Form and function: What makes α-synuclein toxic?
Changes in shape and structure can turn a well-behaved cellular protein into something altogether more sinister, but in many cases researchers do not know exactly which modifications are at fault. At the 12th International Conference on Alzheimer’s and Parkinson’s Diseases, held March 18-22 in Nice, France, speakers grappled with what makes α-synuclein go rogue. Researchers led by Dennis Selkoe of Brigham and Women’s Hospital, Boston, correlated the presence of pathological variants of α-synuclein with a shift from multimeric forms toward freer monomers. The data address a controversy over whether α-synuclein can form higher-order structures that protect against aggregation. Meanwhile, Paola Picotti at ETH Zurich debuted a method of determining the aggregation state of α-synuclein in complex mixtures.
To study pathological changes, researchers first have to know the native state of α-synuclein. Scientists had long believed the molecule existed as a disordered monomer, but this view was challenged in 2011 when Selkoe and a group led by Gregory Petsko, who is now at Weill Cornell Medical College in New York City, reported the isolation of α-synuclein tetramers with an -helical structure from red blood cells, neural cell cultures, and bacterial cells (see Aug 2011 news and commentary; Wang et al., 2011). Tetramers do not aggregate into amyloid-like fibrils, and might in fact represent the principal physiologic species, the researchers proposed. The results stirred debate, with other scientists reporting that they were unable to replicate the findings, albeit not using the same methods (see Feb 2012 news and commentary).
What Does Physiological α-Synuclein Look Like? Possible properties based on recent publications. [Image courtesy of Ulf Dettmer, BWH Boston.]
Since then, however, several other groups have independently turned up evidence for multimeric forms of α-synuclein (Trexler and Rhoades, 2012; Westphal and Chandra, 2013; Gould et al., 2014). Two independent groups, one led by Subhojit Roy at the University of California, San Diego, and the other by Nobel laureate Thomas Sdhof at Stanford University, Palo Alto, California, reported that α-synuclein multimers corral and dock synaptic vesicles, suggesting a physiological role for the structures (see Oct 2014 news). In one of these synaptic studies (from the Sdhof lab), multimers were described only on membranes, while cytosolic α-synuclein remained monomeric. New work from Sdhof and first author Jacqueline Burr, who is also now at Weill Cornell, extends the findings, reporting that mutations in α-synuclein which interfere with membrane binding promote aggregation of the protein (see Burr et al., 2015).
In Nice, Selkoe emphasized that tetramers remain stable only in crowded molecular environments, falling apart upon cell lysis (see Dettmer et al., 2013; Luth et al., 2015). This may explain why most isolation methods fail to detect the higher-order structures, he said. At AD/PD, first author Ulf Dettmer said, “Alpha-synuclein multimerization is best studied in intact cells.” Dettmer described how he studied a human cerebral cortex biopsy removed from an otherwise healthy epileptic surgery patient. By finely mincing the material without lysing cells, and then adding a cross-linker to the intact brain bits, Dettmer was able to recover abundant α-synuclein tetramers. In his hands, these structures associated with cytosolic fractions, whereas monomeric α-synuclein appeared in both cytosol and membrane fractions. “We propose that an intact neuron maintains a physiological equilibrium of monomers and multimers,” Dettmer said.
In addition, the researchers traced the protein’s ability to form multimers to KTKEGV, six amino acids that repeat at least six times in the α-synuclein sequence. The known familial Parkinson’s mutation E46K replaces the glutamic acid in this sequence with lysine. Introducing this mutation lowered the amount of α-synuclein multimers present in cells, Dettmer said. Making the same mutation in a second of the repeat domains dropped multimers further, and three such mutations virtually
abolished them, he added.
Does this relate to pathology? In a poster at AD/PD, Victoria von Saucken of Selkoe’s group showed that neuroblastoma cells expressing E46K-like and other mutations in the KTKEGV-repeat motifs accumulated aggregates of α-synuclein and died more quickly than control cells. Dettmer examined E46K and four additional missense mutations known to cause familial ParkinsonÕs disease: A53T, A30P, H50Q, and G51D. Like E46K, each mutation shifted the balance of α-synuclein in mouse brain from multimeric toward monomeric forms. Human neuronal cells expressing α-synuclein protein containing either a single or all five familial PD mutations built up more aggregated species due to tetramer destabilization, according to a poster presented by Tim Bartels of Brigham and Women’s.
The finding implies that stabilizing α-synuclein tetramers could help slow disease, Dettmer said in his talk. Similar strategies have been used to treat peripheral transthyretin amyloidosis (see Aug 2011 news; Dec 2013 news). One such drug, tafamidis, is currently approved for use in Europe and Japan, though not in the United States. Outside of this tetramer-stabilization approach, however, the debate about a native tetramer is not crucial to targeting pathologic, aggregated forms of α-synuclein, pharma researchers told Alzforum.
Acceptance of the multimeric model seems to be growing in the field. At AD/PD, a poster from Woon Ki Lim and Hae Ja Shin of Pusan National University in South Korea described a mass spectrometry analysis of α-synuclein that concluded that the protein forms highly ordered structures consistent with tetramers. The controversy now may be shifting to the questions of where multimers occur and what role they play. Another poster, from Harutsugu Tatebe and colleagues at Kyoto Prefectural University of Medicine in Japan reported no evidence of tetramers in human cerebrospinal fluid. To Dettmer’s mind, this finding is consistent with the tetramers being unstable outside of cells.
“The majority of data from many labs would suggest that in brain, soluble α-synuclein is monomeric but self-assembles on membranes,” Sreeganga Chandra at Yale University, New Haven, Connecticut, wrote to Alzforum. “However, it is possible to purify the tetramer from blood, though it is labile and will disassemble to monomers. So maybe this ‘controversy’ is just comparing brain versus blood.” The neuronal cell data from Dettmer et al. suggest otherwise. Chandra noted that she was not at AD/PD and could not comment on the latest data presented there. “The data on α-synuclein multimers cannot be ignored anymore,” Roy wrote to Alzforum. “In particular, a concept is emerging that monomers and multimers are in dynamic equilibrium, and the multimeric state helps the protein associate with vesicles. It also seems that shifting this equilibrium away from multimers favors aggregation of the protein.”
Not everyone is convinced, however. “I don’t see evidence that α-synuclein in its native state exists predominantly as a tetramer, but we cannot rule out the possibility that it can form oligomers upon interaction with membrane,” Hilal Lashuel at the Swiss Federal Institute of Technology (EPFL), Lausanne, told Alzforum. He had not heard the latest talks. Others, including Sdhof, see evidence for multimers, but not specifically for tetrameric forms. These scientists, too, have not yet seen the new data.
If the structure of α-synuclein determines the protein’s toxicity, researchers would want a quick and simple way to measure its state in body fluids. Picotti from the ETH said in her talk that an ideal method would allow researchers to analyze complex mixtures without labeling or purification. She developed a technique in which she first briefly digested mixtures with proteinase K before quenching the enzyme. This cuts only one or two sites on a protein, typically the most exposed, and the sites exposed vary depending on how the protein folds. Then the mixtures undergo tryptic digestion and mass spectrometry. Compared to standard mass spec results, the resulting spectra lack one or two peaks that correspond to the cleavage sites snipped by proteinase K (see Feng et al., 2014).
Picotti used this method to compare α-synuclein monomers to aggregated _-sheet forms. In each spectrum, she saw two unique sites that were missing in the other one. The unique sites served as markers for each conformation. “When we saw this, we got pretty excited,” she told the audience. The method may allow researchers to follow the kinetics of aggregation in vivo and to analyze modulating factors, she suggested. She told Alzforum she would like to apply the technique to the α-synuclein tetramers isolated by Selkoe’s group, as well, to see if her method can distinguish this species. In theory, the regions where subunits interface would be protected from proteolysis, potentially providing a unique signal from this form. She is also investigating whether the α-synuclein spectra could serve as a biomarker of disease in people. Other scientists said that this technique is innovative but not readily applicable for clinical purposes.
-Madolyn Bowman Rogers
Retrieved 27 November 2018 from https://www.alzforum.org/news/conference-coverage/form-and-function-what-makes-synuclein-toxic
