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Title: Non-toxic concentrations of α-synuclein exacerbate Parkinson's disease-like cell death by inducing mitochondrial dysfunction
Author: Williamson, Sally Joanne Mary
ISNI:       0000 0004 2727 8184
Awarding Body: University of Edinburgh
Current Institution: University of Edinburgh
Date of Award: 2008
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α-Synuclein (α-syn), is a self-aggregating protein that has been identified as a pathologically important component in a number of diseases, such as Parkinson’s disease (PD). PD, a progressive neurological disorder affecting 1 in 500 people, results in motor dysfunction following the loss of dopaminergic neurones of the nigrastriatal pathway. A pathological hallmark of PD is the presence of α-syn containing Lewy bodies and Lewy neurites. Although α-syn has been linked to PD by both histology and genetic studies on familial PD, neither the physiological function nor the pathophysiological role of α-syn in PD has been fully elucidated. This thesis examines the cellular responses to exogenously applied recombinant α-syn under normal and disease-like conditions. Within this thesis large-scale expression and purification of α-syn was successfully established, reproducibly producing large quantities of pure recombinant α-syn that was utilised within in vitro experiments. In SHSY-5Y neuroblastoma cells, α-syn (10 and 30 μM) significantly decreased NAD(P)H levels after 48 h incubation, indicative of either cell death or disruption to energy metabolism of the cells. However, α-syn (0.1 - 30 μM) did not induce cell death, as determined by the LDH assay, even when the cells were exposed for 48 h. Therefore our studies show that under normal, physiological conditions, α-syn is not inherently toxic, but does result in a decrease of total cellular energy levels. The mitochondrial toxin, 1-methyl-4-phenylpyridinium ion (MPP+), induced cell death in SHSY-5Y cells that was both concentration- and time-dependent. α-Syn (30 μM) significantly exacerbated MPP+-induced cell death in this model of PD. This suggests that while α-syn is normally non-toxic, under PD-like conditions it can exacerbate the cell death process. We identified that α-syn (30 μM) significantly increased cytosolic Ca2+ levels in a time-dependent manner as well as increasing the levels of the apoptotic mediator, cytochrome c (cyt c). The release of cyt c from the mitochondria into the cytosol is indicative of mitochondrial dysfunction and pore formation within mitochondrial membranes. However, α-syn-induced increase in cytosolic Ca2+ was not blocked by the mitochondrial pore inhibitor, cyclosporine A. This suggests that α-syn effects were not mediated through the mitochondrial pore usually associated with dysfunction and cyt c release. α-Syn therefore releases cyt c and Ca2+ by a separate mechanism, such as the formation of α-syn protofibril pores. This was further compounded by data that showed that α-syn (30 μM) significantly decreased mitochondrial membrane potential after 48 h incubation. The loss of the mitochondrial membrane potential coincided with a decrease in NAD(P)H. These data would therefore suggest that physiologically α-syn induces a low, non-toxic effect on the mitochondrial membrane. Under pathological conditions similar to PD however, this mitochondrial stress mediated by α-syn acts to exacerbate cell death.
Supervisor: Jones, Paul. ; Sharkey, John. Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available
Keywords: a-synuclein ; Parkinson's disease