High-content analysis for ethanol-induced mitochondrial and neuronal toxicity in human iPSC-derived neurons

Abstract

Ethanol is the most frequently abused drug in the world with a worldwide mortality of three million deaths each year (5.3% in 2016). Excessive ethanol consumption can adversely affect several organs, where it can lead to cellular and mitochondrial toxicity. The brain is particularly susceptible to ethanol toxicity. The exact mechanism by which ethanol affects the brain is still unknown. Dopaminergic neurons are believed to play a role, especially in the rewarding and reinforcing (i.e. addictive) effects of alcohol. Thus, model systems enabling to detect early toxicity in human dopaminergic neurons may help to discover potential treatment strategies. In this study, I used human induced pluripotent stem cells (iPSCs) from control individuals and from patients with alcohol use disorder (AUD) to generate neural progenitor cells (NPCs) and neuronal cultures containing dopaminergic neurons. I developed a high-content analysis (HCA)-based assay, dubbed Mitochondrial Neuronal Health (MNH) assay, to quantitatively assess mitochondrial neuronal health in iPSCderived neurons following ethanol exposure. Acute and chronic exposure of ethanol on iPSC-derived neurons led to decreased mitochondrial membrane potential (MMP) and decreased neuronal branching complexity in a dose-dependent manner. Ethanol toxicity was similarly observed in control neurons and in neurons derived from individuals with AUD. Finally, I adapted the MNH assay to perform a proof-of-concept compound screening to identify modulators of ethanol-induced neurotoxicity. I found that the two drugs disulfiram and baclofen, which are used to treat AUD, and lithium caused neurotoxicity. In contrast, the spasmolytic drug flavoxate had a positive effect on mitochondrial neuronal health.