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available at www.sciencedirect.com
www.elsevier.com/locate/brainres
Research Report
Inhibition of cerebral ischemia/reperfusion-induced injury by adenovirus expressed C-terminal amino acids of GluR6 Ting Li a,1 , Hong-Min Yu c,1 , Ya-Feng Sun a , Yuan-Jian Song a , Guang-Yi Zhang a , Dong-Sheng Pei a,b,⁎ a
Research Center for Biochemistry and Molecular Biology, Xuzhou Medical College, Xuzhou 221002, China Laboratory of Biological Cancer Therapy, Xuzhou Medical College, Xuzhou 221002, China c Laboratory Center of Department of Public Health, Xuzhou Medical College, Xuzhou 221002, China b
A R T I C LE I N FO
AB S T R A C T
Article history:
GluR6 kainate receptor subunit is largely expressed in hippocampus of brain regions and
Accepted 1 September 2009
plays an important role in brain ischemia/reperfusion-mediated neuronal cell death. Our
Available online 9 September 2009
previous researches have shown that cerebral ischemia/reperfusion could facilitate the assembly of GluR6 and postsynaptic density protein 95(PSD95) as well as mixed lineage
Keywords:
kinase 3(MLK3) and further induce the activation of c-Jun NH2-terminal kinase 3(JNK3),
GluR6
leading to neuronal death of hippocampal CA1. Here, we show that over-expression of C-
Over-expression
terminal amino acids of GluR6 can interrupt the combination of GluR6 with PSD95, inhibit
JNK
the assembly of GluR6·PSD-95·MLK3 signaling module, suppress the activation of JNK3 and
Ischemia
the downstream signaling pathway. Thus, our results imply that over-expression of Cterminal amino acids of GluR6 induce neuroprotection against ischaemic brain injury in rat hippocampal CA1 region via suppressing proapoptosis signaling pathways, which can be an experimental foundation for gene therapy of stroke. © 2009 Elsevier B.V. All rights reserved.
1.
Introduction
Excitotoxicity is regarded as the main mechanism leading to neuronal death in stroke. The ionotropic glutamate receptors, which are involved in the neuronal excitotoxicity, are pharmacologically divided into N-methyl- D -aspartate (NMDA),α-amino-3-hydroxy-5-methyl-4-isoxazole propionate (AMPA) and kainate (KA) receptors. Since NMDA and AMPA receptors have been widely researched, the physiological roles of KA receptors have begun to be delineated. KA receptors
have five different subunits identified: GluR5, GluR6, GluR7, KA1, and KA2, which are expressed in distinct patterns in different areas of the hippocampus (Bureau et al., 1999). GluR6 is largely expressed in brain regions involved in learning and memory, for example the CA1 and CA3 of the hippocampus (Darstein et al., 2003). Fisahn (2005) concluded that the KAR GluR6 was involved in mediating kainate-induced excitation in the hippocampal network. GluR6 kainate receptor subunit is a major subunit which can take part in native heteromeric complexes by co-assembly with other kainate receptor
⁎ Corresponding author. Laboratory of Biological Cancer Therapy, Xuzhou Medical College, 84 West Huai-hai Road, Xuzhou, Jiangsu, 221002, P.R. China E-mail address:
[email protected] (D.-S. Pei). Abbreviations: Ad, adenovirus; GluR6, glutamate receptor 6; KA, kainic acid; AMPA, α-amino-3-hydroxy-5-methyl-4-isoxazole propionate; JNK, c-Jun N-terminal kinase; MAPK, mitogen-activated protein kinase; MKK4/7, mitogen-activated kinase kinase 4/7; MLK3, mixed lineage kinase 3; NMDA, N-methyl-D-aspartate; PSD-95, postsynaptic density protein 95; SD, Sprague-Dawley 1 The first two authors contribute equally to this work. 0006-8993/$ – see front matter © 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.brainres.2009.09.002
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subunits (Paternain et al., 2000; Martin et al., 2007). Each GluR6 subunit is composed of a long extra-cellular N-terminal glutamate-binding pocket domain, which includes S1 region in cooperation with the S2 region, three trans-membrane domains (TM1, TM3 and TM4), a re-entrant membrane loop (M2), and of C-termini on the cytoplasmic side (Kornreich
et al., 2007). Studies have shown that GluR6 C terminus could be disturbed to influence assembling of the downstream proteins. When the C-terminal region of GluR6 is deleted, the protein is not associated with the synapse-associated protein PSD95/SAP90 (Strutz-Seebohm et al., 2006). The last four residues of the GluR6 C terminus (E-T-M-A-oh) were
Fig. 1 – Over-expression of C-terminal amino acids of GluR6 improves the survival SD rat hippocampal CA1 neurons induced by 15 min of ischemia followed by 5 days of reperfusion. (A) Example of cresyl violet-stained sections of the hippocampi of sham operated rats (a, e), rats subjected to 15 min of ischemia followed by 5 days of reperfusion (b, f), rats subjected to 15 min of ischemia followed by 5 days of reperfusion with administration of the Ad-GFP (c, g) and Ad-GluR6c-GFP 5 days before ischemia (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 than 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. (B) Cell density was expressed as the number of cells per 1 mm length of the CAI pyramidal cells counted under a light microscope. Data were the mean ± S.D.(n = 6). (a) P < 0.05 versus sham; (b) P < 0.05 versus Ad-GFP groups.
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shown to be responsible for the association with the PDZ1 domain of PSD95 (Savinainen et al., 2001). Our previous researched have shown that KA receptor GluR6 subunit played an important role in brain ischemia/reperfusion-mediated neuronal cell death, via assembling postsynaptic density protein (PSD-95) as well as mixed lineage kinase 3 (MLK3) and activating the downstream signaling pathway (Tian et al., 2005). It is reported in our previous studies that cerebral ischemia/reperfusion facilitated the association of PSD-95 with GluR6 and MLK3 and further induced the activation of c-Jun NH2-terminal kinase 3(JNK3) (Gu et al., 2001; Tian et al., 2005), MLK3 could directly bind and activate mitogen-activated kinase kinase 4(MKK4) and mitogen-activated kinase kinase 7 (MKK7), which in turn could phosphorylate and activate JNKs (Wang et al., 2004). JNK3, belonging to the mitogen-activated protein kinase (MAPK) family, is found predominately in neurons. Studies show that JNK3 were activated and implicated in neuronal degeneration in response to ischemic insult (Gu et al., 2001). JNK3 exerts its proapoptotic actions mainly through the two different possible pathways. On one hand, the activated JNK3 translocates into the nucleus and phosphorylates the transcription factor c-Jun. The activated JNK may enhance the expression of Fas ligand(Fasl), which could ultimately contribute to Fas receptor-mediated neuronal death. On the other hand, partial activated JNK remains in cytosol and regulates the activation of Bcl-2 family members. It is possible that activated JNK may increase mitochondrial membrane permeability, via regulating the activation of some Bcl-2 family members, and the subsequent release of apoptogenic factors, which could ultimately contribute to mitochondria-mediated neuronal death (Guan et al., 2006). Based on the above, brain ischemia/reperfusion can activate GluR6, facilitate its assembly with PSD95 and MLK3, further activate the JNK3 signaling pathway and lead to the neuronal death of hippocampal CA1. In this study, we wonder whether it could perturb the combination of GluR6 and PSD95 as well as MLK3, via applying the recombinant Ad(adenovirus)-GluR6c(C-terminal amino acids of GluR6)-GFP constructs.
2.
Results
2.1. Over-expression of GluR6c increases neuronal survival after cerebral ischemia/reperfusion We examined whether Ad-GluR6c-GFP could be expressed in Sprague–Dawley rat hippocampal CA1. Ad-GluR6c-GFP was administrated to adult Sprague–Dawley rats through hippocampal CA1 injection. After 5 days, rats were perfusion-fixed with paraformaldehyde, followed by preparation of coronal sections 5 μm thick using a microtome and their fluorescence of hippocampi CA1 neuronal was visualized by fluoroscope. CA1 treated with Ad-GluR6c-GFP exhibited strong fluorescence, indicating GluR6c-GFP was expressed (data not shown). AdGluR6c-GFP was administrated to the adult Sprague–Dawley rats through hippocampal CA1 injection 5 days before 15 min ischemia. After 5 days of reperfusion, hippocampal CA1 neuronal death was detected by histological methods. Rats were perfusion-fixed with paraformaldehyde and cresyl violet staining was used to examine the survival of CA1 pyramidal
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cells of hippocampus. Results from histology indicated that normal CA1 pyramidal cells showed round and pale stained nuclei (Fig. 1Aa and e), while ischemia-induced dead cells showed pyknotic nuclei (Fig. 1Ab and Af). Administration of AdGluR6c-GFP 5 days before cerebral ischemia significantly decreased neuronal degeneration (Fig. 1Ad and Ah), at the same time, as a control, Ad-GFP did not show any protection against the degeneration induced by ischemia and 5 days reperfusion (Fig. 1Ac and Ag). The numbers of surviving pyramidal cells in CA1 region of sham group, ischemia insulted group, Ad-GFP and Ad-GluR6c-GFP treated group were 208.7 ± 16.8, 43.3 ± 6.1, 37.3 ± 7.6, 118.0 ± 14.0, respectively (Fig. 1B).
2.2. Over-expression of GluR6c suppresses the assembly of the GluR6·PSD-95·MLK3 signaling module Since cerebral ischemia/reperfusion can facilitate the assembly of GluR6·PSD-95·MLK3 signaling module, we wonder if over-expression of GluR6c could disturb the interaction of GluR6 and PSD-95 as well as MLK3. In our previous study, we proved that the interaction of GluR6 and MLK3 with PSD-95 reached its peak level at 6 h reperfusion after 15 min ischemia. Then, Ad-GluR6c-GFP was administrated 5 days before 15 min ischemia, immunoprecipitation (IP) and immunoblotting (IB) were used to examine the association of GluR6 and MLK3 with PSD-95 after 6 h reperfusion. As shown in Fig. 2, the interaction of GluR6 and MLK3 with PSD-95 increased after 15 min ischemia followed by 6 h reperfusion. Administration of AdGluR6c-GFP 5 days prior to ischemia diminished the increased interaction of GluR6, MLK3 and PSD-95, while the protein levels of GluR6, PSD-95 and MLK3 have no remarkable variation. On the contrary, the same dose of Ad-GFP did not significantly affect the association of GluR6, MLK3 and PSD-95.
2.3. Over-expression of GluR6c decreases the phosphorylation of MLK3, MKK4/7 and JNK3 Our previous study indicated that MLK3, an upstream kinase of JNK, could be activated via the combination with PSD95 as well as GluR6. To demonstrate whether downstream proteins of GluR6 were also affected by the effect of over-expression of GluR6c, Ad-GluR6c-GFP was administrated to observe the variation of phosphorylated MLK3, MKK4/7 and JNK3. In our previous study, we found that the phosphorylation of JNK3 peaked at 3 days after ischemia. As indicated in Fig. 3A, B, C, D, results of Western blotting revealed that the injection of AdGluR6c-GFP suppressed p-MLK3, p-MKK4, p-MKK7 and p-JNK3, while the protein expression of MLK3, MKK4, MKK7 and JNK3 kept unchanged.
2.4. JNK-mediated nucleic and mitochondrial pathways were involved in the neuroprotective effects of GluR6c Over-expression On one hand, the activation of JNK3 can activate nucleus substrate c-jun and may enhance the expression of Fasl, which can ultimately contribute to Fas receptor-mediated neuron death. On the other hand, partial activated JNK remains in cytosol and regulates the activation of non-nuclear substrates including Bcl-2 family members and facilitate the release of
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apoptogenic factors, which could ultimately contribute to mitochondria-mediated neuron death. As shown in Fig. 4A, B, results of Western blotting revealed that the administration of Ad-GluR6c-GFP significantly decreased the phosphorylation of c-Jun, Bcl-2 and the expression of FasL, while the protein levels of c-Jun, Bcl-2 and Fas have no remarkable variation. The same dose of Ad-GFP also has no remarkable change, compared to the group of 6 h reperfusion after 15 min ischemia.
3.
Fig. 2 – Effects of pretreatment with Ad-GluR6c-GFP on the ischemia/reperfusion-induced increased interactions of GluR6 and MLK3 with PSD-95. (A–C) Reciprocal co-IP analysis of interactions of GluR6 and PSD-95 with MLK3. Sample proteins were immunoprecipitated (IP) with anti-GluR6 or anti-PSD95 or anti-MLK3 antibodies and then blotted (IB) with anti-GluR6 or anti-PSD95 or anti-MLK3 antibody. (D)Data are the mean ± SD and were expressed as fold versus sham. a P < 0.05 versus sham; bP < 0.05 versus reperfusion groups (n = 4 rats).
Discussion
Our previous studies have heavily reported that brain ischemia/reperfusion could activate the excitative glutamate receptors GluR6, facilitate the assembly with PSD95 and MLK3, further activate JNK signaling pathway, leading to the neuronal death. In order to save neurons from ischemia/ reperfusion-induced death, we tried ways to perturb the combination of GluR6 with PSD95, such as the injection of GluR6 antisense oligodeoxynucleotides (Pei et al., 2005), the reduction of GluR6 S-nitrosylation (Yu et al., 2008) and the administration of Tat-GluR6-9c (a GluR6 C-terminus containing peptide conjugated to tat peptide) (Pei et al., 2006 ). In this study, we reported for the first time that over-expression of GluR6c by recombinant Ad-GluR6c-GFP could inhibit the binding of GluR6 with PSD-95 and prevent the activation of the downstream JNK signaling pathway, resulting in the protection against rat ischaemic brain injury. GluR6 is largely expressed in brain regions and exert vital effect in ischemia/reperfusion-induced neuronal death. Genetic deletion of GluR6 has revealed important roles in synaptic transmission and plasticity in the hippocampus (Contractor et al., 2000; Huettner, 2001). Prevention of GluR6 and its downstream signaling pathway can be valid to reduce ischemic brain injury. GluR6 antisense oligodeoxynucleotides (ODNs) cannot only inhibit the expression of GluR6 but also prevent the assembly of the GluR6-PSD-95-MLK3 signaling module (Pei et al., 2005). Tat-GluR6-9c, a GluR6 C-terminus containing peptide conjugated to tat peptide, could interfere in the interaction of GluR6 with PSD95 and decrease the assembly of the GluR6-PSD95-MLK3 signaling module (Pei et al., 2006). Brain ischemia/reperfusion-induced damage could be alleviated by lessened S-nitrosylation of GluR6 and reductive assembly of the GluR6-PSD-95-MLK3 signaling module[14]. Actinfilin, a synaptic member of the BTB-Kelch protein family, is able to bind to GluR6. Since actinfilin acts as a substrate adaptor, binding Cullin 3 (Cul3) and linking GluR6 to the E3 ubiquitin–ligase complex, GluR6 can be ubiquitinated and decreased by actinfilin overexpression (Salinas et al., 2006). From the above, we know that GluR6 can be regulated and it promote our choice of GluR6c (C-terminal amino acids of GluR6). It is reported that over-expression of GluR6 appears to produce seizures and spontaneous non-synaptic bursting (Telfeian et al., 2000). However, in the current research, we promote the over-expression of GluR6c, but not GluR6. Evidence has shown that GluR6c completely lost the ability to conduct ions when activated by kainate or glutamate (Motazacker et al., 2007). So the over-expression of GluR6c will not act as GluR6, and will not activate the downstream signaling pathway, but only occupy the PDZ1 region of
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PSD95. The C-terminus of GluR6 plays an important role in the organization and electrophysiological properties of the receptor. It is reported that PKA could phosphorylate the C-terminal tail of GluR6 and lead to potentiation of whole cell response (Kornreich et al., 2007).When the C-terminal region of GluR6 is deleted, the protein will not associate with PSD95, which might result in reduced targeting of the receptor to the synapse (Strutz-Seebohm et al., 2006). It is well known that PSD-95 plays an important role in signal transduction by linking special receptors to downstream signal-transducing proteins and regulating the signal enzymes activity. Our recent study have shown that neuronal apoptosis is prevented by overexpression of PDZ1, which inhibited the binding of GluR6 with PSD-95(Hu et al., 2009). It was reported that PSD-95 binds GluR6 via its PDZ1 domain, while PSD-95 associates with MLK but not nNOS (Garcia et al., 1998). Evidence has also provided that PSD-95 links GluR6 to JNK by anchoring MLK2 or MLK3 (Savinainen et al., 2001). JNK appears to be another particularly important protein of this apoptotic signaling pathway. JNK can be activated by its upstream kinases with phosphorylation and in turn phosphorylates the transcription factor c-Jun and mitochondrial Bcl-2 family members (Kallunki et al., 1996; Liu et al., 1996). It is reported that JNK3 knockout mice exhibit resistance to neuronal degeneration and seizure induced by kainic acid (Yang et al., 1997). In previous studies, inhibition of JNK via overexpression of the JNK binding domain of JIP-1 (JBD) can prevent apoptosis in sympathetic neurons (Harding et al., 2001). In summary, we show that over-expression of the GluR6c in hippocampal CA1 region interrupts the assembly of GluR6PSD-95-MLK3 signaling module, inhibits the phosphorylation of JNK and the downstream signaling pathway, leading to the increased survival of neurons. Most of all, over-expression of the GluR6c protects neuronal cell from ischemic brain damage, which provides an experimental foundation for gene therapy of stroke.
4.
Experimental procedures
4.1.
Antibody and reagents
Goat polyclonal anti-GluR6 (sc-7618), mouse monoclonal antip-JNKs (sc-6254), rabbit polyclonal anti-MLK3 (sc-13072), rabbit
Fig. 3 – Effects of pretreatment with Ad-GluR6c-GFP on the ischemia/reperfusion-induced increased MLK3-MKK4/7-JNK3 signaling module in hippocampal CA1 region. (A) Immunoblotting (IB) analysis of the protein levels of MLK3 or p-MLK3 with anti-MLK3 or anti-p-MLK3 antibodies. Phosphorylation of MKK4/7 and the protein levels of MKK4/7 were examined by IB with anti-p-MKK4/7 antibody and anti-MKK4/7 antibodies. (C) Phosphorylation of JNK3 was examined by IP with anti-JNK3 antibody followed by IB with antibody against p-JNKs. The protein levels of JNK3 were examined by IB with anti-JNK3 antibodies.(B, D) Data are the mean ± SD and were expressed as fold versus sham. a P < 0.05 versus sham; bP < 0.05 versus reperfusion groups (n = 4 rats).
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donkey anti-goat IgG were from Sigma. pAdTrack-CMV vector and pAdEasy-1 plasmid were kept in our laboratory. Other chemicals used in our experiment were all acquired from Sigma.
4.2.
Recombinant adenoviral vectors
Recombinant Ad (adenovirus)-GluR6c(C-terminal amino acids of GluR6)-GFP constructs were produced according to standard techniques. The pAd Track CMV vector is bicistronic, expressing both GFP and the GluR6c domain. Briefly, GluR6c(852-908 amino acid of GluR6) was generated by polymerase chain reaction of the appropriate GluR6c coding region to incorporate flanking BglΠ and HindШ sites followed by ligation into the Ad shuttle vector pAdTrack-CMV digested with BglΠ and HindШ. The resultant plasmid is linearized by digesting with restriction endonuclease PmeІ, and subsequently cotransformed into Escherichia coli. BJ5183 cells with an adenoviral backbone plasmid pAdEasy-1. Recombinants are selected for kanamycin resistance, and recombination confirmed by restriction endonuclease analyses. Finally, the linearized recombinant plasmid is transfected into adenovirus packaging cell lines HEK 293 cells, human embryonic kidney cells. Recombinant adenoviruses are typically generated within 7 to 12 days and purified and tittered.
4.3.
Fig. 4 – Effects of pretreatment with Ad-GluR6c-GFP on the ischemia/reperfusion-induced increased p-c-Jun, p-Bcl-2 and FasL. (A) Phosphorylation of c-Jun or Bcl-2 and the protein levels of c-Jun or Bcl-2 were examined by IB with anti-p-c-Jun or anti-p-Bcl-2 antibody and anti-c-Jun or anti-Bcl-2 antibodies. Western blot analysis was performed to detect the protein levels of Fas L and Fas. (B) Bands were scanned and the intensities were represented as fold versus sham control. Data are the mean ± SD from four independent experiments (n = 4). aP < 0.05 versus sham; bP < 0.05 versus reperfusion groups.
polyclonal anti-p-c-Jun(sc-16312-R), rabbit polyclonal anti-Fas (sc-716), rabbit polyclonal anti-c-Jun (sc-1694) were purchased from Santa Cruz Biotechnology. Rabbit polyclonal anti-FasL (#4273), rabbit polyclonal anti-MKK7(#4172), rabbit polyclonal anti-p-MKK7(#4171) and Rabbit polyclonal anti-p-MLK3(#2811) were obtained from Cell Signaling Biotechnology. Rabbit polyclonal anti-GluR6 antibody (#06-309) was obtained from Upstate Biotechnology. Mouse monoclonal anti-PSD95 (p-246) was bought from Sigma. The secondary antibodies used in our experiment were goat anti-mouse IgG, goat anti-rabbit IgG and
Ischemic model
Adult male Sprague–Dawley rats weighing 220–250 g were used. The experimental procedures were approved by the local legislation for ethics of experiments on animals. Transient brain ischemia was induced by four-vessel occlusion (4-VO) as described before (Pulsinelli and Brierley, 1979). Briefly, rats were anesthetized with chloral hydrate (300 mg/ kg, i.p.) and both vertebral arteries were occluded permanently by electrocautery. On the following day, both carotid arteries were occluded with aneurysm clips to induce cerebral ischemia. After 15 min of the occlusion, the aneurysm clips were removed for reperfusion. The rats which lost their righting reflex within 30 s, and their pupils were dilated and unresponsive to light, were selected for the experiments. The rats with seizures were discarded. An EEG was monitored to ensure isoelectricity after carotid artery occlusion. Rectal temperature was maintained at about 37 °C during the whole procedure. Sham control were performed using the same surgical procedures except for occlusion of the carotid artery.
4.4. Adenovirus-mediated GluR6c-GFP or GFP gene transfer 10 μl Ad-GluR6c-GFP or Ad-GFP (1010 pfu) were administrated to the rats 5 days before ischemia through hippocampi CA1 injection (anteroposterior, 3.6 mm; lateral, 2.0 mm; depth, 4.0 mm from bregma).
4.5.
Sample preparation
Rats were decapitated immediately after different time of reperfusion and then the hippocampal CA1 were isolated and quickly frozen in liquid nitrogen. The hippocampal CA1 were
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homogenized in ice-cold homogenization buffer containing 50 mM 3-(Nmorpholino) propanesulfonic 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, 20 mM sodium pyrophosphate, 20 mM β-phosphogrycerol, 1 mM p-nitrophenyl phosphate (PNPP), 1 mM benzamidine, 1 mM phenylmethylsulfonyl (PMSF) and 5 μg/ml each of leupeptin, aprotinin, pepstatin A. The homogenates were centrifuged at 800×g for 10 min at 4 °C. Protein concentration was determined by the methods of Lowry et al. (1951). Samples were stored at −80 °C until use. When we examined p-Bcl-2 and Bcl-2, 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 800g for 10 min at 4°C. The pellets were discarded, and supernatants were centrifuged at 17,000g for 20 min at 4°C to obtain 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.
4.6.
Nuclear extraction
When we examined p-c-Jun and c-Jun, the homogenates were centrifuged at 800g for 10 min at 4 °C. Supernatants as 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 centrifuged at 12,000g 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 until used.
4.7.
Immunoprecipitation
Samples (400 μg of protein) were diluted four-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 μl protein A sepharose CL-4B at 4 °C, and then centrifuged to remove proteins that had adhered nonspecifically to protein A. The supernatants were incubated with 1–2 μg 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 buffer for three-times. Bound proteins were eluted by boiling at 100 °C for 5 min in SDS-PAGE loading buffer and then isolated by centrifuge. The supernatants were used for immunoblot analysis.
4.8.
Western blot analysis
Proteins (100 μg) were separated on 7.5% polyacrylamide gels and then electrotransferred onto nitrocellulose membrane. After blocking for 3 h with 3% bovine serum albumin (BSA) in
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Tris-buffered saline with 0.1% Tween-20 (TBST), membranes were incubated over night 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 with NBT/BCIP color substrate. The densities of the bands on the membrane were scanned and analyzed with LabWorks image analysis software (UVP Upland, CA, USA).
4.9.
Histological assessment
Rats were perfusion-fixed with 4% paraformaldehyde in 0.1 M sodium phosphate buffer (pH 7.4) under anesthesia after 5 days of reperfusion. Brains were removed quickly and further fixed with the same fixation solution overnight at 4 °C overnight. Post-fixed brains were embedded by paraffin, followed by preparation of coronal sections (5 μm thick) using a microtome. The paraffin embedded brain sections were deparaffinized with xylene and rehydrated with 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.
4.10.
Toxicity observation
The animal weight was measured and there were no significant differences among the groups. No gross abnormalities were noticed in Ad-GluR6c-GFP treated rats. In addition, there were no significant pathologic alterations in liver, lung, kidney, spleen or heart as revealed by H&E histological staining.
4.11.
Statistical evaluation
Data were presented as means ± S.D. from at least six independent rats. Statistical analysis of the results was performed by one-way analysis of variance (ANOVA) followed by the Duncan's new multiple range method. P-values < 0.05 were considered significant.
Acknowledgments This work was supported by a grant from the Project of the National Natural Science Foundation of China (No. 30800309). President Special Grant of Xuzhou Medical College (08KJZ02) and Natural Science Research Funds of Jiangsu Province (No. BK2006536 and BK2006035).
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