Two distinct β-sheet structures in Italian-mutant amyloid-beta fibrils: a potential link to different clinical phenotypes
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Most Alzheimer’s disease (AD) cases are late-onset and characterized by the aggregation and deposition of the amyloid-beta (Aβ) peptide in extracellular plaques in the brain. However, a few rare and hereditary Aβ mutations, such as the Italian Glu22-to-Lys (E22K) mutation, guarantee the development of early-onset familial AD. This type of AD is associated with a younger age at disease onset, increased β-amyloid accumulation, and Aβ deposition in cerebral blood vessel walls, giving rise to cerebral amyloid angiopathy (CAA). It remains largely unknown how the Italian mutation results in the clinical phenotype that is characteristic of CAA. We therefore investigated how this single point mutation may affect the aggregation of Aβ1–42 in vitro and structurally characterized the resulting fibrils using a biophysical approach. This paper reports that wild-type and Italian-mutant Aβ both form fibrils characterized by the cross-β architecture, but with distinct β-sheet organizations, resulting in differences in thioflavin T fluorescence and solvent accessibility. E22K Aβ1–42 oligomers and fibrils both display an antiparallel β-sheet structure, in comparison with the parallel β-sheet structure of wild-type fibrils, characteristic of most amyloid fibrils described in the literature. Moreover, we demonstrate structural plasticity for Italian-mutant Aβ fibrils in a pH-dependent manner, in terms of their underlying β-sheet arrangement. These findings are of interest in the ongoing debate that (1) antiparallel β-sheet structure might represent a signature for toxicity, which could explain the higher toxicity reported for the Italian mutant, and that (2) fibril polymorphism might underlie differences in disease pathology and clinical manifestation.
KeywordsAmyloid-beta peptide E22K mutation Secondary structure Fibril polymorphism Tropism β-sheet conformation
Atomic force microscopy
Attenuated total reflectance
Cerebral amyloid angiopathy
Familial Alzheimer’s disease
Fourier transform infrared
Nuclear magnetic resonance
Transmission electron microscopy
The conversion of native and functional peptides or proteins into higher ordered, toxic aggregates, and eventually to amyloid fibrils, is characteristic of many human proteinopathies . Amyloid fibrils deposit extra- or intracellularly and are implicated in neurodegenerative disorders and systemic amyloidoses . The defining molecular unit of these amyloid fibrils is the cross-β spine that originates from extended β-sheets composed of β-strands that are arranged perpendicular to the fiber axis .
Although the cross-β characteristic is a common structural feature, amyloid fibrils show a great structural variety and can differ in their underlying structure, symmetry, width, twist periodicity, and curvature [4, 5, 6]. This structural polymorphism can have several molecular origins. First, fibril polymorphs can differ in the number of protofilaments (the minimal fibrillar entities) . Second, distinct orientations and modes of association of protofilaments and patterns of inter-residue interactions determine how protofilaments are oriented [8, 9, 10, 11]. Third, variations in the underlying protofilament substructure can contribute to fibril polymorphism [12, 13]. Despite the highly conserved arrangement of fibrils in a cross-β manner along the elongation axis, fibrils can thus display considerable heterogeneity and structural polymorphism.
The biological relevance of fibril polymorphism is not yet fully understood, but it is notable that fibril polymorphism has been reported for several disease-related proteins. Substantial evidence indicates that different fibril morphologies exert different toxicities in vitro and could be related to differences in disease pathology and progression in vivo, or could underlie the preference of amyloid to deposit in specific cellular locations (i.e., tropism) [13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23]. However, the link between fibril polymorphism and clinical subtypes of amyloidoses is still lacking.
One of the disease-related proteins for which fibril polymorphism has been reported is the amyloid-beta (Aβ) peptide . The Aβ peptide is one of the underlying causes of Alzheimer’s disease (AD) [25, 26], inclusion body myositis , dementia with Lewy bodies , and cerebral amyloid angiopathy (CAA) [29, 30, 31]. Whereas Aβ in AD is primarily deposited in the brain parenchyma, and CAA is related to cerebrovascular amyloid deposition. Aβ deposition in vessel walls makes them prone to rupture and narrows their lumina to the point of occlusion, resulting in secondary lesions associated with CAA, such as intracerebral and subarachnoid bleeding, multiple infarcts, and periventricular edema . CAA is, however pathophysiologically related to AD and found with high prevalence in AD patients (80–90 %) .
CAA is a common clinical symptom of early-onset familial AD (FAD), in which disease symptoms occur earlier in life compared to the more prevalent sporadic, late-onset AD . Most mutations that are recognized to cause FAD are concentrated in the amyloid precursor protein (APP) either within or around the Aβ domain. Mutations clustering near the Aβ N-terminus were shown to alter Aβ production and enhance the kinetic of fibril and intermediate aggregate species formation [35, 36], while mutations located at the Aβ C-terminus were only shown to affect the release of Aβ by favoring acceleration of the production [37, 38, 39, 40]. Interestingly, mutations reported within residues 21–23 of Aβ are implicated in increasing Aβ production, enhancing Aβ aggregation kinetic and/or delaying Aβ clearance [41, 42, 43, 44, 45, 46].
Carriers of the Italian E22K , Iowa D23N , or Dutch E22Q  Aβ mutation predominantly display the clinical phenotype characteristic of CAA. In contrast, the Flemish A21G mutation results in both significant amyloid accumulation in brain blood vessels and parenchymal amyloid plaques , whereas Arctic E22G carriers develop progressive dementia, typical of AD, but without the severe CAA that characterizes other mutations in this region .
In this study, we monitored the aggregation of wild-type (WT) and Italian-mutant E22K Aβ1–42 under different experimental conditions, and structurally characterized the resulting fibrils. We provide evidence that, under near-physiological conditions, E22K Aβ1–42 spontaneously forms fibrils comprising stable antiparallel β-sheets, in contrast to WT fibrils that are composed of parallel β-sheets, similar to most amyloid fibrils described in the literature . Moreover, to the best of our knowledge, this is the first experimental demonstration that the underlying β-sheet arrangement of Italian-mutant Aβ fibrils is altered upon a change in pH, and that inter-conversion of the corresponding fibril polymorphs occurs.
These results are interesting in light of the emerging view that (1) antiparallel β-sheet structure may be of importance in the pathology of AD [5, 51], and that (2) fibril polymorphism may be implicated in in vivo differences in terms of disease pathology and age of disease onset. We suggest that the antiparallel β-sheet conformation of E22K Aβ fibrils, and maybe other CAA-related Aβ peptides, predisposes them to mainly deposit in blood vessel walls, resulting in CAA.
Materials and methods
Reagents and chemicals
WT Aβ1–42 was purchased from American Peptide Co. (Sunnyvale, CA, USA), and E22K and D23N Aβ1–42 were purchased from JPT (JPT Peptide Technologies, Germany). Dimethyl sulfoxide (DMSO 99.9 % purity), hexafluoropropan-2-ol (HFIP), Thioflavin T (ThT), uranyl acetate, and pepsin were obtained from Sigma-Aldrich (St. Louis, MO, USA). Antibodies (6E10, 4G8, 12F4) were from Covance (Emeryville, CA, USA). Horseradish peroxidase-conjugated anti-mouse antibodies were purchased from Millipore (Billerica, MA, USA). Supersignal West Pico Chemiluminescent Substrate and ECL plus Western blot detection system were obtained from Thermo Fisher Scientific (Biotechnology, Rockford, IL, USA) and GE Healthcare (Piscataway, NJ, USA), respectively.
Aβ sample preparation
Aβ peptides were dissolved in cold HFIP at a concentration of 2 mg/mL and incubated at room temperature (25 °C) for 1 h. HFIP was evaporated under nitrogen flow and residual HFIP was removed under vacuum using a Speed Vac (Thermo Savant). Prior to incubation, peptides were dissolved in DMSO at a final concentration of 5 mM and then diluted to a final concentration of 100 μM in TBS (Tris-buffered saline: 20 mM Tris/HCl, pH 7.4, 100 mM NaCl) or in 10 mM HCl pH 2.0. Peptides were incubated at 37 °C under quiescent conditions. Fibrillar samples were centrifuged for 30 min at 16,100g prior to analysis by Fourier transform infrared (FTIR) spectroscopy and hydrogen/deuterium exchange mass spectrometry (HDX-MS).
Transmission electron microscopy
Aβ samples (5 µL of a 100 µM concentration) were adsorbed to carbon-coated Formvar 400-mesh copper grids (Agar Scientific) for 1 min. The grids were washed and stained with 1 % (w/v) uranyl acetate. Samples were studied with a JEOL JEM-1400 microscope (JEOL Ltd., Tokyo, Japan) at 80 kV. Transmission electron microscopy (TEM) images are representative of three independently prepared Aβ solutions.
Atomic force microscopy
Atomic force microscopy (AFM) images were acquired using a VEECO Dimension 3100 atomic force microscope (Bruker), operated in tapping mode in air using silicon cantilevers with a resonance frequency of 325 kHz, a spring constant of 46 Nm−1, and a tip radius of 10 nm (µMASCH, NSC15/no Al). Images were collected at a scan rate of 1 Hz. Each fibrillar sample (5 µL of a 100 µM concentration) was deposited for 15 min onto freshly cleaved mica surfaces to enable adsorption. The samples were rinsed with ultrapure water (5 × 200 µL) and left to dry in air before imaging.
Dot blot analysis with Aβ-region specific antibodies
Aβ aggregation was monitored by spotting 1 µg of Aβ onto a nitrocellulose membrane at several incubation times. The membranes were blocked for 1 h at 4 °C in 5 % non-fat dry milk in TBS-Tween 20 buffer and then incubated (24 h, 4 °C) with mouse monoclonal Aβ-region specific antibodies 6E10, 4G8, or 12F4 (all diluted 1:3000 in 0.5 % non-fat dry milk in TBS-Tween 20 buffer). Horseradish peroxidase-conjugated anti-mouse antibody (1:2000 in 0.5 % non-fat dry milk in TBS-Tween 20 buffer, 4 °C, 1 h) was used as secondary antibody. Detection was carried out using the Supersignal West Pico Chemiluminescent Substrate and the ECL® Western blot kit. Images were recorded and analyzed using the ImageQuant 400 gel imager and ImageQuant TL software (GE Healthcare).
Secondary structure and HDX measurements using ATR-FTIR spectroscopy
Attenuated total reflectance FTIR (ATR-FTIR) spectra were recorded on an Equinox 55 IR spectrophotometer (Bruker Optics, Ettlingen, Germany). A quantity of 2 µg of Aβ was spread on the diamond surface (2 × 2 mm) of the internal reflection element and was washed with excess milli-Q water to eliminate salts. Excess water was evaporated under nitrogen flow. Each spectrum represents the mean of 128 repetitions, recorded at a resolution of 2 cm−1. The ATR-FTIR data were analyzed using Kinetics software (SFMB, Brussels, Belgium) and processed for baseline correction and subtraction of the water vapor contribution. Spectra were smoothed at a final resolution of 4 cm−1 by apodization of their Fourier transform by a Gaussian line and intensities were normalized to the intensity of the major β-structure peak around 1630 cm−1. All depicted spectra were deconvolved using a Lorentzian deconvolution factor with a full width at half height (FWHH) of 20 cm−1, a Gaussian apodization factor with FWHH of 16.67 cm−1 to obtain a resolution enhancement factor of 1.2. Deconvolution increases the resolution of the spectra in the amide I region which is most sensitive to the secondary structure of proteins. Next, curve fitting was performed on the non-deconvolved ATR-FTIR spectra to determine the secondary structure content of each sample. The proportion of a particular structure is computed to be the sum of the area of all the fitted bands (having their maximum in the frequency region where that structure occurs) divided by the area of all the Lorentzian bands (having their maximum between 1700 and 1600 cm−1). They were chosen by Kinetics software based on the shape of the most deconvolved spectrum (α-helices and random coil: 1637–1662 cm−1, turn: 1662–1682 cm−1, β-sheet: 1613–1637 cm−1, and 1682–1689 cm−1). Curve fitting has a tendency to overestimate the β-sheet content. Combining curve fitting analysis and hydrogen/deuterium exchange on Aβ fibrils (7 days) enhances the prediction of secondary structure and allow us to determine exchange dynamics. The decay of the NH-associated amide II band (1520–1580 cm−1) was used to monitor the exchange of the amide group. Results were analyzed as previously described . This information is of macroscopic nature and is related to the compactness of the amyloid fold.
Thioflavin T fluorescence
Fibril formation was probed using ThT fluorescence as described previously . Briefly, 5 µM of ThT was freshly dissolved in 50 mM glycine/NaOH pH 8.5 and 4.5 µg of Aβ was added to 1 mL ThT solution. ThT fluorescence emission was recorded between 460 and 560 nm (excitation wavelength of 450 nm). All measurements were recorded on a LS55 fluorimeter (PerkinElmer Instruments) at 25 °C with slit widths of 5 nm.
X-ray fiber diffraction
Aβ fibrils (8 mg/mL) were aligned between wax-tipped capillaries and allowed to air dry . The partially aligned fiber was placed on a goniometer head and data were collected using a Rigaku rotating anode source (CuKα) and Saturn 944+ CCD detector. The diffraction patterns were examined and measured using Clearer .
HDX-MS coupled to pepsin proteolysis
A volume of 25 μL of amyloid fibrils of WT and E22K Aβ1–42, grown for 21 days in TBS (100 μM monomeric concentration), were collected by centrifugation at 16,100g for 30 min at 4 °C. A volume of 24 μL of supernatant was removed and the pellet was resuspended with 24 μL D2O. Labeling was carried out for 15 min at room temperature. Fibril samples were recovered by centrifugation at 16,100g for 30 min at 4 °C. Since then, samples and solutions were held on ice. A volume of 24 μL of D2O supernatant was removed and fibrils were dissolved in 20.4 μL of 100:0.5 (v/v) H2O/HCOOH containing pepsin (1:5, enzyme/substrate, w/w). After 20 s, 3.6 μL of 100:0.5 (v/v) MeCN/HCOOH was added [85:15:0.5 (v/v/v) H2O/MeCN/HCOOH final concentration] allowing fibril dissolution .
For HDX on monomers of WT and E22K Aβ1–42, the dried peptides were first dissolved in DMSO at a concentration of 2 mM and subsequently resuspended in D2O (final concentration: 80 μM) and exchange was allowed for 45 min. One μL of the exchanged monomeric peptide was added to 20.4 μL of 100:0.5 (v/v) H2O/HCOOH containing pepsin (1:5, enzyme/substrate, w/w). After 20 s, 3.6 μL of 100:0.5 (v/v) MeCN/HCOOH was added [85:15:0.5 (v/v/v) H2O/MeCN/HCOOH final concentration].
HDX was then evaluated by electrospray ionization mass spectrometry (ESI–MS). The deuterium content of the samples was analyzed by ESI–MS on a Q-TOF Ultima API spectrometer (Waters/Micromass). Samples were electrosprayed from gold-coated glass capillaries (Thermo Fisher). Capillary and cone voltages applied were 1.8 kV and 50 V, respectively. The same dead time (1 min) after mixing in protic solvent was used for all experiments. All measurements were performed in triplicate and mass spectra presented are averages of 20 s acquisition.
E22K Aβ1–42 forms fibrils with an antiparallel β-sheet conformation
Distinct HDX and secondary structure contributions for WT and E22K Aβ1–42 fibrils
HDX-MS coupled to pepsin proteolysis
Secondary structure (%)
Exchanged amide protons (%)
Pepsin-induced Aβ fragments
Exchanged amide protons
Protected NH/total (%)
WT Aβ1-42 fibrils
30 ± 5
14.0 ± 0.5
7.3 ± 0.7
3.1 ± 0.5
E22K Aβ1-42 fibrils
50 ± 5
14.3 ± 0.4
9.6 ± 0.8
3.4 ± 0.3
The distinct underlying conformations of WT and E22K Aβ1–42 fibrils were further marked by different sensitivities for staining with ThT, a dye commonly used to detect amyloid fibrils [53, 74]. Whereas WT Aβ1–42 fibrils displayed a high ThT fluorescence intensity, the ThT fluorescence of E22K Aβ1–42 fibrils was significantly lower (Figs. 1e, 2c). Lower levels of ThT fluorescence could be indicative of a lower affinity of Italian-mutant fibrils for ThT, as shown previously for the Japanese ΔE22-Aβ1–39 mutant, and/or of less accessibility of the dye to potential binding sites .
As neither ATR-FTIR nor ThT fluorescence spectroscopy provided information on the relative positioning in the three-dimensional space, we resorted to X-ray diffraction. The latter revealed that both fibril types displayed meridional (4.7 Å) and equatorial (9.6–9.7 Å) reflections, corresponding to the distance between β-strands within one β-sheet, and to the distance between β-sheets, respectively (Fig. 2d). These reflections are consistent with the cross-β diffraction pattern that is characteristic for amyloid fibrils . However, the orientation obtained was not sufficient to distinguish between parallel and antiparallel fibril structures. Furthermore, it is difficult to establish antiparallel β-sheet signatures from X-ray fiber diffraction due to a systematic absence of even numbered repeats arising from the 21 helix .
ATR-FTIR and fluorescence spectroscopy data indicate that WT and E22K Aβ1–42 fibrils contain different β-sheet organizations. Further analysis was then performed to gain more detailed understanding of their structural differences.
The central region is more exposed in E22K than in WT Aβ1–42 fibrils
HDX patterns of fibrils were measured using ATR-FTIR spectroscopy to gain insight into the H-bonded β-sheet network. Hydrogen atoms exchange most easily when not involved in H-bonds and/or when they are not buried in the fibril core. Estimates of the secondary structure contributions, based on the analysis of IR spectra (Fig. 2b), indicated that E22K Aβ1–42 fibrils contain slightly less β-sheet and more intrinsic disorder and turn contributions compared to WT fibrils (Table 1). Accordingly, E22K Aβ1–42 fibrils demonstrated a higher HDX ratio than WT fibrils: 50 (±5) % of amide hydrogens were exchanged by deuterium compared to 30 (±5) % for WT fibrils, during a time lapse of 1 h (Table 1). The difference in total HDX between WT and mutant fibrils corresponds to the backbone of eight amino acids, but the specific region responsible for this difference cannot be derived from this dataset.
A change in pH reveals different β-sheet conformations for E22K Aβ1–42 fibrils
The antiparallel β-structured fibrils that have been recently reported for the Iowa mutant D23N Aβ1–40 peptide were suggested to be thermodynamically metastable . In contrast, Italian-mutant fibrils displayed a high β-index ratio for several months that reached approximately 60 % of its original value after 1 year of incubation under near-physiological conditions (TBS pH 7.4), with the majority of the fibrils still retaining the antiparallel β-sheet conformation (Fig. S3).
Next, E22K antiparallel fibrils were first grown at neutral pH (TBS pH 7.4) and 37 °C, and the fibril pellet (obtained after centrifugation for 30 min at 16,100g) was then redissolved in 10 mM HCl pH 2.0. This pH jump lead to a significant decrease in the β-index ratio from 0.21 ± 0.01 to 0.08 ± 0.01, corresponding to parallel β-structured fibrils, indicating that an antiparallel-to-parallel conversion occurred and demonstrating that the inter-conversion of fibril polymorphs is possible. This decrease of the β-index ratio occurred within the dead time of the experiment (~30 min).
Amyloid oligomers have emerged as the culprit species responsible for potent toxicity activities [78, 79]. However, despite the amount of data published supporting their role(s) in neuronal degeneration and cell death, the molecular mechanism of toxicity is still puzzling . Even so, oligomers exist in equilibrium with fibrils and therefore the kinetic, thermodynamic, and physiological factors affecting the formation, metabolism, and activity of one of these assemblies also affect the others. Aβ fibrils are inherently stable but are not inert and neither, non-toxic end product deposits. Although the toxicity of oligomeric species is much more pronounced [81, 82], Aβ fibrils do have a significant established effect on neuronal viability compared to unaggregated peptide . By themselves, fibrils present direct detrimental effects in particular contexts (e.g., obstructive vascular amyloid, mechanical effect on membrane cells) [83, 84, 85] and can induce a neuroinflammatory response triggered by the activation of microglial cells  which is currently thought to contribute to AD progress .
In this manuscript, we report that the E22K mutation of Aβ oligomers shows a similar antiparallel β-sheet structural arrangement which is retained upon fibril conversion. This observation raises the question if indeed an antiparallel β-sheet can be regarded as a structural fingerprint for toxicity. Interestingly, in the particular case of FAD-related Aβ1–42 mutants, in vitro studies have demonstrated that neurotoxicity correlates well with the ability of these peptides to aggregate [88, 89].
Antiparallel β-sheet structure: a key organization in amyloid aggregates?
The impact of antiparallel β-sheet structure on amyloids is still under debate, but evidence emerges that this structural signature can be attributed to several higher toxicity intermediates or off-pathway aggregates of the amyloid formation pathway. Streltsov and co-workers  reported the first crystal structure of oligomers of the p3 fragment, the N-terminally truncated Aβ variant, and provided evidence for their antiparallel arrangement. Later, the research team of Eisenberg revealed the structure of an off-pathway, cylindrical oligomer of a segment of the amyloid-forming protein αB-crystallin that resembled a β-barrel composed of six antiparallel β-strands . We and other groups demonstrated that amyloidogenic proteins, such as Aβ, pass through an antiparallel β-sheet structured state, corresponding to oligomers, before undergoing a transition in structure to parallel β-sheet fibrils [69, 70, 71, 91, 92, 93]. Recently, the group of Tycko demonstrated that Aβ1–40 containing the Iowa D23N mutation, resulting in a predominant vascular phenotype, can assemble into antiparallel β-sheet structured fibrils that are thermodynamically metastable and have a ribbon-like appearance . As these aggregates were all described to be transient and toxic, the antiparallel β-sheet arrangement has been suggested to represent a unique toxic signature .
In this study, we report that an antiparallel β-sheet signature is shared by oligomers and fibrils comprising the Italian-mutant E22K Aβ1–42 peptide (Figs. 1, 2). The antiparallel β-sheet fibrils are formed spontaneously under near-physiological conditions and display a high β-index ratio for at least 1 year (Fig. S3). The Italian-mutant Aβ peptide can thus potentially provide a major source of neurotoxicity, either in the form of soluble and diffusible oligomers that are considered the main toxic agents in AD [79, 94, 95], or in its fibrillar form as a trigger of neuroinflammation . One may speculate that, together with the imbalance between the production and clearance of the mutated peptide, this unique structural signature may be linked to the early-onset and aggressive progression of the associated FAD. Accordingly, the Italian Aβ mutant shows increased pathogenicity compared to the WT Aβ peptide and is up to tenfold more toxic to cerebrovascular smooth muscle cells  and PC12 cells in vitro [88, 89], supporting its role in CAA.
The Italian Aβ mutant can form antiparallel and parallel β-sheet fibrils
Amyloid fibrils share the cross-β spine motif, but their quaternary arrangement can differ due to distinct non-polar interactions; i.e., Van der Waals forces and aromatic packing, and polar interactions, i.e., electrostatic and H-bonding interactions. It is conceivable that substitution of a glutamic acid to a lysine at position 22 within the Aβ sequence may profoundly affect the pattern of interactions that determines its fibrillar structure. The residue at position 22 is part of the central Aβ region that poses the critical limiting step leading to the formation of the β-hairpin, the ordered β-turn-β structural organization characteristic of Aβ monomers within the fibril [89, 98, 99]. Based on solid state NMR data, Masuda and co-workers [77, 100] suggested previously that intermolecular β-sheet contacts in E22K Aβ1–42 fibrils are key events driven by this turn region.
The results obtained at pH 7.4 give more insight into the possible effects on the fibril structure due to the charge alteration in this central Aβ region. First, we demonstrate that the E22K mutation results in differences in IR spectra that reflect distinct H-bonding organizations of WT and E22K Aβ1–42 fibrils, i.e., parallel and antiparallel β-sheet arrangements respectively. The differences in structure of WT and E22K Aβ1–42 fibrils were further marked by different ThT fluorescence intensities and HDX behaviors (Figs. 1, 2, 3). Second, the E22K mutation most likely induces changes in the pattern of stabilizing electrostatic interactions, as it can interfere with salt bridges that have been suggested to occur within the monomeric unit of the fibrillar structure, e.g., between D23 and K28 [13, 17], but potentially also between K16 and E22 , and E22 and K28 . Differences in electrostatic interactions could underlie the structural alterations demonstrated for E22K fibrils at different pH values (Fig. 4). Third, the E22K mutation may potentially influence conserved hydrophobic contacts within the monomer unit composing the fibril, e.g., between F19 and G38 , or affect the interdigitation of β-sheets that is responsible for the steric zipper interface underlying the cross β structure . The steric hindrance induced by the large side chain of K22 may result in changes in the exposure of side chains to the outer fibril surface and contribute to packing polymorphism. Accordingly, the E22K mutation induced changes in solvent accessibility around the central region, as assessed by HDX measurements (Table 1; Fig. 3).
Furthermore, we provide evidence that structural alterations occur for E22K Aβ fibrils in different environmental growing conditions. At neutral pH, the antiparallel β-sheet fibrillar structure is favored. In contrast, the E22K Aβ1–42 peptide forms fibrils with a parallel β-sheet arrangement at low pH (Fig. 4). To the best of our knowledge, this is the first experimental demonstration that a mutated form of the full-length Aβ peptide can form amyloid fibrils with two different β-sheet structures in a pH-dependent manner. This structural diversity might be due to neutralization of charges of amino acid side chains involved in key fibril contacts, as discussed in the previous section. Moreover, our results show inter-conversion of antiparallel-to-parallel β-sheet Italian-mutant fibrils when changing from neutral to acidic pH conditions. Recently, Tycko and co-workers  demonstrated the evolution of a mixture of two Aβ fibril polymorphs to the thermodynamically most stable polymorph. It remains to be discovered whether the inter-conversion of Italian-mutant fibril polymorphs demonstrated in this work is due to internal structural rearrangement, or whether the antiparallel β-sheet fibrils are destabilized upon a decrease in pH and are rebuilt in a parallel β-sheet arrangement.
A detailed molecular understanding of the intermolecular interactions that dictate the quaternary structure of Italian-mutant Aβ is still lacking, but the results presented here suggest that differences in the H-bonding pattern, electrostatic interactions, and balance of multiple side chain contacts may underlie fibril polymorphism for this Aβ mutant.
Pathophysiological relevance of different β-sheet architectures
It has been suggested that Aβ fibril polymorphism may have biological significance. Fibril polymorphism could underlie in vitro differences in neurotoxicity, or in vivo differences in disease pathology and progression in different individuals/cell types and/or types of amyloidosis (i.e., tropism) [13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 104]. Recently, the first detailed look at the architecture of amyloid fibrils from patient brains demonstrated that two AD patients with distinct clinical histories possessed Aβ fibrils with a different underlying structure . Moreover, Prusiner and co-workers showed that mice inoculated with brain homogenates from an Arctic AD case exhibited a pathology that could be distinguished from mice inoculated with Swedish or sporadic AD samples. This was seen by differential accumulation of Aβ isoforms and distinct morphology of cerebrovascular Aβ deposition . It now becomes clear that several mutations in the central Aβ region that are associated with CAA, including the Italian E22K and Iowa D23N mutations, can result in the formation of Aβ fibrils with an antiparallel β-sheet structure (Fig. 2b). This unique structural signature might predispose them to deposit in cerebral blood vessels, rather than mainly accumulating in plaques, as seen for WT Aβ. It remains to be solved how these in vitro observations of structural differences can translate into differences in vivo, but one possibility might be through distinct interactions with receptors responsible for Aβ clearance across the blood–brain barrier.
In conclusion, the Italian-mutant Aβ peptide forms oligomers and fibrils in vitro that share the antiparallel β-sheet organization. Our results are particularly interesting in light of the ongoing debate that suggests that the antiparallel β-sheet signature might provide the potential detrimental toxic effect of Aβ. Moreover, this is the first study that experimentally demonstrates structural plasticity for E22K Aβ fibrils. The structural differences of WT and E22K Aβ fibrils observed in vitro might be a direct implication for their in vivo differences: WT and E22K Aβ deposition in extracellular plaques and cerebral blood vessel walls, respectively, and therefore associated with late- and early-onset AD, respectively.
EH is supported by a FWO doctoral fellowship. NvN is supported by the VIB and the Flemish Hercules Foundation. KB is supported by a grant from the Internationale Stichting Alzheimer Onderzoek (ISAO), an Odysseus II award from FWO and a UTWIST fellowship. RS and SD are postdoctoral researchers for the National Fund for Scientific Research (F.R.S.-FNRS, Belgium), VR is senior research associate for the National Fund for Scientific Research (F.R.S.-FNRS, Belgium), and is supported by grants from the F.R.S.-FNRS (Belgium), the SAO-FRA (Belgium), and the Foundation Van Buuren (Belgium).
- 35.Hori Y, Hashimoto T, Wakutani Y, Urakami K, Nakashima K, Condron MM, Tsubuki S, Saido TC, Teplow DB, Iwatsubo T (2007) The Tottori (D7N) and English (H6R) familial Alzheimer disease mutations accelerate Abeta fibril formation without increasing protofibril formation. J Biol Chem 282:4916–4923CrossRefPubMedGoogle Scholar
- 37.Maruyama K, Tomita T, Shinozaki K, Kume H, Asada H, Saido TC, Ishiura S, Iwatsubo T, Obata K (1996) Familial Alzheimer’s disease-linked mutations at Val717 of amyloid precursor protein are specific for the increased secretion of Abeta 42(43). Biochem Biophys Res Commun 227:730–735CrossRefPubMedGoogle Scholar
- 38.Ancolio K, Dumanchin C, Barelli H, Warter JM, Brice A, Campion D, Frebourg T, Checler F (1999) Unusual phenotypic alteration of beta amyloid precursor protein (betaAPP) maturation by a new Val-715 → Met betaAPP-770 mutation responsible for probable early-onset Alzheimer’s disease. Proc Natl Acad Sci USA 96:4119–4124PubMedCentralCrossRefPubMedGoogle Scholar
- 40.Zhou L, Brouwers N, Benilova I, Vandersteen A, Mercken M, Van Laere K, Van Damme P, Demedts D, Van Leuven F, Sleegers K, Broersen K, Van Broeckhoven C, Vandenberghe R, De Strooper B (2011) Amyloid precursor protein mutation E682K at the alternative β-secretase cleavage β-site increases Aβ generation. EMBO Mol Med 3:291–302PubMedCentralCrossRefPubMedGoogle Scholar
- 41.Vandersteen A, Masman MF, De Baets G, Jonckheere W, van der Werf K, Marrink SJ, Rozenski J, Benilova I, De Strooper B, Subramaniam V, Schymkowitz J, Rousseau F, Broersen K (2012) Molecular plasticity regulates oligomerization and cytotoxicity of the multipeptide-length amyloid-β peptide pool. J Biol Chem 287:36732–36743PubMedCentralCrossRefPubMedGoogle Scholar
- 43.Natté R, Maat-Schieman ML, Haan J, Bornebroek M, Roos RA, van Duinen SG (2001) Dementia in hereditary cerebral hemorrhage with amyloidosis-Dutch type is associated with cerebral amyloid angiopathy but is independent of plaques and neurofibrillary tangles. Ann Neurol 50:765–772CrossRefPubMedGoogle Scholar
- 45.Nilsberth C, Westlind-Danielsson A, Eckman CB, Condron MM, Axelman K, Forsell C, Stenh C, Luthman J, Teplow DB, Younkin SG, Naslund J, Lannfelt L (2001) The ‘Arctic’ APP mutation (E693G) causes Alzheimer’s disease by enhanced Abeta protofibril formation. Nat Neurosci 4:887–893CrossRefPubMedGoogle Scholar
- 47.Bugiani O, Giaccone G, Rossi G, Mangieri M, Capobianco R, Morbin M, Mazzoleni G, Cupidi C, Marcon G, Giovagnoli A, Bizzi A, Di Fede G, Puoti G, Carella F, Salmaggi A, Romorini A, Patruno GM, Magoni M, Padovani A, Tagliavini F (2010) Hereditary cerebral hemorrhage with amyloidosis associated with the E693K mutation of APP. Arch Neurol 67:987–995CrossRefPubMedGoogle Scholar
- 75.Cloe AL, Orgel JP, Sachleben JR, Tycko R, Meredith SC (2011) The Japanese mutant Aβ (ΔE22-Aβ(1–39)) forms fibrils instantaneously, with low-thioflavin T fluorescence: seeding of wild-type Aβ(1–40) into atypical fibrils by ΔE22-Aβ(1–39). Biochemistry 50:2026–2039PubMedCentralCrossRefPubMedGoogle Scholar
- 83.Qiang W, Yau WM, Schulte J Fibrillation of β amyloid peptides in the presence of phospholipid bilayers and the consequent membrane disruption. Biochim Biophys Acta 1848:266–276Google Scholar
- 84.Canale C, Seghezza S, Vilasi S, Carrotta R, Bulone D, Diaspro A, San Biagio PL, Dante S Different effects of Alzheimer’s peptide Aβ(1–40) oligomers and fibrils on supported lipid membranes. Biophys Chem 182:23–29Google Scholar
- 85.Cecchi C, Stefani M The amyloid-cell membrane system. The interplay between the biophysical features of oligomers/fibrils and cell membrane defines amyloid toxicity. Biophys Chem 182:30–43Google Scholar
- 86.Doens D, Fernandez PL Microglia receptors and their implications in the response to amyloid β for Alzheimer’s disease pathogenesis. J Neuroinflammation 11:48Google Scholar
- 87.Heneka MT, Golenbock DT, Latz E Innate immunity in Alzheimer’s disease. Nat Immunol 16:229–236Google Scholar
- 88.Murakami K, Irie K, Morimoto A, Ohigashi H, Shindo M, Nagao M, Shimizu T, Shirasawa T (2002) Synthesis, aggregation, neurotoxicity, and secondary structure of various Abeta 1–42 mutants of familial Alzheimer’s disease at positions 21–23. Biochem Biophys Res Commun 294:5–10CrossRefPubMedGoogle Scholar
- 89.Murakami K, Irie K, Morimoto A, Ohigashi H, Shindo M, Nagao M, Shimizu T, Shirasawa T (2003) Neurotoxicity and physicochemical properties of Abeta mutant peptides from cerebral amyloid angiopathy: implication for the pathogenesis of cerebral amyloid angiopathy and Alzheimer’s disease. J Biol Chem 278:46179–46187CrossRefPubMedGoogle Scholar
- 92.Sandberg A, Luheshi LM, Söllvander S, Pereira de Barros T, Macao B, Knowles TP, Biverstål H, Lendel C, Ekholm-Petterson F, Dubnovitsky A, Lannfelt L, Dobson CM, Härd T (2010) Stabilization of neurotoxic Alzheimer amyloid-beta oligomers by protein engineering. Proc Natl Acad Sci USA 107:15595–15600PubMedCentralCrossRefPubMedGoogle Scholar
- 105.Watts JC, Condello C, Stöhr J, Oehler A, Lee J, DeArmond SJ, Lannfelt L, Ingelsson M, Giles K, Prusiner SB (2014) Serial propagation of distinct strains of Aβ prions from Alzheimer’s disease patients. Proc Natl Acad Sci USAGoogle Scholar
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