Alpha-synuclein phosphorylation enhances eosinophilic cytoplasmic inclusion formation in SH-SY5Y cells.
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.
Smith
Wanli W
WW
Department of Psychiatry, The Johns Hopkins University School of Medicine, Baltimore, Maryland 21205, USA.
Margolis
Russell L
RL
Li
Xiaojie
X
Troncoso
Juan C
JC
Lee
Michael K
MK
Dawson
Valina L
VL
Dawson
Ted M
TM
Iwatsubo
Takashi
T
Ross
Christopher A
CA
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*
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)