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.
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.
|Effective start/end date||01.01.15 → 31.12.17|
In 2015, UN member states agreed to 17 global Sustainable Development Goals (SDGs) to end poverty, protect the planet and ensure prosperity for all. This project contributes towards the following SDG(s):