Cellular models of alpha‐synuclein toxicity and aggregation

Delenclos, Marion; Burgess, Jeremy D.; Lamprokostopoulou, Agaristi; Outeiro, Tiago F.; Vekrellis, Kostas; McLean, Pamela J.

doi:10.1111/jnc.14806

Cited by 100 publications

(71 citation statements)

References 123 publications

(150 reference statements)

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“…Our studies show that a-syn-Vc is expressed at levels that are significantly less than Vn-α-syn in various cellular systems using different methods of protein delivery, and that only a fraction of Vn-a-syn participates in the formation of complexes that give rise to the fluorescence signal. This discrepancy in protein expression levels between the two fragments has been consistently observed in previous studies (Bartels et al, 2019;Cai et al, 2018;Delenclos et al, 2019;Eckermann et al, 2015;Gonçalves et al, 2016;Gustafsson et al, 2018;Herrera & Outeiro, 2012;Lázaro et al, 2016Lázaro et al, , 2014Moussaud et al, 2015;Prasad et al, 2018;Zondler et al, 2014), but rarely discussed or taken into account in the interpretation of the results with the BiFC assays. Despite our attempts to express both proteins under a single Tet-On inducible promoter we could not overcome these differential expression issues.…”

Section: Discussionsupporting

confidence: 56%

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: Discussionsupporting

confidence: 56%

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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“…The available cellular models range from powerful yeast cells, to neuronal and non‐neuronal mammalian cell lines (human and non‐human), primary neuronal cultures and, more recently, patient‐derived iPS cells, to name just a few (Delenclos et al, ; Marvian, Koss, Aliakbari, Morshedi, & Outeiro, ). Different aSyn expression systems have also been employed, enabling transient or stable expression of either wild‐type or PD‐associated mutant forms of aSyn (Delenclos et al, ; Lazaro, Pavlou, & Outeiro, ; Vasili, Dominguez‐Meijide, & Outeiro, ). The use of cellular models affords numerous advantages, such as the ease of use and manipulation, both genetically and pharmacologically, the low maintenance costs, and the reduced ethical constraints.…”

Section: Cell Models Of Asyn Toxicity and Aggregationmentioning

confidence: 99%

Synucleinopathies: Where we are and where we need to go

Brás

Dominguez-Meijide

Gerhardt

et al. 2020

Journal of Neurochemistry

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All authors contributed equally to this manuscript. This artic le is relat ed to the Speci al Issue "Synuc lein" Abbreviations: A30P, aSyn substitution of an alanine for a proline at position 30; A53E, aSyn substitution of an alanine for a glutamic acid at position 53; A53T, aSyn substitution of an alanine for a tyrosine at position 53; ALP, autophagy-lysosomal pathway; APLP1, Aβ precursor-like protein 1; aSyn, alpha-synuclein; BiFC, bimolecular fluorescence complementation; CMA, Chaperone-mediated autophagy; CNS, central nervous system; co-IP, co-immunoprecipitation; Cryo-EM, cryo-electron microscopy; CSF, cerebrospinal fluid; Cu, copper; DLB, dementia with Lewy bodies; E46K, aSyn substitution of a glutamic acid for a lysine at position 46; ENS, enteric nervous system; ER, endoplasmic reticulum; Fe, iron; FRET, fluorescence resonance energy transfer; G51D, aSyn substitution of a glycine for a aspartic acid at position 51Abstract Synucleinopathies are a group of disorders characterized by the accumulation of inclusions rich in the a-synuclein (aSyn) protein. This group of disorders includes Parkinson's disease, dementia with Lewy bodies (DLB), multiple systems atrophy, and pure autonomic failure (PAF). In addition, genetic alterations (point mutations and multiplications) in the gene encoding for aSyn (SNCA) are associated with familial forms of Parkinson's disease, the most common synucleinopathy. The Synuclein Meetings are a series that has been taking place every 2 years for about 12 years. The Synuclein Meetings bring together leading experts in the field of Synuclein and related human conditions with the goal of discussing and advancing the research. In 2019, the Synuclein meeting took place in Ofir, a city in the outskirts of Porto, Portugal. The meeting, entitled "Synuclein Meeting 2019:Where we are and where we need to go", brought together >300 scientists studying both clinical and molecular aspects of synucleinopathies. The meeting covered a many of the open questions in the field, in a format that prompted open discussions between the participants, and underscored the need for additional research that, hopefully, will lead to future therapies for a group of as of yet incurable disorders. Here, we provide a summary of the topics discussed in each session and highlight what we know, what we do not know, and what progress needs to be made in order to enable the field to continue to advance. We are confident this systematic assessment of where we stand will be useful to steer the field and contribute to filling knowledge gaps that may form the foundations for future therapeutic strategies, which is where we need to go.

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“…There is controversy about which forms of proteins are toxic in neurodegenerative disorders. While some studies attribute toxicity to the aggregated form, other studies suggest aggregation to be a beneficial process that protects the cell from intermediate or misfolded toxic proteins [138,139]. Neurodegenerative diseases such as Alzheimer's disease (AD) and Parkinson's disease (PD) are also discussed as potential prion-like diseases for which misfolded proteins are known to propagate and convert other proteins into pathological forms [140,141].…”

Section: Targeted Proteolysis Via Lysosomes or The Ermentioning

confidence: 99%

Applying Antibodies Inside Cells: Principles and Recent Advances in Neurobiology, Virology and Oncology

et al. 2020

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To interfere with cell function, many scientists rely on methods that target DNA or RNA due to the ease with which they can be applied. Proteins are usually the final executors of function but are targeted only indirectly by these methods. Recent advances in targeted degradation of proteins based on proteolysis-targeting chimaeras (PROTACs), ubiquibodies, deGradFP (degrade Green Fluorescent Protein) and other approaches have demonstrated the potential of interfering directly at the protein level for research and therapy. Proteins can be targeted directly and very specifically by antibodies, but using antibodies inside cells has so far been considered to be challenging. However, it is possible to deliver antibodies or other proteins into the cytosol using standard laboratory equipment. Physical methods such as electroporation have been demonstrated to be efficient and validated thoroughly over time. The expression of intracellular antibodies (intrabodies) inside cells is another way to interfere with intracellular targets at the protein level. Methodological strategies to target the inside of cells with antibodies, including delivered antibodies and expressed antibodies, as well as applications in the research areas of neurobiology, viral infections and oncology, are reviewed here. Antibodies have already been used to interfere with a wide range of intracellular targets. Disease-related targets included proteins associated with neurodegenerative diseases such as Parkinson's disease (α-synuclein), Alzheimer's disease (amyloid-β) or Huntington's disease (mutant huntingtin [mHtt]). The applications of intrabodies in the context of viral infections include targeting proteins associated with HIV (e.g. HIV1-TAT, Rev, Vif, gp41, gp120, gp160) and different oncoviruses such as human papillomavirus (HPV), hepatitis B virus (HBV), hepatitis C virus (HCV) and Epstein-Barr virus, and they have been used to interfere with various targets related to different processes in cancer, including oncogenic pathways, proliferation, cell cycle, apoptosis, metastasis, angiogenesis or neo-antigens (e.g. p53, human epidermal growth factor receptor-2 [HER2], signal transducer and activator of transcription 3 [STAT3], RAS-related RHO-GTPase B (RHOB), cortactin, vascular endothelial growth factor receptor 2 [VEGFR2], Ras, Bcr-Abl). Interfering at the protein level allows questions to be addressed that may remain unanswered using alternative methods. This review addresses why direct targeting of proteins allows unique insights, what is currently feasible in vitro, and how this relates to potential therapeutic applications.Therapeutic antibodies are valuable drugs, which mostly act outside of cells.Reaching the numerous drug targets that reside inside cells by antibodies is possible in vitro and allows unique insights compared with other methods.Applying antibodies inside cells for therapeutic purposes has been explored in animal models and promises specific therapeutic benefits in neurobiology, virology and oncology.

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Cellular models of alpha‐synuclein toxicity and aggregation

Cited by 100 publications

References 123 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

Synucleinopathies: Where we are and where we need to go

Applying Antibodies Inside Cells: Principles and Recent Advances in Neurobiology, Virology and Oncology

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