Final Activity Report Summary - STAGGY (Development of a yeast model to study aggregation of human tau and a-synuclein) Aggregation of alpha-SYN (SYN) in Lewy Bodies is considered a key pathogenic issue of the Parkinson's disease. We used a yeast model to study features of SYN pathophysiology. We screened for yeast deletion mutants with mislocalisation and enhanced inclusion formation of SYN. This study led us to identify of several yeast orthologs and homologs of human proteins involved in vesicular transport and to demonstrate the involvement of SYN phosphorylation, N-terminal acetylation and lipid raft interaction for its traffic and toxicity in yeast. We identified the casein kinase CK1 and CK2 as responsible for SYN phosphorylation as well as transacetylases that modulate the SYN-membrane interaction. SYN was found to associate with lipid rafts, a phenomenon dependent on the ergosterol content. We also found that deletion of genes responsible for proteasomal activity increases SYN toxicity. The screening of a human hippocampal cDNA library let us to identify several proteins that when co-expressed alter the SYN-induced toxicity and its localization in the cell. In addition, we demonstrate that of the different SYN proteins tested only the A30P mutant is subject to ubiquitination. Although A30P SYN is normally cytoplasmic protein we found that it is targeted to the plasma membrane in a time-dependent manner. A delay in A30P transport to the plasma membrane is due to it weaker membrane and lipid rafts binding. Overexpression of A30P-SYN in strains with deletion of genes responsible for vesicular transport shows inclusion formation, as in case of wild type SYN (WT-SYN). A30P-SYN is also transported via endocytosis from plasma membrane to the vacuole but it does not affect vacuolar fusion, in contrast to wild type SYN. We found that SYN-toxicity is directly proportional to the property of the protein to bind to forming vesicles. This correlates with rate of phosphorylation, which appears to mark SYN for endocytosis. We also found that both WT-SYN and the A53T mutant dramatically shortened the chronological lifespan of yeast, whereas the A30P did not. The shortened lifespan coincided with a pronounced increase of ROS and typical markers for apoptotic cell death. However, necrosis is also enhanced in those cultures. Moreover, our data indicated that the induction of apoptotic markers depended on the presence of functional mitochondria and that they cannot be relieved by deletion of genes involved in yeast apoptosis such as caspase YCA1. Humanised yeast cells also recapitulated robustly the important aspects of a tauopathy, i.e. hyper-phosphorylation, conformational change and self-aggregation. In the present study we illustrated the role of Mds1 and Pho85 on phosphorylation and aggregation of the tau mutants and demonstrate the importance of the epitopes AD2 and PG5. Finally, we showed that also in yeast mitochondrial dysfunction and oxidative stress are important determinants for tau filament formation.