Dataset Information

Aggregation and neurotoxicity of recombinant ?-synuclein aggregates initiated by dimerization.

ABSTRACT:

Background

Aggregation of the ?-Synuclein (?-Syn) protein, amyloid fibril formation and progressive neurodegeneration are the neuropathological hallmarks of Parkinson's Disease (PD). However, a detailed mechanism of ?-Syn aggregation/fibrillogenesis and the exact nature of toxic oligomeric species produced during amyloid formation process are still unknown.

Results

In this study, the rates of ?-Syn aggregation were compared for the recombinant wild-type (WT) ?-Syn and a structurally relevant chimeric homologous protein containing an inducible Fv dimerizing domain (?-SynFv), capable to form dimers in the presence of a divalent ligand (AP20187). In the presence of AP20187, we report a rapid random coil into ?-sheet conformational transformation of ?-SynFv within 24 h, whereas WT ?-Syn showed 24 h delay to achieve ?-sheet structure after 48 h. Fluorescence ANS and ThT binding experiments demonstrate an accelerated oligomer/amyloid formation of dimerized ?-SynFv, compared to the slower oligomerization and amyloidogenesis of WT ?-Syn or ?-SynFv without dimerizer AP20187. Both ?-SynFv and ?-Syn pre-fibrillar aggregates internalized cells and induced neurotoxicity when injected into the hippocampus of wild-type mice. These recombinant toxic aggregates further converted into non-toxic amyloids which were successfully amplified by protein misfolding cyclic amplification method, providing the first evidence for the in vitro propagation of synthetic ?-Syn aggregates.

Conclusions

Together, we show that dimerization is important for ?-Syn conformational transition and aggregation. In addition, ?-Syn dimerization can accelerate the formation of neurotoxic aggregates and amyloid fibrils which can be amplified in vitro. A detailed characterization of the mechanism of ?-Syn aggregation/amyloidogenesis and toxicity is crucial to comprehend Parkinson's disease pathology at the molecular level.

SUBMITTER: Roostaee A

PROVIDER: S-EPMC3764494 | biostudies-literature | 2013 Jan

REPOSITORIES: biostudies-literature

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Publications

Aggregation and neurotoxicity of recombinant α-synuclein aggregates initiated by dimerization.

Roostaee Alireza A Beaudoin Simon S Staskevicius Antanas A Roucou Xavier X

Molecular neurodegeneration 20130122

<h4>Background</h4>Aggregation of the α-Synuclein (α-Syn) protein, amyloid fibril formation and progressive neurodegeneration are the neuropathological hallmarks of Parkinson's Disease (PD). However, a detailed mechanism of α-Syn aggregation/fibrillogenesis and the exact nature of toxic oligomeric species produced during amyloid formation process are still unknown.<h4>Results</h4>In this study, the rates of α-Syn aggregation were compared for the recombinant wild-type (WT) α-Syn and a structural ...[more]

PMID: 23339399

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Project description:Aggregation of ?-Synuclein (?S) protein to amyloid fibrils is a neuropathological hallmark of Parkinson's disease (PD). Growing evidence suggests that extracellular ?S aggregation plays a pivotal role in neurodegeneration found in PD in addition to the intracellular ?S aggregates in Lewy bodies (LB). Here, we identified and compared a diverse set of molecules capable of mitigating protein aggregation and exogenous toxicity of ?SA53T, a more aggregation-prone ?S mutant found in familial PD. For the first time, we investigated the ?S anti-amyloid activity of semi-synthetic flavonoid 2', 3', 4' trihydroxyflavone or 2-D08, which was compared with natural flavones myricetin and transilitin, as well as such structurally diverse polyphenols as honokiol and punicalagin. Additionally, two novel synthetic compounds with a dibenzyl imidazolidine scaffold, Compound 1 and Compound 2, were also investigated as they exhibited favorable binding with ?SA53T. All seven compounds inhibited ?SA53T aggregation as demonstrated by Thioflavin T fluorescence assays, with modified fibril morphology observed by transmission electron microscopy. Ion mobility-mass spectrometry (IM-MS) was used to monitor the structural conversion of native ?SA53T into amyloidogenic conformations and all seven compounds preserved the native unfolded conformations of ?SA53T following 48 h incubation. The presence of each test compound in a 1:2 molar ratio was also shown to inhibit the neurotoxicity of preincubated ?SA53T using phaeochromocytoma (PC12) cell viability assays. Among the seven tested compounds 2-D08, honokiol, and the synthetic Compound 2 demonstrated the highest inhibition of aggregation, coupled with neuroprotection from preincubated ?SA53T in vitro. Molecular docking predicted that all compounds bound near the lysine-rich region of the N-terminus of ?SA53T, where the flavonoids and honokiol predominantly interacted with Lys 23. Overall, these findings highlight that (i) restricted vicinal trihydroxylation in the flavone B-ring is more effective in stabilizing the native ?S conformations, thus blocking amyloidogenic aggregation, than dihydroxylation aggregation in both A and B-ring, and (ii) honokiol, punicalagin, and the synthetic imidazolidine Compound 2 also inhibit ?S amyloidogenic aggregation by stabilizing its native conformations. This diverse set of molecules acting on a singular pathological target with predicted binding to ?SA53T in the folding-prone N-terminal region may contribute toward novel drug-design for PD.