title: 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.
authors.affiliation: Instituto de Medicina Molecular, Faculdade de Medicina da Universidade de Lisboa, Lisboa, Portugal.
authors.forename: Sandra
authors.initials: S
authors.lastname: Tenreiro
authors.affiliation: Instituto de Medicina Molecular, Faculdade de Medicina da Universidade de Lisboa, Lisboa, Portugal.
authors.forename: Madalena M
authors.initials: MM
authors.lastname: Reimão-Pinto
authors.affiliation: Instituto de Medicina Molecular, Faculdade de Medicina da Universidade de Lisboa, Lisboa, Portugal.
authors.forename: Pedro
authors.initials: P
authors.lastname: Antas
authors.affiliation: Instituto de Medicina Molecular, Faculdade de Medicina da Universidade de Lisboa, Lisboa, Portugal.
authors.forename: José
authors.initials: J
authors.lastname: Rino
authors.affiliation: Department of Genetics and Cell Physiology, Institute of Experimental Biology, University of Wroclaw, Wroclaw, Poland.
authors.forename: Donata
authors.initials: D
authors.lastname: Wawrzycka
authors.affiliation: Instituto de Medicina Molecular, Faculdade de Medicina da Universidade de Lisboa, Lisboa, Portugal; Instituto de Tecnologia Química e Biológica, Universidade Nova de Lisboa, Oeiras, Portugal.
authors.forename: Diana
authors.initials: D
authors.lastname: Macedo
authors.affiliation: Instituto de Medicina Molecular, Faculdade de Medicina da Universidade de Lisboa, Lisboa, Portugal.
authors.forename: Rita
authors.initials: R
authors.lastname: Rosado-Ramos
authors.affiliation: Department of Cell and Developmental Biology, Alexander Silberman Institute of Life Sciences, Hebrew University of Jerusalem, Jerusalem, Israel.
authors.forename: Triana
authors.initials: T
authors.lastname: Amen
authors.affiliation: Department of Cell and Developmental Biology, Alexander Silberman Institute of Life Sciences, Hebrew University of Jerusalem, Jerusalem, Israel.
authors.forename: Meytal
authors.initials: M
authors.lastname: Waiss
authors.affiliation: Instituto de Medicina Molecular, Faculdade de Medicina da Universidade de Lisboa, Lisboa, Portugal.
authors.forename: Filipa
authors.initials: F
authors.lastname: Magalhães
authors.affiliation: Instituto de Tecnologia Química e Biológica, Universidade Nova de Lisboa, Oeiras, Portugal; Instituto de Biologia Experimental e Tecnológica, Oeiras, Portugal.
authors.forename: Andreia
authors.initials: A
authors.lastname: Gomes
authors.affiliation: Instituto de Tecnologia Química e Biológica, Universidade Nova de Lisboa, Oeiras, Portugal; Instituto de Biologia Experimental e Tecnológica, Oeiras, Portugal.
authors.forename: Cláudia N
authors.initials: CN
authors.lastname: Santos
authors.affiliation: Department of Cell and Developmental Biology, Alexander Silberman Institute of Life Sciences, Hebrew University of Jerusalem, Jerusalem, Israel.
authors.forename: Daniel
authors.initials: D
authors.lastname: Kaganovich
authors.affiliation: Instituto de Medicina Molecular, Faculdade de Medicina da Universidade de Lisboa, Lisboa, Portugal; Instituto de Fisiologia, Faculdade de Medicina da Universidade de Lisboa, Lisboa, Portugal; Department of NeuroDegeneration and Restorative Research, Center for Nanoscale Microscopy and Molecular Physiology of the Brain, University Medical Center Göttingen, Göttingen, Germany.
authors.forename: Tiago Fleming
authors.initials: TF
authors.lastname: Outeiro
mesh_terms: D001343:Autophagy; D006801:Humans; D008954:Models, Biological; D010300:Parkinson Disease / Q000378:metabolism*; D010766:Phosphorylation; D012441:Saccharomyces cerevisiae / Q000378:metabolism*; D051844:alpha-Synuclein / Q000378:metabolism*
chemical_list: D051844:alpha-Synuclein