Single Particle Characterization of Iron-induced Pore-forming α-Synuclein Oligomers

Marcus Kostka,Tobias Högen,Karin M Danzer,Johannes Levin,Matthias Habeck,Andreas Wirth,Richard Wagner,Charles G Glabe,Sabine Finger,Udo Heinzelmann,Patrick Garidel,Wenzhen Duan,Christopher A Ross,Hans Kretzschmar,Armin Giese

doi:10.1074/jbc.m709634200

Abstract

Aggregation of alpha-synuclein is a key event in several neurodegenerative diseases, including Parkinson disease. Recent findings suggest that oligomers represent the principal toxic aggregate species. Using confocal single-molecule fluorescence techniques, such as scanning for intensely fluorescent targets (SIFT) and atomic force microscopy, we monitored alpha-synuclein oligomer formation at the single particle level. Organic solvents were used to trigger aggregation, which resulted in small oligomers ("intermediate I"). Under these conditions, Fe(3+) at low micromolar concentrations dramatically increased aggregation and induced formation of larger oligomers ("intermediate II"). Both oligomer species were on-pathway to amyloid fibrils and could seed amyloid formation. Notably, only Fe(3+)-induced oligomers were SDS-resistant and could form ion-permeable pores in a planar lipid bilayer, which were inhibited by the oligomer-specific A11 antibody. Moreover, baicalein and N'-benzylidene-benzohydrazide derivatives inhibited oligomer formation. Baicalein also inhibited alpha-synuclein-dependent toxicity in neuronal cells. Our results may provide a potential disease mechanism regarding the role of ferric iron and of toxic oligomer species in Parkinson diseases. Moreover, scanning for intensely fluorescent targets allows high throughput screening for aggregation inhibitors and may provide new approaches for drug development and therapy.

Highlights

All common neurodegenerative diseases are characterized by the formation and deposition of fibrillar aggregates of specific proteins, such as tau protein and A␤ in Alzheimer disease
Initial evidence for a central role of ␣-syn in the pathogenesis of Parkinson disease (PD) came from the discovery of point mutations in the ␣-syn gene in families with familial PD (3, 4)
␣-syn has been identified as the major component of Lewy bodies and in Lewy neurites, which are characteristic deposits of aggregated protein in PD, dementia with Lewy bodies, and Lewy body variant of Alzheimer disease, and as the major component of the glial cytoplasmic inclusions that characterize multiple system atrophy (5, 6)

Summary

Introduction

All common neurodegenerative diseases are characterized by the formation and deposition of fibrillar aggregates of specific proteins, such as tau protein and A␤ in Alzheimer disease, prion protein in prion diseases, and ␣-synuclein (␣-syn)4 in Parkinson disease (PD), dementia with Lewy bodies, and multiple system atrophy (1). We used ␣-syn labeled with fluorescent dyes to monitor the aggregations process with a single particle approach that has been used to detect huntingtin and prion protein oligomers (40, 42, 43).

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Conclusion