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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.

SUBMITTER: Tenreiro S

PROVIDER: S-EPMC4014446 | biostudies-literature | 2014 May

REPOSITORIES: biostudies-literature

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Phosphorylation modulates clearance of alpha-synuclein inclusions in a yeast model of Parkinson's disease.

Tenreiro Sandra S Reimão-Pinto Madalena M MM Antas Pedro P Rino José J Wawrzycka Donata D Macedo Diana D Rosado-Ramos Rita R Amen Triana T Waiss Meytal M Magalhães Filipa F Gomes Andreia A Santos Cláudia N CN Kaganovich Daniel D Outeiro Tiago Fleming TF

PLoS genetics 20140508 5

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 yea ...[more]

PMID: 24810576

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Project description:Parkinson´s disease (PD) is characterized by the presence of proteinaceous inclusions called Lewy bodies that are mainly composed of α-synuclein (αSyn). Elevated levels of oxidative or nitrative stresses have been implicated in αSyn related toxicity. Phosphorylation of αSyn on serine 129 (S129) modulates autophagic clearance of inclusions and is prominently found in Lewy bodies. The neighboring tyrosine residues Y125, Y133 and Y136 are phosphorylation and nitration sites. Using a yeast model of PD, we found that Y133 is required for protective S129 phosphorylation and for S129-independent proteasome clearance. αSyn can be nitrated and form stable covalent dimers originating from covalent crosslinking of two tyrosine residues. Nitrated tyrosine residues, but not di-tyrosine-crosslinked dimers, contributed to αSyn cytotoxicity and aggregation. Analysis of tyrosine residues involved in nitration and crosslinking revealed that the C-terminus, rather than the N-terminus of αSyn, is modified by nitration and di-tyrosine formation. The nitration level of wild-type αSyn was higher compared to that of A30P mutant that is non-toxic in yeast. A30P formed more dimers than wild-type αSyn, suggesting that dimer formation represents a cellular detoxification pathway in yeast. Deletion of the yeast flavohemoglobin gene YHB1 resulted in an increase of cellular nitrative stress and cytotoxicity leading to enhanced aggregation of A30P αSyn. Yhb1 protected yeast from A30P-induced mitochondrial fragmentation and peroxynitrite-induced nitrative stress. Strikingly, overexpression of neuroglobin, the human homolog of YHB1, protected against αSyn inclusion formation in mammalian cells. In total, our data suggest that C-terminal Y133 plays a major role in αSyn aggregate clearance by supporting the protective S129 phosphorylation for autophagy and by promoting proteasome clearance. C-terminal tyrosine nitration increases pathogenicity and can only be partially detoxified by αSyn di-tyrosine dimers. Our findings uncover a complex interplay between S129 phosphorylation and C-terminal tyrosine modifications of αSyn that likely participates in PD pathology.

Dataset Information

Phosphorylation modulates clearance of alpha-synuclein inclusions in a yeast model of Parkinson's disease.

Publications

Phosphorylation modulates clearance of alpha-synuclein inclusions in a yeast model of Parkinson's disease.

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