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

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.

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    • "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. "
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    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 · 4.24 Impact Factor
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    • "A slight increase of [Ca 2+ ] i both in the pre-as well as the post-synaptic compartment could account for all the neurophysiological phenomena described above. Indeed, the formation of ion-permeable pores by Fe-induced a-syn oligomers has already been shown in previous studies (Kayed et al. 2004; Danzer et al. 2007; Tsigelny et al. 2007; Kostka et al. 2008). Based on these studies, an enhanced calcium influx has been claimed to be the primary toxic event mediating neuronal cell loss in PD. "
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    ABSTRACT: Aggregated α-synuclein (α-syn) is a characteristic pathological finding in Parkinson's disease and related disorders, such as dementia with Lewy bodies. Recent evidence suggests that α-syn oligomers represent the principal neurotoxic species; however, the pathophysiological mechanisms are still not well understood. Here, we studied the neurophysiological effects of various biophysically-characterized preparations of α-syn aggregates on excitatory synaptic transmission in autaptic neuronal cultures. Nanomolar concentrations of large α-syn oligomers, generated by incubation with organic solvent and Fe(3+) ions, were found to selectivity enhance evoked α-amino-3-hydroxy-5-methylisoxazole-4-propionate (AMPA)-receptor, but not NMDA-receptor, mediated synaptic transmission within minutes. Moreover, the analysis of spontaneous AMPA-receptor-mediated miniature synaptic currents revealed an augmented frequency. These results collectively indicate that large α-syn oligomers alter both pre- and post-synaptic mechanisms of AMPA-receptor-mediated synaptic transmission. The augmented excitatory synaptic transmission may directly contribute to nerve cell death in synucleinopathies. Indeed, already low micromolar glutamate concentrations were found to be toxic in primary cultured neurons incubated with large α-syn oligomers. In conclusion, large α-syn oligomers enhance both pre- and post-synaptic AMPA-receptor-mediated synaptic transmission, thereby aggravating intracellular calcium dyshomeostasis and contributing to excitotoxic nerve cell death in synucleinopathies.
    Journal of Neurochemistry 03/2011; 117(5):868-78. DOI:10.1111/j.1471-4159.2011.07254.x · 4.24 Impact Factor
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    • "Baicalein also attenuated 6-hydroxydopamine-and MPTP-induced neurotoxicity in cells and mice (Cheng et al. 2008; Im et al. 2005; Lee et al. 2005; Mu et al. 2009; Wu et al. 2006) and inhibited methamphetamine-induced loss of dopamine transporter in mouse striatum (Wu et al. 2006). Our previous study provided some initial data that baicalein can inhibit E46K αsyn-induced LDH release in PC12 cells (Kostka et al. 2008), suggesting an effect on toxicity. However, definitive evidence for an effect on toxicity was not provided, and mechanisms by which baicalein protects cells against mutant α-syn are unknown. "
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    ABSTRACT: The E46K is a point mutation in alpha-synuclein (alpha-syn) that causes familial Parkinsonism with Lewy body dementia. We have now generated a cell model of Parkinsonism/Parkinson's disease (PD) and demonstrated cell toxicity after expression of E46K in the differentiated PC12 cells. E46K alpha-syn inhibited proteasome activity and induced mitochondrial depolarization in the cell model. Baicalein has been reported to inhibit fibrillation of wild type alpha-syn in vitro, and to protect neurons against several chemical-induced models of PD. We now report that baicalein significantly attenuated E46K-induced mitochondrial depolarization and proteasome inhibition, and protected cells against E46K-induced toxicity in a cell model of PD. Baicalein also reduced E46K fibrilization in vitro, with a concentration-dependent decrease in beta sheet conformation, though it increased some oligomeric species, and decreased formation of E46K alpha-syn-induced aggregates and rescued toxicity in N2A cells. Taken together, these data indicate that mitochondrial dysfunction, proteasome inhibition and specific aspects of abnormal E46K aggregation accompany E46K alpha-syn-induced cell toxicity, and baicalein can protect as well as altering aggregation properties. Baicalein has potential as a tool to understand the relation between different aggregation species and toxicity, and might be a candidate compound for further validation by using in vivo alpha-syn genetic PD models.
    Journal of Neurochemistry 07/2010; 114(2):419-29. DOI:10.1111/j.1471-4159.2010.06752.x · 4.24 Impact Factor
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