Different Effects of α-Synuclein Mutants on Lipid Binding and Aggregation Detected by Single Molecule Fluorescence Spectroscopy and ThT Fluorescence-Based Measurements

Ruf, Viktoria; Nübling, Georg; Willikens, Sophia; Shi, Song; Schmidt, Felix; Xiong, Chengjie; Bötzel, Kai; Kamp, Frits; Giese, Armin

doi:10.1021/acschemneuro.8b00579

Cited by 47 publications

(65 citation statements)

References 61 publications

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“…Similarly, one would expect an increased BiFC signal when using proteins with disease-specific point mutations such as A53T, that promote a-syn aggregation. Although several a-syn PDassociated mutants have been shown to promote a-syn oligomerization and/or fibril formation in vitro (de Oliveira & Silva, 2019;Ruf et al, 2019), in cells (M. B. Fares et al, 2014;Khalaf et al, 2014;Lei, et al, 2019) and in vivo (Mbefo et al, 2015;Paumier et al, 2013), Lázaro et al reported identical Venus BiFC signals from WT a-syn and the PD-linked mutants A30P, E46K, H50Q, G51D and A53T (Lázaro et al, 2014).…”

Section: Discussionmentioning

confidence: 99%

Monitoring alpha-synuclein oligomerization and aggregation using bimolecular fluorescence complementation assays: what you see is not always what you get

Frey

AlOkda

Lashuel

2020

Preprint

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Abbreviations:a-syn: a-synuclein; a-syn-V: a-synuclein-full length Venus; Vn-a-syn: n-terminal fragment of Venus fused to the amino-terminus of a-syn; a-syn-Vc: c-terminal fragment of Venus fused to the carboxy-terminus of a-syn; BiFC: bimolecular fluorescent complementation; PD: Parkinson´s disease; SEC: size exclusion chromatography Abstract Bimolecular fluorescence complementation (BiFC) was introduced a decade ago as a method to monitor alpha-synuclein (α-syn) oligomerization in intact cells. Since then, several α-syn BiFC cellular assays and animal models have been developed, including mouse, rat and Drosophila models, based on the assumption that an increase in the fluorescence signal correlates with increased α-syn oligomerization or aggregation. Despite the increasing use of these assays and models in mechanistic studies, target validation and drug screening, there have been no reports that 1) validate the extent to which the BiFC fluorescent signal correlates with α-syn oligomerization at the biochemical level; 2) provide a structural characterization of the oligomers and aggregates formed by the BiFC fragments; or 3) investigate the extent to which the oligomers by the fluorescent complex resemble oligomers that form on the pathway to α-syn fibrillization. To address this knowledge gap, we first analyzed the expression level and oligomerization properties of the individual constituents of α-syn-Venus, one of the most commonly used BiFC systems, in HEK-293 cells using multiple approaches, including size exclusion chromatography, semiquantitative Western blot analysis, in-cell crosslinking, immunocytochemistry and sedimentation assays. Next, we investigated the biochemical and aggregation properties of α-syn upon co-expression of both BiFC fragments. Our results show that 1) the two BiFC fragments are not expressed at the same level since C-terminal-Venus fused to α-syn (a-syn-Vc) was present in very low abundance; 2) the fragment with Nterminal-Venus fused to α-syn (Vn-a-syn) exhibited a high propensity to form oligomers and higher-order aggregates; 3) the expression of either or both fragments does not result in the formation of α-syn fibrils, cellular inclusions or the accumulation of pS129 immunoreactive αsyn aggregates. Furthermore, our results suggest that only a small fraction of Vn-a-syn is involved in the formation of the fluorescent BiFC complex and that some of the fluorescent signal may arise from the association or entrapment of a-syn-Vc in Vn-a-syn aggregates. The fact that the N-terminal fragment exists predominantly in an aggregated state also indicates that one must exercise caution when using this system to investigate α-syn oligomerization in cells or in vivo. Altogether, our results suggest that oligomerization, aggregation and cell-tocell transmission assays and model systems based on a-syn BiFC systems should be thoroughly characterized at the biochemical level to ensure that they reproduce the process of interest and measure what they are intended to measure.

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Section: Discussionmentioning

confidence: 99%

Monitoring alpha-synuclein oligomerization and aggregation using bimolecular fluorescence complementation assays: what you see is not always what you get

Frey

AlOkda

Lashuel

2020

Preprint

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“…It is also reported that αSyn interacts with membrane lipid components, which could be targets for αSyn toxicity [40–42]. In vitro studies showed that A30P and G51D mutations of αSyn had decreased lipid binding, whereas A53T and H50Q mutations did not differ from WT αSyn in their lipid binding [43–46]. Therefore, a universal mechanism of enhanced toxicity that applies to all αSyn mutants still remains elusive, and each mutation may have different and multiple mechanisms for their toxic effects.…”

Section: Discussionmentioning

confidence: 99%

E46K mutant α-synuclein is more degradation resistant and exhibits greater toxic effects than wild-type α-synuclein in Drosophila models of Parkinson's disease

Sakai¹,

Suzuki²,

Ueyama³

et al. 2019

PLoS ONE

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Parkinson’s disease (PD) is one of the most common neurodegenerative diseases, which is characterized by progressive motor dysfunction as well as non-motor symptoms. Pathological and genetic studies have demonstrated that α-synuclein (αSyn) plays key roles in the pathogenesis of PD. Although several missense mutations in the αSyn gene have been identified as causes of familial PD, the mechanisms underlying the variance in the clinical phenotypes of familial PD caused by different mutations remain elusive. Here, we established novel Drosophila models expressing either wild-type (WT) αSyn or one of five αSyn mutants (A30P, E46K, H50Q, G51D, and A53T) using site-specific transgenesis, which express transgenes at equivalent levels. Expression of either WT or mutant αSyn in the compound eyes by the GMR-GAL4 driver caused mild rough eye phenotypes with no obvious difference among the mutants. Upon pan-neuronal expression by the nSyb-GAL4 driver, these αSyn-expressing flies showed a progressive decline in locomotor function. Notably, we found that E46K, H50Q, G51D, and A53T αSyn-expressing flies showed earlier onset of locomotor dysfunction than WT αSyn-expressing flies, suggesting their enhanced toxic effects. Whereas mRNA levels of WT and mutant αSyn were almost equivalent, we found that protein expression levels of E46K αSyn were higher than those of WT αSyn. In vivo chase experiments using the drug-inducible GMR-GeneSwitch driver demonstrated that degradation of E46K αSyn protein was significantly slower than WT αSyn protein, indicating that the E46K αSyn mutant gains resistance to degradation in vivo . We therefore conclude that our novel site-specific transgenic fly models expressing either WT or mutant αSyn are useful to explore the mechanisms by which different αSyn mutants gain toxic functions in vivo .

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“…Expression and purification were performed as previously described [19,20]. Briefly, the pET5α/αSynuclein (136 TAT) plasmid (wt‐plasmid by Philipp Kahle, LMU Munich; 136‐TAC/TAT‐mutation by Matthias Habeck) was transformed into BL21(DE3) Escherichia coli (New England Biolabs, Frankfurt am Main, Germany).…”

Section: Methodsmentioning

confidence: 99%

Potential sources of interference with the highly sensitive detection and quantification of alpha‐synuclein seeds by qRT‐QuIC

Ruf

Shi

Schmidt³

et al. 2020

FEBS Open Bio

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Parkinson’s disease (PD) is a progressive neurodegenerative disease which is histologically characterized by loss of dopaminergic neurons in the substantia nigra and deposition of aggregated alpha‐synuclein (aSyn) in the brain. The detection of aSyn in well accessible fluids has been one of the central approaches in the development of biomarkers for PD. Recently, real‐time quaking‐induced conversion (RT‐QuIC) has been successfully adapted for use with aSyn seeds. Here, we systematically analysed parameters potentially impacting the reliability of this assay by using quantitative real‐time quaking‐induced conversion (qRT‐QuIC) with in vitro‐formed aSyn seeds. Seeds diluted in cerebrospinal fluid (CSF) accelerated the seeding reaction and slightly increased the sensitivity without affecting specificity. Repeated freeze–thaw cycles decreased the apparent lag times of seeds diluted in ddH2O but did not alter the seeding activity of seeds diluted in CSF. High levels of artificial contamination with blood resulted in prolonged apparent lag times, while sensitivity and specificity were unaffected. Altogether, qRT‐QuIC with aSyn seems to be robust concerning sensitivity and specificity in our model system, but quantitative interpretation might be limited under certain conditions.

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Different Effects of α-Synuclein Mutants on Lipid Binding and Aggregation Detected by Single Molecule Fluorescence Spectroscopy and ThT Fluorescence-Based Measurements

Cited by 47 publications

References 61 publications

Monitoring alpha-synuclein oligomerization and aggregation using bimolecular fluorescence complementation assays: what you see is not always what you get

Monitoring alpha-synuclein oligomerization and aggregation using bimolecular fluorescence complementation assays: what you see is not always what you get

E46K mutant α-synuclein is more degradation resistant and exhibits greater toxic effects than wild-type α-synuclein in Drosophila models of Parkinson's disease

Potential sources of interference with the highly sensitive detection and quantification of alpha‐synuclein seeds by qRT‐QuIC

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