Single particle characterization of iron-induced pore-forming alpha-synuclein oligomers.
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.
Kostka
Marcus
M
CNS Research, Boehringer Ingelheim Pharma GmbH & Co. KG, CNS Research, Birkendorferstrasse 65, 88397 Biberach, Germany. Marcus.Kostka@boehringer-ingelheim.com
Högen
Tobias
T
Danzer
Karin M
KM
Levin
Johannes
J
Habeck
Matthias
M
Wirth
Andreas
A
Wagner
Richard
R
Glabe
Charles G
CG
Finger
Sabine
S
Heinzelmann
Udo
U
Garidel
Patrick
P
Duan
Wenzhen
W
Ross
Christopher A
CA
Kretzschmar
Hans
H
Giese
Armin
A
D052160:Benzothiazoles; D004594:Electrophysiology / Q000379:methods; D044950:Flavanones / Q000737:chemistry; D005456:Fluorescent Dyes / Q000737:chemistry; D005786:Gene Expression Regulation; D006801:Humans; D007501:Iron / Q000737:chemistry*; D008051:Lipid Bilayers; D018625:Microscopy, Atomic Force; D018613:Microscopy, Confocal; D008954:Models, Biological; D010300:Parkinson Disease / Q000378:metabolism; D011485:Protein Binding; D012997:Solvents / Q000737:chemistry; D013844:Thiazoles / Q000737:chemistry; D051844:alpha-Synuclein / Q000737:chemistry*
D052160:Benzothiazoles; D044950:Flavanones; D005456:Fluorescent Dyes; D008051:Lipid Bilayers; D012997:Solvents; D013844:Thiazoles; D051844:alpha-Synuclein; C009462:thioflavin T; C006680:baicalein; D007501:Iron