From the Department of Dementia Research, National
Institute for Longevity Sciences, 36-3 Gengo, Morioka, Obu, Aichi
474-8522, Japan, § Department of Pediatrics, The University
of Arizona, Steele Memorial Children's Research Center, Tucson,
Arizona 85724, and ¶ Department of Neurobiology, Tottori
University, Faculty of Medicine, Yonago, Tottori 683, Japan
Received for publication, October 25, 2000, and in revised form, December 12, 2000
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ABSTRACT |
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Niemann-Pick type C (NPC) disease is
characterized by an accumulation of cholesterol in most tissues and
progressive neurodegeneration with the formation of neurofibrillary
tangles. Neurofibrillary tangles are composed of paired helical
filaments (PHF), a major component of which is the hyperphosphorylated
tau. In this study we used NPC heterozygous and NPC homozygous mouse
brains to investigate the molecular mechanism responsible for tauopathy
in NPC. Immunoblot analysis using anti-tau antibodies (Tau-1, PHF-1,
AT-180, and AT-100) revealed site-specific phosphorylation of tau at
Ser-396 and Ser-404 in the brains of NPC homozygous mice.
Mitogen-activated protein kinase, a potential serine kinase known to
phosphorylate tau, was activated, whereas other serine kinases such as
glycogen synthase kinase-3 Niemann-Pick type C
(NPC)1 disease is an
autosomal recessive disorder characterized by an accumulation of
cholesterol in most tissues and progressive neurodegeneration marked by
premature neuronal death (1). The most prominent cellular feature of NPC is the accumulation of low density lipoprotein-derived cholesterol due to a defect in the sorting/trafficking of cholesterol from lysosomes and late endosomes (1, 2). Neuropathologic examination has
revealed neuronal distension, swollen axons, and polymorphorous cytoplasmic bodies that react with the cholesterol binding reagent, filipin. Since low density lipoprotein-derived cholesterol is inaccessible to the brain and nervous system, the accumulation of
cholesterol in neurons must be derived from an additional source. Recent studies using NPC fibroblasts have concluded that endogenously synthesized cholesterol can contribute to cholesterol accumulation as a
result of the circulation of cholesterol between the plasma membrane and endosomal/lysosomal compartments (3).
The gene responsible for NPC, referred to as the Niemann-Pick C1 gene
(NPC1), was mapped to a region of chromosome 18 in both human and mice and subsequently cloned (4). Although the function of
NPC1 remains undefined, studies demonstrate a crucial role for this
protein in cholesterol metabolism (5-7). NPC mice share many of the
pathophysiological abnormalities observed in patients with NPC (8),
including the accumulation of cholesterol in tissues and
neurodegeneration marked by decreased Purkinje cell numbers. Use of the
murine model for NPC has provided important insights into the role of
NPC1 in cholesterol metabolism (4). In a previous study it was reported
that NPC mice are asymptomatic at birth, with the earliest definitive
symptoms of the disease apparent by 4-6 weeks of age and death ensuing
by 10-15 weeks of age (8).
It has been shown that the brains of NPC patients with
neurodegeneration have neurofibrillary tangles (NFTs) without amyloid deposits (9-11). Interestingly, the presence of NFTs, which are composed of paired helical filaments (PHF), is one of the diagnostic hallmarks of Alzheimer's disease (AD) (12). A major component of PHF
is tau, which is a microtubule-associated protein (13, 14). It has
previously been shown that the phosphorylation of tau prevents it from
binding to microtubules (15-19). Although the phophorylation of tau in
AD is the subject of intense investigation, the molecular mechanism
responsible for this altered regulation remains to be defined. In this
context, it is interesting to note that a perturbation in cholesterol
metabolism and NFT formation without amyloid deposits coexist in the
brains of NPC patients. This may indicate that a disturbance in
cholesterol metabolism is responsible for tauopathy. We have recently
demonstrated using cultured neurons that cholesterol deficiency results
in axonal degeneration associated with microtubule depolymerization and hyperphosphorylation of tau (20). Therefore, it is important to
investigate the molecular mechanisms underlying tau phosphorylation associated with perturbed cholesterol metabolism in NPC brains.
Studies on the phosphorylation state of tau in the brains of NPC mice
have not been performed. Evidence indicates that the defect in NPC1
function together with the perturbation of cholesterol metabolism may
be an important tool for elucidating the pathways involved in the
modulation of tau phosphorylation, NFT formation, and
neurodegeneration. In this work, we have determined the phosphorylation state of tau in brains of BALB/c mice carrying the genetic mutation in
NPC1. Our results demonstrate that tau is
hyperphosphorylated at Ser-396 and Ser-404. The elevation of tau
phosphorylation in NPC ( NPC Mice--
BALB/c mice carrying the genetic mutation for NPC1
were obtained from The Jackson Laboratory (Bar Harbor, MA). These
heterozygous mice were bred to acquire NPC (+/+), NPC (+/-), and NPC
( Antibodies--
The monoclonal antibody Tau-1 was obtained from
Chemicon International (Temecula, CA). The monoclonal antibody PHF-1
was kindly provided by Dr. P. Davies (Albert Einstein College of
Medicine). The monoclonal antibodies AT-100 and AT-180 were purchased
from Innogenetics (Ghent, Belgium). The rabbit polyclonal anti-NPC1 antibody was generated by immunizing rabbits with a MAP-peptide conjugate and purified from serum using peptide-specific affinity chromatography as previously described (21). Rabbit polyclonal anti-phospho-MAPK, rabbit polyclonal anti-phospho-independent-MAPK, and
monoclonal anti-phospho-GSK-3 Protein Preparation--
Mouse tissues were homogenized in 10 volumes of Tris-saline (50 mM Tris-HCl, pH 7.4, 150 mM NaCl), containing protease inhibitors (CompleteTM) and
phosphatase inhibitors (10 µM NaF and 1 mM
orthovanadate) using a motor-driven Teflon homogenizer. The homogenates
were centrifuged at 3,000 × g for 10 min at 4 °C,
and supernatants were saved for biochemical analyses. Protein
concentrations were determined using the bicinchoninic acid protein
assay kit (Pierce). Aliquots of supernatant containing equal amounts of
protein were subjected to sodium dodecylsulfate-polyacrylamide gel
electrophoresis for immunoblot analysis.
Heat and Alkaline Phosphatase Treatment--
Supernatants
containing equal amounts of protein were heated to 95 °C for 10 min
and clarified by centrifugation at 20,630 × g for 15 min. To each of these clarified heat-stable supernatants was added the
same volume of saturated ammonium sulfate, and the mixtures were kept
at 0 °C for 1 h. The 20,630 × g pellets were suspended in 50 mM Tris-HCl buffer, and alkaline
phosphatase treatment was performed essentially as described elsewhere
(22).
Immunoblot Analysis--
Proteins separated using
SDS-polyacrylamide gel electrophoresis were electrophoretically
transferred onto a polyvinylidene difluoride membrane (Millipore,
Bedford, MA). Nonspecific binding was blocked with 5% fat-free milk in
phosphate-buffered saline containing 0.1% Tween 20. The blots were
then incubated with primary antibodies overnight at 4 °C. For the
detection of both monoclonal and polyclonal antibodies, appropriate
peroxidase-conjugated secondary antibodies were used in conjunction
with SuperSignal chemiluminescence (Pierce) to obtain images saved on film.
Lipid Analysis--
The concentration of cholesterol and
phospholipid in samples were determined using enzymatic methods.
Cholesterol was determined using a cholesterol determination kit, LTCII
(Kyowa Medex, Tokyo), whereas phospholipid was determined using a
phospholipid determination kit, PLB (Wako, Osaka, Japan).
Histological Analyses--
The brains isolated from NPC (+/+)
and NPC ( Electron Microscopy--
The cerebellum of NPC (+/+) and NPC
( The expression of NPC1 in the cerebrums, cerebellums, and livers
of NPC (+/+), NPC (+/ and cyclin-dependent kinase 5 were inactive. Morphological examination demonstrated that a number of
neurons, the perikarya of which strongly immunostained with PHF-1,
exhibited polymorphorous cytoplasmic inclusion bodies and
multi-concentric lamellar-like bodies. Importantly, the accumulation of
intracellular cholesterol in NPC mouse brains was determined to be a
function of age. From these results we conclude that abnormal
cholesterol metabolism due to the genetic mutation in NPC1
may be responsible for activation of the mitogen-activated protein
kinase-signaling pathway and site-specific phosphorylation of tau
in vivo, leading to tauopathy in NPC.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
/
) mice was accompanied by activation of
MAP kinase (MAPK). The activation of MAPK may result from decreased
levels of cholesterol in cellular compartments due to defects in
NPC1-mediated cholesterol trafficking.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
/
) mice used for the study. The genotypes of the mice were
determined from genomic DNA isolated from tail-snip DNA using a
polymerase chain reaction-based method and oligonucleotide primers
described previously (4). Mice used in this study ranged from 6 to 12 weeks of age.
antibody were purchased from New
England Biolabs (Beverly, MA). Rabbit anti-p35 antibody, which reacts
with the p35 and p25 regulatory subunits of
cyclin-dependent kinase 5, was obtained from Santa Cruz
Biotechnology (Santa Cruz, CA). Fluorescein isothiocyanate-conjugated
anti-mouse IgG and biotinylated goat anti-mouse IgG were purchased
from Vector (Burlingame, CA).
/
) mice were fixed in 10% formalin and embedded in
paraffin for histological analysis. The tissue sections were washed
four times with phosphate-buffered saline (PBS) and stained with a
filipin solution for 1 h at room temperature (RT) before washing
and mounting for microscopy. The fixed cells were examined using a UV
filter. For immunohistochemistry, the sections were washed in PBS,
incubated with 5% normal goat serum in PBS for 1 h at RT,
followed by incubation with primary antibody against PHF-1 (1:20
dilution) for 1 h at RT. The sections were then washed in PBS
three times and incubated with fluorescein isothiocyanate-conjugated
anti-mouse IgG (1:5,000 dilution) or biotinylated goat anti-mouse IgG
(1:200 dilution) for 1 h at RT. Fluorescein isothiocyanate-labeled
sections were washed in PBS and observed using a fluorescence
microscope. Biotin labeled sections were washed in PBS five times and
visualized with 3',3'-diaminobenzidine chloride.
/
) mice were washed with PBS and fixed with 2.0% glutaraldehyde
in 0.1 M cacodylate buffer, pH 7.4, containing 0.8%
saccharose for 3 h. The cerebellum were washed in 0.1 M cacodylate buffer containing 0.8% saccharose overnight
at RT and then incubated with 1% osmium tetroxide in the same buffer
for 3 h. After dehydration, the cells were embedded in epoxy
resin. Ultrathin sections were doubly stained with 2% uranyl acetate
for 10 min and with a lead-staining solution for 5 min and observed
using a transmission electron microscope (JEOL JEM-1200EX).
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
), and NPC (
/
) mice were determined using
immunoblot analysis. Consistent with previous results (21), NPC1 was
determined to be a 180-kDa protein in the livers of NPC (+/+) and NPC
(+/
) mice (Fig. 1). NPC (+/
) livers
expressed approximately half the amount of NPC1 that NPC (+/+) livers
expressed, whereas NPC (
/
) livers expressed no detectable NPC1 due
to a truncation mutation. The lower band seen in the livers was a
nonspecific one, because it was also detected using preimmune serum
(data not shown). In the cerebrums and cerebellums of NPC (+/+) and NPC
(+/
) mice, NPC1 was determined to be a 165-kDa protein, whereas NPC1
was not present in samples from the NPC (
/
) mice (Fig. 1). As with
the liver, NPC (+/
) cerebrums and cerebellums expressed approximately
intermediate levels of NPC1. Immunoblot analysis of each sample using
preimmune serum or anti-NPC1 in the presence of immunizing peptide
resulted in the detection of no bands corresponding to the above
molecular weights, indicating that these lower molecular weight bands
represent NPC1. The amino acid sequence based on the cDNA of NPC1
predicts a molecular mass of 142 kDa. The difference in the apparent
molecular mass, i.e. 165 kDa in the brain and 180 kDa in the
liver, is likely due to altered glycosylation of NPC1 (23).
View larger version (20K):
[in a new window]
Fig. 1.
Immunoblot analysis of NPC1 in NPC (+/+), NPC
(+/ ), and NPC (
/
)
mouse tissues. An equivalent amount of postnuclear supernatant
protein was prepared from cerebrum, cerebellum, and liver for
immunoblot analysis according to the procedures described under
"Experimental Procedures." Proteins were separated using 7.5%
SDS-polyacrylamide gel electrophoresis, transferred onto an Immobilon
membrane, and probed with anti-NPC1. Film was used to obtain images of
the protein bands using enhanced chemiluminescence.
The concentrations of cholesterol in the cerebrum, cerebellum, and
liver were not significantly different in 6-week-old NPC(+/+), NPC(+/), and NPC(
/
) mice (Fig.
2a). However, by 10-12 weeks of age, NPC(
/
) mice had a significantly increased concentration of
cholesterol in the cerebrum (2.5-fold) and cerebellum (2.8-fold) compared with NPC (+/+) mice. As reported previously, the liver of NPC
(
/
) mice had an approximate 10-fold increase in the amount of
cholesterol compared with NPC (+/+) mice (24-26) (Fig. 2b). Moreover, the concentration of phospholipids in the liver of 10-12 week old NPC (
/
) mice was 1.8-fold higher than those in NPC (+/
)
and NPC (+/+) mice of the same age. The concentrations of phospholipids
in the cerebrum and cerebellum were similar between the three
genotypes.
|
In direct support of the increased amount of cholesterol measured in
10-12-week-old NPC (/
) mice, filipin staining demonstrated an
accumulation of cholesterol in neurons, particularly in the Purkinje
cells of a NPC (
/
) mouse (Fig.
3b, arrows), that
was not observed in the neurons from a NPC (+/+) mouse (Fig.
3a, arrowheads). Moreover, an electron micrograph
of cerebellum sections from a 12-week-old NPC (
/
) mouse shows
polymorphorous cytoplasmic bodies, loosely packed multi-concentric
lamellar-like structures in a Purkinje cell, and a smaller type of
neuron (Fig. 3, c and d). These features are
characteristic of the intracellular accumulations of cholesterol in the
brains of patients with NPC (27).
|
Several well characterized antibodies to tau were used to examine its
presence in various tissues of mice from 10 to 12 weeks of age. The
immunoblot analysis of tau using Tau-1 antibody, which recognizes
dephosphorylated sites of tau at four closely located serine residues,
Ser-195, Ser-198, Ser-199, and Ser-202 (28), shows that tau in the
cerebrum, cerebellum, and liver was found to have apparent molecular
masses between 50 and 70 kDa (Fig. 4a). For samples derived from
NPC (/
) and NPC (+/
) mice, the main bands immunoreactive to Tau-1
appear to exhibit slower electrophoretic mobility than the bands for
samples derived from NPC (+/+) mice. (Fig. 4, a and
b). These bands of tau exhibiting slower mobility are known
to be characteristic of phosphorylated tau. The increased level of tau
phosphorylation in NPC (
/
) mice was marked in the cerebellum, which
is the most commonly affected region in NPC brains. Thus, we focused on
the cerebellum to examine the phosphorylation state of tau. The
antibodies used were the site-specific phospho-dependent antibodies PHF1, AT-180, and AT-100, which recognize the phosphorylated tau epitopes Ser-396/Ser-404, Thr-231, and Ser-214/Thr-217,
respectively. Only the upper migrating band representing tau was
strongly reactive to PHF-1 when samples from the cerebellum of NPC
(
/
) mice were analyzed. This was not observed when samples from the
cerebella of NPC (+/
) and NPC (+/+) mice were analyzed (Fig.
4b), indicating that the Ser-396/Ser-404 sites were highly
phosphorylated in NPC (
/
) mice. In contrast, the intensity of the
band reactive to AT-100 and AT-180 was stronger for NPC (+/+) mice than
for NPC (+/
) and NPC (
/
) mice. However, the mobility of the
AT-100- and AT-180-reactive band for NPC (
/
) mice was reduced to
that of the band for NPC (+/+) and NPC (+/
) mice (Fig.
4b).
|
Alkaline phosphatase treatment, which induces protein
dephosphorylation, revealed a detailed pattern of the tau isoforms with significant shifts in electrophoretic mobility (Fig. 4c).
After dephosphorylation, tau from the cerebellum was resolved into
three major isoforms with apparent molecular masses of 62, 58, and 52 kDa (Fig. 4c). These three isoforms were detected in samples
derived from each genotype. Importantly, the heat-stable supernatant of samples from the cerebellum of NPC (/
) and NPC (+/
) mice showed that the bands immunoreactive with Tau-1 exhibited a slower
electrophoretic mobility than those from NPC (+/+) mice (Fig.
4c, open triangles).
Immunohistochemical analysis using PHF-1 was performed to determine
whether tau phosphorylation was also evident in brain slices. Some of
the cerebellum granular neurons (Fig.
5a) and cerebrum cortical
neurons (Fig. 5c) in the brain slices of NPC (/
) mice at
10 weeks of age were shown to be PHF-1 immunopositive, whereas those in
the brain slice of NPC (+/+) mice were immunonegative (Fig.
5b), lending support to the results shown in Fig.
4b. We also performed electron microscopy to determine
whether filaments with PHF exist in the neurons of the cerebrum and
cerebellum. However, we could not detect PHF formation in these cells
(data not shown).
|
To determine the molecular basis for increased tau phosphorylation, the
expression and the phosphorylation state of several well known
tau-directed protein kinases, including MAPK, GSK-3, and p25, were
determined. Immunoblot analysis using the anti-phospho-MAPK antibody,
which recognizes only an activated form of MAPK, revealed that brains
from 12-week-old NPC (
/
) mice have an 11-fold increase in MAPK
activity compared with the brains of 12-week-old NPC (+/
) and NPC
(+/+) mice (Fig. 6a). The
overall level of MAPK was similar for each of the three genotypes (Fig.
6a). Immunoblot analysis using the anti-phospho-GSK-3
antibody showed no alteration in the amount of the active GSK-3
in
the brains from the three genotypes (Fig. 6a). Immunoblot
analysis using the anti-p35 and p25 antibodies also showed no
alteration in the conversion of p35 to p25 from the brains of the three
genotypes (Fig. 6a). Together, these results indicate that
neither GSK-3
nor p25 are responsible for increased tau
phosphorylation. In contrast, markedly increased levels of MAPK
activity, in conjunction with increased phosphorylation of tau, suggest
that the MAPK pathway may be responsible for tauopathy in NPC.
Additionally, NPC(
/
) mice have increased MAPK activity in the
cerebellum (3-fold) compared with NPC(+/+) and NPC(+/
) mice at 6 weeks of age (Fig. 6b); however, cholesterol accumulation was not found (Fig. 2a).
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DISCUSSION |
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The mechanism by which NPC1 deficiency, associated with altered
cholesterol metabolism, contributes to tauopathy remains undefined. In
the present study, we have shown for the first time that (i) the
accumulation of intracellular cholesterol in NPC mouse brains was a
function of age, (ii) the levels of tau phosphorylation at
Ser-396/Ser-404 are markedly increased, (iii) the increase in tau
phosphorylation is marked in the cerebellum, which is the most commonly
affected region of the brain in NPC, and (iv) MAPK is activated in the
brains of NPC (/
) mice.
Consistent with previous studies (1, 5, 24), our results have
demonstrated that the concentration of cholesterol measured in the
brains of NPC (/
) mice at 6 weeks of age were not elevated compared
with the brains of NPC (+/+) mice. However, using both biochemical and
morphological analysis of NPC(
/
) mice, we demonstrate that the
concentration of cholesterol measured in the brains of NPC (
/
) mice
10-12 weeks of age were elevated, similar to other organs (24-26).
Cholesterol accumulation has previously been shown to occur as a result
of lipoprotein-cholesterol uptake via the coated-pit pathway (25). But
a recent study suggests that NPC cells can accumulate cholesterol in
the absence of low density lipoprotein, indicating that endogenously
synthesized cholesterol and plasma membrane cholesterol can contribute
to the accumulation of lysosomal cholesterol (3). In any event, our
results indicate that the accumulation of cholesterol is
age-dependent. Cholesterol accumulation is detected in 10- to 12-week-old mice but not detected in 6-week-old (this study) or
7-week-old mice, as shown in other studies (24).
There are several possible mechanisms for the increased phosphorylation
of tau at the PHF-1 epitope: (i) a decrease in the level of cholesterol
at specific cellular compartments due to a defect in cholesterol
trafficking, (ii) the accumulation of cholesterol and other lipids in
the lysosomal/late endosomal compartment, or (iii) the direct result of
a defect in NPC1 function. Based on the results of our study and those
presented previously by other investigators, the evidence indicates
that a decrease in the level of available cholesterol due to a defect
in cholesterol trafficking is responsible for tau phosphorylation in
the brains of NPC (/
) mice. Previous studies show that, despite an
accumulation of cellular cholesterol, the rate of cholesterol synthesis
and the expression of low density lipoprotein receptors is not
down-regulated (24). Moreover, the esterification of excess cholesterol
at the endoplasmic reticulum is delayed as a result of cholesterol not
being able to gain access to pools responsible for maintaining intracellular cholesterol homeostasis (26, 29). Consistent with this
hypothsis, our results suggest that additional cellular compartments
may be cholesterol-deficient and, therefore, directly responsible for
inducing tau phosphorylation and microtubule depolymerization in the
axons of cultured neurons (20).
Cholesterol depletion of plasma membrane caveolae have been shown to
cause the activation of MAPK (30). Consistent with these results, we
have found that MAPK is activated in cholesterol-deficient neurons and
that NPC fibroblasts have significantly decreased levels of caveolae
cholesterol (data not shown). In the present study, we demonstrate that
MAPK activity is elevated in the brains of NPC (/
) mice not only at
10-12 weeks of age but also at 6 weeks of age, indicating that the
activation of MAPK precedes the accumulation of cholesterol. Since tau
is known to be a substrate for MAPK and that Ser-396/Ser-404, but not
Ser-214, Thr-217, or Thr-231 sites, are phosphorylated by MAPK
(31-33), the assumption that MAPK is responsible for tau
phosphorylation in NPC (
/
) brains would well explain our present
results that tau was phosphorylated at Ser-396 and Ser-404
(demonstrated as PHF-1-immunopositive) and was not phosphorylated at
Ser-214 and Thr-217 (demonstrated as AT-100-immunopositive) nor at
Thr231 (demonstrated as AT-180-immunopositive). These results indicate
that a decrease in the amount of cholesterol available at particular
cellular compartments may be directly responsible for the stimulation
of MAPK activity and the subsequent promotion of site-specific
phosphorylation of tau in the brains of NPC (
/
) mice and NPC
patients. The mechanisms underlying time discrepancy in the activation
of MAPK and the elevation of tau phosphorylation, however, remain
unclear. But there are several studies demonstrating the relationship
between the kinetics of kinases and tau phosphorylation and/or NFT
formation. It has been shown that activation of the MAPK cascade is
most pronounced during early stages of AD (34), suggesting that
activation of MAPK may precede tauopathy found in AD. It has also been
suggested that responsible kinases including MAPK work at different
stages or varying durations in Alzheimer's disease (32). In addition, tau antigenicity and its behavior has been shown to be influenced by
the sequential and convergent influences of multiple kinases (35).
These lines of evidence suggest that tau phosphorylation is modulated
by a complex mechanism and only allows us to argue that activation of a
given kinase may precede tau phosphorylation. However, direct evidence
is indeed required to elucidate the precise kinetics by which
responsible kinases, including MAPK and phosphatases, induce tau
phosphorylation and contribute to NFT formation in vivo.
The remaining question to be answered is why tau is site-specifically
phosphorylated at Ser-396/Ser-404 sites in the brains of NPC (/
)
mice, whereas other sites, including Ser-214, Thr-217, and Thr-231 are
relatively dephosphorylated compared with the brains of NPC (+/+) and
NPC (+/
) mice. The molecular mechanism underlying this regulation
remains unclear, and additional studies are necessary. Our finding
suggests that MAPK, but neither GSK-3
nor
cyclin-dependent kinase 5, modulates the phosphorylation of tau in a site-specific manner. Other proteins, including tau-directed kinases and phosphatases, may also be involved in the regulation of tau
phosphorylation and dephosphorylation in the brains of NPC (
/
)
mice. Consistent with alterations in the activity of specific kinases,
our studies have shown that the expression of specific protein kinases
and phosphorylated state of caveolin-1 and annexin II are increased in
the livers of NPC (
/
) mice (36).
The association between cholesterol deficiency and the promotion of tau
phosphorylation may provide clues to the pathogenesis of tauopathy in
AD, because possession of the allele 4 of apolipoprotein E, which is
a key molecule regulating cholesterol metabolism in the central nervous
system, has been found to be a strong risk factor for the development
of AD (37-39). Recently, we have demonstrated that oligomeric amyloid
-protein reduces the intracellular cholesterol content in
neurons,2 which may result in
tau phosphorylation (20). Taken together, these findings may suggest
that cholesterol plays a critical role in the tauopathy of AD and
NPC.
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FOOTNOTES |
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* This work was supported by Longevity Sciences Research Grant 8A-1, grants from Research on Brain Science from the Ministry of Health and Welfare, Japan, CREST (Core Research for Evolutional Sciences and Technology), Japan, and Ono Medical Research Foundation and Life Science Foundation of Japan, and by National Institutes of Health Grant DK56732.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: Dept. of Dementia
Research, National Institute for Longevity Sciences, 36-3 Gengo, Morioka, Obu, Aichi 474-8522, Japan. Tel.: 81-562-46-2311; Fax: 81-562-44-6594; E-mail: michi@nils.go.jp.
Published, JBC Papers in Press, January 4, 2001, DOI 10.1074/jbc.M009733200
2 J.-S. Gong, K. Yanagisawa, and M. Michikawa, unpublished results.
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ABBREVIATIONS |
---|
The abbreviations used are:
NPC, Niemann-Pick
type C;
AD, Alzheimer's disease;
NFT, neurofibrillary tangle;
PHF, paired helical filaments;
ECL, enhanced chemiluminescence;
MAP, mitogen-activated protein;
MAPK, MAP kinase;
GSK-3, glycogen
synthase kinase-3
;
PBS, phosphate-buffered saline;
RT, room
temperature.
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