Functions of parkin in Parkinson’s disease

总结 在帕金森氏病中丢失的黑质多巴胺能（DA）神经元（即A9 DA神经元） 疾病（PD）具有自主起搏动作电位，大量轴突分支， 表达GIRK 2，但不表达钙结合蛋白。尽管在分化方面取得了重大进展， 人类胚胎干细胞（hESC）到中脑DA神经元，一直难以产生 A9型DA神经元，特别是来自人诱导多能干细胞（iPSC）。我们 开发了一种改进的基于底板的方法来区分患者特异性iPSC， 中脑DA神经元表达A9型细胞的适当标记物，并表现出 钙通道依赖性自主起搏活动 输入。这些iPSC衍生的DA神经元在移植到6- 100个细胞时延长了精细的神经纤维。 OHDA损伤大鼠并恢复运动缺陷。我们已经产生了同基因对， 通过修复患者细胞中的帕金突变和通过将帕金突变引入iPSC， 对照细胞。使用这些同基因iPSC，我们将研究parkin突变如何在机制上 在三种不同的制剂中破坏多巴胺能传递的精确性：单层 培养物、脑类器官和6-OHDA损伤的大鼠脑中的移植物。三种新方法 能让我们大致了解帕金森病患者的大脑状况这项研究将在 对parkin细胞功能的机制理解与其在PD中的作用之间存在差距 与运动症状直接相关的病理生理学。结果将刺激 帕金森病的疾病修饰疗法的发展。

Summary Nigral dopaminergic (DA) neurons (i.e. A9 DA neurons) that are lost in Parkinson’s disease (PD) have autonomous pacemaking action potentials, massive axon arborization, and expression of GIRK2, but not calbindin. Despite the significant progress in the differentiation of human embryonic stem cells (hESCs) to midbrain DA neurons, it has been difficult to generate A9 type DA neurons, particularly from human induced pluripotent stem cells (iPSCs). We developed an improved floorplate-based method to differentiate patient-specific iPSCs to midbrain DA neurons that expressed appropriate markers for A9 type cells and exhibited calcium channel-dependent autonomous pacemaking activities independent of glutamatergic inputs. These iPSC-derived DA neurons extended elaborate neuronal fibers when grafted to 6- OHDA-lesioned rats and restored locomotor deficits. We have generated isogenic pairs of iPSCs by repairing parkin mutations in patient cells and by introducing parkin mutations to control cells. Using these isogenic iPSCs, we will study how parkin mutations mechanistically disrupt the precision of dopaminergic transmission in three different preparations: monolayer cultures, brain organoids, and graft in 6-OHDA-lesioned rat brains. The three novel approaches will enable us to approximate the situation in the brains of PD patients. The study will bridge the gap between mechanistic understanding of the cellular function of parkin and its role in PD pathophysiology that is directly linked to the motor symptoms. The results will stimulate the development of disease-modifying therapies of Parkinson's disease .