Annular Protofibrils Are a Structurally and Functionally Distinct Type of Amyloid Oligomer

Abstract

Amyloid oligomers are believed to play causal roles in several types of amyloid-related neurodegenerative diseases. Several different types of amyloid oligomers have been reported that differ in morphology, size, or toxicity, raising the question of the pathological significance and structural relationships between different amyloid oligomers. Annular protofibrils (APFs) have been described in oligomer preparations of many different amyloidogenic proteins and peptides as ring-shaped or pore-like structures. They are interesting because their pore-like morphology is consistent with numerous reports of membrane-permeabilizing activity of amyloid oligomers. Here we report the preparation of relatively homogeneous preparations of APFs and an antiserum selective for APFs (αAPF) compared with prefibrillar oligomers (PFOs) and fibrils. PFOs appear to be precursors for APF formation, which form in high yield after exposure to a hydrophobic-hydrophilic interface. Surprisingly, preformed APFs do not permeabilize lipid bilayers, unlike the precursor PFOs. APFs display a conformation-dependent, generic epitope that is distinct from that of PFOs and amyloid fibrils. Incubation of PFOs with phospholipids vesicles results in a loss of PFO immunoreactivity with a corresponding increase in αAPF immunoreactivity, suggesting that lipid vesicles catalyze the conversion of PFOs into APFs. The annular anti-protofibril antibody also recognizes heptameric α-hemolysin pores, but not monomers, suggesting that the antibody recognizes an epitope that is specific for a β barrel structural motif. Amyloid oligomers are believed to play causal roles in several types of amyloid-related neurodegenerative diseases. Several different types of amyloid oligomers have been reported that differ in morphology, size, or toxicity, raising the question of the pathological significance and structural relationships between different amyloid oligomers. Annular protofibrils (APFs) have been described in oligomer preparations of many different amyloidogenic proteins and peptides as ring-shaped or pore-like structures. They are interesting because their pore-like morphology is consistent with numerous reports of membrane-permeabilizing activity of amyloid oligomers. Here we report the preparation of relatively homogeneous preparations of APFs and an antiserum selective for APFs (αAPF) compared with prefibrillar oligomers (PFOs) and fibrils. PFOs appear to be precursors for APF formation, which form in high yield after exposure to a hydrophobic-hydrophilic interface. Surprisingly, preformed APFs do not permeabilize lipid bilayers, unlike the precursor PFOs. APFs display a conformation-dependent, generic epitope that is distinct from that of PFOs and amyloid fibrils. Incubation of PFOs with phospholipids vesicles results in a loss of PFO immunoreactivity with a corresponding increase in αAPF immunoreactivity, suggesting that lipid vesicles catalyze the conversion of PFOs into APFs. The annular anti-protofibril antibody also recognizes heptameric α-hemolysin pores, but not monomers, suggesting that the antibody recognizes an epitope that is specific for a β barrel structural motif. Many age-related neurodegenerative diseases are characterized by the accumulation of amyloid deposits derived from a variety of misfolded proteins (1Dobson C.M. Protein Pept. Lett. 2006; 13: 219-227Crossref PubMed Scopus (142) Google Scholar). These diseases typically have both sporadic and inherited forms, and in many cases the mutations associated with the familial forms are in the gene encoding the protein that accumulates or in genes directly related to its production, processing, or accumulation (2Hardy J. Biochem. Soc. 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In the atomic force microscope prefibrillar oligomers (PFOs) 3The abbreviations used are: PFO, prefibrillar oligomer; APF, annular protofibrils; αAPF, anti-APF; PFT, pore-forming toxin; MTT, (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide; AD, Alzheimer disease; TBS-T, Tris-buffered saline-Tween; ELISA, enzyme-linked immunosorbent assay. 3The abbreviations used are: PFO, prefibrillar oligomer; APF, annular protofibrils; αAPF, anti-APF; PFT, pore-forming toxin; MTT, (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide; AD, Alzheimer disease; TBS-T, Tris-buffered saline-Tween; ELISA, enzyme-linked immunosorbent assay. appear as spherical particles of ∼3–10 nm. PFOs appear at early times of incubation and disappear as mature fibrils appear (16Harper J.D. Wong S.S. Lieber C.M. Lansbury P.T. Chem. Biol. 1997; 4: 119-125Abstract Full Text PDF PubMed Scopus (622) Google Scholar, 17Lashuel H.A. Hartley D. Petre B.M. Walz T. Lansbury Jr., P.T. Nature. 2002; 418: 291Crossref PubMed Scopus (1117) Google Scholar, 18Anguiano M. Nowak R.J. Lansbury Jr., P.T. Biochemistry. 2002; 41: 11338-11343Crossref PubMed Scopus (253) Google Scholar). At longer times of incubation PFOs appear to coalesce to form curvilinear beaded strings that have been called protofibrils and ring-shaped, pore-like structures referred to as annular protofibrils (APFs) (17Lashuel H.A. Hartley D. Petre B.M. Walz T. Lansbury Jr., P.T. Nature. 2002; 418: 291Crossref PubMed Scopus (1117) Google Scholar). APFs appear to be formed from the circularization of PFO subunits. A similar spectrum of PFOs and APFs has been observed for many types of amyloids, such as α-synuclein (19Conway K.A. Harper J.D. Lansbury Jr., P.T. Biochemistry. 2000; 39: 2552-2563Crossref PubMed Scopus (688) Google Scholar), islet amyloid (20Janson J. Ashley R.H. Harrison D. McIntyre S. Butler P.C. Diabetes. 1999; 48: 491-498Crossref PubMed Scopus (513) Google Scholar), and non-disease associated “neoamyloids” (21Bucciantini M. Giannoni E. Chiti F. Baroni F. Formigli L. Zurdo J. Taddei N. Ramponi G. Dobson C.M. Stefani M. Nature. 2002; 416: 507-511Crossref PubMed Scopus (2145) Google Scholar). Although PFOs, APFs, and fibrils have been observed for many different types of amyloidogenic proteins and peptides (22Lashuel H.A. Lansbury Jr., P.T. Q. Rev. Biophys. 2006; 39: 167-201Crossref PubMed Scopus (338) Google Scholar), their structures, interrelationships, and contributions to disease pathogenesis are not entirely clear. Insoluble fibrils and small soluble pieces of fibrils known as fibrillar oligomers appear to have a distinct and mutually exclusive underlying structure than PFOs because they display generic epitopes that are recognized by distinct conformation-dependent monoclonal antibodies (23Hrncic R. Wall J. Wolfenbarger D.A. Murphy C.L. Schell M. Weiss D.T. Solomon A. Am. J. Pathol. 2000; 157: 1239-1246Abstract Full Text Full Text PDF PubMed Scopus (159) Google Scholar, 24O'Nuallain B. Wetzel R. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 1485-1490Crossref PubMed Scopus (300) Google Scholar) and antisera (25Kayed 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 (3424) Google Scholar, 26Kayed R. Head E. Sarsoza F. Saing T. Cotman C.W. Necula M. Margol L. Wu J. Breydo L. Thompson J.L. Rasool S. Gurlo T. Butler P. Glabe C.G. Mol. Neurodegener. 2007; 2: 1-11Crossref PubMed Scopus (570) Google Scholar). It is not yet known whether APFs represent a unique conformation or whether they are structurally related to PFOs or fibrils. So far APFs have only been defined morphologically as pore-like structures and have been observed in preparations of PFOs and in fibril-containing preparations (27Hafner J.H. Cheung C.L. Woolley A.T. Lieber C.M. Prog. Biophys. Mol. Biol. 2001; 77: 73-110Crossref PubMed Scopus (299) Google Scholar, 28Shtilerman M.D. Ding T.T. Lansbury Jr., P.T. Biochemistry. 2002; 41: 3855-3860Crossref PubMed Scopus (258) Google Scholar, 29Ding T.T. Lee S.J. Rochet J.C. Lansbury Jr., P.T. Biochemistry. 2002; 41: 10209-10217Crossref PubMed Scopus (322) Google Scholar). Familial mutations associated with inherited forms of Parkinson and Alzheimer diseases increase the of APFs, suggesting that their is related to activity (17Lashuel H.A. Hartley D. Petre B.M. Walz T. Lansbury Jr., P.T. Nature. 2002; 418: 291Crossref PubMed Scopus (1117) Google Scholar, H.A. Hartley D.M. Petre B.M. Wall Walz T. Lansbury Jr., P.T. J. Mol. Biol. 2003; PubMed Scopus Google Scholar). the between APFs and pore-forming it has been that APFs permeabilize (22Lashuel H.A. Lansbury Jr., P.T. Q. Rev. Biophys. 2006; 39: 167-201Crossref PubMed Scopus (338) Google Scholar). is a activity of prefibrillar amyloid oligomers C.G. R. 2006; PubMed Scopus Google Scholar) and PFOs are a precursor to annular formation, the of APFs is an for the of oligomers because annular is also a and they the pathological of Aβ APFs has been by a of homogeneous preparations of annular structures and the of a of from in Here we report the preparation of relatively homogeneous of APFs that have the pore-like morphology have used preparations to their and membrane-permeabilizing and as an for the preparation of an antiserum that recognizes APFs, compared with monomers, PFOs, and fibrils. APFs are and do not into fibrils or PFOs of APFs also membrane-permeabilizing activity compared with the prefibrillar oligomer precursors to APF with a hydrophobic-hydrophilic the conversion of PFOs into APFs. Incubation of PFOs with lipid vesicles results in a loss of the prefibrillar oligomer specific epitope and the of an annular APFs display a unique conformation-dependent epitope that is distinct from PFOs and fibrils. antibody recognizes mature heptameric from suggesting that APFs may form structures. Aβ and islet amyloid peptides by a as described (11Burdick D. Soreghan B. Kwon M. Kosmoski J. Knauer M. Henschen A. Yates J. Cotman C. Glabe C. J. Biol. Chem. 1992; 267: 546-554Abstract Full Text PDF PubMed Google Scholar). a from from and of of annular protofibrils from different peptides and proteins by PFOs as the that as described R. Glabe C.G. 2006; PubMed Scopus Google Scholar). APFs by different PFOs to in a in the to for of the and exposure of spherical oligomers to interface. of to a of PFOs, and the with a for for a the in a of The morphology of annular preparations by of the and and for in The with for and with a microscope The of APFs by of to a of in Protein Sci. 2: PubMed Scopus Google Scholar). the spectrum in the a at nm. with a and of of APFs from as described an with of APFs at the of and and used for at in small of in a the from the to and and in The of the antiserum (αAPF) with and and with of different amyloid aggregates PFOs, and as for at times with for at with The The times with and of αAPF antiserum in in and to for at The times with TBS-T, and of in in and for at times with and with from the the with of and at nm. the as described as to a to at with in at for times for with TBS-T, and for at with αAPF antiserum in in or in in times for with and with in in for at times with and with from Aβ APFs at and pore-forming at in with an of before and at and to αAPF antibody used for as described for and formed at by the of as described R. Y. B. McIntire T.M. Milton S.C. Glabe C.G. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, Y. R. A. Glabe C. J. 2006; PubMed Scopus Google Scholar). lipid a in in a the The by with a of in from by to with for after the by used as to and the The as and to the and at a of by a by by an of the as described (25Kayed 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 (3424) Google Scholar). with Aβ APFs and PFOs. incubation for at the an to the in of APF in from the for and cases The an with a of before The for and the and The a with a of before The for and the Alzheimer disease and The from and at a in with before in or with and with in to with for not in with and for with at in several αAPF primary antibody with and a in in and in with before and of in of the a of in the the with of and for used to of α-hemolysin by α-hemolysin with at oligomers at in and at with in ELISA, with or protein and with antibody antibody (αAPF) and and of the of Aβ APFs, we to for in relatively homogeneous APFs are morphologically defined (17Lashuel H.A. Hartley D. Petre B.M. Walz T. Lansbury Jr., P.T. Nature. 2002; 418: 291Crossref PubMed Scopus (1117) Google Scholar), we used to the of that of PFOs APFs as reported and that exposure of to an the of annular structures observed not also the of of spherical PFOs with in and the in the of large of relatively homogeneous annular structures The APFs have ranging from to with a or they are they appear to be up of spherical oligomeric as incubation for the APFs beaded and a The APFs also appear to at longer incubation to a APFs of similar morphology, and also by the with and α-synuclein and amyloid fibrils observed by after incubation of APFs for up to either with or with that APFs are and do not to fibrils not also the structure of APFs by The spectrum that APFs and of APFs have a pore-like morphology and amyloid oligomers have been reported to permeabilize we the of APFs Surprisingly, APFs have membrane-permeabilizing activity compared with the PFOs that are used as precursors in annular APFs display activity with a of at but is than the corresponding PFOs that a of at the annular preparations PFOs it is that the membrane-permeabilizing activity of the annular preparations may be to PFOs than APFs. a number of reports have that PFOs are more toxic than amyloid fibrils to compared the of APFs and PFOs and that the APFs are toxic the membrane-permeabilizing the of APFs is than that of the PFOs oligomers used to of the of APFs and PFOs. and the as described APFs or PFOs. PFOs are more toxic than APFs different morphology and activity of APFs and PFOs that there may be underlying that for whether be recognized by conformation-dependent antibodies that are specific for different structural antisera by with preparations of APFs and characterized the of the for different of Aβ and amyloidogenic proteins and peptides by The antisera (αAPF) annular preparations and display with and fibrillar The αAPF also with prefibrillar oligomer but it is not whether epitopes or the that prefibrillar fibrillar oligomer preparations formed APFs. and antisera generic conformation-dependent epitopes that are to we whether αAPF recognizes APFs from protein and αAPF recognizes APFs from α-synuclein and islet amyloid and does not the forms of that αAPF also recognizes generic antiserum recognizes conformation-dependent generic epitopes that are of specific APFs and proteins and peptides by for islet amyloid and APFs. proteins and also compared the of αAPF, and antisera by and to whether they have unique or of APFs, PFOs, and fibrils between PFOs, and APFs with αAPF The of prefibrillar oligomer and with αAPF may of the antisera or the of of PFOs and fibrils in that APFs. The is consistent with reports that APFs in preparations of Aβ aggregates (17Lashuel H.A. Hartley D. Petre B.M. Walz T. Lansbury Jr., P.T. Nature. 2002; 418: 291Crossref PubMed Scopus (1117) Google Scholar). annular are only recognized by and with antibody is The immunoreactivity is consistent with the that spherical PFOs in preparations of APFs The of prefibrillar oligomer and the of immunoreactivity of with αAPF that there is relatively or immunoreactivity the for antisera are different of the Aβ of APFs a at with αAPF that do not with either or that APFs display an unique epitope that is not recognized by either or of or aggregates is observed with αAPF recognizes a unique epitope associated with APFs, we with αAPF antisera to its in disease specific of either of that αAPF does not plaques and that are features of disease is evidence that αAPF does not with amyloid fibrils. also of and and not specific immunoreactivity not These results are whether annular protofibrils in because they may be the of by to amyloid plaque deposits in both and amyloid plaque deposits specific of amyloid deposits observed for αAPF APF at an or with is we whether the conversion of PFOs to APFs be more whether incubation with catalyze conversion and αAPF immunoreactivity as an for their that incubation of PFOs with the loss of immunoreactivity with a corresponding increase in αAPF immunoreactivity that PFOs to APFs more and that the of PFOs with their conversion into annular pores, which may for their to permeabilize of morphologically that are from PFOs, pore-forming by the conformation conversion of into heptameric M. E. Biophys. 2003; PubMed Scopus Google Scholar). have reported that a of α-hemolysin and the heptameric Y. R. Milton S.C. A. Glabe C.G. 2007; PubMed Scopus Google Scholar). compared the of α-hemolysin pores, and with and αAPF antibodies In to αAPF antibody the mature heptameric and only the misfolded antibody the of that the antibody is Incubation of α-hemolysin with and of the heptameric in a that αAPF immunoreactivity is more that may in These results that APFs represent a distinct of amyloid oligomer that has a unique underlying structural that APFs from several types of with that relatively homogeneous of APFs are in to exposure of PFOs to an with or a interface. that the structural APF is by of the Although and are the of conversion incubation of PFOs with lipid the of PFOs with to a loss of that the of APFs the of the may be a in the loss of Aβ amyloid oligomers are and display a of and a B. Kosmoski J. Glabe C. J. Biol. Chem. 1994; 269: 28551-28554Abstract Full Text PDF PubMed Google Scholar). The β in amyloid fibrils also display an where the between the S. R. Thompson M.J. M. C. D. Nature. 2007; PubMed Scopus Google Scholar). β in amyloid oligomers may at the interface. oligomers are to the they with the oligomers to form a that into the in which the of the the and the forms a A similar has been for the in which the of to the conformation and to the of M. E. Biophys. 2003; PubMed Scopus Google Scholar). Although the morphology of APFs is similar to pores, we that the annular preparations membrane-permeabilizing activity than their corresponding PFO The of APFs is also compared with PFOs but not as as the membrane-permeabilizing of membrane-permeabilizing activity of the preformed is similar to the of and activity of preformed α-hemolysin R. S. A. J. T. J. Cell Biol. PubMed Scopus (258) Google Scholar). from aggregate in the of and are or into the the APFs, are of into the and the The of APFs and may not only the to permeabilize and the of but also structural as a specific antiserum by with Aβ APFs. a for APFs compared with monomers, PFOs, and fibrils. PFOs are with lipid the αAPF immunoreactivity in a and the immunoreactivity suggesting a precursor relationship between the epitopes recognized by the the conformation-dependent antibodies we and have described (23Hrncic R. Wall J. Wolfenbarger D.A. Murphy C.L. Schell M. Weiss D.T. Solomon A. Am. J. Pathol. 2000; 157: 1239-1246Abstract Full Text Full Text PDF PubMed Scopus (159) Google Scholar, 24O'Nuallain B. Wetzel R. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 1485-1490Crossref PubMed Scopus (300) Google Scholar, 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 (3424) Google Scholar, 26Kayed R. Head E. Sarsoza F. Saing T. Cotman C.W. Necula M. Margol L. Wu J. Breydo L. Thompson J.L. Rasool S. Gurlo T. Butler P. Glabe C.G. Mol. Neurodegener. 2007; 2: 1-11Crossref PubMed Scopus (570) Google Scholar), the αAPF antisera recognizes a generic epitope that is of the specific the αAPF antisera also recognizes the heptameric α-hemolysin The antibody is conformation-dependent because the α-hemolysin and the are not with These results that the is structurally related to APFs and that the αAPF antibodies a unique structure associated with β have reported that the α-hemolysin is recognized by antibody and that the heptameric is only Y. R. Milton S.C. A. Glabe C.G. 2007; PubMed Scopus Google Scholar). The that immunoreactivity is as αAPF of PFOs with is consistent with a relationship that is similar to the relationship between the α-hemolysin and its the heptameric that are known to the conversion of α-hemolysin to pores, increase αAPF The of APFs the from PFO precursors at of the between reports of by N. E. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar, R. R. J. Biol. PubMed Scopus Google Scholar, R. Biophys. 2007; PubMed Scopus Google Scholar) and that PFOs permeabilize evidence of R. Y. B. McIntire T.M. Milton S.C. Glabe C.G. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, Y. R. A. Glabe C. J. 2006; PubMed Scopus Google Scholar). APFs have a size and but they do not they have been formed in the of the the heptameric In PFOs to the and form APFs of size, which of are have to and permeabilize specific they form heptameric with It has been that phospholipids vesicles Aβ fibrils into protofibrils or prefibrillar oligomers E. M. W. P. D. R. B. J. F. J. PubMed Scopus Google Scholar). phospholipids vesicles also catalyze the conversion of prefibrillar oligomers into annular that with phospholipids also the of amyloid fibrils to annular Although prefibrillar oligomers appear to be more toxic in S. Casamenti F. Bucciantini M. Luheshi L.M. Taddei N. Chiti F. Dobson C.M. Stefani M. J. Neurosci. 2006; 26: 8160-8167Crossref PubMed Scopus (207) Google Scholar, 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 (3424) Google Scholar), the for conversion the of is toxic in The of monoclonal antibodies that may to the question of which species is toxic in results that APF is related to their to form membrane-permeabilizing are in and the but they are in The proteins also form β their permeabilize and R.H. R.E. K. K.A. K. Wong W. J. Smith A.I. J. M.A. J.C. Science. 2007; PubMed Scopus Google Scholar, M.A. P. Science. 2007; PubMed Scopus Google Scholar). activity is unlike APFs, which permeabilize as a of a generic β The of in may be a of their generic and the in suggesting that the has been The structural relationship between APFs and pore-forming that the to permeabilize may be a of β and that may represent a mechanism of pathogenesis in amyloid-related degenerative diseases.