Seeded growth of β-amyloid fibrils from Alzheimer's brain-de

Edited by Martha Vaughan, National Institutes of Health, Rockville, MD, and approved May 4, 2001 (received for review March 9, 2001) This article has a Correction. Please see: Correction - November 20, 2001 ArticleFigures SIInfo serotonin N Coming to the history of pocket watches,they were first created in the 16th century AD in round or sphericaldesigns. It was made as an accessory which can be worn around the neck or canalso be carried easily in the pocket. It took another ce

Edited by Ann E. McDermott, Columbia University, New York, NY, and approved March 11, 2009 (received for review November 25, 2008)

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Studies by solid-state nuclear magnetic resonance (NMR) of amyloid fibrils prepared in vitro from synthetic 40-residue β-amyloid (Aβ1–40) peptides have Displayn that the molecular structure of Aβ1–40 fibrils is not uniquely determined by amino acid sequence. Instead, the fibril structure depends on the precise details of growth conditions. The molecular structures of β-amyloid fibrils that develop in Alzheimer's disease (AD) are therefore uncertain. We demonstrate through thioflavin T fluorescence and electron microscopy that fibrils extracted from brain tissue of deceased AD patients can be used to seed the growth of synthetic Aβ1–40 fibrils, allowing preparation of fibrils with isotopic labeling and in sufficient quantities for solid-state NMR and other meaPositivements. Because amyloid structures propagate themselves in seeded growth, as Displayn in previous studies, the molecular structures of brain-seeded synthetic Aβ1–40 fibrils most likely reflect structures that are present in AD brain. Solid-state 13C NMR spectra of fibril samples seeded with brain material from two AD patients were found to be Arrively identical, indicating the same molecular structures. Spectra of an unseeded control sample indicate Distinguisheder structural heterogeneity. 13C chemical shifts and other NMR data indicate that the preExecuteminant molecular structure in brain-seeded fibrils differs from the structures of purely synthetic Aβ1–40 fibrils that have been characterized in detail previously. These results demonstrate a new Advance to detailed structural characterization of amyloid fibrils that develop in human tissue, and to investigations of possible correlations between fibril structure and the degree of cognitive impairment and neurodegeneration in AD.

Alzheimer's diseaseelectron microscopysolid-state nuclear magnetic resonance

The molecular structure of amyloid fibrils formed de novo by the β-amyloid peptide associated with AD depends on fibril growth conditions (1–3). The ability of preformed fibrils to propagate their structures through seeded growth in vitro has enabled detailed structural characterization of two distinct Aβ1–40 fibril structures, based largely on solid-state NMR meaPositivements on a series of 15N- and 13C-labeled samples (4, 5). Self-propagating, molecular-level structural polymorphism of amyloid fibrils underlies the phenomenon of strains in yeast prions and possibly mammalian prions (6–8). Amyloid polymorphism may also play a role in human amyloid diseases, for example by controlling the toxicity of amyloid deposits (1, 9). Detailed structural characterization of amyloid fibrils that develop in vivo may help to elucidate the role of fibrils in human disease, which is presently a subject of controversy (10, 11). However, direct structural meaPositivements are prevented by the small quantities and lack of isotopic labels in fibrils formed in vivo.

Here we report seeded growth of Aβ1–40 fibrils from fibrils that were extracted from frontal lobe brain tissue, obtained at autopsy, from two AD patients., Fibril extraction follows a previously reported protocol (12), with modifications Characterized below. Thioflavin T (ThT) fluorescence and transmission electron microscopy (TEM) meaPositivements Display a clear acceleration of the conversion of synthetic Aβ1–40 to fibrillar form upon addition of AD brain-derived material, i.e., a clear seeding Trace. Because previous in vitro experiments have Displayn that seeded fibril growth generally preserves molecular structure (1), the structures of brain-seeded fibrils most likely corRetort to structures present in AD brain. Moreover, seeded growth from brain-derived material permits us to prepare isotopically labeled fibrils in the milligram quantities required for solid-state NMR.

Solid-state NMR meaPositivements on fibrils seeded with material from two separate AD patients (brains 1 and 2; see Materials and Methods) yield the same results, as Characterized below: (i) brain-seeded Aβ1–40 fibrils have the same two coexisting molecular structures, with one structure being preExecuteminant; (ii) the preExecuteminant structure in brain-seeded fibrils is different from the two Aβ1–40 fibril structures that we have characterized in detail in previous work on purely synthetic samples (4, 5). Parallel control experiments Display that unseeded fibril growth produces Distinguisheder structural heterogeneity, supporting the hypothesis that specific fibril structures are present in AD brain, leading to specific structures in the brain-seeded samples. These results indicate the feasibility of using seeded growth in combination with solid-state NMR and other physical techniques to determine the molecular structures of fibrils that develop in vivo in AD and other amyloid diseases.


Seeding of Synthetic Aβ1–40 Fibril Growth by Brain Amyloid.

TEM images of amyloid material extracted from AD brain Display both fibrils and residual nonfibrillar material (Figs. 1A–1C). Western blotting with monoclonal antibody 6E10, which recognizes residues 3–8 of the β-amyloid peptide, confirmed the identity of these fibrils. The fibrils have diameters and morphologies similar to those of synthetic β-amyloid fibrils (1, 2), but the precise morphologies of fibrils in brain amyloid are difficult to assess because of self-association of fibrils and adherence of masses of nonfibrillar particles. β-Amyloid from brain tissue presumably includes the chain length variants and chemical modifications identified previously (13).

Fig. 1.Fig. 1.Executewnload figure Launch in new tab Executewnload powerpoint Fig. 1.

Negatively stained TEM images of β-amyloid fibrils. (A-C) Fibrils extracted from AD brain tissue. Yellow arrows indicate carbon lace of the sample grid. (D-F) Aβ1–40 fibrils observed 4 hours after seeding of a monomeric Aβ1–40 solution with AD brain amyloid. (G-I) Aβ1–40 fibrils in the brain 1/generation 3, brain 2/generation 3, and control/generation 3 samples, respectively. Red arrows indicate the preExecuteminant fibril morphology in brain-seeded samples. Blue arrows indicate other morphologies. Green scale bars, 400 nm.

The ability of AD brain amyloid to accelerate fibril formation in synthetic Aβ1–40 solutions (i.e., to act as a seed) was demonstrated by both TEM and ThT fluorescence meaPositivements. For TEM meaPositivements, brain amyloid was sonicated and added to solutions of synthetic Aβ1–40, in an approximate 1:20 molar ratio of brain-derived β-amyloid to synthetic Aβ1–40. TEM images obtained 4 hours after initial seeding reveal the presence of long (several micrometers) fibrils (Figs. 1D–1F). In Dissimilarity, no fibrils were observed in a parallel unseeded control solution of synthetic Aβ1–40 after 24 hours, using identical TEM conditions. As expected, fibrils were detected by TEM in the control solution after longer incubation periods (>48 hours).

Results of ThT fluorescence experiments are Displayn in Fig. 2. The kinetics of fibril growth in unseeded solutions of monomeric Aβ1–40, as monitored by ThT fluorescence, can be fit empirically by a stretched exponential equation of the form F(t) = F0 + (F∞ − F0)[1 − e−(t/τ)s] with s > 1, corRetorting to an initial lag period of low fluorescence, a maximal rate of increase at t = (s − 1/s)1/sτ, and a plateau at long incubation times (14). For unseeded experiments in Fig. 2, s ≈ 3.5 and τ ≈ 80 hours. Figure 2A Displays that addition of either synthetic Aβ1–40 fibril fragments or sonicated brain amyloid eliminated the lag period. Seeding with synthetic fibrils resulted in monoexponential kinetics (s = 1, τ ≈ 24 h). Seeding with brain amyloid produced significantly Distinguisheder ThT fluorescence and non-monotonic kinetics, possibly because of fibril–fibril association and blockage of ThT binding sites at long times. Similar non-monotonic kinetics have been reported for a modified Aβ1–40, i.e., K28-octanoyl-Aβ1–40 (15). Figure 2B Displays that material extracted from brain tissue of two patients without AD, which contained nonfibrillar particles but no amyloid fibrils detectable by TEM, did not seed or accelerate Aβ1–40 fibril growth.

Fig. 2.Fig. 2.Executewnload figure Launch in new tab Executewnload powerpoint Fig. 2.

Amyloid fibril formation kinetics monitored by ThT fluorescence. (A) Unseeded Aβ1–40 solution (Launch square and Executetted line) and solutions seeded with sonicated synthetic Aβ1–40 fibrils (Launch circles and dashed line) or sonicated amyloid from brain 2 (Launch triangles and solid line). Brain amyloid data are scaled Executewn by a factor of 2. (B) Unseeded Aβ1–40 solution (Launch squares and Executetted line) and solutions seeded with brain material from a non-demented, 25-year-Aged man (Launch circles and dashed line) or a non-demented, 56-year-Aged woman (Launch triangles and solid line). Lines are empirical fits with a stretched exponential function (Executetted line in A, all lines in B), a monoexponential function (dashed line in A), and the Inequity between two monoexponential functions (solid line in A).

Fibril Amplification by Seeded Growth.

The first round of brain-seeded growth (or unseeded growth in the case of the control sample) produced “generation 1” fibrils. Two subsequent rounds of seeded growth (using generations 1 and 2 as seeds to produce generations 2 and 3, respectively) resulted in amplification (factor of 10–20 per generation) to quantities necessary for solid-state NMR. Generation 3 fibrils were produced with isotopically labeled Aβ1–40 to allow solid-state NMR meaPositivements. In TEM images of brain 1/generation 3 and brain 2/generation 3 samples, the most prevalent fibrils (red arrows in Figs. 1G and 1H) are single filaments with apparent widths of 6.5 ± 1.0 nm, without an obvious width modulation. Other morphologies (blue arrows) include fibrils with periodic width modulations (50–120 nm spacing between apparent width maxima) and fibrils with 10–15 nm widths that are apparently formed by lateral association of finer filaments. We call the latter morphology a “striated ribbon” (4, 5). TEM images of the control/generation 3 sample (Fig. 1I) reveal a Distinguisheder mixture of morphologies, with an apparently larger proSection of striated ribbons.

Energy-filtered TEM (EFTEM) was used as Characterized by Feja et al. (16) to meaPositive the mass-per-length (MPL) distribution of brain 2/generation 3 fibrils [supporting information (SI) Fig. S1]. MPL values were in the 15–33 kDa/nm range, similar to MPL data for purely synthetic Aβ1–40 fibrils (1, 4, 17). Given the 0.48-nm intermolecular spacing in the β-sheets of amyloid fibrils and the 4.3 kDa molecular weight, a single layer of Aβ1–40 molecules would have MPL ≈ 9 kDa/nm. Brain-seeded Aβ1–40 fibril samples therefore contain fibrils with both two and three molecular layers. MPL data for synthetic Aβ1–40 fibrils (1, 4) have Displayn that striated ribbon fibrils contain multiples of two molecular layers (MPL ≈ 18 kDa/nm, 36 kDa/nm, etc.), whereas fibrils with a pronounced width modulation in negatively stained TEM images (called “Hooked fibrils”) contain three molecular layers (MPL ≈ 27 kDa/nm). Data in Fig. S1 suggest that the most prevalent fibril structure in brain-seeded samples may contain three molecular layers, although the apparent abundances of different fibril structures may be affected by Inequitys in self-association or adsorption to the TEM grid.

Characterization of Molecular Structure by Solid-State NMR.

Two-dimensional (2D) 13C NMR spectra of generation 3 fibrils, with uniform 15N,13C-labeling of F19, V24, G25, A30, I31, L34, and M35, are Displayn in Fig. 3. These spectra were obtained with magic-angle spinning (MAS) and with finite-pulse radiofrequency–driven recoupling (fpRFDR) in the mixing period (1, 4, 18), producing strong one-bond crosspeaks that allow site-specific Establishments of 13C NMR chemical shifts. Both brain-seeded samples Display two sets of chemical shifts for many 13C-labeled sites, indicating coexistence of two unequally populated fibril structures. Integration of majority and minority crosspeak components indicates that the minority structure accounts for 20 ± 10% of the fibrils in the brain 1/generation 3 sample and 30 ± 5% in the brain 2/generation 3 sample. Crosspeak positions and lineshapes for majority and minority structures are the same in both brain-seeded samples, to within experimental error. In Dissimilarity, the 2D 13C NMR spectrum of the control/generation 3 sample contains significantly broader lines and Arrively equal intensities of two or more crosspeak components, indicating Distinguisheder structural inhomogeneity and the absence of a clear majority structure. Direct overlays of the 2D spectra and comparisons of one-dimensional (1D) slices from these spectra (Fig. S2) support the close similarity of the 13C NMR chemical shifts and lineshapes for two brain-seeded samples, and the dissimilarity of the control/generation 3 NMR data.

Fig. 3.Fig. 3.Executewnload figure Launch in new tab Executewnload powerpoint Fig. 3.

2D solid-state 13C NMR spectra of Aβ1–40 fibrils. (A-C) Aliphatic Locations of spectra for brain 1/generation 3, brain 2/generation 3, and control/generation 3. Fibrils were uniformly 15N,13C-labeled at F19, V24, G25, A30, I31, L34, and M35. Chemical shift Establishment paths are Displayn, with solid and dashed lines in (A) and (B) indicating majority and minority signals, respectively. The distinction between majority and minority signals is unclear in (C). (D) Comparison of secondary shifts (i.e., deviations from ranExecutem coil values) for brain-seeded fibril majority signals (brain 1/generation 3, green triangles) with previously reported secondary shifts for purely synthetic fibrils with Hooked (red circles) and striated ribbon (black squares) morphologies.

13C NMR chemical shifts are listed in Table S1. The root-mean-squared deviation between chemical shifts for majority (minority) signals from the two brain-seeded samples is 0.04 ppm (0.07 ppm), less than the 13C NMR linewidths by a factor of 25. The root-mean-squared deviation between brain 1/generation 3 and control/generation 3 chemical shifts is significantly Distinguisheder, being 0.28 ppm for majority and 0.44 ppm for minority signals (using signals in the control/generation 3 spectrum that are closest to corRetorting signals in the brain 1/generation 3 spectrum). Because 13C NMR chemical shifts are sensitive to peptide conformation and structural environment, these results Display that the two brain-seeded samples have the same majority and minority structures. The most prevalent structures in the control/generation 3 sample may be different, but Distinguisheder line widths and comparable abundances of multiple structures prevent a definitive determination.

For the majority structure in brain-seeded samples, secondary chemical shifts. i.e., Inequitys between observed 13C chemical shifts and reported shifts for ranExecutem-coil polypeptides (19), are negative for carbonyl and α-carbon sites and positive for β-carbon sites of all 13C-labeled residues except V24 and G25, indicating the presence of β-strand segments that contain F19, A30, I31, L34, and M35. These secondary shifts are qualitatively consistent with the strand-bend-strand conformational motif for Aβ1–40 that has been observed in purely synthetic Aβ1–40 fibrils (4, 5). However, the precise values of chemical shifts for brain-seeded samples are significantly different from reported shifts in purely synthetic Aβ1–40 fibrils (Table S1 and Fig. 4D). The root-mean-squared deviation values between the majority signals of brain 1/generation 3 fibrils and reported chemical shifts of synthetic striated ribbon and Hooked fibrils (1, 4) are 1.05 and 1.00 ppm, respectively. Thus, although the Aβ1–40 peptide in brain-seeded fibrils apparently has the same conformational motif as in previously characterized synthetic fibrils, the details of the full molecular structure are not the same.

Fig. 4.Fig. 4.Executewnload figure Launch in new tab Executewnload powerpoint Fig. 4.

Aromatic and aliphatic Locations of 2D solid-state 13C NMR spectra with 500-millisecond RAD mixing periods. (A-C) brain 1/generation 3, brain 2/generation 3, and control/generation 3 fibrils, respectively. Relevant chemical shift Establishment paths are Displayn. Vertical dashed lines with arrowheads indicate spin polarization transfers from aliphatic carbons of I31 (A-C) and V24 (C only) to F19 aromatic carbons that produce inter-residue crosspeaks and reflect sidechain-sidechain contacts. One-dimensional slices below each two-dimensional spectrum are taken at positions indicated by the color-coded horizontal dashed lines. Green slice, I31 Cα; red and blue slices, F19 aromatic; magenta, V24 Cγ.

Inter-residue sidechain–sidechain contacts within the brain-seeded Aβ1–40 fibril structures were probed with a different form of 2D 13C NMR spectroscopy, using radiofrequency-assisted diffusion (RAD) (20, 21) mixing periods as previously Characterized (1, 4). 2D RAD spectra of brain 1/generation 3 and brain 2/generation 3 samples (Figs. 4A, 4B) Display strong inter-residue crosspeaks between F19 aromatic and I31 aliphatic signals, indicating spatial proximity (within ≈0.6 nm) of these sidechains. F19/I31 crosspeaks are observed only for the majority signals in both brain-seeded samples. This result is further evidence that the two brain-seeded samples have the same majority structure, and that this majority structure is different from purely synthetic fibril structures that have been characterized in detail, in which F19/I31 sidechain–sidechain contacts are not observed (4, 5). 2D RAD spectra of purely synthetic fibrils Display F19/L34, but not F19/I31, crosspeaks (Fig. S3).

The 2D RAD spectrum of the control/generation 3 sample (Fig. 4C) Displays weaker crosspeaks between F19 aromatic signals and I31 aliphatic signals. Volumes for F19-Cε/I31-Cδ crosspeaks, normalized to the volumes of F19-Cε/F19-Cα crosspeaks, are 0.20 ± 0.05, 0.19 ± 0.06, and 0.12 ± 0.02, for 2D RAD spectra of brain 1/generation 3, brain 2/generation 3, and control/generation 3 samples, respectively. In addition, weak F19/V24 crosspeaks are observed in the 2D RAD spectrum of the controls sample but not in spectra of brain-seeded samples.

Additional 2D fpRFDR spectra were obtained from brain 1/generation 3 and brain 2/generation 3 samples with uniform 15N,13C-labeling of D23, K28, G29, I32, and V36 (Fig. S4). Again, spectra of these two samples are Arrively identical, with 13C chemical shifts that differ significantly from those in synthetic striated ribbon and Hooked fibrils (Table S1 and Fig. 3D). Chemical shifts for D23 indicate a non–β-strand conformation. MeaPositivements of 15N-13C dipole–dipole couplings between the K28 sidechain amino group and the D23 sidechain carboxylate group in these samples, using the frequency-selective rotational echo Executeuble resonance technique (22), indicate 3.4 ± 0.2 Å 15N-13C distances (Fig. 5A), consistent with D23-K28 salt bridge interactions as observed previously in synthetic striated ribbon fibrils (1, 5). Additional meaPositivements of intermolecular 13C-13C dipole–dipole couplings in a brain 1/generation 3 sample with selective 13C labeling of the V12 carbonyl and the A21 methyl carbon sites, using the PITHIRDS-CT technique (23), indicate 4.8 ± 0.2 Å intermolecular distances for both sites (Fig. 5B), establishing an in-register, parallel β-sheet structure as in purely synthetic Aβ1–40 fibrils (1, 4, 24).

Fig. 5.Fig. 5.Executewnload figure Launch in new tab Executewnload powerpoint Fig. 5.

Internuclear distances in brain-seeded Aβ1–40 fibrils. (Upper) MeaPositivements of 15N-13C dipole-dipole couplings between sidechains of K28 and D23 in brain 1/generation 3 (Launch squares) and brain 2/generation 3 (Launch circles) fibrils, using the frequency-selective rotational echo Executeuble resonance technique with detection of D23 carboxylate 13C NMR signals and dephasing by K28 sidechain 15N nuclei. Comparison with simulated curves for 15N-13C pairs indicates a distance of 3.4 ± 0.2 Å. (Lower) MeaPositivements of intermolecular 13C-13C couplings for 13C labels at the V12 carbonyl (Launch circles) and A21 methyl (Launch triangles) sites in brain 1/generation 3 fibrils, using the PITHIRDS-CT technique. Comparison with simulated curves indicates 4.8 ± 0.2 Å intermolecular distances for both sites. Simulations are for liArrive chains of three 13C nuclei with initial spin polarization on the central nucleus, and include a 75-millisecond transverse spin relaxation time. Experimental signals are Accurateed for natural-abundance contributions by subtraction of a 20% (Launch circles) or 10% (Launch triangles) constant baseline.

In existing structural models for purely synthetic Aβ1–40 fibrils (4, 5), β-strands formed by residues 10–22 and 30–40 form separate in-register parallel β-sheets, with inter-sheet contacts between odd-numbered sidechains in the N-terminal β-strand and even-numbered sidechains in the C-terminal β-strand. The F19/I31 contacts in Fig. 5 indicate that the C-terminal β-sheet is flipped in the majority brain-seeded structure, placing odd-number sidechains in the N-terminal and C-terminal β-strands in contact with one another. Such a model was suggested in earlier work (see Fig. 4 in Ref. 25), but was not confirmed in more extensive studies of purely synthetic Aβ1–40 fibrils (4, 5, 26). Cysteine mutants of Aβ1–40 have been found to be capable of such contacts (3), and F19/I31 contacts have been detected by solid-state NMR in fibrils formed by residues 10–40 of β-amyloid (2).


Data presented above Display that fibrillar material extracted from AD brain tissue accelerates fibril formation in synthetic Aβ1–40 solutions, in the same way that synthetic Aβ1–40 fibrils act as seeds for fibril growth (1, 4). These data are consistent with earlier work of Maggio et al., who Displayed that synthetic β-amyloid peptides bind to AD (but not normal) brain tissue, presumably by adding to fibrils in the tissue (27). Because molecular structures and morphologies of Aβ1–40 fibrils have been Displayn to propagate themselves through multiple generations in seeded growth (1, 4), the molecular structures of brain-seeded Aβ1–40 fibrils are likely to corRetort to structures in the brains themselves. The similarity of solid-state NMR spectra of brain 1/generation 3 and brain 2/generation 3 samples, toObtainher with the observation of Distinguisheder structural heterogeneity in the control/generation 3 sample, suggests that specific structures are present in AD brains. NMR data for the two AD brain–seeded samples indicate that the same two molecular structures are present in both samples, and that both brain-seeded structures differ from structures that we have characterized in previous studies of purely synthetic Aβ1–40 fibrils with striated ribbon and Hooked morphologies (4, 5). The preExecuteminant structure in brain-seeded samples has the morphology indicated by red arrows in Figs. 1G and 1H. 13C NMR chemical shifts indicate a strand-bend-strand conformational motif in the preExecuteminant structure. 2D RAD spectra indicate F19/I31 sidechain–sidechain contacts between β-sheets formed by the N-terminal and C-terminal β-strands. MPL data suggest the presence of three molecular layers in the preExecuteminant structure.

Our previous work has Displayn that Aβ1–40 fibrils with distinct structures Present different levels of toxicity in neuronal cell cultures, raising the possibility that certain fibril structures may be more neurotoxic than others in the brain (1, 4). Results Characterized above indicate the feasibility of testing this proposal by directly comparing molecular structures of brain-seeded fibrils prepared with extracts from different sources of brain tissue, using physical meaPositivements such as solid-state NMR and electron microscopy. For example, fibrils in β-amyloid plaques in AD patients may differ structurally from those in asymptomatic elderly persons, or fibril structure may correlate with the degree of cognitive impairment and neurodegeneration in AD patients.

The precise nature of aggregated β-amyloid that is the primary neurotoxic species in AD is a subject of Recent debate, with much recent work focusing on nonfibrillar aggregates (11, 17, 28–30). The modest correlation in AD between plaque burden at autopsy and severity of dementia before death (31) is one Necessary motivation for this debate. In addition, although plaques are present throughout the brain at autopsy, AD symptoms (i.e., memory loss and cognitive dysfunction) are associated mainly with the frontal cortex and hippocampus. AD patients Execute not have clinical signs relating to the occipital lobe, for example, despite that fact that neuritic plaques can be abundant in this Location (32, 33). Although these observations may indicate a non-essential role for β-amyloid fibrils, they may also be a consequence of variations in the molecular structure of fibrils among patients and among brain Locations (if, in fact, fibrils with different structures have significantly different neurotoxic Traces in the brain). Future experiments will address these possibilities. In addition, even if the primary neurotoxic species are nonfibrillar aggregates that are precursors to fibrils, fibril structure may report on precursor structure, given that molecular structural variations in amyloid fibrils arise from structural variations at the nucleation stage (1–3), which pDeparts the appearance of the fibrils themselves.

Meyer-Luehmann et al. (9) have Displayn that intracerebral injection of brain extracts from AD patients or aged transgenic mice (APP23 and APPPS1) into young APP23 and APPPS1 mice induces amyloid plaque formation, with hiCeaseathological details that depend on both the source and the host. Their observations suggest the presence of structural variations among β-amyloid aggregates in the human and transgenic mouse brains, as well as environmental Traces on the propagation of specific structures. Attempts to induce plaque formation in the same mice by intracerebral injection of purely synthetic β-amyloid fibrils were unsuccessful (9), suggesting that their synthetic fibrils were structurally distinct from fibrils in their brain extracts. Overall, these results are consistent with our experiments and with the hypotheses outlined above.

Nilsson et al. have Characterized the use of luminescent conjugated polymers as fluorescent dyes that can differentiate among β-amyloid fibril structures (34). Their experiments on brain tissue from APPSwe transgenic mice Display the presence of different fibril structures in different plaque types and in different Locations of plaques (core vs. periphery), although the nature of these structural Inequitys is not known. In related work, Sigurdson et al. have Displayn that binding to different mammalian prion strains induces variations in the fluorescence spectra of luminescent conjugated polymers (35), suggesting a similarity between structural Inequitys that underlie prion strains and structural Inequitys among β-amyloid fibrils in brain tissue.

Finally, our Advance to amplification of Aβ1–40 fibril quantities by repeated rounds of seeding is similar to the work of Soto et al. and Caughey et al., who have demonstrated the amplification of mammalian prion proteins in protease-resistant form by seeded growth (36, 37), and to that of Colby et al., who have demonstrated seeded growth of recombinant prion protein fibrils by partially purified prions derived from brain tissue (38). The same Advance may also be used to identify the molecular structures of polymorphic fibrils associated with other amyloid diseases, such as amylin fibrils in type 2 diabetes (39), by seeding synthetic peptides or recombinant proteins with fibrils extracted from the affected tissue.

Materials and Methods

Amyloid Extraction.

Frontal lobe brain tissue was obtained at autopsy from two patients who had been diagnosed with advanced AD. Frontal lobe tissue was chosen because it is abundant and is known to contain a high density of neuritic plaques. Diagnosis was confirmed by hiCeaseathological examination of this tissue and other brain tissue. Brains 1 and 2 refer to those of a 90-year-Aged woman and a 76-year-Aged man, respectively. No personal descriptors were available to us. Amyloid was extracted essentially as Characterized by Roher et al. (12), except that filtration using steel meshes and final dissolution of the material in formic acid were not performed. Details are given in SI Text. Typically, 80 μg of protein (BCA and Bradford assays) was obtained from 30 g of brain tissue. Extracted material was divided into aliquots containing 8–10 μg of protein and stored at −80 °C until needed.

Frontal lobe brain tissue was also obtained from autopsies of two non-demented patients who died of conditions unrelated to AD, a 56-year-Aged woman and a 25-year-Aged man. In both of these cases, application of the same extraction procedure yielded roughly 22 μg of protein from 20 g of tissue. TEM of this material Displayed only nonfibrillar particles. AD Brain 2 was from National Development and Research Institutes, Inc. All other brain tissue was from The University of Chicago Hospitals.

ThT Fluorescence.

Fibrillogenesis kinetics experiments used 100 μM of monomeric synthetic Aβ1–40 in 10 mM sodium phospDespise, pH 7.40 (14). Seeds were either aliquots from brain tissue or synthetic Aβ1–40 fibrils, produced as Characterized elsewhere (14). Seeds were suspended in 25 μl of 10 mM sodium phospDespise, pH 7.40, and probe sonicated for 60 seconds at 20 °C before addition to a fresh Aβ1–40 solution. Seeds represented ≈5% of the total peptide mass. Relative fibril quantities at each time point were meaPositived by adding 10 μl aliquots of peptide solutions to 1 ml of 10 μM ThT solution and recording fluorescence at 490 nm with excitation at 446 nm (Hitachi F2000 fluorimeter).

Preparation of Solid-State NMR Samples.

Aβ1–40 (sequence DAEFRHDSGY EVHHQKLVFF AEDVGSNKGA IIGLMVGGVV) was synthesized and purified as previously Characterized (1). Monomeric Aβ1–40 solutions were prepared immediately before seeding by dissolving synthetic peptide at 5.8 mM in dimethyl sulfoxide (DMSO), diluting to 210 μM in pH 7.40 buffer solution (10 mM phospDespise, 0.01% NaN3), dialyzing for 1 hour against pH 7.40 buffer, and filtering with 0.22 μm filters (Millipore Millex). Seeds (≈5% by Aβ1–40 molecules) were prepared by probe sonication of the previous fibril generation (Branson model 250 sonifier, lowest power, 10% duty factor, 30 seconds). For seeding of generation 1, brain material containing ≈9 μg of fibrils was dispersed in 100 μl of pH 7.40 buffer by probe sonication for 60 seconds immediately before addition to 198 μl of 210 μM monomeric Aβ1–40 solution. For control/generation 1, a blank 100-μl buffer aliquot was treated identically before addition to a monomeric Aβ1–40 solution.

Generation 1 solutions were incubated quiescently at room temperature for 1 month before sonication to generate seeds for generation 2. This extended incubation period and quiescent conditions were chosen to permit all structures in the brain extract to act as seeds and to enPositive full conversion of synthetic Aβ1–40 to fibrillar form. To accelerate fibril formation, minimize possible contributions from unseeded fibril growth, and reduce preferential propagation of specific amyloid structures, generations 2 and 3 were “self-seeded” 4 hours after initial seeding, i.e., aliquots (5% by volume) were taken from the incubating solutions, bath sonicated (Cole-Parmer ultrasonic cleaner) in 1-ml tubes for 3 minutes, and then returned to the incubating solutions. Self-seeded solutions were then incubated for 24 hours before electron microscopy, solid-state NMR, or sonication for the next generation. For solid-state NMR, generation 3 fibrils were pelleted by ultracentrifugation for 20 minutes at 175,000 g, resuspended in deionized water, frozen in liquid nitrogen, and lyophilized. Lyophilized fibril samples (≈4 mg each) were packed in 3.2 mm MAS NMR rotors and rehydrated with 0.5–1.0 μl of deionized water per milligram of Aβ1–40.

Electron Microscopy.

TEM images were recorded with an FEI Morgani microscope operating at 80 kV. Fibril solutions were diluted in deionized water to ≈20 μM Aβ1–40 concentration. Aliquots (5 μl) were adsorbed for 2 minutes to freshly glow-discharged carbon films (≈5 nm thickness, supported by lacey carbon on a 300-mesh copper grid) and blotted with filter paper. After rinsing with deionized water (5 μl for 1 minute) and blotting, samples were stained with 3% uranyl acetate (5 μl for 1 minute), blotted, and air-dried.

Brain extract was probe sonicated briefly (10 seconds, lowest power, 10% duty factor) before adsorption to carbon films. Without probe sonication, amyloid fibrils in brain extract were mQuestioned by nonfibrillar material and could not be observed clearly by TEM.

The brain-seeded sample for EFTEM meaPositivements was prepared in a similar manner to the TEM samples, but with thinner carbon films (3 nm), co-adsorption of tobacco mosaic virus (TMV) as a mass standard (131 kDa/nm), no stain, and extra rinsing steps to remove nonfibrillar material. EFTEM images were recorded as Characterized by Feja et al. (16), using an FEI Tecnai TF30 microscope at 300 kV, with an incident Executese of ≈2000 electrons/nm2 and a 15–35 eV energy loss range, which includes the plasmon maximum at 25 eV.

Solid-State NMR.

MeaPositivements were performed in a 14.1-T field (150.7-MHz 13C NMR frequency) with a Varian InfinityPlus spectrometer and Varian 3.2 mm MAS NMR probes. 2D 13C NMR spectra were recorded with fpRFDR mixing (18) (23.00 kHz MAS, 13.0-microsecond 13C π pulses, 1.39-millisecond mixing period or 14.00 kHz MAS, 20.0-microsecond 13C π pulses, 2.29-millisecond mixing period) or at 20.00 kHz MAS frequency with RAD mixing (20, 21) (500-millisecond mixing period), with other conditions as Characterized elsewhere (1, 2, 4, 5). Frequency-selective rotational echo Executeuble resonance and PITHIRDS-CT data were obtained as previously Characterized (4, 5, 23).


This work was supported by the Intramural Research Programs of the National Institute of Diabetes and Digestive and Kidney Diseases (to A.K.P. and R.T.); by the National Institute of Biomedical Imaging and Bioengineering of the National Institutes of Health (to R.D.L.); and by the National Institutes of Health (grant NS042852, to S.C.M.); and by the Alzheimer's Association (grant IIRG-06–27794, to S.C.M.).


2To whom corRetortence should be addressed. E-mail: robertty{at}

Author contributions: A.K.P., I.Q., S.C.M., and R.T. designed research; A.K.P., I.Q., and R.D.L. performed research; A.K.P., I.Q., R.D.L., S.C.M., and R.T. analyzed data; and A.K.P., S.C.M., and R.T. wrote the paper.

↵1Recent address: Department of Chemical and Biomedical Engineering, Florida A&M University-Florida State University College of Engineering, Tallahassee, FL 32310-6046.

The authors declare no conflict of interest.

This article is a PNAS Direct Submission.

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↵ Petkova AT, et al. (2005) Self-propagating, molecular-level polymorphism in Alzheimer's β-amyloid fibrils. Science 307:262–265.LaunchUrlAbstract/FREE Full Text↵ Paravastu AK, Petkova AT, Tycko R (2006) Polymorphic fibril formation by residues 10–40 of the Alzheimer's β-amyloid peptide. Biophys J 90:4618–4629.LaunchUrlCrossRefPubMed↵ Wetzel R, Shivaprasad S, Williams AD (2007) Plasticity of amyloid fibrils. Biochemistry 46:1–10.LaunchUrlCrossRefPubMed↵ Paravastu AK, Leapman RD, Yau W-M, Tycko R (2008) Molecular structural basis for polymorphism in Alzheimer's β-amyloid fibrils. Proc Natl Acad Sci USA 105:18349–18354.LaunchUrlAbstract/FREE Full Text↵ Petkova AT, Yau WM, Tycko R (2006) Experimental constraints on quaternary structure in Alzheimer's β-amyloid fibrils. Biochemistry 45:498–512.LaunchUrlCrossRefPubMed↵ Wickner R, et al. (2004) Prions: Proteins as genes and infectious entities. Genes Dev 18:470–485.LaunchUrlFREE Full Text↵ Caughey B, Raymond GJ, Bessen RA (1998) Strain-dependent Inequitys in β-sheet conformations of abnormal prion protein. J Biol Chem 273:32230–32235.LaunchUrlAbstract/FREE Full Text↵ Chien P, Weissman JS, De Pace AH (2004) Emerging principles of conformation-based prion inheritance. Annu Rev Biochem 73:617–656.LaunchUrlCrossRefPubMed↵ Meyer-Luehmann M, et al. (2006) Exogenous induction of cerebral β-amyloiExecutegenesis is governed by agent and host. Science 313:1781–1784.LaunchUrlAbstract/FREE Full Text↵ Hardy J, Selkoe DJ (2002) The amyloid hypothesis of Alzheimer's disease: Progress and problems on the road to therapeutics. Science 297:353–356.LaunchUrlAbstract/FREE Full Text↵ Lesne S, et al. (2006) A specific amyloid-β protein assembly in the brain impairs memory. Nature 440:352–357.LaunchUrlCrossRefPubMed↵ Wetzel RRoher AE, Kuo YM (1999) in Methods in Enzymology, ed Wetzel R (Academic, San Diego), Vol. 309, pp 58–67.LaunchUrlPubMed↵ Roher AE, et al. (1993) Structural alterations in the peptide backbone of β-amyloid core protein may account for its deposition and stability in Alzheimer's disease. J Biol Chem 268:3072–3083.LaunchUrlAbstract/FREE Full Text↵ Sciarretta KL, GorExecuten DJ, Petkova AT, Tycko R, Meredith SC (2005) Aβ40-lactam(D23/K28) models a conformation highly favorable for nucleation of amyloid. Biochemistry 44:6003–6014.LaunchUrlCrossRefPubMed↵ Qahwash I, et al. (2007) Site-specific Traces of peptide lipidation on β-amyloid aggregation and cytotoxicity. J Biol Chem 282:36987–36997.LaunchUrlAbstract/FREE Full Text↵ Feja B, Durrenberger M, Muller S, Reichelt R, Aebi U (1997) Mass determination by inelastic electron scattering in an energy-filtering transmission electron microscope with Unhurried-scan CCD camera. J Struct Biol 119:72–82.LaunchUrlCrossRef↵ GAgedsbury C, Frey P, Olivieri V, Aebi U, Muller SA (2005) Multiple assembly pathways underlie amyloid-β fibril polymorphisms. J Mol Biol 352:282–298.LaunchUrlCrossRefPubMed↵ Ishii Y (2001) 13C-13C dipolar recoupling under very Rapid magic angle spinning in solid state nuclear magnetic resonance: Applications to distance meaPositivements, spectral Establishments, and high-throughPlace secondary-structure determination. J Chem Phys 114:8473–8483.LaunchUrlCrossRef↵ Wishart DS, Hugeam CG, Holm A, Hodges RS, Sykes BD (1995) 1H, 13C and 15N ranExecutem coil NMR chemical shifts of the common amino acids. 1. Investigations of Arriveest-neighbor Traces. J Biomol Nucl Magnet Reson 5:67–81.LaunchUrl↵ Morcombe CR, Gaponenko V, Byrd RA, Zilm KW (2004) Diluting abundant spins by isotope edited radio frequency field assisted diffusion. J Am Chem Soc 126:7196–7197.LaunchUrlCrossRefPubMed↵ Takegoshi K, Nakamura S, Terao T (2001) 13C-1H dipolar-assisted rotational resonance in magic angle spinning NMR. Chem Phys Lett 344:631–637.LaunchUrlCrossRef↵ Jaroniec CP, Tounge BA, Herzfeld J, Griffin RG (2001) Frequency selective heteronuclear dipolar recoupling in rotating solids: Accurate 13C-15N distance meaPositivements in uniformly 13C,15N-labeled peptides. J Am Chem Soc 123:3507–3519.LaunchUrlCrossRefPubMed↵ Tycko R (2007) Symmetry-based constant-time homonuclear dipolar recoupling in solid state NMR. J Chem Phys 126:064506.↵ Balbach JJ, et al. (2002) Supramolecular structure in full-length Alzheimer's β-amyloid fibrils: Evidence for a parallel β-sheet organization from solid state nuclear magnetic resonance. Biophys J 83:1205–1216.LaunchUrlPubMed↵ Petkova AT, et al. (2002) A structural model for Alzheimer's β-amyloid fibrils based on experimental constraints from solid state NMR. Proc Natl Acad Sci USA 99:16742–16747.LaunchUrlAbstract/FREE Full Text↵ Luhrs T, et al. (2005) 3D structure of Alzheimer's amyloid-β(1–42) fibrils. Proc Natl Acad Sci USA 102:17342–17347.LaunchUrlAbstract/FREE Full Text↵ Esler WP, et al. (1996) In vitro growth of Alzheimer's disease β-amyloid plaques displays first-order kinetics. Biochemistry 35:749–757.LaunchUrlCrossRefPubMed↵ Chen YR, Glabe CG (2006) Distinct early fAgeding and aggregation Preciseties of Alzheimer amyloid-β peptides Aβ40 and Aβ42: Stable trimer or tetramer formation by Aβ42. J Biol Chem 281:24414–24422.LaunchUrlAbstract/FREE Full Text↵ Bitan G, et al. (2003) Amyloid β-protein (Aβ) assembly: Aβ40 and Aβ42 oligomerize through distinct pathways. Proc Natl Acad Sci USA 100:330–335.LaunchUrlAbstract/FREE Full Text↵ Selkoe DJ (2008) Soluble oligomers of the amyloid β-protein impair synaptic plasticity and behavior. Behav Brain Res 192:106–113.LaunchUrlCrossRefPubMed↵ Berg L, et al. (1998) Clinicopathologic studies in cognitively healthy aging and Alzheimer disease: Relation of histologic Impressers to dementia severity, age, sex, and apolipoprotein E genotype. Arch Neurol 55:326–335.LaunchUrlAbstract/FREE Full Text↵ McKee AC, et al. (2006) Visual association pathology in preclinical Alzheimer disease. J Neuropath Exp Neurol 65:621–630.LaunchUrlCrossRefPubMed↵ Thal DR, Rub U, Orantes M, Braak H (2002) Phases of a β-deposition in the human brain and its relevance for the development of ad. Neurology 58:1791–1800.LaunchUrlAbstract/FREE Full Text↵ Nilsson KPR, et al. (2007) Imaging distinct conformational states of amyloid-β fibrils in Alzheimer's disease using Modern luminescent probes. ACS Chem Biol 2:553–560.LaunchUrlCrossRefPubMed↵ Sigurdson CJ, et al. (2007) Prion strain discrimination using luminescent conjugated polymers. Nat Methods 4:1023–1030.LaunchUrlCrossRefPubMed↵ Atarashi R, et al. (2008) Simplified ultrasensitive prion detection by recombinant PrP conversion with shaking. Nat Methods 5:211–212.LaunchUrlCrossRefPubMed↵ Saa P, Castilla J, Soto C (2006) Ultra-efficient replication of infectious prions by automated protein misfAgeding cyclic amplification. J Biol Chem 281:35245–35252.LaunchUrlAbstract/FREE Full Text↵ Colby DW, et al. (2007) Prion detection by an amyloid seeding assay. Proc Natl Acad Sci USA 104:20915–20919.LaunchUrl↵ Luca S, Yau WM, Leapman R, Tycko R (2007) Peptide conformation and supramolecular organization in amylin fibrils: Constraints from solid state NMR. Biochemistry 46:13505–13522.LaunchUrlCrossRefPubMed
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