Assessment of Ethanol-Induced Toxicity on iPSC-Derived Human Neurons Using a Novel High-Throughput Mitochondrial Neuronal Health (MNH) Assay

Abstract

Excessive ethanol exposure can cause mitochondrial and cellular toxicity. In order to discover potential counteracting interventions, it is essential to develop assays capable of capturing the consequences of ethanol exposure in human neurons, and particularly dopaminergic neurons that are crucial for the development of alcohol use disorders (AUD). Here, we developed a novel high-throughput (HT) assay to quantify mitochondrial and neuronal toxicity in human dopaminergic neuron-containing cultures (DNs) from induced pluripotent stem cells (iPSCs). The assay, dubbed mitochondrial neuronal health (MNH) assay, combines live-cell measurement of mitochondrial membrane potential (MMP) with quantification of neuronal branching complexity post-fixation. Using the MNH assay, we demonstrated that chronic ethanol exposure in human iPSC-derived DNs decreases MMP and neuronal outgrowth in a dose-dependent manner. The toxic effect of ethanol on DNs was already detectable after 1 h of exposure, and occurred similarly in DNs derived from healthy individuals and from patients with AUD. We next used the MNH assay to carry out a proof-of-concept compound screening using FDA-approved drugs. We identified potential candidate compounds modulating acute ethanol toxicity in human DNs. We found that disulfiram and baclofen, which are used for AUD treatment, and lithium caused neurotoxicity also in the absence of ethanol, while the spasmolytic drug flavoxate positively influenced MNH. Altogether, we developed an HT assay to probe human MNH and used it to assess ethanol neurotoxicity and to identify modulating agents. The MNH assay represents an effective new tool for discovering modulators of MNH and toxicity in live human neurons.

High-content analysis of neuronal morphology in human iPSC-derived neurons

“…However, the HCA protocol described here can also be reliably applied to iPSC-derived neurons obtained using other differentiation approaches. For example, we successfully used this protocol for dopaminergic neurons obtained by exposing iPSCs to specific growth factors and small molecules ( Inak et al., 2021 ; Zink et al., 2020 ).

Figure 1 Neurons differentiated from human induced pluripotent stem cells (iPSCs) Upper panel, step-by-step protocol for the NGN2 induction from the engineered iPSC line BIHi005-A-24.

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“…The HCA pipeline can also be applied to neurons differentiated from patient-derived iPSCs to possibly unveil disease-specific neuronal defects ( Inak et al., 2021 ). In combination with the assessment of live mitochondrial activity ( Zink et al, 2022 ), this protocol can also enable the identification of modulators of human neuronal and mitochondrial toxicity ( Zink et al., 2020 ).…”

“…The approach can be applied to patient-derived iPSCs to identify patient-specific defects and possible therapeutic strategies. For complete details on the use and execution of this protocol, please refer to Zink et al. (2020) and Inak et al.…”

“…For complete details on the use and execution of this protocol, please refer to Zink et al. (2020) and Inak et al.…”

Genome Editing in iPSC-Based Neural Systems: From Disease Models to Future Therapeutic Strategies

“…Moreover, the pooled setup does not allow the interrogation of non-cell-autonomous phenotypes, in which the relevant phenotype is not physically coupled to the cell in which a gene is perturbed, such as excitation of target cells and interactions between neurons and glia, among others. Screens for non-cell-autonomous or complex functional or morphological phenotypes typically require arrayed approaches that allow setting a multitude of different readouts, such as high-content imaging (Bolognin et al, 2019 ; Yamaguchi et al, 2020 ) measurements of electrophysiological activity (Daily et al, 2017 ), and various biochemical assays (Medda et al, 2016 ; Zink et al, 2020 ). However, arrayed screens necessitate automated platforms for high-throughput measurement of the phenotypes of interest, and handling, culturing and analysis of hundreds of plates, a major limiting factor for most academic laboratories with limited capacity.…”