Single Point Mutations Induce a Switch in the Molecular Mechanism of

Letters pubs.acs.org/acschemicalbiology

Single Point Mutations Induce a Switch in the Molecular Mechanism of the Aggregation of the Alzheimer’s Disease Associated Aβ42 Peptide Benedetta Bolognesi,†,⊥ Samuel I. A. Cohen,† Pablo Aran Terol,† Elin K. Esbjörner,† Sofia Giorgetti,‡ Maria F. Mossuto,§ Antonino Natalello,∥ Ann-Christin Brorsson,† Tuomas P. J. Knowles,† Christopher M. Dobson,*,† and Leila M. Luheshi*,† †

Department of Chemistry, University of Cambridge, Lensfield Road, Cambridge, CB2 1EW, United Kingdom Department of Biochemistry, University of Pavia, via Taramelli, 27100, Pavia, Italy § Ospedale San Raffaele, Via Olgettina 60, 20132 Milan, Italy ∥ Department of Biotechnology and Biosciences, University of Milano-Bicocca, Pizza della Scienza 2, 20126 Milan, Italy ‡

S Supporting Information *

ABSTRACT: Single point mutations in the Alzheimer’s disease associated Aβ42 peptide are found to alter significantly its neurotoxic properties in vivo and have been associated with early onset forms of this devastating condition. We show that such mutations can induce structural changes in Aβ42 fibrils and are associated with a dramatic switch in the fibrildependent mechanism by which Aβ42 aggregates. These observations reveal how subtle perturbations to the physicochemical properties of the Aβ peptide, and the structural properties of fibrils that it forms, can have profound effects on the mechanism of its aggregation and pathogenicity.

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aggregation.13 In order to understand how the intrinsic physicochemical properties of the Aβ42 peptide contribute to determining the precise molecular mechanism by which Aβ42 forms aggregates, including neurotoxic oligomers, we investigate the intrinsic aggregation behavior of two single point mutants, which have been shown in our previous Drosophila studies to modulate dramatically the in vivo neurotoxicity of Aβ42.7 We find that these mutations bring about drastic switches between distinct Aβ42 aggregation mechanisms and are also associated with changes in the properties of the fibrils that are ultimately formed. We first analyzed the aggregation behavior of the Aβ42 peptide carrying the ‘arctic’ mutation, E22G, which is associated with an inherited early onset form of AD.14,15 We determined initially whether or not the aggregation behavior of the E22G Aβ42 peptide is dominated by primary or by secondary (fibrildependent) nucleation processes, by measuring its aggregation kinetics, under quiescent conditions, in the presence and absence of fibrillar seeds (Figure 1a). We followed the kinetics of fibril formation using ThT fluorescent measurements, which we verified to be linearly related to the total mass of fibrils in our recent study on wild type Aβ42.13 In the same context, we have also shown that the mass concentration of fibrillar species, as measured by ThT, is approximately equal to the total aggregate mass concentration. Addition of 1% fibrils preformed

n addition to adopting their functional conformations, peptides and proteins are inherently prone to self-assemble into highly ordered structures known as amyloid fibrils1 that possess a characteristic cross-β core,2 in which an array of βstrands is stabilized by a network of hydrogen bonds.3 While this cross-β structure is, in principle, a state accessible to all polypeptide sequences, the propensity of different proteins to form amyloid fibrils varies widely depending on their physicochemical properties.4,5 The formation and in vivo deposition of such structures is closely linked to the onset and progression of neurological disorders such as Alzheimer’s disease (AD) and is known to be modulated by a wide range of extrinsic cellular factors.6 We have shown, however, by combining computational analysis, a Drosophila model of Aβ42 neurotoxicity and biophysical measurements of Aβ aggregation, that the relative propensity to aggregate in vivo correlates strongly with the intrinsic properties of the peptides and proteins themselves, such as charge and hydrophobicity.7,4,5 Over the last two decades, the study of the pathological behavior of several Aβ42 variants has increased dramatically our understanding of the relationship between amino acid sequence and pathological outcome.8,9 An important feature of fibril formation by Aβ42 is the concomitant formation of oligomeric intermediates, some of which are highly neurotoxic and are now thought to be primarily responsible for the neurodegeneration observed in AD.10−12 It has been shown recently that a variety of distinct mechanisms, involving a range of both primary and secondary (i.e., fibril dependent) processes, can be involved in the formation of these neurotoxic oligomers during Aβ 42 © 2013 American Chemical Society

Received: April 25, 2013 Accepted: November 7, 2013 Published: November 7, 2013 378

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Figure 1. Fitting of the aggregation kinetics of E22G Aβ42 and E22G/I31E Aβ42. Seeded (orange) and unseeded (light blue) aggregation reactions for 0.5 μM E22G Aβ42 (a) and 5 μM E22G/I31E Aβ42 (d). Global fits of E22G Aβ42 (b) and E22G/I31E Aβ42 (e) aggregation kinetics measured at different peptide concentrations. Double logarithmic plot of the variation of the time of half completion with the initial monomer concentration for E22G Aβ42 (c) and E22G/I31E Aβ42 (f). (g) Global mechanisms of the aggregation process and corresponding predicted slopes of the double logarithmic plot. The scaling exponents for monomer dependent secondary process and primary nucleation have been reported for the case of a second order reaction in the monomer concentration. (h) A comparison of the slopes of the two variants with that of the WT Aβ42.11

0.03). Strikingly, the exponent of the double logarithmic relationship between the half-time of the reaction and the monomer concentration for E22G Aβ42 (−0.54 ± 0.05) is almost identical to that found for wild type Aβ42 aggregation under conditions of high shear (−0.62 ± 0.04),13 where a secondary process, which is zeroeth-order in the monomer concentration (here, filament fragmentation), dominates the aggregation kinetics as observed for E22G Aβ42 (Figure 1b,c). Thus, the introduction of the E22G Aβ42 mutation results in a switch in the kinetic process of Aβ42 aggregation from a monomer dependent to a monomer independent secondary nucleation dominated process, consistent with a mechanism in which existing aggregates acts as the major source of new nuclei. We have found in a previous study that the introduction of a second mutation, I31E, into the E22G Aβ42 peptide results in an essentially complete rescue of its neurotoxicity in Drosophila and significantly reduces its propensity to form neurotoxic prefibrillar aggregates in vitro but not its ability to form deposits in the brain.7,16 We therefore investigated the effects of the I31E mutation on the aggregation mechanism of E22G Aβ42 by measuring the aggregation kinetics of this peptide in a range of concentrations where a lag phase in the formation of ThTbinding amyloid structures is clearly observable. First, a comparison of the seeded and unseeded in vitro aggregation kinetics for the I31E/E22G Aβ42 peptide confirms that its aggregation kinetics are again dominated by a secondary process (Figure 1d, orange vs blue curve). Unlike the case of the E22G peptide itself, however, the analysis of the unseeded kinetics (10−50 μM) (Figure 1e) reveals that there is a very strong monomer dependence of the secondary process that

from E22G Aβ42 accelerates the aggregation of the soluble peptide to the extent that the reaction rate has reached its limiting value before the unseeded reaction has formed a detectable quantity of ThT-positive aggregates. This behavior indicates that the kinetics of aggregation of E22G Aβ42, in common with wild type Aβ42,13 are dominated by secondary processes that depend on the presence of fibrillar aggregates, as no significant primary nucleation occurs in an unseeded reaction on a time scale that is sufficient for the seeded reaction to reach completion. The aggregation kinetics were then measured for a range of concentrations (1−5 μM) of E22G Aβ42 at which, in the absence of seeds, a measurable lag phase in the formation of ThT-binding amyloid structures was clearly observable. The data were then fitted globally using integrated rate laws that describe the process of amyloid fibrillar growth8 (Figure 1 b). An important feature of this type of analysis is that it predicts a power law relationship between the half-time of the reaction and the initial monomer concentration, the exponent of which is determined by the reaction order of the secondary processes that dominate the aggregation kinetics of Aβ42 (Figure 1g).8 The result for E22G Aβ42 (−0.54 ± 0.05) (Figure 1c) reveals that these secondary processes are zero order with respect to the initial monomer concentration, that is, that the concentration of monomeric Aβ42 molecules in solution is not rate limiting for the proliferation of new aggregates. This finding is of particular interest as a similar analysis carried out for the wild type Aβ42 peptide under similar quiescent conditions reveals a very different result.11 In this case, the rate determining step, and thus the proliferation of amyloid aggregates, is strongly dependent on the monomer concentration (slope = −1.33 ± 379

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Figure 2. Properties of amyloid fibrils of the mutational variants. (a) FTIR spectrum of fibrils of E22G Aβ42 (continuous line) and I31E/E22G Aβ42 (dashed line). The arrow points at the disordered region (∼1653 cm−1). All the experimental data were fitted to a set of Gaussian curves (S.I.) (b) Relative secondary structure content of E22G Aβ42 and I31E/E22G Aβ42 as determined by curve fitting. (c) Time of appearance of soluble fragments during the tryptic digestion of E22G Aβ42 and I31E/E22G Aβ42 fibrils.

Figure 3. Fibrillar architecture. (a) Aggregated samples of E22G Aβ42 (top) and E22G/I31E Aβ42 (bottom) as imaged by AFM after prolonged periods (5 days). The expanded areas indicate the characteristic appearance of species from each sample. (b) Height distributions of the species detected by AFM for E22G Aβ42 (top) and E22G/I31E Aβ42 (bottom). (c) Dot-Blot assay showing binding of B10 antibody to isolated fibrils of I31E/E22G AB42 and E22G AB42.

dominates the kinetics of aggregation of I31E/E22G Aβ42 kinetics of aggregation (Figure 1e,f). Remarkably, the exponent

of the double logarithmic plot of the reaction half-time vs monomer concentration for I31E/E22G Aβ42 is −1.31 ± 0.06, 380

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lower elastic moduli than more ordered species implying that, consistent with their lower β-sheet content (Figure 2), their constituent monomers are held together by weaker interactions,3 leaving them more prone to fragmentation or to disruption by extrinsic factors such as proteases. Conversely, the more extensive hydrogen bonded core and the higher levels of interfilament organization in I31E/E22G Aβ42 are more likely to result in a more stable fibrillar state.18,19,23 Correspondingly, we found that the I31E/E22G Aβ42 fibrils are significantly more resistant to trypsin digestion than those of the E22G Aβ42 peptide. Residues 17−28, which NMR studies have shown to be within the core of wild type Aβ42 fibrils,24,25 remained resistant to proteolysis by trypsin for greater than one hour for I31E/E22G Aβ42 fibrils, whereas this same fragment is released from E22G Aβ42 fibrils after only 20 min of tryptic digestion (Figure 2c). The monomer dependent secondary process determining the aggregation kinetics of I31E/E22G Aβ42 involves both the monomeric form of the peptide and the surface of fibrils and is, therefore, like that of WT Aβ42, attributable to surfacedependent nucleation. We also tested, therefore, whether or not the fibrils of the two variants differed in their surface properties by evaluating their ability to bind to the conformation specific camelid antibody B10,26,27 which has been shown to interact specifically with negatively charged fibrillar surfaces.26,27 We found that E22G Aβ42, but not I31E/ E22G Aβ42, fibrils bind strongly to B10 in a dot blot assay, indicating that the changes in their core structural characteristics propagate to changes in the properties of their exposed surfaces (Figure.3c), and hence could affect radically the ability of fibrils to act as efficient catalysts for the nucleation of new oligomers. Such a change in the templating properties of fibrils is likely to contribute strongly to the switch between the dominant nucleation mechanisms described in this study. In summary, we have shown that the structural changes in the fibrillar state of the Aβ42 peptide that are observed to occur upon introduction of single point mutations can be accompanied by changes in the dominance of the microscopic processes by which these aggregates are themselves formed. This work highlights the vital importance of future investigations to determine the detailed microscopic molecular mechanisms that underlie the aggregation of peptides and proteins associated with neurodegenerative diseases, as well as the effect of perturbations to these mechanisms, if we are to intervene rationally and successfully in such processes to prevent or even reverse the devastating symptoms to which they give rise.

that is, within experimental error, identical to that of the wild type Aβ42 peptide (−1.33 ± 0.3).7 Thus, the introduction of the I31E mutation into the E22G Aβ42 peptide results in a dramatic increase in the monomer dependence of its aggregation kinetics such that this mechanism reverts to that of the wild type Aβ42 peptide, where the formation of new nuclei occurs predominantly through the interaction of monomers on the surface of pre-existing fibrils.7 We note that the common feature in the aggregation kinetics of all the Aβ42 variants examined so far is their dependence on secondary phenomena (i.e., that the formation of fibrillar aggregates drives the generation of new aggregates). We therefore set out to characterize the properties of the fibrillar aggregates formed by the differently toxic Aβ42 variants. We find that the I31E mutation affects profoundly the morphology of the E22G Aβ42 fibrils,16 and we have investigated in greater depth whether or not these mutations induce differences in the structural properties of fibrils that can influence the manner in which they participate in further aggregation processes. Fourier transform infrared (FTIR) spectroscopy demonstrates that E22G Aβ42 peptide forms fibrils with a significantly lower β-sheet content (38.1 ± 1.8%) than that of fibrils formed by the I31E/E22G Aβ42 peptide (50.8 ± 6.8%) as indicated more clearly by a less intense band at 1628 cm−1 in the spectrum of the former (Figure 2a). Furthermore, E22G Aβ42 fibrils have a significantly higher content of disordered structure, (21.4% ± 3.4) relative to I31E/E22G Aβ42 fibrils (10.2% ± 2.1), as shown most clearly by a more intense band in the spectrum at 1650 cm−1 (Figure 2a,b).17 This result is likely to arise from substitution of E22 with a flexible glycine residue, as this lower β-sheet content is not observed in wild type Aβ42 fibrils (Supporting Information Figure 1). This mutation also leads to loss of E22 charge, another factor that may concur with the destabilization of the fibrillar β-sheet structure. Upon introduction of the second mutation, Ile31 to Glu31, the structure of the fibrils reverted to a more wild-type-like state, with a more prominent β-core, despite the presence of Gly22. A negative charge at residue 31 may help stabilizing the fibrillar βsheet through the reestablishment of electrostatic interactions within the low dielectric constant core region. This fundamental difference in the extent of the β-sheet core of the fibrils formed by E22G and I31E/E22G Aβ42 fibrils may offer an explanation for the dramatic difference in their ability to nucleate aggregation, as a more extensive hydrogen-bonded cross-β core is associated with greater stability of the fibrillar state.3,18,19 We next investigated, using atomic force microscopy (AFM), whether or not differences in the cross-β core of E22G and I31E/22G fibrils correlate with changes in their morphology. E22G Aβ42 forms a heterogeneous mixture of roughly spherical aggregates (aspect ratio 2.5, defined in Figure 3 as thin fibrils) which vary in height between 2 and 4 nm. By contrast I31E/E22G Aβ42 forms long, straight, well-ordered fibrillar structures that fall into one of three distinct height clusters of 3.2, 4.2 (thin fibrils) and 7.0 nm (thick fibrils), consistent with the packing of two, three, or four protofilaments into the fibrils (Figure 3)16,20−22 This observation suggests that the I31E/E22G fibrillar species have a much greater degree of interfilament organization than the more heterogeneous species formed by E22G Aβ42. Less-ordered protofibrillar-like species similar to those formed by E22G Aβ42 have been shown previously to have

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METHODS

Peptides. Synthetic Aβ42 and its mutated variants were purchased from Bachem. Briefly, after TFA/HFIP treatment, the peptides were dissolved directly in 20 μM ThT and 50 mM NaH2PO4 at pH 7.4, and allowed to aggregate at 29 °C. Thioflavin T fluorescence measurecments were performed with a Fluostar Optima plate reader. Fitting of the Aggregation Kinetics. The aggregation kinetics were fitted by means of a recently published approach where a single master equation accounts for each microscopic process involved in the aggregation reaction.13 Fourier Transform Infrared Spectroscopy. Aβ42 fibrils (1 mg mL−1) were directly deposited on the bio-ATR cell of a Bruker Equinox 55 system and FTIR spectra between 1580 and 1720 cm−1 were recorded following buffer evaporation. Limited Proteolysis. Trypsin was added to Aβ42 fibrils at a 1:700 concentration, and aliquots were taken at every time point for 381

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inhibition of trypsin activity with 1% TFA and subsequent characterization of the digested fragments through laser desorption/ionization. Atomic Force Microscopy. A JPK Nano Wizard II microscope was used to image samples of 10 μM aggregate peptide deposited on freshly cleaved mica. Further details for all methods are described in the Supporting Information.

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(11) Haass, C., and Selkoe, D. J. (2007) Soluble protein oligomers in neurodegeneration: Lessons from the Alzheimer’s amyloid [β]peptide. Nat. Rev. Mol. Cell Biol. 8, 101−112. (12) Ono, K., Condron, M. M., and Teplow, D. B. (2009) Structureneurotoxicity relationships of amyloid β-protein oligomers. Proc. Natl. Acad. Sci. U.S.A. 106, 14745−14750. (13) Cohen, S. I., Linse, S., Luheshi, L. M., Hellstrand, E., White, D. A., Rajah, L., Otzen, D. E., Vendruscolo, M., Dobson, C. M., and Knowles, T. P. (2013) Proliferation of toxic Aβ42 oligomers occurrs through secondary nucleation. Proc. Natl. Acad. Sci. U.S.A. 110, 9758− 9763. (14) Nilsberth, C., Westlind-Danielsson, A., Eckman, C. B., Condron, M. M., Axelman, K., Forsell, C., Stenh, C., Luthman, J., Teplow, D. B., Younkin, S. G., Näslund, J., and Lannfelt, L. (2001) The ’Arctic’ APP mutation (E693G) causes Alzheimer’s disease by enhanced Aβ protofibril formation. Nat. Neurosci. 4, 887−893. (15) Johansson, A. S., Berglind-Dehlin, F., Karlsson, G., Edwards, K., Gellerfor, P., and Lannfelt, L. (2006) Physiochemical characterization of the Alzheimer’s disease-related peptides A β 1−42 Arctic and A β 1−42wt. FEBS J. 273, 2618−2630. (16) Brorsson, A. C., Bolognesi, B., Tartaglia, G. G., Shammas, S. L., Favrin, G., Watson, I., Lomas, D. A., Chiti, F., Vendruscolo, M., Dobson, C. M., Crowther, D. C., and Luheshi, L. M. (2010) Intrinsic determinants of neurotoxic aggregate formation by the amyloid β peptide. Biophys. J. 98, 1677−1684. (17) Barth, A. (2007) Infrared spectroscopy of proteins. Biochim. Biophys. Acta Bioenergy 1767, 1073−1101. (18) Kodali, R., Williams, A. D., Chemuru, S., and Wetzel, R. (2010) Aβ(1−40) forms five distinct amyloid structures whose β-sheet contents and fibril stabilities are correlated. J. Mol. Biol. 401, 503−517. (19) Shammas, S. L., Knowles, T. P., Baldwin, A. J., Macphee, C. E., Welland, M. E., Dobson, C. M., and Devlin, G.L.. (2011) Perturbation of the stability of amyloid fibrils through alteration of electrostatic interactions. Biophys. J. 100, 2783−2791. (20) Fandrich, M., Schmidt, M., and Grigorieff, N. (2011) Recent progress in understanding Alzheimer’s β-amyloid structures. Trends Biochem. Sci. 36, 338−345. (21) Petkova, A. T., Ishii, Y., Balbach, J. J., Antzutkin, O. N., Leapman, R. D., Delaglio, F., and Tycko, R. (2002) A structural model for Alzheimer’s β-amyloid fibrils based on experimental constraints from solid state NMR. Proc. Natl. Acad. Sci. 99, 16742−16747. (22) Paravastu, A. K., Leapman, R. D., Yau, W. M., and Tycko, R. (2008) Molecular structural basis for polymorphism in Alzheimer’s βamyloid fibrils. Proc. Natl. Acad. Sci. U.S.A. 105, 18349−18354. (23) Mossuto, M. F., Bolognesi, B., Guixer, B., Dhulesia, A., Agostini, F., Kumita, J. R., Tartaglia, G. G., Dumoulin, M., Dobson, C. M., and Salvatella, X. (2010) The non-core regions of human lysozyme amyloid fibrils influence cytotoxicity. J. Mol. Biol. 402, 783−96. (24) Olofsson, A., Sauer-Eriksson, A. E., and Ohman, A. (2006) The solvent protection of Alzheimer amyloid-β-(1−42) fibrils as determined by solution NMR spectroscopy. J. Biol. Chem. 281, 477− 483. (25) Luhrs, T., Ritter, C., Adrian, M., Riek-Loher, D., Bohrmann, B., Döbeli, H., Schubert, D., and Riek, R. (2005) 3D structure of Alzheimer’s amyloid-β(1−42) fibrils. Proc. Natl. Acad. Sci. U.S.A. 102, 17342−17347. (26) Haupt, C., Morgado, I., Kumar, S. T., Parthier, C., Bereza, M., Hortschansky, P., Stubbs, M. T., Horn, U., and Fändrich, M. (2011) Amyloid fibril recognition with the conformational B10 antibody fragment depends on electrostatic interactions. J. Mol. Biol. 405, 341− 348. (27) Habicht, G., Christian Haupt, C., Friedrich, R. P., Hortschansky, P., Sachse, C., Meinhardt, J., Wieligmann, K., Gellermann, G. P., Brodhun, M., Götz, J., Halbhuber, K. J., Röcken, C., Horn, U., and Fändrich, M. (2007) Directed selection of a conformational antibody domain that prevents mature amyloid fibril formation by stabilizing Aβ protofibrils. Proc. Natl. Acad. Sci. U.S.A. 104, 19232−19237.

ASSOCIATED CONTENT

S Supporting Information *

Additional figure and detailed methods. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected]. *E-mail: [email protected]. Present Address ⊥

Centre for Genomic Regulation, UPF, 08003 Barcelona, Spain

Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work has been supported by the Alzheimer’s Research Trust (B.B. and C.M.D.). We thank M. Fändrich for the generous gift of the B10-AP antibody domain.

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REFERENCES

(1) Chiti, F., and Dobson, C. M. (2006) Protein misfolding, functional amyloid, and human disease. Annu. Rev. Biochem. 75, 333− 366. (2) Sunde, M., Serpell, L. C., Bartlam, M., Fraser, P. E., Pepys, M. B., and Blake, C. C. (1997) Common core structure of amyloid fibrils by synchrotron X-ray diffraction. J. Mol. Biol. 273, 729−739. (3) Knowles, T. P., Fitzpatrick, A. W., Meehan, S., Mott, H. R., Vendruscolo, M., Dobson, C. M., and Welland, M. E. (2007) Role of intermolecular forces in defining material properties of protein nanofibrils. Science 318, 1900−1903. (4) Chiti, F., Stefani, M., Taddei, N., Ramponi, G., and Dobson, C. M. (2003) Rationalization of the effects of mutations on peptide and protein aggregation rates. Nature 424, 805−808. (5) Dubay, K. F., Pawar, A. P., Chiti, F., Zurdo, J., Dobson, C. M., and Vendruscolo, M. (2004) Prediction of the absolute aggregation rates of amyloidogenic polypeptide chains. J. Mol. Biol. 341, 1317−1326. (6) De Strooper, B. (2010) Proteases and proteolysis in Alzheimer disease: A multifactorial view on the disease process. Physiol. Rev. 90, 465−494. (7) Luheshi, L. M., Tartaglia, G. G., Brorsson, A. C., Pawar, A. P., Watson, I. E., Chiti, F., Vendruscolo, M., Lomas, D. A., Dobson, C. M., and Crowther, D. C. (2007) Systematic in vivo analysis of the intrinsic determinants of amyloid β pathogenicity. PLoS Biol. 5, 2493−2500. (8) Murakami, K., Irie, K., Morimoto, A., Ohigashi, H., Shindo, M., Nagao, M., Shimizu, T., and Shirasawa, T. (2003) Neurotoxicity and physicochemical properties of Aβ mutant peptides from cerebral amyloid angiopathy: Implication for the pathogenesis of cerebral amyloid angiopathy and Alzheimer’s disease. J. Biol. Chem. 278, 46179−46187. (9) Perálvarez-Marín, A., Mateos, L., Zhang, C., Singh, S., CedazoMínguez, A., Visa, N., Morozova-Roche, L., Gräslund, A., and Barth, A. (2009) Influence of residue 22 on the folding, aggregation profile, and toxicity of the Alzheimer’s amyloid β peptide. Biophys. J. 97, 277−285. (10) Walsh, D. M., Klyubin, I., Fadeeva, J. V., Cullen, W. K., Anwyl, R., Wolfe, M. S., Rowan, M. J., and Selkoe, D. J. (2002) Naturally secreted oligomers of amyloid β protein potently inhibit hippocampal long-term potentiation in vivo. Nature 416, 535−539. 382

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