Identification of a specific α-synuclein peptide (α-Syn 29-40) capable of eliciting microglial superoxide production to damage dopaminergic neurons

Shijun Wang,Hui Nie,Mingri Guo,Jau-Shyong Hong,Tessandra Stewart,Belinda Wilson,Chun-Hsien Chu,Li Yang,Wei Zhang,Jing Zhang,Lulu Jiang

doi:10.1186/s12974-016-0606-7

Abstract

BackgroundMisfolded α-synuclein (α-Syn) aggregates participate in the pathogenesis of synucleinopathies, such as Parkinson’s disease. Whereas much is known about how the various domains within full-length α-Syn (FL-α-Syn) contribute to the formation of α-Syn aggregates and therefore to their neurotoxicity, little is known about whether the individual peptides that can be generated from α-syn, possibly as intermediate metabolites during degradation of misfolded α-Syn aggregates, are neurotoxic themselves.MethodsA series of synthesized α-Syn peptides, corresponding to the locus in FL-α-Syn containing alanine 30, substitution of which with a proline causes a familial form of Parkinson’s disease, were examined for their capacity of inducing release of microglial superoxide. The neurotoxicity of these peptides was measured according to their influence on the ability of neuroglial cultures deficient in gp91phox, the catalytic unit of NADPH oxidase (Nox2), or wild-type cultures to take up 3H-labeled dopamine and on the number of tyrosine hydroxylase-staining-positive neurons. Western blots and confocal images were utilized to analyze membrane translocation of p47phox and p67phox, phosphorylation of p47phox and Erk1/2 kinase, and binding of α-Syn peptides to gp91phox. Activation of brain microglia in mice injected with α-Syn peptides was demonstrated by immunostaining for major histocompatibility complex (MHC)-II along with qPCR for Iba-1 and MHC-II.ResultsWe report α-Syn (29-40) as a specific peptide capable of activating microglial Nox2 to produce superoxide and cause dopaminergic neuronal damage. Administered to mice, this peptide also activated brain microglia to increase expression of MHC-II and Iba-1 and stimulated oxidation reaction. Exploring the underlying mechanisms showed that α-Syn (29-40) peptide triggered Nox2 to generate extracellular superoxide and its metabolite H2O2 by binding to the catalytic unit gp91phox of Nox2; diffusing into cytosol, H2O2 activated Erk1/2 kinase to phosphorylate p47phox and p67phox and further activated Nox2, establishing a positive feedback loop to amplify the Nox2-mediated response.ConclusionsCollectively, our study suggests novel information regarding how α-Syn causes neuronal injury, possibly including mechanisms involving abnormal metabolites of α-Syn aggregates.

Highlights

Misfolded α-synuclein (α-Syn) aggregates participate in the pathogenesis of synucleinopathies, such as Parkinson’s disease
To determine the effects of alanine 30 to proline-mutated α-synuclein (A30P) mutation on the ability of α-Syn to induce dopaminergic neuronal damage via microglia activation, we first compared the effects of WT or A30P FL-α-Syn on DA uptake in neuron-glia cultures, which consist of approximately 40 % neurons, 50 % astrocytes, and 10 % microglia [18]
As demonstrated previously [16], FL-α-Syn aggregates featuring the A30P substitution are more toxic to dopaminergic neurons than WT, leading to an even lower ability of neurons to take up dopamine (DA), and resulting in fewer surviving THpositive cells, which represent dopaminergic neurons in the cultures (Fig. 1a, b)

Summary

Introduction

Misfolded α-synuclein (α-Syn) aggregates participate in the pathogenesis of synucleinopathies, such as Parkinson’s disease. Aggregation of α-synuclein (α-Syn) in the central nervous system (CNS) is a fundamentally important pathological process involved in the development and progression of synucleinopathies including Parkinson’s disease (PD) [1]. Our previous experiments supported the notion that the A30P or A53T forms of the full-length protein (FL-α-Syn) activated microglia more potently than their corresponding wild type [16] and led to greater dopaminergic (DA) neuronal damage. This finding suggests that specific sequences of amino acids in α-Syn could be essential to enhanced neurotoxicity by activating microglia more effectively

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