In Vivo Cross-linking Reveals Principally Oligomeric Forms of α-Synuclein and β-Synuclein in Neurons and Non-neural Cells

Aggregation of alpha-synuclein (alpha Syn) in neurons produces the hallmark cytopathology of Parkinson disease and related synucleinopathies. Since its discovery, alpha Syn has been thought to exist normally in cells as an unfolded monomer. We recently reported that alpha Syn can instead exist in cells as a helically folded tetramer that resists aggregation and binds lipid vesicles more avidly than unfolded recombinant monomers (Bartels, T., Choi, J. G., and Selkoe, D. J. (2011) Nature 477, 107-110). However, a subsequent study again concluded that cellular alpha Syn is an unfolded monomer (Fauvet, B., Mbefo, M. K., Fares, M. B., Desobry, C., Michael, S., Ardah, M. T., Tsika, E., Coune, P., Prudent, M., Lion, N., Eliezer, D., Moore, D. J., Schneider, B., Aebischer, P., El-Agnaf, O. M., Masliah, E., and Lashuel, H. A. (2012) J. Biol. Chem. 287, 15345-15364). Here we describe a simple in vivo cross-linking method that reveals a major similar to 60-kDa form of endogenous alpha Syn (monomer, 14.5 kDa) in intact cells and smaller amounts of similar to 80- and similar to 100-kDa forms with the same isoelectric point as the 60-kDa species. Controls indicate that the apparent 60-kDa tetramer exists normally and does not arise from pathological aggregation. The pattern of a major 60-kDa and minor 80- and 100-kDa species plus variable amounts of free monomers occurs endogenously in primary neurons and erythroid cells as well as neuroblastoma cells overexpressing alpha Syn. A similar pattern occurs for the homologue, beta-synuclein, which does not undergo pathogenic aggregation. Cell lysis destabilizes the apparent 60-kDa tetramer, leaving mostly free monomers and some 80-kDa oligomer. However, lysis at high protein concentrations allows partial recovery of the 60-kDa tetramer. Together with our prior findings, these data suggest that endogenous alpha Syn exists principally as a 60-kDa tetramer in living cells but is lysis-sensitive, making the study of natural alpha Syn challenging outside of intact cells.