JBC Papers in Press. Published on August 23, 2007 as Manuscript M705282200
The latest version is at http://www.jbc.org/cgi/doi/10.1074/jbc.M705282200
-1-
The β-propensity of Tau determines aggregation and synaptic loss
in inducible mouse models of Tauopathy
Katrin Eckermann1, Maria-Magdalena Mocanu1, Inna Khlistunova1, Jacek Biernat1,
Astrid Nissen1, Anne Hofmann1, Kai Schönig2, Hermann Bujard2, Andreas Haemisch4,
Eckhard Mandelkow1, Lepu Zhou3, Gabriele Rune3, Eva-Maria Mandelkow1*
1
Max-Planck-Unit for Structural Molecular Biology
Notkestrasse 85, 22607 Hamburg, Germany
2
Center of Molecular Biology (ZMBH), University of Heidelberg
Im Neuenheimer Feld, 69120 Heidelberg, Germany
3
Dept. of Neuroanatomy, University of Hamburg Medical School
Martinistr. 20, 20146 Hamburg, Germany
4
Central Animal Facility, University of Hamburg Medical School
Martinistr. 20, 20146 Hamburg, Germany
Neurofibrillary lesions are characteristic for
a group of human diseases, named
tauopathies, which are characterized by
prominent intracellular accumulations of
abnormal filaments formed by the
microtubule-associated protein Tau. The
tauopathies are accompanied by abnormal
changes in Tau protein, including
pathological conformation, somatodendritic
mislocalization, hyperphosphorylation, and
aggregation, whose interdependence is not
well understood. To address these issues we
have created transgenic mouse lines in
which different variants of full-length Tau
are expressed in a regulatable fashion,
allowing one to switch the expression on and
off at defined time points. The Tau variants
differ by small mutations in the hexapeptide
motifs that control Tau's ability to adopt a
β-structure conformation and hence to
aggregate. The "pro-aggregation" mutant
ΔK280, derived from one of the mutations
observed in frontotemporal dementias,
aggregates avidly in vitro, whereas the
"anti-aggregation"
mutant
ΔK280/PP
cannot aggregate because of two β-breaking
prolines. In the transgenic mice, the proaggregation Tau induces a pathological
conformation and pre-tangle aggregation,
even at low expression levels, the antiaggregation mutant does not. This
illustrates that abnormal aggregation is
primarily controlled by the molecular
structure of Tau in vitro and in the
organism. Both variants of Tau become
mislocalized
and
hyperphosphorylated
independently of aggregation, suggesting
that localization and phosphorylation are
mainly a consequence of increased
concentration. These pathological changes
are reversible when the expression of Tau is
switched off. The pro-aggregation Tau
causes a strong reduction in spine synapses.
Tau is a microtubule-associated protein that
was initially identified by its ability to drive
microtubule (MT) assembly in vitro (1). It
binds to tubulin via its repeat domain and
adjacent sequences (Fig. 1) and thereby
promotes MT nucleation and stabilization in
neurons, particularly in axons. Thus Tau plays
a crucial role in modulating MT dynamics and
supporting axonal transport (2-4). The protein
is encoded by a single gene on chromosome
17, alternative splicing leads to 6 isoforms
ranging from 352 to 441 amino acids. They
differ by the presence or absence of two Nterminal inserts (exons 2 and 3) and by the
second of four pseudo-repeats of 31 amino
acids in the C-terminal half (exon 10). The
efficacy of Tau in binding to MTs is influenced
by the degree of phosphorylation and the
exclusion/inclusion of exon 10. The majority
of phosphorylation sites are serine or threonine
residues followed by prolines (SP/TP motifs)
which lie in the domains flanking the repeats
and can be phosphorylated by several prolinedirected kinases. There are other non-SP/TP
sites whose phosphorylation causes the
detachment of Tau from microtubules. This
includes S214 and the KXGS motifs within the
repeats (S262, S293, S324, S356) (5). Most of
these sites are "hyperphosphorylated" in
Alzheimer's disease (6). Tau belongs to the
Copyright 2007 by The American Society for Biochemistry and Molecular Biology, Inc.
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Running title: β-structure and aggregation in mouse models of Tauopathy
*Address correspondence to: Eva-Maria Mandelkow, Email: mandelkow@mpasmb.desy.de
-2-
Whereas AD is characterized by the
coexistence of two types of protein aggregates
(amyloid fibers composed of Aβ, and PHFs
composed of Tau), there are several
neurodegenerative disorders dominated by Tau
aggregates and hence termed "Tauopathies"
(10,11). The main link between AD and other
Tauopathies is the presence of aggregated Tau
protein. Several transgenic mouse models have
been developed in the past to unravel the
underlying mechanisms of Tau pathology in
AD and other Tauopathies (12-23). The
principle behind most models was the
overexpression of Tau, assuming that this
would accelerate aggregation. An alternative
was the overexpression or activation of
kinases, assuming that this would lead to
hyperphosphorylation and hence aggregation
of Tau (18,24,25). The expression of human
Tau isoforms met with limited success with
regard to Tau aggregation, presumably because
of the high solubility of Tau and the short
lifetime of mice. However, the approaches
could be improved by: i) Tau mutants with a
higher aggregation potential, e.g. P301L or
P301S: (20,26,27) P301S: (21,22,28), ii)
combining Tau expression with enhanced Aβ
activity, by using APP or PS mutations
(19,29), or iii) expressing several Tau isoforms
analogous to human brain (16). With these
tools, part of the Tauopathies and AD-like
pathology can be approximated in mice, such
as
missorting,
hyperphosphorylation,
aggregation
(ranging
from
pretangle
accumulations of Tau to full neurofibrillary
tangles), and age-dependent spreading of the
pathology in the appropriate brain areas. These
studies revealed that high concentrations of
(mutant) Tau can generate Tauopathy, that Aβ
exacerbates the degeneration, and that
Tauopathy leads to synaptic dysfunction and
behavioral deficits.
Nevertheless, several issues remain poorly
understood. This includes the mode of Tau
toxicity and the relationship between Tau
levels, Tau conformation, phosphorylation, and
aggregation. We set out to test these questions
with new transgenic mouse models designed
on the basis of our previous experience with
Tau properties in vitro: (1) We use a
regulatable expression system (Tet-Off) which
allows one to switch the Tau expression on and
off at defined time points and avoids artefacts
of expression at early age. (2) We use in
parallel two variants of Tau which are similar
with regard to microtubule interactions but
opposite with regard to aggregation. The first
variant is full-length Tau (hTau40, 2N4R)
carrying the FTDP-17 mutation ΔK280 in the
hexapeptide motif of the second repeat. The
deletion of K280 facilitates the formation of ßstructure and strongly promotes Tau
aggregation ("pro-aggregation mutant") (8,30).
The second variant contains the same ΔK280
mutation and two additional proline mutations,
one in each of the two hexapeptide motifs
(I277P and I308P). They disrupt β-structure
and thus abrogate aggregation ("antiaggregation" mutant). For both types of
mutants we used a Tet-Off inducible system
which combines the tetracycline-responsive
transactivator and the CaMKIIα-promoter (31)
for the temporal and regional expression of
Tau. The temporal control of the Tet-Off
system allows us to study age-dependent
effects and reversibility of Tau pathology. The
CaMKIIα-promoter enables us to achieve a
preferential expression of the transgene in the
entorhinal region and hippocampus so that the
spatial distribution of the human Tau mutants
in our model is similar to that in AD. With
these mouse models we show that pathological
conformation, aggregation, and loss of
synapses depend mainly on the β-propensity of
the protein. By contrast, hyperphosphorylation
and somatodendritic missorting are observed
with both Tau mutants, indicating that this
depends mainly on Tau concentration but not
on aggregation. All changes can be reversed by
switching off the Tau expression. Thus,
strategies to prevent Tau aggregation should
also protect neuronal integrity.
Downloaded from www.jbc.org by Eckhard MANDELKOW PHD on August 24, 2007
class of natively unfolded proteins, it contains
very little secondary structure, has a
hydrophilic and basic character, and is highly
soluble (7,8). In spite of this, Tau aggregates
into abnormal "paired helical filaments" in
Alzheimer's disease which can coalesce into
"neurofibrillary tangles". The aggregation is
based on short hexapeptide motifs in repeats
R2 and R3 which are prone to form β-structure
(8). Tau is normally confined to axons, but in
diseased neurons it becomes missorted to cell
bodies and dendrites. Thus, the three most
visible changes in Alzheimer Tau are its
hyperphosphorylation,
aggregation,
and
mislocalization. The progressive spreading of
neurofibrillary tangles correlates well with
cognitive deficits and neuronal loss in AD (9).
-3EXPERIMENTAL PROCEDURES
Animals: The activator mouse line (CaMKIIαtTA mice) in which the tTA transgene is under
control of the calcium/calmodulin kinase IIa
(CaMKIIα) promoter was a gift from Dr. E.
Kandel, Columbia University, New York, (31).
For the generation of responder mice we used
the bidirectional plasmid carrying the
bidirectional tetO responsive CMV promoter
(Ptet-bi), followed by both a Tau mutant in one
direction and luciferase reporter sequences in
the other. The double transgenic mice were
generated by crossing the activator and
responder mice, resulting in double transgenic
progeny constitutively expressing both
transgenes, the mutant Tau and luciferase. The
expression can be switched on by withdrawal
of doxycyclin, and switched off by addition of
doxycycline (200 µg/ml in the drinking water)
(Tet-Off system). All animals were handled
according to standards and guidelines based on
the German Animal Welfare Act. The
offsprings of mice were screened by PCR
using the primer pair 5’-AAT GAG GTC GGA
ATC GAA GG-3’ / 5’-TAG CTT GTC GTA
ATA ATG GCG G-3’ for activator genes and
the two primers pairs 5’-GAC CTT CCG CGA
GAA CGC CAA A-3’ / 5’-AAG AAC AAT
CAA GGG TCC CCA-3’ and 5’-CCA GCC
TGG AAG ACG AAG CT-3’ / 5’-GCG GAA
GAC GGC GAC TTG GG-3’ for responder
transgenes.
Luciferase reporter gene assay: Luciferase
activity was used to filter out positive founder
animals to ensure that the integration site is not
within a silencer region. To make sure that the
effects in switched-off animals are not due to
Tissue preparation: In order to quantify the
species of Tau and the extent of Tau
aggregation, two methods were used for the
preparation of the tissue, the sarkosyl
extraction method (33) and the sequential
extraction with "reassembly buffer" (RAB),
"radio immunoprecipitation assay buffer"
(RIPA) and 70 % formic acid (FA) (14). For
both protocols, the brain was dissected into the
cortex, the hippocampus and the rest of the
brain.
RAB-RIPA-FA extraction: The tissue was
homogenized in 2 volumes RAB (0.1 M MES
pH 7, 1 mM EGTA, 0.5 mM MgSO4, 0.75 M
NaCl, 0.02 M NaF, 1mM PMSF and protease
inhibitors (Complete Mini, Roche) using a
DIAX 900 homogenizer (Heidolph) and
centrifuged for 20 minutes at 50,000 x g and 4
°C. The RAB supernatants were collected. The
pellets were suspended in 2 volumes RIPA (50
mM TRIS pH 8, 150 mM NaCl, 1 % NP-40, 5
mM EDTA, 0.5 % sodium deoxycholate, 0.1
% SDS) and centrifuged as above. The RIPA
supernatants were also collected. The pellets
were resuspended in 0.6 volumes 70% formic
acid and again centrifuged as above. The
supernatants were dialyzed against 50 mM
TRIS pH 7.4, 1 mM dithiothreitol, 0.1 mM
PMSF and used for immunoblot analysis
together with the RAB and RIPA supernatants.
Sarkosyl extraction: The tissue was
homogenized (DIAX 900, Heidolph) in 3
volumes (for cortex) and 10 volumes (for
hippocampus) of buffer H (10 mM TRIS pH
7.4, 0.8 M NaCl, 1 mM EGTA, 10 % sucrose,
1 mM PMSF, protease inhibitors). The
homogenate was kept on ice for 20 minutes
and centrifuged for 20 minutes at 27,200 x g
and 4°C. The pellets were homogenized a
second time in the same volumes of buffer H
and centrifuged as above. The supernatants
from both centrifugation steps were combined
(S1+S2). The pellets (P2) were resuspended in
3 volumes of buffer III (0.5 M NaCl, 5 mM
Downloaded from www.jbc.org by Eckhard MANDELKOW PHD on August 24, 2007
Transgenic constructs: The bidirectional
transcription unit encoding the human longest
Tau CNS isoform with deletion of lysine 280
(hTau40/ΔK280) was generated by PCR based
site-directed
mutagenesis
and
PCR
amplification. This cDNA was inserted at the
ClaI and SalI restriction sites into the pBI-5
bidirectional expression vector carrying the
reporter luciferase gene (32). The resultant
plasmid DNA was digested with XmnI and
AseI enzymes and 5.75 kb fragment containing
the Tet-operon–responsive element (tetO),
bidirectional
CMV
promoter
(Ptet-bi),
hTau40/ΔK280, and luciferase sequences, was
used for the introduction by microinjection into
fertilized DBA eggs.
silenced integration site but due to the
switched off Tau gene, fibroblasts from mouse
ear tissue were cultivated, transfected with 400
ng plasmid encoding the reverse transactivator
protein (rtTA) and induced with 1 mg/ml
doxycycline. 18 hours later cells were
harvested, lysed and luciferase activity was
measured with the Promega kit according to
the manufacturers protocol. Non-induced
fibroblasts served as a reference.
-4EGTA, 50 mM Na-PIPES, 50 mM NaF, 1 mM
Na3VO4, 5 mM DTT, 5 mM Na2H2PO7, 500
µM PMSF, 1 µM microcystin, 10 µg/ml
leupeptin, aprotinin and pepstatin A). The
combined supernatants S1+S2 were mixed
with 1% N-lauroylsarcosinat (sarcosyl) and 1%
mercaptoethanol, incubated for at least 1 hour
at 37°C and centrifuged for 35 min at 150,000
x g at room temperature. The supernatants S3
were collected. The pellets (P3) were
resuspended in 0.5 volumes TBS (10 mM
TRIS, 154 mM NaCl). All fractions (S1+S2,
P2, S3 and P3) were used for immunoblot
analysis.
Immunohistochemistry:
Brains
from
hTau40ΔK280 Tg mice and control animals
were either immersion-fixed in 3.5%
phosphate-buffered
formaldehyde
(29
mM NaH2PO4*H2O, 45.8 mM Na2HPO4 pH
7,0), or they were from animals that had been
perfused
transcardially
with
4%
paraformaldehyde in PBS (Biochrom AG).
Histological
staining
and
immunohistochemistry were performed on 5
µm paraffin sections. Paraffin was removed
from the sections by immersing them in xylole
substitute (Fluka), followed by sequential
incubation in a descending series of ethanol.
Endogenous peroxidase activity was blocked
Antibodies: K9JA (pan-Tau antibody from
DAKO, A0024, recognizes human and mouse
Tau), MC-1 (pathological Tau conformation)
and PHF-1 (phospho-S396+S404) (both gifts
from P. Davies, Albert Einstein College), 12E8
(phosphorylated KXGS motifs, pS262, pS356,
gift from P. Seubert, Elan Pharma). pS199,
pT231,
pS214
and
pS46
recognize
phosphorylation at single SP or TP sites
(custom-made, Eurogentec), HT-7 (PerBio)
and T14 (Zymed) are human Tau specific.
AT180 (pT231+pS235) and AT8 (pS202+
pT205) recognize dual phosphorylation sites
(PerBio).
Synapse count: Animals were perfused
transcardially with 4% paraformaldehyde in
phosphate-buffer (50 mM NaH2PO4, 50 mM
Na2HPO4 pH 7.4), and analysis of synapses
was performed as described (34). Briefly, 400
µm hippocampal slices after fixation in 1%
glutaraldehyde, 1% paraformaldehyde in 0.1M
phosphate buffer were postfixed in 1% OsO4,
dehydrated in ethanol and embedded in Epon
820. Blocks were trimmed to contain only the
stratum pyramidale and radiatum of the CA1
region. Ultrathin sections were cut on a
Reichert-Jung OmU3 ultramicrotome. The
sections were stained with uranyl acetate,
followed by lead citrate. The spine synapse
density was calculated using stereological
methods as described (35).
Downloaded from www.jbc.org by Eckhard MANDELKOW PHD on August 24, 2007
Immunoblot analysis: Fractionated tissue
extracts were dissolved in sample buffer
containing 2% β-mercaptoethanol and 2.5 %
SDS and boiled. Equal volumes of the samples
were separated on 8-10 % SDS-PAGE gels and
transferred onto polyvinylidenfluoride (PVDF)
membranes using blotting buffer (38.4 mM
TRIS, 31.2 mM glycine, 0.024 % SDS, 20 %
methanol) in a semi-dry chamber. After
blocking with TBS + 5% non-fat milk + 0.1%
Tween-20, the membranes were incubated with
various primary antibodies overnight at 4°C
and subsequently incubated with peroxidaseconjugated secondary antibodies (DAKO, antimouse and anti-rabbit 1:5,000) for 1 hour at
room temperature. Bound antibodies were
detected
using
the
enhanced
chemiluminescence
system
ECL
Plus
(Amersham Biosciences). The intensity of
immunolabeling was quantitatively analyzed
with the imaging system LAS 3000 (Fuji). In
some cases PVDF membranes were stripped
(62.5 mM TRIS pH 6.8, 2% SDS, 0.7% βmercaptoethanol for 30 minutes at 60°C) and
used several times with different primary
antibodies.
using 0.6% H2O2 in 100% ethanol as an
intermediate step. Epitope retrieval was done
dependent on the primary antibodies and
performed in citrate buffer (1.8 mM citric acid,
8.2 mM sodium citrate pH 6) for 5 minutes at
95°C. The sections were incubated with
primary antibodies diluted in TBS/Triton (50
mM TRIS pH 7.6, 145 mM NaCl, 0.1% Triton
X-100) for 1 hour at room temperature. All
following steps were done using the Vectastain
Elite ABC Kit Universal (Vector Laboratories)
according to the manufacturer's protocol. 3,3’diaminobenzidine (DAB) plus solution
(DAKO) served as substrate for the
peroxidase. In some experiments the sections
were counterstained with Mayer's Hemealaun
(MERCK). Sections were dehydrated in an
ascending series of ethanol and xylol substitute
and mounted in Permount (Fisher Scientific).
Gallyas silver staining was done on 5 µm
paraffin sections as described (9).
-5RESULTS
Inducible transgenic mice
Tau pre-tangle
transgenic mice
aggregation
in
the
The use of the longest human Tau isoform as a
transgene made it possible to discriminate
between the human Tau protein (Mr ~67 kDa)
and the three endogenous mouse Tau isoforms
(45 - 55 kDa, Fig. 2A,C) which represent the
three 4-repeat isoforms typical of adult rodent
brain (40). This allowed the reliable
quantification of the ratio of human to
We investigated the solubility of Tau in the
two transgenic mouse lines hTau40ΔK280 and
hTau40ΔK280/PP (pro- and anti-aggregation
mutants). Across all ages the majority of the
human pro-aggregation Tau was recovered in
the soluble fractions (Fig. 2A+B). A minor part
of Tau was detected in the sarkosyl-insoluble
fractions, but this fraction clearly increased
with age, from 2% of the total human Tau at 4
months to 13% at 8 months. In very old mice
(>24 months Tau expression) we found up to
18% (Fig. 2C). We next analyzed the
endogenous mouse Tau in the different
fractions (sarkosyl soluble and insoluble). Like
the human Tau, endogenous mouse Tau was
present mainly in the soluble fractions, but
small amounts (~ 1-9 % of total mouse Tau)
were detected in the sarkosyl-insoluble pellets.
As a control, non-transgenic animals had no
detectable insoluble Tau (Fig. 2B). We
conclude that the additional level of human
Tau caused a time-dependent aggregation, and
that both mouse and human Tau partially coaggregated in the insoluble fractions of
transgenic animals.
We compared these results with the solubility
of Tau in transgenic mice expressing the antiaggregation mutant hTau40ΔK280/PP. This
protein was recovered entirely in the soluble
fractions, even at 24 months of age, and there
was no detectable sarkosyl-insoluble fraction.
The same was true for the endogenous mouse
Tau (Fig. 2A, B). This shows that elevation of
Tau as such is not sufficient for inducing
aggregation; the additional requirement is that
Tau must allow a β-conformation in the
hexapeptide motifs to support self-propagating
assembly. We further conclude that the
endogenous mouse Tau, which contains the
hexapeptide motifs in a wild-type form and
therefore is in principle capable of aggregation
(41), cannot persuade the exogenous antiaggregation Tau to aggregate. It is remarkable
that the behavior of Tau in the transgenic mice
corresponds exactly to the behavior in vitro,
except that the time scales are longer.
Downloaded from www.jbc.org by Eckhard MANDELKOW PHD on August 24, 2007
We generated two inducible transgenic mouse
models with animals expressing hTau40 (the
longest isoform in human CNS, 2N4R)
carrying the deletion mutation ΔK280 (Fig.
1A). This mutation is known from a case of
FTDP-17 (36) and strongly increases the
tendency of Tau to aggregate into PHFs in
vitro because it enforces the β-structure around
the hexapeptide motif in repeat 2 ("proaggregation mutant"). By contrast the
additional point mutations I277P (in R2) and
I308P (in R3) abrogate Tau's ability to form
PHFs ("anti-aggregation mutant") because the
prolines act as β-breakers (8,30). We used a
transgenic mouse where the tetracycline
transactivator (tTA) gene (37) was placed
downstream of the CaMKIIα promoter in
order to restrict the expression of tTA protein
to the forebrain (31). The choice of the
CaMKIIα promoter, rather than the Thy-1
promoter used in earlier studies, (12,27,38)
was dictated by the need to avoid the
expression of Tau in the spinal cord where it
would impair axonal traffic and cause motor
neuron defects (15,39). The second mouse
contained
the
responder
tau
cDNA
downstream of the bidirectional tTA
responsive promoter Ptetbi-1 in order to enforce
a tTA-dependent Tau expression. The two
types of mice harboring the Tau or activator
transgenes were bred to generate bigenic
offspring. Expression was repressed by
doxycyclin during gestation and for 6 weeks
postnatally to avoid any disturbances during
development to adulthood. As expected,
transgenic Tau protein expression in bigenic
mice was restricted to the forebrain with
highest levels in the hippocampus, the cortex,
the striatum and the olfactory bulb, and
occurred predominantly in neurons (Fig. 1B).
endogenous mouse Tau by densitometry of the
bands on gels or western blots with one panTau antibody. The expression ratio of total
human Tau over endogenous Tau in transgenic
mice varied from 1 to 3 fold for both mutants.
The relatively low overexpression ensures that
other Tau-induced changes are minimized.
-6Similar results were obtained when Tau was
sequentially extracted with the different
buffers RAB, RIPA, and formic acid described
previously (14). In the case of the proaggregation mouse line, most of the protein
was soluble and detected in the first two
fractions RAB and RIPA, but in older animals
(> 5.5 months) it began to appear in the
insoluble fraction FA. Here, too, mouse Tau
co-aggregated with human Tau in pre-tangles.
By contrast, the anti-aggregation mutant
remained soluble at all extraction stages (data
not shown).
Early pathological hallmarks of human Tau
Immunohistochemistry with antibody MC-1
revealed an abnormal conformation of Tau,
especially in the hippocampus and cortex
where it labelled apical dendrites and cell
bodies (Fig. 4A). The MC-1 staining started
very early at 3 months of gene expression and
increased with advancing age. By contrast, no
conformationally changed human Tau was
detected in the brains of anti-aggregation mice
(Fig. 4B). Similarly, we noticed MC-1 positive
Tau species in the soluble fractions of the
sarkosyl extraction or in RAB/RIPA fractions
but not in the insoluble fractions of the proaggregation mutant mice. No MC-1 positive
For the pro-aggregation mutant mice, an
increase in Tau phosphorylation was initially
observed at 3 months after induction of Tau
expression with the antibody 12E8 (Fig. 5).
This antibody recognizes two KXGS motifs in
the repeat domain (pS262, pS356) whose
phosphorylation causes the detachment of Tau
from microtubules (5,44). On immunohistochemically stained sections of the proaggregation mutant mice we found slight
phosphorylation at S262 after 3 months of
gene expression, increasing at later stages (6
months) with high levels in the apical dendrites
of the hippocampal cells (Fig. 5A, B). Only a
few cell bodies were stained in this area.
Phosphorylated Tau species were also detected
in anti-aggregation mutant mice, although at
lower levels (Fig. 5C).
In addition, in the pro-aggregation mutant
mouse Tau was highly phosphorylated at many
single SP or TP sites which occur mainly in
regions flanking the repeat domain, including
the site S214 (a non-SP site). The
phosphorylation was initially detected at 3
months and remained stable with age. We
observed phosphorylation at S46, S199, S214,
T231, or at the AT180 epitope (phosphoT231+S235) with different intracellular
localization of the phosphorylated Tau species.
An antibody against pT231 showed that most
of the phosphorylated Tau was localized in the
cell bodies of the pyramidal layer (Fig. 6A). In
some cases we found also the proximal
processes of the cells stained. Control animals
showed no staining with the phosphorylationdependent
antibodies.
The
dual
phosphorylation
at
T231/S235
was
investigated with the antibody AT180. Similar
to the single phosphorylation at T231 we found
most of the AT180-Tau species in the cell
bodies of the pyramidal cells (Fig. 6C, 9A).
Substantial staining was observed with an
antibody recognizing pS199. Most of the
phosphorylated Tau was localized in the apical
dendrites and the cell bodies of both the
hippocampal and the cortical neurons (Fig. 6E,
9C). The distribution of phosphorylated Tau
(pS199) was not restricted to certain areas but
rather occurred in the whole hippocampus and
cortex. By contrast, immunohistochemical
staining of phosphorylated S46 revealed a
completely different pattern; it was almost
exclusively present in the stratum lucidum of
Downloaded from www.jbc.org by Eckhard MANDELKOW PHD on August 24, 2007
The formation of neurofibrillary tangles in AD
is preceded by a "pretangle" stage
characterized by a translocation of Tau from
the axon to the somatodendritic compartment,
a "pathological" conformation of Tau detected
by certain antibodies, e.g. MC1, Alz-50 (42),
and elevated phosphorylation at several sites
(6,43). Analogous observations were made in
the transgenic mice expressing the proaggregation mutant Tau. Elevated human Tau
(detected by the pan-Tau antibody K9JA) was
seen in the dendritic and cell body layers of the
hippocampus such as stratum radiatum,
molecular layer lacunosum, pyramidal cells of
CA1 to CA3, granule cells of the dentate
gyrus, hilus cells and subiculum (Fig. 3A,B).
Human Tau was likewise expressed in the
cortex of these animals (Fig. 3C). By contrast,
in control animals, the mouse Tau appeared
pronounced only in the stratum lucidum, the
location of axonal mossy fibers (Fig. 3E). The
anti-aggregation human Tau had generally a
similar distribution as the pro-aggregation
mutant, except that it was lower in the
dendrites of the hippocampus, especially the
stratum radiatum (Fig. 3D).
species could be detected in anti-aggregation
mice (which contain only soluble fractions).
-7the hippocampus (CA3 mossy fibers),
indicating a mostly axonal distribution of this
phospho-Tau species (Fig. 6G). A similar
axonal distribution in the mossy fibers was
seen for pS214 in the pro-aggregation mutant
(Fig. 6I). This residue, together with pT212,
creates the epitope of antibody AT100, one of
the most characteristic antibodies for
Alzheimer Tau (45,46), yet AT100 reactivity
was not seen at all in our mouse models.
We compared the phosphorylation of proaggregation and anti-aggregation Tau at
different SP/TP sites. The anti-aggregation
mutant showed a similar phosphorylation at
T231, S199, S214, S46 or the AT180 epitope
although the phosphorylation was mostly
weaker than in the pro-aggregation Tau mice
(Fig. 6B, D, F, H, J). In contrast to these single
phosphoepitopes
we
did
not
find
phosphorylation at the dual sites of AT8 or
PHF-1 in anti-aggregation mutant mice, nor
Gallyas silver staining, even in very old mice.
Overall, these observations suggest that the
elevation of Tau leads to increased early
phosphorylation at various single SP/TP sites
in the regions flanking the repeats, combined
with a gradual increase of conformationally
altered pre-tangle Tau. However, the
appearance of silver-stained tangles correlates
Reversibility of Tau pathology
One of the major aims of this study was to
determine if and to what extent the Tauinduced pathological changes are reversible
(pathological
conformation,
missorting,
hyperphosphorylation, and aggregation). This
was investigated by first switching on the Tau
expression by withdrawal of doxycycline, then
switching the expression off by addition of
doxycyclin. Switching off the Tau gene
expression led to an almost complete reduction
of human Tau levels in the mouse brains
within 6 weeks (Fig. 7C+D). Interestingly, this
decrease was more pronounced in the soluble
than the insoluble fractions. Human Tau
disappeared from the dendrites in the stratum
radiatum and stratum lacunosum-moleculare
(Fig. 7A, B). Cell layers became completely
free of human Tau. Solely in the stratum
lucidum, some Tau remained after switching
off the human Tau gene, presumably due to
endogenous mouse Tau.
Switching off the human Tau gene expression
resulted in the complete reversibility of the
pathological conformational change of the
protein, the pre-tangle aggregation, as well as a
reversal of the hyperphosphorylated state of
human Tau. MC1 positive Tau became
undetectable after switching off the Tau gene
for 6 weeks and disappeared independently of
the age of the transgenic animals (Fig. 9G, H).
Similar results were obtained for the cases of
the phosphorylation-dependent antibodies
pS46, pS199, pS214, pT231, and AT180.
While the staining at these sites became
pronounced after 6-8 months of Tau
expression, the switching off of the Tau gene
led
to
complete
clearance
of
the
phosphorylated Tau from dendrites and cell
bodies at all ages of the mice (Fig. 9A-F). The
reversibility of the phosphorylation was
furthermore independent of the previous
intracellular localization of the Tau species
(cell bodies, dendrites) detected with the
different antibodies. This coincides with the
overall clearance of Tau protein from the
hippocampus after switching off the Tau gene.
Biochemical analysis of the MARK target site
serine 262 and other KXGS motifs showed a
complete reversibility of human Tau
phosphorylation at these sites (Fig. 8C+D).
Downloaded from www.jbc.org by Eckhard MANDELKOW PHD on August 24, 2007
We investigated the phosphorylation of Tau at
many other sites which are considered to be
relevant for later stages of Tau aggregation and
tangle formation. In 1-18 month old proaggregation mice no phosphorylation of Tau at
the dual sites pT212/pS214 (AT100),
pS202/pT205 (AT8), or pS396/pS404 (PHF1),
as well as the single site S422.
Immunohistochemical studies with these and
other antibodies revealed that in most of our
transgenic animals, Tau did not undergo
neurofibrillary tangle formation. Consistent
with that we found very little Tau species
stainable by Gallyas silver or Thioflavin S,
even though we detected routinely Tau in the
sarkosyl pellet, considered to be mostly pretangle aggregates. Only in very old mice, after
longer and higher Tau expression (> 24
months) we found phosphorylation of proaggregation mutant Tau at the epitopes of AT8
and PHF-1 as well as tangles stained by
Gallyas silver (Fig. 6K-N). This indicates that
the pre-tangles eventually convert to tangle
pathology if the concentration of mutant Tau
becomes high enough.
closely with hyperphosphorylation at the dual
sites AT8 and PHF1 in very old mice (Fig. 6).
-8The upper Tau band (corresponding to human
Tau) disappeared independently of the length
of Tau gene expression. However, the lower
bands
(corresponding
to
endogenous
phosphorylated mouse Tau) did not vanish
upon switching off the expression of human
Tau, indicating that some of the mouse Tau
still remained phosphorylated at KXGS motifs.
This reversibility was confirmed in the
immunohistochemical
staining.
Tau
phosphorylated at the MARK target sites was
no more detectable in neuronal cells after
switching off the human Tau gene for 6 weeks
(Fig. 8A+B).
Loss of synapses
DISCUSSION
Tau protein undergoes several pathological
changes in Alzheimer's disease and other
Tauopathies, most notably aggregation,
hyperphosphorylation, and missorting to the
somatodendritic compartment. The linkage
between these properties is not well understood
at present. The purpose of this study was to
separate out the effects of aggregation. To this
end, we created two new transgenic mouse
models based on the structural properties of
Tau. In the first model we expressed hTau40,
the longest isoform in human CNS, with the
deletion mutation ΔK280 known from FTDP17 (36). This mutation, located in the
hexapeptide motif of repeat R2, is a potent
enhancer of Tau aggregation since it increases
the propensity of Tau for β-structure (hence
termed "pro-aggregation" mutant), (30). In the
The transgenes were expressed via the
CaMKIIα promoter, for two reasons: First, the
expression of Tau was directed to the forebrain
in order to achieve a realistic model for AD
and other Tauopathies. The second aim was to
avoid interference with spinal cord and motor
neuron pathology. In some earlier mouse
models, Tau was expressed under the Thy-1 or
prion promoters (12,14,15,26,27,38) which
resulted in motor neuron defects and precluded
the analysis of cognitive deficits. The likely
cause is that the elevation of Tau tends to
inhibit axonal traffic by interfering with
microtubule-based traffic (39,49), which
becomes most noticeable in the case of long
axons such as the motor neurons (50).
These features were combined with regulatable
expression using the Tet-Off system, whereby
the transgene is expressed only in the absence
of doxycycline (37). This served a threefold
purpose: First, during brain development, Tau
undergoes major changes, notably a strong
upregulation in neurons, redistribution from
ubiquitous to axonal, reduction of the
embryonically high level of phosphorylation,
and diversification of isoforms by alternative
splicing (2,51). However, AD is a disease of
advanced age, and therefore it is preferable to
allow normal development into adulthood
before switching on transgenic Tau. The
second purpose is to allow Tau expression and
abnormal changes to take place, and then to
switch the expression off in order to observe
recovery of the brain. Thirdly, the Tet-off
system enables controlling the level of
exogenous Tau via the doxycycline
concentration. This allows one to test how the
age of onset of Tau expression or the Tau
concentration affects pathology.
Generation of the tau transgenic mouse models
are valuable tools for the research of the
neurodegenerative diseases. The principle
behind most models was the overexpression of
Downloaded from www.jbc.org by Eckhard MANDELKOW PHD on August 24, 2007
The loss of synapses is known to precede
neuronal degeneration in AD (47,48). We
therefore performed electron microscopy
experiments in order to count the total number
of synapses and the fraction of spine-synapses
in the CA1 region of the hippocampus (Fig.
10). Transgenic animals expressing the proaggregation mutant hTau40Δ280 showed a
moderate but significant decline in the number
of synapses, particularly of spine-synapses by
(~40% after 13 months). The effect was less
pronounced with the anti-aggregation mutant
mice (~20%). This suggests that the decline of
synapses is due to a combination of Tau
aggregation and other effects (possibly
transport inhibition (49)).
second variant ("anti-aggregation mutant") we
inserted two additional point mutations into the
two hexapeptide motifs (I277P and I308P) in
order to block β-structure and thus aggregation
(8). Apart from aggregation the two variants
show no significant differences, e.g. they have
similar affinities to microtubules and contain
the same phosphorylatable residues. Therefore
this pair of Tau variants makes it possible to
observe effects that are solely due to
aggregation.
-9-
(a) Tau conformation: A major parameter
that decides whether or not Tau will aggregate
is the propensity for β-structure around the
hexapeptide motifs in the repeat domain.
Enhancing this conformation will lead to faster
aggregation, whereas abrogating β-structure
will prevent aggregation. The kinetics of
aggregation in mouse brains is much slower
than in vitro, but the behavior in vitro is
surprisingly predictive of the behavior in vivo.
With regard to the pro-aggregation mutation
ΔK280, our results agree well with other
mouse models which made use of other FTDP17 mutations, notably P301L, because in both
cases the rate of aggregation is enhanced in
vitro and in vivo (19,20,26,27,50). On the
other hand, aggregation can be blocked
completely by destroying the β-propensity of
the hexapeptide motifs in Tau by inserting
prolines. This illustrates that even in a complex
organism the mechanistic explanation of
aggregation is rather simple.
(b) Tau concentration: The rate of
aggregation is modified by the concentration
(as expected for a self-assembling system).
Therefore, in order to observe Tau aggregation
within the limited life span of a mouse, it is
important to encrease the Tau concentration
(52,53). However, the level of exogenous Tau
can be kept lower if one uses a Tau species
with a higher β-propensity, as is the case with
the ΔK280 mutation used here, or for the
P301L or P301S mutations used by others.
Compared to these mouse models (20,22,50),
our level of Tau expression is lower. This
would explain why our mice do not reach the
full neurofibrillary tangle state (except for
beginnings at very old age), why we do not
observe pronounced AT8 and PHF1 staining
(which accompanies tangles), and why there is
synapse loss but no neuronal loss.
(c) Tau composition: A further modifier of
the rate of aggregation is the type of expressed
Tau. In general, shorter isoforms (e.g. the fetal
form hTau23) aggregate more readily because
the domains outside the repeats tend to slow
down aggregation (3,4). This is demonstrated
by several mouse models (13,14,54), and our
cell models expressing the repeat domain alone
underscores this finding (55). The advantage of
using the longest human isoform hTau40 (2N4R), in spite of the slower rate of aggregation,
is that it is more easily distinguished from the
endogenous adult mouse isoforms where the
corresponding 2N-4R form has a low
abundance (50).
(d) Stages of aggregation: Aggregation
becomes detectable in several stages, initially
in the pre-tangle state by staining with
conformation-dependent antibodies, e.g. MC1,
Alz50 (42,56) in stained sections or by a
sarkosyl-insoluble pellet by biochemical
extraction (33). At this point, PHFs may still
be difficult to detect, and most aggregates are
still in a pre-tangle oligomeric state. PHFs
would be expected to gradually assemble and
coalesce into neurofibrillary tangles (detectable
by Gallyas silver staining); however, since we
use the "slow" hTau40 isoform, the
aggregation in our models proceeds only to the
pre-tangle state in the case of the proaggregation Tau (with some exceptions for
very old mice, >24 months gene expression),
while no sign of any aggregation is seen with
the anti-aggregation Tau at any time. To
observe pronounced tangle formation at earlier
time points one would require higher Tau
concentrations (19-22) or more amyloidogenic
Tau variants (e.g. the repeat domain only with
pro-aggregation mutations).
(e) Tau co-aggregation: The question of
whether endogenous mouse Tau co-assembles
with exogenous human Tau has been a matter
of debate (12,18,57). In our case, we find that
mouse Tau is clearly incorporated into the pretangle aggregates, provided that the exogenous
Downloaded from www.jbc.org by Eckhard MANDELKOW PHD on August 24, 2007
one of the Tau isoforms, based on the
assumption that a higher level of Tau would
accelerate
aggregation.
An
alternative
approach was the overexpression or activation
of kinases, assuming that this would lead to
hyperphosphorylation and hence aggregation
of Tau (18,24,25). The expression of human
Tau isoforms met with limited success with
regard to Tau aggregation, presumably because
of the high solubility of Tau, short lifetime of
the animals (compared with the pathological
process), and other factors. However, the
approaches could be improved by: i) using Tau
mutants with a higher aggregation potential,
e.g. P301L: (20,26,27) P301S: (21,22,28), ii)
combining Tau expression with enhanced Aβ
activity, by using APP or PS mutations
(19,29), or iii) expressing several Tau isoforms
analogous to human brain (16). Compared to
other mouse models of tauopathy carrying tau
transgene with P301L and P301S mutations
(Table 1) our results can be summarized as
follows:
-10Tau suffices to initiate the aggregation (e.g. by
carrying pro-aggregation mutations). This is
consistent with our earlier observation that
normal mouse Tau does not differ significantly
from normal human Tau in terms of assembly
properties, and that both types of Tau can be
co-polymerized in suitable conditions (41).
(g) Tau phosphorylation: A major discussion
point in the field is the question whether
phosphorylation primes Tau for aggregation.
Our data leave this possibility open: in both
types of mice, Tau variants become
phosphorylated at a number of sites, especially
single sites (e.g. S262 and S214, both of which
potently detach Tau from MT, as well as S199,
S46, and S231), but the effect is more
pronouced with pro-aggregation mutants. The
transition from pre-tangle to tangle pathology
in old pro-aggregation mice correlates with a
higher
level
and
new
quality
of
phosphorylation, notably the appearance of
dual-phosphorylation sites characteristic of
AD-Tau, recognized by antibodies AT8
(epitope
pS202+pT205),
AT100
(pT212+pS214), and PHF1 (pS396+pS404)
(16,60,61). It appears that the pairwise
phosphorylation is indicative of advanced
stages of aggregation (Fig. 6N). It is also
noteworthy that the phosphorylation at KXGS
sites (recognized by antibody 12E8) or pS214
rises at an early time point, consistent with the
notion that the cell responds to increased Tau
by detaching it from microtubules via
phosphorylation in the repeat domain and/or
S214. This could be followed by the (early)
(h) Synapses vs. Tau aggregation: Loss of
synapses is considered as one of the early signs
of AD (47,48), and therefore it is notable that
there is a moderate but significant loss of
synapses in the mice. It is particularly visible
at the level of spine synapses (40%) in the
CA1 region and appears to be partly related to
aggregation, since it is most pronounced with
the pro-aggregation Tau mutant. This suggest
that there is toxicity caused by pre-tangle Tau
species, perhaps oligomers, which precede
outright tangle formation. An additional cause
may be the inhibition of intraneuronal traffic,
due to the obstruction of the microtubule tracks
(49), or other toxic effects of Tau (62,63). Note
that both pro- and anti-aggregation mutants
bind equally well to microtubules and therefore
have the same capacity of inhibiting transport.
This would explain why the anti-aggregation
mutant also shows some synapse loss.
(i) Reversibility of Tau-induced changes:
Nearly all Tau-induced pre-tangle changes are
reversible when the expression of Tau is
discontinued. This means that the pathways for
Tau degradation, probably via proteasomes
(64), are still intact. Therefore, in contrast to
the inducible Tau mouse model of SantaCruz
et al. (20) we did not observe a time point
beyond which the changes in Tau became
irreversible. A possible explanation is that our
Tau expression levels were too low so that the
mice did not develop the irreversible tangle
state within the life span tested so far. These
observations are also consistent with those of
Oddo et al. (29) where the Aβ-induced pretangle pathology disappears upon Aβ
immunization, but fully formed tangles remain.
In conclusion, in this study we attempted to
test to what extent the observations on Tautransgenic mice can be explained by Tau's
biophysical properties. With regard to
aggregation, the correlation is remarkably good
if one allows for appropriate scaling of the
reaction rates (from hours in vitro to days in
cell models to months in mice). Other
pathological
changes
(phosphorylation,
missorting) correlate only partly with
aggregation and could be explained by changes
in Tau interaction partners (e.g. imbalance of
the
phosphorylation
potential,
or
overwhelming of the cell's sorting machinery).
However, Tau aggregation clearly enhances
Downloaded from www.jbc.org by Eckhard MANDELKOW PHD on August 24, 2007
(f) Tau missorting: Another controversial
issue has been the relationship between
missorting of Tau into the somatodendritic
compartment and aggregation. In our models,
we clearly observe missorting of both mutant
Tau variants. The extent and the distribution of
missorted Tau differs somewhat, which may
depend on cell type and level of expression.
But the dominant feature is that missorting
occurs when Tau levels are increased,
independently of aggregation. In other words,
there is no evidence to assume that the
missorting of Tau is caused by aggregation.
This is best explained in terms of the general
behavior of Tau in cells: In principle, Tau
diffuses readily into the soma and dendrites
when it becomes elevated (e.g. by expression
or microinjection) (58) so that the normal
axonal
sorting
machinery
becomes
overwhelmed (59).
conformational change visualized by antibody
MC1, and later by aggregation.
-11synaptic decay, the likely cause of cognitive
deficits in AD and in mice (23,65). This
reinforces the view that a reduction of
abnormal protein aggregation is a means to
combat the disease.
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-13FOOTNOTES
Acknowledgements: We acknowledge the excellent technical assistance of O. Petrova, D. Drexler
and K. Skokann. We thank the animal care takers (A. Kurzawa, N. Lüder, S. Noster) at Univ. of
Hamburg Medical School for their dedicated help in breeding. We are grateful to Dr. P. Davies (Albert
Einstein College, Bronx, USA) and Dr. P. Seubert (Elan Pharma, South San Francisco, USA) for
providing antibodies MC1, PHF-1, and 12E8, and to Dr. E. Kandel for the transactivator (CamKIIαtTA) construct. This research was supported in part by grants from MPG and DFG.
Keywords: Alzheimer's disease; beta-structure, protein aggregation; Tau protein; transgenic mice;
Downloaded from www.jbc.org by Eckhard MANDELKOW PHD on August 24, 2007
Abbreviations:
AD = Alzheimer's disease
FTDP-17 = frontotemporal dementia with parkinsonism linked to chromosome 17;
NFT = neurofibrillary tangle;
MT = microtubule;
PHF = paired helical filament;
-14FIGURE LEGENDS
Fig. 1: Generation of inducible transgenic mice expressing the longest human Tau isoform with
pro- and anti-aggregation mutations.
(A) Illustration Tau mutant transgenes hTau40ΔK280 (pro-aggregation) and hTau40ΔK280/PP (antiaggregation). (B) The CaMKIIα promoter directed the transgene expression to the forebrain (mainly
hippocampus, cortex and striatum, shown on sagittal paraffin sections stained with pan-Tau antibody
K9JA). Scale bar (B) 1 mm.
Fig. 3: Somatodendritic mislocalization of human Tau in pro- and anti-aggregation mice.
Coronal paraffin sections of hippocampus stained with Ab K9JA recognizing human and mouse Tau.
(A) Pro-aggregation hTau40ΔK280 animal. Tau is prominent in the stratum radiatum (sr), the
molecular layer lacunosum (ml), the stratum lucidum (sl) and the hilus cells (hc). Less Tau is detected
in the pyramidal cell (pc) layer of CA1 to CA3 and the granular cell layer (gc). (B,C), magnifications
of the CA1 region and the cortex emphasizing the dendritic compartmentalization of Tau. (D) Antiaggregation hTau40ΔK280/PP animal. The somatodendritic localization of mutant Tau is less
pronounced. (E) Endogenous Tau in a control animal is mainly visible in the mossy fibers of the CA3
region, indicating a normal axonal distribution. Scale bars (A,D,E) 500 µm; (B,C) 50 µm.
Fig. 4: Pathological conformation in pro-aggregation but not anti-aggregation mutant mice.
Paraffin sections of hippocampus of pro-aggregation (A) and anti-aggregation mice (B) stained with
conformation-dependent MC-1 antibody. (A) Tau with pathological conformation in cell bodies and
dendrites of pyramidal layer and stratum radiatum (MC-1 staining starting at 3 months). (B) No MC-1
staining in anti-aggregation mice.
Fig. 5: Human Tau is highly phosphorylated at KXGS motifs in the repeat domain.
(A) Paraffin sections of pro-aggregation mouse: Weak phosphorylation at S262/S356 as seen by 12E8
antibody. (B) At 6 months, phosphorylation is strongly enhanced and appears in most apical dendrites
of CA1 (arrowheads) and in the cell body layer. (C) The anti-aggregation mutant shows
phosphorylation at S262 to a lesser extent. Sections were counterstained with haemalaun.
Fig. 6: Single site and double-site phosphorylation at SP/TP motifs in pro- and anti-aggregation
mice.
Panels show paraffin brain sections stained with different antibodies against phosphorylated SP or TP
motifs. Overall, phosphorylation of single sites is elevated at early stages and is more pronounced in
pro-aggregation than in anti-aggregation mice. By contrast, dual-site phosphorylation (characterized
by AD-diagnostic antibodies AT8 and PHF1) tends to be elevated at late stages and only in proaggregation mice. Special cases: AT180 (dual site but early stage in both types of mice), MC-1
(conformation specific, early, but only in pro-aggregation mice).. Comparison of pro-aggregation
(A,C,E,G,I) and anti-aggregation mice (B,D, F,H,J). Scale bars 50 µm.
(A, B) pT231 is localized in the periphery of cell bodies and in proximal processes of hippocampal
CA3 neurons in pro- and anti-aggregation mice. (C, D) AT180 staining (p231+p235) localizes to the
cell bodies of pyramidal cells. This dual site is an "early" site, in contrast to others, see below. (E, F)
pS199 staining reveals localization in the apical dendrites of pyramidal CA3 neurons. (G, H) p46
staining of Tau is localized only in the stratum lucidum of the CA3 region, in contrast to other SP/TP
sites. The pyramidal cell bodies are free of staining, indicating an axonal rather than somatodendritic
Downloaded from www.jbc.org by Eckhard MANDELKOW PHD on August 24, 2007
Fig. 2: Tau aggregation increases in pro-aggregation but not in anti-aggregation mice.
Western blot analysis using the pan-Tau antibody K9JA shows soluble and sarkosyl-insoluble Tau
species. Human Tau (arrow, upper band) can be distinguished from mouse Tau (arrowhead, lower
bands). (A) hTau40ΔK280 accumulates in the sarkosyl-insoluble fractions with increasing age of the
animals. (B) hTau40ΔK280/PP does not show up in the sarkosyl-insoluble fractions. (C) Comparison
of two aged mice (24 months gene expression), one with full tangle pathology (starred lanes, left) with
upward shift of human and mouse Tau in SDS gel due to hyperphosphorylation, the other showing
only pre-tangle aggregates and no shift in SDS gel.
-15distribution. (I, J) The non-SP/TP site S214 is distributed similar to pS46 with most of the
phosphorylated Tau species in the mossy fibers of CA3.
(K, L, M, N) Example of aged pro-aggregation mouse, demonstrating that prolonged gene expression
(24 months) leads from pre-tangles to NFT pathology. (K) AT8 staining in CA3 region, (L) PHF-1
staining, (M) MC-1 staining, and (N) Gallyas staining in the cortex. demonstrates that prolonged gene
expression leads from pre-tangles to NFT pathology.
Fig. 8: Phosphorylation of pro-aggregation Tau at S262 is abolished after switching off Tau
expression.
(A, B) Paraffin sections of brains of pro-aggregation animals with continous or interrupted Tau
expression, stained with 12E8 antibody. (A) Pro-aggregation Tau phosphorylated at S262 shows a
dendritic distribution within the hippocampus. (B) Suppression of the Tau gene expression for 6 weeks
leads to clearance of dendrites from pS262-Tau. Scale bars 50 µm.
(C) Biochemical analysis by differential extraction (RAB, RIPA, FA) of Tau from the brain of a
transgenic animal with 8 months continous expression is strongly phosphorylated at S262 in all three
fractions (lanes "8"). Interruption of the Tau expression for 6 weeks decreases the phosphorylation at
S262 to the limit of detection (lanes "8 off"). (D) The quantitative analysis demonstrates the Tau gene
suppression and the reversibility of the phosphorylation in switched off animals.
Fig. 9: Reversibility of pathological conformation and hyperphosphorylation of Tau after
switching off expression of pro-aggregation Tau.
Paraffin hippocampal sections of transgenic animals expressing hTau40ΔK280 were stained with
different antibodies, recognizing the phosphorylation state (pS199, pT231, AT180, pS46) or a
pathological conformation (MC1). (A, C, E, G) illustrates continous gene expression, (B, D, F, H)
shows switch-off for the last 6 weeks. (A-F) Tau phosphorylation at sites AT180, pS199, and pS46
disappears from hippocampal cells after switch-off. (G, H) The conformational change seen by the
MC1-antibody and its somatodendritic localization is fully reversible. Scale bars 50 µm.
Fig. 10: Reduction of spine-synapses in transgenic mice.
Brains of transgenic and control animals were embedded, cut and stained for electron microscopy. The
numbers of synapses and spine-synapses were counted in 2 serial sections. Pro-aggregation mutant
Tau mice showed a significant reduction in spine-synapses (-40%, black bar on right), antiaggregation mice a 20% reduction (grey bar). The loss of total synapses was less conspicuous but
showed a clear trend (-20% or -15%, resp., left). Values are shown as mean ± SE.
Downloaded from www.jbc.org by Eckhard MANDELKOW PHD on August 24, 2007
Fig. 7: Somatodendritic mislocalization is reversed and Tau levels are decreased after switching
off expression of pro-aggregation Tau.
(A, B) Paraffin sections of a pro-aggregation mouse with 10 months Tau expression (A) and of a
mouse with 8.5 months Tau expression and subsequent switch-off for 1.5 months (B). Brain sections
after switching off Tau expression are devoid of hTau40ΔK280 protein. The somatodendritic
localization of the Tau (Fig. 7A) is fully reversible in 6 weeks. The remaining protein is localized
mainly in the stratum lucidum of CA3 (small insert in B).
(C) Western blots of soluble and insoluble fractions of the same animals show significant reduction in
human pro-aggregation Tau expression whereas endogenous mouse Tau levels remain stable.
(D) Quantification of western blots indicates the disappearance of hTau40ΔK280 to control levels.
Figure 1
A. human tau mutants
I1 I2
P1
P2
1
2
3 4
1
B. sagittal brain section
ht40
441
280
pro-aggregation
VQI I NK VQI VYK
anti-aggregation
VQPI NK VQPVYK
(ΔK280)
(ΔK280/I277P/I308P)
280
K9JA
6 mo
1 mm
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Figure 2
A. ht40ΔK280
K9JA
mo
human
mouse
soluble tau
wt
4 6 8
B. ht40ΔK280/2P
sarkosylinsoluble tau
wt
4
6
8
soluble tau
wt 6
sarkosylinsoluble tau
7 10 wt 6 7 10
C. ht40ΔK280
sarkosylsoluble tau insoluble tau
wt 24 24 wt 24 24
mo
human
mouse
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Figure 3
A. ht40ΔK280
D. ht40ΔK280/2P
CA2
CA1
pc
pc
sr
DG
hc
sl
CA3
CA4
pc
500 µm
K9JA
10 mo
C. Cortex
50 µm
10 mo
K9JA
500 µm
13 mo
E. wild type
K9JA
500 µm
14 mo
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B. CA1
K9JA
ml
gc
Figure 4
14 mo
500 µm
MC1
10 mo
500 µm
MC1
B. ht40ΔK280/2P
A. ht40ΔK280
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Figure 5
CA1
50 µm
CA1
12E8
C. 6 mo
B. 6 mo
A. 3 mo
ht40ΔK280/2P
ht40ΔK280
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Figure 6
A. Pro
B. Anti
C. Pro
D. Anti
13 mo, CA3
13 mo, CA3
13 mo, CA1
13 mo, CA1
50 µm
pT231
E. Pro
F. Anti
13 mo, CA1 13 mo, CA1
50 µm
H. Anti
13 mo, CA3 13 mo, CA3
50 µm
pS46
I. Pro
50 µm
J. Anti
13 mo, CA3 13 mo, CA3
50 µm
pS214
K. Pro
L. Pro
M. Pro
N. Pro
24 mo, CA3
24 mo, CA3
24 mo, CA3
24 mo, Cortex
MC1
Gallyas
AT8
PHF-1
50 µm
50 µm
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pS199
G. Pro
AT180 (pT231/pS235)
Figure 7
A. 10 mo
B. 1.5 mo OFF C. K9JA
sarkosylinsoluble tau
soluble tau
wt 9
mo
CA3
CA3
9 10 10
off
off
9 10 10
off
off
wt
human
mouse
D.2.000
[a.u.]
1.500
400
[a.u.]
600
200
500
10
10 m
m
of
f
of
f
9m
9m
f
10
10 m
m
of
f
of
w
t
0
0
9m
CA1
w
t
9m
K9JA
50 µm
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1.000
Figure 8
A. 8 mo
B. 1.5 mo OFF
soluble tau
C. 12E8
ht40
RAB
wt
8
insoluble tau
RIPA
8 wt
off
8
FA
wt
8
off
8
8
off
p-human
p-mouse
of
f
8m
t
8m
w
of
f
8m
8m
w
t
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of
f
50 µm
8m
CA1
400
300
200
100
0
8m
12E8
[a.u.]
2.000
1.500
1.000
500
0
w
t
D.
Figure 9
A. 10 mo ON
B. 1.5 mo OFF C. 8 mo ON
AT180
CA1
E. 9 mo ON
pS199
F. 1.5 mo OFF G. 10 mo ON
CA3
50 µm
MC1
CA1
50 µm
H.1.5 mo OFF
CA1
50 µm
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pS46
50 µm
D. 1.5 mo OFF
25
20
15
10
5
total synapses
ht40ΔK280/2P
ht40ΔK280
wt
spine synapses
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0
number of synapses per 6,4 µm³
Figure 10