Dataset Information

Elucidating the aggregation number of dopamine-induced ?-synuclein oligomeric assemblies.

ABSTRACT: Conventional methods to determine the aggregation number, that is, the number of monomers per oligomer, struggle to yield reliable results for large protein aggregates, such as amyloid oligomers. We have previously demonstrated the use of a combination of single-molecule photobleaching and substoichiometric fluorescent labeling to determine the aggregation number of oligomers of human ?-synuclein, implicated in Parkinson's disease. We show here that this approach is capable of accurately resolving mixtures of multiple distinct molecular species present in the same sample of dopamine-induced ?-synuclein oligomers, and that we can determine the respective aggregation numbers of each species from a single histogram of bleaching steps. We found two distinct species with aggregation numbers of 15-19 monomers and 34-38 monomers. These results show that this single-molecule approach allows for the systematic study of the aggregation numbers of complex supramolecular assemblies formed under different aggregation conditions.

SUBMITTER: Zijlstra N

PROVIDER: S-EPMC3907248 | biostudies-literature | 2014 Jan

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

Elucidating the aggregation number of dopamine-induced α-synuclein oligomeric assemblies.

Zijlstra Niels N Claessens Mireille M A E MM Blum Christian C Subramaniam Vinod V

Biophysical journal 20140101 2

Conventional methods to determine the aggregation number, that is, the number of monomers per oligomer, struggle to yield reliable results for large protein aggregates, such as amyloid oligomers. We have previously demonstrated the use of a combination of single-molecule photobleaching and substoichiometric fluorescent labeling to determine the aggregation number of oligomers of human α-synuclein, implicated in Parkinson's disease. We show here that this approach is capable of accurately resolvi ...[more]

PMID: 24461019

Similar Datasets

Project description:BackgroundAggregation of α-synuclein (α-syn) is a prominent feature of Parkinson's disease (PD) and other synucleinopathies. Currently, α-syn seed amplification assays (SAAs) using cerebrospinal fluid (CSF) represent the most promising diagnostic tools for synucleinopathies. However, CSF itself contains several compounds that can modulate the aggregation of α-syn in a patient-dependent manner, potentially undermining unoptimized α-syn SAAs and preventing seed quantification.MethodsIn this study, we characterized the inhibitory effect of CSF milieu on detection of α-syn aggregates by means of CSF fractionation, mass spectrometry, immunoassays, transmission electron microscopy, solution nuclear magnetic resonance spectroscopy, a highly accurate and standardized diagnostic SAA, and different in vitro aggregation conditions to evaluate spontaneous aggregation of α-syn.ResultsWe found the high-molecular weight fraction of CSF (> 100,000 Da) to be highly inhibitory on α-syn aggregation and identified lipoproteins to be the main drivers of this effect. Direct interaction between lipoproteins and monomeric α-syn was not detected by solution nuclear magnetic resonance spectroscopy, on the other hand we observed lipoprotein-α-syn complexes by transmission electron microscopy. These observations are compatible with hypothesizing an interaction between lipoproteins and oligomeric/proto-fibrillary α-syn intermediates. We observed significantly slower amplification of α-syn seeds in PD CSF when lipoproteins were added to the reaction mix of diagnostic SAA. Additionally, we observed a decreased inhibition capacity of CSF on α-syn aggregation after immunodepleting ApoA1 and ApoE. Finally, we observed that CSF ApoA1 and ApoE levels significantly correlated with SAA kinetic parameters in n = 31 SAA-negative control CSF samples spiked with preformed α-syn aggregates.ConclusionsOur results describe a novel interaction between lipoproteins and α-syn aggregates that inhibits the formation of α-syn fibrils and could have relevant implications. Indeed, the donor-specific inhibition of CSF on α-syn aggregation explains the lack of quantitative results from analysis of SAA-derived kinetic parameters to date. Furthermore, our data show that lipoproteins are the main inhibitory components of CSF, suggesting that lipoprotein concentration measurements could be incorporated into data analysis models to eliminate the confounding effects of CSF milieu on α-syn quantification efforts.

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 (α-syn) aggregation in the both laboratory animal and human. In this study, exposure to high METH doses increased the expression of α-syn and the small ubiquitin-related modifier 1 (SUMO-1). Therefore, we hypothesized that SUMOylation of α-syn is involved in high-dose METH-induced α-syn aggregation. We measured the levels of α-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 α-syn SUMOylation on α-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 α-syn by transfecting a lentivirus containing the sequence of WT α-syn or K96/102R α-syn into SH-SY5Y cells and injecting an adenovirus containing the sequence of WT α-syn or K96/102R α-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 α-syn, although the expression of α-syn and SUMO-1 are increased. One possible cause is the reduction of UBC9 level. The increase in α-syn SUMOylation by UBC9 overexpression relieves METH-induced α-syn overexpression and aggregation, whereas the decrease in α-syn SUMOylation by SUMO-1 silencing exacerbates the same pathology. Furthermore, mutations in the major SUMOylation acceptor sites of α-syn also aggravate α-syn overexpression and aggregation by impairing degradation through the UPS and the ALP in vitro and in vivo. These results suggest that SUMOylation of α-syn plays a fundamental part in α-syn overexpression and aggregation induced by METH and could be a suitable target for the treatment of neurodegenerative diseases.

Project description:Positional isomers of zinc-nitrobenzoate complexes possessing pyridine -3-(or-4-) carboxamide are used for a comparative theoretical and experimental study to understand their utility as model complexes to understand the role of metal-to-ligand charge transfer in aggregation-induced emission (AIE). Among the five different model zinc complexes, four of them are non-ionic, and one is an ionic complex. The frontier molecular energy levels of different combinations of the positional isomeric complexes and the absorption maximum were ascertained by density functional theory calculations. The PolyQ value obtained from solid samples of each complex is different. Shifts in the emissions to higher wavelengths than the expected emission for the S1 to S0 transition were observed due to aggregations. The highest value of PolyQ among the complexes was 13.56% observed for emission at 439 nm (λex = 350) of the non-ionic complex, namely, (di-aqua)bis(pyridine-3-carboxamide)di(2-nitrobenzoato)zinc(II) monohydrate. Close resemblance in emission lifetime decay profiles of the solid samples of those complexes and the respective solutions of those complexes in dimethyl sulfoxide with or without water showed a common trend, suggesting aggregation-induced emission in each case. Aggregation-induced emission caused by adding water in dimethyl sulfoxide solution of each complex showed an initial increase without a shift in the emission wavelength followed by a quenching with a shift of the respective emission peak to a short wavelength. Dynamic light scattering studies showed an increase in the average particle sizes upon an increase in the concentration of water. This indicated initial participation of water molecules to form aggregates with the complexes, favoring an increase in the AIE intensity. Aggregation of each complex changes with the concentration of water, and an increase in the concentration of water beyond a characteristic limit causes lowering of the emission intensity to the short wavelength.

Dataset Information

Elucidating the aggregation number of dopamine-induced ?-synuclein oligomeric assemblies.

Publications

Elucidating the aggregation number of dopamine-induced α-synuclein oligomeric assemblies.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets