Washington University

NIH Research Projects · FY 2025 · 2021-08

Project Summary Perhaps the defining feature of the eukaryotic cell is its organization into biochemically distinct compartments known as organelles. While the biochemical functions of individual organelles are often well known, how cells regulate the copy numbers, sizes, and subcellular positions of its diverse organelles in a coordinated fashion and how organelles interact to produce integrated physiological outputs remain one of the grand challenges in cell biology. The goal of my research program is to discover the quantitative principles governing how cells regulate systems-level organelle dynamics to coordinate metabolism, growth, and proliferation. To achieve this goal, my research strategy will proceed along two directions. In the first direction, I will quantitatively determine how cells coordinate systems-level organelle dynamics with cellular growth demands. Specifically, I will quantify and build a mathematical model of the relationship between cellular organelle composition and cell growth. The model will be calibrated from data obtained by simultaneously visualizing all major metabolic organelles using our machine learning-based hyperspectral imaging platform, exerting chemical biological control over cell growth and proliferation rates, and genetically perturbing key organelle biogenesis, organization, and interaction factors. In the second direction, I will determine how cells coordinate systems-level organelle dynamics and gene expression to control metabolism during growth and proliferation. I will categorize single cells according to their organelle content and systematically measure the temporal correlations in their expression of genes whose products execute organelle-specific functions. I will concomitantly measure the metabolomic profile of these cells sorted by organelle content. I will then combine these measurements to develop a mathematical model that quantitatively captures the connection between gene expression and metabolism as mediated by the cell's organelle makeup. I will subsequently test predictions of this model by systematically tuning organelle interaction strengths by modulating the expression of organelle biogenesis factors and organelle contact sites. Successful investigations along these two directions will yield mechanistic insight into how to untangle the complex interdependencies between organelle dynamics, metabolism, and cell growth and proliferation. A systems- level understanding of how organelle composition and interactions coordinate metabolism to control cellular growth and development will lay a rigorous foundation into future investigations into how the cell actively shapes its organelle composition to match biochemical supply with physiological demand through, how this plasticity is leveraged in health by multicellular organisms to provide the metabolic flexibility needed to develop its myriad cell types, but also in disease by allowing for multiple routes to metabolic pathologies in cancer, diabetes, and aging.

NIH Research Projects · FY 2025 · 2021-08

CONTE CENTER SUMMARY Neuroactive steroids (NAS) offer novel directions for psychiatric therapeutics. The prototypical NAS is allopregnanolone (AlloP), which under the formulation of brexanolone, has recently been approved by FDA for treatment of women with postpartum depression; a second, orally-active NAS has had a successful Phase 3 study for postpartum depression and a Phase 2B study for men and women with major depression. While AlloP is a potent and effective enhancer of GABAA receptors (GABAARs), it is not presently clear that the antidepressant effects of NAS are mediated solely by GABAergic effects, and recent data indicate that other mechanisms including effects on cellular stress and inflammatory pathways could also be involved. Members of our center have extensive experience studying the medicinal chemistry and mechanisms underlying the effects of AlloP-like NAS. In this Conte Center proposal we will leverage novel NAS compounds to probe varied molecular, synaptic, network and behavioral effects of NAS as clues to their therapeutic mechanisms and potential. Our center proposal is driven by unique NAS analogues synthesized in our Chemistry Core and involves three complementary and intertwined projects that will pursue three specific goals. First, we will test the hypothesis that selective actions of NAS on a class of GABAARs underlies effects on hippocampal electrical activity, network function and behavior in novel mouse lines and mouse models of postpartum and major depression. Second, we will examine the role of non-GABAA ion channels in mediating hippocampal and behavioral effects of NAS focusing on novel NAS that modulate NMDA glutamate receptors and low voltage activated calcium channels. These studies will use unique photoaffinity labeling approaches to elucidate sites of actions of NAS on NMDA receptors and other targets. Other studies will examine effects of NAS analogues on cellular function, neural network function, and behavior. Third, we will test the role of intracellular targets engaged by NAS on hippocampal circuits and behavior, focusing on the roles of neuroinflammation and cellular stress pathways. Our center is uniquely positioned to traverse the exploration of antidepressant NAS from molecules to sites of action and effects on dysfunctional circuits to identify potentially novel agents and targets for psychiatric therapeutic development.

NIH Research Projects · FY 2026 · 2021-08

Most individuals with rapid eye movement (REM) sleep behavior disorder (RBD) develop additional neurological symptoms and are subsequently diagnosed with overt synucleinopathies, including dementia with Lewy bodies (DLB), Parkinson’s disease (PD), and multiple system atrophy (MSA), indicating that RBD represents a prodromal stage of synucleinopathy. RBD therefore offers a window of opportunity to intervene with neuroprotective treatments at the earliest stages of disease when treatment is most likely to be effective. Recognizing the importance of early intervention, key federal agencies focused on neurodegenerative disease have proposed high priority recommendations for prodromal aspects of synucleinopathies, including specifically RBD, to prepare for clinical trials. The North American Prodromal Synucleinopathy (NAPS) Consortium began in 2018 to plan for neuroprotective clinical trials in RBD. The NAPS Consortium, currently at 10 sites, has thus far enrolled 215 participants with polysomnogram-confirmed RBD, and has successfully performed comprehensive and standardized assessments and biofluids collection. The North American Prodromal Synucleinopathy Consortium for RBD, Stage 2 (NAPS2) program represents an integrated expansion of NAPS to support a longitudinal, prospective study of RBD, to address key gaps currently prohibiting neuroprotective clinical trials in RBD. NAPS2 will establish enhanced infrastructure to support long-term research in prodromal synucleinopathies. We will institute 8 Cores—Administrative; Clinical; Biofluid; Neuroimaging; Polysomnogram (PSG); Genetics; Data Management and Statistics (DMS); and Recruitment, Education, and Outreach (REO)—to augment our protocol and to support a Project to predict phenoconversion to overt synucleinopathy. NAPS2 will prospectively assess >300 participants with RBD for comprehensive clinical evaluation and collection of PSG/neurophysiological, biofluid (blood and cerebrospinal fluid), genetic, and neuroimaging (MRI and DaTscan) biomarkers. The overarching goal of NAPS is to enable neuroprotective clinical trials to prevent or delay synucleinopathies. Toward this goal, the NAPS2 aims are: 1) to conduct research on RBD as a prodromal manifestation of DLB, PD, and MSA; 2) to expand our cohort of RBD participants and add matched control participants for longitudinal, standardized collection of clinical, PSG, genetic, biofluid, and neuroimaging data; 3) to analyze collected data against longitudinal clinical outcomes to refine scales and develop biomarkers to optimally design clinical trials; 4) to share data, samples, and methods for use by the scientific community; 5) to interact with NIH, other scientific groups on RBD and overt synucleinopathies, industry partners, patients, and other groups; and 6) to prepare for large-scale clinical trials. Ultimately, synucleinopathy biomarkers and neuroprotective treatments developed in the RBD population could be applied to the larger population at risk for synucleinopathies, to delay or prevent DLB, PD, and MSA.

NIH Research Projects · FY 2025 · 2021-08

ABSTRACT In recent decades, the primary setting for oncology treatment has shifted from the hospital inpatient unit to the outpatient clinic, leaving nearly 4.6 million family caregivers responsible for most of the day-to-day, round-the- clock cancer care provided in the United States. Cancer caregiving often occurs in a highly stressful emotional and social context, leaving caregivers vulnerable to significant, potentially long-lasting, adverse effects. Although palliative care teams are ideally positioned to help family caregivers cope with the numerous stressors they encounter, there is little evidence on which to base cancer caregiver support in the outpatient palliative care clinic. To address the pressing need for research on caregiver interventions in this setting, an interdisciplinary team of investigators will conduct a multisite, randomized trial of problem-solving therapy for family caregivers of individuals with cancer who are receiving outpatient palliative care. Potential barriers and facilitators to the adoption of problem-solving therapy in both rural and urban clinics will also be examined, resulting in a greater understanding of the context in which the therapy will ultimately be delivered if shown to be effective. The study’s specific aims are as follows: 1) determine the effect of problem-solving therapy on psychological distress among family caregivers of patients with cancer receiving outpatient palliative care; 2) measure the effect of problem-solving therapy on positive aspects of caregiving among family caregivers of patients with cancer receiving outpatient palliative care; and 3) identify potential barriers and facilitators to the adoption of problem-solving therapy for family caregivers in outpatient palliative oncology. At the conclusion of the study, investigators will broadly disseminate findings regarding the effectiveness of problem-solving therapy, and they will be well positioned to promote family caregiver wellbeing through the routine and sustained delivery of problem-solving therapy in outpatient palliative cancer care.

NIH Research Projects · FY 2025 · 2021-08

Project Summary Transposable elements (TEs) comprise roughly half of the human genomes, and some TE subfamilies can even contain hundreds of thousands of copies, such as LINE and SINE elements. Highly enriched transcriptional factor binding sites in the TEs sequence enable TEs the huge regulatory potential to the host genome. Mounting evidence suggests some of TEs escaped from epigenetic silencing and actively involved in multiple biological processes of host genome. TEs are significant contributors to the origin of vertebrate long non-coding RNAs, and some TEs are also found to play roles as promoters in early development and some terminally differentiated tissues. Our recent study found that domesticated rodent-specific TEs can play roles as promoters to initiate the tissue-specific transcription of more than 300 genes during mouse tissue differentiation. However, how the domesticated TEs-derived promoters in the human genome to regulate the gene transcription in distinct tissues and cell types, is not clearly characterized. For example, we do not know how many genes can be transcribed by TEs-derived promoters in particular human tissues; we have no idea about the usage of TEs-derived promoters in the different cell types from the same tissue; finally, how domesticated TEs in the human genome created novel tissue-specific expression pattern of conserved protein- coding genes, is still mystified. Thus, in this proposed project, we will focus on investigating the tissue- and cell type-specific gene transcription controlled by the domesticated TEs-derived promoters in the human genome. Leveraging the big data generated by large consortiums, e.g., ENCODE, Roadmap Epigenomics, GTEx, and Human Cell Atlas, we will perform a systematic survey of the usage of domesticated TEs-derived promoters in the human genome. Firstly, we will identify the TEs that were domesticated as promoters of protein-coding genes and non-coding genes in the human genome, and further characterize the tissue-level expression pattern of domesticated TEs-derived transcripts, by using our established transcripts assemble pipeline to analyze the tissue bulk RNA-seq data generated by ENCODE and GTEx. Secondly, we will investigate the cell-type-specific expression pattern of domesticated TEs-derived transcripts, by reconstructing the single-cell RNA-seq data analysis with novel bioinformatics analysis tool. Finally, we will apply comparative-genomics approaches to create the expression matrix of orthologous TEs-derived protein- coding genes across multi-species, and construct the phylogenetic trees to explore the expression pattern changes of TEs-derived protein-coding genes during evolution.

NIH Research Projects · FY 2024 · 2021-08

Tauopathies may occur by familial mechanisms in which mutations in the MAPT gene are dominantly inherited causing frontotemporal lobar degeneration (FTLD-tau) or by sporadic mechanisms in which MAPT haplotypes are associated with increased disease risk (e.g. progressive supranuclear palsy and corticobasal degeneration). MAPT mutations and risk haplotypes have been proposed to drive disease pathogenesis through proteoforms that contain 3-microtubue binding domain repeats (3R tau), 4R tau, or both. However, the mechanisms by which tauopathies occur remains poorly understood. We propose that MAPT mutations drive tau aggregation and neuronal dysfunction through altered proteostasis. In preliminary studies, we have shown that induced pluripotent stem cell derived-neurons expressing MAPT mutations exhibit changes in tau turnover compared to isogenic, control neurons, and we observed differences in the turnover of specific tau proteoforms in mutant neurons. Neurons expressing MAPT mutations exhibit enlarged lysosomal structures and secondary elevation of lysosomal enzymes, markers of lysosomes that are unable to properly degrade their contents. Correction of the mutant allele was sufficient to restore these lysosomal defects. This suggests that altered tau kinetics may be due to defects in the endolysosomal pathway. Thus, a unifying feature by which MAPT mutations drive tauopathy is through disrupted proteostasis. The objective of this study is to extend our preliminary findings to manipulate tau proteoforms using genetic or molecular methods to define the mechanisms by which tau proteoforms disrupt proteostasis in tauopathies. We hypothesize that tau proteoforms are sufficient to destabilize proteostasis and to result in the accumulation of tau in vulnerable brain regions. To test this hypothesis, we will determine the extent to which MAPT mutations cause impaired tau phenotypes and proteostasis characteristic of tauopathy. We will also generate a systematic genetic interaction map to elucidate connections between MAPT mutations, proteostasis, and associated therapeutic targets. Together, this study will reveal novel mechanisms underlying tauopathy that are driven by specific tau proteoforms and whether therapeutics designed to block specific tau proteoforms impact pathologic events.

NIH Research Projects · FY 2025 · 2021-08

Abstract Sepsis is a frequently encountered critical care syndrome leading to 250, 000 deaths annually, making it one of the leading causes of mortality in the U.S. Despite over 75 randomized controlled trials (RCTs), no treatments have shown a survival benefit and currently there are no approved therapies for sepsis. The multitude of negative RCTs has been attributed to the clinical and biological heterogeneity subsumed within the non- specific clinical definition of sepsis. The failed therapies in the context of vast heterogeneity makes sepsis ideal for precision medicine-based approaches where targeted therapies are used in biologically-informed subgroups. Acute respiratory distress syndrome (ARDS) is another critical care syndrome with significant heterogeneity. In secondary analyses of five ARDS RCTs, we consistently identified two phenotypes, the hyperinflammatory and hypoinflammatory phenotypes, with divergent clinical outcomes, biomarker profiles and differential responses to therapies. Crucially, in an RCTs that only recruited sepsis-associated ARDS, the corresponding two phenotypes emerged again. Our preliminary data, in a separate cohort of 587 patients with sepsis, also identified these two phenotypes. In this proposal, we will test the hypothesis that the molecular phenotypes previously identified in ARDS are also evident in sepsis, and that they represent distinct biologic subtypes characterized by differences in circulating inflammatory responses. Using latent class analysis with a composite of clinical and biomarker data, we will seek phenotypes in four independent cohorts of sepsis (>4000 patients). We will develop clinically implementable models to classify the phenotypes using previously described algorithmic pipelines. Heterogeneous treatment effect in the phenotypes will be sought in CLOVERS, a multicenter RCT comparing fluid resuscitation strategies in severe sepsis. In a prospective cohort, we will study novel techniques such as next-generation sequencing, mass cytometry and functional immune responses in stimulated peripheral blood mononuclear cells to better understand the biological and immunological characteristics of the phenotypes and identify phenotype-specific treatable traits. We will also study the phenotypes longitudinally to evaluate their temporal stability. This proposal represents an independent niche of research for my group, which has the requisite experience and expertise to successfully deliver this program. The culmination of the program will potentially lead to several highly impactful discoveries with important implications for sepsis care. We anticipate identifying robust and reproducible phenotypes of sepsis in multiple cohorts, with phenotype-specific response to therapies. We will develop practical models that can identify phenotypes at the bedside. We will comprehensively map the biological and immunological profiles of the phenotypes to identify treatable traits that may enable precision-based approaches in sepsis. Finally, the finding of equivalence of sepsis and ARDS phenotypes would represent a paradigm-changing discovery, leading to a novel classification system of critical illness agnostic of syndromic diagnosis.

NIH Research Projects · FY 2025 · 2021-08

Project Summary Background: Traditional scientific training prepares investigators to conduct scientific research, but does not adequately prepare early-career investigators to lead and manage their own labs. Yet, when early-career researchers launch their independent research laboratories, they are tasked with creating a productive, rigorous, and supportive research environment. Good leadership and management practices can cultivate an effective environment and the success of the lab. Conversely, poor management and leadership can result in high turnover, reduced output, and lapses in scientific rigor and responsible conduct of research. Therefore, education and mentoring is needed to foster leadership and management best practices. Participants and Program Aims: The overall goal of this program, Leadership and Management Practices for Scientists (LAMPS), is to develop a nationally accessible training and mentoring program for early-career faculty researchers launching their independent labs. The program develops essential leadership and management skills and provides practical tools and a personalized Lab Manual. We will apply findings from leadership and management research, utilize novel training and mentoring methods, and engage members of the research community to obtain feedback and encourage their support of LAMPS. The program aims to advance diversity by recruiting and fostering the careers of members of historically underrepresented groups in the biomedical sciences, and by increasing inclusive leadership and mentoring practices among LAMPS scholars. Methods and Evaluation: Over the course of 5 years we will: 1) build an innovative, collaborative digital learning environment to deliver a scientific leadership and management curriculum and scalable mentoring, 2) pilot test and refine the program 3) recruit and provide the program to 100 early-career researchers per year, 4) implement program outreach, dissemination, and sustainability plans, and 5) evaluate all research education program components and track the career development of LAMPS scholars. Team: An outstanding team of experts in industrial-organizational psychology, research ethics, diversity and inclusion, and biology will develop, implement, and evaluate the LAMPS program. Innovation and Impact: The LAMPS program is highly innovative and is grounded in research conducted by our team and effective pedagogical methods. It is informed by relevant biomedical stakeholders and includes a novel approach to peer mentoring for professional development. The novel and scalable digital learning environment will be open to all junior faculty researchers at institutions across the nation. Practical tools related to leadership and management will drive application of best practices in the laboratory, strengthening scholars' ability to conduct impactful research. In turn, LAMPS will create a lasting positive impact in the biomedical research community.

NIH Research Projects · FY 2025 · 2021-08

PROJECT SUMMARY Evidence in adults and youth suggests that sleep disturbances precede and predict the onset of depression and may serve as an important marker of depression. Various types of sleep disturbances, including disruptions of sleep continuity and abnormal sleep architecture, have been identified as markers of depression in adults, but have been virtually unexplored as markers of early childhood depression. Early childhood depression symptoms have become widely recognized as a significant public health concern as they are associated with functional impairments and heightened risk for depression across the lifespan. However, these symptoms often go unreported and underdiagnosed because they are difficult for caregivers and clinicians to identify. Research is needed to identify markers of early childhood depression as early as age 3 years or before, to improve our capacity for the earliest possible identification of young children struggling with, and at risk for, depression. This K23 proposal will examine sleep disturbances as a plausible marker of early childhood depressive symptoms in a prospectively examined sample of young children, oversampled for familial risk for depression. This proposal leverages an exceptional opportunity to add measures to a NIMH-funded study following a large, high-risk cohort of mother-infant dyads annually from birth through age 3. This K23 proposal will add comprehensive measures of sleep (i.e., sleep diaries, actigraphy, and polysomnography) at age 3 years to examine a) which specific features of objectively measured sleep (during both day and night) at age 3 best correlate with early childhood depression symptoms, b) whether misalignments in circadian rhythm – “chronodisruptions” – are associated with early childhood depression symptoms, and c) whether sleep disturbances in infancy and toddlerhood precede the onset of depression symptoms at age 3. This project has the potential to greatly inform our understanding of both the sequalae of sleep disturbances in early childhood, as well as identify a plausible marker of early childhood depression. Completion of the proposed project will provide the applicant with needed training and expertise in 1) the collection and analysis of PSG data in early childhood, 2) the developmental psychopathology of early childhood depression, 3) the quantification of chronodisruption in early childhood, and 4) advanced statistical analysis techniques. 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 the role of sleep disturbances in the development of psychopathology across early childhood.

NIH Research Projects · FY 2025 · 2021-08

ABSTRACT/PROJECT SUMMARY This proposal employs novel methods to identify key determinants and consequences of concurrent HIV infection and regular cannabis use. We will acquire extensive phenotype data from peripheral and brain markers of immune activation, brain structure, and neuropsychological performance (NP) in persons living with HIV (PLWH) receiving combination anti-retroviral therapy (cART) (80 regular cannabis users and 80 non-users) and HIV uninfected (HIV-) controls (80 regular cannabis users and 80 non-users). Our overall hypothesis is that cannabis use leads to increases in inflammation in the peripheral and brain compartments. We also hypothesize that phenotypic signatures due to regular cannabis use and HIV will be delineated through NP and brain volumetrics. In Aim 1 we hypothesize that regular cannabis use will increase both peripheral and brain immune indices in PLWH on cART. In Aim 2 we hypothesize that regular cannabis use will lead to a worsening of NP and reductions in brain volumetrics in both PLWH on cART and HIV- controls. This proposal will provide key insights into the effects of regular cannabis and HIV on peripheral and brain markers of immune function and NP in PLWH and HIV- controls. These insights are critical for cure strategies and ongoing HIV treatment initiatives.

NIH Research Projects · FY 2024 · 2021-07

The long-term goal of this project is to define the function(s) of the cap-specific N6, 2'-O-dimethyladenosine (m6 Am) present at the transcription start site of eukaryotic mRNAs. We and others recently identified the cellular mRNA methyltransferase responsible for methylation of the cap-proximal adenosine at the N6 position as phosphorylated carboxy-terminal domain interacting factor 1 (PCIF1). PCIF1 binds the phosphorylated C- terminal domain of host RNA polymerase II to selectively modify the cap proximal A, following the sequential methylation of the cap-structure by the guanine-N7-methylase and ribose-2'-O methylase. The functional significance of the cap-proximal m6Am modification is uncertain with published literature reaching different conclusions regarding mRNA stability and translation. Viruses that replicate in the cytoplasm such as the negative-strand RNA virus vesicular stomatitis virus (VSV) also contain this cap-proximal m6Am modification on mRNA synthesized in infected cells despite the absence of a viral encoded N6, 2'-O-dimethyltransferase. In preliminary data we have found that PCIF1 is relocalized to the cytoplasm in VSV infected cells and methylates VSV mRNA. The 5 VSV mRNAs are well characterized and we have developed tools necessary to define how m6Am influences the function of each of those mRNAs. Our preliminary data shows that neither mRNA stability nor mRNA translation is impacted by the loss of m6Am, and that in 293T and Hela cells in culture, virus replication is unaffected under basal conditions. Pretreatment of cells with interferon, however, demonstrates that loss of PCIF1 results in the further translational suppression of viral mRNA and a more pronounced reduction in viral growth. This PCIF1 dependent phenotype suggests that one function of this cap-proximal m6Am is to discriminate self from non-self mRNA. The mRNA cap has been hypothesized to have emerged with eukaryotic evolution, when PCIF1 is first detected, to replace the Shine-Dalgarno sequence for directing ribosomes to mRNAs and to protect mRNAs from digestion by 5' exoribonucleases thus providing an early method for distinguishing self- versus foreign mRNAs. It is likely that extant viruses have evolved in the face of this RNA methylation to evade the eukaryotic self-defense system. To further probe the role of PCIF1 modification of viral RNA we generated a PCIF1 -/- mouse providing an additional unique reagent to mechanistically dissect the role of m6Am of viral mRNA in vivo. Capitalizing on this preliminary data we will use genetic, biochemical, cell biological and virological approaches both in cell-culture and in vivo to dissect the role of m6Am and PCIF1 mediated mRNA methylation. Our underlying hypothesis is that PCIF1 modification of mRNA contributes to distinguishing self from non-self mRNA, and that viruses have coopted PCIF1 to ensure efficient replication.

NIH Research Projects · FY 2025 · 2021-07

The gastrointestinal (GI) tract is a large surface lined by a single layer epithelium which is exposed to trillions of microbes and innocuous substances from the diet. The largest collection of immune cells in the body underlies this single layer epithelium and monitors the luminal contents to maintain tolerance to dietary and commensal antigens in the steady-state while retaining the ability to rapidly induce immunity to pathogens during infection. While great progress has been made in elucidating the role(s) of specific immune cell subsets, cytokines, and other factors promoting tolerance or immunity, the processes intrinsic to the gut that enable the immune system to switch from an overwhelmingly tolerogenic tone in the steady-state to inflammatory responses during infection remains a gap in our understanding. Recently, we have uncovered that inhibiting goblet cell associated antigen passages (GAPs) in the small intestine (SI) rapidly shifts the immunologic tone away from tolerance and promotes the rapid induction of inflammatory Th17 responses in the absence of infection or injury. We hypothesize that the inhibition of GAPs is a physiologic response to enteric infection, which in and of itself, promotes the generation of Th17 cells and inflammatory cytokines and shifts the tone of the immune system away from tolerance toward immunity. By studying this process in the absence of enteric infection or injury we can disentangle contributions of the pathogen and injury to the inflammatory response from intrinsic properties of the gut ecosystem promoting the switch from a tolerogenic to pro-inflammatory state. Understanding intrinsic properties of the gut that allows the rapid generation of protective responses could provide new approaches to treat enteric infections and provide insight into the pathogenesis of chronic inflammatory diseases of the gut. We hypothesize that when SI GAPs are inhibited, other pathways take over driving the development and/or expansion of Th17 cells specific for dietary, microbial, and/or self antigens, which shifts the tone of the immune system to provide enhanced protection during enteric infection and/or injury. To explore this hypothesis we propose the following specific aims: In aim 1 we will identify the early events resulting in Th17 expansion following SI GAP inhibition, in aim 2 we will define the origins and specificities of the Th17 cells that expand when SI GAPs are inhibited in aim 3 we will determine if the inhibition of SI GAPs is protective in models of enteric infection and whether inappropriate inhibition of SI GAPs potentiates intestinal inflammatory disease.

NIH Research Projects · FY 2025 · 2021-07

Project Summary/Abstract Urologic Chronic Pelvic Pain Syndrome (UCPPS) is a debilitating condition afflicting 10 million men and women in the U.S. UCPPS encompasses two highly prevalent chronic urologic pain disorders, interstitial cystitis/bladder pain syndrome (IC/BPS) in men and women, and chronic prostatitis/chronic pelvic pain syndrome (CP/CPPS) in men. UCPPS prominently manifests as debilitating symptoms characterized by urinary urgency, frequency, and pain. Unfortunately, the biological mechanisms contributing to UCPPS symptoms remain unclear. This delays diagnosis and limits therapeutic development. Using a mass spectrometry-based metabolomics approach, we recently found that elevated urinary etiocholanolone sulfate (Etio-S) levels identify a high symptom score subgroup of female UCPPS patients. We hypothesize that elevated Etio-S is symptomatic of a more general perturbation of related steroids. We also hypothesize that this represents just one of multiple UCPPS subgroups arising from biochemically distinctive etiologies. In this study, we will use state-of-the-art targeted and untargeted metabolomics approaches to discern UCPPS- associated biochemical signatures in human specimens from the Multidisciplinary Approach to the Study of Chronic Pelvic Pain (MAPP) research network. Rigorous chemical identification will connect these results to specific physiological processes, providing the basis for new diagnostic and therapeutic approaches. Targeted metabolite assays of samples from these studies will be used to identify treatment-responsive subgroups in clinical trials, improving UCPPS patient care. An experienced interdisciplinary research team of physicians, analytical chemists, and mathematical data scientists has been assembled to ensure the rigor and clinical validity of this effort.

NIH Research Projects · FY 2025 · 2021-07

Project Summary Lewy body diseases (LBDs) are highly heterogeneous neurodegenerative disorders including Parkinson's disease (PD), Parkinson's disease dementia (PDD), and dementia with Lewy bodies (DLB). LBDs are characterized by the abnormal aggregation of the protein α-synuclein in neuronal cell bodies (Lewy Body) and neurites which are currently considered to be the common cause of the diseases. Lewy Body deposition starts in the caudal brainstem of PD but in the neocortex of DLB cases. The regional differences of initial α-synuclein deposition correlate with neuronal loss in the corresponding regions - dopamine neurons in the substantia nigra (SN) and neurons of unknown identity in the neocortex, and the unique clinical manifestations with a predominant motor symptom in PD whereas early dementia in DLB. Why some particular neurons and brain regions are affected at the disease onset, whereas the neighboring cells and regions not? This is a fundamental question in the field of neurodegenerative diseases that this proposal will address via novel genomics technologies and bioinformatics tools. In an initial pilot study, using single nucleus RNA-sequencing (snRNA-seq) analyses, we identified a novel disease-associated astrocyte (DAA) subpopulation and demonstrated that DAA contributed to increased inflammation, amyloid pathology, and neurodegenerative disease pathogenesis whereas parenchymal astrocytes had compromised functionality in both AD and PD brains. Additionally, we identified three microglia subpopulations that were similar to but with marker gene expression profiles distinct from the conventional resting (M0), M1, and M2 activated microglia. We observed deficient microglia functionality shared across all microglia subpopulations and uniquely up-regulated inflammatory pathways in PD suggesting common and PD-specific mechanisms of neurodegeneration. These data provide us with an exclusive opportunity to analyze the relationships between these glia subpopulations and selective regional and neuronal vulnerability in different diseases. In Aim 1, we will identify vulnerable neuronal types in the frontal cortex (FC) and SN of patients with PD, PDD, and DLB. In Aim 2, we will test the hypothesis that astrocyte/microglia dysfunction underlies the mechanism of the selective regional vulnerability of LBD. In Aim 3, we will test the hypothesis that dysregulated interactions between neurons and astrocytes/microglia underlie the mechanism of the selective neuronal vulnerability of LBD. Our study will provide deep insights into the molecular mechanisms of selective neuronal and regional vulnerability in LBDs. Besides, our study will provide molecular biomarkers and tools for neuron cell-type-specific protection and targeted astrocyte/microglia subpopulation isolation and manipulation. Furthermore, our study will provide molecular biomarkers for distinguishing PD, PDD, and DLB, which is very important but a considerable challenge today.

NIH Research Projects · FY 2025 · 2021-07

Project Summary This proposal outlines a 5-year research and career development plan designed to support the candidate’s trajectory towards an independent academic career. The proposed research project, which focuses on acute myeloid leukemia (AML)-associated MYC mutations and their key target genes in AML cells, will capitalize on critical expertise and resources available at Washington University School of Medicine. The career development plan and didactic work will provide the candidate with a variety of skills that will enable a successful transition to independence. This proposal is founded on our recent observation that a unique set of recurrent MYC missense mutations are significantly enriched in the dominant clones of normal karyotype AML patients who have very long first remissions (>5 years) with chemotherapy only; in our series, 6/6 MYC mutations co- occurred with NPM1 mutations (NPMc), suggesting a unique form of cooperativity. Based on our preliminary findings, we hypothesize that: 1) AMLs overexpressing WT or mutant MYC proteins are biologically different; 2) each MYC mutation may have unique effects on its transcriptional targets; and 3) MYC mutations may cooperate with NPMc mutations. To address these hypotheses, we propose the following specific aims: Specific Aim 1: We will examine the function of AML-associated MYC mutations alone, or in cooperation with NPMc. We have developed a doxycycline-inducible system that allows us to regulate Myc expression in hematopoietic stem and progenitor cells (HSPCs) that do or do not contain the Npmc mutation. Using this system, we will evaluate how WT or mutant Myc, with or without Npmc, impacts AML development, progression, and AraC responsiveness both in vitro and in vivo. We are also developing a more physiologic, conditional knock-in model of MYCT58N (a recurrent mutation both in AML and B cell malignancies) that will allow us to explore the influence of physiologic doses of mutant Myc and Npmc on AML pathogenesis and AraC sensitivity. Specific Aim 2: We will define the transcriptional outputs and genomic binding sites of WT vs. mutant Myc proteins in AMLs arising in WT vs. Npmc mice. To identify Myc dysregulated genes, we will use scRNA- seq to determine the transcriptional profiles of AMLs (obtained in Aim 1) initiated by WT vs. mutant Myc over- expression, either alone or in combination with Npmc. We will also perform ChIP-seq studies to characterize the genomic binding sites of WT vs. mutant Myc, and perform an integrated analysis of these datasets to define the relative contributions of direct vs. indirect effects of Myc on transcriptional outputs in AML cells. By combining the results of the functional studies (Aim 1) with the expression and DNA binding studies (Aim 2), we hope to better understand how Myc mutations alter transcription in hematopoietic cells, and how Npmc affects this output. If these studies are successful, they may provide important insights on MYC-mediated transformation, refine our current predictive tools for AML risk stratification, identify pathways involved in AraC sensitivity, and allow us to create new approaches for the treatment of AML patients.

NIH Research Projects · FY 2025 · 2021-07

ABSTRACT TGF? is an essential regulator of articular chondrocyte/cartilage homeostasis. However, reduced/absent TGF? receptor (Tgfbr2) expression with aging, joint injury, and in osteoarthritis (OA) prevents the use of TGF?1 as a clinical treatment for OA. Therefore, the goal of this proposal is to identify the key genes, pathways, and potential therapeutic targets that are regulated by TGF?1. Our preliminary data shows that TGF?1 regulates chondrocyte homeostasis and anabolic biosynthesis through stimulation of glucose uptake, glycolysis and anabolic Hexosamine Biosynthetic Pathway (HBP). Specifically, we show that TGF?, via TAK1 signaling, induces the HBP through upregulation of 3 key genes/targets: i) Glut1, the major enzyme involved in glucose uptake; ii) Gfpt2 (glutamine-fructose-6-phosphate amidotransferase-2, the rate limiting enzyme of HBP), and iii) Slc25a1, the key mitochondrial citrate transport protein that provides a source of cytoplasmic Acetyl CoA necessary for production of UDP-GlcNAc. UDP-GlcNAc is the terminal metabolite in the HBP pathway and is required for matrix synthesis of hyaluronic acid and glycosaminoglycans (GAGs). Our mass spectrometry (MS) data establish that TGF?1 enhances the production of UDP-GlcNAc and increases the proportion of carbons in UDP-GlcNAc derived from radiolabeled glucose. Moreover, our RNA-seq data and additional in vitro data identify Igf1 as a critical downstream target of TGF?1 since the induction of glucose metabolism, glycolytic gene expressions, glucose uptake, HBP, and proteoglycan production is abolished in in TGF?1 treated articular chondrocytes with Igf1r gene deletion. In contrast, Igf1 over-expression mimics the effect of TGF?1 on glucose metabolism as well as cartilage anabolism and homeostasis. Collectively, these novel findings indicate the existence of a TGF?/IGF1 signaling axis in chondrocytes, and that modulation of this axis may be a promising therapeutic strategy to treat OA. Two Specific Aims are proposed. Specific Aim 1 will define the upregulation of Hexosamine Biosynthesis Pathway (HBP) as a key mechanism involved in TGF?-mediated homeostasis of articular cartilage. Complementary in vitro and in vivo genetic approaches targeting Tgfbr2, Tak1, Glut1, Gfpt2 and Slc25a1 as well as HPLC-MS will be used to establish regulation of the HBP as an essential anabolic pathway necessary for articular chondrocyte homeostasis. Specific Aim 2 will utilize Igf1r loss-of-function and Igf1 gain-of-function models in vitro and in vivo to establish Igf1 signaling as a downstream effector of TGF? regulation of glucose metabolism and articular cartilage homeostasis. In summary, the proposed studies will define TGF?/IGF1 as a novel pathway axis in regulation of glucose metabolism, HBP, and articular chondrocytes homeostasis in the context of OA. This work will enhance our understanding of mechanisms regulating OA and provide novel targets for innovative therapeutic approaches.

NIH Research Projects · FY 2025 · 2021-07

The long term goal of this study is to understand how severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causal agent of coronavirus disease 2019 (COVID-19), enters cells and how to block that process through the use of therapeutics. Like other enveloped viruses, SARS-CoV-2 cell entry begins with engagement at the cell-surface and is completed on release of the viral contents following membrane fusion. During the process of cell entry, the SARS-CoV-2 spike protein (S) engages the cellular receptor, angiotensin converting enzyme (ACE2). Proteolytic activation of S is required to activate the fusion machinery which can be achieved by cell surface or endosomal proteases positing a model of cell surface and endosomal entry routes that depend on engagement of different host-cell molecules that vary among cell types. To interrogate the entry pathway of SARS-CoV-2 we developed a set of unique tools that permit application of single virion imaging approaches to track productive entry routes in an unbiased way and to help identify host factors coopted during viral entry. This imaging is facilitated by the use of a chimeric vesicular stomatitis virus (VSV) in which its glycoprotein gene (G) was replaced with the spike (S) gene of SARS-CoV-2. Inhibition of VSV-SARS-CoV-2 infection with monoclonal antibodies, soluble receptor and small molecule inhibitors correlates closely with inhibition of a clinical isolate of SARS-CoV-2, corroborating that the chimera is an effective BSL2 surrogate to study SARS-CoV-2 S-mediated entry. This permits us to genetically modify a core protein of the VSV ribonucleoprotein core to render the particles visible by fluorescent microscopy. By combining this imaging approach, with genetic, chemical and biological perturbations, we will map the entry routes of VSV-SARS-CoV-2 and then examine the effect of those perturbations on infection of cells with a clinical isolate of SARS-CoV-2. We will use this approach to determine how countermeasures currently in clinical trials including monoclonal antibodies, soluble ACE2, and two small molecule inhibitors apilimod and nafamostat block entry. Using genome-wide loss-of-function screens we will also interrogate the requirements for entry of SARS-CoV-2, under native and perturbed conditions to uncover new host proteins that are coopted during entry as potential additional targets for therapeutic intervention. Successful completion of this work will define the entry pathways that lead to productive SARS-CoV-2 infection, inform the mechanism by which multiple molecules in clinical development interfere with that process and unearth new host factors that are coopted during the entry pathway.

NIH Research Projects · FY 2025 · 2021-07

PROJECT SUMMARY Accurate assessment of risk is a top priority in oncology due to the population burden of cancer. Breast cancer is the leading cancer diagnosis among women worldwide and accordingly has the longest and broadest focus on risk prediction. Most traditional prediction models only utilize baseline factors known to be associated with breast cancer risk. The intrinsic heterogeneity between and within patients over time are reflected in part, by the time-varying covariate trajectories, which may provide important information for the prediction of breast cancer risk. The accumulation of cancer risk over life, well documented for breast cancer, is ideally suited to methods that incorporate time-varying covariates. The objective of this proposal is to provide novel statistical models that can incorporate patient heterogeneity in a personalized, dynamic manner leading to a more accurate risk prediction scheme. The proposed algorithms encompass innovative functional approaches to comprehensively characterize the changing pattern of the longitudinal trajectories by a set of outcome- independent/unsupervised and outcome-dependent/supervised features. The set of individual-specific features will contain information on the observed time-varying ‘pattern’ rather than one-time exposure in existing methods, leading to a higher predictive power. The dynamic prediction models will be built in a stepwise fashion, starting with a single time-varying covariate, and extended to the multivariate settings, to accommodate multiple time-varying covariates. In addition to contributions in prediction, the temporal change/trajectory of risk factors can add insights to pathways operating on risk of breast cancer. To develop viable preventive strategies, understanding the causal mechanisms whereby an exposure affects such dynamic trajectories (or mediators), to then in turn produce the breast cancer outcome is crucial, as these will provide insights into pathways that can better target breast cancer prevention and intervention trials. Given our ability to characterize the dynamic trajectories, we are positioned to fill in this gap and assess their intermediate role on the breast cancer pathway. In the two-year extension, we propose to develop a computationally efficient causal mediation framework to quantify the extent to which the effect of risk factors on breast cancer risk is mediated through BMI, hormone type and duration, mammographic density, and the whole mammogram image trajectory and the extent it is through other pathways. Successful completion of the proposed project will provide a transparent, robust, and reproducible statistical basis for inferences with the potential to shift the current paradigm leading to new pathways that can be targeted in breast cancer prevention and intervention trials to expand capacity for precision prevention.

NIH Research Projects · FY 2025 · 2021-07

This proposal, from the Neuroscience Program in Washington University’s Division of Biology and Biomedical Sciences (DBBS), is a new application to the Jointly Sponsored Ruth L. Kirschstein National Research Service Award Institutional Predoctoral Training Program in the Neurosciences. The overarching goals of our Neuroscience program are to equip our trainees with a firm foundation in nervous system function and dysfunction, the ability to identify problems and design strategies to address them critically and rigorously, and the skills required to perform, present, and mentor others in research. The strengths of our current training program include a strong and evolving curriculum to address critical areas of modern neuroscience and the skills necessary for success in any neuroscience career, a focus on improving diversity of students in neuroscience and retaining diverse students in the program, a collegial and collaborative atmosphere, broad institutional support, multiple neuroscience-related opportunities for community outreach and teaching and a supportive administrative structure that facilitates all aspects of the educational process, from recruitment of students to thesis defense and beyond. This proposal builds on these features with ongoing and future initiatives aimed at improving quantitative, experimental and statistical thinking, facilitating interdisciplinary and/or advanced training in areas relevant to a student’s research, modernizing curriculum delivery, providing evidence-based ethics training to address well-publicized problems of rigor and reproducibility, and assessing the impact of these initiatives and modifying their implementation as needed. We are requesting 11 slots for students in their 1st and 2nd years. Students will emerge from this program with a stronger foundation in experimental and statistical thinking, ethics and methods to improve rigor and reproducibility. Faculty in the program will also benefit from exposure to emerging methods and approaches in these areas.

NIH Research Projects · FY 2025 · 2021-07

Enter the text here that is the new abstract information for your application. This section must be no longer than 30 lines of text. The mission of the Cell and Molecular Biology (CMB) program at Washington University in St. Louis is to provide rigorous interdisciplinary training to PhD students in four related disciplines in order to prepare them for successful careers in the biomedical workforce. It will provide students with the fundamental concepts and methods of cellular and molecular biology, quantitative training, critical thinking and communication skills, and other core competencies. The goal of our renowned and committed faculty is to provide students with a firm foundation in rigorous research design via an approach that includes high- quality mentoring and fostering career development in a vibrant student-focused research environment. The objective of the CMB training program is to enable our students to pursue careers at the vanguard of scientific research and education by helping them establish a broad scientific knowledge and professional skills, rich career preparation guidance and resources, and an interdisciplinary network of colleagues. The CMB training program will support 25 trainees for 2 years. Half of the trainees will be in year 2 and the other half will be in year 3 of the program. These are pivotal years in the training path towards successful independent research. The CMB program is highly integrated and will have a profound impact on the training of more than just our training grant-supported students. Measurable outcomes include PhD completion rates, time-to-degree, program reviews, longitudinal tracking of student development and self-efficacy in core competencies. Most importantly, the pinnacle of success of the CMB training program is the ability of our students to achieve their career ambitions in the biomedical workforce. This proposal builds on our successes by introducing both proven and innovative educational features including five new initiatives: 1) Updated curriculum, including a novel skills competency course; 2) Improved mentor training processes; 3) improved admissions and trainee appointment processes; 4) Enhanced training in rigor and reproducibility in core courses and thesis committee meetings; 5) Earlier and more directed focus on career exploration and experiences. The CMB program employs a continuous improvement process informed by internal and external reviewers and evidence-based assessments to develop and implement effective new training methods.

NIH Research Projects · FY 2025 · 2021-07

Project Summary Autism Spectrum Disorder (ASD) is a neurodevelopmental disorder that impacts approximately 1.5% of children in the United States. Individuals with ASD experience deficits in social communication or restricted interests and repetitive behavior; but the severity and patterns vary greatly and convey lifelong impairment for some. It is unclear how the presentation of ASD changes from early childhood into adolescence or adulthood. The causes of ASD are also unknown, though substantial evidence supports the contribution of both genes and environmental factors. These gaps in knowledge exist because US studies to date have lacked the sample size, depth of data collection, or appropriate life course timing to address these questions. The Study to Explore Early Development (SEED) is now able to address these prior limitations. SEED is a large case- control study of children ages 2-5 years and their families, implemented across eight states over three phases. SEED collected detailed data on children's core ASD symptoms, cognitive status, and presence of co- occurring conditions in early childhood, along with extensive risk factors related to maternal health and the perinatal environment as well as genomics. The SEED sample includes 2044 children with ASD, 1950 children with non-ASD developmental disabilities (DD), and 2285 population control children (POP), making this the largest etiologic study of ASD in the US. Recent ancillary studies - the SEED Teen Pilot and SEED COVID studies -- will soon add data on adolescent health and the consequences of the pandemic, respectively, for some SEED participants. The work proposed here, SEED Follow-up Studies (SEED FU), will maximize the impact of extant SEED data through analyses that characterize ASD phenotypes and assess the potential interplay between genetic and modifiable risk factors. SEED FU will also facilitate new data collection in middle childhood, adolescence and early adulthood to characterize changes in ASD phenotype across developmental stages, and the associated health, educational, and service needs across the early life course. These data will further enable prospective analyses of associations between early life factors and later childhood through early adulthood outcomes. Studying risk factors in relation to life course phenotypic subgroups may also help elucidate etiologies previously masked in ASD case-control studies. The MO SEED Team in combination with the SEED Network's collaborative infrastructure and extensive extant data resources, will ensure the successful implementation of the SEED FU Study in Missouri and contribute to success across the network. SEED is well-powered for making significant contributions to our understanding of the complex autism phenotype and identifying factors associated with ASD risk in the population. The knowledge gained by SEED FU will greatly advance our ability prevent adverse developmental outcomes and to support individuals with ASD and their families to ensure optimal wellbeing through early adulthood.

NIH Research Projects · FY 2025 · 2021-07

Project Summary/Abstract G protein-coupled receptors (GPCRs) are the largest class of receptors in the human genome and can signal through multiple transducers, including heterotrimeric G protein and β-arrestins. Opioid receptors are GPCRs whose role in pain sensation has made them primary drug targets for pain medications such as oxycodone and morphine. However, opioids have exceptionally high abuse potential and often cause fatal side effects such as respiratory arrest and death. The magnitude of these problems has led to a search for opioid alternatives to treat pain and related conditions. Activation of opioid receptors activates downstream effectors, including multiple G proteins (Gi1, Gi2, Gi3, GoA, GoB, Gz, and Ggustducin) and β-arrestins (β-arrestin1 and β-arrestin2). A major gap is an incomplete understanding of how opioid receptors recognize different transducers and the functional effects of signaling through each pathway. Kappa opioid receptor (KOR) has displayed promising therapeutic potential because of its novel analgesic activity –drugs that target KOR do not lead to addiction or cause death due to overdose as observed from mu opioid receptor agonists. Selective KOR antagonists have also been pursued in clinical trials for the treatment of addiction and depression. The research in my lab is driven by the overall hypothesis that large-scale structural determination studies of receptor-ligand/transducer complexes will provide molecular-level insights into opioid receptor signaling, and facilitate the design and optimization of novel ligand scaffolds that could be further developed into new drugs with desired behavior profile. By combining X- ray crystallography, Cryo-EM, and molecular pharmacology, we will elucidate fundamental mechanisms of KOR ligand selectivity, receptor activation and signaling. To do so, we will pursue the following main directions: (i) identify structural determinants of ligand selectivity between different opioid receptors types, (ii) identify the molecular basis for different G protein subtypes recognition, and (iii) identify structural features responsible for arrestin-bound activating states. The long-term goal is to develop receptor-specific and pathway-selective probes using structure-based drug discovery and study the function of individual opioid receptor signaling in vivo.

NIH Research Projects · FY 2025 · 2021-07

Project Summary Alphaviruses are mosquito-transmitted, positive-strand enveloped RNA viruses of the Togaviridae family that cause global disease in humans. At present, no antiviral agents or licensed vaccines exist for the treatment or prevention of any alphavirus infections. We recently used a genome-wide CRISPR/Cas9-based screen to identify the cell surface molecule LDLRAD3 as a novel, highly conserved entry receptor for Venezuelan equine encephalitis virus (VEEV), an emerging pathogen capable of causing fatal neuroinvasive disease in humans and other vertebrate animals. Gene editing of mouse or human LDLRAD3 resulted in reduced VEEV infection of neuronal cells, and reciprocally, ectopic expression of LDLRAD3 resulted in increased infection. LDLRAD3 bound directly to VEEV virions and enhanced virus attachment and internalization into cells. Genetic studies indicated that domain 1 (D1) of LDLRAD3 is necessary and sufficient to support VEEV infection. We hypothesize that engagement of LDLRAD3 by VEEV will explain how infection, tissue targeting, and disease pathogenesis occurs. The primary goals of this collaborative, interactive project between the Diamond, Fremont, and Whelan laboratories are to define the precise mechanism(s) by which LDLRAD3 facilitates alphavirus entry into cells, to gain high-resolution structural insight as to how LDLRAD3 engages the spike proteins on the virion, and to determine the cell-type specific role of LDLRAD3 in VEEV pathogenesis in vivo. The experiments in this proposal will define fundamental aspects of VEEV biology that enhance our understanding of infection and cell tropism. This information may facilitate the development of small molecules or biologicals that disrupt LDLRAD3 interaction with VEEV spike proteins, which could form the basis of future therapeutics that ameliorate disease of this emerging and highly pathogenic alphavirus.

NIH Research Projects · FY 2025 · 2021-07

The spatial specificity, temporal dynamics, and context dependence of neuromodulator-induced intracellular signals are essential to explain neuromodulator function. However, although the identity of many signaling molecules downstream of neuromodulator receptors are known, the nature and functions of these features are poorly understood. The long-term goal is to uncover the cellular and subcellular specificity, the temporal dynamics, and the context-dependence of neuromodulator-induced intracellular signals. The overall objective here is to determine the features and synaptic functions of acetylcholine (ACh)-mediated protein kinase A (PKA) activity in the hippocampus. The central hypothesis is that ACh regulates PKA with spatial, temporal, and context-dependent specificity that is essential to synaptic plasticity. The rationale for this project came from multiples lines of evidence. First, Gαq-coupled muscarinic ACh receptors (mAChRs) elevate PKA activity. Second, PKA activity demonstrates rich spatial, temporal, and context-dependent features. Third, perturbations of the spread and duration of PKA activity alter cellular and behavior functions, illustrating the importance of its spatiotemporal dynamics. Finally, mAChRs and PKA are both powerful regulators of synaptic plasticity. The central hypothesis will be tested in both acute hippocampal slices and head-fixed mice, with three specific aims to determine the subcellular compartments (Aim 1), the temporal dynamics (Aim 2), and the context- dependence (Aim 3) of PKA activation by ACh and the roles of these features for synaptic plasticity. To determine the features of mAChR-mediated PKA activity, optogenetics will be used to induce ACh release, and ACh level and PKA activity will be measured with novel biosensors and two-photon fluorescence lifetime imaging microscopy (2pFLIM). To determine the contribution of these features to synaptic plasticity, subcellular compartment-targeted, light-activated actuators will be used to perturb PKA activity with spatial and temporal precision, and electrophysiology will be used to measure synaptic transmission. The proposed research is innovative because conceptually, it goes beyond the identity of molecules to revealing their actions, goes beyond static snapshots to revealing signaling dynamics, and goes beyond knowing the involvement of a signal to revealing their contributions. Methodologically, the research employs cutting-edge technology to induce neuromodulator release, and to measure and perturb intracellular signals with spatial and temporal precision – these approaches will find widespread application in cellular signaling beyond neuromodulator research. The proposed research is significant because it will offer explanatory power on how features, and not just identity of intracellular signals, shape cellular physiology and behavior. These results will reveal new principles of neuromodulator action, and provide insights into how molecular mechanisms general behaviorally relevant features. In the long run, these results will help design better therapies that target the relevant features in neurological and psychiatric disorders.

NIH Research Projects · FY 2025 · 2021-07

The long-term goal of the proposed work is to determine mechanisms that control gene regulatory networks to generate a normal endowment of mature nephrons. Formation of the proper complement of nephrons requires a balance between self-renewal and differentiation of progenitor cells. Disruption of this balance leads to renal hypoplasia and chronic kidney disease. Soon after progenitor cells begin to differentiate, chromatin must undergo significant changes to set up gene expression patterns for formation of distinct cell types along the nephron. Defects in formation of specific renal epithelial cell types leads to glomerular disease and an inability of tubules to perform normal physiological functions, such as maintaining salt and water balance. A major mechanism by which cells respond to signals to direct gene expression to a particular fate or lineage is through the concerted action of chromatin remodeling complexes and tissue restricted transcription factors. These proteins act on regulatory regions in the genome to establish control of gene expression at the correct time and place. While individual factors that control gene expression in nephron progenitor cells have been defined, how these proteins cooperate to control gene expression in the developing kidney has not been explored. The identification and characterization of the regulatory elements where these factors act is also a major challenge in the field. We discovered that Sall1 plays a pivotal role in these developmental decisions by interacting with two distinct chromatin remodeling complexes, the Nucleosome Remodeling and Deacetylase (NuRD) complex and the Switch/Sucrose Non-Fermentable (SWI/SNF) complex. We propose that the integrated actions of Sall1, Six2, NuRD and SWI/SNF controls nephron progenitor cell gene expression to attain a dynamic balance between self-renewal and differentiation, and establishes the epigenetic modifications required for formation of specific cell lineages in the mature kidney. This novel paradigm will be tested in three Aims. In Aim 1, we will perform Hi-ChIP to identify enhancer-promoter contacts in uncommitted and induced progenitors, and define changes in mutant cells. Aim 2 will determine how genomic binding of Sall1, Six2, NuRD and SWI/SNF are affected in mutant nephron progenitor cells. Epigenomic editing will be used to demonstrate causal relationships between gene expression and binding of these factors at regulatory regions. In order to translate our findings to human development and disease, in Aim 3 we will test our findings in the mouse from Aims 1 and 2 in human iPS cell-derived nephron progenitor cells. These studies will illuminate mechanisms that underlie human genetic syndromes and sporadic birth defects, such as renal hypoplasia, which commonly cause childhood kidney failure. Our findings will also provide insight into how to reprogram kidney cells from a differentiated state back to a progenitor state, to promote regeneration of mature tubular epithelial cells in order to correct nephron deficits.