Akademik Veri Yönetim Sistemi
Hacettepe University Journal of the Faculty of Pharmacy, cilt.33, sa.1, ss.59-74, 2013 (Scopus)
Gaucher disease is caused by defects in the activity of the lysosomal enzyme, glucocerebrosidase (EC 3.2.1.45). Since lysosomes ultimately are responsible for turning over macromolecules transported to them from both outside (endocytosis and phagocytosis) and inside the cell (macroautophagy-lysosomal system), we explored the possibility that the glucosylceramide and glucosylsphingosine accumulation resulting from Gaucher disease may disrupt the normal autophagic fusion process and/ or autophagic flux exacerbating the clinical phenotype. To discriminate between an induction of autophagy and defective maturation/fusion of autophagosomes to autolysosomes, versus autophagic flux (turnover of cargo in the autolysosome), LC3B-II levels and proteolysis of internalized fluorescent DQ-BSA in lysosomes (with the use of high throughput instrument Cellomics KSR) were monitored in Gaucher fibroblasts carrying the most prevelant mutations L444P and N370S. Increased levels of LC3B-II relative to LC3B-I and GAPDH demonstrated an increase in the number of autophagosomes and/or a decrease rate of lysosomal turn-over (flux) in fibroblasts of patients which were correlated with the phenotypes. Lower turnover of the DQ-BSA in patient fibroblasts indicated that the flux rate was decreased in Gaucher disease. The decrease of lysosomal flux was more significant in the Type II, acute neurophathic form of L444P mutation. We can conclude that L444P and N370S mutations decreased the the rate of turn-over of autophagy and other substrates, including LC3B-II, in the lysosome and that the decreases roughly correlated with the clinical severity.