Dataset Information

?-Synuclein-Confocal Nanoscanning (ASYN-CONA), a Bead-Based Assay for Detecting Early-Stage ?-Synuclein Aggregation.

ABSTRACT: ?-Synuclein fibrils are considered a hallmark of Parkinson's disease and other synucleinopathies. However, small oligomers that formed during the early stages of ?-synuclein aggregation are thought to be the main toxic species causing disease. The formation of ?-synuclein oligomers has proven difficult to follow, because of the heterogeneity and transient nature of the species formed. Here, a novel bead-based aggregation assay for monitoring the earliest stages of ?-synuclein oligomerization, ?-Synuclein-Confocal Nanoscanning (ASYN-CONA), is presented. The ?-synuclein A91C single cysteine mutant is modified with a trifunctional chemical tag, which allows simultaneous fluorescent labeling with a green dye (tetramethylrhodamine, TMR) and attachment to microbeads. Beads with bound TMR-labeled ?-synuclein are then incubated with a red dye (Cy5)-labeled variant of ?-synuclein A91C, and EtOH (20%) to induce aggregation. Aggregation is detected by confocal scanning imaging, below the equatorial plane of the beads, which is known as the CONA technique. On-bead TMR-labeled ?-synuclein and aggregated Cy5-labeled ?-synuclein from the solution are quantitatively monitored in parallel by detection of fluorescent halos or "rings". ?-Synuclein on-bead oligomerization results in a linear increase of red bead ring fluorescence intensity over a period of 5 h. Total internal reflection fluorescence microscopy was performed on oligomers cleaved from the beads, and it revealed that (i) oligomers are sufficiently stable in solution to investigate their composition, consisting of 6 ± 1 monomer units, and (ii) oligomers containing a mean of 15 monomers bind Thioflavin-T. Various known inhibitors of ?-synuclein aggregation were used to validate the ASYN-CONA assay for drug screening. Baicalein, curcumin, and rifampicin showed concentration-dependent inhibition of the ?-synuclein aggregation and the IC₅₀ (the concentration of the compound at which the maxiumum intensity was reduced by one-half) were calculated.

SUBMITTER: Perez-Pi I

PROVIDER: S-EPMC6534341 | biostudies-literature | 2019 May

REPOSITORIES: biostudies-literature

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α-Synuclein-Confocal Nanoscanning (ASYN-CONA), a Bead-Based Assay for Detecting Early-Stage α-Synuclein Aggregation.

Pérez-Pi Irene I Evans David A DA Horrocks Mathew H MH Pham Nhan T NT Dolt Karamjit S KS Koszela Joanna J Kunath Tilo T Auer Manfred M

Analytical chemistry 20190418 9

α-Synuclein fibrils are considered a hallmark of Parkinson's disease and other synucleinopathies. However, small oligomers that formed during the early stages of α-synuclein aggregation are thought to be the main toxic species causing disease. The formation of α-synuclein oligomers has proven difficult to follow, because of the heterogeneity and transient nature of the species formed. Here, a novel bead-based aggregation assay for monitoring the earliest stages of α-synuclein oligomerization, α- ...[more]

PMID: 30964656

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Project description:Methamphetamine (METH) is an illegal and widely abused psychoactive stimulant. METH abusers are at high risk of neurodegenerative disorders, including Parkinson's disease (PD). Previous studies have demonstrated that METH causes alpha-synuclein (?-syn) aggregation in the both laboratory animal and human. In this study, exposure to high METH doses increased the expression of ?-syn and the small ubiquitin-related modifier 1 (SUMO-1). Therefore, we hypothesized that SUMOylation of ?-syn is involved in high-dose METH-induced ?-syn aggregation. We measured the levels of ?-syn SUMOylation and these enzymes involved in the SUMOylation cycle in SH-SY5Y human neuroblastoma cells (SH-SY5Y cells), in cultures of C57 BL/6 primary mouse neurons and in brain tissues of mice exposure to METH. We also demonstrated the effect of ?-syn SUMOylation on ?-syn aggregation after METH exposure by overexpressing the key enzyme of the SUMOylation cycle or silencing SUMO-1 expression in vitro. Then, we make introduced mutations in the major SUMOylation acceptor sites of ?-syn by transfecting a lentivirus containing the sequence of WT ?-syn or K96/102R ?-syn into SH-SY5Y cells and injecting an adenovirus containing the sequence of WT ?-syn or K96/102R ?-syn into the mouse striatum. Levels of the ubiquitin-proteasome system (UPS)-related makers ubiquitin (Ub) and UbE1, as well as the autophagy-lysosome pathway (ALP)-related markers LC3, P62 and lysosomal associated membrane protein 2A (LAMP2A), were also measured in SH-SY5Y cells transfected with lentivirus and mice injected with adenovirus. The results showed that METH exposure decreases the SUMOylation level of ?-syn, although the expression of ?-syn and SUMO-1 are increased. One possible cause is the reduction of UBC9 level. The increase in ?-syn SUMOylation by UBC9 overexpression relieves METH-induced ?-syn overexpression and aggregation, whereas the decrease in ?-syn SUMOylation by SUMO-1 silencing exacerbates the same pathology. Furthermore, mutations in the major SUMOylation acceptor sites of ?-syn also aggravate ?-syn overexpression and aggregation by impairing degradation through the UPS and the ALP in vitro and in vivo. These results suggest that SUMOylation of ?-syn plays a fundamental part in ?-syn overexpression and aggregation induced by METH and could be a suitable target for the treatment of neurodegenerative diseases.

Dataset Information

?-Synuclein-Confocal Nanoscanning (ASYN-CONA), a Bead-Based Assay for Detecting Early-Stage ?-Synuclein Aggregation.

Publications

α-Synuclein-Confocal Nanoscanning (ASYN-CONA), a Bead-Based Assay for Detecting Early-Stage α-Synuclein Aggregation.

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