Purification of α-Synuclein from Human Brain Reveals an Instability of Endogenous Multimers as the Protein Approaches Purity

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