Analysis of Mitochondrial Dysfunction by Microplate Reader in hiPSC-Derived Neuronal Cell Models of Neurodegenerative...

Mitochondria are responsible for many vital pathways governing cellular homeostasis, including cellular energy management, heme biosynthesis, lipid metabolism, cellular proliferation and differentiation, cell cycle regulation, and cellular viability. Electron transport and ADP phosphorylation coupled with proton pumping through the mitochondrial complexes contribute to the preservation of mitochondrial membrane potential (ΔΨ_m). Importantly, mitochondrial polarization is essential for reactive oxygen species (ROS) production and cytosolic calcium (Ca²⁺) handling. Thus, changes in mitochondrial oxidative phosphorylation (OXPHOS), ΔΨ_m, and ATP/ADP may occur in parallel or stimulate each other. Brain cells like neurons are heavily reliant on mitochondrial OXPHOS for its high-energy demands, and hence improper mitochondrial function is detrimental for neuronal survival. Indeed, several neurodegenerative disorders are associated with mitochondrial dysfunction. Modeling this disease-relevant phenotype in neuronal cells differentiated from patient-derived human induced pluripotent stem cells (hiPSCs) provide an appropriate cellular platform for studying the disease pathology and drug discovery. In this review, we describe high-throughput analysis of crucial parameters related to mitochondrial function in hiPSC-derived neurons. These methodologies include measurement of ΔΨ_m, intracellular Ca²⁺, oxidative stress, and ATP/ADP levels using fluorescence probes via a microplate reader. Benefits of such an approach include analysis of mitochondrial parameters on a large population of cells, simultaneous analysis of different cell lines and experimental conditions, and for drug screening to identify compounds restoring mitochondrial function.