Given a little coaxing, monomers of α-synuclein huddle into non-toxic, soluble multimers, according to recent findings. As described September 27 in Scientific Reports, arachidonic acid, the most abundant fatty acid in the brain, provides the incentive. Researchers led by David Klenerman at the University of Cambridge in England reported these multimers sport α-helices and dissociate without hassle. Unlike fatty acid-deprived counterparts that form β-sheet-rich fibrils, the multimers appeared benign to neurons. The researchers proposed that in the cramped quarters of the membrane-rich synapse, fatty acids tip the balance from harmful, rigid fibrils of α-synuclein toward the friendlier, soluble species.
“Collectively, these intriguing data describe the in vitro reconstitution of metastable α-helical α-synuclein multimers that have similar properties to those we have described in experiments on neurons, erythrocytes, and other cells,” wrote Dennis Selkoe of Brigham and Women’s Hospital in Boston in a comment to Alzforum (see full comment below).
Like other amyloidogenic proteins, α-synuclein is known for forming fibrils. These form Lewy body inclusions found in people with Parkinson’s disease and other synucleinopathies. However, researchers have long tried to discern what configuration the native soluble protein takes. Over decades a consensus gradually built that the soluble α-synuclein was a monomer lacking secondary structure. In 2011, two studies turned this view upside down. Researchers in Selkoe’s lab, and in the labs of Quyen Hoang of Indiana University School of Medicine in Indianapolis and Thomas Pochapsky, Dagmar Ringe, and Gregory Petsko at Brandeis University in Waltham, Massachusetts, claimed that native α-synuclein predominantly existed as a soluble tetramer with α-helical structure. Further, they proposed that toxic fibrils are only formed by the less common disordered monomers (see Aug 2011 news; Wang et al., 2011).
Other investigators could not corroborate these findings. A collaboration among six different research groups—including Hilal Lashuel’s at École Polytechnique Fédérale de Lausanne, Switzerland, and Eliezer Masliah’s, then at the University of California, San Diego—maintained that a disordered monomer is the normal physiological form (Feb 2012 news). Still others had a slightly different take. In two separate studies, researchers in Tom Südhof’s lab at Stanford University and Subhojit Roy’s lab at the University of California, San Diego reported that in healthy neurons, α-synuclein resides in synaptic compartments as a multimer that comingles with membranes and helps cluster synaptic vesicles (see Oct 2014 news). What’s more, preventing α-synuclein’s association with membranes promoted its aggregation into insoluble, neurotoxic forms, claimed Südhof and colleagues (see Burré et al., 2015; Apr 2015 conference coverage).
Fat-Free Fibrils. Without ARA (top), a mix of α-synuclein monomers, oligomers, and fibrils form (all in left panel). Centrifugation removes fibrils (supernatant, middle; pellet, right). With ARA, only oligomers form (bottom at two magnifications). [Image courtesy of Iljina et al., Scientific Reports, 2016.]
Now the new evidence. Given α-synuclein’s physiological role within membrane-rich synaptic compartments, first author Marija Iljina and colleagues wondered how α-synuclein would behave in the presence of fatty acids, which are found in cell membranes and can be enzymatically released from the lipid bilayer (see Rossetto et al., 2006). Because arachidonic acid (ARA) is the most abundant fatty acid in the gray matter of the brain, Iljina decided to test it on α-synuclein first. The researchers mixed 35μM of fluorescently labeled, full-length α-synuclein with 1μM ARA, and monitored protein aggregation via fluorescence resonance energy transfer. In the presence of ARA, α-synuclein rapidly oligomerized. An apparent tetramer was the most predominant species, but larger species, ranging up to 20-mers, appeared after a few hours. Without ARA, few multimers formed, and only after vigorous shaking of the sample. Shaking breaks apart growing fibrils to increase the number of nucleation “seeds” for oligomerization.
To investigate the conformation of α-synuclein in the samples, the researchers used circular dichroism spectroscopy, which detects different secondary structures. This revealed that α-synuclein transformed from a random coil into an α-helical shape shortly after the addition of ARA, and remained in that state for at least 24 hours. In contrast, β-sheets formed in samples lacking the fatty acid. A closer look at the samples under the electron microscope revealed that α-synuclein mixed with the fatty acid formed large oligomers of various shapes and sizes, while α-synuclein without ARA consisted of a mixture of monomers, smaller oligomers, and insoluble fibrils that could be recovered by centrifugation. Iljina speculated that oligomers formed in the absence of the fatty acid were precursors to fibrils.
Interestingly, multimers formed with ARA appeared to be less stable than those formed without ARA—they were easier prey for the proteasome and proteinase K, and also dissociated more readily when diluted. When the fatty acid was removed, larger oligomers dissolved, leaving only smaller ones. These retained their α-helical structure. The researchers speculated this is because they still bound ARA. This could explain why small oligomers, such as the tetramers identified by Selkoe and Hoang, can be isolated from intact cells, Iljina said. Selkoe agreed that was possible.
The researchers found that α-helical, multimeric species also formed at physiological concentrations of α-synuclein (2μM) and ARA (10μM). However, the fatty acid had less effect on the oligomerization of α-synuclein harboring the PD-associated A30P, A53T, or E46K mutations.
These in vitro findings meshed nicely with those from the previous tetramer papers as well as those pointing to α-synuclein multimerization in the presence of synaptic vesicles. Iljina pointed out that the story could be different in the expanse of the cytosol, where fatty acids such as ARA are less abundant. There, it is unclear which species would predominate, although the survival of ARA-formed oligomers after washing suggests they could be present in the cytosol as well as in the synaptic compartments.
Finally, the researchers compared the effects of different α-synuclein oligomers on neurons and microglia. Those generated without ARA triggered the abundant release of reactive oxygen species from cortical neurons, and also caused cell death, while those generated with ARA did neither. The ARA-less oligomers also stimulated a microglial cell line to pump out more TNFα, an inflammatory cytokine.
Although data from Südhof’s and other labs suggested that α-synuclein multimers are primarily associated with membranes, other researchers have found the multimers (in particular, the tetramer) in the cytosol. How might the new data shed light on that discrepancy? ARA, like other membrane-associated fatty acids, can be released from the cell membrane. Hence, cytosolic tetramers may have ARA bound to them. Jacqueline Burré, who reported that α-synuclein multimers help cluster synaptic vesicles while in Südhof’s lab, speculated that α-synuclein, which is highly enriched on synaptic vesicles, may detach from them when arachidonic acid releases from synaptic phospholipids. “This remains to be tested,” she wrote. Burré now runs her own lab at Weill Cornell Medical College in New York.
Selkoe proposed a similar idea, suggesting that tetramers form on membranes but can then detach into the cytosol and can be recovered from there (see Dettmer et al., 2013). “Perhaps in the process of tetramerization and detachment from membranes, a bound lipid component stabilizes the helical multimer, akin to the role of ARA in the in vitro experiments of Iljina et al.,” he wrote.
Pochapsky welcomed further evidence of multimeric α-synuclein. “The concept of α-synuclein as spaghetti soup in the cytosol, in the presence of so many possible nucleating agents and chaperones (including, as we now see, ARA) really needs to give way to the reality that α-synuclein can assume different forms under different conditions,” he wrote (see full comment below).
The idea that α-synuclein exists as a multimer stabilized by fatty acids makes biological sense, commented Petsko, now of Weill Cornell in New York. “We have to find an explanation not only for the fact that a lot people get PD, but also for the fact that most people don’t,” he said. “It makes more sense that α-synuclein can adopt a structure, and that it does something.” Petsko, who recently reported that caspase-1 cleavage of α-synuclein promotes its toxic aggregation, pointed out that there are likely several fatty acids or other molecules capable of stabilizing α-synuclein multimers, as well as multiple pathways that might bungle their formation and cause disease, such as mutations and/or truncations (see Wang et al., 2016). Critics of the α-synuclein tetramer hypothesis did not respond in time for this article.
“The central lesson overall is that altered tetramer-to-monomer equilibria may represent the ultimate upstream event in the initiation of α-synuclein misfolding and human synucleinopathies, coming well before any ‘pathogenic spread’ of abnormal oligomers,” commented Selkoe. “Stabilizing endogenous tetramers/multimers thus presents an entirely new therapeutic approach for PD, dementia with Lewy bodies, multiple system atrophy, and other α-synuclein disorders.”
The strategy has precedent. Tafamidis, approved in the EU for treating familial amyloid polyneuropathy, stabilizes tetramers of transthyretin, preventing the protein from forming fibrils (see Aug 2011 news).
Ilgina and colleagues plan to look at how other fatty acids affect the structure of α-synuclein, and proposed that the acids could work as therapeutic treatment. However, the solution may not be straightforward. A previous study reported that the configuration of α-synuclein in the presence of another brain fatty acid—docosahexanoic acid (DHA)—depended on concentration. A little DHA stabilized soluble multimers, while too much triggered formation of fibrils (see De Franceschi et al., 2011).—Jessica Shugart
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- Form and Function: What Makes α-Synuclein Toxic?
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