Genetic Parkinson disease (PD) has been associated with mutations in PINK1, a gene encoding a mitochondrial kinase implicated in the regulation of mitochondrial degradation. While the studies so far examined PINK1 function in non-neuronal systems or through PINK1 knockdown approaches, there is an imperative to examine the role of endogenous PINK1 in appropriate human-derived and biologically relevant cell models. Here we report the generation of induced pluripotent stem (iPS) cells from skin fibroblasts taken from three PD patients with nonsense (c.1366C>T; p.Q456X) or missense mutations (c.509T>G; p.V170G) in the PINK1 gene. These cells were differentiated into dopaminergic neurons that upon mitochondrial depolarization showed impaired recruitment of lentivirally expressed Parkin to mitochondria, increased mitochondrial copy number and upregulation of PGC-1α, an important regulator of mitochondrial biogenesis. Importantly, these alterations were corrected by lentiviral expression of wild-type PINK1 in mutant iPS cell-derived PINK1 neurons. In conclusion, our studies suggest that fibroblasts from genetic PD can be reprogrammed and differentiated into neurons. These neurons exhibit distinct phenotypes that should be amenable to further mechanistic studies in this relevant biological context.

Under resting conditions, Pink1 knockout cells and cells derived from patients with PINK1 mutations display a loss of mitochondrial complex I reductive activity, causing a decrease in the mitochondrial membrane potential. Analyzing the phosphoproteome of complex I in liver and brain from Pink1(-/-) mice, we found specific loss of phosphorylation of serine-250 in complex I subunit NdufA10. Phosphorylation of serine-250 was needed for ubiquinone reduction by complex I. Phosphomimetic NdufA10 reversed Pink1 deficits in mouse knockout cells and rescued mitochondrial depolarization and synaptic transmission defects in pink(B9)-null mutant Drosophila. Complex I deficits and adenosine triphosphate synthesis were also rescued in cells derived from PINK1 patients. Thus, this evolutionary conserved pathway may contribute to the pathogenic cascade that eventually leads to Parkinson's disease in patients with PINK1 mutations.

Background:The Parkinson disease-related proteins PINK1 and Parkin initiate mitophagy of damaged mitochondria. Results: Endogenous Parkin is not sufficient to induce mitophagy due to PINK1-dependent ubiquitination of Parkin. Conclusion: Mitophagy is detectable only with supraphysiological levels of Parkin and differs between fibroblasts and iPSderived neurons. Significance: Stresses the importance of future studies in Parkinson disease-relevant tissue, i.e., dopaminergic neurons.

Vacuolar protein sorting 35 (VPS35) is a core component of the retromer complex, crucial to endosomal protein sorting and intracellular trafficking. We recently linked a mutation in VPS35 (p.D620N) to familial parkinsonism. Here, we characterize human VPS35 and retromer function in mature murine neuronal cultures and investigate neuron-specific consequences of the p.D620N mutation. We find VPS35 localizes to dendritic spines and is involved in the trafficking of excitatory AMPA-type glutamate receptors (AMPARs). Fundamental neuronal processes, including excitatory synaptic transmission, AMPAR surface expression and synaptic recycling are altered by VPS35 overexpression. VPS35 p.D620N acts as a loss-of-function mutation with respect to VPS35 activity regulating synaptic transmission and AMPAR recycling in mouse cortical neurons and dopamine neuron-like cells produced from induced pluripotent stem cells of human p.D620N carriers. Such perturbations to synaptic function likely produce chronic pathophysiological stress upon neuronal circuits that may contribute to neurodegeneration in this, and other, forms of parkinsonism.

Mutations in the PTEN-induced putative kinase 1 (PINK1), a mitochondrial serine-threonine kinase, and Parkin, an E3 ubiquitin ligase, are associated with autosomal-recessive forms of Parkinson disease (PD). Both are involved in the maintenance of mitochondrial integrity and protection from multiple stressors. Recently, Parkin was demonstrated to be recruited to impaired mitochondria in a PINK1-dependent manner, where it triggers mitophagy. Using primary human dermal fibroblasts originating from PD patients with various PINK1 mutations, we showed at the endogenous level that (i) PINK1 regulates the stress-induced decrease of endogenous Parkin; (ii) mitochondrially localized PINK1 mediates the stress-induced mitochondrial translocation of Parkin; (iii) endogenous PINK1 is stabilized on depolarized mitochondria; and (iv) mitochondrial accumulation of full-length PINK1 is sufficient but not necessary for the stress-induced loss of Parkin signal and its mitochondrial translocation. Furthermore, we showed that different stressors, depolarizing or non-depolarizing, led to the same effect on detectable Parkin levels and its mitochondrial targeting. Although this effect on Parkin was independent of the mitochondrial membrane potential, we demonstrate a differential effect of depolarizing versus non-depolarizing stressors on endogenous levels of PINK1. Our study shows the necessity to introduce an environmental factor, i.e. stress, to visualize the differences in the interaction of PINK1 and Parkin in mutants versus controls. Establishing human fibroblasts as a suitable model for studying this interaction, we extend data from animal and other cellular models and provide experimental evidence for the generally held notion of PD as a condition with a combined genetic and environmental etiology.