Separation and determination of alpha‐synuclein monomeric and oligomeric species using two electrophoretic approaches

Abstract

Parkinson's disease (PD) is a frequent degenerative disorder that is diagnosed based on clinical symptoms. When the first symptoms appear, more than 70% of the dopaminergic cells are already lost. Therefore, it is of utmost importance to have reliable biomarkers to diagnose much earlier PD. In this context, alpha-synuclein (aSyn) is a protein of high interest because of its tendency to form oligomers and amyloid fibrils. The oligomeric forms seem to play a critical pathological role in PD. To date, most of studies aiming at detecting and quantifying aSyn oligomers were performed by immunoassays, mainly by ELISA using specific antibodies. In this study a capillary gel electrophoresis (CGE) coupled with fluorescence detection method was developed to detect and quantify the oligomeric forms of aSyn formed in vitro. All the results obtained were supported by SDS-PAGE analysis, a widely used and well-known technique but exhibiting a main drawback since it is not an automated technique. The repeatability and the intermediate precision of the method were evaluated, as well as the stability of the labeled and non-labeled aSyn samples. After careful screening and optimization of various labeling reagents, 4-fluoro-7-nitrobenzofurazan (NBD-F) was selected and used to establish a calibration curve with monomeric fluorescently-labeled aSyn. Finally, the method was used to study the effect of doxycycline on the oligomerization process. Altogether, our results show that CGE is a very promising automated technique to analyze aSyn monomers, as well as small oligomers.

α-Synuclein Oligomer Detection with Aptamer Switch on Reduced Graphene Oxide Electrode

“…To date, various efforts have been made to develop new techniques to sensitively detect α-Syn oligomers as a biomarker for PD diagnosis. These techniques include methods 2 of 11 based on mass spectrometry [17,18], capillary electrophoresis [19,20], nuclear magnetic resonance spectroscopy [21,22], fluorescence spectroscopy [23,24], and surface plasmon resonance [25]. Many of these techniques require costly and complex instruments and suffer from time-consuming and multistep sample preparation.…”

Rapid Structural, Kinetic, and Immunochemical Analysis of Alpha-Synuclein Oligomers in Solution

“…In addition to these approaches, previous studies have employed microcapillary electrophoresis (MCE) techniques , to analyze protein aggregation products; these works were primarily focused on the observation of fibrillar species rather than oligomers or were nonquantitative. , While MCE techniques generally provide advantages in terms of resolution relative to μFFE, , they pose challenges in the quantitation of rare species due to the injection of a finite volume of sample. In contrast, the steady-state, continuous fractionation process intrinsic to μFFE prevents sample bleaching during data acquisition and enhances sensitivity through the use of arbitrary exposure times in conventional epifluorescence microscopy.…”

Doxycycline interferes with tau amyloid aggregation abolishing its associated neuronal toxicity

“…Recent results from our group and others [17,23,51–53] have shed light regarding the ability of tetracyclines in interfering with the toxicity and seeding of aggregate-prone proteins. In the present work, we extend these studies to tau.…”