Effect of protease cleavage on de novo aggregation of alpha-synuclein

The main signs of Parkinson's disease (PD) result from a loss of dopaminergic cells in the substantia nigra. Intracellular inclusions consisting of aggregated alpha-synuclein are the pathological hallmark of PD, but also of other neurodegenerative diseases such as dementia with Lewy bodies (DLB) and multiple systemic atrophy (MSA). Although the pathogenesis of PD is still unknown, there is a growing body of evidence suggesting that alpha-synuclein aggregates play a key role in cell death and neurodegeneration. Immunohistochemical studies indicate that specific alpha-synuclein aggregates (Lewy bodies) also contain Matrix-Metalloproteases (MMPs), and that MMPs and tissue inhibitor of metal proteases (TIMP) are up-regulated in brains of PD cases. Only recently, a characteristic pattern of alpha-synuclein fragmentation by MMPs has been described. Based on these findings we studied the influence of MMP cleavage on alpha-synuclein de novo aggregation in vitro.

We present data from a novel approach to study protein aggregation based on Fluorescence Correlation Spectroscopy (FCS) and Scanning for Intensely Fluorescent Targets (SIFT). FCS uses a confocal microscope setup to focus a laser beam to a focus volume of approximately one femtoliter. Avalanche photo diodes detect photons emitted by fluorophores moving in and out of this focus by diffusion. SIFT uses a mobile focus to scan the sample for highly fluorescent targets. Using this approach we studied the effect of MMPs on the de novo aggregation of alpha-synuclein. In these experiments, we used ethanol to trigger the aggregation process of alpha-synuclein labelled with fluorescent dyes.

We could demonstrate that high concentrations of proteases (75ng/ml) result in degradation of alpha-synuclein. Under these conditions, no aggregates were formed. When very low concentrations of proteases (750fg/ml) were used, no difference of the aggregation levels were found in comparison to control measurements. However, when we used intermediate concentrations (75pg/ml) of MMPs in our aggregation assay, we detected a strong increase in alpha-synuclein aggregation.

These data suggest that fragments produced by limited proteolysis stimulate alpha-synuclein aggregation and that MMPs may influence the pathogenesis of Parkinson's disease by influencing alpha-synuclein aggregation.