title: Alpha-synuclein phosphorylation enhances eosinophilic cytoplasmic inclusion formation in SH-SY5Y cells.
abstract: Parkinson's disease (PD) is a neurodegenerative disorder characterized by selective loss of dopaminergic neurons and the presence of Lewy bodies. Previous reports have shown that alpha-synuclein deposited in brain tissue from individuals with synucleinopathy is extensively phosphorylated at Ser-129. Here, we investigate the role of phosphorylation of alpha-synuclein in the formation of inclusions involving synphilin-1 and parkin using site-directed mutagenesis to change Ser-129 of alpha-synuclein to alanine (S129A) to abolish phosphorylation at this site. Coexpression of wild-type alpha-synuclein and synphilin-1 in human neuroblastoma SH-SY5Y cells yielded cytoplasmic eosinophilic inclusions with some features resembling Lewy bodies, whereas coexpression of S129A alpha-synuclein and synphlin-1 formed few or no inclusions. Moreover, coexpression of parkin with alpha-synuclein and synphilin-1 formed more ubiquitinated inclusions, but these inclusions decreased with expression of S129A alpha-synuclein instead of wild-type alpha-synuclein. Coimmunoprecipitation assays revealed a decreased interaction of S129A alpha-synuclein with synphilin-1 compared with wild-type alpha-synuclein. Expression of S129A alpha-synuclein instead of wild-type alpha-synuclein also decreased the association of synphilin-1 and parkin and subsequently reduced the parkin-mediated ubiquitination of synphilin-1 and the formation of ubiquitinated inclusions. Treatment of SH-SY5Y cells with H(2)O(2) increased alpha-synuclein phosphorylation and enhanced the formation of inclusions formed by coexpression of alpha-synuclein, synphilin-1, and parkin, whereas treatment with the casein kinase 2 inhibitor 5,6-dichloro-1-beta-d-ribofuranosylbenzimidazole had the opposite affect. These results indicate that phosphorylation of alpha-synuclein at S129 may be important for the formation of inclusions in PD and related alpha synucleinopathies.
authors.affiliation: Department of Psychiatry, The Johns Hopkins University School of Medicine, Baltimore, Maryland 21205, USA.
authors.forename: Wanli W
authors.initials: WW
authors.lastname: Smith
authors.forename: Russell L
authors.initials: RL
authors.lastname: Margolis
authors.forename: Xiaojie
authors.initials: X
authors.lastname: Li
authors.forename: Juan C
authors.initials: JC
authors.lastname: Troncoso
authors.forename: Michael K
authors.initials: MK
authors.lastname: Lee
authors.forename: Valina L
authors.initials: VL
authors.lastname: Dawson
authors.forename: Ted M
authors.initials: TM
authors.lastname: Dawson
authors.forename: Takashi
authors.initials: T
authors.lastname: Iwatsubo
authors.forename: Christopher A
authors.initials: CA
authors.lastname: Ross
mesh_terms: D002352:Carrier Proteins / Q000378:metabolism; D047390:Casein Kinase II / Q000037:antagonists & inhibitors; D045744:Cell Line, Tumor; D003593:Cytoplasm / Q000378:metabolism / Q000648:ultrastructure*; D004004:Dichlororibofuranosylbenzimidazole / Q000494:pharmacology; D004801:Eosine Yellowish-(YS); D006801:Humans; D006861:Hydrogen Peroxide / Q000494:pharmacology; D016631:Lewy Bodies / Q000648:ultrastructure*; D009154:Mutation; D009419:Nerve Tissue Proteins / Q000378:metabolism; D018384:Oxidative Stress; D010766:Phosphorylation; D013194:Staining and Labeling; D025801:Ubiquitin / Q000378:metabolism; D044767:Ubiquitin-Protein Ligases / Q000378:metabolism; D051844:alpha-Synuclein / Q000235:genetics / Q000378:metabolism*
chemical_list: D002352:Carrier Proteins; D009419:Nerve Tissue Proteins; C118958:SNCAIP protein, human; D025801:Ubiquitin; D051844:alpha-Synuclein; D004004:Dichlororibofuranosylbenzimidazole; D006861:Hydrogen Peroxide; D044767:Ubiquitin-Protein Ligases; C111567:parkin protein; D047390:Casein Kinase II; D004801:Eosine Yellowish-(YS)