Liu P, Reed MN, Kotilinek LA, Grant MK, Forster CL, Qiang W, Shapiro SL, Reichl JH, Chiang AC, Jankowsky JL, Wilmot CM, Cleary JP, Zahs KR, Ashe KH. Quaternary Structure Defines a Large Class of Amyloid-β Oligomers Neutralized by Sequestration. Cell Rep. 2015 Jun 23;11(11):1760-71. Epub 2015 Jun 4 PubMed.

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  1. This is a nice piece of work and I commend the authors on their systematic analyses. From my own experience, I know how challenging working with oligomers and conformational antibodies can be. Amyloid formation in vitro and in vivo is very complex; thus, understanding the structural basis for amyloids is very important for basic biology and drug design. This is the first study that clearly reveals differences between two types of amyloid oligomers. Although we have known that antibodies A11 and OC recognize oligomers that are conformationally distinct (Kayed et al. 2007; Kayed et al., 2010), and follow distinct assembly pathways (Glabe 2008 and Krishnan et al., 2012), their distinguishing characteristics, formation in vivo, and toxicity were unknown.

    The out-of-register oligomers recognized by A11 do not form parallel β-sheets found in Aβ fibrils in vivo. They are highly toxic in vitro and in vivo, and these dynamic structures exert toxicity via multiple mechanisms, such as synaptic dysfunction, pore formation, and tau aggregation. 

    Recently, we demonstrated that only A11-binding oligomers can cross-seed other amyloidogenic proteins. The results from this paper may explain this phenomenon and provide structural bases that can account for the formation of different amyloid oligomers in AD and other neurodegenerative diseases (Guerrero-Muñoz et al.,  2014). 

    Important models for the out-of-register structures are available (Laganowsky et al., 2012, and Liu et al. 2012). This study suggests that therapeutic approaches, antibodies, and designed small molecules specifically targeting these structures may be beneficial for AD and other amyloid diseases.

    Finally, this paper lays the groundwork for similar studies on other proteins implicated in neurodegenerative diseases, including α-synuclein, tau, TDP43, and others. Therefore, more methods and reagents will be needed in order to reveal the different amyloid structures. 

    References:

    Kayed R, Head E, Sarsoza F, Saing T, Cotman CW, Necula M, Margol L, Wu J, Breydo L, Thompson JL, Rasool S, Gurlo T, Butler P, Glabe CG. Fibril specific, conformation dependent antibodies recognize a generic epitope common to amyloid fibrils and fibrillar oligomers that is absent in prefibrillar oligomers. Mol Neurodegener. 2007;2:18. PubMed.

    Kayed R, Canto I, Breydo L, Rasool S, Lukacsovich T, Wu J, Albay R, Pensalfini A, Yeung S, Head E, Marsh JL, Glabe C. Conformation dependent monoclonal antibodies distinguish different replicating strains or conformers of prefibrillar Aβ oligomers. Mol Neurodegener. 2010;5:57. PubMed.

    Glabe CG. Structural classification of toxic amyloid oligomers. J Biol Chem. 2008 Oct 31;283(44):29639-43. PubMed.

    Krishnan R, Goodman JL, Mukhopadhyay S, Pacheco CD, Lemke EA, Deniz AA, Lindquist S. Conserved features of intermediates in amyloid assembly determine their benign or toxic states. Proc Natl Acad Sci U S A. 2012 Jul 10;109(28):11172-7. PubMed.

    Guerrero-Muñoz MJ, Castillo-Carranza DL, Krishnamurthy S, Paulucci-Holthauzen AA, Sengupta U, Lasagna-Reeves CA, Ahmad Y, Jackson GR, Kayed R. Amyloid-β oligomers as a template for secondary amyloidosis in Alzheimer's disease. Neurobiol Dis. 2014 Nov;71:14-23. Epub 2014 Aug 15 PubMed.

    Laganowsky A, Liu C, Sawaya MR, Whitelegge JP, Park J, Zhao M, Pensalfini A, Soriaga AB, Landau M, Teng PK, Cascio D, Glabe C, Eisenberg D. Atomic view of a toxic amyloid small oligomer. Science. 2012 Mar 9;335(6073):1228-31. PubMed.

    Liu C, Zhao M, Jiang L, Cheng PN, Park J, Sawaya MR, Pensalfini A, Gou D, Berk AJ, Glabe CG, Nowick J, Eisenberg D. Out-of-register β-sheets suggest a pathway to toxic amyloid aggregates. Proc Natl Acad Sci U S A. 2012 Dec 18;109(51):20913-8. PubMed.

    View all comments by Rakez Kayed

  2. Elucidating the identity(ies) of the specific Aβ assembly (assemblies) that is (are) most toxic to synapses and, hence, cause memory/cognitive dysfunction in AD remains a major research goal. Even though the role of Aβ in neuronal damage leading to dementia has been questioned recently (e.g., Herrup, 2015; Musiek and Holtzman, 2015), considerable evidence accumulated in the past 15 years indicates that soluble, diffusible Aβ oligomers are likely the proximal synaptotoxins in AD. However, the term “oligomer” constitutes a rather loose definition comprising assemblies ranging from dimers all the way to dodecamers and higher molecular mass species. Which of these oligomers is more closely associated with disease has baffled researchers and caused substantial controversy (Benilova et al., 2012). 

    Early studies in postmortem AD brain detected the presence of Aβ dodecamers (Gong et al., 2003), likely the same species subsequently termed Aβ*56 when detected in Tg mouse brains (Lesné et al., 2006) and verified in AD brains (Lesné et al., 2013). On the other hand, smaller assemblies (dimers/trimers) have also been found to accumulate in AD brains, to be potently toxic to neurons, and to disrupt memory (e.g., Klyubin et al., 2008; Shankar et al., 2008; Lesné et al., 2013).

    This new study represents an important step toward solving the mystery of which Aβ species represents the most relevant target for diagnostics and therapeutics. By performing an extensive and careful analysis of the immunoreactivity of two different conformational antibodies—A11 and OC, which recognize different Aβ aggregates—in brain tissue from four different transgenic mouse models of AD, the authors have identified soluble and freely diffusible Aβ oligomers, which they termed type 1 AβOs, as the species associated with cognitive deficits in mice. Interestingly, they found that accumulation of type 2 AβOs, which are spatially restricted to the immediate vicinity of dense core amyloid plaques, does not affect memory in one transgenic mouse model investigated.

    Further investigation of type 1 AβOs indicated that they comprise Aβ*56 and, possibly, other higher molecular mass species. This is an interesting result in light of other literature findings. We performed intracerebroventricular (icv) injections of high- or low-molecular mass oligomers isolated by size-exclusion chromatography under native-like conditions, and found that low molecular mass oligomers (ranging in mass from the equivalent of dimers to tetramers) caused loss of synaptophysin immunoreactivity and persistent memory impairment in mice (Figueiredo et al., 2013). On the other hand, high molecular mass oligomers (ranging from about 50 to 150 kDa) did not affect synaptophysin immunoreactivity and caused transient memory impairment, which was fully recovered by two weeks after icv injection. Interestingly, another recent study (Baker-Nigh et al., 2015) reported that high (but not low) molecular mass oligomers accumulate in AD forebrain, and may be involved in cholinergic dysfunction in AD.

    An intriguing question arising from the combined results of Liu et al. (2015) and our previous findings (Figueiredo et al., 2013) is whether high molecular mass oligomers (i.e., Aβ*56 and higher) cause permanent or transient memory impairment in mice. Although this issue has not been directly addressed in the study by Liu et al., the finding that accumulation of type 1 AβOs is associated with progressive, age-dependent memory deficits in transgenic mice would argue that such oligomers cause persistent memory damage. Our previous results, by contrast, indicated that high molecular mass oligomers do not induce structural damage to synapses or persistent cognitive impairment.

    The answer to this apparent contradiction may lie in the experimental paradigms used in the two studies. We used icv injections of synthetic Aβ oligomers into the brains of wild-type Swiss mice, whereas Liu et al. have examined the presence of oligomers in the brains of transgenic mice that express human APP variants leading to elevated Aβ levels. It is thus possible that, even though the impact of Aβ*56 and other high molecular mass oligomers might be transient, their constant production in the brains of Tg mice leads to persistently elevated levels, whereas injected oligomers may be cleared and/or detoxified in the brains of wild-type mice after a few days.

    Yet another issue is the seemingly differential sensitivity of different brain regions to oligomer toxicity. As pointed out by Liu et al., one possibility is that the rate of activity-dependent Aβ production varies in different brain regions, thus favoring kinetic pathways leading to either type 1 (more neurotoxic) or type 2 (more benign) AβOs. Another possibility that remains open, however, is that different brains regions differentially express proteins involved in AβO binding to neurons and/or in the toxic signaling triggered by oligomers. In support of the latter possibility, we recently found that Aβ oligomers injected into the lateral ventricles of cynomolgus monkeys accumulated differently in distinct brains regions (Forny-Germano et al., 2014), in a pattern not clearly related to the distance from the site of injection.

    It would be interesting to investigate the quaternary structures of type 1 and type 2 AβOs using biophysical/biochemical techniques, to complement the antibody-reactivity results of Liu et al.  Although this appears a challenging task, it may pay off in terms of improved understanding of the roles of distinct Aβ assemblies and toward development of effective diagnostics and therapeutics for AD.

    References:

    Herrup K. The case for rejecting the amyloid cascade hypothesis. Nat Neurosci. 2015 Jun;18(6):794-9. PubMed.

    Musiek ES, Holtzman DM. Three dimensions of the amyloid hypothesis: time, space and 'wingmen'. Nat Neurosci. 2015 Jun;18(6):800-6. PubMed.

    Benilova I, Karran E, De Strooper B. The toxic Aβ oligomer and Alzheimer's disease: an emperor in need of clothes. Nat Neurosci. 2012 Jan 29;15(3):349-57. PubMed.

    Gong Y, Chang L, Viola KL, Lacor PN, Lambert MP, Finch CE, Krafft GA, Klein WL. Alzheimer's disease-affected brain: presence of oligomeric A beta ligands (ADDLs) suggests a molecular basis for reversible memory loss. Proc Natl Acad Sci U S A. 2003 Sep 2;100(18):10417-22. PubMed.

    Lesné S, Koh MT, Kotilinek L, Kayed R, Glabe CG, Yang A, Gallagher M, Ashe KH. A specific amyloid-beta protein assembly in the brain impairs memory. Nature. 2006 Mar 16;440(7082):352-7. PubMed. RETRACTED

    Lesné SE, Sherman MA, Grant M, Kuskowski M, Schneider JA, Bennett DA, Ashe KH. Brain amyloid-β oligomers in ageing and Alzheimer's disease. Brain. 2013 May;136(Pt 5):1383-98. PubMed. Correction.

    Klyubin I, Betts V, Welzel AT, Blennow K, Zetterberg H, Wallin A, Lemere CA, Cullen WK, Peng Y, Wisniewski T, Selkoe DJ, Anwyl R, Walsh DM, Rowan MJ. Amyloid beta protein dimer-containing human CSF disrupts synaptic plasticity: prevention by systemic passive immunization. J Neurosci. 2008 Apr 16;28(16):4231-7. PubMed.

    Shankar GM, Li S, Mehta TH, Garcia-Munoz A, Shepardson NE, Smith I, Brett FM, Farrell MA, Rowan MJ, Lemere CA, Regan CM, Walsh DM, Sabatini BL, Selkoe DJ. Amyloid-beta protein dimers isolated directly from Alzheimer's brains impair synaptic plasticity and memory. Nat Med. 2008 Aug;14(8):837-42. PubMed.

    Liu P, Reed MN, Kotilinek LA, Grant MK, Forster CL, Qiang W, Shapiro SL, Reichl JH, Chiang AC, Jankowsky JL, Wilmot CM, Cleary JP, Zahs KR, Ashe KH. Quaternary Structure Defines a Large Class of Amyloid-β Oligomers Neutralized by Sequestration. Cell Rep. 2015 Jun 23;11(11):1760-71. Epub 2015 Jun 4 PubMed.

    Figueiredo CP, Clarke JR, Ledo JH, Ribeiro FC, Costa CV, Melo HM, Mota-Sales AP, Saraiva LM, Klein WL, Sebollela A, De Felice FG, Ferreira ST. Memantine rescues transient cognitive impairment caused by high-molecular-weight aβ oligomers but not the persistent impairment induced by low-molecular-weight oligomers. J Neurosci. 2013 Jun 5;33(23):9626-34. PubMed.

    Baker-Nigh A, Vahedi S, Davis EG, Weintraub S, Bigio EH, Klein WL, Geula C. Neuronal amyloid-β accumulation within cholinergic basal forebrain in ageing and Alzheimer's disease. Brain. 2015 Jun;138(Pt 6):1722-37. Epub 2015 Mar 1 PubMed.

    Forny-Germano L, Lyra e Silva NM, Batista AF, Brito-Moreira J, Gralle M, Boehnke SE, Coe BC, Lablans A, Marques SA, Martinez AM, Klein WL, Houzel JC, Ferreira ST, Munoz DP, De Felice FG. Alzheimer's disease-like pathology induced by amyloid-β oligomers in nonhuman primates. J Neurosci. 2014 Oct 8;34(41):13629-43. PubMed. Correction.

    View all comments by Sergio Ferreira

  3. Dr. Ferreira provides a nice summary of our study, links it to several interesting findings from his group, and offers his thoughtful suggestions on future directions.

    First, we would like to point out that the concept of type 1 and type 2 Aβ oligomers (Aβo) is defined by their spatial, temporal, and structural relationships, with Aβ fibrils constituting dense-core plaques. The molecular size of type 1 and type 2 Aβo has been unclear yet. To our knowledge, research on the size of brain-derived type 1 Aβo is quite limited thus far besides that the conformation-sensitive A11 antibodies detect Aβo in brains of transgenic APP mice and AD patients of no smaller than hexamers (~25 kDa) under denatured western blot conditions (Lesne et al., 2006; Lesne et al., 2013). Unmasking the complete gamut of structural epitopes A11 antibodies recognize is critical to unveil the structural features, including the size, of type 1 Aβo. Out-of-register, anti-parallel-β sheet is one such epitope (Laganowsky et al., 2012; Liu et al., 2012), but it is highly likely there are others.

    It is an interesting finding that Aβo influence cognitive function in a size-dependent manner. Though the size of type 1 and type 2 Aβo is not fully understood yet, a couple of observations from previous reports and this paper seem to support the findings of Dr. Ferreira and colleagues: 1) Aβ*56, a oligomeric assembly belonging to the type 1 Aβo, causes a transient spatial reference memory decline in healthy rats, when purified and injected into the animals (Lesne et al., 2006); 2) type 2 Aβo, showing their sizes no smaller than a 60 kDa globular protein in non-denatured size exclusion chromatography, lead to a transient work memory impairment in healthy rats, when type 2 Aβo-containing brain extracts were applied to animal brains (Liu et al., 2015). 

    Nonetheless, one of the key points we would like to deliver from this paper is that, in situ, spatial distribution is a key factor in determining the role of Aβo in neurological function. Should the sequestration of type 2 Aβo around plaques be disrupted, the benign Aβo would turn toxic, though in a different species. The distinct ex situ and in situ effects of type 2 Aβo add another layer of complexity to oligomer study, and suggest the importance and necessity of understanding the in situ effects of Aβo in future investigation.

    What remains unsolved, however, is the spatial distribution of Aβo in AD brain. Do the findings in transgenic mouse models reflect what occurs in AD? This is the question we are currently addressing.

    Finally, we agree that it is important to dissect the quaternary structures of type 1 and type 2 Aβo. We are developing methods to isolate individual Aβo species from a highly heterogeneous brain environment while maintaining their native conformations. We must also bear in mind that certain oligomeric species may fail to be discretely isolated because of the heterogeneous and metastable nature of Aβo. Biochemical/biophysical analyses would have to be carried out on a spectrum of conformationally analogous species.

    References:

    Laganowsky A, Liu C, Sawaya MR, Whitelegge JP, Park J, Zhao M, Pensalfini A, Soriaga AB, Landau M, Teng PK, Cascio D, Glabe C, Eisenberg D. Atomic view of a toxic amyloid small oligomer. Science. 2012 Mar 9;335(6073):1228-31. PubMed.

    Lesné S, Koh MT, Kotilinek L, Kayed R, Glabe CG, Yang A, Gallagher M, Ashe KH. A specific amyloid-beta protein assembly in the brain impairs memory. Nature. 2006 Mar 16;440(7082):352-7. PubMed. RETRACTED

    Lesné SE, Sherman MA, Grant M, Kuskowski M, Schneider JA, Bennett DA, Ashe KH. Brain amyloid-β oligomers in ageing and Alzheimer's disease. Brain. 2013 May;136(Pt 5):1383-98. PubMed. Correction.

    Liu C, Zhao M, Jiang L, Cheng PN, Park J, Sawaya MR, Pensalfini A, Gou D, Berk AJ, Glabe CG, Nowick J, Eisenberg D. Out-of-register β-sheets suggest a pathway to toxic amyloid aggregates. Proc Natl Acad Sci U S A. 2012 Dec 18;109(51):20913-8. PubMed.

    Liu P, Reed MN, Kotilinek LA, Grant MK, Forster CL, Qiang W, Shapiro SL, Reichl JH, Chiang AC, Jankowsky JL, Wilmot CM, Cleary JP, Zahs KR, Ashe KH. Quaternary Structure Defines a Large Class of Amyloid-β Oligomers Neutralized by Sequestration. Cell Rep. 2015 Jun 23;11(11):1760-71. Epub 2015 Jun 4 PubMed.

    View all comments by Karen Hsiao Ashe

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