J. Biochem. 121, 179-188 (1997)
Physiology and Pathology of Tau Protein Kinases in Relation to
Alzheimer's Disease
Kazutomo Imahori1 and Tsuneko Uchida
Mitsubishi Kasei Institute of Life Sciences, 11 Minamiooya, Machida, Tokyo 194
Received for publication, November 27, 1996
Alzheimer's disease (AD) is characterized by neuronal cell death and two kinds of deposits,
neurofibrillary tangles (NFT) and senile plaques. The main component of NFT is paired
helical filaments (PHF), which mainly consist of hyperphosphorylated tau protein. Tau
protein kinases I and II were found as candidate enzymes responsible for hyperphos
phorylation of tau to induce the formation of PHF. Since prior phosphorylation of tau by
TPKII strongly enhanced the action of TPKI, it was thought that TPKII was involved in the
formation of PHF-tau in concert with TPKI. After cloning, TPKI was found to be identical
with glycogen synthase kinase 3ƒÀ (GSK3A), while TPKII consists of a novel 23kDa protein
activator and a catalytic subunit that is identical with cyclin-dependent kinase 5 (CDK5).
The phosphorylation sites on tau by TPKI and TPKII could account for the most, but not all,
of the major phosphorylation sites of fetal tau and PHF-tau. An antibody for a site
specifically phosphorylated by TPKI (Ser413) could identify all three neurofibrillary
lesions in the AD brain, and double staining for either TPKI or TPKII and NFT in the brain
of Down's syndrome patients clearly demonstrated that TPKI and TPKII are both associat
ed with NFT in vivo, suggesting that the level of TPKI or TPKII is elevated in AD brain by
some mechanism. On the other hand, the levels of both TPKs change developmentally, being
high in the neonatal period when the phosphorylation of fetal tau proceeds actively,
suggesting that the TPKI/TPKII cooperative system has an important physiological role in
the formation of neural networks. In AD brain, aberrant accumulation of amyloid-ƒÀ
protein (A,6) occurs ahead of the accumulation of PHF in NFT. When a primary culture of
embryonic rat hippocampus was treated with 20ƒÊM AƒÀ, induction of TPKI, extensive
phosphorylation of tau and then programmed cell death were observed, indicating that
TPKI induced by AƒÀ phosphorylates tau, followed by disruption of axonal transportation
and finally cell death. By using a yeast two hybrid system, TPKI was found to interact with
pyruvate dehydrogenase (PDH), which is a key enzyme in the glycolytic pathway. PDH was
phosphorylated in vitro by TPKI to reduce the activity converting pyruvate into acetyl
CoA, which is required for acetylcholine synthesis. In a primary culture of rat hippocampal
cells treated with AƒÀ, PDH was inactivated in inverse relation to the activation of TPKI,
resulting in accumulation of pyruvate or lactate, energy failure induced by the disturbance
of glucose metabolism, and a shortage of acetylcholine owing to deficiency of acetyl-CoA, all
of which are characteristic of AD brain. In cholinergic neurons such as those of the septum,
non-aggregated A,8, specifically AƒÀ (1-42), not AƒÀ (1-40), caused a shortage of acetylcholine
by activation of TPKI and inactivation of PDH without cell death.
Key words: Alzheimer's disease, amyloid-ƒÀ protein, phosphorylation of tau, pyruvate
dehydrogenase, tau protein kinases I and II.
Alzheimer's disease (AD) is a heterogeneous group of dementias that share common clinical symptoms, involving progressive cognitive impairments. This form of senile
dementia is characterized by two kinds of pathological
deposits in specific areas of the brain, called senile plaques
and neurofibrillary tangles (NFTs), and finally by severe
atrophy of the brain resulting from the extensive loss of
neuronal cells. The relationships among these three charac
teristics have not yet been established, and further patho
logical and biochemical evidence, is required to clarify the
etiology of Alzheimer's disease.
Senile plaques contain extracellular deposits of ƒÀ-amy
loid protein (ƒ¿ƒÀ) associated with degenerating nerve
processes known as dystrophic neurites. Initial deposits are
non-fibrillar (a diffuse plaque), but are progressively trans
formed into fibrils, giving rise to the characteristic amyloid
1 To whom correspondence should be addressed. Tel: +81-427.24
6222, Fax: +81-427-29-1252
Abbreviations: Af, amyloid-ƒÀ protein or J4-amyloid protein; Ach,
acetyl choline; AD, Alzheimer's disease; CDK5, cyclin dependent
kinase 5; DS, Down's syndrome; FITC, fluorescein isothiocyanate;
GSK3ƒ¿(ƒÀ), glycogen synthase kinase 3ƒ¿(ƒÀ); MAPK, mitogen
activated protein kinase; MARK, microtubule affinity regulating
kinase; NFT, neurofibrillary tangle; NT, neuropil thread; NP,
neuritic plaque; PDH, pyruvate dehydrogenase; PHF, paired helical
filament; TPK, tau protein kinase.
Vol. 121, No. 2, 1997 179
180 K. Imahori and T. Uchida
plaques. Neurofibrillary tangles form inside neuronal cells that die during the course of the disease and consist
primarily of abnormal paired helical filaments (PHFs). However, large numbers of senile plaques are found in some cognitively normal individuals, suggesting that not only their presence, but also the coexistence of NFTs is required for dementia. On the other hand, large numbers of NFTs in cerebral cortex and hippocampus closely correlate with the degree of dementia in Alzheimer's disease (1). Thus, the accumulation of neurofibrillary lesions may represent a final pathway that leads to neuronal cell death and neurodegeneration. Therefore, our attention was first directed to the formation and accumulation of PHF.
PHF consists of hyperphosphorylated tau (PHF-tau) (2, 3), and contains a small amount of ubiquitin (4). Tau is one of the microtubule-associated proteins and is specifically localized in the neuron. In the normal neuron, tau has the physiological role of promoting the assembly and stability of axonal microtubules, which are involved in intraneuronal transport (5, 6). During the course of Alzheimer's disease, tau becomes hyperphosphorylated at multiple sites and integrated into PHFs, losing its physiological functions. No protein kinase phosphorylating tau to give PHF-like state was known at that time. Such an enzyme, if found, could serve as a probe for investigating the mechanism of the formation and accumulation of PHF, as wwell as the changes of cellular metabolism involved in the degenerative process leading to neuronal death. Therefore, our initial goal was identification and characterization of the candidate kinase.
Identification of a candidate kinase for hyperphos
phorylation of tau leading to a PIIF-like stateThe characteristics of PHF-tau described in early reports
were an increase of apparent molecular weight (Mr) on SDS-gel owing to hyperphosphorylation (3), and the acquisition of immunoreactivity with anti-PHF polyclonal antibody (2) or the loss of immunoreactivity with anti-non
phosphorylated tau monoclonal antibody (tau-1), in addition to high resistance to proteolysis (7). Thus, three criteria were set up for identifying a candidate kinase as mentioned above: i.e., it must be capable of (1) phosphorylating tau, (2) inducing the mobility shift of tau on SDSpolyacrylamide gel electrophoresis, and (3) forming a PHF epitope on the phosphorylated tau. These criteria proved to be reasonable, because PHF-like structure was later shown to be constructed from highly phosphorylated whole tau
protein (A68) alone (8). Protein kinase activity meeting all three criteria was found in the microtubule proteins fraction of rat or bovine brain extracts and partially purified (9). This kinase is a serine/threonine kinase, which phosphorylates tau to form a PHF epitope, easily binds to tau, and does not depend on second messengers, such as cyclic AMP, cyclic GMP, Ca2+, and calmodulin. The phosphorylation catalyzed by this kinase was stimulated by tubulin under conditions of microtubule formation. This novel enzyme, different from known protein kinases, was designated as tau protein kinase [EC 2.7.1.135] by the Nomenclatture Committee of the International Union of Biochemistry (10). It is abbreviated as TPK.
On further purification, tau protein kinase was separated into two kinds of activities with different modes of phosphorylation of tau. These were purified to homogeneity and designated as TPKI and TPKII, respectively (11).
Relation between TPKI and TPKII
The two enzymes differed in the mode of phosphorylation
of tau. TPKI can phosphorylate native tau isolated from
normal brain, which is already phosphorylated to some
extent, but not completely dephosphorylated tau. In con
trast with TPKI, TPKII can phosphorylate both. TPKI can
induce an apparent molecular weight shift of tau and form
a PHF epitope by phosphorylating native tau, but TPKII
can not do so. Apparently, TPKI converts native tau to
PHF-tau, whereas TPKII seems to have no relation to PHF
formation (11). When dephosphorylated tau is initially
phosphorylated by TPKII, however, TPKI can then phos
phorylate it as well as native tau to generate a PHF epitope,
indicating that prior phosphorylation of tau by TPKII
enhances phosphorylation by TPKI (12). TPKII may
regulate the phosphorylation state of tau not only in normal
brain, but also in Alzheimer's disease brain, in concert with
TPKI.
Chemical nature of TPKI and TPKII
TPKI consists of a single polypeptide of 45kDa. Se
quence analyses of its cDNA proved that TPKI is identical
with "glycogen synthase kinase 3,8 (GSK3ƒÀ)" (13), one of
two isoforms of GSK3 that are encoded by different genes
(14). Mammalian GSK3ƒÀ is homologous to the product of
the Drosophila gene termed shaggy (sgg) or zeste-white 3
(zw-3), and is reported to rescue the sgg/zw-3 mutant
phenotype connected with wingless (wg) signal transduc
tion (15) and with Notch signalling (16). Furthermore,
GSK3 was recently shown to be required for ventral
differentiation (17). Therefore, mammalian GSK3 is also
considered to be involved in embryogenesis and neurogene
sis.
TPKII is a complex composed of two subunits, a catalytic
subunit of 30kDa and a regulatory subunit of 23kDa. Since
TPKII extensively phosphorylated histone H1 and MAP2
as well as tau, this enzyme was expected to be similar to the
cdc2 enzyme. The catalytic subunit of TPKII was proved by
sequence analysis of the cDNA to be identical with a "cdc2 -related kinase , PSSALRE/Cdk5" (18), which was
first found as one of 7 novel cdc2-related kinases cloned
from a human cell line (19). TPKII is an active complex of
CDK5 with a 23kDa protein; CDK5 monomer alone is
inactive (20). Therefore, the 23kDa protein (p23), not
cyclin, is regarded as a CDK5 activator in neuronal cells.
Cloning and sequencing of the 23kDa subunit revealed it to
be a C-terminal region of a novel protein with a molecular
weight of 34kDa (20). The sequence of the 34kDa protein,
p34, contains that of p23 and an additional 98 amino acids
at the N-terminus of p23, suggesting that p34 is a precursor
of the regulatory subunit, p23 (21). Certainly, no active
complex of CDK5 with p34 was detected in bovine brain
extract. However, it might be argued that p34 itself is a
CDK5 activator and p23 is the proteolytic derivative,
because recombinant p34 can be reconstituted with bacter
ially synthesized CDK5 to result in high kinase activity
(22). In the purification process of TPKII, enzymatic
activity is often localized in the high-molecular region.
These results suggest that TPKII may form a larger active
complex in vivo.
Our results on p23 are in good agreement with those on
the regulatory subunit, p25 of neuronal cdc2-like kinase
(nclk) (23), formerly called bovine proline-directed kinase
J. Biochem.
Pathology of Tau Protein Kinases in Alzheimer's Disease 181
(BPDK), which was purified from bovine brain extract as a
neurofilament kinase. These enzymes, TPK II and nclk, are
considered to be identical.
Phosphorylation of tau with TPKI and TPKII
The phosphorylation sites in tau protein by TPKI and
TPKII were first examined chemically. Protease digests of
tau maximally phosphorylated by the kinase in the pres
ence of [ƒÁ-32P]ATP, were separated by HPLC using a C4
column. In the case of TPKI, tau which had first been fully
phosphorylated with TPKII and cold ATP was used. The
sequences of phosphopeptides were determined using an
improved protocol in a gas-phase sequencer (24), and the
phosphorylation sites of tau by TPKII were identified as
Ser202, Thr205, Ser235, and Ser404 (25), while those by
TPKI were Ser199, Thr231, Ser396, and Ser413 (number
ing of amino acids is based on the longest human tau) (26)
(Table I and Fig. 1). We omitted ambiguous sites which
exhibited very low levels of phosphorylation or which were
localized in the extreme C-terminal region of long peptide
fragments.
Phosphorylation sites by TPKII have a common sequence
of SerPro or ThrPro, indicating that TPKII is a proline
directed kinase, though not all Ser/ThrPro sequences in tau
protein, were phosphorylated. Although TPKI/GSK3ƒÀ is
also regarded as a member of the class of proline-directed
kinases, one of the 4 TPKI sites, Ser413, is non-proline
directed site.
As a next step, we aimed to prepare antibodies specific
for these phosphorylation sites of TPKI and TPKII by using
chemically synthesized phosphopeptides as antigens, and to
confirm that TPKI and TPKII act at the phosphorylation
sites determined chemically (12, 27). Antigen dodecapep
tides containing phosphoserine or phosphothreonine in the
center were synthesized by the use of a phenyl group (28)
or a cyclohexyl group (in the case of aromatic or sulfur
containing amino acids) (29) as a protecting group for the
phosphate group. In addition, antibodies raised against
peptides phosphorylated at Ser262 or Ser422 were prepar
ed in the method described above. Each phosphopeptide
was named based on the species and the number of the
phosphorylated amino acid, e.g., PS199 or PT205. The
obtained antibodies against the phosphorylated 4 TPKI
sites, 4 TPKII sites, Ser262 and Ser422 were specific for
the corresponding phosphopeptide, and did not cross-react
with other phosphopeptides. Human recombinant tau
(htau40) phosphorylated by TPKII, reacted with all anti
bodies specific for the 4 TPKII sites, anti-PS202, anti-
TABLE I. Phosphorylation sites of tau in vivo (A) and those generated by various kinases reported so far as candidates to act in PHF formation (B).
aCited from f. 33. bCited from Ref. 34. cCited from Ref. 38. dSer205 was reported as a PHF-site by others (35, 36).
Fig. 1. Phosphorylation sites on tau by TPKI and II. The primary structure of the longest human tau is presented here. •«:
Phosphorylation sites by TPKI and TPKII are indicated by 1 and 2, respectively. •ª: Phosphorylation sites in PHF-tau (33). Rn: 31 or 32 amino
acid repeat region. •¡: 18 amino acid microtubule -binding element.
Vol. 121, No. 2, 1997
182 K. Imahori and T. Uchida
PT205, anti-PS235, and anti-PS404, confirming the results
of chemical analysis. TPKI in the presence of TPKII
incorporated phosphate into tau at 4 TPKI sites, Ser199,
Thr231, Ser396, and Ser413, inducing the largest shift-up
of apparent molecular weight of tau. Unexpectedly, TPKI
slightly phosphorylated tau at Ser262, but not at Ser422.
Accordingly, immunoblots of recombinant tau phosphor
ylated by TPKI and/or TPKII with these antibodies specific
for phosphorylation sites, clearly confirmed the 4 TPKI and
4 TPKII sites identified from the chemical analysis, as well
as the enhancement of TPKI activity after prior phosphor
ylation of tau with TPKII, that is, the TPKII/TPKI co
operative phosphorylation system (27). This immunoblot
method is advantageous to detect low levels of phosphor
ylation and changes of phosphorylation state in vitro and in
vivo (24).
These 8 TPKs sites are localized in the amino and
carboxyl-terminal flanking regions of the microtubule
binding domain of tau, as shown in Fig. 1. The microtubule
binding domain consists of the tandem 31 or 32 amino acid
repeat region, which includes 4 repeats in adult tau and 3
repeats in fetal tau (30). The sequences flanking the repeat
were indicated by binding studies to enhance the affinity of
this domain for microtubules, though they do not bind
directly with microtubules (31). In particular, the proline
rich region upstream of the repeats, which contains a
cluster of Ser/ThrPro phosphorylation sites, appears to be
essential for tubulin binding in vivo (32). Thus, phosphor
ylation by TPKI and TPKKKII markedly reduces the tubulin
assembly-promoting activity (26). Through its moduration
of the extent of phosphorylation of tau, TPKII appears to be
involved in the regulation of microtubule dynamics in
normal brain. In addition, TPKII sites, particularly in the
proline-rich region, are phosphorylated to a two to three
fold higher level at postnatal week 1 as compared with the
level in adult rat, suggesting that TPKII is developmentally
regulated in rat brain (12).
Recently, phosphorylation sites in PHF-tau from the
brain of Alzheimer's disease patients, in fetal tau from
neonatal rat brain (33) and in adult tau from adult rat brain
(34) have been determined by identifying phosphopeptides
by means of ion spray mass spectrometry followed by
sequencing of the ethanethiol-modified peptides, as shown
in Table I and Fig. 1. The degree of phosphorylation
increases in the order of normal adult tau <fetal tau
<PHF-tau. Nineteen sites identified in PHF-tau are all
localized in the N. and C-terminal regions of the micro
tubule binding domain except for Ser262. The extent of
phosphorylation at each site varies differently. Major
phosphorylation sites shown by double circles in Table I are
10 sites of the 19 sites. All the TPKI and TPKII sites except
Ser205 are included in these major sites. Ser205 was
identified as a PHF site by other workers (35, 36), in
accordance with our result. Three non-proline-directed
sites, Ser198, Ser400, and Ser409 could be phosphorylated
by TPKI/GSK3ƒÀ if Ser202, Ser404, and Ser413 were first
phosphorylated, as in fetal tau. Ser262 is considered to be
very important for microtubule-binding because it is locat
ed in the first repeat, having the highest affinity for
microtubules compared with the other three repeats. The
possibility that TPKI/GSK3ƒÀ is responsible for phosphor
ylation of tau at Ser262 in vivo still remains, because
TPKI/GSK3ƒÀ certainly phosphorylates a trace amount of
tau at Ser262 in vitro and this would be enhanced somewhat
in the presence of TPKII/(CDK5+p23) (27), and further,
the activity of GSK3ƒ¿, another isoform of GSK3ƒÀ, to
phosphorylate tau at Ser262, Ser324, and Ser356 is strong
ly stimulated in the presence of heparin (37). However, it
seems most probable at present that p110/MARK is the
kinase that phosphorylates Ser262 (38), because PKA,
another candidate, phosphorylates tau so weakly. Thus,
most of the phosphorylation sites in fetal tau and in
PHF-tau are modified by TPKI/TPKII, but one of the
major sites, Ser422, in PHF-tau is not notably phosphor
ylated by TPKI alone or by the TPKI/TPKII cooperative
system (27). Thus, it is reasonable to consider that other
kinases are also involved in the hyperphosphorylation of
tau to induce PHF-tau. Considering that all tau molecules in
PHF are not homogeneously hyperphosphorylated and that
the non-proline-directed sites that distinguish PHF-tau
from fetal tau are mostly minor sites, it is questionable
whether just phosphorylation at a crucial site or several
sites is essential for PHF formation. Nevertheless some
hyperphosphorylation, particularly in the two flanking
regions of the microtubule-binding domain, seems to be
essential at least for dissociation from the microtubules, in
addition to the initiation of self-assembly of PHF-tau.
Physiological roles of TPKI and TPKII
No information was available on the function of TPKI/
GSK3ƒÀ and TPKII/(CDK5 + p23) in normal brain when we
started our studies. We first demonstrated that TPKII sites
in tau are maximally phosphorylated at the first week
postnatally in rat brain (12). Phosphorylation sites in fetal
tau were mostly modulated by the TPKI/TPKII coopera
tive system as described above. We then examined de
velopmental change of both kinases in rat brain.
Northern blot analysis showed that CDK5 was expressed
predominantly in brain, though it is also ubiquitous in other
tissues, while p34, the precursor of the regulatory subunit
p23, is expressed specifically in brain (21). Other groups
also showed, using in situ hybridization of embryonic
mouse tissues, that p35 expression was restricted to the
central nervous system (CNS) (22), but CDK5 was ex
pressed in both the CNS and the peripheral nervous system
(39), indicating that the localizations of the two subunits
are slightly different.
Expression of p34 mRNA rapidly increased during
gestation day 15 to 18, reached the highest level just at
birth, remained abundant for 2 weeks in neonatal brain,
and then decreased gradually to about 10% of the maximum
in aged brain. Expression of CDK5 mRNA also changed
developmentally in a manner similar to p34 mRNA expres
sion, except that it remained at about 50% of the maximal
level in aged brain. In contrast, TPKII activity develop
mentally changed just in parallel with expression of p34
mRNA, but not of CDK5 mRNA , suggesting that it is controlled by p34 expression. Immunohistochemical
studies and in situ hybridization showed that both CDK5
and p34 are detectable from gestational day 12, when
proliferation of preneuronal cells comes to an end, and
CDK5 is expressed only in postmitotic neurons, but not in
glial cells or mitotically active cells (22). Therefore,
TPKII/(CDK5+p23) is involved in neurogenesis, but not
in cell division, particularly in sprouting for neural network
formation, which is a common feature of fetal brain (40, 41)
J. Biochem.
Pathology of Tau Protein Kinases in Alzheimer's Disease 183
and Alzheimer's disease brain (42). Recently, the subcell
ular localization of CDK5 and p34 proteins was examined
by indirect immunocytochemistry and confocal micros
copy, using cultured primary neurons derived from em
bryonal day 17-18 rat cortices (43). It was found that both
CDK5 and p34 are present in the growth cones of develop
ing neurons. Furthermore, expression of dominant-nega
tive mutants of CDK5 inhibited neurite outgrowth, which
was rescued by co-expression of the wild-type CDK5 and
p34. These observations suggest that TPKII/(CDK5 + p34)
plays a critical role in neurite outgrowth during neuronal
differentiation.
Developmental change of TPKI in rat brain was ex
amined by immunoblot analysis using monoclonal antibody
(T1.7) raised against TPKI (44) and polyclonal antibody
(C-1) raised against a C-terminal peptide of TPKI/GSK3ƒÀ
(45), which specifically recognized GSK3ƒÀ, but not
GSK3ƒ¿. The level of TPKI changed developmentally in a
similar way to that of TPKII, that is, it increased from
gestational day 12 (E12) to birth, was maintained at a high
level for 2 weeks after birth, and decreased in aged brain
(45), though TPKI reached the maximum level several
days later than TPKII. Immunohistochemistry with rabbit
antibody C-1 revealed that staining intensity in embryos
from E9 to E10 increased in both presumptive neural and
non-neural tissues. A further increase in staining, however,
was asssciatedd only with differentiating central and periph
eral neurons (44). In hippocampus and neocortex of 3 to
11-day-old rats, TPKI immunoreactivity was observed in
young axonal tracts and growing neuropil, where phos
phorylated Ser396 was also detected, while it declined to a
low level in a sections of adult brain, where phosphorylated
Ser-immunoreactivity was undetectable (46). These find
ings indicate that TPKI is also involved in the processes
leading to differentiation of neuronal cells and in the
extension of neuronal processes. Therefore, the TPKI/
TPKII cooperative system may play an immportant physi
ological role in the formation of neural networks.
Involvement of TPKs in PHF formation
In order to confirm that both TPKs are involved in PHF
formation, we carried out the following experiments. As
described above, about 19 sites are phosphorylatable in
PHF-tau (33) and 10 of them are major sites. Among these
sites, Ser413 is unique because it could be phosphorylated
only by TPKI, but not by any other kinase tested so far. In
other words, phosphorylation of Ser413 is the fingerprint of
TPKI. Probably Thr205 is the fingerprint of cyclin-depen
dent kinase. Both fingerprints are found in PHF. As the
next step, in order to show that TPKs are operating in PHF
formation, we examined the staining of AD brain using
anti-PS413 and anti-PT205, to detect the fingerprints of
TPKI and TPKII, respectively. Twelve AD brains and nine
control brains with no evidence of Alzheimer-like changes
were examined by staining for PS413 and PT205 epitopes
in the CAI subfield of the hippocampus, where neurode
generative change in AD brain is most prominent. In serial
vibrotome sections of formalin-fixed hippocampus, anti
PS413 and anti-PT205 identified three major lesions
associated with neurofibrillary degeneration (i. e., NFT,
NP, and NT) involving pyramidal neurons of the CAI
region in all twelve AD cases (47). In control experiments,
preabsorption of antisera with PS413 or PT205 abolished
immunostaining. On the other hand, the brains of non-de
mented individuals were faintly stained and the number of
stained neurons was small, being in agreement with the
previous report (48). These results suggested strongly that
both TPKs are operating in AD brain to generate their
specific epitopes.
Up-regulation of TPKs in AD brain
The preferential appearance of epitopes of TPKI and
TPKII in AD brain suggested up-regulation of these kinases
in AD brain. In order to test this, we tried to raise antibody
against TPKI/GSK3ƒÀ. We obtained antibodies against
synthetic peptides representing carboxyl-terminal frag
ments of rat TPKI/GSK3ƒÀ and its isoform GSK3ƒ¿, where
the difference in homology between these isoforms is most
marked (45). After brief microwave heating, intense
staining of pyramidal neurons of focal groups in AD brain
and their surrounding neuropil made a vivid contrast with
the weaker, diffuse reaction of the neighboring cells (Fig.
2A). The same region of the age-matched control brains was
also stained, but only faintly, and the number of stained
cells was small (Fig. 2B). Similar strong staining was
observed in the prefrontal cortex, entorhinal cortex, and
amygdala, but not in the cerebellum, which lacks AD
pathology. Preabsorption of the anti-serum with TPKI
peptide completely inhibited the immunostaining, but
preabsorption with GSK3ƒ¿-peptide had no effect (47).
Similar experiments were carried out on the brain of a
Down's syndrome (DS) patient (49). In this experiment,
colocalization of TPKI/GSK3ƒÀ was clearly revealed by
double immunostaining for TPKI/GSK3ƒÀ (labeled with
FITC) and tau-1 (labeled with Texas red). The size and
shape of the areas of strong TPKI labeling were quite
similar to those of tau-1-positive NFT. Neuropil threads
and degenerating neurites of senile plaque were also TPKI
positive in DS brain (49) (Fig. 2C).
As mentioned earlier, TPKII consists of two subunits and
the larger one is identical with CDK5. We therefore stained
AD brain by using commercially available anti-CDK5
antibody. The results obtained were very similar to those in
the case of TPKI. Double staining clearly indicated that
tau-l-positive NFT were also positive for CDK5. Neuropil
threads were negative for CDK5 in contrast with the case of
TPKI (49) (Fig. 2D). However our results clearly indicate
that the level of TPKI or TPKII is elevated in AD brain.
Induction of TPKI and neuronal cell death
The above results suggested to us that some factor(s)
causing induction of TPKs in neuronal cells may appear in
AD brain. There is a long-standing hypothesis that aberrant
accumulation of AƒÀ occurs in AD brain and is associated
with NFT formation and neuronal death (50, 51). In order
to test whether AƒÀ might be the putative inducer, we
adopted an in vitro approach. Primary cultures of embry
onic rat hippocampus were used for this purpose. This
tissue was treated with or without 20ƒÊM AƒÀ. At this
concentration, AƒÀ molecules aggregated and the solution
was turbid. After 3h, the tissue was stained by anti-TPKI
antibody and also by anti-PS396 antibody. Both antibodies
stained AƒÀ-treated cells strongly, whereas non-treated
cells were scarcely stained (27, 52). This suggests that in 3
h, AƒÀ induced TPKI, which in turn phosphorylated tau
proteins. The activity of TPKI was assayed quantitatively
Vol. 121, No. 2, 1997
184 K . Imahori and T. Uchida
Fig. 2. Immunocytochemistry of anti-TPKs in the human hippocampal CAI subfield. (A) AD brain stained with anti-TPKI
antibody. (B) Control brain with slight AD-like changes stained with anti-TPKI antibody. In pyramidal cells, the cell body, apical dendrite , basal neurite and proximal axon are stained. (C) Staining of Down's
syndrome brain with anti-TPKI antibody (top) , tau-1 (middle), and double exposure (bottom) . (D) Staining of Down's syndrome brain
with anti CDK5 antibody (top) , tau-1 and double exposure (bottom). Tau-I stained dephosphorylated NFT . Overlapping of blue and red
gave yellow.
J. Biochem.
Pathology of Tau Protein Kinases in Alzheimer's Disease 185
after immunoprecipitation with anti-TPKI . The specific activity of TPKI was elevated to more than two times the con
trol by the treatment with AƒÀ. As shown in Table I there are
several candidate kinases which might be involved in PHF
formation through phosphorylation of tau . We examined whether some of them, such as MAPK and GSK3a
, might also be induced by AƒÀ treatment. However neither of them
showed any increase in activity. Thus , TPKI appears to be
primarily involved in AƒÀ-induced phosphorylation of tau.
In parallel with the enzyme assay, we examined the
survival of the cells. After incubation for 24 h , the survival of the cells were reduced to less than 10% of that in the
control culture (culture without AƒÀ), suggesting neurotox
icity of AƒÀ, as reported by several authors (53, 54). In
order to clarify the nature of TPKI activation, we synthe
sized both sense and antisense oligonucleotides of the TPKI
gene based on the cDNA sequence (52). Sense or antisense
oligonucleotide was added to an incubation mixture con
taining A6. After incubation for 3 h with antisense oligonu
cleotide, the activity of TPKI was suppressed to the same
level as that of the control. On the other hand, sense
oligonucleotide had no ability to suppress TPKI activity
(52). Antisense oligonucleotide was also effective to rescue
the cells from death. After 24 h, survival of the cells
incubated with both AƒÀ, and antisense oligonucleotide was
about 80% of the control while sense oligonucleotide had no
effect on cell survival (52). These results indicate that (1)
AƒÀ induces net synthesis of TPKI and (2) TPKI thus
expressed acts to kill the cell. When actinomycin D or
cycloheximide was added to the medium containing A13 to
inhibit protein biosynthesis, cell death was prevented.
Thus, it was suggested that rat hippocampal neurons
succumb to programmed cell death triggered by AƒÀ and
that TPKI participates in this process in some way.
It is clear that induction of TPKI phosphorylates tau on
the one hand and kills the cell on the other. However, an
anti-phosphopeptide antibody such as anti-PS396 stained
the cell body uniformly, not just special structures such as
NFT. Nevertheless, the cells died. Thus, PHF formation is
not requisite for cell death, but phosphorylation of tau
protein itself results in cell death. Tau protein is a kind of
microtubule-associated protein, forming cross bridges
between microtubule molecules and stabilizing them. Thus,
phosphorylation near or at the three or four repeats
sequence inhibits tau functions by disfavoring binding of
tau with tubulin, which results in disruption of axonal
transportation and finally cell death.
Inactivation of pyruvate dehydrogenase by TPKI/
GSK3ƒÀ
The phosphorylation of tau protein may lead to cell death
via some indirect way such as destabilization of micro
tubules. On the other hand, since TPKI is an isoform of
GSK, it is conceivable that TPKI might interact with some
key enzyme of the glycolytic pathway. Thus, we searched
for other substrates of TPKI using a yeast two-hybrid
system. We constructed the fusion protein plasmid be
tween TPKI and GAL4 DNA-binding domain. This hybrid
protein was employed to screen a human brain cDNA
library tagged by GAL4 transcriptional activation domain.
By screening 4 million transformants, we obtained clones
encoding pyruvate dehydrogenase (PDH). Then we ex
amined whether TPKI could phosphorylate PDH (55). As
expected, incubation of porcine heart PDH complex with
purified TPKI resulted in ATP-dependent phosphorylation
of PDH. It is quite striking that, in parallel with this
phosphorylation, the activity of PDH decreased. Other
kinases, including TPKII, failed to inactivate PDH.
Since these results were obtained in an in vitro system we
examined the situation in an in vivo system using primary
cultures of rat hippocampal neurons, as used in the above
experiments. Since PDH exists inside mitochondria we first
confirmed that TPKI is present in mitochondria, both
biochemically and morphologically (56). On treatment with
AƒÀ, TPKI activity in mitochondria increased rapidly,
reached the maximum at 6 h after AƒÀ exposure and then
leveled off. PDH was inactivated in inverse proportion to
the AƒÀ-induced TPKI activity (Fig. 3A). After 12h, the
remaining activity of PDH was only 20% of the control.
When radioactive orthophosphate was present in the incu
bation mixture, a 6h AƒÀ-treatment resulted a 2-3-fold
increase of radioactive phosphate in PDH. Moreover, when
TPKI synthesis was blocked by the antisense oligonucleo
tide as described above, PDH activity showed a 2-3-fold
increase, corresponding to the reduction in the amount of
TPKI by the antisense oligonucleotide (55). Inactivation of
PDH should result a reduction of in the level of acetyl-CoA,
which is a key substance for ATP synthesis through the
TCA cycle. The level of ATP in AƒÀ-treated cells was indeed
remarkably lowered.
Fig. 3. Activation of TPKI/GSK3ƒÀ, inactiva
tion of PDH, and inhibition of Ach synthesis in
septum cells cultured with amyloid 8. (A)
Dependency of TPKI activation and PDH inactiva
tion on the concentration of AƒÀ (1-42). The
activity was measured after 12h incubation with
various concentrations of AƒÀ (1-42) as shown on
the ordinate. (B) Effects of AƒÀ (1-42) and AƒÀ (1
40) on the suppression of Ach synthesis. Intracel
lular level of Ach was determined by HPLC with
an electrochemical detector system after 12h
culture with AƒÀ.
Vol. 121, No. 2, 1997
186 K. Imahori and T. Uchida
The above results are extremely significant since they
can explain many phenomena observed in AD patients.
1) Since PDH complex is essential to energy formation,
especially in brain, where energy is supplied by glu
cose, abnormal activation of TPKI will severely reduce
brain energy, as reported for AD brain.
2) In AD brain, a 44% reduction in cerebral metabolic rate
of glucose and a fourfold increase of lactate production
were observed, though the cerebral blood flow and
cerebral metabolic rate of oxygen were found to be
unaltered (57, 58). Those results can be well under
stood if PDH activity is blocked.
3) In postmortem studies, PDH activity was found to be
greatly decreased in AD brain (59-61).
These considerations lead to the following hypothesis for
AD pathogenesis in terms of the pathological function of
TPKI. AƒÀ interacts with neurons and activates TPKI. The
activated TPKI causes extensive phosphorylation of tau
and destabilizes microtubules, resulting in impaired axonal
transport and neuronal death. On the other hand, the
activated TPKI in mitochondria will phosphorylate and
inactivate PDH, resulting in dysfunction of glucose metabo
lism, which also contributes to neuronal death through
energy failure.
Inhibition of acetylcholine synthesis by TPKI
Acetyl-CoA is also important for acetylcholine (Ach)
production, and in cholinergic neurons, the inactivation of
PDH will cause a reduction of Ach synthesis, which will
impair release of Ach and synapse-forming ability. Thus,
we examined the relation between PDH inactivation and
Ach synthesis, using primary cultures of cholinergic neu
rons such as rat septum. As expected, Ach level in the
cultured cells declined to 14% relative to the control after
AƒÀ treatment for 24 h. Under this condition AƒÀ did not
affect choline acetyltransferase or choline esterase activity
in cultured rat septum, indicating that the decreased level
of acetyl-CoA consequent upon inactivation of PDH caused
the reduction of Ach synthesis.
In this system, aggregation of AƒÀ to a fibrillary state was
not necessary and a concentration of 10 nM was enough to
cause a significant increase in TPKI activity and a signifi
cant reduction in the intracellular Ach level, when AƒÀ (1
42) was used. On the other hand, AƒÀ (1-40) was completely
ineffective at this concentration (62) (Fig. 3B). AƒÀ (1-42)
was not toxic for the cells under this condition. Soluble AƒÀ
appears specifically to activate mitochondrial TPKI and to
inactivate PDH, without accelerating tau phosphorylation.
It is reported that, in AD brain, there is a severe selective
loss of cholinergic neurons, mostly due to the decline of Ach
level (59, 63). Considering that a specific increase in
soluble AƒÀ (1-42) occurs in AD brain before amyloid
plaque formation, soluble AƒÀ (1-42) may act as an acti
vator of TPKI in the pathogenesis of AD.
Conclusion
We set out to identify the enzyme responsible for the
hyperphosphorylation of tau protein, which is believed to
trigger the formation of PHF structure. We purified two
candidate enzymes, tau protein kinases I and II, which could
convert tau protein into a PHF-like state. The sites phos
phorylated by these kinases were confirmed to be among
the phosphorylated sites of PHF. Although TPKI and II
have different substrate specificity, they operate in a
concerted manner. Unless stripped tau protein has been
first phosphorylated by TPKII, TPKI cannot react with it.
This suggested that TPKII behaves as regulator of TPKI. In
addition TPKII itself is a kind of regulatory enzyme. TPKII
consists of two subunits and the catalytic subunit (30kDa)
is inactive in the absence of the other subunit (23kDa).
TPKI and II cannot account for all the phosphorylated
sites of PHF. Nevertheless these kinases seem to play a
crucial role in PHF formation. First, although about 19 sites
are phosphorylated in PHF, they are not equally phos
phorylated. Among them, about 10 sites are regarded as
major phosphorylation sites. TPKI and II can act at most of
them. In addition, one of the major sites, Ser413, could be
phosphorylated only by TPKI, so far as tested.
Involvement of TPKs in NFT or PHF formation was
confirmed by immunohistochemistry. Antibody raised
against an antigen-peptide containing the fingerprint amino
acid in the center stained NFT very strongly. In addition,
antibody raised against TPKI stained pyramidal cells in the
CA1 subfield of AD brain, but scarcely stained those of the
normal control. These facts indicate that TPKI is up
regulated in AD brain. Since similar results were obtained
for CDK5, it is conceivable that TPKII is also up-regulated
in AD brain. If TPKs were activated, there should be some
factor in AD brain which induces or activates TPKs. We
consider that AƒÀ is a likely candidate. Indeed, when
hippocampal cells were cultured with AƒÀ, the activity of
TPKI increased to more than two times the control, which
in turn resulted in phosphorylation of tau proteins.
Since tau protein binds to and stabilizes microtubules its
moderate phosphorylation might be necessary to make
microtubules flexible, as required for plasticity. This may
explain the physiological function of TPKs, but hyperphos
phorylation of tau would result in neuronal pathogenesis by
converting tau protein into PHF-like structure.
TPKI phosphorylates not only tau proteins, but also
pyruvate dehydrogenase, which results in its inactivation.
Since PDH is a key enzyme in the glycolytic pathway its
inactivation will have many serious consequences. It will
result in failure of the TCA cycle and of ATP production,
which, of course, is very harmful to the cells. Accumulation
of lactate will occur, resulting in acidosis, as is actually seen
in AD brain. However the most serious effect will be the
inhibition of acetylcholine synthesis. This would affect
cholinergic neurons, and might easily lead to loss of
memory and cognitive ability, which is characteristic of
AD.
In summary, TPKs, which were isolated and character.
ized as the mediators of the hyperphosphorylation of tau
protein accounted for not only PHF formation, at least in
part, but also cell death, which characterizes AD brain. In
addition, our finding that TPKI can inactivate PDH by
phosphorylation could also explain the poor energy metabo
lism, accumulation of lactate and shortage of acetylcholine,
which are well known symptoms of AD. Thus, TPKs may be
the key to the pathogenesis of AD from the biochemical
point of view.
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