PROJECT SUMMARY / ABSTRACT Cocaine abuse continues to be a national crisis, with the cost of addiction to illegal drugs in the United States related to criminal activity, lost work productivity, and medical care totaling more than $740 billion per year. In particular, African-Americans, who make up 13.4% of the U.S. population, disproportionately bear the burden of cocaine use and overdoses. Multiple studies have shown that prolonged use of cocaine affects the gut microbiota, which in turn alters hypothalamus-pituitary-axis activity and emotional behavior. Cocaine-related studies have shown that drugs of abuse trigger preferential proliferation of gram-positive Firmicutes and reduction of gram- negative Bacteroidetes, results that are consistent with microbiota associations with drug addiction. Accumulating evidence also shows that gut microorganisms influence the bidirectional communication along the gut- brain axis, which indicates the crucial role of the gut microbiota on cocaine. Since evidence exists for the influence of commonly abused drugs on the microbiota gut-brain axis, the transverse colon, the distal ileum, the basal ganglia, and the pituitary and adrenal glands may have potential roles as biomarkers, specifically in overdose cases, due to the damage to these tissues caused by cocaine abuse. In this proposal, we will address the impact that cocaine has on the microbiota of the gut-brain axis. Our approach will allow for evaluation of microbial signatures in microbiota gut-brain axis-associated tissues using specimens extracted from cocaine overdose cadavers, an approach that previous studies have not used but could provide information that helps determine how people respond differently to cocaine, allowing researchers to better examine the potential adverse effects of microbes. Specifically, we will test the hypothesis that there is a significant association between gut microbiota and the gut-brain axis in cocaine overdose cases from three Southern regions - Alabama, Florida, and Tennessee. To test this hypothesis, we will (1) determine whether the gut microbiome that is associated with the transverse colon and distal ileum mediates the relationship between cocaine use and lethal drug intoxication in African Americans from Southern regions of the United States; (2) determine whether the gut microbiome that is associated with the basal ganglia and the pituitary and adrenal glands mediates the relationship between cocaine use and lethal drug intoxication in African Americans from Southern regions of the United States;; and (3) validate a mathematical model of specific microbial biomarkers in single-blind studies using African-American tissues to increase confidence and demonstrate reproducibility. These processes will be evaluated and validated to provide a basis for new diagnostics as well as therapeutic targets to prevent intentional and unintentional cocaine overdose with the long-term objective of restoring gut microbiome homeostasis. In addition, the hands-on experience that the undergraduate students gain conducting phenol-chloroform extractions, polymerase chain reaction, and the workshop at the Freeman Ranch Body Farm at Texas State University in this project will provide them with insights into whether an academic career in biomedical sciences might be an avenue they want to further pursue.

PROJECT NARRATIVE In this proposal, we will test the impact of cocaine use on the microbiota of selected tissues associated with the gut-brain axis. Knowing that imbalances in microbial composition or activity arising from chronic cocaine use influences health and is implicated in various diseases (e.g., leaky gut and mood disorders), our studies will be executed in tissues extracted from African-American overdose cases from three Southern regions of the United States. Since the gut microbiome plays major roles in maintaining human health, our studies will contribute fundamental knowledge to enhancing the well-being of individuals with cocaine abuse disorders.