in a new sample, on a different MRI scanner, and with dimensional developmental characteristics are critical next steps for moving the field forward toward (a) the development of a clinically-useful, presymptomatic test for identifying ultra-high risk infants who would benefit from very early intervention in infancy, (b) efficient studies of presymptomatic intervention strategies in individuals at ultra-high risk, and (c) the development of future presymptomatic tests for use in the general (not just HR) population.

NIH Research Projects · FY 2026 · 2024-03

PROJECT SUMMARY The objective of this proposal is to define the mechanism and functional role of innate immune signaling events during base damaging cancer chemotherapy. These agents induce base damage on RNA as well as DNA. We have recently discovered that cells elicit an RNA-dependent innate immune response upon encountering alkylating agents, which represent one of the most commonly used systemic chemotherapies for cancer treatment. Our preliminary data demonstrates that alkylating agents induce a DAMP (damage associated molecular pattern) response, akin to the signaling observed during viral infection, which is known to impact downstream cellular events such as apoptosis. In contrast to other types of DNA damaging agents, this pathway depends on the RNA-associated DAMP receptor RIG-I and requires spliceosomal activity. Furthermore, we have found that ASCC1, which encodes a protein with a metal-independent RNA phosphoesterase domain, associates with intronic RNAs upon damage and is required for the induction of the DAMP response during alkylation. These and other data strongly suggest a model where spliceosome-associated processing of damaged RNA activates DAMP signaling, and may have functional consequences for tumor cell fate upon base damage. In this proposal, we will determine the mechanism by which ASCC1 functions with its associated partner proteins to gain access to damaged nascent RNAs and process them as part of this response, and determine the function consequence of this signaling pathway (Aim 1). In turn, we will characterize the RNA structures that activate RIG-I during alkylation stress and reconstitute RIG-I signaling with purified components (Aim 2). Together, these studies will shed light on a hitherto undescribed mechanism by which a nuclear RNA quality control pathway is connected to innate immune signaling.

NIH Research Projects · FY 2026 · 2024-03

Project Abstract Over 297,790 new cases of invasive breast cancer diagnoses and 43,170 deaths from metastatic breast cancer (mBC) are expected in the USA this year (SEER database). Despite advances in the treatment of localized disease, the 5-year overall survival of patients with mBC remains at a dismal 29%. Current therapeutic strategies primarily focus on tumor cells and neglect the important role the tumor stroma plays in cancer progression. Underscoring the importance of the stromal compartment, stromal gene signatures can predict clinical outcomes for breast cancer patients7. The most common site of breast cancer metastasis is the bone and unfortunately, once the disease metastasizes to the bone, it is considered incurable and treatment is mainly palliative8. In the bone, the tumor stroma consists of a wide array of cell types including fibroblasts, adipocytes, nerve cells, endothelial cells, immune cells, chondrocytes, osteocytes, osteoblasts, and osteoclasts. Nearly all of these stromal cell types has been implicated in tumor progression by directly supporting tumor cell growth, survival, modulating the immune response to tumors, and/or driving bone loss that ignites a vicious cycle that supports further tumor growth in the bone8. One important stromal cell that supports primary tumor progression is the senescent cell, which was recently described as an emerging cancer hallmark9. Indeed, we demonstrated that senescent fibroblasts drive tumor growth10 and create immunosuppressive environments11 in the primary tumor setting through expression of p38MAPKα-dependent senescence associated secretory phenotype (SASP) factors. Whether the same mechanisms are active in the metastatic setting remains an open question. We have found that p38MAPKα inhibition (p38i) reduces mBC progression by targeting the metastatic stromal compartment, but not tumor cells, in numerous preclinical models. In this proposal, we will examine how p38MAPKα activation in the metastatic stroma contributes to tumor progression and modifies the immune response to allow mBC to progress. We will utilize genetic and pharmacologic tools to identify the cell targets of p38i, determine if a new stromal cell that we have identified, the senescent metastatic-associated cancer associated fibroblast (smeCAF) contributes to cancer-induced bone loss, and determine if smeCAFs modify the host immune response within the bone. Our goal is to understand how p38i alters the stromal compartment to decrease mBC progression so that this knowledge can be leveraged to develop combinatorial strategies to deploy p38i, senolytics (drugs that kill senescent cells), and immunotherapy to limit mBC progression.

NIH Research Projects · FY 2026 · 2024-03

PROJECT SUMMARY / ABSTRACT On a population level, digital mental health interventions effectively reduce depression and anxiety symptoms. However, middle-aged and older adults with depression and/or anxiety and coexisting chronic pain have not been adequately represented in digital mental health studies and are a significant population because: 1) chronic pain reduces the effectiveness of stand-alone mental health treatment unless a person’s pain is simultaneously addressed; 2) the prevalence of chronic pain increases with age; 3) physical and mental health related disability in these age groups have unique downstream effects (lost workforce, effects on dependent children, strain on caregiver availability); and 4) use of technology in these age groups is widespread and growing, but these users have unique digital health needs and preferences. This proposal is a partnership between Washington University and Wysa, an established mental health app company that delivers cognitive behavioral therapy, mindfulness training, and sleep tools using chatbot technology and human coaches. Wysa has developed an app specifically for people with mental illness and coexisting chronic pain (Wysa for Chronic Pain (WCP)) which addresses depression and anxiety via the intermediate mechanisms of behavioral activation, pain acceptance, and sleep quality. However, the app is not yet designed to meet the usability needs of middle-aged and older adults with chronic pain. The goals of this proposal are to: 1) refine an established digital mental health intervention (WCP) for middle-aged and older adults with depression and/or anxiety and coexisting chronic pain, and 2) determine its effectiveness. The central hypothesis is that incorporating just-in-time adaptive interventions (JITAIs) and other usability-related adaptations will improve app engagement and, subsequently, depression and anxiety symptoms in this target population. Using a human-centered design approach, the Behavioral Intervention Technology (BIT) model will be leveraged to link established behavioral change theory with technology-related usability and engagement factors. Study activities will follow the Discover, Design / Build, and Test (DDBT) framework. Aim 1 is to identify stakeholder- informed contextual determinants of engagement with WCP by the target population. Members of the target population with varying levels of technological literacy will participate in semi-structured interviews and usability testing. Next, in a series of micro-randomized trials, adaptations designed to increase engagement with WCP (e.g., JITAIs) will be created, tested, and iteratively refined (Aim 2). Finally, in a pragmatic randomized clinical trial including 4,000 commercial Wysa users, the real-world effectiveness of the package of adaptations to improve depression and anxiety symptoms within 12 weeks will be determined in a hybrid type 1 effectiveness- implementation study (Aim 3). This proposal responds to PAR-22-154’s call to leverage an existing partnership between WU and a theory-driven, well-established digital health platform (Wysa) to rapidly develop and test JITAIs and their effect on digital engagement in people with clinically significant functional impairment.

NIH Research Projects · FY 2025 · 2024-03

Huntington’s disease (HD) is a devastating and invariably fatal neurodegenerative disease caused by an abnormal expansion of polyglutamine repeat in the protein called huntingtin (Htt). HD is characterized by progressive loss of selective neurons in the striatum and cortex, but the precise mechanisms underlying neuronal dysfunction and death are not fully understood, and no disease-modifying treatment is currently available. Thus, there is an urgent need to identify critical therapeutic targets and establish effective neuroprotective treatments for HD. One of the major challenges in developing effective treatments lies in the complexity of the brain and our incomplete mechanistic understanding of the disease, The brain comprises diverse cell types, each with distinct vulnerabilities in HD, which complicates treatment approaches. Understanding the individual responses of these brain cell types to the treatment would be crucial for maximizing the effectiveness of the treatment. Transcriptional dysregulation is one of the early molecular abnormalities found in the course of HD and is thought to play a central role in disease pathogenesis. Accumulating evidence suggests that genome-wide perturbations of DNA methylation may drive alterations in gene expression in HD. The fundamental objective of this proposal is to determine the effect of a DNA demethylating agent on gene expression, behavior, and pathology of HD in vivo using two different mouse models. The molecular impact of the treatment on individual cell types in the brain, including neurons and glial cells, in particular, in the striatum, the most vulnerable brain region in HD, will be determined. The underlying hypothesis is that treatment with the hypomethylating agent protects HD brain by inhibiting specific DNA methylation-mediated transcriptional alterations critical for neuronal function and survival, thereby attenuating disease progression. Importantly, therapeutic manipulation of DNA methylation has not been clinically used for neurodegenerative diseases, including HD. To test our hypothesis, the following specific aims will be pursued: 1) Determine the impact of a DNA methylation inhibitor on altered gene expression in individual cell types in HD mouse brains and 2) Determine the effectiveness of the DNA methylation inhibitor on behavior and pathology of HD mice. Given that both neurons and glia contribute to HD pathogenesis, it is crucial to understand the transcriptional responses of individual brain cell types to the treatment. The impact of the demethylating agent in multiple cell types in the striatum of HD model mice will be determined by innovative single-nucleus RNA-seq analysis. If successful, the proposed study will lay the foundation for the development of a new class of disease-modifying treatment for HD. It will also provide a mechanistic understanding of neuroprotection in vivo by this manipulation.