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