Targeting α-synuclein oligomers by protein-fragment complementation for drug discovery in synucleinopathies

Abstract

Objective Reducing the burden of alpha-synuclein oligomeric species represents a promising approach for disease-modifying therapies against synucleinopathies such as Parkinson's disease and dementia with Lewy bodies. However, the lack of efficient drug discovery strategies that specifically target alpha-synuclein oligomers has been a limitation to drug discovery programs. Research design and methods Here we describe an innovative strategy that harnesses the power of bimolecular protein-fragment complementation to monitor synuclein–synuclein interactions. We have developed two robust models to monitor alpha-synuclein oligomerization by generating novel stable cell lines expressing alpha-synuclein fusion proteins for either fluorescent or bioluminescent protein-fragment complementation under the tetracycline-controlled transcriptional activation system. Main outcome measures A pilot screen was performed resulting in the identification of two potential hits, a p38 mitogen-activated protein kinase inhibitor and a casein kinase 2 inhibitor, demonstrating the suitability of our protein-fragment complementation assay for the measurement of alpha-synuclein oligomerization in living cells at high-throughput. Conclusions The application of the strategy described herein to monitor alpha-synuclein oligomer formation in living cells with high-throughput will facilitate drug discovery efforts for disease-modifying therapies against synucleinopathies and other proteinopathies.

Investigation of Endocytic Pathways for the Internalization of Exosome-Associated Oligomeric Alpha-Synuclein

“…Despite growing evidence for the role of these extracellular vesicles as carriers of αsyn, little is known about the mechanism of entry into neighboring cells. In this present study we took advantage of a recently generated stable cell line (Moussaud et al, 2015) to purify exosomes containing αsyn oligomers and investigate internalization into H4 neuroglioma recipient cells. Briefly, in this SL1SL2 cell line, two h αsyn proteins fused to N- or C-terminal halves of a luciferase reporter can reconstitute the enzymatic activity of gaussia luciferase when αsyn-αsyn interactions occur, thus providing a readout for the presence of αsyn oligomeric species.…”

“…A stable human H4 neuroglioma cell line coexpressing human αsyn tagged with either the amino-terminal (SL1) or the carboxy-terminal fragments (SL2) of Gaussia princeps luciferase was generated and described previously (Moussaud et al, 2015). SL1SL2 cells were maintained at 37°C in a 95% air/5% CO 2 humidified incubator in Opti-MEM supplemented with 10% FBS.…”

“…We previously described a cell based assay where exosome–associated αsyn oligomers could be produced and monitored using a highly sensitive bioluminescence protein complementation assay (Danzer et al, 2012). Taking advantage of this new technology, we purified exosomes from conditioned media of a newly generated stable cells line (Moussaud et al, 2015) secreting αsyn oligomers and investigated endocytic pathway(s) required for their uptake. An understanding of these events will clarify the therapeutic potential of enzymes that regulate protein trafficking and degradation in synucleinopathies.…”

The neural chaperone proSAAS blocks α-synuclein fibrillation and neurotoxicity

“…In separate experiments, the final product of fibrillation was assessed by centrifugation and SDS/PAGE to determine extent of fibrillation. In these experiments, the majority of α-synuclein remained in the supernatant for both proSAAS 1-180 and α-crystallin, but proSAAS 1-157 caused the majority of α-synuclein to pellet ( assay (35,36) and quantified oligomerization in SH-SY5Y cells. Importantly, this assay may not distinguish between native or misfolded oligomers, although the maturation time required to form the Venus fluorophore suggests that a very stable interaction is required, on the order of hours (37).…”

Cellular models of alpha‐synuclein toxicity and aggregation

“…). More recently (c) protein complementation assay (PCA) approaches have been used where synuclein tagged with either N‐ or C‐ terminal portions of a split reporter (fluorescent or bioluminescent) is expressed and the reporter signal used as a proxy for dimeric or higher order oligomeric species (Moussaud et al, ). In order to label endogenous α‐synuclein aggregates, PLA can be employed (d) where proximity‐dependent rolling circle amplification of oligonucleotide‐labeled antibodies generates a signal to mark α‐synuclein aggregates (Roberts et al, )…”