17 February 2012. Researchers created a stir last year when they claimed α-synuclein is not a random, unfolded monomer in its native form, as was widely believed, but is, in fact, a tetramer comprising subunits with α-helical structure. Working independently, two research groups came to that conclusion. One, led by Dennis Selkoe at Brigham and Women’s Hospital, Boston, Massachusetts, studied human α-synuclein; the other, led by Dagmar Ringe, Gregory Petsko, and Thomas Pochapsky at Brandeis University, Waltham, also in Massachusetts, and Quyen Hoang at Indiana University School of Medicine, Indianapolis, studied human recombinant protein made in bacteria. The reports sparked controversy and called for independent verification (see ARF related news story on Bartels et al., 2011, and Wang et al., 2011, and related comments).
A collaboration of six research groups led by Hilal Lashuel at the École Polytechnique Fédérale de Lausanne, Switzerland, has since tried to repeat some of the original experiments. In a Journal of Biological Chemistry paper released online February 8, the researchers published a counterclaim that α-synuclein from numerous sources, including the human central nervous system, human red blood cells, and Escherichia coli, exists predominantly as a disordered monomer. First author Bruno Fauvet and colleagues used a variety of techniques, including native and denaturing electrophoresis, size exclusion chromatography, and oligomer-specific ELISA, to detect synuclein tetramers, but concluded that the protein is monomeric. Using circular dichroism spectroscopy to determine secondary structure, they determined that α-synuclein from human red blood cells adopts a disordered conformation inconsistent with α-helical structure. The scientists used a cell-permeable crosslinker called disuccinimidyl suberate to trap any native oligomers of α-synuclein before subjecting them to further analysis. That method revealed primarily dimers with a small amount of a larger species that migrates on denaturing gels corresponding to a 57 kDa protein. “It is not clear if the band observed at ~57 kDa corresponds to an α-synuclein trimer or tetramer,” wrote the authors. “You have to wonder how relevant these crosslinked species are,” Lashuel told Alzforum. The researchers could not purify enough material to conduct analytical ultracentrifugation (AUC), which Selkoe’s group had used. In AUC proteins, sedimentation is based primarily on mass with little influence by charge or secondary structure that can complicate electrophoretic- and size exclusion-based analysis. But even at that, the method has been questioned because hemoglobin, a major component of red blood cells, is the same size as a synuclein tetramer.
Alzforum contacted numerous researchers in the field to see if there is any consensus over α-synuclein structure. No one had succeeded in reproducing the original data. “In my lab, we specifically investigated if α-synuclein mutations would disrupt tetramer formation. However, we were unable to prove a tetramer for wild-type α-synuclein; therefore, we gave up,” Christian Haass, University of Munich, Germany, wrote to Alzforum by e-mail. “However, these are negative data; therefore, you never know,” he cautioned. On the tetrameric synuclein from E. coli, Roland Riek at the Swiss Federal Institute of Technology, Zurich, Switzerland, told Alzforum that their analysis (based on size exclusion chromatography and multi-angle light scattering) indicates the protein is a monomer. Virginia Lee at the University of Pennsylvania, Philadelphia, also told Alzforum that her group tried in vain to detect synuclein tetramers. Others preferred to speak without attribution.
Looking at the various techniques involved, it is apparent that the devil is in the details. In an interview with Alzforum, Selkoe acknowledged that the methodology can be tricky. “The protein is difficult to purify and easy to denature,” he said. “The problem is that you need to keep the protein in its native state at all times, and there are various treatments that can denature it,” he said. Selkoe also noted that the purification protocol Lashuel and colleagues used was not identical to his own (see detailed comment below). Haass told Alzforum that his group tried the Selkoe lab protocol. Lee told ARF that her group has not tried to purify α-synuclein from red blood cells, the source Selkoe’s group used for many of their experiments, but has tried from a variety of other sources (see full comment below).
Selkoe believes the controversy primarily revolves around the specific techniques that various labs use in their analyses. He added that his lab now uses a more efficient crosslinking agent that traps most of the protein in a tetrameric conformation. “We feel very comfortable that everything we have reported is correct, and we believe that our revised crosslinking protocol should allow others to see what we see.” (See full comment below.) “We are happy to continue the dialogue with Hilal and others,” he told Alzforum.
So what gives? Is most α-synuclein an unfolded monomer or an α-helical tetramer? The debate matters. Misfolded variants of the protein are the principal component of the Lewy body inclusions in damaged and dying neurons in Parkinson’s disease and other synucleinopathies. The native structure is fundamental to α-synuclein misfolding and aggregation, and potentially to therapeutics that could limit that process. As Michael Lee, University of Minnesota, Twin Cities, pointed out in an e-mail to Alzforum, the Selkoe and Hoang groups concluded that the tetramer does not form fibrils; therefore, the shape α-synuclein takes in cells is more than a biophysical puzzle.
“We shouldn’t lose sight of the most important aspect of this controversy, which is that α-synuclein can, and does, take on different forms in response to the local environment, and likely occupies multiple states in vivo,” wrote Thomas Pochapsky (see full comment below). “I suspect that the tetrameric form may represent a stable non-toxic ‘storage’ of soluble α-synuclein at high concentrations, as one might find in neurons or erythrocytes,” he wrote.
“Looking at these reports, it is tempting to conclude that only one of the views is correct,” wrote Michael Lee (see full comment below). However, as is often the case in science, “resolution of the issues will likely require additional studies,” he added. Alzforum welcomes comments on the subject.—Tom Fagan.
Fauvet B, Mbefo MK, Fares MB, Desobry C, Michael S, Ardah MT, Tsika E, Coune P, Prudent M, Lion N, Eliezer D, Moore DJ, Schneider B, Aebischer P, El-Agnaf OM, Masliah E, Lashuel HA. Α-synuclein in the central nervous system and from erythrocytes, mammalian cells and E. coli exists predominantly as a disordered monomer. J Biol Chem. 2012 Feb 9. Abstract