Mitochondrial and Clearance Impairment in p.D620N VPS35 Patient‐Derived Neurons

Abstract

A BS TRACT: Background: VPS35 is part of the retromer complex and is responsible for the trafficking and recycling of proteins implicated in autophagy and lysosomal degradation, but also takes part in the degradation of mitochondrial proteins via mitochondria-derived vesicles. The p.D620N mutation of VPS35 causes an autosomal-dominant form of Parkinson's disease (PD), clinically representing typical PD. Objective: Most of the studies on p.D620N VPS35 were performed on human tumor cell lines, rodent models overexpressing mutant VPS35, or in patient-derived fibroblasts. Here, based on identified target proteins, we investigated the implication of mutant VPS35 in autophagy, lysosomal degradation, and mitochondrial function in induced pluripotent stem cell-derived neurons from a patient harboring the p.D620N mutation. Methods: We reprogrammed fibroblasts from a PD patient carrying the p.D620N mutation in the VPS35 gene and from two healthy donors in induced pluripotent stem cells. These were subsequently differentiated into neuronal precursor cells to finally generate midbrain dopaminergic neurons. Results: We observed a decreased autophagic flux and lysosomal mass associated with an accumulation of α-synuclein in patient-derived neurons compared to controls. Moreover, patient-derived neurons presented a mitochondrial dysfunction with decreased membrane potential, impaired mitochondrial respiration, and increased production of reactive oxygen species associated with a defect in mitochondrial quality control via mitophagy. Conclusion: We describe for the first time the impact of the p.D620N VPS35 mutation on autophago-lysosome pathway and mitochondrial function in stem cell-derived neurons from an affected p.D620N carrier and define neuronal phenotypes for future pharmacological interventions.

Parkinson’s disease‒associated VPS35 mutant reduces mitochondrial membrane potential and impairs PINK1/Parkin-mediated mitophagy

“…Mitochondrial quality control mechanisms that maintain Δψ m , such as mitochondrial fragmentation 52 or the removal of depolarized mitochondria through mitophagy 29,53 , are thus essential and are likely affected in VPS35 D620N cells, which leads to the observed accumulation of damaged and fragmented mitochondria under steady state conditions. Importantly, while this manuscript was in preparation, another study was published showing that p.D620N-mutant VPS35 patient-derived dopaminergic neurons also exhibit mitochondrial impairments, including a reduction in Δψ m under steady state, and present defects in CCCP-induced mitophagy, thereby con rming our results 54 . Moreover, depletion of VPS35 in neuroblastoma cells also causes reduced basal Δψ m and an increase in mitochondrial ssion at steady state 15 .…”

A novel autophagy-related genes prognostic risk model and validation of autophagy-related oncogene VPS35 in breast cancer

“…The retromer is able to sustain lysosome structure stability and normal lysosome function, which participates in regulating autophagy process [ 28 ]. Moreover, there is a co-expression network between VPS35 and several proteins involving in autophagy process [ 29 ]. More importantly, VPS35 is implicated in both the activity of Wnt signaling pathway and the endocytosis process [ 30 – 32 ].…”

Gene-corrected Parkinson’s disease neurons show the A30P alpha-synuclein point mutation leads to reduced neuronal branching and function

“…For the image acquisition, 10 to 12 Z-stack images per coverslip were acquired using Zeiss spinning disk confocal microscope from four independent directed neuronal differentiations. The custom image analysis algorithms for the morphological analysis of neurology morphology, neurite morphology and skeletonisation, nuclei segmentation and TH neuron segmentation have been previously described 40,41 .…”

“…The neurons were cultured on the plate for an additional 10 days and recorded at d55. The recording of the MEA plate has been previously described 41 . Briefly, the MEA plate was recorded using the Axion Integrated Studio (AxIS) software, version 2.1 (Axion Biosystems, Atlanta, GA, USA) on the Axion Maestro (Axion Biosystems, Atlanta, GA, USA).…”