 |
INTRODUCTION |
Neuronal development involves morphogenetic changes of neurons
associated with complex regulatory mechanisms, which coordinate at the
levels of gene expression and post-translational modifications. Current
evidence supports the view that neuronal microtubule-associated proteins (MAPs)1 determine
the microtubule rearrangements underlying neuronal morphogenesis (1).
This process can be achieved through the regulation of the expression
of particular MAP isoforms at specific subcellular locations and at
distinct developmental stages as well as through the modification of
MAPs by phosphorylation and dephosphorylation (2).
Among the neuronal MAPs, tau protein has attracted a particular
interest due to its polar distribution in the axon as compared with the
somatodendritic compartment, as well as its developmentally regulated
expression and phosphorylation (3, 4). Tau is one of several MAPs that
regulate the assembly and stabilization of the microtubule network (5).
Multiple isoforms of tau are generated from a single gene by
alternative splicing, leading to the developmentally regulated
expression of different isoforms (6). Phosphorylation provides tau with
further molecular diversity; the function of tau as a
microtubule-binding protein is regulated by the phosphorylation of
specific residues (7). In general, an increase in tau
phosphorylation correlates inversely with its ability to bind and
stabilize microtubules. Thus, phosphorylation of tau contributes an
additional mechanism to control the balance between microtubule
dynamics and stabilization in developing axons (8, 9). Several
developmental studies have already shown that phosphorylated tau is
present in neurons at a high level only during the period of intense
neuritic outgrowth and that it becomes barely detectable during the
period of neurite stabilization and synaptogenesis (10, 11).
Phosphorylated tau is essential during brain development to maintain a
certain degree of microtubule instability, thus affecting the growth of neurites.
Tau phosphorylation at specific residues not only occurs during normal
brain development but also during pathological conditions such as
Alzheimer's disease (AD). In normal brain, the equilibrium between tau
phosphorylation and dephosphorylation modulates the stability of the
axonal cytoskeleton and thereby the axonal morphology (12). However,
the breakdown of this equilibrium under pathological conditions results
in tau dysfunction, which is considered to be one of the critical
events leading to neuronal degeneration (13). It has been shown that
hyperphosphorylation of tau reduces its affinity for microtubules and
can contribute to the self-association of tau and the formation of
neurofibrillary tangles, one of the major histopathological
hallmarks of AD (14). The phosphorylation is interpreted as abnormal in
the sense that this kind of tau phosphorylation has never been observed
in normal aged human brain (15). Surprisingly, the phosphorylation of
tau in neurofibrillary tangles has been found to be very similar to a
transient hyperphosphorylation of tau that occurs during early
development of the brain (8, 9, 16-18). Therefore, the biochemical
pathways that play a role during early brain development are likely to
be reactivated in AD brains. Tau is phosphorylated in vitro
by several protein kinases, including cyclic AMP-dependent
protein kinase (19), calcium/calmodulin-dependent protein
kinase II (20), protein kinase C (21), casein kinase I (22), casein
kinase II (21), and proline-directed protein kinases such as MAP kinase
(23), glycogen synthase kinase-3
(24). and
cyclin-dependent kinase 5 (Cdk5) (3, 25-27). Of these
kinases, Cdk5 has been found to phosphorylate tau at sites implicated
in AD pathology (13, 28).
Cdk5 is an active enzyme in postmitotic neurons; the activation of Cdk5
requires its association with the neuronal activators, p35 or p39
(29-31). p35 was the first activator identified for Cdk5 (29, 30).
Subsequently, an additional Cdk5 activator, p39, was identified based
on its sequence homology to p35 (31). p35 and p39 share 57% amino acid
identity (31). Cdk5 has been shown to play an important role in the
development of the nervous system, including neuronal migration and
neurite outgrowth (32-35). Insights into the critical function of
Cdk5/p35 in brain development have been gained from gene targeting
experiments (32, 35). Cdk5
/
mice display extensive defects in all
brain areas (35), whereas p35
/
mice display defects mostly
confined to the forebrain (32). This difference in the phenotypic
severity suggests the functional importance of another Cdk5 activator,
p39, in brain development. Although p39
/
mice have no obvious
abnormalities in the brain, p35
/
p39
/
double knockout mice
exhibit phenotypes identical to those of Cdk5
/
mice (36). These
findings suggest that p35 and p39 are major activators of Cdk5 in the
brain and that their coexistence as Cdk5 activators may contribute to
the in vivo activation of Cdk5 in a region-specific or
developmentally regulated manner, as proposed by Wu et al.
(37).
Developmental regulation of tau phosphorylation is critical in
maintaining the balance between microtubule plasticity and stability in
developing axons. The phosphorylation pattern of tau in AD brains
closely resembles that of tau in embryonic brains (8, 9, 16-18). Thus,
the developing brain is a useful experimental system to study the
mechanisms that control tau phosphorylation. Although Cdk5 has been
found to phosphorylate tau in vitro, its in vivo
roles remain to be examined. Here, we show that Cdk5 in association
with p39 is involved in the in vivo phosphorylation of tau
during brain development and that its phosphorylation reduces the
ability of tau to bind to microtubules. In addition, Cdk5/p35 and
Cdk5/p39 were found to exhibit different abilities for tau phosphorylation, with the higher activity in Cdk5/p39. Considering the
temporal and spatial expression patterns of p35 and p39 reported here,
the role of tau phosphorylation by Cdk5 is discussed with regard
to the regulation of microtubule stability during brain development.
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EXPERIMENTAL PROCEDURES |
Chemicals and Antibodies--
Polyclonal antibodies to p35
(C-19) and Cdk5 (C-8) were purchased from Santa Cruz Biotechnology,
Inc. (Santa Cruz, CA). An affinity-purified rabbit polyclonal antibody
against p39 was kindly provided by Dr. Tsai (Howard Hughes Medical
Institute and the Department of Pathology, Harvard Medical School,
Boston, MA). The phosphorylation-dependent tau antibody,
AT-8, was obtained from Endogen (Woburn, MA). The
phosphorylation-independent mouse monoclonal TAU-5 antibody was
purchased from BIOSOURCE International, Inc.
(Camarillo, CA). An antibody against
-tubulin was purchased from
Sigma. Cell culture reagents were purchased from Invitrogen.
Animals--
The gene-targeting strategy and generation of
Cdk5
/
and p35
/
mice have been reported previously (35, 38).
Mouse lines of Cdk5 and p35 mutants, as well as wild-type controls,
were maintained in a C57BL/6 × 129/SvJ hybrid background.
Conception was ascertained by the presence of a vaginal plug. The first
24 h following conception was considered day 0 of embryonic
development (E0), and the first 24 h following birth was
considered day 0 postpartum (P0). Mouse embryonic development takes
~20 days. The genotypes of the mutants were determined by performing
Southern blot and/or PCR analysis on genomic DNA isolated from tail
biopsies as described earlier (35, 38). Animal care and use practices
conformed to the NIH Guide for Care and Use of Laboratory Animals.
Preparation of Tissue Samples at Different Developmental
Stages--
Embryonic and postnatal mouse brains were dissected on ice
into various brain regions: cerebral cortex, cerebellum, brain stem,
and spinal cord. For Northern blot analysis, total RNA was extracted
from the various brain regions of wild-type mice with TRIzol reagent
(Invitrogen), as recommended by the manufacturer. Total RNA
concentration and purity were determined by UV absorbance at 260 and
280 nm. For Western blot analysis, each tissue from wild-type,
p35
/
, and Cdk5
/
mice was washed in ice-cold PBS and homogenized
in 10 volumes of lysis buffer at 4 °C. The lysis buffer consisted of
50 mM Tris-HCl (pH 7.5) containing 150 mM NaCl,
5 mM EDTA, 1% Triton X-100, 1 mM
dithiothreitol, 1 mM phenylmethylsulfonyl fluoride, 1 µg/ml aprotinin, 1 µg/ml leupeptin, 20 mM
-phosphoglycerate, 10 mM sodium fluoride, 1 mM sodium vanadate, and 1 µM okadaic acid.
Following a 30-min incubation on ice, insoluble material was removed by
centrifugation at 4 °C, and the protein concentration of the
supernatant was determined as described (39). For Cdk5 kinase activity
assay, the brain extracts were prepared with the same lysis buffer
except with a NaCl concentration of 50 mM to allow Cdk5
association with p35 and p39.
Northern Blot Analysis--
Each sample (20 µg of total RNA)
was electrophoresed in a 1% agarose-formaldehyde gel and blotted onto
a nylon membrane (Schleicher & Schuell). After UV cross-linking, the
membrane was prehybridized for more than 3 h at 42 °C in the
presence of 50% formamide, 10× Denhardt's solution, 5× SSPE, 0.1%
SDS, 10% dextran sulfate, and 100 µg/ml boiled fragmented salmon
sperm DNA. 32P-Labeled probes were added to the
prehybridization buffer, and the membrane was incubated overnight at
42 °C. After hybridization, the membrane was washed two times in 2×
SSC, 0.1% SDS at 42 °C for 10 min, and two times in 0.1× SSC,
0.1% SDS at 65 °C for 20 min. The washed membrane was exposed to
x-ray film with double intensifying screens at
80 °C. After
stripping the probe, the same membrane was used for hybridization with
a new probe. The expression levels of Cdk5, p35, and p39 mRNAs were
quantified by measuring the optical densities of specific bands using
an image analysis system with NIH Image software, version 1.62.
For detection of p35 mRNA, a 924-bp fragment of mouse p35 cDNA
containing the entire coding region was used as a probe as previously
described (40). A 275-bp fragment used as a p39 probe (nucleotides
891-1165 of the full-length mouse p39 cDNA) was generated by
reverse transcription-PCR using the following primers:
5'-CAACGAGATCTCCTACCCGCTC-3' and 5'-TCATAGTCCAGTGCTTGGCTCC-3'. This
fragment excludes the region of homology between p35 and p39 cDNAs,
thus minimizing the probability of cross-hybridization. A 560-bp
fragment used as a Cdk5 probe (nucleotides 331-890 of the full-length
mouse Cdk5 cDNA) was produced by reverse transcription-PCR using
the following primers: 5'-AGCTGCAATGGTGACCTGGACC-3' and
5'-TCCTCTGCTGAGATGCGCTGCA-3'. As an internal control, a 433-bp fragment
used as a mouse glyceraldehyde-3-phosphate dehydrogenase probe was
generated as described previously (41).
Western Blot Analysis--
Equal amounts of protein for each
experiment were separated by SDS-PAGE before being transferred onto a
nitrocellulose membrane. The membranes were blocked in 1× PBS
containing 5% skim milk and 0.05% Tween 20 and incubated with primary
antibodies overnight at 4 °C. Incubation with peroxidase-conjugated
anti-mouse or rabbit IgG (1:10,000) was performed at room temperature
for 60 min. A signal was detected by enhanced chemiluminescence
(Pierce), and relative optical densities of the bands were quantified
as described above.
For detection of p35, p39, and Cdk5 protein, membranes were incubated
with anti-p35 antibody (1:1,000), anti-p39 antibody (1:1,000), and
anti-Cdk5 antibody (1:1,000). The phosphorylation state of tau was
examined using the AT-8 antibody (diluted to 5 µg/ml), which
recognizes tau only when Ser-202 and Thr-205 (numbering based on
longest human brain tau isoforms) are phosphorylated (42). To determine
total tau levels, the phosphorylation-independent monoclonal antibody
TAU-5 (1:5,000) was used (43). The data obtained with the AT-8 antibody
were normalized to total tau levels on the stripped and reprobed
membranes. For reuse, the membranes were stripped for 30 min at
50 °C in 63 mM Tris-HCl (pH 6.8) containing 100 mM 2-mercaptoethanol and 2% SDS. For the detergent
extraction assay, the amount of tau was determined with the TAU-5 antibody.
Cdk5 Kinase Activity Assay--
Different regions of mouse brain
were lysed in ice-cold lysis buffer as described above. The
supernatants (brain extracts) were collected after centrifugation at
10,000 × g for 30 min at 4 °C and
immunoprecipitated with either anti-Cdk5 (C-8), anti-p35 (C-19), or
anti-p39 antibodies. The Cdk5 immunoprecipitate was prepared by
incubation of 300 µl of the lysate (corresponding to 300 µg of
protein) with anti-Cdk5 antibody (3 µg) overnight at 4 °C followed
by further incubation with 25 µl of Protein A-agarose beads (50%
slurry in lysis buffer; Santa Cruz Biotechnology) for 3 h at
4 °C. For the preparation of p35 or p39 immunoprecipitates, 500 µl
of the lysate (corresponding to 1 mg of protein) was incubated with
either anti-p35 antibody (3 µg) or anti-p39 antibody (3 µg) as
described above. The immunoprecipitates were washed twice with the
lysis buffer and twice with a kinase buffer consisting of 50 mM Tris-HCl (pH 7.4), 5 mM MgCl2, 1 mM EDTA, 1 mM EGTA, and 1 mM
dithiothreitol and resuspended in 60 µl of the kinase buffer. Kinase
activity assays were performed using either histone H1 or bacterially
expressed human tau as a substrate as described previously (44). Tau
was purified from the heat-stable supernatant of an
Escherichia coli lysate by phosphocellulose
column chromatography (45, 59). Briefly, a total volume of 50 µl of
kinase assay mixture was used, containing 50 mM Tris-HCl
(pH 7.4), 1 mM EDTA, 1 mM EGTA, 1 mM dithiothreitol, 5 mM MgCl2, 0.5 mM microcystin LR, 10 µl of the immunoprecipitate, and
either 10 µg of histone H1 or 5 µg of tau protein. The
phosphorylation reaction was initiated by the addition of 0.1 mM [
-32P]ATP and incubated at 30 °C for
60 min. The reaction was stopped by the addition of SDS-PAGE sample
buffer and boiled immediately for 5 min. Samples were separated by
SDS-PAGE on a 15% polyacrylamide gel, and autoradiography was used to
detect the phosphorylation of histone H1 or tau. To quantify the Cdk5
levels in the p35 and p39 immunoprecipitates, 10 µl of each
immunoprecipitate was immunodetected by Western blotting using an
anti-Cdk5 antibody.
Cell Culture and Detergent Extraction--
Primary cultures of
cerebellar neurons were prepared as described previously (4). Briefly,
cerebella were dissected from E17 wild-type, p35
/
, and Cdk5
/
mouse brains; dissociated by trituration; counted; and plated at 1 × 105 cells/cm2 onto six-well culture plates
previously coated with polyethylenimine (2 µg/ml in 0.1 M
boric acid buffer, pH 7.4). After 1 h in 10% horse
serum-containing medium, the cells were maintained with Dulbecco's
modified Eagle's medium/F-12 with N2 supplement, 50 units/ml
penicillin G, and 50 mg/ml streptomycin. Cultures were maintained in a 37 °C incubator with 5% CO2. The cells
were used for experiments on the 7th day in culture.
To investigate the effect of tau phosphorylation on its association
with microtubules, a Triton X-100 extraction assay was utilized to
separate the detergent-insoluble cytoskeletal component from the
detergent-soluble cytosolic component. The fractionation of cell
lysates was performed by a modification of a previously reported
procedure (46). Briefly, cultures were washed with prewarmed (37 °C)
PBS followed by washing with prewarmed (37 °C) microtubule
stabilization buffer (0.1 M PIPES, pH 6.8, 1 mM
MgCl2, 2 mM EGTA, 30% glycerol, 1 mM dithiothreitol, 1 mM phenylmethylsulfonyl fluoride, 1 µg/ml aprotinin, 1 µg/ml leupeptin, 20 mM
-phosphoglycerate, 10 mM sodium fluoride, 1 mM sodium vanadate, and 1 µM okadaic acid)
and incubated at 37 °C for 10 min in the same buffer containing 0.1% Triton X-100. The lysates were centrifuged, and the
detergent-soluble supernatants were collected. The supernatants were
incubated in a boiling water bath for 5 min following the addition of
2× SDS sample buffer. The remaining cellular pellets were solubilized in 2× SDS sample buffer, sonicated, and incubated in a boiling water
bath as above. The relative protein concentration of these samples was
determined by densitometry of a Coomassie Blue-stained SDS-gel. Equal
protein amounts were subsequently electrophoresed on 10%
SDS-polyacrylamide gels, and immunodetection was performed with a TAU-5 antibody.
 |
RESULTS |
Regional Differences in Tau Phosphorylation during Brain
Development--
Tau is predominantly localized in axons and has a
specific function in the formation of the axonal cytoskeleton. A study
that demonstrated the temporal and spatial expression patterns of tau mRNA in the rat brain suggested that its expression appeared to coincide with the region-specific onset of axogenesis (47). The onset
of tau mRNA expression and its subsequent decrease in the brain
stem occurred earlier than those in the cerebral cortex (47). Whereas
studies have demonstrated the developmental regulation of tau
phosphorylation in the brain, its spatial regulation remains less
clear. To address this question, we examined the developmental changes
in the expression and phosphorylation of tau in different brain
regions, the cerebral cortex and brain stem, using the
phosphorylation-dependent AT-8 antibody and
phosphorylation-independent TAU-5 antibody. It was found that the
developmental phosphorylation of tau was also regulated in a spatial
manner. Tau phosphorylation in the cerebral cortex was detected at E15,
increased to a peak level at P7, decreased by P14, and could not be
detected at P28 (Fig. 1A).
Interestingly, tau phosphorylation in the brain stem peaked earlier at
P0, decreased by P7, and was undetectable at P14 and P28 (Fig.
1B). These results indicate that there are differences in
tau phosphorylation in specific brain regions at different developmental stages. It was also noted that the rapid down-regulation of phosphorylation coincided with the transition from immature to
several adult isoforms of tau, with a regional difference between the
cerebral cortex and brain stem (Fig. 1), consistent with the previous
report (47).

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Fig. 1.
Regional differences in tau phosphorylation
during brain development. Brain lysates were prepared from
cerebral cortex (A) and brain stem (B) of
wild-type mice on days 15 (E15) and 17 (E17) of embryonic development
and days 0 (P0), 7 (P7), 14 (P14), and 28 (P28) postpartum; the first
24 h following conception was considered E0, and the first 24 h following birth was considered P0. Developmental changes in tau
phosphorylation were examined by SDS-PAGE and Western blotting with
phosphorylation-dependent (AT-8) and -independent (TAU-5)
tau antibodies. The amount of protein loaded on the gels was determined
by reprobing the same membrane with an anti- -tubulin antibody. The
rapid down-regulation of phosphorylation of tau coincided with the
transition in the composition of the isoforms. Note that the
down-regulation in the brain stem precedes that seen in the cerebral
cortex.
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|
Expression Patterns of Cdk5, p35, and p39 in the Developing Mouse
Brain--
Although Cdk5 has been shown to phosphorylate tau in
vitro, its contribution to a temporal and spatial regulation of
tau phosphorylation remains to be determined. Moreover, the fact that
the phenotypic abnormalities in p35
/
mice involved fewer brain
regions and were of lesser magnitude as compared with those in
Cdk5
/
mice, suggested the functional importance of another Cdk5
regulatory subunit, p39, in the developing brain and led us to examine
the possible differences in the temporal and spatial expression of Cdk5
and its regulatory subunits, p35 and p39, during brain development. We
have determined their mRNA levels in various brain regions at E15,
E17, P0, P7, P14, and P28. Northern blot analysis revealed bands of
~4.4, 2.4, and 1.6 kb for p35, p39, and Cdk5, respectively (Fig.
2). Interestingly, analysis of various
brain regions at different developmental stages revealed distinct p35
and p39 expression patterns. The p35 mRNA level was higher during
embryonic development in all regions of the brain and then decreased
postnatally. A significant difference was observed in the developmental
expression pattern of p39 in the cerebral cortex as compared with other
brain regions. In contrast to p35, the p39 mRNA level in the
cerebral cortex was markedly lower before birth and then gradually
increased to its peak level at P7. Thereafter, this high level was
maintained throughout the period analyzed up to P28. In the cerebellum,
brain stem and spinal cord, the p39 mRNA was maintained at high
levels during embryonic development and then gradually decreased
postnatally. Thus, except in the cerebral cortex, the developmental
expression pattern of p39 in these brain regions was similar to that of
p35. The Cdk5 mRNA expression was maintained at high levels in all regions of the brain throughout the period analyzed.

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Fig. 2.
Developmental expression patterns of p35 and
p39 in various brain regions. A, total RNA was prepared from
various brain regions of wild-type mice on days 15 (E15) and 17 of
embryonic development and days 0 (P0), 7 (P7), 14 (P14), and 28 (P28)
postpartum and subjected to Northern blot analysis. The p35 mRNA
level was higher during embryonic development in all regions of the
brain. The developmental expression pattern of p39 was similar to that
of p35 in the cerebellum, brain stem, and spinal cord. However, in the
cerebral cortex, the p39 expression pattern was inverse to that of p35.
B, quantitative results indicate the optical densities of
p35, p39, and Cdk5 mRNA relative to that of
glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA,
expressed as a percentage of the value for the P0 group, and are shown
as the mean ± S.D. from three independent experiments.
|
|
Furthermore, we have determined the regional distribution patterns of
p35 and p39 at different developmental stages and also observed a
prominent difference in the regional distribution pattern of p39 in the
embryonic and postnatal brain (Fig. 3).
The expression of p35 mRNA was most prominent in the cerebral
cortex as compared with other regions of the brain throughout brain
development. In contrast, the regional distribution pattern of p39
mRNA changed after birth. At E15, the p39 mRNA level was higher
in the cerebellum, brain stem, and spinal cord but very low in the
cerebral cortex. This pattern of expression continued at P0. The
regional distribution pattern of p39 mRNA shifted by P14, with the
highest expression level now in the cerebral cortex, similar to that of
p35. The protein levels of p35, p39, and Cdk5 correlated with their
mRNA levels in a temporally and spatially regulated manner as shown by Western blot analysis of wild-type mouse brain regions (Fig. 4). We have found that the p39 protein
level was up-regulated in the p35
/
mouse brain, whereas the Cdk5
level was unaffected, suggesting a possible compensatory mechanism in
the absence of p35 (Fig. 4). This finding was consistent with a
previous report by Ko et al. (36).

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Fig. 3.
Regional distribution patterns of p35 and p39
at different developmental stages. A, total RNA was prepared
from various brain regions of wild-type mice on day 15 of embryonic
development (E15) and days 0 (P0) and 14 (P14) postpartum and subjected
to Northern blot analysis. The expression of p35 was most prominent in
the cerebral cortex throughout brain development. In contrast, the
regional distribution pattern of p39 changed after birth; the p39
mRNA level was first higher in embryonic (E15) and perinatal (P0)
cerebellum, brain stem, and spinal cord and then higher in postnatal
cerebral cortex (P14). B, quantitative results indicate the
optical densities of p35, p39, and Cdk5 mRNA relative to that of
glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA
and are shown as the mean ± S.D. (n = 3).
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Fig. 4.
Compensatory increase in p39 protein in the
absence of p35. Brain lysates from the different regions of
wild-type (WT) and p35 / (KO) mice at the 15th
day of embryonic development (A) and the 14th day postpartum
(B) were examined by SDS-PAGE and Western blotting with
anti-p35, anti-p39, and anti-Cdk5 antibodies. Note that the p39 protein
level was up-regulated in the p35 / mouse brain.
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|
Different Substrate Preference of Cdk5/p35 and
Cdk5/p39--
It was of interest to see if the different
distribution patterns of p35 and p39 correlated with different effects
on Cdk5 activity in the developing brain. To address this question, the substrate preference of Cdk5/p35 and Cdk5/p39 was examined using the
different substrates, histone H1 and tau protein. The kinase activities
for these substrates were determined with either p35 or p39
immunoprecipitates from brain extracts of P14 mouse cerebral cortex.
The Cdk5 kinase assay revealed that Cdk5/p35 phosphorylated histone H1
more than did Cdk5/p39, whereas the kinase activity using tau as a
substrate was higher with Cdk5/p39 than with Cdk5/p35 (Fig.
5). The p35 and p39 immunoprecipitates
used in this assay did not contain the same amount of Cdk5. The p35
immunoprecipitate contained about 3 times as much Cdk5 than did the p39
immunoprecipitate (Fig. 5A). Thus, we cannot conclude that
Cdk5/p35 preferentially phosphorylates histone H1 as compared with
Cdk5/p39, because a larger amount of Cdk5 may phosphorylate any
substrate better than the smaller amount. However, these results
clearly indicate that the smaller amount of Cdk5 phosphorylates tau
better in the presence of p39 than it does in the presence of p35,
suggesting that p39 preferentially activates Cdk5 phosphorylation of
tau as compared with p35. To confirm this observation, Cdk5
immunoprecipitates from different brain regions of wild-type and
p35
/
mice at E17 and P14 were subjected to Cdk5 kinase assay using
either histone H1 or tau as a substrate (Fig.
6). Cdk5 immunoprecipitates from wild-type mouse brain consisted of Cdk5/p35 and Cdk5/p39, whereas those
from p35
/
mouse brain contained only Cdk5/p39. The kinase activity
using histone H1 as a substrate in p35
/
mouse brain was less than
20% in all brain regions tested as compared with wild-type mouse brain
(Fig. 6, A and B); however, p35
/
mouse brain
exhibited 40-80% of the kinase activity of wild-type mouse brain when
using tau as a substrate (Fig. 6, C and D). These
results further indicated that Cdk5/p39 preferentially phosphorylates tau. Regional distribution of the kinase activity using tau as a
substrate varied between embryonic (Fig. 6C) and postnatal
brain (Fig. 6D). Higher activity was observed in embryonic
cerebellum, brain stem, and spinal cord. However, this activity pattern
changed postnatally with the highest activity now found in the cerebral cortex, consistent with the temporal and spatial expression pattern of
p39 protein (Fig. 4).

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Fig. 5.
Differences in substrate preference of
Cdk5/p35 and Cdk5/p39 complexes. A, 1 mg of brain lysate from 14th day postpartum mouse cerebral cortex was
immunoprecipitated with anti-p35 or anti-p39 antibody and then
subjected to Cdk5 kinase assay using either histone H1 or tau as a
substrate. Autoradiography was used to detect the phosphorylation of
histone H1 and tau. These immunoprecipitates were also examined to
determine the amount of the associated Cdk5 by Western blotting
(WB) with an anti-Cdk5 antibody. B and
C, quantitative results are shown by normalizing the kinase
activity to the amount of Cdk5 in each immunoprecipitate and represent
the mean ± S.E. from three independent experiments. These data
were analyzed using the Student's t test and considered to
be significantly different when p < 0.05 (*).
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Fig. 6.
Developmental change in the regional
distribution of Cdk5/p39 kinase activity. Lysates from different
brain regions of wild-type (WT) and p35 / (KO)
mice at the 17th day of embryonic development (E17) and the 14th day
postpartum (P14) were immunoprecipitated with an anti-Cdk5 antibody
(C-8) and subjected to the kinase assay using either histone H1 or tau
as a substrate. Autoradiography was used to detect the phosphorylation
of histone H1 and tau. Quantitative results indicate the relative Cdk5
kinase activity expressed as a percentage of the value for the cerebral
cortex of a wild-type mouse and are shown as the mean ± S.E. from
three independent experiments. A, Cdk5 kinase activity using
histone H1 in the E17 mouse brain. B, Cdk5 kinase activity
using histone H1 in the P14 mouse brain. C, Cdk5 kinase
activity using tau in the E17 mouse brain. D, Cdk5 kinase
activity using tau in the P14 mouse brain. Note that Cdk5 in the
p35 / mouse brain shows a higher kinase activity using tau as a
substrate, as compared with that using histone H1, with a distinct
regional distribution during brain development.
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Involvement of Cdk5 in the in Vivo Phosphorylation of
Tau--
Although Cdk5 has been reported to phosphorylate tau
in vitro (25-27), its ability to phosphorylate tau in
vivo remains to be determined. To address this question, we have
examined the phosphorylation state of tau in p35
/
and Cdk5
/
mouse brains using a phosphorylation-dependent tau
antibody, AT-8 (Fig. 7). This antibody
recognizes the sites on tau that Cdk5 has been shown to phosphorylate
in vitro. A decrease in AT-8 immunoreactivity was seen in
Cdk5
/
mouse brain but not in p35
/
mouse brain. It is of
interest to note that there was no difference in AT-8 immunoreactivity
in the cerebral cortex where the p39 protein level was very low, which
is consistent with a previous observation that AT-8 immunoreactivity
was observed in the brain stem of E18 rats but not in the cerebral
cortex (10). These results indicate that Cdk5, especially in
association with p39, is involved in the in vivo
phosphorylation of tau during brain development.

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Fig. 7.
In vivo phosphorylation of tau by
Cdk5/p39 during brain development. Brain lysates were prepared
from different brain regions of wild-type, p35 / , and Cdk5 /
mouse embryos on the 15th day of embryonic development. The samples
were examined by SDS-PAGE and Western blotting using the
phosphorylation-dependent tau antibody, AT-8, and the
phosphorylation-independent tau antibody, TAU-5. A, decrease
in AT-8 immunoreactivity was observed in Cdk5 / mouse brain but not
in p35 / mouse brain. Note that there was no difference in the
immunoreactivity in the cerebral cortex, where the p39 protein level
was very low. B, to quantitatively determine the
phosphorylation state of tau, the data obtained with the AT-8 antibody
for wild-type, p35 / , and Cdk5 / mice were normalized to total
tau levels in the samples. The data are presented as a densitometric
ratio of AT-8 immunoreactivity to TAU-5 immunoreactivity from three
independent experiments. These data were analyzed using the Student's
t test and considered to be significantly different when
p < 0.05 (*).
|
|
Effect of Tau Phosphorylation by Cdk5 on Its Binding Affinity for
Microtubules--
As shown above, Cdk5 was found to be involved in the
in vivo phosphorylation of tau and, therefore, may regulate
tau function in axonal development. Tau function is regulated through
its association with microtubules. To investigate the physiological
role of tau phosphorylation by Cdk5, the ability of tau to bind to
microtubules was examined by a detergent extraction assay using
cultured cerebellar neurons from E17 wild-type, p35
/
, and Cdk5
/
mouse embryos (Fig. 8). The
detergent-insoluble cytoskeletal component was separated from the
soluble cytosolic component to determine the amount of tau associated
with microtubules. Although tau was present both in the soluble
component and in the insoluble component, tau in the insoluble
component migrated faster than tau in the soluble component, suggesting
that tau in the insoluble component was phosphorylated to a lesser
extent than tau in the soluble component. In addition, the amount of
tau in the insoluble component (microtubule-associated tau) was
greater in cultured neurons from Cdk5
/
mice than those from
wild-type and p35
/
mice. Concomitantly, the soluble component from
the Cdk5
/
mouse neurons contained a lesser amount of tau. There was
no apparent difference in the amount of tau associated with
microtubules between wild-type and p35
/
mouse neurons. Thus, these
results suggest that tau phosphorylation by Cdk5/p39 reduces the
ability of tau to bind to microtubules.

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Fig. 8.
Reduced ability of tau phosphorylated by
Cdk5/p39 to bind to microtubules. Cultured cerebellar neurons from
wild-type, p35 / , and Cdk5 / mouse embryos were subjected to the
Triton X-100 extraction assay on the 7th day after plating, and the
detergent-insoluble cytoskeletal component was separated from the
soluble cytosolic component to determine the amount of tau associated
with microtubules. An equal amount of protein from each component was
subjected to SDS-PAGE and Western blotting using the
phosphorylation-independent tau antibody, TAU-5. A, the
amount of tau in the detergent-insoluble component (associated with
microtubules) was greater in cultured neurons from Cdk5 / mice as
compared with those from wild-type and p35 / mice. Concomitantly,
the soluble component from Cdk5 / mouse neurons contained a lesser
amount of tau. B, the amounts of tau in the soluble
component (upper panel) and the insoluble
component (lower panel) were quantitatively
determined by densitometry and presented as the relative level of TAU-5
immunoreactivity to that from the wild-type mouse neurons. All values
are shown as the mean ± S.E. from three independent
experiments.
|
|
 |
DISCUSSION |
The functions of tau as a microtubule-associated protein are
modulated by its phosphorylation state. The phosphorylation of tau may
be essential during brain development to maintain a certain degree of
microtubule instability, thus affecting neurite outgrowth. This study
reports that Cdk5/p39 is involved in the in vivo
phosphorylation of tau during brain development, as evidenced by the
observation that tau phosphorylation at Ser-202 and Thr-205 was
decreased in Cdk5
/
mouse brain but not in p35
/
mouse brain.
Cdk5/p35 and Cdk5/p39 exhibited different preferences for tau as a
substrate. The ability of Cdk5 to phosphorylate tau was higher when in
association with p39 rather than in association with p35. Furthermore,
during brain development, the p39 expression and the Cdk5/p39 activity as a tau kinase were varied in a spatial manner. Higher expression and
kinase activity were observed in embryonic cerebellum, brain stem, and
spinal cord, but their distribution changed postnatally, with the
highest expression and activity in the cerebral cortex. Additionally,
the phosphorylation of tau by Cdk5/p39 reduced the affinity of tau for
microtubules. These results described here suggest that the in
vivo phosphorylation of tau by Cdk5 may regulate microtubule
dynamics in a region-specific and developmentally regulated manner.
Cdk5 displays ubiquitous tissue distribution (48); however, its kinase
activity has been found mainly in brain where its regulatory subunits,
p35 and p39, are predominantly expressed (29, 49, 50). Insight into the
critical functions of Cdk5 in brain development has been gained from
gene targeting experiments for Cdk5, p35, and p39 genes. It is of
interest to note that there are prominent differences in the phenotypic
severity of these three mice (32, 35, 36, 51). Cdk5
/
mice display
late embryonic and/or perinatal lethality and extensive defects in the
central nervous system (35), whereas, p35
/
mice are viable and
display defects mostly confined to the forebrain, with cortical lamination defects similar to those observed in Cdk5
/
mice (32). In
the cerebellum, the typical foliation and tripartite layering are
observed in p35
/
mice but not in Cdk5
/
mice (32, 35). In the
brain stem, p35
/
mice show no apparent abnormalities, whereas
Cdk5
/
mice display migration defects in facial branchiomotor and
inferior olive neurons (51). In the spinal cord, abnormal motor neurons
with ballooned perikarya, characteristic of chromatolytic changes, are
observed in Cdk5
/
mice but not in p35
/
mice (32, 35). In
comparison, p39
/
mice do not show any noticeable defects in the
brain, whereas p35
/
p39
/
double knockout mice display a
phenotype similar to that of Cdk5
/
mice (36). Our results presented
here clearly demonstrate that p39 expression is higher during embryonic
development in the cerebellum, brain stem, and spinal cord, where the
pathological defects are mostly absent in p35
/
mice. However, the
level of p39 expression is very low in the embryonic cerebral cortex,
where p35
/
mice have lamination defects similar to those observed
in Cdk5
/
mice. These results suggest that Cdk5/p39 may compensate
for a lack of Cdk5/p35 activity in p35
/
mice, indicating the
overlapping roles of p35 and p39. Furthermore, the fact that p39
/
mice do not show any noticeable abnormalities is also compatible with
our observation that the p35 expression level is high in all regions of
the brain during embryonic development, suggesting the compensatory
role of p35 in the absence of p39.
It has not been clear, however, whether p35 and p39 can each confer a
distinct function to Cdk5. The specific subcellular localization of p35
and p39 has been reported; p39 is both cytosolic and
membrane-associated, whereas p35 is mainly membrane-associated, suggesting a distinct role of each regulatory subunit to determine the
subcellular localization of the Cdk5 complex (52). Our observation that
the developmental expression pattern of p39 in the cerebral cortex
appears to be the inverse to that of p35 may be related to a hypothesis
that each regulatory subunit targets Cdk5 to different substrates
during brain development. We show here that Cdk5/p35 and Cdk5/p39
exhibit different preference for tau as a substrate; p39 preferentially
activates Cdk5 phosphorylation of tau relative to p35. The
preference of Cdk5/p39 for tau was also confirmed by the observation
that p35
/
mouse brain showed higher Cdk5 activity using tau as a
substrate as compared with histone H1. Furthermore, p35
/
mouse
brain, which contains the up-regulated amounts of p39 protein, does not
show any differences in tau phosphorylation as compared with wild-type
mouse brain. In contrast, a significant reduction of tau
phosphorylation is observed in Cdk5
/
mouse brain, except in the
cerebral cortex, where the p39 expression level is very low. These
results indicate that Cdk5/p39 is involved in the in vivo
phosphorylation of tau during brain development, whereas the remaining
AT-8 immunoreactivity in Cdk5
/
mouse brain suggests the
contribution of other protein kinases. Thus, the regulatory subunits,
p35 and p39, appear to play important roles in determining the
substrate specificity of Cdk5 as well as its subcellular localization.
To gain insight into the functional consequences of tau phosphorylation
by Cdk5/p39, the ability of tau to bind to mictotubules was examined
using cultured cerebellar neurons from wild-type, p35
/
, and
Cdk5
/
mice. Cultured cerebellar neurons were used, because the p39
expression level is higher in the embryonic cerebellum. A detergent
extraction assay revealed that the amount of tau associated with
microtubules was greater in cultured neurons from Cdk5
/
mice than
those from wild-type and p35
/
mice. Moreover, cerebellar neurons
from p35
/
mice showed no difference in the amount of tau associated
with microtubules as compared with those from wild-type mice. These
results suggest that tau phosphorylation by Cdk5/p39 reduces the
ability of tau to bind to microtubules. The observations that the
regional distribution in the p39 expression level and the Cdk5/p39
activity as a tau kinase are drastically changed during the
perinatal period further suggest that Cdk5/p39 may contribute to a
developmental regulation of tau phosphorylation and its microtubule
binding ability. Phosphorylated tau has been shown to be less efficient
than dephosphorylated tau in promoting microtubule assembly, whereas
the dephosphorylated tau promotes significantly more rapid and more
extensive polymerization of microtubules (53). Thus, tau
phosphorylation by Cdk5/p39 in the developing brain may provide the
microtubules with more dynamic properties to allow microtubule
rearrangements during axonal growth in a region-specific and
developmentally regulated manner. It has been previously described that
the extent of Cdk5 kinase activity is coincident with neurite outgrowth
in cultured cerebellar neurons (54). Moreover, distinct roles of p35
and p39 have been observed in neurite outgrowth (55). Although both p35
and p39 induced neurite outgrowth in cultured hippocampal neurons, only
antisense p39 prevented basic fibroblast growth factor-induced neurite
outgrowth. This observation suggests that p39 is required for neurite
outgrowth, consistent with our present observations.
A transient hyperphosphorylation of tau during the early development of
the brain has been found to be very similar to the abnormal
phosphorylation of tau in AD brains (9, 17, 18). The
hyperphosphorylation of tau in mature neurons may contribute to its
loss of microtubule binding ability and aggregation into neurofibrillary tangles. Cdk5 has been proposed to be implicated in the
hyperphosphorylation of tau observed in AD brains in association with
p25 but not with p35 (27). p25 is generated from p35 upon neurotoxic
insults activating the Ca2+- dependent cysteine protease,
calpain (45, 56, 57). This proteolytic conversion gives p25 altered
biological properties as compared with p35. p25 is more stable and has
a different subcellular localization, leading to mislocalization and
prolonged activation of Cdk5 (45, 56, 57). Treatment of cultured
neurons with amyloid-
peptide, a primary constituent of the amyloid
plaques in AD brains, causes the conversion of p35 to p25, concomitant with the activation of calpain (56). Furthermore, recent in vitro and in vivo studies demonstrated that the
conversion of p35 to p25 promotes phosphorylation of tau at Ser-202 and
Thr-205 (57-59). These results suggest that Cdk5/p25 may be involved
in the abnormal phosphorylation of tau in AD brains. Thus, it seems that Cdk5 not only acts as a physiological tau kinase in association with p39 but also as a pathogenic tau kinase in association with p25.
Recently, p39 has also been found to be a substrate for calpain (60).
Similar to the conversion of p35 to p25, calpain-mediated cleavage of
p39 generates the C-terminal fragment p29, with biological properties
that cause the deregulation of Cdk5 (60). It will be important to
examine whether Cdk5/p29 may act as a pathogenic tau kinase in certain
pathological conditions. Cdk5 may display diverse functions through
tau phosphorylation in developing brain as well as in pathogenic brain
depending on its association with different partners, p35/p25 or
p39/p29.