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Cellosaurus publication CLPUB00553

Publication number CLPUB00553
Authors Larsen S.
Title Phenotypic characterization of cellular models of VPS35-associated mutation in Parkinson's disease.
Citation Thesis PhD (2019), University of Luxembourg, Luxembourg
Web pages https://hdl.handle.net/10993/40938
Abstract Parkinson's disease was first described more than 200 years ago and yet the aetiology of the disease is not fully understood. Cellular phenotypes include mitochondrial impairment, lysosomal clearance dysfunction with alpha-synuclein accumulation and intracellular trafficking alterations. In most cases PD develops sporadically and the causes are yet unknown. However, in 10% of the cases, PD is inherited and the cause is genetic. Currently, 23 PARK loci have been identified within genes causing PD. Here, we focus on PARK17, where a point mutation leading to an amino acid exchange p.D620N in VPS35 has been found to cause an autosomal-dominant form of PD. Fibroblasts from a patient carrying the p.D620N mutation and from two age and gendermatched control were derived from a skin biopsy. Functional analyses of these fibroblasts revealed that mitochondrial membrane potential (MMP) was decreased in the patient cells compared to controls without any alteration of mitochondrial morphology. Next, the fibroblasts were reprogrammed into induced pluripotent stem cells (iPSCs), that were characterised in detail. The iPSCs were first differentiated in small molecule neuronal precursor cells (smNPCs), where we analysed mitochondrial function and lysosomal clearance capacity. We found no significant difference in MMP between patient and control smNPCs. However, we observed a decreased autophagic flux and lower levels of mature cathepsin D protein, a lysosomal hydrolase responsible for the degradation of a-synuclein. Nonetheless, we found no difference in a-synuclein protein level. In order to study the impact of p.D620N on PD-related neuronal phenotypes we differentiated the smNPCs in this more disease-relevant cell population. We generated a neuronal culture enriched in dopaminergic neurons. We found that the mitochondrial network was fragmented with smaller mitochondria and less branching. Mitochondria had lower MMP and increased intra-mitochondrial reactive oxygen species (ROS) levels. In addition, mitochondrial respiration was impaired which resulted in lower production of ATP. After CCCP treatment, mitophagy was induced in the patient neurons to the same level as in the control neurons. However, while in the control neurons the autophagosomes containing mitochondrial fragments were successfully cleared, in the patient neurons they accumulated and were not cleared properly. In line with this observation, the autophagic flux was decreased and late endosome/lysosomal mass was decreased. This decreased autophagic flux was accompanied by an increase of alpha-synuclein protein levels. We further wanted to pinpoint how p.D620N VPS35 caused mitochondrial impairment in patient neurons. Accumulation of a-synuclein has been shown to induce similar mitochondrial alterations. Therefore, we measured alpha- synuclein protein levels in the mitochondrial and the cytosolic fraction and found that it was increased in both fractions to the same ratio. However, when we knocked down alpha-synuclein to the levels of the controls, it was not sufficient to rescue the decreased MMP and increased ROS level in the patient neurons. We conclude that alpha-synuclein accumulation in the mitochondrial fraction was not sufficient to cause the observed phenotype. Recently, an increased LRRK2 kinase activity was identified in p.D620N VPS35 mutant monocytes and erythrocytes from patients, as well as brains from homozygous mice. Therefore, we hypothesized that pathological LRRK2 kinase activity may induce mitochondrial impairment. We measured the phosphorylation levels of Rab10, one of LRRK2 kinase substrates, however, no significant difference between patient and control neurons was observed. In this study, we show for the first time that p.D620N VPS35 causes PD- related cellular phenotypes in patient-derived neurons. The patient- derived neurons displayed impaired mitochondria and lysosomal clearance, with accumulation of alpha-synuclein.
Cell lines CVCL_YD64; LCSBi001-A