Duke University

NIH Research Projects · FY 2025 · 2023-09

ABSTRACT The U.S. is facing a biomedical workforce crisis, starting with attrition from STEM majors in undergraduate training. The result is a lack of innovative solutions to advance health outcomes. Cancer remains a significant cause of morbidity and mortality. The prevalence of cancer diagnoses and survivors in the U.S. was more than 19 million in 2020, with 9.9 million cancer-related deaths worldwide. Duke University is uniquely positioned to address this critical problem by building a robust pipeline of future academic cancer researchers. The Duke Cancer Institute (DCI) is nationally recognized for its research programs in the areas of cancer biology; cancer prevention and control; cancer risk detection and interception; immune- oncology; neuro-oncology; precision cancer medicine and investigational therapeutics; and radiation oncology and imaging. Our recently established Office of Physician Scientist Development (OPSD) offers a sustainable structure for mentorship, professional development, and research funding. By leveraging these institutional structures, we are well positioned to support the development, implementation, and evaluation of a program that links resources across the training spectrum to introduce undergraduate students to varied cancer research career opportunities. To address the cancer research workforce gap, we propose the Duke Preparing Research scholars In bioMEdical sciences (PRIME): Cancer Research Program, which will recruit cohorts of undergraduate students and provide them with research skills-building and mentored research experiences in cancer research. We will provide evidence-based professional development activities and sustained engagement through virtual platforms and the opportunity for program alumni to return in peer-to-peer mentor roles. The Duke PRIME: Cancer Research Program will achieve our long-term goal, addressing barriers that limit the recruitment and retention of a cancer research workforce through early exposure to intensive research experiences, an innovative didactics curriculum, and peer networking. The program focuses on 1) providing a mentored summer research intensive experience for undergraduates; 2) providing a parallel OPSD PRIME Academy enrichment curriculum to enhance understanding of FDQFHU research concepts and career paths in research; and 3) developing a peer networking model with sustained engagement throughout the academic year. Successful implementation of the proposed program will lead to a scalable model for development of a robust pipeline for a diverse biomedical research workforce across disciplines.

NIH Research Projects · FY 2025 · 2023-09

Leptomeningeal (LM) metastases occur in a wide variety of hematologic and solid malignancies, including leukemia, breast cancer (BC), and lung cancer. When LM metastases arise, they are almost always rapidly fatal, causing severe neurologic symptoms and death within weeks to months. The molecular mechanisms that enable LM metastasis have been poorly understood, and there are currently few targeted interventions to prevent or treat this deadly disease complication. Our lab recently made the seminal discovery of a direct cell trafficking pathway between the vertebral and calvarial bone marrow (BM) and the adjacent CNS LM. We initially demonstrated this pathway in acute lymphoblastic leukemia (ALL) mouse models, showing that ALL cells invade the central LM by migrating along the abluminal surface of emissary blood vessels that bridge the vertebral and calvarial BM and subarachnoid spaces. These emissary blood vessels, whose basement membrane is highly enriched in the extracellular matrix molecule laminin, pass from the BM through apertures in the vertebral or calvarial bone to enter the LM. ALL cells crawl along the outside of this emissary vasculature by binding laminin via cell surface integrin α6 laminin receptors, circumventing the blood brain barrier (BBB) to efficiently metastasize to LM by this perivascular route. Subsequent work has shown that this direct cell trafficking pathway between BM and LM is also used by immune cells to rapidly respond to CNS inflammation, although whether this pathway is important for tumor-immune responses is unknown. It is also unknown whether continued tumor integrin α6 interactions within the LM membranes, which highly express laminin, are important to sustain tumor growth in the LM microenvironment. Our new data in mouse breast cancer (BC) LMD models show that solid tumors can enter the LM through this novel BM-to-meninges perivascular migration pathway and suggest that the high affinity laminin receptor, α6 integrin, is a critical target to prevent breast cancer LMD. These data also demonstrate a crucial role for perivascular macrophages in promoting BC LMD. Our proposal aims to further our understanding of the interplay between laminin-rich emissary vessels, meninges, tumor cells, and immune cells in LM metastasis, in order to expose novel approaches to augment therapeutic responses in the “sanctuary” of the LM. Through cutting-edge spatial transcriptomic analyses and real-time in vivo imaging approaches, our work will also create an unprecedented understanding of the tumor microenvironment of the LM niche, and how this laminin-rich environment contributes to disease survival and proliferation. Finally, we will seek to translate these discoveries into clinical practice through an understanding of how integrin α6 blockade can be used to prevent and treat LMD in preclinical models of BC LM metastasis. Our approach represents a shift in the treatment paradigm for LMD, away from minimally effective cytotoxic therapies toward molecularly targeted exploitation of microenvironment-based vulnerabilities.

NIH Research Projects · FY 2025 · 2023-09

PROJECT SUMMARY/ABSTRACT We propose to develop fully autonomous “self-driving laboratories” to rapidly engineer enzymes for broad applications in biomedicine and biocatalysis. Our approach mimics the methodology of a protein engineering researcher with an AI layer that builds an understanding of protein sequence-structure-function and plans experiments to test specific protein design hypotheses, and a robotic system that experimentally tests designed proteins by synthesizing genes, expressing proteins, and performing biochemical measurements of enzyme activity. Seamless integration between the intelligent agent and experimental automation enables fully autonomous design-test-learn cycles to understand and optimize the sequence-function landscape. Self- driving laboratories will revolutionize the fields of biomolecular engineering and synthetic biology by automating highly inefficient, time consuming, and laborious protein engineering campaigns, enabling rapid turnaround, and allowing researchers to focus efforts on important downstream applications.

NIH Research Projects · FY 2025 · 2023-09

Project Summary / Abstract Bronchopulmonary dysplasia (BPD) is the most common respiratory sequelae of prematurity and is often fatal. Montelukast is FDA-approved and already shows promise in small neonatal trials. Because of limited pharmacokinetic (PK)/pharmacodynamics (PD) research, the optimal montelukast dose for premature infants to avoid BPD is unknown. We propose the Pharmacokinetic and pharmacodynamic study of montelukast in infants with developing BPD (PRISM) trial, a dose-escalating open-label pharmacokinetic, safety and preliminary efficacy trial in premature infants (born < 29 weeks’ gestation) at high risk for BPD (requiring positive pressure ventilation between 29-33 weeks). Our central premise is that re-purposing a non-steroid anti-inflammatory agent will be safe and show preliminary efficacy in preventing BPD. Our objective is to perform population-specific PK/PD analyses under FDA regulatory guidance to identify the optimal montelukast dose to use in a definitive phase III trial. The trial will begin with a low-dose (0.75 mg/kg/day) cohort, and will proceed sequentially to the medium- (1.5 mg/kg/day) and high-dose (2.25 mg/kg/day) cohorts after safety reviews. Dosing will last 4 weeks involving 90 infants (30/cohort) at up to 8 experienced neonatal sites. A comparator cohort of placebo-treated NICHD-trial infants with similar age and disease severity will serve as historical controls. The primary outcome will be the drug clearance (CL/F), which is the key determinant of maintenance dosing. Secondary outcomes will include safety (determined by total adverse events) and exposure-response relationships between montelukast and change in BPD risk over the treatment period. Our first aim will be to characterize the PK of montelukast in premature infants. We hypothesize that the clearance in the infants ≤ 28 weeks post-menstrual age (PMA) (i.e. PMA = gestational age + postnatal age) will be at least 30% reduced compared to infants > 28 weeks. Our second aim is to characterize the safety profile of montelukast in premature infants; we hypothesize that the montelukast adverse event rate will not increase with montelukast exposure. Lastly, we aim to determine preliminary efficacy of montelukast in premature infants; we hypothesize that the change in moderate-severe BPD risk calculated from a population PK/PD linear regression model will decrease by 30% with increasing montelukast AUC0-24.The PRISM PK/PD results will be submitted to the FDA, and will help determine the optimal dose of montelukast to reduce BPD. The impact of the PRISM trial could be monumental in the field of neonatology.

NIH Research Projects · FY 2025 · 2023-09

ABSTRACT Head and neck squamous cell carcinoma (HNSCC) represents the sixth most common cancer worldwide. The Global Cancer Observatory predicts a 30% increase in annual incidence by 2030 with approximately 1.08 million new cases/year. Radiation therapy (RT) plays an integral role in treating HNSCC; however, head and neck RT is associated with significant toxicity in the oral mucosa. This toxicity, termed radiation-induced oral mucositis (RIOM), can lead to opioid use, reduced oral intake/poor nutritional status, and the need for treatment breaks, all of which are correlated with worse outcomes for patients with HNSCC. As current treatment options for RIOM are limited, there is an unmet need to develop novel radioprotectors that will widen the therapeutic window of head and neck RT. Our long-term goal aims to develop novel therapeutic strategies that will prevent or reduce RIOM without sacrificing tumor control. The overall objective of this application is to define the role of p53 in regulating RIOM. The p53 gene is mutated in >80% of human papillomavirus negative HNSCC, yet it remains wild type in adjacent normal tissues. The p53 protein plays a critical role in regulating various cellular responses to stress such as cell death, cell survival, metabolic adaptation, and maintenance of genomic integrity. In normal oral epithelium, RT markedly increases the level of p53 protein as well as its transcriptional target and negative regulator Mdm2. However, how p53 affects damage and recovery of the oral epithelium following irradiation remains poorly understood. Based on our preliminary data generated from p53 knockout mice, we hypothesize that the response of p53 to acute DNA damage plays a crucial role in promoting the regeneration of oral epithelium following severe radiation injury. Therefore, treatment with Mdm2 inhibitors that enhance p53-dependent signaling specifically in cells harboring a functional p53 protein before and during RT will ameliorate acute injury of p53 wild-type oral epithelium without decreasing the therapeutic response of p53 mutant HNSCC. We will test this hypothesis through both loss-of-function and gain-of-function approaches to modulate the response of p53 to radiation using genetically engineered mouse models and small molecule Mdm2 inhibitors. The impact of Mdm2 inhibition on RIOM will be evaluated in normal and tumor-bearing mice. Successful completion of the proposed study will provide mechanistic insights into the crucial role of p53 in promoting the regeneration of oral epithelium following acute radiation injury. Our findings will provide a proof-of-concept to support clinical development of Mdm2 inhibitors as radioprotectors for RIOM to widen the therapeutic window of RT for treating p53 mutant HNSCC.

NIH Research Projects · FY 2024 · 2023-09

Project Summary/Abstract Temporal lobe epilepsy (TLE) is a common and commonly devastating form of human epilepsy that lacks preventive or disease modifying therapy. An estimated 30% suffer recurrent seizures despite symptomatic treatment with anticonvulsants. One cause of TLE is status epilepticus (SE). Defining the molecular mechanisms by which SE induces TLE promises to identify molecular targets for therapies. We therefore conducted extensive target validation experiments which revealed TrkB-PLCγ1 as a druggable molecular target that can prevent TLE in adult mice. The goal of our drug discovery program is to develop small molecule inhibitors for TrkB-PLCγ1 signaling to treat TLE. With the support of Blueprint Neurotherapeutics Network (BPN), we identified multiple compounds within distinct series with demonstrated in vivo efficacy for inhibition of TrkB-PLCγ1 signaling in mouse brain. Our Specific Aims are to benchmark, expand, and optimize novel small molecule inhibitors of TrkB-PLCγ1 signaling. Successful completion of the work proposed will identify a leading series for entry to the Discovery Phase of BPN program.

NIH Research Projects · FY 2024 · 2023-09

Abstract Brain function is based on neural systems that comprise a combination of long-range recurrent connections among many brain regions and local circuits to perform specific computations within each region. Our goal is to understand how a full neural system mediates specific brain functions. We address this general question through investigation of a specific visual-motor behavior in non-human primates: visual guidance of smooth pursuit eye movements. The neural system for pursuit includes extrastriate visual area MT, the smooth eye movement region of the frontal eye fields (FEFSEM), the dorsolateral pontine nucleus (DLPN) and nucleus reticularis tegmenti pontis (NRTP) in the brainstem, and the floccular complex of the cerebellum. The major deliverable of this project is an understanding of the neural system for smooth pursuit eye movements in terms of the features of a canonical visual-motor/sensory-motor circuit. Our first aim will ask how signals are transformed in the cortico-cortical pathways between MT and FEFSEM. We will record multiple signal units simultaneously in both structures while varying the degree of correlation in small patches of moving dots to control the reliability of visual motion signals. In addition to asking how signals are transformed between the two areas, we will use noise correlations between simultaneously recorded neurons in MT and FEFSEM to constrain the architecture of their interconnections. Our second aim will reveal the nature of the visual-motor transformation in a cortico-ponto-cerebellar pathway from MT and FEFSEM to the floccular complex of the cerebellum. We will complement existing data from MT and the pons by recording in FEFSEM and the floccular complex under conditions that will complete our understanding of the representation and processing of visual- motor gain and expectations of target speed. Our third aim will explore recurrent connections from the floccular complex of the cerebellum to the motor cortex for pursuit, FEFSEM. We will stimulate in the floccular complex during both fixation and steady-state tracking while recording from neurons in FEFSEM. The proposed experiments will extend our knowledge of the operation of the sensory-motor circuit for pursuit, place it in the context of the architecture of a canonical visual-motor/sensory-motor system, and reveal what transformations occur in local circuits versus in long-range connections between nodes of the full system.

NIH Research Projects · FY 2025 · 2023-09

Summary Cellular mRNAs are susceptible to a variety of chemical modifications that play important roles in regulating gene expression and cellular function. The most abundant internal mRNA modification is m6A, which occurs when adenosine residues become methylated. m6A influences nearly every aspect of the mRNA life cycle and is critical for a variety of physiological processes, such as gametogenesis, neurodevelopment, learning and memory, immune regulation, and stem cell proliferation and differentiation. Additionally, m6A has emerged as an important RNA regulatory mechanism during cancer progression: altered levels of m6A readers, writers, and erasers are observed in several human cancers, and methylation of oncogenes and tumor suppressors has been shown to impact their expression and promote cancer development. Thus, there is a great need both for understanding how m6A is regulated in cells as well as for developing drugs that target the m6A methyltransferase machinery. However, a major limitation has been the lack of simple, cost-effective methods for detecting changes in m6A methylation in cells in a manner that is compatible with high-throughput screening (HTS). Here, we overcome this barrier by developing a novel genetically encoded m6A sensor which provides a simple fluorescent readout for m6A methylation in living cells. Aim 1 will optimize the m6A sensor system and generate cellular tools to facilitate using the system for diverse applications. Aim 2 will develop the m6A sensor into a HTS-compatible system and demonstrate its utility for drug discovery efforts by performing a pilot HTS designed to identify m6A methyltransferase inhibitors. Aim 3 will create molecular tools incorporating the system and measure the performance of the m6A sensor in vivo. Altogether, these studies will provide a much-needed tool for detecting m6A dynamics in cells and will develop an optimized system for HTS-based studies of m6A methylation. Our technology is likely to have an immediate impact both for basic investigations of m6A biology in cancer as well as drug discovery efforts aimed at identifying novel m6A methyltransferase inhibitors.

NIH Research Projects · FY 2025 · 2023-09

ABSTRACT. Infertility is a problem that affects one out of five couples, yet the cause of sterility is unknown in 30% of cases. One threat to viable gamete development is activity of a subset of transposons known as endogenous retroviruses (ERVs). ERVs have the ability to self-replicate and sometimes mobilize, creating new genomic insertions within the same cell or even neighboring cells. Previous studies have demonstrated that, in Drosophila, ERV activation within the follicle cells of developing eggs causes sterility. The current explanation for ERV-triggered sterility postulates that ERVs migrate from the follicle cells into the oocyte and insert themselves into new genomic loci, damaging DNA integrity. However, our lab has found no evidence of novel ERV insertions being made into the oocyte genome, suggesting sterility may arise from uncharacterized mechanism(s). Here, we propose an alternative explanation for transposon-triggered sterility: we hypothesize that ERV derepression induces sterility through activation of an aberrant immune response. We found that depletion of key immune pathway components rescued fertility despite ERV hyperactivity. These data provide evidence that transposon-triggered sterility may be due to immune activation rather than DNA damage within the oocyte. This proposal seeks to investigate how the immune system recognizes ERVs and whether that immune response can cause sterility. Ultimately, these findings may provide an alternative explanation for ERV-triggered sterility as well as advance our understanding of how ERVs impact human health.

NIH Research Projects · FY 2024 · 2023-09

ABSTRACT. Black women in the United States are three to four times more likely to die from pregnancy- related causes than White women, a disparity that has persisted for the past five decades. Gestational diabetes (GDM) and hypertensive disorders of pregnancy (HDP) disproportionately develop in the pregnancies of Black women and negatively impact cardiometabolic health in the years following pregnancy. Gendered racism, a hybrid phenomenon of racism and sexism that interlocks to create a unique form of oppression, is a chronic stressor experienced by Black women and contributes to ongoing health disparities. However, the empirical evidence on the health implications of gendered racism is still nascent. Psychological resilience has been found to protect against adverse pregnancy outcomes, yet Black women benefit less from such resilience assets. A more culturally relevant understanding of resilience incorporating the individual-level (psychological resilience and spirituality) and family-level (family support) will be explored in this study. The overall purpose of this study is to examine the relationships between gendered racism experiences, resilience, and high-risk pregnancies among Black women. Data from the National Longitudinal Study of Adolescent to Adult Health will be used to assess experiences of gendered racism, high-risk pregnancy, and resilience among Black women with at least one pregnancy (N = 2,153). The aims are to: Aim 1. Identify latent subgroups of women with different gendered racism experiences determined by four indicators (structural sexism, interpersonal sexism, structural racism, and interpersonal racism). Aim 2. Determine the relationship of history of a high-risk pregnancy (GDM/HDP, cases vs controls) with gendered racism experiences characterizing the identified latent subgroups, covarying for chronic hypertension, diabetes (type 1 or 2), body mass index, level of education, and income. Aim 3. Describe the association of history of a high-risk pregnancy and gendered racism experiences with sources of resilience (individual and family) during adulthood, covarying for chronic hypertension, diabetes (type 1 or 2), body mass index, level of education, and income. Latent profile analysis will be used to identify subgroups for Black women with different gendered racism experiences. Bivariate and covariate-adjusted multinominal logistic regression approach will be used to determine the association between high-risk pregnancy and gendered racism subgroup experiences. Covariate-adjusted multiple regression will be conducted to examine the relationship of high-risk pregnancy and gendered racism subgroup experiences with resilience. This study uses an innovative methodological approach that integrates both interpersonal and structural gendered racism to quantity the multifaceted nature of oppression experienced by Black women along with a culturally relevant conceptualization of resilience. The findings will enhance the understanding of the health consequences of gendered racism and inform future research on multi-level interventions to combat systemic inequalities and foster resilience.

NIH Research Projects · FY 2025 · 2023-09

PROJECT SUMMARY/ABSTRACT Older dialysis patients commonly have geriatric syndromes which contribute to increased healthcare utilization and poor quality of life. Currently, identification and management of geriatric syndromes are not included in dialysis care. Adding geriatric syndrome management into routine care for dialysis patients could result in significant improvements in quality of life and healthcare utilization. Barriers to integration of geriatric evaluation and management into dialysis clinics (e.g., time, personnel, costs) can be minimized through the development of innovative geriatric care models that leverage the existing dialysis unit interprofessional team and workflow. We will pilot a new dialysis care model that includes a centralized geriatric team that uses information from the Geriatric screen for OLder Dialysis patients (GOLD) to develop individualized recommendations for geriatric syndrome management based on the patient’s priorities. The GOLD is a self-administered screening battery of validated instruments to screen for a range of geriatric syndromes (cognitive impairment, depression, falls, mobility disability, social support needs, and malnutrition). The multidisciplinary dialysis team will integrate the recommendations into their care plans with support from the centralized geriatric team. Ultimately, this geriatric care model could be seamlessly integrated into dialysis clinic structure. The objective of this application is to optimize the care model with key stakeholder input and conduct a pilot randomized controlled trial (RCT) to obtain evidence critical to inform a definitive RCT. We propose three aims: 1) iteratively refine GOLD to achieve acceptable agreement with a geriatrician evaluation, 2) iteratively refine the geriatric care model to ensure acceptability and feasibility to key stakeholders, and 3) conduct a pilot RCT (n=100) to evaluate the geriatric care model’s impact on geriatric syndrome management and inform design of a larger RCT. For Aim 1, we will have participants complete the GOLD and undergo geriatric evaluation. We will assess agreement of each GOLD instrument with its corresponding geriatric evaluation, and if target agreement is not achieved, we will iteratively modify and re-test the GOLD instrument. For Aim 2, we will refine the care model using experience-based co- design with a stakeholder advisory board and multiple phases of feedback from study participants. After patients complete the care model, these patients and their dialysis clinicians will assess acceptability and feasibility of the care model through surveys and interviews. The care model will be iteratively refined until specific metrics of success are achieved. For Aim 3, patients will be randomized to receive the geriatric care model or usual care; geriatric syndrome management measured by patient report and chart abstraction will be compared at 3 months. We will also assess patient reported outcomes, physical function, and health care utilization at intervals up to 12 months to assess their adequacy for inclusion as outcomes in a subsequent definitive RCT. Upon completion, we will have key preliminary data for a large RCT testing a novel dialysis geriatric care model. Overall, this application will address the significant problem of geriatric syndromes in the older dialysis population.

NIH Research Projects · FY 2024 · 2023-09

1 PROJECT SUMMARY/ABSTRACT 2 The increasingly limited availability of affordable housing paired with a shortage in provision of federal rental 3 assistance among eligible families means millions of low-income, U.S. children live in unstable and/or substand- 4 ard-quality housing – conditions which have been linked to a range of adverse child health and development 5 outcomes. The lack of affordable housing in low-poverty neighborhoods further contributes to families living in 6 crowded housing conditions and/or residing in neighborhoods that expose children to stressful, unsafe environ- 7 ments. Although infants and toddlers experience the highest rates of childhood poverty and spend a substantial 8 portion of their time at home, there exists little evidence assessing the impact of adverse housing and neighbor- 9 hood conditions specifically on the health and development of this age group. Moreover, as less than 25% of 10 eligible, low-income families with young children receiving housing assistance, there is need to understand 11 whether other anti-poverty programs can help improve housing conditions for these children and their families. 12 The proposed research seeks to address these knowledge gaps and provide scientific evidence to inform 13 the design of anti-poverty programs that aim to improve early housing and neighborhood environments for low- 14 income children. By using data from Baby’s First Years (BFY) – a randomized control trial evaluating the impact 15 of a monthly, unconditional cash transfer (UCT) to low-income, U.S. mothers and their young children (NICHD 16 R01HD087384) – this research will provide a rich understanding of the housing and neighborhood ecosystems 17 low-income infants and toddlers (0-3 years) inhabit and how these environments impact their healthy develop- 18 ment. Aim 1 estimates the causal impacts of the UCT on housing affordability, composition, stability, and quality 19 using a multivariate linear regression model. Aim 2 conducts latent class analysis using the control sample to 20 identify configurations of housing and neighborhood ecosystems experienced by BFY participants and then eval- 21 uates how these configurations influence young children’s (0-3 years) physical health, use of medical care, be- 22 havioral and socioemotional development, and language acquisition. Aim 3 investigates the mediating role of 23 housing and neighborhood conditions on young children’s healthy development using the same outcome 24 measures as Aim 2. 25 This fellowship application is designed to provide the PI with training in advanced statistical methods 26 critical for conducting policy and public health research, as well as opportunities to maximize the policy relevance 27 of her work, gain further clinical experience, and build professional skills critical to success as an academic 28 scholar. Her proposed research, training plan, and highly qualified, interdisciplinary mentorship team will ensure 29 she is well-positioned to achieve her long-term goal of becoming an independent physician-scientist dedicated 30 to using rigorous, policy-driven research to improve the health and well-being of children and their families.

NIH Research Projects · FY 2025 · 2023-09

ABSTRACT This application is in response to the NOSI of Promoting Fundamental and Applied Research in Inflammation Resolution, in particular to NIEHS’ interest in inflammation resolution related to environmental exposure. It is increasingly recognized that the immune response to an inflammatory stimulus involves specialized pro- resolving mediators (SPMs) that orchestrate the lung’s return to homeostasis by resolving cellular and tissue inflammation. However, little data in humans are available concerning the effects of PM2.5, a ubiquitous air pollutant, on SPMs and inflammation resolution. This is in marked contrast to the large body of literature on the proinflammatory response to PM2.5. Here we hypothesize that PM2.5 impairs cellular biosynthesis and kinetics of SPMs, leading to compromised resolution of inflammation in the airway. As airway inflammation is a hallmark of asthma, it is highly plausible but yet to be confirmed that individuals with asthma are less capable of resolving pollution-induced inflammation. No data are available to support a sex-specific hypothesis on inflammation resolution, despite the known sex-difference in proinflammatory responses to air pollution. Hence, we further hypothesize the effects of PM2.5 on inflammation resolution differ between people with and without asthma and between men and women. We propose to test these hypotheses in a translational study framework by leveraging an existing panel study of air pollution health effects. Our approach comprises of ex vivo cell culture experiments focusing on molecular mechanisms of SPM biosynthesis and resolution kinetics (Aim 1) and a panel study aiming to examine SPM-PM2.5 relationships in vivo (Aim 2) and to examine potential SPMs mediation of the PM2.5 effects on clinical outcomes (Aim 3). To maximize the translatability of the mechanistic findings in Aim 1, we will use primary airway epithelial cells collected from among the panel study participants and will use composition-characterized PM2.5 collected in London, UK, where participants reside. In Aim 2 panel study, 40 participants with and 40 without asthma will be measured 4 times longitudinally for SPMs in nasal fluid and induced sputum, representing the first portal of PM2.5 entry and the lung, respectively. Detailed personal PM2.5 doses and internal doses (biomarkers) of source-specific PM2.5 constituents hours to days prior to SPM measurements will be associated with sputum and nasal SPM concentrations. We anticipate to see differences by asthma and sex, respectively, in the time-concentration profile. In Aim 3, by leveraging the panel study’s rich dataset on health outcomes of clinical relevance, we will examine the mediating effects of SPMs on the exposure- outcome associations at the key time-points of inflammatory and resolution responses identified in Aims 1 and 2. Taking all together, we anticipate to link molecular mechanisms regulating SPM biosynthesis with resolution kinetics and clinically-relevant functional and inflammatory responses to PM2.5. The study will generate real-life data to better understand the role of SPM in resolving pollution-induced inflammation in the airways of asthmatics versus non-asthmatics and those of men versus women. 1

NIH Research Projects · FY 2024 · 2023-09

Project Abstract Older adults with Alzheimer's disease and related dementias (ADRD) have a 2-fold increased risk of clinical bone fracture, and 33% higher rate of morbidity and mortality following fracture. Our prior study showed clinical fractures were reduced by 18% among those prescribed an acetylcholinesterase inhibitor (AchEI). With both cognitive and non-cognitive benefits, AchEIs such as donepezil would be valuable in a fracture prevention program, for older adults with ADRD, with multiple complementary and synergistic components. However, the pathways by which AchEIs reduce fracture risk represent a significant gap in knowledge. Before incorporating AchEIs into a multicomponent program, the specific effects of AchEIs on bone metabolism must be understood. The objective of this application is to measure the effect of ADRD treatment with AchEIs on fracture risk factors including bone mineral density (BMD), bone turnover markers, and bone quality. Our central hypothesis is that AchEIs reduce fracture risk through direct effects on bone metabolism via stimulation of osteoblastic bone formation and reduction in osteoclastic bone resorption. We will recruit adults aged >50 years from the Memory Disorders Clinic with mild to moderate ADRD (N = 45) who will be randomized 2:1 to either the AchEI donepezil 10 mg daily or placebo, respectively. From this biomarker-diagnosed cohort of older adults with mild to moderate ADRD, we will address the following Specific Aims: 1) Determine change over 12-months in Bone Mineral Density measured by dual x-ray absorptiometry associated with the initiation of donepezil; 2) Determine change over 6- and 12-months in Bone Turnover measured by (A) the Bone Resorption Marker C-telopeptide (CTX) and (B) the Bone Formation Marker Procollagen 1 intact N-terminal Pro-peptide (P1NP) associated with the initiation of donepezil; 3) Determine change over 12-months in Bone Quality measured by Trabecular Bone Score associated with the initiation of donepezil. In addressing this significant area, the current application focuses on several NIA priorities including multiple comorbidities and care for adults with ADRD. The proposed study is innovative in its comprehensive, prospective assessment of bone metabolism among adults with biomarker- based diagnosis of ADRD initiating AchE.

NIH Research Projects · FY 2024 · 2023-09

A cancer diagnosis requires patients and their intimate partners to communicate effectively to navigate illness- related challenges. Research indicates that couples who use effective communication strategies have better individual psychological adjustment and higher relationship satisfaction. However, many couples have difficulty communicating about cancer-related issues which can lead to poorer individual, relationship, and patient health outcomes. While dyadic interventions to improve couple communication have proven efficacious, they are often time intensive and have limited reach. The challenges of recruiting couples into dyadic interventions are well-documented, with low recruitment rates especially among underserved couples. Micro-interventions, which consist of brief educational materials and short activities delivered via text message or a mobile application, have significant potential to increase reach and participation in diverse groups of couples by increasing flexibility and reducing barriers to access. Previous studies have found micro-interventions to be effective in promoting health behavior change in a wide range of individuals and in enhancing dyadic functioning in community couples. To date, there are no existing micro-interventions that have been developed and tested in couples coping with cancer. Therefore, the proposed project aims to develop, and pilot test a text-messaging micro-intervention focused on improving communication skills for couples coping with advanced gastrointestinal (GI) cancer. The content of the dyadic micro-intervention will be adapted from existing, empirically validated couple-based interventions developed for cancer and non-cancer couples. Aim 1 focuses on developing the micro-intervention through focus groups and interviews with couples coping with advanced GI cancer and community advisors representative of minority groups, along with user experience testing with the target population. The data collected will be used to increase content relevance, acceptability, feasibility, and cultural sensitivity of the micro-intervention. Aim 2 will be a randomized pilot test of the dyadic micro-intervention to assess feasibility and acceptability. Aim 3 will examine pre-to-post intervention outcomes as measured by improvements in relationship satisfaction and constructive communication. This project is in line with the National Cancer Institute’s priority research area of cancer survivorship and has the potential to increase health equity in cancer by increasing participation of underserved couples. The proposed project will provide training in 5 key areas for the applicant: (1) psycho-oncology and cancer control, (2) theories and didactics related to the development, implementation, and dissemination of behavioral interventions in the context of cancer, (3) mixed research methods specific to development of psychosocial interventions, (4) health equity, diversity, and inclusion training, and (5) professional development. The unique and extensive resources available at Duke University Medical Center, including the Duke Cancer Institute, will aid and enhance training, scientific productivity, and successful completion of the proposed project.

NIH Research Projects · FY 2024 · 2023-09

PROJECT ABSTRACT Recognizing the significant impact of structural racism on kidney health in minority populations, the NIH is creating a new Consortium focused on Interventions that Address Structural Racism to Reduce Kidney Health Disparities. The Consortium will bring together research teams from six Intervention Sites to conduct community-engaged intervention studies to address structural racism and reduce disparities across the spectrum of kidney health and disease. We propose to serve as the Research Coordinating Center (RCC) for the Consortium. The Duke RCC will leverage extensive institutional resources and expertise at Duke University to provide administrative leadership and research coordination to ensure seamless operations of the Consortium and successful conduct of Consortium studies; support rigorous data collection, data management, and data analysis for Consortium studies; and foster research collaboration, capacity-building, and workforce diversification in kidney health equity research. Three closely integrated cores will lead the core activities of the RCC. The Administrative and Coordination Core will provide administrative, operational, and logistical support to the Consortium, including coordinating meetings, developing a secure web-based communications and collaboration platform, supporting program management and regulatory submissions, administering an opportunity pool for ancillary studies, and continually evaluating and enhancing Consortium operations. The Data Management and Analysis Core will contribute to the final design of Consortium studies, provide data monitoring for multi-site Consortium studies, and support statistical analysis for multi-site Consortium studies and pooled analyses of common data elements across the Consortium. The Collaboration and Community Engagement Core will foster a vibrant and collaborative research community within and beyond the Consortium by facilitating discussion at Consortium meetings and workshops, supporting dissemination of Consortium results to the broader research and lay communities, integrating selected NIH career development awardees in relevant Consortium activities, and exposing early-career trainees from underrepresented groups to kidney- focused structural racism scholarly work in order to successfully implement health equity research and interventions. Together, the multidisciplinary RCC team will ensure the successful completion of multiple intervention trials targeting disparities in kidney health and build a diverse and sustainable community of researchers and community partners focused on improving kidney health in marginalized populations.

NIH Research Projects · FY 2024 · 2023-09

Abstract Chronic primary pain conditions (CPPCs) such as fibromyalgia and temporomandibular disorder (TMD) constitute a significant healthcare problem that affects over 100 million, predominately female, Americans. The origin of CPPCs is linked to genetic and environmental factors that enhance catecholamine tone. An estimated 2 in 3 patients with CPPCs have variants in the gene encoding catechol-O-methyltransferase (COMT; an enzyme that metabolizes catecholamines) that result in low COMT activity and increased catecholamine levels. Pain in these individuals is enhanced by stressful events (eg, motor vehicle collision) resulting in increased release of catecholamines from sympathetic nerves. Our lab has shown that catecholamines drive pain via activation of peripheral beta-adrenergic receptor 3 (Adrb3) and downstream mediators that regulate neuronal excitability and immune responses. Catecholamine signaling has also been shown to alter the expression of microRNAs (miRNAs), which are small non-coding RNAs that negatively regulate mRNA targets. However, the role of miRNA dysregulation in CPPC pathophysiology remains understudied and unclear. Preliminary data from our case- control study reveal that patients with TMD have decreased levels of miR-374. We replicated this finding in an animal model of CPPCs where mice with low COMT activity exposed to stress exhibited pain and decreased levels of miR-374. In the same mice, 5 miR-374 mRNA targets that were dysregulated in patients with TMD (ATXN7, CRK, HIF1A, NUMB, and TGFBR2) were also dysregulated in adipose and spinal cord tissues, where they are predicted to influence immune signaling and pain. HIF1A, NUMB, and TGFBR2 were upregulated in adipose from female mice, while ATXN7 and TGFBR2 were downregulated in spinal cord from male mice. These findings point to new RNA targets that may play an important role in pain related to heightened catecholamine tone, yet mechanistic studies are needed to determine their causal role. Thus, the objective of this proposal is to directly test the relationship between miR-374, its mRNA targets, pain, and inflammation. My central hypothesis is that catecholamine activation of Adrb3 reduces levels of miR-374, leading to dysregulation of mRNAs that promote inflammation and chronic pain in a sex hormone-dependent manner. I will use primary adipocytes and neurons to measure 1) miR-374 binding to mRNA targets using a luciferase reporter system and 2) the effects of Adrb3 activation and sex hormones on miR-374 and mRNA target expression using qPCR. In our CPPC mouse model, I will also measure 3) the effects of synthetic miR-374 overexpression and antagonism on pain and cytokine production, and 4) miR-374 and mRNA target expression in distinct cell types using combined RNAscope and immunohistochemical methods. These results will elucidate our understanding of epigenetic mechanisms contributing to CPPCs and identify novel targets for improved treatment options for those with these conditions. The proposed training plan will promote development of new in vitro and in vivo techniques and foster career advancement in a highly supportive and collaborative environment at Duke University.

NIH Research Projects · FY 2026 · 2023-09

PROJECT SUMMARY Alzheimer’s disease (AD) is a severe neurodegenerative illness that destroys cognitive abilities causing memory impairment, difficulties with speech and language, behavioral changes, functional decline, and significant impairment. AD affects an estimated 13.8 million people in the United States and 50 million worldwide, imposing a significant economic burden and global health crisis. While many researchers are working towards developing a cure for the disease, there is currently no objective, point-of-care diagnostic test for the early diagnosis of AD, significantly limiting screening efforts for clinical studies and delaying treatment for patients suffering from early AD pathology. The current standard methods for AD diagnosis involve expensive PET imaging methods that irradiate the patient and cerebrospinal fluid biomarker tests, which are invasive and require a lumbar puncture. Recently, exosomes (30-150 nm extracellular vesicles) have been identified as a possible tool for AD diagnosis, attributed to their ubiquitous presence in biofluids, their ability to pass through the blood-brain-barrier, and their rich library of AD-relevant physiological information present in the molecular cargo they carry, making them prime candidates for use as biomarkers. However, the clinical application of exosomes is hindered by slow, inefficient techniques for exosome isolation and the absence of standardized exosomal biomarker detection and analysis. Thus, an automated, highly sensitive, fast, and efficient system that can isolate exosomes from biofluids and analyze the miRNAs and proteins they contain will significantly improve early-stage AD diagnosis efforts. In this R01 project, we will develop an Automated High-purity Exosome isolation-based AD diagnostics system (AHEADx) to address the limitations of current technologies. The proposed AHEADx platform includes two units: (1) a rapid (<1 min), high-yield (>90%), and high-purity (>90%) acoustic Bessel beam-based separation unit to isolate and enrich exosomes from whole blood, and (2) a rapid (<6 mins), highly sensitive photonic PCR and immuno-PCR (~1 copy/µL for nucleic acids and ~5 copies/µL for proteins, respectively) utilizing a plasmonic nanopillar array to enable on-chip thermocycling and multiplexed exosomal screening of combined panels of AD-relevant biomarkers. Our rapid and precise AHEADx platform will provide a simple, minimally invasive liquid biopsy to detect molecular AD biomarkers with ultrahigh accuracy and sensitivity in early-stage AD patients, allowing for an effective diagnostic method of AD screening for earlier treatment before the onset of severe symptoms of the disease and enable long-term studies on AD development and progression. Additionally, the proposed technology will accelerate the discovery of new exosomal miRNA and protein AD biomarkers and help to elucidate the mechanisms in which exosome trafficking and transport contribute to AD pathology. With these advantages, our AHEADx platform can potentially exceed current clinical standards in AD diagnostics, addressing a significant need in the field and providing a compelling platform for earlier, accurate, and sensitive detection of AD and long-term studies on the effects of novel AD therapeutics.

NIH Research Projects · FY 2024 · 2023-09

ABSTRACT Atherosclerotic cardiovascular disease (ASCVD) is the leading cause of death in the United States (U.S.)(1). Statins reduce the risk of cardiovascular events by 30% in those with prior history of ASCVD (2). Despite being inexpensive and well-tolerated, however, statins are underused in clinical practice, particularly in patients who identify as Black. A large-scale intervention to improve evidence-based statin initiation among individuals with ASCVD is needed, particularly for the Black population. To meet this need, we aim to conduct a multi-center, pragmatic, cluster randomized, controlled trial to assess the impact of a provider- and patient-directed intervention to increase the prescription of, and adherence to, appropriate statin therapy among those with ASCVD (ACHIEVE: A Controlled trial to improve use of High IntEnsity statins for Vascular protEction). The intervention will be pharmacist-led, embedded in commercial payer systems, partnering with the National Committee for Quality Assurance (NCQA), and with a particular emphasis on improving statin use in the Black population. Eligible patients and their clinicians will be identified through commercial payer records. The pharmacist will then work with the patient and the clinician to overcome barriers and to achieve evidence- based statin therapy and patient adherence to the medication. As with all pragmatic trials that operate outside of the traditional clinic-based recruitment model, the optimal method to reach and engage both clinicians and patients in this trial is uncertain. Our objective for this planning grant is to determine whether our strategies for clinician and patient recruitment yield a high enough degree of engagement, including among Blacks, to justify moving forward with the larger trial. Our specific aims are: (1) To evaluate our strategy for recruitment and engagement of providers to participate in this multifaceted intervention to improve statin prescription and adherence, with the goal of recruiting at least 25% of identified providers. (2) To evaluate our strategy for recruitment and engagement of patients to participate in this multifaceted intervention to improve statin prescription and adherence, with the goal of recruiting at least 30% of identified patients overall and within the Black patient population. To our knowledge, this approach of engaging providers and their patients through commercial payer networks has not been tried on the scale envisioned for the ACHIEVE trial. Understanding whether we can recruit clinicians and patients in this setting will benefit not only the ACHIEVE program, but also future pragmatic trials across therapeutic areas.

NIH Research Projects · FY 2024 · 2023-09

PROJECT SUMMARY/ABSTRACT Older dialysis patients commonly have geriatric syndromes which contribute to increased healthcare utilization and poor quality of life. Currently, identification and management of geriatric syndromes are not included in dialysis care. Adding geriatric syndrome management into routine care for dialysis patients could result in significant improvements in quality of life and healthcare utilization. Barriers to integration of geriatric evaluation and management into dialysis clinics (e.g., time, personnel, costs) can be minimized through the development of innovative geriatric care models that leverage the existing dialysis unit interprofessional team and workflow. We will pilot a new dialysis care model that includes a centralized geriatric team that uses information from the Geriatric screen for OLder Dialysis patients (GOLD) to develop individualized recommendations for geriatric syndrome management based on the patient’s priorities. The GOLD is a self-administered screening battery of validated instruments to screen for a range of geriatric syndromes (cognitive impairment, depression, falls, mobility disability, social support needs, and malnutrition). The multidisciplinary dialysis team will integrate the recommendations into their care plans with support from the centralized geriatric team. Ultimately, this geriatric care model could be seamlessly integrated into dialysis clinic structure. The objective of this application is to optimize the care model with key stakeholder input and conduct a pilot randomized controlled trial (RCT) to obtain evidence critical to inform a definitive RCT. We propose three aims: 1) iteratively refine GOLD to achieve acceptable agreement with a geriatrician evaluation, 2) iteratively refine the geriatric care model to ensure acceptability and feasibility to key stakeholders, and 3) conduct a pilot RCT (n=100) to evaluate the geriatric care model’s impact on geriatric syndrome management and inform design of a larger RCT. For Aim 1, we will have participants complete the GOLD and undergo geriatric evaluation. We will assess agreement of each GOLD instrument with its corresponding geriatric evaluation, and if target agreement is not achieved, we will iteratively modify and re-test the GOLD instrument. For Aim 2, we will refine the care model using experience-based co- design with a stakeholder advisory board and multiple phases of feedback from study participants. After patients complete the care model, these patients and their dialysis clinicians will assess acceptability and feasibility of the care model through surveys and interviews. The care model will be iteratively refined until specific metrics of success are achieved. For Aim 3, patients will be randomized to receive the geriatric care model or usual care; geriatric syndrome management measured by patient report and chart abstraction will be compared at 3 months. We will also assess patient reported outcomes, physical function, and health care utilization at intervals up to 12 months to assess their adequacy for inclusion as outcomes in a subsequent definitive RCT. Upon completion, we will have key preliminary data for a large RCT testing a novel dialysis geriatric care model. Overall, this application will address the significant problem of geriatric syndromes in the older dialysis population.

NIH Research Projects · FY 2025 · 2023-09

PROJECT SUMMARY/ABSTRACT Humans have a uniquely developed ability to impose internal goals on how they interact with their environment. Referred to as “cognitive control”, this capacity includes two core components: (1) the ability to focus attention on currently goal-relevant stimulus features and responses (a “task set”) while ignoring task-irrelevant features (cognitive stability); and (2) the ability to switch to a different task set when circumstances change (cognitive flexibility). Crucially, to thrive in a dynamic environment, we need to continuously adapt our levels of cognitive stability and flexibility to suit changing demands. E.g., when cooking a meal, needs for stability (e.g., a strong task-focus when slicing onion) and flexibility (e.g., rapid shifting between recipe reading and stovetop monitoring) change frequently over time. The strategic regulation of stability and flexibility is thus fundamental for success in everyday life, and is in fact severely impaired in many clinical conditions. However, the underlying neurocognitive mechanisms are poorly understood. This is due to the fact that, while there are large literatures on cognitive stability (in the shape of conflict-control studies) and flexibility (in the shape of task-switching studies), these processes have been either investigated in isolation, conflated, or not interrogated in terms of their dynamic adaptation. The present proposal seeks to overcome these barriers to progress by combining a novel task protocol that assesses simultaneous and independent adaptive shifts in stability and flexibility with computational modeling, functional magnetic resonance imaging (fMRI), and intracranial electro- encephalography (iEEG). Our overall goal is to characterize the neurocognitive mechanisms of concurrent, strategic control over cognitive stability and flexibility. We triangulate this goal via three aims: Aim 1 seeks to establish the first computational model of concurrent stability and flexibility regulation by fitting and simulating behavioral data from protocols with time-varying demands on stability and flexibility (Studies 1 and 2). Our working model consists of two independent reinforcement learners making trial-by-trial predictions about forthcoming demands on stability (conflict- likelihood) and flexibility (switch-likelihood), which in turn modulate distinct within-trial drift-diffusion model parameters. Aim 2 employs the winning model to determine the neural mechanisms mediating these adjustments in stability and flexibility. Building on a large prior literature, we use complementary fMRI (Study 3) and iEEG (Study 4) approaches to test specific neuroanatomical hypotheses about the respective roles of the lateral prefrontal, posterior parietal, and anterior cingulate cortex, as well as the basal ganglia, in supporting the proactive adaptation of stability and flexibility to time-varying demands. Finally, Aim 3 will use fMRI to characterize the neural reinstatement of context- appropriate stability and flexibility settings when they are applied reactively, i.e., in response to specific demand- predicting stimuli (Study 5). Together, these complementary aims represent the first systematic investigation into the computational and neural mechanisms underlying the concurrent regulation of cognitive stability and flexibility. This innovative project will significantly advance our understanding of the neurocomputational bases of cognitive control, and lay the groundwork for identifying potential failure modes of stability and flexibility regulation in clinical conditions.

NIH Research Projects · FY 2025 · 2023-09

Project Summary/Abstract Temporal lobe epilepsy (TLE) is a common and commonly devastating form of human epilepsy that lacks preventive or disease modifying therapy. An estimated 30% suffer recurrent seizures despite symptomatic treatment with anticonvulsants. One cause of TLE is status epilepticus (SE). Defining the molecular mechanisms by which SE induces TLE promises to identify molecular targets for therapies. We therefore conducted extensive target validation experiments which revealed TrkB-PLCγ1 as a druggable molecular target that can prevent TLE in adult mice. The goal of our drug discovery program is to develop small molecule inhibitors for TrkB-PLCγ1 signaling to treat TLE. With the support of Blueprint Neurotherapeutics Network (BPN), we identified multiple compounds within distinct series with demonstrated in vivo efficacy for inhibition of TrkB-PLCγ1 signaling in mouse brain. Our Specific Aims are to benchmark, expand, and optimize novel small molecule inhibitors of TrkB-PLCγ1 signaling. Successful completion of the work proposed will identify a leading series for entry to the Discovery Phase of BPN program.

NIH Research Projects · FY 2024 · 2023-09

The complement systems of humans and laboratory animals (mice and rats) show striking species differences in function, particularly with regards to the classical pathway of complement activation. Indeed, studies since the 1950’s have documented markedly reduced classical pathway function in mice as compared to rats and humans. In preliminary data, we confirm and extend these findings using model immune complexes (ICs) formed by monoclonal antibodies to platelet factor 4 (PF4) and heparin (KKO ICs) or polyclonal rabbit anti-dinitrophenol (DNP) antibodies to DNP conjugated with keyhole limpet hemocyanin (DNP ICs). Specifically, we show: 1) robust C3 activation by KKO and DNP ICs in whole blood and plasma from mice, rats and humans 2) classical pathway requirements for complement activation by KKO ICs in rats and human, but alternative pathway requirements in mice 3) incorporation of alternative pathway proteins into KKO ICs in mice, but not rats or humans 4) comparable requirements for the alternative pathway among four murine strains and 5) differential expression of alternative pathway proteins by proteomics in mice as compared to rats and humans. Based on these preliminary data and published observations, we will test the overall hypothesis that impaired classical pathway activity in mice is counterbalanced by the alternative pathway C3b feedback cycle. To test this hypothesis, we propose the following two aims: 1) Comparative studies of human, mouse, and rat complement systems. In this aim, we will test that hypothesis that genetic variation in murine classical pathway proteins contributes to impaired classical pathway function. We will apply molecular approaches to compare genetic sequences and perform structural homologies of classical pathway proteins in mice v rats and humans. In other studies, we will investigate the plasma proteome of mice, rats, and humans to examine protein/immune complex interactions, co-expression of complement and complement-associated proteins and correlate complement protein levels with functional responses to KKO and DNP ICs. 2) Functional studies of murine and human complement systems. In this aim, we will test the hypothesis that mice utilize the C3b feedback cycle of the alternative pathway as a compensatory response to low classical pathway activity. To test this hypothesis, we will use purified human and mouse complement proteins to demonstrate reduced impaired classical C3 convertase activity and normal/heightened activity of the C3b feedback cycle in mice, examine the effects of interchanging human and mouse complement proteins, and identify potential murine inhibitors of classical pathway activation from mouse serum. Together, these studies are expected to delineate the molecular and functional basis of divergent complement pathway responses in mice and humans. We expect these studies to generate preliminary data for an expanded application to elucidate the molecular and structural divergence of murine complement responses with the long term goal of developing translationally relevant animal models.

NIH Research Projects · FY 2025 · 2023-09

ABSTRACT Developing a healthy functioning brain depends on the coordination of neuronal circuit formation by glial cells called astrocytes. Previous work has shown that astrocytes, born from the same neural stem cells after the end of neurogenesis, require contact with neurons to mature morphologically, functionally, and transcriptionally. Astrocytic expression of the cell adhesion molecule neuroligin 2 (Nlgn2) is necessary for neuronal contact- dependent astrocyte morphogenesis and synaptogenesis. These findings led us to investigate how neuronal contact mediates astrocyte morphogenesis and synaptogenic functions. To address this question, we performed multiplexed indexed T7 chromatin immunoprecipitation (MintChIP) sequencing to measure histone modifications across the astrocyte genome. These experiments found that the chromatin modification landscape changes significantly over postnatal astrocyte development. Interestingly, when we measured histone modifications in astrocytes from Nlgn2 KO mice, we found a substantial decrease in the H3K4me1 and H3K4me3 modifications compared to wildtype astrocytes. Based on these preliminary findings, this proposal will test the hypothesis that epigenetic histone modifications are the mechanistic link between neuronal contact and astrocyte transcriptional maturation. Specifically, aim 1 will test how histone modifications and transcriptional maturation change when astrocytes are cultured with or without neurons and whether inhibiting histone-modifying enzymes is sufficient to prevent astrocyte transcriptional maturation. Aim 2 will investigate the molecular mechanisms of Nlgn2-dependent astrocyte transcriptional maturation. In particular, we will test the hypothesis that the chromatin remodeler Chd8 is required for Nlgn2-dependent H3K4 methylation and that preventing this H3K4 methylation through astrocyte-specific Chd8 knockout will prevent astrocyte transcriptional maturation. The successful completion of these aims will determine the extent to which histone modifications regulate astrocyte maturation and further our understanding of the molecular mechanism responsible for Nlgn2-induced gene expression changes in astrocytes. These findings will also be highly relevant to future work in neurodevelopmental disorders as astrocyte-regulated synaptogenesis, and epigenetic regulation of gene expression are emerging as prominent aspects of conditions like autism spectrum disorders. Finally, this project will provide an excellent training experience for Mr. Justin Savage through its intersection of epigenetics and glial cell biology.

NIH Research Projects · FY 2024 · 2023-09

Abstract Transposons are mobile pieces of DNA that comprise significant proportions of eukaryotic genomes, including around 45% of the human genome and 30% of the fly genome. While most transposons have lost the ability to jump into new locations in the genome, several in each organism maintain the ability to mobilize. New transposon insertions are particularly consequential if they are generated in germline cells. Germline insertions are subsequently present in every cell of the organism they develop into and can be passed on to future generations. Thus, germline cells employ multilayered mechanisms to repress transposons. These repressive mechanisms are essential to largely suppress transposon for development of germline cells into mature eggs and sperm because transposon activity can contribute to sterility. Since these repressive mechanisms like the piRNA pathway render transposition events so rare, they are difficult to detect and our understanding of how transposons mobilize in the germline remains incomplete. Namely, the host factors that transposons employ to generate new insertions and the time point when they make new insertions during sperm maturation are undefined. To find potentially active transposons in the male germline, I have sequenced circular DNA from Drosophila testes with the piRNA pathway depleted. Disrupting the piRNA pathway allowed for transposon activation and revealed that the nomad transposon generates the most circular DNA in the testes. Circular DNA is a transposition intermediate of LTR retrotransposons and its presence can indicate the likelihood of a transposon being able to still make new insertions. With the knowledge that nomad is the most active transposon in the male germline, I propose to use this transposon to study how transposons mobilize in the germline. I hypothesize that transposons utilize host factors from the alt-EJ DNA repair pathway to achieve stage specific mobilization during spermatogenesis. I will utilize circular nomad DNA as a readout to find host factors required for its production and transposition reporters for nomad to precisely identify when it generates new insertions.