NIAAA - National Institute on Alcohol Abuse and Alcoholism Grants

NIAAA - National Institute on Alcohol Abuse and Alcoholism

Excessive alcohol consumption is thought to be the third leading preventable cause of death in the United States. But these estimates of the deleterious effect of alcohol are largely based off self-reports of alcohol consumption. Concerningly, because we and others have shown that clinical self-reports are unreliable, while others have shown that the estimates of daily alcohol consumption by CDC are off by at least three-fold, the true cost of excessive drinking may be much higher. In fact, it is possible that excessive alcohol consumption is the leading preventable cause of death. We have developed a methylation sensitive digital PCR (MSdPCR) test called the Alcohol T Score (ATS) that quantifies chronic alcohol consumption. In several peer reviewed studies, we have shown that the ATS significantly outperforms the carbohydrate deficient transferrin (CDT) test, the current industry gold standard, in predicting chronic heavy alcohol consumption (HAC; ≥ 6 drinks/day for 8 weeks) and strongly predicts death in those with severe coronary heart disease. We recently examined an epidemiologically sound sample of DNA and data from 4910 subjects (3:1 mix of lung cancer cases and controls) who participated in the Prostate, Lung, Colorectal and Ovarian (PLCO) Cancer Screening Trial with the ATS. We found that the ATS was a powerful predictor of mortality in smokers with (n=92) and without lung cancer (n=402). In fact, in both groups, the effects of alcohol consumption on mortality greatly outweighed those of smoking. Taken together with our prior findings, these new findings strongly suggest that heavy drinking, not smoking is the largest preventable cause of mortality in the United States. Unfortunately, this sample was largely White and nonsmoking, which limits generalizability of the results. Therefore, in this high-risk R43 study, we propose to extend our ATS determinations to all non-smoking subjects in this group (n=612) and all of those of non-White ancestry (n=985) from our existing cohort of 5000 subjects from the PLCO cohort. We hypothesize that the ATS will predict mortality broadly across both sexes, and in all ancestries whether or not they smoke. This project is innovative because heavy alcohol consumption (HAC) is often missed by clinicians. It is high impact because HAC may be a significant, if not the largest preventable cause of death in the United States. The team is well prepared to conduct the research including statisticians and a leading expert on DNA methylation. It is highly feasible because the assay already exists and we have both the DNA and the data. As a direct result of this research, we will determine the frequency and effect of HAC on a broad epidemiologically sound, cross section of the American public. As an indirect outcome, we will demonstrate the potential use case for conducting this rapid, easy to perform MSdPCR test as part of medical assessments of those presenting for lung or perhaps any other cancer screening while gathering needed data for eventual approval of the ATS by the FDA as a test for HAC.

NIAAA - National Institute on Alcohol Abuse and Alcoholism

Scaling up AUD Information Resources for Provider Education: The Behavioral Health Counseling Competencies AUD Training Suite Project Summary The alcohol treatment gap is a multi-dimensional phenomenon that requires intervention related to public awareness, access to specialty care, and improvement in the quality and quantity of the broader, behavioral health workforce. The current proposal targets the latter via the development of a training product designed to increase provider knowledge and skill related to alcohol misuse and AUD. This Phase I/II Fast Track STTR proposal will create a didactic and interactive simulation training that will facilitate provider use of the content and tools within NIAAA’s key dissemination resource - Rethinking Drinking. The product will incorporate two additional elements. First, content delivery will be grounded in an NIAAA-funded training program targeting four core competencies of behavioral health counseling: 1) developing a behavior change relationship, 2) providing psychoeducation, 3) setting and monitoring behavior change goals, and 4) providing behavioral skills training. Second, training technology will include proprietary conversation-simulation technology that has a 15-year track record in changing provider behaviors. The team includes the small business applicant, the primary academic partner, and the conversation simulation company. Additional content expertise will be provided by a panel of subject matter experts. The team are established independent investigators, small business award recipients, and long-standing collaborators, which enhances the feasibility and probability of success in meeting the following Specific Aims. Phase I will establish feasibility and technical merit via the development, expert panel review, and end user piloting of the first of the four-module training program – Alcohol and you, establishing a behavior change relationship. Phase II will develop Module 2 How much is too much? providing psychoeducation, Module 3 Thinking about change, goal setting and monitoring, and Module 4 Setting drinking boundaries, providing behavioral skills training and conduct a pilot randomized trial (N = 55) comparing the training suite to a dissemination as usual control using a pre-post, standardized patient design. Commercialization will occur throughout the award period and particularly in year three. The proposed project merges team expertise and technical support to create a novel training product that can increase the quality and quantity of the behavioral health workforce, thereby contributing to reductions in the alcohol treatment gap.

NIAAA - National Institute on Alcohol Abuse and Alcoholism

PROJECT SUMMARY Alcohol use disorder (AUD) represents a significant public health crisis in the U.S, affecting over 10% of the population above the age of 12. Despite its prevalence, only 8% of AUD patients receive any treatment, with only 2% receiving pharmaceutical interventions. Current standard treatments, including cognitive-behavioral therapy, naltrexone, acamprosate, and disulfiram, demonstrate limited efficacy and unacceptably high relapse rates. This substantial treatment gap highlights the urgent need for novel therapeutic approaches. Emerging research suggests serotonergic psychedelics, including dimethyltryptamine (DMT), show promise in treating addiction with larger and longer-lasting effects than conventional treatments. Compared to LSD and psilocybin, DMT offers distinct advantages over other psychedelics, including higher efficacy for activation of intracellular 5- HT2A, tolerance development with repeated use, and potentially the strongest subjective experience. This makes DMT particularly suitable for regular dosing regimens and suggests significant potential for therapeutic applications. However, DMT faces significant pharmacokinetic challenges when administered orally. It is rapidly degraded by monoamine oxidase (MAO), resulting in no psychotropic effects and extensive conversion to inactive metabolites. This necessitates alternative administration routes like inhalation or infusion, which introduce variability or patient distress. Remedi Inc. is pioneering an innovative approach by co-formulating DMT with CX157, a proprietary reversible and selective MAO-A inhibitor (RIMA). Unlike irreversible MAOIs, CX157 reversibly inhibits MAO-A, mitigating serious side effect risks. CX157's selectivity for MAO-A over MAO-B also improves tolerability. Additionally, this co-formulation reduces abuse potential, as CX157 creates subjectively unpleasant side effects at high doses. Unlike harmine, a commonly used MAOI, CX157 is not a P450 substrate, reducing pharmacokinetic variability across patients. Preliminary data also demonstrates that CX157 is consistently reversible, enabling recovery of MAO activity within 12 hours in humans. This Phase I project aims to demonstrate feasibility for this oral combination therapeutic approach through 1) demonstrating CX157 enables oral bioavailability in a rat model, where comprehensive pharmacokinetic profiles will be characterized. This study will also include quantification of the head twitch response (HTR). 2) Demonstrate efficacy in reducing ethanol consumption in a validated rat model of AUD and characterize synaptogenesis in rats orally administered CX157/DMT. Success metrics include achieving appropriate pharmacokinetic parameters and significant reduction in alcohol consumption in animal models. Commercialization of this DMT/CX157 product could transform AUD treatment by providing the first orally bioavailable administration of DMT with stable consistent dosing and a strong safety profile. Success with this novel approach has the potential to significantly impact public health by addressing the substantial unmet need in AUD treatment.

NIAAA - National Institute on Alcohol Abuse and Alcoholism

PROJECT SUMMARY / ABSTRACT Hazardous alcohol consumption remains a significant public health issue in the U.S., responsible for over 140,000 deaths annually. Despite its profound impact, only about 10% of individuals diagnosed with alcohol use disorder (AUD) receive any form of treatment, underscoring a substantial treatment gap. This SBIR Phase I proposal seeks to address this gap by enhancing the "Step Away Alcohol Manager" (SAAM), a mobile health (mHealth) intervention developed by Here and Now Systems, LLC (HNS). SAAM builds on the success of the Step Away app, which has demonstrated significant reductions in alcohol use —50-60% reductions in four pilot trials. However, sustaining long-term user engagement remains a challenge, with only 40% of users continuing to engage with the app after six months and some modules being used infrequently. This project aims to integrate cutting-edge artificial intelligence (AI) and biometric technologies to enhance personalization, improve engagement, and increase the app’s overall effectiveness in promoting long-term behavior change. Specifically, we will incorporate a multi-agent generative AI (GenAI) and large language model (LLM) to provide users with empathic, real-time conversations and strategies to manage drinking-related challenges such as difficulty sleeping, stress, and cravings. The AI will initiate interactions during users' high-risk times for drinking and during daily check-ins, providing proactive and responsive support. Additionally, we will integrate facial scan technology which will monitor users’ stress levels, sleep quality, and overall health in real time. This biometric data will be used to tailor feedback and enhance the relevance of the AI-driven interactions, further personalizing the user experience. The Phase I project has three specific aims: (1) Develop SAAM with advanced AI-driven conversational capabilities and real-time biometric feedback. The chatbot will deliver evidence-based responses based on trusted sources, ensuring it adheres to scientifically sup ported interventions. (2) Conduct end-user testing with 20 participants who meet AUDIT criteria for hazardous drinking to evaluate the app’s content, usability, and the quality of the personalized messages generated by the A I. We will use validated tools such as the Client Satisfaction Questionnaire (CSQ) and System Usability Scale (SUS), with benchmarks of 24+ on the CSQ and 70+ on the SUS. We will also utilize qualitative research methodology to determine themes related to SAAM’s strengths and limitations, which will be used to inform needed modifications. (3) Refine SAAM based on feedback from these reviews, addressing any usability issues, content gaps, and engagement barriers to create a polished, user-friendly app ready for Phase II evaluation. Through this Phase I project, SAAM has the potential to significantly expand access to effective, evidence-based interventions for alcohol use disorder by leveraging advanced AI and biometric technologies to provide real-time, personalized support. This innovative approach will address the critical public health need for more accessible and engaging alcohol treatment solutions, offering a novel, scalable intervention with the potential for widespread adoption.

NIAAA - National Institute on Alcohol Abuse and Alcoholism

Abstract: Alcohol consumption adversely affects up to 28% of hospitalized patients and contributes to a loss of 133 million disability-adjusted life years and 5.3% of worldwide deaths each year. As alcohol use has substantial health and economic impact, much attention has been directed toward the numerous adverse health outcomes in patients with unhealthy alcohol use. Alcohol use disorder (AUD) occurs on a spectrum from mild to severe and is precipitated by binge drinking and heavy alcohol use. We and others have shown that AUD is a modifiable perioperative risk factor and is present in up to 18% of surgical patients. Despite its significant clinical impact, AUD is often overlooked in the design of perioperative care plans. Although AUD affects 9% of the US population, less than one in ten individuals with AUD receives any treatment. Barriers to treatment are multiple and include scarce care availability, limited access, and social stigma. Although anesthesiologists routinely provide guideline-concordant treatment for non-operative medical conditions such as coronary artery disease, AUD-specific care is rarely provided, even to high-risk patients. Thus, there is a compelling opportunity to integrate AUD screening and treatment into routine perioperative care. Our central hypotheses are that AUD health services initiated in the perioperative period will 1) leverage the significant resources made available to perioperative care in the US, 2) forge novel synergistic alliances between previously disconnected healthcare settings, and 3) break down barriers to access to care for broad populations. A multi-institutional team of anesthesiologists, biostatisticians, psychologists, and psychiatrists with expertise in AUD treatment and novel clinical trial designs will lead the Leveraging Alcohol Use Disorder Screening for Treatment in Routine Perioperative Care: AllUsCare proposal and proposes three aims: 1) Leverage our existing EHR-integrated Alcohol Use Disorders Identification Test-Concise (AUDIT-C) screening tool to increase provision of AUD-specific perioperative care, 2) Conduct a two-center prospective observational cohort study to assess patient acceptability of interventions, feasibility of outcome data collection, and optimum outcome measures for a future pragmatic trial, and 3) Optimize perioperative AUD intervention bundles most likely to be effective in a future pragmatic randomized factorial cluster trial. This R34 planning grant will lay the groundwork for identifying the most effective health service intervention bundles in surgical patients at high risk for AUD. To maximize inclusion of broad populations, we will conduct our study in two centers that serve inner-city and rural populations and will deliberately include patients aged 12 years and older. At the conclusion of AllUsCare, we will have established the research team, designed an AUD intervention bundle most likely to be effective in a future multi-center pragmatic trial, demonstrated a single-IRB governed uniform data collection and entry process, and confirmed the acceptability and feasibility of the future pragmatic trial.

NIAAA - National Institute on Alcohol Abuse and Alcoholism

Project Summary/Abstract American Indian and Alaska Native (AIAN) communities experience significant alcohol-related problems, further propagated by obstacles to beginning effective, high-quality treatment for alcohol use disorder (AUD). Precision medicine approaches to AUD treatment may make treatment more efficient and effective by individually tailoring AUD treatments to common etiological and maintenance mechanisms underlying AUD. For example, recent precision medicine efforts have identified that individuals who drink primarily in the context of reward (i.e., drinking for enhancement and social interaction) may respond better to naltrexone, whereas those who drink primarily in the context of relief (i.e., drinking to relieve negative physical and affective states) may respond better to acamprosate. However, extant measures used to classify reward and relief drinking tendencies do not account for the contextual factors associated with these constructs in AIAN peoples. Identifying relevant measures of reward and relief drinking tendencies in AIAN peoples may inform precision medicine research and reduce burden related to alcohol problems in AIAN. The proposed work will utilize a multi-method approach grounded in a community-engaged research framework to develop a tailored measure of reward and relief drinking by refining and expanding on several existing measures of reward and relief drinking and assess the construct validity of the newly developed measure in AIAN peoples. First, we will recruit AIAN people with AUD (N=20) to examine the content validity of three existing measures of reward and relief drinking and use cognitive interviewing methods to refine existing items and/or develop additional relevant items. Second, we will administer these items and any participant-identified additional items to a geographically varied sample (N=100) of AIAN with AUD. Using these data, we will then examine structural validity by identifying the hierarchical contributions of individual items to respective reward and relief drinking factors by conducting nonparametric item response theory (IRT) analyses. Lastly, we will utilize the refined measure to identify reward and relief drinking subgroups and examine comparative associations with convergent constructs to assess external validity. Ultimately, the proposed research will produce a construct-valid measure of reward and relief drinking informed by the lived experiences of AIAN peoples with AUD. The tailoring of an AIAN-specific measure of reward and relief drinking will play a direct role in improving alcohol problems in this population.

NIAAA - National Institute on Alcohol Abuse and Alcoholism

Project Summary Alcohol use disorder (AUD) is a prevalent, affecting over 10% of Americans, with only a fraction of individuals seeking treatment. Chronic alcohol exposure induces maladaptive changes in neuronal physiology, particularly within neural circuits that mediate reward and consumption behaviors, hallmarks of AUD that render treatment difficult and increases the likelihood of relapse. Despite substantial progress in understanding neurotransmitter- mediated changes in AUD, the role of neuropeptides such as orexin (hypocretin) remains underexplored. Orexin is a hypothalamic neuropeptide that contributes to a number of physiological conditions, including regulation of arousal, feeding, and stress. Orexinergic neurons from the lateral hypothalamic area (LHA) innervate a number of brain regions implicated in AUD, such as the ventral tegmental area (VTA) and nucleus accumbens (NAc). In my preliminary data, I have found that chronic voluntary EtOH consumption leads to activation of orexinergic neurons in the LHA in male and female C57BL/6J mice. Furthermore, I have demonstrated that I can track orexinergic activity in a free-moving mouse while it is consuming EtOH through the use of fiber photometry and the orexin peptide sensor, OxLight1. This proposal aims to investigate the impact of alcohol dependence on orexinergic circuits, particularly the LHA-VTA pathway, and its role in excessive alcohol consumption. Using cutting-edge tools, including the OxLight1 biosensor for real-time orexin monitoring, optogenetics for pathway- specific neuronal silencing, and whole-cell patch-clamp electrophysiology, this study will elucidate the functional changes in orexinergic activity in male and female mice following chronic intermittent ethanol (CIE) exposure. The innovative approach includes sex-specific and within subject analyses and behaviorally guided experiments to address critical gaps in the field. Aim 1 of this proposal will determine how silencing LHA orexinergic neurons projecting to the VTA modulates voluntary alcohol consumption in ethanol-dependent mice. Aim 2 of this proposal will assess functional adaptations in the LHA orexinergic circuits to regions implicated in AUD following ethanol dependence and their impact on voluntary alcohol consumption. The expected outcomes will reveal how chronic alcohol exposure alters orexinergic signaling, contributing to excessive drinking and relapse. These findings will enhance our understanding of AUD's neural mechanisms, identify potential sex differences, and detect therapeutic strategies targeting orexinergic pathways. This research holds significant translational potential, advancing efforts to mitigate the global burden of AUD.

NIAAA - National Institute on Alcohol Abuse and Alcoholism

Project Summary/Abstract This project proposes to investigate the underlying neural mechanisms within the nucleus accumbens (NAc) driving cue-elicited reward seeking during aversion-resistant alcohol drinking (ARD), defined as drinking despite negative consequences or “compulsive alcohol drinking”, using optogenetics and simultaneous in-vivo electrophysiology and optogenetics in male and female Long-Evans rats. Environmental reward predicting cues provide a source of motivation for reward-seeking which may be heightened following extended alcohol use, resulting in maladaptive, ARD, a defining feature of alcohol use disorder (AUD). Despite the well- established role of the NAc in mediating cue-elicited reward seeking, motivation, and ARD, the neural signature within the NAc during cue and alcohol access in ARD is not known, which greatly limits prevention and treatment of AUD. The NAc is innervated by glutamatergic projections from the paraventricular nucleus of the thalamus (PVT). This projection is thought to be relevant for processing motivational conflict and preventing unproductive reward seeking, while sparing normal affective behavior. However, the PVT-to-NAc pathway’s role in modulating NAc encoding during ARD or how the activity of PVT-to-NAc neurons alter ARD is not known. Thus, the central hypotheses of this project are 1) the NAc encodes cue and outcome information related to ARD. Specifically, neural activity in the NAc responds to reward (alcohol access) predictive cues and to alcohol rewards in a discriminative stimulus task and that over continued alcohol use, this activity increases in correlation with the development of ARD and 2) the neuronal activity in the NAc is causally related to an excitatory projection from the PVT that acts to prevent ARD initially, but erodes over time resulting in ARD. This project will assess the neural mechanisms of cue-elicited reward seeking and ARD during a discriminative stimulus task using both optogenetic circuit manipulations and simultaneous optogenetics and awake-and- behaving electrophysiology recordings. The training plan for this project is curated in an ideal research environment in the Department of Neuroscience at the University of Minnesota which will provide training in cutting edge neuroscience techniques, professional development, and research ethics that will amass an ideal training experience to facilitate my career as an independent alcohol research scientist. The specific research hypotheses are 1) Optogenetic inhibition of PVT-to-NAc neurons at cue presentation and alcohol reward will cause ARD in otherwise aversion-sensitive rats. 2) Optogenetic excitation of PVT-to-NAc at cue presentation and alcohol reward will reduce ARD, causing aversion-sensitivity, in otherwise ARD rats. 3) Ensembles of neurons within the NAc encode cue and alcohol rewards during ARD. This encoding is facilitated by the PVT- to-NAc projection whereby PVT-to-NAc inhibition will increase NAc encoding of cues and alcohol rewards and PVT-to-NAc excitation will reduce this encoding within the NAc. Completion of the proposed work will elucidate understanding of the neural mechanisms of ARD and will aid in the prevention and treatment of AUD.

NIAAA - National Institute on Alcohol Abuse and Alcoholism

PROJECT SUMMARY The ability to adaptively and appropriately respond to changes in the environment is a crucial behavior. It is cognitive flexibility that enables this adaptive responding. An inability to adaptively shift behavior (cognitive inflexibility) despite negative outcomes is observed in various psychiatric disorders and is a key diagnostic criterion of alcohol use disorder (AUD). The long-term objective of this work is to elucidate how chronic alcohol and stress promote AUD through cognitive inflexibility. In humans, AUD is accompanied by aberrant frontal cortex function alongside deficits in cognitive flexibility. A significant body of work, including that from our lab, has demonstrated impaired cognitive flexibility in animal models following chronic alcohol which can be exacerbated by stress exposure. However, the frontal cortical neuroadaptations that mediate these alcohol and stress induced cognitive impairments remain unresolved. This is a critical gap given that stress is a key risk factor in perseverative relapse for individuals with AUD. My central hypothesis is that functional network changes across the dorsomedial frontal cortex underlie cognitive flexibility impairments following repeated alcohol and stress exposure. Moreover, I hypothesize that changes in modulatory norepinephrine inputs to this region are critical contributors to these flexibility deficits. To test these hypotheses, I will investigate the mechanisms underlying the negative impact of chronic alcohol and stress on cognitive flexibility using a novel attentional set shifting task. In Aim 1 I will use in vivo calcium imaging to investigate alcohol- and stress- associated changes in dorsomedial frontal cortex dynamics underlying behavioral impairment. In Aim 2, I will determine whether chronic alcohol and stress functionally disrupts noradrenergic regulation of the dorsomedial frontal cortex using in situ hybridization readouts and pharmacological interventions to recover cognition. These proposed studies will provide ample training in cutting-edge techniques and facilitate my professional development in the alcohol research field. At the same time, this work will advance our understanding of the impact of long-term alcohol and stress exposure on cortical function and identify potential mechanisms for therapeutic interventions.

NIAAA - National Institute on Alcohol Abuse and Alcoholism

PROJECT ABSTRACT Fetal alcohol spectrum disorders (FASD) can occur in a person when they are exposed to alcohol before birth and are characterized by impairments in physical, cognitive, and behavioral functions. Common signs and symptoms associated with FASD include low body weight, abnormal facial features, poor coordination, hyperactive behaviors, difficulty with attention, and difficulty in school. There is also growing research that individuals with FASD have sensory processing deficits that can impact self-regulation and adaptive behavior. Sensory enriched occupational therapy intervention may improve the neurological sensory processing deficits experienced by individuals with FASD, which may lead to lifelong improvements in self-regulation and adaptive behavior. Here, we propose a Phase 1 clinical trial to assess the following: 1) characterize changes in brain white matter microstructure of sensory integration in 6 to 8-year old children with FASD following intervention; 2) characterize changes in sensory processing, self-regulation, and adaptive behavior; and 3) develop a conceptual model of mediation, whereby a sensory enriched intervention alters brain sensory white matter networks, leading to improvements in sensory processing, self-regulation, and adaptive behavior in children with FASD. We predict that children with FASD who receive a sensory enriched intervention will demonstrate positive changes in white matter neural architecture when compared to controls, and that we will see improvements in sensory processing, self-regulation, and adaptive behaviors. This two phase R61/R33 will allow us the time to adapt and assess the study procedures and intervention to inform the development of future larger clinical trials.

NIAAA - National Institute on Alcohol Abuse and Alcoholism

PROJECT SUMMARY/ABSTRACT Adolescent brain development makes individuals particularly vulnerable to the negative and long-lasting consequences of adolescent alcohol drinking. Adolescent alcohol use is associated with delayed maturation of the prefrontal cortex, leading to cognitive dysfunction and a loss of inhibitory control. The rodent medial prefrontal cortex (mPFC) is functionally homologous to the human dorsolateral PFC, each having similar layered structures and circuitry. Importantly, rodent models of adolescent drinking show similar mPFC-related deficits as humans, including reduced behavioral flexibility, increased impulsivity, impaired working memory, and abnormal social interaction. These long-lasting mPFC neuronal and behavioral effects are believed to be driven by changes in mPFC glutamate transmission. Despite this, the mechanisms underlying the long-term consequences of adolescent alcohol drinking on PFC glutamate function remain not well understood. Astrocytes are a compelling mechanism through which adolescent alcohol drinking can produce long- lasting changes in PFC function into adulthood. Astrocytes continue to mature into young adulthood in rodents, making them developmentally vulnerable to the effects of alcohol. Additionally, they mediate synapse formation and elimination during development to assemble the neural circuits that last into adulthood, as well as regulate glutamate uptake and release at synapses to optimize neuronal function and prevent glutamate excitotoxicity. Thus, changes in astrocytes following adolescent alcohol drinking are likely driving the long-term changes in mPFC glutamate function. In this proposal, we will examine how adolescent ethanol drinking (AIE) produces long-term changes in astrocyte distance from synaptic contacts, and their regulation of glutamate synapses in mPFC prelimbic (PL) layer 2/3 and 5 in male and female mice. We will also examine the astrocyte-specific mechanisms that are affected by AIE in adulthood and pharmacologically target these mechanisms to examine if they contribute to acute ethanol regulation of PL glutamate synapses. We have intentionally designed this project to maximize its interdisciplinary nature. We will label PL astrocytes using viral infusions, as well as employ immunofluorescence staining methods to label synaptic contacts and characterize layer 2/3 and 5 astrocytes. This will greatly complement our electrophysiology experiments, which will examine astrocyte regulation of PL layer 2/3 and 5 glutamate synapses in water and AIE mice of both sexes. We will then extend analysis to the transcription level to identify astrocyte-specific mechanisms that are altered by AIE. I will learn single-nucleus RNA sequencing to identify if subgroups of astrocytes that populate layers 2/3 and 5 are differentially affected by AIE. My mentors and consultants will help me establish these sequencing techniques in Dr. Florence Varodayan’s laboratory. Collectively, this work will provide insight into the long-term changes in PFC function following adolescent alcohol drinking.

NIAAA - National Institute on Alcohol Abuse and Alcoholism

Project Summary This interdisciplinary project addresses the pressing public health challenge of alcohol use disorder (AUD). AUD affects approximately 14% of adults in the United States alone, with profound societal and individual detrimental consequences. Despite its prevalence, effective treatments for AUD are limited, highlighting the urgent need for a better understanding of the underlying molecular mechanisms of this disorder. This project investigates the role of neurexin 3 (NRXN3) in AUD using rhesus macaque and mouse models. Our laboratory has shown that NRXN3, a presynaptic transmembrane protein, is downregulated in the prefrontal cortex of high ethanol consuming non-human primates. Given the central role of this protein in synaptic transmission and plasticity, processes known to underlie AUD-related behaviors, investigating its role in mediating the effects of alcohol in brain function is paramount. However, the specific role of NRXN3 in AUD remains poorly understood, with only a handful of published GWAS studies linking single nucleotide polymorphisms with different risk propensities for addiction. Here, we aim to elucidate the NRXNs' landscape in the cortex following chronic ethanol use and protracted abstinence, with particular focus on a specific transcript/isoform, Nrxn3β, and its role in synaptic plasticity. It also explores manipulating Nrxn3β transcripts in one of the major inhibitory neurons in the cortex, parvalbumin (PV) neurons, to modulate excitatory outputs to reward and motor centers and reduce ethanol intake. By employing cutting-edge molecular profiling techniques, advanced immunohistological analyses, and in vivo manipulation, we will examine changes in Nrxns’ expression and excitatory/inhibitory synaptic properties in key brain regions implicated in AUD, such as the prefrontal cortex. This research holds significant intellectual significance as it seeks to advance our understanding of the neurobiological basis of AUD and identify novel therapeutic targets. By uncovering the precise mechanisms through which Nrxn3β influences synaptic plasticity and alcohol-related behaviors, we aim to inform the development of targeted interventions to mitigate the adverse effects of AUD and improve treatment outcomes for affected individuals.

NIAAA - National Institute on Alcohol Abuse and Alcoholism

PROJECT SUMMARY/ABSTRACT Early life stress is a prominent risk factor for initiation of alcohol use and increased lifetime alcohol consumption. Both adolescent stress and alcohol use increase the risk for developing depression during adulthood, and both exposures negatively influence the neuroimmune system, resulting in a sensitized or exaggerated response to a neuroimmune challenge. However, in rodent models, the interactive effects of alcohol and stress co-exposure are not widely studied. Because there is a bidirectional relationship among adolescent stress and alcohol use, it is critical for these exposures to be studied independently as well as simultaneously to capture synergistic or opposing effects. Therefore, one overall goal of the current proposal is to utilize an exposure paradigm that captures both the independent effects of adolescent intermittent ethanol (AIE) and chronic corticosterone (CORT) consumption in the drinking water, a commonly used model to mimic the downstream effects of one primary component of chronic stress by a tractable means. One component of the neuroimmune system that sits at the nexus between chronic stress and ethanol exposure is the inflammasome, nucleotide-binding oligomerization domain-like (NOD-like) receptor protein 3 (NLRP3). The NLRP3 inflammasome is highly expressed in the resident immune cell of the brain, microglia, and NLRP3 inflammasome expression is increased following both stress and ethanol exposures, causing a sensitized cytokine response upon challenge. Increased cytokine expression has been implicated in dopaminergic dysfunction, such as decreased dopamine availability and downregulated dopamine receptor expression, which may explain AIE- or stress-induced dysfunction of reward and motivation pathways as well. Therefore, the overall goal of the proposed studies is to determine the independent and interactive effects of AIE and CORT on 1) neuroimmune function, measured by NRLP3 expression and microglial activation state, and 2) changes to the reward and motivational systems including behavioral measures such as sucrose preference for anhedonia and effort-based decision making (EBDM) for motivation, as well as dopaminergic dysfunction such as expression of ΔFosB, D1, D2, and DAT. Specifically, whether the NLRP3 inflammasome plays a mechanistic role as a neuroimmune target for future treatment. Overall, this proposal will provide essential scientific and professional development, so enabling me to reach my goal of becoming an independent researcher.

NIAAA - National Institute on Alcohol Abuse and Alcoholism

While extensive research highlights how poverty can negatively impact health—including by increasing alcohol-related harms—little is known about how women’s economic experiences affect their use of alcohol and other substances around the time of pregnancy. This gap in the evidence is especially notable given that pregnancy is a high-risk period where any type of insecurity can have long-lasting implications. More than a quarter of U.S. births occur in families living below the poverty line, and half of pregnant women cite financial worries as a major source of stress. Low income is also a risk factor for fetal alcohol spectrum disorder (FASD) and numerous adverse birth outcomes that may involve stress-mediated alcohol use. Identifying how pregnancy-specific alcohol consumption patterns are influenced by sources of insecurity (e.g., food, housing, transportation, healthcare costs) is therefore a critical priority—with implications for screening, targeted prevention, and counseling in prenatal care, as well as other key healthcare system interventions. The objective of this K99/R00 proposal is to provide Dr. Emilie Bruzelius with the training and mentorship needed to begin investigating how economic stressors biologically and behaviorally mediate prenatal alcohol use and related birth outcomes. In the K99 phase, Aim 1 will first address a persistent methodological challenge—alcohol inaccurate reporting during pregnancy—by using biologically verified data from an external cohort (the HEALthy Brain and Child Development Study [HBCD]) to correct prenatal consumption estimates in two national surveys (the Pregnancy Risk Assessment Monitoring System [PRAMS] and the Behavioral Risk Factor Surveillance System [BRFSS]). Aim 2 will then use these corrected data to characterize trends by income and examine how specific domains contribute to observed patterns. In the R00 phase, Aims 3 and 4 will assess how insecurity changes, as proxied by access to refundable tax credits (Aim 3) and nutrition assistance (Aim 4)—are associated with differences in alcohol use patterns and related birth outcomes. Consistent with NIAAA’s priorities, these aims will contribute to our understanding of the factors influencing alcohol misuse during pregnancy to inform health systems strategies for improving pregnancy-related alcohol health. Complementing these research aims is a training plan that enhances the applicant’s background in epidemiologic methods and substance use health services research through new training in (1) perinatal alcohol epidemiology, neurodevelopment and FASD, (2) determinants of health, (3) advanced causal inference techniques, and (4) career development. This project will also lay the foundation for a future R01 application using the HBCD cohort to longitudinally examine how broad-based support programs can be better designed to support child health and neurodevelopment by addressing maternal substance use pathways. Collectively, these training and research activities, along with the support of an exceptional mentorship team, will prepare Dr. Bruzelius to launch an independent research career focused on improving alcohol-related health during pregnancy and across generations.

NIAAA - National Institute on Alcohol Abuse and Alcoholism

SUMMARY Individuals with alcohol use disorder (AUD) are more likely to suffer from chronic pain conditions and to report moderate to severe pain compared with AUD-free subjects. Importantly, pain is a significant risk factor for relapse back to alcohol use, and a reduction in pain is associated with reduced relapse. These clinical data strongly suggest that effective pain management during AUD treatment can facilitate abstinence. Our long- term goals are to delineate the mechanisms underlying the impact of chronic alcohol consumption on pain processing and to identify molecular targets for intervention strategies that selectively target pain in the context of AUD. We have established a behavioral paradigm that models the effects of chronic alcohol on nociception and persistent pain. Our preliminary data indicate that repeated cycles of chronic alcohol consumption and withdrawal resulted in alcohol-induced hypersensitivity (AIH). Moreover, chronic alcohol consumption delayed recovery from tactile hypersensitivity induced by a transient nerve injury. We postulate that the observed AIH is driven by maladaptive changes in neural processing that establish a vulnerability to chronic pain. The goal of this exploratory project is to investigate the spinal mechanisms of AIH. In a preliminary experiment, we found that AIH is attenuated by intrathecal administration of gabazine, an antagonist of gamma-aminobutyric acid (GABA) type A receptors (GABAAr). This observation suggests that chronic alcohol consumption leads to maladaptive changes that disrupt spinal inhibitory signaling – a mechanism implicated in hypersensitivity across a broad spectrum of rodent pain models, including neuropathic pain and morphine-induced hyperalgesia. Moreover, the chloride transporter KCC2 has been identified as a key contributor to this mechanism and a druggable chronic pain management target. Based on our preliminary data, we hypothesize that chronic alcohol consumption leads to tactile hypersensitivity through dysregulation of spinal inhibitory signaling. In Aim 1, we will determine whether chronic alcohol consumption leads to excitatory actions of the normally inhibitory neurotransmitters GABA and glycine (Gly) using behavioral testing and patch clamp electrophysiology. In Aim 2, we will identify mechanisms of dysregulated spinal inhibition following chronic alcohol consumption. We will combine targeted analysis of the contribution of KCC2 to AIH and a global approach using single-nucleus RNAseq and quantitative phosphoproteomic analyses of the effects of alcohol in the dorsal horn, which will identify potential signaling pathways contributing to altered KCC2 function and additional potential spinal mechanisms underlying AIH. Impact: Increased mechanistic understanding of the spinal effects of chronic alcohol consumption will inform efforts to identify pain management strategies tailored to AUD.

NIAAA - National Institute on Alcohol Abuse and Alcoholism

Project Summary/Abstract Parents play a critical role in their adolescent children’s alcohol use decision-making. However, the relative importance and underlying mechanisms of various alcohol-specific parenting (e.g., alcohol provision, attitudes, and rules) and general parenting factors (e.g., monitoring, support) remains unclear. This knowledge gap continues to impede the advancement of theories and practices in parent-based alcohol prevention programs, reducing their potential to effectively mitigate widespread adolescent drinking and its consequences. Examining alcohol-specific cognitions (e.g., alcohol expectancies, normative beliefs) as mediators between parenting and adolescent drinking is essential for addressing this gap, as altering these cognitions may be the most efficient and effective way to influence adolescent alcohol use—supported by the empirically validated Reasoned Action Approach. However, prior studies exploring these indirect relationships have yielded inconsistent findings. Indeed, alcohol-specific cognitions comprise diverse constructs with varying levels of importance in alcohol use decision-making, yet their relative importance remain improperly studied. To address these gaps, the proposed study outlines three specific aims based on a comprehensive systematic review and meta-analysis of empirical studies targeting U.S. adolescents aged 10 to 18. Aim 1 is to conduct a meta-analysis to synthesize evidence on the influence of parenting (including both alcohol-specific and general parenting factors) and alcohol-specific cognitions (alcohol-related behavioral, normative, and control beliefs) on adolescent alcohol use, and to explore whether these relationships vary by participant and study characteristics. Aim 2 is to use dominance analysis (a more advanced method for assessing predictor relative importance) to rank the relative importance of various parenting factors as well as alcohol-specific cognitions in predicting adolescent alcohol use. Aim 3 is to identify the mechanisms through which parenting factors influence adolescent drinking via alcohol-specific cognitions by testing meta-analytic structural equation models (MASEMs). Core research processes will involve developing and registering a review protocol, searching scientific databases and grey literature, screening studies for eligibility, extracting qualitative and quantitative data (e.g., correlations) to produce a meta-analysis database, assessing included studies’ risk of bias, performing dominance analysis and MASEM to test our specific aims, and composing publications adhering to PRISMA 2020 guidelines. The meta-analytic approach enables us to integrate the massive and disparate empirical evidence, thereby enhancing our findings’ generalizability and clarifying subgroup differences. Our novel approach also stands to advance theoretical understandings beyond prior meta-analyses by exploring indirect effects and predictor relative importance. Our findings will offer valuable insights for evaluating and streamlining parenting programs—a foundational step toward overcoming barriers to the effectiveness and expansion of these prevention efforts.

NIAAA - National Institute on Alcohol Abuse and Alcoholism

Project Summary/Abstract Fetal alcohol spectrum disorders are a group of physical and mental defects caused by fetal exposure to alcohol, including the most severe form known as fetal alcohol syndrome (FAS). A diagnosis of FAS requires severe growth deficiency, but little is known about the mechanism by which exposure to alcohol affects the fetal skeleton. Our previous work demonstrated that normal skeletal development in mice requires peripheral innervation by sensory nerves through NGF-TrkA signaling. Furthermore, inhibition of NGF-TrkA signaling during development results in skeletal deficits in mice that mirror those observed in rodents exposed to alcohol during development as well as in children with FAS. Thus, the main objective of this study is to determine how prenatal alcohol exposure in mice affects the innervation of bone during endochondral ossification. Our overall hypothesis is that disruption of the neurotrophic NGF-TrkA signaling axis due to alcohol exposure negatively affects the innervation of fetal bone, which negatively impacts bone mass and length. This hypothesis will be directly tested in two specific aims. First, we will characterize skeletal innervation in mice exposed to alcohol during development. Here, alcohol will be administered to three strains of pregnant reporter mice by alcohol vapor exposure. Histological examination of these mice will be performed throughout skeletal development to quantify NGF expression, osteoprogenitor pool population, innervation, and vascularization. In addition, dorsal root ganglia will be harvested to quantify neuronal number and type. Next, we will determine if increasing NGF-TrkA signaling is sufficient to mitigate alcohol-induced skeletal deficits. Here, pregnant mice will be exposed to alcohol vapor starting 9 days after vaginal plug. Either NGF, gambogic amide, or vehicle (control) will be administered every third day after embryonic day 9 until birth. Bones will be harvested at postnatal day 7, 21, and 56 for analysis by microCT, static and dynamic histomorphometry, histology, and immunohistochemistry. Lumbar spine will be harvested to analyze the size, number, and type of DRG neurons. This study will explore a completely novel paradigm regarding the pathobiology of the skeletal abnormalities observed in children with fetal alcohol spectrum disorders. Importantly, the results will make a significant contribution towards understanding the effects of alcohol on fetal bone and may provide a curative strategy to address one of the major complications associated with FAS.

NIAAA - National Institute on Alcohol Abuse and Alcoholism

Project Summary/Abstract Most US women of childbearing age (86%) are mothers who will develop a maternal identity reflecting their own beliefs and behaviors related to motherhood. Theory and research suggest that many women look to their social groups for cues about how to be a mother. Identifying with a social group has powerful effects on human behavior, including an influence over choices about alcohol use and risky drinking, though this work has largely targeted a social cuing effect related to exposure to alcohol cues, leaving ambiguity about their relevance for the downstream consequences of explicitly linking social group identification and alcohol use. The current study is an opportunity to understand whether a contemporary cultural phenomenon, the “wine mom,” which conveys drinking as a socially acceptable and potentially necessary aspect of motherhood, leverages the same neural mechanisms of alcohol salience as have been identified in other established social groups, not only to increase the salience of alcohol cues (Aim 1), but to determine whether such salience effects may interact with maternal identity to predict real life consequences. Specifically, this work will test whether associating alcohol with maternal identity, predicts concurrent (Aim 2) and longitudinal (Aim 3) maternal drinking behaviors. Moreover, because mothers comprise the majority of primary caregivers, increasing links between alcohol and the maternal identity ma also affect the mother-infant relationship via maternal parenting dynamics. As such, this project also includes a test of whether associating alcohol with maternal identity is linked to concurrent (Aim 2) and longitudinal (Aim 3) observations of negative and positive parenting behaviors when mothers interact with their toddlers. This project will test effects for mothers of young children given the critical importance of maternal behaviors for offspring development during this period. This project leverages both cross-sectional and longitudinal data, both experimental and correlational components, and a multi-trait, multi- method design. Maternal identity and drinking behaviors will be assessed via self-report, alcohol salience will be tested at the level of neural activity using event-related potentials (ERPs), and parenting behaviors will be assessed in the laboratory through observations of mother-toddler interactions. Results provide pilot data regarding the potential for the “wine mom” movement to impact maternal function with downstream consequences for maternal drinking and child development. This work can also offer a proof of concept for a larger and much-needed longitudinal study of the unfolding “mom wine” movement for maternal and child development. Consistent with the aims of the NIH to enhance maternal health and understand social influences on drinking behaviors, the long-term goal of this area of work is to provide information about the social dynamics of “mom wine” culture and offer insight into the need for potential intervention in the future.

NIAAA - National Institute on Alcohol Abuse and Alcoholism

While data linking chronic heavy drinking to accelerated cognitive aging and early onset dementia risk are compelling, epidemiological data probing the effects of less severe patterns of drinking unaccompanied by alcohol use disorder (AUD; here, moderate drinking (MD)) produce inconsistent findings. Outcomes from the sparse body of human laboratory research lend little clarity regarding the long-term neurobiobehavioral (NBB) outcomes of alcohol consumption in MDs, who constitute the majority of older drinkers. In this innovative R21 application, we propose a framework for understanding differential risk and seek to clarify its core tenets. We assert that the heterogeneity of reported outcomes derive, in part, from varying degrees of unaccounted for individual differences in vulnerability to alcohol’s effects on cognitive aging processes. More specifically, we suggest that alcohol-related impairment/decline occurs among MDs who are differentially vulnerable to the interaction of alcohol’s neurobiological effects and cognitive aging processes. With continued drinking, this heightened sensitivity to alcohol’s neurobiological effects exacerbates cognitive aging processes, eventuating in alcohol-related cognitive impairment/decline for this subgroup of MDs, i.e., vulnerable MDs. In contrast, moderate drinking is not expected to compromise cognitive status for others, i.e., resilient MDs, even when controlling for typical intake. While the premise is appealing, the constructs are ill-defined. In the absence of empirically vetted operational definitions that avoid tautologies and lacking preliminary data, the longitudinal designs needed for rigorous evaluation are premature. Thus, we propose a proof-of-concept study designed to interrogate the framework’s key requisite conditions to the extent permitted within R21 constraints. Using a blinded w/in Ss placebo-controlled design (N= 60 (30 each sex) aged 65-80), our intent is to derive performance differences between alcohol/placebo conditions (i.e., deriving individual estimates of acute alcohol sensitivity in behavior & neurophysiology), explore variability in sensitivity and correlates thereof, and interrogate the relationship between sensitivity and performance on a neuropsychological battery. Our intent is not to predict late-onset AUD or drinking problems. Instead, we query relationships between alcohol sensitivity and cognitive vulnerability. We note that the predicted interaction may be modulated by numerous factors. Here, we explore if healthy MDs who carry risk for accelerated cognitive decline exhibit greater sensitivity. We anticipate this subgroup to exhibit stronger negative relationships between alcohol sensitivity and performance- suggesting its relevance in longitudinal study. Extant data and feasibility concerns led to our defining “at-risk” based on subjective cognitive complaints. Acute sensitivity provides a starting point but is unlikely to account for all variability. Thus, we will probe how variables such as age, substance use history, family history (AUD /dementia), and other factors impact outcomes. Critically, regardless of specific outcomes, this proof-of-concept study constitutes a significant step in establishing a framework for systematic research in this critical domain.

NIAAA - National Institute on Alcohol Abuse and Alcoholism

Abstract College students drink more often and in larger quantities, experience more consequences, and are more likely to receive a DSM diagnosis of an alcohol use disorder (AUD) compared to their non-college same-aged peers. Studies also show seniors in college drink more often, engage in more high-intensity drinking, and experience more consequences compared to earlier on in college, indicating drinking rates are not waning as students progress through college. These data suggest 1) students drink in a high-risk manner throughout college, and 2) to best combat the development of chronic drinking problems, efforts targeting high-risk drinking students across all years of college are warranted. Research has shown the benefits of personalized normative feedback for diverse samples to reduce drinking, but there remain unanswered questions about whether these are sufficient for reducing the development of chronic drinking problems and whether their efficacy can be strengthened by using personalized biomarker information. The proposed research will attempt to address these questions by examining the feasibility of adding an innovative brief personalized transdermal alcohol biomarker feedback component (TAC feedback) to an existing efficacious personalized feedback intervention, eCHECKUP TO GO (eCTG), for high-risk college students. Our study will deliver the eCTG + TAC feedback to high-risk drinkers of all class standings (freshman, sophomores, juniors, seniors) and compare their risky drinking, alcohol related consequences, and AUD symptoms at 6-month post-intervention relative to eCTG only as the active control. Our team has published research showing transdermal alcohol concentration (TAC) biomarkers (rise rates and peak intoxication) predict consequences independently, while adjusting for self- reported drinking. We recently pilot-tested the feasibility integrating TAC biomarkers into our intervention work on a small student sample (n= 11). The students wore the TAC sensors for two weeks, we extracted their TAC biomarker data, and met with them individually to deliver intervention sessions. The motivational interviewing- based personalized feedback sessions provided students with TAC information and visual representations for varying levels of risks using alcohol biomarker data collected from drinking events in the previous two weeks. Afterwards, we assessed students’ willingness to engage in behavioral strategies to modify their TAC biomarkers (i.e., rise rates and peak intoxication) in future drinking events and all indicated a willingness to do so. Although the sample was small, the pilot provides preliminary evidence to implement the TAC feedback, and warrant conducting the proposed research. Aim 1: will conduct a feasibility assessment for the TAC feedback component. Aim 2: will assess the effects of the eCTG + TAC at 6- months post-baseline relative to an eCTG alone. To the extent that the research is successful, it will provide data to warrant conducting a larger clinical efficacy trial in a future R01 study.

NIAAA - National Institute on Alcohol Abuse and Alcoholism

Significant alterations of brain connectivity in alcohol use disorders (AUDs) may result from disturbances of signal integration at the synaptic level in the gray matter. In addition, defective brain connectivity in AUDs may be affected by alcohol abuse-induced myelin disturbances along white matter (WM) myelinated axons, since myelin critically supports fast signal propagation and contributes to axon maintenance. However, a major source of signal propagation anomalies may also depend on changes in and around nodes of Ranvier (NRs), myelin-bare regions of myelinated axons, that are essential to the regeneration of action potentials (signals) conveyed by axons, and thus to connectivity. However, morphological and molecular alterations at NRs that may contribute to abnormal connectivity in AUDs still remain mostly unexplored despite the well- known centrality of NRs to signal propagation. This proposal will determine whether alcohol abuse in humans with AUDs, and in experimental rats, leads to alterations of NR morphology and of distribution of extracellular matrix molecules (ECMMs) that are crucial to assembly and maintenance of NRs. In a different psychiatric disorder, depression, and in animals with chronic stress, we have found that levels of two glial NR proteoglycans and neurofascin 186, and in NR length are changed in the PFC of an animal model and in human subjects with depression. Some glial disturbances in AUDs and its animal models are shared with stress and depression while others differ. However, AUDs more globally affect various brain regions, so we hypothesize that structural (reduced NR length) and molecular changes in the NR’s environment will be found in AUDs in both prefrontal and occipital cortical WM. However, we hypothesize that overexpression of some NR-associated proteoglycans in PFC, but not in the OCC, WM will increase while their suppression will decrease alcohol drinking in experimental rats. The hypotheses will be tested according to the following specific aims: Specific aim 1 : We will test the hypothesis that alcohol intake in rats significantly reduces extent of astrocyte processes and NR length, and alters levels of ECMMs and their partners at NRs in OFC and OCC WM. Specific aim 2: We will test the hypothesis that diagnosis of AUD in humans is associated with shorter NRs, reduced astrocyte processes contacting NRs and altered levels of astrocyte-related ECMMs and partner axonal proteins in OFC and OCC WM, but not in the WM of the occipital cortex. Specific aim 3: We will test the hypothesis that suppression of astrocytic ECMMs in OFC but not in OCC, results in decreased alcohol intake behavior in rats. If this research supports the hypothesis that glial-derived proteoglycans and axonal components of the NR environment are involved in cellular and behavioral pathology related to alcohol intake, future research in rodents will target the mechanisms and intracellular pathways that regulate NR components in brain circuits involved in AUDs, and the ability of alcohol- intake-reducing treatments to reverse WM neuropathology and behavioral effects of AUDs.

NIAAA - National Institute on Alcohol Abuse and Alcoholism

Abstract Rejuvenation Technologies Inc. (RTI) seeks to continue advancing the translation of a telomere extension biologic, TERT mRNA, which is delivered to the liver intravenously (i.v.) using hepatocyte-targeted lipid nanoparticles (LNPs) for the treatment of alcoholic hepatitis (AH). AH is an acute form of alcoholic liver disease (ALD) that represents a dire unmet medical need, as mortality within 1 month of presentation is 25–50%. Most AH patients exhibit advanced fibrosis/cirrhosis, contributing to acute-on-chronic liver failure. The only definitive treatments are steroids, which are ineffective at reducing patient mortality. However, there is strong evidence that short telomeres play a casual role in AH. Telomeres, the protective DNA tips of chromosomes shorten with each cell division; chronic liver injury due to excessive alcohol consumption drives compensatory hepatocyte proliferation resulting in rapid telomere shortening, hepatocytic senescence, and the secretion of senescence- associated secretory phenotype factors that activate hepatic stellate cells, causing them to become fibrogenic. Short telomeres limit the ability of hepatocytes to divide and the liver to regenerate, ultimately leading to liver failure. RTI’s team has invented a breakthrough treatment addressing shortening telomeres in AH patients comprising 1) the telomere-extending biologic telomerase (TERT) mRNA and 2) an LNP vehicle that delivers mRNA to the liver with high efficiency, transfecting >99% of hepatocytes at very low doses (0.05 mg/kg), with high transfection even in cirrhotic livers. Upon delivery to the cells, TERT mRNA is translated to functional TERT protein, and both are degraded within days. During this brief treatment, telomeres are extended sufficiently to reverse years of telomere shortening. After the TERT degrades, telomeres resume shortening normally. TERT mRNA does not immortalize cells, and TERT is not an oncogene. RTI’s TERT mRNA LNPs are a highly effective, low risk, revolutionary biologic to improve and extend the lives of AH patients. A prior NIAAA Phase II SBIR and an INTERACT meeting on AH with the FDA has led to this proposed Commercialization Readiness Pilot (CRP) that will culminate in an IND submission. The specific aims are: 1) Pharmacokinetic (PK) study of mRNA and select lipids in mice to measure rate of clearance; 2) Pre-treatment evaluation in mice; 3) GMP manufacturing, genotoxicity studies, and IND-enabling GLP toxicology studies in NHPs; and 4) Preparation of IND data package and FDA submission.

NIAAA - National Institute on Alcohol Abuse and Alcoholism

Men remain disproportionately impacted by HIV, and while pre-exposure prophylaxis (PrEP) is a proven prevention strategy, its effectiveness is undermined by nonadherence linked to alcohol and illicit drug use. Digital platforms, widely used by men to connect, are associated with higher alcohol and illicit drug use, presenting both challenges and opportunities for intervention. This study explores the intersection of alcohol misuse and PrEP adherence among men, with a focus on the influence of digital platforms. This study uses qualitative methods, specifically semi- structured interviews and thematic analysis, to:(1) examine how digital platforms impact alcohol and illicit drugs use behaviors and their effects on PrEP care continuum among men who misuse alcohol, and; (2) assess the acceptability and feasibility of leveraging these platforms to deliver harm-reduction messaging for alcohol and illicit drug use moderation management to improve PrEP adherence among men who misuse alcohol in Kentucky. Preliminary findings from the parent study “Alcohol Misuse, Gut Microbial Dysbiosis and PrEP Care Continuum: Application and Efficacy of SBIRT Intervention”, (R01 AA030485) suggest men prefer harm- reduction approaches over abstinence for managing substance use, indicating potential receptivity to digital interventions. By exploring the feasibility and acceptability of integrating moderation management into messaging, this study aims to enhance PrEP adherence and inform the development of innovative, digitally delivered harm-reduction strategies tailored to this population. The findings will contribute to the future design of scalable, community-focused solutions to address overlapping public health challenges of alcohol misuse, illicit drug use, and HIV prevention.

NIAAA - National Institute on Alcohol Abuse and Alcoholism

PROJECT SUMMARY In the United States, up to 1 in 20 school age children have a diagnosable fetal alcohol spectrum disorder (FASD), which manifests in diverse phenotypes such as cognitive and behavioral deficits, craniofacial anomalies, and organ malformations. Importantly, up to 30% of patients with FASDs are thought to have a congenital heart defect (CHD). Despite the significant impacts of PAE on the developing cardiovascular system, the molecular and cellular mechanisms underlying EtOH-induced CHDs remain elusive along with any biomarkers that could be used to stratify individuals with FASDs by cardiovascular disease risk. In a zebrafish model of embryonic alcohol exposure (EAE), we confirmed a defect in cardiomyocyte (CM) and anterior endoderm (AE) migration that causes abnormal cardiac cone and heart tube development, resulting in ventricular structural abnormalities that persist through adulthood. Inhibition of PDGFRA and PI3K signaling through pharmacologic and genetic strategies sensitizes embryos to EtOH-induced defects in CM midline migration, suggesting a role for these pathways in failed cardiac cone fusion and downstream CHD formation. Following EAE, adults develop signs of progressive cardiomyopathy and diastolic dysfunction, which are worse in individuals with a documented CHD. RNA-sequencing of adult male and female EAE ventricles reveal consistently dysregulated gene expression, including a downregulation of gene candidates ankrd9, eya4, and slc25a33, which may contribute to the progressive cardiac dysfunction observed in EAE adults. In Specific Aim 1, we will test the hypothesis that abnormal cell migration and altered PI3K signaling in CM and AE cell populations causes failed cardiac cone fusion and CHDs. We will utilize a zebrafish model of EAE to identify changes in CMs and AE through advanced live imaging, single cell RNAseq, spatial transcriptomics, and an evaluation of PDGFRA and PI3K signaling. In Specific Aim 2, we will test the hypothesis that newly identified cardiac-expressed biomarker candidates ankrd9, eya4, and slc25a33 can modulate CHD and adult cardiomyopathy risk in individuals with FASDs. Loss of function mutants generated through CRISPR-Cas9 will be assessed for embryonic and adult cardiovascular phenotypes in the presence and absence of EAE through ISH, ICH, live imaging, and echocardiography. Taken together, the proposed studies will provide fundamental insights into the molecular mediators of CHDs and adult cardiac dysfunction following EAE.