Abstract
α-synuclein plays a key role in the pathogenesis of Parkinson’s disease (PD); its deposits are found as amyloid fibrils in Lewy bodies and Lewy neurites, the histopathological hallmarks of PD. Amyloid fibrillation is a progressive polymerization path starting from peptide/protein misfolding and proceeding through the transient growth of oligomeric intermediates widely considered as the most toxic species. Consequently, a promising approach of intervention against PD might be preventing α-synuclein build-up, misfolding and aggregation. A possible strategy involves the use of small molecules able to slow down the aggregation process or to alter oligomer conformation favouring the growth of non-pathogenic species. Here, we show that oleuropein aglycone (OleA), the main olive oil polyphenol, exhibits anti-amyloidogenic power in vitro by interacting with, and stabilizing, α-synuclein monomers thus hampering the growth of on-pathway oligomers and favouring the growth of stable and harmless aggregates with no tendency to evolve into other cytotoxic amyloids. We investigated the molecular basis of such interference by both biophysical techniques and limited proteolysis; aggregate morphology was monitored by electron microscopy. We also found that OleA reduces the cytotoxicity of α-synuclein aggregates by hindering their binding to cell membrane components and preventing the resulting oxidative damage to cells.
Highlights
Luana Palazzi[1], Elena Bruzzone[2], Giovanni Bisello[1], Manuela Leri[2,3], Massimo Stefani[2], Monica Bucciantini2 & Patrizia Polverino de Laureto[1] α-synuclein plays a key role in the pathogenesis of Parkinson’s disease (PD); its deposits are found as amyloid fibrils in Lewy bodies and Lewy neurites, the histopathological hallmarks of PD
No variation in the retention time (RT 35.5 min) of Syn when eluted in the presence of oleuropein aglycone (OleA) was seen in reverse phase (RP)-HPLC; we noticed the presence of a new peak at RT 51.2 min (Fig. 1c, highlighted with two stars), indicating increased hydrophobicity, likely due to some chemical modifications or change in the aggregation state of this species
We show that OleA interferes with Syn fibrillation in a dose-dependent manner, with the optimal 1:10 Syn/OleA ratio; the inhibition is due, apparently, to OleA interaction with Syn monomeric and oligomeric species, an effect similar to that reported for other polyphenols[64,65]
Summary
Subconfluent SH-SY5Y cells grown on glass coverslips were treated for 48 h with Syn aggregates at a 5.0 μM final concentration and washed with PBS. The cells were incubated for 1 h at room temperature with a rabbit polyclonal anti-Syn antibody (Abcam, Cambridge UK) diluted 1:500 in blocking solution and washed with PBS for 30 min under stirring. The cells were washed twice in PBS and once in water to remove non- bound Abs. Cell fluorescence was imaged using a confocal TCS SP5 scanning microscope (Leica) equipped with a HeNe/Ar laser source for fluorescence measurements. The results were compared using Student’s t-test between two groups, *P < 0.05; **P < 0.01; ***P < 0.001 versus untreated cells and °P < 0.05; °°P < 0.01; °°°P < 0.001 versus cells treated with aggregates grown in the absence of OleA
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