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.
Mesh Terms:
Carrier Proteins, Casein Kinase II, Cell Line, Tumor, Cytoplasm, Dichlororibofuranosylbenzimidazole, Eosine Yellowish-(YS), Humans, Hydrogen Peroxide, Lewy Bodies, Mutation, Nerve Tissue Proteins, Oxidative Stress, Phosphorylation, Staining and Labeling, Ubiquitin, Ubiquitin-Protein Ligases, alpha-Synuclein
Carrier Proteins, Casein Kinase II, Cell Line, Tumor, Cytoplasm, Dichlororibofuranosylbenzimidazole, Eosine Yellowish-(YS), Humans, Hydrogen Peroxide, Lewy Bodies, Mutation, Nerve Tissue Proteins, Oxidative Stress, Phosphorylation, Staining and Labeling, Ubiquitin, Ubiquitin-Protein Ligases, alpha-Synuclein
J. Neurosci.
Date: Jun. 08, 2005
PubMed ID: 15944382
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