α-Synuclein occurs physiologically as a helically folded tetramer that resists aggregation

Abstract

Parkinson's disease is the second most common neurodegenerative disorder. Growing evidence indicates a causative role of misfolded forms of the protein α-synuclein in the pathogenesis of Parkinson's disease. Intraneuronal aggregates of α-synuclein occur in Lewy bodies and Lewy neurites, the cytopathological hallmarks of Parkinson's disease and related disorders called synucleinopathies. α-Synuclein has long been defined as a 'natively unfolded' monomer of about 14 kDa (ref. 6) that is believed to acquire α-helical secondary structure only upon binding to lipid vesicles. This concept derives from the widespread use of recombinant bacterial expression protocols for in vitro studies, and of overexpression, sample heating and/or denaturing gels for cell culture and tissue studies. In contrast, we report that endogenous α-synuclein isolated and analysed under non-denaturing conditions from neuronal and non-neuronal cell lines, brain tissue and living human cells occurs in large part as a folded tetramer of about 58 kDa. Several methods, including analytical ultracentrifugation, scanning transmission electron microscopy and in vitro cell crosslinking confirmed the occurrence of the tetramer. Native, cell-derived α-synuclein showed α-helical structure without lipid addition and had much greater lipid-binding capacity than the recombinant α-synuclein studied heretofore. Whereas recombinantly expressed monomers readily aggregated into amyloid-like fibrils in vitro, native human tetramers underwent little or no amyloid-like aggregation. On the basis of these findings, we propose that destabilization of the helically folded tetramer precedes α-synuclein misfolding and aggregation in Parkinson's disease and other human synucleinopathies, and that small molecules that stabilize the physiological tetramer could reduce α-synuclein pathogenicity.

Figure 1

Western blot analysis of lysates of M17D, HeLa, HEK293 and COS-7 cells, mouse cortex and human RBCs probed for endogenous αSyn. A: Blue Native PAGE. B: Clear Native PAGE. The band just below the main ∼55-60 kDa RBC species (B, lane 6) may represent an alternatively spliced form of αSyn. . Arrow marks a possible dimeric species. C: Left: SDS-PAGE/Western blot (antibody C20) analysis of cell lysates without crosslinking. Right: Proteins were crosslinked in intact living cells with membrane permeable DSS (M17D, HeLa, HEK 293, COS-7) or in RBC lysate with water soluble BS³ and then run on SDS-PAGE.

Figure 2

Sizing analyses of αSyn from human RBCs. A: Representative large-angle dark-field STEM image of purified αSyn from human RBC. A few representative particles are circled. As an internal size standard, tobacco mosaic virus (TMV) helical rod was included during EM specimen preparation. B: Mass histogram (bin size = 5 kDa) of 1,000 automatically selected αSyn particles. C: Sedimentation equilibrium AUC of purified, native RBC αSyn. Upper panel shows the individual experimental analyses fitting an ideal single-species model to the equilibrium data obtained at 12k, 16k, and 20k RPM for 1.1 mg/ml αSyn solution. The fitting yielded a molecular weight of 57,753 Da (SD: +/- 655.199) with a root mean square deviation of 0.004533. Lower panel shows an overlay of the residuals of data and theoretical fit for the three different speeds.

Figure 3

A: CD-spectra of native tetrameric αSyn (isolated under non-denaturing conditions from human RBC) before vs. after addition of PC/PS SUV (protein/lipid 1:500). B: CD spectra of recombinant αSyn monomer purified from E. coli alone and with addition of PC/PS SUV (protein/lipid 1:500). C: SPR sensorgram of equal protein concentrations of αSyn recombinant monomer vs. endogenous tetramer injected on a L1 chip covered with a PC/PS membrane. D: Amyloid-type aggregation kinetics of recombinant αSyn monomer vs. native RBC tetramer monitored by ThT fluorescence; average values from 3 independent experiments (error bars = SD; some SD for RBC-derived αSyn are smaller than the symbol size). AU, arbitrary units.