Phosphorylation of Ser-129 Is the Dominant Pathological Modification of α-Synuclein in Familial and Sporadic Lewy Body Disease

Abstract

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 ␣-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 ␣-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 ␣-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 ␣-synuclein is the key event responsible for the formation of Lewy bodies in various Lewy body diseases.A number of neurodegenerative diseases, including Parkinson disease (PD), 4 dementia with Lewy bodies (DLB), and multiple system atrophy (MSA) are defined histologically by the presence of Lewy bodies (LBs), intracellular protein aggregates that have a range of morphologies, from cytoplasmic spheres to neuritic threads also referred to as Lewy neurites (LNs). A number of proteins have been identified in LBs largely by immunohistochemical staining of brain, although the two most common are ubiquitin and ␣-synuclein (1-4). The invariable presence of ␣-synuclein in LBs suggests that it plays a key role in the etiology of such diseases ("synucleinopathies"). Point mutations in the synuclein gene as well as multiplication of the gene in familial cases of PD lead to autosomally dominant familial forms of PD (5-9). As in sporadic PD, LBs are also found in the brains of individuals with familial PD suggesting that clues about the pathogenic role of synuclein lie within the LB.Because ␣-synuclein is a relatively abundant neuronal protein, and LBs are found in diseased brain, we hypothesized that the formation of the abnormal LB structures results from specific modifications to this protein. We therefore analyzed the specific forms of ␣-synuclein that are found in LBs is...

α-Synuclein interacts with the switch region of Rab8a in a Ser129 phosphorylation-dependent manner

“…Phosphorylation of αS at S129 is the most abundant posttranslational modification in Lewy bodies (Anderson et al, 2006;Fig. 8.…”

“…4A) is the most abundant post-translational modification of αS in PD (Anderson et al, 2006;Fujiwara et al, 2002). Titration of pS129-αS to Rab8a(GDP) revealed that, at the same molar ratio, Rab8a caused stronger NMR signal attenuation in pS129-αS than in wild-type αS (Figs.…”

N‐terminal acetylation is critical for forming α‐helical oligomer of α‐synuclein

“…3 They found the protein to have a molecular mass consistent with the presence of an additional acetyl group, 3 and it has been known for several years that AS purified from both normal and pathological brain tissue is N-terminally acetylated. 5 Wang et al purified from E. coli an AS construct that included an additional 10 residue N-terminal fragment 4 which may mimic the physical consequences of N-terminal acetylation. Both manuscripts suggested that the mild, non-denaturing conditions used for purification may also preserve the native tetramer structure.…”

The phosphorylation state of Ser-129 in human α-synuclein determines neurodegeneration in a rat model of Parkinson disease

“…Phosphorylation at Ser-129 is a specific marker of all ␣-synucleinopathy lesions including PD (16). Alteration of Ser-129 to the negatively charged aspartate to mimicphosphorylationandSer-129phosphorylationbyGprotein-coupled receptor kinase 2 significantly enhanced ␣-syn toxicity in the Drosophila model of Parkinson disease (17).…”