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Iljina M, Tosatto L, Choi ML, Sang JC, Ye Y, Hughes CD, Bryant CE, Gandhi S, Klenerman D. Arachidonic acid mediates the formation of abundant alpha-helical multimers of alpha-synuclein. Sci Rep. 2016 Sep 27;6:33928. PubMed.
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Co-Director, Brigham and Women's Hospital's Ann Romney Center for Neurologic Diseases
α-Synuclein Conformation: The Felicity of Helicity
Over the last 2½ decades, hundreds of papers have been written about the conformational states of α-synuclein, most all of them analyzing the recombinant protein in vitro, with few attempted analyses of physiological αSyn in cells. The universal consensus until five years ago was that αSyn exists biologically as a "natively unfolded" monomer, and this continues to be the broadly held assumption in the field (Fauvet et al., 2012). As a result of this strong conviction, new information suggesting that αSyn can also exist in vivo in α-helically folded multimers, principally sizing as tetramers, has gained limited acceptance. But a growing number of labs has provided evidence for the existence of the latter forms in living cells and sometimes after non-denaturing purification (Bartels et al., 2011; Wang et al., 2011; Dettmer et al., 2013; Westphal and Chandra, 2013). One telling example is the purification of αSyn from human brain under native conditions: a five-step protocol retained some α-helical multimers until the final step of full purification, which then yielded just the 14 κDa monomer (Luth et al., 2015). This result was interpreted as suggesting the loss of an unknown “stabilizing co-factor (e.g., a small lipid)” that keeps tetramers/multimers together within cells and until they are fully purified. Supportive of this interpretation was evidence that helical multimers in cells are immediately depolymerized to monomers upon cell lysis, unless the cells are lysed in a highly concentrated solution that retains ‘molecular crowding’ (see Fig. 5D in Dettmer et al., 2013).
This elegant new study provides, among other insights, an explanation for the aforementioned findings. The authors used single molecule FRET to show that incubation of the recombinant unfolded monomer with arachidonic acid (ARA) allowed the formation of α-helical multimers whose apparent size distribution peaked at 4 monomer units and whose conformation was α-helical by CD. The ARA-mediated multimers were distinct from oligomers formed by incubating αSyn alone; the authors showed previously and also here that the latter process leads to β-sheet-rich oligomers and fibrils that are neurotoxic. In contrast, the ARA-induced helical multimers resisted fibril formation and were not neurotoxic, and they produced much less pro-inflammatory activation of microglia than the β-sheet-rich oligomers (Fig. 4e,f). The initial experiments were done at supraphysiological concentrations of the two components, but they were repeated at 2 μM aSyn and 10 μM ARA, yielding smaller numbers of α-helical multimers whose apparent size again distributed around a peak of 4 monomer units (Fig. 5b, d). While the latter concentrations are in a physiological range for brain tissue, one needs to confirm that micelles of ARA were not present at 10 μM. However, the authors used 100 κDa spin filters to separate the multimers (which should be tetramers or greater to be retained) from excess free ARA that flowed through, and CD confirmed that soluble α-helical multimers were still present in the retentate (Fig. 5d).
Collectively, these intriguing data describe the in vitro reconstitution of metastable helical α-synuclein multimers that have closely similar properties to those we have described in experiments on neurons, erythrocytes, and other cells (Bartels et al., 2011; Dettmer et al., 2013), as the authors pointed out. The physiological nature of such assemblies is supported by the findings of Iljina et al. and by our description of highly similar tetramers of β- and γ-synuclein, which are not implicated in PD pathogenesis (Dettmer et al., 2013). In accord, all familial PD missense mutations have been shown to decrease the tetramer:monomer ratio in intact neural cells, and amplifying the E46K mutation by placing it into the two adjacent KTKEGV repeat motifs abrogates multimers, increases free monomers, and leads to neurotoxicity and round cytoplasmic inclusions (Dettmer et al., 2015). Thus, specific residues in the imperfect KTKEGV repeat motifs of aSyn help provide a structural basis for tetramer/multimer formation (Dettmer et al., 2015).
Together, the emerging information supports a model in which unfolded αSyn monomers, after their synthesis on the ribosome, contact the outsides of vesicles (e.g., synaptic vesicles) and fold into α-helical monomers, as was first found to occur in vitro long ago (Davidson et al., 1998). We hypothesize that the membrane-folded monomers can assemble into an energetically favored tetramer (and perhaps multimers of tetramers) that can detach from the membrane into the aqueous cytosol, where they are principally recovered (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.
Regarding function, there is evidence that αSyn multimers in living neurons can occur on the surface of synaptic vesicles, clustering them and attenuating their diffusion to the plasma membrane for exocytosis (Wang et al., 2014). Tetramer-abrogating mutations can prevent this, suggesting that the dynamic equilibrium between helical tetramers and unfolded monomers helps regulate this function. Any genetic or environmental factors that shift the equilibrium toward excess free monomers could both alter this function and predispose to the formation of β-sheet-rich oligomers that would exert cytotoxicity over time.
Relevant to this debate about αSyn native state, Theillet et al. (Theillet et al., 2016) recently used solution-phase in-cell NMR to report that unfolded, bacterial αSyn, when electroporated into mammalian cells, showed no signs of undergoing membrane interactions, oligomerization, aggregation, targeted degradation, or interaction with other cytosolic proteins. They interpreted this lack of change as indicating that “αS is disordered in mammalian cells.” An explanation for the apparent discrepancy between the various studies mentioned above and Theillet et al. arises from the methods chosen to address which forms of αSyn exist in intact cells. Complexes larger than monomers and membrane-bound forms of αS are difficult to detect via solution NMR due to their lower tumbling rates and significant line broadening, which together cause markedly decreased NMR signal/noise ratios. Thus, it is problematic to use this method to attempt to prove their non-existence. By the authors’ estimate, up to 20 percent of their electroporated αSyn could form multimers that they would not detect by NMR, as also pointed out in an accompanying commentary (Alderson and Bax, 2016). When we followed the electroporation protocol of Theillet et al. but assessed the assembly state of the introduced αSyn by intact-cell crosslinking, we observed αSyn multimers similar to those we have described for endogenous αSyn. We believe that acute electroporation of large amounts of αSyn and analysis by in-cell solution NMR may not be a quantitatively sensitive way to resolve dynamic metastable interactions of αSyn in vivo.
In summary, intact-cell crosslinking (Bartels et al., 2011; Dettmer et al., 2013), fluorescence protein complementation (Wang et al., 2014; Dettmer et al., 2015), and FRET (Burré et al., 2014) are three independent methods that have been used to observe the existence of αSyn tetramers/multimers in cells. This line of research has now been substantially enhanced by the impressive in vitro reconstitution of α-helical multimers by Iljina et al., providing an exciting advance by which to study their assembly, disassembly and biological properties in much more detail. The central lesson overall is that altered tetramer-to-monomer equilibria may represent the ultimate upstream event in the initiation of αSyn misfolding and human synucleinopathies, coming well before any "pathogenic spread" of abnormal oligomers. Stabilizing endogenous tetramers/multimers thus presents an entirely new therapeutic approach for PD, dementia with Lewy bodies, multiple system atrophy, and other αSyn disorders.
References:
Alderson TR, Bax A. Parkinson's disease: Disorder in the court. Nature. 2016 Feb 4;530(7588):38-9. Epub 2016 Jan 25 PubMed.
Bartels T, Choi JG, Selkoe DJ. α-Synuclein occurs physiologically as a helically folded tetramer that resists aggregation. Nature. 2011 Aug 14;477(7362):107-10. PubMed.
Burré J, Sharma M, Südhof TC. α-Synuclein assembles into higher-order multimers upon membrane binding to promote SNARE complex formation. Proc Natl Acad Sci U S A. 2014 Oct 7;111(40):E4274-83. Epub 2014 Sep 22 PubMed.
Davidson WS, Jonas A, Clayton DF, George JM. Stabilization of alpha-synuclein secondary structure upon binding to synthetic membranes. J Biol Chem. 1998 Apr 17;273(16):9443-9. PubMed.
Dettmer U, Newman AJ, Luth ES, Bartels T, Selkoe D. In vivo cross-linking reveals principally oligomeric forms of α-synuclein and β-synuclein in neurons and non-neural cells. J Biol Chem. 2013 Mar 1;288(9):6371-85. Epub 2013 Jan 14 PubMed.
Dettmer U, Newman AJ, Soldner F, Luth ES, Kim NC, von Saucken VE, Sanderson JB, Jaenisch R, Bartels T, Selkoe D. Parkinson-causing α-synuclein missense mutations shift native tetramers to monomers as a mechanism for disease initiation. Nat Commun. 2015 Jun 16;6:7314. PubMed.
Dettmer U, Newman AJ, von Saucken VE, Bartels T, Selkoe D. KTKEGV repeat motifs are key mediators of normal α-synuclein tetramerization: Their mutation causes excess monomers and neurotoxicity. Proc Natl Acad Sci U S A. 2015 Aug 4;112(31):9596-601. Epub 2015 Jul 7 PubMed.
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 central nervous system and from erythrocytes, mammalian cells, and Escherichia coli exists predominantly as disordered monomer. J Biol Chem. 2012 May 4;287(19):15345-64. Epub 2012 Feb 7 PubMed.
Luth ES, Bartels T, Dettmer U, Kim NC, Selkoe DJ. Purification of α-synuclein from human brain reveals an instability of endogenous multimers as the protein approaches purity. Biochemistry. 2015 Jan 20;54(2):279-92. Epub 2014 Dec 23 PubMed.
Theillet FX, Binolfi A, Bekei B, Martorana A, Rose HM, Stuiver M, Verzini S, Lorenz D, van Rossum M, Goldfarb D, Selenko P. Structural disorder of monomeric α-synuclein persists in mammalian cells. Nature. 2016 Feb 4;530(7588):45-50. Epub 2016 Jan 25 PubMed.
Wang L, Das U, Scott DA, Tang Y, McLean PJ, Roy S. α-synuclein multimers cluster synaptic vesicles and attenuate recycling. Curr Biol. 2014 Oct 6;24(19):2319-26. Epub 2014 Sep 25 PubMed.
Wang W, Perovic I, Chittuluru J, Kaganovich A, Nguyen LT, Liao J, Auclair JR, Johnson D, Landeru A, Simorellis AK, Ju S, Cookson MR, Asturias FJ, Agar JN, Webb BN, Kang C, Ringe D, Petsko GA, Pochapsky TC, Hoang QQ. A soluble α-synuclein construct forms a dynamic tetramer. Proc Natl Acad Sci U S A. 2011 Oct 25;108(43):17797-802. Epub 2011 Oct 17 PubMed.
Westphal CH, Chandra SS. Monomeric synucleins generate membrane curvature. J Biol Chem. 2013 Jan 18;288(3):1829-40. Epub 2012 Nov 26 PubMed.
View all comments by Dennis SelkoeBrandeis University
The formation of helix-rich α-synuclein (αSyn) multimers in the presence of arachidonic acid (ARA, a common fatty acid found in brain tissue) described by Iljina et al. in their Sci. Rep. article is both welcome further evidence for such species, and, frankly, a little embarrassing for those of us who have previously published experimental evidence for their existence. The authors performed some fairly simple yet elegant experiments showing conclusively that helix-rich, non-toxic, and easily dissociated αSyn multimers form with much different characteristics than the αSyn oligomers formed in the absence of ARA. The first thing that crossed my mind upon reading this article was “Why didn’t I think of that?” The second was “Why are we still arguing about this?” It is admittedly difficult to drop a long-cherished viewpoint, but the concept of αSyn 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 αSyn can assume different forms under different conditions. Clinging to an all-or-nothing view of αSyn behavior is blinding us to multiple possibilities for PD prophylaxis/treatment.
One other important aspect of this work to note is that apparently ARA needs to be above the critical micelle concentration in order for the multimers to form, but upon dilution of ARA, they remained relatively stable (there was some increase in oligomer formation, and some loss of material, but the helicity of the remaining multimers was maintained). This indicates (as we have previously suggested) that the multimers are kinetic, rather than thermodynamic, products, and likely the result of interaction between cell membrane components and bound αSyn. Furthermore, it appears that the lipid is a component of the multimers, not just a catalyst for their formation. In other words, the method of introducing αSyn into an experimental system will affect what form of αSyn is observed.
View all comments by Thomas PochapskyWeill Cornell Medicine
The paper is interesting and agrees with our previous studies where we reported multimerization of α-helical α-synuclein in the presence of liposomes and brain membranes that mediate SNARE-complex formation, and the protection of membrane-bound α-synuclein from aggregation and thus cellular toxicity (Burré et al., 2014; Burré et al., 2015). Overall, it has been becoming clear in the last couple of years that there are more species of α-synuclein in a neuron than previously anticipated, which exist in various equilibria. Along this line, it would be helpful for the α-synuclein field to distinguish these various α-synuclein species by using separate terms for pathologically relevant species that form β-sheets (e.g., oligomers) versus physiologically functional species that are α-helical and do not aggregate (e.g., we have previously introduced the term “multimers”). In this vein, it is confusing that the authors use the term “aggregate formation” in presence of ARA in Fig. 1a/b/d, but discuss at the same time that α-synuclein in presence of ARA is α-helical and is at least partially protected from fibril formation (Fig. 1c).
We and others have reported that α-helical multimers of α-synuclein play a role in clustering of synaptic vesicles in the presynaptic terminal (Diao et al., 2013; Wang et al., 2014). The physiological role of ARA-induced multimers of α-synuclein remains to be determined, in particular because α-synuclein is highly enriched on synaptic vesicles and may not encounter much cytosolic ARA under normal physiological conditions. Possibly, α-synuclein multimers may come off synaptic vesicles upon releasing ARA from synaptic vesicle phospholipids, but this remains to be tested.
A few things I would have loved to see or am surprised about:
References:
Burré J, Sharma M, Südhof TC. α-Synuclein assembles into higher-order multimers upon membrane binding to promote SNARE complex formation. Proc Natl Acad Sci U S A. 2014 Oct 7;111(40):E4274-83. Epub 2014 Sep 22 PubMed.
Burré J, Sharma M, Südhof TC. Definition of a molecular pathway mediating α-synuclein neurotoxicity. J Neurosci. 2015 Apr 1;35(13):5221-32. PubMed.
Diao J, Burré J, Vivona S, Cipriano DJ, Sharma M, Kyoung M, Südhof TC, Brunger AT. Native α-synuclein induces clustering of synaptic-vesicle mimics via binding to phospholipids and synaptobrevin-2/VAMP2. Elife. 2013;2:e00592. PubMed.
Wang L, Das U, Scott DA, Tang Y, McLean PJ, Roy S. α-synuclein multimers cluster synaptic vesicles and attenuate recycling. Curr Biol. 2014 Oct 6;24(19):2319-26. Epub 2014 Sep 25 PubMed.
Burré J, Sharma M, Südhof TC. α-Synuclein assembles into higher-order multimers upon membrane binding to promote SNARE complex formation. Proc Natl Acad Sci U S A. 2014 Oct 7;111(40):E4274-83. Epub 2014 Sep 22 PubMed.
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