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- W2042846220 abstract "It is established that fibrillar α-synuclein is the major component of Lewy bodies, the hallmark pathology of Parkinson's disease. However, the physiological conformation of non-pathological α-synuclein is unknown. In order to determine the native structure of α-synuclein, we analyzed physiological samples with non-denaturing, high-resolution technologies. Murine RBC and plasma, human CSF, cellular extracts of HEK293 and HEK293 transfected with inducible WT or A53T α-synuclein, as well as recombinant α-synuclein, were subjected to non-denaturing gradient gel electrophoresis (NDGGE) followed by Western blotting. We optimized the NDGGE technique in order to accurately determine the hydrated diameter of native α-synuclein by performing extended electrophoresis (20–40 hrs) on custom gradient gels. Analyses of recombinant α- synuclein at high concentrations (micrograms) revealed that the majority of the protein migrated at 40–45 kDa, which is suggestive of an oligomeric structure. In contrast, low concentrations of α-synuclein (nanograms) resolved in a smeared migration pattern that likely represented α-synuclein in numerous conformations and/or oligomers of various hydrated diameters. Interestingly, the smear resolved to a sharp 45-kDa band when crude cell lysate was incorporated. We next determined that the major species of the endogenous α-synuclein expressed in HEK293 migrated at ∼14 kDa, suggesting a compact monomer as the main physiological form, while a minor population was present at ∼45 kDa. Furthermore, compared with the WT, the presence of the mutant A53T α-synuclein in the cells consistently resulted in an increased 14 kDa population. Finally, we demonstrated that the main species of α-synuclein in mouse plasma, RBC and human CSF had hydrated diameters of ∼45 kDa, similar to the postulated oligomeric species. We analyzed physiological α-synuclein by NDGGE. In contrary to prior reports, we demonstrate that endogenous α-synuclein of HEK293 existed primarily as compact monomers. Interestingly, α-synuclein in inducible HEK293, plasma and RBC appeared to have hydrated diameters consistent with oligomers; although mutant A53T cells did have increased monomeric α-synuclein. Current studies are expanding these analyses to include primary neuronal cultures and additional biochemical technologies (cross-linking, 2D-gels, analytical ultracentrifugation, and light scattering). Further determination of the broad physiological conformation of α-synuclein may enable target identification and subsequent drug development." @default.
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- W2042846220 date "2012-07-01" @default.
- W2042846220 modified "2023-09-27" @default.
- W2042846220 title "P3-043: Analysis of physiological α-synuclein by non-denaturing gel electrophoresis" @default.
- W2042846220 doi "https://doi.org/10.1016/j.jalz.2012.05.1261" @default.
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