Order can come from chaos. Take α-synuclein, a protein implicated in Parkinson’s disease (PD) and long believed to exist in native form as a monomeric random coil. In the August 14 Nature online, researchers led by Dennis Selkoe at Brigham and Women’s Hospital, Boston, report that the protein actually predominates as a tetramer with an α-helical secondary structure. The tetramer shows little propensity to aggregate, suggesting that it must dissociate before toxic oligomers of α-synuclein assemble in vivo. That could have implications for the study of synuclein and the treatment of PD. Selkoe told ARF that the result were unexpected. “I think the biggest surprise is that synuclein folds into a helical formation, and that is essentially the opposite of what has been taught for years,” he said. Other researchers are not so convinced, however, and the findings seem to be generating some controversy in the field.

The idea that α-synuclein is a monomer with little secondary structure comes from studies of recombinant protein expressed in bacteria or human cell lines (see, e.g., Weinreb et al., 1996; Eliezer et al., 2001). While there were hints that the protein could be larger, including migrations on acrylamide gels and gel filtration chromatography columns slower than usual for a monomeric protein of its size, researchers ascribed those properties to an unfolded protein of large dynamic radius, said Selkoe. “Because many labs repeated the initial findings, people really bought into the idea that the protein was a monomer,” he said.

But when first author Tim Bartels joined Selkoe’s lab to study native forms of α-synuclein, he noticed something curious. On non-denaturing gel electrophoresis, α-synuclein from mouse frontal cortex, human red blood cells, and a variety of cell lines, including M17D dopaminergic neuroblastoma cells, did not behave as a 14.5 kDa α-synuclein monomer, but as a 55-60 kDa protein indicative of a tetramer. To confirm this, Bartels used a cell-permeable crosslinker to covalently stabilize potential oligomers, and then ran the protein on denaturing gels. It ran as a tetramer. Two-dimensional denaturing electrophoresis indicated that this tetramer had the same isoelectric point, that is, the same net charge, as monomers, indicating the tetramer comprises four identical subunits.

The use of native electrophoresis is one of the sticking points raised by researchers in the field who reviewed this work. Hilal Lashuel, who studies α-synuclein at the Swiss Federal Institute of Technology, Lausanne, told ARF that everyone knows that recombinant α-synuclein, whether denatured by boiling or not, runs at around 60 kDa on native gels and size exclusion chromatography. “A good experiment I recommend to those planning to repeat the work is to run the denatured recombinant protein side by side with their cell extracts as a control in these assays,” he told ARF. He also pointed out that while almost all the synuclein Bartels extracted from human red blood cells migrated as a 60 kDa protein on native gels, 90 percent of the protein ran as a monomer on denaturing gels, even after crosslinking, suggesting tetramers represent a minor species. An alternative explanation is that crosslinking is inefficient.

“Clear native gels are certainly not the be all and end all of molecular size determination,” agreed Selkoe, which is why Bartels used a variety of sophisticated biophysical techniques to back up the electrophoresis experiments. Scanning transmission electron microscopy (STEM) and sedimentation analytical ultracentrifugation, an older but highly accurate technique for determining the oligomeric structure of native proteins, also predicted the protein is a tetramer. Lashuel pointed out that the STEM data reflect a sample that is heterogeneous. At least two major species are observed, 30 kDa an 55 kDa, which is inconsistent with the analytical ultracentrifugation results, which show a single ideal species.

“I think these researchers have done a very careful and thorough job,” said Gregory Petsko, from Brandeis University, Waltham, Massachusetts. Petsko, together with Dagmar Ringe and Thomas Pochapsky, also at Brandeis, and Quyen Hoang at the Indiana University School of Medicine, Indianapolis, have evidence that recombinant α-synuclein expressed in bacteria also exists as a tetramer. Their findings will be published in the Proceedings of the National Academy of Sciences USA in the coming weeks.

Petsko told ARF that what is really important about the Selkoe group’s work is the discovery that the tetramer is stable and does not readily form amyloid. Bartels used circular dichroism spectroscopy to study the tetramer from red blood cells and 3D5 neuroblastomas, and found to his surprise that the spectra were characteristic of a protein with α-helical structure. This is in stark contrast to many studies that found the monomer was unstructured. Furthermore, thioflavin T-based assays showed that the tetramer did not form amyloid over a 10-day period, unlike recombinant monomers which began to aggregate around day 4, and continued to form amyloid for the next six days. “In terms of disease, this suggests that the tetramer, which is the predominant species, does not aggregate, and either needs other factors to make it aggregate or, more likely, it needs to disassemble first,” said Selkoe.

That could have important implications for therapeutic approaches. “If the hypothesis that α-synuclein adopts a defined tetrameric structure stands the test of time, which I believe it will, based on the data I’ve seen, then this structural insight represents a new paradigm for developing tetramer stabilizing drugs to prevent α-synuclein aggregation linked to Parkinson’s disease,’’ wrote Jeffery Kelly, from Scripps Research Institute, in an e-mail to ARF. “Numerous biotechnology and pharmaceutical companies will look at this opportunity very carefully,” he added. Kelly has pioneered the same strategy to tackle familial amyloid polyneuropathy, a rare disease caused by misfolding of mutant transthyretin, a protein that migrates from the liver to other tissues. A committee of the European Medicines Agency recently recommended approval of a small molecule arising from that work that stabilizes transthyretin tetramers, preventing them from dissociating into amyloidogenic monomers (see ARF related news story).

Petsko stressed that the synuclein tetramer is not necessarily the physiological form. But he agreed that if it is the physiological form, then stabilizing the tetramer could be a viable therapeutic strategy. “It gives us options that we would not have thought of before,” he said.

In fact, other researchers in the field doubted the physiological relevance, stressing that membrane-bound α-synuclein is the most important. On that point, Bartels found that the tetramer binds lipids with around 10-fold more potency than the monomer, and Selkoe believes it is much more likely that the tetramer is the more physiologically relevant.

Time will tell how important this ordered, tetrameric form of synuclein is. Meanwhile, researchers are concerned that this new order may bring its own form of chaos. Several labs, both academic and private, have tried to reproduce these findings since they were first presented in public, according to some researchers, and so far there are no reports of success, though it could be early days yet. “I am not rejecting the possibility that α-synuclein exists as a structured oligomer. In fact, it would be much more interesting if it did,” said Lashuel, but he added that “it is of critical importance and in the interest of everyone, including Parkinson’s disease patients and their families, to investigate and reproduce this data quickly.”—Tom Fagan.

Reference:
Bartels T, Choi JG, Selkoe D. a-Synuclein occurs physiologically as a helically folded tetramer that resists aggregation. Nature. 2011 Aug 14. Abstract

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Comments on News and Primary Papers

  1. The study of Bartels et al. on α-synuclein is very interesting and provocative, and has the potential to further the understanding of the functional role of α-synuclein. However, given that the study was performed primarily with α-synuclein derived from red blood cells (RBCs), it will be important to investigate if the behavior of α-synuclein is similar in the central nervous system, where the lipid content in the plasma membranes is rather different. It is worth noting that utilizing native gels, the monomer of α-synuclein can be readily identified in human brains. The behavior of α-synuclein in RBCs might be unique to the peripheral compartments. It will be also important to determine if other investigators are able to reproduce such results in RBCs versus the CNS utilizing the scanning transmission electron microscopy approach.

    View all comments by Eliezer Masliah
  2. Reply to comments by Eliezer Masliah and Hilal Lashuel

    Comments by Dr. Lashuel within the ARF news story and the added comment by Dr. Masliah do not fully reflect what we actually published in the paper. First, Dr. Lashuel suggested that one should run the denatured recombinant protein side by side with cell extracts. That is just what we did, and showed in Supp. Fig. S5 A and B. These two figures demonstrate the distinct migration of the cell-derived α-synuclein versus both recombinant α-synuclein and denatured cell-derived α-synuclein. Also, Dr. Lashuel chooses not to mention the higher percentages of cellular α-synuclein we observed to migrate as tetramers versus monomers upon crosslinking of intact cells other than red blood cells (RBC). In any event, such in-vivo crosslinking is not fully efficient, as the news story points out.

    Dr. Masliah appears to overlook the considerable data on α-synuclein in cell types other than RBC in our paper, most importantly, the native (α-helically folded) α-synuclein purified from human neuroblastoma cells, which was sized by scanning transmission electron microscopy (STEM) at ~55,000 Daltons (Fig. S7), a value indistinguishable from the STEM sizing of the RBC α-synuclein, addressing what he is inquiring about. There are several additional data about α-synuclein from cells other than RBC in the article, for example, in Fig. 1, Fig. S5A, Fig. S6, Fig. S8, and Fig. S9. In addition, even though much of our work was done on the abundant, native α-synuclein found endogenously in fresh RBC, the resulting findings should still have more relevance to other human cell types (including neurons) than the bacterially expressed protein extensively used heretofore. Indeed, we report a post-translational modification (N-acetylation) that we documented in RBC by mass spectrometry (Fig. S4) that was not shown to exist on α-synuclein previously, and that is not present on bacterially expressed α-synuclein.

    Finally, it seems unlikely to us that "several labs, both academic and private, have tried to reproduce these findings since they were first presented in public, according to some researchers" as the data were only presented publicly a few months before the article appeared, and the multiple biochemical and biophysical approaches we used to purify, characterize, and size the native cellular form of α-synuclein over more than two years will understandably take time and effort to reproduce. To this end, we have provided very extensive details about our methods to readers via the nature Protocol Exchange website, as we mentioned in the paper (see Methods and Nature Protocol Exchange), and we are actively communicating with other labs (including Dr. Lashuel's) to assist them in any way we can to reproduce the findings.

  3. These are interesting observations presented by Bartels et al. The data suggest that equilibrium may exist between a tetrameric aggregation-incompetent α-synuclein species, and an aggregation-competent monomer. Shifting the equilibrium to the monomeric form would be expected to promote the formation of amyloid and Lewy bodies. Consistent with their notion that certain oligomeric forms of α-synuclein resist aggregation, we have also previously identified aggregation-incompetent, non-toxic assemblies in certain "pathology-free" regions of the A53T transgenic mouse brain (Tsika et al., 2010). The size of the α-synuclein assemblies documented in that study ranged from ~225 to 50 kDa. This size estimate was obtained by size exclusion chromatography, and is an interpretation based on the elution profile of globular protein standards. It is also important to note that we were likely detecting the most stable species under the specific conditions used for brain extracts (100,000 x g centrifugation of protein soluble in 1 percent Triton X-100). It is unclear whether the tetrameric species documented by Bartels et al. are stable under these extraction conditions. It will be of future interest to determine if such tetrameric species identified by Bartels et al. exist in human neurons, and if this particular form of α-synuclein is the predominant species in healthy brain.

    View all comments by Joseph Mazzulli
  4. What I find most compelling about this work is the purification of helical α-synuclein from a cellular source. It is a long-standing hypothesis, based on conserved protein domains, that the active form of synuclein should be helical, but it has been difficult to directly demonstrate this species in vivo. Evidence that the native form is tetrameric, and that the tetramer has higher lipid affinity than monomer, is also intriguing. Identification of this conformer should help facilitate efforts to understand the normal function of α-synuclein.

    It remains to be seen whether the helical tetramer is the predominant cellular species in brain neurons, an important question with relevance to Parkinson’s disease, which is not addressed directly by this study. I personally favor the idea that α-synuclein in the living neuron exists in conformational equilibrium, and that this equilibrium is influenced by phospholipids, fatty acids, and other environmental factors. Maintenance of this equilibrium might regulate synuclein function, while its perturbation could lead to protein misfolding and aggregation. Future experiments will hopefully illuminate these issues.

    View all comments by Julia George
  5. Dr. Selkoe’s group has, in my opinion, made a breakthrough on the protein chemistry of α-synuclein. The finding that this protein indeed exists natively as a structured oligomer is very exciting and opens the field up for new experiments to elucidate its molecular biology and potentially also for new strategies for therapy development. I would like to add a few thoughts on biophysics to the ongoing discussion on this forum.

    First, it does not come as a surprise that α-synuclein indeed natively adopts α-helical secondary structure, because secondary structure prediction algorithms predict substantial helix propensity in the N-terminal and central regions of the 140-residue isomer. This is in contrast to typical intrinsically unstructured proteins (IUPs) for which little ordered secondary structure (only “coil”) is predicted. The fact that α-synuclein previously has been deemed as unstructured has in this regard been somewhat of an enigma (to me). The present results clarify the issue very nicely.

    Second, I still think that the stoichiometry of the present oligomer (tetramer) might not be the last word, although in particular, the sedimentation equilibrium experiments provide strong support for a tetramer. A full structure determination by crystallography or nuclear magnetic resonance might eventually confirm the tetramer. However, the stoichiometry is, at present, not that important. What is important is that a native oligomeric folded state has been identified.

    Third, it is not surprising that the STEM indicates some heterogeneity, as oligomers actually can be expected to dissociate in diluted samples under STEM. (I would actually have expected to see more monomeric species, but maybe these cannot be detected.) The apparent presence of a 30-kDa species is presumably due to such dissociation and possibly also an indication that the native tetramer is a dimer of dimers (containing two different monomer interaction surfaces).

    Fourth, the question has been raised whether α-synuclein really adopts the folded oligomeric state also in the central nervous system. I think that this, in fact, is very likely, and that the described oligomer indeed is a native conformation in all cell types. I think so not only because the authors test a number of cell lines including neuroblastoma cells and mouse cortex tissue, but also because it would appear strange from a biological point of view if the physiologically relevant species in the human brain should resemble test-tube recombinant peptides rather than the natively folded state of the protein found in cell lines and tissue samples.

    View all comments by Torleif Hard
  6. We thank Julia George and Torleif Hard for their helpful comments and agree with both comments.

    The four points outlined by Dr. Hard are compelling and make sense, and they reflect our own views about α-synuclein based on the new information.

    View all comments by Dennis Selkoe
  7. Reply to Selkoe Comment
    In Dr. Selkoe's comment above, he writes: "First, Dr. Lashuel suggested that one should run the denatured recombinant protein side by side with cell extracts. That is just what we did, and showed in Supp. Fig. S5 A and B. These two figures demonstrate the distinct migration of the cell-derived α-synuclein versus both recombinant α-synuclein and denatured cell-derived α-synuclein."

    It would be great if the authors could provide more details about the exact conditions they used to generate the data shown in this figure, more specifically how they prepared the denatured α-synuclein in Figs. S5 A and B. Which denaturing agents or methods were used? How stable is the denatured protein and reproducibility of the data? This information is not in the manuscript, supplementary materials, or figure legend, and is essential for reproducing this particular experiment.

    Also, it would be great if the authors could explain the discrepancy between the data in Fig. 1B and supplementary Fig. S5. In figure 1B, they indicated, as quoted below from Nature paper, that monomeric α-synuclein runs below the 14 kDa molecular weight marker in a clear native gel, whereas in Fig. S5, they showed that it migrates at a much higher molecular weight in the same gel system.

    The better resolution CN-PAGE without Coomassie dye also revealed small amounts of apparent monomers running below the 14 kDa molecular weight markers (Fig. 1B, lanes 1-4,6).

    View all comments by Hilal Lashuel

References

News Citations

  1. Amyloid-Blocking Drug Poised for Approval for Rare Disease

Paper Citations

  1. . NACP, a protein implicated in Alzheimer's disease and learning, is natively unfolded. Biochemistry. 1996 Oct 29;35(43):13709-15. PubMed.
  2. . Conformational properties of alpha-synuclein in its free and lipid-associated states. J Mol Biol. 2001 Apr 6;307(4):1061-73. PubMed.
  3. . α-Synuclein occurs physiologically as a helically folded tetramer that resists aggregation. Nature. 2011 Sep 1;477(7362):107-10. PubMed.

Further Reading

Papers

  1. . α-Synuclein occurs physiologically as a helically folded tetramer that resists aggregation. Nature. 2011 Sep 1;477(7362):107-10. PubMed.

Primary Papers

  1. . α-Synuclein occurs physiologically as a helically folded tetramer that resists aggregation. Nature. 2011 Sep 1;477(7362):107-10. PubMed.