Impact of mutant VPS35 on human dopaminergic neurons

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The overall hypothesis in this application is that impaired cellular trafficking plays a key role in the pathogenesis of Parkinson disease (PD); and that this global insult will have more pronounced effects in more vulnerable neurons. PD is the second most common neurodegenerative disorder and pathologically associated with the death of midbrain DA neurons. These neurons are characterized by a unique anatomical complexity, which is combined with high bioenergetic demands due to intense trafficking processes and may make DA neurons particularly vulnerable to stress. Mutations in the gene vacuolar protein sorting 35 (VPS35) were recently linked to dominantly inherited PD, a rare monogenetic form that is clinically indistinguishable from idiopathic PD. VPS35 has been shown to regulate vesicle sorting and degradation. In a collaborative study between the applicant and the laboratory of the selected host institute, our teams showed that VPS35 binds and traffics excitatory neurotransmitter receptors and governs their surface levels and functional fate. The goal of this application is to extend our preliminary study and to establish and characterize a human DA neuronal model using patient-derived induced pluripotent stem cells (iPSCs) harboring a VPS35 mutation to examine the impact of this mutation. The following three specific aims will be addressed: (i) Establish a cell culture of human DA neurons derived from iPSC lines (patient and isogenic control) amenable to live-cell assays. These cultures will contain mature, identifiable DA neurons in co-culture with excitatory cortical neurons. (ii) Assess these neurons for morphological and physiological phenotypes. Axonal arborisation and neurite outgrowth of identified DA neurons will be assessed by confocal microscopy, and intrinsic excitability, channel function, and action potential firing will be assessed by whole cell patch-clamp technique. (iii) Basal and pharmacologically stimulated DA release will be measured by HPLC. In additional to novel pathophysiological insights into PD, the ultimate value of this project will be an extensively characterized unique model of human disease that is highly amenable to experimental manipulations and available for the screening of new therapeutic compounds.