Pathophysiological effects of alpha-synuclein on SNARE complex proteins in models of alpha-synucleinopathies

Abstract: Accumulation and spread of alpha-synuclein (α-syn) aggregates are central to the disease pathogenesis of Parkinson’s disease and dementia with Lewy bodies, collectively known as α-synucleinopathies. Native α-syn is a monomeric presynaptic protein that can act as a molecular chaperone for the soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) complex assembly. The aim of this thesis was to investigate the pathophysiological effect of different α-syn species on SNARE protein distribution and to study the involvement of extracellular vesicles (EVs) in the propagation of α-syn pathology.In paper I, the co-localization between α-syn and the SNARE proteins VAMP-2, SNAP-25 and syntaxin-1, was analyzed in primary cortical neurons from transgenic (tg) human A30P α-syn and wild type (wt) mice using proximity ligation assay (PLA). The results demonstrated that SNARE proteins co-localized with total α-syn mainly in neuronal processes, and with A30P α-syn predominantly in the cell soma. In paper II, we investigated how altered molecular properties of α-syn could affect its cellular processing. Different α-syn constructs were expressed in SH-SY5Y cells and the culture medium was analyzed for free-floating α-syn, as well as for α-syn within the EV fraction. Modifications in the N-terminal increased the EV secretion and enhanced the cell-to-cell transfer of α-syn. In paper III, the synaptic α-syn species of the A30P tg mouse brain were biochemically characterized and their effect on SNARE protein distribution was analyzed with western blot and PLA. We found that synaptosomal α-syn aggregates were mainly composed of non-phosphorylated human A30P α-syn. A decrease of intact SNARE complexes was observed in the tg A30P synaptosomes and in the prefrontal cortex, even though the total levels of SNARE proteins were unchanged in A30P compared to wt mice.In paper IV, we studied the effect of α-syn monomers and α-syn preformed fibrils (PFFs) on SNARE protein distribution in wt primary neurons, using PLA. Both short- and long-term exposure to α-syn monomers or PFFs altered the co-localization of SNARE proteins. Promoting the long-term uptake of α-syn by using a protein delivery reagent, further increased SNARE protein redistribution. In contrast, a PFF-induced SNARE protein redistribution was not observed when lysosomal degradation was inhibited. Interestingly, addition of EVs from monomer- and PFF-treated astrocytes also affected SNARE protein distribution in recipient neurons. Taken together, the results from this thesis indicate that synaptic α-syn aggregates and EV-associated α-syn could be promising therapeutic targets in the α-synucleinopathies.