Abstract
β-Sheet-rich aggregates of α-synuclein (αSyn) are the hallmark neuropathology of Parkinson’s disease and related synucleinopathies, whereas the principal native structure of αSyn in healthy cells - unfolded monomer or α-helically folded oligomer - is under debate. Our recent crosslinking analysis of αSyn in intact cells showed that a large portion of endogenous αSyn can be trapped as oligomers, most notably as apparent tetramers. One challenge in such studies is accurately quantifying αSyn Western blot signals among samples, as crosslinked αSyn trends toward increased immunoreactivity. Here, we analyzed this phenomenon in detail and found that treatment with the reducible amine-reactive crosslinker DSP strongly increased αSyn immunoreactivity even after cleavage with the reducing agent β-mercaptoethanol. The effect was observed with all αSyn antibodies tested and in all sample types from human brain homogenates to untransfected neuroblastoma cells, permitting easy detection of endogenous αSyn in the latter, which had long been considered impossible. Coomassie staining of blots before and after several hours of washing revealed complete retention of αSyn after DSP/β-mercaptoethanol treatment, in contrast to a marked loss of αSyn without this treatment. The treatment also enhanced immunodetection of the homologs β- and γ-synuclein and of histones, another group of small, lysine-rich proteins. We conclude that by neutralizing positive charges and increasing protein hydrophobicity, amine crosslinker treatment promotes adhesion of αSyn to blotting membranes. These data help explain the recent report of fixing αSyn blots with paraformaldehyde after transfer, which we find produces similar but weaker effects. DSP/β-mercaptoethanol treatment of Western blots should be particularly useful to quantify low-abundance αSyn forms such as extracellular and post-translationally modified αSyn and splice variants.
Highlights
The synuclein family consists of a, b- and c-Synuclein, small soluble proteins with a molecular weight below 15 kDa. aSyn is by far the best known and most studied among the three, due to its strong link with Parkinson’s disease (PD), the second most common human neurodegenerative disease
A recent publication, has challenged this view by demonstrating that clear and strong detection of total endogenous aSyn from cultured cells is facilitated by fixation of proteins on blotted membranes with low concentrations of paraformaldehyde (PFA) [12], a method previously applied to the study of hemoglobin [13]
We speculated that unmodified aSyn may be washed off of Western blot membranes, whereas crosslinker modification might prevent this loss
Summary
The synuclein family consists of a-, b- and c-Synuclein (aSyn, bSyn, cSyn), small soluble proteins with a molecular weight below 15 kDa. aSyn is by far the best known and most studied among the three, due to its strong link with Parkinson’s disease (PD), the second most common human neurodegenerative disease. A recent publication, has challenged this view by demonstrating that clear and strong detection of total endogenous aSyn from cultured cells is facilitated by fixation of proteins on blotted membranes with low concentrations of paraformaldehyde (PFA) [12], a method previously applied to the study of hemoglobin [13]. The authors suggest that this treatment prevents washing off of synuclein from PVDF or nitrocellulose membranes. We applied their method in a previous study of aSyn crosslinking, and it enabled meaningful Western blot comparisons of monomeric and oligomeric aSyn species without vs with crosslinking [12]. We observed the same enhancement of monomer immunodetection with the synuclein homologs b- and cSyn, as well as with another group of small, lysine-rich proteins, the histone family, suggesting that this new method may benefit work outside the field of aSyn research
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