Aggregation of alpha-synuclein (ASYN) in Lewy bodies and Lewy neurites is the typical pathological hallmark of Parkinson's disease (PD) and other synucleinopathies. Furthermore, mutations in the gene encoding for ASYN are associated with familial and sporadic forms of PD, suggesting this protein plays a central role in the disease. However, the precise contribution of ASYN to neuronal dysfunction and death is unclear. There is intense debate about the nature of the toxic species of ASYN and little is known about the molecular determinants of oligomerization and aggregation of ASYN in the cell. In order to clarify the effects of different mutations on the propensity of ASYN to oligomerize and aggregate, we assembled a panel of 19 ASYN variants and compared their behaviour. We found that familial mutants linked to PD (A30P, E46K, H50Q, G51D and A53T) exhibited identical propensities to oligomerize in living cells, but had distinct abilities to form inclusions. While the A30P mutant reduced the percentage of cells with inclusions, the E46K mutant had the opposite effect. Interestingly, artificial proline mutants designed to interfere with the helical structure of the N-terminal domain, showed increased propensity to form oligomeric species rather than inclusions. Moreover, lysine substitution mutants increased oligomerization and altered the pattern of aggregation. Altogether, our data shed light into the molecular effects of ASYN mutations in a cellular context, and established a common ground for the study of genetic and pharmacological modulators of the aggregation process, opening new perspectives for therapeutic intervention in PD and other synucleinopathies. The accumulation of aggregated proteins in the brain is common across several neurodegenerative disorders. In Parkinson's disease (PD), the protein alpha-synuclein (ASYN) is the major component of aggregates known as Lewy bodies. It is currently unclear whether protein aggregates are protective or detrimental for neuronal function and survival. The present hypothesis is that smaller aggregated species, known as oligomers, might constitute the toxic forms of ASYN. Several mutations in ASYN cause familial forms of PD. In the laboratory, artificial mutations have been designed to enable the study of the aggregation process. However, different studies relied on the use of different model systems, compromising the interpretation of the effects of the mutations. Here, we addressed this by (i) assembling a panel of 19 ASYN variants and (ii) by performing a systematic comparison of the effects of the mutations in mammalian cell models. Interestingly, our study enabled us to correlate oligomerization and aggregation of ASYN in cells. Altogether, our data shed light into the molecular determinants of ASYN aggregation, opening novel avenues for the identification of modulators of ASYN aggregation, which conceal great hopes towards the development of strategies for therapeutic intervention in PD and other synucleinopathies.

Parkinson disease is the second most common neurodegenerative disease. The molecular hallmark is the accumulation of proteinaceous inclusions termed Lewy bodies containing misfolded and aggregated α-synuclein. The molecular mechanism of clearance of α-synuclein aggregates was addressed using the bakers' yeast Saccharomyces cerevisiae as the model. Overexpression of wild type α-synuclein or the genetic variant A53T integrated into one genomic locus resulted in a gene copy-dependent manner in cytoplasmic proteinaceous inclusions reminiscent of the pathogenesis of the disease. In contrast, overexpression of the genetic variant A30P resulted only in transient aggregation, whereas the designer mutant A30P/A36P/A76P neither caused aggregation nor impaired yeast growth. The α-synuclein accumulation can be cleared after promoter shut-off by a combination of autophagy and vacuolar protein degradation. Whereas the proteasomal inhibitor MG-132 did not significantly inhibit aggregate clearance, treatment with phenylmethylsulfonyl fluoride, an inhibitor of vacuolar proteases, resulted in significant reduction in clearance. Consistently, a cim3-1 yeast mutant restricted in the 19 S proteasome regulatory subunit was unaffected in clearance, whereas an Δatg1 yeast mutant deficient in autophagy showed a delayed aggregate clearance response. A cim3-1Δatg1 double mutant was still able to clear aggregates, suggesting additional cellular mechanisms for α-synuclein clearance. Our data provide insight into the mechanisms yeast cells use for clearing different species of α-synuclein and demonstrate a higher contribution of the autophagy/vacuole than the proteasome system. This contributes to the understanding of how cells can cope with toxic and/or aggregated proteins and may ultimately enable the development of novel strategies for therapeutic intervention.

Parkinson disease is associated with the progressive loss of dopaminergic neurons from the substantia nigra. The pathological hallmark of the disease is the accumulation of intracytoplasmic inclusions known as Lewy bodies that consist mainly of post-translationally modified forms of α-synuclein. Whereas phosphorylation is one of the major modifications of α-synuclein in Lewy bodies, sumoylation has recently been described. The interplay between α-synuclein phosphorylation and sumoylation is poorly understood. Here, we examined the interplay between these modifications as well as their impact on cell growth and inclusion formation in yeast. We found that α-synuclein is sumoylated in vivo at the same sites in yeast as in human cells. Impaired sumoylation resulted in reduced yeast growth combined with an increased number of cells with inclusions, suggesting that this modification plays a protective role. In addition, inhibition of sumoylation prevented autophagy-mediated aggregate clearance. A defect in α-synuclein sumoylation could be suppressed by serine 129 phosphorylation by the human G protein-coupled receptor kinase 5 (GRK5) in yeast. Phosphorylation reduced foci formation, alleviated yeast growth inhibition, and partially rescued autophagic α-synuclein degradation along with the promotion of proteasomal degradation, resulting in aggregate clearance in the absence of a small ubiquitin-like modifier. These findings suggest a complex interplay between sumoylation and phosphorylation in α-synuclein aggregate clearance, which may open new horizons for the development of therapeutic strategies for Parkinson disease.

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. Parkinson’s disease is characterized by loss of dopaminergic neurons in midbrain and the presence of αSyn protein inclusions. Human αSyn mimics the disease pathology in yeast resulting in cytotoxicity and aggregate formation. αSyn is abundantly phosphorylated at serine S129 and possesses four tyrosines (Y39, Y125, Y133, and Y136) that can be posttranslationally modified by nitration or phosphorylation. The consequence of each of these possible modifications is still unclear. Nitration as consequence of oxidative stress is a hallmark for neurodegenerative diseases. Here, we addressed the molecular mechanism, how tyrosine posttranslational modifications affect αSyn cytotoxicity. Tyrosine nitration can contribute to αSyn toxicity or can be part of a cellular salvage pathway when di-tyrosine-crosslinked dimers are formed. The Y133 residue, which can be either phosphorylated or nitrated, determines whether S129 is protectively phosphorylated and αSyn inclusions are cleared. This interplay with S129 phosphorylation demonstrates a dual role for C-terminal tyrosine residues. Yeast flavohemoglobin Yhb1 and its human counterpart neuroglobin NGB protect cells against cytotoxicity and aggregate formation. These novel insights into the molecular pathways responsible for αSyn cytotoxicity indicate NGB as a potential target for therapeutic intervention in PD.

The budding yeast Saccharomyces cerevisiae represents an established model system to study the molecular mechanisms associated to neurodegenerative disorders. A key-feature of Parkinson’s disease is the formation of Lewy bodies, which are cytoplasmic protein inclusions. Misfolded α-synuclein is one of their main constituents. Expression of α-synuclein protein in yeast leads to protein aggregation and cellular toxicity, which is reminiscent to Lewy body containing human cells. The molecular mechanism involved in clearance of α-synuclein aggregates is a central question for elucidating the α-synuclein-related toxicity. Cellular clearance mechanisms include ubiquitin mediated 26S proteasome function as well as lysosome/vacuole associated degradative pathways as autophagy. Various modifications change α-synuclein posttranslationally and alter its inclusion formation, cytotoxicity and the distribution to different clearance pathways. Several of these modification sites are conserved from yeast to human. In this review, we summarize recent findings on the effect of phosphorylation and sumoylation of α-synuclein to the enhanced channeling to either the autophagy or the proteasome degradation pathway in yeast model of Parkinson’s disease.