Molecular Structure of b-Amyloid Fibrilsin Alzheimer’s Disease Brain TissueJun-Xia Lu,1 Wei Qiang,1 Wai-Ming Yau,1 Charles D. Schwieters,2 Stephen C. Meredith,3 and Robert Tycko1,*1Laboratory of Chemical Physics, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda,

MD 20892-0520, USA2Division of Computational Bioscience, Center for Information Technology, National Institutes of Health, Bethesda, MD 20892-5624, USA3Department of Pathology and Department of Biochemistry and Molecular Biology, The University of Chicago, Chicago, IL 60637, USA*Correspondence: robertty@mail.nih.gov

http://dx.doi.org/10.1016/j.cell.2013.08.035

SUMMARY

In vitro, b-amyloid (Ab) peptides form polymorphicfibrils, with molecular structures that depend ongrowth conditions, plus various oligomeric and pro-tofibrillar aggregates. Here, we investigate structuresof human brain-derived Ab fibrils, using seeded fibrilgrowth from brain extract and data from solid-statenuclear magnetic resonance and electron micro-scopy. Experiments on tissue from two Alzheimer’sdisease (AD) patients with distinct clinical historiesshowed a single predominant 40 residue Ab (Ab40)fibril structure in each patient; however, the struc-tures were different from one another. A molecularstructural model developed for Ab40 fibrils fromone patient reveals features that distinguish in-vivo-from in-vitro-produced fibrils. The data suggestthat fibrils in the brain may spread from a singlenucleation site, that structural variations maycorrelate with variations in AD, and that structure-specific amyloid imaging agents may be an impor-tant future goal.

INTRODUCTION

Aggregation of b-amyloid (Ab) peptides in brain tissue is the likely

cause of Alzheimer’s disease (AD) (Tanzi and Bertram, 2005;

Yankner and Lu, 2009). The most abundant Ab aggregates in

AD brain tissue are amyloid fibrils, the end point of the aggrega-

tion process, which are contained in neuritic plaques, diffuse

amyloid, and vascular amyloid. A variety of other aggregates

have also been identified, including Ab protofibrils and soluble

oligomers of various sizes. In vitro structural studies, using

solid-state nuclear magnetic resonance (NMR) (Benzinger

et al., 1998; Bertini et al., 2011; Lansbury et al., 1995; Paravastu

et al., 2008; Petkova et al., 2005, 2006; Qiang et al., 2011), elec-

tron microscopy (Goldsbury et al., 2005; Meinhardt et al., 2009;

Zhang et al., 2009), and other techniques (Kheterpal et al., 2001;

Kodali et al., 2010; Luhrs et al., 2005; Olofsson et al., 2007; Torok

et al., 2002), show that Ab fibrils are highly polymorphic, with

molecular structures that depend on aggregation conditions.

C

Detailed structural models for fibrils formed in vitro have been

developed from experimental data, showing that fibril poly-

morphs can differ in specific aspects of peptide conformation

and interresidue interactions, as well as overall structural sym-

metry (Bertini et al., 2011; Luhrs et al., 2005; Paravastu et al.,

2008; Petkova et al., 2006). Studies of certain nonfibrillar (Ahmed

et al., 2010; Chimon et al., 2007; Lopez del Amo et al., 2012) and

protofibrillar (Qiang et al., 2012; Scheidt et al., 2011) Ab aggre-

gates, also formed in vitro, provide evidence for peptide confor-

mations similar to those in fibrils, but with reduced structural

order and different supramolecular organizations.

Molecular structures of Ab aggregates that develop in human

brain tissue have not been characterized in detail but evidence

exists that structural variations may be biomedically important:

(1) Structurally distinct fibrils can have different levels of toxicity

in neuronal cell cultures (Petkova et al., 2005); (2) Propagation of

exogenous Ab amyloid within transgenic mouse brains depends

on the source of the exogenous Ab-containing material (Langer

et al., 2011; Meyer-Luehmann et al., 2006; Stohr et al., 2012);

(3) The binding stoichiometry of amyloid imaging agents, devel-

oped for positron emission tomography (PET), can differ sub-

stantially between Ab fibrils formed in vitro and fibrils in AD brain

tissue (Mathis et al., 2003). A precedent for the importance of

molecular structural variations in a neurodegenerative disease

is provided by transmissible spongiform encephalopathies

(TSEs), in which variations in the molecular structures of

mammalian prion protein (PrP) aggregates within brain tissue

produce distinct, self-propagating TSE ‘‘strains’’ (Bessen and

Marsh, 1994; Safar et al., 1998; Stohr et al., 2012). Analogous

variations in the molecular structures of yeast prion fibrils pro-

duce prion strains in yeast (Toyama and Weissman, 2011).

Solid-state NMR has been an especially powerful structural

probe of Ab aggregates formed in vitro (Ahmed et al., 2010; Ben-

zinger et al., 1998; Bertini et al., 2011; Chimon et al., 2007; Lans-

bury et al., 1995; Lopez del Amo et al., 2012; Paravastu et al.,

2008; Petkova et al., 2005, 2006; Qiang et al., 2012, 2011;

Scheidt et al., 2011). Since solid-state NMR measurements

require milligram-scale quantities of 15N- and 13C-labeled fibrils,

direct measurements on Ab aggregates from brain tissue are not

possible. However, in vitro studies show that Ab fibrils grown

from seeds (i.e., short fibril fragments, produced by sonication

of existing fibrils) retain the molecular structures of the seeds

(Kodali et al., 2010; Paravastu et al., 2008; Petkova et al.,

ell 154, 1257–1268, September 12, 2013 ª2013 Elsevier Inc. 1257

2005). Thus, if amyloid-containing material is extracted from

brain tissue and used as the source of seeds, seeded fibril

growth can be used to amplify and label structures that develop

in the human brain (Paravastu et al., 2009).

In this paper, we describe studies of fibril structures from brain

tissue of two AD patients (patients I and II) with distinct clinical

histories and neuropathology, using simplified amyloid extrac-

tion and seeding protocols. Solid-state NMR and electronmicro-

scopymeasurements on 40 residue Ab (Ab40) fibrils seeded with

extract from two brain regions of patient I and three brain regions

of patient II indicate that each patient developed a single

predominant fibril structure. However, the predominant struc-

tures from the two patients are clearly different, as indicated

by both NMR chemical shifts and fibril morphologies in electron

microscopy. We develop a full molecular structural model for

Ab40 fibrils from patient I, which represents the first detailed,

experimentally determined structure of any brain-derived Ab

aggregate.

Given that Ab aggregation is inherently polymorphic, the struc-

tural specificity of brain-seeded Ab40 fibrils indicated by our data

is remarkable. Possible implications for processes by which

fibrils develop within the human brain, for the role of Ab fibrils

and the significance of their structures in AD, and for future

developments of structure-specific imaging agents and aggre-

gation inhibitors are discussed below.

RESULTS

A Single Predominant Ab40 Fibril Structure fromPatient IPatient I, a female who died at age 72, had tentative clinical diag-

noses of Lewy body dementia (LBD) and primary progressive

aphasia. Autopsy revealed neuritic Ab plaques, diffuse amyloid,

neurofibrillary tangles, and mild atrophy of frontal and parietal

lobes indicative of AD, but few Lewy bodies. Starting with 1 g

of tissue that contains abundant plaques (Figure 1A), our amyloid

extraction protocol (see Extended Experimental Procedures)

produced approximately 10 mg of amyloid-enriched material.

Fibril fragments in this material were visible by transmission elec-

tron microscopy (TEM) (Figure 1B). After addition of 0.8 mg of

synthetic 15N,13C-labeled Ab40, long fibrils developed within

several hours (Figures 1C and 1D). The fibrils have constant

apparent diameters of 7 ± 1 nm, do not self-associate into thicker

bundles, and appear morphologically homogeneous. Fibrils

seeded with extract from occipital lobe tissue (Figures 1C and

1D) are indistinguishable from fibrils seeded with extract from

temporal/parietal lobe tissue (Figure 1E). Brain-seeded fibrils

from patient I are morphologically distinct from ‘‘striated ribbon’’

and ‘‘twisted’’ Ab40 fibrils grown in vitro (without brain material)

in our laboratory (Paravastu et al., 2008), as well as in vitro fibrils

described by others (Goldsbury et al., 2005; Kodali et al., 2010;

Meinhardt et al., 2009).

Control experiments, detected by thioflavin T fluorescence

and by TEM, showed rapid fibril growth when brain tissue from

AD patients was used, but not when identical experiments

were performed with non-AD tissue (Figure S1 available online).

Thus, seeding of fibril growth is attributable to fibrils in the AD

brain tissue, not to other components of brain tissue that survive

1258 Cell 154, 1257–1268, September 12, 2013 ª2013 Elsevier Inc.

the extraction protocol. Compared with protocols described

previously (Paravastu et al., 2009), our current protocols involve

fewer extraction steps, preserve more components of the brain

tissue, and permit NMR samples to be created in a single seed-

ing step from smaller quantities of tissue.

Two-dimensional (2D) solid-state NMR spectra of Ab40 fibrils

seeded with extract of occipital lobe tissue (Figures 1F and 1G)

show remarkably sharp cross-peak signals, with a single set of15N and 13C NMR chemical shifts for each 15N,13C-labeled resi-

due (0.7–1.2 ppm and 1.3–1.8 ppm FWHM linewidths for 13C and15N signals, respectively). Because chemical shifts are exqui-

sitely sensitive tomolecular structural variations, the observation

of a single set of shifts implies a single predominant fibril struc-

ture, consistent with the morphological homogeneity in TEM

images. Two-dimensional spectra of fibrils seeded with extract

of temporal/parietal lobe tissue (Figures 1H and 1I; Figure S2F)

are nearly identical, apart from differences in signal-to-noise ra-

tio due to differences in sample sizes. Chemical shifts in spectra

of these brain-seeded fibrils are significantly different from shifts

reported previously for fibrils grown in vitro (Bertini et al., 2011;

Paravastu et al., 2008; Petkova et al., 2005). The high signal-

to-noise ratio in Figure 1F allows us to place an upper limit of

10% on the mass of minor structural components relative to

the predominant fibril structure in the occipital-seeded sample.

Different Ab40 Fibril Structures from Brain Tissue ofPatients I and IIPatient II, a female who died at age 80, had a clinical diagnosis of

probable AD. Autopsy revealed severe AD, including gross atro-

phy of the brain, loss of neurons, gliosis in the hippocampus and

cortex, neurons with granulovacuolar degeneration and Hirano

bodies, neuritic plaques, and neurofibrillary tangles. Brain-

seeded Ab40 fibrils prepared from occipital lobe (Figures 2A

and 2B) and frontal lobe (Figure 2C) tissue of patient II show an

apparent periodic twist in TEM images (minima and maxima in

apparent width approximately equal to 11 nm and 5 nm; 85 ±

10 nm distance between minima) that is absent from images of

fibrils from patient I (Figures 1C–1E). Solid-state NMR spectra

again show a single set of chemical shifts, indicating a single pre-

dominant fibril structure that is the same in samples seeded with

occipital lobe, frontal lobe, or temporal lobe tissue (Figures 2D–

2G, S2G, and S2H) However, chemical shifts for brain-seeded

fibrils from patients I and II are obviously different (Figures 2D

and 2E). Signals with the chemical shifts of fibrils from patient I

are undetectable in spectra of fibrils from patient II, and vice

versa. These data indicate that patients I and II developed struc-

turally distinct fibrils in their brains.

From the 2D spectrum in Figure 2D, the mass of any minor

structural components must be less than 10% relative to the pre-

dominant fibril structure. Although the twisted morphology in

TEM images of fibrils derived from brain tissue of patient II is

similar to the morphology of certain Ab40 fibrils grown in vitro

(Paravastu et al., 2008), the NMR chemical shifts are significantly

different (Figure S6).

Structural Model for Ab40 Fibrils from Patient IThe high quality of 2D solid-state NMR spectra of Ab40 fibrils

derived from brain tissue of patient I encouraged us to pursue

A B

C D E

F H

G I

Figure 1. Evidence for a Single Predominant

Ab40 Fibril Structure in Brain Tissue of

Patient I

(A) Light microscope image of cortical tissue from

patient I, with immunohistochemical staining for

b-amyloid using monoclonal antibody 6E10.

Amyloid deposits are red or pink.

(B) Negatively stained TEM image of extract from

occipital lobe tissue before addition of monomeric

Ab40. Fibril fragments are circled in red.

(C and D) TEM images of isotopically labeled Ab40

fibrils, recorded 24 hr after addition of monomeric

Ab40 to sonicated extract from occipital lobe tis-

sue. Ab40 was uniformly 15N,13C-labeled at F19,

V24, G25, A30, I31, L34, and M35.

(E) TEM image of fibrils prepared by seeding with

sonicated extract from temporal/parietal lobe

tissue.

(F and G) 2D 13C-13C and 15N-13C solid-state NMR

spectra of Ab40 fibrils, seeded with extract from

occipital lobe tissue. Black lines and labels show

site-specific cross-peak assignments.

(H and I) 2D 13C-13C and 15N-13C solid-state NMR

spectra of Ab40 fibrils, seeded with extract from

temporal/parietal lobe tissue, with the same black

lines as in (F) and (H).

See also Figures S1 and S2.

a full molecular structure determination. We prepared fibril sam-

ples with a variety of isotopic labeling patterns (Table 1). A uni-

formly 15N,13C-labeled sample (sample A) was generated by

seeding recombinant Ab40 with extract from occipital lobe tis-

sue. Samples with other labeling patterns (samples C–G) were

generated by seeding synthetic Ab40 with aliquots of samples

whose spectra appear in Figure 1 (sample B). NMR chemical

Cell 154, 1257–1268, Sep

shifts are consistent in 2D spectra of all

samples (Figures 3 and S2), indicating

that all samples contained the same fibril

structure.

From 2D and 3D NMR spectra, we

obtained unique assignments of all 15N

and 13C chemical shifts (Table S1). As

shown in Figure 3, all residues in the

Ab40 sequence contribute strong and

sharp signals to the solid-state NMR

spectra, except H14 and E22. Because

dynamically disordered segments of a

protein assembly are generally absent

from such solid-state NMR spectra

and statically disordered segments yield

broad signals, it appears that the

entire Ab40 sequence participates in the

ordered and relatively rigid molecular

structure.

We obtained structural restraints from a

combination of solid-state NMR and elec-

tron microscopy measurements. Struc-

tural restraints from solid-state NMR,

summarized in Table S2, include: (1)

conformation-dependent 13Cand 15Nchemical shifts (Figure 4A),

from which backbone torsion angle predictions (Table S1) were

derived using the TALOS+ program (Shen et al., 2009); (2) inter-

molecular 13C-13C magnetic dipole-dipole couplings, measured

with the PITHIRDS-CT technique (Tycko, 2007), which imply an

in-register parallel b sheet structure (Figure 4B); (3) 15N-13C

dipole-dipole couplings between K28 Nεand D23 Cg sites,

tember 12, 2013 ª2013 Elsevier Inc. 1259

A B C

D F

E G

Figure 2. Evidence for Different Ab40 Fibril

Structures in Brain Tissue of Patients I and II

(A and B) TEM images of Ab40 fibrils, recorded

36 hr after addition of monomeric Ab40 to soni-

cated extract from occipital lobe tissue of

patient II. Blue arrows indicate minima in the

apparent fibril diameter, associated with a periodic

twist that is absent from fibril images in Figure 1.

Ab40 was uniformly 15N,13C-labeled at F19, V24,

G25, S26, A30, I31, L34, and M35.

(C) TEM image of fibrils prepared by seeding with

sonicated extract from frontal lobe tissue of

patient II.

(D and E) 2D 13C-13C and 15N-13C solid-state NMR

spectra of Ab40 fibrils, seeded with extract from

occipital lobe tissue of patient II (blue contours) or

patient I (red contours). Black lines and labels

show site-specific cross-peak assignments for

spectra from patient II.

(F and G) 2D 13C-13C and 15N-13C solid-state NMR

spectra of Ab40 fibrils, seeded with extract from

frontal lobe tissue of patient II, with the same black

lines as in (D) and (E).

See also Figures S2 and S6.

measured with the fsREDOR technique (Jaroniec et al., 2001),

which indicate a 0.35 ± 0.02 nm distance, consistent with a

D23-K28 salt bridge (Figure 4C). (4) intramolecular dipole-dipole

couplings among backbone amide 15N nuclei and among

backbone carbonyl 13C nuclei, measured with the 15N-BARE

and 13C-BARE techniques (Hu et al., 2012), which serve as

site-specific restraints on backbone conformation (Figures 4D

and S3); (5) interresidue distance restraints from 2D RAD

(Morcombe et al., 2004; Takegoshi et al., 2001), PAR (De Paepe

et al., 2008), band-selective fpRFDR (Bayro et al., 2009; Ishii,

2001), and band-selective TEDOR (Jaroniec et al., 2002) spectra

(Figure S4 and Table S3).

Dark-field TEM images of unstained samples (Figure 4E)

allow quantification of the mass-per-length (MPL) of individual

fibrils (Chen et al., 2009; Goldsbury et al., 2005). Given that

1260 Cell 154, 1257–1268, September 12, 2013 ª2013 Elsevier Inc.

amyloid fibrils contain cross-b structural

motifs with a 0.47–0.48 nm intermolec-

ular distance along the fibril axis

(supported by Figure 4B) and that the

molecular weight of Ab40 is 4.3 kDa, a

single cross-b unit would have MPL

�9.0 kDa/nm. The observed MPL value

of 28 ± 2 kDa/nm (Figure 4F), together

with the observation of a single set of

NMR chemical shifts for all residues,

implies a molecular structure comprised

of three cross-b units, with 3-fold sym-

metry about the long-fibril axis.

Structure calculations were performed

with the Xplor-NIH program (Schwieters

et al., 2006), starting from nine well-

separated Ab40 molecules with random

conformations. In addition to distance

and conformational restraints from the

NMR data, rotational and translational symmetry restraints

were applied, as dictated by the MPL data, the solid-state

NMR spectra, and the fibrillar nature of the Ab40 assemblies.

Low-energy final structures consist of three copies of a

3-fold-symmetric repeat unit, with translational symmetry along

the fibril axis.

Figure 5A shows the repeat unit from the final structure with

the lowest total restraint energy. Repeat units from 20 low-

energy structures (PDB code 2M4J) are superimposed in

Figure 5B. This bundle of structures represents the full range of

final structures that are fully consistent with the experimental

data, defined by the absence of violations of backbone torsion

angle predictions exceeding 7�, the absence of violations of dis-

tance restraints exceeding 0.06 nm, and good agreement with

the 15N- and 13C-BARE data (Figure S3). Structure calculation

Table 1. Summary of Isotopic Labeling Patterns and Solid-State NMR Measurements on Ab40 Fibrils Derived from Patient I

Sample Uniformly 15N,13C-Labeled Residues Selectively 13C-Labeled Sites NMR Measurementsa

A residues 1–40 2D fpRFDR, NCAX, NCOCX, RAD, PAR, and TEDOR;

3D NCACX and NCOCX; 15N- and 13C-BARE

B F19, V24, G25, A30, I31, L34, M35 2D fpRFDR, RAD, NCACX

C F20, D23, V24, K28, G29, A30, I31 2D fpRFDR, RAD, NCACX; fsREDOR

D H14, Q15, A21, V36, G37 2D fpRFDR, RAD, NCACX; 15N- and 13C-BARE

E K16, F19, A21, E22, I32, V36 2D fpRFDR, RAD, NCACX; 15N- and 13C-BARE

F A2 and A21 methyl; V12, F20, I31,

G38 carbonyl

PITHIRDS-CT

G A30 methyl, V18 carbonyl PITHIRDS-CTaThe following abbreviations are used: fpRFDR, finite-pulse radio-frequency-driven recoupling; NCACX, 15N-13Ca-

13Cx chemical shift correlation;

NCOCX, 15N-13CO-13Cx chemical shift correlation; RAD, radio-frequency-assisted diffusion; PAR, proton-assisted recoupling; TEDOR, transferred

echo double resonance; BARE, backbone recoupling; fsREDOR, frequency-selective rotational echo double resonance; PITHIRDS-CT, constant-

time p-pulse recoupling.

statistics appear in Table S4. Figures 5C and 5D show an ideal-

ized representation of the full fibril structure.

Attempts to calculate structures with 2-fold rotational symme-

try, using the same set of experimental restraints and the same

Xplor-NIH protocols, produced no final structures that were fully

consistent with the experimental data.

Key distance restraints for the bundle of structures in Figure 5B

include intramolecular V24-K28, K28-I31, E11-V39, M35-G38,

M35-V39, M35-V40, F19-L34, and H13-V39 distances, as well

as intermolecular F4-V24, R5-V24, D7-S26, S8-V24, A30-V40,

and I32-V39 distances (see Figure S4 and Table S3). I31-V39,

H13-V40, L17-I32, and F19-I32 contacts, observed in 3-fold-

symmetric fibrils formed in vitro (Paravastu et al., 2008), are

not present in Ab40 fibrils from patient I.

Further support for the structure in Figure 5 comes from two

additional experiments. First, we used 2D 15N-13C solid-state

NMR spectra to monitor site-specific hydrogen/deuterium

(H/D) exchange rates during exposure of sample A to D2O buffer

(Figure 6). Only backbone amide sites of A2, F4, D7, S8, G9,

G25, and S26, which are colocalized in the structure, exhibited

significant H/D exchange over an 18 day period. These results

suggest that the N-terminal segment undergoes transient

unfolding events that also expose G25 and S26. (Continual

large-amplitude motions are ruled out by NMR signal strengths

and dipole-dipole couplings in Figures 3, 4B, and S3.) Second,

we measured site-specific enhancements of 15N spin-lattice

relaxation rates induced by paramagnetic CuNa2-EDTA (Fig-

ure S5). The smallest relaxation enhancements were observed

for residues 30–40, consistent with their burial in the fibril core

and with a narrow central pore that excludes the Cu-EDTA

complex.

Comparison with In Vitro Fibril StructuresIn-register parallel intermolecular alignment (Figures 5C and 5D)

also occurs in full-length Ab fibrils formed in vitro without brain

material (Benzinger et al., 1998; Luhrs et al., 2005; Paravastu

et al., 2008; Petkova et al., 2005; Torok et al., 2002), although

metastable intermediates (Qiang et al., 2012) and fibrils formed

by certain Ab fragments (Colletier et al., 2011; Lansbury et al.,

1995) can contain antiparallel cross-bmotifs. Three-fold symme-

C

try occurs in certain in vitro Ab40 fibrils with ‘‘twisted’’ morphol-

ogies (Goldsbury et al., 2005; Paravastu et al., 2008). (Figure 5E),

whereas other in vitro Ab40 and Ab42 fibrils exhibit 2-fold sym-

metry (Bertini et al., 2011; Meinhardt et al., 2009; Petkova

et al., 2006; Zhang et al., 2009) (Figure 5F). D23-K28 salt bridge

interactions have been observed in 2-fold-symmetric (Petkova

et al., 2006), but not 3-fold-symmetric (Paravastu et al., 2008),

Ab40 fibrils formed in vitro. Residues 12–19 form a b strand in

all known Ab40 structures, with sidechains of even-numbered

residues exposed on the exterior surface, whereas residues

30–40 are buried in the fibril core. Close contacts between F19

and L34 sidechains occur in both in vitro Ab40 (Bertini et al.,

2011; Paravastu et al., 2008; Petkova et al., 2006) and in vitro

Ab42 (Ahmed et al., 2010) fibrils.

Novel conformational features in Ab40 fibrils frompatient I may

provide a basis for the development of structure-specific inhibi-

tors or imaging agents. These features include a twist in residues

19–23 that allows sidechains of either F20 or E22 to be buried

within the structure, a kink at G33 that allows sidechains of I32

and L34 to point in opposite directions and make contacts with

different sets of Ab40 molecules, and a bend in glycine residues

37 and 38. In contrast, fibrils formed in vitro by Ab40 and Ab42

contain relatively simple strand-bend-strand conformations, as

in Figures 5E and 5F. A variety of experiments indicate N-termi-

nal disorder in in vitro Ab40 and Ab42 fibrils (Kheterpal et al.,

2001; Luhrs et al., 2005; Paravastu et al., 2008; Petkova et al.,

2005). However, Ab40 fibrils from patient I exhibit strong, sharp

NMR signals (Figure 3) and strong 13C-13C and 15N-15N dipole-

dipole couplings for N-terminal residues (Figures 4B and S3),

indicating structural order.

MPL data for Ab40 fibrils from patient II also indicate 3-fold

symmetry (Figure 4H). F19-L34 sidechain contacts, which are

clear in spectra of samples A and B from patient I (Figure S4C),

are not detected in fibrils from patient II. From chemical

shifts (Figure S6), TALOS+ predicts a continuous b strand in

residues 28–32 in fibrils from patient II, whereas structures in

Figure 5B have a non-b-strand conformation at G29. Thus,

fibrils from patients I and II differ in both peptide backbone

conformation and interresidue interactions, but not overall

symmetry.

ell 154, 1257–1268, September 12, 2013 ª2013 Elsevier Inc. 1261

A B

C

Figure 3. The Entire Ab40 Sequence Is Structurally Ordered in Fibrils from Patient I

(A and B) 2D solid-state 15N-13C NMR spectra of uniformly 15N,13C-labeled Ab40 fibrils. (A) A NCACX spectrum, correlating backbone 15N chemical shifts of a

given residue with 13C chemical shifts of the same residue. (B) A NCOCX spectrum, correlating backbone 15N chemical shifts of a given residue with 13C chemical

shifts of the previous residue. Only the aliphatic 13C regions are shown. Asterisks (B) denote intraresidue cross-peaks and cross-peaks involving sidechain 15N

signals.

(C) 2D 13C-13C spectrum, obtained with 50 ms RAD mixing (left) and 2.94 ms fpRFDR mixing (right). Black labels indicate CO/Ca (left) and Ca/Cb (right) cross-

peaks. Blue labels indicate additional intraresidue cross-peaks, including multiple-bond cross-peaks. Site-specific cross-peak assignments in these 2D spectra

show that residues 1–40 contribute relatively strong, sharp signals, implying structural order.

See also Table S1.

DISCUSSION

Significance of Structural ObservationsPolymorphism is an inherent property of Ab fibril formation

(Colletier et al., 2011; Goldsbury et al., 2005; Kodali et al.,

2010; Meinhardt et al., 2009; Paravastu et al., 2008; Petkova

et al., 2005; Qiang et al., 2011), attributable to the coexistence

of multiple nucleation processes (each leading to a different fibril

structure), the comparably high thermodynamic stabilities of

distinct polymorphs, and the low rates of dissociation of peptide

monomers or soluble species from fibrils (Qiang et al., 2013). It is

therefore surprising that Ab40 fibrils derived from brain tissue of

either patient I or patient II are not polymorphic. There are at least

three possible explanations. (1) The brain tissue environment

1262 Cell 154, 1257–1268, September 12, 2013 ª2013 Elsevier Inc.

permits only one nucleation process. The observation of distinct

fibril structures from patients I and II contradicts this explanation

unless unknown differences in their brain tissue selected

different nucleation processes. (2) Multiple fibril structures are

nucleated, and all but one are eliminated by amyloid clearance

mechanisms. Unknown differences in clearance mechanisms

between patients I and II would be required to account for our

data. (3) The majority of fibrils that persist to the time of death

arise from nucleation of one structure at a single site. This

structure then spreads by physical or biologically-mediated

processes of fragmentation and transport, possibly involving

activated microglia (Majumdar et al., 2008). After transport to a

new site, fibril fragments serve as seeds for the growth of struc-

turally identical fibrils (Langer et al., 2011), as in the experiments

A B

C D

E F

G H

Figure 4. Structural Restraints on Ab40

Fibrils from Patient I

(A) Residue-specific secondary 13CNMR chemical

shifts. Nonglycine residues with positive values for

Cb and negative values for CO and Ca are likely to

have b strand conformations. Open symbols indi-

cate glycine residues.

(B) Intermolecular 13C-13C dipole-dipole couplings

among 13C-labeled methyl sites of A2 or A21 or13C-labeled carbonyl sites of V12, F20, I31, or G38.

Comparison with simulations for linear chains of13C nuclei with various spacings indicates inter-

molecular distances of approximately 0.5 nm for

all labeled sites, implying in-register parallel

b sheets.

(C) 15N-13C dipole-dipole couplings between D23

Cg and K28 Nz sites indicate a 0.35 ± 0.02 nm

interatomic distance, implying salt-bridge in-

teractions between oppositely charged D23 and

K28 sidechains.

(D) 15N-15N dipole-dipole couplings among back-

bone amide nitrogens. Stronger couplings for F20

and A21 indicate non-b strand conformations.

(E) Dark-field TEM image of unstained fibrils.

Mass-per-length (MPL) values are obtained from

integrated intensities within 300 nm 3 50 nm

rectangles, centered on TMV (cyan) or Ab40

(yellow) fibril segments, after subtraction of the

average background intensity from similar rect-

angles on either side (dotted lines). TMV particles

serve as in situ MPL standards.

(F) MPL histogram, with light blue dashed lines

indicating MPL values expected for structures

comprised of 1–5 cross-b units. Dark blue line is a

best-fit Gaussian function, centered at 28.4 ±

0.3 kDa/nm, and with a full-width-at-half-

maximum (FWHM) equal to 12.0 ± 0.8 kDa/nm.

(G) Histogram of errors in MPL values due to noise

in the dark-field images. The best-fit Gaussian

function has FWHM equal to 12.8 ± 0.6 kDa/nm,

showing that random noise accounts for the

width in (F).

(H) MPL histogram for Ab40 fibrils seeded with

extract from occipital lobe tissue of patient II,

with best-fit Gaussian function centered at

29.1 ± 1.2 kDa/nm and FWHM equal to 12.1 ±

0.8 kDa/nm.

Error bars in (B), (C), and (D) are calculated from

the root-mean-squared (rms) noise in the corre-

sponding NMR spectra. See also Figures S3 and

Table S2.

described above. In this scenario, differences between patients I

and II may be due entirely to the stochastic nature of the initial

nucleation event.

Patients I and II differed in clinical history (initial diagnoses of

LBD versus AD) and neuropathology (mild versus severe cortical

atrophy), although both patients developed Ab plaques and

neurofibrillary tangles characteristic of AD. The observation of

distinct Ab40 fibril structures from the two patients, both in

TEM images and in solid-state NMR spectra, suggests that

differences in fibril structure may correlate with differences in

C

disease development. The analogy with TSE strains, in which

self-propagating polymorphisms of PrP aggregates lead to

distinct anatomical patterns of PrP deposition, disease incuba-

tion periods, and symptoms (Bessen and Marsh, 1994; Collinge,

2001; Safar et al., 1998), is obvious. Prior suggestive evidence for

AD strains includes the observation of polymorph-specific differ-

ences in Ab40 fibril toxicity in neuronal cell cultures (Petkova

et al., 2005) and the observation that propagation efficiencies

of exogenous Ab42 fibrils in transgenic mice depend on the

source of the exogeneous material (Meyer-Luehmann et al.,

ell 154, 1257–1268, September 12, 2013 ª2013 Elsevier Inc. 1263

Figure 5. Molecular Structure of Ab40

Fibrils from Patient I

(A) Structure with the lowest total experimental

restraint energy in Xplor-NIH calculations. The

3-fold-symmetric repeat unit is shown, as viewed

along the fibril growth axis. Backbone and

sidechain carbon atoms are gray and green,

respectively.

(B) Superposition of 20 structures that are

consistent with experimental restraints (PDB code

2M4J). Sidechains of the three Ab40 molecules in

the repeat unit are yellow, green, or orange.

(C and D) Two views of the idealized fibril struc-

ture, created by repeating the trimeric unit 18

times with 0.48 nm displacements along the

fibril axis.

(E and F) Structural models for Ab40 fibril poly-

morphs with 3-fold and 2-fold symmetry about the

fibril growth axis, developed previously from solid-

state NMR and electron microscopy measure-

ments on fibrils grown in vitro. Repeat units from

thesemodels are shown, calculated as regularized

averages of structure bundles in PDB codes 2LMP

and 2LMN, respectively.

See also Figures S4 and S6 and Tables S3 and S4.

2006; Stohr et al., 2012). Further experiments are certainly

required before AD strains can be considered a reality.

Role of Fibrils in ADBoth the identity of Ab aggregates that contribute most to neuro-

degeneration in AD and their pathogenic mechanisms are

currently uncertain (Yankner and Lu, 2009). Various experiments

point to nonfibrillar Ab oligomers as neurotoxic species (Krafft

and Klein, 2010; Lesne et al., 2006; Noguchi et al., 2009; Selkoe,

2008). Ongoing fibril growth or conversion of oligomers to fibrils

may be required for neurotoxicity (Jan et al., 2011). Although

mature amyloid fibrils are sometimes described as being

nontoxic in cell cultures, experiments by us (Petkova et al.,

2005; Qiang et al., 2012) and others (Chimon et al., 2007; Walsh

et al., 1999) show that Ab40 fibrils are indeed toxic in cell

cultures. In brain tissue, Ab fibrils may initiate inflammation

(Cameron and Landreth, 2010; Glass et al., 2010), oxidative

damage (Sultana et al., 2009; Tougu et al., 2011), or other path-

ogenic processes.

1264 Cell 154, 1257–1268, September 12, 2013 ª2013 Elsevier Inc.

The severity of cognitive impairment in

AD patients prior to death has been re-

ported not to correlate with plaque den-

sity (Giannakopoulos et al., 1997). At least

one study reached the opposite conclu-

sion (Cummings et al., 1996); the quantity

of brain amyloid has been found to corre-

late with the likelihood of progression

from mild cognitive impairment to AD

(Villemagne et al., 2011). Although

asymptomatic elderly people develop

amyloid plaques, the quantity of amyloid

is generally less than in AD patients

(Aizenstein et al., 2008). Fibrillar oligo-

mers (which may be fibril fragments), but not nonfibrillar oligo-

mers, have been reported to be elevated in AD patients (Tomic

et al., 2009).

Thus, Ab fibrils (along with oligomeric and protofibrillar

species) remain as likely causative or contributing agents in

AD. Our finding of specific fibril structures in AD patients sug-

gests that certain structures may be more pathogenic than

others. The weakness of correlations between total amyloid

deposition and cognitive deficiency in AD patients may be due

in part to differential effects of amyloid deposits comprised of

different fibril structures. Similarly, amyloid deposits in brain

tissue of asymptomatic elderly people may contain relatively

inert structures. It is also conceivable that specific fibril struc-

tures coexist with specific oligomer structures, for example if

the oligomers are ‘‘on-pathway’’ intermediates to fibril formation,

or if the oligomers are produced by degradation of fibrils. Asso-

ciation of specific Ab fibrils with AD may then arise indirectly,

through their association with specific oligomers. Structural

data for fibrils may then represent a window into the properties

60 55 50 45

130

125

120

115

110

105

60 55 50 45 60 55 50 45 60 55 50 45 60 55 50 45

S26S8

G9

G25

A2

D7F4

13C NMR frequency (ppm)

15)

mpp(ycneuqerf

RM

NN

60 50 40 30 20

130

125

120

115

110

105

130

125

120

115

110

105

130

125

120

115

110

105S26

S8 S8

G25

G9

A2

A2

D7 D7F4

15)

mpp(ycneuqerf

RM

NN

13C NMR frequency (ppm)

before H/D exchange 0-12 hr 60-70 hr 426-437 hr after back-exchange

20-60 hr H/D exchange

372-420 hr H/D exchange

after back-exchange

A

B

C

Figure 6. H/D Exchange Measurements on Ab40 Fibrils Derived from Occipital Lobe Tissue of Patient I, Detected through 2D 15N-13C Solid-

State NMR Spectra(A) 2D NCA spectra of uniformly 15N,13C-labeled fibrils, before H/D exchange, after various periods of exchange, and after back-exchange. In all spectra, the

highest contour level is normalized to the strongest cross-peak, and 16 contour levels are shown, decreasing by successive factors of 1.2. In the spectrum at

426–437 hr, arrows indicate positions of resolved or partially resolved cross-peaks that show clear reductions in intensity due to H/D exchange.

(B) 2D NCACX spectra after various periods of exchange and after back-exchange, with contour levels as in (A).

(C) Structure from Figure 5A with residues that exhibit significant H/D exchange colored magenta.

See also Figure S5.

of oligomers, whose molecular structures are particularly chal-

lenging to characterize directly. Further experiments are required

to test these possibilities.

Ab40 and Ab42The 42 residue form of Ab (Ab42) is often considered particularly

important in AD, because of its more rapid aggregation kinetics

in vitro (Bitan et al., 2003; Jarrett et al., 1993), elevated levels in

patients with some disease-associated mutations (Scheuner

et al., 1996), and elevated levels in AD brain tissue (Kuo et al.,

1996). However, depending on experimental conditions, differ-

ences in aggregation kinetics in vitro are not large. Ab40 levels

in humans are still roughly 5-fold larger than Ab42 levels,

even with disease-associated mutations. Direct assessment of

Ab40/Ab42 ratios in Ab aggregates within brain tissue depends

C

on monoclonal antibodies that recognize C-terminal epitopes

(Iwatsubo et al., 1994). For Ab40 fibrils with the structure in Fig-

ure 5A, the C terminus is not accessible to antibodies, suggest-

ing that such assessments may not be reliable. Measurements

on AD brain extract, after dissolution in formic acid, show a

wide range of Ab40/Ab42 ratios (Gravina et al., 1995).

In our hands, polymorphic Ab42 fibrils prepared in vitro do not

seed the growth of Ab40 fibrils. Therefore, our brain-seeded

Ab40 fibrils most likely arise from Ab40 fibrils (not Ab42 fibrils)

in the brain tissue of patients I and II.

Implications for Imaging Agents and InhibitorsIn vivo imaging of Ab amyloid by positron emission tomography,

based on compounds that bind preferentially to Ab fibrils (Clark

et al., 2011; Klunk et al., 2004), has become an important tool

ell 154, 1257–1268, September 12, 2013 ª2013 Elsevier Inc. 1265

in AD research and diagnosis. Existing imaging agents were

developed without detailed molecular structural information for

fibrils that develop in brain tissue. Specific structural differences

between brain-seeded and in vitro fibrils identified above may

facilitate the development of imaging agents with improved

specificity and structural selectivity.

Significant efforts have also been devoted to the development

of inhibitors of Ab aggregation, including both small-molecule

(Yang et al., 2005) and peptide-based (Gordon et al., 2001) inhib-

itors. Compounds that inhibit formation of the specific Ab struc-

tures that develop in AD brain tissue may be particularly valuable

in AD prevention or treatment.

EXPERIMENTAL PROCEDURES

Amyloid Extraction and Seeding

Amyloid-enriched extract was produced by subjecting brain tissue to homog-

enization, ultracentrifugation in 1.2 M and 1.9 M sucrose, DNase I digestion,

and a brief treatment with 1%sodiumdodecyl sulfate to remove residual lipids.

After washing, this material was pelleted and stored at �20�C. Fibrils were

grown at 24�C by adding synthetic or recombinant Ab40, solubilized in

dimethyl sulfoxide, to a 5 mg/ml suspension of sonicated brain extract in

10 mM phosphate buffer (pH 7.4), with a final Ab40 concentration of

100 mM. For solid-state NMR, fibrils (embedded in brain extract) were pelleted,

lyophilized, packed intomagic-angle spinning (MAS) rotors, and rehydrated by

addition of H2O. Full details are given in Extended Experimental Procedures.

Solid-State NMR and Electron Microscopy

NMRmeasurements were performed at magnetic field strengths of 9.4 T, 14.1

T, and 17.5 T using Varian Infinity and InfinityPlus spectrometers and triple-

resonance MAS probes from the group of Dr. Ago Samoson (Tallinn University

of Technology, Estonia) and from Varian. Full details are given in Extended

Experimental Procedures and Table S2.

TEM images of negatively stained and unstained samples were obtained

with an FEI Morgagni microscope, operating at 80 keV. Dark-field images for

MPL determination were obtained and analyzed as described previously

(Chen et al., 2009).

Structure Calculations

Three stages of simulated annealing within Xplor-NIH were used to arrive at

final structures that satisfy all experimental restraints, consisting of 15 distance

restraints per molecule from PITHIRDS-CT, fsREDOR, 15N-BARE, and 13C-

BARE data, 60 torsion angle restraints per molecule from TALOS+ predictions,

50 torsion angle potential surfaces per molecule from 15N-BARE and 13C-

BARE data, and 72 interresidue distance restraints per molecule from 2D

PAR, RAD, band-selective fpRFDR, and TEDOR spectra. Full details are given

in Extended Experimental Procedures and Table S4.

ACCESSION NUMBERS

The Protein Data Bank accession number for the structural model reported in

this paper is 2M4J. The Biological Magnetic Resonance Data Bank accession

number is 19009.

SUPPLEMENTAL INFORMATION

Supplemental Information includes Extended Experimental Procedures, six

figures, and four tables and can be found with this article online at http://dx.

doi.org/10.1016/j.cell.2013.08.035.

ACKNOWLEDGMENTS

Supported by the Intramural Research Programs of the National Institute of

Diabetes and Digestive and Kidney Diseases and the Center for Information

1266 Cell 154, 1257–1268, September 12, 2013 ª2013 Elsevier Inc.

Technology of the National Institutes of Health, and by N.I.H. grant R01

NS042852 (to S.C.M.). We thank Dr. Reed Wickner for assistance with gene

sequencing and Dr. Peter Pytel for assistance with histopathology. Calcula-

tions were performed on the Biowulf Linux cluster at the N.I.H.

Received: May 14, 2013

Revised: July 11, 2013

Accepted: August 19, 2013

Published: September 12, 2013

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