Functions of parkin in Parkinson’s disease

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Summary Nigral dopaminergic (DA) neurons (i.e. A9 DA neurons) that are lost in Parkinson’s disease (PD) have massive axon arborization, autonomous pacemaking activities, 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 genetically-labeled isogenic iPSCs, we will study vulnerabilities of A9 type DA neurons in dopaminergic transmission, oxidative stress, mitochondrial functions, and neuronal morphology at three different levels: monolayer cultures, brain organoids, and graft in 6-OHDA-lesioned rat brains. These novel approaches will enable us to study the impact of parkin mutations on the vulnerabilities of A9 DA neurons using three different preparations to approximate the situation in the brains of PD patients. The study will significantly advance our understanding of the in vivo function of parkin and how it protects against the vulnerabilities of nigral DA neurons. The results will stimulate the development of disease-modifying therapies of