Amyloid-β Protofibrils: Size, Morphology and Synaptotoxicity of an Engineered Mimic

Structural and biochemical studies of the aggregation of the amyloid-β peptide (Aβ) are important to understand the mechanisms of Alzheimer's disease, but research is complicated by aggregate inhomogeneity and instability. We previously engineered a hairpin form of Aβ called Aβcc, which forms stable protofibrils that do not convert into amyloid fibrils. Here we provide a detailed characterization of Aβ42 cc protofibrils. Like wild type Aβ they appear as smooth rod-like particles with a diameter of 3.1 (±0.2) nm and typical lengths in the range 60 to 220 nm when observed by atomic force microscopy. Non-perturbing analytical ultracentrifugation and nanoparticle tracking analyses are consistent with such rod-like protofibrils. Aβ42 cc protofibrils bind the ANS dye indicating that they, like other toxic protein aggregates, expose hydrophobic surface. Assays with the OC/A11 pair of oligomer specific antibodies put Aβ42 cc protofibrils into the same class of species as fibrillar oligomers of wild type Aβ. Aβ42 cc protofibrils may be used to extract binding proteins in biological fluids and apolipoprotein E is readily detected as a binder in human serum. Finally, Aβ42 cc protofibrils act to attenuate spontaneous synaptic activity in mouse hippocampal neurons. The experiments indicate considerable structural and chemical similarities between protofibrils formed by Aβ42 cc and aggregates of wild type Aβ42. We suggest that Aβ42 cc protofibrils may be used in research and applications that require stable preparations of protofibrillar Aβ.


Introduction
Alzheimer's disease (AD) is associated with an imbalance in the production and clearance of the amyloid-b peptide (Ab) followed by Ab aggregation in the brain [1].The aggregation ultimately ends in the formation of insoluble protein fibrils as components of amyloid plaques.Considerable evidence suggests that neurotoxic species are soluble oligomers or protofibrils of Ab that are present on or off aggregation pathways leading to fibril formation [2,3,4,5,6,7,8].The 42-residue Ab 42 fragment is in this regard more aggregation prone than the more prevalent but less active Ab 40 fragment and an increase in the Ab 42 : Ab 40 ratio is also associated with increased neurotoxicity [9].Other evidence suggests that the rate of aggregation, and not only the aggregates that are present, acts to further enhance toxicity [10,11].
Ab can form a multitude of interconverting toxic aggregates both in vitro and in vivo [12,13,14].However, in all cases, aggregate inhomogeneity and instability complicate research on correlations between aggregation, structure and toxicity.Different ways to stabilize intermediate aggregates by chemical cross linking [for instance [6]] or protein engineering [ [15] and work cited therein] have therefore been devised.
We recently engineered a double cysteine mutant of Ab (AbCC) for which aggregation is halted at the protofibrillar state [16], which is suggested to be the penultimate intermediate prior to amyloid fibril formation [13,14,17].Briefly, AbCC was designed to test a structural model of aggregation [18] in which Ab adopts a hairpin conformation in aggregates on the path to fibril formation [18].This model hypothesized that a conformational change in such aggregates results in the formation of seeds for runaway fibril polymerization.AbCC contains a double Ala21Cys/Ala30Cys mutation and a disulfide bond formed between the two cysteines locks the peptide into the hairpin conformation [16].AbCC should therefore, according to the proposed model, form soluble oligomers and protofibrils, but be unable to adopt the cross-b structure present in mature fibrils.We found that this indeed is the case: AbCC can readily form oligomeric species, which do not convert into fibrils unless the intramolecular disulfide bond is broken by reduction [16].Protofibrils of Ab 42 CC are stable towards both dissociation (upon dilution) and fibril formation.
More importantly, protofibrils formed by Ab 42 CC potentially constitute a tool for experiments in which stable protofibrils are required, such as for instance structural studies or immunization trials.Initial studies indicated a number of similarities between wild type and Ab 42 CC protofibrils [16].Here we characterize Ab 42 CC protofibrils in more detail, and compare them to wild type protofibrils, with regard to their morphology and size, surface properties and exposed antibody epitopes, protein binding, and their effect on synaptic activity in neurons.

Protein production and preparation of aggregates
Ab 40 , Ab 42 and Ab 42 CC were produced in E. coli by coexpression with the ZAb3 Ab-binding AffibodyH molecule as described previously [16,19].The peptides were separated from the Affibody binder by denaturation in 7 M guanidinium chloride followed by immobilized metal affinity chromatography (IMAC) under denaturing conditions.Ab 42 CC protofibrils were obtained by separating oligomers with size exclusion chromatography (SEC) under native buffer conditions [16], followed by concentration on a Vivaspin column (GE Healthcare) and heat treatment at 60uC for 10 min.Alternatively, protofibrils also form when guanidinium chloride is removed by dialysis of denatured Ab 42 CC at room temperature against 20 mM sodium phosphate buffer at pH 7.2 with 50 mM NaCl and 5 mM EDTA and a second dialysis for 4 to 6 hours in the same buffer without EDTA.
Protofibrils of wild type Ab 42 were identified in atomic force microscopy (AFM) images of an Ab 42 aggregation reaction mixture.Ab 42 monomer (,100 mM) in 20 mM sodium phosphate buffer at pH 7.2 with 50 mM NaCl was kept at room temperature without shaking.A mixture of Ab 42 protofibrils and smaller oligomers could be observed and distinguished in AFM images recorded after one day of incubation.The same solution was put in 37uC and shaking conditions for one more day in order to produce Ab 42 fibrils for AFM imaging.Fibrils of wild type Ab 40 and Ab 42 for AFM and OC serum dot blot assays were prepared from similar mixtures that were subjected to 37uC and shaking to favor the formation of fibrils.

Atomic force microscopy
Concentrated protofibrils or fibrils of Ab 42 CC, Ab 42 , or Ab 40 were diluted to 0.5 to 1 mM in 20 mM sodium phosphate buffer at pH 7.2 with 50 mM NaCl, and 5 mL solutions were loaded onto freshly cleaved mica.After 1 to 2 min, the mica surface was briefly washed with 100 mL deionized water and air-dried.The samples were imaged immediately in AC-mode using a Cypher AFM instrument (Asylum Research, USA) equipped with NSC36/ Si3N4/AlBs three-lever probes (mMasch).The probes had nominal spring constants of 0.6 to 1.8 N/m and driving frequencies of 75 to 155 kHz.To determine protofibril length distributions, a number of images covering 1 to 2 mm 2 surfaces were scanned and the lengths of particles were measured using a freehand tool in the MFP-3D TM offline section analysis software.The same tool was used to measure cross sections of particles.

Analytical ultracentrifugation
Sedimentation velocity data were collected using the UV-visible optics detector on a Beckman Optima XL-A centrifuge equipped with an An-60Ti 4-cells rotor and double-sector 12 mm Epon centerpieces with quartz windows.The measurements were carried out at 17,000 rpm and 20uC.The Ab 42 CC protofibril concentration was 300 mM (monomer) in 20 mM sodium phosphate buffer at pH 7.2 with 50 mM NaCl and 0.05% NaN 3 .Absorption was recorded at 280 nm and sedimentation profiles were collected every 5 min.Data were analyzed using the SEDFIT program (v 12.52; http://analyticalultracentrifugation.com/default.htm)[20] using continuous distributions of Lamm-equation solutions with a common (unimodal) frictional ratio.The partial specific volume of Ab 42 CC, and the buffer density and viscosity at 20uC, were calculated using the SEDNTERP program v 1.09 (Biomolecular Interaction Technologies Center, University of New Hampshire, Durham, NH; http://bitcwiki.sr.unh.edu/index.php/Main_Page).Measured sedimentation coefficients were corrected to s 20, w values (sedimentation coefficients in water at 20uC).

Nanoparticle tracking analysis (NTA)
Measurements were performed with a NanoSight LM10-HS instrument (NanoSight, Amesbury, UK) equipped with an LM14 viewing unit using a 405 nm laser.Protofibrils of Ab 42 CC were diluted in 20 mM sodium phosphate buffer at pH 7.2 with 50 mM NaCl to a final monomer concentration of 2 mM, and measurements were performed at 20uC for 30 s.The NanoSight NTA 2.2 software was used for data analysis.

Synaptotoxicity
The effect of Ab 42 CC protofibrils on spontaneous synaptic activity was evaluated in an in vitro microelectrode array (MEA) assay [9].Soluble oligomers of Ab 42 were used for comparison.These were prepared as described previously (Ref.[9]; the 10:0 Ab 42 : Ab 40 ratio oligomers), with the modification that a 20 mM sodium phosphate buffer at pH 7.2 with 50 mM NaCl was used to match the Ab 42 CC buffer.Primary hippocampal neurons were dissected from e17 FVB mouse embryos and plated on MEA substrate (Multichannel Systems GmbH, Germany) at a density of 1000 cells mm 22 (500,000 cells per chip).The spontaneous firing of neuronal networks was recorded after 1 to 2 weeks in culture.A temperature controller (Multichannel Systems) was used to maintain the MEA platform temperature at 37uC during the experiments.First, the basal firing rate was recorded for 500 s, then 0.5 mM of either Ab 42 oligomers or Ab 42 CC protofibrils was added to MEA dish and neuronal activity was recorded for the next 500 s.The same amounts of Ab was added two more times to reach final concentration of 1.5 mM.Signals from active electrodes were amplified by means of a MEA1060 amplifier (gain 1200) and digitized by the A/D MC_Card at a sampling rate of 25 kHz.The MC_Rack 3.5.10software (Multichannel Systems) was used for data recording and processing.The raw data were high-pass filtered at 200 Hz, and the threshold for spike detection was set to 5 standard deviations from the average noise amplitude computed during the first 1000 ms of recording.Numbers of spikes detected by every active electrode per time bin of 500 s were normalized to baseline (firing rate in the absence of treatment).The firing rates corresponding to 500 s treatments with 0.5, 1 and 1.5 mM of protofibrils/oligomers were computed and presented as percentage of initial rates.
Use of animals and procedures were approved by the Ethical Committee for Animal Welfare (ECD, Ethische Commissie Dierenwelzijn) of KULeuven and IMEC.Timely pregnant FVB mice were sacrificed with CO 2 , and the embryos were removed immediately thereafter.

Fluorescence spectroscopy
Fluorescence emission spectra of peptide-ANS mixtures were recorded at room temperature on a Varian Cary Eclipse spectrofluorometer using a 0.3 cm path length quartz cuvette and an excitation wavelength of 360 nm.Ab 42 CC monomer samples were obtained as the monomeric fraction in SEC, concentrated and kept frozen until use.Ab 42 CC monomer and protofibril solutions both contained 10 mM peptide in 20 mM sodium phosphate buffer at pH 7.2, with 50 mM NaCl.The ANS concentration was 50 mM.

Binding to serum proteins
Ab 42 CC protofibrils were immobilized on tosyl-activated M280 Dynabeads (Invitrogen) according to the manufacturer's protocol.Briefly, 5 mg of beads were incubated with 100 mg of Ab 42 CC protofibrils in 0.1 M sodium phosphate buffer, pH 7.4 overnight at 37uC to allow covalent binding of Ab 42 CC to the beads.The beads were then washed with PBS buffer with 0.5% Tween-20.As control, glycine was immobilized to the same type of beads.0.5 mg coupled Dynabeads was then incubated with 150 mL human serum (3H Biomedical, Uppsala) for 1 h at 37uC and then washed three times.Bound proteins were eluted using SDS-PAGE sample buffer and separated using SDS-PAGE (4-20% gradient gel from BioRad).The bands were visualized using Acquastain (Acquascience, USA).Separated gel bands were cut, destained in 30% ethanol, trypsin-digested and subjected to mass spectrometry analysis using an Ultraflex II MALDI TOF mass spectrometer (Bruker Daltonics, Bremen, Germany).Proteins were identified using the Mascot search engine (www.matrixscience.com)[22].

Preparation and stability of Ab 42 CC protofibrils
With the terminology used here, oligomers are soluble aggregates that can be separated by size exclusion chromatography.The most abundant of the Ab 42 CC oligomers is a b-sheet containing aggregate with an apparent MW of 100 kDa [16].Protofibrils are much larger aggregates that are clearly rod-like and with an apparent AFM z-height of 3.1 nm, as described below.We previously prepared protofibrils of Ab 42 CC by concentrating the b-sheet-containing oligomers that form when guanidinium chloride solutions are diluted into non-denaturing buffer conditions during size exclusion chromatography [16].A more direct way to obtain Ab 42 CC protofibrils is by removal of guanidinium chloride via dialysis (see Materials and Methods).The biophysical properties of Ab 42 CC protofibrils obtained by these two different methods are not distinguishable.However, the dialysis method results in two to three fold higher final yield of protofibrils while being less laborious.Therefore, the Ab 42 CC protofibrils used in the experiments described below were obtained using the dialysis method.Preparations of Ab 42 CC protofibrils are stable towards dissociation when diluted with buffer, while smaller Ab 42 CC oligomers may dissociate into monomeric species upon dilution (A.Sandberg and A. Dubnovitsky, unpublished).Protofibril preparations are also stable towards dissociation for months at room temperature.

Protofibril size and rod-like morphology revealed by AFM
We used atomic force microscopy (AFM) to study the size and morphology of Ab 42 CC particles on dry mica surface.Protofibrils are obtained in 20 mM sodium phosphate buffer with 50 mM NaCl and sample preparation for AFM in air requires removal of salts.This may be achieved by diluting concentrated protofibril solutions in deionized water before loading them onto mica.However, with such a procedure only smaller Ab 42 CC protofibrils appear to bind the mica.Therefore, special care was taken to minimize exposure to low-salt environment.
Purified protofibrils were diluted in buffer, loaded onto freshly cleaved mica, and briefly washed with water.Prepared in this way, Ab 42 CC protofibrils appear in AFM as unbranched rod-like structures with a well defined average height of 3.1 (60.16) nm and typical lengths in the range 50 to 220 nm (Fig. 1A-B).The largest protofibril is more than 600 nm long (Fig. S1).The protofibril width measured here (AFM z-height) is smaller than widths of wild type Ab protofibrils [23,24] and Ab 42 CC protofibrils [16] observed in electron microscopy with uranyl acetate negative staining (6 to 10 nm), but a 3.1 nm height was also reported in a detailed AFM analysis of protofibrils of wild type Ab 40 [25].
High-resolution scans of single protofibril particles reveal a smooth surface without distinguishing features (Fig. 1C).The shape and size of Ab 42 CC protofibrils are identical to those of wild type Ab 42 protofibrils identified in aggregation reaction mixtures of 100 mM peptide stored at room temperature without shaking (Fig. 1B-D, Fig. S2).The morphology of Ab 42 CC and Ab 42 protofibrils are both very different from the characteristic helical appearance of Ab 40 or Ab 42 fibrils imaged under the same conditions (Fig. 1E-F).(The dimension and morphology of the Ab 40 fibril are such that it may be envisioned as a double helix of 3.1 nm filaments.)Short Ab 42 CC protofibrils appear to be straight in AFM, but smooth curvatures can be observed with increasing particle length.The precise persistence length of the rods was, however, not determined.If protofibrils bound to mica are extensively washed with water, more sharp kinks appear together with multiple brakes in long protofibrils.We characterized the protofibril length distributions in two different samples of Ab 42 CC protofibrils (Fig. 2).One sample was washed only very briefly with water, as above, while the other was washed extensively several times.Extensive washing with water disrupts the protofibrils (on mica) and many particles become shorter than 100 nm (Fig. 2).Interestingly, the two size distributions show a similar fine structure (or periodicity) indicating that protofibril disruption involves dissociation of discrete oligomeric building blocks.

Protofibril size distribution in solution
The size distribution of Ab 42 CC protofibrils in solution was analyzed by non-invasive analytical ultracentrifugation (AUC).Data analysis assuming a continuous c(s) distribution model indicates a distribution of large particles between 2 and 30 S (Fig. 3).A size distribution centered at s 20, w = 18 S with a best-fit frictional ratio f/f 0 of 2.7 to 3.1 suggests the presence of long rodlike particles.For comparison, f/f 0 = 2.3 and s 20, w = 7.6 for human fibrinogen (MW = 330,000 Da), and f/f 0 = 3.6 and s 20, w = 6.4 for myosin (MW = 570,000 Da) [26].
A simple model calculation in which protofibrils rods are assumed to be 3.1 nm in diameter and hydrated suggests that the average s 20,w = 18 S corresponds to a length of 220 to 230 nm, which is somewhat longer than the average length observed with AFM (Fig. 2).Hence, it is likely that sample preparation for AFM measurements results in protofibril breakage, which is also consistent with the observation that washing with deionized water results in shorter protofibrils.A theoretical length distribution derived from the AUC data is shown together with the AFM lengths in Fig 2 .We also studied the size distribution of Ab 42 CC protofibrils in solution using nanoparticle tracking analysis (NTA) using a NanoSight microscope in which laser light scattering allows for tracking of the Brownian motion of individual nanoparticles.The hydrodynamic radius is then determined using the Stokes-Einstein equation based on the mean square speed of a particle.This technique is particularly valuable for analyzing polydisperse nanosized particles [27].The size distribution of Ab 42 CC protofibrils obtained from NTA (Fig 2, solid black line) shows that most particles are found in the range of 100-400 nm.This result is in good agreement with the length distribution calculated from AUC data using a dehydrated particles height obtained from AFM measurements (Fig. 2, dashed grey line).Thus, using two independent methods we demonstrate similar size distribution of Ab 42 CC protofibrils in solution with an average length of 220 to 230 nm.

ANS binding to Ab 42 CC protofibrils reveals hydrophobic surface patches
1-anilinonaphtalene 8-sulfonic acid (ANS) is a fluorescent dye that is widely used to probe the presence of exposed hydrophobic patches or cavities on proteins [28,29].Bolognesi et al. recently showed that toxicity of soluble oligomeric aggregates of different proteins and peptides, including Ab correlates with the presence of hydrophobic cavities as probed by ANS binding.The correlation suggests that hydrophobic surface may be a common feature of pathogenic protein aggregates [30], which may allow them to confer toxicity by direct interactions with membranes and/or membrane proteins.
We analyzed ANS binding to protofibrillar and monomeric species of Ab 42 CC (Fig 4).The increased fluorescence quantum yield of ANS and a blue shift of the emission spectrum from 525 to 500 in the presence of Ab 42 CC protofibrils suggest that hydrophobic ANS-binding sites form on the surface of Ab 42 CC protofibrils and that Ab 42 CC protofibrils are similar to other toxic protein oligomers in this regard [30].Similar, but smaller, changes in the

Ab 42 CC protofibrils share conformational epitopes with wild type Ab oligomers
We previously reported [16] that toxic b-sheet containing oligomers and/or protofibrils of AbCC are recognized by the mAb158 monoclonal antibody, which was selected based on its affinity for protofibrils of wild type Ab [31].AbCC protofibrils are on the other hand not recognized by the A11 serum, which recognizes wild type Ab prefibrillar oligomers as well as oligomers of other peptides [32].However, smaller oligomers of Ab 40 CC with less developed b-sheet content may avoid the protofibrillar state upon further aggregation and instead form aggregates that are indeed recognized by A11 [16].This and other observations led us to suggest that AbCC, like wild type Ab, aggregates along at least two pathways [16].
The question remains, however, whether the aggregation pathways followed by AbCC actually also correspond to wild type aggregation pathways.We used the OC serum to address this issue.OC was obtained following immunization by fibrillar Ab, but OC recognizes an epitope that is common to amyloid fibrils and certain Ab oligomers [21,33].Glabe et al. used the OC serum to show that Ab forms two immunologically distinct types of oligomers along two aggregation pathways: ''pre-fibrillar'' oligomers that are recognized by A11, but not by OC, and ''fibrillar'' oligomers that are recognized by OC, but not by A11 [21,33].
We performed dot blot assays for OC binding to Ab 42 CC protofibrils and monomeric Ab 42 CC that had been immobilized on nitrocellulose membranes.The OC serum recognizes Ab 42 CC protofibrils and wild type Ab 42 fibrils to the same extent in this assay, whereas no binding can be detected to monomeric Ab 42 CC (Fig. 5).The assay is conformation specific because SDS-treated Ab 42 CC protofibrils are not recognized by OC (Fig. 5).These results place Ab 42 CC protofibrils in the same category of aggregated species as the fibrillar oligomers formed along the A11 negative/OC positive aggregation pathway of wild type Ab.

Ab 42 CC protofibrils bind Apolipoprotein E in human serum
The high stability of Ab 42 CC protofibrils makes them potentially useful for studies of biological processes related to AD.To test this possibility we performed a pilot pull-down study to identify protein binders in biological fluids.Using Ab 42 CC protofibrils immobilized on magnetic beads we were able to extract protein ligands from human serum (Fig 6).Interestingly, the strongest (or most prevalent) binder was identified as apolipoprotein E (isoform ApoE4; 67% sequence coverage and Mascot score = 348).Several other proteins were also identified and a complete analysis of the data will be presented elsewhere.ApoE is a ubiquitous cholesterolbinding protein that is linked to Ab biology and plaque deposition and the ApoE4 isoform is a genetic risk factor for AD [1].ApoE has also been shown to interfere with Ab aggregation and to stabilize oligomeric forms [34].The identification of ApoE4 as a binder to AbCC protofibrils in serum therefore supports the relevance of these as an engineered model of wild type Ab protofibrils and suggests that Ab 42 CC protofibrils may be employed in proteomics to identify interacting proteins.

Synaptotoxicity of Ab 42 CC protofibrils
We previously assessed the toxicity of AbCC aggregates by measuring their effect on the rate of apoptosis in SH-SY5Y   CC or monomeric or fibrillar Ab 42 did not, on other hand, show any effects on apoptosis in the studied concentration range.This assay confirmed toxicity, but it would be desirable to monitor the more relevant effects on synaptic activity of living neurons in a more sophisticated assay.Therefore, we analyzed the influence of Ab 42 CC protofibrils on synaptic activity in primary mouse hippocampal neurons cultured on the surface of microelectrode array chips, which allow the recording of spontaneous neuronal firing [9].For comparison in this experiment we used oligomers of wild type Ab 42 prepared as in previous applications of this neuronal activity assay, but in the same phosphate buffer as the Ab 42 CC protofibrils.Treatment with 1.5 mM of either Ab 42 CC protofibrils or wild type Ab 42 oligomers both significantly inhibited spontaneous neuronal activity as compared to buffer-treated culture; the Student's t-test **p,0.0015and *p,0.026,respectively (Fig. 7).The effect is concentration dependent and the toxicity of Ab 42 CC protofibrils is similar to that of the wild type Ab Summary: Ab 42 CC protofibrils as a stable mimic of wild type protofibrils Protofibrils were the first soluble aggregates of Ab to be observed [24,35], and their neurotoxicity was reported shortly thereafter [8].Focus on protofibrils was further motivated by AD genetics since the Arctic Glu22Gly mutation in Ab which is associated with early on-set AD, results in an increased rate of protofibril formation [36].Protofibrils form readily in vitro and they are easily prepared from solubilized Ab by size exclusion chromatography [12].However, they convert into amyloid fibrils; 20 mM samples of Ab 42 form fibrils within a few hours of preparation in physiological buffer at room temperature [37].Protofibrils may be kept at longer times under alkaline conditions [38].However, preparations that are stable at physiological pH would have a range of applications in for instance cell biological assays and immunotherapeutic applications.
AbCC was engineered to form hairpin conformations that are closed by an intramolecular disulfide bond between Cys21 and Cys30, which replace wild type Ala21 and Ala30.The motivation for this particular intramolecular linkage came from the observation of a corresponding hairpin of Ab in complex with an Affibody binding protein [18,39] and from a number of studies that indicate a propensity for such conformations in monomeric Ab [40,41,42,43].We had, in connection to these observations, also suggested that the hairpin form of Ab is present in oligomeric aggregates, and it was subsequently also identified in soluble aggregates [44].
The initial characterization showed that Ab 40 CC and Ab 42 CC form soluble oligomeric and protofibrillar aggregates with properties similar to those formed by wild type Ab [16].The aggregation occurs along two pathways that can be distinguished using the oligomer specific A11 serum and the mAb158 monoclonal antibody, respectively [16].Ab 40 CC has a tendency to form aggregates recognized by the A11 serum.Ab 42 CC, on the other hand, spontaneously aggregates along a pathway that involves formation of anti-parallel b-sheet secondary structure, which is also present in wild type Ab aggregates [45], to form protofibrils that are morphologically indistinguishable from wild type protofibrils when observed in electron microscopy.Aggregates formed along this ''b-sheet'' pathway are recognized by the mAb158 antibody, but not by the A11 serum.We found that they contain SDS-resistant oligomeric building blocks, with the same stoichiometry as in the SDS-stable aggregates of Ab that are directly associated with AD [46], and that they are powerful inducers of apoptosis in the SH-SY5Y neuroblastoma cell line, which is not the case for monomeric or fibrillar peptide species.However, unlike wild type, AbCC cannot form amyloid fibrils unless the intramolecular Cys21-Cys30 disulfide bond is broken by a reduction agent such as TCEP [16].Ab 42 CC, in particular, instead enriches into stable protofibrils.In this work we performed a number of complementary experiments to characterize these protofibrils in more detail.First, we examine Ab 42 CC protofibrils using atomic force microscopy (AFM), analytical ultracentrifugation (AUC), and nanoparticle tracking analysis (NTA) to define their morphology (rod-like) and length (60 to 220 nm on dry mica; even longer in solution).Second, we find that Ab 42 CC protofibrils bind the fluorescent dye ANS and therefore share surface properties that are common to cytotoxic protein aggregates including those of wild type Ab 42 [30].Third, we complement previous studies of A11 serum and mAb158 antibody binding with measurements of OC serum [21] recognition to find that Ab 42 CC protofibrils exhibit the conformational (immunological) properties that also distinguish fibrillar oligomers of wild type Ab 42 from A11 binding prefibrillar oligomers [21,33].Fourth, the biologically relevance of the Ab 42 CC is further strengthened by the observed binding to apolipoprotein E in human serum.Finally, we find that Ab 42 CC protofibrils are not only ''toxic'' but also specifically affect spontaneous synaptic activity in a neuronal cell assay.

Conclusions
We present a detailed characterization of the protofibrils that form when Ab 42 is stabilized in a hairpin conformation in Ab 42 CC.The experiments must not be interpreted as evidence for protofibrils as the most relevant form of biologically active Ab species.Neither can they be seen as a completely unambiguous comparison with wild type protofibrils, because such a comparison is, for instance, complicated by the range of aggregates with different size and morphology that potentially can form in any sample of Ab 42 .Nevertheless, the characteristics of Ab 42 CC aggregates indicate that the major determinant of Ab toxicity is conformation, and that this conformation differs markedly from the cross-b conformation found in fibrillar Ab.In conclusion, based on the multi-faceted coherence between Ab 42 CC and wild type Ab 42 aggregates that we observe here and reported previously [16] we suggest that the protofibrillar form of Ab 42 CC is a close chemical and structural mimic to the protofibrils formed by wild type Ab.

Figure 1 .
Figure 1.Analysis of Ab 42 CC morphology using atomic force microscopy (AFM).(A) AFM image of Ab 42 CC protofibrils on dry mica surface.(B) Average z-heights and cross-sections of Ab 42 CC (black) and wild type Ab 42 (red) protofibrils (grey lines represent measurements of 20 Ab 42 CC protofibrils).(C-F) High magnification AFM images of single protofibrils of Ab 42 CC (C) and wild type Ab 42 (D; identified in aggregation reaction mixtures, Fig. S2), and of amyloid fibrils of Ab 40 (E) and Ab 42 (F).Measured z-heights of particles are indicated in panels C-F.doi:10.1371/journal.pone.0066101.g001

Figure 2 .
Figure 2. Size distribution of Ab 42 CC protofibrils measured using different methods.The blue and red lines/symbols represent data from atomic force microscopy.One sample (blue) was washed briefly with deionized water, while a second sample (red) was washed extensively.The lengths of ca.1500 protofibrils were measured in each case.The gray dashed line reflects an expected distribution corresponding to the analytical ultracentrifugation measurements (Fig.3) assuming that Ab 42 CC protofibrils have a dehydrated diameter of 3.1 nm.The black line represents the distribution of apparent hydrodynamic radius obtained from nanoparticle tracking analysis using a NanoSight microscope.doi:10.1371/journal.pone.0066101.g002

Figure 3 .
Figure 3. Size distribution of Ab 42 CC protofibrils in solution monitored by analytical ultracentrifugation. (A) A subset of the raw sedimentation velocity centrifugation data of 300 mM Ab 42 CC protofibrils at 20uC recorded over a period of 20 h.(B) Sedimentation coefficient distribution of Ab 42 CC protofibrils analyzed using a continuous c(s) distribution model.doi:10.1371/journal.pone.0066101.g003 42 oligomers.Ab 42 CC protofibrils therefore have an effect on synaptic activity that is comparable to what one would anticipate from biologically relevant aggregates used in previous studies of wild type Ab.(But the outcome of the experiment does not exclude the possibility that the Ab 42 oligomers used for comparison are morphologically different from the Ab 42 CC protofibrils.)

Figure 7 .
Figure 7. Effect of Ab 42 CC protofibrils (red) and wild type Ab 42 oligomers (blue) on spontaneous synaptic activity in mouse primary hippocampal neurons grown on a multielectrode array (MEA) chip.Changes in firing rates are normalized to the initial electrical activity in the absence of treatment and compared to buffer-treated neurons: ** -p,0.0015,* -p,0.026(Student's t-test); the difference between Ab 42 oligomers and Ab 42 CC protofibrils is not significant.doi:10.1371/journal.pone.0066101.g007 Figure 4. Ab 42 CC protofibrils expose binding sites for the ANS dye.Fluorescence emission spectra of 50 mM free ANS (red) and of ANS in the presence of Ab 42 CC protofibrils (black) or monomeric Ab 42 CC (green).Peptide concentrations are in both cases 10 mM monomer units.doi:10.1371/journal.pone.0066101.g004neuroblastoma cells.We found that b-sheet containing oligomers and/or protofibrils of Ab 42 CC induced a dose-dependent apoptosis to an extent that equaled, or perhaps exceeded, that of wild type oligomer preparations.Monomeric AbCC or low-molecular weight oligomers of Ab 42