Department of Physiology and Biophysics, State University of New York at Buffalo, Buffalo, New York, USA.
Department of Neurology, Second Affiliated Hospital, College of Medicine, Zhejiang University, Hangzhou, China.
Mov Disord. 2023 Jul;38(7):1273-1281. doi: 10.1002/mds.29434. Epub 2023 May 11.
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 Ca 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.
黑质（A9）多巴胺能（DA）神经元的退化导致了帕金森病（PD）的主要运动症状。Parkin 失活突变与一种罕见的早发性 PD 形式有关，这种 PD 形式是隐性遗传的。
我们生成了具有或不具有 parkin 突变的同源人 A9 DA 神经元，以建立 parkin 突变与人类 A9 DA 神经元功能障碍之间的因果关系。
使用 TALEN（转录激活因子样效应物核酸酶）或 CRISPR/Cas9 介导的基因靶向，我们通过修复源自 PD 患者的 iPSC 中 parkin 的外显子 3 缺失，以及将 PD 相关的 A82E 突变引入源自健康受试者的 iPSC，产生了两对同源的原始诱导多能干细胞（iPSC）。这四条同源 iPSC 系被分化为 A9 DA 神经元，这些神经元依赖 L 型钙通道发射自发起搏动作电位（AP）。
引入正常神经元的 parkin 突变显著降低了起搏 AP 的频率。与此一致的是，同源修复 parkin 突变显著增加了源自患者来源神经元的频率。
这些结果表明 parkin 维持了源自人 iPSC 的 A9 DA 神经元中的强大起搏。该功能对于控制自主运动活动所需的正常 DA 传递至关重要。 © 2023 国际帕金森病和运动障碍协会。
