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

Filsy Samuel,William P Flavin,Sobia Iqbal,Consiglia Pacelli,Sri Dushyaanthan Sri Renganathan,Louis-Eric Trudeau,Edward M Campbell,Paul E Fraser,Anurag Tandon

doi:10.1074/jbc.m115.705095

Abstract

Although trace levels of phosphorylated α-synuclein (α-syn) are detectable in normal brains, nearly all α-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 α-syn structure, function, and intracellular accumulation are poorly understood. Here, co-expression of α-syn and polo-like kinase 2 (PLK2), a kinase that targets Ser-129, was used to generate phosphorylated α-syn for biophysical and biological characterization. Misfolding and fibril formation of phosphorylated α-syn isoforms were detected earlier, although the fibrils remained phosphatase- and protease-sensitive. Membrane binding of α-syn monomers was differentially affected by phosphorylation depending on the Parkinson disease-linked mutation. WT α-syn binding to presynaptic membranes was not affected by phosphorylation, whereas A30P α-syn binding was greatly increased, and A53T α-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 α-syn membrane binding. Finally, the disruption of internalized vesicle membranes was enhanced by the phosphorylated α-syn isoforms, a potential means for misfolded extracellular or lumenal α-syn to access cytosolic α-syn. Our results suggest that the threshold for vesicle permeabilization is evident even at low levels of α-syn internalization and are relevant to therapeutic strategies to reduce intercellular propagation of α-syn misfolding.

Highlights

␣-syn can be modified by multiple covalent post-translational modifications and truncations that may affect both folding and interneuronal transfer by limiting ␣-syn flexibility, modifying membrane association, complex formation, and degradation [9]
Phosphorylated isoforms were generated by co-expressing wild-type (WT) and Parkinson disease (PD) mutant (A30P and A53T) ␣-syn isoforms in Escherichia coli either without or with polo-like kinase 2 (PLK2), which uniquely phosphorylates serine 129 (Ser-129) on ␣-syn [13,14,15,16]
Despite the change in fibril formation, we observed that the relative effects of Ser(P)-129 on ␣-syn membrane binding were similar to its effects on ␣-syn internalization, suggesting that membrane binding is a rate-limiting step in ␣-syn internalization

Summary

Introduction

␣-syn can be modified by multiple covalent post-translational modifications and truncations that may affect both folding and interneuronal transfer by limiting ␣-syn flexibility, modifying membrane association, complex formation, and degradation [9]. Phosphorylation increased the self-assembly of the purified ␣-syn but had differential effects on membrane binding of monomeric ␣-syn and the internalization of aggregated ␣-syn isoforms. To address the role of Ser(P)-129 in ␣-syn fibril formation, the three ␣-syn isoforms and their phosphorylated versions were incubated in neutral pH buffer at 37 °C with constant shaking, and aliquots were periodically removed to assess circular dichroism (CD) spectra and for analysis by electron microscopy (EM).

Results

Conclusion