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

CNS Research, Boehringer Ingelheim Pharma GmbH & Co. KG, CNS Research, Birkendorferstrasse 65, 88397 Biberach, Germany.
Journal of Biological Chemistry (Impact Factor: 4.57). 05/2008; 283(16):10992-1003. DOI: 10.1074/jbc.M709634200
Source: PubMed


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.

21 Reads
  • Source
    • "In addition, their progress can be followed during aggregation. Based on the variation in the brightness of the fluorescence events, growing species can be sorted according to size [19]. If the fluorescence signals are time-correlated with themselves, so-called fluorescence correlation spectroscopy (FCS), the output curve provides information on the diffusion velocity of the labeled species and can be used to follow the oligomer growth and size distributions [20,21]. "
    [Show abstract] [Hide abstract]
    ABSTRACT: Amyloidogenic protein aggregation is a persistent biomedical problem. Despite active research in disease-related aggregation, the need for multidisciplinary approaches to the problem is evident. Recent advances in single-molecule fluorescence spectroscopy are valuable for examining heterogenic biomolecular systems. In this work, we have explored the initial stages of amyloidogenic aggregation by employing fluorescence lifetime correlation spectroscopy (FLCS), an advanced modification of conventional fluorescence correlation spectroscopy (FCS) that utilizes time-resolved information. FLCS provides size distributions and kinetics for the oligomer growth of the SH3 domain of α-spectrin, whose N47A mutant forms amyloid fibrils at pH 3.2 and 37 °C in the presence of salt. The combination of FCS with additional fluorescence lifetime information provides an exciting approach to focus on the initial aggregation stages, allowing a better understanding of the fibrillization process, by providing multidimensional information, valuable in combination with other conventional methodologies.
    International Journal of Molecular Sciences 12/2012; 13(8):9400-18. DOI:10.3390/ijms13089400 · 2.86 Impact Factor
  • Source
    • "Transition metals, such as manganese and iron, have been suggested to be important pathogenetic factors for a number of neurodegenerative diseases, including the most frequent neurodegenerative movement disorder, PD. In particular, metal ions have not only been implicated in mediating oxidative stress, e.g., by catalyzing the Fenton reaction, but have recently also been shown to increase aggregation of asynuclein (Kostka 2008; Uversky et al. 2001). Although PD is now increasingly recognized as a system disorder with a spreading pathology (Braak et al. 2003), which finally affects most regions of the brain, the special vulnerability of the dopaminergic nigrostriatal projections is particularly intriguing. "
    [Show abstract] [Hide abstract]
    ABSTRACT: Transition metals have been suggested to play a pivotal role in the pathogenesis of Parkinson's disease (PD). X-ray microscopy combined with a cryogenic setup is a powerful method for elemental imaging in low concentrations and high resolution in intact cells, eliminating the need for fixation and sectioning of the specimen. Here, we performed an elemental distribution analysis in cultured primary midbrain neurons with a step size in the order of 300 nm and ~0.1 ppm sensitivity under cryo-conditions by using X-ray fluorescence (XRF) microscopy. We report the elemental mappings on the sub-cellular level in primary mouse dopaminergic (DAergic) and non-DAergic neurons after treatment with transition metals. Application of Fe(2+) resulted in largely extracellular accumulation of iron without preference for the neuronal transmitter subtype. A quantification of different Fe oxidation states was performed using XANES analysis. After treatment with Mn(2+) a cytoplasmic/paranuclear localization of Mn was observed preferentially in DAergic neurons, while no prominent signal was detectable after Mn(3+) treatment. Immunocytochemical analysis correlated the preferential Mn uptake to increased expression of voltage-gated calcium channels (VGCC) in DAergic neurons. We discuss the implications of this differential elemental distribution for the selective vulnerability of DAergic neurons and PD pathogenesis. © 2012 International Society for Neurochemistry, J. Neurochem. (2012) 10.1111/jnc.12073.
    Journal of Neurochemistry 10/2012; 124(2). DOI:10.1111/jnc.12073 · 4.28 Impact Factor
  • Source
    • "One possible mechanism of oligomer toxicity is the formation of lipid bilayer permeabilizing pores [14] resulting in cytotoxicity [10]–[12]. Indeed, oligomers dissociated from preformed α-syn fibrils were able to increase the conductance of lipid bilayers [15]. Formation of pores by oligomeric intermediates may be a fundamental mechanism of cell death in a large range of neurodegenerative diseases. "
    [Show abstract] [Hide abstract]
    ABSTRACT: Synucleinopathies such as Parkinson's disease, multiple system atrophy and dementia with Lewy bodies are characterized by deposition of aggregated α-synuclein. Recent findings indicate that pathological oligomers rather than fibrillar aggregates may represent the main toxic protein species. It has been shown that α-synuclein oligomers can increase the conductance of lipid bilayers and, in cell-culture, lead to calcium dyshomeostasis and cell death. In this study, employing a setup for single-channel electrophysiology, we found that addition of iron-induced α-synuclein oligomers resulted in quantized and stepwise increases in bilayer conductance indicating insertion of distinct transmembrane pores. These pores switched between open and closed states depending on clamped voltage revealing a single-pore conductance comparable to that of bacterial porins. Pore conductance was dependent on transmembrane potential and the available cation. The pores stably inserted into the bilayer and could not be removed by buffer exchange. Pore formation could be inhibited by co-incubation with the aggregation inhibitor baicalein. Our findings indicate that iron-induced α-synuclein oligomers can form a uniform and distinct pore species with characteristic electrophysiological properties. Pore formation could be a critical event in the pathogenesis of synucleinopathies and provide a novel structural target for disease-modifying therapy.
    PLoS ONE 08/2012; 7(8):e42545. DOI:10.1371/journal.pone.0042545 · 3.23 Impact Factor
Show more