Impact of mutant VPS35 on human dopaminergic neurons
Molecular Biology and Physiology of Neurons and Glial Cells
Final Report Abstract
The present study demonstrates the successful generation of a new tool to identify living dopaminergic (DA) neurons, the affected cell type in Parkinson disease, and the examination of DA release and stress susceptibility in VPS35 mutant DA neurons. The p.D620N missense mutation in vacuolar protein sorting 35 (VPS35) has been recently linked to dominantly inherited parkinsonism. 1.) A GFP reporter sequence was put under the control of the GIRK2 promoter in a lentiviral vector that enables the identification of living neurons positive for the DA marker tyrosine hydroxylase. These iPSC-derived neurons were analyzed for their ability to fire action potentials by patch clamp electrophysiology. Neurobiotin pipette filling during patching and tyrosine hydroxylase immunofluorescence staining post-fixation confirmed the identity of the examined neurons. 2.) DA neurons were differentiated from isogenic VPS35 wild-type and p.D620N mutant iPSC lines. DA release was markedly increased by KCl-induced membrane depolarization in wild-type but only to a minor degree in p.D620N lines. Further, treatment with the dopamine transporter inhibitor GBR 12909 resulted in increased DA levels in wild-type but not p.D620N neurons. Impaired synaptic neuroreceptor trafficking might cause the observed reduction in DA release and could possibly also affect DA uptake. This might produce chronic pathophysiological stress upon the neurons that need to compensate for the dysfunctional DA release. In-line with this hypothesis is the observation of increased susceptibility to MPP+ mitochondrial toxin found in VPS35 mutant neurons. Degeneration of DA neurons plays an important role in Parkinson disease and there is, therefore, a great need for studies in the affected cell type. We intend to further validate our findings and examine the DA metabolism and electrophysiological characteristics in VPS35 mutant neurons.
