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(Received for publication, May 29, 1997, and in revised form, July 14, 1997)
From the ABSTRACT Lithium is one of the most widely used drugs for
treating bipolar (manic-depressive) disorder. Despite its efficacy, the
molecular mechanism underlying its action has not been elucidated. One
recent study has proposed that lithium inhibits glycogen synthase
kinase-3 and thereby affects multiple cellular functions. Because
glycogen synthase kinase-3 regulates the phosphorylation of tau
(microtubule-binding protein that forms paired helical filaments in
neurons of the Alzheimer's disease brain), we hypothesized that
lithium could affect tau phosphorylation by inhibiting glycogen
synthase kinase-3. Using cultured human NT2N neurons, we demonstrate
that lithium reduces the phosphorylation of tau, enhances the binding
of tau to microtubules, and promotes microtubule assembly through
direct and reversible inhibition of glycogen synthase kinase-3. These results provide new insights into how lithium mediates its effects in
the central nervous system, and these findings could be exploited to
develop a novel intervention for Alzheimer's disease.
INTRODUCTION Paired helical filaments are the major building blocks of
neurofibrillary lesions in Alzheimer's disease brains. Paired helical filaments are composed of hyperphosphorylated central nervous system
tau protein (1-3). Predominantly expressed in axons, tau is a group of
microtubule-associated proteins that normally promote and stabilize the
assembly of microtubules (4-6). Paired helical filament-tau differs
from normal tau by its abnormal hyperphosphorylation, which results in
the decreased tau binding to microtubules. It has been shown that
paired helical filament-tau does not bind to microtubules unless it is
enzymatically dephosphorylated (7, 8). This decreased affinity of
paired helical filament-tau for microtubules, coupled with the reduced
amount of normal tau, may lead to the destabilization of microtubules
and result in impairment of axonal transport, neuronal degeneration,
and the aggregation of paired helical filaments. Therefore the
hyperphosphorylation of tau is believed to be a key event in the
pathogenesis of Alzheimer's disease.
The phosphorylation of tau is regulated by kinases and phosphatases. We
have shown previously that glycogen synthase kinase-3 (GSK-3),1 a serine/threonine
protein kinase, plays an important role in the regulation of tau
phosphorylation (9, 10). When overexpressed in cultured human neurons,
GSK-3 induces an Alzheimer's disease-like hyperphosphorylation of tau.
When GSK-3 activity is down-regulated by insulin or insulin-like
growth factor I, the phosphorylation state of tau is decreased
(10).
GSK-3 was originally identified as a kinase that phosphorylates and
inactivates glycogen synthase, which catalyzes a regulated step in
insulin-mediated glycogen synthesis (11, 12). Insulin induces a
phosphorylation-dependent down-regulation of GSK-3, which
leads to the activation of glycogen synthase and therefore increased
glycogen synthesis.
GSK-3 was later found to have many other cellular functions. For
example, the homologs of GSK-3 play a central role in the development
of diverse organisms including Drosophila,
Dictyostelium, and Xenopus (13-18). During
dorso-ventral axis formation in Xenopus embryogenesis,
inhibition of GSK-3 can lead to increased dorsal tissue (17).
Interestingly, lithium, a drug that has been widely used for the
treatment of bipolar (manic-depressive) disorder, induces similar
effect on Xenopus development, i.e. causing an expansion of the dorsal mesoderm leading to duplication of the dorsal
axis (19, 20). Indeed, a recent study showed that lithium directly
inhibits GSK-3, suggesting that this may be a mechanism whereby lithium
exerts its effect on cell fate determination as well as other cellular
functions (21).
Additionally, lithium has been reported to activate glycogen synthase
and stimulate glycogen synthesis in rat hepatocytes (22). This has also
been proposed (21) to be a result of the inhibition of GSK-3 by lithium
because the inhibition of GSK-3 abrogates the
phosphorylation-dependent inactivation of glycogen synthase, which in turn increases glycogen synthesis.
Despite the remarkable efficacy of lithium in treating bipolar
disorder, the molecular mechanisms underlying its therapeutic actions
have not been fully elucidated. Previous studies have shown that
lithium inhibits the phosphorylation of the mid sized neurofilament
subunit and alters cytoskeletal organization in growing chick sensory
neurons (23, 24). These studies suggest that lithium may exert its
effect on the central nervous system by affecting the neuronal
cytoskeleton. As a neuronal microtubule-associated protein, tau is a
substrate of GSK-3, and the phosphorylation of tau by GSK-3 could be
directly inhibited by lithium and microtubule assembly thereby
affected.
To test this hypothesis, we examined the effects of lithium on tau
phosphorylation and microtubule assembly in cultured NT2N neurons,
which are derived from a human embryonal carcinoma cell line (NTera2/D1
or NT2) following retinoic acid treatment (25, 26). We demonstrate that
lithium reduces the phosphorylation state of tau by a direct and
reversible inhibition of GSK-3 and that this results in an increase in
the binding of tau to microtubules, as well as an augmentation of
microtubule assembly in NT2N neurons.
EXPERIMENTAL PROCEDURES [ To generate NT2N neurons, a human embryonal
carcinoma cell line (NTera2/D1, or NT2) was grown and maintained as
described (26). Briefly, NT2 cells were treated with retinoic acid for 5 weeks and then replated at reduced density. After 10 days,
neuron-like NT2N cells were mechanically and enzymatically separated
from the parent NT2 cells and replated at a density of 1.2 × 106/well on six-well plates previously coated with
poly-D-lysine (10 µg/ml) and Matrigel. The NT2N neurons
were maintained for up to 4-6 weeks in Dulbecco's modified Eagle's
medium with high glucose (DMEM-HG), supplemented with 5% fetal bovine
serum, penicillin/streptomycin, and mitotic inhibitors (1 µM cytosine arabinoside, 10 µM
fluorodeoxyuridine, and 10 µM uridine). 2-4-week-old
NT2N neurons were used for experiments, and each experiment was
repeated at least three times.
Two GSK-3 To determine the effects of
lithium on tau phosphorylation, NT2N neurons were treated with 0-25
mM LiCl for 2 h before being harvested. To examine the
phosphorylation sites in tau affected by GSK-3 and lithium, NT2N
neurons were either infected with GSK-3 NT2N neurons
were washed once with phosphate-buffered saline and lysed in ice-cold
high salt RAB buffer (0.1 M Mes, 0.5 mM MgSO4, 1 mM EGTA, 2 mM
dithiothreitol, and 0.75 M NaCl, pH 6.8) supplemented with
0.1% Triton X-100 and a mixture of protease inhibitors. Cell lysates
were incubated on ice for 10 min, sonicated, and centrifuged for 20 min
at 50,000 × g, 4 °C. The supernatants were
collected and protein concentrations determined using the bicinchoninic
acid method (Pierce). An equal amount of total protein was resolved on
10% SDS-PAGE. Immunoblotting was performed with a panel of site and
phosphorylation-sensitive anti-tau antibodies. The blots were developed
by the enhanced chemiluminescence or 3,3 To determine
the kinase activities of overexpressed GSK-3 NT2N neurons were treated with 10 mM LiCl overnight before they were washed once with
37 °C phosphate-buffered saline and lysed in 37 °C RAB buffer
(0.1 M Mes, 0.5 mM MgSO4, 1 mM EGTA, and 2 mM dithiothreitol, pH 6.8)
supplemented with 0.1% Triton X-100, 20 µM Taxol, 2 mM GTP, and a mixture of protease inhibitors. Cell lysates
were homogenized with 20 strokes in a Dounce homogenizer and
centrifuged for 20 min at 50,000 × g, 25 °C. The
protein concentrations of the soluble fractions were adjusted to be the
same, and the cytoskeletal fractions were resuspended in ice-cold RAB
buffer of the same volume as the corresponding soluble fractions. To enrich tau, an equal fraction of the samples was boiled and clarified by centrifugation. An equal volume was resolved on 10% SDS-PAGE for
immunoblot analysis with T14/46 for tau. For determining the distribution of T3P is rabbit polyclonal anti-tau antiserum. All
the other anti-tau antibodies are mouse MAbs. T14 and T46 are
phosphorylation-independent (27, 28); T1 is
non-phosphorylation-dependent (dephosphorylated residues
189-207, according to the numbering system of the largest central
nervous system tau isoform) (4, 29); PHF1 (phosphorylated serine
396/404) (2, 7, 31-33), PHF6 (phosphorylated threonine 231) (34),
PHF13 (phosphorylated serine 396) (34), T3P (phosphorylated serine 396)
(35), AT8 (phosphorylated serine 202 and threonine 205), AT270
(phosphorylated threonine 181) (36, 37), and 12E8 (phosphorylated
serine 262) (38) are phosphorylation-dependent. T1 was
obtained from Dr. L. Binder, PHF1 from Drs. P. Davis and S. G.
Greenberg, 12E8 from Dr. P. Seubert, and AT8 and AT270 from Innogenetics. The rabbit polyclonal anti-c-Myc was raised against synthetic peptide EQKLISEEDL. The mouse MAb to GSK-3 was purchased from
Transduction Laboratories. The rabbit polyclonal
anti-detyrosinated-tubulin (Glu-tubulin) was obtained from Drs. G. G.
Gundersen and J. C. Bulinski (62). The mouse MAb to RESULTS To examine the effects of
lithium on tau phosphorylation in neuronal cells, NT2N neurons, which
endogenously express tau, were treated with 0, 0.5, 1, 5, 10, or 25 mM LiCl. After 2 h of treatment, cells were lysed and
an equal amount of total protein was resolved on SDS-PAGE for
immunoblot analysis with a panel of anti-tau antibodies. As shown in
Fig. 1, with increasing concentrations of
lithium, T1 immunoreactivity increased and PHF1 immunoreactivity decreased, indicating a reduction of tau phosphorylation. This was
accompanied by an increase in electrophoretic mobility of tau, detected
by T14/46. The quantitative data on T1 and PHF1 immunoreactivities were
normalized to the levels of T14/46 immunoreactivity and are plotted in
Fig. 1B. These results demonstrate that lithium decreases
the phosphorylation of tau in a dose-dependent manner in
the NT2N neurons.
To test whether the effect of lithium on tau
phosphorylation is a result of the inhibition of GSK-3, we first
demonstrated that lithium directly inhibits GSK-3 in NT2N neurons. We
overexpressed WT and a constitutively active mutant (S9A) form of
GSK-3
We also examined the in vivo effects of lithium on tau
phosphorylation in NT2N neurons that overexpressed GSK-3 These experiments indicate that lithium reduces tau phosphorylation by
direct inhibition of GSK-3. The serine 9 residue in GSK-3 To examine the phosphorylation sites
affected by lithium and GSK-3, we treated NT2N neurons with 10 mM LiCl for 2 h or infected the cells with
GSK-3
The phosphorylation status of tau
depends on a balance between kinase and phosphatase activities. A
decrease in kinase activity or an increase in phosphatase activity, or
both, could result in reduced tau phosphorylation. To test whether or
not the effect of lithium on tau phosphorylation requires the
activation of a known phosphatase, we treated NT2N neurons with lithium
in the presence of 0-50 nM calyculin A, a potent inhibitor
of protein phosphatases 1 and 2A (42, 43). By inhibiting these
phosphatases, calyculin A induced a decrease in T1 immunoreactivity in
a dose-dependent manner in cells not treated with lithium
(Fig. 4A). Despite this inhibition of phosphatases by calyculin A, 10 mM LiCl
strongly increased T1 immunoreactivity (Fig. 4A), suggesting
that the effect of lithium on tau phosphorylation is not dependent on
the activation of protein phosphatase 1 or 2A.
We also showed that the inhibition of phosphatases by calyculin A and
the overexpression of GSK-3 had additive effects in inducing
hyperphosphorylation of tau in NT2N neurons. As shown in Fig.
4B, calyculin A induced a dose-dependent
decrease in T1 immunoreactivity in cells infected with LacZ/SFV. A
stronger decrease in T1 was observed when cells infected with
GSK-3 To study further the mechanism of the inhibition of
GSK-3 by lithium, we determined whether the effects of lithium on tau phosphorylation and GSK-3 are reversible. NT2N neurons were treated with 10 mM LiCl for 2 h and subsequently washed and
incubated with lithium-free medium for another 0.5 or 2 h. As
controls, the cells were treated with lithium for 2, 2.5, or 4 h.
Tau phosphorylation was detected by immunoblot analysis with T14/46 and
T1. As shown earlier, lithium inhibits the phosphorylation of tau after
2-4 h of incubation as indicated by an increase in T1
immunoreactivity. This effect was reversed rapidly by the removal of
lithium, since T1 immunoreactivity resumed to its control levels (Fig.
5A). We also demonstrated in
an in vitro kinase assay that the inhibition of GSK-3
activity by lithium is reversible (Fig. 5B). When the GSK-3
kinase assay was performed in the presence of lithium after the
immunoprecipitated GSK-3 was incubated with 10 mM LiCl for 20 min (LiCl+, Wash
The phosphorylation status of tau affects
its affinity for microtubules, and this also may alter the stability of
microtubules (7, 8). We therefore examined whether lithium treatment affects the microtubule binding of tau and microtubule assembly in NT2N
neurons. After overnight treatment by 10 mM LiCl, NT2N neurons were lysed, and soluble (S) and pelletable
cytoskeletal (P) fractions were obtained from the cell
lysates. The tau protein in the cytoskeletal fraction (bound to
microtubules) and in the soluble fraction (unbound to microtubules) was
determined by immunoblotting analysis with T14/46 (Fig.
6A). Shown in Fig.
6B, the ratio of microtubule-bound tau to soluble tau was
increased significantly by lithium treatment. This promotion of tau
binding to microtubules is likely a result of the lithium-induced
decrease in tau phosphorylation.
We also examined the effects of lithium on microtubule assembly by
immunoblotting with antibodies against DISCUSSION Lithium has been used widely for decades to treat bipolar
(manic-depressive) disorder. Despite its remarkable efficacy, the molecular mechanisms underlying its therapeutic actions have not been
elucidated fully. It has been proposed that the inhibition of GSK-3 by
lithium could explain its mechanism of action (21), but this has not
been demonstrated in nerve cells. In the current study, we demonstrated
in neuronal NT2N cells that lithium treatment prevents the
phosphorylation of tau through direct and reversible inhibition of
GSK-3. This provides another clue as to how lithium elicits its effects
on the central nervous system.
One other recent study conducted in non-neuronal COS1 cells transiently
transfected with GSK-3 and tau has shown that lithium treatment
resulted in a decrease in tau immunoreactivity detected by the MAb AT8
(phosphorylated serine 202 and threonine 205) (47). However, this study
did not examine other phosphorylation sites of tau, and the
non-neuronal nature of COS1 cells limits its relevance to the neurons
of the central nervous system. The NT2N neuronal culture system was
used to conduct the current study. We demonstrated that apart from the
AT8 site, lithium affects a series of other sites phosphorylated by
GSK-3, including serine 202/threonine 205, serines 396/404, threonine
181, and threonine 231. The 12E8 site (phosphorylated serine 262, not a
proline-directed serine site), on the other hand, is not regulated by
GSK-3 and not affected by lithium.
We showed previously that insulin and insulin-like growth factor I have
effects similar to those of lithium on tau phosphorylation and the
binding of tau to microtubules in NT2N neurons (10). These effects are
also mediated through the inhibition of GSK-3, although by a different
mechanism that involves signal transduction and
phosphorylation-dependent inactivation of GSK-3. Therefore, lithium is not only an insulin mimic as a stimulator of glycogen synthesis in hepatocytes but also as a regulator of tau phosphorylation in neuronal cells.
Lithium also has been reported to inhibit the phosphorylation of other
cytoskeletal proteins, e.g. in cultured chick sensory neurons, the newly synthesized mid sized neurofilament subunit (23);
and in PC12 cells, chartins, another group of microtubule-associated proteins (48). Although the mechanism underlying these effects is
currently not known, they may be mediated by the inhibition of
GSK-3.
The effects of lithium on microtubule assembly are intriguing. In
cultured chick sensory neurons, lithium blocks initial neurite outgrowth. But, if neurites are allowed to begin extending before exposure to lithium, further outgrowth ceases, and neurites appear frozen at the length achieved before treatment (24, 48). Moreover, lithium treatment does not induce cell death, and the cytoskeletal alterations are completely reversible (24). Lithium has also been
reported to induce rapid degradation of newly synthesized tubulin in
neurons, resulting in a decrease in the fraction of tubulin present in
the unassembled form. However the assembled microtubules are not
affected by this degradation. The dynamic equilibrium of tubulin
polymerization is actually shifted toward assembly, since the
proportion of tubulin present in the assembled microtubule fraction is
increased (49). Our results are consistent with these findings, and
they suggest that the decreased tau phosphorylation and increased tau
binding to microtubules may contribute to this stabilizing effect of
lithium on assembled microtubules. Because neurite elongation is
primarily a function of microtubule dynamics, this microtubule
stabilizing effect, coupled with the increased degradation of nascent
tubulin, may halt further neurite growth and in the meantime preserve
the extant neurites.
One reason why the molecular mechanisms underlying the therapeutic
effects of lithium have been difficult to characterize is that lithium
could affect multiple cellular targets. It has been proposed that
lithium targets key components of signal transduction pathways (50).
One of the prevailing hypotheses is the inositol depletion hypothesis,
which is based on the observation that lithium inhibits inositol
monophosphatase (51, 52). As a result of this inhibition, the cellular
source of inositol could be depleted, and cells could therefore become
unable to generate inositol 1,4,5-trisphosphate. Thus, the inositol
1,4,5-trisphosphate-dependent responses to extracellular stimulation
would be blocked. However, this hypothesis cannot fully explain the
effect of lithium on glycogen synthesis or on cell fate and
development, because treatment with L690,330, a compound that is
approximately 1,000-fold more potent than lithium in inhibiting
inositol monophosphatase (53, 54), does not parallel the dramatic
teratogenic effects of lithium (21). Furthermore, the
membrane-permeable analog of L690,330, termed L690,488, does not affect
tau phosphorylation in NT2N
neurons,2 and thus inhibition
of inositol monophosphatase does not appear to explain the effects of
lithium on tau phosphorylation. The observations that lithium directly
inhibits GSK-3 and decreases tau phosphorylation help to explain these
effects and provide new insights into the pathogenesis and treatment of
bipolar disease.
The hyperphosphorylation of tau and formation of paired helical
filaments are believed to be critical events in the pathogenesis of
Alzheimer's disease and a group of other neurodegenerative diseases
(1, 7, 55-58). The extent and distribution of neurofibrillary lesions
correlate reliably with the degree of dementia in Alzheimer's disease
patients (59-61). We have shown previously that overexpression of
GSK-3 can induce an Alzheimer's disease-like phosphorylation of tau in
neurons (10). It is likely that GSK-3 plays an important role in the
normal and abnormal regulation of tau phosphorylation. By inhibiting
GSK-3 and causing a decrease in tau phosphorylation, it is possible
that lithium could be used as a therapeutic agent to block the
hyperphosphorylation of tau and slow the progression of Alzheimer's
disease. Future studies on the exact mechanism of this inhibition will
facilitate the design of new GSK-3 inhibitors that could be used for
the treatment of Alzheimer's disease and bipolar disease.
FOOTNOTES ACKNOWLEDGEMENTS We thank Drs. M. Goedert, X. He, and H. Varmus for providing GSK-3 cDNAs; and Drs. L. Binder, P. Davis,
S. G. Greenberg, P. Seubert, G. G. Gundersen, and J. C. Bulinski for
the kind gifts of anti-tau and anti-tubulin antibodies. We also thank
Dr. J. Q. Trojanowski for critically reading the manuscript.
REFERENCES
Volume 272, Number 40,
Issue of October 3, 1997
pp. 25326-25332
©1997 by The American Society for Biochemistry and Molecular Biology, Inc.
,,¶
Department of Pharmacology,
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
-32P]ATP and
125I-labeled secondary antibodies were purchased from NEN
Life Science Products and the Semliki Forest virus (SFV) gene
expression system from Life Technologies, Inc. Taxol was obtained from
Dr. V. Narayanan of NCI. Other chemicals were purchased from Sigma.
GSK-3
S9A cDNA and the cDNA construct to make human recombinant tau were obtained from Dr. M. Goedert. GSK-3 WT cDNA was from Drs. X. He and H. Varmus.
in NT2N Neurons Using the SFV Gene
Expression System
/SFV viral constructs were used.
GSK-3 WT is the wild type GSK-3; and GSK-3 S9A is a
constitutively active form of GSK-3, in which the serine 9 residue
was mutated to alanine. A 10-amino acid c-Myc tag (EQKLISEEDL) was
introduced at the COOH termini of both constructs. Another SFV
construct expressing -galactosidase (LacZ) was used as a control. To
overexpress GSK-3 in NT2N neurons, cells were washed once with
DMEM-HG without supplements, and viral stocks diluted in DMEM-HG
(multiplicity of infection = 10) were applied to the cells. After
1 h, the medium containing viruses was replaced by DMEM-HG with
5% fetal bovine serum. The infected NT2N neurons were then incubated
overnight (18 h) before they were harvested. For experiments on the
effects of lithium or calyculin A in infected cells, cells were treated
with 10 mM LiCl for 2 h or with 0-50 nM
calyculin A for 30 min after overnight infection.
/SFV overnight or treated
with 10 mM LiCl for 2 h. To test whether calyculin A
blocks the effects of lithium on tau phosphorylation, cells were
treated with or without 10 mM LiCl for 30 min in the presence of 0-50 nM calyculin A. To determine whether
lithium reversibly decreases tau phosphorylation, NT2N neurons were
treated with 10 mM LiCl for 2 h and subsequently
washed three times with lithium-free medium and incubated for another
0.5 or 2 h. As controls, cells were treated with lithium for 2, 2.5, and 4 h without wash.
-diaminobenzidine method.
Alternatively, for the quantitation of the relative levels of tau
protein, 125I-labeled goat anti-mouse IgG was used as
secondary antibody, and the blots were exposed to PhosphorImager
plates. 10 µg of total protein was loaded for the detection of tau on
immunoblots using the antibodies T14/46, 25 µg for the antibodies T1,
PHF1, T3P, and PHF13, and 50 µg for the antibodies AT8, AT270, PHF6, and 12E8.
, infected NT2N neurons
were lysed in ice-cold lysis buffer (20 mM Pipes, pH 7.0, 10 mM NaCl, 0.5% Nonidet P-40, 0.05% -mercaptoethanol, 5 mM EGTA, 50 mM NaF, 1 mM
Na3VO4, and 1 µM microcystin)
supplemented with a mixture of protease inhibitors. Overexpressed
GSK-3 was immunoprecipitated with a rabbit polyclonal anti-c-Myc
antiserum. The immunoprecipitants were washed twice with lysis buffer
and twice with GSK-3 kinase buffer (20 mM Hepes, pH 7.2, 10 mM MgCl2, 10 mM MnCl2,
1 mM dithiothreitol, 0.2 mM EGTA, and 5 µM ATP). GSK-3 kinase assay was performed at 30 °C for
15 min in 20 µl of GSK-3 kinase buffer supplemented with 4 µg of
recombinant tau and 5 µCi of [-32P]ATP for each
reaction. The reaction was terminated by adding 5 × sample buffer
and boiling for 5 min. The entire reaction was then resolved on 10%
SDS-PAGE, transferred to nitrocellulose membrane, and exposed to a
PhosphorImager plate. GSK-3 kinase activity was represented by the
level of 32P incorporation into tau. The amount of
immunoprecipitated GSK-3 present in each reaction was determined
subsequently by immunoblot analysis with a monoclonal anti-GSK-3
antibody on the same nitrocellulose membrane and used to normalize the
corresponding relative GSK-3 activity. To determine the effect of
lithium on GSK-3 activity, the kinase reactions were performed in the
presence of 0-10 mM LiCl. To determine the reversibility
of the lithium effect, the immunoprecipitated GSK-3 was incubated in
GSK-3 kinase buffer with 10 mM LiCl for 20 min, washed
three times with lithium-free GSK-3 kinase buffer, and then used to
perform kinase assay. As controls, GSK-3 kinase reactions were either
performed in the presence of lithium without wash after 20 min of
lithium incubation, or without lithium incubation or wash.
-tubulin subunits, an equal volume of the unboiled samples was used to perform immunoblot analysis with a mouse monoclonal antibody (MAb) to -tubulin, a rat MAb to Tyr-tubulin, and a rabbit polyclonal anti-Glu-tubulin antiserum. The blots were incubated with
125I-labeled secondary antibodies and exposed to
PhosphorImager plates. Quantitation was performed with the ImageQuant
software (Molecular Dynamics). The ratio of microtubule-bound tau (in
the cytoskeletal fraction) to soluble tau was determined by comparing
the T14/46 immunoreactivities. The ratios of polymerized to soluble
-tubulin, Tyr-tubulin, or Glu-tubulin were determined and plotted in
a similar manner.
-tubulin was
purchased from Amersham (63), and the rat MAb to tyrosinated-tubulin
(Tyr-tubulin) was from Harlen Sera-Lab (30).
Fig. 1.
Lithium reduces tau phosphorylation in NT2N
neurons in a dose-dependent manner. NT2N neurons were
treated with 0-25 mM LiCl for 2 h and then harvested
for immunoblot analysis of tau phosphorylation with T14/46, T1, and
PHF1. Panel A, with increasing concentrations of lithium,
the level of tau phosphorylation was reduced, as indicated by the
increase in T1 and decrease in PHF1 immunoreactivity. This was
accompanied by an increased electrophoretic mobility of tau, detected
by T14/46. Panel B, the T1 and PHF1 immunoreactivities were
quantitated, normalized to the levels of T14/46, and plotted. T1
increased and PHF1 decreased in a dose-dependent manner,
indicating a reduction of tau phosphorylation (n = 3).
[View Larger Version of this Image (42K GIF file)]
in NT2N neurons using the SFV gene expression system. Both
GSK-3/SFV constructs have a c-Myc tag at their COOH termini, and the
overexpressed protein can be immunoprecipitated by a polyclonal
anti-c-Myc antiserum. In Fig.
2A, overexpressed GSK-3 S9A
was immunoprecipitated, and a GSK-3 kinase assay was performed in the
presence of 0-10 mM LiCl. The GSK-3 activities were
assayed by monitoring 32P incorporation into human
recombinant tau. The amount of GSK-3 present in each reaction was
determined by immunoblotting with a MAb to GSK-3 and used to normalize
the relative GSK-3 activities. As shown in Fig. 2A, lithium
directly inhibited the activity of GSK-3 S9A with an
IC50 of 3 mM. Similar results were obtained when GSK-3 WT was overexpressed in NT2N neurons (data not shown). Other monovalent cations (Na+, K+,
Cs+, and NH4+) did not have
a similar effect (data not shown).
Fig. 2.
Lithium reduces tau phosphorylation by direct
inhibition of GSK-3. Panel A, NT2N neurons were infected
with GSK-3 S9A/SFV overnight, and overexpressed GSK-3 was
immunoprecipitated with a polyclonal anti-c-Myc antiserum. The GSK-3
kinase assay was performed in the presence of 0-10 mM
LiCl. GSK-3 activity was monitored by 32P incorporation
into human recombinant tau and normalized by the amount of GSK-3 in
each reaction. Lithium inhibits GSK-3 with a Ki of 3 mM (n = 3) in a dose-dependent
manner. Panel B, NT2N neurons were infected with GSK-3
S9A/SFV, GSK-3 WT/SFV, or LacZ/SFV overnight and treated with or
without 10 mM LiCl for 2 h. Overexpression of GSK-3
(S9A and WT) increased tau phosphorylation, as
indicated by the decrease of T1 and increase of PHF1. Lithium treatment
abolished this increase in tau phosphorylation in cells that expressed
GSK-3 S9A and in cells that expressed GSK-3 WT (S9A+Li
and WT+Li).
[View Larger Version of this Image (36K GIF file)]
WT or
GSK-3 S9A. As shown in Fig. 2B, overexpression of both
GSK-3 constructs increased tau phosphorylation because T1
(dephosphorylated) immunoreactivity decreased and PHF1 (phosphorylated)
immunoreactivity increased compared with control (LacZ). When treated
with LiCl, the increased tau phosphorylation induced by overexpression
of both GSK-3 WT and GSK-3 S9A was abrogated (WT+Li
and S9A+Li), as the changes in T1 and PHF1
immunoreactivities were reversed (Fig. 2B).
is
required for the phosphorylation-dependent inactivation, and mutating this residue to alanine in GSK-3 S9A abolishes the down-regulation of its activity by insulin and other growth factors (10, 39-41). Because the effects of lithium on GSK-3 and tau phosphorylation are not affected by this mutation, the phosphorylation of serine 9 must not be involved in the inhibition of GSK-3 by lithium.
/SFV overnight. Control cells received no treatment. Cell
lysates were resolved on 10% SDS-PAGE, and immunoblot was performed
with a panel of site-specific phosphorylation-sensitive anti-tau
antibodies. The results of a typical experiment are shown in Fig.
3A. The immunoreactivities of
phosphorylated and nonphosphorylated tau were quantitated and
normalized to the levels of T14/46 (total tau), as shown in Fig.
3B. With the overexpression of GSK-3, T1 (dephosphorylated
residues 189-207) immunoreactivity decreased, whereas PHF1
(phosphorylated serine 396/404), T3P (phosphorylated serine 396), PHF13
(phosphorylated serine 396), AT8 (phosphorylated serine 202 and
threonine 205), AT270 (phosphorylated threonine 181), and PHF6
(phosphorylated threonine 231) immunoreactivities increased. Lithium
treatment had the opposite effect on the immunoreactivities of the same
antibodies (Fig. 3, A and B). However,
phosphorylation at the site recognized by 12E8 (phosphorylated serine
262) was not altered by GSK-3 or by lithium. These results demonstrate that lithium and GSK-3 affect the same sites of phosphorylation in tau.
Thus it is likely that lithium exerts its effects on tau phosphorylation through the direct inhibition of GSK-3.
Fig. 3.
Lithium and GSK-3 have opposite effects on
the same phosphorylation sites of tau in NT2N neurons. NT2N
neurons were infected with GSK-3/SFV overnight or treated with 10 mM LiCl for 2 h. Panel A, tau
phosphorylation at multiple sites was determined by immunoblot analysis
with a panel of anti-tau antibodies. The quantitated immunoreactivities
were normalized to T14/46 levels and plotted in panel B. The
expression of GSK-3 increased tau phosphorylation at sites recognized
by T1, PHF1, T3P, PHF13, AT8, AT270, and PHF 6. Lithium treatment
affected all of these sites too, but its effect was the opposite of
that of GSK-3. Neither GSK-3 nor lithium altered the phosphorylation on
the 12E8 site (phosphorylated serine 262).
[View Larger Version of this Image (51K GIF file)]
Fig. 4.
The lithium-induced decrease in tau
phosphorylation is not blocked by the inhibition of phosphatases.
Panel A, NT2N neurons were treated for 30 min with or
without 10 mM LiCl in the presence of 0-50 nM
calyculin A. T1 immunoreactivity was normalized to T14/46 and plotted
(No LiCl, No Calyculin A = 1). Calyculin A alone
induced an increase of tau phosphorylation (decrease in T1), but it did
not block the dephosphorylation of tau induced by lithium (increase in
T1). Panel B, NT2N neurons were infected with GSK-3/SFV
or LacZ/SFV overnight and treated with 0-50 nM calyculin A
for 30 min. The increase of tau phosphorylation induced by
overexpression of GSK-3 was potentiated by calyculin A (LacZ, No
Calyculin A = 1).
[View Larger Version of this Image (25K GIF file)]
/SFV were treated with calyculin A.
), GSK-3 activity
(represented by 32P incorporation into tau) was strongly
reduced compared with the control (no lithium incubation,
LiCl, Wash). When cells were washed
extensively after 20 min of lithium incubation, GSK-3 activity was
recovered (LiCl+, Wash+) (Fig. 5B).
These results indicate that lithium reduces tau phosphorylation in the
NT2N neurons by direct and reversible inhibition of GSK-3 and are in
agreement with a recent in vitro study showing that lithium
reversibly inhibits GSK-3 (47).
Fig. 5.
The effects of lithium on GSK-3 and tau
phosphorylation are reversible. Panel A, NT2N neurons were
treated with 10 mM LiCl for 2 h and subsequently
washed with lithium-free medium and incubated for another 0.5 or 2 h. As controls, cells were treated with lithium without wash for 2, 2.5, or 4 h. Lithium-induced increase in T1 immunoreactivity was
reversed rapidly after removal of lithium. Panel B,
overexpressed GSK-3 was immunoprecipitated, incubated with or without
10 mM LiCl for 20 min (LiCl+ or ). GSK-3
kinase assay was performed in the presence of lithium
(Wash) or after lithium was removed by the wash
(Wash+). Lithium inhibited GSK-3-mediated 32P
incorporation into tau, but the effect was reversed by the removal of
lithium.
[View Larger Version of this Image (55K GIF file)]
Fig. 6.
Lithium enhances tau binding to microtubules
and affects microtubule assembly. NT2N neurons were treated with
or without 10 mM LiCl overnight. Cell lysates were
separated into cytoskeletal fraction (P) and soluble
fraction (S). Panel A, tau, -tubulin (-tub), Tyr-tubulin (Tyr-tub), and Glu-tubulin
(Glu-tub) present in each fraction were determined by
immunoblot analysis. Panel B, the ratios of tau,
-tubulin, Tyr-tubulin, and Glu-tubulin in the cytoskeletal fraction
versus those in the soluble fraction (P/S) were
determined after quantitation and plotted. Lithium treatment
significantly increased tau binding to microtubules and shifted the
microtubule assembly equilibrium toward polymerization (n = 4, *p < 0.01). The distribution
of Glu-tubulin was not altered.
[View Larger Version of this Image (48K GIF file)]
-tubulin, Tyr-tubulin, and
Glu-tubulin. The assembly of microtubules by tubulin polymerization is
a dynamic equilibrium (44). When -tubulin is incorporated into
microtubules, it undergoes post-translational modifications such as
detyrosination to generate the more stable Glu-microtubules (45, 46).
However, when Glu-microtubules are depolymerized, tyrosine residues are
rapidly added back to generate Tyr-tubulin. Therefore, as shown in Fig.
6A (Control), in the normal postmitotic NT2N
neurons, more Tyr-tubulins are found in the soluble fraction (S), whereas 90% of Glu-tubulins are present as
Glu-microtubule polymers in the cytoskeletal fraction (P).
The overall total -tubulin subunits are also distributed with 70%
as microtubule polymers (Fig. 6A, Control). After
lithium treatment, the amounts of soluble -tubulin and Tyr-tubulin
decreased, but the -tubulin and Tyr-microtubule polymers remained
unchanged or slightly increased (Fig. 6A, LiCl). This resulted in a shift of the equilibrium of microtubule
polymerization favoring assembly, since the ratios of -tubulin
polymers to soluble -tubulin subunits increased significantly (Fig.
6B). However, lithium treatment did not alter the
distribution of Glu-tubulin and Glu-microtubules (Fig. 6, A
and B). These effects of lithium on microtubule assembly may
partly be attributed to the increased microtubule stability as a result
of enhanced tau binding.
¶
To whom correspondence should be addressed: Center for
Neurodegenerative Disease Research, Dept. of Pathology and Laboratory Medicine, University of Pennsylvania School of Medicine, HUP/Maloney 3, 3600 Spruce St., Philadelphia, PA 19104. Tel.: 215-662-6427; Fax:
215-349-5909; E-mail: vmylee{at}mail.med.upenn.edu.
1
The abbreviations used are: GSK-3, glycogen
synthase kinase-3; SFV, Semliki Forest virus; WT, wild type; DMEM-HG,
Dulbecco's modified Eagle's medium with high glucose; Mes,
4-morpholineethanesulfonic acid; PAGE, polyacrylamide gel
electrophoresis; Pipes, 1,4-piperazinediethanesulfonic acid; MAb,
monoclonal antibody.
2
M. Hong, unpublished data.
©1997 by The American Society for Biochemistry and Molecular Biology, Inc.
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