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The Journal of Neuroscience, August 15, 2001, 21(16):6026-6035
Attenuated Neurodegenerative Disease Phenotype in Tau Transgenic
Mouse Lacking Neurofilaments
Takeshi
Ishihara,
Makoto
Higuchi,
Bin
Zhang,
Yasumasa
Yoshiyama,
Ming
Hong,
John Q.
Trojanowski, and
Virginia M.-Y.
Lee
Center for Neurodegenerative Disease Research, Department of
Pathology and Laboratory Medicine, University of Pennsylvania School of
Medicine, Philadelphia, Pennsylvania 19104
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ABSTRACT |
Previous studies have shown that transgenic (Tg) mice
overexpressing human tau protein develop filamentous tau aggregates in
the CNS. The most abundant tau aggregates are found in spinal cord and
brainstem in which they colocalize with neurofilaments (NFs) as
spheroids in axons. To elucidate the role of NF subunit proteins in tau
aggregate formation and to test the hypothesis that NFs are
pathological chaperones in the formation of intraneuronal tau
inclusions, we crossbred previously described tau (T44) Tg mice
overexpressing the smallest human tau isoform with knock-out mice
devoid of NFL (NFL /) or NFH (NFH/). Depletion of NF subunit proteins from the T44 mice (i.e., T44;NFL/ and T44;NFH/), in
particular NFL, resulted in a dramatic decrease in the total number of
tau-positive spheroids in spinal cord and brainstem. Concomitant with
the reduction in spheroid number, the bigenic mice showed delayed
accumulation of insoluble tau protein in the CNS, increased viability,
reduced weight loss, and improved behavioral phenotype when compared
with the single T44 Tg mice. These results imply that NFs are
pathological chaperones in the development of tau spheroids and suggest
a role for NFs in the pathogenesis of neurofibrillary tau lesions in
neurodegenerative disorders that contain both NFs and tau proteins.
Key words:
tau; neurofilaments; neurodegeneration; neurofibrillary
pathology; animal models; cytoskeleton
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INTRODUCTION |
Filamentous tau inclusions,
accompanied by extensive neuron loss and gliosis, are the
neuropathological hallmarks of an expanding family of neurodegenerative
diseases that are now collectively known as tauopathies (Lee et al.,
2001 ). Prototypical tauopathies are exemplified by
frontotemporal dementia with parkinsonism linked to chromosome 17 (FTDP-17), amyotrophic lateral sclerosis/parkinsonism-dementia complex
(ALS/PDC), Alzheimer's disease (AD), and progressive supranuclear palsy (PSP). Like other tauopathies, FTDP-17, PSP, AD, and ALS/PDC are
characterized by numerous inclusions formed by aggregated paired
helical filaments (PHFs) and/or straight filaments composed of
aberrantly phosphorylated tau proteins (PHF-tau) in widespread regions
of the CNS (Hong et al., 2000). The discovery of tau gene mutations in FTDP-17 kindreds provides unique opportunities to elucidate the disease mechanisms that underlie FTDP-17 as well as
related brain disorders characterized by abundant insoluble intracellular filamentous tau inclusions, including AD (Clark et al.,
1998; Hong et al., 1998; Hutton et al., 1998; Poorkaj et al., 1998;
Spillantini et al., 1998). The FTDP-17 mutations occur in exons and
introns of the tau gene, and they may cause FTDP-17 by
altering the functions or levels of specific tau isoforms in the CNS
(Hong et al., 1998; Hutton et al., 1998; D'Souza et al., 1999).
To begin elucidating the role of tau inclusions in the pathogenesis of
tauopathies, we generated transgenic (Tg) mice that overexpressed the
shortest human brain tau isoform (T44) in CNS (Ishihara et al., 1999,
2001). As reported previously, the T44 Tg mice acquired age-dependent
CNS pathology similar to the filamentous tau inclusions found in
FTDP-17 and ALS/PDC, including insoluble, hyperphosphorylated
intraneuronal aggregates formed by tau-immunoreactive filaments
(Ishihara et al., 1999). These inclusions, mostly in the form of
spheroids, were abundant in spinal cord neurons, in which they were
associated with axon degeneration and reduced axonal transport in
ventral roots, as well as spinal cord gliosis and motor weakness. Thus,
these Tg mice recapitulate some of, but not all, key aspects of
prototypical tauopathies. For example, tau-positive spheroids are
strongly positive for neurofilaments (NFs) in the spinal cord and
brainstem of T44 Tg mice. Significantly, NF subunit proteins have long
been associated with pathogenic protein aggregates in a number of
neurodegeneration diseases. For example, spheroids in the T44 Tg mice
and in the spinal cord of ALS/PDC patients contain both tau and
NF-immunoreactive filaments, and all three NF subunits (NFL, NFM, and
NFH) are found in neurofibrillary tangles (NFTs) at late stages of the
disease in AD patients (Schmidt et al., 1989; Ishihara et al., 1999).
NFs are also detected in Lewy bodies in which they coexist with
 -synuclein (Tu et al., 1998). Based on these and other observations,
we hypothesize that NFs may play a role as "pathological
chaperones" (Wisniewski and Frangione, 1992) in facilitating the
aggregation of tau in our Tg mice and in the human neurodegenerative
diseases discussed above.
To test this hypothesis, we generated bigenic mice overexpressing tau
protein but lacking NFL (Zhu et al., 1997) or NFH (Elder et al., 1998)
by crossbreeding T44 Tg mice with NFL knock-out (NFL/) or NFH
knock-out (NFH/) mice, respectively. Our studies showed that
depletion of NF subunit proteins, especially NFL, in T44 Tg mice
reduces or delays the pathological phenotype of T44 Tg mice as
demonstrated histologically, biochemically, and clinically. These
results show that NFs could play an important role as pathological
chaperones in the pathogenesis of tau aggregates with both tau and NF
proteins in a number of neurodegenerative tauopathies.
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MATERIALS AND METHODS |
Generation of mice. Tau Tg mice overexpressing the
shortest human brain tau isoform (T44) were generated as described
previously (Ishihara et al., 1999). We have produced stable
transgenic lines (line 7, line 27, and line 43) of T44 mice. Although
the heterozygous line 27 mice had the highest level of Tg tau, they are
not viable beyond 3 months, and homozygous mice generated from each of
the three lines died in utero or within 3 months postnatal.
Therefore, all of the T44 Tg mice used in this study were heterozygotes
from line 7 unless specified.
T44 Tg mice were crossbred with either NFL/ or NFH/ mice to
generate bigenic T44;NFL/ and T44;NFH/ mice. The
characterization of the T44 Tg mice, as well as the NFL/ and
NFH/ knock-out mice, has been well documented previously (Elder et
al., 1998; Ishihara et al., 1999, 2001; Julien, 1999). As a
consequence of crossbreeding between T44 Tg and NFL or NFH knock-out
mice, lines of mice with eight different genotypes were generated,
including wild-type (WT) littermates of T44 Tg mice (Fig.
1). The genotype of these mice was
identified by Southern blot analysis of tail DNA.
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Figure 1.
Analysis of protein expression in cortices.
Western blot analysis of tau, NFs, and -tubulin isolated from
cortices of the different lines of mice. Six-month-old mice from each
group were used. 17026, an anti-recombinant tau antibody that
recognizes both human and mouse tau, shows the expression level of tau
in each line of mice. Endogenous mouse tau protein levels are
comparable in WT, NFL/, and NFH/ mice, and overexpressed human
tau protein levels in T44 Tg, T44 Tg;NFL+/, T44;NFL/,
T44;NFH+/, and T44;NFH/ mice were ~10-fold higher than
endogenous mouse tau. NFH and NFM protein levels were decreased ~85
and 50%, respectively, in the cortices of NFL/ and T44 Tg;NFL/
mice, whereas the level of -tubulin was dramatically increased
compared with WT mice. NFL protein levels were decreased ~30% in
NFH/ and T44 Tg;NFH/ mice without a change in NFM protein
levels. Equal amounts (10 µg for tau; 20 µg for NFs and
-tubulin) of mouse cortical samples were loaded on each gel
lane.
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Immunohistochemical analyses. Tg mice aged 1-15 months were
lethally anesthetized and perfused transcardially using fixative including 70% ethanol in isotonic saline and 4% paraformaldehyde in
PBS as described previously (Ishihara et al., 1999) in accordance with
protocols approved by University of Pennsylvania University Laboratory
Animal Research. Immunohistochemistry were performed on representative
6 µm paraffin sections from brain, spinal cord, and spinal roots.
Three animals from each mouse line and at each specific age were used
for the immunohistochemical studies. Anti-NF antibodies were used to
detect the presence or absence of NF subunit proteins in the CNS of Tg
mice, and sections were also stained with anti-tau antibodies to
localize the tau aggregates (Table 1). In
addition, mouse monoclonal antibodies (mAbs) to - and  -tubulin and antibodies to -internexin and to peripherin were used to assess whether or not these molecules also accumulate in tau
aggregates (Table 1).
Axonal tau pathology in Tg mice was quantified by counting the number
of tau-positive spheroids with a diameter of >5 µm. This
quantitative analysis was performed for 20 lumber spinal cord sections
from each Tg mouse, and the average number of spheroids per section was
used as a representative value. The mean and SD values of the number of
spheroids in three Tg mice at the same age were estimated in each line,
and the difference in the number of spheroids among all of the lines
was examined statistically by one-way ANOVA.
To see colocalization of the cytoskeletal components in the
spheroids, selected sections were also double- and triple-labeled by
immunofluorescence using anti-tau, anti-NF subunit, and anti-tubulin antibodies (Table 1).
Western blot analysis of Tau, NFs, peripherin,
-internexin, and tubulin expressed in the CNS of Tg mice.
Tissues were carefully dissected after mice were lethally anesthetized.
The tissues were homogenized in 2 ml/gm ice-cold high salt reassembly
buffer (RAB) [0.1 M MES, 1 mM EGTA, 0.5 mM
MgSO4, 0.75 M NaCl, 0.02 M NaF, 1 mM PMSF, and 0.1%
protease inhibitor cocktail (100 µg/ml each of pepstatin A,
leupeptin, N-tosyl-L-phenylalanyl cloromethyl ketone, N-tosyl-lisine cloromethyl ketone,
soybean trypsin inhibitor, and 100 mM EDTA), pH
7.0] and centrifuged at 50,000 × g for 40 min at
4°C in the Beckman Instruments (Fullerton, CA) TL-100
ultracentrifuge. The pellets were saved for NF-peripherin-internexin
analyses. Half of the supernatants were saved for tubulin analyses, and the rest were boiled for 5 min, chilled on ice for 5 min, and recentrifuged at 10,000 × g for 20 min at 4°C, and
the supernatant were saved for tau Western blotting. Protein
concentration was then determined for the samples using the BCA assay
kit (Pierce, Rockford, IL). Equal amounts of samples were subsequently
resolved on 7.5% SDS-PAGE gels and transferred onto nitrocellulose
membranes. Quantitative Western blot analyses (three animals from each
line) were performed by using either
[125I]-labeled goat anti-mouse IgG or
[125I]-labeled Protein A (NEN, Boston,
MA) as secondary antibodies as described previously (Ishihara et al.,
1999).
Tau and NF protein solubility in the CNS of Tg mice. To
study the solubility of tau and NF proteins in the mouse CNS, cerebral cortical and spinal cord tissues of 1-, 3-, 6-, 9-, and 12-month-old mice in each group (n = 3) were extracted with RAB to
generate the RAB-soluble fractions as described above. The pellets were rehomogenized with 1 M sucrose-RAB (0.1 M MES, 1 mM EGTA, and 0.5 mM MgSO4, pH 7.0) and
centrifuged at 50,000 × g for 20 min at 4°C to
eliminate myelin and related lipids. The resulting pellets were
extracted with 1 ml/gm radioimmunoprecipitation assay buffer (RIPA) buffer (50 mM Tris, 150 mM NaCl, 1% NP-40, 5 mM
EDTA, 0.5% sodium deoxycholate, and 0.1% SDS, pH 8.0) and centrifuged
to generate RIPA-soluble samples. Finally, the RIPA-insoluble pellets were reextracted with 70% formic acid (FA) to recover the most insoluble cytoskeletal aggregates. Quantitative Western blot analyses were used to determine tau and NF levels in each fraction.
Transmission electron microscopy. To determine the
ultrastructural features of the inclusions, transmission EM (TEM) was
performed in T44, T44;NFL/ mice, as well as WT littermates, at 6 and 12 months of age (n = 6). These mice were deeply
anesthetized and killed by intracardiac perfusion with 10 ml of
0.05% glutaraldehyde and 0.5% paraformaldehyde in 0.1 M cacodylate buffer, pH 7.4, followed by 50 ml
fixative of 2% glutaraldehyde and 2% paraformaldehyde in 0.1 M cacodylate buffer. The L5 segments of the
spinal cord and ventral root were removed and post-fixed in 2% osmium
tetroxide for 60 min at 4°C. After dehydration with graded alcohol
and propylene oxide, the blocks were embedded in Epon-812 and
polymerized at 60°C for 72 hr. Sixty-five nanometer thin sections
were cut and mounted on 200 mesh copper grids, stained with 1% uranyl
acetate in 50% ethanol and bismuth subnitrate, and examined with a
JEM1010 electron microscope (Jeol, Peabody, MA) at 80 kV.
Preembedding immunoelectron microscopy. To determine
components of proteins in the inclusions, preembedding immunoelectron microscopy (immuno-EM) was performed in the T44, T44;NFL/, and age-matched WT mice at 6 and 12 months of age (n = 4).
These mice weredeeply anesthetized and killed by intracardiac perfusion
with 10 ml of 0.05% glutaraldehyde and 0.5% paraformaldehyde in 0.1 M cacodylate buffer, pH 7.4, followed by 50 ml
fixative of 0.2% glutaraldehyde and 2% paraformaldehyde in 0.1 M cacodylate buffer. The L5 segments of the
spinal cord and ventral root were removed and post-fixed in fixative of
4% paraformaldehyde, 0.2% glutaraldehyde, and 0.2 picric acid in 0.1 M cacodylate buffer overnight. The spinal cords
were cut into 50-µm-thick sections with a vibratome, followed by
quenching in 0.1% sodium borohydride in Tris-buffered saline
for 10 min and treatment for another 10 min with 20% ethanol. The
sections were blocked in 5% donor horse serum in PBS with 0.1%
cold-water fish skin gelatin and 1% chicken egg albumin for 60 min and
then incubated with 17026, the recombinant anti-tau antiserum (dilution
of 1:500), in 0.1% bovine serum albumin and PBS overnight at 4°C. By
using diaminobenzidine (DAB) plus the silver-gold enhancement
immuno-EM method, biotinylated goat anti-rabbit IgG (dilution of 1:100;
Vector Laboratories, Houston, TX) secondary antibody was applied for 2 hr at room temperature for each set of sections. After visualizing the
DAB-positive staining labeled by routine immuno-EM methods,
silver-gold intensification was performed by incubating the sections
in silver methenamine developer containing 3% methenamine, 5% silver
nitrate, and 1% sodium tetraborate at 60°C for 10 min as described
previously (Teclemariam-Mesban et al., 1997). The reaction was
stopped with 2% sodium acetate and then stabilized in 3% sodium
thiosulphate for 5 min. Gold toning was obtained by incubating the
sections in 0.1% gold chloride for 5 min, followed by stabilizing with
3% sodium thiosulfate for 5 min. Sections were fixed within 2%
glutaraldehyde in PBS buffer overnight and processed and examined as
described above for TEM.
Tail suspension test. The tail suspension test was performed
as described by Yamamoto et al. (2000). Briefly, mice from each experimental group (n = 4) were suspended by their
tails for 15 sec and videotaped at 5 and 11 months of age. Animals were
assessed for clasping score. The test period was divided into 2 sec
slots. An animal would receive a score of 1 point if they displayed any abnormal movements during the given time slot. An abnormal movement was
defined as dystonic movements of the hindlimbs or a combination of
hindlimbs and forelimbs and trunk, during which the limbs were pulled
into the body in a manner not observed in WT mice.
Antibodies. The antibodies used in this study are
summarized in Table 1.
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RESULTS |
Generation and characterization of bigenic mice
To generate the mice used in these experiments, T44 Tg
mice were crossbred with either NFL/ or NFH/ mice. As a
consequence of crossbreeding, mice with eight different genotypes i.e.,
WT, T44, NFL/, NFH/, T44;NFL+/, T44;NFL/, T44;NFH+/,
and T44;NFH/, were generated (Fig. 1). Western blot analyses were
conducted to determine whether or not the expression of transgenic
human fetal tau or the elimination of NFL and NFH in mice alter the level of expression of the relevant endogenous cytoskeletal proteins in
each of the lines. For example, previous studies have shown that, in
the absence of NFL, there is a downregulation of NFH and NFM levels and
an upregulation of tubulin subunits (Zhu et al., 1997), and our data
(Fig. 1) confirmed reduction in both NFH and NFM (85 and 50%,
respectively) and an increase in -tubulin levels in the NFL/ and
T44;NFL/ mice. Similar to previous studies on NFH/ mice (Elder
et al., 1998), in the absence of NFH, NFL protein levels were
decreased by ~30% in the CNS. However, the endogenous mouse tau
protein levels remained unchanged in cortices of WT, NFL/, and
NFH/ mice (Fig. 1). The expression of human tau in the bigenic
T44;NFL/ and T44;NFH/ mice also has no effect on the levels of
any of the relevant endogenous cytoskeletal proteins, including
peripherin and -internexin (Fig. 1 and data not shown). The same
pattern of protein expression was also observed in the spinal cord of
these mice (data not shown). Thus, the expression of the human fetal
tau transgenic protein was not altered by crossbreeding the T44 mice
with NFL/ and NFH/ mice, nor did the expression of the tau
transgene affect the pattern of relevant endogenous gene expression in
the CNS.
A reduction in the number of spheroids in the CNS of T44 mice
lacking NFs
Because the major pathology found in the T44 mice are spheroids
containing both human tau and NF subunit proteins, we sought to
determine whether or not the removal of NFL or NFH would have any
effect on the number and the composition of the spheroids. As reported
previously (Ishihara et al., 1999) and as shown in Figure
2, the number of tau-positive spheroids
in the spinal cord of the T44 mice increased with age until ~6
months, and they decreased thereafter. The number of spheroids in the
T44;NFH+/ and T44;NFH/ bigenic mice also peak at 6 months of age,
but there is a significant reduction in the total number of spheroids
in the T44;NFH+/ (~80% reduction at the age of 6 months) and
T44;NFH(/) mice (~90% reduction at the age of 6 months) compared
with the single T44 Tg mice (Fig. 2). The T44;NFL/ mice showed an
even greater reduction in the number of spheroids than T44;NFH/
mice. Compared with the T44 Tg mice, there is an ~85% reduction in
the number of spheroids in T44;NFL+/ mice at the age of 6 months.
Notably, almost no spheroids were found in T44;NFL/ mice until
~12 months of age, although dense tau staining was observed in axons
in spinal cord white matter (Fig. 3).
However, the number of spheroids in the T44;NFL/ mice increased
with age, demonstrating that tau aggregates occur over a more prolonged
time period in the absence of NFL. Figure 3 illustrates the progressive
reduction of the total number of spheroids in the spinal cords of T44
(A), T44;NFL+/ (B), and T44;NFL/ (C) mice at 6 months.
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Figure 2.
Quantification of spheroids in the mouse spinal
cord at different ages. Quantification of spheroids is described in
Materials and Methods. The mean value among three mice is shown for the
number of spheroids summarized here. The error bars represent
SEM. Statistical analysis was performed for the mice at the same
age by ANOVA. Significant differences between T44 and other Tg mice are
indicated by asterisks (*p < 0.001;
**p < 0.0001).
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Figure 3.
Comparison of the number of spheroids in T44,
T44;NFL+/, and T44;NFL/ Tg mice. The sections were stained with
17026, a polyclonal antibody to recombinant tau protein. Note the
reduction in the number of tau-positive spheroids in the bigenic mice
relative to the T44 mice at 6 months of age. All photomicrographs are
at the same magnification. Scale bar, 10 µm.
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Because we showed previously that all three NF subunit proteins
coexisted with tau protein in the spheroids of the T44 mice, we
performed triple-labeled indirect immunofluorescence to evaluate the
consequences of elimination one of the three NF subunits on the
composition of the spheroids. In the T44;NFL/ mice, although NFH
was found to colocalize with tau protein in the spheroids, the
intensity of NFH staining was dramatically reduced compared with the
T44 Tg mice (Fig. 4, compare
A-D with E-H), indicating that tau
protein is the major component of spheroids in this mouse. A slight
reduction in the intensity of NFL stain was also observed in spheroids
of T44;NFH/ mice (Fig. 4I-L). The intensity of NFM immunoreactivity in the spheroids also was reduced dramatically in
T44;NFL/ mice and slightly diminished in the T44;NFH/ mice (data not shown). Previous studies also identified - and -tubulin in the tau-immunoreactive spheroids in the T44 mice, and the increase in tubulin subunits observed in the T44;NFL/ bigenic lines (Fig. 1)
also resulted in an increase of - and -tubulin staining in the
spheroids compared with the T44 and T44;NFH/ mice (data not shown).
In contrast, although spheroids also contained -internexin, there
was no difference in the intensity of the staining for this neuronal
intermediate filament protein among all lines of mice, and none of the
spheroids were stained with the anti-peripherin antibody (data not
shown). Thus, the reduction in the intensity of staining described here
reflects the overall change in the neuronal cytoskeleton of the
NFL/ and NFH/ mice (Zhu et al., 1997; Elder et al., 1998).
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Figure 4.
Triple immunofluorescence staining of spheroids in
the spinal cord of 6-month-old T44, 15-month-old T44;NFL/, and
6-month-old T44 Tg;NFH/ mice. The sections were stained with the
T14 mouse anti-human tau mAb (A, E,
I), the rabbit anti-NFL antibody
(B, F, J), and the
DP1.6 rat anti-NFH mAb (C, G,
K). A triple fluorescent filter cube was used to
visualize colocalization of tau, NFL, and NFH (D,
H, L). All photomicrographs are at the
same magnification. Scale bar, 10 µm.
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Electron microscopy detect tau filaments in the absence of
NFs filaments
To determine whether or not tau aggregates contain filaments in
the absence of NFL, which is the backbone of NFs, TEM and immuno-EM were performed in the T44;NFL/ mice. In control WT mice,
NFs were evenly distributed in the normal axon of the spinal cord (Fig.
5A), but less NFs and more
microtubules were found in the NFL/ and T44;NFL/ mice (Fig.
5B,C). TEM studies of the tau
inclusions revealed masses of loosely (Fig. 5D-F) or
tightly packed aggregates (Fig. 5G-I) of randomly
arranged 10-20 nm straight filaments. These aggregates were found in
myelinated (Fig. 5G-L) and unmyelinated axons in peripheral
white matter of the spinal cord (Fig. 5D-F) of
12-month-old T44;NFL/ mice (Fig. 5D-L) but not in
age-matched control WT mice (Fig. 5A). Preembedding
immuno-EM studies showed that the aggregates were immunolabeled by
antibodies to tau (Fig. 5J-L). Different packing densities
of tau filamentous aggregates could represent different stages of
aggregate formation, from loose to tightly packed filamentous
aggregates, or from small to big aggregates. We also observed a shift
in the location of tau aggregates: from the junction of the gray and
white matter in the T44 mice to the peripheral white matter of the
T44;NFL/ mice. Because our previous studies in the T44 mice suggest
that axonal transport was compromised in the presence of tau axonal aggregates, the detection of tau aggregates in peripheral white matter
of the T44;NFL/ mice may indicate a possible endogenous rescue of
axonal transport in the bigenic mice. Together, these results indicate
that filamentous tau aggregates develop without NFL, but the delay in
the formation of aggregates suggests that NFs play an important role as
a pathological chaperone in tau aggregate formation.
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Figure 5.
Tau-rich aggregates in the axons of the peripheral
white matter of spinal cord contain straight tau filaments.
A, NFs are evenly distributed in a spinal cord
myelinated axon of a WT mouse. B, C, Few
NFs (arrowheads) and more microtubules (short
arrows) are seen in the NFL/ (B) and
T44;NFL/ (C) mice. D-F, A
mass of loosely packed disorganized filaments (large
arrow) in a spinal cord unmyelinated axon of a 12-month-old
T44;NFL/ mouse. D-F show the same aggregate at
different magnification. G-I, A mass of tightly packed
disorganized filaments in a myelinated axon of spinal cord in a
12-month-old T44;NFL/ mouse. G-I show the same
aggregate at increased magnification. J-L, Preembedding
immuno-EM labeled the aggregates with antibody 17026 in a 12-month-old
T44;NFL/ mouse. J-L show the same aggregate at
different magnifications. Note that silver enhancement was performed
for 17026 staining. Scale bars: A-C, 500 nm;
D, G, J, 10 µm;
E, H, K, 500 nm;
F, I, L, 100 nm.
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The absence of NFL delayed accumulation of insoluble tau protein in
the CNS of T44;NFL/ bigenic mice
As reported previously, tau protein becomes progressively more
insoluble and hyperphosphorylated with age in the T44 Tg mice as in
human tauopathies (Ishihara et al., 1999, 2001). To determine the
effect of crossbreeding of T44 with NFL/ and NFH/ mice on the
accumulation of insoluble tau, we analyzed the solubility of tau
protein in the different lines by extracting brain and spinal cord
samples using buffers with increasing extraction strengths. The spinal
cord and brain samples from 6- and 12-month-old mice from each group of
single and bigenic mice were sequentially extracted with RAB, RIPA
buffer, and 70% FA. The three fractions were then analyzed by
quantitative Western blotting with antibody 17026. As shown in Figure
6, over 90% of endogenous mouse tau from
both the brain and the spinal cord of the WT mice was primarily RAB soluble, and no tau immunoreactivity was detected in the FA-soluble fraction. At 6 months of age, ~76 and ~74% of total tau protein were RAB soluble, and ~1.2 and ~1.7% were in the FA-soluble
fraction in the brain and spinal cord of the T44 Tg mice, respectively. Although the RAB-soluble tau remained relatively constant (at ~74-80% in each group) in all lines of mice overexpressing human tau protein, the amount of FA-soluble fraction as a percentage of total
tau protein in T44;NFL/ mice was significantly decreased compared
with that of T44 Tg mice at 6 months of age. However, this formic acid
extractable pool of tau in the T44;NFL/ mice increased by 90%
(brain) and 78% (spinal cord) between 6 and 12 months of age, whereas
a similar increase in the other mouse lines was more modest at
~18-43% (Fig. 6C,D). These results are
consistent with the immunohistochemical findings showing that there
were no detectable tau aggregates in the spinal cord of T44;NFL/ mice at 6 months of age but that they appeared at ~12 months of age
(Fig. 2).
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Figure 6.
Accumulation of insoluble tau protein in the CNS
of mice. A, B, Neocortical
(A) and spinal cord (B)
tissues of 6-month-old mice from each group were sequentially extracted
with RAB, RIPA buffer, and 70% FA, and the tau levels were determined
by quantitative Western blot analysis with antibody 17026. C, D, Alteration in the FA-soluble tau as
a percentage of total tau in the neocortex (C)
and spinal cord (D) of T44 tau-overexpressing
mice. The percentage was calculated from quantitative Western blot
analysis at 6 and 12 months of age (n = 3). The
amount of FA-soluble fraction as a percentage of total tau protein in
T44;NFL/ mice was significantly decreased (*p < 0.01) compared with that of T44 mice at 6 month of age, and it
increased by 90.2% (neocortex) and 77.6% (spinal cord) at 12 months
of age, whereas the increase in the other group of mice was
17.5-42.9%.
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To determine whether or not tau proteins in these bigenic mice are also
hyperphosphorylated at the same sites as in PHF-tau in the AD and
ALC/PDC brain, we performed Western blot analysis of RAB- and
FA-soluble tau extracted from the cerebral cortex of Tg mice. Both RAB-
and FA-soluble Tg tau protein in these mice were recognized by the
phosphorylation-dependent antibodies PHF-1 (phosphoserine 396 and 404),
T3P (phosphoserine 396), and AT270 (phosphothreonine 181), indicating
that the phosphorylation state of Tg tau recapitulates that of PHF-tau
found in human tauopathies (including AD and ALS/PDC) and human fetal
tau but is different from that of normal adult human brain tau (Fig.
7). There was no obvious difference in
the extent of phosphorylation between T44 Tg mice and the other T44
overexpressed mice, including T44;NFL+/, T44;NFL/, T44;NFH+/,
and T44;NFH/ mice (Fig. 7).
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Figure 7.
Phosphorylation state of tau in the CNS of
single Tg and bigenic mice. Immunoblots were performed using PHF tau
from AD brain, autopsy-derived normal human adult tau and
autopsy-derived human fetal tau samples, as well as soluble and
insoluble fractions of tau from the neocortex of 12-month-old T44 Tg,
T44 Tg;NFL/, and T44;NFH/ mice. Antibodies 17026 and T14
recognized total tau proteins regardless of the phosphorylation state.
Antibody T1 was specific to nonphosphorylated tau and did not react
with PHF-tau. Phosphorylation-dependent antibodies PHF1, T3P, and AT270
did not recognize normal adult tau but reacted with PHF-tau and fetal
tau, as well as both soluble and insoluble fraction of tau from the
single Tg and bigenic mice.
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Bigenic mice lacking NF subunits showed increased viability,
reduced weight loss, and improved behavioral phenotype
We showed previously a correlation between reduced viability and
increased expression of transgenic tau protein in our three stable tau
Tg lines (Ishihara et al., 1999). For example, the heterozygous mice in
the highest tau-expressing line were not viable beyond 3 months, and
homozygous mice generated from each of the lower expressing lines died
in utero or within 3 months postnatal. Although ~80% of
the heterozygous mice in the lowest expressing T44 line used in this
study survived >3 months, only ~60% lived >1 year (Fig.
8). As a consequence of crossbreeding T44
Tg mice with NF knock-out mice, the impaired viability of these bigenic
mice was reduced. For example, although only 67% of T44 Tg mice lived
for 9 months, the 9 month survival of the T44;NFL/ and T44;NFH/
mice was ~90 or 78%, respectively, whereas the viability of NFL/
and NFH / mice was comparable with that of WT mice at 9 months
(data not shown).
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Figure 8.
Longevity of mice. Approximately 67% of T44 Tg
mice survived until 9 months of age, but after crossbreeding with
NFL/ or NFH/ mice, the survival went up to 90 or 78%,
respectively. Longevity of mice was evaluated using cohorts of pups
weaned at 3-4 weeks old because some mice died before 4 weeks from
poor nursing. WT, n = 30; T44;NFL/,
n = 20; T44;NFH/, n = 18;
T44+/, n = 24; T44+/+, n = 8.
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As reported previously, T44 Tg mice develop progressive motor weakness
as demonstrated by an impaired ability to stand on a slanted surface
and by the clasping of their hindlimbs when lifted by the tail. These
impairments may explain why T44 Tg mice weighed ~30-40% less than
age-matched WT littermates. Crossbreeding T44 Tg mice with NF knock-out
mice reduced the weight loss and abnormal clasping behavior that were
seen in T44 Tg mice. As shown in Figure
9, T44 Tg mice weighed 35% less than WT
littermates at 12 months of age, although no weight loss was observed
after crossbreeding the T44 mice with the NFL/ mice, but
significant albeit moderate (~8%) weight loss was observed when T44
Tg mice were crossbred with NFH/ mice. Crossbreeding T44 Tg mice
with NFL/ mice, but not with NFH/ mice, significantly reduced
the abnormal clasping behavior at 5 and 11 months of age (Fig.
10). For example, the T44 mice had a
clasping score of ~4 at 11 months of age, but the T44;NFL/ mice
had a clasping score of only 2 by the same age, suggesting a dramatic
reduction in hindlimb weaknesses. Together with the change in
viability, our data suggest that the reduction of tau aggregates by
crossbreeding T44 Tg mice with NF knock-out mice partially rescues the
pathological symptoms caused by overexpression of human tau
protein.
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Figure 9.
Weight change of mice. Bar graphs illustrate
average body weight of T44+/: WT, T44;NFL/: NFL/, and
T44;NFH/: NFH/, respectively, at 12 months of age. T44 Tg mice
weighed 35% less than WT littermates, although similar weight loss was
no longer observed after crossbreeding T44 mice with NFL/ mice.
More modest (8%) but significant weight loss was observed in
T44;NFH/ mice (n = 8-12).
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Figure 10.
Clasping phenotype of mice. Mice were videotaped
during a 15 sec tail suspension test at 5 and 11 months of age and
analyzed for clasping score as described in Materials and Methods.
T44;NFL/ mice showed a significantly lower score than T44 Tg mice
at 5 and 11 months. Bar graphs show mean ± SE of five trials.
p values are shown in the graph.
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DISCUSSION |
In the present study, we have provided compelling evidence that NF
subunit proteins act as pathological chaperones that facilitate the
formation of intraneuronal filamentous tau aggregates that cause
neurodegeneration in the T44 Tg mice. To accomplish this, we eliminated
either NFL or NFH by crossbreeding the T44 Tg mice with NFL/ or
NFH/ mice, and our analyses of the T44;NFL/ and T44;NFH/
bigenic mice provide unequivocal evidence supporting a pathogenic role
for NFs in the formation of these filamentous tau lesions. For example,
we showed a dramatic reduction in the number of tau-positive aggregates
in the spinal cord of T44 mice lacking either NFH or NFL, suggesting
that both of these NF subunit proteins play an important role in
facilitating filamentous tau aggregate formation. Furthermore, this
reduction in aggregate number appears to correlate with the overall
improvement in the health of the bigenic mice because increased
survival and reduced weight loss were observed in these mice compared
with the T44 single Tg mice. Finally, the reduction in tau spheroids in
the T44;NFL/ mice also correlated with attenuation of the motor weaknesses observed in the T44 mice, suggesting a pathologic role of
the aggregates in causing motor neuron degeneration.
As discussed above, although NFs have long been associated with a
number of neurodegenerative diseases because NF immunoreactivities have
been detected in well known neuropathologic lesions, including NFTs in
AD (Schmidt et al., 1989, 1990), Lewy bodies in Parkinson's disease
and diffuse Lewy body disease (Nakazato et al., 1984; Trojanowski and
Lee, 1998), as well as in the axonal spheroids in classic ALS
(Nakazato et al., 1984) and in ALS/PDC (Shankar et al., 1989; Ishihara
et al., 1999), the precise role NFs play in the pathogenesis of these
neurodegenerative diseases remains unclear. Studies reported in the
early 1980s implicated NFs as the building blocks of NFTs because NFTs
are immunopositive for NFs and because 10-nm-diameter NFs are the most
abundant cytoskeletal structures in mature neurons (Dahl et al., 1982;
Perry et al., 1985). However, subsequent biochemical and genetic
studies demonstrated unequivocally that tau proteins rather than NFs
are the building blocks of NFTs (Goedert et al., 1989; Lee et al.,
1991; Hutton et al., 1998; Poorkaj et al., 1998). Other studies support
a secondary role for NFs in the pathogenesis of NFTs in AD because NF
immunoreactivity is only detected in NFTs at late stages of the
disease, but NF proteins are not detected in pretangles or in the
majority of the NFTs (Schmidt et al., 1989). In this regard, it is
tempting to speculate that the involvement of NFs at late stages in the neurodegenerative disease mentioned above may be a harbinger of the
final collapse of NFs, the most abundant cytoskeletal structure in
neurons, followed by the trapping of NFs within NFTs and other disease-specific intraneuronal inclusions.
Although NFTs containing both NFs and PHFs are found in neuronal
perikarya, the formation of NF-positive spheroids in the axon hillock
of spinal cord motor neurons of ALS, ALS/PDC, and FTDP-17 patients is
likely to be mediated by different pathogenic mechanism(s).
Significantly, NF-containing spheroids have been detected in the spinal
cord and ventral roots of a number of Tg mouse models overexpressing
the following: (1) different NF subunits (Xu et al., 1993; Wong et al.,
1995); (2) mutant superoxide dismutase 1 (Zhang et al., 1997); and (3)
tau proteins (Ishihara et al., 1999; Spittaels et al., 1999; Probst et
al., 2000). These Tg mice all showed selective axonal degeneration and
motor weaknesses most likely attributable to the interruption of
fast and slow axonal transport by the NF-rich spheroids (Collard et
al., 1995; Zhang et al., 1997; Ishihara et al., 1999). Although the
exact mechanisms for the development of the spheroids in these Tg mice is unknown, it is possible that selective aggregation of NFs occur because of the overexpression of specific transgenes that perturb the
transport of a large number of slow-moving NFs in motor neurons. Regardless of the exact mechanism of pathogenesis, the results of
previous studies in Tg mice point to a role of NFs in spheroid formation and motor neuron degeneration. The production of bigenic mice
overexpressing tau but lacking either NFL or NFH allow the dissection
of the role of each of these NF subunits in the development of tau
pathology in our model of tauopathies.
Our analyses on the T44;NFL/ and T44/NFH/ mice suggest that the
elimination of NFL rather than NFH is more efficacious in reducing the
number of spheroids and ameliorating the phenotypes of the T44 mice.
This observation is reasonable because NFL forms the backbone of intact
NFs and NFL/ mice contain no intact NFs, but they contain reduced
levels of NFH and NFM (Zhu et al., 1997). In contrast, abundant NFs
comprised of NFL and NFM are present in the NFH/ mice (Elder et
al., 1998; Rao et al., 1998; Zhu et al., 1998). Thus, our data suggest
that intact NFs are involved in the coaggregation with tau filaments in
the spheroids. However, it is surprising that the T44;NFH/ mice
also showed a dramatic reduction (fourfold reduction) in the number of
tau-positive spheroids compared with the single T44 mice because
previous studies have shown only a 20% reduction in the total number
of NFs in the NFH/ mice compared with WT mice (Elder et al., 1998).
This observation suggests that, in addition to the filament backbone,
NFH, the sidearms of NFs also participate in coaggregation with tau
filaments. Indeed, the presence of NFH in tau-positive spheroids of
T44;NFL/ mice support an independent role of NFH in tau aggregation formation.
In our previous studies on the T44 Tg mice, we were able to define two
different types of neuronal tau protein aggregates based on different
criteria, including the age of onset, the location within the CNS, and
the protein composition (Ishihara et al., 1999, 2001). The first type
of tau inclusions consists of mostly spheroids found predominantly in
the spinal cord and brainstem. The spheroids contain NFs and other
cytoskeletal proteins appear at ~1 month of age and peak at ~6
months of age. However, a second type of tau aggregates developed in
the hippocampus and associated areas of the T44 mice as they advance in
age to over 18 months. These tau inclusions are not immunopositive for
NFs and appear to comprise only of tau proteins, although ubiquitin
immunoreactivities also are detected (Ishihara et al., 2001). In this
regard, the second type of tau aggregates are more similar to authentic
AD NFTs because the second but not the first are stained by
histochemical dyes such as Congo red, Thioflavin S, and Gallyas Silver.
These hippocampal tau tangles are relative few in number, and we
hypothesize that the development of these tangles is an age-related
phenomenon much like normal aging in humans. Interestingly, although
the absence of NFL in the T44 mice essentially abrogates the first type
of tau inclusions, it has no effect on the development of congophilc
tau tangles in the hippocampus of aged T44;NFL/ mice (data not
shown). Thus, our analyses of the bigenic mice define an NF-dependent
and an NF-independent mechanism for the pathogenesis of tau inclusions.
Although much still needs to be done to elucidate how different types
of tau aggregates develop in different tauopathies, the analyses of tau
Tg mouse models provide important insights into the pathogenesis of
diverse neurodegenerative diseases, including AD, ALS/PDC, and related tauopathies.
| |
FOOTNOTES |
Received March 21, 2001; revised May 11, 2001; accepted May 30, 2001.
This work was supported by grants from the National Institute on Aging.
We thank Drs. Jean-Pierre Julien and Robert Lazzarini for providing the
NFL/ and NFH/ mice, respectively, and the Biomedical Imaging
Core Facility of the University of Pennsylvania for assistance in the
electron microscopic studies.
Correspondence should be addressed to Dr. Virginia M.-Y. Lee,
Department of Pathology and Laboratory Medicine, University of
Pennsylvania School of Medicine, Maloney 3, HUP, 3600 Spruce Street,
Philadelphia, PA 19104-4283. E-mail: vmylee{at}mail.med.upenn.edu.
| |
REFERENCES |
-
Binder LI,
Frankfurter A,
Rebhun LI
(1985)
The distribution of tau in the mammalian central nervous system.
J Cell Biol
101:1371-1378[Abstract/Free Full Text].
-
Carden MJ,
Trojanowski JQ,
Schlaepfer WW,
Lee VM-Y
(1987)
Two-stage expression of neurofilament polypeptides during rat neurogenesis with early establishment of adult phosphorylation patterns.
J Neurosci
7:3489-3504[Abstract].
-
Clark LN,
Poorkaj P,
Wszolek Z,
Geschwind DH,
Nasreddine ZS,
Miller B,
Li D,
Payami H,
Awert F,
Markopoulou K,
Andreadis A,
D'Souza I,
Lee VM-Y,
Reed L,
Trojanowski JQ,
Zhukareva V,
Bird T,
Schellenberg G,
Wilhelmsen KC
(1998)
Pathogenic implications of mutations in the tau gene in pallido-ponto-nigral degeneration and related neurodegenerative disorders linked to chromosome 17.
Proc Natl Acad Sci USA
95:13103-13107[Abstract/Free Full Text].
-
Collard JF,
Cote F,
Julien JP
(1995)
Defective axonal transport in a transgenic mouse model of amyotrophic lateral sclerosis.
Nature
375:61-64[Medline].
-
Dahl D,
Selkoe DJ,
Pero RT,
Bignami A
(1982)
Immunostaining of neurofibrillary tangles in Alzheimer's senile dementia with a neurofilament antiserum.
J Neurosci
2:113-119[Abstract].
-
D'Souza I,
Poorkaj P,
Hong M,
Nochlin D,
Lee VM-Y,
Bird TD,
Schellenberg GD
(1999)
Missense and silent tau gene mutations cause frontotemporal dementia with parkinsonism-chromosome 17 type, by affecting multiple alternative RNA splicing regulatory elements.
Proc Natl Acad Sci USA
96:5598-5603[Abstract/Free Full Text].
-
Elder GA,
Friedrich VL,
Kang C,
Bosco P,
Gourov A,
Tu PH,
Zhang B,
Lee VM-Y,
Lazzarini RA
(1998)
Requirement of heavy neurofilament subunit in the development of axons with large calibers.
J Cell Biol
143:195-205[Abstract/Free Full Text].
-
Goedert M,
Spillantini MG,
Jakes R,
Rutherford D,
Crowther RA
(1989)
Multiple isoforms of human microtubule-associated protein tau: sequences and localization in neurofibrillary tangles of Alzheimer's disease.
Neuron
3:519-526[ISI][Medline].
-
Goedert M,
Jakes R,
Crowther RA,
Cohen P,
Vanmechelen E,
Vandermeeren M,
Cras P
(1994)
Epitope mapping of monoclonal antibodies to the paired helical filaments of Alzheimer's disease: identification of phosphorylation sites in tau protein.
Biochem J
301:871-877.
-
Greenberg SG,
Davies P
(1990)
A preparation of Alzheimer paired helical filaments that displays distinct tau proteins by polyacrylamide gel electrophoresis.
Proc Natl Acad Sci USA
87:5827-5931[Abstract/Free Full Text].
-
Greenberg SG,
Davies P,
Schein JD,
Binder LI
(1992)
Hydrofluoric acid-treated tau PHF proteins display the same biochemical properties as normal tau.
J Biol Chem
267:564-569[Abstract/Free Full Text].
-
Hoffmann R,
Lee VM-Y,
Leight S,
Varga I,
Otvos L
(1997)
Unique Alzheimer's disease paired helical filament specific epitopes involve double phosphorylation at specific sites.
Biochemistry
36:8114-8124[Medline].
-
Hong M,
Zhukareva V,
Vogelsberg-Ragaglia V,
Wszolek Z,
Reed L,
Miller BI,
Geschwind DH,
Bird TD,
McKeel D,
Goate A,
Morris JC,
Wilhelmsen KC,
Schellenberg GD,
Trojanowski JQ,
Lee VM-Y
(1998)
Mutation-specific functional impairments in distinct tau isoforms of hereditary FTDP-17.
Science
282:1914-1917[Abstract/Free Full Text].
-
Hong M,
Trojanowski JQ,
Lee VM-Y
(2000)
Tau-based neurofibrillary lesions.
In: Neurodegenerative dementias: clinical features and pathological mechanisms (Clark CM,
Trojanowski JQ,
eds), pp 161-176. New York: McGraw-Hill.
-
Hutton M,
Lendon CL,
Rizzu P,
Baker M,
Froelich S,
Houlden H,
Pickering-Brown S,
Chakraverty S,
Isaacs A,
Grover A
(1998)
Association of missense and 5'-splice-site mutations in tau with the inherited dementia FTDP-17.
Nature
393:702-705[Medline].
-
Ishihara T,
Hong M,
Zhang B,
Nakagawa Y,
Lee MK,
Trojanowski JQ,
Lee VM-Y
(1999)
Age-dependent emergence and progression of a tauopathy in transgenic mice overexpressing the shortest human tau isoform.
Neuron
24:751-762[ISI][Medline].
-
Ishihara T,
Zhang B,
Higuchi M,
Yoshiyama Y,
Trojanowski JQ,
Lee VM-Y
(2001)
Age-dependent induction of congophilic neurofibrillary tau inclusions in tau transgenic mice.
Am J Pathol
158:555-562[Abstract/Free Full Text].
-
Julien JP
(1999)
Neurofilament functions in health and disease.
Curr Opin Neurobiol
9:554-560[ISI][Medline].
-
Kosik KS,
Orecchio LD,
Binder L,
Trojanowski JQ,
Lee VM-Y,
Lee G
(1988)
Epitopes that span the tau molecule are shared with paired helical filaments.
Neuron
1:817-825[ISI][Medline].
-
Lang E,
Szendrei GI,
Lee VM-Y,
Otvos L
(1992)
Immunological and conformation characterization of a phosphorylated immunodominant epitope on the paired helical filaments found in Alzheimer's disease.
Biochem Biophys Res Commun
187:783-790[ISI][Medline].
-
Lee VM-Y,
Carden MJ,
Trojanowski JQ
(1986)
Novel monoclonal antibodies provide evidence for the in situ existence of a nonphosphorylated form of the largest neurofilament subunit.
J Neurosci
6:850-858[Abstract].
-
Lee VM-Y,
Balin BJ,
Otvos L,
Trojanowski JQ
(1991)
A68: a major subunit of paired helical filaments and derivatized forms of normal Tau.
Science
251:675-678[Abstract/Free Full Text].
-
Lee VM-Y,
Goedert M,
Trojanowski JQ
(2001)
Neurodegenerative tauopathies.
Annu Rev Neurosci
24:1121-1159[ISI][Medline].
-
Matsuo ES,
Shin RW,
Billingsley ML,
Van deVoorde A,
O'Connor M,
Trojanowski JQ,
Lee VM-Y
(1994)
Biopsy-derived adult human brain tau is phosphorylated at many of the same sites as Alzheimer's disease paired helical filament tau.
Neuron
13:989-1002[ISI][Medline].
-
Mawal-Dewan M,
Henley J,
Van de Voorde A,
Trojanowski JQ,
Lee VM-Y
(1994)
The phosphorylation state of tau in the developing rat brain is regulated by phosphoprotein phosphatases.
J Biol Chem
269:30981-30987[Abstract/Free Full Text].
-
Nakazato Y,
Sasaki A,
Hirato J,
Ishida Y
(1984)
Immunohistochemical localization of neurofilament protein in neuronal degenerations.
Acta Neuropathol (Berl)
64:30-36[Medline].
-
Otvos L,
Feiner L,
Lang E,
Szendrei GI,
Goedert M,
Lee VM-Y
(1994)
Monoclonal antibody PHF-1 recognizes tau protein phosphorylated at serine residues 396 and 404.
J Neurosci Res
39:669-673[ISI][Medline].
-
Perry G,
Rizzuto N,
Autilio-Gambetti L,
Gambetti P
(1985)
Paired helical filaments from Alzheimer disease patients contain cytoskeletal components.
Proc Natl Acad Sci USA
82:3916-3920[Abstract/Free Full Text].
-
Poorkaj P,
Bird TD,
Wijsman E,
Nemens E,
Garruto RM,
Anderson L,
Andreadis A,
Wiederholt WC,
Raskind M,
Schellenberg GD
(1998)
Tau is a candidate gene for chromosome 17 frontotemporal dementia.
Ann Neurol
43:815-825[ISI][Medline].
-
Probst A,
Gotz J,
Wiederhold KH,
Tolnay M,
Mistl C,
Jaton AL,
Hong M,
Ishihara T,
Lee VM-Y,
Trojanowski JQ,
Jakes R,
Crowther RA,
Spillantini MG,
Burki K,
Goedert M
(2000)
Axonopathy and amyotrophy in mice transgenic for human four-repeat tau protein.
Acta Neuropathol (Berl)
99:469-481[Medline].
-
Rao MV,
Houseweart MK,
Williamson TL,
Crawford TO,
Folmer J,
Cleveland DW
(1998)
Neurofilament-dependent radial growth of motor axons and axonal organization of neurofilaments does not require the neurofilament heavy subunit (NF-H) or its phosphorylation.
J Cell Biol
143:171-181[Abstract/Free Full Text].
-
Schmidt ML,
Lee VM-Y,
Trojanowski JQ
(1989)
Analysis of epitopes shared by Hirano bodies and neurofilament proteins in normal and Alzheimer's disease hippocampus.
Lab Invest
60:513-522[ISI][Medline].
-
Schmidt ML,
Lee VM-Y,
Trojanowski JQ
(1990)
Relative abundance of tau and neurofilament epitopes in hippocampal neurofibrillary tangles.
Am J Pathol
136:1069-1075[Abstract].
-
Seubert P,
Mawal-Dewan M,
Barbour R,
Jakes R,
Goedert M,
Johnson GV,
Litersky JM,
Schenk D,
Lieberburg I,
Trojanowski JQ,
Lee VM-Y
(1995)
Detection of phosphorylated Ser262 in fetal tau, adult tau, and paired helical filament tau.
J Biol Chem
270:18917-18922[Abstract/Free Full Text].
-
Shankar SK,
Yanagihara R,
Garruto RM,
Grundke-Iqbal I,
Kosik KS,
Gajdusek DC
(1989)
Immunocytochemical characterization of neurofibrillary tangles in amyotrophic lateral sclerosis and parkinsonism-dementia of Guam.
Ann Neurol
25:146-151[ISI][Medline].
-
Spillantini MG,
Murrell JR,
Goedert M,
Farlow MR,
Klug A,
Ghetti B
(1998)
Mutation in the tau gene in familial multiple system tauopathy with presenile dementia.
Proc Natl Acad Sci USA
95:7737-7741[Abstract/Free Full Text].
-
Spittaels K,
Van den Haute C,
Van Dorpe J,
Bruynseels K,
Vandezande K,
Laenen I,
Geerts H,
Mercken M,
Sciot R,
Van Lommel A,
Loos R,
Van Leuven F
(1999)
Prominent axonopathy in the brain and spinal cord of transgenic mice overexpressing four-repeat human tau protein.
Am J Pathol
155:2153-2165[Abstract/Free Full Text].
-
Szendrei GI,
Lee VM-Y,
Otvos L
(1993)
Recognition of the minimal epitope of monoclonal antibody Tau-1 depends upon the presence of a phosphate group but not its location.
J Neurosci Res
34:243-249[ISI][Medline].
-
Teclemariam-Mesbah R,
Wortel J,
Romijin HJ,
Buijs RM
(1997)
A si
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TOP
ABSTRACT
INTRODUCTION
