examined sensitivity to the acetylcholin-
esterase inhibitor aldicarb. Aldicarb causes
paralysis of body movement resulting from
the accumulation of acetylcholine at the
neuromuscular junction (18). Mutations that
reduce synaptic transmission cause resistance
to aldicarb (18). In contrast, mutations that
stimulate synaptic transmission cause hyper-
sensitivity to aldicarb-mediated paralysis (19).
Trimethadione treatment of wild-type animals
caused hypersensitivity to aldicarb-mediated
paralysis (Fig. 3E). The control drug, succin-
imide, did not cause hyperactive motility or
aldicarb hypersensitivity (3). These results
indicate the anticonvulsants stimulate synaptic
transmission in the neuromuscular system that
controls body movement.
Ethosuximide and trimethadione effec-
tively treat absence seizures in humans by
regulating neural activity. A likely target of
ethosuximide is T-type calcium channels,
although it is possible that these compounds
act on multiple targets (20–22). These anti-
convulsants also affected neural activity in
nematodes, and the anticonvulsant and the
life-span extension effects of the compounds
may act through similar mechanisms. The
findings presented here are consistent with
the model that the effect on neural activity
causes the life-span extension, although they
do not exclude the possibility that the drugs
affect neural activity and aging by different
mechanisms. Furthermore, the interactions
with the insulin-signaling mutants suggest
the intriguing possibility that neural activity
regulates aging by both daf-16–dependent
and daf-16–independent mechanisms.
References and Notes
1. L. Guarente, C. Kenyon, Nature 408, 255 (2000).
2. Materials and methods are available as supporting
material on Science Online.
3. S. Hughes, K. Evason, data not shown.
4. B. G. Katzung, Ed., Basic and Clinical Pharmacology
(Appleton and Lange, Upper Saddle River, NJ, ed. 7,
5. R. H. Levy, R. H. Mattson, B. S. Meldrum, E. Perucca,
Eds., Antiepileptic drugs (Lippincott, Williams and
Wilkins, Philadelphia, ed. 5, 2002).
6. P. A. Reddy et al., J. Med. Chem. 39, 1898 (1996).
7. C. Huang, C. Xiong, K. Kornfeld, Proc. Natl. Acad. Sci.
U.S.A. 101, 8084 (2004).
8. D. Gems, D. L. Riddle, Genetics 154, 1597 (2000).
9. D. A. Garsin et al., Science 300, 1921 (2003).
10. J. P. McKay, D. M. Raizen, A. Gottschalk, W. R.
Schafer, L. Avery, Genetics 166, 161 (2004).
11. B. Lakowski, S. Hekimi, Proc. Natl. Acad. Sci. U.S.A.
95, 13091 (1998).
12. C. Kenyon, J. Chang, E. Gensch, A. Rudner, R. Tabtiang,
Nature 366, 461 (1993).
13. D. Gems, S. Pletcher, L. Partridge, Aging Cell 1, 1
14. J. Apfeld, C. Kenyon, Nature 402, 804 (1999).
15. M. Ailion, T. Inoue, C. I. Weaver, R. W. Holdcraft,
J. H. Thomas, Proc. Natl. Acad. Sci. U.S.A. 96, 7394
16. D. L. Riddle, Ed., C. elegans II, Cold Spring Harbor
Monogr. Ser. 33(Cold Spring Harbor Laboratory
Press, Plainview, NY, 1997).
17. C. Trent, N. Tsuing, H. R. Horvitz, Genetics 104, 619
18. K. G. Miller et al., Proc. Natl. Acad. Sci. U.S.A. 93,
19. K. G. Miller, M. D. Emerson, J. B. Rand, Neuron 24,
20. D. A. Coulter, J. R. Huguenard, D. A. Prince, Ann.
Neurol. 25, 582 (1989).
21. J. C. Gomora, A. N. Daud, M. Weiergraber, E. Perez-Reyes,
Mol. Pharmacol. 60, 1121 (2001).
22. N. Leresche et al., J. Neurosci. 18, 4842 (1998).
23. Strains were provided by the Caenorhabditis Genet-
ics Center, which is funded by the NIH National
Center for Research Resources. We thank M. Nonet
for helpful suggestions and aex-3 mutants, S. Hughes
for experimental assistance, and D. Redmond for
assistance with graphics. Support was provided by
the Washington University Alzheimer’s Disease
Research Center (grant no. P50 AG05681) and the
Longer Life Foundation, a Reinsurance Group of
America, Inc. (RGA)/Washington University Partner-
ship (grant no. 1999-001). C.H. was supported by a
Predoctoral Fellowship from the Howard Hughes
Medical Institute. K.K. is a scholar of the Leukemia
and Lymphoma Society.
Supporting Online Material
Materials and Methods
16 September 2004; accepted 16 November 2004
Polymorphism in Alzheimer’s
Aneta T. Petkova,1Richard D. Leapman,2Zhihong Guo,3
Wai-Ming Yau,1Mark P. Mattson,3Robert Tycko1*
Amyloid fibrils commonly exhibit multiple distinct morphologies in electron
microscope and atomic force microscope images, often within a single image
field. By using electron microscopy and solid-state nuclear magnetic
resonance measurements on fibrils formed by the 40-residue b-amyloid
peptide of Alzheimer’s disease (Ab1–40), we show that different fibril
morphologies have different underlying molecular structures, that the
predominant structure can be controlled by subtle variations in fibril growth
conditions, and that both morphology and molecular structure are self-
propagating when fibrils grow from preformed seeds. Different Ab1–40fibril
morphologies also have significantly different toxicities in neuronal cell
cultures. These results have implications for the mechanism of amyloid
formation, the phenomenon of strains in prion diseases, the role of amyloid
fibrils in amyloid diseases, and the development of amyloid-based nano-
Amyloid fibrils are self-assembled filamen-
tous aggregates formed by peptides and
proteins with diverse amino acid sequences
(1). Current interest in amyloid fibrils arises
from their involvement in Alzheimer_s dis-
ease (AD), type 2 diabetes, prion diseases,
and other protein misfolding disorders (2)
and from basic questions about the inter-
actions that stabilize amyloid structures and
the mechanisms by which they form (3).
Recent experiments additionally suggest that
amyloid structures may be a basis for one-
dimensional nanomaterials with possible
technological applications (4, 5).
In transmission electron microscope (TEM)
and atomic force microscope (AFM) images,
amyloid fibrils commonly exhibit multiple dis-
tinct morphologies, often described as twisted
or parallel assemblies of finer protofilaments
(6–8). Two explanations for amyloid polymor-
phism are possible: (i) distinct morphologies
result from distinct modes of lateral associa-
tion of protofilaments without significant vari-
ations in molecular structure (7) or (ii) distinct
morphologies result from significant variations
in molecular structure at the protofilament lev-
el. Here, we report electron microscopy and
solid-state nuclear magnetic resonance (NMR)
data on amyloid fibrils formed by the 40-
residue b-amyloid peptide associated with AD
(Ab1–40) that support the second possibility
and reveal specific molecular-level structural
differences between different fibril morpholo-
gies. The predominant morphology and molecu-
lar structure are sensitive to subtle differences in
fibril growth conditions in de novo preparations
(at fixed pH, temperature, buffer composition,
and peptide concentration), but both morpholo-
gy and molecular structure are self-propagating
in seeded preparations. Different Ab1–40fibril
morphologies also exhibit significantly different
toxicities in neuronal cell cultures.
1Laboratory of Chemical Physics, National Institute of
Diabetes and Digestive and Kidney Diseases, National
Institutes of Health (NIH), Bethesda, MD 20892–
0520, USA.2Division of Bioengineering and Physical
Science, Office of Research Services, NIH, Bethesda,
MD 20892–5766, USA.3Laboratory of Neurosciences,
National Institute of Aging, NIH, Baltimore, MD
*To whom correspondence should be addressed.
R E P O R T S
14 JANUARY 2005 VOL 307SCIENCEwww.sciencemag.org
Amyloid fibrils were prepared from the
human Ab1–40peptide (sequence NH2-
D A E F R H D S G Y
(9, 10). Parent fibrils were grown by incubation
of identical fresh Ab1–40solutions for periods of
21 to 68 days either in vertical dialysis tubes in
an unstirred bath of buffer or in horizontal
polypropylene tubes with gentle circular agita-
tion (Red Rotor orbital mixer, Hoefer Scientific
Instruments, San Francisco, CA), producing
quiescent and agitated parent fibrils, respec-
E V H H Q K L V F F
tively. Daughter fibrils were grown under dial-
ysis conditions by seeding fresh solutions with
sonicated fragments (9.1% of total Ab1–40) of
either quiescent or agitated parents. Daughter
fibrils were used as seeds for granddaughter
fibrils, also grown under dialysis conditions.
Daughter and granddaughter fibrils were grown
for 3 to 8 days.
TEM images of negatively stained Ab1–40
fibrils are shown in Fig. 1 (9). The predominant
morphology for quiescent parents is a fibril
with a periodically modulated width (50- to
200-nm period and 12- T 1-nm maximum
width), commonly ascribed to a periodic
twist (6–8). The predominant morphology
for agitated parents is a filament with no
resolvable twist (5.5- T 0.5-nm width) but
with a pronounced tendency to associate
laterally into dimers or multimers. Morpho-
logical differences are preserved in TEM
images of sonicated seeds and are transmitted
to daughter and granddaughter fibrils, even
though all daughter and granddaughter fibrils
were grown under identical dialysis conditions.
AFM images show similar morphological
differences (fig. S1).
Molecular and supramolecular structures
of amyloid fibrils can be probed by various
solid-state NMR techniques (3, 11–16).
Figure 2 shows two-dimensional (2D) solid-
state13C NMR spectra of parent, daughter,
and granddaughter fibrils with uniform
labeling of F20, D23, V24, K28, G29, A30,
and I31 (9). The patterns of strong one-bond
cross peaks in these spectra are determined
by isotropic13C chemical shifts, which are
sensitive to local structural and conforma-
tional environment (13, 17, 18). Remarkably,
the 2D spectra of quiescent and agitated par-
ent fibrils show pronounced differences in
cross-peak patterns that are transmitted to the
daughter and granddaughter fibrils. Thus, the
morphological differences in Fig. 1 correlate
with underlying differences in structure at the
molecular level. The13C chemical shift dif-
ferences are greater than 1.5 parts per million
(ppm) at multiple backbone and side-chain
Fig. 1. TEM images of amyloid fibrils formed by the Ab1–40 peptide, negatively stained with uranyl
acetate. Parent fibrils were prepared by incubation of Ab1–40 solutions either under quiescent
dialysis conditions or in a closed polypropylene tube with gentle agitation. Daughter and
granddaughter fibrils were grown under dialysis from solutions that were seeded with sonicated
fragments of parent and daughter fibrils, respectively.
Fig. 2. Two-dimensional
spectra of Ab1–40 fibrils,
prepared with13C label-
ing of all carbon sites in
amino acid residues F20,
D23, V24, K28, G29, A30,
and I31. Spectra were
recorded under magic
angle spinning. Regions
enclosed by ellipses con-
tain Cg1/Cd cross peaks of
I31 (red), Cb/Cg cross
peaks of V24 (blue), and
Ca/Cbcross peaks of F20,
D23, V24, K28, and I31
R E P O R T S
www.sciencemag.orgSCIENCE VOL 30714 JANUARY 2005
sites (tables S1 and S2) and are independent
of the protocol used to prepare Ab1–40solu-
tions for parent fibril growth (fig. S2).
The propensities for lateral association
exhibited by quiescent and agitated fibrils
suggest that different amino acid side chains
are exposed on the fibril surfaces, possibly
leading to different biological activities.
Figure 3 shows the results of measurements
of Ab1–40fibril toxicity in cultures of pri-
mary rat embryonic hippocampal neurons
(9). Both quiescent and agitated fibrils are
neurotoxic at Ab1–40concentrations of 10 mM
and above, but the toxicity of quiescent fibrils
is significantly higher than that of agitated
fibrils. Toxicity measurements on nonfibrillar
Ab1–40aggregates, which form in unseeded
solutions at early stages of incubation, show
no significant difference between quiescent
and agitated conditions (fig. S3).
Some specific structural features of qui-
escent and agitated Ab1–40fibrils are indi-
cated in Fig. 4. All amyloid structures
contain the cross-b motif, i.e., b sheets ex-
tending over the length of the fibril, with b
strands roughly perpendicular to and inter-
strand hydrogen bonds roughly parallel to
the long fibril axis (1, 3). Intermolecular
13C-13C nuclear magnetic dipole-dipole cou-
plings (Fig. 4A) indicate intermolecular dis-
tances of 0.55 T 0.05 nm at V12, V39, and
A30, implying in-register, parallel b sheets for
both morphologies as reported previously for
Ab10–35and Ab1–40fibrils prepared with dif-
ferent protocols (12, 14, 15, 19). The15N-13C
dipole-dipole couplings (Fig. 4B) indicate a
0.32 T 0.02-nm distance between side-chain
Cgcarbons of D23 residues and side-chain Nz
nitrogens of K28 residues in agitated Ab1–40
fibrils, consistent with salt bridges between
oppositely charged D23 and K28 side chains.
Weaker D23-K28 couplings are observed in
quiescent fibrils, consistent with longer aver-
age Cg-Nzdistances. Dipole-dipole couplings
between side-chain Cdcarbons of E22 resi-
dues and side-chain Nznitrogens of K16 resi-
dues are observed in quiescent fibrils, possibly
indicating partial occupation of an intermo-
lecular K16-E22 salt bridge, but are absent in
agitated fibrils. Histograms of fibril mass-per-
length (MPL) (Fig. 4C) obtained from scan-
ning transmission electron microscope (STEM)
images (9) show mode values of 21.4 kD/nm
for agitated fibrils Esimilar to earlier Ab1–40
and Ab1–42fibril STEM data (6, 15)^ and
30.3 kD/nm for quiescent fibrils. Given the
0.47- to 0.48-nm spacing between peptide
chains in an ideal cross-b structure, the MPL
of one layer of Ab1–40molecules (4.3 kD mass)
would be 9.1 kD/nm. Figure 4C suggests that
the protofilament (defined here as the ex-
perimentally detected structure with minimal
MPL) contains two molecular layers in agitated
Ab1–40fibrils and three molecular layers in qui-
escent Ab1–40fibrils. Deviations from precise
integer multiples of 9.1 kD/nm may arise from a
nonzero angle q between the interstrand hydro-
gen bonding direction and the long fibril axis,
Established correlations between13C NMR
chemical shifts and secondary structure (13, 17)
and predictions of backbone f and y torsion
angles from the TALOS program (18) sug-
gest that the b strand segments in quiescent
Ab1–40fibrils include residues 10 to 14, 16 to
22, 30 to 32, and 34 to 36, whereas those in
agitated Ab1–40fibrils include residues 10 to
22, 30 to 32, and 34 to 36 (tables S1 and S2).
Figure 4A shows that V12 and V39 partic-
ipate in the parallel, hydrogen-bonded struc-
ture in both polymorphs. The13C chemical
shifts for Q15, D23, G25, and G33 in quies-
cent fibrils and for D23, G25, and G33 in
agitated fibrils suggest non-b strand confor-
mations at these residues. The13C NMR line-
widths (fig. S4) indicate structurally disordered
N-terminal segments in both morphologies,
as previously suggested (13, 14, 19–21). Line-
widths for most CO, Ca, and Cbsites in res-
idues 12 to 39 of quiescent fibrils and residues
10 to 39 of agitated fibrils are 2.5 ppm or less,
indicating overall structural order (13).
Data for agitated Ab1–40fibrils are largely
consistent with our recent model for the
Fig. 4. Molecular structural
features of quiescent (blue)
and agitated (red) Ab1–40
fibrils. (A) Measurements
nuclear magnetic dipole-
dipole couplings for V12 car-
bonyl, A30 methyl, and V39
carbonyl carbons (circles, tri-
angles, and squares, re-
spectively) and numerical
simulations for the indicated
13C-13C distances. Data were
obtained for singly
labeled samples with the
radio frequency–driven re-
coupling solid-state NMR
technique (9, 14). (B) Measurements of15N-13C dipole-dipole couplings for D23 Cg/K28 Nzand
E22 Cd/K16 Nzpairs (triangles and circles, respectively) and numerical simulations for the indicated
15N-13C distances. Data were obtained for samples with uniformly15N- and13C-labeled residues
with the frequency-selective rotational echo double resonance solid-state NMR technique (9, 32).
Error bars are calculated from the root mean square noise in the NMR spectra. (C) MPL histograms
extracted from STEM images of Ab1–40fibrils. Dashed lines indicate MPL values for ideal cross-b
structures with between one and five layers of Ab1–40molecules. a.u., arbitrary units.
Fig. 3. Toxicity of Ab1–40fibrils in cultures of
primary embryonic rat hippocampal neurons,
assayed by counting viable neurons after 24-
hour and 48-hour exposures to Ab1–40at
indicated concentrations in neurobasal medium.
Error bars indicate standard errors on mean
survival values (four culture dishes per condition
and 35 to 145 neurons monitored per dish).
Control is neurobasal medium alone. Vehicle is
supernatant after centrifugation of Ab1–40fibril
solution, added to neurobasal medium in
volume equal to that for 30 mM conditions.
The asterisks and plus symbols indicate con-
ditions where toxicity differences between
quiescent and agitated fibrils are statistically
significant (analysis of variance test, P G 0.01
and P G 0.05, respectively).
R E P O R T S
14 JANUARY 2005 VOL 307SCIENCEwww.sciencemag.org
molecular structure of the Ab1–40protofila- Download full-text
ment (13, 15). STEM,15N-13C coupling, and
chemical shift data for quiescent Ab1–40fibrils
indicate a qualitatively different structure in
both molecular conformation and supramo-
lecular organization. The largest chemical shift
differences, suggesting the largest conforma-
tional differences, occur at Q15 and in residues
22 to 28. Although all residues in agitated
Ab1–40fibril samples exhibit one major set of
13C chemical shifts (with the exception of
D23, V24, and K28, possibly indicating the
coexistence of two distinct D23-K28 salt
bridge geometries), many residues in quies-
cent fibril samples exhibit two sets of13C
chemical shifts, with an approximate 2:1 ratio
of NMR signal intensities (e.g., I31 side-chain
signals in Fig. 2). This observation raises the
possibility that the quiescent Ab1–40protofila-
ment contains two structurally equivalent and
one structurally inequivalent subunits, con-
sistent with Fig. 4, B and C.
We point out four physical and biological
implications of these data. First, the sensi-
tivity of fibril morphology and molecular
structure to growth conditions shows that at
least two distinct fibril nucleation mecha-
nisms exist for Ab1–40. One mechanism,
leading to quiescent fibrils, may be purely
homogeneous. The other mechanism, leading
to agitated fibrils, may depend on the
interface between the peptide solution and
the air or the walls of the sample tube. The
molecular structure of Ab1–40fibrils is not
determined solely by amino acid sequence
and is not purely under thermodynamic
control. Second, the phenomenon of strains
in prion diseases, in which a single prion
protein gives rise to multiple, distinct phe-
notypes, has been attributed to an ability of
both mammalian and yeast prion proteins to
adopt multiple, distinct amyloid-like struc-
tures. Observed differences in proteolysis
patterns (22, 23), resistance to chemical
denaturation (24), seeding efficiencies (25),
and electron paramagnetic resonance signals
(26) support this proposal, but clear con-
nections between morphological variations
and molecular-level structural variations, be-
tween strains and morphological variations,
and between strains and specific features of
molecular structure have not yet been estab-
lished experimentally. The correlations of
amyloid fibril morphology with specific
structural features established by our data,
the demonstration of their self-propagating
nature, and the observation of different neuro-
toxicities for different morphologies further
strengthen the case for a structural origin of
prion strains. Third, the importance of mature
amyloid fibrils as etiological agents in AD
and other amyloid diseases, as opposed to
nonfibrillar oligomers observed at earlier
stages of peptide incubation (27–30), is a
subject of current controversy. One principal
argument against a primary role for mature
fibrils in AD has been the absence of a robust
correlation between the severity of neurolog-
ical impairment and the extent of amyloid
deposition (2, 31). Data in Fig. 3 raise the
possibility that certain amyloid morphologies
may be more pathogenic than others in the
affected organs of amyloid diseases, which
would weaken the correlation between dis-
ease symptoms and total amyloid deposition.
Fourth, amyloid fibrils may prove useful as
structural and chemical templates for self-
assembled, one-dimensional nanomaterials
with novel electronic or optical properties
(4, 5). Because structural uniformity is a
likely prerequisite in such applications, the
self-propagation of molecular structure dem-
onstrated above may be important for reliable
fabrication of amyloid-based nanomaterials.
References and Notes
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Chem. Biol. 4, 119 (1997).
9. Materials and methods are available as supporting
material on Science Online.
10. Single-letter abbreviations for the amino acid resi-
dues are as follows: A, Ala; C, Cys; D, Asp; E, Glu;
F, Phe; G, Gly; H, His; I, Ile; K, Lys; L, Leu; M, Met;
N, Asn; P, Pro; Q, Gln; R, Arg; S, Ser; T, Thr; V, Val;
W, Trp; and Y, Tyr.
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Supporting Online Material
Materials and Methods
Figs. S1 to S4
Tables S1 and S2
15 April 2004; accepted 16 November 2004
Semaphorin 3E and Plexin-D1
Control Vascular Pattern
Independently of Neuropilins
Chenghua Gu,1,2* Yutaka Yoshida,3* Jean Livet,4
Dorothy V. Reimert,1,2Fanny Mann,4Janna Merte,1,2
Christopher E. Henderson,4Thomas M. Jessell,3
Alex L. Kolodkin,1. David D. Ginty1,2.
The development of a patterned vasculature is essential for normal
organogenesis. We found that signaling by semaphorin 3E (Sema3E) and its
receptor plexin-D1 controls endothelial cell positioning and the patterning of
the developing vasculature in the mouse. Sema3E is highly expressed in
developing somites, where it acts as a repulsive cue for plexin-D1–expressing
endothelial cells of adjacent intersomitic vessels. Sema3E–plexin-D1 signaling
did not require neuropilins, which were previously presumed to be obligate
Sema3 coreceptors. Moreover, genetic ablation of Sema3E or plexin-D1 but
not neuropilin-mediated Sema3 signaling disrupted vascular patterning. These
findings reveal an unexpected semaphorin signaling pathway and define a
mechanism for controlling vascular patterning.
The peripheral nervous system and its vascu-
lature develop coordinately in part through
common developmental cues. Semaphorins, a
family of phylogenetically conserved cell-
surface and secreted proteins, control neuronal
cell migration and axon guidance (1–3).
Certain membrane-bound semaphorins bind
directly to receptors of the plexin family
(4–7), but the class 3 secreted semaphorins
(Sema3A to Sema3F, referred to collective-
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