A single cell high content assay detects mitochondrial dysfunction in iPSC-derived neurons with mutations in SNCA

Abstract

Mitochondrial dysfunction is implicated in many neurodegenerative diseases including Parkinson’s disease (PD). Induced pluripotent stem cells (iPSCs) provide a unique cell model for studying neurological diseases. We have established a high-content assay that can simultaneously measure mitochondrial function, morphology and cell viability in iPSC-derived dopaminergic neurons. iPSCs from PD patients with mutations in SNCA and unaffected controls were differentiated into dopaminergic neurons, seeded in 384-well plates and stained with the mitochondrial membrane potential dependent dye TMRM, alongside Hoechst-33342 and Calcein-AM. Images were acquired using an automated confocal screening microscope and single cells were analysed using automated image analysis software. PD neurons displayed reduced mitochondrial membrane potential and altered mitochondrial morphology compared to control neurons. This assay demonstrates that high content screening techniques can be applied to the analysis of mitochondria in iPSC-derived neurons. This technique could form part of a drug discovery platform to test potential new therapeutics for PD and other neurodegenerative diseases.

Induced pluripotent stem cell‐based modeling of mutant LRRK2‐associated Parkinson's disease

“…was also reported for SNCA trp and SNCA A53T mesDA neurons (Little et al, 2018). As these observations hint towards a profound disturbance of mitochondrial function in familial forms of PD, several groups consequently investigated effects of PD risk factors on mitochondrial redox homeostasis and found increased levels of mitochondrial reactive oxygen species (ROS) in mesDA neurons carrying polymorphisms in LRRK2, DJ-1, PINK1, PRKN and SNCA (Burbulla et al, 2017;Chung et al, 2016;Ryan et al, 2013;Su & Qi, 2013;Suzuki et al, 2017), as well as in NPCs with SNCA trp and OPA1 variants (Flierl et al, 2014;Iannielli et al, 2018).…”

“…In summary, alterations in mitochondrial morphology were repeatedly shown not only for LRRK2 variants (Su & Qi, ) but also for mutants of SNCA trp/A53T PINK1, PRKN and OPA1 (Chung et al., ; Iannielli et al., ; Imaizumi et al., ; Little et al., ; Shaltouki et al., ; Zanon et al., ).…”

Using stem cell–derived neurons in drug screening for neurological diseases

“…inhibitor. Interestingly we have used a similar high-content assay to detect decreased mitochondrial membrane potential in iPSC-derived dopaminergic neurons from patients with Parkinson's disease (Little et al, 2018). This suggests that such an assay could be used to identify potential novel therapies for Parkinson's disease too.…”

Modeling Parkinson’s Disease Using Induced Pluripotent Stem Cells

“…A high-throughput analysis showed that SNCA triplication iPSC-induced DA neurons harbored mitochondrial morphological changes and a decrease in mitochondrial membrane potential [33]. A transcriptomic analysis of purified SNCA triplication iPSCderived DA neurons revealed perturbation of gene expression associated with mitochondrial function.…”