Wake Forest University Health Sciences

NIH Research Projects · FY 2026 · 2026-06

PROJECT SUMMARY/ABSTRACT Botulinum neurotoxins (BoNTs) are the most potent biological threats known, with estimated human lethal doses as low as 0.1–10 ng/mL depending on serotype and exposure route. BoNTs act by entering motor neurons and cleaving SNARE proteins essential for neurotransmitter release, resulting in flaccid paralysis and, in severe cases, lethal respiratory failure. Although naturally occurring cases of botulism are rare, the potential for deliberate or accidental exposure presents a serious public health risk. BoNTs are classified as Tier 1 Select Agents due to their extreme potency, ease of dissemination, and capacity to overwhelm health care infrastructure. Currently, the only FDA-approved treatment is an equine-derived immunoglobulin antitoxin administered post-exposure to neutralize circulating toxin. However, antitoxin has a narrow therapeutic window and cannot reverse paralysis once toxin is internalized into neurons—consequently, most patients still require prolonged mechanical ventilation to survive. We recently demonstrated that the FDA-approved voltage-gated potassium channel (VGKC) blocker, 3,4-diaminopyridine (3,4-DAP), reverses symptoms of botulism by increasing acetylcholine release at intoxicated neuromuscular junctions. In rodent and non-human primate models of lethal BoNT exposure, 3,4-DAP improves respiratory function, restores neuromuscular transmission, and prolongs survival. Based on these findings, we are currently funded by the Department of Defense to pursue regulatory approval for 3,4-DAP under the FDA Animal Rule. However, two key limitations restrict its broader clinical use: (1) seizure liability due to CNS penetration, and (2) highly variable blood levels due to polymorphisms in N-acetyltransferase-2 (NAT2), its primary metabolic enzyme. These limitations are primary bottlenecks to its use as a medical countermeasure for botulism exposure under emergency conditions or in austere settings. To address this critical gap, we propose to develop next-generation aminopyridine (AP) analogs that retain the therapeutic efficacy of 3,4-DAP while overcoming its pharmacological and pharmacogenetic liabilities. Our preliminary studies in BoNT-intoxicated rodents demonstrate that structurally diverse AP analogs lacking NAT2-susceptible arylamines can reverse respiratory paralysis in vivo. These studies establish preliminary safety and efficacy for new AP drugs and support the underlying hypothesis that the core AP pharmacophore is amenable to structural modifications that optimize therapeutic benefits. Many of these analogs show efficacy at doses that are safe and well-tolerated in mice. Here, we propose a systematic structure-guided screening and optimization strategy to identify, characterize, and advance AP analogs for eventual regulatory approval.

NIH Research Projects · FY 2026 · 2026-06

PROJECT SUMMARY Background: Compared to the rapid pace of genomic discoveries, the adoption of genomics-informed health applications lags. According to the NHGRI’s Strategic Vision for 2030, there is a critical need for implementation science approaches to bridge the gap between discovery and real-world application. Our recent NIH portfolio analysis and systematic review found that less than 2% of genomics grants and peer-reviewed publications integrate implementation science frameworks. To fully leverage genomic advancements, a workforce trained in applying implementation science to genomics-informed health applications is essential. Goal: The Training Institute for the Implementation of GEnomics Research (TIGER) aims to increase the implementation science capacity for genomics by training investigators across the US. The program will prepare investigators to effectively implement genomics research in real-world settings, ensuring widespread availability of genomics-informed health applications, with an emphasis on populations with limited resources. Approach: Aim 1: Expand and implement TIGER to train a cohort of 32 fellows annually (160 over the five-year grant period). TIGER is a hybrid program combining virtual lectures with an in-person workshop. It is targeted at postdoctoral associates and research faculty applying genomics in the prevention and treatment of disease. Fellows will be paired with mentors to build competencies in implementation science tailored to their research. We will work with the established Genomic Data Science Community Network to recruit a high-quality pool of fellows with a broad range of skills, perspectives, and backgrounds. Aim 2: Continuously evaluate and improve the TIGER program. A robust evaluation plan, guided by a logic model, will track data from fellows, including surveys, assessments, and program metrics, to refine the curriculum and mentorship process. Aim 3: Disseminate TIGER nationally to build a network of genomics implementation science champions. Course materials will be made publicly accessible, and findings will be shared via conferences, publications, and social media. TIGER alumni will be provided with networking opportunities to foster ongoing engagement, and they will be equipped with tools to disseminate TIGER knowledge. Innovation and Impact: TIGER is a pioneering educational initiative designed to address the cross-cutting need for advanced training in the application of implementation science to genomics. This program is significant because it will promote the incorporation of implementation science methods to adopt genomics in real world settings where evidence-based interventions exist but are not commonly applied. We will offer this training using innovative methods for engaging adult learners to build pragmatic skills that can be applied to a range of clinical research and quality improvement programs at learners’ home institutions. Our approach to training delivery is accessible (hybrid design), rigorous, and allows for continuous refinement and improvement through a robust evaluation plan.

NIH Research Projects · FY 2026 · 2026-06

PROJECT SUMMARY Over 19 million older Americans have obesity, which is associated with disability and poor clinical outcomes. Clinical trials by our group and others show that diet-induced weight loss interventions, particularly when combined with exercise, improve body composition and physical and metabolic function over the short-term in older adults with obesity. However, clinical recommendations for obesity treatment in older age are controversial, as weight loss can exacerbate bone and muscle loss and the long-term risks have not been extensively studied. Musculoskeletal losses could increase the risk of osteoporotic fractures, which confer significant morbidity and mortality. Osteoporosis is assessed clinically using dual energy X-ray absorptiometry, but bone mineral density (BMD) measured by this method is often inaccurate in those with obesity and weight loss. Quantitative computed tomography (QCT) offers a more robust method that reduces measurement errors. Further, bone strength – a QCT and finite element modeling assessment of 3D bone morphology, volumetric BMD, and cortical thickness – is a stronger predictor of fracture risk than BMD alone. Nonetheless, the legacy effects of caloric restriction (CR) and exercise (EX) on biomechanical bone strength in older adults remain largely unquantified. Our proposed project leverages our unique access to five NIH-supported trials that randomized older adults (baseline age ≥60; BMI≥27 kg/m2) to CR+EX vs. EX alone at Wake Forest from 2005 to 2014, and the Health Outcomes after Participating in Exercise (HOPE) study (R01 AG056418; PI: Houston), which recently completed a follow- up of these participants ~10 years after they completed the intervention. Long-term follow-up QCT scans acquired for 272 HOPE participants will be analyzed to determine the long-term effects of prior randomization to CR+EX vs. EX alone on hip and lumbar spine bone strength [Aim 1]. We hypothesize that randomization to CR+EX will result in lower bone strength at long-term follow-up versus EX alone (controls). Aim 1 will also examine the effect of exercise modality on bone strength following CR, and we hypothesize that participants randomized to CR with aerobic training will have lower bone strength at long-term follow-up compared to participants randomized to CR with resistance training. Leveraging measures acquired at baseline, end of the intervention, and long-term follow-up, we will determine associations between change in lean mass and physical performance with long-term bone strength [Aim 2]. We will also assay stored blood specimens from three time points to explore associations between changes in biomarkers of bone formation (P1NP) and bone resorption (CTX-1) with long-term bone strength [Aim 3]. This work will be the first randomized, controlled design to test the legacy effects of caloric restriction and exercise interventions on bone strength in older adults. Results will inform obesity treatment guidelines for older adults and may identify new early musculoskeletal screening biomarkers and intervention targets to shift clinical paradigms for fracture prevention.

NIH Research Projects · FY 2026 · 2026-06

Project Summary Qualitative research methods are valuable tools in oncology research because they allow investigators to delve into the human experience of cancer, uncover patient and provider perspectives, and explore complex issues surrounding the disease. Qualitative research is particularly valuable for studying socially and ethically sensitive topics in oncology and enhancing patient- and family-centered care. Yet, many of those conducting oncology research do not have training in qualitative research methods. Thus, the lack of qualitative training coupled with the potential benefits of employing qualitative method in cancer research leaves a knowledge and skill gap that is the focus of the proposed cancer research education program, Qualitative Training Institute for Enhancing Cancer Research (CancerQTI). CancerQTI is a six-month hybrid skills development program to increase knowledge and utilization of state-of-the-science qualitative research methods to address the prevention, screening, treatment, rehabilitation, and continued care of cancer. CancerQTI is designed to provide the foundation for clinician scientists and oncology researchers to use rigorous qualitative research methods to enhance their cancer research programs. The overarching goal of CancerQTI, led by the Qualitative and Patient-Reported Outcomes Shared Resource (Q-PRO) of Atrium Health Wake Forest Baptist Comprehensive Cancer Center, is to increase the use of rigorous qualitative research methods to address the prevention, screening, treatment, rehabilitation, and continued care of cancer. Utilization of qualitative methods in oncology research has the potential to improve outcomes for cancer patients and their families. Program objectives are: (1) Recruit and enroll 25 oncology researchers annually into CancerQTI; (2) Utilize adult learner concepts from Kolb’s Experiential Learning Theory to provide participants with training and knowledge to increase their skills and use of state-of-the-science qualitative methods; (3) Provide mentoring to program participants five months post-training to support participants application of qualitative methods to their own research; (4) Assess long-term outcomes through a participant reporting system to track utilization of qualitative research methods among program participants; and (5) Conduct ongoing course evaluation and refinement to continually improve the program. CancerQTI, a cancer-specific training program for increasing the use of qualitative methods, has the potential to augment the success of research across the cancer care continuum. This state-of-the-science training program will improve the ability of participants to conduct high- quality qualitative research to prevent, diagnose, and treat cancer, ultimately decreasing the burden of cancer. Q-PRO is well-poised to deliver this unique, comprehensive, cancer-focused, national qualitative research training program.

NIH Research Projects · FY 2026 · 2026-06

Project Summary/Abstract Obesity is associated with poor quality of life and reduced lifespan and healthspan – the period of time free of multiple chronic diseases and disability. Given that over one-third of older adults are obese, identifying effective therapies that prevent obesity-related declines in healthspan and lifespan in older adults are urgently needed. Clinical trials by our group and others show that caloric restriction improves physical and metabolic function over the short-term in older adults with obesity. However, the long-term benefits of caloric restriction in this population remain controversial and weight loss is often not recommended because of uncertainty of whether the benefits outweigh the risks (e.g., loss of muscle mass and bone). The field of geroscience aims to address biological aging by targeting the fundamental biology shared by the aging process to prevent or delay common age-related chronic diseases in hopes of extending healthspan and lifespan. Caloric restriction is one such intervention known to alter aging biology to extend healthspan and lifespan in multiple species, including non-human primates; however, the effects of caloric restriction in extending healthspan and lifespan in humans remains unknown. The overall goals of the proposed study are to determine if short-term caloric restriction in older adults with obesity affects biomarkers of biological aging and whether these biomarkers of biological aging are correlated with healthspan. We will determine the effects of randomization to caloric restriction on a consensus derived blood-based biomarker index (Il-6, TNFαR1, GDF-15, cystatin C, CRP, and insulin; primary aim) and multimorbidity and deficit accumulation frailty indices (secondary aim) an average of 10 years after the completion of a caloric restriction intervention. Our primary hypothesis is that randomization to caloric restriction will result in improved biomarkers of biological aging compared to a control condition in older adults with obesity and this improvement will be reflected in better healthspan. We will leverage biospecimens and data from 5 NIH-supported randomized controlled trials conducted under the auspices of the Wake Forest Pepper Center that enrolled older adults (mean age at randomization, 67.3 years) with overweight or obesity (BMI≥27 kg/m2) and randomized them to caloric restriction (n=520) or no caloric restriction (n=446) from 2005 to 2014 to conduct an individual participant-level meta-analysis with sufficient sample size to definitively evaluate the effect of caloric restriction on biological aging. We will also explore the effects of randomization to caloric restriction on cellular senescence and proteomic biomarkers and the underlying molecular mechanisms of caloric restriction, as well as the associations between cellular senescence and proteomic biomarkers on multimorbidity and a deficit accumulation frailty index. The proposed study builds on the Wake Forest’s Pepper Center’s collaborative research focus in geriatric obesity treatment to answer compelling and clinically important questions regarding the efficacy of caloric restriction to slow biological aging and increase healthspan in older adults with obesity in an efficient and cost-effective manner.

NIH Research Projects · FY 2026 · 2026-06

PROJECT SUMMARY/ABSTRACT Botulinum neurotoxins (BoNTs) are highly potent biological neurotoxins produced by Clostridium botulinum, causing botulism, a disease characterized by flaccid paralysis. Structurally, matured BoNTs comprise a ~ 100 kDa heavy chain (HC) and a ~50 kDa light chain (LC) linked by a disulfide bond. The HC consists of a receptor- binding domain (HCC) and a translocation domain (HCN), which mediate neuronal targeting and endosomal escape. At the same time, the LC is a zinc-dependent endopeptidase that disrupts synaptic transmission by cleaving SNARE proteins required for the synaptic fusion event. Despite the extreme toxicity of BoNT, no pharmacological antidote exists. The current treatment is post-exposure prophylaxis with equine-derived Heptavalent Botulinum AntiToxin (HBAT) combined with mechanical ventilation and supportive care. HBAT neutralizes toxins in the circulation but is ineffective once the toxin has been internalized into neurons, an event that marks the onset of clinical symptoms. The combination of its potency and lack of an antidote to reverse its effects after the onset of the symptoms has led to its classification as a Select Agent by the U.S. Centers for Disease Control and Prevention (CDC) due to its potential for bioterrorism. These factors underscore the urgent need to develop an effective antidote to mitigate the threat posed by BoNTs. One major challenge in developing effective antidotes is the existence of many BoNT isoforms, grouped in at least seven different serotypes (indicated by letters A, B, C, D, E, F, and G), each comprising multiple subtypes with varying degrees of amino acid sequence differences. The heterogeneity of the toxin’s molecular target, which influences both the degree of neurotoxicity and susceptibility to neutralization, together with the intracellular localization of the toxic LC metalloprotease, further complicates the design of broadly effective post-symptomatic anti-botulinum therapeutics.To overcome the barriers to developing BoNT antidotes, we propose to create Post-Symptomatic Anti-Botulinum Therapeutics (PSABT), which combine broad-spectrum nanobodies that potently inhibit the catalytic activity of BoNT LC for all subtypes of serotypes A and B, fused to a non-toxic toxin-based neuronal delivery vehicle that mimics the same trafficking pathway as the native toxin, and enables the intracellular delivery of immunotherapies for the treatment of BoNT intoxication. Given that BoNT/A1 and BoNT/B1 are the most long-lasting and prevalent subtypes in botulism cases, which are also present in commercially available products, we propose developing antitoxins targeting all eight subtypes of serotype A and all eight subtypes of serotype B.

NIH Research Projects · FY 2026 · 2026-05

PROJECT SUMMARY C9FTD/ALS is the major genetic cause of frontotemporal dementia (FTD) and amyotrophic lateral sclerosis (ALS). It is characterized by expansion of a G4C2 repeat in the first intron of the C9ORF72 gene. Multiple factors emerging from the mutant C9ORF72 chromatin locus contribute to molecular and cellular pathology, including production of harmful RNA transcript variants (containing the repeat), loss of transcripts encoding C9ORF72 protein, and aberrations at the chromatin level due to unusual structure, modification, or protein interactions. Although the genomic context and transcript landscape at the C9ORF72 locus are complex and influenced by the repeat expansion, the locus may lend well to gene-targeted approaches that can modulate the epigenetic state. A body of literature supports a positive role for DNA methylation at the C9ORF72 locus, such as reducing hallmarks of molecular disease and even improving patient outcomes. Methylation of the repeat expansion sequence, flanking regions, and the promoter has been correlated with reduced production of repeat-containing transcript variants and poly-dipeptide repeats (poly-DPRs) translated from them. Deletion of the C9ORF72 promoter was shown to eliminate detection of a harmful transcript variant, as well as poly-DPRs, and surprisingly preserve most of the expression of the primary C9ORF72 protein coding transcript. We propose to induce methylation of the C9ORF72 promoter and the repeat expansion sequence using CRISPRoff, a CRISPR-based dCas9-methyltransferase fusion system. CRISPRoff can deposit CpG methylation for targeted and heritable gene silencing with little or no reported off-target methylation. CRISPRoff does not induce DNA cleavage and can be delivered transiently. Methylation of the repeat sequence may be allele-selective for the expansion since G4C2 repeat tracts are relatively rare in the human genome. Thus, the potential safety profile of sequence-specific epigenetic editors like CRISPRoff could be favorable for C9FTD/ALS. Site-specific methylation at the C9ORF72 locus will enable investigation of gene expression and regulatory mechanisms to help determine whether epigenetic modulation is a potential candidate for therapeutic intervention. To assess the impact of methylation at the C9ORF72 locus via CRISPRoff, we will employ model patient-derived cell lines and quantify gene expression from the C9ORF72 locus, characterize DNA methylation status with nanopore sequencing, and evaluate the ability of CRISPRoff to correct disease- associated cellular defects, including global gene expression, repeat RNA foci, and mitochondrial dysfunction. If successful, this study will offer new insight into epigenetic regulation of C9ORF72 as well as a potential new therapeutic direction for C9FTD/ALS.

NIH Research Projects · FY 2026 · 2026-05

Project Summary/Abstract: Diet is a main determinant of gut microbial diversity. Our group demonstrated diet shifts breast microbiome populations. In a non-human primate (NHP) model, our group showed that consumption of a Western or Mediterranean diet modulated breast tissue microbiota and microbial-derived metabolite profiles. The shift in the bacterial-produced metabolome was specific to breast tissue and was not observed in circulating plasma samples, suggesting tissue-specific regulation of the metabolite levels. Re-analysis of data grouping subjects by body adiposity along with diet indicates obesity and diet work in tandem to shift the breast microbiome and microbial- derived metabolome. Specifically, we show that % body fat negative correlated with breast tissue hippurate levels in NHP. Hippurate is one of the most abundant microbial–host co-metabolites. Conventional housed mice display 17-fold higher plasma hippurate concentrations than germ-free mice, demonstrating the reliance on bacteria for this metabolite generation. Hippurate exerts its biological signaling through a G-protein coupled protein receptor 109A (GPR109A), and literature indicates that GPR109A activation modifies lipid metabolism and inflammation. Our preliminary data indicates that breast cancer patients with obesity display reduced hippurate concentrations in their non-cancerous breast tissue than lean women, validating our NHP data demonstrating adiposity-associated microbial metabolome perturbations. Moreover, we show exogenous administration of hippurate reduced tumor progression in a syngeneic 4T1.2ER+ and genetic MMTV-PyMT mammary tumorigenesis model. The purpose of this study is to investigate the impact of microbiota-derived metabolite hippurate-mediated GPR109A signaling to reduce obesity-mediated breast cancer risk. We hypothesize that obesity and consumption of a Western diet reduces hippurate bioavailability, decreasing GPR109A receptor activation to promote inflammation, lipid metabolism, and mammary tumorigenesis. Exogenous supplementation of hippurate in Western diet-fed animals will reduce breast cancer risk and improve therapeutic responsiveness to improve outcomes. The following aims address the hypothesis: Aim 1: Investigate whether obesity shifts the bacterial-associated metabolome in breast cancer patients; Aim 2: Determine the impact of microbial-derived metabolite GPR109A receptor signaling on obesity-mediated ER+ breast cancer risk; and Aim 3. Investigate whether GPR109A ligands promotes therapeutic efficacy in metastatic ER+ breast cancer. Successful completion of this proposal will determine the functional relevance of key microbial-processed metabolites to reduce obesity-mediated breast cancer risk. We will also identify potential microbiota-produced metabolites that could be exogenously administered to promote therapeutic efficacy to improve breast cancer outcomes.

NIH Research Projects · FY 2026 · 2026-04

PROJECT SUMMARY/ABSTRACT The eye is the most sensitive organ to chemotoxic injury from sulfur mustard (SM) and chemically related analogs. Long-term ocular prognosis after high-dose SM exposure is poor, with up to 90% of casualties developing a debilitating constellation of corneal symptoms known as mustard gas keratopathy (MGK). MGK typically involves corneal neovascularization, photophobia, dry eye, chronic ocular pain, and corneal ulceration and can lead to progressive corneal decompensation that ultimately results in impaired vision. There is no specific treatment to prevent or reverse MGK. We developed a rabbit SM vapor exposure model to reproducibly study ocular injury progression in response to clinically relevant SM exposures. Using this translational platform, we characterized the dose-dependent effects of SM vapor on the acute lesion and subsequent injury progression. We found that high-dose SM exposure produces an acute lesion that, in turn, activates a variety of pathological adaptive responses in the cornea that culminate in MGK. We hypothesize that understanding the pathological cellular and molecular pathways involved in these pathological adaptive responses represents a potential therapeutic pathway to mitigate or prevent MGK onset. In fact, preliminary studies suggest that treatments that block or reduce acute edema have strong therapeutic effects on corneal injury for at least 12 weeks. Our goal is to use a combination of single-cell RNA sequencing, immunohistochemistry and in situ hybridization to determine the cellular determinants involved in the transition from the acute lesion to MGK. To conduct these studies, we will expose rabbit eyes ex vivo and in vivo to SM vapor, isolate corneal cells at various times after exposure and perform single-cell RNA sequencing analysis to determine (1) which cell types are present in the cornea as the injury progresses and (2) how gene expression patterns change within each cell type as the injury progresses. As a control, we will expose the corneas using a smaller vapor cap, which produces full-thickness corneal lesions but not MGK. If successful, these studies will elucidate cellular changes associated with MGK onset versus corneal healing. They will also identify potential therapeutic targets and therapeutic windows for the comprehensive treatment of vesicating corneal injuries. These data would represent a significant step toward broad-spectrum treatments that reverse corneal pathophysiologies and promote recovery from vesicant-injured eyes.

NIH Research Projects · FY 2026 · 2026-03

SUMMARY While hydrogen peroxide has long been understood as a toxin used by the human immune system to kill infectious organisms, only recently has it become well accepted that it serves as a second messenger in eukaryotes, produced in response to growth factors, cytokines and immune system effectors and modulating downstream signal transduction pathways. Through insights contributed in part by the work of the PI, a family of cysteine-dependent, peroxide-reducing enzymes known as the peroxiredoxins (Prxs) have become widely recognized not just as one of the primary oxidant removal systems in all organisms, but also as key modulators of cell signaling pathways. PI Poole’s work on the enzymology, structures and dynamics of Prxs has contributed greatly to understanding the mechanism and regulation of this widespread and highly abundant family of enzymes. In 2003, PI Poole and collaborator Andy Karplus published a Science paper in which the “floodgate hypothesis” explaining the potential benefits of a peroxide-mediated “off switch” (hyperoxidation or other modification) was proposed; under conditions where peroxide levels begin to rise (e.g. NADPH oxidase activation), Prx inactivation would promote the local accumulation of peroxide near the source, allowing for the oxidation of alternative protein targets. This intriguing perspective remains a topic of debate, while in the meantime several examples of Prx-mediated signaling through redox relays initiated by Prx oxidation have come to light. In fact, the “off switch” of hyperoxidation is not the only modification modulating Prx activity and hyperoxidation sensitivity. Proteomics evidence suggests a number of posttranslational modifications (PTMs) occurring on Prxs, but much remains to be discovered regarding the molecular and biological consequences of the PTMs that may be present and may be associated with disease states. Recently, in collaboration with Ana Denicola in Uruguay, we reported that nitration near the C-terminus of Prx2, caused by peroxynitrite treatment, led to a more resilient (less hyperoxidation prone) peroxidase. As shown by kinetics data and modeling by this same team, a threshold effect due to a change in rate limiting step as peroxide levels rise leads to accumulation of oxidized forms above a peroxide set point which is distinct for each Prx studied. We are also increasingly discovering that Prxs play key metabolic and signaling roles in multiple areas important to disease progression and treatment, including radiation resistance, chemotherapy toxicities and inflammatory signaling. In the next years, we will continue investigations of the contributions of varying levels and posttranslational modifications to modulating the antioxidant activity and signaling capacity of Prx3, the key Prx in mitochondria. Also continuing will be our biophysical and kinetic analyses of Prxs amenable to high resolution studies by X- ray crystallography and NMR. Together, these efforts will address areas important to Prx function, protein oxidation and biomedical applications, enabling further research and biomedical interventions.

NIH Research Projects · FY 2026 · 2026-03

SUMMARY: Dopamine, serotonin, and norepinephrine neurotransmitters are known to be critically involved in process underlying substance use disorder and psychiatric illness, as well as healthy motivated behavior, decision-making, and learning. However, little is known about how these signals coordinate and modulate subjective feeling and motivate behavior as mammals (including humans) navigate the world. Progress has been hindered by a lack of technology that permits fast, real-time, measurements that can discriminate and track dopamine, serotonin, and norepinephrine release simultaneously in areas of the brain where two or more of these neurotransmitters are co-released. A major challenge to current methods (e.g., fast scan cyclic voltammetry) is that the calibration models use to interpret in vivo data are trained in vitro and it is unclear how the background signal changes between these environments and how this affects the measured responses. This proposal capitalizes on (and seeks to radically improve) a technological innovation developed by the principal investigator, which resulted in the first successful colocalized measurements of dopamine and serotonin release with sub-second temporal resolution from the brains of consciously behaving humans. Here, we pursue two specific aims, which seek to develop a computational approach to extend these kinds of measurements to include simultaneous detection of norepinephrine and make these methods available for a larger area of preclinical animal model research and human clinical neuroscience research. In both aims we will be testing the overarching hypotheses that 1) the ‘background’ signal present in fast scan cyclic voltammetry measurements can be quantitatively characterized, mathematically modeled, and therefore subtracted using a model-based approach in in vivo research paradigms; and 2) that the “in vitro bias” in the mathematical models used in model-based electrochemistry can be corrected for if we can obtain a better characterization of the background signals in each of the in vivo, ex vivo, and in vitro conditions. The experiments and analyses proposed will begin to provide much needed clarity on the impact biological ‘interferents’ have on interpreting in vivo fast scan cyclic voltammetry data – currently the only approach amenable to sub-second multi-neurotransmitter detection in humans. We expect to develop mathematical models and calibration methods that can be used to predict and control for unwanted interfering signals while significantly improving detection methods for multi-neurotransmitter detection. Notably, these advances – to be shared via open-source online repositories – would accelerate ongoing efforts in the field aimed at understanding how dopaminergic, serotonergic, and noradrenergic systems coordinate to motivate behavior in humans and pre-clinical model organisms, and thereby provide insight into mechanisms underlying human mental health.

NIH Research Projects · FY 2026 · 2026-02

PROJECT SUMMARY Nutritional consumption of an essential lipid nutrient, the omega-3 docosahexaenoic acid (DHA), is inversely correlated with numerous diseases, yet due to its rarity in western diets an estimated 80% of the US population is nutritionally deficient in DHA. Because DHA is well documented to provide many health benefits, the exciting prospect that dietary lipid augmentation may be an effective therapeutic strategy to prevent and treat disease has been of therapeutic interest. However, effective therapeutic progress has been stymied by critical limitations in the field, including: 1) a fundamental gap in our understanding of how DHA metabolism is regulated in pre- and post-disease states; and 2) a lack of strong pre-clinical models to mechanistically test the effects of DHA metabolism on health. This research team has overcome these barriers by targeting an enzyme, long-chain acyl-CoA synthetase 6 (ACSL6), to generate a novel preclinical model with a ~50% deficit in DHA specifically in neurons independent of dietary manipulations. Preliminary data demonstrate the critical role of lipid metabolism in health evidenced by accelerated disease due to the loss of ACSL6. The team proposes to test the overall hypothesis that ACSL6-mediated DHA metabolism protects against disease. This work will (Aim 1) determine the role that DHA metabolism plays in neurological health, derived the regulatory metabolism that controls the accrual and turnover of neural DHA, and (Aim 3) test the novel hypothesis that DHA- lipid mediators in the membrane-bound state serve as a readily available pool of protective agents. (Aim 2) determine The proposed work will unmask fundamental breakthroughs, resolve long-standing unknowns, and test novel hypotheses regarding the importance of DHA metabolism in pressing aspects of health and disease.

NIH Research Projects · FY 2026 · 2026-02

Project Summary/Abstract Sarcopenia, the progressive loss of muscle mass and function, universally impacts the aging population, contributing to loss of mobility and independence in older adults. With the rapid growth of the aging population, there is an urgent need for effective strategies against sarcopenia. Exercise is the most effective intervention that increases muscle mass and function in younger adults, but its benefits are attenuated in older adults due to diminished responses to exercise training. Over the years, several candidate molecules, including metformin, antioxidants, and resveratrol were combined with endurance exercise (EE) in an attempt to increase training- induced benefits; however, these interventions failed to enhance training responses and, in some cases, reduced the positive effects of exercise. Given metformin’s inhibitory effects on mitochondrial respiration and the ability of antioxidants to scavenge signaling molecules in response to exercise, a logical next step is to combine EE with a candidate molecule that specifically enhances mitochondrial bioenergetics and its downstream pathways. A strong such candidate is unacylated ghrelin (UnAG), whose circulating levels are significantly decreased in older adults and mice. Our pilot data provides evidence that UnAG combined with endurance exercise (UnAG+EE) additively increases mitochondrial bioenergetics, while synergistically improving motor coordination, muscle mass and contractile function. Our data also revealed that UnAG activated both shared (i.e., PGC-1α) and distinct (i.e., mTORC2) pathways compared to those triggered by EE alone, suggesting that UnAG+EE may provide synergistic and complementary benefits. Building on our compelling preliminary data, we hypothesized that the combination of UnAG+EE will enhance mitochondrial bioenergetics and preserve NMJ integrity in older mice, providing synergistic protection for sarcopenia. Our specific aims will focus on the effects of UnAG, EE, and UnAG+EE on mitochondrial bioenergetics and oxidative stress (Aim 1), motor neuron cell numbers and inflammation in the spinal cord and neuromuscular junction (Aim 2), and muscle mass, function, and protein quality in skeletal muscle (Aim 3) in older mice. We will also assess additive or synergistic effects of UnAG+EE on key endpoint measures for each aim. Transcriptomic analysis will be performed to identify differentially expressed genes and pathways influenced by our interventions, which can provide essential information for mechanistic investigations in the future. If our results support the hypothesis, UnAG+EE will be the first combination therapy that shows additive or synergistic effects for sarcopenia. UnAG+EE also has great potential for translation, given the established safety of UnAG and its mimetics in humans. Finally, our proposal aligns directly with the current Strategic Directions of the NIA, “Develop effective interventions to maintain health, well- being, and function, while preventing or reducing the burdens of age-related diseases, disorders, and disabilities.

NIH Research Projects · FY 2026 · 2026-02

PROJECT SUMMARY Aging is characterized by the gradual loss of physiological integrity, and this process may be accelerated in the presence of obesity, increasing susceptibility to disease, frailty, and death. Although the shared molecular pathways involved have not been fully elucidated, adipose tissue dysfunction is likely a key contributor to multisystem functional decline in aging and obesity. Despite growing evidence that β3 adrenergic receptor {β3AR) mediated activation of brown adipose tissue {BAT) may alter pathophysiological pathways implicated in various aging-related diseases including metabolic, cardiovascular, and neurodegenerative diseases, BAT has been largely ignored in aging research. In this highly innovative study, we propose to conduct a randomized, double-blind, placebo-controlled trial to investigate whether treatment with a β3AR agonist (vibegron) can improve energy metabolism, cardiometabolic risk factors, and physical and cognitive function. Vibegron {Gemtesa) was FDA-approved in 2020 for the treatment of overactive bladder and has greater selectivity, potency, and activity at the β3AR than other agonists studied to date. This presents a timely opportunity to explore pharmacological activation of β3ARs as a way to improve multiple health outcomes relevant in aging and obesity. To test our hypothesis, 40 middle-aged and older adults {45-75 yrs) with obesity will be randomized to vibegron {75 mg/day) or placebo for 12 weeks to compare their effects on various bioenergetic, cardiometabolic, physical function, and cognitive outcomes. Specifically, in Aim 1 we will assess the effects of vibegron vs. placebo on energy expenditure, core body temperature, mitochondrial bioenergetics, and thermogenic protein expression. In Aim 2 we will assess the effects of vibegron vs. placebo on glucose and insulin indices, lipid levels, body composition, and body fat distribution. In Aim 3 we will assess the effects of vibegron vs. placebo on self-report and objective measures of lower extremity function, muscle strength and pOY1er, global cognition, memory, executive function, quality of life, and depression. Notable innovations include blood-based bioenergetic profiling to assess systemic mitochondrial function, isolation and characterization of adipose tissue-derived small extracellular vesicles to assess target engagement, and continuous monitoring of core body temperature to assess circadian thermoregulation. In exploratory analyses we will compare the effects of vibegron vs. placebo on the accumulation of health deficits {i.e., frailty) and the preservation of physical and mental abilities {i.e., intrinsic capacity), two integrated measures of phenotypic aging that will provide estimates of the potential for vibegron to impact multisystem functional decline. This unique study will be the first clinical trial to explore the potential to repurpose vibegron for the treatment of aging-related obesity and associated comorbidities. If successful, the results of this study will be used to inform the design of a larger, longer trial to confirm the efficacy of vibegron as a novel treatment to IOY1er risk for multisystem functional decline in both aging and obesity.

NIH Research Projects · FY 2026 · 2026-01

PROJECT SUMMARY/ABSTRACT Patients with brain metastasis of lung cancer have extremely poor prognosis, high mortality rate, and frequent incidence of tumor recurrence. Understanding the pathological mechanism of brain metastasis is urgently needed to develop a novel and effective therapeutic strategy. Published data as well as the results of my own study for brain metastasis of lung cancer indicate that smoking and nicotine significantly increased the incidence and progression of brain metastasis, but the pathological mechanism by which the smoking promotes brain metastasis through modulating brain microenvironment is yet poorly understood. Our preliminary results showed that synaptic formation in brain metastasis region is strongly correlated with poor overall survival of patients with brain metastasis. However, the exact role of neurons in brain metastasis progression remains unclear. The goal of this proposal is to elucidate the mechanism of GABAnergic neuron activation in the brain metastasis in order to develop innovative strategies for the treatment of brain metastasis. I hypothesize that nicotine stimulates microglia to secrete exosomal miR-32-3p which promotes brain metastasis by augmenting GABAergic synaptic formation and hence releasing GABA that serves as metabolic substrate to fuel tumor cell growth. I also hypothesize that inhibiting the GABA transporter of tumor cell suppresses brain metastasis by blocking GABA shunt. In Aim 1, I will clarify the molecular pathway through which nicotine stimulates microglia to secrete exosomal miR32-3p and activates GABAergic neuron. In Aim 2, I will investigate the pathological mechanism by which the activated GABAergic neuron enhances brain metastasis by promoting GABA shunt of tumor cells. Aim 3 is to test the efficacy of inhibitors for GABA transporter on nicotine-stimulated brain metastasis. The K99 phase of the proposed research will be pursued at Wake Forest University (WFU), an interactive cancer biology community, and a wealth of intellectual and technical resources. The training plan, under the mentorship of Dr. Watabe, outlines a comprehensive strategy for acquiring the technical and the professional skills required to complete the proposed research and prepare me for an independent research career. Experienced six members of my mentor team will provide training in new techniques and analyses of data. By taking advantage of Wake Forest‘s exceptional resources for professional development, I will also improve my skills for communication, management, and leadership. The training I obtain at WFU will equip me to lead a laboratory that merges diverse approaches to investigate the mechanisms of initiation and progression to clinically significant brain metastasis and identify innovative strategies for prevention and treatment of brain metastasis.

NIH Research Projects · FY 2025 · 2025-09

The Wake Forest University School of Medicine (WFUSM) Research and Education in Alzheimer’s Disease and Dementias Initiative for Excellence (READDIE; pronounced “ready”) Program is designed to facilitate postbaccalaureate trainees’ entry into and completion of PhD programs in Alzheimer’s Disease and related dementias (ADRD). READDIE leverages our longstanding experience with development of successful programs that prepare post-baccalaureates and undergraduates to attain the PhD, and the unique combination of existing strengths and resources of the WFUSM’s Alzheimer’s Disease Center, Claude D. Pepper Older Americans Independence Center, and Maya Angelou Research Center for Healthy Communities. A one-year training plan will begin in the summer, to allow students to acclimate to the program and select their research mentors and projects prior to the beginning of coursework. Activities during the year will enhance progression towards a PhD program by demonstrating research and academic proficiency prior to fall application dates, with a second year available if needed to solidify science identity and self-efficacy as ADRD researchers. We will expose trainees to varied career paths related to ADRD research, coupled with innovative career counseling to facilitate longterm retention in ADRD research. The READDIE goals are to 1) engage post-baccalaureate scholars in mentored research experiences and related activities to capture and further stimulate enthusiasm for research in ADRD and brain health, and to develop the necessary critical thinking and analytical skills to conduct research that will foster greater science identity and confidence in the research setting; 2) expose scholars to a rigorous graduate research environment with strengths in team science, community engagement, and community health research; 3) equip scholars with key professional and technical skills necessary for graduate study; and 4) enroll scholars into rigorous graduate research programs for successful completion of the PhD or MD/PhD in ADRD research areas. Over the past 10 years, READDIE program faculty have served as advisors for 40% of the WFU Graduate School (WFUGS) Biomedical Sciences PhDs awarded in ADRD/related fields. Research mentors are well-funded (>$500,000 average in ADRD science per investigator) and 55% have formal mentoring training. Specific Measurable Objectives are to have: >95% of READDIE Scholars apply to PhD programs; >75% enroll in PhD or MD/PhD programs related to ADRD; >95% retention in post-READDIE PhD or MD/PhD programs; and 90% retained in ADRD research-intensive or research-related careers. For over 30 years, WFUSM college and post-baccalaureate programs have increased the number of students entering and completing PhD programs at WFUGS and nationally, through dedication and substantial expertise in research skills training, professional development, socialization and continued mentoring, with a demonstrated record of successful implementation and continuous improvement of our training programs.

NIH Research Projects · FY 2025 · 2025-09

PROJECT SUMMARY Diet composition potently modulates trajectories of brain aging. Intake of a "Mediterranean" diet including fruit and vegetables, fish, and healthy fats is associated with reduced risk of Alzheimer's disease and related dementias including vascular dementia. Intake of a "Western" diet high in simple sugars and saturated fat is associated with increased risk of these conditions. Nonhuman primate models of diet composition effects on brain health are critical for providing insight into mechanisms of diet effects on the brain, because of challenges associated with long-term studies of diet manipulations in humans. Our previous work showed Western diet consumption (compared to Mediterranean) increased social isolation and anxiety-related behaviors, increased gray matter volume in an Alzheimer's-related temporal-parietal cortex meta-region of interest, reduced white matter volume, and resulted in lateral temporal transcriptional profiles associated with inflammation in middle- aged female cynomoglus monkeys. However, because cognitive function was not assessed in these monkeys, the interpretation of changes in biomarkers as reflecting a nascent pathological process or, alternatively, a resilient adaptation remains unclear. For example, elevated gray matter volume in monkeys consuming the Western diet may reflect impaired function associated with inflammation, or a reactive change to preserve function and promote resilience in the face of a poor diet. Furthermore, males were not studied in our previous work. To address these critical barriers to progress in the field, we propose to test the impact of Mediterranean vs. Western diet on cognitive function and translational imaging and fluid biomarkers of Alzheimer's disease risk, neuroinflammation, and neurodegeneration in middle-aged male and female cynomolgus monkeys, in a longitudinal design spanning 32 months of diet treatment, corresponding to ~9.5 human years. Our overarching hypothesis is that consumption of Western diet interacts with aging trajectories to exacerbate cognitive impairment with aging and produce pathological brain aging, whereas Mediterranean diet promotes resilient aging, preserved cognitive function, and reduction of markers of Alzheimer's disease risk, including fluid biomarkers of neurodegeneration and neuroinflammation, and PET imaging measures of microtubule stability and neuroinflammation. We expect that within and across diet treatment groups and sexes that elevated markers of neuropathology will predict cognitive decline. This project specifically examines vulnerability of middle-aged monkeys, a time in the lifespan where trajectories of aging take a turn towards successful aging or greater risk of neurodegenerative disease, with immense translational significance.

NIH Research Projects · FY 2025 · 2025-09

PROJECT SUMMARY In 2023, Alcohol Use Disorder (AUD) affected an estimated 28.9 million people, about 10.5% of the American population ages 12 and older. AUD has consistently been a public health crisis for many decades, costing the US economy $249 billion in 2010. The current treatments available are not effective, further warranting continued work on better understanding the mechanisms and circuits involved in AUD-related behaviors. There are many factors that contribute to excessive alcohol use, however, one major component that could be contributing to the development of AUD is stress. While regular stress can drive problematic alcohol consumption, traumatic stress heightens this correlation and increases the probability of AUD development. Despite this interaction between stress and alcohol, the circuitry recruited during stress and its persistent drive on alcohol consumption and negative affect is still largely unknown. The insula is a highly connected region that is associated with crucial roles in emotional processing and cognitive control. The insula participates in the brain’s salience network by conveying interoceptive cues to guide behavior through cortical and subcortical connections. Human imaging studies suggest a decrease in insular volume in alcohol dependent patients, further implicating the insula’s larger role in AUD-related behaviors. We have shown that the insula is important in regulating negative affective states during abstinence and coping behavior during a stressor. To better understand the insular circuitry recruited during stress, we aimed to identify all upstream inputs onto stress- activated insula neurons using a FosTRAP mouse model and a retrograde viral approach with whole-brain light sheet microscopy. Insular stress ensembles receive the densest projections from the basolateral amygdala (BLA), a region involved in modulation of anxiety-like behaviors, alcohol consumption, fear conditioning, and emotions. This proposal aims to characterize the role of the BLA-insula circuit in traumatic stress-induced negative affective states, binge-like, and aversive drinking by using validated preclinical models of PTSD and alcohol drinking, Single Prolonged Stress (SPS) and drinking in the dark (DID), respectively. We will first establish the BLA-insula pathway’s role in stress valence and AUD-related behavior by using chemogenic manipulation during SPS and probing for affective states and alcohol consumption following stress. Next, we will correlate the physiological stress response with neuronal activity in the BLA-insula pathway during withdrawal from alcohol. To complement, we will use in vivo fiber photometry to measure calcium activity in the BLA terminals present in the insula during drinking following stress. We predict valence of traumatic stress is encoded by the BLA-insula pathway, leading to the initiation and maintenance of alcohol consumption, even in aversive conditions. The studies proposed here will provide a more complete perspective on the interaction between stress and the development of AUD, leading to fruitful contribution to better our treatment options.

NIH Research Projects · FY 2025 · 2025-09

PROJECT SUMMARY Alzheimer's disease (AD) is a progressive neurodegenerative disorder marked by memory loss and cognitive deficits. Although reductions in dopamine within cognitive and reward circuits have been seen in post-mortem AD brains, changes in dopamine transmission remain under-researched, particularly in combination with chronic ethanol (EtOH) exposure. Neuropsychiatric symptoms (NPS), such as depression and apathy, are increasingly recognized as early signs of AD and have been linked to the disease's onset and progression. The presence of NPS is associated with a higher risk of transitioning from mild cognitive impairment to AD. Interestingly, NPS are also common in alcohol use disorder (AUD). Dopamine, a crucial regulator of motivation and decision-making, also plays a significant role in learning and memory processes. Deficits in dopamine transmission are strongly linked to apathy and are thought to contribute to other NPS. Dopamine deficits are a known consequence of AUD, and are also thought to occur in AD. Dopamine dysfunction may connect the neuropsychiatric and cognitive symptoms observed in both AD and AUD. The adverse effects of AUD on AD progression are well-established. Preclinical research, including studies from our lab, has shown that chronic EtOH exposure reduces dopamine release and increases its uptake, resulting in hypodopaminergia. This study will examine how dopamine transmission changes throughout progression of AD-associated pathology and the effect of EtOH exposure. Human AD studies have identified decreased dopamine in various neuronal circuits, and correcting these deficits is gaining interest as a method to slow AD symptom progression. Targeting proteins involved in dopamine release and reuptake is one approach, with Synaptogyrin-3 (Syngr-3) being a novel promising candidate. Syngr-3 is a synaptic vesicle protein highly expressed in limbic brain regions. In a mouse model of AD, Syngr-3 levels in the nucleus accumbens (NAc) were found to be reduced. Similarly, there was a negative correlation between EtOH intake and Syngr-3 expression, suggesting that reductions in Syngr- 3 could be a common underlying mechanism of hypodopaminergia in both EtOH and AD. The central hypothesis of this project is that hypodopaminergia contributes to the rapid progression of AD symptoms, and this is exacerbated by EtOH exposure. Genetic manipulation of Syngr-3 levels will be used to document its contribution to hypodopaminergia and AD symptoms.

NIH Research Projects · FY 2025 · 2025-09

PROJECT SUMMARY Among the six percent of active ethanol drinkers who develop an alcohol use disorder (AUD), there is great variability in etiology and relapse rates. Early and accurate identification of individuals at risk of developing an AUD would facilitate effective deployment of personalized interventions. Recent evidence demonstrates that individuals with low cognitive flexibility and a predisposition towards habitual behaviors are at an increased risk for future heavy drinking. However, the underlying molecular mechanisms driving these differences in cognition and the accompanying higher risk for drinking remain unknown. Growing evidence suggests that an epigenetic signature, either inherited or acquired in life, predisposes individuals to risky addictive behaviors. However, cross-sectional studies cannot reliably identify this pre-existing risk and distinguish it from an induced effect of chronic alcohol use. Attempts to identify biomarkers of AUD have focused on easy-access tissues, specifically peripheral blood. While these studies are important, their ability to inform on the specific molecular mechanisms in the brain are limited. To overcome this limitation, we conducted a longitudinal genome-wide DNA methylation (GW-DNAm) analysis of the rhesus macaque dorsolateral prefrontal cortex area 46 (dlPFC-A46) collected before alcohol use. The dlPFC is a component of the executive network and is critical for cognitive function and decision making. With alcohol-naïve dlPFC-A46 samples from 11 male macaques, we identified 3,539 differentially methylated regions (DMRs) that were associated with future ethanol drinking levels (following 12 months of drinking). The genes mapped by these DMRs are enriched in neurogenesis, neuronal differentiation, axonogenesis, synaptic plasticity and glutamatergic neurotransmission. These results suggest that, in an alcohol-naïve state, the variability in DNAm signatures represents a vulnerable neural state primed for future problematic drinking. In this application, we seek to identify more robust molecular signatures that explain pre-existing risk for excessive alcohol consumption by increasing the sample size (n = 54), completing cell-sorting to reduce noise from cellular heterogeneity, and including both males and females. Furthermore, we will include functional studies to better understand the regulatory function of these DMRs and evaluate their impact on gene expression in different cell type populations. This proposal will elucidate, for the first time, the pre-existing neural molecular signatures priming the brain of alcohol-naïve individuals for future risk of excessive alcohol consumption. To increase translatability, we will characterize the methylomics and metabolomics of blood samples collected from the same subjects. In totality, this work will translate behavioral phenotypes into neural markers of risk for AUD and hold promise for parallel discoveries in risk for other disorders involving impaired cognitive flexibility.

NIH Research Projects · FY 2025 · 2025-09

Summary Osteoarthritis (OA) is influenced by factors like joint injury, age, and gender. Chronic pain is the primary debilitating symptom, and current treatments are ineffective and do not reduce disease progression. Understanding OA and developing therapies is challenging because pain does not always correlate with joint damage. Targeting mechanisms that regulate joint tissue integrity and inflammation simultaneously may be necessary to alleviate pain and limit further physical deterioration. Our research identified the role of IRE1α- XBP1 in inflammatory pain and showed that leukocyte-specific IRE1α deficiency reduced pain and inflammatory mediators. We have uncovered that XBP1 transactivates NUPR1, which, when expressed in chondrocytes, leads to apoptosis and cartilage degradation. Furthermore, our studies demonstrate that global deletion of NUPR1 reduced OA pain and structural changes in mice, suggesting that the IRE1α-XBP1-NUPR1 axis regulates inflammation and tissue damage in OA. Thus, we hypothesize that IRE1α-XBP1-NUPR1 drives OA by promoting inflammatory programs in synovial immune cells while fostering joint cartilage damage via catabolic changes in chondrocytes. We will implement our plan through these specific aims: 1. Define how IRE1α-XBP1-NUPR1 signaling in immune cells and chondrocytes differentially contributes to synovial inflammation, cartilage damage, and OA pain. Two knee OA models (an injured model and an aging model) will be used in conditional knockout (cKO) mice lacking IRE1 or NUPR1 exclusively in leukocytes (Vavcre Ern1fl/fl) or chondrocytes (Col2a1cre Ern1fl/fl). Pain related behaviors and structural and molecular changes in the knee will be evaluated over time. 2. Determine how the IRE1α-XBP1-NUPR1 axis programming affects immune cell and chondrocyte function to drive OA joint structural damage, inflammation, and sensory neuron hyperactivity. We will use transgenic reporter mice that exhibit Venus-green fluorescent protein expression upon IRE1α-XBP1 activation to define the anatomical, cellular, and temporal IRE1α activation in the injury-link OA knee and study the functional phenotype of FACS sorted immune cells based on IRE1α activation. Then, we will discern how intrinsic leukocyte or chondrocyte IRE1α (cKO) sculpts the transcriptomic landscape and pathological microenvironment of the OA joint, and how IRE1α dictates leukocyte-chondrocyte interactions via single cell RNA sequencing and interactome bioinformatic analyses. 3. Test the translational hypothesis that inhibiting IRE1α or NUPR1 prevents or limits the progression of OA. We will use pharmacological blockers of IRE1α or NUPR1 to prevent or reverse OA-related pain behaviors and joint structural damage. Our project will establish IRE1α-XBP1-NUPR1 axis as a disease modifying target that would reduce pain and enhance joint functionality. Our unique transgenic mouse lines enable precise dissection of this specific OA molecular mechanism. Our multidisciplinary team is uniquely equipped to successfully complete these studies as we combine expertise in neuroimmunology and pain, cartilage and chondrocyte biology, and genomics coupled with advanced bioinformatic analytical tools.

NIH Research Projects · FY 2025 · 2025-09

Inflammation plays a major role in the progressive pathology of cystic fibrosis (CF), and is generally thought to be a response to increased microbial colonization of CF lungs. However, recent studies involving normal and CFTR-mutant ferrets raised under broad-spectrum antibiotics show robust inflammation in the CF lung despite the absence of bacterial pathogens. Moreover, while the revolutionary class of CFTR modulators improve lung function and reduce exacerbations, they are less successful in mitigating inflammation of the CF lung. These findings raise the possibility that inflammation, and perhaps other pathogenic features of CF, are maintained by elements that emerge in the disease but then drive progression independent of CFTR activity. An analogous scenario may be operating in chronic obstructive pulmonary disease (COPD), where inflammation and disease progression continues despite smoking cessation. In COPD, recent studies have shown a strong correlation (p<10-16) between the emergence of pro-inflammatory, small airway epithelial cells and the disease itself. These pathogenic variants are also present, albeit at low levels, in control patients without COPD and in fetal lung. A similar analysis of CF lungs has revealed them to be inundated by pathogenic stem cell variants highly related to those seen in COPD, along with two novel, hyperinflammatory variants not previously identified in COPD lungs. We hypothesize that these CF stem cell variants play key roles in the progression of CF, and represent pathogenic elements of this disease triggered by, and yet independent of, the CFTR genotype. To test this hypothesis and extend our understanding of the potential significance of these variants in CF disease processes, we will, in three specific aims, 1) identify key inflammatory drivers in the three, hyperinflammatory human CF variants using CRISPR-Cas9-directed mutations and xenograft models, 2) test the dependence of the pro-inflammatory phenotype of these three variants found in CF patients on CFTR activity using gene complementation and CFTR-modulating drugs such as ivacaftor, elexacaftor, and tezacaftor, and 3) exploit our recently developed methods for cloning ferret airway stem cells to determine the dynamics of the pathogenic variants in a ferret conditional model of CF progression. We anticipate that these studies will provide context and insight into the contributions of variant stem cells that dominate CF lungs, assess the impact of the new CF therapeutics on the pathogenic features of these cells, as well as identify nodal genes in the inflammatory signatures of these variants whose suppression could be of therapeutic benefit to these patients.

NIH Research Projects · FY 2025 · 2025-09

PROJECT SUMMARY Alcohol use disorder (AUD) afflicts millions of individuals and their families. Current treatment options and clinical diagnostic tools are limited, necessitating more research on the progression from casual use to AUD in certain individuals. Identifying pre-AUD biomarkers, as is common practice with heart disease and diabetes, could impact millions of lives. However, addiction involves complex overlapping patterns that complicate pinpointing cause and effect, or quantifiable biomarkers, creating an inherent pitfall. Neuronal circuits are the basic functional units that encode behavior. A better understanding of the neurocircuits engaged prior to dependence could shift strategies away from reactive treatments, and towards preventative measures to mitigate risks of developing AUD. Stress is an individualized experience that is highly intertwined with alcohol use and AUD. Could basal stress activation of AUD-associated neurocircuitry reveal unique patterns that predict subsequent AUD-related behavior? The insular cortex (insula) is a key network hub for emotional regulation, salience detection, cognitive control, and sensory processing. Mounting clinical and preclinical evidence suggests activity patterns in the insula correlate with alcohol drinking behavior. We and others have characterized a particularly intriguing projection to the bed nucleus of the stria terminalis (BNST), a critical node for stress-related disorders such as anxiety, depression, and addiction. This circuit is basally quiescent, but highly responsive to stress, binge drinking, and negative affect in abstinence. A relationship between stress-induced insula-BNST activation and subsequent alcohol-related behavior has not been examined. We will test the central hypothesis that stress-induced neuroadaptations in the insula-BNST pathway are associated with future AUD-related behavior. We predict that a group of stress susceptible mice will exhibit high insula-BNST stress response and stress-induced neuroadaptations. These changes are expected to relate to aversion-resistant binge drinking and hyperkatifeia in abstinence, two key features of AUD. Validated mouse models of stress and binge ethanol drinking will be used to test our hypothesis. Aim 1 will use longitudinal strategies to examine the relationship between stress-activated insula-BNST circuitry and binge/aversive ethanol drinking. Aim 2 will identify neuroadaptations within the insula-BNST circuit after stress and alcohol exposure. Aim 3 will use RNA sequencing to compare the insula-BNST transcriptome after stress or binge alcohol exposure. Our goal is to identify overlapping transcriptional signatures in stress and AUD- related behavior. We will mechanistically validate the results with shRNA-mediated knockdown of the top molecular targets in the insula-BNST to determine their role in binge alcohol drinking. Understanding the signatures of AUD-related circuits can bridge the gap between molecular and genetic biomarkers, and broader activity-based digital biomarkers. This conceptually and technically innovative framework will inform improved diagnosis, intervention, and risk assessment before AUD manifests.

NIH Research Projects · FY 2025 · 2025-09

PROJECT SUMMARY Differences in cost-benefit analyses for goal-directed motivation and behavior characterize many substance use disorders (SUD). An important feature of these cost-benefit analyses and decision-making is effort-based decision making (EBDM) which evaluates the reward magnitude and probability in relation to the effort required to obtain the reward. Dopamine (DA) modulates EBDM indicating that increasing or decreasing DA transmission enhances or diminishes (respectively) the willingness to expend effort for rewards. DA has also been shown to be affected by chronic substance use, more specifically tobacco use and opioid use such that over time the production, transmission, absorption, and sensitivity to DA is altered. In line with this, EBDM has been shown to be altered in tobacco use disorder (TUD) but has not been directly investigated in opioid use disorder. For successful recovery, individuals with SUDs must exert effort for non-drug rewards and value non- drug rewards more than drug rewards. But if the endogenous DA system is dysregulated due to substance use, the value of and motivation for non-drug rewards could be hypothetically compromised. This is why it is critical to understand how EBDM might be differentiated in the ability or inability to successfully quit smoking. It is also critical whether these differences in smoking status are unique to the pharmacology of nicotine and tobacco use, or if they are related more broadly to other SUDs such as opioid use. The primary goal of this fellowship proposal is to investigate the neurocomputational basis of EBDM in individuals with TUD, tobacco and opioid use disorder (TOUD), and controls (Aim 1) and characterize the neurocomputational basis of EBDM in a smoking cessation attempt (Aim 2). In both aims I will computationally model effort-based choice behavior to assess the underlying processes that potentially differ between groups such as the systematic use of reward and effort information and learning from the outcomes of previous actions. In Aim 1, I will set the foundation by understanding how EBDM differs between TUD, TOUD, and controls; this will further the understanding of not only how EBDM in TUD but also how EBDM could differ in polysubstance use (TOUD). In Aim 2, I will then further this work by understanding how EBDM is implicated in a smoking cessation attempt to potentially create new pathways for therapies. This 3-year fellowship will be analyzed by leveraging a previously completed dataset and an existing ongoing study. In all, this fellowship will provide training in contemporary computational modeling methods and experimental design that address fundamental questions in computational neuroscience regarding the neurocomputational basis of EBDM. The central findings of this research will advance our understanding of the principles of EBDM in SUDs and a smoking cessation attempt.

NIH Research Projects · FY 2025 · 2025-09

Project Summary/Abstract In 2019, the United States Department of Health and Human Services launched the Ending the HIV Epidemic (EHE) initiative to reduce incident infections by 75% within 5 years and 90% within 10 years. A priority population in the National HIV/AIDS Strategy is people who inject drugs. Concurrently, the overdose crisis remains a public health emergency. While the epidemiology of substance use is constantly evolving, the misuse of opioids, particularly synthetic opioids like fentanyl, continues to drive the crisis. The overdose crisis is part of a syndemic with HIV, hepatitis C (HCV), and opioid use disorder (OUD). North Carolina has been significantly impacted by the opioid syndemic with rates of HIV infections unabated from 2012-2022 and overdose death rates well above the national average. Understanding the opioid syndemic is of public health importance, but this is challenging because no single data source currently observed by the public health surveillance system fully characterizes opioid misuse at relevant spatial and temporal scales. Surveillance data is a valuable resource, but it typically consists of health care encounters for negative health outcomes (e.g. overdose) that only identify the portion of the population who had the event of interest recorded. These data are also increasingly prone to misclassification, or the notion that not everyone captured in the surveillance records belongs to the population who misuse opioids. For example, opioid overdose death counts may include people who use stimulants but overdosed due to fentanyl contamination. When allocating opioid- specific resources, it may not be of interest to include such misclassified individuals in the counts. Novel statistical methods are needed to better leverage existing data and integrate multiple surveillance outcomes while accounting for imperfect detection and misclassification. Additionally, while surveillance data are typically available at the county-level, evaluation of neighborhood accessibility of health services requires estimates at the sub-county level. Novel integration of electronic health record (EHR) data with surveillance data enables high resolution small area estimates of opioid misuse prevalence. There are several methodological challenges that will be overcome with achievement of the following aims: 1) Develop an integrated abundance model that accounts for misclassification in surveillance outcomes to estimate opioid misuse prevalence; 2) Develop methods for small area estimation of opioid misuse prevalence using data at multiple spatial scales including county-level surveillance data and address-level data from the EHR; 3) Assess the current allocation of health services in North Carolina neighborhoods to identify gaps and guide allocation of additional resources. Successful completion of these aims will enhance surveillance of the opioid syndemic by producing accurate small area estimates of opioid misuse prevalence that advance epidemiological understanding of the syndemic and guide public health planning and resource allocation to prevent HIV, HCV, and overdose.