Abstract
Aggregation of α-synuclein (αSyn) in neurons produces the hallmark cytopathology of Parkinson disease and related synucleinopathies. Since its discovery, αSyn has been thought to exist normally in cells as an unfolded monomer. We recently reported that αSyn 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 αSyn 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 ~60-kDa form of endogenous αSyn (monomer, 14.5 kDa) in intact cells and smaller amounts of ~80- and ~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 αSyn. A similar pattern occurs for the homologue, β-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 αSyn exists principally as a 60-kDa tetramer in living cells but is lysis-sensitive, making the study of natural αSyn challenging outside of intact cells.
Highlights
Since its discovery as the first causative gene product for Parkinson disease [3] and the major constituent of Lewy bodies [4], ␣-synuclein (␣Syn)2 has been increasingly implicated as a key pathogenic protein in both sporadic and familial forms of the disorder. ␣Syn has long been thought to occur normally as a natively unfolded monomer of ϳ14.5 kDa [5]
We reasoned that if erythrocytes are rich in ␣Syn, their progenitor cells may be, and we identified the human erythroleukemia cell line Human erythroid leukemia cells (HEL) [15] as our initial system for development of a cross-linking protocol
In Vivo Cross-linking Reveals a Cytosolic 60-kDa Species as the Predominant ␣Syn Form in Intact Cells—To establish the specificity of the ␣Syn bands we detected and to better estimate their cellular levels, we examined a variety of anti-␣Syn antibodies on HEL cells treated with 1 mM DSG versus vehicle alone using the optimized protocol described in the previous section (Fig. 2A)
Summary
Since its discovery as the first causative gene product for Parkinson disease [3] and the major constituent of Lewy bodies [4], ␣-synuclein (␣Syn)2 has been increasingly implicated as a key pathogenic protein in both sporadic and familial forms of the disorder. ␣Syn has long been thought to occur normally as a natively unfolded monomer of ϳ14.5 kDa [5].
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