“Serum Exosome Detection and Monitoring of Alcohol-Related White Matter Brain Pathology-Opportunities to Optimize Treatment and Monitoring of AUD-Related Organ and Tissue Damage

Rhode Island Hospital, Providence RI

Investigators

Abstract

Chronic heavy or binge alcohol consumption damages the brain’s structural and functional integrity. These effects, in large measure, are caused by alcohol’s toxic and degenerative effects on white matter (WM). Consequences include declines in CNS neurobehavioral performance, reinforcement of dysfunctional activities, and multimodal disabilities. Fortunately, growing evidence suggests that WM atrophy in alcohol-related brain degeneration (ARBD) may be partly reversible through abstinence, which could be effected by cognitive-behavioral, brief intervention, or neuro-modulatory strategies that reduce craving. However, to advance this aspect of alcohol use disorder (AUD) research and patient care, efficient, non-invasive monitoring of treatment/intervention outcomes in relation to WM ARBD is needed. Our overarching hypothesis is that WM ARBD is rooted in oligodendrocyte dysfunction caused by oxidative stress, inflammation, and dysregulated signaling through insulin/IGF-1-PI3K-Akt-mTOR-mTORC. These pathophysiological processes reduce mature oligodendrocyte populations and compact myelin, impair the maturation of immature oligodendroglia, and alter sphingolipid metabolism, resulting in sulfatide and sphingomyelin depletion and ceramide accumulation. Using established experimental rat models, we found that abstinence partially reversed these pathologies, and more recently discovered that WM ARBD-related alterations in oligodendrocyte-myelin glycoprotein and lipid profiles can be detected in extracellular vesicles (EVs) isolated from brain and serum. Furthermore, we have preliminary evidence that comparable studies are feasible using human samples. This R21 application leverages the gains achieved through ongoing research by logically extending an important sub-project included in Aim 3 of R01-AA028408. The main goal is to use our existing bank of human AUD and control serum from IRB-approved longitudinal studies in which samples were obtained before and after either brief intervention counseling or short-term baclofen treatment. In addition, we have banked pre- and post-ghrelin treatment serum from a human laboratory study to assess the effects of craving on alcohol consumption. The approaches will include comparative biochemical and molecular analyses of EVs from AUD and control human serum to characterize the effects of ethanol and different treatments on the expression of oligodendrocyte/myelin proteins, sphingolipids, and indices of stress. In addition, we plan to assess the sensitivity and specificity of analyzing total versus oligodendrocyte-derived (O4+) EVs to detect WM ARBD effects and responses to treatment. This R21 proposal has the advantage of collecting relevant human data in parallel with ongoing experimental studies, potentially facilitating earlier clinical translation rather than waiting for the completion of rodent experiments, which are already yielding promising results. Furthermore, the outcomes of this research will expand our insights into the utility of EV-based serum diagnostic aids and generate non-invasive assays for detecting and monitoring WM ARBD and its responses to treatment. Modified SPECIFIC AIMS The main goal of this R21 application is to leverage progress in our ongoing R01AA028408 to investigate the utility of non-invasive bench-to-bedside high-throughput approaches for detecting WM ARBD and responses to treatment in human serum samples. Aim 1 has two segments: Sub-Aim 1a will determine how ARBD shifts the expression of myelin-oligodendrocyte glycoproteins, sphingolipids, and lipid peroxidation markers in extracellular vesicles (EVs) isolated from the serum of chronic heavy drinkers versus controls (light or non-drinkers). Sub-Aim 1b will examine the sensitivity and specificity of results obtained with O4+ (oligodendrocyte specific) versus total EVs to determine if the longitudinal intervention studies in Aim 2 require O4+ EV isolation or could be streamlined using total serum EV samples. Aim 2 is to examine the effects of targeted versus placebo treatment on biomarkers of WM ARBD in EVs isolated from serum. Longitudinal pre- and post-treatment samples are available. Sub-Aim 2a will measure responses to brief intervention counseling. Sub-Aim 2b will measure responses to Baclofen versus placebo. Sub-Aim 2c will measure responses to Ghrelin as an enhancer of craving, to examine the impact of increased alcohol preference behavior on WM ARBD markers in EVs isolated from serum. The studies proposed are feasible due to collaborations with Drs. Lorenzo Leggio (NIDA/NIAAA), Ron Cohen (Brown Alcohol Research Center and the University of Florida), and Chris Kahler (Director, Brown Alcohol Research Center), who supplied serum samples from 509 study participants. The subject populations were well-characterized and participated in IRB-approved studies, including motivational intervention or pharmacological (Baclofen or Ghrelin) protocols. Since the clinical outcomes are known, the results generated under this administrative supplement could be parsed to correlate peripheral biomarkers of oligodendrocyte/myelin degeneration with cognitive-behavioral dysfunction and neuroimaging studies focused on WM ARBD. Aim 1: Characterize oligodendrocyte/myelin protein, sphingolipid, and lipid peroxidation markers in EVs isolated from archival serum samples of AUD and control human study participants. 1a. Perform comparative studies of EVs isolated from the serum of heavy versus light or non-drinker participants. 1b. Determine if AUD/WM ARBD-associated myelin/oligodendrocyte molecular pathologies are better or equally detected in O4+ selected compared with total serum EVs. The results will guide strategies for Aim 2. Aim 2: Examine how targeted treatment of AUD study participants impacts serum EV expression of oligodendrocyte/myelin protein, sphingolipid, and lipid peroxidation markers over time. 2a. Determine if motivational intervention counseling normalizes WM ARBD-related serum EV expression profiles in compliant AUD study participants. 2b. Characterize effects of short-term Baclofen versus placebo on WM ARBD-related EV oligodendrocyte/myelin protein, sphingolipid, and lipid peroxidation markers in AUD study participants. 2c. Determine if Ghrelin-enhanced craving with increased alcohol use exacerbates WM ARBD-related oligodendrocyte/myelin abnormalities in serum-derived EVs. We have established that myelin/oligodendrocyte biomarkers are modulated in WM tissue of experimental chronic alcohol exposure models and in humans with ARBD and that related molecular and biochemical pathologies are detectable in EVs. The planned studies will employ enzyme-linked immunosorbent assays (ELISAs) to compare EV expression of myelin/oligodendrocyte glycoproteins (MAG, MOG, MBP, PDGFRA), sphingolipids (sulfatide, sphingomyelin, ceramide), and lipid peroxidation markers (HNE, MDA, 8-iso-PGF2α). The research conducted under this R21 will utilize technology refined within the past 3 years of funding under R01-AA028408 to logically extend efforts to human studies and fast-forward the implementation of non-invasive serum-based assays for detecting and monitoring WM ARBD, including its progression and responses to treatment. Due to rapid objective non-invasive biochemical/assessments of WM ARBD, the research gains could broadly impact patient care and participation in clinical trials.