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

Abstract

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.

Ubiquitin-dependent proteolysis in yeast cells expressing neurotoxic proteins

“…This depends on α-synuclein phosphorylation (Basso et al, 2013; Tenreiro et al, 2014), and can be interrupted by genetic manipulation ( e.g ., α-synuclein-A30P) (Outeiro and Lindquist, 2003; Dixon et al, 2005; Sharma et al, 2006). Upon high expression levels α-synuclein forms cellular aggregates in a nucleation-dependent manner, which starts at the plasma membrane and eventually leads to cytoplasmic inclusions (Outeiro and Lindquist, 2003).…”

A Longitudinal Study of Total and Phosphorylated α-Synuclein with Other Biomarkers in Cerebrospinal Fluid of Alzheimer’s Disease and Mild Cognitive Impairment

“…We hypothesized that this may reflect the subpopulation of AD subjects with concomitant LB pathology, and found that the mismatch (α-syn–p-tau 181 -Mis) between these two markers both correlated with worse cognitive outcomes, and improved the association of the t-tau/Aβ 42 ratio with these measures when included in the model [17]. Importantly, most α-syn present in LBs is highly phosphorylated, especially at serine 129 (pS129) [15, 20–22], which is thought to alter aggregation and toxicity of α-syn[23, 24]. Notably, the CSF pS129 was significantly higher in PD patients than in healthy controls, and correlated with PD severity[16, 25], suggesting it may serve as a marker of PD pathology independently of total α-syn, which tends to be lower in the CSF of PD patients[18, 26–29].…”

Targeting AMPK Signaling as a Neuroprotective Strategy in Parkinson’s Disease

“…There is some evidence that pS129 reduces αSyn toxicity and promotes its macroautophagic clearance [195]. However, other studies have observed that pS129 increases the toxicity of αSyn and promotes aggregation [171,193].…”