Alrashidi, Haya; (2022) Biochemical characterisation of Parkinson's disease models. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

Parkinson’s disease (PD) is the second most common neurodegenerative disorder. The exact molecular mechanism of disease remains unclear. Several factors are proposed to play part including, but not limited to, decreased activity of mitochondrial complex I and lysosomal glucocerebrosidase enzymes and disrupted cellular antioxidant defence and lysosomal acidification. In addition, there is growing support for a role of organelle crosstalk between mitochondria and lysosome, the disruption of which is proposed to play part in PD pathology. The nature and consequence of this crosstalk remains unclear. The SH-SY5Y neuronal cell line model is commonly used to investigate PD mechanisms and potential therapeutics. However, functional analysis of the suitability of the cell line in its proliferative state or the necessity for differentiation remains unclear. Furthermore, iPSC-derived dopaminergic neurons are another commonly used model for PD and related diseases however, validating their functional dopamine metabolism is important to determine disease mechanism and test potential therapeutics. In this thesis, a host of biochemical tools, including HPLC measurement of neurotransmitter metabolites and enzyme activity assays, were used to elucidate the aforementioned ambiguities. The findings demonstrate that although there are similarities between proliferative and differentiated phenotypes of SH-SY5Y cells, there are also significant differences. Notably, the rate of dopamine turnover and the activity of lysosomal glucocerebrosidase were significantly higher in differentiated SH-SY5Y cells. In contrast, mitochondrial electron transport chain complexes’ activities were similar between the two phenotypes, despite a significant difference in mitochondrial content. Therefore, care should be taken when choosing either phenotype as a PD model. In addition, 4the findings demonstrate that inhibition of either mitochondrial complex I or lysosomal glucocerebrosidase affect both the ratio of pro-cathepsin D/cathepsin D protein expression and enzyme activity. Cathepsin D is one of the most ubiquitous lysosomal enzymes, the state of which can be used as reflection of the degree of lysosomal acidification. This shines a light on the potential involvement of both lysosomal glucocerebrosidase and mitochondrial complex I in maintenance of lysosomal acidification. This could be a consequence of a more dynamic crosstalk between mitochondria and lysosomes than previously thought. Moreover, the work presented provides a method for validation of the dysfunctional dopamine metabolism in iPSC derived dopaminergic neuronal disease models for aromatic amino acid decarboxylase deficiency and PD patients carrying mutations in PINK1. In addition, it provides a proof of concept for the effectiveness of both lentivirus-based gene therapy and levodopa treatment to restore dopamine metabolism in aromatic amino acid decarboxylase deficiency.

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