Halawani, Merfat Mohammed; (2021) Molecular mechanisms of GBA-linked PD. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

Objective: To investigate the effect of GBA heterozygote L444P (wt/L444P) mutation in human dermal fibroblasts and mouse cortical neurons models (MCN) and find if glucocerebrosidase (GCase) deficiency in these cells impair the lysosomal function and induce unfolded protein response (UPR). In addition, to investigate the consequent impairment of alpha synuclein (A-SYN) metabolism in the form of increased intracellular accumulation and extracellular release of fibrillar A-SYN following seeding MCN and differentiated SH-SY5Y neuronal cultures with preformed A-SYN fibrils (PFFs). Background: GBA heterozygous mutations are numerically the most important predisposing factor for developing PD. They contribute to the earlier age of onset and increased cognitive decline in GBA-PD. L444P mutation is proposed to induce unfolded protein response (UPR) and perturbed autophagy pathways. This consequently can lead to impairment of (A-SYN) turnover and eventually result in the loss of substantia nigra dopaminergic cells, the most vulnerable type of cells affected in PD. It is hypothesized that A-SYN pathology can spread through brain regions to reach SNpc thereby supporting the prion-like theory. Fibrillar forms of A-SYN are thought to contribute to this spread as they can induce the endogenous A-SYN to recruit, misfold, and become insoluble aggregates like that observed in Lewy bodies. Methods: Functions of ALP and UPS, protein degradation pathways, were assessed in L444P heterozygous fibroblasts and MCN models. ER stress and UPR investigated in all disease models including fibroblasts, wt/L444P MCN and differentiated SH-SY5Y neurons overexpressing (O/E) L444P GBA. Both differentiated SH-SY5Y treated with the GCase inhibitor (CBE) and wt/L444P mutant MCN were incubated with PFFs to initiate A-SYN pathology. Misfolded and aggregated A-SYN was assessed by western blotting and dot blot. Results: wt/L444P fibroblasts and MCN cellular models showed no impairment of protein quality control systems. There was ER retention of mutant enzyme in wt/ L444P fibroblast but this was not enough to induce UPR in cells. There was also subtle or no alteration in endogenous A-SYN level in wt/L444P MCN and differentiated SH-SY5Y neurons treated with CBE until the addition of another stress such as PFFs seeding. Treating cells with PFFs for 10 days enhanced the recruitment and aggregation of endogenous A-SYN that were phosphorylated at Ser129 with evidence of HMW species. Differentiated SH-SY5Y neurons O/E wildtype SNCA and preloaded with PFFs showed enhanced extracellular release of fibrillar A-SYN. In addition, differentiated cells O/E L444P GBA, but not MCN or fibroblasts, showed evidence of ER stress and UPR. Conclusions: Wt/L444P MCN and differentiated SH-SY5Y dopaminergic neurons treated with CBE showed augmented A-SYN pathology. The increased release of pathogenic A-SYN fibrils was more evident in cells with higher endogenous A-SYN levels and only differentiated SHSY5Y cells O/E L444P GBA showed evidence of UPR.

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