Phosphorylation of Ser-129 is the dominant pathological modification of α-synuclein in familial and sporadic Lewy body disease

A comprehensive, unbiased inventory of synuclein forms present in Lewy bodies from patients with dementia with Lewy bodies was carried out using two- dimensional immunoblot analysis, novel sandwich enzyme- linked immunosorbent assays with modification- specific synuclein antibodies, and mass spectroscopy. The predominant modification of alpha-synuclein in Lewy bodies is a single phosphorylation at Ser-129. In addition, there is a set of characteristic modifications that are present to a lesser extent, including ubiquitination at Lys residues 12, 21, and 23 and specific truncations at Asp- 115, Asp- 119, Asn-122, Tyr- 133, and Asp- 135. No other modifications are detectable by tandem mass spectrometry mapping, except for a ubiquitous N- terminal acetylation. Small amounts of Ser- 129 phosphorylated and Asp- 119- truncated alpha- synuclein are present in the soluble fraction of both normal and disease brains, suggesting that these Lewy body-associated forms are produced during normal metabolism of alpha-synuclein. In contrast, ubiquitination is only detected in Lewy bodies and is primarily present on phosphorylated synuclein; it therefore likely occurs after phosphorylated synuclein has deposited into Lewy bodies. This invariant pattern of specific phosphorylation, truncation, and ubiquitination is also present in the detergent- insoluble fraction of brain from patients with familial Parkinson's disease ( synuclein A53T mutation) as well as multiple system atrophy, suggesting a common pathogenic pathway for both genetic and sporadic Lewy body diseases. These observations are most consistent with a model in which preferential accumulation of normally produced Ser- 129 phosphorylated alpha- synuclein is the key event responsible for the formation of Lewy bodies in various Lewy body diseases.