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Suppression of postsynaptic density protein 95 by antisense oligonucleotides diminishes postischemic pyramidal cell death in rat hippocampal CA1 subfield
Neuroscience Letters 385 (2005) 230–233
Suppression of postsynaptic density protein 95 by antisense oligonucleotides diminishes postischemic pyramidal cell death in rat hippocampal CA1 subfield Xiao-Yu Hou, Guang-Yi Zhang ∗ , De-Guang Wang, Qiu-Hua Guan, Jing-Zhi Yan Research Center for Biochemistry and Molecular Biology, Xuzhou Medical College, University of Science & Technology of China, 84 West Huai-hai Road, Xuzhou, Jiangsu 221002, PR China Received 6 April 2005; received in revised form 4 May 2005; accepted 14 May 2005
Abstract Our previous investigation has shown that postsynaptic density protein 95 (PSD-95) is critical for the Src family kinases-mediated tyrosine phosphorylation of N-methyl-d-aspartate receptor subunit 2A (NR2A) in the postischemic hippocampus. To clarify the roles of PSD-95 in the ischemic brain damage, histological method was performed to examine the effects of PSD-95 antisense oligonucleotides (AS) on the postischemic delayed cell death in rat hippocampus. Transient (15 min) brain ischemia was induced by the four-vessel occlusion method in Sprague–Dawley rats. Five days of reperfusion following brain ischemia (I/R5d) led to hippocampal CA1 pyramidal cell death upward of 90%. Intracerebroventricular infusion of AS (every 24 h for 3 days before ischemia) not only decreased the PSD-95 expression but also increased the number of surviving pyramidal neurons, while missense oligonucleotides (MS) had no effects. To further investigate the mechanisms underlying the neuroprotection of PSD-95 deficiency, the interaction of proline-rich tyrosine kinase 2 (Pyk2) with NR2A as well as autophosphorylation (Tyr402) of Pyk2 were detected. Immunoprecipitation and immunoblot analysis showed that preischemic treatment with AS, but not MS or vehicle, attenuated the I/R6h-induced increases in Pyk2–NR2A association and Pyk2 autophosphorylation. The protein levels of NR2A and Pyk2 had no differences under the above conditions. Our data suggest that the recruitments of ion channels and signaling molecules may be involved in the PSD-95 neurotoxicity in the postischemic hippocampus. © 2005 Elsevier Ireland Ltd. All rights reserved. Keywords: Brain ischemia; Neuroprotection; Postsynaptic density protein 95; N-methyl-d-aspartate receptor; Proline-rich tyrosine kinase 2
Postsynaptic density protein 95 (PSD-95), also known as synapse-associated protein 90 (SAP90), is a predominant kind of scaffold proteins concentrated in glutamatergic postsynaptic density. Although accumulating evidence has indicated that PSD-95 is involved in the ischemic events [1,5–7,15], the exact roles of PSD-95 in the postischemic neuronal cell death have not been elucidated. PSD-95 proteins associate NR2 subunits of N-methyl-daspartate (NMDA) receptors with an array of protein tyrosine kinases (PTKs) including Src family of kinases and prolinerich tyrosine kinase 2 (Pyk2) [18,19]. Src family kinases belong to the family of membrane-associated non-receptor
∗
Corresponding author. Tel.: +86 516 583 4231; fax: +86 516 574 8486. E-mail address: [email protected] (G.-Y. Zhang).
0304-3940/$ – see front matter © 2005 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.neulet.2005.05.054
PTKs. Within nine of Src family members, Src and Fyn kinases are highly expressed in the central nervous system. Electrophysiological studies have shown that NR2A tyrosine phosphorylation by Src family kinases enhances NMDA channel responses [9]. The associations of Src and Fyn with NR2A and NR2A tyrosine phosphorylation were found increased significantly in the postischemic rat hippocampus [5,10]. Knockdown of PSD-95 by antisense oligonucleotides reduced the above increases [7]. These data suggest that PSD95 may be responsible for the excessive activation of NMDA receptor channel activity and thus contributes to the delayed neuronal cell death in the postischemic hippocampus. To prove the hypothesis, the neuroprotective action of PSD-95 suppression by antisense oligonucleotides on the delayed hippocampal cell death after brain ischemia was evaluated by histological method.
X.-Y. Hou et al. / Neuroscience Letters 385 (2005) 230–233
Pyk2, also designated CAK, RAFTK, CADTK or FAK2, may also be involved in the postischemic facilitation of NR2A tyrosine phosphorylation [11]. It has been demonstrated that Pyk2 interacts with NMDA receptors via PSD-95 [18]. In recent experiments, the enhanced associations involving PSD-95, Pyk2 and NR2A as well as autophosphorylation of Pyk2 on Tyr402 in the postischemic hippocampus have been established [5,10,13]. The autophosphorylation of Pyk2 is essential to the enhancement of Pyk2 kinase activity and activation of Src family kinases that in turn induce ion channel function up-regulation and activation of downstream signaling cascades [14]. To reveal the mechanisms underlying PSD-95 neurotoxicity, the effects of PSD-95 antisense oligonucleotides on the binding of Pyk2 to NR2A and Pyk2 autophosphorylation were examined by immunoprecipitation and immunoblot. Adult male Sprague–Dawley rats weighing 250–300 g were purchased from Shanghai Experimental Animal Center, Chinese Academy of Science. End-phosphorothioated antisense oligonucleotides (AS) specifically against PSD-95 (Invitrogen, Japan K.K., Tokyo, Japan) were dissolved in TE (10 mM Tris–HCl, pH 8.0, 1 mM EDTA) and administrated to the rats every 24 h for 3 days by means of unilateral intracerebroventricular (i.c.v.) infusion as described previously [7]. The same doses of end-phosphorothioated missense oligonucleotides (MS) or vehicle (TE) were used as control. The sequences of oligonucleotides (AS: 5 -GAATGGGTCACCTCC-3 ; MS: 5 -CCGCTCTATCGAGGA-3 ) were from Sattler et al. [16]. After pretreated with AS or MS or the same dose of TE, the rats were subjected to 15 min of transient brain ischemia followed by 6 h or 5 days of reperfusion. Transient brain ischemia was induced by the method of four-vessel occlusion with a little modification as indicated before [5]. Shamoperated rats received the same surgical treatment, except for the occlusion of carotid arteries. For histology, the rats subjected to 5 days of reperfusion were perfusion-fixed with 4% paraformaldehyde in 0.1 M phosphate buffer (pH 7.4) under anesthesia. The paraffinembedded brain sections (5 m) were prepared and stained with hematoxylin and eosine. For immunoprecipitation and immunoblot, the rats subjected to 6 h of reperfusion were decapitated, and the hippocampi were removed and rapidly frozen in liquid nitrogen. The frozen tissue samples were homogenized in 1:10 (w/v) ice-cold homogenization buffer containing 50 mM MOPS (3-(N-morpholino)propanesulfonic acid; pH 7.4), 100 mM KCl, 320 mM sucrose, 0.5 mM MgCl2 , 0.2 mM dithiotheitol, and some inhibitors of phosphatase and protease. The homogenates were centrifuged at 800 × g for 10 min at 4 ◦ C and the supernatants were collected. Supernatant proteins (400 g) were used to immunoprecipitate or immunoblot with the indicated antibodies as described formerly [5]. To prove the suppression efficiency of AS on PSD-95 level, immunoblot with mouse monoclonal anti-PSD-95 antibody (clone 7E3-1B8; Oncogene, San Diego, CA, USA)
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Fig. 1. The effect of pretreatment with PSD-95 antisense oligonucleotides (AS) on PSD-95 expression in rat hippocampus. (A) Immunoblot analysis of the protein levels of PSD-95 with anti-PSD-95 antibody. (B) Quantitative representation of the protein levels of PSD-95. Data are mean ± S.D. (n = 3 rats) and expressed as folds vs. sham. a P < 0.05 vs. sham (ANOVA followed by Newman–Keuls test).
was carried out to detect the alteration of PSD-95 expression. As shown in Fig. 1, repeated infusion (i.c.v.) of rats with AS (50 or 100 g) every 24 h for 3 days evidently reduced the protein level of PSD-95, while MS (100 g) or TE did not affect PSD-95 expression. Hematoxylin and eosine staining was performed to show the survival of hippocampal CA1 pyramidal neurons after ischemia. The normal cells showed round cell body and plain stained nuclei (Fig. 2A and E). The neuronal density of the CA1 pyramidal cells was expressed as the number of normal cells per 1 mm length counted under a light microscope (×400) and presented in the Fig. 3. After 5 days of reperfusion following brain ischemia, most of hippocampal CA1 pyramidal neurons were shrunken with pyknotic nucleus and counted as dead cells (Figs. 2B, F and 3). Repeated pretreatment with 50 g of AS before ischemia markedly increased the number of surviving neurons (Fig. 3) and 100 g of AS showed more significant neuroprotection (Figs. 2C, G and 3). Infusion of 100 g MS had no effect on the postischemic cell death (Figs. 2D, H and 3). Above results revealed that PSD-95 is responsible for the delayed hippocampal cell death after ischemia. Previously, both Pyk2–NR2A binding and Pyk2 autophosphorylation have been shown to increase rapidly and sustainedly following brain ischemia and reach a peak level at 6 h of reperfusion [10,13]. To further detect the role of PSD95 in the facilitation of NR2A tyrosine phosphorylation, we inspected the effect of PSD-95 AS pretreatment on these 6 h of reperfusion-induced increases. The Pyk2–NR2A association was inspected by coimmunoprecipitation under non-denatured conditions.
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Fig. 2. Histology analysis of neuroprotection of PSD-95 antisense oligonucleotides (AS) against hippocampal CA1 pyramidal neuronal death induced by 5 days of reperfusion following brain ischemia (I/R5d). Rats were subjected to sham-operation (A and E), I/R5d (B and F) and pretreatment with 100 g of AS (C and G) or MS (D and H) every 24 h for 3 days before ischemia. (A–D) Low-power views of hippocampus sectors in hematoxylin and eosine-stained brain sections. Scale bar = 500 m. (E–H) High-power views of hippocampal CA1 pyramidal cell layer of (A–D), respectively. Scale bar = 50 m. Five of independent experiments (n = 5 rats) were carried out and one typical experiment was presented.
Fig. 3. Quantitative representation of neuroprotection of PSD-95 antisense oligonucleotides (AS) against hippocampal CA1 neuronal death induced by 5 days of reperfusion following brain ischemia (I/R5d). Neuronal density was counted as numbers of normal pyramidal neurons per 1 mm of hippocampal CA1 subfield. Data are mean ± S.D. (n = 5 rats). a P < 0.05 vs. I/R5d groups (ANOVA followed by Newman–Keuls test).
Fig. 4. The effects of pretreatment with PSD-95 antisense oligonucleotide (AS) on the increases in Pyk2–NR2A association and Pyk2 autophosphorylation (phospho-Pyk2) induced by 6 h of reperfusion following brain ischemia (I/R6h) in rat hippocampus. (A) The Pyk2–NR2A association was examined by immunoprecipitation with anti-Pyk2 antibody followed by immunoblot (IB) with anti-NR2A antibody. The phospho-Pyk2 was detected by IB with anti-phospho-Pyk2 (p-Tyr402) antibody. (B) IB analysis of the protein levels of NR2A and Pyk2. (C) Quantitative representation of the Pyk2–NR2A association and phospho-Pyk2. Data are mean ± S.D. (n = 3 rats) and expressed as folds to respective shams. a P < 0.05 vs. respective I/R6h groups (ANOVA followed by Newman–Keuls test).
Supernatant proteins (400 g) were immunoprecipitated with rabbit polyclonal antibody against Pyk2 (Sigma) and then blotted with rabbit polyclonal anti-NR2A antibody (Chemicon, Temecula, CA, USA). The autophosphorylation of Pyk2 were examined by immunoblot with rabbit polyclonal anti-phospho-Pyk2 (p-Tyr402) (Sigma). After 6 h of reperfusion following brain ischemia, the Pyk2–NR2A binding as well as Pyk2 autophosphorylation elevated markedly (Fig. 4A and C). Both of the increases diminished in rats pretreated with 50 g of AS, but not in the same dose of MSor TE-treated groups (Fig. 4A and C). Immunoblot analysis showed that the protein levels of Pyk2 and NR2A had no differences under above conditions (Fig. 4B). The results indicated that PSD-95 is a key factor in the association of Pyk2 with NR2A and activation of Pyk2 after brain ischemia. Excessive openness of two types of calcium channels, NMDA receptors and L-type voltage-gated calcium channels (L-VGCC), is believed to be involved in the pathogenesis of ischemic stroke. However, clinical trials in ischemic stroke with the two calcium channel antagonists have so far failed [4,8]. Recently, there are a number of enthusiasms about the
X.-Y. Hou et al. / Neuroscience Letters 385 (2005) 230–233
functional modulation of the two calcium channels. Tyrosine phosphorylation of the two ion channels is thought to be the main way for the potentiation of ion channel activities [2,9,12]. Our investigations suggest that PSD-95 proteins could bring PTKs including Src, Fyn and Pyk2 kinases into the NMDA receptor complex and enhance the tyrosine phosphorylation of NMDA receptor subunit NR2A during reperfusion following brain ischemia. Besides, PSD-95 may also mediate the increases in tyrosine phosphorylation of l-VGCC ␣1C subunits after brain ischemia [6]. Therefore, PSD-95 plays a crucial role in the overactivation of the two calcium channels after ischemia. In addition to functional enhancement of ion channels, PSD-95 mediates the downstream calcium-dependent nitric oxide production that is involved in the ischemic and excitotoxic neurodegeneration [16]. Furthermore, PSD-95 has been showed to bind kainate receptor GluR6 subunits and mixed lineage kinase (MLK) 2/3. Via PSD-95, kainate receptor activation induces MLK2/3-mediated signaling cascades as well as excitotoxicity [17]. Pei et al. reported that PSD-95 deficiency by antisense oligonucleotides inhibit JNK3 activation through the GluR6·PSD-95·MLK3 module [15]. Some results have suggested an important role for JNK3 in ischemic hippocampal cell death [3,20]. All above results revealed a mechanism involving the ischemia/reperfusion-induced recruitment of ion channels and downstream signal molecules to the PSD-95 signaling complex, which contributes to the enhancement of ion channel function and may be critical to the delayed hippocampal cell death after ischemia. Altogether, PSD-95 is neurotoxic in postischemic rat brain. As a core component in the orchestra of glutamatergic synapse, PSD-95 has the potential to be an effective drug target for the treatment of neurodegenerative disorders resulting from brain ischemia.
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Acknowledgements This study was supported by the grants from the National Natural Science Foundation of China (30330190, 30300069 and 30170220) and the Program for Natural Science Research in Jiangsu Provincial Higher School (02KJB310011 and 01KJB310009). References [1] M. Aarts, Y. Liu, L. Liu, S. Besshoh, M. Arundine, J.W. Gurd, Y.T. Wang, M.W. Salter, M. Tymianski, Treatment of ischemic brain damage by perturbing NMDA receptor-PSD-95 protein interactions, Science 298 (2002) 846–850. [2] K.K. Bence-Hanulec, J. Marshall, L.A. Blair, Potentiation of neuronal L calcium channels by IGF-1 requires phosphorylation of the ␣1 subunit on a specific tyrosine residue, Neuron 27 (2000) 121–131. [3] Q.H. Guan, D.S. Pei, Q.G. Zhang, Z.B. Hao, T.L. Xu, G.Y. Zhang, The neuroprotective action of SP600125, a new inhibitor of JNK,
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on transient brain ischemia/reperfusion-induced neuronal death in rat hippocampal CA1 via nuclear and non-nuclear pathways, Brain Res. 1035 (2005) 51–59. J. Horn, M. Limburg, Calcium antagonists for acute ischemic stroke: a systematic review, Stroke 32 (2001) 570–576. X.Y. Hou, G.Y. Zhang, J.Z. Yan, M. Chen, Y. Liu, Activation of NMDA receptors and L-type voltage-gated calcium channels mediates enhanced formation of Fyn-PSD95-NR2A complex after transient brain ischemia, Brain Res. 955 (2002) 123–132. X.Y. Hou, G.Y. Zhang, J.Z. Yan, Y. Liu, Increased tyrosine phosphorylation of ␣1C subunits of L-type voltage-gated calcium channels and interactions among Src/Fyn, PSD-95 and ␣1C in rat hippocampus after transient brain ischemia, Brain Res. 979 (2003) 43–50. X.Y. Hou, G.Y. Zhang, Y.Y. Zong, Suppression of postsynaptic density protein 95 expression attenuates increased tyrosine phosphorylation of NR2A subunits of N-methyl-d-aspartate receptors and interactions of Src and Fyn with NR2A after transient brain ischemia in rat hippocampus, Neurosci. Lett. 343 (2003) 125–128. J.A. Kemp, R.M. McKernan, NMDA receptor pathways as drug targets, Nat. Neurosci. 5 (Suppl.) (2002) 1039–1042. G. Kohr, P.H. Seeburg, Subtype-specific regulation of recombinant NMDA receptor-channels by protein tyrosine kinases of the src family, J. Physiol. 492 (1996) 445–452. Y. Liu, G.Y. Zhang, C. Gao, X.Y. Hou, NMDA receptor activation results in tyrosine phosphorylation of NMDA receptor subunit 2A (NR2A) and interaction of PYK2 and Src with NR2A after transient cerebral ischemia and reperfusion, Brain Res. 909 (2001) 51–58. Y. Liu, G.Y. Zhang, J.Z. Yan, T.L. Xu, Suppression of Pyk2 attenuated the increased tyrosine phosphorylation of NMDA receptor subunit 2A after brain ischemia in rat hippocampus, Neurosci. Lett. 379 (2005) 55–58. C. Lu, S.L. Chan, W. Fu, M.P. Mattson, The lipid peroxidation product 4-hydroxynonenal facilitates opening of voltage-dependent Ca2+ channels in neurons by increasing protein tyrosine phosphorylation, J. Biol. Chem. 277 (2002) 24368–24375. J. Ma, G.Y. Zhang, Y. Liu, J.Z. Yan, Z.B. Hao, Lithium suppressed Tyr-402 phosphorylation of proline-rich tyrosine kinase (Pyk2) and interactions of Pyk2 and PSD-95 with NR2A in rat hippocampus following cerebral ischemia, Neurosci. Res. 49 (2004) 357–362. S.Y. Park, H.K. Avraham, S. Avraham, RAFTK/Pyk2 activation is mediated by trans-acting autophosphorylation in a Src-independent manner, J Biol. Chem. 279 (2004) 33315–33322. D.S. Pei, Y.F. Sun, Q.H. Guan, Z.B. Hao, T.L. Xu, G.Y. Zhang, Postsynaptic density protein 95 antisense oligodeoxynucleotides inhibits the activation of MLK3 and JNK3 via the GluR6 PSD-95 MLK3 signaling module after transient cerebral ischemia in rat hippocampus, Neurosci. Lett. 367 (2004) 71–75. R. Sattler, Z. Xiong, W.Y. Lu, M. Hafner, J.F. MacDonald, M. Tymianski, Specific coupling of NMDA receptor activation to nitric oxide neurotoxicity by PSD-95 protein, Science 284 (1999) 1845–1848. A. Savinainen, E.P. Garcia, D. Dorow, J. Marshall, Y.F. Liu, Kainate receptor activation induces mixed lineage kinase-mediated cellular signaling cascades via postsynaptic density protein 95, J. Biol. Chem. 276 (2001) 11382–11386. G.K. Seabold, A. Burette, I.A. Lim, R.J. Weinberg, J.W. Hell, Interaction of the tyrosine kinase Pyk2 with the N-methyl-d-aspartate receptor complex via the SH3 domains of PSD-95 and SAP102, J. Biol. Chem. 278 (2003) 15040–15048. T. Tezuka, H. Umemori, T. Akiyama, S. Nakanishi, T. Yamamoto, PSD-95 promotes Fyn-mediated tyrosine phosphorylation of the Nmethyl-d-aspartate receptor subunit NR2A, Proc. Natl. Acad. Sci. U.S.A. 96 (1999) 435–440. H. Tian, G.Y. Zhang, H.C. Li, Q.G. Zhang, Antioxidant NAC, AMPA/KA receptor antagonist DNQX inhibited JNK3 activation following global ischemia in rat hippocampus, Neurosci. Res. 46 (2003) 191–197.
Suppression of postsynaptic density protein 95 by antisense oligonucleotides diminishes postischemic pyramidal cell death in rat hippocampal CA1 subfield Xiao-Yu Hou, Guang-Yi Zhang ∗ , De-Guang Wang, Qiu-Hua Guan, Jing-Zhi Yan Research Center for Biochemistry and Molecular Biology, Xuzhou Medical College, University of Science & Technology of China, 84 West Huai-hai Road, Xuzhou, Jiangsu 221002, PR China Received 6 April 2005; received in revised form 4 May 2005; accepted 14 May 2005
Abstract Our previous investigation has shown that postsynaptic density protein 95 (PSD-95) is critical for the Src family kinases-mediated tyrosine phosphorylation of N-methyl-d-aspartate receptor subunit 2A (NR2A) in the postischemic hippocampus. To clarify the roles of PSD-95 in the ischemic brain damage, histological method was performed to examine the effects of PSD-95 antisense oligonucleotides (AS) on the postischemic delayed cell death in rat hippocampus. Transient (15 min) brain ischemia was induced by the four-vessel occlusion method in Sprague–Dawley rats. Five days of reperfusion following brain ischemia (I/R5d) led to hippocampal CA1 pyramidal cell death upward of 90%. Intracerebroventricular infusion of AS (every 24 h for 3 days before ischemia) not only decreased the PSD-95 expression but also increased the number of surviving pyramidal neurons, while missense oligonucleotides (MS) had no effects. To further investigate the mechanisms underlying the neuroprotection of PSD-95 deficiency, the interaction of proline-rich tyrosine kinase 2 (Pyk2) with NR2A as well as autophosphorylation (Tyr402) of Pyk2 were detected. Immunoprecipitation and immunoblot analysis showed that preischemic treatment with AS, but not MS or vehicle, attenuated the I/R6h-induced increases in Pyk2–NR2A association and Pyk2 autophosphorylation. The protein levels of NR2A and Pyk2 had no differences under the above conditions. Our data suggest that the recruitments of ion channels and signaling molecules may be involved in the PSD-95 neurotoxicity in the postischemic hippocampus. © 2005 Elsevier Ireland Ltd. All rights reserved. Keywords: Brain ischemia; Neuroprotection; Postsynaptic density protein 95; N-methyl-d-aspartate receptor; Proline-rich tyrosine kinase 2
Postsynaptic density protein 95 (PSD-95), also known as synapse-associated protein 90 (SAP90), is a predominant kind of scaffold proteins concentrated in glutamatergic postsynaptic density. Although accumulating evidence has indicated that PSD-95 is involved in the ischemic events [1,5–7,15], the exact roles of PSD-95 in the postischemic neuronal cell death have not been elucidated. PSD-95 proteins associate NR2 subunits of N-methyl-daspartate (NMDA) receptors with an array of protein tyrosine kinases (PTKs) including Src family of kinases and prolinerich tyrosine kinase 2 (Pyk2) [18,19]. Src family kinases belong to the family of membrane-associated non-receptor
∗
Corresponding author. Tel.: +86 516 583 4231; fax: +86 516 574 8486. E-mail address: [email protected] (G.-Y. Zhang).
0304-3940/$ – see front matter © 2005 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.neulet.2005.05.054
PTKs. Within nine of Src family members, Src and Fyn kinases are highly expressed in the central nervous system. Electrophysiological studies have shown that NR2A tyrosine phosphorylation by Src family kinases enhances NMDA channel responses [9]. The associations of Src and Fyn with NR2A and NR2A tyrosine phosphorylation were found increased significantly in the postischemic rat hippocampus [5,10]. Knockdown of PSD-95 by antisense oligonucleotides reduced the above increases [7]. These data suggest that PSD95 may be responsible for the excessive activation of NMDA receptor channel activity and thus contributes to the delayed neuronal cell death in the postischemic hippocampus. To prove the hypothesis, the neuroprotective action of PSD-95 suppression by antisense oligonucleotides on the delayed hippocampal cell death after brain ischemia was evaluated by histological method.
X.-Y. Hou et al. / Neuroscience Letters 385 (2005) 230–233
Pyk2, also designated CAK, RAFTK, CADTK or FAK2, may also be involved in the postischemic facilitation of NR2A tyrosine phosphorylation [11]. It has been demonstrated that Pyk2 interacts with NMDA receptors via PSD-95 [18]. In recent experiments, the enhanced associations involving PSD-95, Pyk2 and NR2A as well as autophosphorylation of Pyk2 on Tyr402 in the postischemic hippocampus have been established [5,10,13]. The autophosphorylation of Pyk2 is essential to the enhancement of Pyk2 kinase activity and activation of Src family kinases that in turn induce ion channel function up-regulation and activation of downstream signaling cascades [14]. To reveal the mechanisms underlying PSD-95 neurotoxicity, the effects of PSD-95 antisense oligonucleotides on the binding of Pyk2 to NR2A and Pyk2 autophosphorylation were examined by immunoprecipitation and immunoblot. Adult male Sprague–Dawley rats weighing 250–300 g were purchased from Shanghai Experimental Animal Center, Chinese Academy of Science. End-phosphorothioated antisense oligonucleotides (AS) specifically against PSD-95 (Invitrogen, Japan K.K., Tokyo, Japan) were dissolved in TE (10 mM Tris–HCl, pH 8.0, 1 mM EDTA) and administrated to the rats every 24 h for 3 days by means of unilateral intracerebroventricular (i.c.v.) infusion as described previously [7]. The same doses of end-phosphorothioated missense oligonucleotides (MS) or vehicle (TE) were used as control. The sequences of oligonucleotides (AS: 5 -GAATGGGTCACCTCC-3 ; MS: 5 -CCGCTCTATCGAGGA-3 ) were from Sattler et al. [16]. After pretreated with AS or MS or the same dose of TE, the rats were subjected to 15 min of transient brain ischemia followed by 6 h or 5 days of reperfusion. Transient brain ischemia was induced by the method of four-vessel occlusion with a little modification as indicated before [5]. Shamoperated rats received the same surgical treatment, except for the occlusion of carotid arteries. For histology, the rats subjected to 5 days of reperfusion were perfusion-fixed with 4% paraformaldehyde in 0.1 M phosphate buffer (pH 7.4) under anesthesia. The paraffinembedded brain sections (5 m) were prepared and stained with hematoxylin and eosine. For immunoprecipitation and immunoblot, the rats subjected to 6 h of reperfusion were decapitated, and the hippocampi were removed and rapidly frozen in liquid nitrogen. The frozen tissue samples were homogenized in 1:10 (w/v) ice-cold homogenization buffer containing 50 mM MOPS (3-(N-morpholino)propanesulfonic acid; pH 7.4), 100 mM KCl, 320 mM sucrose, 0.5 mM MgCl2 , 0.2 mM dithiotheitol, and some inhibitors of phosphatase and protease. The homogenates were centrifuged at 800 × g for 10 min at 4 ◦ C and the supernatants were collected. Supernatant proteins (400 g) were used to immunoprecipitate or immunoblot with the indicated antibodies as described formerly [5]. To prove the suppression efficiency of AS on PSD-95 level, immunoblot with mouse monoclonal anti-PSD-95 antibody (clone 7E3-1B8; Oncogene, San Diego, CA, USA)
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Fig. 1. The effect of pretreatment with PSD-95 antisense oligonucleotides (AS) on PSD-95 expression in rat hippocampus. (A) Immunoblot analysis of the protein levels of PSD-95 with anti-PSD-95 antibody. (B) Quantitative representation of the protein levels of PSD-95. Data are mean ± S.D. (n = 3 rats) and expressed as folds vs. sham. a P < 0.05 vs. sham (ANOVA followed by Newman–Keuls test).
was carried out to detect the alteration of PSD-95 expression. As shown in Fig. 1, repeated infusion (i.c.v.) of rats with AS (50 or 100 g) every 24 h for 3 days evidently reduced the protein level of PSD-95, while MS (100 g) or TE did not affect PSD-95 expression. Hematoxylin and eosine staining was performed to show the survival of hippocampal CA1 pyramidal neurons after ischemia. The normal cells showed round cell body and plain stained nuclei (Fig. 2A and E). The neuronal density of the CA1 pyramidal cells was expressed as the number of normal cells per 1 mm length counted under a light microscope (×400) and presented in the Fig. 3. After 5 days of reperfusion following brain ischemia, most of hippocampal CA1 pyramidal neurons were shrunken with pyknotic nucleus and counted as dead cells (Figs. 2B, F and 3). Repeated pretreatment with 50 g of AS before ischemia markedly increased the number of surviving neurons (Fig. 3) and 100 g of AS showed more significant neuroprotection (Figs. 2C, G and 3). Infusion of 100 g MS had no effect on the postischemic cell death (Figs. 2D, H and 3). Above results revealed that PSD-95 is responsible for the delayed hippocampal cell death after ischemia. Previously, both Pyk2–NR2A binding and Pyk2 autophosphorylation have been shown to increase rapidly and sustainedly following brain ischemia and reach a peak level at 6 h of reperfusion [10,13]. To further detect the role of PSD95 in the facilitation of NR2A tyrosine phosphorylation, we inspected the effect of PSD-95 AS pretreatment on these 6 h of reperfusion-induced increases. The Pyk2–NR2A association was inspected by coimmunoprecipitation under non-denatured conditions.
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Fig. 2. Histology analysis of neuroprotection of PSD-95 antisense oligonucleotides (AS) against hippocampal CA1 pyramidal neuronal death induced by 5 days of reperfusion following brain ischemia (I/R5d). Rats were subjected to sham-operation (A and E), I/R5d (B and F) and pretreatment with 100 g of AS (C and G) or MS (D and H) every 24 h for 3 days before ischemia. (A–D) Low-power views of hippocampus sectors in hematoxylin and eosine-stained brain sections. Scale bar = 500 m. (E–H) High-power views of hippocampal CA1 pyramidal cell layer of (A–D), respectively. Scale bar = 50 m. Five of independent experiments (n = 5 rats) were carried out and one typical experiment was presented.
Fig. 3. Quantitative representation of neuroprotection of PSD-95 antisense oligonucleotides (AS) against hippocampal CA1 neuronal death induced by 5 days of reperfusion following brain ischemia (I/R5d). Neuronal density was counted as numbers of normal pyramidal neurons per 1 mm of hippocampal CA1 subfield. Data are mean ± S.D. (n = 5 rats). a P < 0.05 vs. I/R5d groups (ANOVA followed by Newman–Keuls test).
Fig. 4. The effects of pretreatment with PSD-95 antisense oligonucleotide (AS) on the increases in Pyk2–NR2A association and Pyk2 autophosphorylation (phospho-Pyk2) induced by 6 h of reperfusion following brain ischemia (I/R6h) in rat hippocampus. (A) The Pyk2–NR2A association was examined by immunoprecipitation with anti-Pyk2 antibody followed by immunoblot (IB) with anti-NR2A antibody. The phospho-Pyk2 was detected by IB with anti-phospho-Pyk2 (p-Tyr402) antibody. (B) IB analysis of the protein levels of NR2A and Pyk2. (C) Quantitative representation of the Pyk2–NR2A association and phospho-Pyk2. Data are mean ± S.D. (n = 3 rats) and expressed as folds to respective shams. a P < 0.05 vs. respective I/R6h groups (ANOVA followed by Newman–Keuls test).
Supernatant proteins (400 g) were immunoprecipitated with rabbit polyclonal antibody against Pyk2 (Sigma) and then blotted with rabbit polyclonal anti-NR2A antibody (Chemicon, Temecula, CA, USA). The autophosphorylation of Pyk2 were examined by immunoblot with rabbit polyclonal anti-phospho-Pyk2 (p-Tyr402) (Sigma). After 6 h of reperfusion following brain ischemia, the Pyk2–NR2A binding as well as Pyk2 autophosphorylation elevated markedly (Fig. 4A and C). Both of the increases diminished in rats pretreated with 50 g of AS, but not in the same dose of MSor TE-treated groups (Fig. 4A and C). Immunoblot analysis showed that the protein levels of Pyk2 and NR2A had no differences under above conditions (Fig. 4B). The results indicated that PSD-95 is a key factor in the association of Pyk2 with NR2A and activation of Pyk2 after brain ischemia. Excessive openness of two types of calcium channels, NMDA receptors and L-type voltage-gated calcium channels (L-VGCC), is believed to be involved in the pathogenesis of ischemic stroke. However, clinical trials in ischemic stroke with the two calcium channel antagonists have so far failed [4,8]. Recently, there are a number of enthusiasms about the
X.-Y. Hou et al. / Neuroscience Letters 385 (2005) 230–233
functional modulation of the two calcium channels. Tyrosine phosphorylation of the two ion channels is thought to be the main way for the potentiation of ion channel activities [2,9,12]. Our investigations suggest that PSD-95 proteins could bring PTKs including Src, Fyn and Pyk2 kinases into the NMDA receptor complex and enhance the tyrosine phosphorylation of NMDA receptor subunit NR2A during reperfusion following brain ischemia. Besides, PSD-95 may also mediate the increases in tyrosine phosphorylation of l-VGCC ␣1C subunits after brain ischemia [6]. Therefore, PSD-95 plays a crucial role in the overactivation of the two calcium channels after ischemia. In addition to functional enhancement of ion channels, PSD-95 mediates the downstream calcium-dependent nitric oxide production that is involved in the ischemic and excitotoxic neurodegeneration [16]. Furthermore, PSD-95 has been showed to bind kainate receptor GluR6 subunits and mixed lineage kinase (MLK) 2/3. Via PSD-95, kainate receptor activation induces MLK2/3-mediated signaling cascades as well as excitotoxicity [17]. Pei et al. reported that PSD-95 deficiency by antisense oligonucleotides inhibit JNK3 activation through the GluR6·PSD-95·MLK3 module [15]. Some results have suggested an important role for JNK3 in ischemic hippocampal cell death [3,20]. All above results revealed a mechanism involving the ischemia/reperfusion-induced recruitment of ion channels and downstream signal molecules to the PSD-95 signaling complex, which contributes to the enhancement of ion channel function and may be critical to the delayed hippocampal cell death after ischemia. Altogether, PSD-95 is neurotoxic in postischemic rat brain. As a core component in the orchestra of glutamatergic synapse, PSD-95 has the potential to be an effective drug target for the treatment of neurodegenerative disorders resulting from brain ischemia.
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