Washington University

NIH Research Projects · FY 2025 · 2021-07

This is an application for a new Postdoctoral Training Program in Genomic Medicine (T32). We propose two postgraduate positions per year to support physician-scientists who are engaging in medical genetics research. There are too few medical geneticists to meet the growing need for our services. As a result, much of medical genetics testing, diagnosis, counseling, and management occurs outside of our field and is instead practiced by physicians who receive little specific training. Our goals are therefore twofold: first, to mentor future medical geneticists who are embarking on a career in academic medicine and therefore grow the medical genetics workforce; and secondly, to provide medical genetics training to physician-scientists in other fields who engage in medical genetics-related research, which will enhance the clinical knowledge-base of those specialists who may be most likely to use genetic testing or treatments in their practices. The specific aims of this proposal include: 1) protected mentored research experiences with well-established investigators performing research in medical genetics within the Washington University School of Medicine, 2) obligatory educational programs in laboratory management, scientific rigor, statistics, grantsmanship, responsible conduct of research, and biomedical informatics 3) individualized specific coursework based on the trainees’ areas of investigation, 4) continuous feedback to the trainees, mentors and program leadership, and 5) the development of trainees. Trainees may choose from four focus areas, which represent patient care needs with particularly rapid growth: Cancer, Rare and Undiagnosed Diseases, Gene-Environment Interaction, and Neurodegeneration. The program director, Patricia Dickson, will work closely with co-director Jorge Di Paola to select and oversee training of future leaders in genomic medicine. Major strengths of this program include active medical and laboratory genetics training programs, in-house clinical cytogenetics laboratory, biochemical laboratory, and sequencing capability, a large number of centers and groups performing genomic studies, including Undiagnosed Diseases Network clinical and model organism screening center sites, the McDonnell Genome Institute, the Cancer Atlas Network, the Edison Family Center for Genome Sciences & Systems Biology, and others. Washington University provides an outstanding mentorship environment and infrastructure for genetics research, with an emphasis on collaboration and a superb track record of producing physician-scientists. This proposal will take advantage of the wealth of genetics research, clinical and research training, and mentorship that is thriving on our campus to train those scientists who will bring innovative diagnostics, therapeutics, and data-driven practice to the clinic.

NIH Research Projects · FY 2025 · 2021-07

Project Summary/Abstract Type 2 diabetes (T2D) is characterized by both a loss of insulin sensitivity of target and ultimately, impaired insulin secretion from the pancreatic b-cell. We have identified a novel SWELL1-mediated signaling pathway that regulates both insulin sensitivity and insulin secretion, whereby SWELL1 loss-of-function can both down- regulate insulin signaling in target tissues, and insulin secretion from the pancreatic b-cell. We have identified a small molecule modulator, DCPIB (renamed SN-401), as a tool compound that binds the SWELL1-LRRC8 complex and functions as a molecular chaperone to augment SWELL1 expression, plasma membrane trafficking and signaling. In vivo, SN-401 normalizes glucose tolerance by increasing insulin sensitivity and secretion in murine T2D models. SN-401 also augments glucose uptake into adipose tissue and myocardium, suppresses hepatic glucose production in KKAy mice, and protects against hepatic steatosis in HFD fed mice. We propose that small molecule SWELL1 modulators represent a “first-in-class” therapeutic approach to treat metabolic syndrome and associated diseases by restoring SWELL1 signaling across multiple organ system that are dysfunctional in T2D. Combining recent cryo-EM data of SN-401 bound to its target SWELL1/LRRC8a with molecular docking simulations we have validated a structure-activity relationship (SAR) based approach to generate novel SN-401 congeners with either enhanced or reduced on-target activity. The objectives are: 1. To establish the optimal dosing regimen and mode of administration for the newly synthesized, SAR-inspired SN-401 congeners synthesized to date to achieve a therapeutic effect for T2D; 2. To evaluate for putative beneficial cardiovascular effects; 3. To determine the primary tissue-site(s) of action of SN-401 in vivo; 4. SAR-based compound synthesis to refine and optimize the leads based on in vitro ADMET and selectivity screens, and efficacy studies in vivo. We propose the following specific AIMs AIM#1: Determine optimal dosing regimen, therapeutic effect and target tissue(s) of novel SAR-inspired SN-401 congeners. AIM#2: SAR-directed SN-401 optimization and characterization in vitro and in vivo to identify preclinical lead structures. This proposal seeks to use a validated chemical biology approach to expand a pipeline of novel, bioactive pharmacological SWELL1 signaling modulators for the treatment of T2D, metabolic syndrome and associated diseases to ultimately take into man in the form of a clinical trial for efficacy in humans.

NIH Research Projects · FY 2025 · 2021-07

PROJECT SUMMARY This proposal will determine whether increasing striatal cholinergic interneuron (ChI) activity in the developing mouse brain can prevent dystonia following neonatal brain injury . Dystonic cerebral palsy (CP) due to neonatal brain injury is the most common cause of childhood dystonia and is often medically refractory and functionally debilitating. Yet, its unique pathophysiology remains understudied. Dystonia pathophysiology is more commonly studied in models of rare genetic dystonias which are characterized by striatal ChI hyperexcitability. However, anticholinergic medications are often ineffective for treating dystonia in CP. Determining whether there is striatal cholinergic pathology specific to dystonic CP could yield better targeted treatments. To this end, I have developed a clinically-relevant rodent model of neonatal hypoxic brain injury that displays electrophysiologic markers of dystonia three weeks after injury, mimicking the clinical latency period between neonatal brain injury and dystonia emergence. This latency period allows testing of pre-symptomatic interventions for dystonia prevention. My preliminary data demonstrate increased striatal ChI number in my model but that striatal ChI excitation in young mice during the pre-symptomatic window may be protective against dystonia. In sum, these data suggest that increased striatal ChI number and striatal ChI hyperexcitability may be compensatory mechanisms that are protective against dystonia and, therefore, could be enhanced to prevent dystonia following neonatal brain injury. To test this hypothesis, I propose the following aims: (1) determine whether chemogenetic modulation of striatal ChI activity in young mice after neonatal brain injury changes dystonia severity in adult mice; (2) determine whether chemogenetic modulation of striatal ChI activity in young, otherwise healthy, mice can cause dystonia in adult mice; and (3) determine whether the striatal ChI hyperexcitability observed in genetic dystonias is also present in my model of dystonia following neonatal brain injury. These studies will determine whether pre- symptomatically increasing striatal ChI firing after neonatal brain injury could reduce or prevent dystonia. My long-term career goal is to run a translational research lab focused on preventative treatment development for dystonic CP. I have studied basal ganglia pathophysiology for ten years and have developed a new model of dystonia following neonatal brain injury which will be used for the proposed experiments. However, to complete the proposed research and facilitate my transition to independence, I need additional mentored training in slice electrophysiology (Dr. Steve Mennerick) and chemogenetics (Dr. Jordan McCall). As my physician-scientist advisor, Dr. Joel Perlmutter will provide expertise in dystonia pathophysiology and ensure the translational relevance of my research. The Washington University School of Medicine and Department of Neurology provide a world-renowned research environment and a legacy of passionately and effectively supporting junior faculty. In sum, my proposed research, mentorship team, training plan, and institutional environment pave my path to independence and submission of an R01 on identification of treatment targets for dystonic CP.

NIH Research Projects · FY 2025 · 2021-06

PROJECT ABSTRACT If oxygen supply to the brain does not match its demands, cellular functions are impeded and can lead to cell death. A margin is created between oxygen supply and brain demand by metabolic and hemodynamic reserves. Our preliminary data show that children may have less reserve due to the critical, but costly energy demands of brain growth and maturation. We have shown that lower oxygen supply (hypoxemia) also decreases reserves, and during childhood, may result in impaired brain growth and development. Children with sickle cell anemia (SCA) have chronic hypoxemia due to reduced hemoglobin. Children with SCA have smaller brain volumes and decreased cortical thickness than unaffected children. The intersection of hypoxemia and brain development is poorly understood, impeding our ability to optimize brain development and neurologic outcomes in children with hypoxemia. The goal of this project is to identify physiologic mechanisms of vulnerability and age-dependent consequences of hypoxemia. Our central hypothesis is that the high cerebral metabolic demand in younger children decreases oxygen reserve, resulting in an age-dependent increased risk for impaired brain growth in children with lower oxygen supply. First, we will assess normal developmental changes in metabolic and hemodynamic reserve in 80 healthy children ages 4-21 to determine age-dependence of reserves (Aim 1). Next, we will determine the effects of hypoxemia on metabolic and hemodynamic reserves and the consequence of hypoxemia on brain development. We will compare 40 children with SCA and 40 age and sex-matched controls at baseline and 3 year follow-up imaging, and examine cortical thickness changes in the two cohorts (Aim 2). Finally, we will evaluate whether oxygen reserve increased through hydroxyurea treatment in an SCA cohort impacts long-term brain development. We will utilize a large sickle cell database with 16 years of brain imaging to compare cortical thickness maturation and total brain volumes between treated and untreated children (Aim 3). Determining the age-dependence of hypoxemic vulnerability and its effect on brain development will allow us to personalize treatment strategies for children at high risk for neurodevelopmental injury to be more aggressive during periods of highest vulnerability.

NIH Research Projects · FY 2025 · 2021-06

ABSTRACT During the first 3 years of life (YOL) the infant gut microbiome (GM) rapidly diversifies both in structure and function, concomitant with dietary and environmental transitions. Critically, the GM response to specific external stimuli is patient-specific, complicating individualized risk predictions. Healthy GM maturation includes accruing multiple strains of the same species, which frequently differ in key functions. These functional differences, ac- centuated by horizontal gene transfer (HGT) and de novo mutations, could resolve conflicting associations of the same species with both health and disease. The rationale behind our proposal is that strain- and species- level variation in bacterial functions drives heterogenous GM responses to early-life (EL) dietary and antibiotic perturbations, which explains, in part, individualized developmental trajectories. This proposal pursues two highly complementary Aims: 1) Define strain-resolved functional maturation of the pediatric gut microbiome and 2) Investigate the acute effects of EL antibiotic (ELA) perturbation on strain dynamics, HGT, and micro- biome maturation in preterm neonates and microbiota-humanized mice. Aim 1 will test the hypothesis that EL environmental exposures shape genomic diversification of gut species, causing lasting changes in GM com- munity structure and microbial functions. We will leverage our unique set of 2,436 stools collected over the first 9 YOL from infants variably exposed to dietary and environmental stimuli. By combining culture-enriched meta- genomics, metatranscriptomics, and metabolomics, we will determine taxa-function relationships at the sub-spe- cies level and power statistical models that predict the impact of EL exposures on strain diversification, microbe- function associations, and transcriptional activity. Aim 2 will test the hypothesis that ELAs acutely alter strain dynamics and stimulate HGT and that the GM response to ELA can be predicted from baseline composition and function. Here, we will interrogate 160 stools flanking variable ELA exposure in 80 preterm neonates in the first 4 months of life, combining culture-enriched metagenomics with selective culture and isolate sequencing to char- acterize the preterm `plasmidome' and profile post-ELA strain dynamics and HGT. To identify microbiome-intrin- sic responses to ELA, we will utilize an innovative transgenerational mouse model where germ-free dams receive human, preterm, microbiota that is vertically transferred to their pups, which are treated with parenteral antibiot- ics. We will use the resulting data to predict individual GM responses to specific antibiotics based on composition, resistance gene content, and bacterial functions. Our proposal is innovative because our interdisciplinary re- search team will characterize strain-level bacterial functions to understand the heterogeneity of GM responses to EL perturbations on two pre-existing sets of human specimens; it is significant because it will identify features that predict species-resolved GM-specific responses to EL selection. Our work will advance pediatric microbi- ome research by comprehensively characterizing strain-resolved functional maturation and GM disruption to understand individual variation leading towards a future of personalized, microbiome medicine.

NIH Research Projects · FY 2024 · 2021-06

Osteoarthritis (OA) is painful and debilitating by affecting the synovial joints, and is found in over 12% of the total United States population 25-74 years of age. The prevalence of OA increases significantly with age, with radiographic evidence in over 70% of the population over age 65. In this growing segment of our society, OA is a significant contributor to disability, frailty and social isolation. Despite the tremendous socioeconomic impact of OA, there are no disease-modifying therapies available. OA is distinctively characterized by the progressive, degenerative changes in the morphology, composition, and mechanical properties of articular cartilage. Mechanotransduction in articular chondrocytes is a key component of disease pathogenesis, given the link between direct sensing of the cells’ mechanical environment and the resulting metabolic imbalance of cartilage in OA. We have recently identified the mechanosensitive PIEZO ion channels - in fact a synergy between PIEZO1 and PIEZO2, both expressed in articular cartilage - to underlie chondrocyte mechanotransduction in response to injurious mechanical stress. The overall objective of this study is to define the mechanisms of Piezo-mediated mechanotransduction in chondrocytes more in-depth so that these insights can be leveraged toward the development of disease- modifying approaches in joint-loading-induced injuries, including OA. In addition to our recent discovery of chondrocytic Piezo-mediated mechanotransduction, we found that treatment of chondrocytes with pathophysiologically-relevant concentrations of IL-1α, a pro-inflammatory cytokine, increased Piezo1 gene expression, and that increased expression of Piezo1 was also present in osteoarthritic cartilage from aging pigs and humans. Thus, the Specific Aims of this grant are: (1) to determine the mechanisms of synergistic functioning of Piezo1/2 in chondrocyte mechanotransduction; (2) to deconstruct Piezo-mediated mechanotransduction in chondrocytes under inflammatory conditions; (3) to elucidate the role of Piezo- mediated mechanotransduction in organotypic cartilage explants and in-vivo. Aim 1 will rely on cellular studies. We will explore synergisms of Piezo1/2 at the levels of electrophysiology, channel trafficking, finite element modeling, and ultra-structure, the latter also examining human cartilage from OA vs controls. In Aim 2 primary porcine chondrocytes will be stimulated with IL-1α for deconstruction of Piezo-mediated mechanotransduction. Aim 3 will rely on porcine osteochondral explants and chondrocyte-specific and inducible Piezo1/2-/- mice which we have generated. Various modes of mechanical stress will be applied to cells, explants, and animals, and loss-of-function studies of Piezo-mediated mechanotransduction will be conducted with both mechanistic intent and translational/therapeutic direction. The proposed Aims will extend our initial discovery with mechanistic in- depth studies that will increase our understanding of OA in a non-incremental manner, and this will inspire the development of new Disease-Modifying OA Drugs (DMOADs).

NIH Research Projects · FY 2024 · 2021-06

Project Summary/Abstract Major depression is one of the most common mental health disorders in the United States, affecting approximately 20% of the population at least once in their lifetime. Pharmacologic treatment has been anchored in monoamine reuptake inhibitors; however up to a third of patients find no benefit from these agents. Recently, the development of novel rapid-acting antidepressant treatments has provided improvement of depression symptoms for some patients refractory to traditional pharmacotherapy. Brexanolone, a formulation of the endogenous neurosteroid allopregnanolone (AlloP), is a rapid-acting agent for postpartum depression; however, undesirable side effects limit the feasibility of its widespread use. It is crucial to identify neurophysiologic actions underlying the antidepressant effects of rapid acting agents to inform continued development of safe and effective pharmacotherapies for depression. One proposed mechanism for rapid acting antidepressants is the disinhibition of neural circuits. Here, we focus on hippocampus as a nexus of debilitative cognitive symptoms in depression and other neuropsychiatric illness. We will characterize AlloP- induced changes to neural activity at cellular, circuit, and network levels to identify specific changes to neurophysiology induced by this rapidly acting antidepressant. We hypothesize that low-dose AlloP cultivates paradoxical disinhibition that generates distinct in vivo electrophysiological characteristics of rapid antidepressants. We will test this hypothesis in a series of experimental aims. Aim 1 will test the hypothesis that AlloP disinhibits CA1 through cellular and population measures of activity in vitro. We will test whether sub-sedative concentrations of AlloP disinhibits CA1 pyramidal cells and query the involvement of interneurons in this phenomenon. Aim 2 will investigate the hypothesis that clinically relevant concentrations of AlloP have preferential actions onto hippocampal interneurons compared to CA1 pyramidal neurons. We compare direct actions of AlloP on inhibition of interneurons and pyramidal cells, and probe contributions by GABAAR subpopulations. Aim 3 will identify features of neural oscillations induced by AlloP in vivo at doses relevant for antidepressant effects. Mice will be monitored with video EEG and by depth LFP recordings after administration of drug to probe physiologic markers that differentiate AlloP from comparators and thus may mark antidepressant potential. The results will provide a better understanding of how neurosteroid drugs affect different cell and receptor types to ultimately regulate circuit and network activity. The combination of diverse experimental approaches provides excellent training potential for my scientific training. Coupled with the outstanding clinical training and mentorship provided by Washington University School of Medicine, this proposal will help me achieve my career goal of becoming an independent physician-scientist.

NIH Research Projects · FY 2024 · 2021-06

PROJECT ABSTRACT Lung transplantation is a life-extending therapy for end-stage lung disease, but necessitates life-long immunosuppression with agents that have narrow therapeutic windows and inevitable side effects. Mycophenolic acid (MPA) is the antiproliferative agent of choice. Unlike other agents, routine therapeutic drug monitoring (TDM) is not performed for MPA because it is challenging to measure area under the curve from 0–12 hours (AUC) to accurately assess pharmacokinetics (PK). MPA levels vary widely among transplant recipients, and excess levels are associated with adverse outcomes such as neutropenia, which affects >40% of lung transplant recipients receiving MPA and negatively affects outcomes. This variability is due in part to differences in genes involved in MPA metabolism and affects outcomes. We previously identified a link between single nucleotide polymorphisms in SLCO1B3, a key gene in MPA metabolism, and acute rejection and survival in lung transplant recipients. Although this and other associations are now known, there has been no attempt to incorporate genetic data into TDM strategies for MPA or develop an evidence-based therapeutic range for MPA in lung transplant recipients. We are developing a tool that integrates PK, genetic, and clinical data to predict dose-normalized AUC for lung transplant recipients receiving MPA. This tool will require a single time-point PK measurement and will provide an easily available tool for MPA PK research and clinical management. Unfortunately, target MPA AUC ranges have not been defined due to the lack of routine MPA TDM in lung transplant recipients. We hypothesize that an optimal MPA concentrations provides adequate immunosuppression while preserving immune cell function, and that MPA level variability has a strong genetic component. Therefore, we will achieve three aims in this K01 study. (1) Determine a target therapeutic range for MPA in lung transplant recipients. (2) Identify genetic factors that influence dose-normalized MPA concentration. (3) Establish the relationships between MPA exposure, genetic polymorphisms, and neutrophil function, which are key factors in innate and adaptive immune systems. This will improve clinical practice and outcomes in lung transplant recipients by enhancing drug efficacy and reducing adverse effects. This experimental work is integrated with a comprehensive career development plan that builds on Dr. Tague’s previous training (MD, Master of Science in Clinical Investigation, research in genetics and translational medicine) to become an independent investigator in pharmacogenetics, bioinformatics, statistical genetics, and genetic epidemiology in lung transplantation. The stated goals will be achieved through excellent mentoring, premium coursework, and training in scientific communication. The unique resources and infrastructure of Washington University are ideal for the proposed plan. This K01 will provide the necessary tools to develop into an independent clinician-investigator focused on the pharmacogenetics of lung transplant immunology who can secure R01 funding.

NIH Research Projects · FY 2025 · 2021-06

ABSTRACT Effective responses to viruses, intracellular pathogens and tumors rely on the successful expansion and arming of cytolytic CD8 T cells from a naïve and quiescent T cell repertoire. This process, called CD8 T cell priming, is dependent on a specialized antigen-presenting cell known as conventional dendritic cells (cDCs). Recent studies from our lab have shown that a specific type of dendritic cells, called cDC1, is responsible for CD8 T cell priming and depends on the transcription factor Batf3 for development. In the process of priming CD8 T cells, the cDC1 captures foreign antigens or tumor-specific neo-antigens, and presents these antigens on the MHC-I molecule in order to stimulate the T cell receptor (TCR) of the CD8 T cell. In the absence of cDC1, such as in Batf3-deficient mice, CD8 T cells are not activated against viruses or tumors. In addition, however, full activation of CD8 T cell requires that cDC1 receive signals that program its ability to fully activate CD8 T cells. This process is called `DC licensing', but is currently not fully characterized. Our recent work has shown that current models for DC licensing are incomplete. Specifically, while we have confirmed the requirement for CD40 signaling in response to CD40L provided by a helper cell, we have shown that the expected target of CD40, CD70, does not explain the beneficial effect of DC licensing for tumor rejection. This result implies that additional targets of CD40 are needed to understand DC licensing. Further, it has been thought that CD4 T cells are the exclusive agent in the licensing process. However, these conclusions derived from older studies and less precise methods. In contrast, we developed an Xcr1-Cre deletor strain to delete genes specifically from cDC1, and found unexpected results, that CD4 T cells are not always critical for cDC1 licensing, suggesting that other cells expressing CD40L may be involved. Further, mechanism by which CD40 signaling induces licensing of the cDC1 is unclear. Several mechanisms have been proposed, but our preliminary data excludes the major proposed mechanism, those invoking CD70, as being critical for the effect of licensed cDC1 in priming CD8 T cells. Our proposal will first test (Aim 1) which Batf3-specific genes expressed in cDC1 are important for cDC1 to support CD8 T cells responses. Second (Aim 2), we will determine identify the targets of CD40 signaling in cDC1 that are required for fully activating CD8 T cells. Finally, in Aim 3, we will test the role of cells that we have identified as alternative sources of CD40 ligand, including iNKT cells and γδ T cells.

NIH Research Projects · FY 2025 · 2021-06

NIH Research Projects · FY 2025 · 2021-06

ABSTRACT The γ-aminobutyric acid type A (GABAA) receptor is an ionotropic inhibitory ion channel. Inhibition mediated by GABAA receptors sets the level of activity in the brain, whereas in individual cells it determines the propensity of a cell to fire an action potential in response to excitatory input. The GABAA receptor is a major target for intravenous anesthetics and numerous endogenous compounds including neuroactive steroids. This project will investigate how combinations of endogenous and clinical compounds affect the functioning of the native GABAA receptor, and initiate the onset and offset of anesthesia. Specifically, we will: i) explore the kinetic and energetic aspects of activation and modulation of native synaptic and extrasynaptic GABAA receptors by clinically-relevant compounds; ii) determine the involvement of endogenous neurosteroids in the clinical actions of intravenous anesthetics to test the hypothesis that normal physiological changes in steroid levels modify responses to anesthetics; iii) probe ways for controllable recovery from anesthesia; and iv) test the feasibility of using partial allosteric agonists of the GABAA receptor as safe, mild sedatives.

NIH Research Projects · FY 2025 · 2021-06

PROJECT ABSTRACT Cells choose transcriptional programs, in part, by passing information from molecular sensors to signaling cascades that modulate the activity of DNA-binding transcription factors (TFs). Changes in TF activity lead to changes in the transcription rates of specific genes, which are often the first steps in responding to new information. The molecular machinery responsible for this can be thought of as the cell's control circuits. My research program focuses on developing computational and molecular methods that make it possible to map out a cell's control circuits, to watch them as they respond to new information, and ultimately to rewire them in ways that contribute to human health and well-being. Saccharomyces cere- visiae (yeast) is the ideal organism for developing these methods because of its relatively simple ge- nome, extensive collections of strains engineered for experimental systems biology, and comprehensive datasets for testing and optimizing new methods. The research proposed here focuses exclusively on yeast, but our methods will be immediately applicable to fungal pathogens and ultimately adaptable for model organisms and humans. The first objective of our plan is to optimize methods for determining which genes are regulated by each TF. We will use these methods to produce a map of the TF-target network that goes significantly beyond what is known today, both in accuracy and completeness. Like the map of metabolic reactions, this will be a valuable resource for addressing many scientific questions. Our second objective is to develope methods for inferring the activity levels of all TFs in any sample of cells by analyzing their transcriptomes. One product of this work will be easy-to-use software that will enable other scientists to identify changes in TF activity in any set of yeast transcriptional profiles. Our third objective is to develop methods for identifying proteins that regulate the activities of each TF. This will make it possible to explain the changes in TF activity we observe when stimuli, such as drugs or nutrients, are provided to cells, and to design experiments that test those explanations. Achieving these objectives will make it possible to approach our ultimate goal – to develop a quan- titative model that can predict the transcriptional response to genetic and environmental perturbations. As a concrete benchmark for success, this model should accurately predict the effect on the entire yeast transcriptome when combinations of TFs are simultaneously perturbed (deleted or overex- pressed) under growth conditions for which we have no perturbation data. We can achieve this ambi- tious, long term goal only with stable MIRA funding.

NIH Research Projects · FY 2025 · 2021-06

ABSTRACT Neurons are viewed as the cellular correlate of cognition and only target of clinical therapeutics, in part because manipulating neurons rapidly and directly alters behavior. Yet, the human brain is also made of glial cells, which morphological and genetic complexification is a striking feature of the human brain. Astrocytes, in particular, are now known to orchestrate many neural functions, crystalizing the possibility of a direct astrocyte contribution to cognitive functions and mental health. However, a lack of understanding of the rules that govern astrocytes activity and their involvement in neural circuits has limited our ability to test this idea. Collective work recently showed that astrocytes transduce neuromodulatory information onto synaptic circuits. Specifically, we found that α7 nicotinic acetylcholine receptors (α7nAChRs) on astrocytes regulate the release of the astrocyte transmitter D-serine onto synapses. Neuromodulation, in particular cholinergic signaling, permits behavioral adaptations to changes in the environment, and its alteration is linked to cognitive deficits in schizophrenia. Coincidently, the α7nAChR has focalized major drug development efforts in the past decade to restore the cognitive symptoms of patients with schizophrenia. Here, we will take advantage of this new astrocyte-based α7nAChR pathway to test the role of astrocytes in cognitive functions and pro-cognitive interventions, and elucidate the mechanisms through which neuromodulation is sensed and transduced by astrocytes at the cellular and molecular levels. In doing so, we will test a set of general principles which we hypothesize govern input output fidelity in astrocytes (positional coupling). In Aim 1, we will test the hypothesis that α7nAChRs are located in the immediate vicinity of D-serine pools, directly linking Ca2+ influx through α7nAChR channel activity to the Ca2+-dependent D-serine release machinery. We will conduct fluorescence Ca2+ imaging studies to understand the spatial, temporal and molecular rules of α7nAChR-mediated Ca2+ signals and their relation to D-serine release. We will then map the physical association of α7nAChR and D-serine pools in perisynaptic astrocytic processes, using electron microscopy. In addition, we will conduct single-particle tracking studies to understand how the dynamic distribution of α7nAChR at the surface of astrocytes, with respect to D-serine pools, is influenced by the binding of endogenous and exogenous ligands. In Aim 2, we will generate cell-specific knockout mouse lines to selectively ablate α7nAChR from astrocytes, excitatory neurons or inhibitory neurons in the brain, and canvas the contribution of each cell-types to characteristic behaviors supported by α7nAChRs. We recently showed that an α7nAChR partial agonist tested in Phase-III clinical trials for the treatment of cognitive deficits in patients with schizophrenia, elevates D-serine levels in the mouse brain. Based on our observations that inactivating astrocyte-based α7nAChR signaling leads to specific alterations in D-serine levels and cognitive behavior, we will then test the hypothesis that astrocytes, but not neurons, enable the behavioral efficacy of cognitive enhancers tested in clinical trials, and that D-serine signaling is the circuit actuator of these effects.

NIH Research Projects · FY 2025 · 2021-05

Abstract Despite antiretroviral therapy (ART), HIV-1 infection is not curable due to the presence of viral latent reservoirs primarily in long-lived resting memory CD4+ T cells. The viral latent reservoirs are the major barrier to HIV-1 eradication. The “shock and kill” strategy for HIV-1 eradication involves the use of latency-reversing agents (LRAs) to induce viral gene expression, which renders the infected cells susceptible to viral cytopathic effects or immune clearance. However, in ART-treated patients, the latent HIV-1 resides in cells is resistant to viral- or immune-mediated apoptotic cell death and often carries mutations to escape recognition by T cells or antibodies. Therefore, novel approaches to target immutable components of the virus such as essential viral protein functions are needed. Inflammasome is a critical molecular complex that mediates inflammation and pyroptotic cell death in response to microbial or danger signals. In humans, the physiologic ligand(s) for the CARD8 inflammasome remains unknown. Our studies demonstrate that HIV-1 protease degrades the CARD8 N-terminal domain and releases the C-terminus for inflammasome activation. In HIV-1-infected cells, the viral protease remains inactive as a subunit of viral Gag-Pol polyprotein. After virus budding, it is activated through Gag-Pol dimerization. We show that premature activation of intracellular HIV-1 protease by non-nucleoside reverse transcriptase inhibitors (NNRTI) triggers CARD8 sensing and killing of HIV-infected macrophages and CD4+ T cells. All subtypes of HIV-1 can be sensed by CARD8 despite substantial viral diversity. Our finding suggests that targeted activation of CARD8 inflammasome is a promising strategy to eliminate latent HIV-1 reservoirs. In this proposed study, we will: 1) perform ex vivo assessment and optimization of the CARD8-based “shock and kill strategy” for clearing latent HIV-1 in patient CD4+ T cells; 2) understand the molecular mechanisms of HIV-1 protease- mediated CARD8 inflammasome activation; 3) test CARD8-based “shock and kill” strategy in humanized mouse model of HIV-1 latency. Our proposed study will advance the understanding of the physiological mechanisms of CARD8 inflammasome activation and its role in HIV-1 infection, which will provide critical implications to HIV cure research.

NIH Research Projects · FY 2025 · 2021-05

This proposal builds on an existing infrastructure of the ASSET program (Advancing Secondary Science Education through Tetrahymena), to generate new teaching materials, reach new student populations, and ensure sustainability of the program by transitioning its overall functions from Cornell University in Ithaca, New York to Washington University in St. Louis. Over the past 10 years, ASSET has built a highly successful SEPA program that teaches core biology content to primary, middle, and high school students using a safe, easily grown, and behaviorally complex single-celled organism (viz. Tetrahymena). Tetrahymena provides an ideal platform for teaching basic principles of cell structure and function, genetics, evolution, sex, prey-predator interaction, cell signaling, etc. without engendering in students any of the conflicting reactions often evoked using live vertebrate animals. Additionally, Tetrahymena offers a graphic illustration of the deleterious effects of toxic and/or addictive substances on living cells in real-time, equipping teachers with a powerful tool with which to fight against substance abuse and promote healthy behavior. ASSET provides stand-alone laboratory kits that are easily integrated into existing science/health curricula, along with innovative co-curricular modules that address the intersection between science and society. The program is specifically designed to support all science teachers regardless of whether they teach in rural, suburban, or urban school districts, provides robust on-site and distance teacher development activities and is continuously evaluated for pedagogical effectiveness. This new proposal will greatly expand the program’s current offerings by introducing new materials to existing modules, as well as new modules that address recently identified areas of high programmatic interest to SEPA, specifically, embedding math in P-8 teaching projects; exposing students to research-generated data; and training students in informatics, bioinformatics, and data science. The Co-Directors have extensive experience teaching bioinformatics and helping students interpret research-driven data, while curriculum specialists at Washington University’s Institute for School Partnership are well-positioned to evaluate existing modules to identify opportunities to teach mathematical concepts using examples from biology and student generated data at grade appropriate levels. Finally, the move from Cornell to Washington University addresses an additional area of programmatic interest for SEPA, namely, adapting successful SEPA programs to new areas or with new populations. Through its Institute for School Partnership, Washington University is strongly committed to achieving equity in K-12 education bringing high-quality STEM teaching to >100,000 students in the Midwest through its various teaching programs. Incorporating the ASSET program under its umbrella expands its current activities, introduces ASSET to whole new populations of students, and provides ASSET a safe-haven for continuing its long-term mission to enhance STEM education and, ultimately, the STEM workforce.

NIH Research Projects · FY 2026 · 2021-05

PROJECT SUMMARY The National Action Plan for Combating Antibiotic-Resistant Bacteria identified critical research needs to confront the advancing threat of antimicrobial resistance. These include understanding the nature of microbial communities, determining the effects of antibiotics on these communities, and harnessing these communities to develop strategies for disease prevention. Staphylococcus aureus causes significant morbidity and mortality in healthcare and community settings. S. aureus carriage confers risk for endogenous infection. Previous studies investigated clinical and epidemiological factors associated with S. aureus colonization and infection, although the influence of the skin and nasal microbiota on S. aureus acquisition is unclear. We hypothesize that features of the commensal microbiota may confer susceptibility to, or provide protection against, S. aureus acquisition and development of subsequent infection. Further, in an effort to prevent S. aureus acquisition and infection, decolonization with topical antimicrobials is frequently employed in healthcare and community settings. However, broad-spectrum topical antimicrobial application may disrupt commensal microbiota, thereby promoting the acquisition or enrichment of multidrug-resistant and/or potentially pathogenic microorganisms. The impact of this potential adverse effect remains understudied. In this proposed project, we will analyze a vast and unmatched biorepository of >13,000 skin and nasal swab samples that were longitudinally collected over 24 months from a well-characterized cohort of 476 participants, including 99 pediatric patients with methicillin-resistant S. aureus skin infections and their household contacts, all of whom are at high risk for S. aureus acquisition and infection. The samples were collected during 12 months of longitudinal samplings (the natural history phase) followed by a randomized, 5-day decolonization intervention with topical antimicrobial application, and subsequent longitudinal sampling for 12 months (post-decolonization phase). We will perform metagenomic shotgun sequencing (enabling classification at the species and strain levels), to longitudinally characterize skin and nasal microbial communities in the context of S. aureus colonization and disease. We will compare microbial community features associated with susceptibility or resistance to S. aureus acquisition between household contacts with different phenotypic outcomes (e.g., colonization and infection), and integrate these data with detailed epidemiological data to predict individuals at risk for acquiring S. aureus colonization and/or developing symptomatic infection. We will quantify changes in microbial community structures following topical antimicrobial application, and correlate this disruption with subsequent acquisition and persistence of S. aureus and other pathogens. Finally, we will employ innovative targeted sequence capture to quantify genetic determinants of antimicrobial resistance (the “resistome”) in samples collected before and after decolonization. The essential knowledge generated by this study will optimize preventive strategies for S. aureus colonization and infection and their targeting to susceptible individuals.

NIH Research Projects · FY 2026 · 2021-05

ABSTRACT Permanent disabilities following central nervous system (CNS) injuries result from the failure of injured axons to re-build functional connections. There are currently no therapies to restore mobility and sensation following spinal cord injury or vision after optic nerve damage. The poor intrinsic regenerative capacity of mature CNS neurons is a major contributor to the regeneration failure and remains a major problem in neurobiology. In contrast, peripheral sensory neurons successfully switch to a regenerative state after axon injury. The long-term goal of my research program is to understand the multicellular mechanisms by which injured sensory neurons activate a pro-regenerative program and identify potential targets for future treatment of CNS injuries. Activation of an axon growth program relies in part on the expression of regeneration-associated genes. Because individual gene based approaches have yielded limited success in axon regeneration, we are focusing on epigenomic regulations, which affect globally, yet specifically a combination of multiple genes. Our goal is to uncover how the epigenetic landscape is re-organized in the context of axon injury to enable axon repair. These studies will incorporate cell-type specific epigenomic analyses to study the transcriptional and chromatin conformation changes elicited by peripheral and central axon injury. Axon regeneration is not cell autonomous and is influenced by the environment at the level of the axon injury site and at the level of the cell soma. We have recently discovered that satellite glial cells, the main type of glial cells in sensory ganglia respond to axon injury and contribute to the repair process. We propose to use powerful combinations of tools to pursue an innovative line of research aimed at dissecting the multicellular mechanisms orchestrating axon regeneration and build upon these findings to improve regeneration in CNS models. To achieve this goal, we will determine the intrinsic neuronal mechanisms controlling axon regeneration, focusing on epigenomics studies. We will elucidate the contribution of the microenvironment surrounding neuronal soma to the axon regeneration process, including satellite glial cells and other non-neuronal cells. To determine if findings made in the mouse model system are predictive of human physiology, we will determine the molecular profile of human cells surrounding sensory neurons. Finally we propose to manipulate novel pathways we discover to improve regeneration in two CNS models, spinal cord injury and optic nerve injury. This proposal will use powerful combinations of tools to pursue an innovative line of research aimed at dissecting the multicellular mechanisms orchestrating axon regeneration and build upon these findings to improve regeneration in CNS models.

NIH Research Projects · FY 2025 · 2021-05

Abstract Allergic rhinitis is the most common mucosal allergy. Its cardinal symptoms include excessive sneezing and rhinorrhea, which severely impact our life quality and productivity. Although antihistamines effectively relieved sneezing induced by intermittent mild allergic rhinitis, they are ineffective against persistent moderate/severe allergic rhinitis. The development of new drugs for alleviating allergic sneezing is hindered by a lack of information about the principal nasal sensory neurons that mediate sneezing and their interactions with immune cells. In this proposal, we hypothesize that a highly restricted population of nasal sensory neurons defined by the expression of MrgprC11 detect mast cell mediators in allergic rhinitis and trigger the sneezing reflex. In Aim 1, we will characterize the innervation pattern of MrgprC11-expressing fibers in the nasal mucosa and examine their pathological changes under allergic rhinitis using genetic labeling and axonal tracing approaches. Furthermore, we will determine their physiological responses to a variety of sneeze-inducing molecules using a novel ex vivo calcium-imaging tool. These studies will provide important information on the initial detection of nasal irritants and transduction of sneezing signals. In Aim 2, we will define the role of MrgprC11+ fibers in acute sneezing. We will determine whether ablation of MrgprC11+ neurons attenuates sneezing responses to a variety of nasal irritants and whether selective activation of MrgprC11+ sensory fibers in the nasal mucosa evokes sneezing. These studies will establish whether MrgprC11+ sensory fibers are required for sneezing induced by different sensory stimuli. In Aim 3, we will investigate the neuro-immune interactions between MrgprC11+ nasal sensory fibers and mast cells in allergic rhinitis. We will test whether degranulated mast cells activate MrgprC11+ nasal sensory fibers to induce sneezing in allergic rhinitis. Furthermore, we will determine whether pharmacological silencing of MrgprC11+ sensory fibers is a feasible therapeutic strategy to control sneezing associated with allergic rhinitis. These studies will not only advance our understanding of the neuro-immune interactions that trigger sneezing, but also provide a novel neuronal target for controlling nasal allergic symptoms.

NIH Research Projects · FY 2025 · 2021-05

Abstract Interstitial cystitis/bladder pain syndromes (IC/BPS) is a debilitating condition with loss of bladder control and severe bladder pain on bladder filling. Mechanisms underlying IC/BPS are poorly understood. Compelling studies show that the brainstem via direct projection to the spinal cord can directly modulate nociceptive processing and bladder function. In our preliminary studies, we have identified distinct populations of the brainstem neurons that project to the spinal cord. Here we will determine the precise roles of these distinct brainstem neurons in the pathology of IC/BPS. We will determine if IC/BPS leads to maladaptive changes in these distinct populations. We will use optogenetics, anatomical tracing, in vivo calcium imaging, slice electrophysiology and RNA sequencing to determine if IC/BPS leads to functional changes at the cellular and systems level in these circuits. Do these changes drive IC/BPS? Does reversing these maladaptive changes relieve IC/BPS? Together, this work will reveal the specific roles for this neural circuits in IC/BPS. This rich information can have broad implications for potential new direction in designing safer therapeutic drugs in treatment of the IC/BPS.

NIH Research Projects · FY 2024 · 2021-05

PROJECT SUMMARY The Pathway to Independence Award will equip the candidate with the knowledge and skills to study well-being as a key protective factor against Alzheimer’s Disease and Related Dementias (ADRD). ADRD is a substantial and rapidly-growing public health burden, with no available disease-modifying treatments. However, not everyone with ADRD-related neuropathology experiences symptoms of dementia. Well-being is an understudied but important predictor of cognitive resilience to ADRD neuropathology, given that it has been associated with ADRD risk and is amenable to intervention. The candidate will evaluate multiple well-being predictors of resilience to ADRD pathology (Aim 1). To address questions related to direction of causality, the candidate will examine bidirectional associations between well-being and cognitive decline in older adults with and without ADRD (Aim 2). To increase the generalizability and replicability of findings, both research aims will be conducted in multiple existing longitudinal datasets. To support the candidate in conducting the proposed research, training in three areas is planned: (a) Alzheimer’s Disease and Related Dementias (ADRD), (b) causal modeling of longitudinal observational data, and (c) Integrative Data Analysis. Training will occur under the mentorship of renowned experts in each field (including two personality and health psychologists, an epidemiologist, and a quantitative psychologist). During the K99 period, the candidate will receive training in ADRD that will be applied to both aims of the proposed research and training in causal modeling techniques that will be used in the second aim of the proposed research. During both periods of the award, the candidate will work with the mentor team to build relationships with existing longitudinal studies and develop an Integrative Data Analysis pipeline. The training environment at the Department of Medical Social Sciences, Northwestern Feinberg School of Medicine will be ideal for the proposed training and research, as well as for developing the candidate’s professional skills. Together, the award will help the candidate launch her research career as an independent scientist with unique expertise in well-being, cognitive and physical health across the lifespan, and longitudinal modeling.

NIH Research Projects · FY 2025 · 2021-05

Project Summary Autism spectrum disorder (ASD) is one of the most common and highly inherited of all developmental disorders, with heritability exceeding 0.80. Although remarkable advances in genetics have identified rare de novo mutations in select brain genes, by definition, none of these relate to the pronounced heritability of autism relevant to the vast proportion of cases in the population. Given that heritability has been shown to be a function of additive genetic risk, inferring an impractically large number of genetic targets for intervention, an important strategy is to identify convergent mechanisms of polygenic risk factors by elucidating intermediate phenotypes (i.e., endophenotypes) through which they exert their causal influence. Our group has identified two such candidates that appear highly contributory but not sufficient for ASD: (i) the social behavioral phenotype indexed by quantitative (subclinical) autistic traits in parents (QAT-p); and (ii) variation in social visual engagement (SVE), an eye-tracking measure based on viewing of dynamic social scenes. Both can be ascertained in the first year of life using rapid acquisition methods of less than 20 minutes. Our team’s prior work strongly supports a developmental model in which autism arises from joint additive genetic effects of these and other neurodevelopmental liabilities, suggesting that targeting a discrete group of early-contributing liabilities before autism develops offers the greatest opportunity for high-impact, personalized intervention for children at risk for common, polygenic forms of ASD. The primary objective of this research program is to validate wearable-sensor methodology (bilateral, wrist-worn accelerometers) for quantifying two additional endophenotypes: hyperactivity (HYP, as an early marker of liability to Attention Deficit Hyperactivity Disorder, which is strongly comorbid with autism) and impairment in motor coordination (MOT). We will examine HYP and MOT in three distinct samples. Aim I will study 120 pairs of infant twins first assessed at 6 months of age and then followed at ages 18 and 36 months to evaluate early associations between HYP, MOT, QAT, and SVE, as well as their heritability and ability to predict quantitative variation in autistic traits among the twins. Aim II will study a cohort of 50 toddlers diagnosed with idiopathic ASD to determine whether novel sensor-based metrics of HYP and MOT show differences between the twins and children with ASD. Aim III will study a legacy cohort of 120 infants at elevated risk for autism due to prematurity and/or cerebellar injury, in whom we will explore relationships between cerebellar structure and function in the first year of life and a) sensor-based indices of HYP and MOT, b) quantitative autistic traits, and c) performance-based indices of motor ability at age three years. This project is expected to yield a rich, comprehensive understanding of motor endophenotypes in infants and children and their contributions to autism, with the eventual goal of providing a launching point for future screening and early intervention studies in high-risk children.

NIH Research Projects · FY 2024 · 2021-05

Project Summary Each year in the United States alone, 500,000 infants are born preterm (<37 weeks gestation), putting them at increased risk for neurodevelopmental disabilities, including cerebral palsy and other motor impairments. While specific clinical populations are known to be at increased risk, the likelihood of disability for any individual child cannot currently be accurately predicted based upon clinical risk factors alone, limiting our ability to effectively target therapies and develop new interventions. Prior neuroimaging studies have linked preterm birth to disrupted development of the motor system, encompassing the motor cortex, thalamus, basal ganglia, and cerebellum and associated white matter tracts including the corpus callosum (CC) and corticospinal tract (CST). While aberrant structural and functional connectivity across these regions have been associated with poorer motor outcomes, this has not been investigated across childhood in longitudinal cohorts in a way that allows for individualized outcome prediction. This study proposes to use multiple advanced neuroimaging modalities to statistically model how changes in neonatal structural and functional connectivity within the motor system can predict childhood motor outcomes in children born very preterm (VPT; <30 weeks' gestation). This investigation will leverage a unique, highly valuable, prospective, longitudinal cohort (currently being studied through R01 MH113570) that includes 175 VPT children, including 41 with cerebral palsy and 68 with other motor deficits. We collected state-of-the-art neonatal neuroimaging data for these children, including high- resolution anatomic, functional, and diffusion data. They have also undergone standardized testing of both fine and gross motor function at ages 2, 5, and 9/10 years, with retention rates >80% across assessment waves. Across the three aims of this study, latent growth curve models will be created and compared to determine the individual-level predictive ability of motor system functional connectivity and CC and CST microstructure, both individually and in combination, on motor trajectories through age 10 years. This project would both advance our ability to predict outcomes for individual preterm children into middle childhood and build the applicant's skills in neuroimaging, longitudinal data analysis, and scientific communication in a research environment with clear expertise in these areas. In the process, she would become proficient in the methods necessary for furthering our understanding of the relationships between early brain development and disability in high-risk populations. She would also become prepared to undertake not only strong experimental work, but also care for patients with neurodevelopmental disabilities while effectively integrating her research with disability advocacy. This would pave the way for the applicant to become a successful physician-scientist and child neurologist creating better outcomes for children with neurodevelopmental disabilities.

NIH Research Projects · FY 2024 · 2021-05

PROJECT SUMMARY/ABSTRACT Viral infections are now recognized as risk factors for diseases of progressive pathological forgetting, supporting a new paradigm in neuroimmunology whereby innate immune molecules that function as modulators of a variety of normal CNS functions induce neurodegenerative diseases during host-pathogen responses. Studying virus-mediated cognitive dysfunction through the multidisciplinary prism of immunology, neuroscience, and virology is critical for the identification of novel mechanisms of disease, discovery of neuroimaging tools and therapeutic treatments for a wide range of diseases of memory disorder. In my laboratory we made unanticipated, paradigm-shifting discoveries of the roles of CNS infiltrating mononuclear cells in microglial-mediated synapse elimination, disrupted adult neurogenesis, and generation of neurotoxic astrocytes using novel models of recovery from encephalitogenic flaviviruses, West Nile (WNV) and Zika (ZIKV) viruses. We identified classical complement proteins and cytokine receptor signaling as novel molecular mediators that regulate synapse elimination, neural stem cell (NSC) fates, neuron-microglia and microglia- astrocyte crosstalk within cortical structures that regulate memory formation and maintenance. We have also begun leveraging novel neuroimaging modalities to develop biomarkers that may be used to predict and monitor patients at risk for memory disorders. Our aim is to understand the mechanisms that induce alterations in synaptic connections and methods of repair that contribute to disruption of neuronal networks after recovery from viral infections. Our research program focuses on three broad areas related to the roles and regulation of innate immune molecules involved in spatial learning using novel murine models of post-infectious cognitive dysfunction. First, using genetic, pharmacologic and PET-MRI, we will identify and define molecular interactions between T cells and microglia or neurons that drive the generation and maintenance of resident memory T cells that promote cognitive dysfunction. We will use scRNAseq under BSL3 conditions to screen for genes and pathways to be targeted via cell-specific deletion of cytokine or chemokine receptors, or administration of agents that inhibit or enhance pathways. We will also develop diagnostic tools that employ ABSL3 PET-MRI. Second, we will define how microglia-astrocyte-NSC interactions in the context of recovery from CNS viral infections limit repair and recovery. We will use global and conditional gene targeting in mice to delineate the in vivo roles of cytokines in neural cell types that regulate astrocyte inflammasome activation and its relationship to neuronal and synapse recovery. We will also define innate immune mechanisms that direct and maintain astrogenosis during acute viral infection and recovery using PET-MRI detection of P2X7R, a marker of reactive astrocytes. Finally, will utilize reporter mice/fate mapping, bone marrow chimeras and scRNAseq to delineate the cytokine-mediated roles of myeloid cells in the generation of neurotoxic astrocytes during WNND recovery. Third, we will examine innate immune mechanisms triggered after viral infections that negatively impact cortical connectivity and determine whether neuroimaging can be used to predict and follow this process. Specifically, we will combine genetic approaches with functional optical intrinsic signal imaging of hemoglobin and calcium dynamics to define mechanisms that negatively impact cortical connectivity. Our research program will define new concepts in the molecular neuroimmunological regulation of synapses, T cell and glial interactions, inform studies of related processes throughout the nervous systems, and will likely enhance our understanding of neurodegenerative and other disorders of memory.

NIH Research Projects · FY 2025 · 2021-05

ABSTRACT The extensive family of the Coronins plays an important role in regulating the actin networks that drive cell migration, polarity, cell shape and intracellular trafficking, and thereby contribute to essential biological processes ranging from innate immunity to neuronal signaling. However, these functions and activities have been characterized for the canonical Coronins. In stark contrast, we know next to nothing about the cellular roles and actin cytoskeleton regulation of the structurally distinct class of the tandem Coronins, which comprises Coronin 7 (Coro7), POD-1, and CorB. Our preliminary data show that mammalian Coro7 can control Arp2/3-mediated nucleation, although the mechanism underlying this activity remains elusive. In addition, we show that this actin- regulatory activity can regulate NPF-induced actin networks that drive intracellular membrane transport, including autophagy, a process used by cells to dispose of unwanted organelles, misfolded proteins and toxic aggregates. Using a multidisciplinary approach comprising state-of-the-art biochemical, biophysical, genetic and live-imaging techniques we propose here to elucidate 1) how Coro7 structurally interacts with Arp2/3, 2) how Coro7 mechanistically inhibits Arp2/3-mediated nucleation by nucleation promoting factors, and 3) by other Arp2/3 regulators, and 4) how these actin-regulatory activities of Coro7 regulate the turnover of the branched actin networks that drive autophagy.

NIH Research Projects · FY 2025 · 2021-05

Summary/Abstract: High body fat at midlife, as evidenced by overweight or obese body mass index (BMI), is increasingly understood as a risk factor for Alzheimer’s disease. However, the underlying processes and mechanisms that may underlie this risk remains unknown. With this R01 proposal, we request funding to create a new cohort of cognitively normal 120 midlife individuals, age 40-60 years. We propose to characterize their overweight or obese status using metabolic tests including, an oral glucose tolerance test, fasting plasma insulin, fasting plasma glucose, and hemoglobin A1c measurements. This testing will generate categories of metabolically abnormal overweight or obese (MAOO), metabolically normal overweight or obese, and metabolically normal lean persons. We will evaluate differences between these groups on neuroimaging with the newer classification of Alzheimer’s biomarkers with amyloid (A), tau (T), and neurodegeneration (N), or ATN. Neurodegeneration will be assessed by atrophy on brain MRI as reflected by regional volumes on FreeSurfer. We will also evaluate MR neuroimaging markers for neuroinflammation using a newer method called diffusion basis spectrum imaging (DBSI), developed at the Mallinckrodt Institute of Radiology at Washington University in St. Louis in collaboration with The Charles F. and Joanne Knight Alzheimer’s Disease Research Center (Knight ADRC). Recruitment of participants for this study will be done in conjunction with both the Knight ADRC and the Washington University Center for Human Nutrition. In Aim 1, we hypothesize increased atrophy in MAOO compared to metabolically normal overweight, obese, and lean participants particularly in regions important for AD pathology such as the hippocampus and subregions. With Aim 2, we hypothesize a higher burden of white matter neuroinflammation on DBSI in MAOO compared to other metabolic, overweight or obese and lean groups. In Aim 3, we hypothesize increased amyloid and tau on brain PET in MAOO versus other groups. Related sub-aims will examine sex differences in atrophy and neuroinflammation. We will also investigate sex differences in amyloid and tau deposition across the overweight/obese and lean groups. This approach is not only of interest given the known sex differences in obesity and AD risk but is also recognized by the NIA as important in understanding trends of risk in AD. We will also examine the anatomical distribution of abnormally high body fat by acquiring separate body torso MRI scans at the time of brain MRI scan. This will allow for separate quantification of visceral, abdominal subcutaneous, and liver fat volumes that we will separately relate to the imaging markers in Aims 1-3. By understanding how metabolic abnormalities with, and anatomical distribution of, high body fat relate to brain imaging metrics of ATN and neuroinflammation, we will contribute key understanding to how obesity increases Alzheimer’s risk. These results will have public health implications and inform future studies of interventions to reduce risk for Alzheimer’s by identifying important metrics to utilize for optimization of metabolic and related brain health in overweight and obese persons.