University Of Rochester

NIH Research Projects · FY 2025 · 2022-02

This project describes the aims that will be addressed during the R00 phase in the Department of Pharmacology and Physiology at the University of Rochester. Currently available antidepressants have serious limitations for treating major depressive disorder (MDD), including low response rates, a significant number of treatment resistant patients, and a time-lag before there is a therapeutic response. Notably, ketamine, an NMDA receptor blocker, has demonstrated promise in clinical trials because of its rapid and sustained antidepressant effects. Although its mechanisms of action are still to be elucidated, our previous studies suggest that ketamine first inhibits cortical GABA interneurons, leading to disinhibition of excitatory pyramidal neurons, and subsequently, a glutamate burst, which results in synaptic plasticity and fast antidepressant responses. However, the effects of ketamine seem to be more complex than a simple enhancement of glutamatergic function, since MDD subjects and stressed animals show robust GABAergic deficits in cortical brain areas, which can be reversed by ketamine treatment. In addition, drugs that target the GABA system via α5-containing GABAA receptors (α5-GABAAR) have also been shown to produce fast and sustained behavioral effects in rodents. Therefore, the goal of this project is to extend our previous work and investigate how excitatory and inhibitory neuronal mechanisms interact to promote GABA-mediated plasticity that culminates in ketamine-induced behavioral responses, and explore additional GABAergic compounds relevant to MDD treatment, including α5-GABAAR modulators. The candidate will test the novel hypothesis that, in addition to glutamate-induced plasticity, increased GABA function in the medial prefrontal cortex (mPFC), specifically through α5-GABAAR, is critical for the synaptic and behavioral effects of fast-acting antidepressants. This hypothesis will be investigated by integrating multiple levels of analysis, including pharmacological, molecular, genetic, behavioral and circuit-level approaches. The lab will address the following aims: 1) To investigate cellular and synaptic mechanisms involved in the behavioral actions of α5-GABAAR NAMs and PAMs and, 2) To investigate the role of α5GABAAR in mediating cell type-specific neuronal activity in the mPFC and associated behavioral outcomes. In addition to significant scientific advances in understanding the pathophysiology of depression, this project will guide efforts to develop a new generation of agents to treat MDD.

NIH Research Projects · FY 2026 · 2022-01

Abstract: Current self-tests for HIV rely on detection of antibodies to the virus, and thus are not useful during the early stages of infection. These assays are also not appropriate for patients on antiretroviral therapy who may be at risk for viral rebound, as pre-existing anti-HIV antibodies mask changes in viral load. Conversely, current HIV diagnostics based on detection of viral RNA are sensitive, but too complex and expensive to be appropriate for self-testing. To address this critical need, we will develop the first enzyme-free sample-to-answer HIV viral load self-test, in which a fingerstick volume of blood is passively processed leading to amplification of the isolated HIV genomic RNA. The RNA amplification strategy will rely on hairpin cascade reactions (HCR), a DNA nanotechnology approach with proven single-copy sensitivity in other contexts, in a sessile droplet implementation. The entire assay will be implemented in an inexpensive, disposable, paper-based consumable. In the R61 phase, we propose to: (1) building on methods we have previously validated, optimize passive processing of whole blood coupled with paper-based viral lysis and RNA isolation; (2) complete development of HCR-based amplification of HIV genomic RNA; (3) integrate sample processing and RNA amplification into a single consumable; and (4) obtain feedback on assay design and usability from patients at risk for HIV infection, and develop a pilot manufacturing plan. In the R33 phase, we will (1) scale production of the assay with contract manufacturers in quantities suitable for pilot testing, and (2) complete testing of the assay with a cohort of 1000 patients at-risk for HIV infection, recruited from patients visiting the Emergency Department at the University of Rochester Medical Center.

NIH Research Projects · FY 2026 · 2022-01

Respiratory Syncytial Virus (RSV) is the second leading cause of hospitalization in children worldwide and has been increasing appreciated as a cause of hospitalization and death in the elderly. Recent studies have shown that polyvalent vaccine formulations, mixture of antigens derived from distinct pathogen variants, can induce antibodies to regions conserved between those variants We hypothesize that polyvalent antigen RSV vaccine formulations will increase antibody to regions conserved between the antigens. By drawing on the natural variability that exists among RSV variants, we aim to study the effect that polyvalent RSV G or F antigen formulations have on the immune response. We will test if antibodies are enhanced towards regions conserved between the viral proteins with the polyvalent formulation. Aim 1. Determining the Effect of Polyvalent RSV Vaccine Formulations on Humoral Immunity using Computer Simulations. We hypothesize that RSV polyvalent vaccine formulations consisting of different combinations of G or F-protein antigens will increase the antibody response to conserved regions between the antigens. We will evaluate different vaccine formulations using a computational framework of virus/host- interaction (ssMod.v2). We will test for differences in antibody specificity between polyvalent and monovalent formulations in the framework. Antibody cross-reactivity and protection against RSV challenge will also be evaluated. Aim 2. Comparison of the Host Immune Response to Polyvalent Vaccine Formulations in Mice. We hypothesize that murine immunization with a mRNA-LNP vaccine comprising polyvalent antigen formulations will induce antibodies and immune cells specific to regions conserved between the antigens. mRNA-LNPs will be constructed using cap-1, codon-optimized, structure-stabilized mRNA, encoding G or F from A2 or B1 RSV variants and will be encapsulated using ionizable cationic lipids. Groups of mice will be immunized with either Aim 3. Test if RSV Polyvalent Vaccine Formulation Improves Protection from RSV Disease. We hypothesize that vaccine formulations containing polyvalent mixtures of G or F antigens will increase the extent of protection against RSV disease compared to monovalent formulations. Using different mixtures of mRNA- LNP, we will test the ability of polyvalent vaccines to protect against disease severity using a murine model of RSV challenge. The neutralizing antibody titer of the sera will be tested using a primary human lung epithelial cell RSV-neutralization assay. Monovalent and polyvalent vaccine formulations will be compared by testing for differences between infection and disease severity outcomes.

NIH Research Projects · FY 2026 · 2022-01

Phenotypic plasticity is used by an incredible diversity of organisms, from plants to humans. Its ubiquity attests to its fundamental importance in life. This project addresses the fascinating and understudied question of the mechanistic basis of phenotypic plasticity – i.e., how developmental processes are influenced by environmental cues to cause phenotypic differences -- and, importantly, how those processes evolve. The focus here is on an innovative model, the pea aphid, which exhibits a textbook example of phenotypic plasticity. This species offers an unparalleled opportunity to examine the role of nature and nurture in phenotype determination: it exhibits dramatically different winged and wingless morphs that are induced by environmental conditions in genetically identical, asexual females and controlled by a single genetic locus in males. Thus, strikingly, two dimorphisms, each under different control mechanisms, exist within this single species. The proposed experiments build on the exciting recent discoveries made by the PI about the role of hormones and horizontally transferred genes in the female wing plasticity and about the identification of the wing polymorphism locus in males, which has an insertion containing a duplication of a gene that influences signaling (follistatin) and which is specific to wingless males. The proposed, vigorous research program aims to decipher the molecular mechanisms underlying the function and evolution of plasticity. Experiments on the wing plasticity will examine the regulatory changes that control it, the epigenetic changes that accompany it, and test if horizontally transferred genes are preferentially recruited into the process. Experiments on the genetic male wing dimorphism will use functional and evolutionary studies of the follistatin paralogs to establish how changes in these paralogs underlie male morphological evolution. Studies in females and males will be united with experiments that will test whether or not the more recently derived male dimorphism evolved by genetic accommodation of the female plasticity, hypothesizing that males bypass the environmental signals used by the female plasticity. These studies will provide some of the first insights into the mechanistic basis of genetic accommodation, where trait variation shifts from being caused by “nurture” to “nature”. These experiments will have broad implications for understanding the mechanistic basis and evolution of plasticity, which is significant from a human health perspective because of the numerous plastic traits that influence human health and disease.

NIH Research Projects · FY 2026 · 2022-01

This is a proposal to sustain the University of Rochester Resource-based Center for Bone, Muscle and Orthopaedic Research (ROCSTARR) Program. ROCSTARR is comprised of an Administrative Core that will run the Enrichment Program, and will coordinate and integrate the Histology, Biochemistry and Molecular Imaging (HBMI) and the Biomechanics, Biomaterials and Multimodal Tissue Imaging (B2MTI) Cores, in order to ensure progress of the overall ROCSTARR in an efficient manner. The Core Users Committees, the URMC Shared Resource Laboratories & Facilities, and the meeting structure of the Administrative Core are key elements that organize communication and collaboration among the ROCSTARR investigators, as well as with other faculty in the Center for Musculoskeletal Research (CMSR) and collaborating Departments and Centers within the University. The Specific Aims of the ROCSTARR are: 1) To maintain state-of-the-art resource- based Cores for basic science, translational and clinical musculoskeletal research; and utilize these resources in a cost-effective manner to accelerate the pace of discovery and clinical translation. 2) To continue to foster our highly collaborative environment that serves as a model for translation of basic research to clinical trials, and expands our outreach activities that promote the use of the resources offered by the Core to new external collaborations with the aim of growing our musculoskeletal research base at URMC, nationally and internationally. 3) To: i) maintain our exceptional 42-fold return on investment from Pilot Grants awarded to New/Early Stage Investigators (N/ESI), ii) commence utilization of a new Discovery Fund created with small endowments for research in the prior funding period, and grow it via targeted philanthropy, and iii) sustain our renowned academic Enrichment Programs including the William F. Neuman Visiting Professor series and the Annual Musculoskeletal Research Symposium. 4) To maintain our Sub-Cores that administer: i) human subject research services in concert with the Clinical-Translational Research Institute, ii) biostatistics, computer programing and outcome research resources to grow collaborative networks in Big Data science, surgical outcomes and population health research, and iii) provide new resources for muscle and exercise biology research in animal models and human subjects. And, 5) to expand our highly successful Mentoring Program, which facilitates the advancement of our N/ESIs to NIH R01 funded PIs with national leadership status, and implements the highest standards of scientific rigor in response to NIH’s plan to enhance reproducibility. Thus, building on our remarkable successes during the prior funding period, this P30 Core Center proposes to continue its mission “To advance our faculty members to independence, establishing them as national leaders and the most desirable faculty in their field by the top academic programs in the country, upon which the ultimate success of this Core Program will be judged.”

NIH Research Projects · FY 2026 · 2021-12

ABSTRACT: Use of androgen deprivation therapy (ADT), which causes near-total loss of testosterone, has increased dramatically in elderly prostate cancer patients over the last decade. ADT is associated with a significant increase in bone mineral density (BMD) loss (2-5% annually) and bone fractures, combined with a significant decrease in skeletal muscle mass (2-5% annually) compared to age-matched prostate cancer patients not on ADT and men without cancer. The loss of BMD and muscle mass results in a high prevalence of falls, reduced muscular strength, and decreased balance. Despite the high incidence of ADT-related side effects, treatment options are limited. Bisphosphonate therapy is commonly used for ADT-induced bone loss, but is associated with significant side effects and poor compliance. Vitamin D protects against BMD loss and fractures, but its effects are strongly dose-dependent. Current IOM recommended supplementation (600-800 IU/day) and serum 25-OH levels (20 ng/mL) are inadequate to protect against bone loss in a high risk population such as prostate cancer patients on ADT. In addition, RCT interventions of 400-500 IU/day of vitamin D fail to prevent ADT-induced bone loss. High- dose vitamin D supplementation (e.g., 50,000 IU/week) is a promising intervention that significantly increases 25-OH vitamin D to levels shown to improve BMD in other populations. Vitamin D has also been shown to increase muscular strength, reduce falls, and improve balance. In preparation for this R01, we conducted a Phase II randomized controlled trial (RCT) investigating the feasibility, safety, and preliminary efficacy of HDVD versus placebo for 24 weeks in 59 prostate cancer patients receiving ADT (NCI R21CA185678; PI: Peppone). Compelling evidence from our pilot study showed: 1) 50,000 IU/week of vitamin D (HDVD) was safe with no increase in toxicity versus low-dose vitamin D, 2) compliance was excellent at 94%, and 3) HDVD significantly increased 25-OH vitamin D levels. Those randomized to HDVD lost 1.9% BMD at the total hip versus 3.7% loss in the placebo group (p=0.03). Stratified analyses showed the HDVD group lost 2.3% BMD at the total hip vs 7.1% for the placebo group for those with baseline vitamin D <27 ng/ml (p<0.01). Based on our preliminary data, we propose to conduct a definitive, multi-center, phase III RCT in which 366 prostate cancer patients ≥65 years old starting ADT with vitamin D <27 ng/ml will be randomized to 1) 50,000 IU/week vitamin D or 2) a matching placebo for 52 weeks. All participants will receive a daily supplementation (800 IU vitamin D/1,000 mg calcium) to ensure a minimum of 100% RDA. The primary outcomes is the change in BMD, while secondary outcomes include changes in the clinically relevant outcomes of falls, balance, quality of life and fractures. We also plan to explore the biological pathways of ADT-induced bone loss and response to HDVD using bone biomarkers. If found to be efficacious, HDVD would be a safe, low-cost, widely-available OTC treatment that could revolutionize management of ADT-induced bone loss and change clinical practice paradigms.

NIH Research Projects · FY 2026 · 2021-12

Project Summary/Abstract This proposal represents a five-year research career development program focusing on identifying early predictors of chronic motor control impairments post-stroke. The outlined proposal builds on the candidate’s prior research and experience, and will contribute to the candidate’s development as an independent researcher by supporting her training in the application of neuroimaging, transcranial magnetic stimulation, and advanced biostatistical analysis in post-stroke recovery research. Stroke is the leading cause of adult disability in the US. Of those who receive rehabilitation therapy, approximately 40% have chronically impaired motor function of the upper extremity, contributing to decreased quality of life and increased societal burden of stroke. Currently, clinical rehabilitation focus on repetitive motor exercises, but interventions are not adapted for individual stroke lesion location or predicted trajectory of recovery. Prior studies have identified distinct impairments of motor control that contribute to poor function at the chronic stage and likely depend on neuroanatomical structures damaged by the stroke, but the details of what contributes to development of these impairments and timing of when they emerge remains unclear. In order to create optimal rehabilitation strategies and maximize each individual patient’s rehabilitation potential, we need better methods for predicting and treating these motor control impairments. In this proposal, we aim to use an electromyographic computer interface (ECI) to study motor impairments in the muscle groups that control wrist flexion and extension, and perform a longitudinal study to 1) establish the natural history of distinct motor control impairments post-stroke and 2) identify early neuroimaging, electrophysiologic, and clinical markers that predict specific motor impairments at the chronic stage. We then 3) evaluate the feasibility of using the ECI to provide early, intensive biofeedback therapy to target the predicted impairments of the individual patients. These studies will establish the necessary foundation for future development of precision-medicine therapies in neurorehabilitation. Furthermore, the mentored research and career development plan will provide the applicant with the opportunity for further training in leading longitudinal studies, advanced biostatistical analysis, and functional and structural neuroimaging. The results of this research and career development award will foster the expansion of her clinical research program, which is dedicated to developing novel therapies to treat early motor changes post-stroke.

NIH Research Projects · FY 2025 · 2021-09

PROJECT SUMMARY Dr. Olachi Mezu-Ndubuisi is a neonatologist with a background training as an optometrist, now an Assistant Professor of Pediatrics with an affiliate appointment in the Department of Ophthalmology at University of Wisconsin (UW). She combines these experiences in her research studying Retinopathy of Prematurity (ROP). ROP is a bi-phasic disease of abnormal retinal vascularization in premature infants, characterized by dysregulation of vascular endothelial growth factor (VEGF). ROP has no cure, and existing therapies have adverse systemic effects and long-term deficits. Dr. Mezu-Ndubuisi developed a non-invasive method of visualizing retinal blood vessels in a mouse model of oxygen-induced retinopathy (OIR) using fluorescein angiography. She correlated in vivo retinal vascular changes (arterial tortuosity, venous dilation, and capillary vascularity) to structure and function, and performed histological studies to show unique long-term features of OIR mice, such as prolonged cellular apoptosis, glial activation, and ectopic formation of synapses. Having defined the in vivo and histologic OIR phenotypes, she seeks to use molecular techniques to enhance understanding of VEGF regulation in ROP in order to develop safe and effective therapies. In her preliminary studies, there was a 6-fold increase in total mouse Vegfa164 levels at post-natal day (P) 13, early in Phase 2 ROP, in OIR mice compared to RA mice, and Vegfa164b showed a 1.3-fold higher trend in OIR than RA mice at P10 (peak of retinal vaso-obliteration during hyperoxia). This suggests that an imbalance of VEGF isoforms occurs in ROP. She then administered the pro-angiogenic isoform of VEGF-A165, VEGF-A165a, in sustained release microparticles which resulted in earlier retinal revascularization with improved arterial tortuosity and vein dilation. She next compared topical calcitriol (active form of vitamin D) eyedrop to intraperitoneal (IP) calcitriol and both inhibited retinal angiogenesis later in Phase 2 ROP, but topical calcitriol did not cause growth restriction like IP calcitriol. Dr. Mezu-Ndubuisi hypothesizes that using intravitreal VEGF-A165a early in Phase 2 and topical calcitriol later in Phase 2, would rescue the abnormal OIR in vivo and histologic phenotypes. In this K08, Dr. Mezu-Ndubuisi will determine the most effective and least toxic dose of intravitreal VEGF microparticles or free intravitreal VEGF protein and topical calcitriol in Phase 2 ROP (Aim 1), and then study the molecular mechanisms by which VEGF-A165a and calcitriol regulate angiogenic signaling in ROP (Aim 2). UW provides an excellent academic environment, state-of-the-art facilities, and academic resources dedicated to Dr. Mezu-Ndubuisi’s success. She has strong intra-mural and extra-mural mentoring from well-established NIH investigators and experts in ROP, molecular genetics, angiogenesis, and Vitamin D. Dr. Mezu-Ndubuisi’s career goals and objectives include a detailed training plan to increase her knowledge in biostatistics, biochemistry, histopathology, molecular signaling, and research ethics and laboratory skills. This will enable her successful transition to research independence and clinical translation of her research.

NIH Research Projects · FY 2024 · 2021-09

Pancreatic ductal adenocarcinoma (PDAC) is lethal, with a 5-year survival rate of less than 10%. Radical surgical resection is the only curative option; however, few pancreatic cancer patients have resectable disease at diagnosis. For a subset of patients with borderline resectable tumors, neoadjuvant therapies can downstage the disease and enable surgical resection. Protumor characteristics (i.e., hypoxia, high stromal density, high tissue pressure, and a high number of immunosuppressive cells) reduce the efficacy of neoadjuvant therapies. Stereotactic body radiation therapy (SBRT) is more effective than traditional radiation therapy for downstaging PDAC, but not all tumors are responsive. Traditionally, tumor treatment response is evaluated using anatomical tumor measurements, but this is limited because tumor size often does not correlate with tumor response. To improve this situation, we will establish a fundamentally new tool to image PDAC tumors to augment the available diagnostic imaging. We will advance shear modulus (SM) and vascular perfusion (VP) as surrogate imaging biomarkers for assessing tumor response to neoadjuvant therapies. In a uniquely beneficial approach for a difficult tumor to characterize, this project will combine pre-surgical, intra-surgical, and post-resection imaging with in vivo perfusion assessment and ex vivo pathology. Our extensive pre-clinical results demonstrate that SM and VP are sensitive to changes in protumor characteristics. Therefore, we hypothesize that SM and VP changes are direct diagnostics of the tumor microenvironment and can be used to assess therapeutic efficacy and response. To test this, we will combine shear wave elastography (SWE) with optical fluorescence tomography (OFT) of indocyanine green optical tissue perfusion tracer to evaluate the interplay between SM and Gemcitabine perfusion for different therapies, providing more comprehensive information regarding tumor response. We will develop a new hybrid imaging tool to systematically assess how SM and VP vary during neoadjuvant therapies through two specific aims: In Aim 1, we will perform pre-clinical studies with three progressive PDAC murine models that have different features that recapitulate human disease to evaluate how SM and VP relate to (a) stromal density, (b) the number of immunosupportive cells, and (c) the degree of hypoxia during SBRT, chemotherapy, and chemoradiation therapy. In Aim 2, we will clinically translate this work. We will compare our interventional SWE and OFT imaging to magnetic resonance elastography (MRE) and dynamic-contrast-enhanced magnetic resonance imaging to assess tumor microenvironmental changes during SBRT, chemotherapy, and chemoradiation therapy. We will also perform SWE on excised PDAC to evaluate how SM and VP relates to tumor microenvironment changes. These new imaging features are potential surrogate biomarkers, enabling clinicians to recognize whether treatment succeeds or fails. This practice-changing information will allow for the optimization of neoadjuvant treatment protocols on an individualized patient basis, resulting in more curative surgical candidates.

NIH Research Projects · FY 2025 · 2021-09

Abstract: Alzheimer’s Disease (AD) is thought to be caused by a combination of multiple genetic and environmental factors, and the role of infectious agents has been debated for decades. A recent multi-omic, epidemiological study of large Alzheimer patient cohorts revealed a specific and significant association of human herpesvirus 6A (HHV6A) with AD. However, these studies could not resolve which HHV6A specific genes were involved or how HHV6A might affect cells of the central nervous system (CNS) to exacerbate AD. As HHV6A is mainly present in the brain in its latent form, we asked the question of whether the major latency associated gene U94A, affects host cells functions that are relevant to Alzheimer disease pathology. Using a human cell system, we found that U94A expression impairs the migration and maturation of human glial progenitor cells (OPCs) and leads to synapse loss in human neurons. Preliminary transcriptomic and proteomic analysis of U94A infected cells showed dysregulation of genes involved in cytoskeletal functions and synaptic maturation. In addition, we found that expression of U94A increases accumulation of Aβ and phosphorylation of Tau in cells co-expressing a familial Alzheimer disease (FAD)-linked, mutant APPswe variant. These phenotypes are particularly relevant for the early stages of Alzheimer disease, as mounting evidence suggests that synapse and neurite loss, associated with diffuse demyelination precede cognitive impairment. Based on our published and preliminary data, we propose that the latency gene U94A represents a disease-modifying factor that renders neural cells vulnerable to Alzheimer disease associated risk factors, and exacerbates Alzheimer’s pathology in vitro and in vivo. We propose three Aims in which we test the hypotheses that (i) U94A expression exacerbates Aβ accumulation in iPSC derived neural cells from familial Alzheimer disease (FAD) patients and will exacerbate neuronal and glial cell impairments, (ii) that U94A expression in the context of FAD mutations in mice will exacerbate cellular pathologies in vivo and (iii) that the cellular impairments caused by U94A are sufficient to exacerbate cognitive deficits in familial Alzheimer’s mouse models. This work will provide insight into the potential role of infectious agents in Alzheimer’s pathology and will establish that HHV6A viral latency is not merely a benign state of viral infection, but an important disease modifying factor.

NIH Research Projects · FY 2025 · 2021-09

Abstract To restore high quality, usable vision in patients, it is important to develop regenerative therapies in models that share key features of the human visual system, particularly a fovea, the retinal area specialized for high acuity vision. Pre-clinical testing has been challenging due to both the absence of models of foveal vision loss and the difficulty of demonstrating restored function. Under previous AGI funding, the Advanced Retinal Imaging Alliance at the University of Rochester has recently overcome these challenges to create a pre-clinical testing platform leveraging adaptive optics technology to: 1.) Create localized regions of photoreceptor ablation in the fovea that are axially confined, and 2.) Optically read out restored retinal ganglion cell function by performing cellular scale calcium imaging in the living eye. This system was developed to meet the needs of photoreceptor replacement therapy, which requires photoreceptor loss with preserved host retinal circuitry. Furthermore, a high-resolution in vivo imaging approach is well suited for pre-clinical evaluation of regenerative therapies where the timescales of restored connectivity are unknown and functional integration occurs on the cellular scale. In this proposal, we will use our platform to generate pre-clinical data that will inform future clinical trials of photoreceptor replacement therapy in patients. Functional integration of transplanted photoreceptors with the host retina requires both high-density delivery of high-quality donor photoreceptors and a host retina with the capacity for synaptogenesis. We have assembled a consortium that can explore and optimize both sides of this interaction. In continued collaboration with a team at the University of Wisconsin led by David Gamm, a clinician and expert in photoreceptor replacement therapy, we will evaluate survival and functional integration of transplanted photoreceptor precursors delivered to the sub-retinal space as aggregates or following incorporation into custom biodegradable scaffolds. In collaboration with a team at University of California, Berkeley led by Teresa Puthussery, an expert in retinal remodelling in retinal degeneration models and retinal histology, we will examine the impact of the loss of photoreceptor signalling on inner retina. We will explore whether deafferented cone bipolar cells can remodel and functionally integrate with donor photoreceptors and whether retinal hyperactivity develops in the fovea as it does in rodent. To make meaningful progress toward restoring vision in patients who have lived with vision loss for many years, we will examine how these phenomena develop in the fovea over time and whether the regenerative potential of the host can be improved by therapeutic interventions such as retinoic acid blockers. These studies will allow us to fully characterize our novel photoreceptor ablation model and deploy it with photoreceptor replacement therapies to advance the field toward clinical trials.

NIH Research Projects · FY 2025 · 2021-09

The Central Dogma of molecular biology is that DNA is used to make mRNA, which in turn is used to make proteins. Central to physiology of every live cell, translation of messenger RNA (mRNA) into protein is catalyzed by the ribosome, structurally complex and dynamic macromolecular machine. Dysregulation of translation plays an important role in a number of human diseases including cancer. While some fundamentals of protein synthesis have been revealed, many molecular details of ribosomal translation remain unknown. For example, it is unclear why some mRNAs are translated orders of magnitude more efficiently than the others, and how mRNA structure regulates protein synthesis. My laboratory investigates molecular mechanisms of translation by studying structural dynamics of the ribosome, and the role of mRNA secondary structure in translation regulation. We use single-molecule microscopy and biochemical approaches to address the following questions: (i) How does the small ribosomal subunit move along mRNA in search for the start site for translation initiation in eukaryotes? (ii) How does the intrinsic compactness of mRNA and intramolecular basepairing interactions formed by the 5' and 3' untranslated regions (UTRs) of mRNA regulate the efficiency of protein synthesis in eukaryotes? (iii) How do mRNA stem-loop structures induce ribosome translation pauses, which control expression of a number of proteins in bacteria, eukaryotes and eukaryotic viruses, including Human Immunodeficiency Virus (HIV) and the cause of the COVID-19 pandemic, SARS-CoV-2? (iv) How are structural dynamics of eukaryotic ribosome (in particular, rotational movements between the small and the large ribosomal subunits) converted into the intricate process of protein synthesis? Our studies will substantially contribute to establishing the molecular mechanisms of protein synthesis, and provide the basis for the future development of antiviral and cancer therapies.

NIH Research Projects · FY 2025 · 2021-09

Thyroid eye disease (TED), also referred to as thyroid-associated orbitopathy or ophthalmopathy is the most common orbital pathology. TED is an autoimmune disease that occurs in up to half of patients with Graves’ disease. In TED, the tissues surrounding the eye become inflamed and ultimately remodel to cause protrusion of the eyes, swelling around the eyes, alteration of lid position, and double vision. In the most advanced cases, the expanded tissues compress the optic nerve, causing vision impairment. These clinical manifestations of TED reflect tissue changes triggered by autoantibodies that activate orbital fibroblasts that stimulate proliferation of lipid-laden adipocytes and scar-forming myofibroblasts. Orbital fibroblasts also produce excessive amounts of extracellular matrix composed of hyaluronan and collagen, which further increases the size and stiffness of orbital tissue. There is presently no cure for TED; corticosteroids, radiation therapy, and surgery are routinely used to manage TED symptoms and signs. Teprotumumab, an insulin-like growth factor 1 receptor (IGF1R) blocking antibody has emerged as the first disease-specific treatment for TED. However, a critical knowledge gap that limits our understanding of TED is how autoantibodies activate IGF1R signaling in orbital fibroblasts to promote eye disease. IGF1R can stimulate proliferation and increase myofibroblast formation. The predominant antigen in TED is the thyroid stimulating hormone receptor (TSHR). How IGF1R interacts with TSHR and TED autoantibodies in the disease is a fundamental and unresolved question. One potential mechanism is through the aryl hydrocarbon receptor (AHR). The AHR is a ligand-activated transcription factor that binds synthetic and naturally derived aromatic hydrocarbons. The AHR controls aspects of cell growth, development and the immune system. Evidence suggests that AHR blocks TSHR and IGF1R signaling in vivo and in vitro. However, the mechanism(s) are unclear and whether the AHR regulates these pathways in TED is unknown. One of the most significant risk factors for developing TED is smoking. Smoking activates a transcription factor called hypoxia inducible factor 1 alpha (HIF1a). AHR and HIF1a compete to control cell fate. Smoking may disrupt the HIF1a- AHR balance thereby further increasing IGF1R/TSHR signaling. Central Hypotheses: The AHR blocks IGF1R/TSHR signaling, and loss of this interaction is a primary event in the pathophysiology of TED. Aim 1: Define the molecular pathway(s) by which the AHR blocks orbital fibroblast activation. Aim 2: Determine the role of cigarette smoke exposure in promoting HIF1a and IGF1R signaling while blocking AHR in orbital fibroblasts. Aim 3: Evaluate the ability of the AHR ligands to block TED autoantibody driven TSHR/IGF1R signaling. Impact: Our findings will show that AHR blocks TSHR and IGF1R signaling in TED. Accomplishing the specific aims will establish a molecular mechanism whereby smoking exacerbates IGF1R/TSHR signaling in TED. Further, the experiments should provide critical evidence that activating the AHR pathway is a novel and viable therapeutic target for treating TED.

NIH Research Projects · FY 2024 · 2021-09

PROJECT ABSTRACT This project involves training peer-health coaches to improve the shared-decision making behavior of patients with low-health literacy (LHL). Most patients want to be involved in important health decisions such as taking medications to prevent cardiovascular disease. Yet, patients with LHL often lack the knowledge, confidence, and skills to assume a more active role in these decisions. I am committed to conducting research that will support patients with LHL in becoming equal partners with their clinicians. My commitment is evidenced in this novel intervention that is designed to help LHL patients acquire the knowledge they need to engage in a shared-decision about reducing their cardiovascular disease (CVD) risk; and the agency and confidence they need to do so. This study will provide a foundation for advancing the science of SDM around the primary prevention of CVD with a focus on addressing LHL through trained peers. Moreover, this proposed K-award will springboard to the next steps in my path to an independent investigator. By the end of this award, I will develop expertise in understanding the scope of peers in the SDM process and how they can be used to improve cardiovascular disease outcomes for patients with LHL. This award will span 5 years and will involve a graduated career development plan that includes mentorship, coursework, analyses, publication, and an R-level grant submission. In order to achieve the goals of this research plan and transition to independence, I need formal training and mentorship in implementation science, health literacy & patient communication, and primary prevention of cardiovascular disease. This proposal includes an exceptional team of mentors to help me reach my goals. My primary mentor, Dr. Kevin Fiscella, is a nationally known expert in health care disparities, implementation science, and health care policy. He is experienced in conducting equity- focused primary prevention of cardiovascular disease implementation research in primary care. My Co- Mentor, Dr. Ronald Epstein, is an internationally known expert in patient-provider communication. He is a communication and health services researcher with a focus on pragmatic strategies for improving communication with patients. My goal at the end of this award is to submit an NHLBI grant application (such as in response to NHLBI PA-19-166 (Implementation of shared decision making for HLBS diseases and conditions) to further refine and test the proposed intervention in a broader population.

NIH Research Projects · FY 2025 · 2021-09

PROJECT SUMMARY/ABSTRACT (COMPONENTS A, B and C): Viral acute respiratory infections (ARI) and acute gastroenteritis (AGE) remain major causes of morbidity in children, making vaccine development and implementation a high public health priority. The overall goal of the CDC New Vaccine Surveillance Network (NVSN) is to establish a network of US institutions that develop and implement standard research protocols to answer important questions about the burden of disease and natural history of vaccine preventable diseases, specifically, those causing ARI and AGE. A second goal of population-based NVSN surveillance is to provide accurate estimates of vaccine effectiveness (VE) in preventing pediatric hospitalization or medical care visits. A third goal is the ability to rapidly pivot to study novel agents such as enterovirus-D68 (EV-D68) or SARS-CoV-2, and syndromes such as acute flaccid myelitis (AFM) and multisystem inflammatory syndrome in children (MIS-C). The proposed work (comprising Mandatory Core A, and Optional Components B and C) will enhance the NVSN site in Rochester, NY—one of 2 original members of the now 7-site network. For 20 years we have published important studies on the burden of disease and natural history of AGE due to rotavirus and norovirus, and ARI due to influenza, (RSV), and other viruses. These data helped inform AAP and ACIP on pediatric influenza and rotavirus vaccination, and palivizumab use for RSV prevention. Our specific aims are:  Mandatory Core Component A: Perform prospective, population-based active surveillance for ARIs and AGEs in inpatient and ED settings as well as asymptomatic controls, for children 0-18 years. We will use molecular diagnostics for rotavirus, norovirus, influenza, RSV, PIV, EV-D68, SARS-CoV-2, and other viruses. We will assess VE for influenza, rotavirus, and future SARS-CoV-2 vaccines in preventing pediatric hospitalizations and ED visits. We will delineate the disease burden of ARI and AGE using our unique capture of virtually all pediatric hospitalizations and ED visits in our region. We will assess the impact of future vaccines and other immunoprophylaxis strategies. We will perform active surveillance and epidemiologic studies of acute flaccid myelitis (AFM) syndrome in children. Finally, we will perform and lead epidemiological, implementation and laboratory studies based on population-based NVSN surveillance.  Optional Component B: Implement active prospective population-based ARI/AGE surveillance studies in children seeking medical care in outpatient practices, as the burden of ARI and AGE are likely high.  Optional Component C: Investigate the incidence, spectrum of disease, and risk factors associated with SARS-CoV-2 MIS-C. Our proposed research will provide high impact information to develop sound policies for the prevention of pediatric vaccine-preventable diseases, and to improve the health of US children.

NIH Research Projects · FY 2025 · 2021-08

Protein quality control systems are essential for maintaining neuronal health, with vacuolar dependent pathways playing a primary role in these systems. The endolysosome system is a major contributor to the maintenance of the neuronal proteome, and dysfunction of this system occurs early in the pathogenesis of Alzheimer’s disease (AD) , and likely contributes to the accumulation and mislocalization of tau, which plays a key role in disease pathogenesis. Recent data provide compelling evidence that the stress responsive, multi-domain protein, Bcl-2- associated anthogene 3 (BAG3) plays a role in maintaining protein homeostasis and neuronal health. The expression of BAG3 in specific neuronal populations positively correlates with resistance to the development of tau pathology in AD. Further, our preliminary data indicate that BAG3 is an upstream regulator of vacuolar dependent pathways. The UNDERLYING PREMISE of this proposal is that in neurons, BAG3 plays a critical role in mediating vacuolar dependent pathways, and thus proteostasis and neuronal integrity. The importance of BAG3 in mediating proteostasis is illustrated by the fact that BAG3 not only plays an important role in protecting neurons from the accumulation of pathological tau species, but also likely supports synaptic structure. Nonetheless, our understanding of the mechanisms by which BAG3 regulates vacuolar dependent processes is very limited. Although previous studies have implicated BAG3 as a mediator of autophagy, we are the first to present evidence that BAG3 is a regulator of the endolysosome pathway. Further, our preliminary findings implicate BAG3 as an important modulator of endosomal sorting required for transport (ESCRT) machinery. BAG3 interacts withTBC1D10B, the primary GTPase activating protein (GAP) for Rab35, which facilitates the recruitment of the ESCRT machinery and protein clients to the endosome, as well as mediating other vacuolar dependent processes. The conceptual framework that BAG3 acts upstream of vacuolar pathways through its regulation of Rab35 activity is novel and innovative. The OVERALL HYPOTHESIS of this proposal is that the BAG3-TBC1D10B-Rab35 signaling axis regulates endosome-lysosome function and neuronal health. In the context of this overall hypothesis the specific aims of this proposal are: to test the hypotheses that: (1) the BAG3-TBC1D10B-Rab35 signaling axis regulates ESCRT and the endolysosome pathway, (2) in vivo BAG3 and Rab35 coordinate to mediate neuronal ESCRT/vacuolar processes, and contribute to the maintenance synaptic integrity, and (3) the BAG3-TBC1D10B-Rab35 axis plays a critical role in regulating the clearance of pathological tau species. These studies will be carried out using mouse models and primary neuron cultures. The IMPACT of these studies is that they will provide crucial new insights into the mechanisms by which BAG3 acts as an upstream mediator of the endolysosome systems to maintain a healthy neuron. Overall these studies represent a new, unexplored area of investigation that will increase our understanding of the factors that are essential to maintain a healthy, functional neuronal proteome.

NIH Research Projects · FY 2025 · 2021-08

Abstract Glial progenitor cells (GPCs) pervade the adult human brain, and can give rise to new oligodendrocytes and astrocytes in response to myelin loss; yet they may fail to do so in chronic neuroinflammatory and age- related white matter diseases. Our goal is to identify the transcriptional and epigenetic basis for age-related glial progenitor failure, with the goal of identifying the repressive transcription factors and epigenetic states that restrict progenitor cell expansion and differentiation with age. By targeting these repressive networks, we hope to restore the functional viability of human GPCs, and by so doing prevent the myelin loss that characterizes both aging and those neurodegenerative and inflammatory disorders associated with white matter disease. By so doing, we hope to preserve not only the differentiation competence of the cells, but also their self-renewal, so that myelin- ogenesis may be induced from hGPCs without the progenitor depletion to be expected of strategies designed to trigger terminal oligodendrocytic differentiation. Achieving this in human GPCs, which differ substantially in their biology from mouse, and doing so in vivo, has proven a significant challenge to the field. To this end, we will ask: 1. To what extent is the aging of human ESC-derived GPCs cell-intrinsic and linked to prior cell division, both in vitro and in vivo? What are the transcriptional and epigenetic concomitants to hGPC aging in vivo, and which of these restrict hGPC expansion and differentiation? How do hGPCs, extracted back from neonatally- transplanted human chimeric mouse brains, change in their DNA methylation patterns, their ATAC-Seq-defined patterns of chromatin accessibility, and their consequent RNA expression, over the 2-year lifespan of a mouse? 2. To what extent are the effects of aging on hGPCs a function of the aged brain environment, rather than cell autonomous? In order to define the relationship of hGPC cell age to expansion and myelination competence - and the extent to which the age of the host influences hGPC fate – these experiments will include a set of reciprocal, heterochronic transplants, grafting aged cells into neonates, and new hGPCs into aged brains. 3. In aged GPCs, can genetic knock-down of those repressors implicated in the progression to adult hGPC phenotype restore the transcriptional signature, as well as the expansion and differentiation competence in vivo, of younger hGPCs? Is suppression of MAX, potentially together with a core set of other over-expressed repressors, sufficient to restore MYC-dependent mitotic expansion and host colonization by aged hGPCs? With this work, we expect to establish a granular understanding of the relative roles of cell-intrinsic, expansion- dependent senescence and host context in regulating the proliferation and remyelination competence of human GPCs. Furthermore, if the introduction of young hGPCs into an aged environment allows the selective colonization of the host white matter by those younger hGPCs, the implications may be profound, as disorders as varied as progressive multiple sclerosis and the neurodegenerative disorders might then become potential targets of cell replacement strategies based on the competitive advantages of young over aged glial progenitors.

NIH Research Projects · FY 2025 · 2021-08

Abstract Skeletal muscle wasting (SMW) is a growing burden among cancer survivors and is prognostic of treatment failure, radiotherapy toxicity, and a shorter time to tumor progression related to survival. This is of particular concern for patients with pancreatic ductal adenocarcinoma (PDAC), for whom interventions for severe SMW are now being implemented prior to surgery, radiation and chemotherapy, so that these patients can complete their prescribe treatments. Currently, the mechanism(s) that lead to cancer-related SMW have yet to be elucidated. Furthermore, viable treatment options for patients with this multifactorial syndrome remain undiscovered. A lack of preclinical models that recapitulate human disease is often identified as an obstacle in the development of feasible therapies to treat cancer-related SMW. To this end, our lab developed a murine model of PDAC-related SMW that parallels human pathology and can be longitudinally assessed via Dual Energy X-ray Absorptiometry (DEXA). In this murine model of PDAC we have identified upregulation of pro-inflammatory cytokines, immune cell infiltration, IGFBP-3, and intramuscular adipogenesis as key features in the progression of SMW. In addition, we determined that a single intratumoral injection of IL-12 reduced tumor burden, pro-inflammatory signaling, and SMW, while improving survival out to 50 days. Although a relationship between chronic low grade inflammation and PDAC-related SMW has been suggested, the mechanisms are unknown. Also, the relationship betwe en macrophage associated increases in IGFBP-3 and adipogenesis have yet to be investigated. Thus, the purpose of this proposal is to investigate a novel pathway of SMW and increased intramuscular adipogenesis via pathologic increases in IGFBP-3 by tumor and immune cells. In addition, we seek to determine the efficacy of a multi-dose IL-12 treatment regimen to ameliorate SMW through the reduction of inflammation, intramuscular macrophage infiltration, IGFBP-3 secretion, and adipogenesis in a murine model of PDAC.

NIH Research Projects · FY 2024 · 2021-08

Project Summary/Abstract The overarching goals of this project are to advance knowledge of the complex pathophysiology of distal symmetric polyneuropathies (DSP) and frame future interventions for nutritional neuropathies in Zambia through: 1) assessment of population-based estimates and risk factors for DSP in Zambia’s maize and cassava staple food zones; 2) comparison of population-based estimates and risk factors to previous clinic- based data from the same maize-staple districts; 3) and assessing the odds of specific micronutrient deficiencies among DSP cases and age, gender, and HIV-matched controls from the same districts. To accomplish these goals, Dr. Kvalsund will focus on acquisition of additional expertise and experience in the following key areas: 1) advanced epidemiological methods and analysis; 2) clinical trials methodologies, 3) global health policy; 4) neuropathy outcome assessments; and 5) HIV and nutrition. The needed skills will be achieved through structured mentorship, coursework, and pragmatic clinical trials and health policy experiences. The research-training plan will provide Dr. Kvalsund with the additional proficiency necessary to undertake future intervention research aimed at reducing nutrition-associated neurologic morbidity in Zambia and advancing scientific knowledge of the pathophysiologic mechanisms that generate DSP as a common phenotype. The central hypothesis is that the high rate of DSP previously observed in clinic populations in Zambia is equally prevalent outside healthcare settings owing to common endemic infectious diseases and their neurotoxic treatments, food insecurity and low dietary diversity, recurrent nutritional challenges, specific micronutrient deficiencies and their interactions, and possibly other unidentified latent factors. We hypothesize that folate-deficiency will be highly associated with DSP in Zambian communities, as has been documented in clinic populations, and may warrant a targeted public health intervention given the preventable nature and range of other neurologic morbidities also associated with folate deficiencies. The specific aims are to: 1) Determine population-based DSP estimates in Zambia; 2) Compare and contrast DSP estimates and risk factors with those from clinical settings in Zambia; 3) to consider the relative contributions of urbanicity, agroecological/staple food zone, and specific micronutrient deficiencies on DSP prevalence; and 4) Evaluate for latent environmental or toxico-dietary factors as contributors to DSP in this environment. The research is expected to provide the foundation for future R-01 intervention and observational research and to improve scientific understanding of how diverse etiologies, multiple exposures and interactions lead to a common clinicopathologic DSP phenotype, which remains poorly understood to date.

NIH Research Projects · FY 2025 · 2021-08

Pancreatic ductal adenocarcinoma cancer (PDAC) is the 3rd most common cause of cancer deaths in the United States with a dismal 5-year overall survival of 9%. Surgical intervention is currently the only cure for PDAC, however, 80% of patients are deemed unresectable at presentation due to locally advanced and/or metastatic disease. Existing therapies are often unable to downsize locally advanced PDAC (LAPC) for surgical candidacy, and are also ineffective at providing distal tumor control. Therapeutic approaches capable of both local downstaging and recurrent/metastatic tumor control are desperately needed to improve resectability rates. This application will address the unmet need by translating an innovative combination immunotherapy to the clinic and exploring its effects on LAPC in humans. Our recently published work demonstrated that stereotactic body radiotherapy (SBRT), a less toxic, more effective strategy that focuses higher dose radiation precisely to the tumor, combined with the potent immune cell–stimulating cytokine interleukin-12 (IL-12) encapsulated in polymer microspheres (IL-12MS) resulted in remarkable tumor control and durable cure in preclinical models. Microsphere technology represents an innovative tool that provides a slow, continuous release of cytokine intratumorally while also protecting the labile IL-12 protein from degradation by proteases. The combination of SBRT with IL-12MS not only strongly stimulated the adaptive arm of the immune system including cytotoxic T cells to destroy pancreatic tumor cells, but also had a repolarizing effect on cells of innate immune system converting typically immunosuppressive myeloid cells into ones with immunostimulatory potential. Moving this promising therapy into the clinic, we hypothesize that combined SBRT/IL-12MS therapy is safe and tolerable, and will improve progression-free survival and tumor downstaging to enable resection in LAPC. In Aim 1, we will establish a clinical trial exploring the first-in-human use of SBRT followed by ultrasound- guided IL-12MS delivery in patients with unresectable LAPC. The main objective is to evaluate safety and tolerability, and the secondary objective is to evaluate efficacy and overall outcome. Aim 2 will perform corollary studies on peripheral blood along with baseline and on-study tumor biopsies collected from enrolled patients. These data will address whether SBRT/IL-12MS repolarizes the tumor microenvironment (TME) from an immunologically “cold” tumor to one that is immunologically “hot”. Aim 3 will utilize preclinical modelling to develop a strategy to treat metastatic PDAC using SBRT/IL-12MS therapy. These results are essential in order to expand this therapy into metastatic patients where there are little to no effective treatments. Overall, our proposed application builds on promising preclinical data showing the potential efficacy of combined SBRT/IL- 12MS therapy for patients with LAPC/metastatic lesions. This technologically innovative strategy utilizes a unique strategy of repolarizing the TME to treat this recalcitrant malignancy for which there are few effective therapies.

NIH Research Projects · FY 2025 · 2021-08

ABSTRACT Numerous studies now report associations between air pollution (AP) exposure and neurodevelopmental disorders (NDDs), including autism spectrum disorder, schizophrenia, and attention deficit disorder, all of which share numerous features. My studies of early postnatal (human 3rd trimester brain equivalent) inhalation exposures to concentrated ambient ultrafine (UFP, considered the most reactive component of AP) particles (CAPS) in mice produced numerous neuropathological and behavioral features of these NDDs and of their shared hypothesized mechanisms, including male bias, providing biological plausibility for the epidemiological studies. Additionally, CAPS exposures markedly elevated brain levels of metals and trace elements, including redox metals (Fe, Cu) as well as S, Ca, and Al, findings indicative of brain metal dyshomeostasis. This proposal seeks to test the overarching hypothesis that AP-induced brain metal dyshomeostasis contributes to male- biased NDD phenotypes via production of neuroinflammation and oxidative stress tested in a series of questions designed to accelerate the understanding of mechanisms, and translational relevance of such effects in 5 key integrated questions emanating from these novel, dramatic and unexpected findings: 1) Are toxic trace element contaminants of UFPs a source of CAPS-induced NDD phenotypic features, specifically elevated brain Fe and S (inhaled Fe nanoparticles and/or SO2) both of which are known neurotoxicants via ferroptotic and oxidative stress mechanisms? 2) What accounts for male bias in UFP-induced neurotoxicity? Does it reflect an earlier colonization of male brain by activated microglia and their interactions with Fe uptake? 3) What are the portals of entry of UFPs into brain? We utilize the precocial African spiny mouse with its extended gestational period relative to the altricial C57 mouse in which 3rd trimester occurs postnatally and can include nasal and olfactory uptake to determine whether the African spiny mouse might serve as a more relevant human model. 4) How does nanoparticle processing in brain subsequently influence/modulate toxicity and does it generate toxic or protective mechanisms e.g., alterations in the ferritin cage? 5) Does post-mortem brain tissue from humans that had been diagnosed with NDDs (Neurobiobank) contain exogenous metal nanoparticles as we see, e.g., with Fe located within damaged myelin in corpus callosum after CAPS? These integrated efforts will begin to elaborate mechanisms of AP-induced NDDs and associated sex differences, to define the most relevant mouse model, and to determine the need to regulate air metal levels for public health protection.

NIH Research Projects · FY 2025 · 2021-08

Abstract: The glymphatic system is a network of perivascular spaces that function as a waste clearance system, analogous to the peripheral lymphatic system. Reduced glymphatic function has been a hallmark observation in aging as well as models of Alzheimer's disease, diabetes, hypertension, traumatic brain injury, excess alcohol intake, and chronic unpredictable stress. Preliminary data shows that acute and chronic pain, and one night of light all suppressed glymphatic function. This application will use the murine sparse nerve injury (SNI) model to understand how the brain responds to chronic neuropathic pain. Sleep complaints are prevalent in chronic pain patients, and chronic sleep restriction increases pain sensitivity in mice. Norepinephrine (NE), which disrupts sleep and is released in stressful conditions, suppresses glymphatic function. We hypothesize that increase NE levels in SNI reduce glymphatic function, triggering cytokine accumulation, neuronal excitability, sleep disruption and pain sensitization in a feedforward loop (Aim 1). Traditional analgesics have been shown to relieve pain in models of chronic pain. Our preliminary data show that the same agents restore glymphatic function in SNI mice with no effect on glymphatic functions in control mice. We hypothesize that reducing the severity of pain via analgesia improves glymphatic function by reducing NE levels, which in turn reduces cytokine accumulation and excitability and improves sleep quality (Aim 2.1). Yet, efficacy of modern pharmacology is variable in the patient population, suggesting that while modulation of neural pathways is partially effective, pathology remains. We hypothesize that neuropathic pain induces a CNS maladaptive response involving reduced glymphatic flow, inflammation and waste accumulation. Because both natural and mind-body interventions target multiple facets of glymphatic disruption (sleep, inflammation, cardiovascular disease), we hypothesize that natural supplements (melatonin and eicosapentaenoic acid (an ω-3 fatty acid)) and mind-body interventions (voluntary exercise, improved sleep, and acupuncture) will improve glymphatic disruption in chronic pain (Aims 2.2 and 2.3). The timing of treatment is critical, because the circadian system is integrated into every process in the body including the glymphatic system, the immune system, and chronic pain. We propose that targeting therapeutics to reinforce the rhythm in glymphatic function and clearance will optimize the effect of treatment which can be quantified as an additional decrease in cytokine accumulation and hyperalgesia in SNI (Aim 3). We will time sleep improvements via increased temperature, voluntary exercise, melatonin, and acupuncture, to the endogenous rhythm of CSF distribution - high glymphatic clearance during rest, and low during wakefulness. Aim 3 is unique in that it tests whether efficacy of mind-body therapies, in improving glymphatic function and reducing pain sensitivity, can change based on when during the day they are administered. Overall, this application aims to define whether glymphatic activity may serve as a target for complementary therapeutic approaches and also as a biomarker establishing the efficacy of treatment.

NIH Research Projects · FY 2025 · 2021-08

Abstract Synaptic failure is an important feature of HIV infection of the brain, and a likely key contributor to HIV- associated neurocognitive disorders (HAND). Yet current animal models have proven of limited utility in defining the mechanisms of this process, in part because of the species-specific nature of HIV infection, but also because of the greater complexity of human astrocytes relative to those of mice. To address this issue, we will utilize mice chimeric for both human microglia and human astrocytes, to assess the effects of HIV infection on central neurons. To that end, we will engraft mice with both human glial progenitor cells (hGPCs) and microglia, each derived from embryonic stem cells (hESCs). We have established the methods of generating these human glial chimeras, by the neonatal implantation of hGPCs, which outcompete and ultimately replace the host mouse GPCs, yielding adult chimeras broadly colonized with human astroglia1-5. This process is especially robust in the neostriatum, allowing the glial humanization of regions critically involved in striatal reward and addiction circuits. We have recently extended this approach to include chimerization with hESC-derived microglia, paired with the use of CSF1R null mice lacking host microglia, crossed to NSG SGM3 mice to allow the stable xenograft of hGPCs. The mice are thus chimeric for hGPC-derived astrocytes as well as microglia, in a T- and B-cell deficient background that allows the effects of glial HIV infection on neurons to be isolated, following intracerebral inoculation with HIV-infected microglia. These chimeric human astroglial-microglial (CHAM) mice are especially attractive, since they incorporate the hominid-specific features of human astroglia, which are themselves key components of central synapses. Using this model, we will test the postulate that astrocytes become both structurally and functionally impaired by microglial HIV infection, resulting in the loss of synaptic engagement by affected astrocytes, with consequent dendritic involution and network disruption. By infecting CHAM mice with HIV, and using rabies viral-EGFP to trace striatal dendrites, we will assess the effects of astrocytic HIV infection on the dendritic architecture and synaptic structure of resident medium spiny neurons. In parallel, we will study the effects in CHAM mice of HIV infection complicated by methamphetamine use – a common and disabling comorbidity that suppresses dopaminergic input to the striatum – focusing on the structural and transcriptional responses of human glia to the combination of infection and addiction, as well as on the behavioral effects of that combination. To that end, we will use single cell RNA-Seq to assess the changes in gene expression by human astrocytes and their partnered mouse neurons caused by HIV infection, both alone and together with chronic methamphetamine use, to identify those changes that contribute to the striatal synaptic disruption and behavioral pathology of these mice. Our goal is to test the hypothesis that the HIV-infected striatum, by virtue of astrocytic fiber disengagement from dopaminergic synapses in particular, is especially vulnerable to the effects of amphetamine abuse, while defining the transcriptional basis for the glial pathology underlying that vulnerability.

NIH Research Projects · FY 2024 · 2021-08

ABSTRACT Suicide rates among Hispanics in the United States have increased in the last two decades; coupled with rapid growth of the Hispanic population, suicide among Hispanics is a pressing public health issue. Considering the role of culture in suicide can elucidate unique and modifiable mechanisms for suicide. The Candidate’s long- term research goal is to optimize suicide prevention interventions for Hispanics by targeting cultural determinants of health. Reaching that goal requires mentored training in three domains of suicide prevention research and completion of a research project to produce substantive, foundational results (described below). First, the candidate needs to gain expertise in randomized clinical trials methodology for suicide interventions (Objective 1) to conduct a trial with at risk Spanish-speaking adults in a reliable, valid, and ethical manner, and test target engagement with an experimental therapeutics approach. Second, the candidate needs to gain expertise in optimizing behavioral interventions to impact cultural determinants of health via human-centered design (Objective 2) to deliver a treatment protocol that is culturally-usable and optimized to increase cultural- social engagement. Third, as smartphone technology increases precision to measure mechanisms, the candidate needs to gain expertise in the use of novel smartphone technology for precise assessment of target engagement in clinical trials (Objective 3) to optimally/ethically use this technology in clinical trials research. The principal objective of the proposed research study is to use an experimental therapeutics approach to examine whether a behavioral intervention (SOCIAL ENGAGE; S-ENGAGE) can increase cultural-social engagement (intervention target) and decrease suicide risk (clinical outcome) among Hispanic adults. First, human-centered design approaches will be used to iteratively refine and optimize S-ENGAGE to alter cultural-social engagement via iterative feedback from 5 Spanish-speaking adults with low cultural-social engagement and recent ideation. Second, 60 Spanish-speaking adults who report low cultural-social engagement and recent ideation will be randomized into 10-weeks of S-ENGAGE or an expectancy-matched control. Participants will provide 2-weeks of real-time data via smartphone at baseline, post-treatment, and 3- month follow-up and complete semi-structured interviews. Research Aims are to: (1) Optimize S-ENGAGE for target engagement. (2) Test target engagement: Does S-ENGAGE increase cultural-social engagement among Spanish-speaking adults?; (3) Test clinical impact: Does S-ENGAGE decrease suicide risk among Spanish-speaking adults?; (4) To contextualize findings: What components of S-ENGAGE were most helpful? The resulting findings of this K23 project will function as the basis for a larger R01-funded study powered to examine whether the proposed target (cultural-social engagement) leads to changes in the clinical outcome (suicide ideation). The candidate will build on the skills acquired in this period of mentored career development to contribute to the development and refinement of approaches to suicide prevention for Hispanics.

NIH Research Projects · FY 2025 · 2021-07

The growing medication use in all ages led to the fact that 20% of the US adult population take five or more drugs (polypharmacy). Over 500 medications commonly prescribed in polypharmacy (e.g., antidepressants, urinary antispasmodics, and psycholeptics) possess anticholinergic (AC) properties blocking the muscarinic signaling of neural regulation. Due to the scattered distribution of muscarinic receptors in the body, AC medications have a wide range of side effects. Besides the most severe central toxicity of cognitive impairment, dyskinesia, and psychosis, which can lead to delirium, the most frequent peripheral side effect is dry mouth. Dry mouth is characterized by reduced saliva secretion (hyposalivation), impaired quality of life by causing chewing or swallowing problems, complaints of oral dryness (xerostomia), speaking difficulties, mucosal changes, increased rate of dental caries, and tooth loss. Dry mouth causes increased susceptibility to bacterial colonization and infections in the oral cavity and the upper respiratory tract. However, no data are available on predicting medication-induced dry mouth severity or determining the AC burden from these medications among dental patients. There is a significant research gap in identifying high-risk xerostomia patients in the middle-aged population before reaching older ages when damage to oral health is irreversible. We designed a prospective cohort study with two aims for addressing these questions. In Aim 1., we will evaluate the correlation between AC burden and dry mouth outcomes, including the flow rates of the minor salivary glands (SG) in 90 middle- aged patients (45-64 years). We will determine whether high AC burden, quantified by the AC drug score (ADS) and serum AC activity (SAA) in blood, is associated with more severe dry mouth symptoms, measured at baseline and follow-ups for two years. We will assess dry mouth using saliva flow rates (unstimulated whole saliva and minor SGs) and oral health measures associated with dry mouth, including xerostomia, dental caries, and oral health impact profile. In this aim, we will examine the feasibility of minor SG flow screening as a point- of-care test for dry mouth. In Aim 2., we will explore whether CYP450 genetic polymorphisms predict dry mouth severity. Recent studies reported an increased prevalence of AC side effects in patients with inactive genetic variants of liver cytochrome P450 enzymes responsible for the metabolic clearance of AC drugs. We will analyze DNA from patients’ blood for the genetic variations of CYP2D6 and CYP2C19 enzymes and compare oral health outcomes associated with dry mouth between poor and normo-metabolizing phenotypes. We propose to study whether dry-mouth pharmacogenetics provides evidence for inter-individual variability in oral health outcomes to identify patients with predictable severity of AC medication-induced dry mouth. The overarching goal of our explorative study is to establish clinically relevant associations between AC burden and oral health outcomes, which can support future investigations of potential causal relationships and risk calculations for dry mouth development.