Current Opinion in Colloid & Interface Science 16 (2011) 508–514

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Current Opinion in Colloid & Interface Science

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Assembly of Alzheimer's Aβ peptide into nanostructured amyloid fibrils

Isabel Morgado a, Marcus Fändrich b,⁎a Martin-Luther University Halle-Wittenberg, Institute for Biochemistry and Biotechnology, Kurt-Mothes-Str. 3, 06120 Halle (Saale), Germanyb Max-Planck Research Unit for Enzymology of Protein Folding & Martin-Luther University Halle-Wittenberg, Weinbergweg 22, 01620 Halle (Saale), Germany

⁎ Corresponding author. Tel.: +49 345 5524970; fax:E-mail addresses: isabel.sena-morgado@biochemtec

fandrich@enzyme-halle.mpg.de (M. Fändrich).

1359-0294/$ – see front matter © 2011 Elsevier Ltd. Aldoi:10.1016/j.cocis.2011.06.016

a b s t r a c t

a r t i c l e i n f o

Article history:Received 28 March 2011Accepted 30 June 2011Available online 13 July 2011

Keywords:PrionProtein foldingAggregationInhibitor

The self-assembly of β-amyloid (Aβ) peptide into highly ordered amyloid fibril structures represents one ofthe pathological hallmarks of Alzheimer's disease. This process leads to the transient stabilization of orderedor disordered intermediates, which are thought to act as the main pathogenic culprits in neurodegenerativeamyloid disease. This review describes recent results from different biophysical techniques, ranging fromstructure determination to single-particle methods by which the outgrowth of individual fibrils can befollowed, and it explains their contributions towards understanding the mechanism of assembly. Finally, wewill outline emerging methods and molecules to specifically interfere with the assembly and pathogenicimpact of Aβ peptide.

+49 345 5527282.h.uni-halle.de (I. Morgado),

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© 2011 Elsevier Ltd. All rights reserved.

1. Introduction

Alzheimer's disease (AD) is fundamentally associated with theself-assembly of β-amyloid (Aβ) peptide into specific intermolecularaggregates, termed amyloid fibrils [1–3]. Current research indicatesthat AD pathogenicity may not directly arise from Aβ fibrils butrather from intermediate species occurringwithin this reaction [4,5].Aβ peptide is a naturally occurring peptide fragment of the amyloidprecursor protein (APP). This transmembrane protein is degradedvia a series of proteolytic cleavage steps, ultimately releasing Aβpeptide [2]. Its hydrophobic C-terminus contains part of thetransmembrane region of APP but, due to variations in theproteolytic cleavage site, variable C-terminal ends are known tooccur (Fig. 1). The 40-residue Aβ(1–40) peptide represents the mostabundant Aβ isoform inside the brain; the 42-residue Aβ(1–42)peptide is more aggregation-prone and closely linked to AD [2].Variations in the chemical structure of Aβ also occur at the peptideN-terminus (Fig. 1); for example, truncations and pyroglutamate orisoaspartate modifications have been reported [1]. Moreover, morethan ten single-site mutagenic variants have been identified that aregenetically associated with familial AD (Fig. 1).

The peptide self-assembles via a range of differently structuredintermediates to ultimately form a polymorphic spectrum of highlyordered amyloid fibrils. Recent methodological improvements havesubstantially expanded the available structural information andrevealed important new details. This review will summarize keyfindings from the last three years on the structure of Aβ amyloid fibrilsand Aβ fibrillation intermediates. In a second step, we will describe the

mechanism of Aβ assembly and highlight recently described possibil-ities by which this process can be blocked by targeted interference.

2. Themolecular conformations ofmonomeric Aβ and Aβ aggregates

2.1. The disordered conformation of monomeric Aβ in aqueous solution

Monomeric Aβ represents, at least in principle, the starting point ofAβ self-assembly. It occurs in dilute solutions and in the absence of largeconcentrations of co-solvents or co-solutes. However, its strongpropensity to aggregate complicates experimental studies, specificallywhenworking at high concentrations, prolonged incubation times or inthe presence of certain co-solutes or co-solvents [4–7], such as salts orlipids that were shown to promote Aβ aggregation. This problem isparticularly relevant in cell-culture experiments that involve complexsolution environments, long incubation times or extensive sampleworkup procedures. In fact, some studies refer to the use of ‘monomericAβ’, although robust biophysical evidence to unequivocally demon-strate such properties is not always provided [4].

Long-standing evidence demonstrates that the molecular confor-mation of monomeric Aβ(1–40) peptide is largely disordered inaqueous solutions. Circular dichroism spectroscopic investigation ofsamples of 25 μM Aβ(1–40) in 10 mM Tris buffer, pH 7.4, records apronounced ellipticity minimum at ~197 nm [6,7] and nuclearmagnetic resonance (NMR) spectroscopy shows that bufferedsolutions of Aβ(1–40) and Aβ(1–42) peptides at pH 7.2 producemostly short-range nuclear Overhauser effects and chemical shiftvalues, which indicate the presence of a random extended chainstructure [8]. However, some chemical shift data and hydrogenexchange properties provide evidence for nascently ordered confor-mations that may occur at different structural sites of the peptide [8].

Fig. 1. Chemical variability of the Aβ primary structure. Center: chemical structure and sequence of Aβ(1–40). Upper box: natural occurring single-site variants of Aβ,several of which are associated with FAD. Residue number and alternate residue in the variant are shown. Δ refers to a deletion mutant. Lower box indicates main chainvariations, such as N-terminal pyroglutamate (p) or isoaspartate (i) modifications at residues 3 or 7 or variations at the C-terminal end.

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Monte Carlo simulations also indicated that the conformation ofmonomeric Aβ(1–40) and Aβ(1–42) peptides deviates from a purelydisordered random coil conformation and may adopt a collapsed,though still structurally flexible conformation with micelle-likestructural characteristics [9•]. This conformation is characterized bya preferential burial of the hydrophobic chemical groups of thepeptide C-terminus and of residues 12–21, while the charged aminoacid side chains at residues 22 and 23 and the peptide N-terminus arehighly solvent exposed. Monomeric Aβ shows a nascent predilectiontowards formation of β-sheet structure, which is the highest for thoseresidues that also adopt β-sheet conformation within the fibril [9•].

Increasing the temperature of the simulation from 280 to 325 Kleads to a swelling of the collapsed conformational state, as judged byan increased mean interatomic distances between the residues [9•].However, the basic, micelle-like conformation is retained. A temper-ature-dependent increase of the apparent volume of Aβ(1–42) wasalso reported by a molecular dynamics (MD) simulation of Aβ(1–42)peptide, which investigated a wider temperature range [10].Interestingly, when dissecting out the contributions arising eitherfrom Aβ or from the surrounding watermolecules, a negative intrinsicthermal expansion coefficient (αint≈−0.8×10−3 K−1) was obtainedfor the Aβ portion [10]. These observations testified to the highlyflexible and rubber-like structural properties of monomeric Aβpeptide in aqueous solution.

2.2. The α-helical or β-sheet conformation of Aβ in hydrophobicenvironments

In the presence of more hydrophobic environments, Aβ peptidereadily adopts stable and well-ordered α-helical or β-sheet conforma-tions. The lipid-induced formation of β-sheet structure is observed, forexample, in the presence of a low molar excess of negatively chargedlipid vesicles (lipid/peptide-ratio: 11) [7], sodium dodecyl sulfate (SDS)concentrations below the critical micelle concentration (CMC) [11] and1,2-dihexanoyl-sn-glycero-3-phosphocholine (DHPC) above and im-mediately below its CMC value [12]. By contrast, α-helical secondarystructurewasobservedwithAβ(1–40) peptide in thepresence of a largemolar excess of negatively charged lipid vesicles (lipid/peptide-ratio:110) [7], SDS micelles [13] or 40% trifluoroethanol (TFE) [14]. Whileformation of α-helical structure in hydrophobic environments mightnot be seen as surprising — Aβ peptide is partly derived from the APPtransmembrane region, the peptide helix is not inserted into these

vesicles or micelles. Instead it attaches to their negatively chargedsurfaces. Theα-helical structure is formed by residues 15–24 and 29–35(SDS) or 15–23 and 31–35 (TFE), as observed by NMR spectroscopy[13,14]. Thus, the residues forming the α-helical structure are almostidentical to the sequence segments of Aβ which adopt the β-sheetstructure of mature fibrils [1].

While the ultimate causes which determine the formation of eitherα-helical or β-sheet structure remain to be established, it appears thatlarge quantities of lipids or detergents are more favorable to α-helicalsecondary structure [5], presumably because these conditions isolatethe peptide molecules and thus favor the formation of the intramo-lecular hydrogen bonds of α-helices. By contrast, β-sheet structure isoften promoted at lower lipid or detergent concentrations, wherelipids may serve as mediators of the peptide self-assembly. However,β-sheet structure may also become populated in hydrophobicenvironments upon prolonged incubation conditions [5]. In addition,Aβ peptide can adsorb at the air/water interfaces and it inserts intouncompressed phospholipid monolayers, where a β-sheet conforma-tion is formed that is oriented parallel to the surface [15]. Thesestudies indicated that lipids promote the assembly of Aβ into fibrilsand can lead to the formation of specific structural states that could beimplicated in the disease process. However, numerous other publi-cations have also shown that toxic Aβ aggregates may be formed inthe absence of lipids and detergents.

2.3. The molecular conformation of mature Aβ amyloid fibrils

The terminal end products of the amyloidogenic reaction cascadeare mature amyloid fibrils. Although these fibrils are not currentlybelieved to be the main pathogenic culprits in AD, they are closelyimplicated in cerebral amyloid angiopathy, where large quantities ofAβ fibrils become deposited in cerebral blood vessel walls, causinghemorrhages and stroke. By nature, Aβ fibrils have significantstructural rigidity, as revealed by persistence length measurements(33–108 μm) [16]. Thus, dysfunctions arise from impairments causedin naturally elastic tissues through infiltration by inflexible amyloidfibrils. Aβ fibril structures at atomic resolution are not currentlyavailable, but many details of the fibril architecture have beenrevealed by a battery of biophysical methods.

Amyloid fibrils are defined as linear and highly ordered assemblieswith numerous peptide molecules being stacked along the fibril axis(Fig. 2). The resulting β-sheet structures are, in principle, infinite and

Fig. 2. Structural models of Aβ amyloid fibrils derived from cryo-EM. Surface representation of the reconstructed electron density (gray) and structural models of the cross-β sheetshown (color). Possible orientation of the Aβ molecules (β-strands) indicated, with yellow representing the N-termini and dark red the C-termini. Two oppositely directed Aβmolecules form one molecular layer of the protofilament core. Left: views of cross-sectional slices of the electron density; right: side views. Images are based on original dateobtained by Sachse, et al. [21] and Schmidt et al. [20].

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have their backbone hydrogen bonds oriented parallel to the fibril axis,whereas theβ-strands of thefibril run counter to this direction (‘cross-β’)(Fig. 2). Aβ fibrils are highly polymorphic [17•,18•,19], and each Aβpeptide can form a plethora of different fibril morphologies — evenwithin the same sample [18•]. This polymorphism complicates identifi-cation of systematic differences between Aβ(1–40) andAβ(1–42) fibrils.Although differences between specific Aβ(1–40) and Aβ(1–42) fibrilpreparations have been reported, several properties of Aβ(1–40) andAβ(1–42) fibrils are largely similar. Cryo-EM revealed a commonprotofilament substructure in Aβ(1–40) and Aβ(1–42) fibrils [20•], andsimilar sequence segments appear to be involved in the formation of thecross-β sheet [1]. Most publications report β-strand conformation atsimilar residues, usually involving amino acids 16–20 and 31–36, aparallel β-sheet orientation and a mostly flexible and structurallydisordered conformation at the peptide N-terminus [1]. Therefore,more work is required before Aβ(1–40) and Aβ(1–42) peptide-specificdifferences can be qualified unambiguously.

To explain the fold and structural packing of the Aβ peptide in thefibril, different structural models have been put forward. Most availablemodels assume a U-shaped peptide conformation [1,17•] but structuralreconstruction of an Aβ(1–40) amyloid fibril at sub-nanometer(0.8 nm) resolutionhas revealed anarchitecture different frompreviousproposals [21••]. The electron density was interpreted with a modelwhere the basic structural unit of the protofilament is formed by twooppositely directed Aβ molecules (Fig. 2). This dimeric arrangementmay be retained in Aβ fibril polymorphs that consist of differentcombinationsof two similarly structuredprotofilaments [18•].However,given the significant extent of polymorphism within typical amyloidfibril samples, makes it likely that further fibril morphologies exist,perhaps also encompassing a U-shaped peptide fold.

2.4. The molecular conformation of fibrillation intermediates

The assembly of mature amyloid fibrils occurs via a range of differentfibrillation intermediates. Electron or atomic force microscopy (EM orAFM) provides close-ups of the rough guise of several of these states, butdirect comparisons between different studies are complicated byvariable terminologies [4]. We here distinguish three general groups ofintermediates: non-fibrillar aggregates, protofibrils and annular aggre-gates. This classification is basedon theoverall particle shape, as obtainedfrom EM or AFM. However, it remains arbitrary and does not excludeoverlapping groupings. Many publications describe a general order of

monomersNnon-fibrillar aggregatesNprotofibrils/annular aggregatesNmature fibrils (Fig. 3a). Methods were described to stabilize some ofthese intrinsicallymetastable structures and tomake them accessible fordetailed biophysical analysis. A key technique to investigate theirstructure has been solid-state NMR spectroscopy. Structural data interms of chemical shift measurements are now available for severaldifferent Aβ(1–40) conformers, including Aβ monomers, non-fibrillaraggregates, protofibrils and mature fibrils (Fig. 3b). The obtainedchemical shift data setsprovidedevidenceabout the secondary structuralelements and the residues participating in their formation. However,these solid-state NMR studies investigated only a fraction of the residuesof Aβ peptide (Fig. 3b). Hence our current structural understanding isaffected by significant gaps and as with the mature amyloid fibrils,atomic resolution structures are not currently available.

(i) The first group of intermediates to be described here, the non-fibrillar aggregates, are multimeric assemblies with a non-fibrillar overall structure. These intermediates can be structur-ally diverse, and their sizes range from small-sized oligomers,such as dimers, trimers etc., to particles with considerablemolecular mass and diameter (N50 nm) [4]. They can haveviscoelastic properties [22], and their non-specific shape on EMor AFM images sometimes leads to them being termed‘amorphous aggregates’, without reference to their secondarystructure. In fact, there have been only occasional reports ofrandom coil-like characteristics [23], while many studiesimplied considerable β-sheet content [23–27]. Solid-stateNMR identified the residues forming this β-sheet structure inAβ(1–40) [27] and Aβ(1–42) oligomers [26] and revealedsignificant differences in the measured 13Cα and 13Cβ chemicalshifts from monomeric Aβ(1–40), which shows more randomcoil-like characteristics (Fig. 3b).However, mixed conclusions were obtained regarding theconformational similarity of oligomers and mature fibrils. Inthe case of Aβ(1–40) oligomers, significant structural similarityto fibrils was reported [27], while a study on Aβ(1–42)oligomers rather emphasized the existence of structuraldifferences compared with fibrils [26]. Evidence for structuraldifferences between oligomers and fibrils was also provided byseveral other methods. For example, non-fibrillar aggregatescan provide significant affinity to certain antibodies [28], whilefibril-specific dyes, such as Congo red and Thioflavin T, or fibril-

Fig. 3. Structural assembly of Aβ into amyloid fibrils. (a) Schematic representation of the possible Aβ assembly reaction, indicating the occurrence of different intermediates.(b) Deviation of the measured Aβ(1–40) 13Cα (red) and 13Cβ (blue) chemical shift values and their deviation from random coil values shows the formation of β-sheetstructure [8,17,27,32]. In the plot, β-sheet structure is associatedwith positive values for 13Cβ (blue) and a negative value of 13Cα (red). Green arrows: assignment of β-strandstructure as suggested in the original study. Gray boxes: no chemical shift value reported for the respective amino acid residues. In cases where a study reports two chemicalshift values for a given residue, the more intense signal (or first given value) is shown.

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specific antibody fragments, like B10, are not explicitlyrecognized [24,29]. Hence, non-fibrillar aggregates and fibrilsdiffer in their surface characteristics. Fourier-transform infra-red (FTIR) spectroscopy further indicated differences in theirsecondary structure, and it was concluded that non-fibrillaraggregates may encompass significant antiparallel β-sheetstructure, while fibrils possess parallel β-sheet characteristics[24,25].

(ii) The second group of intermediates to be defined here areprotofibrils. Following their initial description, we refer toprotofibrils as linear aggregates that lack the very high structuralorder of mature amyloid fibrils [29–31]. Protofibrils are oftenthinner than mature fibrils, and they possess a more worm-like,curvilinear morphology, but no highly periodic features, such asa discernible fibril supertwist. Protofibrils, similar to non-fibrillaraggregates, provide only low affinity for typical amyloid dyes[29]. Nevertheless, they encompass a highly ordered β-sheetstructure, which was demonstrated by a variety of techniques,including circular dichroism [31], X-ray diffraction (Braggspacings at 4.62±0.02 and 9.88±0.05 ×102 pm) and FTIR(amide I maximum at 1624 cm−1) [24]. A solid-state NMRstudy of protofibrils trapped by an antibody-derived proteinligand revealed β-strand conformation at residues 16–22 and30–36 (Fig. 3b), whereas the peptide N-terminus presentschemical shift values corresponding to those of a random coil[32•]. Measured 13Cα and 13Cβ chemical shifts related moreclosely to literature values from oligomers than to valuesobtained with mature fibrils, indicating that protofibrils may

structurally correspond to oligomers, while a relatively signifi-cant structural rearrangement must occur when converting intomature fibrils [32•]. Moreover, the β-strands assigned to maturefibrils are often longer than in the case of protofibrils (Fig. 3b).

(iii) The third group of intermediates to be described here areannular aggregates (often also referred to as protofibrils) [5].While their structure has been confirmed by EM and AFM,detail structural information is scarce and different structuralmodels have been put forward [5]. This group of intermediateshas received considerable attention in searching for thepathogenic agent in AD, as it was suggested that Aβpathogenicity might arise from interactions between thefibrillation intermediates and lipid bilayers or cellular mem-branes, resulting in their perforation and ultimately leading tocell death [5]. Ongoing work using the above and newtechnologies are leading to increased understanding of therelevance of each of these intermediate species to AD.

3. The mechanism and kinetics of Aβ fibril formation

Aβ self-assembly into amyloid fibrils occurs from supersaturatedsolutions and at peptide levels above the critical concentration of fibrilassembly [33]. Different biophysical methods have been developed tomonitor the kinetics and mechanism of fibril formation in real time,for instance by using light scattering or fluorescent dyes, such asThioflavin T. The obtained data typically reveal a biphasic kineticprofile, consisting of lag and growth phase [33]. The biphasic nature ofthese data indicated a nucleation polymerization mechanism,

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consisting of a slow formation of fibrillation nuclei (lag phase) and asubsequent fast elongation of fibrils (growth phase). Systematicevaluation of the length of the lag time (length of the lag phase) and ofthe apparent rate constant of fibril growth, which can be obtained byfitting experimental data within the growth phase, indicate anintrinsic relationship between the two phases [34,35•]. However,systematic computational analysis of data obtained with severalamyloidogenic systems argue that a pure nucleation-polymerisationmechanism does not well explain the experimentally observed kineticdata and that additional modulations or amplifications of this processmust occur [35•,36••]. These additional events include, for instance,heterogeneous nucleation reactions and fibril fragmentation events,resulting from sample agitation and shear forces.

MD simulations, carried out with Aβ-derived peptides or coarse-grained model systems, have provided snapshots of the assemblyreaction and depicted possible structures occurring in the course of thisprocess [37,38••]. These studies suggest an early collapse of the peptidesinto nonfibrillar aggregates and amicelle-like architecture of these earlyassemblies [39]. They comprise different peptide conformations thatmay be compatible with β-sheet structure or not [39], and sampledifferent registrations or antiparallel and parallel strand–strand pair-ings, before certain combinationswill prevail [40].While assemblymustbe driven by a lower free enthalpy of the end products compared withthe starting conformations, recent MD simulations revealed that thereaction kinetics can significantly contribute in defining the fibrilassembly pathways and morphologies [38••]; i.e. fibril morphologiesthat are energetically less favorablemay nucleatemore readily and thusaccumulate more preferentially. These data imply that the nucleationbarrier contributes to controlling the population and distribution offibril morphologies under a given set of conditions.

The self-assembly of proteins or polypeptide chains into amyloidfibrils occurs concomitantly with profound changes in the density oftheir structural packing. High-precision volumetric analyses of differentβ-microglobulin states (native, unfolded, protofibrils, mature fibrils)revealed that the transition from acid-unfolded protein to matureamyloid fibrils is associated with a positive change in the partial freevolume [41••]. This change is even larger than the one observed fornative protein folding; i.e. when the polypeptide chain folds up into aglobular conformation. Hence, the average packing density in maturefibrils is lower than in the native protein. Protofibrils, by contrast,showed a negative change of the partial free volume compared with

Fig. 4. Single particle analysis of amyloid fibril growth. (a) Real-time fluorescent observationrepresents 10 μm). (b) Schematic representation of fibril growth from a seed (fibril fragmedistance measurement at the two different ends of the fibril. Fibril extension consists of fast pfibril growth. Image in (a) taken from Ban et al. [43] with permission.

acid-unfolded protein. These findings can be explained by a highlydense packing of specific sequence segments in the intermediates,which leave a considerable part of the protein chain fully solventexposed and hydrated. Maturation into terminal fibril assemblies stripsoff these water molecules, and they are replaced by an assembly thatencompasses significant packing defects [41••]. The observation of asignificant repacking of the polypeptide chain during β-microglobulinfibril formation is consistent with the measurements with Aβ peptidedescribed in the previous section [32•]. They imply a significantremodeling of the polypeptide structure as aggregation progressesfrom protofibrils to mature amyloid filaments.

AFMand total internal reflectionfluorescencemicroscopy have beenused to monitor the assembly of single, surface-immobilized Aβ(1–40)or Aβ(1–42) fibrils under buffered solutions [42••,43]. These real timetechniques enabled the direct visualization and kinetic examination oflate events during fibril elongation (Fig. 4a). They revealed that the twotips of the fibril grow at different speed (Fig. 4b), indicating influence ofthe polar architecture of Aβ amyloid fibrils, which is consistent with theobservation of polarity of many amyloid fibrils by cryo-EM [20•,21••]. Inaddition, a stepwise growth dynamics was found, where bursts of fibrilelongation are interrupted by pause phases featuring little apparentfibril growth (Fig. 4c) [42••]. These single-particle techniques alsorevealed fibril branching events, resulting from the heterogeneousnucleation of new filaments on the surface of an existing fibril [44••].Fibril branching might be a specific property of only some amyloidsystems, as it was found to occur with glucagon but not with Aβ(1–40)amyloid fibrils [44••].

4. Specific modulators of Aβ self-assembly

The self-assembly of Aβ peptide into amyloid fibrils can bemodulated through different environmental factors and externallyadded molecules, suggesting possibilities to generate inhibitors withtherapeutic relevance. The general effectors of aggregation include pHlevel, temperature, pressure, salts, lipids or metal ions [5]. However,recent research has led to the generation of different molecules whicheither target a particular step within the amyloidogenic pathway orwhich bind to a specific conformational state. The chemical structureof these molecules ranges from small-sized organic compounds toglobular proteins. The following description highlights selectedexamples that illustrate their applicative potential.

of the growth of a single Aβ(1–40) amyloid fibril, immobilized on a glass slide (scale barnt), showing the polarity of fibril extension. (c) Schematic representation of a growthhases of fibril extension, which are interrupted by stationary phases with nomeasurable

Fig. 5. Molecules which block specific steps of Aβ fibril formation. (a) Chemical structure of EGCG, which blocks the structural conversion of oligomers to fibrils [45]. EM images ofAβ(1–42) peptide incubated with and without EGCG, as provided by J. Bieschke and E. Wanker (Berlin). (b) Ribbon diagram of the B10 antibody fragment. The complementarydetermining regions are highlighted red, green and blue. Its dimeric form, B10AP, was shown to prevent the formation of mature Aβ(1–40) fibrils by stabilizing their protofibrillarprecursors [24]. EM images of Aβ(1–40) peptide incubated with and without B10AP show differences in morphology. Scale bars represent 100 nm.

• of special interest.•• of outstanding interest.

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The polyphenol (−)-epi-gallocatechine gallate (EGCG) (Fig. 5a),a natural compound derived from green tea, was found to preventthe formation of Aβ fibrils by redirecting the amyloidogenic pathwaytowards non-toxic and non-fibrillar aggregates [45•]. As EGCGinteracts not only with Aβ peptide but also with α-synuclein fromParkinson's disease and other disease-amyloidogenic polypeptidechains, there is the potential that EGCG might be effective inpreventing different types of amyloidosis. A related poly-amyloidspecific affinity was observed for B10 antibody fragment, whichrecognizes a surface pattern that is common to different amyloidfibrils [46]. This antibody fragment was generated to specificallyrecognize amyloid fibril conformations. Indeed, B10 displays strongbinding to Aβ amyloid fibrils, while disaggregated Aβ or non-fibrillarAβ aggregates are not explicitly recognized [24]. Addition of B10AP, arecombinant dimer of B10, to solutions of freshly dissolved Aβpeptide blocked the formation of terminal amyloid fibrils and led tothe accumulation of protofibrillar intermediates [24]. Therefore,B10AP targets a later step within the amyloidogenic cascade thanEGCG (Fig. 5b).

Complementary to these efforts, several other studies were able tospecifically target Aβ monomers or the Aβ sequence. While thesestrategies are only viable with AD and not with other types ofamyloidosis, they do not require any assumption concerning a commonconformational state to be targeted. For example, the artificial bindingprotein ‘ZAβ3 affibody’ was shown to bind monomeric Aβ in a β-turnconformation. Expression of this affibody in Drosophila melanogasterflies, which presented a severe Aβ-dependent phenotype, potentlyneutralized the Aβ-dependent toxicity and lifespan reduction seen inthe flies [47•]. Other inhibitory strategies rest on the concept ofsequence-specific interference by development of peptide-derivedinhibitors complementary to Aβ peptide. For example, the self-association of Aβ can be modulated by its cross-reactivity with otheramyloidogenic peptides. The islet amyloid polypeptide (associatedwithtype 2 diabetes)was found to interact with Aβ peptide, suppressing theformation of thioflavin T-binding fibrillar species and cytotoxicaggregates [48•]. These interactions seem to be mediated by relativelyshort sequence segments that present structural complementarity tothe partner peptide, as identified by membrane-bound peptide arrays.Ultimately, it was possible to use a small dipeptide, which consists of D-tryptophan and α-aminobutyric acid, to interfere with Aβ aggregationin vitro and in vivo [49••]. Treatment of mice, which overexpress humanAPP, with this dipeptide, led to a significant improvement of theircognitive performance, demonstrating that it is possible to ameliorateAβ-dependent learning deficits with rationally designed and chemi-cally synthesized compounds.

5. Conclusions

Different biophysical techniques have provided detail structuralinformation about various Aβ peptide conformers, ranging fromlargely disordered Aβ monomers to mature amyloid fibrils. Cryo-EMprovided images of the fibril structure at sub-nm resolution andsuggested an arrangement of the peptide molecules that was notanticipated by previously existing fibril models. Fibril formation isassociated with a progressive stabilization of β-sheet structure indifferent type of Aβ intermediates, as demonstrated by solid-stateNMR and other techniques. The extension into fibrils follows a step-wise growth kinetics where phases of rapid fibril growth areinterrupted by stationary phases with no measurable fibril extension.The process of amyloid fibril formation can be modulated at specificsteps, suggesting new possibilities for the therapeutic intervention.

One of the challenges of future research will be to investigateamyloidogenic processes not only inside the test tube, but withinnatural cellular environments. For example, it has been shown thatfibril and plaque-forming Aβ peptides can become sequestered andaccumulate at certain subcellular sites and compartments of cellculture models, such as within multivesicular bodies [50]. Moreover, amultitude of factors which occurs inside and outside the cell, is able toinfluence Aβ fibrillogenesis, such as lipids, glycosaminoglycanes anddifferent amyloid-binding proteins [1,2,5]. The development of newinhibitors or amyloidmodifying agents will obviously depend on theireffectiveness in such environments. The promising results obtained inthe different animal model systems outlined above raise hopes thatbetter andmore powerful ways of treating AD patients may arise fromsuch strategies.

AcknowledgmentsThe authors gratefully acknowledge the images and data provided

by T. Ban, J. Bieschke, Y. Goto, M. Schmidt, E. Wanker and M. Zagorski.I.M. and M.F. are supported by DFG, BMBF (ProNet-T3) and thecountry Sachsen-Anhalt (Exzellenznetzwerk Biowissenschaften). Thefunding sources did not influence the writing of this article.

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