doi:10.1093/brain/awh700
Brain (2006), 129, 465–479
Neuroprotection against ischaemic brain injury by
a GluR6-9c peptide containing the TAT protein
transduction sequence
Dong-Sheng Pei,1,2,* Xiao-Tian Wang,1,* Yong Liu,1 Ya-Feng Sun,1 Qiu-Hua Guan,1,2 Wei Wang,2,3
Jing-Zhi Yan,1 Yan-Yan Zong,1 Tian-Le Xu2,3 and Guang-Yi Zhang1,2
1
Research Center for Biochemistry and Molecular Biology, Xuzhou Medical College, Xuzhou,
Department of Neurobiology and Biophysics, School of Life Science, University of Science and Technology of China,
Hefei and 3Institute of Neuroscience, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences,
Shanghai, China
2
Correspondence to: Guang-Yi Zhang, Research Center for Biochemistry and Molecular Biology, Xuzhou Medical College,
84 West Huai-hai Road, Xuzhou, Jiangsu, 221002 P.R. China
E-mail: [email protected]
*These authors contributed equally to this work
It is well documented that N-methyl-D-aspartate and a-amino-3-hydroxy-5-methyl-4-isoxazole propionate
receptors play a pivotal role in ischaemic brain injury. Recent studies have shown that kainate (KA) receptors
are involved in neuronal cell death induced by seizure, which is mediated by the GluR6PSD-95MLK3 signalling
module and subsequent c-Jun N-terminal kinase (JNK) activation. Here we investigate whether GluR6 mediated
JNK activation is correlated with ischaemic brain injury. Our results show that cerebral ischaemia followed by
reperfusion can enhance the assembly of the GluR6PSD-95MLK3 signalling module and JNK activation. As a
result, activated JNK can not only phosphorylate the transcription factor c-Jun and up-regulate Fas L expression
but can also phosphorylate 14-3-3 and promote Bax translocation to mitochondria, increase the release of
cytochrome c and increase caspase-3 activation. These results indicate that GluR6 mediated JNK activation
induced by ischaemia/reperfusion ultimately results in neuronal cell death via nuclear and non-nuclear pathways. Furthermore, the peptides we constructed, Tat-GluR6-9c, show a protective role against neuronal death
induced by cerebral ischaemia/reperfusion through inhibiting the GluR6 mediated signal pathway. In summary,
our results indicate that the KA receptor subunit GluR6 mediated JNK activation is involved in ischaemic brain
injury and provides a new approach for stroke therapy.
Keywords: cerebral ischaemia; glutamate receptor 6 (GluR6); mixed lineage kinase-3 (MLK3); c-Jun N-terminal
kinase (JNK); Tat protein
Abbreviations: AMPA = a-amino-3-hydroxy-5-methyl-4-isoxazole propionate; KA = kainate; IB = immunoblotting;
IP = immunoprecipitation; NMDA = N-methyl-D-aspartate
Received July 14, 2005. Revised October 3, 2005. Accepted October 26, 2005. Advance Access publication December 5, 2005
Introduction
The main mechanism underlying neuronal death in stroke
is excitotoxicity, which is triggered by excessive activation
of glutamate receptors [especially N-methyl-D-aspartate
(NMDA) receptors] and subsequent calcium influx leading
to increased intra-cellular calcium levels. Ionotropic glutamate receptors can be divided into NMDA, a-amino-3hydroxy-5-methyl-4-isoxazole propionate (AMPA) and
kainate (KA) (Seeburg, 1993; Hollmann and Heinemann,
#
1994). Although the bulk of the evidence suggests that
NMDA receptors may play a pivotal role in ischaemiainduced neuronal death, the classical hypothesis on NMDA
receptor–mediated excitotoxicty faces the following challenges: first, the wide distribution of NMDA receptors can
hardly account for the selective neuronal death after transient
cerebral ischaemia (Gill and Lodge, 1997; Weiss and Sensi,
2000). Second, NMDA receptor antagonists confer robust
The Author (2005). Published by Oxford University Press on behalf of the Guarantors of Brain. All rights reserved. For Permissions, please email: [email protected]
466
Brain (2006), 129, 465–479
neuroprotection in animal models of focal (but not global)
cerebral ischaemia; however, severe side-effects of the NMDA
receptor blocker were demonstrated, and therefore, limited its
application in clinical management of neurodegenerative diseases (Olney et al., 1989; Gill and Lodge, 1997). Third, in
contrast to NMDA receptor antagonists, non-NMDA receptor antagonists exhibit a more protective role against neuron
death in hippocampus CA1 subfields induced by transient
global ischaemia (Chung et al., 2000). These findings raise
new questions regarding understanding the function of
NMDA receptors in cerebral ischaemia/reperfusion and reinforce concerns about the potential roles of other glutamate
receptors in mediating neuronal injury during cerebral ischaemia. In contrast to NMDA and AMPA receptors, little is
known about the function of KA receptors in response to
ischaemia. A recent study has shown that c-Jun N-terminal
kinase (JNK)3-deficient mice have strong resistance to the
glutamate-receptor agonist KA-induced seizure and hippocampal neuron apoptosis (Yang et al., 1997). Together with
Mulle’s study (Mulle et al., 1998) showing that GluR6deficient mice exhibit resistance to neurotoxic effects induced
by KA, in turn, there is raised the possibility that the KA
receptor subunit GluR6 may mediate the activation of
JNK3 in response to excitotoxicity of glutamate. Studies
indicated that the motif RLPGKETMA of the carboxylic terminal of GluR6 could bind to the PDZ1 domain of postsynaptic density protein 95 (PSD-95/SAP90) through a
specific interaction (Garcia et al., 1998; Mehta et al., 2001).
A recent study has also shown that MLK3, an upstream kinase
of JNK (Tibbles et al., 1996), could interact with the SH3
domain of PSD-95 (Savinainen et al., 2001). Thus, there
may exist the triplicate complex GluR6PSD-95MLK3,
which could form a signalling module to facilitate JNK activation. Our previous studies demonstrated that JNKs can be
activated during cerebral ischaemia/reperfusion (Gu et al.,
2001; Tian et al., 2003a); however, NMDA and AMPA receptors did not mediate JNK activation (Tian et al., 2003a).
Thus, it renders us to think of the possible contribution
of KA receptors to the activation of JNK in cerebral ischaemia/reperfusion.
In the present study, we show that Tat-GluR6-9c, a GluR6
C-terminus containing peptide conjugated to Tat peptide,
could perturb the interaction of GluR6 with PSD-95 and
suppress the assembly of the GluR6PSD-95MLK3 signalling
module and, therefore, result in blockage of brain injury
caused by a variety of noxious stimuli including cerebral
ischaemia. (Fig. 1A)
Material and methods
Ischaemic model
Adult male Sprague–Dawley rats weighing 200–250 g were used. The
experimental procedures were approved by the local legislation for
ethics of experiments on animals. Transient cerebral ischaemia was
induced by four-vessel occlusion (4-VO) as described before
(Pulsinelli and Brierley, 1979). Briefly, rats were anaesthetized
D.-S. Pei et al.
with chloral hydrate (300 mg/kg, i.p.) and both vertebral arteries
were occluded permanently by electrocautery. Rats were allowed to
recover for 24 h and fasted overnight. On the following day,
both carotid arteries were occluded with aneurysm clips to induce
cerebral ischaemia. After 15 min of the occlusion, the aneurysm
clips were removed for reperfusion. Rats that lost their righting
reflex and whose pupils were dilated and unresponsive to light
were selected for the experiments. Rectal temperature was maintained at 36.5–37.5 C throughout the procedure. Rats with seizures
were discarded. An EEG was monitored to ensure isoelectricity after
carotid artery occlusion. Sham control was performed using the same
surgical procedures except that the carotid arteries were not
occluded.
Administration of peptides
Peptides (100 mg) or control peptides in 10 ml saline were administered to the rats 40 min before or 1 h after ischaemia through cerebral
ventricular injection (anteroposterior, 0.8 mm; lateral, 1.5 mm;
depth, 3.5 mm from bregma).
Sample preparation
Rats were decapitated immediately after different times of reperfusion and then the hippocampi CA1 were isolated and quickly frozen
in liquid nitrogen. The hippocampi were homogenized in an ice-cold
homogenization buffer containing 50 mM 3-(N-morpholino) propanesulphonic acid (MOPS) (Sigma; pH 7.4), 100 mM KCl, 320 mM
sucrose, 50 mM NaF, 0.5 mM MgCl2, 0.2 mM DTT, 1 mM EDTA,
1 mM EGTA, 1 mM Na3VO4 (Sigma), 20 mM sodium pyrophosphate, 20 mM b-phosphoglycerol, 1 mM p-nitrophenyl phosphate
(PNPP), 1 mM benzamidine, 1 mM phenylmethylsulphonylfluoride
(PMSF) and 5 mg/ml each of leupeptin, aprotinin and pepstatin A.
The homogenates were centrifuged at 800 g for 10 min at 4 C.
Supernatants were collected and centrifuged at 100 000 g for 30
min at 4 C. The supernatants were carefully removed and 500 ml
homogenization buffer containing 1% Triton X-100 was added to the
pellets, which then exposed to ultrasound. Protein concentration was
determined by the methods of Lowry et al. Samples were stored at
80 C until use.
When necessary, the hippocampal CA1 was immediately isolated
to prepare mitochondrial fractions. All procedures were conducted
in a cold room. Non-frozen brain tissue was used to prepare mitochondrial fractions because freezing tissue causes release of cytochrome c from mitochondria. The hippocampal CA1 tissues were
homogenized in 1 : 10 (w/v) ice-cold homogenization buffer. The
homogenates were centrifuged at 800 g for 10 min at 4 C. The pellets
were discarded, and supernatants were centrifuged at 17 000 g for
20 min at 4 C to get the cytosolic fraction in the supernatants and the
crude mitochondrial fraction in the pellets. The protein concentrations were determined by the method of Lowry et al.
Nuclei extraction
The homogenates were centrifuged at 800 g for 10 min at 4 C.
Supernatants as the cytosol part were collected and protein concentrations were determined. The nuclear pellets were extracted with
20 mM HEPES, pH 7.9, 20% glycerol, 420 mM NaCl, 0.5 mM
MgCl2,1 mM EDTA,1 mM EGTA, 1 mM DTT and enzyme inhibitors
for 30 min at 4 C with constant agitation. After centrifugation at
12 000 g for 15 min at 4 C, supernatants as nuclear parts were collected and protein concentrations were determined. Samples were
stored at 80 C and were thawed only once.
Neuroprotection against ischaemic brain injury by a GluR6-9c peptide
A
Brain (2006), 129, 465–479
467
D
B
C
Fig. 1 Dissociating effects of Tat peptides on the GluR6–PSD-95
interaction. (A) The hypothesis: the GluR6–PSD-95 complex
(upper panel) may be dissociated using Tat peptides fused to the
COOH-terminus of GluR6 (lower panel), thus reducing the
efficiency of GluR6 mediated signal cascade. (B) Visualization of
intraneuronal accumulation of Tat-GluR6-9c-dansyl (10 mM) but
not Tat38-48-dansyl (10 mM) 30 min after application to
hippocampal cultures. Cultures treated with Tat-GluR6-9c-dansyl
exhibit strong fluorescence while cultures treated with Tat38-48dansyl only have a weak fluorescence similar to background.
(C) Time course of Tat-GluR6-9c-dansyl (10 mM) fluorescence
after application to hippocampal cultures at room temperature.
(D) Co-IP of PSD-95 with GluR6 or NR2A or NR2B subunits in
rat hippocampal membrane fractions treated with Tat peptides.
Data are the mean 6 SD and are expressed as folds versus
control. aP < 0.05 versus control (n = 6 rats).
Immunoprecipitation
Fig. 1 Continued
Tissue homogenates (400 mg of protein) were diluted 4-fold with
50 mM HEPES buffer, (pH 7.4), containing 10% glycerol, 150 mM
NaCl, 1%Triton X-100, 0.5% NP-40 and 1 mM each of EDTA,
EGTA, PMSF and Na3VO4. Samples were preincubated for 1 h
with 20 ml protein A Sepharose CL-4B (Amersham, Uppsala,
Sweden) at 4 C, and then centrifuged to remove proteins adhered
non-specifically to protein A. The supernatants were incubated with
1–2 mg primary antibodies for 4 h or overnight at 4 C. Protein A was
added to the tube for another 2 h incubation. Samples were centrifuged at 10 000 g for 2 min at 4 C and the pellets were washed with
immunoprecipitation (IP) buffer for three times. Bound proteins
were eluted by boiling at 100 C for 5 min in SDS–PAGE loading
468
Brain (2006), 129, 465–479
buffer and then isolated by centrifuge. The supernatants were used
for immunoblot analysis.
Immunoblot
Equal amounts of protein (100 mg/lane) were separated on polyacrylamide gels and then electrotransferred onto a nitrocellulose
membrane (Amersham, Buckinghamshire, UK). After blocking for
3 h in Tris-buffered saline with 0.1% Tween-20 (TBST) and 3%
bovine serum albumin (BSA), membranes were incubated overnight at 4 C with primary antibodies in TBST containing 3%
BSA. Membranes were then washed and incubated with alkaline
phosphatase conjugated secondary antibodies in TBST for 2 h and
developed using NBT/BCIP colour substrate (Promega, Madison,
WI, USA). The densities of the bands on the membrane were scanned
and analysed with an image analyser (LabWorks Software, UVP
Upland, CA, USA).
Hippocampal cell culture
Neurons from hippocampi of fetal Sprague-Dawley rat (18 days
gestation) were prepared as described previously with a little modification. Briefly, hippocampi were meticulously isolated in ice-cold
high-glucose Dulbecco’s modified Eagle medium (h-DMEM,
GibcorBRL, Grand Island, NY, USA). Hippocampal cells were dissociated by trypsinization [0.25% (w/v) trypsin and 0.05%
EDTA in Ca2+- and Mg2+-free Hanks balanced salt solution] at
37 C for 15 min, followed by gentle triturating in plating medium
(h-DMEM supplemented with 10% fetal bovine serum and 10%
horse serum, Gibco BRL). Cells were seeded onto poly-L-lysinecoated wells (Sigma, St. Louis, MO, USA) or coverslips at a density
of 0.8 · 105 cells per cm2 and incubated at 37 C in 5% CO2 atmosphere. After 18–24 h, cells were incubated in Neurobasal Medium
supplemented with B-27 (Gibco BRL) and 0.5 mM glutamine, and
then half-replaced twice every week. Cultures were used after 16 days
in vitro.
Electrophysiological recordings
The electrophysiological recordings were performed in the conventional whole-cell patch-recording configuration under voltageclamp conditions. Patch pipettes were pulled from glass capillaries
with an outer diameter of 1.5 mm on a two-stage puller (PP-830,
Narishige, Tokyo, Japan). The resistance between the recording electrode filled with pipette solution and the reference electrode was
4–6 MV. The standard external solution contained (mM):
NaCl 150, KCl 5, MgCl2 1, CaCl2 2, HEPES 10 and glucose 10.
The pH was adjusted to 7.4 with Tris-base. The osmolarity was
adjusted to 310–320 mOsm/l with sucrose. The ionic composition
of the internal solution medium was (mM): CsCl 140, CaCl2 0.5,
MgCl2 2, EGTA 5, Na2ATP 4 and HEPES 10 with pH adjusted to 7.2,
osmolarity adjusted to 280–300 mOsm/l. Membrane currents were
measured using a patch-clamp amplifier (Axon 200B, Axon Instruments, Foster City, CA, USA), sampled and analysed using a Digidata
1322A interface and a personal computer with Clampex and Clampfit software (Version 9.0.1, Axon Instruments). In most experiments,
60–70% series resistance was compensated. The membrane potential
was held at 60 mV throughout the experiment. All the experiments
were carried out at room temperature (22–25 C).
Histology and immunohistochemistry
Rats were perfusion-fixed with 4% paraformaldehyde in 0.1 M
sodium phosphate buffer (pH 7.4) under anesthaesia after 5 days
D.-S. Pei et al.
of ischaemia/reperfusion. Brains were removed quickly and
further fixed with the same fixation solution at 4 C overnight.
Post-fixed brains were embedded by paraffin, followed by preparation of coronal sections 5 mm thick using a microtome.
The paraffin embedded brain sections were deparaffinized with
xylene and rehydrated by ethanol at graded concentrations of
100–70% (v/v), followed by washing with water. The sections
were stained with 0.1% (w/v) cresyl violet and were examined
with light microscopy and the number of surviving hippocampal
CA1 pyramidal cells per 1 mm length was counted as the neuronal
density.
Immunoreactivity was determined by the avidin–biotin–
peroxidase method. Briefly, sections were deparaffinized with xylene
and rehydrated by ethanol at graded concentrations and distilled
water. High-temperature antigen retrieval was performed in 1 mM
citrate buffer. To block endogenous peroxidase activity, sections were
incubated for 30 min in 1% H2O2. After being blocked with 5% (v/v)
normal goat serum in PBS for 1 h at 37 C, sections were incubated
with rabbit polyclonal antibodies against Fas L (1 : 100) or mouse
nomoclonal antibody against p-c-Jun (1 : 50) at 4 C 2 day. These
sections were then incubated with biotinylated goat-anti-rabbit/
mouse secondar antibody overnight and subsequently with
avidin–conjugated horseradish peroxidase for 1 h at 37 C. Finally,
sections were incubated with peroxidase substrate diaminobenzidine
(DAB) until desired stain intensity developed.
TUNEL staining
TUNEL staining was performed using an ApopTag Peroxidase
In Situ Apoptosis Detection Kit according to the manufacturer’s
protocol with minor modifications. The paraffin-embedded coronal
sections were deparaffinized and rehydrated, and then treated with
protease K at 20 mg/ml for 15 min at room temperature. Sections
were incubated with reaction buffer containing TdT enzyme and at
37 C for 1 h. After washing with stop/wash buffer, sections were
treated with anti-digoxigenin conjugate for 30 min at room temperature and subsequently developed colour in peroxidase substrate.
The nuclei were lightly counterstained with 0.5% methyl green.
Antibody and reagents
The following primary antibodies were used: goat polyclonal antiGluR6 (sc-7618), mouse monoclonal anti-p-JNKs (sc-6254), rabbit
polyclonal anti-MLK3 (sc-13072), mouse monoclonal anti-p-cJun (sc-822), rabbit polyclonal anti-Fas L (sc-6237), rabbit polyclonal anti-Fas (sc-716), rabbit polyclonal anti-c-Jun (sc-1694),
rabbit polyclonal anti-14-3-3 (sc-1019), rabbit polyclonal antiNR2B (sc-9057) and rabbit polyclonal anti-actin (sc-10731) were
purchased from Santa Cruz Biotechnology. Monoclonal antibody
to phosphoserine was obtained from Alexis Biochemicals. Rabbit
polyclonal anti-Bax, rabbit polyclonal anti-cytochrome c, rabbit
polyclonal anti-caspase-3 and rabbit polyclonal anti-p-MLK3 were
obtained from Cell Signal Biotechnology. Mouse monoclonal antiPSD-95 (CP35-100UL) was bought from Oncogene. Monoclonal
antibody of cytochrome c oxidase subunit IV was obtained from
Molecular Probes. Rabbit polyclonal anti-JNK3 antibody (06-749)
was obtained from Upstate Biotechnology. The secondary antibodies
used in our experiment were goat anti-mouse IgG, goat anti-rabbit
IgG and donkey anti-goat IgG. They were from Sigma. ApopTag
Peroxidase In Situ Apoptosis Detection Kit (S7100) was purchased
from Chemicon.
Neuroprotection against ischaemic brain injury by a GluR6-9c peptide
Statistics
Values were expressed as mean 6 SD and obtained from six independent rats. Statistical analysis of the results was carried out by
Student’s t-test or one-way analysis of the variance (ANOVA) followed by the Duncan’s new multiple range method or NewmanKeuls test. P-values <0.05 were considered significant.
Results
Effects of the peptides in vitro and in
cultured hippocampal neurons
In order to interfere with the interaction of GluR6 with
PSD-95, we constructed a peptide comprising the nine
COOH-terminal residues of GluR6 (Arg-Leu-Pro-Gly-LysGlu-Thr-Met-Ala; GluR6-9c), which was conserved in humans
and rodents. Tat protein (Tyr-Gly-Arg-Lys-Lys-Arg-ArgGln-Arg-Arg-Arg), which was obtained originally from the
cell-membrane transduction domain of the human immunodeficiency virus–type 1 (HIV-1), was fused to GluR6-9c and
resulted in a 20-amino acid fusion peptide (Tat-GluR6-9c).
We further examined whether Tat-GluR6-9c could be
delivered into hippocampal neurons. The fluorophore dansyl
chloride was conjugated to Tat-GluR6-9c and to HIV-1 Tat
residues 38 to 48 (Lys-Ala-Leu-Gly-Ile-Ser-Tyr-Gly-Arg-LysLys; Tat38-48) outside of the transduction domain as a control peptide, respectively. These peptides were incubated with
cultured hippocampal neurons and their fluorescence was
visualized by fluoroscope. Neurons treated with TatGluR6-9c-dansyl (10 mM) exhibited strong fluorescence in
the cytoplasm, indicating intracellular peptide uptake (Fig. 1B,
left), whereas cultures treated with Tat38-48-dansyl (10 mM),
lacking the transduction domain, exhibited only background
signal indicating no peptide uptake (Fig. 1B, right). TatGluR6-9c-dansyl accumulation was detectable in neurons
within 10 min of application, peaked from 30 min to
90 min, and remained detectable for 3 h after washing the
peptide from the bath (Fig. 1C).
Next we investigated whether Tat-GluR6-9c could perturb
the assembly of GluR6–PSD-95 complexes by examining its
effects on the co-IP of PSD-95 with GluR6 subunits in vitro.
The membrane protein fraction of rat hippocampal tissue was
incubated with Tat-GluR6-9c or with one of three controls:
Tat38-48, the Tat transduction sequence conjugated to two
alanine residues (Tat-AA), or a Tat-GluR6-9c peptide in
which the COOH-terminal ETMA motif contained four
point mutations (Arg-Leu-Pro-Gly-Lys-Ala-Ala-Asp-Asp;
Tat-GluR6AA) rendering it incapable of binding PSD-95.
Results from co-IP indicated that none of these controls,
each lacking an intact PDZ binding motif, could suppress
the interaction of PSD-95 with GluR6. However, TatGluR6-9c, in which the Glu-Thr-Met-Ala sequence is
unique to the GluR6 COOH-terminus, selectively reduced
the interaction of GluR6 with PSD-95. As a control, the interactions between NR2A or NR2B and PSD-95 were unaffected
(Fig. 1D).
Brain (2006), 129, 465–479
469
Effects of the peptides on the currents of
KA receptors
To investigate whether the peptides would directly affect the
function of KA receptors, the patch-clamp recordings were
used to examine the effect of the peptides on KA-induced
whole-cell currents. As shown in Fig. 2A, extracellular TatGluR6-9c peptide does not directly affect KA receptorsmediated whole-cell currents in cultured hippocampal
neurons (Fig. 2A). Meanwhile, the intracellular Tat-GluR69c peptide also showed no effect on KA receptors-mediated
currents during or after 15 min incubation of the peptides
(Fig. 2B). In order to further confirm the results, we examine
the KA-induced whole-cell currents with the peptides applied
from the internal pipette solution. The results also show that
the currents were not affected by the peptides (Fig. 2C). The
above data indicate that Tat-GluR6-9c peptide will not affect
KA receptors-mediated currents in spite of the fact that it can
interrupt the interactions of GluR6 and PSD-95.
The peptides suppress the assembly of the
GluR6PSD-95MLK3 signalling moduleand
inhibit the activation of MLK3 and JNK3
We make the hypothesis that the assembly of the GluR6PSD95MLK3 signalling module during cerebral ischaemiareperfusion can facilitate the activation of JNK signalling
pathway. A GluR6 c-terminus containing peptide could perturb the interaction of GluR6 with PSD-95 and suppress the
assembly of the GluR6PSD-95MLK3 signalling module,
and subsequently prevent the activation of JNK as well as
neuronal death induced by cerebral ischaemia-reperfusion.
To test our hypothesis, the peptides were administrated to
the adult Sprague–Dawley rats through cerebral ventricular
injection 40 min before ischaemia. Because JNK was selectively activated in the CA1 subfields of the hippocampus, the
CA1 regions were isolated for further examinations (Gu et al.,
2001). After various time of reperfusion, the interactions of
GluR6 and MLK3 with PSD-95 and the phosphorylation of
MLK3, JNK3 and c-Jun were examined, respectively. In our
previous study, we found that the interaction of GluR6 and
MLK3 with PSD-95 reached its peak level at 6 h reperfusion
after 15 min ischaemia (Tian et al., 2005). In order to investigate whether pretreatment of rats with these peptides could
affect the interaction of GluR6 and MLK3 with PSD-95, IP
and immunoblotting (IB) were used to examine the association of GluR6 and MLK3 with PSD-95 after 15 min ischaemia followed by 6 h reperfusion, reciprocal IP experiments
were carried out to confirm the results. As shown in Fig. 3A,
the interaction of GluR6 and MLK3 with PSD-95 increased
after 15 min ischaemia followed by 6 h reperfusion. Administration of Tat-GluR6-9c 40 min prior to ischaemia diminished the increased interaction of GluR6, MLK3 with PSD-95,
meanwhile the protein level of GluR6, PSD-95 and MLK3 were
not altered. Conversely, the same dose of control peptides
Tat-GluR6AA did not affect the increased associations of
GluR6, MLK3 with PSD-95.
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Brain (2006), 129, 465–479
D.-S. Pei et al.
Neuroprotection against ischaemic brain injury by a GluR6-9c peptide
Previous studies indicated that MLK3, an upstream kinase
of JNK, could be activated via GluR6 and PSD-95 (Savinainen
et al., 2001). We then analysed the effects of the peptides on
the activation of MLK3. IB was used to examine the phosphorylation of MLK3. As shown in Fig. 3B, 6 h reperfusion
after 15 min ischaemia resulted in remarkable increase in the
phosphorylation of MLK3. Pretreatment of Tat-GluR6-9c
significantly diminished the increase in the phosphorylation
of MLK3, while the protein level of MLK3 was not changed.
The same dose of control peptides Tat-GluR6AA did not
affect the increased phosphorylation of MLK3. We have
reported that JNK activation was biphasically increased during transient cerebral ischaemia followed by reperfusion (Gu
et al., 2001). To further determine whether the activation of
JNK pathway downstream of MLK3 was affected by the peptides after cerebral ischaemia followed by reperfusion, we
examined the phosphorylation of JNK3 at two peak levels:
30 min and 3 days after ischaemia. Results of western blotting
revealed that activation of JNK3 after 3 days, but not 30 min of
reperfusion was suppressed by application of the peptides
Tat-GluR6-9c (Fig. 3C). Furthermore, similar results were
obtained from c-Jun, one of the substrates of JNK, as compared with that of MLK3 and JNK3 (Fig. 3D). As mentioned
above, our results suggest that MLK3 and JNK3 activation
induced by ischaemia/reperfusion were mediated by
GluR6PSD-95MLK3 signalling module. Tat-GluR6-9c
could perturb the interaction of GluR6 with PSD-95 and,
therefore, inhibit the assembly of the GluR6PSD-95MLK3
signalling module and, as a result, prevent the JNK3 activation
and translocation into the nucleus and subsequently suppress
the phosphorylation of c-Jun. At the same time, results from
immunohistochemistry also confirmed that pretreatment
with Tat-GluR6-9c could significantly diminish the immunoreactivity of p-c-Jun in the nucleus of hippocampal CA1
compared with 6 h reperfusion groups and the control
peptide Tat-GluR6-AA-treated groups (Fig. 3E).
Inhibition of JNK activity by the peptides
diminished the increased expression of
Fas L induced by cerebral ischaemiareperfusion in the hippocampal CA1 region
To investigate whether the Fas receptor-mediated pathway is
involved in the apoptotic profile during cerebral ischaemia/
Brain (2006), 129, 465–479
471
reperfusion, the expression of Fas L and Fas was analysed by
western blot. As indicated in Fig. 4A, the expression of Fas
L was significantly increased 6 h after reperfusion. However,
application of Tat-GluR6-9c could diminish the increasing
expression of Fas L induced by cerebral ischaemia and reperfusion. The same dose of control peptide Tat-GluR6-9c did not
affect the increase on the expression of Fas L. The protein level
of Fas was not affected by Tat-GluR6-AA and control peptide.
As shown in Fig. 4B, the results of immunohistochemistry also
revealed that weak Fas L immunoreactivity was detected in the
cytosol of hippocampal CA1 in the sham group (a, e). On the
contrary, Fas L immunoreactivity was significantly increased
after 6 h reperfusion (b, f) compared with the sham group. No
inhibitory effects of control peptide Tat-GluR6AA on Fas L
immunoreactivity were detected (c, g). However, Fas L
immunoreactivity after ischaemia 6 h reperfusion was significantly inhibited by application of Tat-GluR6-9c (d, h).
Tat-GluR6-9c attenuated the decreased
interaction of Bax and 14-3-3,
phosphorylation of 14-3-3, Bax
translocation, the release of
cytochrome c and neuronal apoptosis
in hippocampal CA1 induced by
cerebral ischaemia-reperfusion
To elucidate the involvement of mitochondria-mediated
pathway in the apoptotic programme during global ischaemia/
reperfusion, phosphorylation of 14-3-3, the interaction of Bax
and 14-3-3, and the expression of Bax and cytochrome c in
mitochondria and cytosol after brain ischaemia was examined
by IB and IP. As indicated in Fig. 5A, results of reciprocal IP
showed that the phosphorylation of 14-3-3 was significantly
increased at 6 h reperfusion after 15 min of ischaemia, but the
protein level of 14-3-3 was not affected at various times after
15 min of ischaemia. Meanwhile, the association of Bax and
14-3-3 keeps decreasing in company with the phosphorylation of 14-3-3. The disassociated Bax would translocate from
cytosol to mitochondria and facilitate cytochrome c release,
which ultimately causes caspase-3 activation and results in
apoptosis.
Recent studies indicated that JNK could phosphorylate
14-3-3 protein and promote Bax disassociate from 14-3-3
Fig. 2 Effects of Tat-GluR6-9c on KA receptors-mediated whole-cell currents in cultured hippocampal neurons. (A) Effects of extracellular
Tat-GluR6-9c peptide on KA receptor-mediated whole-cell currents in cultured hippocampal neurons. (a) KA-induced whole-cell currents
were inhibited by non-NMDA GluR inhibitor CNQX. (b) Representative trace of combined Tat-GluR6-9c and KA-stimulated whole-cell
currents. (c, d) KA-induced peak amplitudes and ratio of steady-state current to peak amplitude had no significant change in the presence
of Tat-GluR6-9c. (B) KA receptors-mediated whole-cell currents were not affected by long time incubation of Tat-GluR6-9c peptide in
cultured hippocampal neurons. KA receptor-mediated whole-cell currents in group of Tat-GluR6-9c (open circles) or control (solid
circles) did not show significant difference during and after 15 min incubation of the peptide. (a) is a representative trace and (b) is the
normalized result. Each point represents the average of six neurons. (C) KA receptors-mediated whole-cell currents were not affected by
the peptides applied from the internal pipette solution in cultured hippocampal neurons. KA receptor-mediated whole-cell currents
in group of GluR6-9c (open circles) or control (solid circles) did not show significant difference during and after 10 min. (a) is a
representative trace and (b) is the normalized result. Each point represents the average of six neurons. All data here are
expressed as mean 6 S.E.M. (KA: 100 mM; GYKI 52466: 30 mM; Tat-GluR6-9c: 50 nM; GluR6-9c: 50 nM).
472
Brain (2006), 129, 465–479
and translocate to mitochondria. Since Tat-GluR6-9c could
inhibit the activation of JNK, we wondered whether the
inhibition of JNK signalling pathway could attenuate the
decreased interaction of Bax and 14-3-3, phosphorylation
of 14-3-3 and subsequently prevent Bax translocation, the
release of cytochrome c and caspase3 activation. As shown
in Fig. 5B, the inhibitory effects of Tat-GluR6-9c on the phosphorylation of 14-3-3 and the association of Bax and 14-3-3
can be observed at 6 hours reperfusion compared with the
Fig. 3 Continued
D.-S. Pei et al.
control peptide Tat-GluR6-AA-treated groups. Similar
inhibitory effects on the Bax translocation can be obtained,
the increased Bax content in the mitochondrial fraction was
attenuated by the pretreatment with Tat-GluR6-9c (Fig. 5C).
In the mitochondrial fraction cytochrome c, immunoreactivity was evident as a single band of molecular mass of
15 kDa. However, it was barely detected in the sham CA1
subregion (Fig. 5D). A significant amount of mitochondrial
cytochrome c was detected in the controls and it decreased
Neuroprotection against ischaemic brain injury by a GluR6-9c peptide
Brain (2006), 129, 465–479
473
IB: p-c-Jun
IB: c-Jun
Fig. 3 Effects of pretreatment with Tat-GluR6-9c on the increased interactions of GluR6 and MLK3 with PSD-95 and phosphorylation of
MLK3, JNK3 and c-Jun. (A) Reciprocal co-IP analysis of interactions of GluR6 and PSD-95 with MLK3. Sample proteins were
immunoprecipitated (IP) with anti-GluR6 or anti-PSD-95 or anti-MLK3 antibodies and then blotted (IB) with anti-GluR6 or anti-PSD-95
or anti-MLK3 antibody. (B) Immunoblotting (IB) analysis of the protein levels of MLK3 and p-MLK3 with anti-MLK3 or anti-p-MLK3
antibodies. (C) Phosphorylation of JNK3 was examined by IP with anti-p-JNKs antibody followed by IB with antibody against JNK3.
The protein levels of JNK3 were examined by IB with anti-JNK3 antibodies. (D) Phosphorylation of c-Jun and the protein levels of c-1 were
examined by IB with anti-p-c-Jun antibody and anti-c-Jun antibodies. Data are the mean 6 SD and were expressed as fold versus respective
sham. aP < 0.05 versus sham; bP <0.05 versus respective reperfusion groups (n = 6 rats). (E) Immunohistochemical staining of
phosphorylation of c-Jun in coronal sections of hippocampus and the inhibitory effect of Tat peptides on phosphorylation of c-Jun. Example
of immunohistochemical staining sections of the hippocampi of sham operated rats (a, e) and rats subjected to 15 min of ischaemia followed
by 6 h of reperfusion (b, f), and rats subjected to 15 min of ischaemia followed by 6 h of reperfusion with administration of 100 mg of
Tat-GluR6AA (c, g), 100 mg of Tat-GluR6-9c 40 min before ischaemia (d, h). Data were obtained from six independent animals in each
experimental group, and the results of a typical experiment are presented. Boxed areas in left column are shown at higher magnification in
right column. a, b, c, d: ·40; e, f, g, h: ·400. Scale bar in D = 200 mm; bar in H =10 mm.
at 6 h reperfusion after ischaemia, corresponding to a marked
increase in the cytosolic fraction at 6 h reperfusion (Fig. 5D).
Moreover, Tat-GluR6-9c also can inhibit the release of cytochrome c to cytosol compared with 6 h reperfusion groups
and the control peptide Tat-GluR6-AA-treated groups
(Fig. 5D). To further validate whether other mitochondrial
protein was released from mitochondria, we examined the
cytochrome c oxidase level in the cytosolic and mitochondrial
fraction using anti-cytochrome c oxidase subunit IV antibody.
The cytochrome c oxidase subunit IV was detected only in the
mitochondrial fraction, not in the cytosolic fraction in sham,
ischaemia and application of peptides, which suggests that
cytochrome c oxidase was not related with the release of
cytochrome from mitochondria.
In line with the demonstrated inhibitory effects on the
mitochondrial signal pathway, Tat-GluR6-9c treatment also
can diminish the activation of caspase-3. The activation of
caspase-3 was confirmed at 6 h reperfusion after ischaemia by
the methods of immunoblot with antibodies recognizing the
activated fragments for caspase-3 (Fig. 5E). Administration of
Tat-GluR6-9c can significantly suppress the activation of
caspase-3 induced by ischaemia/reperfusion.
Since caspase-3 plays a critical role in ischaemic neuronal
apoptosis in the hippocampal CA1 subfield, TUNEL staining
was performed to further explore the role of Tat-GluR6-9c in
ischaemia-induced apoptosis. Rats were pretreated with TatGluR6-9c or Tat-GluR6-AA by cerebral ventriclar injection
40 min before ischaemia. After 3 days reperfusion, rats were
perfusion-fixed with paraformaldehyde and TUNEL staining
was used to examine the apoptosis of CA1 pyramidal cells in
hippocampus. As shown in Fig. 5F, a significant number of
TUNEL-positive cells were observed 3 days after ischaemia
(b, f). Administration of Tat-GluR6-9c 40 min before cerebral
ischaemia significantly decreased TUNEL-positive cells (d, h).
However, as a control, Tat-GluR6-AA did not show any
protection (c, g).
Neuroprotective role of the peptides
on cerebral ischaemia in vivo and KA
stimulation in vitro
To investigate whether pretreatment of Tat-GluR6-9c would
have neuroprotection on ischaemia-induced cell death, adult
S-D rats were subjected to 15 min ischaemia followed by 5 days
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Brain (2006), 129, 465–479
D.-S. Pei et al.
Fig. 4 Effects of pretreatment with Tat-GluR6-9c on the increased protein level of Fas L in hippocampal CA1 region. (A) Effects of
pretreatment with Tat-GluR6-9c on the increase of expression of Fas L induced by 6 h of reperfusion following transient brain
ischaemia (I/R6h) in hippocampal CA1. Western blot analysis was performed to detect the protein levels of Fas L and Fas. Bands
corresponding to Fas L and Fas were scanned and the intensities were represented as fold versus sham control. Data are the mean 6 SD
and were expressed as fold versus respective sham. aP < 0.05 versus sham; bP < 0.05 versus 6 h after 15 min of ischaemia groups
(n = 6 rats). (B) Immunohistochemical staining of expression of Fas L in coronal sections of hippocampus and the inhibitory effect of Tat
peptides on expression of Fas L. Example of immunohistochemical staining sections of the hippocampi of sham operated rats (a, e) and rats
subjected to 15 min of ischaemia followed by 6 h of reperfusion (b, f), and rats subjected to 15 min of ischaemia followed by 6 h of
reperfusion with administration of 100 mg of Tat-GluR6AA (c, g), 100 mg of Tat-GluR6-9c 40 min before ischaemia (d, h). Data were
obtained from six independent animals in each experimental group, and the results of a typical experiment are presented. Boxed areas in
left column are shown at higher magnification in right column. a, b, c, d: ·40; e, f, g, h: ·400. Scale bar in D = 200 mm; bar in H = 10 mm.
reperfusion. Rats were pretreated with Tat-GluR6-9c or TatGluR6AA by cerebral ventricular injection 40 min before ischaemia. After 5 days reperfusion, rats were perfusion-fixed with
paraformaldehyde and cresyl violet staining was used to
examine the survival of CA1 pyramidal cells in hippocampus.
Results from histology indicated that normal CA1 pyramidal
cells showed round and pale stained nuclei (Fig. 6A and E),
while ischaemia-induced dead cells showed pyknotic nuclei
(Fig. 6B and F). Administration of Tat-GluR6-9c 40 min
before cerebral ischaemia significantly decreased neuronal
degeneration (Fig. 6D and H). At the same time, as the control, Tat-GluR6AA did not show any protection against the
degeneration induced by ischaemia and 5 days reperfusion
(Fig. 6C and G). The neuronal density of sham group,
ischaemia insulted group, Tat-GluR6AA and Tat-GluR6-9c
treated group were 205.0 6 27.3, 28.2 6 6.4, 33.7 6 5.1 and
93.4 6 14.6, respectively.
Since Tat-GluR6-9c could perturb the interaction of GluR6
with PSD-95 and in turn protect neurons against the ischaemic damage, we wonder if it could act as a protective trouper in KA induced neuron degeneration. Further study was
explored to investigate the role of Tat-GluR6-9c in KA
induced apoptotic neuronal death. As shown in Fig. 7,
100 mM KA in cultured hippocampal neurons induced severe
apoptotic cell death compared with sham control. However,
administration of Tat-GluR6-9c (50 nM) 1 h before kainic
acid stimulation could attenuate the apoptotic cell death
determined by DAPI staining. On the contrary, TatGluR6AA could not prevent cultured hippocampal neurons
from apoptosis induced by KA stimulation. In order to
exclude the possible role of NMDA receptor, the cultured
hippocampal neurons were pretreated with MK-801, an antagonist of NMDA receptor. The results show that MK-801
failed to rescue neurons from apoptosis induced by KA.
Discussion
Here we report for the first time that the KA receptor subunit
GluR6 plays an important role in ischaemia-induced JNK3
activation and neuronal cell death. Although studies by
Savinainen and colleagues indicated that GluR6, PSD-95
and MLK3 form a signalling module and facilitate MLK3
and JNK phosphorylation and activation (Savinainen et al.,
2001), it is still unclear whether inhibition of the assembly of
GluR6PSD-95MLK3 signalling module could attenuate
Neuroprotection against ischaemic brain injury by a GluR6-9c peptide
JNK activation and brain damage induced by ischaemia/
reperfusion. Our previous studies demonstrated that cerebral ischaemia/reperfusion facilitates the assembly of the
GluR6PSD-95MLK3 signalling module and the activation
of MLK3 and JNK3. In the present study, we construct a
peptide comprising the nine C-terminal amino acids of
GluR6 and examine the effects of the peptide on the associations of GluR6 and MLK3 with PSD-95 and the activation of
MLK3, JNK3 and c-Jun. Results of reciprocal co-IP and IB
showed that pretreatment of the peptide not only diminished
the increased interactions of GluR6 with PSD-95 and MLK3
with PSD-95, but also diminished the interaction of GluR6
with MLK3. These results suggested that the peptides could
competitively bind to the PDZ1 domain of PSD-95 and suppressed the interaction of GluR6 with PSD-95 and MLK3 in
the period of ischaemia/reperfusion. It has been reported that
JNK activation contributed to the delayed neuronal cell death
induced by cerebral ischaemia. As mentioned before, mice
that are deficient in JNK3 are resistant to the hippocampal
neurotoxic events related to administration of the glutamate
receptor agonist kainic acid. However, JNK1 or JNK2 deficient mice are not resistant to either kainic acid-mediated
seizures or neuronal death (Yang et al., 1997). These data
suggested that the different JNK isoforms regulate differential responses to neuronal insults with the JNK3 gene being
involved in glutamate excitotoxicity, an important mechanism underlining ischaemic death. These data also implicate
JNK3 signalling in a brain region that is vulnerable to
global ischaemic conditions. Our previous study suggested
that activation of JNK3 and one of its upstream molecules,
MLK3, which can activate JNK3 through a complex
(Whitmarsh et al., 1998) containing JIP1, MKK7 and JNK3
were related to neuronal death induced by the ischaemia/
reperfusion (Tian et al., 2003a, b). Current study showed
that pretreatment of the peptides could attenuate the activation of MLK3 induced by ischaemia followed by reperfusion.
It is interesting to find out that pretreatment of the peptides
could only attenuate the second peak level of JNK3 with the
30 min activation unaffected, which was consistent with our
previous study indicating DNQX, a selective non-NMDA
receptor antagonist, was inefficacious in the prevention of
30 min JNK3 activation. However, NAC, an antioxidant
reagent, could effectively inhibit JNK3 activation at 30 min
reperfusion, which suggested that 30 min activation of JNK3
was related to the generation of free radicals during the early
reperfusion (Tian et al., 2003a). Furthermore, the capability of
DNQX in the inhibition of 3d JNK3 activation fits well with
our hypothesis that KA receptor subunit GluR6 mediates the
activation of JNK3 in the ischaemia followed by reperfusion.
Since pretreatment of the peptides could inhibit the activation
of MLK3 and JNK3 induced by ischaemia/reperfusion, we
inferred that application of the peptides would also rescue
hippocampal CA1 neurons from degeneration. Results from
our current study showed that the peptides actually had
the ability to prevent hippocampal CA1 neurons from degeneration after cerebral ischaemia/reperfusion. Furthermore,
Brain (2006), 129, 465–479
475
neuroprotective role was also observed after application of
the peptides 1 h after the ischaemic insult (data not shown).
At the same time, the peptides could also protect neurons
against apoptosis induced by KA in vitro. Collectively, our
results indicate that KA receptor is involved in ischaemic
brain injury induced by transient brain ischaemia/reperfusion
and the peptides play a neuroprotective role through
inhibiting the assembly of GluR6PSD-95MLK3 signalling
module.
Recently, it has been demonstrated that Bax plays an essential role in inducing apoptosis in response to stress stimuli, as
revealed by gene disruption of Bax and of Bax and Bak
(Knudson et al., 1995; Lindsten et al., 2000; Wei et al.,
2001; Zong et al., 2001). A substantial proportion of Bax is
bound to 14-3-3 proteins in the cytosol of healthy cells. In
response to stress stimuli, Bax dissociates from 14-3-3 and
redistributes to mitochondria (Nomura et al., 2003). After
translocation to mitochondria, Bax induces cytochrome c
release either by forming a pore by oligomerization in the
outer mitochondrial membrane, or by opening other channels
(Shimizu et al., 1999; Saito et al., 2000; Kuwana et al., 2002).
Recent studies have also shown that 14-3-3 proteins prevent
apoptosis through sequestration of Bax (Samuel et al., 2001;
Nomura et al., 2003). However, JNK can promote Bax translocation to mitochondria through phosphorylation of 14-3-3
proteins (Tsuruta et al., 2004). Since Tat-GluR6-9c could
inhibit the activation of JNK, we suppose that pretreatment
of the peptides should have the ability to inhibit the phosphorylation of 14-3-3 proteins and prevent Bax translocation
to mitochondria and attenuate the release of cytochrome c
and caspase-3 activation. As a matter of fact, results from
western blot and immunohistochemistry show high fidelity
to our hypothesis. At the same time, results from TUNEL
provided strong evidence that the peptide could protect the
hippocampal CA1 neurons from apoptosis. These results
suggest that GluR6 mediated signal pathway is involved in
neuronal apoptosis induced by ischaemia/reperfusion via nonnuclear pathway, i.e. the mitochondria-dependent apoptosis
pathway.
In addition to the non-nuclear pathway, JNK could promote neuronal cell apoptosis also by regulating the activation
of some nuclear substrates such as c-Jun. In fact, studies
suggested that c-Jun plays an important role in neuronal
cell death under in vitro and in vivo conditions (Estus et al.,
1994; Ham et al., 1995). Activated JNK phosphorylates the
transcription factor c-Jun and leads to increase AP-1 transcription activity to modulate transcription of a number of
genes such as Fas ligand (Faris et al., 1998). The increased Fas L
can further activate ASK1 through binding to its receptor Fas
and in turn, the activated ASK1 phosphorylates JNK. Our
results show that pretreatment of Tat-GluR6-9c can diminish
the increased phosphorylation of c-Jun and similar results are
obtained from immunohistochemistry. Moreover, pretreatment of Tat-GluR6-9c can also significantly diminish
the increased expression of Fas L induced by ischaemia and
reperfusion. Taken together, these results suggest that GluR6
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Brain (2006), 129, 465–479
Fig. 5 Continued
D.-S. Pei et al.
Neuroprotection against ischaemic brain injury by a GluR6-9c peptide
Brain (2006), 129, 465–479
477
Fig. 5 Pretreatment with Tat-GluR6-9c attenuates the mitochondrial apoptosis-signalling pathway. (A) Time courses of the
phosphorylation of 14-3-3 and the interaction of Bax and 14-3-3 in hippocampal CA1 derived from sham-treated rats or rats at various time
points after 15 min of ischaemia. (B) Effects of pretreatment with Tat-GluR6-9c on the increased phosphorylation of 14-3-3 and the
decreased interaction of Bax and 14-3-3 induced by transient brain ischaemia followed by 6 h reperfusion (I/R6h) in hippocampal CA1.
(C) Effects of pretreatment with Tat-GluR6-9c on the increased Bax translocation to mitochondria induced by transient brain ischaemia
followed by 6 h reperfusion (I/R6h) in hippocampal CA1. (D) Effects of pretreatment with Tat-GluR6-9c on the redistribution of
cytochrome c in cytosol and mitochondria induced by transient brain ischaemia followed by 6 h reperfusion (I/R6h) in hippocampal CA1.
Cytochrome oxidase 4 (COX4) was strongly expressed in the mitochondrial fraction and did not decrease after ischaemia, but virtually no
immunoreactivity was seen in the cytosolic fraction in both the sham and ischaemic CA1 subregions. (E) Effects of pretreatment with TatGluR6-9c on the increased expression of cleaved caspase-3 in cytosol induced by transient brain ischaemia followed by 6 h of reperfusion
(I/R6h) in hippocampal CA1. Bands corresponding to 14-3-3, Bax, cytochrome c, cytochrome oxidase 4 and activated caspase-3 were
scanned and the intensities were represented as folds versus sham control. Data are the mean 6 SD and were expressed as folds versus
respective sham. aP < 0.05 versus sham; bP < 0.05 versus 6 h after 15 min of ischaemia group (n = 6 rats). (F) Representative hippocampal
photomicrographs of TUNEL staining counterstained with methyl green. Rats were subjected to sham operation (a, e) and 15 min of
ischaemia followed by 3 days of reperfusion (b, f), and to 15 min of ischaemia followed by 3 days of reperfusion with administration of 100
mg of Tat-GluR6AA (c, g), 100 mg of Tat-GluR6-9c 40 min before ischaemia (d, h). Data were obtained from six independent animals and
the results of a typical experiment are presented. Boxed areas in left column are shown at higher magnification in right column.
a, b, c, d: ·40; e, f, g, h: · 400. Scale bar in D = 200 mm; scale bar in H = 10 mm.
mediated signal pathway is involved in neuronal apoptosis
induced by ischaemia/reperfusion via nuclear pathway.
In recent years, more attention was paid to the Tat-peptides
as therapy for ischaemic brain damage. In the recent study, a
peptide comprising the nine C-teminal amino acids of NMDA
receptor was used to treat stroke by perturbing the interactions between NMDA receptor and PSD-95 (Aarts et al.,
2002). Another study showed that a peptide comprising
the JNK binding motif (JBD) of JIP-1 and the Tat transporter
sequence has an extremely potent protective role in vivo
against cerebral ischaemia (Borsello et al., 2003). In general,
ionotropic glutamate receptors can control influx or outflux
of the cations; on the other hand, it also mediates the intracellar signal transduction via the interaction of the receptors
and signal proteins. However, studies indicated that disruption of the NMDA receptor-PSD-95 interaction in hippocampal neurons did not affect the physiological role of the NMDA
receptor (Aarts et al., 2002; Lim et al., 2003). In our study, the
GluR6 C-terminus containing peptides also show neuroprotection in vivo and in vitro without affecting the currents of
KA receptors. On the other hand, the interaction of GluR6
and PSD-95 but not that of NMDA and PSD-95 was affected
by Tat-GluR6-9c, which rendered the peptides relative high
specificity. Thus, these peptides can be used as non-receptor
competitive antagonists to block the glutamate receptormediated downstream signalling cascade and subsequently
prevent delayed neuron death induced by cerebral
ischaemia. In contrast to glutamate receptor antagonists,
one distinct advantage of the peptides was blocking the
receptor-mediated downstream events without affecting the
normal function of the receptors. Second, membrane transduction domain of the HIV-1 was fused to these blocking
peptides, rendering them cell permeable, and the unique
sequence of these peptides ensured the specific interaction
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Brain (2006), 129, 465–479
Fig. 6 Example of cresyl violet-stained sections of the hippocampi
of sham operated rats (A, E) and rats subjected to 15 min of
ischaemia followed by 5 days of reperfusion (B, F), and rats
subjected to 15 min of ischaemia followed by 5 days of reperfusion
with administration of the control peptides (C, G), 100 mg/10 ml
of Tat-GluR6-9c 40 min before ischaemia (D, H). Data were
obtained from six independent animals and the results of a typical
experiment are presented. Boxed areas in left column are shown
at higher magnification in right column. A, B, C, D: ·40; E, F, G, H:
·400. Scale bar in D = 200 mm; scale bar in H = 10 mm.
with corresponding receptors. It is also interesting to investigate the effect of the neuroprotection by combining these
blocking peptides together.
Lack of selective antagonists hampered research on KA
receptors for many years. To date, the physiological and
pathological function of KA receptors still remains unclear.
Recently, with the discovery of selective AMPA receptor and
KA receptor subunit GluR5 antagonists (Paternain et al.,
1995; Clarke et al., 1997), much progress had been made
on understanding the functional properties of KA receptors.
Cumulative studies showed that KA receptors were more
abundant in CA3 than in CA1 subfields of the hippocampus
(Bureau et al., 1999), while studies indicate that degenerating
neurons induced by global ischaemia/reperfusion are typically
found at the CA1 subfields of the hippocampus. Then, a
question raised on how to explain the selective injury in
CA1 during ischaemia, when GluR6 is more abundant in
CA3. The possible explanation about the discrepancy may
be attributed to existence of the anti-apoptotic proteins in
CA3/DG region, since cell survival is determined by a balance
between survival and death signalling pathways. Our previous
study indicated that ERK (Wang et al., 2005), which is considered as anti-apoptotic kinase, are selectively activated in
CA3/DG region but not in CA1. Therefore, these antiapoptotic proteins may inhibit the GluR6–mediated JNK
activation and neuronal cell death in CA3 region of the
D.-S. Pei et al.
Fig. 7 Effects of Tat-GluR6AA and Tat-GluR6-9c on apoptosis-like
cell death induced by KA stimulation. DAPI staining was carried
out at 24 h after KA stimulation. Typical apoptotic cells with
condensed nucleus are marked by arrow heads. Cells of group of
sham control were restored for 24 h. Apoptotic-like cells were
expressed as percent of total cells counted in 10 microscopic
fields (·400) for DAPI staining. (A) Typical photographs showing
DAPI staining of inhibitory effects of Tat-GluR6-9c on KA induced
apoptosis-like cell death. Neurons were treated with 50 nM TatGluR6AA or Tat-GluR6-9c 1 h before KA stimulation or MK-801
20 min before KA stimulation. (B) Quantitative representations
expressed as percentage of total cells counted in 10 microscopic
fields (·400) for DAPI staining. aP < 0.05 versus group of sham,
b
P < 0.05 versus group of KA stimulation. (KA: 100 mM KA +
30 mM GYKI 52466; MK-801: 20 mM)
hippocampus. In our present study, pretreatment of the peptide could prevent GluR6-mediated JNK3 activation and rescue the neurons from degeneration in CA1 subfields in
response to cerebral ischaemia, which indicated that KA
receptor subunit GluR6 participated in the neuronal injury
of CA1 subfields in cerebral ischaemia.
Taken together, our results indicate that cerebral
ischaemia/reperfusion induced the increased assembly of
the GluR6PSD-95MLK3 signalling module and subsequently activated MLK3 and JNK3. GluR6-containing KA
receptors participated in the neuronal cell death induced by
cerebral ischaemia/reperfusion in rat hippocampal CA1
regions. Application of a GluR6 c-terminus containing peptide could suppress the clustering of GluR6 in the postsynaptic regions by competitively binding to the PDZ1
domain of PSD-95 and subsequently inhibit the assembly
of the GluR6PSD-95MLK3 signalling module, which
Neuroprotection against ischaemic brain injury by a GluR6-9c peptide
results in declined activation of MLK3, JNK3 and c-Jun.
Therefore, the peptides could inhibit the increased expression
of Fas L via the nuclear-pathway and attenuate the increased
release of cytochrome c via the mitochondria-dependent nonnuclear-pathway, which ultimately eliminates the activation
of caspase-3. Most important, the peptides have a neuropotective effect on ischaemic brain damage in vivo and on
KA-induced excitotocity in vitro, thus, GluR6 c-terminus containing peptide provides a promising therapeutic approach
for ischaemic brain injury.
Acknowledgement
This work was supported by a grant from the key project
of the National Natural Science Foundation of China
(No. 30330190).
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