Investigation of the effects of carbon-based nanomaterials on A53T alpha-synuclein aggregation using a whole-cell recombinant biosensor

Mohammadi, Soheila; Nikkhah, Maryam; Hosseinkhani, Saman

doi:10.2147/ijn.s144764

Cited by 38 publications

(28 citation statements)

References 58 publications

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“…1); the split Venus fragments fused at the opposite ends of a-syn may influence a-syn stability or clearance. Similar to a-syn, differences in protein expression levels have also been consistently observed when Venus-BiFC has been fused with, amyloid precursor protein (APP) (So et al, 2013) and exon1 of the huntingtin protein (Httex1) (Herrera et al, 2011) but not in the Luciferase-or GFP-based BiFC systems when both complementation constituents are fused to the carboxy-terminus of a-syn (Mohammadi et al, 2017;Moussaud et al, 2015;Outeiro et al, 2008). The large differences in the protein levels of the two Venus BiFC fragments combined with the high aggregation propensity of Vn-a-syn highlight the critical importance of assessing the expression and aggregation state of each component in the Venus BiFC systems as well as other BiFC systems to ensure accurate interpretation of experimental findings.…”

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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“…35 In some investigations, carbon-based nanomaterials also appear to promote amyloid fibril formation, indicating protein specificity as well as fine-tuned importance of the exact physical properties and chemical modifications resulting from the synthesis method used; graphene-based materials can for example vary significantly with respect to size, shape, thickness, and surface charge. 29,36 Studies of how amyloid proteins interact with different nanomaterials in their monomeric form, as well as during the aggregation process, are therefore important to mechanistically understand their function and towards realizing their putative therapeutic potential.…”

Section: Introductionmentioning

confidence: 99%

Graphene oxide sheets and quantum dots inhibit α-synuclein amyloid formation by different mechanisms

et al. 2020

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“…Each one relies on a specific feature of apoptosis pathway (whether intrinsic or extrinsic). However, until the development of the Apaf-1 split luciferase complementary assay, none could be used to monitor apoptosome formation within the cell death signaling pathway [7][8][9].…”

Section: Introductionmentioning

confidence: 99%

Optimization of Experimental Variables Influencing Apoptosome Biosensor in HEK293T Cells

Oladzad

Nikkhah

Hosseinkhani

2020

Sensors

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The apoptotic protease-activating factor 1 (Apaf-1) split luciferase biosensor has been used as a biological tool for the detection of early stage of apoptosis. The effect of doxorubicin in a cell-based assay and the addition of cytochrome c and ATP in a cell-free system have been used to test the functionality of the reporter for the detection of apoptosome formation. Here, our data established a drug- and cytochrome c/ATP-independent way of apoptosis induction relying on the expression of the biosensor itself to induce formation of apoptosome. Overexpression of Apaf-1 constructs led to increased split luciferase activity and caspase-3 activity in the absence of any drug treatment. Caspase-3 activity was significantly inhibited when caspase-9DN was co-overexpressed, while the activity of the Apaf1 biosensor was significantly increased. Our results show that the Apaf-1 biosensor does not detect etoposide-induced apoptosis.

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Investigation of the effects of carbon-based nanomaterials on A53T alpha-synuclein aggregation using a whole-cell recombinant biosensor

Cited by 38 publications

References 58 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

Graphene oxide sheets and quantum dots inhibit α-synuclein amyloid formation by different mechanisms

Optimization of Experimental Variables Influencing Apoptosome Biosensor in HEK293T Cells

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