Sequestration of the Aβ Peptide Prevents Toxicity and Promotes Degradation In Vivo

An engineered protein prevents aggregation of the Aβ peptide and facilitates clearance of Aβ from the brain in a fruit fly model of Alzheimer's disease.


Introduction
Of the neurodegenerative disorders that have been linked to protein misfolding and aggregation [1], Alzheimer's disease (AD) is the most common [2,3].Transgenic animal models have shown that aggregation of the Alzheimer b-peptide (Ab) causes memory impairment [4,5] and cognitive deficits [6] similar to those seen in patients suffering from AD. Ab aggregation precedes neuritic changes [7], and there is a quantitative correlation between the propensities of mutant forms of Ab to aggregate and their neurotoxicity [8].In vitro aggregation of Ab proceeds from the initial association of monomers into oligomeric, but still soluble, assemblies that ultimately form highly structured and insoluble amyloid fibrils [1,9,10,11].Evidence suggests that the primary neurotoxic species are the soluble oligomeric aggregates [4,5,12,13] and that a fundamental building block may be dimeric Ab species [14].However, despite this progress, the details of Ab aggregation in vivo, the structure of toxic aggregates, the mechanism of toxicity, and in particular, the relationship between aggregate formation and peptide clearance are not known.
We set out to investigate a novel approach to study the dynamics of Ab aggregation in vitro and neurotoxicity or degradation in vivo by using a conformation-specific Ab binding protein, the Z Ab3 Affibody [15,16].Affibody molecules are engineered binding proteins, which are selected by phage display from libraries based on the three-helix Z domain [17,18].The Z Ab3 Affibody was selected [15] to bind specifically to Ab monomers with nanomolar affinity (dissociation constant K d <17 nM) [16].It forms a disulfide-linked dimer to which Ab binds and folds by induced fit [19] into a hairpin conformation such that its two aggregation-prone hydrophobic faces become buried within a tunnel-like cavity in the Z Ab3 dimer [16,19].The specificity and well-characterized structural features of Z Ab3 binding to Ab make it an ideal candidate for studying the effects of Ab monomer binding in vivo.We find that the presence of the Affibody molecule, achieved by co-expression, can eliminate Ab neurotoxicity in a fruit fly (Drosophila melanogaster) model of AD [20,21], and we used biochemical and biophysical experiments to identify the molecular mechanism by which this process occurs.

Elimination of Ab Neurotoxicity In Vivo
We first generated Drosophila strains transgenic for Z Ab3 .As Z Ab3 is most effective in binding Ab when it is in its dimeric form, we also generated Drosophila in which two copies of Z Ab3 are connected head-to-tail-(Z Ab3 ) 2 -to enable the disulfide-linked dimer to form more readily.Drosophila transgenic for the wild-type Z domain were used as controls.These three Affibody fly lines were then each crossed with Drosophila transgenic for Ab 42 , Ab 42 E22G [22], or Ab 40 , and the co-expression of both transgenes together in the brain or in the eye was initiated by crossing with appropriate driver flies [20,21].
Expression of Ab 42 E22G in the brain of Drosophila causes rapid neurodegeneration resulting in a drastic reduction in lifespan from 38 (61.8) to 9 (60.5)days, consistent with the findings of previous studies [8].Co-expression of Z Ab3 with Ab 42 E22G, however, increases the lifespan to 20 (60.2) days.Strikingly, if the-head-totail dimer (Z Ab3 ) 2 is co-expressed with Ab 42 E22G, the toxic effects of the peptide are yet further reduced and the lifespan increases to 31 (60.8)days, which is almost as long as in wild-type controls (Figure 1A, Table S1) and indicates that the neurotoxicity of Ab has been almost entirely abolished.Co-expression of the Z domain, which has no affinity for Ab, does not affect Ab 42 E22G  toxicity, demonstrating that the rescue of Ab toxicity in vivo is specific to Z Ab3 .Co-expression of Z Ab3 with wild-type Ab 42 also significantly prolongs the lifespan of these flies (from 28, 60.4, to 32, 60.7, days).Again, the (Z Ab3 ) 2 head-to-tail-dimer is even more effective, completely eliminating the toxicity associated with Ab 42 (lifespan 40, 61.2, days), whereas the Z domain control has no effect (Figure 1B).Expression of the less aggregation-prone Ab 40 has no effect on lifespan, and none of the Affibody molecules or the control significantly affected the lifespan of flies expressing Ab 40 or wild-type Drosophila (Figure 1C and 1D).
The ability of (Z Ab3 ) 2 to abolish the toxic effects of Ab 42 E22G was confirmed physiologically by its ability to abolish the abnormal eye morphology associated with Ab 42 E22G expression in the photoreceptors in the fly (Figure 2).

Clearance of Ab from the Drosophila Brain
To determine the mechanism by which Z Ab3 mediates suppression of Ab toxicity, we assessed the levels of Ab 42 in the brains of flies co-expressing Ab 42 E22G and either Z Ab3 , (Z Ab3 ) 2 , or the Z domain by Western blotting.Fly brains were homogenized in 1% SDS, subjected to electrophoretic separation, and probed using an antibody against the N-terminus of Ab, which detailed structural studies reveal remains exposed in the Ab:Z Ab3 complex [16].SDS soluble Ab can clearly be detected in flies expressing Ab 42 E22G, but it is absent in flies co-expressing Z Ab3 or (Z Ab3 ) 2 (Figure 3A).The specificity of this effect is confirmed by the continued presence of the Ab 42 E22G in flies that co-express the non-binding Z domain.
The Z Ab3 :Ab complex is stable in 1% SDS (B.Macao, unpublished), and Ab remaining in complexes or in SDS insoluble aggregates in the fly brain might therefore not be detectable by Western blot.In order to address this possibility, fly brains expressing Ab 42 E22G with or without (Z Ab3 ) 2 , Z Ab3 or the Z domain were homogenized in 5 M GdmCl, conditions known to dissociate both Ab aggregates and Ab:Z Ab3 complexes.The total level of Ab 42 E22G in these extracts was then measured by a sensitive ELISA assay (Figure 3B).Flies expressing both (Z Ab3 ) 2 and Ab 42 E22G show a 97% (63%) reduction in the concentration of Ab 42 E22G compared to flies co-expressing Ab 42 E22G and the inert Z domain (the most appropriate control for the non-specific effects of expressing a second transgene on the levels of Ab).Decreased Ab 42 E22G levels in the presence of different Affibody constructs correlate well with corresponding reduction in neurotoxicity measured by the survival assay (Figure 1).
The prevention of Ab 42 E22G aggregation by Z Ab3 and (Z Ab3 ) 2 is demonstrated by immunohistochemical detection of Ab 42 E22G in whole mount brain preparations analyzed by confocal microscopy.Flies expressing Ab 42 E22G under the control of the OK107-Gal4 driver, which drives expression in a subset of adult neurons, contain abundant deposits in the brain recognized by the anti-Ab 6E10 antibody, whereas flies co-expressing Ab 42 E22G and (Z Ab3 ) 2 have almost no visible 6E10 immunoreactive deposits (Figure 3C).In good agreement with the results of the ELISA analysis, coexpression of Z Ab3 results in a significant reduction in the burden of aggregates but does not result in their complete removal, whereas co-expression of the Z domain gives levels of Ab deposits similar to those present in flies expressing Ab 42 E22G.
In order to determine whether the presence of Ab 42 E22G had altered the levels of Z Ab3 or (Z Ab3 ) 2 present in the fly brain, brain homogenates were analyzed using either anti-cMyc antibodies to detect Z Ab3 or anti-Affibody antibodies to detect (Z Ab3 ) 2 ; both dimeric Affibody molecules can be observed as 12 kDa dimers under non-reducing conditions.The levels of these Affibody species are not detectably altered in flies co-expressing Ab 42 E22G (Figure 3D) despite the marked reduction of the levels of soluble Ab 42 E22G (Figure 3A).While this experiment suggests that Ab clearance could be occurring without the corresponding clearance of its binding partner Z Ab3 , the quantities seen by Western blot

Author Summary
Alzheimer's disease is thought to be a result of neuronal damage caused by toxic aggregated forms of the Ab peptide in the brain.There is no cure and existing treatments are ineffective in reversing or preventing disease progression.Here we describe a novel strategy that makes use of an engineered ''Affibody'' protein to study the disease and potentially combat its underlying causes.The Affibody occludes the aggregation-prone regions of Ab peptides, preventing their aggregation into toxic forms, and it also acts to dissolve pre-formed Ab aggregates.It is functional in vivo, as its co-expression with Ab peptides in transgenic fruit flies prevents the neuronal damage and premature death that result from expression of Ab peptides alone.Moreover, we show that the origin of this protection is the enhanced clearance of Ab peptides from the brain.These findings open up new opportunities for using engineered binding proteins to probe the origins of Alzheimer's disease and potentially to develop a new class of therapeutic agents.
represent the equilibrium levels of these two proteins, and so would not detect any turnover in Z Ab3 that may also be occurring.
We have established that the reductions in the levels of Ab 42 E22G peptide in the fly brain are not due to altered gene regulation in flies co-expressing Z, Z Ab3 , or (Z Ab3 ) 2 , because the levels of Ab 42 E22G transcription are not significantly reduced in any case (Figure 3E).
In summary, Z Ab3 causes a reduction in Ab 42 E22G levels by actively promoting its clearance from the brain.The clearance does not involve any specific antibody-mediated process, since Drosophila lacks an adaptive immune system [23].In order to determine at which stages of the Ab aggregation process the Z Ab3 Affibody can intervene, we analyzed the effects of Z Ab3 on the dynamic interconversion of monomeric, oligomeric, and fibrillar Ab species in vitro.S1. doi:10.1371/journal.pbio.1000334.g001monomeric Ab.When a molar excess of Z Ab3 is added at different times during the aggregation process, it effectively inhibits all further aggregation (Figure 4B and Figure S3B), indicating that not only does Z Ab3 effectively block aggregation even after its initiation, but also that monomeric Ab is accessible for binding throughout the process of fibril formation.

Kinetics of Amyloid Fibril Dissolution
We noted, however, during the course of the experiments that the ThT fluorescence signal tends to fall after the addition of Z Ab3 at advanced stages of the fibril formation reaction, suggesting that Z Ab3 may also act to reverse the aggregation process (Figure 4D and Figure S3C).To determine the kinetics of fibril dissolution by Z Ab3 in vitro, we set up experiments in which Ab 40 monomers dissociating from pre-formed fibrils are captured by Z Ab3 .We used 15 N-labelled Ab 40 for these experiments so that monomeric Ab 40 in complex with Z Ab3 could be identified by solution nuclear magnetic resonance (NMR) spectroscopy at low micromolar concentrations.The large fibrillar aggregates of 15 N-Ab 40 (Figure 4E) did not generate an observable NMR spectrum even after 24 h of data collection, as expected, due to slow molecular tumbling and no highly mobile residues.The addition of Z Ab3 , however, generated resonances from Z Ab3 -bound monomeric 15 N-Ab 40 , indicating a gradual dissolution of the fibrils (Figure 4F and Figure S4).Only a small fraction of the Ab 40 , however, dissociates from the fibrils over the first three weeks; thereafter the dissolution process becomes very slow, even for fibrils fragmented by sonication (Figure 4G).Still, under these conditions the observed level of dissolution does not represent the equilibrium state, as the pre-formed Ab 40 :Z Ab3 complex is stable in the presence of Ab 40 fibrils (Figure S5).Hence, even though binding of the Z Ab3 Affibody to monomeric Ab 40 can act to dissolve fibrils, the dissociation kinetics are too slow, at least in vitro, for dissolution to be achievable in practice under ambient conditions.

Inhibition and Dissolution of Ab Oligomers
In order to determine the critical issue of whether or not Z Ab3 can prevent the formation of smaller Ab aggregates (oligomers), we examined their formation in vitro by size exclusion chromatography (SEC) in the presence and absence of Z Ab3 (Figure 5A to 5D and Figure S6).Oligomeric species [24] appear within hours in solutions of Ab 42 , prepared by dilution from alkaline conditions [25], where the monomeric species is initially dominant.The partitioning between monomeric and oligomeric Ab then reaches an interim steady state after ,10 h before the onset of the formation of amyloid fibrils (Figure 5A).By contrast, in the presence of the Z Ab3 , oligomer formation is completely inhibited (Figure 5B), a result that can be attributed to the sequestration of Ab 42 within the complex formed with the Affibody.
Isolated Ab 42 oligomers contain elements of well-defined bsheet structure as measured by circular dichroism (CD), but the bsheet content is lower than in mature fibrils (Figure 5E).Their stability is also lower as isolated oligomers dissociate into monomers and convert into amyloid fibrils (Figure 5C).Addition of the Z Ab3 Affibody results in dissolution of the oligomers after a few days (Figure 5D, 5F, and 5G and Figure S7).This is because binding of monomeric Ab acts to shift the dynamic monomeroligomer equilibrium such that the oligomer population is reduced, and NMR (Figure 5H) and SEC analyses (Figure S6) consequently also reveal monomeric Ab 42 in complex with Z Ab3 .

Conclusion
The presence of the Z Ab3 Affibody in vivo results in the effective inhibition of Ab toxicity and the promotion of Ab degradation.These effects can be attributed to the ability of the Z Ab3 Affibody to act in three distinct ways on the Ab aggregation process.First, monomeric Ab will be sequestered by Z Ab3 , the result of which is that toxic Ab aggregates will not be able to form in the brain.Second, if Ab aggregation were to occur, it can be slowed, halted, and even reversed by the action of Z Ab3 on the dynamic Ab monomer-aggregate equilibria.Furthermore, the presence of Z Ab3 not only prevents or reverses Ab aggregate formation, it also promotes clearance from the brain.We envisage that this could occur either by intracellular lysosomal or proteasomal degradation, or alternatively by the secretion and uptake by phagocytic cells of the Z Ab3 :Ab complex.
The results furthermore demonstrate how engineered binding proteins, such as Affibody molecules, that target specific protein conformations can be used to gain important insights into the dynamics of the Ab aggregation process and its toxic consequences both in vivo and in vitro.

Fly Genetics
Drosophila melanogaster transgenic for Ab 40 , Ab 42 , and Ab 42 E22G have been described previously [20].Drosophila transgenic for the Z domain, Z Ab3 , and the (Z Ab3 ) 2 head-to-tail dimer were created by standard p element mediated germ line transformation using pUAST (Brand and Perrimon) as the expression vector.Affibody cDNA was inserted into the multiple cloning site of pUAST using EcoR1 and Xho1, except for (Z Ab3 ) 2 , which was cloned between EcoR1 and Xba1 sites.Each transgene was preceded by the same secretion signal peptide (MASKVSILLLLTVHLLAAQTFAQ), derived from the Drosophila necrotic gene, in order to target its expression to the secretory pathway.Transgenes were injected into w1118 embryos.
Drosophila transgenic for Ab 40 , Ab 42 , and Ab 42 E22G were each crossed with Drosophila transgenic for Z, Z Ab3 , and (Z Ab3 ) 2 to create stable double transgenic stocks.Expression of the transgenes was achieved using the UAS-Gal4 system.UAS-Tg flies were crossed with flies expressing Gal4 under the control of either a neuronal promoter (elavc155 or OK107) or eye specific promoter (gmr).All fly crosses were maintained on standard cornmeal/agar fly food in humidified incubators.Crosses to generate flies expressing Affibody molecules or Ab were performed at 29uC.

Survival Assays
Survival assays were performed as described previously [20].Briefly, 100 flies of each genotype were collected, divided into tubes of 10 flies, and kept at 29uC.The number of live flies was counted every 2-3 days and recorded.Survival curves were calculated using the Kaplan-Meier method, and differences between genotypes were assessed using the log-rank test.

Rough Eye Phenotype
Transgenes were expressed in the eye by crossing with gmr-Gal4 flies.Crosses were performed at 29uC.Flies were collected on the day of eclosion and sputter coated using 20 nM of Au/Pd in a Polaron E5000.SEM images were collected using a Philips XL30 Microscope.

Protein Extraction and Western Blotting
Fifty flies were snap frozen in liquid nitrogen and decapitated for each genotype.Fly heads were homogenized in PBS/1% SDS containing protease inhibitors (Complete, Roche Applied Science, UK).Homogenates were then centrifuged at 12,100 g for 1 min to remove insoluble material, and the supernatants were collected for analysis.Protein concentration in each supernatant was determined using the DC Protein Assay (Biorad).Equal quantities of protein for each genotype were loaded on to 4%-12% Bis/Tris SDS PAGE gels (Invitrogen) for detection of Affibody molecules and 4%-12% Tris/glycine SDS PAGE gels (Invitrogen) for detection of Ab.Electrophoresis was performed under nonreducing conditions, and protein was transferred to nitrocellulose membranes for Western blotting.Z Ab3 was detected using a mouse monoclonal anti-c-Myc antibody (clone 9E10, Abcam), and (Z Ab3 ) 2 was detected using a goat anti-Affibody antibody (Abcam).Ab was detected using a mouse monoclonal anti-Ab antibody directed against the N terminus of Ab (6E10, Signet).All blots were developed using Supersignal West Femto Maximum Sensitivity ECL substrate (Pierce).

Total Ab ELISA
Heads from flies expressing Ab 42 E22G with or without Affibody domains were subjected to mechanical homogenization in 5 M GdmCl, 50 mM Hepes, and 5 mM EDTA followed by 4 min of sonication in a water bath.Homogenates were centrifuged for 7 min at 12,100 g to pellet any GdmCl insoluble material.Supernatants were diluted in 50 mM Hepes and 5 mM EDTA with protease inhibitors to a final concentration of 1 M GdmCl.A sandwich ELISA was performed on the supernatants using biotinylated 6E10 (Signet) and a C terminal Ab x-42 -specific antibody 21F12 (kind gift of D. Schenk, Elan).Protein levels were measured using a Sector Imager (Meso Scale Discovery) and normalized to a percentage of the level obtained for flies expressing Ab 42 E22G alone.

Immunohistochemistry
Flies of all genotypes were crossed with OK107-Gal4 flies (Bloomington Stock No. 854) to drive expression in a subset of neurons that includes, but is not limited to, the mushroom bodies.For each genotype fly brains were dissected in PBS with 0.05% Triton X-100 and fixed in 4% paraformaldehyde for 1 h at room temperature.The brains were then washed three times in PBS/ 0.05% Triton X-100 and blocked in 5% w/v bovine serum albumin in PBS for 1 h at room temperature.Fly brains were incubated overnight in mouse anti-Ab (6E10, Signet) diluted 1:1000 in blocking buffer.After three further washes in PBS/ 0.05% Triton X-100, brains were then incubated in goat antimouse IgG Alexa 546 (Invitrogen) and counterstained with TOTO-3 (Invitrogen) to detect nuclei before mounting in Vectashield (Vectorlabs) anti-fade mounting medium.

Confocal Microscopy
Confocal serial scanning images were acquired at 2 or 4 mm intervals (for high magnification and low magnification images, respectively) using a Nikon Eclipse C1si on Nikon E90i upright stand (Nikon).The image stacks were projected using ImageJ (version 1.42k), and the resulting composite images were processed using Photoshop CS4 software (Adobe Systems).

Transcription Assay
Concentrations of mRNA were determined using quantitative real time PCR (RT-PCR).Twenty-five flies per genotype were collected and snap frozen in liquid N 2 .RNA was extracted from each group of 25 fly heads using TriZol followed by DNAse treatment to remove residual genomic DNA and reverse transcription to produce cDNA.Each sample was subjected to two separate quantitative PCR reactions to detect Ab mRNA and the control gene Actin5c.Real time amplification of cDNA was monitored using SYBR Green fluorescence in a Bio-Rad iQ Cycler.
Protein Samples for Biophysical Analysis Z Ab3 was produced in Escherichia coli and purified as described elsewhere [16].Ab peptides were obtained from a commercial source (rpeptide, Bogart, GA, USA), synthesized in-house, or produced (with an N-terminal methionine) by recombinant coexpression of Ab and Z Ab3 in E. coli [26].Experiments were carried out in 20 mM sodium phosphate, 50 mM NaCl, except for the NMR experiments where NaCl was not included, and pH 7.2.10 mM ThT was added prior to fluorescence measurements.

Ab Fibril Formation
Fibril formation assays were carried out as described previously [16].TEM images were obtained using a LEO 912 AB Omega microscope.CD spectra were recorded on a JASCO J-810 spectropolarimeter.

Ab Fibril Dissolution
Fibrils were prepared from Ab 40 at a concentration of 100 mM with the same set-up and conditions as for the fibril formation assays, but in the absence of ThT.After 3 days of incubation at 37uC, fibrils were isolated by centrifugation at 16,000 g.To remove any residual soluble peptide, fibrils were washed by resuspension in buffer F [20 mM sodium phosphate, pH 7.2, 0.1% sodium azide, complete protease inhibitor (Roche; at the concentration recommended by the manufacturer)], followed by centrifugation.Fibrils were resuspended in buffer F supplemented with 10% D 2 O to a final concentration of 300 mM Ab 40 and investigated by 15 N HSQC NMR with 24 h of data collection on a Varian Inova 900 MHz NMR spectrometer (equipped with a cryogenic probe) or on a Varian Inova 800 MHz spectrometer.The intensity of resonances originating from bound Ab 40 detected in the presence of 325 mM of unlabeled Z Ab3 was followed over time by recording a series of 24 h 15 N HSQC NMR spectra.Five mM of 15 N-Z Ab3 served as an internal concentration reference, assuming identical NMR-sensitivities of the intense resonances of the three C-terminal residues of bound Ab 40 and free Z Ab3 .Sonication was achieved by placing the NMR tube with the fibril sample into a Misonix water bath sonicator for 2 min before acquisition of NMR data.

Ab Oligomer Formation and Dissolution
Oligomer formation was induced by adjusting the pH of alkaline (pH,10.5)solutions of Ab 42 (concentration #100 mM) in 20 mM sodium phosphate and 50 mM sodium chloride to pH 7.2 (with 1 M HCl) [25].The samples were incubated at 21uC and oligomer formation was monitored with SEC and ThT fluorescence.Fifty ml (for analytical runs) or 1 ml (for preparative oligomer isolation) aliquots were injected onto an A ¨KTA Explorer system (GE Healthcare, Uppsala, Sweden) equipped with a Superdex 75 10/300 column, and the elution was monitored by UV absorbance at 220 nm.Preparative oligomer isolation was carried out 4-20 h after induction of oligomer formation and yielded oligomer solutions at 10-20 mM total Ab 42 concentration.The elution volumes of the Z Ab3 :Ab 42 complex and free Z Ab3 were determined in separate runs of the isolated complex or free Affibody, respectively, and conformed to previous SEC studies [19].The amounts of Ab 42 in the monomeric, oligomeric, or Z Ab3 -bound fraction were determined from the elution peak areas obtained by integration using the Unicorn software provided with the chromatography system.The data were normalized by setting to unity the sum of the oligomer and monomer peak areas in the first SEC profiles (at t = 0.2 h for oligomer formation in Figure S6A, and at t = 0.5 h for oligomer dissolution in Figure S6C).The fraction of high molecular weight aggregates that did not enter the column bed was calculated as the difference between unity and the sum of the monomer and oligomer fractions.The fraction of Z Ab3bound Ab 42 shown in Figure 5D was obtained by comparison of the integrated Z Ab3 :Ab 42 /free Z Ab3 peak area with those obtained in calibration runs of free Z Ab3 (set to 0) and Z Ab3 :Ab 42 complex (set to 1) using the same protein concentrations as in the dissolution experiment.The fraction of Ab 42 bound to Z Ab3 was determined by 15 N HSQC NMR employing an internal concentration standard.Samples were incubated at 37uC and data points were recorded every 4 min (Ab 42 ) or 2 min (Ab 42 E22G) with 10 s of orbital shaking preceding the measurement using a FLUOstar OPTIMA reader (BMG) equipped with 440 nm excitation and 480 nm emission filters.Samples analyzed by TEM (in C) were applied to formvar/carbon coated copper grids, stained with 2% (w/v) uranyl acetate, and viewed in a Philips CEM100 transmission electron microscope.  1N-Z Ab3 in the free state do not appear.This demonstrates that Ab 40 does not leave the complex to be incorporated into the fibrils, i.e. the complex is stable in the presence of Ab 40 amyloid fibrils.Moreover, resonances of 15 N-Ab 40 bound to Z Ab3 do not appear in (B), i.e. 15 N-Ab 40 monomers do not dissociate from the fibrils to exchange with unlabeled Ab 40 monomers in the Z Ab3 complex.This finding is in agreement with the high kinetic stability of Ab amyloid fibrils reported in this study.The lifetime of the Ab 40 :Z Ab3 complex was determined as 2.6 (60.3) h at 21uC.Dissociation of the complex cannot therefore be rate-limiting in this experiment.Lifetime determination was carried out by successive recording of the 15 N-HSQC NMR spectrum of 15 N-Z Ab3 : 15 N-Ab 40 complex after addition of an excess of unlabeled Z Ab3 and monitoring the decrease in the intensity of the resonances assigned to bound 15

Supporting Information
Sequestration of the hydrophobic regions of Ab40 and Ab 42 (Figure 4A and Figure S1) allows Z Ab3 to inhibit amyloid fibril formation completely, even that of the extremely aggregationprone Ab 42 E22G variant, as judged by thioflavin T (ThT) fluorescence assays indicative of amyloid fibril formation (Figure 4B-D, Figure S2, and Figure S3).The addition of Z Ab3 to Ab 40 or Ab 42 aggregation reactions has the same effect on the aggregation kinetics as reducing the Ab concentration by the equivalent amount (Figure 4C and Figure S3A), demonstrating that inhibition of fibril formation occurs by sequestration of

Figure 2 .Figure 1 .
Figure 2. Rescue of Drosophila eye morphology.Scanning electron micrographs (SEM) of eyes of flies expressing Ab 42 E22G alone or in combination with the Z domain control or the (Z Ab3 ) 2 Affibody at low and high magnification.A wild-type non-transgenic fly eye is shown for comparison.Scale bar = 100 mm in main pictures and 20 mm in inserts.doi:10.1371/journal.pbio.1000334.g002

Figure 3 .
Figure 3. Clearance of Ab from the Drosophila brain.(A) Electrophoretic (SDS PAGE) analysis of soluble Ab in fly brain extracts.A clear Ab immunoreactive band is seen at 8 kDa (consistent with an Ab dimer [14]) in flies expressing Ab 42 E22G and flies co-expressing Ab 42 E22G and the Z domain.The 8 kDa Ab immunoreactive band is absent in flies co-expressing Ab 42 E22G and either Z Ab3 or (Z Ab3 ) 2 .b-actin immunodetection (bottom row) served as a loading control.(B) ELISA analysis of total (soluble and insoluble) Ab 42 E22G concentration in the brains of flies expressing the different Affibody constructs.The levels of Ab 42 E22G measured in the presence of the different Affibody molecules are expressed as a percentage of the concentration in the Ab 42 E22G alone control.Differences between genotypes were analyzed by ANOVA and post hoc t tests.** p,0.01.(C) Immunohistochemistry and confocal microscopy analysis of Ab 42 E22G aggregates in intact brains from flies expressing Ab 42 E22G alone or in combination with different Affibody constructs.Anti-Ab immunostaining is shown in red, with a nuclear counterstain (TOTO-3) shown in blue.White boxes in brain images to the left are magnified to the right.Ab immunoreactive aggregates are observed as puncta and are abundant in the brains of flies expressing Ab 42 E22G alone or in combination with the Z domain.Immunoreactive Ab deposits are sparse in brains where Z Ab3 is co-expressed with Ab 42 E22G and absent in brains where (Z Ab3 ) 2 is co-expressed with Ab 42 E22G.(D) SDS PAGE analysis of Z Ab3 and (Z Ab3 ) 2 levels in the presence and absence of Ab 42 E22G.Twelve kDa anti-c-Myc immunoreactive bands (consistent with a disulfide linked Z Ab3 dimer) of equal intensity are detected in Z Ab3 -expressing flies in the presence or absence of Ab 42 E22G.Twelve kDa anti-Affibody immunoreactive bands of equal intensity are also detected for the head-to-tail linked (Z Ab3 ) 2 dimer.(E) Quantitative RT-PCR analysis of Ab mRNA levels in flies expressing Ab in combination with different Affibody constructs or the Z domain control.The relative levels of Ab mRNA detected in flies expressing Ab 42 E22G in combination with Z (white), Z Ab3 (red), and (Z Ab3 ) 2 (blue) compared to that detected in flies expressing Ab 42 E22G alone (black) do not differ significantly (n.s., not significant).doi:10.1371/journal.pbio.1000334.g003

Figure 4 .
Figure 4. Inhibition of Ab 40 amyloid fibril formation.(A) Structure of the Z Ab3 Affibody (blue and cyan) in complex with an Ab 40 hairpin (residues 16 to 40; red) [16].White spheres represent buried nonpolar side chains (core) of Z Ab3 .(B-D) Kinetics of Ab 40 amyloid fibril formation monitored by ThT fluorescence using 30 mM Ab 40 with addition of 36 mM Z Ab3 at different times (B and D) or using the specified concentrations of Ab 40 and Z Ab3 (C).Time traces of three or four independent experiments are shown for each condition in (B) and (D).The average of three experiments is shown in (C) with error bars representing maximum and minimum values.Experiments in (B-D) were repeated with Ab 42 (Figure S3).(E) Transmission electron microscopy (TEM) of fibrils prepared for the Ab 40 fibril dissolution assay.Scale bar = 200 nm.(F, top) Up-field region of the 15 N HSQC NMR spectrum of a fibril dissolution sample at 37uC starting from 300 mM 15 N-Ab 40 in fibrils and (middle) 24 h after addition of 325 mM Z Ab3 .The Ab 40 backbone resonances appear as Ab 40 dissociates from fibrils and is

Figure 5 .
Figure 5. Dissolution of Ab oligomers.(A-D) Oligomer formation (A and B; 100 mM total Ab 42 ) and oligomer dissolution (C and D; 20 mM total Ab 42 ) monitored by SEC in the absence or presence of 1.2-fold excess of Z Ab3 .SEC elution profiles were integrated and normalized (see Figure S6 and Materials and Methods).The fraction of high molecular weight (HMW) aggregates was calculated as the difference between unity and the sum of the monomer and oligomer fractions.(E) Normalized CD spectra (MRE, mean residue elliptictiy) of monomeric Ab 42 (black), oligomers (red), and fibrils (blue).b-sheet secondary structure is identified by a distinct minimum at ,215 nm in the spectrum.(F,G) TEM images of oligomeric Ab 42 solutions after isolation and at the endpoint of the dissolution experiment.Scale bar = 100 nm.(H) 15 N HSQC NMR spectrum of an Ab 42 oligomer sample, which has dissociated as a result of the sequestering of monomeric Ab 42 by Z Ab3 (black).Starting from 11 mM 15 N-Ab 42 in oligomeric form (such as

Figure
Figure S1 The Z Ab3 -binding modes of Ab 40 and Ab 42 are identical.15 N-HSQC NMR spectra of Ab 40 (red) and Ab 42 (blue) in the Z Ab3 -bound state.The backbone amide resonances for residues 1 to 39, including all those assigned to the b-hairpin in the core of the complex, coincide.This demonstrates that the mode of binding is identical for Ab 40 and Ab 42 .Buffer, 20 mM sodium phosphate, pH 7.2.Temperature, 21uC.Found at: doi:10.1371/journal.pbio.1000334.s001(0.23 MB TIF) Figure S2 Z Ab3 inhibits fibril formation of Ab 42 and Ab 42 E22G.(A,B) Aggregation time courses of Ab 42 and Ab 42 E22G in the absence (blue) and presence (green and red) of increasing molar equivalents of Z Ab3 monitored by thioflavin T fluorescence.(C) TEM images of the end stage aggregates of Ab 42 in the absence (left) or presence (right) of an equivalent amount of Z Ab3 .Scale bar = 200 nm.Peptides were purchased from Bachem and dissolved in 5 mM NaOH followed by filtration using Centricon YM-10.Solutions were then divided into aliquots and lyophilized.The quantity of peptide in the aliquots was determined by amino acid analysis.Aggregation assay samples in (A) and (B) contained 40 ml of 20 mM Ab 42 or 10 mM Ab 42 E22G in 50 mM Na-phosphate, pH 7.4, and 10 mM Thioflavin T, supplemented with the indicated amount of disulfide linked Z Ab3 .Samples were incubated at 37uC and data points were recorded every 4 min (Ab 42 ) or 2 min (Ab 42 E22G) with 10 s of orbital shaking preceding the measurement using a FLUOstar OPTIMA reader (BMG) equipped with 440 nm excitation and 480 nm emission filters.Samples analyzed by TEM (in C) were applied to formvar/carbon coated copper grids, stained with 2% (w/v) uranyl acetate, and viewed in a Philips CEM100 transmission electron microscope.Found at: doi:10.1371/journal.pbio.1000334.s002(0.80 MB TIF) Figure S3 The Z Ab3 Affibody inhibits fibril formation of Ab 42 by sequestration of monomeric peptide.(A) Aggregation time course of Ab 42 at the specified concentrations of Ab 42 and Z Ab3 .Averages of four experiments are shown with error bars representing estimated standard deviations.(B) Aggregation time course of Ab 42 using 30 mM Ab 42 without (black) or with addition of 36 mM Z Ab3 at the times indicated by the arrows.Averages of four experiments are shown with error bars representing estimated standard deviations.(C) The four individual time traces resulting in the magenta time course in (B).Aggregation was monitored by thioflavin T fluorescence on a FarCyte reader (Tecan) equipped with 440 nm excitation and 480 nm emission filters.The samples contained ,100 ml of the peptide/protein solution in 20 mM Na- Figure S6 Ab 42 oligomer formation and dissolution analyzed by SEC.Elution volumes of monomeric and oligomeric Ab 42 , free Z Ab3 Affibody, and the Z Ab3 :Ab 42 complex on a Superdex 75 10/300 column, with a nominal resolution of 3,000 to 70,000 Da, are indicated.Ab 42 oligomers elute at the void volume (8.3 ml) and Ab 42 fibrils cannot enter the column.(A) A solution of 100 mM Ab 42 was incubated without stirring at 20uC.SEC analysis of samples removed at different times reveals the decrease in concentration of monomeric Ab 42 with time and the transient formation of oligomeric species, followed by formation of HMW aggregates (fibrils).(B) Analysis of an equivalent Ab 42 solution also containing a 1.2-fold excess of the Z Ab3 Affibody shows that the Z Ab3 :Ab 42 remains stable without oligomer or HMW aggregate formation.(C,D) Oligomer dissolution: isolated oligomer Ab 42 fractions isolated subjected to a second incubation followed by SEC analysis.In the absence of Z Ab3 (C), these dissolve on a timescale of several hours and monomeric Ab 42 appears transiently prior to fibril formation.Oligomer dissolution in the presence of an 1.2-fold excess of Z Ab3 (D) results in Z Ab3 :Ab 42 Table S1 Transgenic fly survival (median life span).at: doi:10.1371/journal.pbio.1000334.s008(0.05 MB PDF)