[Downloaded free from http://www.nrronline.org on Friday, July 10, 2020, IP: 158.109.49.69] NEURAL REGENERATION RESEARCH www.nrronline.org PERSPECTIVE Inducing α-synuclein compaction: a solid-state nuclear magnetic resonance, revealing that the NAC domain constitutes the core of the fibril and arranges into a Greek-key β-sheet new strategy for inhibiting α-synuclein motif (Tuttle et al., 2016). This motif was also observed in the structure of two α-syn fibrils determined by cryo-electron microscopy. To inves- tigate if the Greek-key β-sheet motif already exists at the early stages aggregation? of aggregation Carija et al. (2019) conducted a study in which they engineered an artificial disulfide bound between two residues within the NAC domain, which will thermodynamically favor the formation of the Greek-key β-sheet in the mutant protein (α-synCC), if this motif Proteins might misfold during translation and folding or even once they is populated in the initial assemblies (Figure 1). Importantly, they con- are in their native states, due to stochastic fluctuations, destabilizing mu- firmed that the double cysteine mutation did not alter the predicted in- tations or cellular stress. Aberrant protein species are usually detected trinsic aggregation propensity, hydrophobicity and disorder properties and either refolded or cleared by the protein quality control machinery of the sequence, when compared with that of the wild type protein (wt (Ciechanover and Kwon, 2015). When misfolded protein conformers α-syn). On the other hand, the authors verified that, as intended, the cannot be degraded, they tend to self-assemble to form aggregates, a two proteins were conformationally distinct, with α-synCC adopting a characteristic of many neurodegenerative diseases. more compact conformation. This conformational difference impacted Aggregated α-synuclein (α-syn) is the main component of Lewy bod- dramatically the aggregation behavior of the proteins. The impact of the ies and Lewy neurites, proteinaceous inclusions that constitute the ma- jor histopathological hallmarks in Parkinson’s disease, the second most introduced conformational restraint was assayed in different conditions common neurodegenerative disorder after Alzheimer’s disease (Spillatini since α-syn is known to adopt a variety of structures in the aggregated et al., 1997). α-syn is a small protein, highly abundant in the presynaptic state, depending on its microenvironment. These α-syn assemblies terminal of dopaminergic neurons, with a high conformational plas- behave as strains, being able to propagate their structural imprints to ticity. The precise role of α-syn remains unknown, but several functions endogenous α-syn. The existence of such strains might well account have been ascribed to this polypeptide, including the regulation of the for the different pathologies seen in the synucleinopathies and even presynaptic vesicular pool and the protection of nerve terminals against among individuals affected by Parkinson’s disease (Bousset et al., 2013). injury. In terms of structure, it is largely believed that monomeric α-syn In agreement with previous observations, Carija et al. (2019) observed behaves as an intrinsically disordered protein under physiological con- that α-syn aggregation was influenced by pH and ionic strength, orig- ditions in the neuronal cytosol. This unordered conformation shifts to inating strains with distinct conformations, able of being internalized an amphipathic helical structure upon binding to membranes (Uversky by human cells. Furthermore, the aggregation of wt α-syn and α-synCC and Eliezer, 2009). Moreover, it has been reported that α-syn can occur in the same conditions was also different. α-SynCC aggregation was also as an alpha-helical folded tetramer under native conditions (Bartels always lower than the one of wt α-syn, but the impact of the disulfide et al., 2011), but the prevalence of this assembly is still under debate. bridge was particularly significant at acidic pH (5.0). While wt α-syn Although the precise cause of Parkinson’s disease onset remains to be showed the highest aggregation propensity at acidic pH, α-synCC did deciphered, α-syn is seen as one of the most compelling targets for not aggregate at this pH, even after 14 days. Accordingly, human H4 therapeutic intervention. Indeed, interfering with the conversion of the cells incubated with α-synCC presented only small size intracellular above described functional α-syn conformations into misfolded ones inclusions, in contrast to the large aggregates observed for wt α-syn. able to self-assemble and form toxic amyloid aggregates within dopami- The high aggregation of wt α-syn at acidic pH is due to a decrease in nergic neurons appears as one of the most promising strategies to fight its net charge [isoelectric point (pI) = 4.67] and, consequently, in the this devastating disease. electrostatic repulsions that help keeping the protein soluble. However, α-syn primary structure consists of the N-terminal domain, a central contrarily to what was assumed, this is not the only determinant and region known as the non-amyloid β component (NAC), and the C-ter- Carija et al. (2019) demonstrated that, even in this pro-aggregational minal domain (Uversky and Eliezer, 2009). The N-terminal domain conditions, the initial structure of the protein determines its ability to (residues 1–60) contains an imperfect conserved repeat KTKGEV and aggregate. This conformational control on the aggregating properties of it acts as the membrane anchor region of α-syn (Fusco et al., 2014). the proteins was observed in all the assayed conditions, which indicates The NAC domain (residues 61–95) is highly hydrophobic and prone to that is independent of the environment. This effect ultimately relies on aggregation, being responsible for the ability of α-syn to undergo a con- the degree of exposure and flexibility of the hydrophobic and aggrega- formational change from random coil to β-sheet structure. Additionally, tion-prone NAC-domain in any given condition. it is thought that this domain regulates the affinity of α-syn for cellular Remarkably, not only α-synCC was aggregation incompetent, but membranes (Fusco et al., 2014). Finally, the C-terminal domain (residues when mixed with wt α-syn it delayed its aggregation, in a concentra- 96–140) is rich in acidic and disorder-promoting residues, counteracting tion-dependent manner. At equimolar wt α-syn:α-synCC concentra- the tendency to aggregate of the NAC domain. tions, the lag phase was approximately 2 hours longer, but delays were In vitro, the kinetics of α-syn amyloid fibril formation is best de- already observable at substoichiometric rates, suggesting that α-synCC scribed by a nucleation/polymerization reaction that renders the typical binds to higher-order α-syn assemblies, like oligomers, the fibril surface sigmoidal curve, in which an initial lag phase is followed by an expo- or at the fibril ends. At long incubation times, all the samples eventu- nential phase, ending on a plateau. The formation of the nuclei is the ally formed fibrils, but the morphology of the resulting aggregates was rate limiting step of the reaction and accounts for the lag phase. Nuclei highly influenced by the presence of α-synCC, originating smaller and are the smallest stable aggregates for which growth by monomer incor- less ordered aggregates. Because α-syn interaction with membranes ap- poration becomes faster than monomer dissociation. Monomer asso- pears to be involved both in function and disease and the NAC domain ciation to the fibril ends occurs during the elongation phase, until the participates in this process, Carija et al. (2019) tested if the aggregation depletion of monomers leads to a decline in the rate of polymerization of α-synCC in the presence of membranes was also compromised. and, ultimately, to the plateau phase (Uversky and Eliezer, 2009). Much They observed that α-synCC aggregation, which was already low in the effort has been devoted to understanding the structural properties of the absence of lipids, was strongly inhibited by its presence. Therefore, the final amyloid assemblies, especially in the last years, with the emergence authors concluded that having an accessible and flexible NAC domain is of high resolution cryo-electron microscopy. We begin to have atomic also essential for lipid mediated α-syn aggregation. Finally, they evalu- information on the molecular determinants of fibrils structure and ated if the oligomers produced by wt α-syn and α-synCC were toxic for stability. However, we know little on the conformational features of the neuroblastoma cells. The oligomers were generated in conditions report- ensemble of on-pathway and off-pathway oligomeric and proto-fibrillar ed to form toxic species and, while this was true for wt α-syn oligomers, assemblies that populate the early stages of fibrillogenesis. The identifi- α-synCC oligomers did not affect cell viability. Since the morphological cation of these species is extremely important as it may help in the early characteristics of the oligomers did not seem to explain this difference, diagnosis of the disease. Additionally, accumulating evidence suggests as they were mostly similar, the authors proposed that the monomers that the oligomers, rather than the mature fibrils, may be the neurotoxic interact through the NAC domain to form oligomers with a toxic struc- species (Winner et al., 2011). ture, an observation that is also supported by other studies. The lack of a well-defined native structure in monomeric α-syn The data collected by Carija et al. (2019) converge to demonstrate that and the lack of high-resolution structures of α-syn oligomers poses an the Greek-key β-sheet motif does not exist in the early stages of aggrega- obstacle for the use of rational drug design to target the early events tion and, thus, cannot be used for rationally designing therapeutic drugs of α-syn aggregation. Recently, a high-resolution three-dimensional or diagnostic tools, as it was previously proposed. On the other hand, structure of a fibril formed by full-length human α-syn was solved by the authors hypothesized that limiting the exposure of the NAC domain 1897 [Downloaded free from http://www.nrronline.org on Friday, July 10, 2020, IP: 158.109.49.69] Pinheiro F, Ventura S (2019) Inducing α-synuclein compaction: a new strategy for inhibiting α-synuclein aggregation? Neural Regen Res 14(11):1897-1898. doi:10.4103/1673-5374.259608 A B Figure 1 Sequence and structure of the disulfide bridge containing variant (α-synCC). Figure 2 Schematic representation of the effect of α-synuclein (α-syn) (A) Primary sequence of α-synCC, evidencing the N-terminal domain compaction on its aggregation reaction. (in blue), the non-amyloid β component (NAC) domain (in red) and When the non-amyloid β component (NAC) domain (in red) is ex- the C-terminal domain (in green). The NAC domain contains the two posed and flexible, α-syn generates toxic oligomers that evolve into mutated residues in positions 71 (Val to Cys) and 92 (Thr to Cys) (in the formation of amyloid fibrils. α-syn compaction upon incubation black). (B) Cartoon representation of α-synCC three-dimensional with epigallocatechin-3-gallate (ECGC) or nordihydroguaiaretic acid structure with the different regions colored as before. The black line in- (NGDA) or by introduction of an intramolecular disulfide bound leads dicates the formation of the disulfide bond between Cys71 and Cys92. to the production of non-toxic oligomers and a significant decrease in fibrillogenesis. in soluble α-syn could be a good strategy to inhibit aggregation. In fact, it has been observed that naturally occurring interactions between the the new creations are licensed under the identical terms. C-terminal part of the NAC domain and the C-terminal α-syn domain, Open peer reviewers: Panteleimon Giannakopoulos, Hôpitaux Universitaires as well as between the C-terminal and the N-terminal domains, pro- de Genève, Switzerland; Syed Faraz Kazim, Icahn School of Medicine at tect monomeric α-syn from aggregation. When these interactions are Mount Sinai, USA. perturbed, the NAC domain is exposed and aggregation proceeds (Ber- Additional file: Open peer review report 1. toncini et al., 2005). The results of Carija et al. (2019) could explain why some molecules that inhibit α-syn aggregation were found to induce protein compaction. This is the case of epigallocatechin-3-gallate, which References is known to inhibit α-syn fibrillogenesis while altering the course of the Bartels T, Choi JG, Selkoe DJ (2011) alpha-Synuclein occurs physiologically as a reaction towards the formation of off-pathway oligomers that are not helically folded tetramer that resists aggregation. Nature 477:107-110. toxic (Konijnenberg et al., 2016). In line with this hypothesis, the ap- Bertoncini CW, Jung YS, Fernandez CO, Hoyer W, Griesinger C, Jovin TM, pearance of non-toxic oligomers was also observed with α-synCC (Carija Zweckstetter M (2005) Release of long-range tertiary interactions potentiates et al., 2019). Interestingly, dopamine interaction with α-syn originates aggregation of natively unstructured alpha-synuclein. Proc Natl Acad Sci U S A 102:1430-1435. oligomers with significant toxicity and it was observed to promote α-syn Bousset L, Pieri L, Ruiz-Arlandis G, Gath J, Jensen PH, Habenstein B, Madiona extension (Konijnenberg et al., 2016). Another example where α-syn K, Olieric V, Bockmann A, Meier BH, Melki R (2013) Structural and func- compaction and aggregation inhibition appear hand in hand is that of tional characterization of two alpha-synuclein strains. Nat Commun 4:2575. cyclized nordihydroguaiaretic acid analogs (Daniels et al., 2019). These Carija A, Pinheiro F, Pujols J, Bras IC, Lazaro DF, Santambrogio C, Grandori R, molecules induce the compaction of monomeric α-syn in a concentra- Outeiro TF, Navarro S, Ventura S (2019) Biasing the native alpha-synuclein tion-dependent manner, reducing its aggregation. Remarkably, upon conformational ensemble towards compact states abolishes aggregation and nordihydroguaiaretic acid treatment not only α-syn becomes resistant to neurotoxicity. Redox Biol 22:101135. aggregation, but it is able to prevent aggregation of non-incubated α-syn, Ciechanover A, Kwon YT (2015) Degradation of misfolded proteins in neurode- similarly to what Carija et al. (2019) reported for α-synCC and wt α-syn generative diseases: therapeutic targets and strategies. Exp Mol Med 47:e147. (Figure 2). Promoting α-syn compaction was also proposed to explain Daniels MJ, Nourse JB Jr, Kim H, Sainati V, Schiavina M, Murrali MG, Pan B, why arginine hinders α-syn aggregation, both in vitro and in living cells Ferrie JJ, Haney CM, Moons R, Gould NS, Natalello A, Grandori R, Sobott (Ghosh et al., 2018). These evidences together with the study of Carija et F, Petersson EJ, Rhoades E, Pierattelli R, Felli I, Uversky VN, Caldwell KA, et al. (2019) Cyclized NDGA modifies dynamic alpha-synuclein monomers al. (2019) we discuss here prompts the development of therapies aimed preventing aggregation and toxicity. Sci Rep 9:2937. to reduce the exposure and flexibility of the NAC domain and opens Fusco G, De Simone A, Gopinath T, Vostrikov V, Vendruscolo M, Dobson CM, new avenues for the treatment of Parkinson’s disease. Veglia G (2014) Direct observation of the three regions in alpha-synuclein that determine its membrane-bound behaviour. Nat Commun 5:3827. Francisca Pinheiro, Salvador Ventura* Ghosh S, Kundu A, Chattopadhyay K (2018) Small molecules attenuate the Institut de Biotecnologia i Biomedicina, Universitat Autònoma de interplay between conformational fluctuations, early oligomerization and Barcelona, Bellaterra, Spain; Departament de Bioquímica i Biologia amyloidosis of alpha synuclein. Sci Rep 8:5481. Konijnenberg A, Ranica S, Narkiewicz J, Legname G, Grandori R, Sobott F, Na- Molecular, Universitat Autònoma de Barcelona, Bellaterra, Spain talello A (2016) Opposite Structural effects of epigallocatechin-3-gallate and *Correspondence to: Salvador Ventura, PhD, dopamine binding to alpha-synuclein. Anal Chem 88:8468-8475. salvador.ventura@uab.es. Spillatini MG, Schmidt ML, Lee VM, Trojanowski JQ, Jakes R, Goedert M (1997) orcid: 0000-0002-9652-6351 (Salvador Ventura) Alpha-synuclein in Lewy bodies. Nature 388:839-840. Received: March 27, 2019 Tuttle MD, Comellas G, Nieuwkoop AJ, Covell DJ, Berthold DA, Kloepper KD, Accepted: May 5, 2019 Courtney JM, Kim JK, Barclay AM, Kendall A, Wan W, Stubbs G, Schwieters CD, Lee VM, George JM, Rienstra CM (2016) Solid-state NMR structure of a pathogenic fibril of full-length human alpha-synuclein. Nat Struct Mol Biol doi: 10.4103/1673-5374.259608 23:409-415. Copyright license agreement: The Copyright License Agreement has been Uversky VN, Eliezer D (2009) Biophysics of Parkinson’s disease: structure and signed by both authors before publication. aggregation of alpha-synuclein. Curr Protein Pept Sci 10:483-499. Plagiarism check: Checked twice by iThenticate. Winner B, Jappelli R, Maji SK, Desplats PA, Boyer L, Aigner S, Hetzer C, Loher T, Peer review: Externally peer reviewed. Vilar M, Campioni S, Tzitzilonis C, Soragni A, Jessberger S, Mira H, Consiglio Open access statement: This is an open access journal, and articles are dis- A, Pham E, Masliah E, Gage FH, Riek R (2011) In vivo demonstrastion that tributed under the terms of the Creative Commons Attribution-NonCommer- alpha-synuclein oligomers are toxic. Proc Natl Acad Sci U S A 108:4194-4199. cial-ShareAlike 4.0 License, which allows others to remix, tweak, and build upon the work non-commercially, as long as appropriate credit is given and P-Reviewers: Giannakopoulos P, Kazim SF; C-Editors: Zhao M, Yu J; T-Editor: Jia Y 1898