Prion-like behaviour and tau-dependent cytotoxicity of pyroglutamylated amyloid-β.

Abstract

Extracellular plaques of amyloid-β and intraneuronal neurofibrillary tangles made from tau are the histopathological signatures of Alzheimer's disease. Plaques comprise amyloid-β fibrils that assemble from monomeric and oligomeric intermediates, and are prognostic indicators of Alzheimer's disease. Despite the importance of plaques to Alzheimer's disease, oligomers are considered to be the principal toxic forms of amyloid-β. Interestingly, many adverse responses to amyloid-β, such as cytotoxicity, microtubule loss, impaired memory and learning, and neuritic degeneration, are greatly amplified by tau expression. Amino-terminally truncated, pyroglutamylated (pE) forms of amyloid-β are strongly associated with Alzheimer's disease, are more toxic than amyloid-β, residues 1-42 (Aβ(1-42)) and Aβ(1-40), and have been proposed as initiators of Alzheimer's disease pathogenesis. Here we report a mechanism by which pE-Aβ may trigger Alzheimer's disease. Aβ(3(pE)-42) co-oligomerizes with excess Aβ(1-42) to form metastable low-n oligomers (LNOs) that are structurally distinct and far more cytotoxic to cultured neurons than comparable LNOs made from Aβ(1-42) alone. Tau is required for cytotoxicity, and LNOs comprising 5% Aβ(3(pE)-42) plus 95% Aβ(1-42) (5% pE-Aβ) seed new cytotoxic LNOs through multiple serial dilutions into Aβ(1-42) monomers in the absence of additional Aβ(3(pE)-42). LNOs isolated from human Alzheimer's disease brain contained Aβ(3(pE)-42), and enhanced Aβ(3(pE)-42) formation in mice triggered neuron loss and gliosis at 3 months, but not in a tau-null background. We conclude that Aβ(3(pE)-42) confers tau-dependent neuronal death and causes template-induced misfolding of Aβ(1-42) into structurally distinct LNOs that propagate by a prion-like mechanism. Our results raise the possibility that Aβ(3(pE)-42) acts similarly at a primary step in Alzheimer's disease pathogenesis.

Full text

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LETTER
doi:10.1038/nature11060
Prion-like behaviour and tau-dependent
cytotoxicity of pyroglutamylated amyloid-b
Justin M. Nussbaum1*, Stephan Schilling2*, Holger Cynis2, Antonia Silva1, Eric Swanson1, Tanaporn Wangsanut1, Kaycie Tayler3,
Brian Wiltgen3, Asa Hatami4, Raik Ro ¨nicke5, Klaus Reymann5, Birgit Hutter-Paier6, Anca Alexandru7, Wolfgang Jagla7,
Sigrid Graubner7, Charles G. Glabe4, Hans-Ulrich Demuth2,7& George S. Bloom1,8
Extracellularplaquesofamyloid-bandintraneuronalneurofibrillary
tangles made from tau are the histopathological signatures of
Alzheimer’sdisease.Plaquescompriseamyloid-bfibrilsthatassemble
from monomeric and oligomeric intermediates, and are prognostic
indicators of Alzheimer’s disease.Despite the importance of plaques
to Alzheimer’s disease, oligomers are considered to be the principal
toxic forms of amyloid-b1,2. Interestingly, many adverse responses to
amyloid-b,suchascytotoxicity3,microtubuleloss4,impairedmemory
and learning5, and neuritic degeneration6, are greatly amplified by
tau expression. Amino-terminally truncated, pyroglutamylated (pE)
forms of amyloid-b7,8are strongly associated with Alzheimer’s
disease, are more toxic than amyloid-b, residues 1–42 (Ab1–42) and
Ab1–40, and have been proposed as initiators of Alzheimer’s disease
pathogenesis9,10. Here we report a mechanism by which pE-Ab may
trigger Alzheimer’s disease. Ab3(pE)–42co-oligomerizes with excess
Ab1–42to form metastable low-n oligomers (LNOs) that are struc-
turally distinct and far more cytotoxic to cultured neurons than
comparable LNOs made from Ab1–42alone. Tau is required for
cytotoxicity, and LNOs comprising 5% Ab3(pE)–42plus 95% Ab1–42
(5% pE-Ab) seed new cytotoxic LNOs through multiple serial dilu-
tionsintoAb1–42monomersintheabsenceofadditionalAb3(pE)–42.
LNOs isolated from human Alzheimer’s disease brain contained
Ab3(pE)–42, and enhanced Ab3(pE)–42formation in mice triggered
neuronlossandgliosisat3months,butnotinatau-nullbackground.
We conclude that Ab3(pE)–42confers tau-dependent neuronal death
and causes template-induced misfolding of Ab1–42into structurally
distinctLNOsthatpropagatebyaprion-likemechanism.Ourresults
raise thepossibility thatAb3(pE)–42actssimilarly ata primary stepin
Alzheimer’s disease pathogenesis.
pE-Ab peptides contain an amino-terminal pyroglutamate, whose
modification from glutamate is catalysed by glutaminyl cyclase (QC;
alsoknownasQPCT)10.ThemostprominentpE-Abspeciesinvivoare
Ab3(pE)–40, Ab3(pE)–42, Ab11(pE)–40and Ab11(pE)–42(ref. 8; Supplemen-
tary Fig. 1), with Ab3(pE)–42being most abundant11. pE-Ab is more
cytotoxic12and aggregates more rapidly13,14than conventional
amyloid-b,andQCactivityandpE-Ablevelsareincreasedseveral-fold
in Alzheimer’s disease brain10. Alzheimer’s disease mouse models also
indicatearoleforpE-Abininitiatingpathology:oraladministrationof
a QC inhibitor led to improved memory and learning, and reduced
levels of pE-Ab and conventional amyloid-b10. These data imply that
pE-Ab potentiates the neurotoxicity of conventional amyloid-b, but
leaveopentheissueofmolecularmechanisms.Toaddressthatissue,we
compared oligomerization of Ab3(pE)–42, Ab1–42, and mixtures of the
peptides in vitro, and analysed responses of primary cultured neurons
and glial cells (Supplementary Fig. 2) to the oligomers.
At5mMpeptide,5%pE-AbaggregatedfasterthanAb3(pE)–42orAb1–42
alone, based on thioflavin T fluorescence shifts15(Supplementary
Fig. 3). The ratio of optical densities at 450nm versus 490nm
(OD450nm/OD490nm)forAb3(pE)–42roseandpeakedmorerapidlythan
for Ab1–42, but peaked at a ,25% lower level. The fastest rise in the
*These authors contributed equally to this work.
1Department of Biology, University of Virginia, Charlottesville, Virginia 22904, USA.2Probiodrug AG, 06120 Halle (Saale), Germany.3Department of Psychology, University of Virginia, Charlottesville,
Virginia 22904, USA.4Department of Biochemistry and Molecular Biology, University of California at Irvine, Irvine, California 92697, USA.5Deutsches Zentrum fuer Neurodegenerative Erkrankungen, c/o
Leibniz-Institut fuer Neurobiologie, 39118 Magdeburg, Germany.6JSW Life Sciences GmbH, A-8074 Grambach, Austria.7Ingenium Pharmaceuticals GmbH, 82152 Munich/Martinsried, Germany.
8Department of Cell Biology, University of Virginia, Charlottesville, Virginia 22904, USA.
Viability (% control)
100
80
60
40
20
0.1 μM
0.5 μM
1.0 μM
P < 0.01
Aβ1–42
Aβ3(pE)–42
Oligomerized
separately
Oligomerized
together
5% Aβ3(pE)–42, 95% Aβ1–42
b
Vehicle
95% Aβ1–42
5% Aβ3(pE)–42
0
42 μm
a
WT glia
WT
neurons
Tau KO
neurons
Aβ1–42
Aβ3(pE)–42
Figure 1 | Tau-dependent cytotoxicity of oligomers formed by co-
incubationofAb3(pE)–42andAb1–42. Primarymousewild-type(WT)andtau-
knockout (KO) forebrain neurons, and secondary cultures of wild-type mouse
glia were treated for 12h with Ab1–42, Ab3(pE)–42, or 5% Ab3(pE)–42plus 95%
Ab1–42, which were oligomerized for 24h at 5mM before dilution into culture
media.a,Cellswereexposedtocalcein-AMandimagedlivebyepifluorescence
microscopy to assay viability16. Extensive death and detachment of cells were
observedonlyforwild-typeneuronstreatedwithAb3(pE)–42orthe5%Ab3(pE)–42
plus 95% Ab1–42. b, Following peptide treatment, cell viability was analysed by
the XTT plate reader assay17. Note the robust cytotoxicity of Ab3(pE)–42
containing solutions at concentrations as low as 0.5mM, unless Ab3(pE)–42and
Ab1–42wereincubatedseparatelyduringoligomerization(P,0.01;yellowstars
signifystatisticalsignificanceoftheindicatedbargraphsversusvehiclecontrols;
black stars signify statistical significance between the indicated bar graph pairs;
mean6standarderrorofthemean(s.e.m.),n59replicatesfrom3independent
experiments).
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OD450nm/OD490nmratiowasfor5%pE-Ab,whichpeakedsimilarlyto
Ab3(pE)–42. Ab3(pE)–42, Ab1–42and 5% pE-Ab thus oligomerized by
different pathways.
To test whether distinct biological activities were coupled to these
oligomerization differences, we compared cytotoxicity of the peptides
towards cultured neurons or glia using calcein-AM and fluorescence
microscopy16. Twelvehours of Ab1–42exposure had little effect on cell
viabilityforwild-typeortau-knockoutneurons,orwild-typeglialcells
(Fig. 1a). Contrastingly, most wild-type neurons died and detached
from the substrate after exposure to Ab3(pE)–42or 5% pE-Ab. Tau-
knockout neurons and wild-type glia, which express little tau, were
resistant to Ab3(pE)-42and 5% pE-Ab.
Cytotoxicity dose dependence was examined by incubating wild-
type neurons for 24h in oligomers comprising 0.1, 0.5 or 1mM
peptides, and using the 2,3-bis-(2-methoxy-4-nitro-5-sulphophenyl)-
2H-tetrazolium-5-carboxanilide (XTT) reduction assay17(Fig. 1b). Cells
wereunaffectedbyAb1–42,butAb3(pE)–42and5%pE-Abhadsubstan-
tialcytotoxicityat0.5mMandevenmoreat1.0mM.Cytotoxicityof5%
pE-Ab required Ab3(pE)–42and Ab1–42to incubate together for 24h
before being added to cells. When they were incubated separately for
24handmixedtogetherata1:19molarratioimmediatelybeforebeing
applied to cells, they were not cytotoxic. A small amount of Ab3(pE)–42
canthusmarkedlyenhancethecytotoxicityofalargeexcessofAb1–42,
provided the two peptides oligomerize together.
Evidence for hybrid oligomers came from immunoprecipitation of
various forms of amyloid-b using aggregation-dependent M64, which
does not recognize Ab3(pE)–42(see Supplementary Fig. 4 for character-
izationofallanti-amyloid-bantibodiesused,includingM64).Immuno-
precipitations were analysed on dot blots using 4G8, which equally
recognizesAb3(pE)–42andAb1–42,andanti-pE-Ab,whichdoesnotreact
with Ab1–42. M64 immunoprecipitated oligomers made from Ab1–42
or 5% pE-Ab, but it did not immunoprecipitate Ab3(pE)–42oligomers,
normonomers ofeitherpeptide (Fig.2a).Because anti-pE-Ab reacted
with material immunoprecipitated out of 5% pE-Ab, M64 pulled
down hybrid peptide oligomers. Ab3(pE)–42accounted for ,16% of
theamyloid-bingel-filteredcytotoxicoligomersafter3hofoligomer-
ization,andsteadilydroppedto,8%by24h(Fig.2b).Ab3(pE)–42thus
acts as a template that initiates formation of cytotoxic oligomers.
Cytotoxicitywassensitivetooligomerizationtime(Fig.2c).Baseline
cytotoxicitywas observed atalltime pointsforAb1–42,andfor5%pE-
Ab solutions in which Ab3(pE)–42and Ab1–42oligomerized separately.
Pure Ab3(pE)–42killed ,50% of the cells after 24h of oligomerization,
butwasvirtuallynon-toxicat0handafter96hofoligomerization.The
most cytotoxic solutions were 5% pE-Ab, in which the constituent
peptides co-oligomerized for 24h. These solutions killed ,60% of
the cells within 24h, and lower but robust cytotoxicity was observed
at 96h. Even the 0h co-oligomers of 5% pE-Ab exhibited low, signifi-
cant cytotoxicity. Co-incubated mixtures of 5% Ab3(pE)–42and 95%
0
15
10
5
20
48 24 12
3
Per cent Aβ3(pE)–42 (of total)
Oligomerization time before
fractionation (h)
3
b
a
IP: M64
(5X)
Aβ1–42 oligomers
Aβ3(pE)–42 oligomers
5% Aβ3(pE)–42 + 95% Aβ1–42
oligomers
Aβ1–42 monomers
Aβ3(pE)–42 monomers
4G8
Anti-pE-Aβ
(5× load)
15.4 ng
(corrected to 1×)
101 ng 162 ng
0 h 24 h 96 h
0
20
100
40
60
80
Viability (% control)
Aβ1–42
Aβ3(pE)–42
5% Aβ3(pE)–42, 95% Aβ1–42
Oligomerized
together
c
P < 0.01
P < 0.01
Oligomerized
separately
Figure 2 | Ab3(pE)–42and Ab1–42form metastable, cytotoxic, hybrid
oligomers. a, Ab3(pE)–42and Ab1–42were incubated together at a 1:19 molar
ratio (5% pE-Ab) for 24h at 1mM total amyloid-b, and were then
immunoprecipitated (IP) with M64, a rabbit monoclonal antibody that
specifically recognizes residues 3–7 (EFRH) of Ab1–40oligomers or fibrils.
Additional samples that were immunoprecipitated included otherwise
identically treated oligomers made from pure Ab3(pE)–42or Ab1–42, and
monomeric versions of the two peptides. Immunoprecipitated oligomers were
converted to monomers by lyophilization, solubilization with HFIP and
dilution into PBS, and along with the other samples were dot blotted onto
nitrocellulose and analysed using 4G8, a mouse monoclonal antibody that
recognizesAb3(pE)–42andAb1–42equallywell,andanantibodythatspecifically
recognizes pE-Ab (see Supplementary Fig. 4 for characterization of all
antibodies used here). Quantification of the dot blots using a LI-COR Odyssey
imaging station indicated that the oligomers that were immunoprecipitated
from the mixed peptide solution contained both Ab3(pE)–42and Ab1–42, at a
molarratioof,1:10.b,Solutionscontaining5%pE-Ab3(pE)–42and95%Ab1–42
were incubated for the indicated times, and then were fractionated by gel
filtration. At each time point, fractions that eluted at 12.5ml, where most
cytotoxicity resided (see Fig. 3b) were immunoprecipitated using anti-human
amyloid-b (N), an amino-terminal-specific antibody that does not react with
pE-Ab (data not shown). The immunoprecipitates were then lyophilized, re-
solubilized with HFIP, and quantitatively analysed on dot blots with 4G8 and
anti-pE-Ab using the LI-COR Odyssey. The time-dependent decrease in the
Ab3(pE)–42content of the immunoprecipitated oligomers implies that Ab3(pE)–
42initiated formation of hybrid peptide oligomers. c, Ab3(pE)–42and Ab1–42
oligomerized for 0, 24 and 96h either separately or together as 1:19 mixtures,
and then were added to primary wild-type neuron cultures for 24h at a final
concentration of 1mM total amyloid-b. Following peptide treatment, cell
viability was analysed by the XTT plate reader assay17. The most cytotoxic
species observed were the hybrid oligomers after 24h of oligomerization
(P,0.01;yellowstarssignifystatisticalsignificanceoftheindicatedbargraphs
versus vehicle controls; black stars signify statistical significance between the
indicated bar graph pairs; mean6s.e.m., n56 or 9 replicates from 3
independent experiments for panel b or c, respectively).
RESEARCH LETTER
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Page 3

Ab1–42canthereforeformoligomerswhosecytotoxicityisbothgreater
and more enduring than oligomers formed by Ab3(pE)–42alone.
To identify the co-oligomer size(s) that were cytotoxic, amyloid-b
solutions were oligomerized for various times from 0–96h before
fractionation by gel filtration. Total amyloid-b in all fractions was
determined using 4G8 dot blots that, as shown in Fig. 3a (for 5%
pE-Ab) and Supplementary Fig. 5 (for Ab1–42and Ab3(pE)–42), illus-
trate the full fractionation range of the column but exclude most void
volumefractions.PresumptivemonomericAb1–42dominatedinitially
and persisted at 3h, but was nearly undetectable after 12h. The 3h
time point also marked the appearance of Ab1–42oligomers, which
graduallyincreasedinsizeoverthenext93h.Ab3(pE)–42and5%pE-Ab
oligomerized differently. Putative monomers were present at 0h for
both samples, when slightly larger species, LNOs that possibly corre-
sponded to dimers or trimers (Supplementary Fig. 6), were also pre-
sent.Thesepersistedasthemainspeciesfor24hforAb3(pE)–42andfor
nearly 72h for 5% pE-Ab, and later time points were dominated by
largeraggregatesthatelutedinvoidvolumefractions.Cytotoxicitywas
assayed for individual fractions of 5% pE-Ab that oligomerized for
24h (Fig. 3b). Most cytotoxicity was associated with the possible
dimers/trimers that eluted at 12.5ml, which at 425nM peptide killed
more than 60% of the cells. Low cytotoxicity was also observed at
554nM peptide for the larger oligomers that eluted at 8.5ml.
The marked enhancement of Ab1–42cytotoxicity by Ab3(pE)–42
suggested a prion-like templating mechanism of Ab1–42misfolding
initiated by Ab3(pE)–42. To test that hypothesis, 5% pE-Ab that
oligomerized for 24h was diluted into 19 volumes of monomeric
Ab1–42.A24hincubationofthismixtureyielded‘serialpassage1’,which
was followed by two equivalent, sequential dilutions into monomeric
Ab1–42toyieldserialpassages2and3.Agraduallossofcytotoxicitywas
observed with successive passages, but evenpassage3, which contained
only 0.000625% Ab3(pE)–42, killed ,50% of the neurons within 24h
(Fig. 3c). Serially passaged gel-filtration samples contained abundant
material that eluted at 12.5ml in passages 1–3, despite the progressive
dilution of Ab3(pE)–42(Fig. 3d). Ab3(pE)–42can therefore template
formation of metastable, cytotoxic LNOs fromexcess Ab1–42, yielding
potent bioactivity that can be serially passaged multiple times into
monomeric Ab1–42without further addition of Ab3(pE)–42.
One possible explanation for why Ab1–42LNOs were inert is that
they lacked sufficient properly sized oligomers. Accordingly, we
altered the oligomerization protocol from 5mM peptide for 24h at
37uC to 10mM peptide for 30min at 4uC to obtain abundant Ab1–42
oligomersthatelutedat12.5ml(Fig.3e).TheseLNOswerenotcytotoxic
(Fig.3f),implyingthattheywerestructurallydistinctfromtheputative
dimers/trimersinitiatedbyAb3(pE)–42.Thiswasconfirmedbydotblots
using M87, a conformation-sensitive anti-amyloid-b antibody, to
compare the putative dimers/trimers used for the cytotoxicity assays
shown in Fig. 3f. We first lyophilized aliquots of all the amyloid-b
solutions, resuspended them with hexafluoroisopropanol (HFIP) to
restore them to monomers, and then analysed them using 4G8.
When parallel samples that were not lyophilized but were otherwise
identical were analysed using M87, immunoreactivity was approxi-
mately twice as strong with LNOs made from Ab1–42versus those
made from 5% pE-Ab (Supplementary Fig. 7). Cytotoxic LNOs of
5% pE-Ab are thus structurally distinct from comparably sized
LNOs of Ab1–42.
a
5% Aβ3(pE)–42, 95% Aβ1–42 oligomerization (h)
0 3 12 24 48 72 96
7.5
9.5
10.5
11.5
12.5
14
8.5
Ve (ml)
b
Viability (% control)
Peptide concentration (nM)
ND D
507 554 452
ND ND
425
100
80
60
40
20
P < 0.01
14
7.5 9.5
10.5
Ve (ml)
11.5
12.5
8.5
0
e
Aβ1–42
5% Aβ3(pE)–42
95% Aβ1–42
7.5
9.5
10.5
11.5
12.5
14
8.5
f
Viability (% control)
100
Aβ1–42, 1 μM
unfractionated
5% Aβ3(pE)–42, 1 μM
unfractionated
Ve = 12.5 ml
40
20
60
80
0
P < 0.01
7.5
9.5
10.5
11.5
12.5
14
Aβ1–42
8.5
Serial passage 1
Serial passage 2
Serial passage 3
d
c
Viability (%) control)
5% Aβ3(pE)–42, 95% Aβ1–42
P < 0.01
5% pE
0.25% pE
0.0125% pE
0.000625% pE
Serial
passage 1
Serial
passage 3
Oligomerized
separately
Oligomerized
together
Ve (ml)
Aβ1–42, 0.44 μM
Aβ1–42, 0.22 μM
Ve = 12.5 ml
5% Aβ3(pE)–42, 0.36 μM
Ve = 12.5 ml
5% Aβ3(pE)–42, 0.18 μM
Ve = 12.5 ml
Ve (ml)
80
60
40
20
0
passage 2
Serial
Figure 3 | The cytotoxic species are low-n, prion-like oligomers. a, Gel-
filtration chromatography was used to fractionate 5% pE-Ab after
oligomerization at 5mM at 37uC for 0–96h. The resulting fractions were then
converted to monomers using HFIP and analysed on dot blots using
monoclonal antibody 4G8. Note the metastable oligomers with an average
elution volume(Ve) of 12.5ml. b, Isolated gel-filtration fractionsfrom the24h
time point were added to wild-type neuron cultures for 24h, after which the
cells were assayed for cell viability using XTT17. Robust cytotoxicity was
associated only with the Ve512.5ml fraction, although the Ve58.5ml
fraction had low, but statistically significant cell killing activity (P,0.01;
mean6s.e.m., n59 replicates from 3 independent experiments). ND, not
detected. c, Cytotoxic hybrid oligomers made by co-incubating a 1:19 ratio of
Ab3(pE)–42:Ab1–42for 24h at 5mM were diluted into a 19-fold molar excess of
freshly dissolved, monomeric Ab1–42, which was then incubated at 5mM for
another 24h to yield serial passage 1. Two further iterations of this strategy
yielded serial passages 2 and 3. The starting material and its serially passaged
derivatives were added to wild-type neurons at 1mM peptide for 24h, after
whichcellswereanalysedusingtheXTTassayforcellviability17.Onlyagradual
loss of cytotoxicity was observed with each successive serial passage. d, Each
serially passaged sample, as well as otherwise identically prepared oligomers
madefrom pure Ab1–42, were fractionatedbygelfiltrationandanalysedondot
blots with 4G8. Note that all serially passaged samples contained metastable
LNOs of Ve512.5ml, which were absent from the pure Ab1–42samples.
e, Ab1–42(10mM) that was oligomerized for 30min at 4uC, and 5% Ab3pE–42
plus 95% Ab1–42(5mM) that was oligomerized for 24h at 37uC were
fractionated by gelfiltration andanalysed on dot blotsexactly using4G8. Note
the isolation of fractions with Ve512.5ml from both preparations. f, Wild-
type neurons were assayed for viability using the XTT plate reader assay17
following 24h of exposure to the indicated amyloid-b preparations. Note the
minimal cytotoxicity of unfractionated Ab1–42and Ab1–42with Ve512.5 ml
(P,0.01, mean6s.e.m., n59 replicates from 3 independent experiments).
P,0.01; yellow stars signify statistical significance of the indicated bar graphs
versus vehicle controls; black stars and blue stars signify statistical significance
between the indicated bar graph pairs; blue mean6s.e.m., n59 replicates
from 3 independent experiments for panels b, c and f.
LETTER RESEARCH
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Several lines of evidence demonstrate in vivo relevance for the data
described so far. First, we identified LNOs containing Ab3(pE)–42in
three out of three Alzheimer’s disease samples, based on gel filtration
of human brain extracts followed by dot blots of resulting fractions
with anti-pE-Ab and M87. In contrast, only one of three age-matched
samples with normal neuropathological diagnoses was positive for
Ab3(pE)–42(Fig. 4a and Supplementary Fig. 8). Second, we crossed
TBA2.1 mice18into a tau-knockout background19. By 3 months,
TBA2.1 mice accumulated small amounts (40–100ngg21brain
weight) of Ab3(pE)–42, which formed primarily intraneuronal aggre-
gates, and was associated with massive hippocampal neuron loss and
gliosis18. Knocking out tau provided almost complete protection
against neuron loss and glial activation (Fig. 4b). Additional in vivo
data are shown in Supplementary Fig. 9. Long-term potentiation
(LTP) of mouse hippocampal neurons in slice cultures was potently
and equally inhibited by oligomers made from 5% Ab3(pE)–42or 100%
Ab3(pE)–42, whereas Ab1–42oligomers had no effect on LTP. 1%
Ab3(pE)–42provoked mild, but statistically insignificant LTP impair-
ment (Supplementary Fig. 9a). To evaluate the effects of increased
Ab3(pE)–42in animal models, we crossed mice with neuron-specific
expression of human b-amyloid precursor protein (APP) harbouring
Swedish and London mutations (hAPPSL)20, with mice expressing
human QC21. Nine-month-old double (hAPPSL/hQC) and single
(hAPPSL)transgenicmicewereindistinguishableintermsofinsoluble
and soluble Abx–42levels, but the double transgenics had approxi-
mately twofold more insoluble Ab3(pE)–42and approximately ninefold
more soluble Ab3(pE)–42than single transgenics (Supplementary Fig.
9b).FurtheranalysisofthesolubleAbx–42bytheA4assay22revealedan
approximatelyeightfoldexcessofoligomersinthedoubleversussingle
transgenics (Supplementary Fig. 9c). Double transgenics performed
more poorly in Morris water maze tests (Supplementary Fig. 9d) and
had reduced hippocampal immunoreactivity for the synapse marker,
synaptophysin (Supplementary Fig. 9e). Finally, peri-hippocampal
injection of 5% pE-Ab at 5mM into APPSwDI/NOS22/2Alzheimer’s
disease model mice23led 3–5 months later to the presence of plaques
containing both pE-Ab and conventional amyloid-b. Comparable
plaques were rarely seen in sham-injected Alzheimer’s disease mice
or in wild-type mice injected with 5% pE-Ab (Supplementary Fig. 9f).
These collective in vivo results emphasize the physiological signifi-
cance of the companion biochemical and cultured cell results.
Our studies provide new insights into Alzheimer’s disease patho-
genesis by demonstrating that hypertoxic amyloid-b oligomers can be
triggered by small quantities of a specifically truncated and post-
translationally modified version of amyloid-b. Although some pre-
vious studies demonstrated that pE modification of amyloid-b
considerably enhances its aggregation kinetics13,14,24, toxicity12,18,25
and resistance to degradation12, a mechanistic explanation for the
unique properties of pE-Ab has been lacking until now. Prior studies
suggestcoincidentappearanceofAb3(pE)–42withdevelopmentorpro-
gression of human Alzheimer’s disease26,27. Co-localization of QC and
Ab3(pE)–42 was found in cored plaques of vulnerable regions in
Alzheimer’s disease, and evidence was provided for axonal transport
of Ab3(pE)–xfrom QC-rich neuronal populations of the entorhinal
cortex and locus coeruleus28. As LNOs containing Ab3(pE)–42 are
reasonably stable (Fig. 3a), they might initiate tau-dependent
cytotoxicity intracellularly during axonal transport29or extracellularly
following release at remote hippocampal synapses30of projection
neurons28. The Ab3(pE)–42-induced formation of toxic mixed oligomers
provides a rationale for these previous observations, and the tau-
dependent cytotoxicity of 5% pE-Ab establishes a new functional con-
nectionbetweenamyloid-bandtauinAlzheimer’sdiseasepathogenesis.
METHODS SUMMARY
FulldescriptionsofthioflavinTassays,cellculture,cellviabilityassays,procedures
foroligomerization of amyloid-b peptides andtheir fractionation by gel-filtration
chromatography,productionandspecificityofrabbitmonoclonalanti-amyloid-b
antibodies, immunoprecipitation, dot blots and western blots, generation of
hAPPSL/hQC transgenic mice, LTP measurements of mouse hippocampal slice
cultures,peri-hippocampalinjectionof5%pE-AbintoAlzheimer’sdiseasemodel
mice, cultured cell and brain immunohistochemistry, and collection of human
brain extracts are provided in Supplementary Methods.
Received 16 May 2011; accepted 16 March 2012.
Publishedonline2May;corrected30May2012(seefull-textHTMLversionfordetails).
1.Gandy, S. et al. Days to criterion as an indicator of toxicity associated with human
Alzheimer amyloid-b oligomers. Ann. Neurol. 68, 220–230 (2010).
Walsh, D. M. & Selkoe, D. J. Ab oligomers—a decade of discovery. J. Neurochem.
101, 1172–1184 (2007).
Rapoport,M.,Dawson,H.N.,Binder,L.I.,Vitek,M.P.&Ferreira,A.Tauisessentialto
b-amyloid-inducedneurotoxicity.Proc.NatlAcad.Sci.USA99,6364–6369(2002).
King, M. E. et al.Tau-dependent microtubule disassembly initiated by pre-fibrillar
b-amyloid. J. Cell Biol. 175, 541–546 (2006).
2.
3.
4.
AD
(62-98)
AD
(17-01)
AD
(10-02)
Normal
(15-02)
Normal
(47-97)
Normal
(03-00)
Anti-pE-Aβ
M87
Anti-pE-Aβ
M87
Anti-pE-Aβ
M87
Anti-pE-Aβ
M87
Anti-pE-Aβ
M87
Anti-pE-Aβ
M87
8.4 9.2
10.0 10.8
11.6
12.4
13.2
14.0 14.8 15.6
a b
100 μm
GFAP
+
Hem.
Hem.
Aβ3(pE)–x
WT TBA2.1
TBA2.1
tau KO
Ve (ml)
Figure 4 | Ab3(pE)–42in vivo. a, Cytosol obtained from human Alzheimer’s
disease (AD) and similarly aged normal brains (Supplementary Fig. 9) were
fractionatedbygelfiltration,andanalysedbydotblottingwithanti-pE-Aband
M87. Note the appearance of pE-Ab in LNO fractions, including those that
eluted at 12.4ml, especially in the Alzheimer’s disease samples. b, Three-
month-old TBA2.1 mice generating Ab3(pE)–42
(arrows), massive astrogliosis (GFAP) and neuron loss (Hem., haematoxylin
nuclear staining), none of which are evident in comparably aged wild-type
(WT) mice or TBA2.1/tau-knockout19(KO) hybrids.
18show amyloid-b deposits
RESEARCH LETTER
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5. Roberson, E. D. et al. Reducing endogenous tau ameliorates amyloid b-induced
deficits in an Alzheimer’s disease mouse model. Science 316, 750–754 (2007).
Jin, M. et al. Soluble amyloid b-protein dimers isolated from Alzheimer cortex
directly induce Tau hyperphosphorylation and neuritic degeneration. Proc. Natl
Acad. Sci. USA 108, 5819–5824 (2011).
Mori,H.,Takio,K.,Ogawara,M.&Selkoe,D.J.Massspectrometryofpurifiedamyloid
b protein in Alzheimer’s disease. J. Biol. Chem. 267, 17082–17086 (1992).
Saido,T.C.etal.Dominantanddifferentialdepositionofdistinctb-amyloidpeptide
species, AbN3(pE), in senile plaques. Neuron 14, 457–466 (1995).
Jawhar,S.,Wirths,O.&Bayer,T.A.Pyroglutamateamyloid-b(Ab):ahatchetmanin
Alzheimer disease. J. Biol. Chem. 286, 38825–38832 (2011).
10. Schilling, S. et al. Glutaminyl cyclase inhibition attenuates pyroglutamate Ab and
Alzheimer’s disease-like pathology. Nature Med. 14, 1106–1111 (2008).
11. Tabaton, M. et al. Soluble amyloid b-protein is a marker of Alzheimer amyloid in
brain but not in cerebrospinal fluid. Biochem. Biophys. Res. Commun. 200,
1598–1603 (1994).
12. Russo,C.etal.Pyroglutamate-modifiedamyloidb-peptides—AbN3(pE)—strongly
affect cultured neuron and astrocyte survival. J. Neurochem. 82, 1480–1489
(2002).
13. Schilling,S.etal.OntheseedingandoligomerizationofpGlu-amyloidpeptides(in
vitro). Biochemistry 45, 12393–12399 (2006).
14. Schlenzig, D. et al. Pyroglutamate formation influences solubility and
amyloidogenicity of amyloid peptides. Biochemistry 48, 7072–7078 (2009).
15. Levine, H. III. Thioflavine T interaction with synthetic Alzheimer’s disease
b-amyloid peptides: detection of amyloid aggregation in solution. Protein Sci. 2,
404–410 (1993).
16. Wang,X.M.etal.AnewmicrocellularcytotoxicitytestbasedoncalceinAMrelease.
Hum. Immunol. 37, 264–270 (1993).
17. Scudiero, D. A. et al. Evaluation of a soluble tetrazolium/formazan assay for cell
growth and drug sensitivity in culture using human and other tumor cell lines.
Cancer Res. 48, 4827–4833 (1988).
18. Alexandru, A. et al. Selective hippocampal neurodegeneration in transgenic mice
expressing small amounts of truncated Ab is induced by pyroglutamate–Ab
formation. J. Neurosci. 31, 12790–12801 (2011).
19. Tucker,K.L.,Meyer,M.&Barde,Y.A.Neurotrophins arerequiredfornervegrowth
during development. Nature Neurosci. 4, 29–37 (2001).
20. Rockenstein, E., Mallory, M., Mante, M., Sisk, A. & Masliaha, E. Early formation of
matureamyloid-bproteindepositsinamutantAPPtransgenicmodeldependson
levels of Ab1–42. J. Neurosci. Res. 66, 573–582 (2001).
21. Jawhar, S. et al.Overexpression of glutaminyl cyclase, the enzyme responsible for
pyroglutamate Ab formation, induces behavioral deficits, and glutaminyl cyclase
knock-out rescues the behavioral phenotype in 5XFAD mice. J. Biol. Chem. 286,
4454–4460 (2011).
22. Tanghe,A.etal.Pathologicalhallmarks,clinicalparallels,andvaluefordrugtesting
in Alzheimer’s disease of the APP[V717I] London transgenic mouse model. Int. J.
Alzheimers Dis. 2010 (2010).
23. Wilcock, D. M. et al. Progression of amyloid pathology to Alzheimer’s disease
pathologyinanamyloidprecursorproteintransgenicmousemodelbyremovalof
nitric oxide synthase 2. J. Neurosci. 28, 1537–1545 (2008).
6.
7.
8.
9.
24. He,W.&Barrow,C.J.TheAb3-pyroglutamyland11-pyroglutamylpeptidesfound
insenileplaquehavegreaterb-sheetformingandaggregationpropensitiesinvitro
than full-length Ab. Biochemistry 38, 10871–10877 (1999).
25. Wirths,O.etal.Intraneuronalpyroglutamate-Ab3–42triggers neurodegeneration
and lethal neurological deficits in a transgenic mouse model. Acta Neuropathol.
118, 487–496 (2009).
26. Gu ¨ntert, A., Dobeli, H. & Bohrmann, B. High sensitivity analysis of amyloid-b
peptidecompositioninamyloid depositsfromhuman and PS2APP mousebrain.
Neuroscience 143, 461–475 (2006).
27. Piccini, A. et al.b-Amyloid is different in normal aging and in Alzheimer disease. J.
Biol. Chem. 280, 34186–34192 (2005).
28. Hartlage-Ru ¨bsamen, M. et al. Glutaminyl cyclase contributes to the formation of
focal and diffuse pyroglutamate (pGlu)-Ab deposits in hippocampus via distinct
cellular mechanisms. Acta Neuropathol. 121, 705–719 (2011).
29. Vossel, K. A. et al. Tau reduction prevents Ab-induced defects in axonal transport.
Science 330, 198 (2010).
30. Wilcox, K. C., Lacor, P. N., Pitt, J. & Klein, W. L. Ab oligomer-induced synapse
degeneration in Alzheimer’s disease. Cell. Mol. Neurobiol. 31, 939–948 (2011).
Supplementary Information is linked to the online version of the paper at
www.nature.com/nature.
Acknowledgements The authors are grateful for support from the following sources:
the Alzheimer’s Association (grant 4079 to G.S.B.); the Owens Family Foundation
(G.S.B.); the Cure Alzheimer’s Fund (G.S.B., C.G.G.); NIH/NIGMS training grant T32
GM008136,whichfundedpartofJ.M.N.’sPhDtraining;NIH/NIAgrantR01AG033069
(C.G.G.); and the German Federal Department of Science and Technology grant
03IS2211F (H.-U.D.). Funding for the UCI-ADRC was provided by NIH/NIA grant P50
AG16573. We also thank H. Dawson and M. Vitek of Duke University for providing the
tau-knockout mice. This work fulfilled part of the requirements for the PhD earned by
J.M.N. at the University of Virginia. The technical assistance of A. Spano, H.-H. Ludwig,
E. Scheel and K. Schulz is gratefully acknowledged.
Author Contributions J.M.N. performed most of the biochemical and cell biological
experiments; S.S. was the principal force behind the experiments involving hAPPSL/
hQC and TBA2.1/tau-knockout mice, and was aided by B.H.-P. and H.C.; A.S. and T.W.
fractionated and analysed human brain extracts; E.S., K.T. and B.W. performed the
peri-hippocampal injection experiments; A.H. and C.G.G. produced and characterized
the M64 and M87 antibodies; R.R. and K.R. performed the electrophysiology
experiments; A.A., W.J. and S.G. performed and analysed the immunohistochemical
experiments on TBA2.1 and tau-knockout/TBA2.1 mice; G.S.B. and H.-U.D. initiated
and directed the project; G.S.B.was the principal writer of the paper; all of the authors
participated in the design and analysis of experiments, and in editing of the paper.
Author Information Reprints and permissions information is available at
www.nature.com/reprints. The authors declare no competing financial interests.
Readers are welcome to comment on the online version of this article at
www.nature.com/nature. Correspondence and requests for materials should be
addressed to G.S.B. (gsb4g@virginia.edu) or H.-U.D.
(Hans-Ulrich.Demuth@probiodrug.de).
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FULL METHODS
Thioflavin T Assays
Peptides were diluted from monomeric solutions in hexafluoroisopropanol (HFIP) into
PBS to a final peptide concentration of 5 µM and then were incubated at 37° C for the indicated
times. At each time point, samples of 100 µl each were placed in 96-well black-walled plates
(Corning) taken, 5 µl of 200 µM thioflavin-T (Sigma-Aldrich) were added to each well, which
were then assayed for fluorescence (450 nm excitation, 490 nm emission) using a SPECTRAmax
Gemini EM fluorescent plate reader (Molecular Devices).

Cell culture

embryos1 trypsinization and mechanical disruption. Cells were plated plated on a poly-L lysine
Neurons were extracted from the forebrains of E17-E20 C57BL/6 or Tau KO mouse
(Sigma-Aldrich) coated substrate in Neurobasal medium supplemented with Glutamax, glucose,
B27 supplement, penicillin/streptomycin (Invitrogen) and Cosmic Calf Serum (Thermo
Scientific). Four hours after plating, the medium was removed and replaced with otherwise
identical medium lacking cosmic calf serum. Cells were grown for 7-8 days prior to Aβ
treatment. Glial cells were extracted from whole brain homogenates of 2 day-old pups by
trypsinization and plated in Dulbecco’s modified Eagle’s medium supplemented with 10%
Cosmic Calf Serum and gentamycin. Following 7 days of growth, cultures were shaken to
remove loosely attached cells, split into fresh medium by trypsinization, and grown for 3-4 days
prior to Aβ treatment.

Cell Viability Assays
For light microscopic assays (as in Figure 1), primary neurons grown on poly-L lysine
coated glass cover slips were exposed to 1 µM calcein AM (Invitrogen) for 15 minutes at 37° C
after exposure to Aβ. Cells were then quickly washed in PBS and inverted onto slides for
imaging. Live cells were imaged using epifluorescence illumination with a YFP filter set
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mounted on a Zeiss Axiovert 100 inverted microscope. Images were captured by a Hamamatsu??  
9100-­‐13??  ImagEM??  cooled??  EMCCD.

For quantitation of cell viability primary neurons grown in black-walled 96-well tissue
culture plates (Corning) were assayed using Cell Proliferation Kit II (Roche) according to the
vendor’s instructions. This method measured reduction of XTT (sodium 3 ́-[1-(phenyl-
aminocarbonyl)-3,4-tetrazolium]-bis(4-methoxy-6-nitro) benzene sulfonic acid hydrate) by live,
but not dead cells, and spectrophotometric detection of the reduction product using an iMark
Microplate Absorbance Reader (Bio-Rad) to measure absorbance at 450nm. A reference reading
at 690 nm was subtracted from all measurements. The mean from a control condition, in which
all cells were killed, was also subtracted. Complete cell death was induced by addition of 10 µl
of 1M HCl at the same time as peptide treatment. Immediately prior to addition of XTT reagent,
10 µl of 1M NaOH was added to normalize pH. Unpaired, two-tailed t-tests were used to judge
statistical significance.

Aβ oligomers
Peptides were synthesized in 50 µmol scale on an automated Symphony synthesizer
(Rainin) applying a Fmoc-strategy. Aβ1-42 was synthesized on Fmoc-Ala-NovaSyn ® TGA resin
(Merck Biosciences). Gly-25 and Ser-26 were incorporated using isoacyl dipeptide Boc-
Ser(Fmoc-Gly)-OH (4 eq.). Amino acid coupling was achieved using HOBt (4 eq.) / DIPCDI
(4.4 eq.) for 2 × 45 min. The resulting 26-O-Isoacyl-β-amyloid(x-42) was purified by RP-HPLC
after deprotection. Subsequently, the depsipeptides were dissolved in 0.1 M ammonium
bicarbonate (pH 7.4) for 1 hour to initiate isoacyl conversion. The reaction was monitored by
analytical RP-HPLC. Analytical HPLC analysis was performed on a 4.6 × 150 mm Source 5RPC
column (5 µm; GE Healthcare) with a gradient made of solvent A (0.1% NH4OH in H2O at pH
9) and solvent B (acetonitrile / solvent A 60:40). In case of all N-terminal pyroglutamated
peptides the pGlu was incorporated as Boc-pGlu-OH.

Lyophilized, synthetic Aβ peptides were dissolved to 1 mM in 1,1,1,3,3,3-hexafluoro-2-
propanol (HFIP; Sigma-Aldrich). Oligomerization was initiated by adding the dissolved peptides
directly to neurobasal medium at final peptide concentrations of 1-10 µM, evaporating the HFIP
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using an air stream, and incubating the solutions at 37° C or 4° C for the indicated times.
Supplemental cell culture reagents were added following oligomerization, and when applicable,
after subsequent fractionation by gel filtration chromatography. Peptide-containing media were
added to cultures to achieve the noted final concentrations of total Aβ.

Rabbit anti-Aβ monoclonal antibodies
Rabbit monoclonal antibodies M64 and M87 were made under contract to Epitomics
(Burlingame, CA) using fibrillar Aβ1-42 as an antigen and immunizing New Zealand white rabbits
as previously described for preparing OC polyclonal serum2. Pools of hybridomas
(approximately 10,000) were screened for those expressing antibodies against Aβ1-42 fibrils, and
prefibrillar oligomers or monomeric Aβ, and 120 pools giving at least a 3-fold higher absorbance
than background in ELISA assays were selected for further analysis. Secondary screening
consisted of probing blots of a medium density array of 130 different preparations of fibrils,
prefibrillar oligomers and monomers of Aβ1-42, Aβ1-40, islet amyloid polypeptide, polyQ40,
overlapping 15 residue peptide segments of Aβ and amyloid-forming random peptides, and by
immunohistochemistry on human AD brain tissue. Pools giving a unique pattern of
immunoreactivity on the array or on immunohistochemistry were selected for cloning and further
characterization by western blotting and ELISA. Both M64 and M87 recognize aggregated, but
not monomeric Aβ, and their epitopes are 3EFRH6 and 3EFRHD7, respectively
(Suppplementary Fig. 4).

For epitope mapping, a peptide array (PepSpotsTM) consisting of a series of overlapping
10 mers from the -4 position of the Aß sequence to residue 46 covalently bonded via the
carboxyl terminus to a cellulose membrane was prepared by JPT Peptide Technologies, GmbH,
Berlin, Germany and used according to the manufacturer’s recommendations. Membranes were
incubated with 100 ng/ml of primary antibody and then 1 ug/ml goat anti-rabbit secondary
conjugated with alkaline phosphatase and visualized with TMB substrate (Promega, Madison,
WI).

For aggregation kinetics, Aβ1-40 was dissolved in a 1:1 mixture of water and acetonitrile,
aliquoted into 0.3 mg samples and lyophilized overnight. Prep A: 33 µL of 10% SDS was added
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to 0.3 mg lyophilized Aβ1-40 to obtain a 2 mM Aβ1-40 solution. This solution was then boiled for
10 minutes and diluted with 1.5 ml of water to obtain a 47 uM Aß solution, which was incubated
at room temperature without stirring for 7 days. Prep B: 0.3 mg lyophilized Aβ1-40 was
dissolved in 200 µl of HFIP and HFIP was evaporated under a stream of air to leave a dry film of
peptide in the centrifuge tube. This film was then dissolved in 33 µl of 100 mM NaOH to yield a
2 mM solution. This solution was incubated at room temperature for 15 minutes and then diluted
with 1.5 ml 20 mM sodium phosphate buffer to yield a final concentration of 47 uM Aβ. This
prep was then incubated at room temperature without stirring for 7 days. Prep C: 200 µl HFIP
was added to 0.3 mg Aβ1-40. This solution was allowed to incubate for 15 minutes at room
temperature in a centrifuge tube with the cap closed. Following the incubation, 700 µl of water
was added to the tube to yield a 47 uM Aβ solution in 22% HFIP. This prep was then kept in a
centrifuge tube with a perforated top under a fume hood at room temperature with stirring at 180
RPM for 7 days. Prep D: 33 µL 100 mM NaOH was added to 0.3 mg lyophilized Aβ1-40 to yield
a 2 mM solution. This solution was then incubated at R/T for 15 minutes. Following the
incubation, the solution was diluted with 1.5 mL of 10 mM HEPES/ 100 mM NaCl to yield a 47
uM solution and incubate at room temperature for 7 days without stirring.

Oligomer fractionation and immunoprecipitation
1 ml oligomer solutions of synthetic peptides were fractionated on a 30 x 0.8 cm
Superdex-75 column (GE Healthacare) using 50 mM ammonium acetate or neurobasal medium
(when added to cultures) as the mobile phase. Fractions of 1 ml each were collected, and native
samples from each fraction were lyophilized and re-solubilized with HFIP to restore peptides to
the monomeric state, as described earlier in the "Aβ oligomers" section. The monomeric
peptides were then applied directly onto nitrocellulose using a BioDot (Bio-Rad) dot-blot
manifold. Gel filtration fractions of human brain extracts (Figure 4a) were blotted directly onto
nitrocellulose, without prior lyophilization and re-solubilization with HFIP. All dot blots were
imaged on a Odyssey (LI-COR Biosciences) imaging station, and a standard curve of known
monomeric Aβ concentration was used for quantitation of gel filtration fractions of synthetic
peptides. Signal intensity was linear from a few ng to a few hundred µg of peptide, with the
exact range depending on which primary anti-Aβ antibody was used. Unpaired, two-tailed t-tests
were used to judge statistical significance for dot blot samples that were compared quantitatively.
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The mouse monoclonal antibodies, 4G8 and anti-human amyloidβ (N), were purchased from
Covance and Immuno-Biological Laboratories (clone 82E1), respectively. Immunoprecipitations
were performed by binding purified M64 or anti-human amyloidβ (N) IgG to protein A or
protein G magnetic beads (New England Biolabs) according to the manufacturers instructions.
Following antibody binding, peptides in tissue culture media were incubated with the beads and
then washed with PBS before collection of the beads using a magnet. Peptides were removed
from beads by addition of HFIP, which also returned them to the monomeric state.

The 23.6 ml (30 x 1.0 cm) Superdex-75 column was calibrated with both globular protein
standards (Sigma-Aldrich: aprotinin, MW 6500; horse heart cytochrome c, MW 12400; bovine
erythrocyte carbonic anhydrase, MW 29000; bovine serum albumin, MW 66000; and blue
dextran, MW 2000000) and globular dextran standards (Pharmacosmos: MWs 4400, 9890,
21400, 43500, 66700 and 123600). Equations relating elution volumes to MW were generated by
plotting Kav versus MW for each MW standard, where Kav = (Ve-Vo)/Vt-Vo), and Ve = elution
volume, Vo = void volume of the column and Vt = total column volume. Elution volumes were
determined by measuring absorbance at 280 nm for proteins and by a colorimetric assaay for
dextrans3

Tau-KO/TB2.1 Animals
The tau-KO mouse line Mapttm1(EGFP)Klt/J (Stock No. 004779) was purchased from The
Jackson Laboratory (JAX, Maine, USA). These mice have a C57BL/6 x 129S4/SvJae hybrid
background and were generated by knock-in of the EGFP coding sequence into the first exon of
the MAPT gene4. Homozygotes are viable, fertile, normal in size and do not display any gross
physical or behavioral abnormalities. No MAPT gene product is detected, while cytoplasmic
EGFP signal is detected in the CNS during development and at an adult age (JAX strain
datasheethttp://jaxmice.jax.org/strain/004779.html, The Tau-KO line was used in crossbreeding
experiments with TBA2.1 mice (genetic background C57Bl/6/DBA hybrids) yielding double
homozygous mice (Tau-KO/TB2.1) and control genotype combinations. TBA2.1 mice over
expresses the peptide AβQ3-42 driven by the Thy-1 promotor. The regulatory element flanks the
coding sequence for a fusion protein consisting of the pre-pro-peptide of murine thyrotropin
releasing hormone (TRH, Thyroliberin), fused to the N terminus of the modified human Aβ
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polypeptide, AβQ3–42. Prohormone convertase (PC) cleavage within the trans-Golgi and secretory
vesicles liberates the N-truncated Aβ species5.

Immunohistochemical Studies
For immunohistochemistry, mice were deeply anesthetized, transcardially perfused with
phosphate-buffered saline (PBS) and brains were removed. Tissue was then immersion-fixed in
IHC zinc fixative (BD Pharmingen), dehydrated, embedded in low-melting-point paraffin (DCS
Innovative Diagnostic Systems) and sectioned at 8 µm on a rotating microtome. After
deparaffinization, sections were incubated with the primary antibodies overnight at 4°C. The
following antibodies were used in this study: glia-specific antibody: GFAP (rabbit polyclonal,
Z0334; DAKO Cytomation, Glostrup, Denmark, dilution 1:5,000), pE3-Aβ-specific antibody:
Pyro-Glu Abeta (rabbit polyclonal, 218003; Synaptic Systems, Göttingen, Germany, dilution
1:500). For immunodetection, a biotinylated rabbit-specific IgG (Vector Laboratories,
Burlingame, CA, USA) was used, followed by diaminobenzidine staining (Vectastain ABC-Kit,
Vector Laboratories). 3,3’-diaminobenzidine (ImmPACT DAB Peroxidase Substrate, Vector
Laboratories) was used for visualization.

Injection of AD model mice with 5% pE-Aβ and immunmohistochemical analysis
Mice were anesthetized with isoflurane and mounted in a stereotaxic apparatus (David
Kopf Instruments, Tujunga, CA, USA). The scalp of each animal was retracted and the skull was
adjusted to place bregma and lambda in the same horizontal plane. Small burr holes were drilled
at the appropriate injection sites. Injectors (23 gauge) were inserted bilaterally at the following
positions relative to bregma (mm): AP -2, ML ±1.5, DV: -2.3. Solutions of co-oligomerized 5%
Aβ3(pE)-42 plus 95% Aβ1-42, or 0.1M PBS was infused into the hippocampus (1 µl/side; 0.1
µl/min). Infusion was followed by a ten minute diffusion period, during which injectors
remained in place. A dental cement cap (Harry J. Bosworth Company, Skokie, IL, USA) was
used to secure the incised scalp and protect the infused area. Mice were transcardially perfused
with 0.1 M PB followed by 4% paraformaldehyde at 8 or 18 weeks following the infusion
procedure. Brains were post-fixed in paraformaldehyde for 24 hours.

Analysis of hAPPSL/hQC mice
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Transgenic APPSL mice6 (C57BL/6xDBA background) were intercrossed with human QC
transgenic mice7 (C57Bl/6 background) for generation of double transgenic mice and littermate
control animals. Mice were housed in individually ventilated cages (IVCs) under a constant
light-cycle (12 hours light/dark). Normal tap water was available to the animals ad libitum.
Animals were housed in individual ventilated cages (IVCs) on standardized rodent bedding.
Each cage contained a maximum of five mice. The room temperature during the study was
maintained at 24°C and the relative humidity was maintained between 40 to 70 %. Animals were
housed under a constant day/night cycle (12 hours light/dark). Dried, pelleted standard rodent
chow (Altromin®) and normal tap water were available to the animals ad libitum.

The Morris water maze task was conducted in a black circular pool of 100 cm diameter.
Tap water was filled and a temperature of 22 ± 1°C was maintained. During the whole test
session the platform was located in the southwest quadrant of the pool. Each mouse had to
perform three trials on four consecutive days. A single trial lasted for a maximum of one minute.
During this time, the mouse had the chance to find the hidden, diaphanous target. If the animal
did not find the platform the investigator guided to or placed the mouse on it at the end of each
trial. After each trial, mice were allowed to rest on the platform for 10–15 sec. For the
quantification of escape latency (the time [second] - the mouse needed to find the hidden
platform and therefore to escape from the water), of pathway (the length of the trajectory [meter]
to reach the target) and of the abidance in the goal quadrant a computerized tracking system was
used. Monitoring stops when the mouse sits on the platform for more than 2 sec. At least one
hour after the last trial on day 4, mice had to fulfill a so-called probe trial (PT). During the probe
trial (PT), the platform was removed from the pool and the number of crossings over the former
platform position and the abidance in this quadrant was measured.

After the last behaviour test animals were sacrificed and blood, CSF and brains were
collected. Therefore, mice were sedated by standard inhalation anesthesia. Following blood
sampling, mice were transcardially perfused with physiological (0.9%) saline. Thereafter, brains
were removed and hemisected. The hemisected brain regions were frozen immediately and
stored at -80°C until shipment to the sponsor. The right hemisphere of all mice was prepared for
histology.
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To perform ELISA analysis, brain tissue was homogenized in TBS (20 mM Tris, 137
mM NaCl, pH 7.6) containing protease inhibitor cocktail (Complete Mini, Roche, Switzerland),
sonicated and centrifuged at 75,500 x g for 1 hour at 4°C. The supernatant was stored at –80°C
and Aβ peptides were sequentially extracted with TBS/1% Triton X-100 (TBS/triton fraction),
2% SDS in distilled water (SDS fraction), and 70% formic acid (formic acid fraction). The
combined SDS and FA fractions were considered as the insoluble pool of Aβ. Aβx–42 and Aβ3(pE)–
42 specific sandwich ELISAs (IBL-Hamburg, Germany) were performed according to the
manufacturer’s manual. In addition, the TBS fractions were applied to oligomer concentration
analysis, which is based on matrix-based isolation of aggregates, dissociation and
immunodection8. Briefly, using a proprietary sample enrichment protocol, only the aggregated
Aβ was isolated from TBS fractions. Each sample was then disaggregated to allow detection of
monomeric Aβ using an immunoassay based on an europium fluorescent beads coupled to the
4G10 antibody (N-terminal) and magnetic beads coupled to the antibodies 1F8 and 2H12 (C-
terminal) recognizing Aβ1-40 and Aβ1-42, respectively. The europium fluorescence intensity was
measured using Time Resolved Fluorescence (TRF). The signal/noise cutoff value for all
experiments was 2.0, equaling two times the background signal from buffer alone. The mean of
three determinations from one TBS sample was calculated and applied for comparison of the
experimental groups (Figure 5).

The right hemispheres of all mice were fixed by immersion in freshly prepared 4%
paraformaldehyde/PBS (pH 7.4) for one hour at room temperature. Thereafter brains were
transferred to a 15% sucrose PBS solution for 24 hours to ensure cryoprotection. On the next day
brains were frozen in isopentane and stored at -80° C until used for histological analysis. 15
cryo-sections per layer (altogether 5 layers), each 10µm thick were sagittally cut (Leica CM
3050S). Brain levels were chosen according to the morphology atlas “The Mouse Brain” from
Paxinos and Franklin (2nd edition). The cut of the five levels started with a random slice
corresponding to figure 105 (total appearance of the dentate gyrus), then sampling was continued
uniformly and systematically, always retaining 15 slices per level in series and discarding 100
µm in between the levels. Plaque load was determined with 6E10 primary antibody (Covance,
SIG-39320; 1:1000 dilution) directed against the human amyloid peptide (amino acids 1-16) as
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well as pGlu Abeta (anti-Abeta N3pE; antibody provided by Probiodrug) clone 6 and
ThioflavinS (Sigma ®) staining against beta-sheet structures in a double incubation.

For analysis of synaptic density, hippocampal CA3 and dentate gyrus regions were
quantitated in sections stained with synaptophysin monoclonal antibody (Chemicon; 1:5000
dilution). The number of synapses was quantified in 9 images per animal and region, whereas
three 1000-fold magnified images were recorded from the granular layer of the dentate gyrus
(medial blade) regions. Object count was classified into constituent and cohesive synapses, total
area of cohesive synapses was divided by the mean measured size of constituent synapses in the
image, the outcome was added to the number of constituent synapses and built the number of
total synapses.


Electrophysiological analysis of hippocampal slice cultures

(Institute breeding stock) as described previously9. Briefly, both hippocampi were isolated and
Hippocampal slices (400 µm thick) were prepared from 4-months-old male C57/Bl6 mice
transferred into a pre-chamber containing 8 ml permanently carbogen-gasified artificial
cerebrospinal fluid (ACSF), to allow Aβ application. The lyophilized Aβ peptides were dissolved
in HFIP to 1 mM. The solution was aliquoted, HFIP was evaporated and the peptide stored at -
80°C. Then, Aβ was dissolved (100 µM) in dimethylsulfoxide (DMSO; Sigma, St. Louis, MO),
sonicated and diluted to 20µM in F12/DMEM (without glutamine, Biochrom). Peptide-
containing media were added to the pre-chamber to achieve the final concentrations of total Aβ.
Slices were transferred into a submerged-type recording chamber and were allowed to recover
for at least 30 min before the experiment started. The chamber was constantly perfused with
artificial cerebrospinal fluid (ACSF) at a rate of 2.5 ml/min at 33±1 °C.

Synaptic responses were elicited by stimulation of the Schaffer collateral–commissural
fibers in the stratum radiatum of the CA1 region using lacquer-coated stainless steel stimulating
electrodes. Glass electrodes (filled with ACSF, 1–4 MΩ) were placed in the apical dendritic
layer to record field excitatory postsynaptic potentials (fEPSPs). The initial slope of the fEPSP
was used as a measure of this potential. The stimulus strength of the test pulses was adjusted to
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30% of the EPSP maximum. During baseline recording, single stimuli were applied every
minute. Once a stable baseline had been established, long-term potentiation was induced by
applying 100 pulses at an interval of 10 ms and a width of the single pulses of 0.2 ms (strong
tetanus) three times at 10 min intervals.

Human brain extracts
??  
Frozen brain tissue was obtained from the Institute for Memory Impairment and
Neurodegenerative Diseases, and was collected from individuals enrolled in the UC Irvine
Alzheimer Disease Center in full compliance with institutional review board (IRB), state of
California and US federal regulations. Soluble lysates were prepared from frozen frontal cortex
(Boradman’s B11) as previously described10. Briefly, frozen tissues were weighted, diced and
homogenized in freshly prepared ice-cold PBS, 0.02% NaN3, pH 7.4 with protease inhibitor
cocktail (4:1 PBS volume/brain wet weight). The samples were ultracentrifuged at 100,000 x G
for 1 hr at 4oC. The PBS soluble fraction was collected, the protein concentration normalized and
aliquoted and stored at –80oC for further testing.

Mouse husbandry
??  
All experiments involving mice were performed with full approval of the Institutional
Animal Use and Care Committee of the University of Virginia; the German animal protection
law §11; TVA 55.2-1-54-2531-135-07 allowance to Ingenium GmbH, Bavaria, Germany; the
ethics committee of the German federal state of Sachsen-Anhalt to the Institute for
Neurobiology, Magdeburg (performed in accordance with the European Communities Council
Directive 86/609/EEC); or in conformity with the Austrian Animal Experiments Act BGBl. Nr.
501, especially Part III. § 11 and Part V. § 15 und § 16.


References??  ??  
18.??   Alexandru,??   A.??   et??   al.??   Selective??   hippocampal??   neurodegeneration??   in??   transgenic??   mice??  
expressing??   small??   amounts??   of??   truncated??   Abeta??   is??   induced??   by??   pyroglutamate-­‐Abeta??  
formation.??  J.??  Neurosci.??  31,??  12790-­‐12801??  (2011).??  
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