Parkinson’s disease (PD) is a neurodegenerative disorder characterized by protein inclusions mostly composed of aggregated forms of α-synuclein (α-Syn) and by the progressive degeneration of midbrain dopaminergic neurons (mDANs), resulting in motor symptoms. While other brain regions also undergo pathologic changes in PD, the relevance of α-Syn aggregation for the preferential loss of mDANs in PD pathology is not completely understood yet. To elucidate the mechanisms of the brain region-specific neuronal vulnerability in PD, we modeled human PD using human-induced pluripotent stem cells (iPSCs) from familial PD cases with a duplication (Dupl) of the α-Syn gene (SNCA) locus. Human iPSCs from PD Dupl patients and a control individual were differentiated into mDANs and cortical projection neurons (CPNs). SNCA dosage increase did not influence the differentiation efficiency of mDANs and CPNs. However, elevated α-Syn pathology, as revealed by enhanced α-Syn insolubility and phosphorylation, was determined in PD-derived mDANs compared with PD CPNs. PD-derived mDANs exhibited higher levels of reactive oxygen species and protein nitration levels compared with CPNs, which might underlie elevated α-Syn pathology observed in mDANs. Finally, increased neuronal death was observed in PD-derived mDANs compared to PD CPNs and to control mDANs and CPNs. Our results reveal, for the first time, a higher α-Syn pathology, oxidative stress level, and neuronal death rate in human PD mDANs compared with PD CPNs from the same patient. The finding implies the contribution of pathogenic α-Syn, probably induced by oxidative stress, to selective vulnerability of substantia nigra dopaminergic neurons in human PD.

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Parkinson’s disease (PD) is neuropathologically characterized by the loss of dopaminergic neurons and the deposition of aggregated alpha synuclein (aSyn). Mounting evidence suggests that neuritic degeneration precedes neuronal loss in PD. A possible underlying mechanism could be the interference of aSyn with microtubule organization in the neuritic development, as implied by several studies using cell-free model systems. In this study, we investigate the impact of aSyn on microtubule organization in aSyn overexpressing H4 neuroglioma cells and midbrain dopaminergic neuronal cells (mDANs) generated from PD patient-derived human induced pluripotent stem cells (hiPSCs) carrying an aSyn gene duplication (SNCADupl). An unbiased mass spectrometric analysis reveals a preferential binding of aggregated aSyn conformers to a number of microtubule elements. We confirm the interaction of aSyn with beta tubulin III in H4 and hiPSC-derived mDAN cell model systems, and demonstrate a remarkable redistribution of tubulin isoforms from the soluble to insoluble fraction, accompanied by a significantly increased insoluble aSyn level. Concordantly, SNCADupl mDANs show impaired neuritic phenotypes characterized by perturbations in neurite initiation and outgrowth. In summary, our findings suggest a mechanistic pathway, through which aSyn aggregation interferes with microtubule organization and induces neurite impairments.