Parkin Maintains Robust Pacemaking in Human Induced Pluripotent Stem Cell-Derived A9 Dopaminergic Neurons.

Abstract

The degeneration of nigral (A9) dopaminergic (DA) neurons results in cardinal motor symptoms that define Parkinson's disease (PD). Loss-of-function mutations in parkin are linked to a rare form of early-onset PD that is inherited recessively. We generated isogenic human A9 DA neurons with or without parkin mutations to establish the causal relationship between parkin mutations and the dysfunction of human A9 DA neurons. Using TALEN (transcription activator-like effector nuclease)- or CRISPR/Cas9-mediated gene targeting, we produced two isogenic pairs of naivetropic induced pluripotent stem cells (iPSCs) by repairing exon 3 deletions of parkin in iPSCs derived from a PD patient and by introducing the PD-linked A82E mutation into iPSCs from a healthy subject. The four lines of isogenic iPSCs were differentiated to A9 DA neurons, which fired spontaneous pacemaking action potentials (AP) dependent on L-type Ca2+ channels. The frequency of the pacemaking APs was significantly reduced by parkin mutations introduced to normal neurons. Consistent with this, isogenic repair of parkin mutations significantly increased the frequency from that observed in patient-derived neurons. The results show that parkin maintains robust pacemaking in human iPSC-derived A9 DA neurons. The function is critical to normal DA transmission required for controlling voluntary locomotor activities. © 2023 International Parkinson and Movement Disorder Society.