Smith, Laura Jane;
(2022)
Biochemical properties of mutant GBA on alpha-synuclein metabolism.
Doctoral thesis (Ph.D), UCL (University College London).
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Abstract
Mutations in the GBA1 gene are numerically the greatest risk factor for Parkinson disease (PD). The GBA1 gene encodes for the lysosomal hydrolase enzyme, glucocerebrosidase (GCase), and mutations often lead to lysosomal impairment. The lysosome is important in the turnover of alpha-synuclein, the key hallmark for PD. The E326K mutation is one of the most common GBA1 mutations and is proposed to have a minimal effect on GCase activity. Common mutations, L444P and N370S, are well documented loss of function mutations. It was hypothesised that GBA1 mutations predispose patients to PD through influencing the relationship between GCase and alpha-synuclein, by altering the structural stability of GCase. The effect of the E326K mutation was characterised in human dermal fibroblasts, and demonstrated that it does not induce loss of GCase function, endoplasmic reticulum (ER) retention or ER stress, unlike L444P. Functional assays and proteolytic digestion analysis of recombinant GCase protein revealed minimal changes in stability across all mutants. This was corroborated in undifferentiated and differentiated SH-SY5Y dopaminergic neuron-like cell lines over-expressing GCase variants. Undifferentiated SH-SY5Y also demonstrated a drastic increase in lipid droplet number and a modest increase of insoluble alpha-synuclein aggregates in E326K mutants. Differentiated SH-SY5Y neurons and human midbrain dopamine neurons differentiated from induced pluripotent stem cells were treated with alpha-synuclein pre-formed fibrils (PFF) to accelerate alpha-synuclein pathology. No change in aggregation was demonstrated by Homogeneous Time Resolved Fluorescence® (HTRF®) analysis in E326K neurons compared to control. In conclusion, the work presented here demonstrates that the E326K mutation behaves differently to common loss of function mutations, however lipid dyshomeostasis and alpha-synuclein pathology is still evident.
| Type: | Thesis (Doctoral) |
|---|---|
| Qualification: | Ph.D |
| Title: | Biochemical properties of mutant GBA on alpha-synuclein metabolism |
| Open access status: | An open access version is available from UCL Discovery |
| Language: | English |
| Additional information: | Copyright © The Author 2022. Original content in this thesis is licensed under the terms of the Creative Commons Attribution-NonCommercial 4.0 International (CC BY-NC 4.0) Licence (https://creativecommons.org/licenses/by-nc/4.0/). Any third-party copyright material present remains the property of its respective owner(s) and is licensed under its existing terms. Access may initially be restricted at the author’s request. |
| UCL classification: | UCL > Provost and Vice Provost Offices > School of Life and Medical Sciences > Faculty of Brain Sciences UCL > Provost and Vice Provost Offices > School of Life and Medical Sciences > Faculty of Brain Sciences > UCL Queen Square Institute of Neurology > Clinical and Movement Neurosciences UCL > Provost and Vice Provost Offices > School of Life and Medical Sciences UCL UCL > Provost and Vice Provost Offices > School of Life and Medical Sciences > Faculty of Brain Sciences > UCL Queen Square Institute of Neurology |
| URI: | https://discovery.ucl.ac.uk/id/eprint/10144868 |
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