Virginia Commonwealth University

NIH Research Projects · FY 2024 · 2024-08

Alzheimer’s disease (AD) and other neurodegenerative diseases will have a global impact on health outcomes for people in the coming decades. Without a cure or preventative treatment, it is likely that the economic costs to the U.S. and other countries will be prohibitive, with estimates in the billions of dollars. Thus, understanding the pathophysiology of the disease process will be integral for future therapeutic treatments. The presence of senile plaques and neurofibrillary tangles (NFTs) are hallmarks of Alzheimer’s disease (AD). However, only NFTs have been shown to correlate with the severity of the associated dementia. NFTs have been shown to consist of aggregates of phosphorylated tau protein. Tau normally functions to stabilize microtubules in neuronal processes, but in diseased brain cells tau becomes part of insoluble multi-protein NFT complexes as well as soluble pathogenic oligomers. The progression of the disease follows a well described path initially displaying NFT-associated neurodegeneration in a brain region called the entorhinal cortex followed by neurodegeneration in a downstream brain region - the hippocampus. Importantly, these brain regions are crucial for the formation of long-term memories and are likely responsible for the memory loss observed in AD patients. This proposal will investigate the effect of trans-synaptic spread of pathogenic tau from entorhinal cortical neurons to downstream neurons in the hippocampus. Our preliminary evidence demonstrates that not all neurons in the hippocampus accumulate pathogenic tau and that specific subtypes of neurons in the hippocampus are particularly vulnerable. The experimental design will involve adeno-associated viral vector transfection, immunofluorescence to monitor tau trans-synaptic spread, and the use of cellular electrophysiology to assess the impact that pathogenic tau spread has on vulnerable hippocampal neurons. Importantly, we will also determine the effect of pathogenic tau on network properties of CA1 pyramidal neurons when tau is expressed in select vulnerable neurons. By understanding the resultant changes produced by pathogenic tau at the cellular and network level, it may be possible to develop therapies to counteract these changes at early stages of AD.

NIH Research Projects · FY 2025 · 2024-08

The Virginia Advancing Cancer Control Engaged Research Through Transformative Solutions (VA-ACCERT) Center is an academic-public partnership between an interdisciplinary team of investigators from the Virginia Commonwealth University, the Macon & Joan Brock Virginia Health Sciences at Old Dominion University, and Virginia’s U.S. Department of Housing and Urban Development-administered public housing communities. The goal and cross-cutting theme of the VA-ACCERT Center is to improve dissemination and implementation of health promotion and cancer prevention services within income-based communities and across the state-wide public housing system in an effort to close the gaps in cancer health outcomes among certain populations. The VA-ACCERT Center will utilize two essential theoretical models: the Collective Efficacy Mechanism of Action Model to build social capital, community empowerment, and collective efficacy and elements from the Social Cognitive Theory and Social Ecological Model to inform multi-level behavioral change. Community members will engage with the VA-ACCERT Center through these frameworks to aid in the cultural adaptation and delivery of evidence-based intervention practices, and their participation may include serving on the Housing Collaborative Community Advisory Board, as Co-Leads and participants in Center-supported research projects, and/or serving as peer-to-peer health advisors. Led by a Multiple Principal Investigator triad with demonstrable experience in conducting community-engaged cancer control research, the VA-ACCERT Center includes four interdependent components: a large, five-year, multi-level, interventional Social Drivers of Health Research (SDOH) Project designed to improve healthy dietary patterns and reduce sedentary lifestyle through gains in low and moderate-to-vigorous physical activity; a Community Responsive Research Projects initiative that will support the design, implementation, evaluation, and dissemination of four research projects; a Research Methods, Measures, and Data Management Core that will provide integral qualitative and quantitative research services spanning the data management, study coordination, measure development, study or methods adaptation, ethical considerations, and data sharing for all Center-supported research initiatives; and an Administrative Core supporting essential internal and external communications and coordination, guided by several advisory entities. In addition, VA-ACCERT leadership will be engaged in national Consortium-wide efforts involving four other National Cancer Institute-funded ACCERT Centers and a Central Coordination Center. The VA-ACCERT Center will also be a hub for engaging early-career investigators in community-based, multi-level science research. These investigators will be equipped with the skills and support to shape new directions in cancer prevention efforts within income-based housing communities. Ultimately, the VA-ACCERT Center will disseminate and promote its findings and best practices for wide-scale adoption and sustainability across income-based housing communities locally and nationally.

NIH Research Projects · FY 2025 · 2024-08

Modified Project Summary/Abstract Section Drinking water instead of sugar-sweetened beverage (SSBs) can help prevent obesity and dental caries, two of the most prevalent pediatric diseases, disproportionately impacting marginalized youth. Adequate hydration also impacts cognitive function, essential for academic success. Federal policies require public schools to provide free drinking water and place restrictions on SSB availability. Yet, these policies alone have not optimized student hydration practices and numerous barriers to healthy hydration remain. An urban Virginia school district has partnered with our team to optimize hydration. This district has newly installed water bottle refill stations (“hydration stations”) and a model hydration policy. Yet challenges with policy implementation and inadequate drinking vessel access presented major barriers to hydration. To address this concern, we used a participatory approach to build capacity and gather in-depth information on community strengths and needs related to hydration. Grounded in participatory research methods and informed by extensive formative work, we developed a multi-faceted program that aligns with the social-ecological framework to optimize hydration, builds on district strengths, and addresses gaps. We propose to conduct a stepped wedge, cluster randomized controlled trial to test the effectiveness of the intervention within 12 randomly selected, matched Title I elementary schools serving predominately Black and Latino students from lower income backgrounds who all receive free meals (N=~6400 students). This intervention was designed collaboratively with key stakeholders, partners with teacher and student ambassadors, and includes social marketing, behavioral reinforcement, and education and outreach. Students and staff receive refillable water bottles and promotional materials encouraging water consumption over SSBs. Teachers receive training on the hydration policy and hydration lessons to integrate into their curriculum. School-wide assessments of: 1) hydration station usage (primary outcome), 2) water bottle usage and beverage selection at lunch, and 3) beverage intake will be conducted. Longitudinal assessments of anthropometrics and dental caries will be conducted with a randomly selected subset of 3rd grade students (N=406). We will also obtain data on beverage sales, NSLP participation, and academic outcomes, and conduct a systematic evaluation of sustainability. Consistent with the stepped wedge design, concurrent assessments will be conducted at 6 timepoints in all 12 schools over 3 years, with all baseline (T0 [timepoints prior to the intervention]) and post (T1-T6 [timepoints after receiving the intervention]) data used in primary effectiveness analyses. Results will directly inform school-based interventions addressing these urgent public health needs and guide policy mandates regarding access to and promotion of water within the school setting.

NIH Research Projects · FY 2025 · 2024-07

Project Summary/Abstract Epidermal growth factor receptor (EGFR), a receptor tyrosine kinase (RTK), is an oncogenic driver that is frequently overexpressed or mutated in non-small cell lung cancer (NSCLC). Many EGFR inhibitors are used to treat NSCLC, but drug resistance is common. Necitumumab (Neci), an EGFR-directed monoclonal antibody, is approved for a subset of NSCLC harboring wild type (WT) EGFR but its efficacy is very limited. Patients whose tumors harbor mutated EGFR often derive significant benefit from EGFR tyrosine kinase inhibitors (TKIs), but resistance invariably develops. Mechanism of resistance to EGFR inhibitors is complex and not fully known. Our objective in this project is to evaluate a new agent for overcoming drug resistance in NSCLC using preclinical models. The project builds on our recent discovery that a recombinant human protein, PEPDG278D, induces the degradation of EGFR and HER2 by binding to their extracellular domain. HER2 is closely related to EGFR and a key contributor to drug resistance in NSCLC. PEPDG278D is an enzymatically inactive mutant of human peptidase D, also known as prolidase important for collagen metabolism. We also find that PEPDG278D induces the degradation of mutated EGFR and HER2 that occur in NSCLC and strongly inhibits NSCLC cells and tumors resistant to current EGFR inhibitors. We envision that targeted degradation of EGFR and HER2 is more effective than only inhibiting the activation or activity of their tyrosine kinase, as kinase-independent functions are also important for their oncogenic signaling. Our overall hypothesis is that NSCLC cells and tumors resistant to current EGFR inhibitors are vulnerable to targeted degradation of EGFR and HER2 by PEPDG278D and that PEPDG278D induces the degradation of not only WT EGFR and HER2 but also a broad spectrum of clinically relevant mutants. We will test the hypothesis in three specific aims: 1) to assess PEPDG278D for inhibition of oncogenic signaling and antitumor activity in NSCLC cells and tumors expressing WT EGFR, and to compare PEPDG278D to Neci; 2) to determine if PEPDG278D inhibits the oncogenic signaling of a broad spectrum of EGFR and HER2 mutants in NSCLC cells by inducing their degradation and to compare PEPDG278D to osimertinib (Osi) which is a third generation EGFR TKI and the preferred EGFR TKI for NSCLC patients; and 3) to determine if Osi-resistant NSCLC cells and tumors are sensitive to PEPDG278D and if combining the two agents enhances therapeutic outcome. PEPDG278D and Osi complement each other mechanistically in targeting EGFR mutants. The proposed research is significant, because it addresses drug resistance in NSCLC which remains a major clinical problem. We expect 1) to show that PEPDG278D is therapeutically superior to Neci and Osi; 2) to establish the novel concept that inducing the degradation of EGFR and HER2 is an effective strategy for overcoming drug resistance in NSCLC. These findings may generate strong enthusiasm for clinical evaluation of PEPDG278D in NSCLC and stimulate research of other EGFR and HER2 degraders.

NIH Research Projects · FY 2026 · 2024-07

PROJECT SUMMARY / ABSTRACT Postural tachycardia syndrome (POTS) is a common, disabling disorder among adolescents, interfering with their schoolwork, physical and social development. It occurs most often in adolescent females post- menarche. The combination of orthostatic symptoms such as dizziness, nausea, weakness, or brain fog with a 40 bpm rise in heart rate in the first 10 minutes of a tilt table test without a drop in blood pressure defines the disorder. Two striking and nearly universal features of POTS are (1) its onset after a major threat such as an infection or trauma and (2) its association with many co-morbid disorders such as migraine headache, fibromyalgia, functional gastrointestinal disorders like irritable bowel syndrome, and others that commonly fall under the classification of chronic overlapping pain conditions (COPC). Although we and others have repeatedly reported these curious associations, no study or pathophysiologic theory has attempted to include either of these observations in a model for the fundamental etiology of POTS. Here we propose that the brain region responsible for the orchestration of “life or death” response to an acute major threat, the periaqueductal gray region (PAG) in the midbrain, does not properly reset after a threat has passed (explaining the observation that POTS onset follows a threat) and continues in a chronic state of threat readiness. We further hypothesize that this chronic state impacts the two major areas of known control by the PAG: cardiovascular autonomic regulation, resulting in POTS, and pain signaling from end-organs, resulting in COPC’s. Such PAG dysregulation likely occurs through the vagus nerve, known to be abnormal in POTS. We will test this hypothesis in 3 aims comparing 60 adolescent and young adult females with POTS to 60 healthy female subjects, 40 of whom will just had an infection severe enough for a hospital admission. In aim 1, we will study the natural history to understand whether POTS and COPC’s flare simultaneously and are typically associated with a preceding identifiable threat, and whether the healthy controls after infection develop any limited symptoms of POTS or COPC that eventually recede. In aim 2 we will image the PAG both at rest and during a “looming threat task” which probes the PAG’s responsiveness to threat. We expect the PAG to be more threat-responsive in the POTS subjects and in the healthy subjects immediately in the wake of the infection compared to subjects without POTS or recent infection. Aim 3 will consist of assessing cardiovascular vagal function through heart rate variability (HRV). We expect that more severe POTS subjects will show lower HRV in association with greater PAG threat readiness, and that changes in PAG threat readiness will be associated with changes in HRV. This project is the first proposal of a unifying hypothesis explaining all the major features of POTS, and the potential for new treatment approaches.

NIH Research Projects · FY 2024 · 2024-07

Compared to non-Hispanic Whites (NHW), African Americans (AAs) are 20% more likely to get colorectal cancer (CRC) and 40% more likely to die from CRC. The negative impacts of health inequity on CRC incidence and mortality are undeniable, though it is becoming clear that ancestry-specific tumor biology also plays a significant role. A greater understanding of ancestry specific biology on CRC etiology is required if we are to fully elucidate its true contribution to cancer disparity, ATG16L1 is critical for LC3 lipidation and autophagosome formation. ATG16L1 isoforms show ancestry specific expression in CRC tissue. Alanine homozygosity at residue 300 of ATG16L1 (A300A) is more predominant in NHWs and threonine homozygosity at the same residue (T300T) is more predominant in AA’s. In CRC, the A300A genotype is associated with reduced metastasis and increased overall survival and the T300T genotype is associated with increased risk of CRC. Using isogenic CRC cells, we found that chemotherapies that enhance autophagy show more therapeutic efficacy in T300T cells than more established cytotoxic based chemotherapies which have more cell killing efficacy in the A300A isoform. Collectively, these data suggest that isoform status of ATG16L1 promote aggressive CRC and represent a key pivotal biomarker to stratify CRC treatment response in relation to autophagic function. Our hypothesis is “that the ATGL16L T300T isoform is a prognostic indicator of response in AA CRC patients and represents a predictive treatment biomarker for treatment decisions and help to reduce disparate outcomes.” Specific Aim 1. Determine whether ATG16L1 isoforms differentially promote aggressive CRC growth and metastasis in vivo. Using isogenic male and female ATG16L1 T300T and ATG16L1 A300A mice we will chemically induce colitis-associated tumors using azoxymethane (AOM) and dextran sodium sulfate (DSS).10 Following AOM / DSS exposure, we will determine tumor number, size, and location in T300T and A300A mice. Male and female Apc min/+ mice that are ATG16L1 T300T will be cross-bred with ATG16L1 A300A mice.11 We will isolate tumor epithelial cells and stromal fibroblasts from tumors arising from transgenic mice over time, define alterations in their molecular biology and establish novel cell lines.12,13 This aim will inform on the biological mechanisms promoting aggressive tumor growth and metastasis in patients with the different ATG16L1 isoforms. Specific Aim 2. Determine whether the T300T isoform predicts tumor response and improves outcome to combination-treatment regimens that target autophagy in pre-clinical isogenic mouse tumor models. The response of male and female murine colorectal tumors using cells isolated from Aim 1 will be defined in either T300T or A300A mice, to drug combinations that utilize autophagy to kill, or where autophagy reduces drug efficacy. This will define the importance of T300T or A300A isoform expression with respect to both the tumor epithelial cells and to the autophagic competency of the tumor microenvironment. This aim will inform on why AA CRC patients have poorer responses to standard of care therapies resulting in worse outcomes.

NIH Research Projects · FY 2024 · 2024-07

Multiple Sclerosis (MS) is a progressive inflammatory demyelinating disease that affects the central nervous system (CNS), and it is considered the most common non-traumatic debilitating neurologic disease in young adults. Based on recent advances in the understanding of the disease mechanisms underlying MS, it has become evident that clinical relapse and progressive accumulation of disability are driven by different pathological processes. Importantly, current therapies for MS mostly target the chronic inflammatory component mediating clinical relapse, and they are effective in modifying the disease course and in managing symptoms. Relapse-independent disease progression, on the other hand, is to a large extent driven by the degeneration of chronically demyelinated and hence more vulnerable axons. Hence, myelin restoration has emerged as a promising strategy toward a regenerative treatment for MS. At present, however, we are only at the very beginning of developing practical approaches with the potential to promote myelin regeneration in vivo in the human brain, and therapeutic agents that limit progressive disease mechanisms and promote CNS repair remain an important unmet challenge in MS clinical practice. Thus, there is a critical need to broaden the scope of druggable targets to advance the design of myelin regenerating therapies for MS. To address this need, we propose here to explore the role of a novel glutamate-driven signaling pathway that is initiated in maturing oligodendrocytes (OLGs), the myelinating cells of the central nervous system (CNS), through activation of the sodium-dependent glutamate transporter GLT-1, also known as excitatory amino acid transporter 2 (EAAT2) or solute carrier family 1 member 2 (SLC1A2). In MS, and particularly in progressive MS, there is evidence that this pathway is impaired, thereby limiting efficient myelin repair. Importantly, our preliminary data provide compelling evidence that loss of GLT-1 in maturing OLGs limits myelin repair in the cuprizone model that reflects features of particularly progressive stages of MS. In light of the glutamate homeostasis regulating role of GLT-1 in astrocytes, characterizing mechanisms downstream of GLT-1 activation in maturing OLGs represents a critical step toward the identification of potential therapeutic targets. In this context, our preliminary data point toward a critical role of the small GTPase RhoB and its unique functional roles in regulating endosomal and myelin protein trafficking mechanisms. By characterizing the role of a promising new signaling cascade and by investigating a molecular mechanism targeting more mature OLGs rather than OLG progenitor cells, our studies address major gaps in the field. Notably, there is increasing evidence for an involvement of Rho GTPase signaling and protein targeting in CNS myelin repair, and Rho GTPases emerge as promising therapeutic targets. Thus, in the long-term, the proposed studies are anticipated to provide novel insight into the molecular mechanisms modulating CNS myelin regeneration and their potential for therapeutic intervention toward stimulating CNS myelin repair.

NIH Research Projects · FY 2024 · 2024-07

Project Summary Marginal zone lymphomas (MZL), the second most common subtype of indolent lymphomas, is a B-cell malignancy where the tumor microenvironment plays an important role in its pathogenesis. Furthermore, it lacks reliable preclinical models including cell lines, which has hampered rational drug development for this common indolent lymphoid tumor. The phosphoinositide 3-kinase (PI3K), protein kinase B (Akt), mammalian target of rapamycin (mTOR) pathway is one of the frequently deregulated pathways in human cancer and is an essential component of the B-cell receptor signaling pathway that is important to the pathogenesis of B-cell malignancies. Interestingly, PI3K/AKT/mTOR (PAM) pathway inhibitors such as copanlisib and capivasertib have shown strong clinical activity in patients with relapsed or refractory MZL. The tumor suppressor gene (TSG) phosphatase and tensin homolog (PTEN) negatively regulate PI3K signaling, while the TSG liver kinase B1 (LKB1), also known as STK11 negatively regulates mTOR signaling. Prior studies using heterozygous global knockout of PTEN and hypomorphic mutation that decreases LKB1 expression in genetically engineered mouse model (GEMM) lead to formation of indolent lymphomas that were not well characterized, in addition to other types of malignancies such as pheochromocytomas, prostate, breast and pancreatic carcinomas. Therefore, we have generated novel GEMM where we have restricted the activation of the PAM pathway to B cells using the Cre/loxP system with CD19-Cre in order to create a more cell type-specific tumor model and more accurately mimic the underlying human disease, with the long-term goal of using these models for evaluating targeted and immunotherapies for human MZL. We will test the following hypotheses: Aim 1: To study the impact of PI3K/AKT/mTOR pathway activation in the development of marginal zone lymphomas (MZL) using novel genetically engineered mice. Aim 2: To determine the optimal drug combination for targeting the PI3K/AKT/mTOR pathway in MZL. These studies will be the first to demonstrate the possibility of modeling human MZL in mice, using a model with a functional immune system. This novel model will have a tremendous impact on our understanding of the underlying biology of the disease and on our ability to design future rationale therapies for this incurable malignancy.

NIH Research Projects · FY 2025 · 2024-07

PROJECT SUMMARY Down syndrome (DS), or Trisomy 21, is the most frequent chromosomal abnormality in humans, resulting from a complete or partial extra copy of chromosome 21. Clinically, DS is identified after birth through the recognition of specific physical characteristics such as flat nasal bridge and midface, decreased muscle tone, clinodactyly, and short neck with excess skin at the back. Additionally, children and adults with DS have a higher likelihood of gastrointestinal dysfunction that markedly affects quality of life. While our understanding of the underlying cognitive and other central effects in DS have been studied for many years, our understanding of the gastrointestinal effects in these patients in still quite rudimentary. Gut motility is controlled by the interdependent actions of enteric neurons (ENS), interstitial cells of Cajal, and smooth muscle cells. Investigation of the enteric nervous system in the Ts65Dn mouse model of DS (a well-established mouse model of Trisomy 21) demonstrates that these mice have ENS neurons throughout the bowel, including in distal colon, and that longitudinal migration of enteric neural crest–derived cells during development is normal. However, adult Ts65Dn mice demonstrate reduced colonic motility. Our overarching hypothesis is that downregulation of ionic conductances, specifically, smooth muscle voltage-gated calcium channels by increased oxidative stress results in reduced contractility. In this proposal, we will test this hypothesis in the adolescent (PND 25) and young adult (PND65) Ts65Dn and their disomic littermates. In the first specific aim, we will conduct isometric tension recordings of distal smooth muscle strips in response to neuronal stimulation, acetylcholine induced contractions and calcium influx. In the second specific aim, we will test the hypothesis that trisomy 21 results in altered voltage-gated calcium currents (VGCCs) in smooth muscle in the distal colon, leading to altered smooth muscle motility. We propose to test all aspects of this hypothesis at levels spanning from in vitro gastrointestinal motility assays, to VGCCs functional alterations on individual smooth muscle cells in adolescence and in adults. This comprehensive approach will enhance validation and interpretation of findings through comparisons across these systems, enhancing scientific rigor. Our closely integrated, multidisciplinary, research plan is intended to establish for the first time a generalized framework to understand the mechanisms underlying karyotype-induced changes of bowel smooth muscle function in adolescence and in adulthood using a well-established mouse model of DS. Because these mechanisms are potentially shared across other gastrointestinal abnormalities (e.g. Hirschsprung’s disease), these studies may define a common thread spanning multiple diseases. These newly- defined mechanisms could then be targeted for therapeutic intervention by, e.g., manipulating VGCCs directly or through intervention of newly-defined downstream pathways altered by these interactions.

NIH Research Projects · FY 2025 · 2024-07

Project Summary Illicit drug use is pervasive, yet treatment rates remain disproportionately low. Individuals with substance use disorder (SUD) encounter numerous obstacles to engagement in treatment. Addressing the specific needs of groups least likely to receive treatment requires integrating their perspectives into user-centered design. Between 2015 and 2020, the rate of fatal opioid overdoses in Virginia increased from 10.8 to 25.1 deaths per 100,000, with significantly greater increased mortality for African Americans. There are racial disparities in rates of prescriptions for medications for opioid use disorder and completion of office-based treatment programs. To better understand the needs and barriers faced by adults with SUD in underserved, high-poverty communities in Richmond, VA. In the initial phase of our project, the Virginia Patient Engagement Resource Center (PERC) is set to establish and sustain a patient advisory panel. Comprising approximately 15 members from Richmond and the surrounding region, this panel will include people in recovery as well as peer recovery navigators. Employing diverse engagement strategies, we aim to create an inclusive, accessible, and productive structure, drawing lessons from existing literature and fostering authentic engagement. Collaborating with the panel, our focus will be on building their capacity as consultants to researchers striving to enhance SUD treatment and research. We will implement a consultation process co-developed with the panel to facilitate discussions with researchers regarding their grant proposals, ongoing research projects, and/or emerging research outcomes. The impact of the panel on both its members and participating researchers will be systematically evaluated. Moving into the second phase, the PERC will adopt a community- based participatory (CBPR) approach to investigate patient experiences. The CBPR approach recognizes the importance of engaging stakeholders equitably across all facets of research and is particularly important in addressing the needs of vulnerable and hard-to-reach populations often excluded from health research. Research on patient experiences will include a survey of individuals not yet in recovery as well as qualitative, participatory, arts-based research that aims to empower participants to explore and articulate their experiences. Data and insights gleaned from the research and collaboration with stakeholders will inform the design of responsive interventions or tailoring of existing evidence-based strategies. In the third phase, we will plan and conduct two pilot research projects focused on increasing treatment initiation and retention. Testing strategies proposed by stakeholders, our goal is to establish a proof of concept regarding the impact of patient panels on SUD intervention efforts. Our broader objective is to gather multiple perspectives on the feasibility and accessibility of SUD service delivery to inform program modifications tailored to the needs of underserved patients, ultimately improving treatment initiation and retention rates.

NIH Research Projects · FY 2025 · 2024-07

Despite aggressive use of combination antiretroviral therapy, HIV infection is associated with cognitive and neurobehavioral impairment, collectively termed neuroHIV. HIV and opiates are independently associated with blood brain barrier (BBB) dysfunction, alterations in inflammatory signaling, and cognitive and motor deficits. The HIV viral protein Tat is thought to mediate much of the HIV-associated damage within the brain. This proposal will use both a Tat transgenic mouse model and a murine tropic HIV-infected mouse model. The Tat transgenic mouse model expresses HIV-1 Tat driven by a GFAP promotor, which limits expression to the CNS. The two models will be used to examine the effects of HIV and morphine in vivo. Our central hypothesis is that opiates compromise BBB function and contribute to neuropathology through complex mechanisms which include enhancing paracellular flux, while paradoxically decreasing net flux of antiretrovirals across the barrier, increasing infiltration of monocytes, and increasing inflammatory signals within the brain. We will address our hypotheses with the following specific aims. Aim 1. Define the effects of opiates ± HIV-1 Tat and HIV infection on BBB integrity and function and on region-specific impact on antiretrovirals and morphine concentrations within the brain. Aim 2. Characterize regional differences in the interplay between opiates ± HIV/HIV-1 Tat and ARVs on macrophage infiltration into the CNS and on proinflammatory cytokine production the CNS. These studies will define and relate the effects of opiates and/or HIV-1 on regional drug accumulation, BBB integrity, drug metabolism/efflux, and immune cell trafficking into and inflammatory signals within the brain. Better understanding of this dynamic interplay, with specific focus on antiretroviral brain concentrations, will improve the current therapeutic approaches for the HIV patients who use opioids (for licit or illicit use). The long-term goal is to understand how opiates limit ARV therapeutic efficacy within the brain in the setting of HIV and to identify targets for therapeutic development to eliminate the negative effects of opiates/HIV on neurocognitive outcomes.

NIH Research Projects · FY 2025 · 2024-07

Despite progress in human papillomavirus (HPV) vaccination uptake, about 3 million women are still diagnosed with precancerous cervical lesions each year in the US. Most spontaneously regress but yearly follow up and invasive prophylactic care (e.g., endocervical curettage and cervical biopsy) is still required as it is not clear which may progress to cervical cancer. Women with more persistent HPV infections and precancerous lesions, as well as a higher incidence of cervical cancer are disproportionately impacted by these invasive prophylactic procedures and their sequelae (e.g., pain, bleeding, scarring, infertility, future miscarriages, and preterm births). The vaginal microbiome (VMB) has been implicated in sustaining HPV infection and development of precancerous lesions. HPV-positive women, and women with precancerous lesions are more likely to have a suboptimal VMB with high taxonomic diversity with depletion of Lactobacillus species (both features of vaginal inflammation and dysbiosis) and are also associated with worse prognosis for precancerous lesions. My preliminary data shows that an optimal, Lactobacillus predominant VMB is protective against the risk of a precancerous cervical lesion for some but not all women. Since the VMB is susceptible to the host micro and macro environments, it is plausible that these differences override the protective effect of the VMB in some women. At the micro level, differences in species-specific metabolic profiles might be contributing to lesion prognosis by disturbing vaginal homeostasis. At the macro level, psychological stress, which is experienced and perceived differently across individuals, may influence the VMB through a cortisol-mediated pathway which has been biologically implicated in VMB dynamics and known to become dysregulated in some women in response to stress. In this K01 application, I will test the hypothesis that daily experiences of stress and corresponding cortisol responses drive changes in the VMB that, in turn, influence differential regression of precancerous lesions. I will measure VMB changes and psychological (ecological momentary assessments), and physiological stress (salivary cortisol) among 180 women diagnosed with a cervical lesion, assessed during the critical time between their routine pap screen and abnormal follow-up. The proposed mentorship, training and research will give me specific critical skills in bioinformatics, statistical and molecular biology to interrogate plausible mechanisms through which the VMB might underlie differential etiologies for pre-cancerous cervical lesions. The training described in this proposal will prepare me to become an independent translational investigator leading to the identification of practical solutions to improve cervical health outcomes.

NIH Research Projects · FY 2025 · 2024-07

Abstract/Summary The stratum corneum contains unique species of the sphingolipid ceramide and that unique constellation is essential for formation of the epidermal permeability barrier and is altered in diseases such as atopic dermatitis. These ceramides are composed of fatty acids linked to a set of sphingoid backbones. The species of sphingoid backbones found in the epidermis are highly unique. The premise of this application is that the unique sphingoid backbones found in the epidermis are essential for formation of a fully functioning permeability barrier and that alterations to that composition contributes to compromised barrier function in skin diseases. These concepts have not previously been experimentally addressed. The sphingoid backbones are generated by the serine palmitoyltransferase (SPT) complex, a hetero-oligomeric enzyme composed of four subunits. Three of these four subunits have alternate isoforms. Depending on which isoforms are expressed, SPT generates distinct sphingoid backbones. Our preliminary data demonstrates that during keratinocyte differentiation there is a dramatic increase in the SPT subunits expected to generate the unique sphingoid bases of the epidermis. The ability to genetically alter the expression of the epidermal SPT subunits provides an avenue to directly test the role of epidermal-specific sphingoid backbones in development of the epidermal permeability barrier. We will utilize both organoid culture systems and the intact animal to test this concept. Additionally, we will utilize an animal model of atopic dermatitis to examine the role of these SPT subunits in the etiology of that disease. Through collaboration between experts in keratinocyte biology and skin disorders (Dr. Paller and the Northwestern SBDRC) and lipidomics/the SPT complex (Drs. Wattenberg and Cowart at Virginia Commonwealth), we will fill key gaps in understanding the epidermal lipid barrier through this regulatory mechanism

NIH Research Projects · FY 2026 · 2024-07

PROJECT SUMMARY Background: Collective migration is a type of cell migrationwhere groups of cells move together in a coordinated fashion that is essential for development and disease progression, including wound healing or metastasis. During collective migration, a few cells at the front (i.e. leader cells) define the leading edge, integrate signals from the surrounding environment and send signals to neighboring (follower) cells. Recent findings in our lab demonstrate in order for collective migration to occur, leader cells must first arrive (or polarize) at the front edge to begin the collective migration cascade. However, it is largely unknown how leader cells interpret signals from the microenvironment via sensing of mechanical forces and how cell junction forces contribute to leader cell activation and collective migration. Thus, there is much interest to understand leader cell mechanotransduction and the signaling mechanisms used to drive collective migration. Hypothesis: Our central hypothesis is that dynamic extracellular matrix cues activate leader cell mechanics via both cell-matrix and cell-cell contacts, are required to initiate and sustain directional collective migration. Goals: This project is divided into 3 main goals: 1. Investigate the effect of biomechanical cues to activate leader cells and directional collective migration 2. Elucidate which and how leader cell mechanics are responsible for leader cell development, and 3. Investigate the functional role of forces at cell contacts and how cell junctional forces contribute to collective migration Study Design: We will combine microfluidic lab-on-a-chip devices which can modulate multiple microenvironment features, and investigate how mechanical cues effect leader cell development and directional collective migration. We will also incorporate FRET-based tension sensors to our microfluidic platform so we can quantify changes in cell-cell load during leader cell driven collective migration in real-time. To understand how cell-matrix interactions effect leader cell development, we will measure matrix deformation rate as exerted by leader cells on the surrounding environment. Furthermore, we will perform series of targeted knockdowns and rescue experiments to investigate our ability to disrupt, prevent, or revive collective migration by disrupting leader cell signaling. These studies will reveal how microenvironment cues, cell-matrix, and cell- cell interactions contribute to leader cell development and mechanics that is essential for collective migration. Impact: Understanding the development of collective migration, and the role of leader cells in driving collective migration will pave the way for accelerated understanding of biological processes where collective migration is fundamental to its success.

NIH Research Projects · FY 2026 · 2024-06

Fentanyl and its analogues are increasingly responsible for drug overdose deaths in the United States and worldwide. As a potent µ-opioid receptor (MOR) agonist, fentanyl is anticipated to exacerbate the pathobiology of neuroHIV and HIV-associated neurocognitive disorder (HAND) via complex mechanisms involving direct peripheral and CNS effects. Unlike typical opioids, fentanyl appears to also target non-opioid receptors causing chest wall muscle rigidity, laryngospasm, and rapid death, and pronounced neuroimmune dysregulation. Our preliminary data show that non-opioid receptor targets, especially α1-, α2-, and possibly β-adrenergic receptors (ARs), widely expressed by astroglia in the striatum, are critical for the unique pathophysiologic effects of fentanyl. We find that acute fentanyl (i) exposure differentially alters the firing rates of dopamine receptor D1- and D2-expressing (D1 and D2) medium spiny neurons (MSNs), while sustained exposure (ii) nearly eliminates spontaneous activity in MSNs if co-cultured with astroglia and (iii) increases inflammatory chemokines and oxyradicals, and that (iv) many of fentanyl's sustained effects are likely mediated by α1AR-expressing astro- and microglia. Xylazine, an α2AR agonist, which is increasingly combined with fentanyl to create “tranq”, is highly excitotoxic to MSNs in mixed glial co-cultures and appears to have much more severe pathophysiologic effects in neuroHIV models than the prototypical opioid morphine, or than fentanyl alone. We hypothesize that, in addition to its effects at MORs and unlike typical opioids, fentanyl exacerbates HIV-induced MSN dysfunction and injury through actions at αARs and that its unique pathological effects are mediated by α1AR-, α2AR- (especially with xylazine), but not β2AR-, expressing astro- and microglia. This hypothesis will be tested in the following Aims: Aim 1 will identify the MOR-, α1 and α2AR, and βAR mechanisms by which fentanyl (with some comparisons to morphine) and Tat and infectious and replicative- deficient HIV cause D1 and D2 MSN excitotoxicity and synaptodendritic injury in vitro. The role of MORs, α1ARs, and α2ARs will be assessed in studies of MSN-mixed-glial co-cultures and human MSN iPSCs using confocal microscopy and whole-cell and perforated-patch physiologic approaches. Aim 2 will determine the MOR- and α1 and α2-AR mechanisms by which fentanyl and HIV interact to disrupt D1 and D2 MSN synaptodendritic structure and function in control and Tat tg mice in vivo and using whole-cell neurophysiologic approaches in ex vivo slices. Aim 3 will determine the MOR- and α1 and α2AR mechanisms by which fentanyl and xylazine (“tranq”) combinations and Tat/HIV interact to disrupt D1 and D2 MSN synaptodendritic structure and function in vitro and in vivo/ex vivo. This project will systematically explore and compare novel interactions of fentanyl with HIV using well-established models used previously to characterize the effects of the prototypical MOR-agonist opioid morphine in neuroHIV. If confirmed, our findings will establish a conceptual framework for understanding how fentanyl, its synthetic analogues and xylazine impact neuroHIV.

NSF Awards · FY 2024 · 2024-06

The IEEE International Conference on Mobile Ad-Hoc and Smart Systems (MASS) is a premier annual forum for sharing original, novel ideas in mobile ad-hoc and smart systems. The proposed travel grant aims to provide financial support to 10-15 graduate or undergraduate students who will attend IEEE MASS 2024, which will be held in Seoul, South Korea between September 23-25, 2024. Reasons to support this request include providing the students with the opportunity to: o Interact with top researchers within their research domain; o Learn, listen, and exchange ideas with other students working in their research domain to build future collaborations; o Learn what is considered state-of-the-art in design, implementation, analysis, evaluation, and deployment of computer systems and applications of mobile ad-hoc and smart systems.; and o Attend topic specific workshops held in the conference. This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.

NIH Research Projects · FY 2025 · 2024-06

Project Summary Signal transducers and activators of transcription (Stats) are transcription factors with critical roles in the immune and endocrine systems. While there is considerable knowledge of Stat- mediated gene control, we know far less about lineage-restricted Stat functions. Understanding cell-specific actions is necessary for progress in diseases impacted by Stat dysfunction – including cancer, infertility, metabolic disorders, immunodeficiency, and chronic inflammation. We have evidence that Stat5B is pivotal for mast cell function. Like several other Stat proteins, Stat5B forms dimers and tetramers. We find that Stat5B dimers and tetramers have some similar and some opposing functions. Dimers and tetramers promote cytokine secretion. But while Stat5B dimers promote mast cell histamine production and cell migration, tetramers appear to inhibit both functions. These data are consistent in vitro and in a model of systemic anaphylaxis. Therefore, our hypothesis is that Stat5B dimers and tetramers have distinct effects on mast cell function, with tetramers often limiting the effects of dimers. Understanding this fundamental biology has considerable translational potential. Therefore, we propose creating several strains of mice that allow lineage-specific deletion of Stat5B or selective expression of a Stat5B mutant that can form protein dimers but not tetramers. These mice will make possible detailed studies of Stat5B, allowing us to understand how this protein contributes to the function of specific cell types.

NIH Research Projects · FY 2025 · 2024-06

PROJECT SUMMARY Periodontal disease (PD) is an inflammatory condition in which immune dysregulation causes inflammation and eventually results in tissue destruction. The onset of PD is caused by the outgrowth of pathobiont organisms that activate chronic immune stimulation. A numerous and complex variety of bacteria participate in the etiology of PD. One such member of the pathobiont community in disease is Selenomonas sputigena, the subject of this proposal. Numerous studies have reported that S. sputigena is associated with disease. Despite this, no other published research has directly investigated how the bacterium causes PD. Recently, our group has published the first ever characterization of how S. sputigena promotes inflammation in gingival keratinocytes. This study reports that S. sputigena activates the secretion of a broad range of important pro-inflammatory mediators. Importantly, this prior study also displays that S. sputigena stimulates immune cell recruitment; of which is an essential hallmark of PD etiology. In preliminary data reported here, we show that S. sputigena invades gingival epithelial cells (GECs). Here, we propose to elucidate i) the mechanism by which S. sputigena is endocytosed by GECs, ii) the biochemical and endosomal characteristics of the S. sputigena-enclosing vesicle, and iii) the intracellular lifecycle of the organism, including the time frame of bacterial viability and subsequent exocytosis. We hypothesize that S. sputigena is endocytosed by GECs via a clathrin-dependent mechanism and is ultimately trafficked to the nuclear membrane in a late endosome-resembling multivesicular body. Afterwards, we hypothesize that the organism survives within the endosome via inhibition of lysosomal fusion and exits the cell via ESCRT-dependent exocytosis. We propose two integrated Aims to investigate this hypothesis. In Aim 1, to investigate initial invasion, we will directly modulate clathrin-dependent endocytosis via pharmacological inhibitors. Also, in Aim 1 we will utilize immunofluorescence microscopy to observe S. sputigena vesicular trafficking during invasion. Last, we will perform transmission electron microscopy (TEM) in order to identify defining features of the S. sputigena-enclosing vesicle. In Aim 2 we will identify how long S. sputigena remains alive inside the cell. Also, we will investigate exocytosis of the S. sputigena-enclosing vesicle via immunofluorescence microscopy and, in so doing, identify a mechanism by which the bacterium may escape to re-infect naïve cells. Finally, we will infect a three-dimensional epithelial tissue model with S. sputigena to investigate tissue invasion via cell to cell spread. The data resulting from these Aims will elucidate the intracellular life cycle of S. sputigena in GECs. Thorough characterization of this process will establish the foundation through which to identify specific interactions between the bacterium and intracellular constituents. In this manner, the precise mechanism by which S. sputigena stimulates inflammation in GECs can be investigated and novel therapeutic targets can be considered. Understanding how S. sputigena causes PD will further the collective knowledge of how periopathogens interact with the host and will progress the current understanding of how the members of the complex plaque biofilm cause disease. Last, researching host-pathogen interactions in S. sputigena will allow for experiments that investigate bacterial community interactions between S. sputigena and more robustly characterized periopathogens like Porphyromonas gingivalis.

NIH Research Projects · FY 2025 · 2024-06

Multiple BTB-ZF family transcription factors are essential for immune system development and/or essential immune system functions. Some BTB-ZF transcription factors function, essentially, as “toggle switches” that control key decision points during development. For example, the commitment of lymphocytes into the T or B cell lineage requires LRF and commitment to the CD4 or CD8 T cell lineage requires ThPOK. Loss of expression of the necessary BTB-ZF gene results in loss of cell lineage identify and dedifferentiation. Other BTB-ZF family members control essential or unique effector functions of lymphocytes. Zbtb20, for example, defines the function of natural IL-10 producing Tregs that are necessary for intestinal homeostasis. Bcl6 is required for both the development of T follicular helper cells and germinal center B cells and Zbtb32 is required for the proliferative burst of NK cells in response to viruses. In this application, we show that the function of PLZF, the BTB-ZF transcription that controls the phenotype and innate like effector functions of invariant natural killer (NKT) cells, requires Yin Yang 1 (YY1). YY1 is a multi-faceted transcriptional regulator that belongs to the Polycomb protein family. YY1 is ubiquitously expressed and is important for multiple, diverse biological processes. YY1 functions to regulate gene transcription directly, via chromatin modifications, via direct interactions with other transcription factors and even by impacting higher order functions such as by 3D chromatin looping or phase separation. The goal of this application is to discriminate between different possible mechanisms by which YY1 controls PLZF. In doing so, we anticipate that we will significantly advance the understanding of the regulation of BTB- ZF master regulator transcription factors. We anticipate that we will also learn more about how the ubiquitously expressed, multi-faceted transcription regulator YY1 controls discrete biological functions in different cell types. Advances in these areas would fill substantial knowledge gaps and have the potential to impact both the understanding of immune responses and, most likely, other biological systems where BTB-ZF genes play important roles. Overall, our application seeks to understand how a lineage specific transcription factor (PLZF) is controlled by a ubiquitously expressed co-factor (YY1) resulting in lymphocyte subset discrete effector functions.

NIH Research Projects · FY 2025 · 2024-06

Medicaid expansion covers over 18 million low-income patients, including those experiencing an intensive care unit (ICU) stay during serious illness. More than half of ICU survivors have new or worsening problems with cognitive, physical, and mental health. Further, experiences of critical illness worsen health outcomes. This study applies the NIMHD’s Research Framework to understand how sociocultural environment, the built environment, and the health system domains of influence at the patient, interpersonal and community-level, differentially shape ICU survivorship among patients covered by Medicaid expansion. Such knowledge will enable state and federal policymakers and clinicians to provide improved coordination of medical and social services for Medicaid members at the greatest risk of poor health outcomes. This work will leverage the resources of a well-established partnership between Virginia Medicaid and Virginia Commonwealth University (VCU) and as well as complementary methods and data sources to better understand how health determinants shape patients’ recovery after an ICU stay. Aim 1 assess how patient level and community-level sociocultural, built environment and health system domains are associated with post-ICU hospital free days among Medicaid patients in Virginia using Virginia Medicaid claims data and publicly available community level-data on community resource and health system access. Aim 2 uses qualitative research methods to expand beyond what is available in claims data by interviewing 25 Medicaid patients to explore their perspectives on how health determinants shape their experiences during and after ICU stays at VCU Health System, the largest safety net provider in Virginia. Aim 3 examine whether including health determinants improves machine learning models predicting hospital readmissions after an ICU stay using electronic health record data from VCU Health System. The proposed research is supported by Virginia Medicaid, aligning with the agency’s commitment to improving care for complex patients. Ultimately this work will provide much-needed evidence to policymakers exploring disparities in recovery after serious illness across the sociocultural environment, built environment and health system domains. This application describes an excellent research and training environment for the proposed work, supported by a collaborative multi-disciplinary mentorship team. This proposal will not only explore a novel research area, the recovery of Medicaid patients after serious illness, but also provide integrated and robust clinical and research training in health-policy translation, qualitative methods and community-engaged research and machine learning to a physician-scientist dedicated to practicing critical care medicine and pursuing a faculty position with a focus on improving health outcomes through health policy.

NIH Research Projects · FY 2025 · 2024-06

PROJECT SUMMARY/ABSTRACT We aim to evaluate a novel approach for effective treatment of nitrogen mustard (NM)-induced corneal injury in rat and rabbit models. NM is a bi-functional analogue of sulphur mustard (SM). Ocular exposure of NM causes a range of complications including corneal ulceration, corneal opacity, chronic inflammation, corneal neovascularization, and even blindness. Therapeutic interventions to treat vesicants induced ocular injury has not been identified yet, which highlights the need to identify effective treatment options for vesicants-induced corneal injury. Dexamethasone sodium phosphate (DSP) eye drop is FDA approved drug for the treatment of ocular inflammation associated with various agents. Studies have reported beneficial effects of dexamethasone eye drops in vesicants-induced corneal injury in the rabbits. However, due to the rapid tear turnover and clearance of instilled drops and poor ocular bioavailability, frequent administration is necessary which results in poor patient compliance. To address these problems, we developed biodegradable nanoparticles of DSP with poly(lactic-co-glycolic acid) (PLGA) or poly(lactic acid) (PLA), that can be administered by subconjunctival (SCT) injection after NM exposure and provide sustained release of DSP, improving patient’s compliance and increasing therapeutic efficacy. We hypothesize that long-lasting PLA-DSP-NP (3 months in vitro drug release) could be effective in preventing corneal ulceration, corneal NV, corneal opacity, suppressing inflammatory and angiogenic biomarkers in both immediate (0 h post NM exposure) and delayed treatment (24 h post NM exposure). In Aim 1, we plan to investigate the dose-dependent efficacy of PLA-DSP-NP dosed at both 0 h and 24 h post NM exposure in the corneal injury rat model. In Aim 2, we will evaluate the efficacy of PLA-DSP-NP at a selected dose after immediate (0 h post NM exposure) and delayed treatment (24 h post NM exposure) in a more translational NM-induced corneal injury rabbit model.

NIH Research Projects · FY 2026 · 2024-06

PROJECT SUMMARY/ABSTRACT Cigarette smoking and nicotine dependence account for more than 7 million deaths per year worldwide. A major challenge for treatments targeting smoking cessation is relapse: Most people who attempt to quit return to smoking within months. When individuals stop smoking, being in environments where they used to smoke, like bars, coffee shops, or bus stops, can trigger cravings for nicotine and lead to a return to smoking. This is known as context-induced relapse, where context refers to the physical space or social setting associated with drug use. These associative memories between contexts and nicotine are thought to be formed and retrieved by the hippocampus and dependent on nicotinic acetylcholine receptors (nAChRs), specifically β2 containing-nAChRs (β2*-nAChRs). However, the specific neuronal mechanisms underlying these nicotine-context associations in the hippocampus and their modulation by β2*-nAChRs are not yet fully understood. Studying the involvement of the hippocampus in learning these associations presents challenges, as it requires tracking the activity of the same neurons over extended periods of time (days to weeks). To address this, I propose to use large-scale imaging techniques that allow for long-term recording of neural activity in awake and behaving mice, combined with computational tools to uncover the underlying neural dynamics. My central hypotheses are 1) that the formation of nicotine-context memories in the hippocampus requires the binding of nicotine to β2*-nAChRs 2) this binding, also enables hippocampal neurons to form stable memory representations of the nicotine-context with repeated learning and 3) that stable memory traces are re-activated during context re-exposure leading to relapse. To test these hypotheses, I will combine a novel behavior paradigm to assess context-induced relapse behavior using virtual-reality in mice with two-photon imaging of large populations of hippocampal neurons. With this technique, I will track activity of the same neurons during nicotine-context learning and memory retrieval. Further, I will integrate this activity imaging with genetic techniques and pharmacological manipulations to causally examine the role of β2*-nAChRs. Through this comprehensive approach, my research aims to provide a detailed understanding of how nicotine-driven memory representations in the hippocampus contribute to context-induced relapse, at a spatial and temporal scale that has not been achieved before. This mechanistic understanding of neural computations in the hippocampus will potentially help identify novel therapeutics and relapse-prevention strategies. To achieve these goals, I will receive mentorship and support from a group of experts in various fields, including hippocampal memory processes (Dr. Mark Sheffield), nicotinic receptor function (Dr. Daniel McGehee), task design and animal behavior (Dr. Rick Bevins), genetic techniques (Dr. Xiaoxi Zhuang) and computational analysis of large-scale neural recordings (Dr. Jason MacLean). This mentored training will facilitate my transition to become an independently funded investigator, where I will continue to study the neurobiological mechanisms underlying drug-dependence and relapse.

NIH Research Projects · FY 2025 · 2024-06

A pathogenic immune response targeting aquaporin-4 (AQP4) causes neuromyelitis optica spectrum disorder (AQP4 NMOSD), a disabling neurological illness that leads to blindness and paralysis. Recent advances in treatment for AQP4 NMOSD have improved its prognosis but are associated with increased risk of serious infections. As an autoimmune neurological illness with a well-defined autoantigen, AQP4 NMOSD should be amenable to a cure by antigen-specific ablation or tolerization. However, preclinical testing of antigen-specific tolerizing therapy for AQP4 NMOSD remains hampered by the lack of an animal model that recapitulates the autoimmunity and the pathology in the same animal. Current animal models of AQP4 NMOSD require adoptive transfer which introduces confounding variables that are difficult to resolve. The objective of this proposal is to develop a model that recapitulates the autoimmunity and the pathology of AQP4 NMOSD in the same animal and thereby develop an animal model suitable for preclinical testing of antigen-specific tolerization therapies. Aim 1.1 will establish anti-AQP4 autoimmunity in the AQP4 null (AQP4.lacZ) mouse. Aim 1.2 will test two methods to restore AQP4 expression in the AQP4.lacZ mouse: 1) AAV-mediated AQP4 transduction and 2) inducible (tamoxifen) and conditional (GFAP promoter) Cre recombinase system to restore AQP4 expression in the AQP4.lacZ mouse. Aim 1.3 will test the hypothesis that restoring AQP4 expression following immunization against AQP4 in the AQP4 null mouse will result in AQP4-directed autoimmunity and pathology in the same animal. The successful completion of the proposed research is expected to deliver a new animal model that more fully recapitulates the autoimmunity and the pathology of AQP4 NMOSD, and thereby overcome the shortcomings of the current models. Once this model is developed, it will make a positive impact by facilitating preclinical studies aimed at developing antigen-specific ablation/tolerization therapies as cure for AQP4 NMOSD.

NIH Research Projects · FY 2025 · 2024-06

PROJECT SUMMARY. Opioid use disorder (OUD) is a critical problem that contributes to the spread of HIV and may intrinsically worsen neuroHIV pathology. Although the prevalence of antiretroviral therapy (ART) has improved the lifespan and quality of life of persons infected with HIV (PWH), the viral protein HIV-Tat is still present in the central nervous system (CNS) of many PWH on ART. Further, almost half of PWH still experience cognitive deficits and worsened substance misuse outcomes. Opioids can interact with HIV and HIV-Tat to promote inflammation and exacerbate neuronal damage and dysfunction, leading to increased behavioral and cognitive deficits, via µ-opioid receptors (MOR) expressed on different cell types. Medium spiny neurons (MSNs) within the striatum are uniquely vulnerable to the combined effects of opioids and HIV/Tat, and associated with opioid and HIV/Tat deficits in motivation and reward. The spiraling inflammation between astro- and microglia is driven by astroglial CCL5-CCL2 inflammatory signaling and is critical to opioid exacerbation of HIV neuropathology. However, the role of MOR on each cell sub-type are unknown. We hypothesize that the neuroinflammatory response to combined opioid and HIV/Tat, resulting in increased neuronal damage and aberrant behavior is largely mediated by MOR activation on astroglia. Aim 1 during the K99 phase will characterize the impact of astroglial MOR on opioid and HIV-Tat-induced astroglial pathology and dysfunction in vitro. MOR astroglial-null astroglia will be used to assess neuroinflammation and glutamate buffering, and MSN survival and morphology. and assessed for opioid reward and in vivo astroglial calcium transients while freely behaving and awake. Aim 2 (K99) will delineate the loss of MOR activation on astroglia on opioid and HIV-Tat-induced astroglial pathology and dysfunction in vivo. Tat-tg mice will be crossed with MOR astroglial- null mice and assessed for reward and in vivo astroglial calcium transients while freely behaving and awake. Dysfunction will be correlated with neuroinflammation and neuropathology. The above mentored training in genetically encoded indicators and in vivo calcium imaging combined with advance career development training will prepare Dr. Nass for the independent R00 phase and be essential in her pursuit of an independent research program investigating the mechanisms of substance misuse and neurovirology-induced behavioral dysfunction. Aim 3 (R00) will determine if astroglial activation is required for opioid and HIV-Tat-induced astroglial destabilization and MSN neuropathology. Tat-tg mice with attenuated astroglial function will be assessed as described in Aims 1 and 2. The proposed studies will further our understanding of the mechanisms by which MOR signaling on specific cell sub-type mediates opioid exacerbation of HIV/Tat- induced inflammation and neuropathology within the reward circuitry and identify possible targets for therapeutic interventions.

NIH Research Projects · FY 2026 · 2024-05

SUMMARY Regulated exocytosis is a conserved eukaryotic process that is essential for numerous cellular functions, making it a prime target for exploitation by both infectious and non-infectious diseases. Indeed, various malignancies, viruses, bacteria, and protozoa commandeer this pathway to promote disease progression. Numerous studies have interrogated the molecular players of regulated exocytosis that are modulated in cancer formation, including the conserved second messengers, calcium (Ca2+) and sphingosine-1-phosphate (S1P). However, far less is known about the mechanisms that intracellular bacterial pathogens employ to exploit this process. Human granulocytic anaplasmosis is an emergent disease caused by the obligate intracellular vacuolar-adapted bacterium, Anaplasma phagocytophilum (Ap). Ap is a tick-transmitted pathogen that exhibits an unusual tropism for neutrophils. A primary function of neutrophils is to destroy microorganisms through degranulation, a form of regulated exocytosis. Intriguingly, both Ap-infected neutrophils and ticks exhibit prolonged degranulation and increased exocytosis, respectively. We recently published that the Ap- containing vacuole (ApV) resembles a host multivesicular body (an organelle that undergoes regulated exocytosis) and that late-stage ApVs mobilize to and fuse with the plasma membrane to release Ap into extracellular milieu for reinfection. Our preliminary results indicate that intracellular Ca2+ concentration ([Ca2+]i), sphingosine kinase 1 (SK1 [produces S1P]), and S1P are increased late in Ap infection and that inhibition of Ap protein synthesis prevents these phenomena as well as ApV exocytosis. These data suggest that Ap manipulates regulated exocytosis for egress. We also uncovered a potential mechanistic role for the Ap effector, P130, in this process. P130 is upregulated during late Ap infection and localizes to the cytosolic face of the ApV membrane. We discovered that P130 interacts with host calcium and integrin-binding protein 1 (CIB1), which, to date, has been indirectly implicated in regulated exocytosis. CIB1 also localizes with P130 on exocytosing ApVs, further supporting its role in Ap dissemination. In Aim 1, we will interrogate the relevance of [Ca2+]i and S1P to ApV exocytosis and infectious progeny release using in vitro and in vivo approaches. In Aim 2, we will identify the P130 domains necessary for its interaction with CIB1 and its promotion of ApV exocytosis as well as determine the relevance of CIB1 to ApV exocytosis in vitro and in vivo. This work will advance our fundamental understanding of the mechanisms that intracellular bacterial pathogens utilize to facilitate their release and potentially define novel roles for CIB1 in host cell biology and infectious disease.