Washington University

NIH Research Projects · FY 2025 · 2021-04

PROJECT SUMMARY In the progression from middle to older-age, healthy adults typically experience improvements in their emotional functioning, such as increases in positive emotion and greater expertise in managing emotions. However, not everyone shows these age-related improvements, and the mechanisms that give rise to emotional functioning changes across adulthood are still poorly understood. The primary goal of this project is to examine the critical factors that promote positive emotional development in normative aging, and to test whether depression history might moderate this process as a key trait individual difference marker. To this end, we test our proposed Value- Based Cognitive Control Model of Emotion Regulation in ADulthood (VBCC-MERiAD). The VBCC-MERiAD framework suggests a novel insight: that interactions between reward motivation and cognitive control play a central role in understanding both the normative trajectory of emotional functioning in older adults, and conversely, why and how individuals with depression histories may get “off track”. We focus on effectively upregulating positive emotion, given that older adults prioritize positive emotion goals, and because depression is characterized by blunted reward processing. Our primary hypothesis is that positive emotion regulation (ER) abilities will rely upon the integrity of fronto-striatal circuitry (i.e., activity and connectivity between the lateral prefrontal cortex and nucleus accumbens / ventral striatum). Engagement of this circuit is predicted to reflect the utilization of reward motivation as a means of engaging cognitive control (i.e., to update and maintain ER goals). Across three Specific Aims, we propose to characterize the mechanisms of ER in middle-aged and older adults (35-74), focusing on neural and behavioral indicators of motivation and cognitive control that predict daily emotional functioning, and potential dysregulation in individuals with depression history. To achieve these aims, we will employ a multi-method design involving functional neuroimaging measures, laboratory behavioral assessments, and experience sampling methods. The sample (N=220) will include an ethnically/racially diverse set of adults (66% women) of ages 35-74, equally subdivided into two groups: healthy controls and people with depression histories. A state-of-the art neuroimaging protocol will assess brain activity associated with different ER strategies, and test for linkages with reward-motivated cognitive control. The comprehensive laboratory assessments will include diagnostic interviewing, self-report measures, cognitive functioning batteries, and a standardized ER task with measures of autonomic reactivity and behavioral coding of emotion. The experience sampling protocol will provide a naturalistic, ecologically valid assessment of participants’ emotional experiences, goals and regulatory strategies. The proposed research will dramatically extend our understanding of both normative and dysfunctional age-related change in emotional function, by identifying mechanisms that promote positive ER in late adulthood. In so doing, we will lay the foundation for new interventions to improve quality of life for healthy older adults and preventative therapeutic targets for individuals with depression history.

NIH Research Projects · FY 2025 · 2021-04

PROJECT SUMMARY Significance: Neural circuit assembly requires activity-dependent refinement of circuit architecture (e.g. plasticity) to produce stereotyped behavior. Neurons are particularly susceptible to functional and structural plasticity during early developmental windows called critical periods. It is clear that failure to terminate critical period plasticity adversely affects mature circuit function in both animal models and humans (e.g. autism and epilepsy), yet the mechanisms that close critical periods are largely unknown. This Pathway to Independence Award proposal seeks to understand the cellular and molecular mechanisms that promote critical period closure, and to define how critical periods shape circuit architecture to ensure proper locomotor behavior. Candidate and environment: Dr. Ackerman was trained in molecular genetics and developmental neuroscience in the laboratory of Dr. Kelly Monk at WashU School of Medicine, where she used forward and reverse genetic strategies to uncover regulators of myelination (NS087801). She then joined the laboratory of the renowned neurobiologist Dr. Chris Doe (UO, HHMI/NAS). Here, she defined a novel critical period of plasticity in the developing Drosophila motor circuit, and uncovered a series of astrocyte-derived molecular regulators of critical period closure (NS098690). In this proposal, Dr. Ackerman will extend her current skills in molecular genetics, live imaging, and circuit analysis to include training in electrophysiology and single cell RNAseq (scRNAseq), two completely new techniques for her. Further, she will use two model systems (fly and zebrafish) to determine how these novel, astrocyte-derived factors restrict motor circuit plasticity (Aim 1), to define how the critical period contributes to motor circuit connectivity, function, and behavior (Aim 2), and to determine how motor circuit plasticity is developmentally constrained in vertebrates (Aim 3). Career development: In addition to continued mentorship by Dr. Doe, the candidate has assembled an exceptional team of mentors and collaborators from the University of Oregon and beyond. During the mentored phase, the candidate will train in NMJ electrophysiology from Dr. Dion Dickman (USC) in order to define how the level of activity experienced by motor neurons during the critical period shapes motor output and behavior. This training is essential for future studies of motor circuit function in the candidate's own lab. Further, she has gathered a local team of advisors from the zebrafish community, Dr. Judith Eisen and Dr. Adam Miller, who have a combined 40 years of experience in zebrafish motor circuits. Drs. Eisen and Miller will facilitate training in scRNAseq, and will provide critical career development advice from the complementary perspectives of a seasoned (Dr. Eisen) and recently-established (Dr. Miller) principal investigator. Funding of this proposal will equip Dr. Ackerman with the unique skillset required to launch a robust and successful research program that pushes the boundaries of our understanding of circuit plasticity, from molecules to behavior.

NIH Research Projects · FY 2025 · 2021-04

Abstract Artificial intelligence (AI) and other computationally intensive methods have the potential to revolutionize cancer imaging research and patient care. The broad adoption of these technologies depends on the availability development of imaging informatics tools to assist users in managing massive data sets, generating well-curated annotations, and accessing scalable computing resources. The Integrative Imaging Informatics for Cancer Research (I3CR) Center has played a lead role in implementing cancer imaging informatics technology, with a focus on expanding the widely used open source XNAT informatics platform to better support computational workflows in cancer imaging. I3CR has also developed knowledge management tools to better track data processing and analysis, including tools for orchestrating and tracking container-based computing pipelines. As result of this work, XNAT has emerged as the most widely used imaging informatics platform in cancer research. It is deployed in over 200 organizations across academia and industry and has been adopted across a wide range of research contexts, including preclinical imaging, multi-site clinical trials, and clinical translation. As the I3CR informatics platform has matured and been widely adopted, the Center is now evolving to the next phase of development to more broadly sustain the platform. In the work proposed here, we will sustain the I3CR’s ongoing engineering initiatives and expand its outreach efforts. The Center’s sustainment activities will build on and extend the I3CR platform’s expansive set of data and knowledge management capabilities, with a focus on addressing key emergent needs within our user base. In Aim 1, we will continue to develop the XNAT-based data management platform, including adding standards-based clinical interfaces, developing a cohort discovery service with natural language processing support, implementing a task automation service, and extending its container-based computing service to support high performance computing and cloud computing environments. In Aim 2, we will implement a suite of integrations with complementary image analysis and data sharing platforms, including The Cancer Image Archive and the NCI Imaging Data Commons. In Aim 3, we will expand the I3CR’s outreach initiatives to ensure broad and effective adoption of the I3CR informatics platforms. The Center’s training program will utilize the XNAT Academy education platform to host a series of online training programs directed at specific audiences including developers, data scientists, integrators, and system administrators. A suite of supporting cloud services will be implemented assist the community in adopting and developing on the I3CR platform.

NIH Research Projects · FY 2025 · 2021-04

PROJECT SUMMARY Children with sickle cell anemia (SCA) suffer from cognitive decline, even when unaffected by stroke. The pathophysiology of cognitive dysfunction in SCA is poorly understood. Understanding the mechanism and trajectory of injury and degree of reversibility is necessary to prevent lifelong disability in this vulnerable population. Furthermore, current screening tools are inadequate as transcranial Doppler ultrasound and structural brain MRI only screen for risk of stroke and evaluate for presence of irreversible infarction. The long- term goal of this proposal is to determine the mechanism of brain injury with MR measures of oxygen metabolism, structural connectivity, and functional connectivity, and develop neuroimaging biomarkers for cognitive dysfunction in SCA. Children with SCA experience cerebral metabolic stress, as measured by increased oxygen extraction fraction (OEF). OEF peaks within the deep white matter, co-localizing with the brain region at greatest risk for stroke in SCA. Increased metabolic stress is associated with disrupted connectivity within specific functional brain networks, and the nodes of networks with diminished functional connectivity in SCA are anatomically- contiguous, clustered and aligned with the region of elevated OEF in the white matter. However, OEF decreases and executive function abilities improve with a single red blood cell transfusion in children with SCA, suggesting some aspects of cognitive dysfunction, potentially driven by alterations in FC, are acutely reversible as metabolic stress is attenuated in SCA. The central hypothesis of this proposal is that isolated disruption of functional connectivity caused by increased metabolic stress will be acutely reversible; however, disruption of functional connectivity mediated by structural connectivity will be irreversible. Treatment of the former may improve cognitive function, while treatment of the latter may mitigate progressive cognitive decline. In order to test her hypothesis, Dr. Fields will obtain longitudinal measures, separated by three years, of cognitive testing and brain MRIs to measure OEF, structural connectivity and functional connectivity in control, non-transfused SCA, and transfused SCA participants. The transfused participants will undergo cognitive testing and brain MRI before and after red blood cell transfusion at study entrance. Using this data, she will test her central hypothesis by completing the following specific aims: 1) Determine if disruption of the structural and functional connectome is reversible with transfusion of RBCs in SCA, 2) Determine the impact of increased OEF on the development of the structural and functional connectome, and 3) Determine if MR metrics of metabolic stress, SC and FC predict aberrant cognitive trajectories. Completion of these aims will provide insight into the pathophysiology of cognitive dysfunction in SCA, and allow the definition and development of biomarkers for reversible neurologic injury, which can potentially guide treatment effect, and improve outcomes in this vulnerable population.

NIH Research Projects · FY 2025 · 2021-04

Responding to PAR 18-497: Sleep disorders and circadian clock disruption in Alzheimer’s disease and other dementias of aging, the overall goal of this study is to elucidate the brain molecular mechanisms underlying the bi-directional links between AD and sleep disruption in older adults. Sleep disruption predisposes to AD, while AD results in substantial sleep disruption. However, the brain molecular mechanisms underlying these bi-directional relationships remain obscure. Studies relating sleep disruption to the human transcriptome have generally involved adults without AD, examined blood rather than brain, and cannot distinguish sleep effects on AD from AD effects on sleep. We propose an innovative, cross-species translational approach to overcome these gaps. We will integrate transcriptome and proteome profiles from well-characterized human brains to identify promising gene associations with sleep fragmentation, AD pathology, and cognitive impairment, with validation of causal genes through which sleep fragmentation leads to AD pathology and cognitive impairment, and vice versa, based on genetic manipulation in Drosophila. In compelling pilot studies, we showed that sleep fragmentation in older humans is associated with a greater risk of incident AD. We have also defined a preliminary set of human genes that are significantly differentially expressed in prefrontal cortex in association with sleep fragmentation and both AD pathology and cognitive impairment. Moreover, from among these human candidate genes, we have successfully deployed ethologically relevant Drosophila paradigms to validate conserved genetic modifiers of sleep fragmentation, sleep-related cognitive plasticity, and both Tau and Aß neurotoxicity. This study will collect new human lateral orbitofrontal cortex RNA sequencing data from 500 well-characterized older decedents from the Rush Memory and Aging Project (R01AG17911), integrate this with available proteomic data, and relate this to actigraphic sleep metrics, cognitive testing, and post-mortem indices of Aß and Tau pathology to identify candidate brain- expressed genes underlying the bi-directional links between sleep fragmentation, AD pathology and cognition. To move beyond descriptive associations, we will use Drosophila genetics to validate causal roles for these genes in A/Tau-induced neurodegeneration, cognitive plasticity, and sleep. Finally, leveraging available genomic data, we will confirm these causal relationships, observed in Drosophila, in humans using Mendelian randomization. Leveraging broad expertise, and augmenting human brain transcriptomics and proteomics with causal validation in Drosophila, this study will provide an in-depth description of the key bi-directional molecular mechanisms linking sleep fragmentation, AD pathology, and cognitive impairment. These genes will represent novel targets for further drug development to prevent the deleterious impact of sleep fragmentation on AD pathology and cognition, and to treat sleep fragmentation in adults with AD, with the potential to have a sustained impact on the health of millions of older Americans at risk for, or suffering from, AD.

NIH Research Projects · FY 2025 · 2021-04

Most CNS neurons express GABAA receptors (GABAARs), which mediate inhibition. GABAARs are comprised of 5 subunits. Two of these are α and two are β subunits. The fifth is usually γ2 or δ. Although the fifth is not required for gating, in recombinant receptors the presence of the γ2 or δ subunit dramatically alters biophysical and pharmacological properties. In native cells, the fifth subunit is thought to mark specific roles in tonic (δ subunit) and phasic (γ2 subunit) inhibition. We do not understand the physiological contributions of receptor subclasses defined by these subunits, although emerging evidence suggests that receptor subclasses have distinct roles in mental functions, and therapeutic drugs target one or the other subclass to produce different psychoactive effects. For instance, neurosteroids, which may have δ-selective actions, are emerging antidepressants. No antagonist exists to separate δ receptors from γ2 receptors, so many questions about their respective contributions remain. We were compelled by the shortcomings of previous approaches and the historical advantages of selective antagonism to create mouse lines with a point mutation in either γ2 or δ, which endows resistance to the non-competitive GABAAR antagonist picrotoxin. Preliminary data show the potential utility of these tools. Here, we test the overarching hypothesis that δ receptors mediate δ-driven disinhibition in cortical areas including the hippocampus. We will explore the role of δ receptors in cell classes known to express δ and that may offer a substrate for δ-driven disinhibition. Finally, we will test the impact of δ receptors in circuits of the hippocampus and thalamus important for neuropsychiatric illness, with the hypothesis that δ-driven disinhibition drives γ oscillations responsible for aspects of cognition, and δ receptors separately drive sleep spindles in thalamocortical circuits. Our recent results have already altered prevailing views and allow us to interrogate roles of receptor subpopulations in cellular and network function. Our approach will guide rational drug development aimed at inhibition.

NIH Research Projects · FY 2025 · 2021-04

Project Summary Now in its 13th year, the Psychiatric Genomics Consortium is perhaps the most innovative and productive experiment in the history of psychiatry. The PGC unified the field and attracted a cadre of outstanding scientists (802 investigators from 157 institutions in 41 countries). PGC work has led to identification of ~500 genetic loci in the 11 psychiatric disorders we study. Our work has led to 320 papers, many in high-profile journals (Nature 3, Cell 5, Science 2, Nat Genet 27, Nat Neurosci 9, Mol Psych 37, Biol Psych 25). As summary statistics are freely available, psychiatric disorders often feature prominently in papers by non-PGC investigators. To advance discovery and impact, we propose to continue the work of the PGC across 11 disorder groups. Considerable new data are coming in the next five years. We thus can rapidly and efficiently increase our knowledge of the fundamental basis of major psychiatric disorders. Aim 1: we will continue to advance genetic discovery for severe psychiatric disorders in all working groups, systematically interface with large biobank studies to ensure maximal comparability, and aggressively promote new studies of individuals with psychiatric disorders from diverse ancestries to increase discovery and improve fine-mapping. Aim 2: most studies analyze common variation (Aim 1), rare CNV (Aim 2), and rare exome/genome resequencing results (via collaboration) in isolation: we will apply an integrative framework to rigorously evaluate the contributions of all measured types of genetic variation on risk for psychiatric disorders. Aim 3: we will move beyond classical case-control definitions to a more biologically-based and nuanced understanding by enabling large trans-diagnostic studies, convene trans-disciplinary teams to use genetics to address unresolved questions about the nature of psychiatric disorders, and to promote large studies of the severest cases seen in psychiatric practice (leveraging the global reach of PGC investigators). Aim 4: we will work to maximize the impact of our work via translational efforts: close collaborations with neuroscience consortia to understand the biological implications of our findings; work to identify modifiable causal risk factors; and work to robustly predict clinical outcomes and identify patient subsets. Aim 5: we will increase impact of our work by extending and formalizing outreach to different communities (including pharma and biotech), via digital media (Twitter, Facebook, Wikipedia), and by developing, distributing, and updating resources/educational material for patients, families, and medical professionals. We will convene a Scientific Advisory Board to ensure we respond positively to those invested in our results Successful completion of this body of work will greatly advance knowledge of the genetic basis of psychiatric disorders with potentially major nosological and treatment implications. These goals are consistent with a core mission of the NIMH, and the central idea of the PGC: to convert the family history risk factor into biologically, clinically, and therapeutically meaningful insights.

NIH Research Projects · FY 2025 · 2021-04

Project Summary Recreational cannabis use is becoming increasingly common in the United States, even within vulnerable populations. Amidst growing concerns surrounding the possible adverse consequences of chronic cannabis use, there is evidence that cannabis use disorder (CUD) is genetically correlated with susceptibility to several behavioral (e.g., lower educational achievement) and psychiatric (e.g., schizophrenia) outcomes, thus bringing into question prior causal claims. The most aggressively contested discussion surrounds the role of cannabis use and CUD in the etiology of schizophrenia (SCZ) and psychotic illness. While there is now an abundance of evidence supporting shared genetic influences, studies also outline the psychotomimetic effects of especially high potency forms of tetrahydrocannabinol (THC). A systematic search for pleiotropic variants that undergird this comorbidity between CUD and SCZ can not only provide insights into shared biology, but also outline avenues for identifying subgroups of individuals at greatest risk. This Mentored Research Scientist Development Award (K01) proposes a research plan that leverages some of the largest currently available genome-wide association study (GWAS) datasets to (a) conduct a cross-disorder GWAS of CUD with SCZ, and to contrast it with findings from a similar cross-disorder analysis of cannabis use with SCZ, to identify loci of convergent and divergent effect; (b) to test for a causal relationship using a genetically-informed approach and harness curated `omics data from human and rodent models of cannabis exposure and SCZ, to fine-map significant loci and further prioritize causal variants for biological plausibility; and (c) to utilize polygenic risk scores derived from these cross-disorder analyses to identify associations with first-episode psychosis, cannabis-induced psychosis, and childhood psychosis-proneness in independent samples. These research aims are founded on four key training objectives that will enhance the applicant's career goal of becoming an NIH-funded independent investigator who works at the interface of addictions and psychiatric illness. These training objectives include (a) a deep understanding of the clinical effects of acute and chronic exposure to cannabis, (b) integrative bioinformatics approaches for post-GWAS annotation, including cross-species data (c) an appreciation of the neurobiology underlying the comorbidity between cannabis and SCZ, and (d) career development towards leadership and mentorship positions. The applicant builds upon her current funding and training directed at advanced statistical genetics to addressing comorbidity by adding on novel facets relating more broadly to multi- omics data integration and more specifically to the unique yet ubiquitous comorbidity between cannabis and SCZ. Together, this training and research plan will produce some of the first insights into the shared genetic etiology underlying CUD and SCZ and provide opportunities for functionally targeted future studies, with the ultimate objective of producing therapeutic alternatives that can partially mitigate the serious personal costs of chronic cannabis use in SCZ patients.

NIH Research Projects · FY 2025 · 2021-04

Project Summary Quantitative imaging, where a numerical/statistical feature is computed from a patient image, is emerging as an important tool for diagnosis and therapy planning. Several new and improved quantitative imaging (QI) methods, which include reconstruction, analysis, and estimation methods are thus being developed. There is an important and timely need to optimize the QI methods on the underlying clinical quantitative task, as sub-optimal methods would yield quantitative values that are unreliable, and thus have limited clinical value. Performing this evaluation with patient imaging data is highly desirable, but the unreliability or unavailability of a gold standard for most patient studies makes evaluation impractical or impossible. To enable evaluation of imaging methods with patient data, several no-gold-standard evaluation (NGSE) techniques have been developed, but mostly in the context of detection tasks. More recently, similar NGSE techniques for quantitative tasks have been developed by us and others. We have demonstrated the efficacy of our NGSE technique in ranking segmentation methods for diffusion MR and reconstruction methods for quantitative SPECT. Our goal in this project is to take steps towards translating this mathematical concept to a clinical tool. Existing NGSE techniques make assumptions that may not hold in several QI applications, require large amounts of patient images that are often unavailable, and have been validated using only computational studies. To address these issues, we propose to develop and comprehensively validate a novel generalized Bayesian NGSE framework. This framework will be a generalized Bayesian approach that will reflect clinical scenarios accurately and not require multiple patient studies. The framework will be validated using new anthropomorphic physical phantom and patient data in addition to realistic and validated simulation studies. For clinical translation, it is also necessary to demonstrate the efficacy of the framework in answering an important clinical question. The clinical question we choose is that of using the NGSE framework to determine the optimal segmentation method to compute volumetric features from PET for early prediction of therapy response in patients with non-small cell lung cancer (NSCLC). Answering this question will help address a critical, urgent and unmet need for strategies to personalize the treatment of NSCLC, a disease with high morbidity and mortality rates. The proposed NGSE framework is well poised to accelerate the clinical translation of new and improved QI methods by enabling their evaluation with patient data. The framework will have multiple high-impact applications such as in determining the optimal QI method for measuring biomarkers to monitor cancer-treatment response, diagnose cardiac/neurodegenerative diseases, and conduct imaging- based dosimetry. Thus, developing this NGSE framework has the potential to significantly impact QI-based clinical decision making.

NIH Research Projects · FY 2025 · 2021-04

PROJECT SUMMARY / ABSTRACT Bacterial adhesion to the urinary tract epithelium is a critical step in establishing urinary tract infections. During infection of the mammalian bladder (cystitis), uropathogenic Escherichia coli (UPEC) are well described to employ type 1 pili, bearing the tip adhesin FimH, to bind oligomannose-decorated uroplakins that coat the luminal surfaces of superficial bladder epithelial cells. However, less detail is known about host-pathogen interactions in the kidney that enable initiation of upper-tract UTIs, including pyelonephritis and renal abscess. We have found that type 1 pili, previously thought to be essential only in cystitis, also mediate establishment of pyelonephritis and the initiation of renal abscesses in C3H mice. Furthermore, in an in vitro model of UPEC binding to renal collecting duct epithelium, we identified a candidate renal epithelial receptor for FimH, namely the mannosylated cell-junctional protein desmoglein-2 (Dsg2). This protein is expressed throughout the nephron but most highly in collecting duct epithelium, and bears typical N-linked mannose-containing glycans as well as cadherin family-specific O-linked mannosylation. In this project, we will test the central hypothesis that desmoglein-2 is an epithelial receptor for FimH that mediates establishment of UPEC pyelonephritis and can bind FimH in gut and exfoliated bladder. First, we will use multiple genetic and pharmacologic systems to interrogate the importance of FimH binding to mannosylated Dsg2 in recently published, optimized mouse models of UPEC pyelonephritis. Among these systems will be newly generated C3H mice carrying renal epithelial-specific deletion of Dsg2. Next, we will quantify the binding affinity of the FimH lectin domain to the purified extracellular domain of human DSG2 by SPR, and co-crystallize the relevant FimH and DSG2 domains to reveal the structural basis for the DSG2-FimH interaction. Controls in these experiments will include FimHQ133K, which carries a mutation that abrogates mannose binding; mannosides, high-affinity small-molecule inhibitors of FimH binding; enzymatic pre-treatment of purified protein and kidney tissue sections to eliminate N- or O-linked glycans; and monoclonal antibodies generated against the key DSG2 peptides mediating interaction with FimH. Third, desmoglein-2 is also expressed widely on other epithelial cell types (in both humans and mice), raising the added possibility that it binds FimH in other niches relevant to UTI pathogenesis. These include the bladder after exfoliation (a rapid response to initial UPEC infection that eliminates the primary FimH receptor) and the colon (which serves as a UPEC reservoir to seed recurrent UTI). Therefore, we will use mouse and human tissue sections, an in vivo gut colonization model, and additional new conditional Dsg2 knockout mice to investigate whether Dsg2 can serve as a FimH receptor in these tissues. At the conclusion of these studies, we will have specified a novel receptor for type 1 pili, elucidated the structural basis of FimH interaction with desmoglein-2, and defined the functional roles of this pharmacologically targetable interaction in multiple UTI-relevant niches.

NIH Research Projects · FY 2025 · 2021-04

PROJECT SUMMARY Chikungunya virus (CHIKV) is a mosquito-transmitted, positive-strand enveloped alphavirus that causes global disease in humans. At present, no antiviral agents or licensed vaccines exist for the treatment or prevention of any alphavirus infections. While age, immune status, and pre-existing chronic illness are associated with increased risk of severe CHIKV infection, the role of acquired factors in disease progression is poorly understood. Our preliminary data suggests that the microbiota regulates CHIKV infection, dissemination, and musculoskeletal inflammation and disease through a previously undefined axis by which signals from gut bacteria and bile acids instruct innate immune cell responses to control CHIKV infection of monocytes in circulation and monocyte migration to affected joint tissues. We hypothesize that specific gut bacteria and their microbial constituents modulate CHIKV pathogenesis by regulating antiviral type I IFN and inflammatory responses in pDCs and monocytes. In the absence of these microbial signals, CHIKV disseminates widely, and arthritis ensues after joint infiltration by immune cells. This proposal combines investigators with expertise in alphavirus pathogenesis and immunity (Diamond) and the study of the gut microbiota in disease (Handley, Stappenbeck, and Fischbach). Using a suite of transgenic mice and microbiome reconstitution experiments paired with detailed virological and immunological analyses, we will address the following key questions: (a) which immune cells coordinate the rapid systemic IFN response following CHIKV infection (e.g., pDCs) (b) what immune cues limit viral infection in circulating immune cells (e.g., monocytes)? (c) how does the gut microbiota regulate pDC IFN production and trafficking of circulating immune cells? and (d) which constituents (e.g., metabolites) of the microbiota regulate antiviral and inflammatory responses? Through these detailed mechanistic studies, we expect to link the microbe-derived constituents of specific commensal bacteria with innate antiviral responses that modulate alphavirus infection, dissemination, joint disease, and possibly transmission. Beyond enhancing our understanding of acquired determinants of alphavirus pathogenesis, the findings of this proposal could inform more generally our understanding of how the gut microbiota shapes innate immune responses to limit infection and pathogenesis of other viruses.

NIH Research Projects · FY 2025 · 2021-03

Project Summary/Abstract Rift Valley fever virus (RVFV) is a phlebovirus that belongs to the Phenuiviridae (formerly Bunyaviridae) family of negative-sense RNA viruses. As an emerging mosquito-borne virus, the significance of RVFV is highlighted by its designation as a NIAID Category A pathogen and its inclusion on the WHO's Blueprint of Priority Diseases. Recently, the Coalition for Epidemic Preparedness Innovations (CEPI) has also included RVFV as a part of their emerging infectious diseases vaccine program, further emphasizing the potential impact of RVFV on the global health and economy. While RVFV is endemic throughout sub-Saharan Africa, competent mosquito vector species are found in North America, highlighting the potential for emergence of RVFV in non-endemic countries, including the United States. During outbreaks, RVFV causes severe disease in livestock, including sheep and cattle, which dramatically impact the socioeconomic framework in resource limited settings. Humans are spill- over hosts, where infections can result in severe consequences, including hepatic necrosis, hemorrhagic fever, encephalitis, and retinal vasculitis. Despite its significance to human health and the potential to negatively impact the socioeconomic fabric of resource-limited countries where the virus is endemic, there is a lack of safe and efficacious prophylactic and therapeutic treatment options. This gap is in part due to our lack of knowledge on host factors that contribute to RVFV infection. To address this need, we conducted a genomic screen that defined several critical factors, including a potential entry factor, which we will characterize by a multidisciplinary approach. In support, we provide compelling preliminary data, including in vitro validation in host factor sufficient and deficient cells, transcomplementation studies, direct interaction between RVFV glycoprotein Gn and the host proteins in vitro, inhibition of the entry factor by endogenous ligands in vitro in multiple cell lines from evolutionarily distinct hosts, and preliminary results of protection from RVFV infection in two conditional knock out mouse models. Importantly, we have generated many key reagents, including most cell lines and proteins, and knock-out mice supporting the feasibility. Importantly, this work will be performed by highly productive and collaborative investigators with expertise in every aspect of the proposed studies, including biochemistry, RVFV pathogenesis, immunology, proteomics, structural biology, and virology. Completion of the proposed studies will define novel host or entry factors for RVFV in target cells with tissue-specific relevance. As a specific receptor for RVFV has not previously been identified, these studies will provide important information for design of therapeutic interventions to prevent RVFV infection and disease. At the completion, we expect to fill a key gap in the field and to provide novel targets for therapeutic development.

NIH Research Projects · FY 2025 · 2021-03

Development of small molecule inhibitors of metabolic enzymes as broad spectrum anthelmintic drugs Abstract Parasitic intestinal nematodes including hookworms, roundworm and whipworms, infect over two billion people worldwide, causing significant morbidity, perpetuation of poverty, and loss of life. Characterization of nematode genomes provides fundamental molecular information essential for accelerating basic and translational research, which is a public health priority due to the limited number of currently available effective drugs and increasing drug resistance. In this proposal, we will pursue post-genomic drug discovery studies to develop small molecule drugs as novel therapeutics to treat infections caused by these devastating parasites. We have established an extensive omics/bioinformatics database for human nematode parasites spanning the major taxonomic clades of Nematoda. Using systems biology and evolutionary principles, we reconstructed metabolic networks for 56 diverse nematode parasites and identified chokepoint enzymes, i.e. metabolic enzymes that uniquely consume a specific substrate or generate a unique product. This led to our central hypothesis that compounds that inhibit conserved chokepoint enzymes have a strong potential for broad control of diverse nematodes. To test this, we identified conserved targets and initial inhibitors with potential for broad-spectrum activity, for which phenotypic screening of parasites at the extremes of the phylogeny have validated our predictions. Furthermore, we established a unique database of nematode-specific molecular features among the chokepoint enzyme targets and experimentally established that active-site differences in the nematode enzymes relative to their human orthologs can rationally guide the design of selective inhibitors. The compounds with the best activity in our phenotypic screens are inhibitors predicted to target three known enzyme classes (CPT, mTOR/PI3K, and PDE). To confirm the putative nematode target(s), we will express nematode proteins and implement biochemical enzyme inhibition assays, employ affinity-based labeling techniques, and test for activity against target knockdown worms (Aim 1). By leveraging parasite-specific active- site features of the confirmed protein targets, we will use a X-ray structure-based drug design (SBDD) to optimize lead inhibitors of the three identified target classes (Aim 2). Optimized lead compounds most effective against the human hookworm Ancylostoma ceylanicum and the whipworm Trichuris muris in vitro will be tested in vivo for their pan-intestinal efficacy in hamster and mouse animal models of nematode infection (Aim 3). Our preliminary results, combined with this proposed research, are highly significant since they provide a better understanding of metabolic functions essential for nematode survival, which can be targeted for drug discovery. The rational targeting of metabolic chokepoint enzymes as anthelminthic agents is innovative, as is the concept of utilizing specific pan-phylum conserved targets to develop anthelmintic drug or drugs with broad spectrum efficacy against nematodes. Collectively, this work has high potential to provide one or more new small molecule therapeutics with broad spectrum activity against parasitic nematode infections.

NIH Research Projects · FY 2025 · 2021-03

Staphylococcus aureus is an aggressive antibiotic-resistant human bacterial pathogen. S. aureus is a leading cause of infectious disease morbidity, mortality, and hospital-associated infection in the U.S., and a formidable health threat worldwide. The only durable solution to mitigate S. aureus disease is the successful development of a universal vaccine. In spite of the clinical burden of human S. aureus infection, and considerable molecular knowledge of disease pathogenesis gleaned from experimental systems, there is a striking paucity of knowledge regarding the host immune response in human S. aureus infection. This fact has remained the single most significant shortcoming of prior vaccine approaches, as the scientific premise for the inclusion of vaccine antigens has not been based on known correlates of human immunity to S. aureus. The investigators’ studies to date in the pediatric population strongly suggest that the adaptive immune response to S. aureus is templated early in life when initial exposure to the organism occurs, amplifying the importance of understanding the development of the human adaptive immune response to S. aureus during infancy and early childhood. The proposed project addresses current knowledge gaps through a multifaceted analysis of the natural development of protective immunity to S. aureus, leveraging novel insight on the role of S. aureus α-toxin (Hla) as a virulence factor that dampens the antigen-specific T cell response in the host. Coupled with the observation that the serologic response to Hla is a correlate of long-term protective immunity to S. aureus in children, these findings suggest that neutralizing Hla may simultaneously afford disease protection and preclude modulation of host immunity by S. aureus. To this end, a cohort-based approach for comparative analysis of human immunity in the context of normal, healthy childhood development, as well as in the setting of S. aureus disease, will be performed. The context of this exposure is expected to elicit protective immunologic responses or maladaptive responses, which we hypothesize will be discernable through paired analyses of healthy and infected subjects as a function of development. These studies will be the first to leverage high-dimensionality mass cytometry (CyTOF)-based analysis of the human T cell response to S. aureus, characterizing both cellular differentiation and the functional response. Paired with multiplex analysis of the developing human antibody response to S. aureus virulence factors, the biorepository generated through the proposed study will support a comparative analysis of adaptive immunity in healthy infants and young children relative to that observed in patients who manifest both local and invasive S. aureus infection. The proposed multi-disciplinary team is uniquely positioned to conduct the first highly-focused, hypothesis-driven approach to examination of the development of human immunity to S. aureus. This research will inform our understanding of the natural development of the host response to S. aureus, providing an essential foundation for the strategic design and implementation of a S. aureus vaccine capable of eliciting population-level immunity.

NIH Research Projects · FY 2025 · 2021-03

Project Summary/Abstract Transcription factors act as specifying agents of cell differentiation during development by binding to DNA enhancer sequences and activating them to control developmental gene expression. Enhancer activation is typically associated with the removal of nucleosomes, which decorate eukaryotic genomes and normally wrap roughly 150 base pairs of DNA in a highly stable configuration. A persistent puzzle of developmental gene regulation is how TFs bind and activate their target enhancers when they are initially wrapped in nucleosomes, which typically inhibit TF binding. One hypothesis posits that a special class of “pioneer factors” are able to bind their targets in the context of nucleosomal wrapping and displace the nucleosomes they bind to activate and expose the enhancer for downstream TF binding. However, it has been exceedingly difficult to confirm the presence of nucleosome binding “pioneer activity” in vivo, leaving the developmental roles of pioneer factors in question. We recently used high-resolution epigenome profiling to identify instances of nucleosome binding by pioneer factors that were enriched at enhancers with suboptimal motif binding sequences, presenting the intriguing possibility that pioneer activity is a mechanism to ensure the fidelity of enhancer activation at sites that are vulnerable to natural fluctuations in the local chromatin environment. Pioneer factors often function in early development, which maintains high fidelity despite natural variation in chromatin structure that is sensitive to the metabolic state of the cell. Therefore, pioneer factors may play a direct role in insulating developmental transitions against metabolic variance. However, the potential roles of pioneer factors in developmental fidelity and buffering against metabolic heterogeneity have not been uncovered to date. In this proposal, I will use a controlled pioneer factor expression system to study how pioneer factor-driven developmental changes are buffered against deliberate chromatin and metabolic perturbations. In Aim 1, I will test the hypothesis that pioneer activity facilitates developmental fidelity by observing development after genetically enforcing chromatin barriers to pioneer factor binding and inactivating the nucleosome binding pioneer activity of a specific pioneer factor. In Aim 2, I will use a model system of metabolic control of development to understand how pioneer factor binding responds to metabolic changes, and how specific pioneer factor-enhancer activation events underlie different developmental outcomes in response. These Aims will uncover mechanistic explanations for the disparity between variance in gene regulatory processes on the molecular level and the precision of cell fate outcomes on the developmental level, and my findings will be of direct consequence to diseases such as cancer where extreme heterogeneity overwhelms the checks and balances on cell fate. A K99/R00 Award will be instrumental in addressing these questions and furnishing me with high level training in new methods and biological theory that will prepare me to continue to pursue major research avenues related to pioneer factor and chromatin control of development in my future independent career.

NIH Research Projects · FY 2025 · 2021-03

PROJECT SUMMARY/ABSTRACT GM1-gangliosidosis is a rare, fatal, neurodegenerative genetic disease caused by the deficiency of β- galactosidase enzyme activity and characterized clinically by a wide range of variable neurovisceral, ophthalmological and dysmorphic features. There are currently no effective therapies for GM1-gangliosidosis and only symptomatic treatments are available. In preclinical models, adeno-associated viral (AAV) gene therapy that restores the β-galactosidase enzyme activity is the most promising therapy for delaying symptom onset, reducing storage in the brain and peripheral tissues, and increasing lifespan. These impressive results have provided the foundation for AAV gene therapy clinical trials. One of the major challenges for developing treatments for GM1-gangliosidosis is the difficulty in the evaluation of treatment efficacy due to the small and heterogeneous patient population as well as slow progression in non-infantile patients. Recently we used liquid chromatography-tandem mass spectrometry (LC-MS/MS) to identify a pentasaccharide (referred to as H3N2b) that is elevated > 20-fold in patient urine, plasma, and cerebrospinal fluid (CSF), and in the central nervous system (CNS) of the GM1-gangliosidosis cat. The CNS H3N2b levels in the GM1-gangliosidosis cat are reduced in response to AAV-treatment. H3N2b has potential as a pharmacodynamics/response biomarker for assessment of AAV-treatment efficacy in GM1-gangliosidosis. The goal of this proposal is to validate LC-MS/MS methods for determination of H3N2b in human urine, plasma, CSF, which will be used to assess AAV gene therapy treatment efficacy in a clinical trial. The aims of this application are 1) validation of LC-MS/MS methods for quantification of H3N2b in human plasma, urine, and CSF; 2) assessment of H3N2b in samples collected from GM1-gangliosidosis natural history study; and 3) application of H3N2b for assessment of treatment efficacy of AAV gene therapy. The proposed work will provide a much-needed tool for assessing therapeutic efficacy.

NIH Research Projects · FY 2025 · 2021-03

PROJECT SUMMARY/ ABSTRACT: Antibiotic-resistant bacterial infections that are no longer sensitive to our life saving antibiotic arsenal are a looming catastrophe and like the recent COVID-19 crisis, will have dire consequences for human health if we are not prepared. This proposal leverages basic science findings for development of antibiotic-sparing medicines with impact on treatment for most pathogens designated threats to human health by the CDC. Projects 1 and 2 target multi-drug resistant (MDR) Gram-negative pathogens that express adhesive pili required for colonization and infection in the host habitats involved in acute and chronic/recurrent urinary tract infections (UTIs) and catheter-associated UTIs (CAUTIs), including MDR Acinetobacter, carbapenem-resistant Enterobacteriaceae (CRE) and extended-spectrum beta-lactamase (ESBL)-producing Enterobacteriaceae. Project 2 expands on this list to include other Gram-negative pathogens of concern. Since UTIs account for ~10% of antibiotic use in humans, the development of antibiotic-sparing therapeutics will not only allow treatment of antibiotic-resistant infections, but by reducing the use of current antibiotics, will decrease selective pressures for resistance. Project 1 is focused on neutralizing bacterial pilus adhesins using glycomimetics designed in CORE 1 and mAbs developed in CORE 2 that will block critical interactions between bacterial adhesins and their host ligands. Glycomimetics have shown great promise in neutralizing chaperone/usher pathway (CUP) adhesins in vivo to treat disease. For example, mannosides, which neutralize uropathogenic E. coli (UPEC) adhesin FimH, are potent therapeutics for treating and preventing UTI, since FimH is required by UPEC to colonize the bladder. In collaboration with GlaxoSmithKline a mannoside has been selected to proceed into Phase 1a/1b clinical trials, thus validating the potential of this strategy. Therapeutic mAbs have not yet been fully harnessed for treating infectious diseases. With antibiotic resistance on the rise, it is time to apply this strategy. Project 1 will also target a sortase-assembled pilus adhesin of Gram-positive enterococci, which causes CAUTIs and is often MDR. Project 2 will use similar tools to focus on the CUP machinery that assembles the Gram-negative adhesins in Project 1 at the tip of pilus fibers. Project 3 will target all Gram- positive species identified by the CDC as significant threats by furthering the development of GmPcides, a novel family of ring-fused 2-pyridone compounds that are bactericidal against a broad spectrum of Gram- positive species. The COREs will be fully integrated with the Scientific Projects providing computational and synthetic medicinal chemistry in the development of small molecule therapeutics (CORE 1) and the application of high throughput mAb generation against bacterial proteins (CORE 2). The combined knowledge, expertise and successes of the Leaders of the Projects and Cores will lead to the development of antibiotic-sparing therapeutics for treatment of the growing number of antibiotic-resistant pathogens to stave off the return to the pre-antibiotic era when common infections were essentially untreatable.

NIH Research Projects · FY 2025 · 2021-02

Project Summary/Abstract Dentists are responsible for approximately 10% of outpatient antibiotic prescriptions in the United States. Many of these antibiotic prescriptions are unnecessary and place patients at risk of adverse drug events, including allergic reactions and infections with drug resistant organisms. However, improving antibiotic prescribing practices in dental settings is challenging. Dental clinics often lack the resources to design and implement policies to evaluate dental antibiotic prescribing practices and design interventions to improve prescribing practices. My long-term goal is to oversee the development of electronic clinical decision support tools (CDST) to facilitate optimal antibiotic prescribing among dentists. I envision creating, pilot testing, and disseminating CDSTs that can be embedded within electronic health record systems, including electronic dental record systems, to improve adherence to science-based practices while making the lives of clinicians easier. This specific proposal aims to design and implement a novel web-based application “app” that can be accessed by dentists on mobile phones or websites. Our first aim is to design the CDST based on focus group feedback from dental students, residents, and faculty at the University of Illinois College of Dentistry (UIC- COD). Our second aim is to pilot test the app using clinical vignettes and validated technology survey questions. The third aim is to evaluate the effectiveness and implementation of our CDST within UIC-COD using a dissemination and implementation research framework. We will propose to measure changes in antibiotic knowledge and confidence in antibiotic decision making before and after implementation of the CDST using paired surveys. We will also use surveys to measure provider satisfaction with the CDST. We will track CDST usage (implementation metrics) using Google analytics. After completion of this project, we plan to test our CDST in community dentists via the National Institute for Dental and Craniofacial Research Dental Practice Based Research Network and apply for grant funding to embed this CDST into electronic dental record systems.

NIH Research Projects · FY 2025 · 2021-02

The brain changes of Alzheimer disease (AD) start many years before the onset of cognitive symptoms. Most models of AD propose a stepwise progression of brain pathology starting with amyloid plaque deposition, then tau tangle formation, then neurodegeneration. Cerebrospinal fluid (CSF) and imaging biomarkers have been developed that allow detection of AD brain pathology in living individuals, but these modalities have significant drawbacks that limit their widespread use. Over the last three years, there has been rapid development of blood-based biomarkers that accurately detect AD brain pathology. In this study, we propose to study some of the most promising blood-based biomarkers for three types of AD brain pathology: amyloid (Aβ42/Aβ40), tau (phosphorylated tau [pTau] isoforms), and neurodegeneration (neurofilament light chain protein [NfL]). The research team has developed immunoprecipitation-mass spectrometry assays for plasma Aβ42/Aβ40 and pTau isoforms that will be further optimized and automated as part of the proposed project. The Knight Alzheimer Disease Research Center cohort will be studied, and has available data on plasma and CSF NfL, clinical dementia diagnosis, performance on cognitive tests, health history, amyloid PET, tau PET, structural brain volumes by MRI, genetic markers, numerous CSF biomarker measures, discovery proteomics data, and autopsy reports. Approximately 1,700 matched pairs of banked plasma and CSF samples from ~1,000 individuals will be examined, which is similar in size to recent major studies of cognitive outcomes as a function of biomarker combinations. The correlation of the blood-based measures with better established CSF and imaging measures will be evaluated. Biomarkers of amyloid, tau and neurodegeneration will be used independently and in combination within a modality (blood-based, CSF or imaging) to predict the risk for current or future symptomatic AD. For all analyses, the effects of individual characteristics (including age, sex, years of education, APOE ε4 genotype, polygenic risk score, race, and medical comorbidities) will be evaluated to identify factors that modify the expression of symptoms associated with biomarker levels. We hypothesize that the combination of plasma Aβ42/Aβ40, pTau isoforms and NfL will perform better than amyloid PET in predicting risk for current or future symptomatic AD. Because blood tests are well-accepted by patients, physicians, and researchers, an accurate blood test for symptomatic AD would likely be widely used and could be a game-changer in improving AD research, accelerating clinical trials and enabling more accurate diagnoses in the clinic.

NIH Research Projects · FY 2025 · 2021-02

PROJECT SUMMARY/ABSTRACT Antipsychotic-induced weight gain (AIWG) is of significant public health importance in mentally ill populations, potentially addressable with personalized, precision medicine. Antipsychotic medications increase body weight, thereby increasing cardiometabolic risk (CMR) conditions like type 2 diabetes and cardiovascular disease, conditions associated with accelerated cellular aging. This has contributed to a 10 to 15-year mortality gap between mentally ill individuals and the general population. Antipsychotic medications are commonly used at all ages, but are associated with differential patterns of fat gain, whereby children gain more and older adults gain less. Numerous genome-wide association studies (GWAS) have identified key genetic factors associated with AIWG, but are limited by the use of indirect measures of body fat, like weight or body mass index (BMI), that are less well correlated with metabolic disease risk. Additionally, existing research does not fully address age-related differences in AIWG. In response to NIH PA-17-088 “Secondary Analyses of Existing Cohorts, Data Sets and Stored Biospecimens to Address Clinical Aging Research Questions,” we propose a novel approach applying population-based genetics, existing biospecimen with linked clinical data including precisely-measured adiposity and insulin sensitivity, and advanced molecular tools to identify and functionally validate key genetic determinants of AIWG and CMR across the age-span. This approach leverages 1) existing population-level data from large biobanking initiatives and epidemiological studies inclusive of approximately 15,000 individuals with genetic and relevant phenotypic data, 2) existing clinical and biospecimen data from NIH funded randomized clinical trials or RCTs characterizing the metabolic effects of antipsychotics in children, adults and older adults with direct and precise measures of body fat, together with data from approximately 600 individuals with genetic data and additional biomarkers of metabolic risk, and 3) CRISPR based in vitro drug exposure, followed by cellular functional assays to characterize molecular mechanisms impacted by antipsychotic. Additional sources of existing data will be available upon funding, including data on approximately 3000 individuals from large industry funded RCTs, data on up to 250,000 individuals from the Psychiatric Genetics Consortium (PGC, see letter of support), and data from more than 2,000 individuals from the Dutch Bipolar Cohort Study (see letter of support) will also be used for independent validation and replication. This study will combine unbiased genomic methods, including array-based genotyping, GWAS and GWAS meta-analysis, CRISPR-based gene inhibition/activation screens (CRISPRi/a), and functional molecular and cellular studies on prioritized variants of interest, combined with unique clinical data to identify genetic factors and generate predictive models of weight related physiological changes associated with accelerated aging. This combined set of molecular techniques will allow us to build on known genetic associations, while discovering new genes and genetic variants that are associated with the greatest risk for treatment-related fat gain in younger and older patients. This project will contribute to the development of a precision-based treatment algorithm that can accurately predict and prevent AIWG and cardiometabolic risk in youth, young, middle-aged, and older adults. The results from this study will also importantly contribute to publicly available datasets, and motivate future collection of similar data necessary for further validation of our results.

NIH Research Projects · FY 2025 · 2021-02

SUMMARY Pulmonary fibrosis is the result of a poorly understood, dysregulated cellular response that is difficult to diagnose and treat. A common form, idiopathic pulmonary fibrosis (IPF), has a progressive, downhill course. There are no well-established molecular biomarkers for diagnosis, treatment, or disease activity. Clinicians currently depend on changes in chest computed tomography (CT) and pulmonary function to monitor patients. Moreover, there are only two approved drug therapies, and treatment is not guided by molecular biomarkers. Lung CCR2+ (C-C motif chemokine receptor 2) inflammatory monocytes and their pathologic progeny, interstitial macrophages, are strongly associated with the experimental development of lung fibrosis, elevated in the lungs of patients with pulmonary fibrosis, and produce profibrotic factors. Fibrosis is significantly attenuated in Ccr2 null mice and by deletion of CCR2+ progeny macrophages, strongly supporting a role for CCR2+ cells in human disease. This proposal aims to utilize a molecular, positron-emission tomography (PET)-based diagnostic to detect CCR2- mediated inflammation in the lungs of patients with fibrosis and to develop targeted therapies. Our multidisciplinary group has established that a peptide-based radiotracer, 64Cu-DOTA-ECL1i, identifies CCR2+ monocytes in animal models and has acceptable dosimetry in our recent human Phase 0/1 trial of PET/CT imaging. The known relationship of CCR2+ cells to pulmonary fibrosis and the clinical challenges of managing patients with IPF, make this disease particularly suited for evaluating the radiotracer. Therefore, we have used multiple mouse models of lung fibrosis to show that increased 64Cu-DOTA-ECL1i lung uptake correlates with CCR2+ cell infiltration and fibrosis. Our data also show that the radiotracer detects decreases in lung uptake in bleomycin-induced fibrosis after blockade of interleukin-1b, a mediator of fibrosis expressed in CCR2+ cells, and treatment with anti-fibrotic drug, pirfenidone. Pilot CCR2-PET imaging of patients with IPF show increased lung signal, particularly in regions of subpleural fibrosis. We propose to use 64Cu-DOTA-ECL1i PET imaging to evaluate modulation of CCR2+-specific inflammation during the course of fibrotic lung disease in animal models, validate the detection of CCR2 cells in human lung tissue, and assess the potential for monitoring patients. We hypothesize that 64Cu-DOTA-ECL1i detects the CCR2+ cell inflammatory process associated with pulmonary fibrosis and can be used to monitor disease activity. Specific aims are: (1) In mouse fibrosis models, assess the change in the 64Cu-DOTA-ECL1i PET/CT uptake relative to inflammation and fibrosis upon treatment with clinical anti-fibrotic drugs and following molecular targeting with CCR2 antagonists, and (2) In patients with IPF, assess the relationship between PET uptake, CT imaging, and clinical status, then validate the relationship of PET uptake with CCR2-mediated inflammation and pro-fibrotic gene expression in lungs removed after transplant. Together, the aims provide a platform to obtain detailed information related to the underpinnings of CCR2+ cell imaging in IPF and the interpretation of human studies that may lead to targeted molecular therapies for IPF.

NIH Research Projects · FY 2025 · 2021-02

Project Summary Control of the internal organization of cells is essential to the form and function of all organisms. This ability rests largely on an intricate protein filament network called the cytoskeleton which serves as a scaffolding structure to pattern cellular contents in space and time. Unlike human-made scaffolding structures, the cytoskeleton is highly dynamic and is able to change its configuration in response to developmental and environmental signals, allowing cells to adapt to changing conditions. Thus, the cytoskeleton is like an ever- changing structural “diagram”, and the goal of my research program is to understand how these diagrams are generated to execute essential cellular activities that underlie growth, development and physiology. Our research focuses on mechanisms for the creation, maintenance and restructuring of the microtubule cytoskeleton using the cortical microtubule array of Arabidopsis thaliana as an experimentally tractable system. We use a multidisciplinary approach and benefit from an extensive network of close collaborators with whom we freely share reagents and ideas. These advantages have allowed us to address previously intractable questions about cytoskeleton structure and function. Here, we will build on our recent progress to focus on four major goals: Goal 1) characterize new regulatory mechanisms that specifically tune the microtubule severing activity of katanin to uncover how various internal and external signals influence the assembly and disassembly of diverse microtubule structures through katanin. Goal 2) elucidate the structural dynamics that determine the functional diversity of MAP65 microtubule crosslinking proteins to gain insight into how evolution selected particular MAP65 sequences for specialized functions and to enable the creation of new tools to manipulate microtubule function in plants and animals. Goal 3) uncover the structure and mechanism of action of a new class of microtubule minus-end regulators to understand how TOG domains, which are typically associated with microtubule plus-end tracking proteins, have been repurposed to recognize and stabilize microtubule minus-ends. Goal 4) develop a new microfluidics chip platform to analyze the complex relationships between sets of microtubule regulatory proteins to obtain a clear and integrated picture of how concurrent molecular activities dynamically pattern microtubule structures. Together, these synergistic projects will provide a mechanistically detailed picture of the inner workings of complex microtubule structures and advance our understanding of their functions in cell biology and disease.

NIH Research Projects · FY 2025 · 2021-02

PROJECT SUMMARY Callous-unemotional behavior (CU behavior), characterized by atypically low empathy, prosociality and guilt, represents a serious impairment in moral development associated with severe and persistent conduct problems, violent crime, social rejection, and substance use disorders. Alarmingly, CU behaviors have been historically difficult to treat. By age 3, CU behaviors are reliably measurable, predict CU into late childhood, and are already associated with greater conduct and social problems. Despite this evidence, very few studies have examined precursors of CU behaviors (i.e., emerging CU) or identified risk and protective factors during infancy and toddlerhood, when morality develops rapidly and thus may be most malleable. This knowledge may identify more precise risk and protective processes underlying emerging CU that may be targeted to prevent a highly impairing psychosocial trajectory. Consistent with NIMH Strategic Objective 2 to “chart mental illness trajectories to determine when, where, and how to intervene,” this K23 application aims to identify neural correlates and environmental, child dispositional, and parenting risk and protective factors for emerging CU behavior across infancy and toddlerhood. To accomplish these aims, this proposal leverages an exceptional opportunity to add measures to an NIH-funded study following a large, high-risk cohort of mother-infant dyads annually from birth through age 3. The applicant will add critical measures to this parent study including observational and parent- report assessments of emerging CU, an event-related potential (ERP) task, and experimenter-child interactions to assess children's dispositions. This proposal will use ERPs to characterize neural markers of CU behavior and examine whether aspects of children's environments (early life adversity) and dispositions (low affiliation) measured in infancy predict maladaptive trajectories of emerging CU through age 3. Further, it will test whether low parenting warmth is implicated in these risk trajectories and could thus be a protective factor targeted in treatment. Findings will inform the developmental neurobiology of emerging CU behavior and elucidate promising early prevention and treatment targets. To execute this proposal, the training plan in this application addresses the applicant's need for training in 1) ERP assessment methods; 2) the developmental psychopathology of CU behavior; and 3) longitudinal design and analysis. A rich training environment and a multidisciplinary team of mentors in each of these areas is detailed. The described research and training activities will enable the candidate to become an independent scientist investigating neural and psychosocial risk for aberrant moral development and its role in the onset and maintenance of early childhood psychopathology.

NIH Research Projects · FY 2025 · 2021-02

Project Summary The overarching goal of this study is to identify new mechanisms that preserve neuronal function with age. As the world's aging population steadily increases, the number of diagnoses for neurodegenerative disease and dementia is projected to more than double within the next 30 years, underscoring our immediate need to understand the cellular and molecular basis of brain aging. Atrophy of the connections that mediate neuronal communication leads to aberrant activity within neural circuits in the aging brain. How changes in activity modify the properties of aging neurons is not yet clear. The brain adapts to neuronal activity in part via the induction of new gene expression programs encoding critical cell-type-specific mediators of circuit plasticity. Whether re-engaging the regulators of these gene programs in aging brains can ameliorate declining neuronal function remains unknown. The bHLH-PAS transcription factor NPAS4 constitutes a major regulator of activity-dependent gene programs in both mice and humans. NPAS4 integrates into the NuA4/TIP60 acetyltransferase protein complex, a transcriptional co-activator and DNA repair complex, which has been linked to learning and memory in invertebrates. Intriguingly, activity-dependent elements targeted by NPAS4 transiently acquire a chromatin mark of DNA damage signaling upon neuronal activation (γH2AX), raising the possibility that NPAS4 may function at these sites to help repair damage resulting from activity-driven transcription. In preliminary data, I discovered that Npas4 knockout mice die prematurely with signs of cell stress in the hippocampus. This study will examine the hypothesis that the newly identified NPAS4:NuA4 complex has evolved a protective role to promote the sustained functionality of neurons by maintaining transcriptional control and genome stability at activity-dependent gene loci. I will examine age-dependent changes to Npas4 regulation and activity- dependent gene induction across neuronal cell types (Aim 1, K99) and identify critical gene targets of this complex in activated neurons (Aim 2, K99). During the R00 phase, I will expand upon these ideas to explore a novel role for this activity-dependent protein complex in the repair of directed DNA damage at enhancers and promoters, and will examine how this directed DNA repair activity changes with age (Aim 3, K99). In the long term, I will leverage the datasets, and new skills in bioinformatics and neurobiology acquired during the K99 training period, to identify new mechanisms and molecules that preserve cell-type-specific function in the nervous system. My ultimate goal is to design targeted strategies to slow or reverse decline in the neuronal subtypes most susceptible to age-dependent diseases.

NIH Research Projects · FY 2026 · 2021-01

Project Abstract The phagocytosis of apoptotic cells or “efferocytosis, represents a fascinating and complex challenge for phagocytes. These specialized cells must simultaneously process the various components of ingested cellular debris, produce anti-inflammatory mediators, and maintain their own homeostasis. Recently, we uncovered new insights suggesting that solute carrier (SLC) proteins within phagocytes play a crucial role in regulating phagocyte homeostasis, thereby influencing inflammatory responses in tissues. During our current project period, we observed a rapid modulation (within just 30 minutes) in the expression of numerous Slc genes and their downstream regulators during efferocytosis. Notably, it revealed an array of metal ion- responsive genes, including a significant number that are regulated by zinc. In Aim1, we will delve into the intriguing relationship between zinc and efferocytic responses, focusing on specific zinc-induced genes and their roles in inflammation and tissue repair. Additionally, we conducted an unbiased CRISPR/Cas9- mediated screen targeting the SLCome and other transporters, leading to the identification of brakes/negative regulators of efferocytosis. The loss of these regulators significantly enhances efferocytosis. In Aim 2, we will closely examine several SLC and ABC transporters to elucidate their functions in macrophages and various disease models. Collectively, we anticipate that these studies will yield groundbreaking insights into macrophage and SLC biology, potentially unveiling novel protein targets for modulating inflammation and advancing therapeutic strategies.