Stabilization of α-synuclein oligomers using formaldehyde

Ruesink, Harm; Reimer, Lasse; Gregersen, Emil; Moeller, Arne; Betzer, Cristine; Jensen, Poul Henning

doi:10.1101/623538

Cited by 3 publications

(3 citation statements)

References 34 publications

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“…The SEC chromatogram revealed two elution peaks: at 8.5 mL and 14.5 mL corresponding to oligomers and monomers in agreement to what has previously been reported (Figure 3C). [21,31,37]. As shown in Figure 4A, we identified a cluster of ThT + events averaging 5.5 events per trace at the first elution peak in the SEC chromatogram, which corresponds to α-syn oligomers, according to previous publications.…”

Section: Coupling Single Molecule Detection On Size Exclusion Chromatography Demonstrates That Isolation Of α-Syn Oligomers Is Efficient supporting

confidence: 52%

“…Preformed aggregates generated from recombinant proteins have been utilised, but even this material can be difficult to standardise between laboratories. [21,30,37,47] One object of debate in using preformed aggregates has been the relevance of using α-syn fibrils compared to oligomers. Indeed, during the self-association process, both in vitro and in vivo, α-syn is thought to change from a mainly unstructured monomer to form small, soluble oligomers (consisting of 30-50 monomers).…”

Section: Introductionmentioning

confidence: 99%

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Single molecule fingerprinting reveals different amplification properties of α-synuclein oligomers and preformed fibrils in seeding assay

Lau

Magnan

Hill

et al. 2021

Preprint

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The quantification of α-synuclein (α-syn) aggregates has emerged as a promising biomarker for synucleinopathies. Assays that amplify and detect such aggregates have revealed the presence of seeding-competent species in biosamples of patients diagnosed with Parkinsons disease. However, multiple species such as oligomers and amyloid fibrils, are formed during the aggregation of α-synuclein and these species are likely to co-exist in biological samples and thus it remains unclear which species(s) are contributing to the signal detected in seeding assays. To identify which species can be detected in seeding assays, recombinant oligomers and preformed fibrils were produced and purified to characterise their individual biochemical and seeding potential. Here, we used single molecule spectroscopy to track the formation and purification of oligomers and fibrils at the single particle level and compare their respective seeding potential in an amplification assay. Single molecule detection validates that size-exclusion chromatography efficiently separates oligomers from fibrils. Oligomers were found to be seeding-competent but our results reveal that their seeding behaviour is very different compared to preformed fibrils in our amplification assay. Overall, our data suggest that even a low number of preformed fibrils present in biosamples are likely to dominate the response in seeding assays.

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Section: Coupling Single Molecule Detection On Size Exclusion Chromatography Demonstrates That Isolation Of α-Syn Oligomers Is Efficient supporting

confidence: 52%

Section: Introductionmentioning

confidence: 99%

Single molecule fingerprinting reveals different amplification properties of α-synuclein oligomers and preformed fibrils in seeding assay

Lau

Magnan

Hill

et al. 2021

Preprint

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“…Proteins were separated on precast polyacrylamide gels (Bio-Rad) and transferred to Hybond (Amersham). For detection of α-synuclein, proteins were cross-linked to membranes using 0.4% paraformaldehyde ( 51 , 52 ). Chemiluminescent detection was performed with enhanced chemiluminescence (Amersham).…”

Section: Methodsmentioning

confidence: 99%

The AKT modulator A-443654 reduces α-synuclein expression and normalizes ER stress and autophagy

Gandelman

Dansithong

Kales

et al. 2021

Journal of Biological Chemistry

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Accumulation of α-synuclein is a main underlying pathological feature of Parkinson’s disease and α-synucleinopathies, for which lowering expression of the α-synuclein gene ( SNCA ) is a potential therapeutic avenue. Using a cell-based luciferase reporter of SNCA expression we performed a quantitative high-throughput screen of 155,885 compounds and identified A-443654, an inhibitor of the multiple functional kinase AKT, as a potent inhibitor of SNCA . HEK-293 cells with CAG repeat expanded ATXN2 (ATXN2-Q58 cells) have increased levels of α-synuclein. We found that A-443654 normalized levels of both SNCA mRNA and α-synuclein monomers and oligomers in ATXN2-Q58 cells. A-443654 also normalized levels of α-synuclein in fibroblasts and iPSC-derived dopaminergic neurons from a patient carrying a triplication of the SNCA gene. Analysis of autophagy and endoplasmic reticulum stress markers showed that A-443654 successfully prevented α-synuclein toxicity and restored cell function in ATXN2-Q58 cells, normalizing the levels of mTOR, LC3-II, p62, STAU1, BiP, and CHOP. A-443654 also decreased the expression of DCLK1, an inhibitor of α-synuclein lysosomal degradation. Our study identifies A-443654 and AKT inhibition as a potential strategy for reducing SNCA expression and treating Parkinson’s disease pathology.

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Structural and Biophysical Characterization of Stable Alpha-Synuclein Oligomers

Vaikath

Sudhakaran

Abdi

et al. 2022

IJMS

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The aggregation of α-synuclein (α-syn) into neurotoxic oligomers and fibrils is an important pathogenic feature of synucleinopatheis, including Parkinson’s disease (PD). A further characteristic of PD is the oxidative stress that results in the formation of aldehydes by lipid peroxidation. It has been reported that the brains of deceased patients with PD contain high levels of protein oligomers that are cross-linked to these aldehydes. Increasing evidence also suggests that prefibrillar oligomeric species are more toxic than the mature amyloid fibrils. However, due to the heterogenous and metastable nature, characterization of the α-syn oligomeric species has been challenging. Here, we generated and characterized distinct α-syn oligomers in vitro in the presence of DA and lipid peroxidation products 4-hydroxy-2-nonenal (HNE) and 4-oxo-2-nonenal (ONE). HNE and ONE oligomer were stable towards the treatment with SDS, urea, and temperature. The secondary structure analysis revealed that only HNE and ONE oligomers contain β-sheet content. In the seeding assay, both DA and ONE oligomers significantly accelerated the aggregation. Furthermore, all oligomeric preparations were found to seed the aggregation of α-syn monomers in vitro and found to be cytotoxic when added to SH-SY5Y cells. Finally, both HNE and ONE α-syn oligomers can be used as a calibrator in an α-syn oligomers-specific ELISA.

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Stabilization of α-synuclein oligomers using formaldehyde

Cited by 3 publications

References 34 publications

Single molecule fingerprinting reveals different amplification properties of α-synuclein oligomers and preformed fibrils in seeding assay

Single molecule fingerprinting reveals different amplification properties of α-synuclein oligomers and preformed fibrils in seeding assay

The AKT modulator A-443654 reduces α-synuclein expression and normalizes ER stress and autophagy

Structural and Biophysical Characterization of Stable Alpha-Synuclein Oligomers

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