Title: Aromatic Small Molecules Remodel Toxic Soluble Oligomers of Amyloid β through Three Independent Pathways
Abstract: In protein conformational disorders ranging from Alzheimer to Parkinson disease, proteins of unrelated sequence misfold into a similar array of aggregated conformers ranging from small oligomers to large amyloid fibrils. Substantial evidence suggests that small, prefibrillar oligomers are the most toxic species, yet to what extent they can be selectively targeted and remodeled into non-toxic conformers using small molecules is poorly understood. We have evaluated the conformational specificity and remodeling pathways of a diverse panel of aromatic small molecules against mature soluble oligomers of the Aβ42 peptide associated with Alzheimer disease. We find that small molecule antagonists can be grouped into three classes, which we herein define as Class I, II, and III molecules, based on the distinct pathways they utilize to remodel soluble oligomers into multiple conformers with reduced toxicity. Class I molecules remodel soluble oligomers into large, off-pathway aggregates that are non-toxic. Moreover, Class IA molecules also remodel amyloid fibrils into the same off-pathway structures, whereas Class IB molecules fail to remodel fibrils but accelerate aggregation of freshly disaggregated Aβ. In contrast, a Class II molecule converts soluble Aβ oligomers into fibrils, but is inactive against disaggregated and fibrillar Aβ. Class III molecules disassemble soluble oligomers (as well as fibrils) into low molecular weight species that are non-toxic. Strikingly, Aβ non-toxic oligomers (which are morphologically indistinguishable from toxic soluble oligomers) are significantly more resistant to being remodeled than Aβ soluble oligomers or amyloid fibrils. Our findings reveal that relatively subtle differences in small molecule structure encipher surprisingly large differences in the pathways they employ to remodel Aβ soluble oligomers and related aggregated conformers. In protein conformational disorders ranging from Alzheimer to Parkinson disease, proteins of unrelated sequence misfold into a similar array of aggregated conformers ranging from small oligomers to large amyloid fibrils. Substantial evidence suggests that small, prefibrillar oligomers are the most toxic species, yet to what extent they can be selectively targeted and remodeled into non-toxic conformers using small molecules is poorly understood. We have evaluated the conformational specificity and remodeling pathways of a diverse panel of aromatic small molecules against mature soluble oligomers of the Aβ42 peptide associated with Alzheimer disease. We find that small molecule antagonists can be grouped into three classes, which we herein define as Class I, II, and III molecules, based on the distinct pathways they utilize to remodel soluble oligomers into multiple conformers with reduced toxicity. Class I molecules remodel soluble oligomers into large, off-pathway aggregates that are non-toxic. Moreover, Class IA molecules also remodel amyloid fibrils into the same off-pathway structures, whereas Class IB molecules fail to remodel fibrils but accelerate aggregation of freshly disaggregated Aβ. In contrast, a Class II molecule converts soluble Aβ oligomers into fibrils, but is inactive against disaggregated and fibrillar Aβ. Class III molecules disassemble soluble oligomers (as well as fibrils) into low molecular weight species that are non-toxic. Strikingly, Aβ non-toxic oligomers (which are morphologically indistinguishable from toxic soluble oligomers) are significantly more resistant to being remodeled than Aβ soluble oligomers or amyloid fibrils. Our findings reveal that relatively subtle differences in small molecule structure encipher surprisingly large differences in the pathways they employ to remodel Aβ soluble oligomers and related aggregated conformers. IntroductionA central tenet of protein folding is that a given amino acid sequence encodes a single folded structure (1Anfinsen C.B. Science. 1973; 181: 223-230Crossref PubMed Scopus (5079) Google Scholar). By analogy, one would expect that a given protein sequence would encode a single misfolded structure (e.g. a single amyloid fibril conformation). Instead, each protein sequence encodes numerous aggregated isoforms that possess unique secondary and tertiary structures (2Frost B. Ollesch J. Wille H. Diamond M.I. J. Biol. Chem. 2009; 284: 3546-3551Abstract Full Text Full Text PDF PubMed Scopus (168) Google Scholar, 3Chien P. Weissman J.S. Nature. 2001; 410: 223-227Crossref PubMed Scopus (133) Google Scholar, 4Chien P. DePace A.H. Collins S.R. Weissman J.S. Nature. 2003; 424: 948-951Crossref PubMed Scopus (86) Google Scholar, 5Tanaka M. Chien P. Naber N. Cooke R. Weissman J.S. Nature. 2004; 428: 323-328Crossref PubMed Scopus (666) Google Scholar, 6Tessier P.M. Lindquist S. 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The development of several conformation-specific antibodies capable of selectively detecting aggregated conformers ranging from intermediates (e.g. soluble oligomers (32Kayed R. Head E. Thompson J.L. McIntire T.M. Milton S.C. Cotman C.W. Glabe C.G. Science. 2003; 300: 486-489Crossref PubMed Scopus (3405) Google Scholar, 33Lambert M.P. Viola K.L. Chromy B.A. Chang L. Morgan T.E. Yu J. Venton D.L. Krafft G.A. Finch C.E. Klein W.L. J. Neurochem. 2001; 79: 595-605Crossref PubMed Scopus (297) Google Scholar, 34Zameer A. Kasturirangan S. Emadi S. Nimmagadda S.V. Sierks M.R. J. Mol. Biol. 2008; 384: 917-928Crossref PubMed Scopus (67) Google Scholar), fibrillar oligomers (21Wu J.W. Breydo L. Isas J.M. Lee J. Kuznetsov Y.G. Langen R. Glabe C. J. Biol. Chem. 2010; 285: 6071-6079Abstract Full Text Full Text PDF PubMed Scopus (129) Google Scholar), and annular protofibrils (35Kayed R. Pensalfini A. Margol L. Sokolov Y. Sarsoza F. Head E. Hall J. Glabe C. J. Biol. 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Herein, we demonstrate that diverse aromatic small molecules utilize three independent pathways to remodel mature Aβ soluble oligomers into benign conformers with highly dissimilar biochemical properties.DISCUSSIONOur findings illuminate multiple pathways employed by small molecules to remodel soluble oligomers of Aβ42 (Fig. 8). Indeed, even in the absence of small molecule antagonists, we have reported previously that Aβ soluble oligomers are not committed to a single nucleation pathway (12Ladiwala A.R. Lin J.C. Bale S.S. Marcelino-Cruz A.M. Bhattacharya M. Dordick J.S. Tessier P.M. J. Biol. Chem. 2010; 285: 24228-24237Abstract Full Text Full Text PDF PubMed Scopus (260) Google Scholar). Instead, soluble oligomers can mature into multiple Aβ conformers (fibrillar intermediates and non-toxic oligomers) that possess highly dissimilar biochemical properties, and each conformer can be formed in a specific manner via simple changes in agitation. This multifaceted capacity of Aβ soluble oligomers to mature into different isoforms is consistent with our findings that small molecules can remodel soluble oligomers into conformers with significantly different sizes and structures. That only four of the small molecules investigated in this work remodel mature Aβ fibrils (whereas all seven remodel soluble oligomers) highlights the conformational plasticity of soluble oligomers to evolve into diverse conformers.Our results for Class I molecules confirm that converting soluble oligomers into large, unstructured aggregates is a common pathway for remodeling toxic oligomers. We reason that it is simpler to promote nonspecific intermolecular interactions between Aβ peptides within soluble oligomers than it is to promote specific ones necessary for fibril formation or to prevent all possible intermolecular interactions. Studies of small molecule inhibitors of protein aggregation also support our findings because many such antagonists promote alternative, unstructured aggregates rather than preventing condensation of protein monomers (30Wang H. Duennwald M.L. Roberts B.E. Rozeboom L.M. Zhang Y.L. Steele A.D. Krishnan R. Su L.J. Griffin D. Mukhopadhyay S. Hennessy E.J. Weigele P. Blanchard B.J. King J. Deniz A.A. Buchwald S.L. Ingram V.M. Lindquist S. Shorter J. Proc. Natl. Acad. Sci. U.S.A. 2008; 105: 7159-7164Crossref PubMed Scopus (51) Google Scholar, 44Necula M. Kayed R. Milton S. Glabe C.G. J. Biol. Chem. 2007; 282: 10311-10324Abstract Full Text Full Text PDF PubMed Scopus (595) Google Scholar, 45Ehrnhoefer D.E. Bieschke J. Boeddrich A. Herbst M. Masino L. Lurz R. Engemann S. Pastore A. Wanker E.E. Nat. Struct. Mol. Biol. 2008; 15: 558-566Crossref PubMed Scopus (1098) Google Scholar).Nevertheless, we found that a Class II molecule (methylene blue) selectively converts soluble oligomers into fibrils, which is consistent with previous work (38Necula M. Breydo L. Milton S. Kayed R. van der Veer W.E. Tone P. Glabe C.G. Biochemistry. 2007; 46: 8850-8860Crossref PubMed Scopus (193) Google Scholar). Importantly, methylene blue fails to remodel amyloid fibrils within 24 h or accelerate aggregation of freshly disaggregated Aβ within 4 h (longer times not tested). Thus, the primary activity of methylene blue is to accelerate the conversion of Aβ oligomers into fibrillar conformers. Because a key structural difference between Aβ soluble oligomers and fibrils is the presence of β-sheets, it is possible that Class II molecules facilitate mature self-stacking interactions between residues necessary for β-sheet formation. Alternatively, Class II molecules may accelerate conversion of soluble oligomers into fibrils by inhibiting nonspecific interactions sampled within Aβ soluble oligomers that govern assembly of non-toxic oligomers.The conformational specificity of methylene blue to remodel Aβ soluble oligomers into fibrils makes it and related variants potential drug candidates to evaluate the hypothesis that Aβ soluble oligomers are the primary cellular insult in Alzheimer disease. The therapeutic activity of many small molecules for remodeling toxic Aβ oligomers is limited because they do not cross the blood-brain barrier. In contrast, methylene blue crosses the blood-brain barrier, and possesses many other desirable drug properties, including high solubility and low toxicity (46Peter C. Hongwan D. Küpfer A. Lauterburg B.H. Eur. J. Clin. Pharmacol. 2000; 56: 247-250Crossref PubMed Scopus (266) Google Scholar, 47Küpfer A. Aeschlimann C. Wermuth B. Cerny T. 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Bale S.S. Marcelino-Cruz A.M. Bhattacharya M. Dordick J.S. Tessier P.M. J. Biol. Chem. 2010; 285: 24228-24237Abstract Full Text Full Text PDF PubMed Scopus (260) Google Scholar). Because piceid is identical to resveratrol except for the 3-O-β-glucosidic moiety, it appears that the sugar moiety is an important determinant of its disaggregation activity. Future structure-activity analysis is necessary to resolve whether the stilbenoid backbone is important for the disaggregation activity of piceid and whether other phenolic glycosides possess similar disaggregation activity.Are there additional pathways by which aromatic small molecules remodel toxic soluble oligomers into non-toxic assemblies? Indeed, we expect that several other pathways are possible. For example, soluble oligomers could be converted into various types of fibrillar conformers other than fibrils or precipitated into large aggregates without structural changes (thereby reducing toxicity due to increased size). Moreover, some aromatic small molecules modify polypeptides covalently (52Zhu M. Rajamani S. Kaylor J. Han S. Zhou F. Fink A.L. J. Biol. Chem. 2004; 279: 26846-26857Abstract Full Text Full Text PDF PubMed Scopus (379) Google Scholar, 53Meng X. Munishkina L.A. Fink A.L. Uversky V.N. Biochemistry. 2009; 48: 8206-8224Crossref PubMed Scopus (104) Google Scholar) and are likely to remodel mature soluble oligomers through unique pathways not considered here.A puzzling finding of our work is that Class IA polyphenols selectivity remodel Aβ soluble oligomers and fibrils into indistinguishable, unstructured aggregates with identical dose dependence. A simple explanation would be that soluble oligomers and fibrils possess common structural features recognized by Class IA molecules. However, we were unable to demonstrate a single biochemical property shared by these two conformers. We find that Aβ soluble oligomers are A11-positive, OC-negative, ThT-negative, SDS-soluble, highly toxic and largely unstructured, whereas fibrils possess the “opposite” properties (i.e. they are A11-negative, OC-positive, ThT-positive, SDS-insoluble, mildly toxic and β-sheet rich). Because polyphenols are suspected to antagonize π-stacking interactions between aromatic side chains in amyloids (54Gazit E. FASEB J. 2002; 16: 77-83Crossref PubMed Scopus (924) Google Scholar), it may be that such side chains stack in soluble oligomers (despite that they lack mature β-sheets detectable by circular dichroism) as they do in Aβ fibrillar oligomers and fibrils (21Wu J.W. Breydo L. Isas J.M. Lee J. Kuznetsov Y.G. Langen R. Glabe C. J. Biol. Chem. 2010; 285: 6071-6079Abstract Full Text Full Text PDF PubMed Scopus (129) Google Scholar, 55Margittai M. Langen R. Q. Rev. Biophys. 2008; 41: 265-297Crossref PubMed Scopus (142) Google Scholar). Such stacking interactions between aromatic side chains of Aβ may render soluble oligomers and fibrils sensitive to being remodeled by polyphenols. It is also possible that Class IA molecules recognize other structural motifs that may be common to soluble oligomers and fibrils, such as sheet-like structures in both prefibrillar oligomers (α-pleated (56Armen R.S. DeMarco M.L. Alonso D.O. Daggett V. Proc. Natl. Acad. Sci. U.S.A. 2004; 101: 11622-11627Crossref PubMed Scopus (116) Google Scholar), anti-parallel (57Cerf E. Sarroukh R. Tamamizu-Kato S. Breydo L. Derclaye S. Dufrêne Y.F. Narayanaswami V. Goormaghtigh E. Ruysschaert J.M. Raussens V. Biochem. J. 2009; 421: 415-423Crossref PubMed Scopus (361) Google Scholar), or disordered (58Manavalan P. Johnson W.C. Nature. 1983; 305: 831-832Crossref Scopus (357) Google Scholar, 59Gursky O. Aleshkov S. Biochim. Biophys. Acta. 2000; 1476: 93-102Crossref PubMed Scopus (130) Google Scholar) sheets) and fibrils (parallel (55Margittai M. Langen R. Q. Rev. Biophys. 2008; 41: 265-297Crossref PubMed Scopus (142) Google Scholar) or anti-parallel (60Tycko R. Sciarretta K.L. Orgel J.P. Meredith S.C. Biochemistry. 2009; 48: 6072-6084Crossref PubMed Scopus (117) Google Scholar) β-sheets), although additional analysis is needed to test these speculative hypotheses.A striking finding of our work is that non-toxic oligomers are more resistant to being remodeled by diverse aromatic small molecules than all other Aβ conformers, including highly stable Aβ fibrils. A possible explanation is that non-toxic oligomers lack aromatic stacking interactions, which allows them to more effectively evade the remodeling activity of the small molecules studied in this work. This hypothesis would also predict that substituting aromatic amino acids for non-aromatic ones would desensitize soluble oligomers to the remodeling activity of polyphenols. However, it is also possible that Aβ non-toxic oligomers are simply more stable than other Aβ conformers (61Lee S. Fernandez E.J. Good T.A. Protein Sci. 2007; 16: 723-732Crossref PubMed Scopus (102) Google Scholar), which would also explain their increased resistance to being remodeled by diverse aromatic molecules.It is notable that two oligomeric forms of a non-disease associated protein (HypF) have been reported to also possess unique toxicities (11Campioni S. Mannini B. Zampagni M. Pensalfini A. Parrini C. Evangelisti E. Relini A. Stefani M. Dobson C.M. Cecchi C. Chiti F. Nat. Chem. Biol. 2010; 6: 140-147Crossref PubMed Scopus (444) Google Scholar). Similar to Aβ soluble and non-toxic oligomers reported here, the N-terminal domain of HypF (denoted HypF-N) forms two oligomeric forms, one of which is toxic. Chiti and co-workers (11Campioni S. Mannini B. Zampagni M. Pensalfini A. Parrini C. Evangelisti E. Relini A. Stefani M. Dobson C.M. Cecchi C. Chiti F. Nat. Chem. 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We expect that future site-specific structural studies of Aβ toxic and non-toxic oligomers will illuminate the structural differences between these oligomers that confer their unique toxicities and susceptibilities to being remodeling by aromatic small molecules. IntroductionA central tenet of protein folding is that a given amino acid sequence encodes a single folded structure (1Anfinsen C.B. Science. 1973; 181: 223-230Crossref PubMed Scopus (5079) Google Scholar). By analogy, one would expect that a given protein sequence would encode a single misfolded structure (e.g. a single amyloid fibril conformation). Instead, each protein sequence encodes numerous aggregated isoforms that possess unique secondary and tertiary structures (2Frost B. Ollesch J. Wille H. Diamond M.I. J. Biol. Chem. 2009; 284: 3546-3551Abstract Full Text Full Text PDF PubMed Scopus (168) Google Scholar, 3Chien P. Weissman J.S. Nature. 2001; 410: 223-227Crossref PubMed Scopus (133) Google Scholar, 4Chien P. 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The development of several conformation-specific antibodies capable of selectively detecting aggregated conformers ranging from intermediates (e.g. soluble oligomers (32Kayed R. Head E. Thompson J.L. McIntire T.M. Milton S.C. Cotman C.W. Glabe C.G. Science. 2003; 300: 486-489Crossref PubMed Scopus (3405) Google Scholar, 33Lambert M.P. Viola K.L. Chromy B.A. Chang L. Morgan T.E. Yu J. Venton D.L. Krafft G.A. Finch C.E. Klein W.L. J. Neurochem. 2001; 79: 595-605Crossref PubMed Scopus (297) Google Scholar, 34Zameer A. Kasturirangan S. Emadi S. Nimmagadda S.V. Sierks M.R. J. Mol. Biol. 2008; 384: 917-928Crossref PubMed Scopus (67) Google Scholar), fibrillar oligomers (21Wu J.W. Breydo L. Isas J.M. Lee J. Kuznetsov Y.G. Langen R. Glabe C. J. Biol. Chem. 2010; 285: 6071-6079Abstract Full Text Full Text PDF PubMed Scopus (129) Google Scholar), and annular protofibrils (35Kayed R. Pensalfini A. Margol L. Sokolov Y. Sarsoza F. Head E. Hall J. Glabe C. J. Biol. 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