gms | German Medical Science

Introduction: The 140 amino acid protein alpha-Synuclein (aSyn) plays a central role in the pathogenesis of PD. Point mutations or duplications and triplications of the synuclein gene SNCA lead to familiar forms of Parkinsonism. Five point mutations have been described to date: A30P, E46K, H50Q, G51D, A53E, and A53T. Differences in the clinical presentation of patients with certain mutations suggest that there might be different molecular mechanisms acting in the pathogenesis of PD.

Objectives: The known point mutations of aSyn display a suitable model to characterize the impact of single amino acid exchanges on the aggregation and lipid binding properties in a comparative manner. In this study, all aSyn mutants known so far were purified as recombinant protein and analyzed with different methods for a better understanding of the molecular mechanisms playing a role in the pathogenesis of PD.

Material and methods: Real time quaking induced conversion (RT-QuIC) was applied to monitor fibril growth via ThT-fluorescence. Using single particle fluorescence techniques, metal ion induced aggregation and interaction of fluorescently labeled recombinant aSyn with lipid membranes was analyzed. Lipid interaction propensities were further analyzed by CD spectroscopy and ITC.

Results: Compared to wt aSyn, the mutants revealed different aggregation propensities with A30P showing a high tendency to form metal induced oligomers, whereas A53T displayed only low amounts of oligomer formation. Inversely, A53T is able to rapidly build up amyloid fibrils, reaching a plateau within few hours, while A30P fibril formation displayed a very long lag phase and never reached a plateau within 200 hours of incubation. Addition of trivalent metal ions under these conditions led to an inhibition of fibril formation with prolonged lag phases. Furthermore, different binding affinities to bind to lipid membranes were found for different mutants.

Conclusions: The different behaviour of different mutants under the same assay conditions suggests that there might be differing mechanisms on the molecular level finally all leading to Parkinson syndromes.

These insights are of relevance for a better understanding of the pathogenesis of PD and provide a promising link for the development of prevention or new therapy strategies.