Phosphorylation Modulates Clearance of Alpha-Synuclein Inclusions in a Yeast Model of Parkinson's Disease

Alpha-synuclein (aSyn) is the main component of proteinaceous inclusions known as Lewy bodies (LBs), the typical pathological hallmark of Parkinson's disease (PD) and other synucleinopathies. Although aSyn is phosphorylated at low levels under physiological conditions, it is estimated that ∼90% of aSyn in LBs is phosphorylated at S129 (pS129). Nevertheless, the significance of pS129 in the biology of aSyn and in PD pathogenesis is still controversial. Here, we harnessed the power of budding yeast in order to assess the implications of phosphorylation on aSyn cytotoxicity, aggregation and sub-cellular distribution. We found that aSyn is phosphorylated on S129 by endogenous kinases. Interestingly, phosphorylation reduced aSyn toxicity and the percentage of cells with cytosolic inclusions, in comparison to cells expressing mutant forms of aSyn (S129A or S129G) that mimic the unphosphorylated form of aSyn. Using high-resolution 4D imaging and fluorescence recovery after photobleaching (FRAP) in live cells, we compared the dynamics of WT and S129A mutant aSyn. While WT aSyn inclusions were very homogeneous, inclusions formed by S129A aSyn were larger and showed FRAP heterogeneity. Upon blockade of aSyn expression, cells were able to clear the inclusions formed by WT aSyn. However, this process was much slower for the inclusions formed by S129A aSyn. Interestingly, whereas the accumulation of WT aSyn led to a marked induction of autophagy, cells expressing the S129A mutant failed to activate this protein quality control pathway. The finding that the phosphorylation state of aSyn on S129 can alter the ability of cells to clear aSyn inclusions provides important insight into the role that this posttranslational modification may have in the pathogenesis of PD and other synucleinopathies, opening novel avenues for investigating the molecular basis of these disorders and for the development of therapeutic strategies.
Author Summary Protein aggregation is a common hallmark in neurodegenerative disorders, but is also associated with phenotypic plasticity in a variety of organisms, including yeasts. Alpha-synuclein (aSyn) forms aggregates that are typical of synucleinopathies, and is phosphorylated at S129, but the significance of phosphorylation in the biology and pathophysiology of the protein is still controversial. Exploring the power of budding yeast, we found phosphorylation reduced aSyn toxicity and inclusion formation. While inclusions formed by WT aSyn were homogeneous, those formed by S129A aSyn were larger and heterogeneous. Interestingly, clearance of aSyn inclusions was reduced in cells expressing S129A aSyn, correlating with deficient autophagy activation. The finding that phosphorylation alters the ability of cells to clear aSyn inclusions provides novel insight into the role phosphorylation may have in synucleinopathies, and suggests posttranslational modifications might constitute switches cells use to control the aggregation and clearance of key proteins, opening novel avenues for the development of therapeutic strategies for these devastating disorders.