Effects of Serine 129 Phosphorylation on α-Synuclein Aggregation, Membrane Association, and Internalization

Abstract

Although trace levels of phosphorylated ␣-synuclein (␣-syn) are detectable in normal brains, nearly all ␣-syn accumulated within Lewy bodies in Parkinson disease brains is phosphorylated on serine 129 (Ser-129). The role of the phosphoserine residue and its effects on ␣-syn structure, function, and intracellular accumulation are poorly understood. Here, co-expression of ␣-syn and polo-like kinase 2 (PLK2), a kinase that targets Ser-129, was used to generate phosphorylated ␣-syn for biophysical and biological characterization. Misfolding and fibril formation of phosphorylated ␣-syn isoforms were detected earlier, although the fibrils remained phosphatase-and protease-sensitive. Membrane binding of ␣-syn monomers was differentially affected by phosphorylation depending on the Parkinson disease-linked mutation. WT ␣-syn binding to presynaptic membranes was not affected by phosphorylation, whereas A30P ␣-syn binding was greatly increased, and A53T ␣-syn was slightly lower, implicating distal effects of the carboxyl-on amino-terminal membrane binding. Endocytic vesicle-mediated internalization of pre-formed fibrils into non-neuronal cells and dopaminergic neurons matched the efficacy of ␣-syn membrane binding. Finally, the disruption of internalized vesicle membranes was enhanced by the phosphorylated ␣-syn isoforms, a potential means for misfolded extracellular or lumenal ␣-syn to access cytosolic ␣-syn. Our results suggest that the threshold for vesicle permeabilization is evident even at low levels of ␣-syn internalization and are relevant to therapeutic strategies to reduce intercellular propagation of ␣-syn misfolding.

Selective imaging of internalized proteopathic α-synuclein seeds in primary neurons reveals mechanistic insight into transmission of synucleinopathies

“…Other pathogenic species, particularly oligomers, have also been postulated to directly permeabilize membranes or form membrane-spanning pores (43)(44)(45). It has recently been demonstrated that phosphorylation of exogenous ␣-syn species leads to an increase in lysosomal rupture following endocytosis, which may be a relevant mechanism of pathological ␣-syn transmission (46). Using sonicated pffs generated from unmodified recombinant ␣-syn, we did not observe endocytosis-independent mechanisms of uptake in our studies.…”

Phosphorylation induces distinct alpha-synuclein strain formation

“…While Engelborghs et al found that on the strength of polo-like kinase 2 to target Ser129 specifically, pS129 exhibit no influence on fibrillization kinetics of α-Syn16. Recently, co-expression of α-Syn with polo-like kinase 2 in E. coli , was used to prepare pS129 α-Syn, with resulting earlier fibril formation of pS129 α-Syn compared to WT α-Syn17. The inconsistency of the outcome may partially result from the S87A mutation and the efficiency of the enzyme.…”

“…The main component of the pathological lesions is extensively phosphorylated α-Syn at serine 129 (pS129 α-Syn)14, and pS129 α-Syn may play a critical role in synucleinopathy pathogenesis. Phosphorylation at Ser129 can regulate α-Syn fibril formation14151617 and enhance α-Syn toxicity both in vitro and in vivo 18192021. As the main form of α-Syn in the pathological process, pS129 α-Syn may be associated with multiple strain formation in vivo .…”

A Longitudinal Study of Total and Phosphorylated α-Synuclein with Other Biomarkers in Cerebrospinal Fluid of Alzheimer’s Disease and Mild Cognitive Impairment

“…We hypothesized that this may reflect the subpopulation of AD subjects with concomitant LB pathology, and found that the mismatch (α-syn–p-tau 181 -Mis) between these two markers both correlated with worse cognitive outcomes, and improved the association of the t-tau/Aβ 42 ratio with these measures when included in the model [17]. Importantly, most α-syn present in LBs is highly phosphorylated, especially at serine 129 (pS129) [15, 20–22], which is thought to alter aggregation and toxicity of α-syn[23, 24]. Notably, the CSF pS129 was significantly higher in PD patients than in healthy controls, and correlated with PD severity[16, 25], suggesting it may serve as a marker of PD pathology independently of total α-syn, which tends to be lower in the CSF of PD patients[18, 26–29].…”