Rutgers Biomedical And Health Sciences

NIH Research Projects · FY 2026 · 2024-09

Project Summary/Abstract Mu opioids, such as fentanyl and oxycodone, remain in the mainstream for moderate-to-severe pain management. However, they also produce many side-effects, leading to the development of opioid use disorder, a main cause of the opioid epidemic and climbing opioid overdose death. The actions of mu opioids are primarily mediated through the mu opioid receptors encoded by the single-copy mu opioid receptor gene, OPRM1, that undergoes extensive alternative splicing, creating multiple splice variants. One set of OPRM1 variants, exon 7- associated full-length 7TM C-terminal variants (E7 variants), contain a unique intracellular C-terminal tail sequence with 30 amino acids encoded by E7 that are abundant and highly conserved from rodents to humans. Culminative evidence has indicated that E7 variants contribute to several mu opioid-induced adverse side-effects. First, truncating E7-enocded C-terminal tails in mice (mE7M-B6) attenuated mu opioid-induced tolerance, reward, and respiratory depression without effect on analgesia. Second, similarities and dissimilarities in several phenotypes between mE7M-B6 and β-arrestin2-KO mice suggest that E7 variants involve β-arrestin2-dependent and β-arrestin2-independent mechanisms. Third, a mouse model in which only a single E7 variant, mMOR-1O, is expressed (mMOR-1O-KI) showed enhanced morphine tolerance and reward, complementing those from mE7M-B6 mice. Finally, a vivo-morpholino antisense oligonucleotide (ASO) and a newly developed rabbit monoclonal antibody (RabmAb) targeting E7 variants diminished morphine tolerance and reward in mice. Together, these observations strongly support our central hypothesis that Oprm1 E7 variants mediate several adverse effects associated with clinically used mu opioids, such as tolerance, reward, and respiratory depression, and targeting E7 variants can diminish these adverse effects but maintain mu opioid analgesic potency via other Oprm1 7TM variants. These observations also provide compelling rationales for further investigating the role of Oprm1 E7 variants in various actions of mu opioids using ASOs and antibodies as proposed in this application with four independent, but integrated specific aims. Aim 1 is to characterize ASOs and RabmAbs that target mouse Oprm1 E7 variants in both in vitro cell models and in vivo C57BL/6J (B6) mice, providing the necessary information for designing experiments in Aims 2 & 3. Aims 2 & 3 are to investigate the effects of the selected ASO and RabmAb on mu opioid-induced tolerance, reward, addiction, and respiratory depression in B6 mice under naive (Aim 2) and chronic pain conditions (Aim 3), respectively. Aim 4 is to develop and characterize a nanobody or an ASO targeting human/monkey exon O (E7 homolog) sequences and investigate the effects of the nanobody or ASO on mu opioid abuse potential in rhesus monkeys. The proposed studies promise to yield significant findings in establishing the E7 variants as therapeutic targets and provide the groundwork for developing a therapeutic medication that combines clinically used mu opioids with an inhibitor (nanobody or ASO) to mitigate the adverse effects of mu opioids in pain management.

NIH Research Projects · FY 2025 · 2024-09

PROJECT SUMMARY The US is currently going through an opioid crisis, and while Medication Assisted Treatments such as buprenorphine (BUP) have proved highly effective at stabilizing the neurobiology underlying acute withdrawal, they have been less effective at preventing longer-term relapse and adherence. This may be due to the fact that they do not fully engage the neural processes sub-serving the emotional control of sensitized negative mood and reward sensitivity during stress- and opioid-cue provocation, respectively. In contrast while the alpha2 agonist, guanfacine, may attenuate stress-provoked opioid craving by mediating top-down prefrontal control over sensitized dysphoria, the behavioral intervention, Mindfulness Oriented Recovery Enhancement (MORE) may reduce opioid cue-provoked craving by mediating top-down prefrontal control over hedonic dysregulation. Furthermore, while both interventions separately may prove effective as longer-term adjunctive therapies, they may offer greater efficacy together, providing a unique medication/behavioral combination able to target both stress and reward provocation mechanisms. To optimally test this hypothesis, a staged approach is proposed to first confirm the efficacy of both GXR and MORE, independently and combined (R61), prior to elucidating underlying neural mechanisms (R33). Using a 2 X 2 design, N=80 OUD individuals on Buprenorphine (BUP) will be randomized to either 6-weeks of Guanfacine extended release (GXR; 3mgs, n=40) or placebo (PBO; n=40). Half of all participants in each group will then receive either weekly MORE, or a Support Group (SG) control, creating four intervention groups (Control Grp: PBO+SG, n=20); (GXR Grp: GXR+SG, n=20); (MORE Grp: PBO+ MORE, n=20); (Combined Grp: GXR+MORE, n=20). A pre- and post- laboratory study will be conducted before and after six weeks of intervention where participants will be randomly exposed to 3 personalized guided imageries (stress, opioid cue, neutral). Subjective measures of opioid craving, anxiety, mood, stress, emotional reappraisal, and heart rate will be collected before and after imagery exposure. Following milestone completion, an identical design is proposed in N=144 individuals, where participants will be exposed to imageries in the scanner (R33). On the basis of prior research, it is hypothesized in that GXR will attenuate opioid craving and improve emotion regulation during stress, while MORE will demonstrate the same effects during opioid cue exposure. GXR and MORE will also demonstrate additive or synergistic improvements compared with each intervention alone (R61). The effects of GXR on opioid cue- and MORE on stress-provoked opioid seeking will be explored. In the R33 component, it is hypothesized that GXR will improve regulatory and affective brain function during stress, and MORE will improve regulatory and reward function during opioid cue exposure. Combined GXR and MORE may improve regulatory function in an additive or synergistic manner (R33). Findings will help elucidate the efficacy and neural mechanisms underpinning a novel integrated pharmaco-behavioral therapy for OUD individuals maintained on BUP.

NIH Research Projects · FY 2024 · 2024-09

PROJECT SUMMARY Drug overdose is now a leading cause of death among U.S. adolescents, with mortality increasing in recent years with the proliferation and involvement of illicitly manufactured fentanyl (IMF). In parallel, nonfatal adolescent drug overdoses treated in emergency department (ED) settings have also increased. In contrast to fatal overdoses, nonfatal overdoses mainly involve non-opioid drugs, including psychostimulant and other psychotropic drugs that adolescents may use with a prescription or misuse from illicit sources with high risk of IMF adulteration. To reduce morbidity and mortality from adolescent drug overdose, we need to better understand the epidemiology of specific drug involvement in nonfatal overdoses, focusing on drugs commonly containing IMF. Overdoses treated in acute care (ED) settings represent critical opportunities for risk assessment and intervention, as mortality is significantly elevated in the period after a nonfatal event. However, limited research suggests that receipt of comprehensive behavioral health care after overdose is exceedingly low in adolescents, related to the lack of pharmacological treatments for substance use disorders, limited access to developmentally appropriate behavioral therapies, and high rates of co-occurring mental illness and suicide symptoms that complicate treatment decisions. Thus, we need to address the large gaps in evidence on the type and timing of treatment adolescents receive following a nonfatal overdose and understand the relationship between follow-up care and risk of repeat overdose. To generate evidence that can be used to inform effective prevention and treatment strategies, this proposal’s overall goal is to identify individual and clinical care factors associated with adolescent overdose risk. The specific aims are to: (1) estimate the prevalence of adolescent nonfatal drug overdose and identify high-risk demographic and clinical subgroups, stratifying by drug involvement (e.g., fentanyl) and overdose intent; (2) examine receipt of behavioral health treatment (type, timing) after nonfatal drug overdose and variation in treatment receipt by demographic and clinical characteristics; and (3) estimate incidence, critical high-risk periods, and risk factors for repeat overdoses, including the relationship with behavioral health care received after the initial event. Our central hypothesis is that drug overdose patterns differ by adolescent characteristics (e.g., sex, age group, mental health diagnoses) and that follow-up care is limited but impacts the risk of repeat overdoses. To achieve these goals, the study team will leverage national longitudinal Medicaid claims data (2016-2022) comprising over 400,000 overdose events in adolescents aged 12-18 years. Our proposal offers a valid and timely approach to yield novel, generalizable, and up-to-date evidence on overdose risk and treatment in a vulnerable adolescent population. Findings will generate actionable estimates to inform targeted prevention and treatment strategies as well as a future R01 application to integrate health systems and structural factors in research on the effectiveness of behavioral health treatment modalities on reducing risk of subsequent fatal and nonfatal overdose events.

NIH Research Projects · FY 2024 · 2024-09

Project Summary Cardiometabolic diseases contribute to more than 1 in 4 deaths in U.S. adults, including Asian Americans. Psychosocial stress is believed to be a key risk factor leading to elevated risks of cardiometabolic diseases, beyond traditional factors such as poor diet and physical inactivity. However, there is limited knowledge of how psychosocial stress affects cardiometabolic health among Asian Americans, especially older foreign-born Asian American who disproportionately encounter psychosocial stress due to racism, language barriers, and low socioeconomic status. Notably, a lack of knowledge of key biopsychological mechanisms through which psychosocial stress affects cardiometabolic health hampers public health efforts to develop effective interventions to promote cardiometabolic health in the understudied Asian American population, which is projected to reach 46 million by 2060. To address these important scientific gaps, the proposed study is to investigate key biopsychological processes through which daily psychosocial stress affects cardiometabolic health among 300 older foreign-born Chinese Americans. We will use a prospective design and harness the power of ecological momentary assessment and biospecimen sampling to achieve three specific aims. We propose to: (Aim 1) determine the mediation effects of biopsychological processes (daily negative emotion, saliva cortisol secretion, daily sleep quality) on the associations between psychosocial stressors (e.g., social isolation) and cardiometabolic health (e.g., blood pressure, Hemoglobin A1C); (Aim 2) investigate micro-level day-to-day associations among psychosocial stressors, biopsychological processes, and blood pressure; and (Aim 3) examine the moderation roles of sociocultural factors (e.g., acculturation, social cohesion) on the longer-term effects of psychosocial stressors on cardiometabolic health. Completion of the proposed study will provide new knowledge to facilitate the understanding of key psychosocial stressors and related biopsychological processes contributing to poor cardiometabolic health in older Chinese Americans. As a result, this project will guide the design of future culturally-tailored, time-sensitive, personalized psychosocial interventions that aim to promote cardiometabolic health in this population.

NIH Research Projects · FY 2025 · 2024-09

Project Summary This proposed study, aligned with RFA-AG-24-025, aims to address how interpersonal processes and social network characteristics of aging Black and Latino sexual minority men (SMM) facilitate or impede health behaviors affecting cognitive performance. Little is known about the mechanisms of health behavior change within social networks over time in aging Black and Latino SMM. Our proposal will address critical gaps in knowledge that have prevented the development of effective social network interventions to promote social and physical activities and maintain cognitive health among aging Black and Latino SMM.This will be achieved by using respondent-driven sampling (RDS) to establish a cohort of 400 aging Black and Latino SMM in Essex, Bergen, and Hudson counties, New Jersey in order to assess the following specific aims: 1) Cross-sectionally elucidate the association between interpersonal (intersectional stigma and social support) and social network characteristics and health behaviors (social and physical activities) among aging Black and Latino SMM; 2) longitudinally examine interpersonal and social network characteristics (network stability, density, centrality) that act to promote or impede social and physical activities; and 3) longitudinally examine variability in interpersonal and social network characteristics and their time-lagged relationships with subsequent trends in social and physical activities and cognitive performance. Our research will establish critical knowledge that will improve the understanding of pathways and processes of interpersonal and social network influences on health behaviors associated with cognitive performance. It will provide important new information for the future development of strengths-based interventions intended to boost the ability of aging Black and Latino SMM to leverage protective social network effects.

NIH Research Projects · FY 2025 · 2024-09

Project Summary Our overarching goal is to identify and assess policy and community-level interventions that counteract the effects of SRD within neighborhoods, schools, and peer networks, on adolescent substance use and mental health outcomes. We will leverage data from The Context Study, a large (n= 7,174), seven wave panel study of public-school children in three racially diverse, predominantly rural counties in North Carolina. At wave one, all 6th, 7th, and 8th graders attending a public school in the three counties were eligible; the final wave occurred when participants were in 10th-12th grade. Context includes extensive panel data on peer networks and comprehensive residential geocodes for participants. We have linked these data to students’ school and criminal records, Census, county tax-value property assessments, Google Maps, and the American Community Survey to construct measures of SRD. Guided by the NC State Health Improvement Plan to increase health equity and empirical evidence, we will use Context data, group model building, and an agent-based model (ABM) to project the long-term impact of policy and community-level interventions (e.g., resource distribution, social integration, and school discipline policies) that target SRD and its consequences on adolescent substance use and mental health. Our specific aims are: Aim 1: Develop a detailed theoretical model of the systems rooted in SRD that drive adolescent substance use and mental health outcomes and determine how these may be addressed through policy and community-level interventions. We will use a community-based systems dynamics approach to convene a group model building workshop with diverse community stakeholders to develop a causal loop diagram of the system and identify a set of community co-created candidate policy and community-level interventions that could modify the system and reduce the impact of SRD. Aim 2: Use ABM to compare the expected long-term outcomes of various types and combinations of candidate policy and community-level interventions to address SRD and its effects on adolescent substance use and mental health. We will use information from the group model building workshops and leverage national models and a representative synthetic population to develop an ABM to examine spatial and temporal effects of SRD on adolescent health. We will validate the model on Context data, conduct sensitivity analyses to evaluate the robustness of model results to uncertainty in model structures and parameters, and project the impact of candidate interventions. Aim 3: Determine key implementation factors of candidate policy and community-level interventions. For selected interventions, we will conduct focus groups and in-depth interviews with community stakeholders to gain insight on implementation factors including feasibility, acceptability, and scalability. We will analyze these data and develop intervention maps of the most promising candidate interventions. Identifying and projecting systems- level interventions that disrupt adolescent exposure to SRD has strong potential to move the field beyond individually focused solutions and ultimately support strategies to reduce adolescent health inequities.

NIH Research Projects · FY 2025 · 2024-09

PROJECT SUMMARY Due to organ shortage, livers are transplanted in order of recipient medical urgency; however, ethical principles dictate avoidance of futile transplantation. The most common cause of death early after liver transplant (i.e. futility) relates to consequences of an immune system which is frail or dysfunctional prior to transplant. We have previously identified the Liver Immune Frailty Index (LIFI), a pre-transplant biomarker panel that predicts risk of early post-transplant death. In our discovery cohort of 279 liver transplant recipients, pre- transplant measurement of LIFI, based on HCV IgG, and plasma Fractalkine, and MMP3, can discriminate risk of death within one-year following liver transplant with a c-statistic of 0.84 and a false-positive rate of 4%. LIFI- low corresponds to 1.4% one-year mortality compared with 58.3% for LIFI-high. Multicenter validation is necessary to advance the LIFI as an objective clinical index to predict the risk of liver transplant futility. Mechanistically, our data suggest that immune frailty may relate to skewed intracellular energetics and immune cell exhaustion. These alterations likely result from severe cirrhosis-associated immune dysfunction. In preliminary studies, LIFI was determined at the time of transplant. Pre-transplant immune dysfunction is likely a fluid process and may also be responsible for decompensation and death on the waitlist. Based on this, we hypothesize that patients listed for LT exhibit both progressive pre-LT immune dysfunction (immune frailty) and cirrhosis-related physical frailty, which predisposes patients to wait-list and early post-LT mortality. The cumulative effect is detectable with serum biomarkers of immune dysfunction. To evaluate this, we have established the Liver Immune Frailty Evaluation (LIFE) Consortium, comprised of six centers whose volume encompasses almost 10% of annual US liver transplants. Plasma, PBMCs, radiographic, and clinical data will be collected from waitlisted patients at LIFE centers. In Aim 1, we will perform multicenter validation and refinement of the LIFI. In Aim 2, we will longitudinally assess biomarkers of immune dysfunction and AI- driven body composition analysis (physical frailty) among waitlisted patients who either receive liver transplant or who expire due to severe decompensation prior to or early post-liver transplant. We will also expand our understanding of the development of immune frailty through targeted multiomic assessment. This study serves as a direct extension and complement to our ongoing evaluation of severe pre-transplant immune dysfunction, currently supported by a NIDDK K08 Award. Outcomes from this study will advance LIFI as a novel objective index for pre-transplant patient assessment, and will expand understanding of immune frailty development. IMPACT: LIFI is an innovative index that provides crucial insight into liver transplant candidate immune function and risk of post-transplant mortality, and no alternative pre-transplant clinical indices provide this key data. Validation and pre-transplant correlation of LIFI is critical to improve transplant timing, prevent wasting of livers in high-risk patients, and identify therapeutic targets to reverse immune frailty and improve outcomes.

NIH Research Projects · FY 2024 · 2024-09

ABSTRACT A hallmark of the oral bacterium Aggregatibacter actinomycetemcomitans’ biology and infectivity are its ability to form tenacious biofilms. Biofilm formation in the organism is attributed to attachment via surface proteins and exopolysaccharides (EPS). The EPS of A. actinomycetemcomitans (Aa), also referred to as PNAG, is often overlooked as a virulence factor of bacterial pathogenesis and only considered for its role in biofilm formation. PNAG plays a critical role in the evasion from macrophages, oral colonization, and bone loss. However, very little information is available for the regulation of PNAG production in Aa. In other PNAG-producing bacteria, such as E. coli, the synthesis of PNAG is regulated by the second messenger, cyclic-di-GMP or the post- translational carbon storage regulator CsrA. Aa does not produce cyclic-di-GMP due to the absence of the genes responsible for its synthesis in the Aa genome and the role of CsrA is unknown in Aa. In the absence of such regulation, in anaerobic environment, Aa could produce excessive amount of PNAG which is a highly energy consuming process and can potentially reduce fitness. A quorum sensing two-component system, QseBC, functions as a global regulator of virulence in Aa but does not regulate PNAG genes AapgaABCD. Thus, there is a knowledge gap regarding regulation of PNAG in Aa. In search of novel mechanisms of regulation, we have identified two proteins, AaFlp-1 (a unique Aa pili protein) and AaDcuB (an ABC succinate transporter involved in fumarate respiration), both of which impacted PNAG production. Succinate in an anaerobic environment contributes to dysbiosis of the oral microbiota and chemotaxis. Further, exogenous addition of succinate has been shown to accelerate periodontal disease and Aa’s ability to expel succinate through fumarate respiration and AaDcuB might be critical in disease initiation. These two proteins, apparently unrelated, appear to control PNAG production either independently or in a coordinated manner. Our overarching hypothesis is that AaFlp-1 and AaDcuB proteins regulate the PNAG production in Aa either directly or indirectly. Such impact by AaFlp-1 and AaDcuB has not been reported in other PNAG-producing bacteria such as E. coli or S. epidermidis and these two proteins offer a unique opportunity to study the novel regulatory mechanism(s) operational in Aa. Our immediate goal is to test the overarching hypotheses that AaFlp-1 and AaDcuB proteins regulate the PNAG production in Aa and that the production of PNAG is unique in Aa. The following aims are proposed: Specific Aim 1. Decipher the unique crosstalk mechanism between AaFlp-1 and PNAG production in Aa. Hypothesis: AaFlp-1 is a signal for the induction of PNAG production in Aa. Specific Aim 2: Determine the mechanism by which AaDcuB regulates the production of PNAG in Aa. Hypothesis: AaDcuB protein impacts the production of PNAG through the fumarate respiration.

NIH Research Projects · FY 2024 · 2024-09

Summary Short-chain fatty acids (SCFAs) are metabolites produced by the fermentation of fibers by bacteria in the gut and play an important role in the maintenance of health and disease prevention in a variety of disorders, including metabolic, autoimmune, and neurological diseases. The sufficient production of SCFAs in the gut and their efficient absorption by intestinal epithelial cells and transport into systemic circulation are important for disease prevention. Although the effect of prebiotics and probiotics on the production of SCFAs in the gut has been extensively studied, their effect on the absorption and transport of SCFAs has not been well studied. Low-grade intestinal inflammation is one of the causes of reduction in the expression of SCFAs-transporter genes and the transport of SCFAs into the systemic circulation. We found that the levels of intestinal inflammation biomarker are increased in MS patients compared to healthy donors (HDs), and the levels of the SCFA acetate are lower in the blood but higher in fecal samples in MS patients compared to HDs, suggesting insufficient absorption and transport of SCFA acetate in MS patients. We hypothesize that MS-associated gut dysbiosis may trigger and/or exacerbate the inflammation in the gut and subsequently reduce the expression of SCFAs-transporter genes and their transport into the system circulation. To examine this hypothesis, germfree mice will be transplanted with fecal samples collected from MS patients and HDs, and examined for the expression of SCFA-transporters and tight junction proteins, intestinal permeability, and concentration of SCFAs in fecal and blood samples. Further, we will examine the development of experimental autoimmune encephalomyelitis (EAE), an animal model of MS, in the human microbiota-transplanted mice in Aim-1. We found that the abundance of B. massiliensis was significantly associated with increased levels of acetate, butyrate, and propionate in the blood. B. massiliensis is a newly isolated bacterium from healthy human fecal samples, and its effect on health has not been explored yet. Of interest, the culture supernatant of B. massiliensis and its outer membrane vesicles (OMVs) up-regulated the expression of SCFAs-transporter genes. We also found that A. hallii, SCFA-producing bacterium, is reduced in the gut of MS patients and that intake of high-fiber diets (HFD) can increase the colonization of SCFAs-producing bacteria. Therefore, we hypothesize that treatment with A. hallii or HFD can increase the production of SCFAs in the gut and increased colonization of B. massiliensis could promote the transport of SCFAs into the blood circulation. We will test this hypothesis in Aim-2. In this study, we will elucidate a novel mechanism of how the gut microbiome impacts health and modulates disease, and identify a synbiotic intervention with HFD and B. massiliensis as a beneficial treatment for autoimmune diseases including MS.

NIH Research Projects · FY 2025 · 2024-09

PROJECT SUMMARY/ABSTRACT Career Goal: My long-time career goal is to become an independent investigator studying the effects of health care policies on access and use of maternal and infant health care services among low-income populations. With additional skills in claims data, subject matter expertise, and causal inference methods, I plan to build a career focused on generating evidence to improve maternal and child health outcomes and reduce disparities in the United States. Career Development: I will pursue the following training aims during the mentored (K99) phase of the award: (1) clinical maternal and infant health content expertise; (2) claims data analysis skills; (3) advanced causal methods, and; (4) professional development. Research Project: Extending postpartum Medicaid has the potential to address poor maternal health in the United States, with additional implications for infant care use and health.1 Approximately 63% of maternal deaths in the U.S. occur postpartum, and more than half are considered to be preventable.2 However, despite high postpartum healthcare needs, pregnancy- related Medicaid ends after 60 days postpartum. Medicaid pays for almost half of all births in the US and disproportionately covers births to the low-income population and people of color.3–5 There has recently been increased interest in postpartum Medicaid, including an American Rescue Plan (ARP) option to extend Medicaid one-year postpartum.6 Prior to this, the March 2020 Families First Coronavirus Response Act (FFCRA) prevented Medicaid disenrollment during the pandemic, thereby extending postpartum Medicaid eligibility, which can inform current postpartum Medicaid policies under consideration. Using the Rhode Island All-Payer claims data, this proposal's Specific Aims are: K99/1) To evaluate the effects of the FFCRA on coverage and maternal and infant care in the postpartum period, R00/2) To evaluate the heterogeneous effects of the FFCRA on outcomes by race and ethnicity, R00/3) To evaluate how the COVID-19 pandemic affected the effects of extended postpartum Medicaid eligibility on care use. Mentorship: I have assembled a highly accomplished team of experts at Brown University to provide mentorship and guidance as I transition to research independence, comprised of Dr. Trivedi, Professor in the Department of Health Services, Policy and Practice and Department of Medicine; Dr. Steenland, Research Assistant Professor of Population Studies in the Population Studies and Training Center; Dr. Vivier, Professor of Health Services, Policy and Practice and Professor of Pediatrics and Emergency Medicine; Dr. Wilson, Professor and Chair of the Department of Health Services, Policy and Practice, and Professor of Medicine; Dr. Savitz, Professor of Epidemiology and Professor of Obstetrics and Gynecology and Pediatrics; and Dr. Tuuli, Chace-Joukowsky Professor and Chair of Obstetrics & Gynecology. Future Directions: The training and research in this proposal will provide me with a unique set of expertise and skills to prepare me to reach research independence and submit an R01 application to examine the effects of state variation in postpartum Medicaid policies using claims data.

NIH Research Projects · FY 2025 · 2024-09

Project Summary/ Abstract American Indians are at higher risk for Alzheimer’s disease and Alzheimer’s disease related dementia (AD/ADRD) compared to the general population, yet dementia onset and frequency in this population are understudied. American Indians have a disproportionate burden of diabetes and vascular brain injury along with evidence of health disparities. Diabetes increases the risk of vascular disease, which accelerate Alzheimer’s disease-related processes coexisting in mixed forms of dementia. Intraindividual cognitive variability (IICV), a measure of variability in neuropsychological test performance within a person at a single timepoint, may be a novel, low-cost, noninvasive biomarker of neurodegeneration and early dementia. We aim to characterize associations between IICV, longitudinal changes in magnetic resonance imaging (MRI) measures related to AD/ADRD, and diabetes among older American Indians. For this we will use data from the Strong Heart Study (SHS) and Cerebrovascular Disease and its Consequences in American Indians (CDCAI) Study, calculating IICV measures for memory, executive function and processing speed, and multidomain cognition for 403 American Indians (age 70-95) who had diabetes assessments at three different timepoints over the course of ~27 years (SHS Time 0, CDCAI time 1 and CDCAI time 2); neuropsychological and neuroimaging data at two time points, with an interval of ~7 years (CDCAI time 1 and CDCAI time 2), and dementia adjudication at time 2. We will accomplish this work through the following Specific Aims: (1) Examine (a) whether IICV at time 1 is associated with neurodegenerative-related changes in MRI in critical brain regions (entorhinal, hippocampal, whole brain atrophy), in American Indians, and (b) whether diabetes moderates this relationship. We expect (a) that IICV will be positively associated with neurogenerative-related MRI changes, and (b) a stronger association between IICV and MRI findings in those with diabetes at midlife (time 0) or later life (time 1 and 2); (2) Examine (a) whether IICV at time 1 is associated with vascular brain injury in American Indians (WMH volume progression), and (b) whether diabetes moderates this relationship. We expect (a) that IICV will be positively associated with WMH progression, and (b) a stronger association between IICV and MRI findings in those with diabetes; and (3) Examine whether (a) IICV at time 1 is associated with cognitive decline and dementia adjudication at time 2, and (b) whether diabetes moderates these associations. We expect (a) that increased IICV will be associated with an increased likelihood of cognitive decline, MCI, and dementia and (b) a stronger association in those with diabetes. The validation of IICV as a marker of future AD/ADRD risk will allow for earlier noninvasive identification and mitigation of risk in American Indians. Results from this study are expected to advance the understanding of how IICV relates to neuropathology, dementia, and type 2 diabetes among aging American Indians, and is expected to serve as preliminary data to optimize the design of a future R01 dedicated to investigating the utility of IICV as early marker of AD in minority populations.

NIH Research Projects · FY 2024 · 2024-09

Summary Mitochondrial Lon is an ATP-powered protease that plays a pivotal role in regulating mitochondrial proteostasis, metabolism and cell stress responses. Biallelic mutations in the LONP1 gene cause a broad spectrum of rare developmental diseases presenting during early development, which include CODAS syndrome- characterized by cerebral, ocular, dental, auricular and skeletal anomalies, profound neurological dysfunction and depletion of mitochondrial DNA (mtDNA). This project aims at filling a fundamental knowledge gap pertaining to the mechanistic impact of Lon binding to mtDNA and its role in regulating mtDNA integrity and expression. We will employ isogenic patient-derived induced pluripotent cell (iPSCs) with homozygous mutations in the LONP1 gene causing CODAS syndrome c.2161C>G, (p.Arg721Gly), or severe neurologic dysfunction c.2282 C>T, (p.Pro761Leu). Using iPSC-derived cell types expressing Lon-WT, Lon-R721G and Lon-P761L we will: (1) characterize differences in mitochondrial protein turnover, energetics and cell stress responses; (2) determine mtDNA-binding by wild-type and mutant Lon proteins using chromatin immunoprecipitation with sequencing (ChIP-seq); and (3) analyze the biogenesis of mtRNA transcripts using single-molecule fluorescence in situ hybridization (smFISH). The innovative approach of smFISH will allow us to determine differences in mtRNA synthesis, half-lives (i.e., degradation rates) and spatial localization within mitochondria. This project will provide new mechanistic insights into the importance of Lon in regulating mtDNA maintenance, transcription and translation, which has broader implications for its critical roles during normal physiology and common diseases such as cancer, neurodegeneration and cardiac dysfunction.

NIH Research Projects · FY 2024 · 2024-09

Project Summary In the mammalian brain, action, perception, and cognition arise from the coordination of many neurons working in concert. For decades, neuroscientists have observed these multi-neuron patterns of neural activity, and developed complex ‘population codes’ to describe how a pattern correlates with a behavior. These population codes make up our core understanding of how the brain works. However, simply observing activity cannot distinguish between activity that drives a behavior (i.e., causality) vs that which only reports that a behavior happened (i.e., correlation). Even modern optogenetic or chemogenetic approaches struggle to assess causality in complex interconnected systems, like the mammalian cortex, and are incapable of testing the importance of a pattern of activity, where information is encoded in the timing and relative firing rates of neurons. This has led to calls for revised methodologies to study causality in neural systems. Here we leverage a unique and novel approach, that we developed, to test the causal link between neural activity and specific actions. This new approach enables the previously impossible task of recreating population activity de novo, writing different numbers of action potentials into adjacent cells with millisecond precise timing. We will use this technique to ask a fundamentally different class of questions than ever previously possible and create a framework determining the causal role of population codes on behaviors. Vector Optogenetics is the most advanced form of optogenetic stimulation yet, and the only approach able to reproduce population activity. Evolving from multiphoton optogenetics, vector optogenetics allows a user to specify not just which cells are activated but how many action potentials they will fire and when – anywhere within the field of view of a conventional two-photon microscope and in a behaving mouse. In this proposal, we use this technique to address several critical questions in motor cortex. Exploring, quantitatively, the nuance of what patterns of activity drive specific behaviors, and other cells. We explore possible important features of a population code (e.g., identity of activated cells, relative firing rates, synchrony, or sparsity of a pattern), as well as popular theories of the importance of ‘low dimensional’ activity spaces. By combining vector optogenetics with electrophysiology in the striatum, we further explore how patterns of activity drive downstream nuclei – addressing questions about inter-area communication and the initiation of actions. My experience as a systems neuroscientist, combined with being the inventor of these optical approaches, makes me ideally suited to execute this plan, and overcome any obstacle. This proposal promises to revolutionize how we study and validate models of cortical interaction and neural coding. Far from being limited to motor control alone, these findings will have far-reaching implications – helping to create next-generation neural prosthetics, treat complex neurological diseases, and change the way we study complex systems.

NIH Research Projects · FY 2025 · 2024-09

SUMMARY: According to the World Health Organization, approximately 15 million children are born prematurely each year. Many of these infants end up spending days to weeks in a neonatal intensive care unit (NICU). Infants who are born prematurely are often exposed to noise and light levels that affect auditory and visual development. Subsequently, these children can have long-term impairments in cognition, visuospatial processing, hearing, and language. We have developed a rodent model of NICU exposure to light and sound using the Mongolian gerbil (Meriones unguiculatus), which has a low frequency human-like audiogram and is altricial. To simulate preterm infancy the eyes and ears will be opened prematurely, followed by exposure to the NICU-like sensory environment throughout the gerbil’s cortical critical period of auditory development. Here, natural eye-opening closes the critical period ~ P18, and early eye opening simulates the effect of preterm birth, by closing the critical period precociously (Mowery et al., 2016). This motivated the core hypothesis that early eye opening induces precocious closure of auditory development through feed forward peripheral visual excitatory input onto cross modal synapses located in auditory cortex. Recent research has provided preliminary validation for the effect of early light and noise exposure on long-term brain development (Gay et al., 2023). We will test this hypothesis with three aims. The first aim will track electrophysiological development of inhibition within the auditory cortex after NICU (early eye/ear opening) or noise only (early ear opening) exposed neonates, juveniles, and adults. The second aim will track the development of physiological peripheral measures for auditory (auditory brainstem response) and visual (visual evoked potentials) function in NICU (early eye/ear opening) or noise only (early ear opening) exposed neonates as they develop into juveniles and then adults. Histological measurements of the white and grey matter along the visual and auditory neuraxes will be generated and correlated with any impairments to peripheral physiology thresholds of each animal in AIM 2. The third aim will behaviorally test measures of auditory and visual based decision-making impairments in NICU (early eye/ear opening) or noise only (early ear opening) exposed animals after they have become adults. Together, the findings from this project will introduce a new animal model of the NICU preterm infant, with which mitigative and treatment-based approaches to early light and sound exposure can be ethically carried out by the research community. We hope to establish this animal model to create a bridge between clinical pediatric physician researchers and the animal research community to advance clinical treatments and care for the NICU-exposed preterm infant.

NIH Research Projects · FY 2026 · 2024-09

ABSTRACT Nearly one third of the world’s population lacks access to adequate food, and over three billion people cannot afford a healthy diet. Nutrition insecurity - the lack of consistent access, availability, and affordability of foods and beverages that promote well-being and prevent disease - leads to both undernutrition and obesity/overweight: a ‘dual burden’ associated with high rates of non-communicable disease, particularly in low- and middle-income countries (LMICs). This is especially true in informal urban settlements, where our work has shown that a lack of formal infrastructure and access to services exacerbates underlying social and economic barriers to eating well. Adolescence is an optimal time to intervene to reduce nutrition insecurity and malnutrition. However, interventions seldom target adolescents, nor are they tailored to the lived experiences of adolescents or to the broader systems that influence adolescent food choices and nutrition security. The overarching goal of this project is to reduce nutrition insecurity and malnutrition among adolescents in LMICs. To do this we employ an innovative combination of participatory ‘systems thinking’ methods, quantitative empirical data, and modeling. We will work in informal and formal settlements in Nairobi, Kenya so that we can characterize differences in the food systems between them, and understand how these differences, and other individual-, household- and community-level factors, influence adolescents’ food choices. We will (Aim 1) physically mapping the food environments of 10 schools (2 per neighborhood in each of 5 neighborhoods) and use a participatory systems science toolkit that includes cognitive mapping and group-based modeling to understand how adolescents make decisions around food. In parallel, we will quantify the influence of nutrition insecurity on dietary patterns, malnutrition, and food preferences (Aim 2) by conducting surveys (using standardized measures of food and water insecurity and the perceived food environment) with adolescents and their primary caregivers as well as repeated 24-hour dietary recalls, and physical measurements (body mass index, micronutrient status, and hemoglobin) with adolescents (n=700) at three time points. Food preferences will be assessed by conducting a discrete choice experiment with the adolescents included in our sample. The data from Aims 1 and 2 will then be used to co-design food systems interventions to improve adolescents’ food choices, nutrition security, and malnutrition outcomes and rank them using stakeholder-derived multi-criteria decision analysis (Aim 3). We will work with local policymakers and community leaders to prioritize interventions with the potential to transform LMIC food systems that are experiencing urban growth based on their feasibility and likely impact. This project will address an urgent need to develop double-duty interventions that address the root cause of undernutrition and overweight/obesity among adolescents in LMICs.

NIH Research Projects · FY 2025 · 2024-09

PROJECT SUMMARY/ABSTRACT Tobacco use remains a leading cause of premature disease and death in the United States, including those attributable to cancer. In a landmark racketeering lawsuit filed by the US Department of Justice in 1999, several major tobacco manufacturers were found guilty of consumer fraud, which included deceptive tactics such as lying to consumers about the deadly effects of cigarettes, manipulating cigarettes to make them more addictive, and marketing their products to young people. To help remedy these actions, the court ordered the manufacturers to run a series of “corrective statements” in several communication channels in the US, including television, newspapers, and signage displays in stores at the point-of-sale. After nearly two decades of tobacco industry litigation to delay the corrective statement requirement in retail settings – one of the industry’s most valuable marketing channels – a 2022 court order finally mandated that the signs be posted from October 1, 2023 through June 30, 2025, a 21-month period. More than 200,000 retailers that have marketing agreements with the affected manufacturers – approximately 2 in 3 retailers nationwide – are required to display these large, eye-catching signs that inform consumers about the health risks of tobacco and the tobacco industry’s history of deception. While this historic retail-based intervention has the potential to positively impact public health, critical questions remain about the nature of its implementation, reach, and effects on consumers, including impacts on health equity. For example, it is unknown how implementation features of the signage (e.g., presence, number, placement) may vary by store type and neighborhood; if, and to what extent, consumers notice and retain the corrective messages; and whether exposure impacts tobacco knowledge, beliefs, intentions and behaviors (e.g., quit attempts). The finite window of intervention delivery offers a unique and time-sensitive opportunity to answer these questions, which may inform future large-scale tobacco information dissemination efforts, as well as identify subpopulations who may benefit from supplemental health education programs or other services. The specific aims of the study are to: (1) Characterize both implementation and reach by collecting detailed information about corrective statement implementation features through a retailer audit study. Supplemental analyses will calculate the prevalence/density of retailers required to display the statements across the United States. (2) Use eye- tracking methodologies to explore attention to the corrective statements (i.e., reach and impact) in the context of real-world exposures. (3) Field longitudinal surveys of adults and youth to examine reach and impact of the statements among a nationally representative sample, including reported exposures, message perceptions, and impact on tobacco knowledge, attitudes, intentions and behaviors.

NIH Research Projects · FY 2025 · 2024-09

Abstract. The maintenance of a differentiated cell type (i.e., cell identity), a feature established by cell type- specific transcriptional programs, is critical for cell function and tissue homeostasis. Yet, despite acquiring a specific function, the differentiated cell remains a plastic entity which retains the ability to functionally reprogram itself in response to incoming signals while simultaneously maintaining its identity. To maintain this plasticity, the cell relies on transcription factor (TF) networks that access, interpret and implement genomic information. Upon receiving and processing an incoming signal (whether developmental, environment or damage), the cell responds by rewiring its TF network. In some instances, the processing of the incoming signal rewires the TF network and destabilizes cell identity leading to a transitional cell state with reduced function known as senescence. Originally described as a stable proliferative arrest, senescence has recently emerged as a transitional cell state heavily linked to the aging process and development of diseases such as osteoarthritis (OA), cancer and fibrosis. The resolution of this transitional state can lead to outcomes linked with disease development, including cell death, stabilization of senescence or disease states (Graphical Abstract). With this understanding, we hypothesize that transitional senescence states represent critical intermediates that could be manipulated for therapeutic applications. Given the overarching contribution of senescent cells to pathological processes such as aging, cancer and fibrosis, manipulating senescence states has tremendous potential for restoring cellular and tissue function across many diseases. The chief focus of our research program is to define and manipulate the gene regulatory networks that dictate the transition through senescence states. To achieve this goal, we employ an unbiased approach that involves the generation of TF network models from bulk and single-cell time-series high-throughput sequencing epigenomic data from primary human cells representing various tissues undergoing senescence-associated transitions linked to disease development. Using the TF network model as a logical structure to validate and guide the manipulation of senescence states, we target critical nodes using reverse genetics, genome editing and pharmacological approaches, and confirm key findings in human samples using senescent cell isolation methods. Over the next 5 years, the main goals of our research program are: 1) To generate TF network models of primary human chondrocytes (to model OA) and hepatic stellate cells (HSCs, to model fibrosis) undergoing replicative and cytokine-induced senescence (RS and CIS), 2) To test the prognostic potential of senescence-linked epigenomic signatures using publicly available human datasets, and 3) To develop strategies to restore the function of senescent cells by modulation of their TF network. The overall vision of the research program is to design and implement strategies to reprogram senescent cells across multiple diseases. If successful, this research program will pave the way toward a new era of cell therapy in which diseased cells could recover their function through TF network reprogramming.

NIH Research Projects · FY 2025 · 2024-09

PROJECT SUMMARY Creating a pathway for trainees to enter the clinical and translational science (CTS) workforce, including students from backgrounds often underrepresented in STEM and CTS, requires early engagement while in training. Addressing the needs of trainees at the forefront of their CTS education and stimulating CTS interest in historically underrepresented student populations to foster the development of a diverse CTS workforce are central to the efforts of the New Jersey Alliance for Clinical and Translational Science (NJ ACTS), the first and only CTSA Hub in the state of New Jersey. Our R25 grant application, leveraging the resources of the parent UM1 Award, proposes the creation of the NJ ACTS Clinician Research Experiential Summer Training (CREST) Program involving: A) a mentored research and interprofessional training in clinical and translational science component for medical and pharmacy students enrolled at Rutgers who are prime for early intervention as the number of physician-scientists are in decline and there are limited nationwide programs for training pharmacist-scientists; and B) an outreach component to stimulate interest, engage, and foster CTS for Rutgers undergraduates participating in the NJ Educational Opportunity Fund (EOF) program for students from educationally and economically disadvantaged backgrounds, and for medical students at City University of New York (CUNY), whose majority of graduates go on to practice primary care in underserved communities. In component A, 10 medical and pharmacy students will participate in mentored research and interprofessional training in clinical and translational science each summer. This comprehensive 10-week program provides students with hands-on research training using state-of-the-art, case-based learning along with career development workshops and formal training in translational science. The objective of CREST Component A is to immerse medical and pharmacy students in research experiences that advance analytical and technical skills and stimulate their pursuit of advanced research training. Component B broadens the impact of the CREST program and benefits a diverse population of students. In this component, medical and pharmacy CREST participants will serve as “ambassadors” who will meet in hybrid (in-person/virtual) sessions with Rutgers EOF undergraduates to discuss their experience and paths to medical and pharmacy school, and to share their enthusiasm for and the importance of their CTS research. Component B will also offer the opportunity for 1st and 2nd year CUNY medical students to complete the online/asynchronous translational science digital badge. After completing the badge, these students will be offered the opportunity to attend in a virtual format the research presentations from students in Component A. The objective of CREST Component B is to foster interest and educate undergraduate and medical students from underrepresented backgrounds about the CTS process. Ultimately, CREST will build a well-trained and diverse workforce prepared to address challenges in clinical and translational science and improve public health.

NIH Research Projects · FY 2025 · 2024-09

The overarching goal of this T32 Predoctoral Training Program is to prepare future generations of translational researchers to leverage the principles of translational science to identify, effectively disseminate, and implement new approaches to address current health challenges. The objective of this training program is to build upon the rich history of collaborative research and education among our alliance universities to develop a personalized “precision” training program that instills in our early career biomedical researchers the knowledge, skills, and abilities needed to effectively translate the information gleaned in the laboratory, clinic, and community to real-world advances that improve health. All NJ ACTS Elements, Modules, and Workgroups will provide services and platforms for training our T32 trainees and facilitate their nimble access to content and method experts in a breadth of disciplines, including team science, informatics, biomarker discovery, community engagement, health policy. Uniquely, ASCENT transcends the curriculum encountered in traditional predoctoral programs by focusing on the knowledge and expertise needed to conduct translational team research across disciplines while building social and leadership skills. Our Specific Aims are to: 1) Develop the accessible and flexible ASCENT program with a focus on the precision training of predoctoral translational researchers in the knowledge, skills and abilities needed to efficiently and effectively advance the diagnosis and treatment of disease, modify behavior to prevent or reduce the impact of disease, and assure equity in the research of disease and the access to healthcare; 2) Identify and recruit a cadre of outstanding translational research and translational science faculty to act as Preceptors and instructors for our trainees and assure that they have appropriate skills to be effective mentors for trainees with diverse scientific interest and backgrounds, both during their time in the ASCENT program and as they transition to the next step of their research careers; 3) Recruit and retain a diverse and exceptional cohort of trainees, committed to a career in translational research and provide personalized training in the principles underlying each step along the spectrum of translational research so they will complete the program with the knowledge, skills and abilities, as well as the key characteristics, of a translational scientist; 4) Promote and assure a safe and inclusive learning environment, where each trainee is respected as an individual, their learning is tailored to their previous and current research interest, career aspirations, and personal situations, and where they recognize their role as scientists to conduct and present their research with integrity, respect, clarity and transparency for the good of society; and 5) Provide strong leadership, support and oversight, with comprehensive evaluation and assessment of the faculty, students and programmatic activities, to assure that the ASCENT program fulfills the mission and expectations of NCATs, the CTSA and NJ ACTS.

NIH Research Projects · FY 2024 · 2024-09

PROJECT SUMMARY/ABSTRACT Tobacco use remains a leading cause of premature disease and death in the United States, including those attributable to cancer. In a landmark racketeering lawsuit filed by the US Department of Justice in 1999, several major tobacco manufacturers were found guilty of consumer fraud, which included deceptive tactics such as lying to consumers about the deadly effects of cigarettes, manipulating cigarettes to make them more addictive, and marketing their products to young people. To help remedy these actions, the court ordered the manufacturers to run a series of “corrective statements” in several communication channels in the US, including television, newspapers, and signage displays in stores at the point-of-sale. After nearly two decades of tobacco industry litigation to delay the corrective statement requirement in retail settings – one of the industry’s most valuable marketing channels – a 2022 court order finally mandated that the signs be posted from October 1, 2023 through June 30, 2025, a 21-month period. More than 200,000 retailers that have marketing agreements with the affected manufacturers – approximately 2 in 3 retailers nationwide – are required to display these large, eye-catching signs that inform consumers about the health risks of tobacco and the tobacco industry’s history of deception. While this historic retail-based intervention has the potential to positively impact public health, critical questions remain about the nature of its implementation, reach, and effects on consumers, including impacts on health equity. For example, it is unknown how implementation features of the signage (e.g., presence, number, placement) may vary by store type and neighborhood; if, and to what extent, consumers notice and retain the corrective messages; and whether exposure impacts tobacco knowledge, beliefs, intentions and behaviors (e.g., quit attempts). The finite window of intervention delivery offers a unique and time-sensitive opportunity to answer these questions, which may inform future large-scale tobacco information dissemination efforts, as well as identify subpopulations who may benefit from supplemental health education programs or other services. The specific aims of the study are to: (1) Characterize both implementation and reach by collecting detailed information about corrective statement implementation features through a retailer audit study. Supplemental analyses will calculate the prevalence/density of retailers required to display the statements across the United States. (2) Use eye- tracking methodologies to explore attention to the corrective statements (i.e., reach and impact) in the context of real-world exposures. (3) Field longitudinal surveys of adults and youth to examine reach and impact of the statements among a nationally representative sample, including reported exposures, message perceptions, and impact on tobacco knowledge, attitudes, intentions and behaviors.

NIH Research Projects · FY 2024 · 2024-09

Project Summary/Abstract This proposal requests funding for the purchase of a Bruker timsTOF HT mass spectrometer (timsHT MS) coupled with an Evosep One HPLC in order to meet the increasing demand for high-performance proteomics instrumentation at Rutgers University Biomedical and Health Sciences (RBHS)-Newark Campus. The Center for Advanced Proteomics Research (CAPR) at RBHS-New Jersey Medical School in Newark will benefit from this instrument, enhancing service quality. With ever-increasing service needs from NIH-funded researchers, the existing Lumos Orbitrap MS has been exhausted, resulting in a long delay for large-scale proteomics studies. This wait time impedes the progress of NIH-funded investigations. Therefore, adding a timsHT MS at CAPR will speed up the research momentum of Rutgers' NIH-supported researchers. Experienced CAPR scientists will operate the new timsHT MS to conduct wide-scale proteomics studies for a major user group of 15 NIH-funded researchers and others. These investigations aim to analyze more samples quickly and identify more low-abundant proteins, post-translational modifications, and their positional isomers. To achieve these objectives, a high-end MS that can offer sensitivity, speed, and time-saving technology to distinguish isobaric peptides and robustness for boosting instrument uptime. The timsHT MS has a unique blend of engineering and software functionalities that will enable sensitive proteomics studies to meet three unmet needs. Firstly, deep proteome coverage will be achieved through dual Trapped Ion Mobility Spectrometry (TIMS), which facilitates ~30- fold time-focusing of low-level peptide ions, and software that is aware of collision cross-section for in-depth peptide identification and quantification. Secondly, high sensitivity without compromise on speed and resolution will be achieved through Parallel Accumulation - Serial Fragmentation technology, which facilitates MS/MS acquisition at 150 Hz, enabling speedy studies of moderate-level samples with short LC gradients. Thirdly, instrument uptime and robustness will be maximized through an orthogonally positioned ion source to cut impurities in the mass analyzer, a dual-TIMS funnel to filter contaminating gas, and an improved focus on low-level peptide ions. The NIH-funded purchase of this shared timsHT MS will empower CAPR to overcome the time limitations of existing equipment and offer complementary MS technologies to support RBHS-Newark researchers and those from neighboring institutions in carrying out their research goals on NIH-funded grants.

NIH Research Projects · FY 2025 · 2024-09

ABSTRACT Piezo2 is a non-selective Ca2+-permeable cation channel activated by mechanical force. It is expressed in peripheral sensory neurons of the dorsal root ganglia (DRG) and it plays important roles in gentle touch and injury-induced mechanical pain. Piezos are large proteins, with 38 transmembrane domains and their membrane footprint is larger than that of any other known ion channels. This suggests that membrane lipids could significantly impact the behavior of these channels, but exploration of the effects of membrane lipids on Piezo function is in its early stages. The goal of this proposal is to gain insights into how lipids regulate Piezo channels, building upon our recent identification of membrane lipids that specifically inhibit Piezo2 activity while not affecting Piezo1. The proposal is based on our recent finding that the TMEM120A protein robustly inhibits Piezo2, but not Piezo1 channels. Structural studies of TMEM120A indicate that this protein shows similarity to a lipid modifying enzyme ELOVL7, with a CoA molecule bound to each monomer. We hypothesized that TMEM120A inhibits Piezo2 by modifying cellular lipid composition. Our preliminary data show that expressing TMEM120A increases levels of lipids in the Kennedy pathway of triglyceride synthesis, with the most robust changes in phosphatidic acid (PA) and lysophosphatidic acid (LPA) with saturated acyl chains. Intracellular PA delivery through the whole cell patch pipette inhibited Piezo2, but not Piezo1 activity. Generating endogenous PA in the plasma membrane by optogenetic activation of phospholipase D inhibited Piezo2, but not Piezo1 activity. Intracellular delivery of LPA, as well as incubation with carbacyclic PA (ccPA), a non-metabolizable analogue of PA and LPA inhibited Piezo2 channels. Our data identify PA and LPA as endogenous inhibitors of Piezo2 channels. In our proposal we aim to identify the molecular determinants of the effects of PA and LPA (Aim1), investigate their roles in regulation of Piezo2 activity by signaling pathways (Aim 2), and tests in vivo effectiveness of this lipid regulation on Piezo2 mediated sensory processes (Aim 3). Overall, we identified novel lipid regulators of Piezo2 channels, that do not affect Piezo1. Our data will provide important insight into the regulation of Piezo2 by membrane lipids, and the knowledge gained from this study has the potential to be utilized in the future development of Piezo2 inhibitors for treating various pain conditions.

NIH Research Projects · FY 2025 · 2024-08

In humans, exposure to Mycobacterium tuberculosis (Mtb) results in varied infection and disease outcomes. 5- 10% of individuals infected with Mtb manifest disease, with evidence of infection in the remaining majority evinced only as positivity of IGRA (interferon gamma release assay). Given that the risk of developing reactivation TB is ~10% per lifetime in immunocompetent LTBI, it is thought the anti-TB immunity developed in this population can control the infection rather effectively. Another group, termed resisters, are defined as persons who remain IGRA-negative despite prolonged heavy exposure to Mtb. Resisters, compared to LTBI subjects, exhibit an interferon (IFN)γ-independent T cell response. The non-IFNγ T cell response to Mtb antigens (Ags), in conjunction with class switch recombination and the relatively high binding affinity Abs developed in resisters, suggests that this population has been infected with and has established an adaptive immune response to Mtb. Our Brazil household contact (HHCs) study of pulmonary TB showed differential IGRA response in the exposed HHCs despite having similar exposure to the infectious index case. Approximately 45% of HHCs were found to be IGRA negative. We defined the resister IGRA negative group as “Resistant to IGRA Conversion” (RIC) and the IGRA positive group as IGRA Converters (IC). Our preliminary data show that IFNγ-independent adaptive T cell immunity is engaged in RIC-HHCs and peripheral blood mononuclear cells from RIC-HHCs restrict Mtb growth. Further, preliminary data points to GM-CSF-secreting T cells as a likely component of the IFNγ-independent T cell response. GM-CSF signaling enhances IL-1β production and given that IL-1β is critical to anti-bacterial immunity in TB, we hypothesize that stable GM-CSF secreting memory T cells contribute to the RIC phenotype and the cytokine may also have a significant role in IC-HHCs. Accumulating evidence suggests that antibodies (Abs) play a role in regulating infection and disease outcomes in individuals exposed to Mtb, including the RIC- and IC-HHCs. We showed that Tfh cells (the CD4 T helper cell that is essential for the development of the B cell and Ab responses) from LTBI subjects harbor phenotypically distinct subsets that have been linked to discrepant B cell-regulatory functions in varied immunological systems. We thus posit that varied functions of Tfh cells in individuals exposed to Mtb may influence infection outcome by directing the development of distinct Ab responses. Comparative characterization of anti-TB immunity in RIC-HHCs versus IC-HHCs is a major goal of this proposal. The proposed studies will likely illuminate the IFNγ-independent T cell mediated immunological mechanisms that define the RIC phenotype, and of protective mechanisms mediated by the Tfh cell-B cell-Ab axis--this may lead to the development of novel vaccine targets. We will reconsent HHCs, retest them for their IGRA status and then recruit to the study the ones who have maintained their original IGRA phenotype. Our aims are: i) Elucidate the IFN-γ-independent T cell response in RIC-HHC in comparison to IC- HHC; ii) Characterize the B cell, Ab, and cTfh responses of RIC-HHCs and IC-HHCs.

NIH Research Projects · FY 2023 · 2024-08

Project Summary An overactive Parathyroid Hormone Receptor 1 (PTHR1) and its downstream signaling pathway (Gsα) are involved in several bone diseases including hyperparathyroidism, Jansen’s metaphyseal chondrodysplasia (due to a constitutively active PTHR1) or Fibrous Dysplasia-McCune-Albright Syndrome (due to a constitutively active Gsα) which, in the case of the genetic diseases, can cause major developmental and lifelong problems with their skeletons. In all three situations, the only therapies available are surgical or palliative. Protein Kinase A (PKA) is an enzyme whose activity is dependent on the activity of Gsα and cellular levels of cAMP. It is regulated by PTH binding to PTHR1 in osteoblasts. We have generated an inducible, osteoblast-specific mouse model of hyperactive PKA by collagen 1-directed deletion of the regulatory subunit, Prkar1a, and found a high bone turnover phenotype in skulls, long bones, vertebrae and caudal bones of the tail that mimics the PTHR1 diseases. We and others have shown that PTH action on the osteoblast through PKA controls gene expression, in part, through inhibition by phosphorylation of salt-inducible kinases (SIKs). These kinases tonically phosphorylate cAMP-regulated transcriptional coactivators (CRTC1, 2 and 3) and histone deacetylases 4/5 (HDAC4/5), sequestering them in the cytoplasm. Upon PTH action, PKA-mediated SIK inhibition causes CRTC2 and HDAC4/5 phosphorylation levels to decrease with subsequent nuclear translocation of CRTC2/3 and HDAC4/5 which mediates transcription of the osteoclastogenic gene, Rankl, regulation of Mmp13 and suppression of Sost transcription. SIK inhibition, deletion or knockdown mimics the effects of PTH. We have recently tested several agents for possible activation of the SIKs and resultant inhibition of PTH-induced Rankl transcription in differentiating mouse calvarial osteoblasts and found a small molecule, 9117014, as a “putative” SIK activator, to fit these goals. From these data of cells in culture and preliminary data in vivo, we have developed the hypothesis that small molecule activators of SIKs will reverse the unwanted effects of the PTH signaling pathway in bone. Consequently, the specific aims of this resubmitted R21 application are to 1) determine if activation of SIKs by a small molecule will inhibit PTH regulated gene expression in osteoblasts, by a. performing dose responses on PTH-stimulation of Rankl, b. knocking down SIK2/3, c. testing the effects on other PTH-regulated genes, 2) assess if a small molecule activator of SIKs will inhibit a high bone turnover phenotype in mice, by a. injecting a developmental model of high bone turnover, b. injecting an adult model of high bone turnover, c. injecting an adult model of hyperparathyroidism, and conducting complete bone analyses. In so doing, the results would be of high impact and may lead to further preclinical studies that could form the basis for the first disease-modifying oral treatments for patients with hyperparathyroidism, Jansen’s metaphyseal chondrodysplasia or Fibrous Dysplasia-McCune-Albright Syndrome.

NIH Research Projects · FY 2026 · 2024-08

PROJECT SUMMARY The advances in cancer survival rates greatly enhance the awareness of side effects of cancer therapy and long- term life quality after cancer. Primary ovarian insufficiency (POI) and infertility are major side effects in young female cancer patients. As the female gonad, the ovary contains various stages of follicles as its functional unit. Each follicle consists of a central germ cell oocyte and surrounding somatic cells. Primordial follicles are at the earliest stage and remain quiescent for months or decades to establish ovarian reserve, a marker of female reproductive life span. Our research teams have demonstrated that commonly used chemotherapeutic drugs, including doxorubicin (DOX), cisplatin (CDDP), and cyclophosphamide (CPA), primarily induce DNA damage and apoptosis of the oocytes in primordial follicles, resulting in the entire primordial follicle death, POI, infertility, and endocrine disorders. Mechanistically, the gonadotoxic anti-cancer agents activate DNA damage response (DDR)-TAp63-related signaling to trigger oocyte apoptosis. However, the underlying mechanism remains largely unknown. Using wild type (WT) mice and a genetically modified mouse model with oocyte-specific deletion of all three c-Jun NH2-terminal kinase (JNK) genes (Jnk1/2/3), our preliminary studies demonstrated that: (1) JNK was selectively activated in primordial follicle oocytes following DOX treatment; (2) a selective JNK inhibitor, SP600125, blocked DOX-induced activation of TAp63 and follicle atresia; (3) oogenic deletion of JNK didn’t affect normal ovarian functions but prevented the induction of apoptotic genes and primordial follicle atresia following DOX treatment; and (4) pharmacological inhibition or genetic deletion of JNK blocked TAp63 activation induced by CDDP and CPA, the other two gonadotoxic anti-cancer agents. Based on these preliminary data and the fact that JNK has been found to pro-oncogenic in certain types of cancer such as leukemia, our central hypothesis is that JNK critically regulates DDR-TAp63-related apoptotic signaling in primordial follicle oocytes following treatment with gonadotoxic anti-cancer agents; and pharmacological inhibition of JNK prevents chemotherapy-induced POI and infertility without compromising anti-cancer efficacy of chemotherapy. We will use DOX as a representative gonadotoxic anti-cancer agent to test our hypothesis. In Aim 1, we will investigate the mechanism by which JNK regulates DDR-related apoptotic signaling in primordial follicle oocyte following DOX treatment. In Aim 2, we will determine the efficacy of pharmacological inhibition of JNK against DOX treatment on protecting ovarian reserve in a young leukemic female mouse model. Completion of these two Specific Aims will allow us to (1) elucidate the molecular mechanisms of oogenic JNK in contributing to DDR-TAp63-related apoptotic signaling in primordial follicle oocytes following treatment with gonadotoxic chemotherapeutic agents, and (2) capitalize on the cell type-dependent pro- or anti-apoptotic effects of JNK to identify selective JNK inhibitors that preserve ovarian reserve, fertility, and ovarian endocrine functions in young female cancer survivors without interfering with the efficacy of chemotherapy.