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