Glukozilseramidaz-Beta Gen Mutasyonlarının Dopaminerjik Nöronlarda α-sinüklein Yıkım Mekanizmaları Üzerindeki Etkilerinin Araştırılması

Abstract

Glucosylceramidase-Beta (GBA1) Gene Mutations are the most important risk factor identified for Parkinson's Disease (PD). Clinically, 'mild' GBA1 mutations (such as N370S) associated with non-neuropathic Gaucher Disease (GD) and 'severe' GBA1 mutations (such as L444P and D409H) associated with neuropathic GD are known to have different phenotypic effects of developing PD, age at disease onset, prognosis, and symptoms, in terms of severity. One of the main pathological features of PD is the presence of oligomeric/fibrillar α-synuclein-positive inclusions, known as Lewy bodies, in dopaminergic neurons. In this study, we hypothesized that GBA1 mutations cause accumulation of α-synuclein by affecting the crosstalk between cellular protein degradation mechanisms and cause neurodegeneration. Accordingly, Type I GD patient (N370S/N370S genotype) and Type III GD patients (L444P/L444P and D409H/D409H genotypes), heterozygous obligatory carriers whom are the family members of these patients (N370S/-, L444P/- and D409H/- genotypes), idiopathic PD patient without GBA1 mutation, PD patient with heterozygous GBA1 mutation (N370S/-) and healthy individuals. Primary fibroblasts obtained from individuals were reprogrammed into induced pluripotent stem cells (IPSCs) and then differentiated into dopaminergic neurons. It was aimed to investigate the ubiquitin-proteosome system (UPS), chaperone-mediated autophagy (CMA) and macroautophagy mechanisms which are responsible for the degradation of α-synuclein and thereby the accumulation and release of α-synuclein in these cells. Monomeric α-synuclein accumulated in D409H/D409H GBA1 mutant cells and PH N370S/- cells, while oligomeric αsynuclein and extracellular release of α-synuclein were found to be increased in 'severe' GBA1 (D409H and L444P) homozygous mutant cells and PH N370S/- cells. It was determined that ER stress and total ubiquitination rates were significantly increased in cells with 'severe' (L444P and D409H) homozygous GBA1 mutations and cells from PD patients. A significant decrease in proteosome activities was detected in all GBA1 mutant cells compared to control cells. Dysfunction in the CMA mechanism in cells with a 'mild' (N370S) homozygous GBA1 mutation was detected. When the macroautophagy mechanism in iPSC derived dopaminergic neurons was examined, it was determined that the formation of autophagosomes decreased in the cells of individuals with the N370S/N370S and D409H/D409H genotypes, and there were defects in autophagosome-lysosome fusion in the cells of the individuals with the L444P/L444P genotype. When iPD and PD N370S/- patients were compared, it was found that there were defects in both autophagosome formation and autophagosome-lysosome fusion in PD N370S/- cells. 'Severe' GBA1 mutations affect more ER stress and macroautophagy mechanism in cells by triggering oligomeric α-synuclein accumulation and release, while 'mild' GBA1 mutations affect monomeric α-synuclein accumulation and CMA mechanism responsible for monomeric α-synuclein degradation. These results may explain the molecular basis of the clinical PD data ralated to 'mild' and 'severe' GBA1 mutations.