Heterozygous mutations in the glucocerebrosidase (GBA) gene represent the most common risk factor for Parkinson’s disease (PD), a disease in which midbrain dopaminergic neurons are preferentially vulnerable. However, the mechanisms underlying this association are still unknown, mostly due to the lack of an appropriate model of study. In this thesis, we aimed at elucidating the role of heterozygous GBA mutations in PD using a specific human induced pluripotent stem cell (hiPSC)-based model of disease. First we developed a protocol for the efficient differentiation of hiPSCs into dopaminergic cultures, and extensively characterized the derived dopaminergic neurons which expressed multiple midbrain relevant markers and produced dopamine. Next we screened a clinical cohort of PD patients to identify carriers of GBA mutations of interest. Using for the first time hiPSCs generated from PD patients heterozygous for a GBA mutation (together with idiopathic cases and control individuals) we were able to efficiently derive dopaminergic cultures and identify relevant disease mechanisms. Upon differentiation into dopaminergic neuronal cultures, we observed retention of mutant glucocerebrosidase (GCase) protein in the endoplasmic reticulum (ER) with no change in protein levels, leading to upregulation of ER stress machinery and resulting in increased autophagic demand. At the lysosomal level, we found a reduction of GCase activity in dopaminergic neuronal cultures, and the enlargement of the lysosomal compartment in identified dopaminergic neurons suggesting a decreased capacity for protein clearance. Together, these perturbations of cellular homeostasis resulted in increased release of α-synuclein and could likely represent critical early cellular phenotypes of Parkinson's disease and explain the high risk of heterozygous GBA mutations for PD.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:647642 |
| Date | January 2014 |
| Creators | Ribeiro Fernandes, Hugo José |
| Contributors | Wade-Martins, Richard |
| Publisher | University of Oxford |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Source | http://ora.ox.ac.uk/objects/uuid:7027574c-dda4-4752-9010-4c573bd0b2aa |
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