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The Journal of Neuroscience, October 1, 2001, 21(19):7551-7560
-Amyloid Induces Neuronal Apoptosis Via a Mechanism that
Involves the c-Jun N-Terminal Kinase Pathway and the Induction of Fas
Ligand
Yoshiyuki
Morishima1,
Yukiko
Gotoh1,
Janine
Zieg1,
Tamera
Barrett2,
Hiromichi
Takano3,
Richard
Flavell4,
Roger J.
Davis2,
Yasufumi
Shirasaki3, and
Michael E.
Greenberg1
1 Division of Neuroscience, Children's Hospital, and
Department of Neurobiology, Harvard Medical School, Boston,
Massachusetts 02115, 2 Howard Hughes Medical Institute,
Program in Molecular Medicine, Department of Biochemistry and Molecular
Biology, University of Massachusetts Medical School, Worcester,
Massachusetts 01605, 3 New Products Research Laboratories
III, Daiichi Pharmaceutical Co., Ltd., Tokyo 134-8636, Japan, and
4 Howard Hughes Medical Institute, Section of
Immunobiology, Yale University School of Medicine, New Haven,
Connecticut 06510
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ABSTRACT |
Elevated levels of 
-Amyloid (A) are present in the brains of
individuals with either the sporadic or familial form of Alzheimer's disease (AD), and the deposition of A within the senile plaques that
are a hallmark of AD is thought to be a primary cause of the cognitive
dysfunction that occurs in AD. Recent evidence suggests that A
induces neuronal apoptosis in the brain and in primary neuronal
cultures, and that this A-induced neuronal death may be responsible
in part for the cognitive decline found in AD patients. In this study
we have characterized one mechanism by which A induces neuronal
death. We found that in cortical neurons exposed to A, activated
c-Jun N-terminal kinase (JNK) is required for the phosphorylation and
activation of the c-Jun transcription factor, which in turn stimulates
the transcription of several key target genes, including the death
inducer Fas ligand. The binding of Fas ligand to its receptor Fas then
induces a cascade of events that lead to caspase activation and
ultimately cell death. By analyzing the effects of mutations in each of
the components of the JNK-c-Jun-Fas ligand-Fas pathway, we
demonstrate that this pathway plays a critical role in mediating
A-induced death of cultured neurons. These findings raise the
possibility that the JNK pathway may also contribute to A-dependent
death in AD patients.
Key words:
Alzheimer's disease; -amyloid; c-Jun N-terminal
kinase; JNK; c-Jun; Fas; Fas ligand; apoptosis; neuronal cell death
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INTRODUCTION |
Alzheimer's disease (AD) is a
neurodegenerative disorder that is characterized by senile plaques,
neurofibrillary tangles, and neuronal loss (Yankner, 1996
; Selkoe,
1999). A common feature of AD is the accumulation of -amyloid
(A), 39- to 43-amino acid peptides derived from the amyloid
precursor protein (APP). A peptides aggregate to form fibrillar
deposits that are the principal component of senile plaques. The
importance of A in the pathogenesis of AD is suggested by several
findings. Notably, mutations in APP or presenilin, two proteins that
are implicated in familial forms of AD, lead to an increase in the
amyloidogenic form of A (Selkoe, 1999). In addition, fibrillar A,
but not soluble A, is specifically toxic to cultured neurons
in vitro (Yankner et al., 1990; Yankner, 1996). The evidence
that A accumulation is a determining factor in AD makes it important
to determine the mechanism by which A induces neuronal cell death.
Recent studies have shown that in AD brains and in cultures of neurons exposed to A, the dying cells display the characteristics of apoptosis (Anderson et al., 1996; Estus et al., 1997; Stadelmann et
al., 1999). A-induced apoptosis has been suggested to involve oxidative stress and the perturbation of intracellular calcium homeostasis and to be protein synthesis-dependent (Takashima et al.,
1993; Pike et al., 1996; Yankner, 1996; Imaizumi et al., 1999; Selkoe,
1999). However, the specific intracellular signaling pathways by which
A triggers cell death are not yet well defined.
Several features of the c-Jun N-terminal kinase (JNK) pathway suggested
that this pathway might mediate A-induced apoptosis. The JNK pathway
is activated by oxidative stress, raising the possibility that A
might also activate the JNK cascade. In addition, activation of the JNK
pathway triggers the induction of gene transcription. Thus the protein
synthesis dependence of A-induced apoptosis might reflect a
requirement for JNK-dependent transcription.
Activated JNK phosphorylates and activates several transcription
factors, c-Jun, activating transcription factor 2 (ATF2), and
Elk-1 (Ip and Davis, 1998). Recent evidence suggests that JNK and its
target c-Jun play an important role in triggering neuronal apoptosis
(Xia et al., 1995; Yang et al., 1997; Herdegen et al., 1998; Luo et
al., 1998; Behrens et al., 1999; Le-Niculescu et al., 1999). The
inhibition of JNK or c-Jun activity in nerve growth factor
(NGF)-deprived PC12 cells or sympathetic neurons inhibits apoptosis.
Likewise, in JNK3
/ or c-Jun mutant
mice, hippocampal neurons are highly resistant to kainic acid-induced
cell death (Yang et al., 1997). These studies indicate that the JNK
pathway mediates NGF withdrawal- and kainic acid-induced neuronal
apoptosis. The precise mechanism by which activation of the JNK cascade
leads to apoptosis is not known. However, it was demonstrated recently
that Fas ligand transcription is activated on neurotrophic factor
withdrawal by a JNK-dependent mechanism (Le-Niculescu et al., 1999).
Once induced, Fas ligand contributes to cell death in an autocrine or
paracrine manner by activating its receptor Fas, which in turn leads to
activation of a caspase cascade and the demise of the cell.
In the present study, we show that A induces the death of cortical
neurons via the activation of c-Jun and the induction of the Fas ligand
death cascade in a JNK-dependent manner. These findings raise the
possibility that this signaling cascade may contribute to neuronal cell
death in AD patients.
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MATERIALS AND METHODS |
Materials. Antibodies were obtained from the
following sources: anti-phospho-specific JNK and anti--galactosidase
(-Gal) antibodies from Promega (Madison, WI); anti-phospho-specific
c-Jun (Ser-73) and anti-JNK3 antibodies from Upstate Biotechnology
(Lake Placid, NY); anti-JNK1 antibody from PharMingen (San Diego, CA); anti-c-Jun, anti-Fas, anti-caspase-8, and anti-Fas-associated death-domain-containing protein (FADD) antibodies from Santa Cruz Biotechnology (Santa Cruz, CA); anti-Fas ligand antibody from Transduction Laboratories (Lexington, KY); anti-TuJ1 antibody from
Babco (Richmond, CA); and anti-Fas antibody SM1/23 from Alexis. The
A peptides A25-35, A35-25, and A1-40 were obtained from Bachem (Torrance, CA). The Fas-Fc fusion protein was from R & D
Systems (Minneapolis, MN), and the caspase-8 inhibitor
IETD-fluoromethyl ketone (fmk) was from Calbiochem (La Jolla,
CA). The caspase-8 assay kit was from Clontech (Cambridge, UK). Kainic
acid, anisomycin, and potassium cyanide (KCN) were from Sigma (St.
Louis, MO). The MTS reagent
(3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium) was obtained from Promega.
Plasmid constructs. Dominant negative (DN)-SEK-1, DN-c-Jun
Flag
169, the JNK interacting protein (JIP) binding domain
(JBD) of JIP-1, cytomegalovirus (CMV)-LacZ, and
CMV-enhanced green fluorescent protein constructs used in this
study have been described previously (Xia et al., 1995; Whitmarsh et
al., 1998; Brunet et al., 1999). DN-FADD (Chinnaiyan et al., 1996) was
kindly provided by Dr. V. M. Dixit (Genentech, San Francisco, CA).
Animals. Long-Evans rats were obtained from Charles River
(Wilmington, MA) for primary neuronal cultures. The JNK3 wild-type and
knock-out mice from strain C57BL/6 were described previously (Yang et
al., 1997). gld and lpr mice from the C3H/HeJ
background and mice of strain ddY were obtained from Japan SLC.
Cell culture conditions. Primary cortical and hippocampal
neurons were cultured from embryonic day 17-18 rat fetuses or from embryonic day 16-17 mouse fetuses as described previously (Xia et al.,
1996). The neurons were plated on glass coverslips coated with
poly-D-lysine or polyethyleneimine in 24-well plates at a density of 200,000 cells per well for nontransfection experiments and
400,000 cells per well for transfection experiments. Neurons were
cultured in DMEM containing 10% calf serum for 1 d, and the medium was changed to serum-free DMEM with B27 supplement (Life Technologies, Gaithersburg, MD). The experiments were performed after
4-6 d in culture.
Treatment of neurons with A peptides. One millimolar
stock solutions of A25-35, A35-25, and A1-40 were prepared
in sterile water and stored at 
20°C. To prepare aggregated A
peptides, the same volume of PBS was added to the stock solution and
incubated at 37°C for 3-5 d. The soluble form of A1-40 was
prepared by adding PBS without subsequent incubation. Preaggregated
A peptides were added to the cultures at 25 µM and
incubated for 24-48 hr for the apoptosis assays.
Immunoblotting and immunocytochemistry techniques. For
immunoblotting, neurons treated with A were washed with cold PBS, solubilized with SDS sample buffer, and then sonicated for 10 sec. The
lysates were boiled for 5 min and centrifuged for 15 min. The
supernatants were subjected to SDS-PAGE. Equal amounts of lysate
protein were run on a 10% SDS polyacrylamide gel and transferred to
nitrocellulose membranes. Nitrocellulose blots were blocked with 3%
bovine serum albumin (BSA) in Tris-buffered saline containing 0.1%
Triton X-100 (TBST) and then incubated with primary antibodies
[anti-phospho-specific JNK, 1:5000; anti-JNK3, 1:1000; anti-JNK-1,
1:1000; anti-phospho-specific c-Jun (Ser-73), 1:5000; anti-c-Jun,
1:1000; anti-Fas ligand, 1:500; anti-Fas, 1:500; anti-caspase-8, 1:500;
anti-FADD, 1:500; or anti-TuJ1, 1:1000] in TBST containing 3% BSA.
Immunoreactivity was detected by sequential incubation of horseradish
peroxidase-conjugated secondary antibody (1:15,000; Calbiochem) and ECL
reagents (DuPont, Billerica, MA).
For -Gal staining, cells were fixed in 4% paraformaldehyde and 2%
sucrose, permeabilized with 0.1% Triton X-100, and blocked with 3%
BSA. Cells were incubated with anti--Gal antibodies (1:300) and then
incubated with the Cy3-conjugated secondary antibody (1:500, Jackson
ImmunoResearch, West Grove, PA). For Fas ligand staining, cells were
fixed in 4% paraformaldehyde and 2% sucrose and treated with cold
MeOH. Cells were incubated with anti-Fas ligand antibody (1:500),
biotin-conjugated secondary antibody (1:300; Calbiochem), and
Cy3-labeled streptavidin (1:500; Jackson ImmunoResearch). Nuclei were
stained with Hoechst 33258 (2.5 µg/ml; Sigma).
Transfection and apoptosis assay. Using the calcium
phosphate transfection method (Xia et al., 1996; Brunet et al., 1999), cortical neurons were transfected with 2-4 µg of the construct of
interest along with 0.3 µg of CMV-EGFP plasmid and 0.7 µg of CMV-LacZ plasmid. Two days after transfection, neurons were treated with A25-35 for 24 hr, or 1 d after transfection, cells were treated with A1-40 for 48 hr. The transfected neurons were fixed in
4% paraformaldehyde and 2% sucrose, and immunostaining for -Gal
was performed as described above. Nuclei were stained with Hoechst
33258. Apoptotic neurons among the -Gal-positive neurons were
counted in a blinded manner.
Cell death was determined under a fluorescence microscope after
staining with Hoechst 33258 by counting the number of cells containing
condensed or fragmented nuclei in two or three fields (100-200 cells)
per well in a blinded manner. Data are expressed as percentage of
apoptotic cells of the total cells counted. In some experiments, cell
viability was assessed by measuring the ability of cellular
dehydrogenase enzymes to reduce the MTS reagent leading to the
formation of a color formazan dye. The data are expressed as percentage
of loss of cell viability compared with the control condition.
Reverse transcriptase-coupled PCR analysis. Total RNA was
isolated from cortical neurons using the RNAzole B reagent (Tel-Test). First-strand cDNA was synthesized by extension of oligo-dT primers with
SuperScript II reverse transcriptase (Life Technologies) in a mixture
containing 3 µg of total RNA according to the manufacturer's protocol. PCR amplifications were performed in a 25 µl reaction volume containing 0.2 mM dNTPs, 1.5 mM
MgCl2, 1.8 U of ExpandTaq polymerase (Roche
Molecular Biochemicals, Indianapolis, IN), and 0.4 µM of
each primer. The PCR products were separated by 1% agarose gel
electrophoresis and visualized by ethidium bromide staining. Primers
used were as follows: rat Fas ligand sense,
5'-GCTCTGGTTGGAATGGGGTTAG-3', and antisense,
5'-ATAGACCTTGTGGCTTAGGGGC-3'; rat Fas sense,
5'-CAAGGGACTGATAGCATCTTTGAGG-3', and antisense,
5'-TCCAGATTCAGGGTCACAGGTTG-3' (Le-Niculescu et al., 1999); rat S100
sense, 5'-GGATGTCTGAGCTGGAGAAG-3', and antisense, 5'-ACTCCTGGAAGTCACACTCC-3' (Freeman et al., 1994); mouse FasL sense,
5'-CAGCAGTGCCACTTCATCTTGG-3', and antisense,
5'-TTCA-CTCCAGAGATCAGAGCGG-3'; mouse Fas sense,
5'-GAGAATTGCT-GAAGACATGACAATCC-3', and antisense, 5'-GTAGTTTTCACTC-CAGACATTGTCC-3' (Lee et al., 1999);
and mouse -actin sense, 5'-TGGATTCCTGTCGCATCCATGAAAG-3', and
antisense, 5'-CACTCC-AGAGATCAAAGCAGTTC-3' (Le-Niculescu et al.,
1999).
Caspase-8 activity assay. Fluorometric caspase-8 activity
assays were performed according to the manufacturer's manual. In brief, cell lysates were prepared at 24 hr after A25-35 or kainic acid treatment. The lysates were incubated with the fluorogenic substrate IETD-7-amino-4-trifluoromethyl coumarin at 37°C for 1 hr. Proteolytic cleavage of the substrate by caspase-8 emitted a
fluorescent signal that was measured with a fluorometer (excitation, 400 nm; and emission, 505 nm).
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RESULTS |
A activates the JNK pathway in cortical neurons
To study the mechanism of A-induced cell death, we exposed
primary cultures of rat cortical neurons to A or control peptide for
24-48 hr and assessed in a blinded manner the effect on cortical neuron survival by visually monitoring nuclear condensation and neural
process fragmentation. This approach is essentially identical to that
used previously by Yankner et al. (1990) to model AD in vitro. As described previously, the fibrillar form of A peptide (A1-40), but not the soluble form of the peptide, effectively induced the death of cortical neurons within 48 hr (Fig.
1A; Estus et al., 1997;
Imaizumi et al., 1999). In addition, 10-50 µM
concentrations of a smaller A peptide (A25-35), but not a
control peptide with the amino acid sequence inverted (A35-25),
were found to efficiently induce the death of cortical neurons within
24 hr (Fig. 1B,C). The A25-35 peptide is a
fragment of A that has previously been shown to mimic the effects of
the A1-40 (Yankner et al., 1990; Imaizumi et al., 1999).
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Figure 1.
A25-35 and A1-40 promote
neuronal cell death. A, Neurotoxicity of A1-40. Rat
primary cortical neurons were exposed to 50 µM fibrillar
(Fibril) or soluble A1-40 for 72 hr. Cells
were fixed, nuclei were stained with Hoechst 33258, and apoptotic
neurons were scored in a blinded manner as those cells that displayed
condensed or fragmented nuclei. Experiments were performed at least
three times, and data represent mean ± SEM of four wells from a
representative experiment. B, Time course of
A25-35-induced neuronal apoptosis. Cultured neurons were treated
with 25 µM A25-35 for the indicated times, and
apoptotic neurons were scored as described in A.
C, Concentration-dependent neurotoxicity of A25-35
and control peptide, A35-25. Cortical neurons were exposed to
A25-35 or A35-25 at various concentrations for 24 hr. Apoptotic
neurons were scored as described in A.
Cont, Control.
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To test the hypothesis that A-induced apoptosis of cortical neurons
might involve the JNK signaling pathway, we asked whether the treatment
of these neurons with A leads to activation of components of the JNK
pathway. JNK pathway activation was assessed by Western blotting using
phosphorylation site-specific antibodies that recognize the
phosphorylated and activated form of JNK or c-Jun (Fig.
2). By Western blotting with the
anti-phospho-JNK antibodies, it was possible to detect the two isoforms
of JNK, 46 and 54 kDa. The anti-phospho-c-Jun antibodies specifically recognize c-Jun that is phosphorylated at Ser-73. The phosphorylation of c-Jun at Ser-73 has previously been shown to contribute to the
induction of the transcriptional activity of c-Jun (Behrens et al.,
1999). As shown in Figure 2A-D, exposure of cortical
neurons to A25-35 enhanced the phosphorylation of JNK and c-Jun in
a time- and concentration-dependent manner. Western blotting with antibodies that recognize JNK1, JNK3, and c-Jun regardless of their
state of phosphorylation indicated that the overall levels of these
proteins was not affected by the addition of A to cortical neurons.
(Fig. 2A,C,D). The small change in the level of
phosphorylated JNK was detected in multiple experiments and is
statistically significant, as shown in Figure 2B. In
addition, direct measurement of JNK activity revealed a consistent
twofold increase in cells treated with A25-35 (data not shown).
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Figure 2.
A activates c-Jun and JNK in cortical neurons.
A, Time course of A-induced JNK and c-Jun
phosphorylation. Rat cortical neurons were treated with 25 µM A25-35 for the indicated number of hours.
Whole-cell extracts were resolved by SDS-PAGE and immunoblotted with
the antibodies directed against phospho-JNK or phospho-c-Jun (Ser-73).
The total amount of JNK1, JNK3, and c-Jun protein was assessed using
antibodies that recognize these proteins regardless of their
phosphorylated state. B, Quantification of the
phospho-JNK data (46 kDa isoform) from A. The densities
of the bands were determined with a dual-wavelength Flying-spot
scanner. *Statistical significance (p < 0.05) as assessed by the Wilcoxon test. C,
Concentration-dependent activation of the JNK pathway by A25-35.
Cortical neurons were treated with A25-35 at various concentrations
for 8 hr. Western blotting analysis was done as described in
A. D, A1-40 activates the JNK
pathway. Cortical neurons were incubated with 25 µM
A1-40 or A25-35 for the indicated times. Western blotting
analysis was done as described in A. E,
A25-35 and fibril A1-40 induce JNK phosphorylation in neurons.
Cortical neurons were treated with PBS (lane 1), 25 µM A25-35 (lane 2), 25 µM A35-25 (lane 3), 25 µM fibrillar A1-40 (lane 4), or
25 µM soluble A1-40 (lane 5) for 8 hr.
Western blotting analysis was performed using anti-phospho-specific
c-Jun and anti-c-Jun as described in A.
C, Control.
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Given that the anti-phospho-JNK and anti-phospho-c-Jun antibodies
recognize the activated forms of these proteins, these findings indicate that A treatment of cortical neurons leads to the
activation of c-Jun and raises the possibility that the activation of
the JNK pathway may be relevant to A-induced cell death in AD.
A25-35 peptide (25 µM) activation of JNK and c-Jun
occurred within 4 hr of peptide addition, was sustained for at least 24 hr (Fig. 2A), and was found to precede the onset of
apoptosis (Fig. 1B). This finding suggested that the
activation of c-Jun might mediate A induction of cortical neuron
apoptosis. The possibility that the JNK-c-Jun pathway is critical for
A-induced neuronal apoptosis was supported by the observation that
the concentrations of A (25-50 µM) that
induce JNK and c-Jun phosphorylation (Fig. 2C) are similar
to the concentration of A (10-50 µM) that
are required to induce cell death (Fig. 1C). In addition,
like A25-35, exposure of cortical neurons to the fibrillar form of
A1-40, but not the soluble form, led to an induction of c-Jun
phosphorylation (Fig. 2E). The control peptide
A35-25 had no effect on c-Jun phosphorylation (Fig.
2E).
The JNK pathway is involved in A-induced apoptosis
To determine whether the activation of the JNK pathway is required
for A-induced apoptosis, we introduced into cortical neurons various
dominant interfering forms of components of the JNK signaling pathway
(DN-SEK-1 and the JBD of JIP-1) and assessed their effect on
A-induced apoptosis. DN-SEK-1 is a catalytically inactive form of
SEK-1, the protein kinase that phosphorylates and activates the JNKs.
When DN-SEK-1 is expressed at high levels in cells, it has been shown
to block JNK activation (Xia et al., 1995; Luo et al., 1998). JIP-1 is
a scaffolding protein that is thought to bind and localize components
of the JNK signaling pathway to specific subcellular regions (Whitmarsh
et al., 1998; Yasuda et al., 1999). A fragment of JIP-1 termed the JBD
has been shown to bind to JNKs and to inhibit their ability to
phosphorylate substrates such as c-Jun (Dickens et al., 1997). The JBD
of JIP-1 thus functions as an inhibitor of the JNK signaling pathway
(Dickens et al., 1997). We have previously shown that the expression of DN-SEK-1 or the JBD of JIP-1 in NGF-differentiated PC-12 cells effectively inhibits NGF withdrawal-induced apoptosis (Xia et al.,
1995; Dickens et al., 1997). Therefore, we tested the ability of these
JNK signaling pathway inhibitors to block A-induced cortical neuron apoptosis.
We transfected cortical neurons with plasmids encoding DN-SEK-1 or the
JBD of JIP-1, treated the cells with 25 µM A25-35 for
24 hr or A1-40 for 48 hr, and performed a blind analysis of cell
survival and apoptosis in the transfected neurons. Of the A25-35-
or A1-40-treated cortical neurons that were transfected with an
empty vector, approximately half of the neurons underwent apoptosis
(Fig. 3A-D). By contrast, in
neurons transfected with DN-SEK-1 or the JBD of JIP-1, the number of
neurons undergoing A25-35- or A1-40-induced apoptosis was
significantly decreased (Fig. 3A-C). The inhibitory effects
of the various dominant interfering forms of JNK signaling pathway
components were found to be specific to A, inasmuch as these JNK
pathway inhibitors had little effect on the basal level of apoptosis
detected in cortical neurons exposed to the vehicle control. Taken
together, these findings suggest that A induction of the SEK1-JNK
signaling cascade is required for A-induced cortical neuron
apoptosis.
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Figure 3.
Dominant negative mutants of SEK-1 and c-Jun and
the JBD of JIP-1 prevent A-induced apoptosis in cortical neurons.
Cortical neurons were cotransfected with 4 µg of an empty vector,
DN-SEK-1, the JBD of JIP-1, or DN-c-Jun Flag169 and 0.7 µg of
CMV-LacZ and 0.3 µg of CMV-EGFP. Two or 1 d after the
transfection, the neurons were treated with 25 µM
A25-35 for 24 hr or 25 µM A1-40 for 48 hr,
respectively. The cells were fixed and immunostained with anti--Gal
antibody. Nuclei were stained with Hoechst 33258. Transfected cells
were identified as those that express green fluorescent protein. From
the transfected cells, the apoptotic neurons were identified as those
displaying condensed and fragmented nuclei. Data represent mean ± SEM of four wells from a typical experiment. *p < 0.05, significantly different from vector (ANOVA and Dunnett's test).
Each experiment was repeated two or three times. A,
Effect of DN-SEK-1 on A25-35 toxicity. B, Effect of
DN-SEK-1 on A1-40 toxicity. C, Effect of the JBD of
JIP-1 on A25-35 toxicity. D, Effect of DN-c-Jun
Flag169 on A25-35 toxicity.
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c-Jun transcriptional activity is required for A induction
of apoptosis
A primary function of the JNK signaling pathway is the
phosphorylation of key transcriptional regulators such as c-Jun, Elk1, and ATF2 at sites that lead to their activation. One of the best characterized targets of JNK is the c-Jun proto-oncogene. The exposure
of cells to stress or the withdrawal of neurotrophic factors such as
nerve growth factor leads to the phosphorylation of c-Jun at Ser-63 and
Ser-73 within the c-Jun transactivation domain. The phosphorylation of
c-Jun at these two sites potently induces the ability of c-Jun to
activate transcription. As shown in Figure 2, A promotes c-Jun
phosphorylation at Ser-73, suggesting that A treatment may activate
c-Jun. To determine whether A-induced phosphorylation of c-Jun is
critical for A induction of apoptosis, we blocked c-Jun function in
cortical neurons and assessed the effect on cortical neuron death.
DN-c-Jun Flag169 is a form of c-Jun that lacks the c-Jun
transactivation domain but is still capable of forming dimers and
binding to the c-Jun DNA-binding element. When overexpressed in cells,
DN-c-Jun Flag169 effectively competes with endogenous c-Jun for
binding to the c-Jun DNA regulatory element and thereby inhibits
c-Jun-dependent transcription (Ham et al., 1995; Xia et al., 1995). We
found that the introduction of DN-c-Jun Flag169 into cortical
neurons led to a significant decrease in the number of neurons
undergoing A-dependent cell death (Fig. 3D). These
findings strongly suggest that A induces cell death by a
c-Jun-dependent mechanism that requires the activation of particular
c-Jun target genes.
-amyloid-induced death is attenuated in cortical neurons from
JNK3 knock-out mice
To further investigate the involvement of the JNK signaling
pathway for A-induced apoptosis, we tested the ability of A to
induce the death of cortical neurons derived from JNK3 knock-out mice
that bear a mutation leading to the loss of JNK3 expression. We first
verified by Western blotting that neurons prepared from the JNK3
knock-out mice did not express JNK3 (Fig.
4). We then found that A25-35 is
slightly less effective at inducing c-Jun phosphorylation at Ser-73 in
the JNK3/ neurons compared with
wild-type neurons, consistent with the absence of A25-35 induction
of JNK3 activity in the JNK3/ neurons
(Fig. 4). Although the level of phosphorylation of c-Jun was
reproducibly reduced in the JNK3/
neurons, A25-35 was still capable of inducing some phosphorylation of c-Jun, suggesting that the continued presence of JNK1, JNK2, and
perhaps other kinases in JNK3/ neurons
are capable of phosphorylating c-Jun. A25-35 treatment of cortical
neurons induced the phosphorylation and activation of the JNK3
activator SEK1 in both wild-type and
JNK3/ neurons, indicating that, like
the wild-type neurons, JNK3/ neurons
are still capable of responding to A25-35 (data not shown).
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Figure 4.
Effect of A on the expression levels of JNK1
and JNK3 and on the phosphorylation of c-Jun in cortical neurons from
wild-type and JNK3/ mice. Whole-cell extracts
from wild-type and JNK3/ cortical neurons
treated with or without 25 µM A25-35 (8 hr) were
prepared, and equal amounts of proteins were resolved by SDS-PAGE. The
levels of JNK1, JNK3, phosphorylated c-Jun, and total c-Jun were
examined by immunoblotting. WT, Wild type;
KO, knock-out.
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When primary cortical neurons obtained from wild-type or
JNK3/ mice were treated with 25 µM A25-35 for 24 hr, we found that A25-35 was
less effective at inducing apoptosis in the
JNK3/ neurons than in wild-type
neurons. Approximately 55% of the cortical neurons from wild-type mice
treated with A were apoptotic, whereas only 35% of A-treated
JNK3-deficient neurons were undergoing apoptosis (Fig.
5A). Similar findings were
obtained when hippocampal neurons from wild-type or
JNK3/ mice were exposed to A25-35
and when wild type and JNK3/ cortical
neurons were exposed to A1-40 (Fig. 5B,C). By contrast, the number of neurons undergoing apoptosis was similar when wild-type or JNK3/ neurons were treated with
vehicle control, indicating that the decrease in apoptosis detected in
JNK3/ neurons specifically reflects a
decrease in A-mediated cell death.
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Figure 5.
JNK3-deficient neurons are resistant to
A-induced apoptosis. A, B, Cortical
(A) and hippocampal (B)
neurons were cultured from wild-type and JNK3/
mice embryos and treated with 25 µM A25-35 for 24 hr.
Cells were fixed, and nuclei were stained with Hoechst 33258. Apoptotic
neurons were scored in a blinded manner. Data represent mean ± SEM of four wells from a typical experiment. *p < 0.05, significantly different from wild type (ANOVA and Dunnett's
test); n = 4. C, Wild-type and
JNK3/ cortical neurons were treated with 25 µM A1-40 for 48 hr, and apoptotic neurons were
counted as described in A. D, Wild-type
and JNK3/ cortical and hippocampal neurons were
treated with 25 µM A25-35 as described in
A. Cell viability was assayed by metabolic integrity
(MTS assay). Data represent mean ± SEM (n = 4). *p < 0.05, significantly different from wild
type (Student's t test). KO, Knock-out;
WT, wild type.
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In addition to determining the percentage of wild-type or
JNK3/ neurons undergoing apoptosis by
assessing the number of cells containing fragmented or condensed
nuclei, we obtained a measure of cell viability within the neuronal
cultures by measuring cellular bioreducing activity using the MTS
reagent (see Materials and Methods). When the MTS assay was used to
measure the viability of A-treated cortical or hippocampal neurons
from wild-type or JNK3/ mice,
significantly more viable neurons were detected in the cultures
prepared from JNK3/ mice compared with
cultures derived from wild-type mice (Fig. 5D). This
biochemical analysis of apoptosis confirms the findings obtained by
visual quantification of apoptotic nuclei and supports the conclusion
that A promotes neuronal apoptosis by a JNK3-dependent mechanism.
A leads to Fas ligand induction
Because the ability of A to induce apoptosis is dependent on
the ability of A to activate JNK3- and c-Jun-dependent
transcriptional events, we next sought to identify JNK3 and c-Jun
targets that might mediate A-induced apoptosis. Several potential
JNK3 and c-Jun targets have recently been identified whose
transcription might directly or indirectly be induced on A
activation of the JNK3-c-Jun signaling pathway. These potential
targets include Fas ligand, its receptor Fas, the p53 tumor suppressor
gene, and tumor necrosis factor-
(TNF-).
Previous studies have shown that survival factor withdrawal leads to
the induction of Fas ligand mRNA and protein in cerebellar granule
neurons and PC12 cells (Le-Niculescu et al., 1999). In addition,
inhibition of the interaction of Fas ligand with its receptor Fas leads
to a reduction in apoptosis (Faris et al., 1998a; Ishiyama et al.,
1998; Kasibhatla et al., 1998; Brunet et al., 1999; Le-Niculescu et
al., 1999; Kolbus et al., 2000). Consistent with the idea that Fas
ligand is a target of the JNK3-c-Jun signaling pathway, the Fas ligand
gene has c-Jun binding sites within its promoter (Faris et al.,
1998a,b; Kasibhatla et al., 1998).
To determine whether the Fas ligand gene is a target of A-dependent
c-Jun activation, we first asked whether the exposure of cortical
neurons to A affects the expression of Fas ligand. Treatment of
cortical neurons with A25-35 led to a small but reproducible
increase in the level of the Fas ligand mRNA but not the S100 control
mRNA, as assessed by reverse transcriptase-coupled PCR (RT-PCR; Fig.
6A). The increase in
Fas ligand transcription was observed at 8 or 16 hr after A25-35
treatment, indicating that it occurred subsequent to JNK3 and c-Jun
activation. The A-induced increase in the Fas ligand mRNA level was
accompanied by an increase in Fas ligand protein, as indicated by
Western blotting and immunofluorescence microscopy using anti-Fas
ligand antibodies (Fig. 6B,C). Quantification of
these results indicated a statistically significant induction of the
Fas Ligand protein in response to A induction (Fig.
6B). In addition, like A, the glutamate receptor
agonist kainic acid, which has previously been shown to induce
JNK3-dependent neuronal apoptosis (Yang et al., 1997), was also found
to induce Fas ligand mRNA and protein levels (Fig.
6B,C).
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Figure 6.
A upregulates Fas ligand expression in cortical
neurons. A, A induction of Fas ligand
(FasL) mRNA. Cortical neurons were treated with 25 µM A25-35, and total RNA was extracted at the
indicated times. Expression of Fas ligand and S100 mRNAs was examined
by semiquantitative RT-PCR. B, Induction of Fas ligand
protein level by A and kainic acid. Cortical neurons were treated
with 25 µM A25-35 or 200 µM kainic
acid, and total cell extracts were prepared at the indicated times. The
level of Fas ligand was determined by immunoblotting using anti-Fas
ligand antibodies and quantified using the Wilcoxon test
(*p < 0.05). C, Immunostaining
using anti-Fas ligand (FasL) antibody. Neurons were
treated with A25-35 or kainic acid (KA) for 16 hr
and fixed. The cells were incubated with anti-Fas ligand antibody,
followed by anti-mouse biotin-conjugated secondary antibody and then
Cy3-labeled streptoavidin and Hoechst 33258. D, Cortical
neurons derived from wild-type and JNK3/ mice
were treated with 25 µM A25-35, and total RNA was
extracted at 24 hr. Expression of Fas ligand and actin mRNAs was
examined by semiquantitative RT-PCR. WT, Wild type;
KO, knock-out.
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To determine whether A induction of the Fas ligand mRNA level is
dependent on A activation of the JNK signaling pathway, we assessed
whether A was capable of inducing the level of Fas ligand mRNA
expression in neurons from JNK3/ mice.
Cortical neurons obtained from wild-type and
JNK3/ neurons were left untreated or
treated with A. RNA was extracted from the cells, and the level of
Fas ligand mRNA was determined by RT-PCR analysis (Fig.
6D). Whereas a significant induction of Fas ligand
mRNA was detected in A25-35-treated wild-type neurons, no increase
in the level of the Fas ligand mRNA was observed when neurons from
JNK3/ mice were exposed to A25-35.
These findings indicate that A induction of the Fas ligand mRNA
requires JNK3 and suggest that A induces the expression of this gene
by triggering the activation of JNK3, which in turn phosphorylates and
activates c-Jun. Activated c-Jun may then bind to the promoter of the
Fas ligand gene, leading to an increase in transcription.
A-induced apoptosis is dependent on Fas ligand and
Fas signaling
Given that binding of Fas ligand to Fas has previously been shown
to induce apoptosis in lymphoid cells and in neuronal cells deprived of
critical survival factors (Faris et al., 1998a,b; Kasibhatla et al.,
1998; Brunet et al., 1999; Le-Niculescu et al., 1999), we next asked
whether A induction of cortical neuronal apoptosis is mediated by
the Fas ligand-Fas signaling complex. Previous studies have shown that
binding of Fas ligand to Fas triggers the formation of a death-inducing
signaling complex (DISC), which consists of Fas ligand, Fas, the
adapter protein FADD, and caspase-8. The interaction of caspase-8 with
DISC leads to caspase-8 proteolysis and activation. This then triggers
a cascade of caspase activation that promotes the demise of the cell by
digestion of critical cellular constituents (Nagata, 1997).
To test whether the Fas ligand-Fas pathway plays a critical role in
A-induced toxicity, we used several approaches to interfere with Fas
ligand-Fas function. The first approach was to block Fas signaling by
incubating cortical neurons with a soluble Fas-Fc protein or an
interfering anti-Fas antibody. Previous studies have shown that soluble
Fas-Fc protein effectively competes with membrane-bound Fas for Fas
ligand and thereby blocks the effects of Fas ligand (Faris et al.,
1998a; Kasibhatla et al., 1998; Brunet et al., 1999; Le-Niculescu et
al., 1999). Likewise, the interfering anti-Fas antibody (SM1/23) blocks
the binding of Fas ligand to Fas (Ishiyama et al., 1998). We found that
the incubation of cortical neurons with Fas-Fc or the neutralizing
anti-Fas antibody, but not with control IgG, resulted in a significant
reduction in A25-35-induced cell death, as determined by both
visualization of apoptotic cells and the MTS biochemical assay (Fig.
7A-C). The interfering
anti-Fas antibody also inhibited kainic acid-induced apoptosis (Fig.
7D). The inhibitory effects of the anti-Fas antibody were
specific, inasmuch as this antibody did not significantly block
KCN-induced neuronal cell death (Fig. 7E). KCN-induced cell
death has previously been shown to occur by necrosis rather than
apoptosis (Shimizu et al., 1996) and presumably occurs by a Fas
ligand-independent mechanism. Taken together, these experiments
indicate that A induces cortical neuron apoptosis via JNK3- and
c-Jun-dependent activation of the Fas ligand-Fas signaling
pathway.
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Figure 7.
A induces apoptosis by a Fas
ligand-Fas-dependent mechanism. A, B,
Cortical neurons were pretreated with 15 µg/ml Fas-Fc or control IgG
for 2 hr, and then 25 µM A25-35 was added, and
apoptosis was determined by scoring cells with condensed and fragmented
nuclei (A) or using the MTS assay
(B). *p < 0.05, significantly different from IgG (ANOVA and Dunnett's test or
Student's t test). C-E, Cortical
neurons were incubated with 2.5 µg/ml anti-Fas antibody or control
IgG for 2 hr and then treated with 25 µM A25-35
(C), 200 µM kainic acid
(D), or 5 mM KCN
(E). Cell death was assessed by the MTS assay.
*p < 0.05, significantly different from IgG
(Student's t test).
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We further assessed the importance of the Fas ligand-Fas signaling
complex for A-induced cortical neuronal death by examining the
efficacy with which A induced the death of neurons isolated from
mice that have mutations in either Fas ligand or Fas. gld and lpr mice carry mutations that lead to a loss of function
of Fas ligand and Fas genes, respectively. We treated cortical neurons from wild-type, gld, and lpr mice with 25 µM A25-35 or A1-40 and scored viability
of the neurons 24-72 hr after A addition using the MTS assay.
Whereas A induced the death of a large percentage of the wild-type
neurons (60%), A was quite ineffective at inducing the death of
neurons (15%) from gld and lpr mice (Fig.
8A). By contrast, the
viability of neurons from wild-type, gld, and lpr mice was comparable when the neurons were grown in the absence of A
or treated with KCN, which induces necrotic cell death (data not
shown). These findings provide further evidence that the Fas ligand-Fas signaling complex is a critical mediator of A-induced neuronal death. We also examined whether the downstream signaling component of the Fas ligand-Fas signaling complex, caspase-8, is
activated in response to A. As shown in Figure 8B,
A or kainic acid treatment induced a twofold elevation in caspase-8
activity in rat and mouse neurons. The caspase-8 activity was
completely inhibited in the presence of a specific caspase-8 inhibitor,
Z-IETD-fmk.
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Figure 8.
The Fas ligand-Fas pathway is required for
A-induced apoptosis. A, Cortical neurons were
prepared from wild-type, gld, and lpr
mice embryos and treated with 25 µM A25-35 for 24 hr
or 25 µM A1-40 for 72 hr. Cell viability was assessed
by the MTS assay. Data represent mean ± SEM
(n = 5). *p < 0.05, significantly different from wild type (ANOVA and Dunnett's test).
B, Neurons from rat and mouse were treated with 25 µM A25-35 and the caspase-8 inhibitor, Z-IETD-fmk,
or with 200 µM kainic acid. Caspase-8 activity was
assessed by a fluorometric assay kit. Data represent mean ± SEM
(n = 6 for rats; n = 4 for
mice). C, Cortical neurons were cotransfected with 4 µg of an empty vector or DN-FADD and 0.7 µg of CMV-LacZ and 0.3 µg of CMV-EGFP. After 2 d, the cells were treated with 25 µM A25-35 for 24 hr. The cells were fixed and
immunostained with anti--Gal antibody. Nuclei were stained with
Hoechst 33258. Transfected cells were identified as those that
expressed green fluorescent protein, and of these cells, apoptotic
neurons were scored as those that display condensed and fragmented
nuclei. Data represent mean ± SEM of four wells from a typical
experiment. D, Neurons were pretreated with DMSO or 100 µM caspase 8 inhibitor Z-IETD-fmk for 1 hr, and then 25 µM A25-35 was added, and apoptosis was determined by
the MTS assay. Data represent mean ± SEM of seven wells from a
typical experiment. *p < 0.05, significantly
different from vector or control (ANOVA and Dunnett's test or
Student's t test).
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We then determined whether downstream components of the Fas ligand-Fas
signaling pathway are required for A-induced apoptosis. A dominant
interfering form of FADD (DN-FADD), which lacks 80 N-terminal amino
acids of the death effector domain responsible for interaction with
caspase-8, was introduced into neurons. Because DN-FADD is still able
to associate with Fas, DN-FADD inhibits the interaction of Fas with
endogenous FADD and blocks Fas signaling (Chinnaiyan et al., 1996).
Caspase-8 activity was blocked by incubating cortical neurons with the
selective caspase-8 inhibitor Z-IETD-fmk, before A
treatment. Consistent with the data by Ivins et al. (1999), the
inhibition of FADD or caspase-8 function significantly reduced the
number of neurons undergoing A-induced apoptosis. (Fig.
8C,D).
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DISCUSSION |
The accumulation of A in the brains of AD patients has been
implicated as a cause of the neuronal loss that occurs in Alzheimer's disease. However, the mechanisms by which A induces neuronal death
are not well understood. In this report we provide evidence that A
induces the activation of c-Jun in a JNK-dependent manner. JNK3 appears
to promote apoptosis by phosphorylating and activating the
transcription factor c-Jun. Our data show that A induces a small but
consistent phosphorylation of JNK and a relatively large increase in
phosphorylation of c-Jun. The discrepancy between a small JNK
activation and a robust c-Jun phosphorylation could reflect the fact
that JNK activity is constitutively high in neurons compared with other
cell types. These results further suggest that the induction of JNK
alone is not sufficient for c-Jun phosphorylation and that there are
other kinases contributing to the induction of c-Jun activity. However,
it is clear that JNK activity is important for A induction of
neuronal death, inasmuch as the disruption of JNK3 function leads to a
marked decrease in the percentage of neurons undergoing A-induced
neuronal apoptosis. In neurons from JNK3 knock-out mice, A induction
of c-Jun phosphorylation is partially blocked, again suggesting that
kinases other than JNK may be involved in c-Jun phosphorylation. In
addition, A induction of neuronal apoptosis is inhibited when c-Jun
function is blocked either by the expression of dominant interfering
forms of c-Jun (this study) or by a targeted disruption of the c-Jun gene (Kihiko et al., 1999). Although a variety of JNK3-c-Jun targets have been identified, we find that Fas ligand specifically plays a role
in A-induced neuronal apoptosis. A was found to induce Fas ligand
expression in a JNK3-dependent manner. In addition, inhibition of Fas
ligand and Fas function led to a decrease in A-induced apoptosis.
Taken together, these findings implicate the JNK-c-Jun-Fas
ligand-Fas signaling cascade in A-mediated death of cultured neurons.
Several recent observations also provide initial support for the
possibility that the JNK3-c-Jun-Fas ligand-Fas pathway may mediate
cell death in AD. First, in AD brains, JNK3 immunoreactivity is
co-localized with ALZ-50 antigen, a marker for early neurofibrillary degeneration (Mohit et al., 1995), suggesting that JNK3-expressing neurons are highly vulnerable in AD brains. Second, JNK activation is
detected in degenerating neurons in AD brains (Shoji et al., 2000; Zhu
et al., 2001). Third, c-Jun is expressed at high levels specifically in
apoptotic neurons that are detected in the AD brain (Anderson et al.,
1994, 1996; Marcus et al., 1998). Finally, Fas expression is
upregulated in the neurons of AD brains (de la Monte et al., 1997,
1998; Seidl et al., 1999). Taken together, this correlative
evidence suggests a role for the JNK-c-jun-Fas Ligand-Fas signaling
cascade in AD, although additional experimentation will be required to
confirm that this pathway mediates the neuronal cell death that occurs
in AD.
A number of observations suggest that the JNK3-c-Jun-Fas ligand-Fas
signaling pathway also mediates neuronal cell death that is initiated
by apoptotic stimuli other than A. The JNK pathway has been shown to
play an important role in dopamine-induced apoptosis in a cellular
model of Parkinson's disease (PD) (Luo et al., 1998). In addition,
several recent reports suggest a significant role for Fas ligand and
Fas in neurodegenerative disease. In an animal model of focal cerebral
ischemia, Fas ligand and Fas are induced in neurons undergoing
apoptosis, and the infarct volume after brain ischemia in
lpr mice is significantly reduced compared with that in
wild-type mice (Matsuyama et al., 1995; Herdegen et al., 1998;
Martin-Villalba et al., 1999). Furthermore, in several
neurodegenerative diseases such as PD and Down syndrome, the
upregulation of Fas expression was detected specifically in neurons
that were dying (de la Monte et al., 1997, 1998; Seidl et al., 1999).
Thus it is possible that the JNK3-c-Jun-Fas ligand-Fas pathway may
be a common mediator of cell death in a variety of neurodegenerative diseases.
With respect to AD, it is important to note that A toxicity is not
completely abolished in JNK3-deficient neurons or gld and
lpr neurons. In addition, although c-Jun phosphorylation is reproducibly decreased in JNK3 knock-out cells, significant
phosphorylation remains. This suggests that in addition to the
JNK3-c-Jun-Fas ligand-Fas signaling pathway, there are other
pathways that contribute to A induction of apoptosis. It is likely
that the other members of the JNK family (i.e., JNK1 and JNK2) play a
role in A-induced apoptosis. Consistent with this possibility, we
have found that JNK1-deficient neurons are also resistant to the
death-promoting effects of A (data not shown). In addition to Fas
ligand, there are likely to be other targets of c-Jun that contribute
to its ability to mediate A-induced apoptosis. One possible target
is the death cytokine TNF-, because the expression of TNF- is
known to be regulated by JNK and c-Jun (Ishizuka et al., 1997;
Hoffmeyer et al., 1999). Finally, c-Jun is most likely just
one of several JNK substrates that contribute to A-induced
apoptosis. The phosphorylation of Bcl-2 by JNK suppresses the
prosurvival activity of Bcl-2 (Chang et al., 1997; Maundrell et al.,
1997; Yamamoto et al., 1999). In addition, p53 and c-Myc, two potent inducers of apoptosis, are substrates of JNK. The phosphorylation of
p53 and c-Myc by JNK leads to the induction of the proapoptotic function of these two proteins, consistent with the possibility that
p53 and c-Myc might be mediators of the apoptotic effect of JNKs (Milne
et al., 1995; Fuchs et al., 1998; Noguchi et al., 1999). It will be
important to determine whether A induction of JNK activity leads to
Bcl-2, p53, and c-Myc phosphorylation and whether the phosphorylation
of these proteins then contributes to A-induced cell death.
It will also be of interest to establish the mechanism by which
extracellular A induces intracellular JNK activation. The fibrillar
form of A appears to bind to the surface of neurons through multiple
receptors. A has been shown to bind to a variety of proteins,
including the A precursor protein (Lorenzo et al., 2000), an
endoplasmic reticulum amyloid peptide binding
protein/L-3-hydroxyacyl-coenzyme A dehydrogenase (Yan et al.,
1997, 1999), and the receptor for advanced glycation end products,
which mediates oxidative stress (Yan et al., 1996). Whether one or
several of these A-binding proteins mediates A induction of JNK
activation is not known.
A reasonable possibility is that one or several A receptors promote
apoptosis via a JNK-dependent pathway, whereas the other A-binding
proteins contribute to A-mediated apoptosis through distinct
signaling pathways. One such pathway could involve the action of
calpain, p25, and cdk5 (Patrick et al., 1999; Lee et al., 2000). In a
recent study, it was demonstrated in cultured neurons that A induces
the Ca2+-dependent activation of calpain
I, which then cleaves p35, the regulatory subunit of cdk5, to p25,
leading to constitutive cdk5 activation. Because p25 and cdk5
overexpression leads to the apoptosis of cultured neurons, it is likely
that one way A induces cell death is through the activation of p25
and cdk5. It remains to be determined whether there is cross-talk
between JNKs and cdk5 that is important for A induction of
apoptosis. Further characterization of the multiple pathways that
mediate the A-dependent apoptosis of cultured neurons, and the
importance of these pathways in AD, is likely to provide new avenues
for treating this disease.
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FOOTNOTES |
Received Sept. 15, 2000; revised July 16, 2001; accepted July 16, 2001.
This research was supported by a grant to M.E.G. from Daiichi
Pharmaceutical Co., Ltd. We thank Dr. Vishva M. Dixit for kindly providing plasmids of DN-FADD. We thank all members of the Greenberg laboratory for helpful discussions and support, particularly Dr. Anne Brunet.
Correspondence should be addressed to Michael E. Greenberg, Division of
Neuroscience, Children's Hospital, and Department of Neurobiology,
Harvard Medical School, Boston, MA 02115. E-mail: michael.greenberg{at}tch.harvard.edu.
Dr. Gotoh's present address: Institute of Molecular and Cell
Bioscience, University of Tokyo, Tokyo 113-0032, Japan.
| |
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TOP
ABSTRACT
INTRODUCTION
