β-Sheet-rich α-synuclein (αS) aggregates characterize Parkinson's disease (PD). αS was long believed to be a natively unfolded monomer, but recent work suggests it also occurs in α-helix-rich tetramers. Crosslinking traps principally tetrameric αS in intact normal neurons, but not after cell lysis, suggesting a dynamic equilibrium. Here we show that freshly biopsied normal human brain contains abundant αS tetramers. The PD-causing mutation A53T decreases tetramers in mouse brain. Neurons derived from an A53T patient have decreased tetramers. Neurons expressing E46K do also, and adding 1-2 E46K-like mutations into the canonical αS repeat motifs (KTKEGV) further reduces tetramers, decreases αS solubility and induces neurotoxicity and round inclusions. The other three fPD missense mutations likewise decrease tetramer:monomer ratios. The destabilization of physiological tetramers by PD-causing missense mutations and the neurotoxicity and inclusions induced by markedly decreasing tetramers suggest that decreased α-helical tetramers and increased unfolded monomers initiate pathogenesis. Tetramer-stabilizing compounds should prevent this.

A switch in the conformational properties of α-synuclein (αS) is hypothesized to be a key step in the pathogenic mechanism of Parkinson’s disease (PD). Whereas the beta-sheet-rich state of αS has long been associated with its pathological aggregation in PD, a partially alpha-helical state was found to be related to physiological lipid binding; this suggests a potential role of the alpha-helical state in controlling synaptic vesicle cycling and resistance to β-sheet rich aggregation. N-terminal acetylation is the predominant post-translational modification of mammalian αS. Using circular dichroism, isothermal titration calorimetry, and fluorescence spectroscopy, we have analyzed the effects of N-terminal acetylation on the propensity of recombinant human αS to form the two conformational states in interaction with lipid membranes. Small unilamellar vesicles of negatively charged lipids served as model membranes. Consistent with previous NMR studies using phosphatidylserine, we found that membrane-induced α-helical folding was enhanced by N-terminal acetylation and that greater exothermic heat could be measured upon vesicle binding of the modified protein. Interestingly, the folding and lipid binding enhancements with phosphatidylserine in vitro were weak when compared to that of αS with GM1, a lipid enriched in presynaptic membranes. The resultant increase in helical folding propensity of N-acetylated αS enhanced its resistance to aggregation. Our findings demonstrate the significance of the extreme N-terminus for folding nucleation, for relative GM1 specificity of αS-membrane interaction, and for a protective function of N-terminal-acetylation against αS aggregation mediated by GM1.

Despite two decades of research, the structure-function relationships of endogenous, physiological forms of α-synuclein (αSyn) are not well understood. Most in vitro studies of this Parkinson's disease-related protein have focused on recombinant αSyn that is unfolded and monomeric, assuming that this represents its state in the normal human brain. Recently, we have provided evidence that αSyn exists in considerable part in neurons, erythrocytes and other cells as a metastable multimer that principally sizes as a tetramer. In contrast to recombinant αSyn, physiological tetramers purified from human erythrocytes have substantial α-helical content and resist pathological aggregation into β-sheet rich fibers. Here, we report the first method to fully purify soluble αSyn from the most relevant source, human brain. We describe protocols that purify αSyn to homogeneity from non-diseased human cortex using ammonium sulfate precipitation, gel filtration, and ion exchange, hydrophobic interaction and affinity chromatographies. Crosslinking of the starting material and the partially purified chromatographic fractions revealed abundant αSyn multimers, including apparent tetramers, but these were destabilized in large part to monomers during the final purification step. The method also fully purified the homologue β-synuclein, with a similar outcome. Circular dichroism spectroscopy showed that purified, brain-derived αSyn can display more helical content than the recombinant protein, but this result varied. Collectively, our data suggest that purifying αSyn to homogeneity destabilizes native, α-helix-rich multimers that exist in intact and partially purified brain samples. This finding suggests the existence of a stabilizing co-factor (e.g., a small lipid) present inside neurons that is lost during final purification.