Tau Filament Formation in Transgenic Mice Expressing P301L
Tau*
Jürgen
Götz
,
Feng
Chen,
Robi
Barmettler, and
Roger
M.
Nitsch
From the Division of Psychiatry Research, University of
Zürich, 8008 Zürich, Switzerland
Received for publication, July 21, 2000, and in revised form, September 15, 2000
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ABSTRACT |
Mutations in the microtubule-associated protein
tau, including P301L, are genetically coupled to hereditary
frontotemporal dementia with parkinsonism linked to chromosome 17. To
determine whether P301L is associated with fibril formation in mice, we expressed the longest human tau isoform, human tau40, with this mutation in transgenic mice by using the neuron-specific mouse Thy1.2
promoter. We obtained mice with high expression of human P301L tau in
cortical and hippocampal neurons. Accumulated tau was
hyperphosphorylated and translocated from axonal to somatodendritic compartments and was accompanied by astrocytosis and neuronal apoptosis
indicated by terminal deoxynucleotidyl transferase-mediated biotinylated dUTP nick end-labeling staining. Moreover, P301L tau formed abnormal filaments. Electron microscopy of
sarcosyl-insoluble protein extracts established that the filaments had
a straight or twisted structure of variable length and were ~15 nm
wide. Immunoelcecton microscopy showed that the tau filaments were
phosphorylated at the TG3, AT100, AT8, and AD199 epitopes in
vivo. In cortex, brain stem, and spinal cord, neurofibrillary
tangles were also identified by thioflavin-S fluorescent microscopy and
Gallyas silver stains. Together, our results show that expression of
the P301L mutation in mice causes neuronal lesions that are similar to
those seen in human tauopathies.
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INTRODUCTION |
Hereditary frontotemporal dementia with parkinsonism linked to
chromosome 17 is a group of neurodegenerative diseases characterized by
early behavioral changes accompanied by subsequent cognitive and motor
disturbances. More than a dozen families were identified, with diverse
but overlapping clinical features. Pathological changes include
selective frontotemporal atrophy, neuronal loss, gliosis, and
spongiosis in several brain areas in addition to abundant filamentous
inclusions composed of hyperphosphorylated tau protein in neurons and,
to some extent, in glial cells (1).
Tau is an axonal, microtubule-associated phosphoprotein in normal adult
brain (2). Tau has tubulin-polymerizing activities in vitro
(3); it establishes short cross-bridges between axonal microtubules and
thereby supports functions in intracellular trafficking including
axonal transport (4). In neurons affected by tauopathy, tau is
hyperphosphorylated and is located not only in axons but also in cell
bodies and dendrites (5, 6). Results from in vitro studies
suggest that disease-causing mutations in the tau gene result either in
the reduced ability of tau to interact with microtubules or in
increased ratios of four-repeat
(4R)1 to three-repeat tau
caused by quantitative changes in the splicing in of exon 10. (7, 8).
To demonstrate that human tau can form filaments in mouse brains and to
reproduce aspects of the human pathology, including neurofibrillary
tangle formation, in transgenic mice, we expressed the longest isoform
of human tau with the frontotemporal dementia with parkinsonism linked
to chromosome 17 causing mutation P301L in neurons by using a
neuron-specific promoter. Tau accumulated mainly in neurons of the
neocortex and hippocampus; it was hyperphosphorylated at distinct sites
and formed filaments similar to those present in human tauopathies.
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EXPERIMENTAL PROCEDURES |
Constructs and Transgenic Mice--
By a PCR-mediated approach,
the human pathogenic mutation P301L was introduced into the cDNA
encoding the longest human brain tau isoform. This isoform contains
exons 2 and 3 as well as four microtubule-binding repeats
(2+3+4R, human tau40). To be able to
discriminate P301L tau transgenic from wild-type tau transgenic mice, a
silent mutation was introduced into the P301L construct that destroys a
diagnostic SmaI restriction site. The cDNA was conferred
with a Kozak consensus sequence and was subcloned into a murine Thy.1.2
genomic expression vector (Dr. Herman van der Putten, Novartis, Basel,
Switzerland). Vector sequences of this construct (named pR5) were
removed before microinjection. Transgenic mice were produced by
pronuclear injection of B6D2F1 × B6D2F1 embryos. Founders were
identified by PCR analysis of lysates from tail biopsies using two
different primer pairs. Founder animals were intercrossed with C57BL/6
mice to establish lines.
Transgenic mice were screened with oligonucleotides tau-I (O-100,
5'-GGAGTTCGAAGTGATGGAAG-3') and tau-K (O-101,
5'-GGTTTTTGCTGGAATCCTGG-3') and yielded an amplification product of 500 base pairs. A restriction digest of the amplification product by
SmaI confirmed the presence of the P301L transgene. Ten
independent transgenic lines were generated, four of which had
comparable expression levels as determined by immunoblot analysis.
Antibodies--
Antibody HT7 (Innogenetics Inc.; amino acids
159-163, diluted 1:400) was used to detect human tau specifically;
tau-1 (Roche Molecular Biochemicals) was used to detect both human and
murine tau on immunoblots; AT8 (Innogenetics Inc.; diluted 1:20) was used to detect tau phosphorylated at epitopes serine 202 and threonine 205; AT100 (Innogenetics Inc.; diluted 1:100) was used to detect tau
phosphorylated at serine 212 and threonine 214; AT180 (Innogenetics Inc.; diluted 1:50) was used to detect tau phosphorylated at threonine 231 and serine 235; 12E8 (Dr. Peter Seubert, Elan Pharmaceuticals; diluted 1:100) was used to detect tau phosphorylated at serines 262 and
356; conformation-dependent antibody TG3 (Dr. Peter Davies; diluted 1:20) was used to detect tau phosphorylated at threonine 231 and serine 235; PHF1 (Dr. Peter Davies; diluted 1:50) was used to
detect tau phosphorylated at serine 396 and serine 404; AD2 (Dr.
Chantal Mourton-Gilles; diluted 1:500) was used to detect tau
phosphorylated at serine 396 and serine 404; AD199 (Dr. A. Delacourte;
diluted 1:1000; Ref. 9) was used to detect tau phosphorylated at serine
199; MC1 (Dr. Peter Davies; diluted 1:10) was used to detect the
conformational ALZ50 epitope; and rabbit anti-glial fibrillary
acidic protein IgG (Sigma, catalog no. G-9269; diluted 1:400) was used
to detect activated astrocytes. For peroxidase and diaminobenzidine
stainings, secondary antibodies were obtained from Vector Laboratories
(Vectastain ABC kits PK-6101 and PK-6102). For immunofluorescence,
secondary antibodies were obtained from Molecular Probes (ALEXA-FLUOR series).
Immunoblot Analysis--
Brains from transgenic and control mice
(aged 3 weeks to 8 months) were weighed and Dounce homogenized in 2.5%
(v/v) perchloric acid in phosphate-buffered saline (PBS), allowed to
stand on ice for 30 min, and centrifuged for 10 min at 10,000 × g. The supernatants were dialyzed against 50 mM
Tris-HCl (pH 7.4), 1 mM dithiothreitol, and 0.1 mM phenylmethylsulfonyl fluoride and used for immunoblot analysis as described, using equal amounts except for ALZ17, for which
only half the amount of extract has been used (10). Ponceau staining of
the membranes was included to confirm loading of comparable amounts of protein.
Sarcosyl extractions were done as described (10). In brief, brain
tissue of 8-month-old pR5 transgenic and control mice were homogenized
in 10 volumes of buffer consisting of 10 mM Tris-HCl (pH
7.4), 0.8 M NaCl, 1 mM EGTA, and 10% sucrose.
An Alzheimer's disease (AD) brain sample was included as control. The
homogenate was spun for 20 min at 20,000 × g. The
supernatant was brought to 1% N-lauroylsarcosinate (Fluka,
no. 61744), and incubated 1 h at room temperature while shaking.
After a 1-h spin at 100,000 × g, the
sarcosyl-insoluble pellets were resuspended in 50 mM Tris-HCl (pH 7.4) and stored at 4 °C. This material was used for both immunoblot analysis and electron microscopy.
Samples were run on 10% SDS-polyacrylamide gels and
electrophoretically transferred to a nylon membrane (Hybond-ECL,
Amersham Pharmacia Biotech). Residual protein-binding sites were
blocked by incubation with 5% semifat dried milk in Tris-buffered
saline (TBS) and 0.1% Tween 20 for 1 h at room temperature,
followed by a 3-h incubation at room temperature with the primary
antibody in TBS and 0.1% Tween 20, 1% semifat dried milk, and 0.02%
sodium azide. Antibodies Tau-1 and HT7 were diluted 1:5000 and 1:250, respectively. After four washes for a total of 30 min in TBS, the
membrane was incubated with a horseradish peroxidase-linked sheep
anti-rabbit Ig (Amersham, NA931) at 1:5000 dilutions for 1 h at
room temperature, followed by a 30-min wash in TBS. The membrane was
then incubated for 1 min in ECL reagent (Vector Laboratories), excess
liquid was removed, and the membrane was exposed to x-ray films. For
reuse, membranes were stripped for 30 min at 50 °C in 100 mM 2-mercaptoethanol and 2% SDS in 62.5 mM
Tris (pH 6.8).
Immunohistochemistry and TUNEL Staining--
Brains from
3-4-month-old pR5 transgenic mice and an equal number of control mice
(nontransgenic and wild-type human tau transgenic ALZ17 mice; Ref. 11)
were used for immunohistochemical analysis. Animals were perfused
transcardially with 4% paraformaldehyde in saline and sodium phosphate
buffer (pH 7.4). Immunohistological, hematoxylin-eosin, and combined
Holmes and Luxol stainings were done on 4-µm paraffin sections from
brain and spinal cord by using standard published procedures (12). Some
of the sections were pretreated with 5 µg/ml proteinase K in TBS or
PBS at 37 °C for 2.5 min for signal enhancement. Sections were
dehydrated in an ascending series of ethanol and flat embedded between
glass slides and coverslips in Eukitt (Kindler). Sections were stained
with thioflavin-S and silver impregnated by Gallyas (13) and modified Bielschowsky (14) protocols (15).
TUNEL Staining--
To detect cells undergoing apoptosis, the
peroxidase in situ cell death detection kit (Roche
Molecular Biochemicals, no. 1684817) was used. In brief,
paraffin-embedded sections were rehydrated, treated with 5 µg/ml
proteinase K in PBS for 10 min at 37 °C, washed with ice-cold PBS
four times, incubated in 3% H2O2 in methanol for 5 min at room temperature, and washed again. For a positive control, sections were incubated in 100 µg/ml DNase I (Roche
Molecular Biochemicals, no. 104132) in 20 mM Tris-HCl (pH
8.0) and 10 mM MgCl2 for 10 min at room
temperature. Then, the labeling solution containing the enzyme terminal
deoxynucleotidyl transferase (POD kit) was diluted 1:10 and added to
the sections for 30 min at 37 °C. For a negative control, the enzyme
was omitted from the labeling solution. Sample sections were washed,
blocked with 2% bovine serum albumin, and incubated with convert
solution (POD kit), washed again, and incubated in 1:10 diluted
diaminobenzidine solution (Pierce, no. 1856090) for 10 min at room
temperature to visualize the DNA breaks. Sections were dehydrated and
mounted in Eukitt. To correlate numbers of HT7-positive neurons with
glial fibrillary acidic protein-positive astrocytes and TUNEL
reactivity, positive cells in an area of 0.7 × 0.6 mm of the
somatosensory cortex S1 and the motor cortex M1 were counted in 10 sections per animal.
Electron Microscopy and Immunogold Electron
Microscopy--
Resuspended sarcosyl-insoluble material obtained from
brains of transgenic mice, control mice, and an AD patient were placed directly on carbon-coated, 300-mesh grids, stained with 2%
phosphotungstic acid, and analyzed by electron microscopy (see below).
Samples were also processed for immunogold electron microscopy and
incubated with the primary antibody in PBS and 0.1% gelatin for 90 min
at room temperature. Antibody AT100 was used at 1:100 dilutions, antibody AT180 at 1:5 dilutions, rabbit antiserum AD199 at 1:50 dilutions, and hybridoma supernatant TG3 undiluted. As negative controls, filament preparations from AD brain and transgenic mice were
incubated with
-synuclein-specific antiserum PER4 as well as an
antibody against a PR8 influenza virus surface antigen. In addition,
controls were included, in which the primary antibody had been omitted.
Incubations were placed on Formvar- and carbon-coated, 300-mesh grids
and allowed to evaporate partially. Grids were washed twice in PBS and
0.1% gelatin and incubated with the respective secondary antibody
conjugated to 6 nm Au (Sigma) for 30 min in a humid chamber at room
temperature. After two washes in PBS and 0.1% gelatin and one in
water, the grids were blotted, stained with 2% phosphotungstic acid,
and allowed to air dry. Micrographs were recorded at an operating
voltage of 80-100 kV and at nominal magnifications of × 88,000 and × 175,000 on a Philips CM12 electron microscope.
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RESULTS |
Overexpression of Human tau--
The neuron-specific elements of
the mouse Thy1.2 promoter were used to express the longest human
four-repeat tau isoform containing the two amino-terminal exons 2 and 3 (2+3+4R), along with human pathogenic mutation
P301L (pR5 construct). Ten founders were obtained, four of which were
used for further analysis on the basis of their expression levels: By
immunoblot analysis of similar amounts of perchloric acid-soluble
protein extracts, as judged by Ponceau stainings, a strong
immunoreactive 66-kDa protein that corresponded to the transgenic tau
band was identified in animals of some pR5 lines; it reacted with the
anti-tau antibody Tau-1 that recognizes both human and murine tau (Fig. 1A), as well as with antibody
HT7 against human, but not murine, tau (Fig. 1B). Soluble
tau brain tissue levels for line pR5-183 were ~70% of endogenous tau
(Fig. 1A, lane 4, upper transgenic band representing human tau compared with lower
bands representing the endogenous murine tau isoforms). As
expected, transgenic tau was absent from the wild-type brain protein
extracts (Fig. 1B, lane 8). Amounts of sarcosyl-insoluble
tau (Fig. 1C) were generally higher in the pR5-183 (Fig.
1C, lane 1) than in our transgenic mice that expresses human
tau without the mutation (ALZ17 line; Ref. 11; Fig. 1C, lane
3). Protein extracts from pR5-183 mice were therefore subsequently
used for electron microscopy. The transgenic mutant tau in pR5-183 mice
was hyperphosphorylated, as indicated by several
phosphorylation-dependent anti-tau antibodies (data not
shown).

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Fig. 1.
Immunoblot analysis of tau protein in brains
from mice of pR5 lines. A, tau protein extracted with
perchloric acid from brains of different 3-month-old mice of pR5 lines
(lanes 1-7; lane 4, line pR5-183) and a
wild-type control mouse (lane 8) was analyzed by
immunoblotting using anti-tau antibody Tau-1. This antibody recognizes
murine and human tau. B, the blot was stripped and reprobed
with HT7, a human tau-specific antibody. The arrow points to
the human tau band. C, sarcosyl-insoluble tau from the
brains of 8-month-old mice of line pR5-183 (lane 1), a
wild-type control mouse (lane 2), and a wild-type human tau
transgenic ALZ17 mouse (lane 3) analyzed by immunoblotting
using antibody HT7. The arrow points to the human tau
band.
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To determine the distribution and localization of tau in brain, a
3-month-old mouse of the pR5-183 line was analyzed by
immunohistochemistry using antibody HT7. Expression levels of human tau
were high in hippocampus, fornix fimbriae, amygdala, spinal cord, and
cortex (Fig. 2A), weaker in
brain stem and striatum, and not detectable in olfactory bulb and
cerebellum. A sagittal section of the hippocampus showed numerous
HT7-positive pyramidal neurons in the CA1 region but not in CA3. The
mossy fiber projections of the hippocampus were strongly stained,
whereas staining intensity was somewhat weaker in dentate gyrus granule
cells (Fig. 2, A and C). In CA1 pyramidal neurons
human tau was present in axons but accumulated also in cell bodies and
apical dendrites (Fig. 2B). In brain stem, HT7-positive
cells were identified, in addition to axonal spheroids, and cells that
expressed tau were strongly stained, with granular accumulation of tau
(Fig. 2D). In addition, a subset of pyramidal cells in the
cerebral cortex was HT7 immunoreactive. As for CA1 pyramidal cells, we
found granular immunostaining of cell bodies and dendrites (Fig.
2E). In comparison with wild-type human tau transgenic ALZ17
mice, staining of cell bodies and dendrites of the dentate gyrus
granule cells was less pronounced in pR5-183 mice, as determined with
antiserum AD199 (Fig. 2, F and G) or antibody HT7
(data not shown). In other brain areas, staining patterns were
comparable. Neurofibrillary tangles were identified in spinal cord,
brain stem, and cortical layers 5 and 6 by thioflavin-S fluorescent
microscopy (Fig. 2H) and Gallyas silver stains (Fig. 2I).

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Fig. 2.
Tau immunoreactivity in brain from a
3-month-old mouse of the pR5 line reveals high expression of human
tau. A, sagittal section of the hippocampus shows
HT7-positive pyramidal neurons in CA1. Staining is less intense for
dentate gyrus (dg) granule cells. Also note strong
immunostaining of the mossy fiber (mf) projection in sector
CA3 of the hippocampus. B, CA1 pyramidal neurons accumulate
human tau in cell bodies and apical dendrites. C, the mossy
fiber network is intensely stained, revealing transport of human tau
into the axon. D, In brain stem, HT7-positive cells are
found with granular accumulation of tau, in addition to axonal
spheroids. E, in cerebral cortex, a subset of pyramidal
cells is HT7-immunoreactive. Note the intense, granular immunostaining
of cell bodies and dendrites. No immunoreactivity was observed in
control mice. F and G, staining of cell bodies
and dendrites of dentate gyrus granule cells with antiserum AD199 is
less pronounced in pR5-183 mice (F) compared with wild-type
human tau transgenic ALZ17 mice (G). H and
I, neurofibrillary tangles are identified in several brain
areas including spinal cord by thioflavin-S fluorescent microscopy
(H) and Gallyas silver stains (I). Scale
bars: A, F, and G, 100 µm;
B-E, 20 µm.
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Abnormal Phosphorylation and Conformation of tau--
To determine
the phosphorylation status of tau in 3-month-old pR5 mice, we used a
panel of phosphorylation- and conformation-dependent anti-tau antibodies. Antibody TG3, a phosphorylation- and
conformation-dependent antibody, stained pyramidal neurons
of the CA1 region of the hippocampus of transgenic mice (Fig.
3A) but not controls (Fig.
3B). TG3 stained numerous pyramidal neurons in cortices of
transgenic (Fig. 3C) but not control (Fig. 3D)
mice. The conformation-dependent antibody MC1 that detects
the ALZ50 epitope of AD tau stained pyramidal neurons of the CA1 region
and the cortex of transgenic (Fig. 3, E and G)
but not control (Fig. 3, F and H) mice. The same
staining pattern was obtained with antibody AT180, which detects tau
phosphorylated at threonine 231 and serine 235 (Fig. 3,
I-M). Antibody AD199 directed against phosphorylated serine
199 stained hippocampal and cortical neurons moderately, antibody AT8
directed against serines 202 and 205 stained only weakly and was mainly
restricted to cortical neurons, whereas antibodies AD2 and PHF1
directed against serines 396 and 404 did not detect any neurons.
Staining with antibodies 12E8 and AT100 revealed similar patterns in
transgenic and control mice (data not shown).

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Fig. 3.
Tau phosphorylation and conformational
changes in P301L tau transgenic mice. Antibody TG3, a
phosphorylation- and conformation-dependent antibody,
stains pyramidal neurons of the CA1 region of the hippocampus of
transgenic mice (A), but not controls (B). Also
in cortex, numerous pyramidal neurons are stained by this antibody in
transgenic mice (C), but not controls (D). The
conformation-dependent antibody MC1 stains pyramidal
neurons of the CA1 region and the cortex of transgenic mice
(E and G) but not controls (F and
H). The same staining pattern is obtained with
phosphorylation-dependent antibody AT180 (I-M).
Scale bars: A-D, 30 µm; E-M, 30 µm.
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Astrocytosis and Apoptosis--
To detect activated astrocytes, we
used an antibody against glial fibrillary acidic protein. This
antibody revealed activated astrocytes in the amygdala and in cortical
areas of transgenic mice that contained numerous tau-positive neurons
(Fig. 4A). No such activated
astrocytes were observed in control amygdala and cortex (Fig.
4B). Also, no astrocyte activation was found in the hippocampus of transgenic mice (data not shown). To identify cells that
underwent apoptosis, we used TUNEL stains and found many TUNEL-positive
neurons in the somatosensory cortex that contained numerous
tau-positive neurons (Fig.
5A). By contrast, only very few TUNEL-positive cells were present in wild-type mice, and staining was faint compared with transgenic mice (Fig. 5B). As
negative control, we omitted the POD convert solution (Fig.
5C), and, as a positive control, we pretreated sections with
DNase I (Fig. 5D). No TUNEL-positive neurons were identified
in the hippocampus of transgenic mice (data not shown). We correlated
numbers of HT7-positive neurons with glial fibrillary acidic
protein-positive astrocytes and TUNEL reactivity in the somatosensory
cortex and counted per visual field 50 (±14) HT7-positive neurons, 19 (±6) activated astrocytes, and 7 (±3) dark TUNEL-positive cells in transgenic brain compared with no HT7-positive neurons, 5 (±2) activated astrocytes, and 3 (±3) faint TUNEL-positive cells in the
wild-type control (Fig. 4C). To correlate astrocytosis and tau expression in different brain areas, we compared in transgenic brain motor cortex M1 and somatosensory cortex S1 and counted per
visual field 14 (±4) HT7-positive neurons and 5 (±1) activated astrocytes in the M1 cortex compared with 50 (±14) HT7-positive neurons and 19 (±6) activated astrocytes in the S1 cortex (Fig. 4D).

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Fig. 4.
A, activated astrocytes are detected in
those cortical areas of transgenic mice that contain numerous
tau-positive neurons. B, no such activated astrocytes are
found in control cortex. C and D, In
the somatosensory cortex S1, 50 (±14) HT7-positive neurons, 19 (±6)
activated astrocytes, and 7 (±3) dark TUNEL-positive cells are present
per visual field in transgenic brains, compared with no HT7-positive
neurons, 5 (±2) activated astrocytes, and 3 (±3) faint TUNEL-positive
cells in wild-type controls (C). Astrocytosis and tau
expression are positively correlated in different brain areas of
transgenic mice: per visual field, 14 (±4) HT7-positive neurons and 5 (±1) activated astrocytes are present in the M1 cortex, compared with
50 (±14) HT7-positive neurons and 19 (±6) activated astrocytes in the
S1 cortex (D).
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Fig. 5.
TUNEL staining reveals neurons undergoing
apoptosis in transgenic mice. A and B, in
cortical areas of transgenic mice that contain numerous tau-positive
neurons, many TUNEL-positive neurons are identified (A),
which are absent from controls (B). Arrows point
to some positive neurons. C and D, negative
control without reagent (C) and positive control, pretreated
with DNase I (D).
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Tau Filaments--
We prepared sarcosyl protein extracts for
electron microscopy analyses in parallel from brain tissue of an AD
patient, 8-month-old pR5 transgenic mice, and control mice and found
tau filaments in both AD and transgenic mouse brains. The tau filaments
in AD brain had a width of ~20 nm (Fig.
6, A and B), and
those in transgenic mice were ~15 nm wide and significantly shorter
(Fig. 6C). No such filaments were present in any of the
control mice (data not shown). To identify phosphoepitopes of tau, we
incubated the filaments with a panel of
phosphorylation-dependent antibodies. Immunogold electron
microscopy of extracts obtained from AD (Fig. 6, D and F) and pR5 transgenic (Fig. 6, E and
G) brains using TG3 (Fig. 6, D and E)
or AT8 (Fig. 6, F and G) identified several 6-nm, gold-decorated filaments. These filaments were also stained by the
antibodies AT100 and AD199 (data not shown). In AD brains, tau
filaments consist of two structurally distinct parts, the core and the
fuzzy coat. The known, darkly stained space between the filaments and
the gold particles typically corresponds to the known fuzzy coat of the
filaments (10, 16). No filaments were identified or decorated by
gold-labeled antibodies in negative controls, proving the specificity
of these antibodies. In particular, we found no neurofilaments,
with a width of 10 nm and an axial periodicity of 21 nm in the
preparation (17).

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Fig. 6.
Neuronal tau filaments in 8-month-old P301L
tau transgenic brain revealed by electron microscopy and immunogold
electron microscopy of sarcosyl extracts. A and
B, tau filaments in an extract obtained from an AD brain
have a width of ~20 nm. C, Tau filaments in an extract
obtained from the pR5 brain are shorter and have a width of ~15 nm
(arrows). No such filaments were identified in sarcosyl
extracts of control mice. D and E, immunogold
electron microscopy of extracts obtained from AD (D) and pR5
brain (E) using phosphorylation- and
conformation-dependent antibody TG3 identifies 6-nm,
gold-decorated filaments. F and G, immunogold
electron microscopy of extracts obtained from AD (F) and pR5
brain (G) using phosphorylation-dependent antibody
AT8 also identifies 6-nm, gold-decorated filaments. Scale
bars: A, 400 nm; B and C, 80 nm;
D--G, 80 nm.
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DISCUSSION |
The results of this study show that transgenic expression of human
P301L mutant tau under control of the murine Thy1.2 genomic expression
vector leads to the formation of sarcosyl-insoluble, 15-nm-wide tau
filaments. By comparison, tau filaments in frontotemporal dementia with
parkinsonism linked to chromosome 17 patients with the P301L mutation
(Dutch family 1) consist of 15-nm-wide, slender, twisted filaments with
variable periodicity, in addition to a few straight filaments (1), and
the tau filaments obtained from AD brains consist of 20-nm-wide, paired
helical or straight filaments. The filaments in our P301L tau
transgenic mice have the same width as those in the human disease
associated with the P301L mutation (Dutch family 1). They were,
however, shorter than filaments enriched from AD brains, which we
included as positive control because of the unavailability of a brain
sample from Dutch family 1. One likely explanation for the prevalence
of short filaments in our mice is that the longest tau isoform
expressed in our mouse contained two calpain recognition motifs for
proteolytic degradation that may favor the formation of shorter
filaments. In addition to the P301L mutation, the filament formation in
our mice may also be related to high expression levels achieved by
using the murine Thy1.2 expression vector, as shown by immunoblot
analyses. A possible contribution of high expression levels to tau
filament formation in our mouse lines is supported by the concentration dependence of filament assembly in vitro that resembles a
nucleation-dependent process (18, 19). Together, the
failure of previous attempts to model tau filament formation in
transgenic mice may be related to either the absence of disease-causing
mutations in the expression constructs or low expression levels (11,
20-25) or both.
In our mice, the murine Thy1.2 promoter directed expression of tau
mainly to hippocampal and cortical neurons. Brain regions, which in
human disease are spared from tau pathology, such as the cerebellum,
did not express detectable levels of tau, as determined by
immunofluorescence. As in human tauopathies (26), tau accumulated not
only in axons but also in cell bodies and dendrites. To establish phosphoepitopes that are related to tau aggregation and filament formation, we used a panel of phosphorylation-dependent
antibodies. In brains from patients of Dutch family 1, phosphorylation-dependent antibodies AT8, AT100, AT180,
AT270, PHF1, and 12E8 stained numerous deposits in several brain
regions, including the cortex, the dentate gyrus, and the CA1 region of
the hippocampus. These deposits were mainly of the pretangle type and
located in the perinuclear region and cell body and sometimes extended
to the apical dendrites of neurons (1). In our model, AT100 and 12E8
did not discriminate wild-type from transgenic mice. AT8 staining was
weak in transgenic mice and mainly restricted to a subset of cortical
neurons, where it was found not only in cell bodies but also in
dendrites. AT100 and PHF1 did not stain any neuron at up to 6 months of
age. Age may account for this apparent difference between human disease and the mouse model, especially because filaments obtained from 8-month-old transgenic mice were AT100- and AT8-positive. AT180 and
also the conformation-dependent antibodies MC1 and TG3,
which were not included in the human study, revealed strong
somatodendritic staining in both cortical and hippocampal pyramidal
neurons of transgenic mice. Antibodies MC1 and TG3 recognize a distinct
pathological conformation of the tau molecule in AD. In normal
autopsy-derived brain tissue, tau is not stained by these antibodies.
Our data indicate that transgenic tau underwent a conformational change favoring filament formation. Indeed, by immunogold electron microscopy of sarcosyl-insoluble tau protein, the AT8, AD199, AT100, and TG3
epitopes were identified on tau filaments in our P301L transgenic mice.
For comparison, in the human study only one antibody, AT8, was used
that labeled tau filaments (1). The distance of the gold particles from
the core of the filament is similar to that reported for AD filaments
(10). The space between the filaments and the gold particles
corresponds to the known fuzzy coat of the filaments (10, 16). Taken
together, tau in the nonfilamentous and filamentous states is
hyperphosphorylated at several sites, which are also
hyperphosphorylated in human tauopathies.
In Dutch family 1, neurological examination of brains revealed gliosis
and severe neuronal loss in frontal and temporal cortex and variable
loss in the parietal cortex, whereas the hippocampus showed only mild
to moderate neuronal loss and gliosis (1). These data are consistent
with our findings. Despite comparable levels and phosphorylation of
human tau in hippocampus and cortex of transgenic mice, we found
evidence for apoptosis and astrocytosis in cortical areas and in the
amygdala but not in the hippocampus. Astrocytosis was positively
correlated with levels of human tau expression, as shown for the M1 and
S1 cortices. Therefore, we cannot exclude the possibility that this
pathology progresses and will include the hippocampus with advanced
aging in older mice.
Together, our data show that the P301L mutation in combination with
high expression levels can cause the formation of abnormal tau
filaments in neurons in mice. Filament formation was also reported
recently in mice expressing 2
3
4R tau, along
with the human pathogenic mutation P301L, under control of the mouse
prion protein promoter. These mice show an advanced neurological
phenotype, likely reflecting differences between
2
3
4R and 2+3+4R tau
(27). In our P301L mice, filaments were phosphorylated at distinct
epitopes, and their formation was accompanied by astrocytosis and
apoptosis. These data indicate that P301L is a key pathogenic factor,
and they underscore its pathophysiological role in frontotemporal dementia with parkinsonism linked to chromosome 17. Moreover, our
results suggest the use of these mice, either alone or in combination
with a pathogenic APP mutation, for the study of both the
pathophysiology and the prevention of tau filament formation in
neurodegenerative diseases.
 |
ACKNOWLEDGEMENTS |
We thank Daniel Schuppli, Eva Moritz, Yves
Santini, and James Opoku for excellent technical assistance. We thank
Dr. Thomas Baechi for providing the electron microscopy facility and
members of his laboratory, in particular Ruth Keller and Hans-Peter
Gautschi, for assistance. We thank Dr. Peter Davies for antibodies TG3, MC1, and PHF1, Dr. A. Delacourte for antiserum AD199, Dr. Peter Seubert
(Elan Pharmaceuticals) for antibody 12E8, and Drs. Michel Goedert and
Ross Jakes for rabbit anti-
-synuclein antiserum PER4.
 |
FOOTNOTES |
*
The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in
accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
To whom correspondence should be addressed: Division of Psychiatry
Research, University of Zürich, August Forel Strasse 1, 8008 Zürich, Switzerland. Tel: 41-1-634-8873; Fax: 41-1-634-8874; E-mail: goetz@bli.unizh.ch.
Published, JBC Papers in Press, September 29, 2000, DOI 10.1074/jbc.M006531200
 |
ABBREVIATIONS |
The abbreviations used are:
4R, four-repeat;
3R, AD, Alzheimer's disease;
TUNEL, terminal deoxynucleotidyl
transferase-mediated biotinylated dUTP nick end-labeling;
PBS, phosphate-buffered saline;
TBS, Tris-buffered saline;
S1, somatosensory
cortex 1;
M1, motor cortex 1;
CA, cornu ammonis.
 |
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