- Email: [email protected]
threonine protein phosphatase 1 mRNA in the vulnerable regions of gerbil brain
Neuroscience Letters 325 (2002) 115–118 www.elsevier.com/locate/neulet
Transient forebrain ischemia induces expression of serine/threonine protein phosphatase 1 mRNA in the vulnerable regions of gerbil brain Takashi Horiguchi a,*, Hiroshi Shima b, Sadao Suga a, Masahiro Ogino a, Katsuyoshi Shimizu a, Shigeo Toya a, Minako Nagao c, Takeshi Kawase a b
a Department of Neurosurgery, Keio University School of Medicine, Tokyo, Japan Division of Biochemical Oncology and Immunology, Institute for Genetic Medicine, Hokkaido University, Sapporo, Japan c Biochemistry Division, National Cancer Center Research Institute, Tokyo, Japan
Received 10 January 2002; received in revised form 8 March 2002; accepted 10 March 2002
Abstract Apoptosis is thought to be implicated in delayed neuronal cell death following transient forebrain ischemia. Recently, apoptosis in neurons induced by an inhibitor of serine/threonine (ser/thr) protein phosphatases (PPs) has been reported. In this study, we investigated the effect of transient forebrain ischemia on the expression of ser/thr PPs in the brain of Mongolian gerbils. At 24 h after 5-min bilateral carotid artery occlusion, Northern blotting analysis revealed the increase of PP1 mRNA expression in the vulnerable CA1 region of the hippocampus and striatum, but not in the cortex and CA3 region. In contrast, the protein level of PP1 detected by Western blotting analysis decreased in all regions. We conclude that the inhibition in PPs expression in the vulnerable regions may affect cell death after transient forebrain ischemia. q 2002 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Protein phosphatase; Global ischemia; Delayed neuronal death; Vulnerability; Protein synthesis
Transient forebrain ischemia results in neuronal cell death in vulnerable regions of the brain, including the CA1 sector of the hippocampus and the dorsolateral striatum [8,15]. Alterations of protein phosphorylation in neuronal cells may play a key role in development of delayed neuronal death (DND). Olah et al. showed transient elevations of protein kinase C (PKC) activities at 24 h after 5-min forebrain ischemia in the CA1 sector but not in the CA3 region [14]. Recent studies have revealed that ischemia induced phosphorylation of mitogen-activated protein (MAP) kinase elicits DND [7]. On the contrary, dual specificity protein phosphatase (PP) PAC-1 has been reported to inactivate MAP kinase [19]. Wiessner showed PAC-1 mRNA expression after cerebral ischemia in the CA3 and dentate gyrus, but not in the CA1 area [20]. Recently, Runden et al. demonstrated that inhibition of serine/threonine (ser/thr) PPs by the algal toxin okadaic acid led to * Corresponding author. Department of Physiology and Pharmacology, Wake Forest University School of Medicine, Medical Center Boulevard, Winston-Salem, NC 27157-1083, USA. Tel.: 11-336-716-4367; fax: 11-336-716-0237. E-mail address: [email protected] (T. Horiguchi).
neuronal degeneration through the MAP kinase pathway [16]. Okadaic acid induced neuronal cell apoptosis has been documented previously [13] and numerous studies have supported the connection of apoptotic cell death with DND [2]. Although the results of these studies suggest strongly that ser/thr PPs are involved in signal transduction of cellular degenerative processes, the correlation between cerebral ischemia and ser/thr PPs is still unknown. In this study, we investigated the effect of transient forebrain ischemia on the expression of ser/thr PPs in gerbil brain. Two PP1 isotypes (1a and 1g1) [17] were examined in the present study. Under 1–2% halothane anesthesia in an oxygen/nitrous oxide mixture (30/70%), forebrain ischemia was induced in 80 male Mongolian gerbils (60–70 g) by bilateral carotid artery occlusion for 5 min with Sugita’s temporary clips. During the period of ischemia, rectal and temporal muscle temperature was maintained at 38 8C. At the end of the 5 min of ischemia, the carotid occluders were removed, allowing recirculation. For Northern and Western blotting analysis, under deep anesthesia, gerbils were sacrificed after 24 h ischemia, and their brains were dissected out quickly and
0304-3940/02/$ - see front matter q 2002 Elsevier Science Ireland Ltd. All rights reserved. PII: S03 04 - 394 0( 0 2) 00 24 4- 6
116
T. Horiguchi et al. / Neuroscience Letters 325 (2002) 115–118
Fig. 1. The effect of transient forebrain ischemia on the mRNA expression of each PP in the cortex, striatum, and CA1 and CA3 regions in the hippocampus of Mongolian gerbil brains. The specific bands of mRNAs of PP1a (A) and PP1g1 (B) are obtained. Lanes 1–4 are control brains and lanes 5–8 are postischemic brains. Lanes 1 and 5 are the cortex, lanes 2 and 6 are the striatum, lanes 3 and 7 are the CA1 region, and lanes 4 and 8 are the CA3 region. Note the stronger inductions of mRNA are observed especially in the vulnerable regions. Co, cortex; St, striatum; Con, control; Pos, postischemia.
frozen with liquid nitrogen. Under a surgical microscope, the specimens of parietal cortex, the striatum, and the CA1 and CA3 regions of hippocampus were removed on crushed ice. The above procedure was carried out without the induction of ischemic insults to provide a control group. Northern blotting analysis was carried out according to the method of Sasaki et al. [17]. Briefly, after total RNA was prepared by the single-step total RNA isolation method, 10 mg samples of total RNAs were electrophoresed and transferred to a membrane, and hybridized with 32P-labeled PP1a and 1g1 probes. The Western blotting analysis was performed according to the method of Shima et al. [18]. Briefly, the cytosolic fraction was harvested and each 10 mg of sample was electrophoresed and transferred to membranes. The membranes were incubated with rabbit polyclonal antibodies against the C-terminal of the two rat PP1 isotypes, and with peroxidase conjugated anti-rabbit IgG. Each of the bands, which were visualized with DuPont NEN(r) chemiluminescence, was analyzed with a computer-assisted image analyzer (Luzex-F). In this method, the amount of each band was expressed as the value obtained by multiplying the area by the mean density. The results of Northern blotting analysis revealed the increases in PP1a and 1g1 mRNA 24 h after transient fore-
brain ischemia (Figs. 1 and 2). It was particularly noteworthy that induction of these isotypes was stronger in the CA1 area and striatum than in the CA3 area and cortex. Western blotting analysis revealed that there is no remarkable change in expression of PP1a 24 h after ischemia, in contrast, the expression of PP1g1 tended to decrease in all regions (Figs. 3 and 4). This is the first study that examined the relationship
Fig. 2. The results of semiquantitative analysis of Fig. 1. The values of the area multiplied by the mean density of each band (designated as Area*Dens) were calculated with a computerassisted image analyzer.
T. Horiguchi et al. / Neuroscience Letters 325 (2002) 115–118
117
Fig. 3. The effect of transient forebrain ischemia on the protein expression of each PP in the cortex, striatum, and CA1 and CA3 regions in the hippocampus of Mongolian gerbil brains. The specific bands of PP1a (A) and 1g1 (B) are obtained. Lanes 1–4 are control brains and lanes 5–8 are postischemic brains. Lanes 1 and 5 are the cortex, lanes 2 and 6 are the striatum, lanes 3 and 7 are the CA1 region, and lanes 4 and 8 are the CA3 region. Note that PP1g1 has a tendency to decrease in all subfields contrary to the results of Northern blotting analysis 24 h after ischemia. On the other hand, PP1a has no remarkable change of expression in all regions 24 h after ischemia. Co, cortex; St, striatum; Hippo, hippocampus; Con, control; Pos, postischemia.
between PP1 and DND. We interpret the discrepancy of the expression between PP1 mRNA and protein in the vulnerable regions as possibly being associated with selective neuronal damage. The precise role of PP1a and PP1g1 in the brain has not been clarified. Hashikawa et al. reported localization of PP1g1 in the distal dendrites of cerebellar Purkinje cells [3]. The pharmacological studies revealed that the long-term depression (LTD) in the CA1 and cerebellum were inhibited by a PP1 inhibitor [6,11]. For sustaining LTD, the activation of alpha-amino-3-hydroxy-5methyl-4-isoxazolepropionic acid (AMPA) receptors plays a key role [9]. According to these studies, we believe the essential role of PP1g1 in the signal transduction through AMPA receptors in not only normal but also ischemic brain. The underlying mechanisms involved in alterations in PP1 mRNA and protein expression are unclear. The close relationship between selective vulnerability to ischemia and the persistent depression of protein synthesis is well known [1]. Transient global ischemia induced accumulation of mRNA without protein synthesis in vulnerable areas has been obtained in not only PPs but also with other gene products [12]. Therefore, the depressed protein synthesis may be the one possible explanation of the increases in PP1 mRNA. However, we speculate that the increases of PP1g1 mRNA, especially in the more vulnerable regions, may be a reaction to the hyperphosphorylated state.
Although the reduction of PP1g1 expression in protein levels was observed in the cortex and CA3 region, the expression in mRNA level does not change in the same region after ischemia. Another equally important mechanism to explain our data is the translocation of PP1 after transient ischemia. A similar change of protein kinases (PKs) has been reported in hippocampal cells after ischemia [4,14]. Hu et al. observed translocation of Ca 21/calmodulin-
Fig. 4. The results of semiquantitative analysis of Fig. 3. The values of the area multiplied by the mean density of each band (designated as Area*Dens) were calculated with a computerassisted image analyzer.
118
T. Horiguchi et al. / Neuroscience Letters 325 (2002) 115–118
dependent protein kinase II (CaM kinase II) to the synaptic junction after cerebral ischemia and concluded that persistent translocation of CaM kinase II in the vulnerable ischemic CA1 area is related to DND [4]. They emphasized events occurring in the nerve terminal and synaptic junctions after ischemia. Recent studies have revealed that changes in the activity of ser/thr PKs, e.g. PKC, or CAM kinase II, may contribute to neuronal death after ischemic insults [4,14]. However, little is known about ser/thr PPs in the ischemic brain. Morioka et al. demonstrated the preservation of immunoreactivity for PP2B (Calcineurin) in CA1 region. Since the activity of CAM kinase II was depressed in their study, they concluded that the difference between those enzymes might play a key role in DND [10]. However, the activities of each PP are regulated by a combination of targeting and regulatory subunits and by specific inhibitors [5]. We believe that the function of PP1 in a phosphorylation equilibrium is different from PP2B. In conclusion, our results suggest that PP1s may play a key role in DND. This suggestion is largely in agreement with pharmacological data demonstrating the crucial role of PPs in cell survival [13,16]. Further investigation to elucidate the cardinal role of PP1s may serve the clue to clarify the molecular mechanisms in ischemic neuronal injury. [1] Bodsch, W., Takahashi, K., Barbier, A., Grosse, O.B. and Hossmann, K.A., Cerebral protein synthesis and ischemia, Prog. Brain Res., 63 (1985) 197–210. [2] Hara, A., Niwa, M., Nakashima, M., Iwai, T., Uematsu, T., Yoshimi, N. and Mori, H., Protective effect of apoptosisinhibitory agent, N-tosyl-l-phenylalanyl chloromethyl ketone against ischemia-induced hippocampal neuronal damage, J. Cereb. Blood Flow Metab., 18 (1998) 819–823. [3] Hashikawa, T., Nakazawa, K., Mikawa, S., Shima, H. and Nagao, M., Immunohistochemical localization of protein phosphatase isoforms in the rat cerebellum, Neurosci. Res., 22 (1995) 133–136. [4] Hu, B.R. and Wieloch, T., Persistent translocation of Ca 21/ Calmodulin-dependent protein kinase II to synaptic junctions in the vulnerable hippocampal CA1 region following transient ischemia, J. Neurochem., 6 (1995) 277–284. [5] Hunter, T., Protein kinases and phosphatases: the yin and yang of protein phosphorylation and signaling, Cell, 80 (1995) 225–236. [6] Ito, M. and Karachot, L., Protein kinases and phosphatase inhibitors mediating long-term desensitization of glutamate receptors in cerebellar Purkinje cells, Neurosci. Res., 14 (1992) 27–38.
[7] Kindy, M.S., Inhibition of tyrosine phosphorylation prevents delayed neuronal death following cerebral ischemia, J. Cereb. Blood Flow Metab., 13 (1993) 372–377. [8] Kirino, T., Delayed neuronal death in the gerbil hippocampus following ischemia, Brain Res., 239 (1982) 57–69. [9] Linden, D.J., Dickinson, M.H., Smeyne, M. and Connor, J.A., A long-term depression of AMPA currents in cultured cerebellar Purkinje neurons, Neuron, 7 (1991) 81–89. [10] Morioka, M., Fukunaga, K., Yasugawa, S., Nagahiro, S., Ushio, Y. and Miyamoto, E., Regional and temporal alterations in Ca 21/calmodulin-dependent protein kinase II and calcineurin in the hippocampus of rat brain after transient forebrain ischemia, J. Neurochem., 58 (1992) 1798–1809. [11] Mulkey, R.M., Herron, C.E. and Malenka, R.C., An essential role for protein phosphatases in hippocampal long-term depression, Science, 261 (1993) 1051–1055. [12] Nowak Jr., T.S., Localization of 70 kDa stress protein mRNA induction in gerbil brain after ischemia, J. Cereb. Blood Flow Metab., 11 (1991) 432–439. [13] Nuydens, R., de Jong, M., Van Den Kieboom, G., Heers, C., Dispersyn, G., Cornelissen, F., Nuyens, R., Borgers, M. and Geerts, H., Okadaic acid-induced apoptosis in neuronal cells: evidence for an abortive mitotic attempt, J. Neurochem., 70 (1998) 1124–1133. [14] Olah, Z., Ikeda, J., Anderson, W.B. and Joo, F., Altered protein kinase C activity in different subfields of hippocampus following cerebral ischemia, Neurochem. Res., 15 (1990) 515–518. [15] Pulsinelli, W.A., Brierley, J.B. and Plum, F., Temporal profile of neuronal damage in a model of transient forebrain ischemia, Ann. Neurol., 11 (1982) 491–498. [16] Runden, E., Seglen, P.O., Haug, F.M., Ottersen, O.P., Wieloch, T., Shamloo, M. and Laake, J.H., Regional selective neuronal degeneration after protein phosphatase inhibition in hippocampal slice cultures: evidence for a MAP kinase-dependent mechanism, J. Neurosci., 18 (1998) 7296–7305. [17] Sasaki, K., Shima, H., Kitagawa, Y., Irino, S., Sugimura, T. and Nagao, M., Identification of members of the protein phosphatase 1 gene family in the rat and enhanced expression of protein phosphatase 1a gene in rat hepatocellular carcinomas, Jpn. J. Cancer Res., 81 (1990) 1272–1280. [18] Shima, H., Hatano, Y., Chun, Y.S., Sugimura, T., Zhang, Z., Lee Ernest, Y.C. and Nagao, M., Identification of PP1 catalytic subunit isotypes PP1g1, PP1d, and PP1a in various rat tissues, Biochem. Biophys. Res. Commun., 192 (1993) 1289–1296. [19] Ward, Y., Gupta, S., Jensen, P., Wartmann, M., Davis, R.J. and Kelly, K., Control of MAO kinase activation by the mitogen-induced threonine/tyrosine phosphatase PAC1, Nature, 367 (1994) 651–654. [20] Wiessner, C., The dural specific phosphatase PAC-1 is transcriptionally induced in the rat brain following transient forebrain ischemia, Brain Res. Mol. Brain Res., 28 (1995) 353–356.
Transient forebrain ischemia induces expression of serine/threonine protein phosphatase 1 mRNA in the vulnerable regions of gerbil brain Takashi Horiguchi a,*, Hiroshi Shima b, Sadao Suga a, Masahiro Ogino a, Katsuyoshi Shimizu a, Shigeo Toya a, Minako Nagao c, Takeshi Kawase a b
a Department of Neurosurgery, Keio University School of Medicine, Tokyo, Japan Division of Biochemical Oncology and Immunology, Institute for Genetic Medicine, Hokkaido University, Sapporo, Japan c Biochemistry Division, National Cancer Center Research Institute, Tokyo, Japan
Received 10 January 2002; received in revised form 8 March 2002; accepted 10 March 2002
Abstract Apoptosis is thought to be implicated in delayed neuronal cell death following transient forebrain ischemia. Recently, apoptosis in neurons induced by an inhibitor of serine/threonine (ser/thr) protein phosphatases (PPs) has been reported. In this study, we investigated the effect of transient forebrain ischemia on the expression of ser/thr PPs in the brain of Mongolian gerbils. At 24 h after 5-min bilateral carotid artery occlusion, Northern blotting analysis revealed the increase of PP1 mRNA expression in the vulnerable CA1 region of the hippocampus and striatum, but not in the cortex and CA3 region. In contrast, the protein level of PP1 detected by Western blotting analysis decreased in all regions. We conclude that the inhibition in PPs expression in the vulnerable regions may affect cell death after transient forebrain ischemia. q 2002 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Protein phosphatase; Global ischemia; Delayed neuronal death; Vulnerability; Protein synthesis
Transient forebrain ischemia results in neuronal cell death in vulnerable regions of the brain, including the CA1 sector of the hippocampus and the dorsolateral striatum [8,15]. Alterations of protein phosphorylation in neuronal cells may play a key role in development of delayed neuronal death (DND). Olah et al. showed transient elevations of protein kinase C (PKC) activities at 24 h after 5-min forebrain ischemia in the CA1 sector but not in the CA3 region [14]. Recent studies have revealed that ischemia induced phosphorylation of mitogen-activated protein (MAP) kinase elicits DND [7]. On the contrary, dual specificity protein phosphatase (PP) PAC-1 has been reported to inactivate MAP kinase [19]. Wiessner showed PAC-1 mRNA expression after cerebral ischemia in the CA3 and dentate gyrus, but not in the CA1 area [20]. Recently, Runden et al. demonstrated that inhibition of serine/threonine (ser/thr) PPs by the algal toxin okadaic acid led to * Corresponding author. Department of Physiology and Pharmacology, Wake Forest University School of Medicine, Medical Center Boulevard, Winston-Salem, NC 27157-1083, USA. Tel.: 11-336-716-4367; fax: 11-336-716-0237. E-mail address: [email protected] (T. Horiguchi).
neuronal degeneration through the MAP kinase pathway [16]. Okadaic acid induced neuronal cell apoptosis has been documented previously [13] and numerous studies have supported the connection of apoptotic cell death with DND [2]. Although the results of these studies suggest strongly that ser/thr PPs are involved in signal transduction of cellular degenerative processes, the correlation between cerebral ischemia and ser/thr PPs is still unknown. In this study, we investigated the effect of transient forebrain ischemia on the expression of ser/thr PPs in gerbil brain. Two PP1 isotypes (1a and 1g1) [17] were examined in the present study. Under 1–2% halothane anesthesia in an oxygen/nitrous oxide mixture (30/70%), forebrain ischemia was induced in 80 male Mongolian gerbils (60–70 g) by bilateral carotid artery occlusion for 5 min with Sugita’s temporary clips. During the period of ischemia, rectal and temporal muscle temperature was maintained at 38 8C. At the end of the 5 min of ischemia, the carotid occluders were removed, allowing recirculation. For Northern and Western blotting analysis, under deep anesthesia, gerbils were sacrificed after 24 h ischemia, and their brains were dissected out quickly and
0304-3940/02/$ - see front matter q 2002 Elsevier Science Ireland Ltd. All rights reserved. PII: S03 04 - 394 0( 0 2) 00 24 4- 6
116
T. Horiguchi et al. / Neuroscience Letters 325 (2002) 115–118
Fig. 1. The effect of transient forebrain ischemia on the mRNA expression of each PP in the cortex, striatum, and CA1 and CA3 regions in the hippocampus of Mongolian gerbil brains. The specific bands of mRNAs of PP1a (A) and PP1g1 (B) are obtained. Lanes 1–4 are control brains and lanes 5–8 are postischemic brains. Lanes 1 and 5 are the cortex, lanes 2 and 6 are the striatum, lanes 3 and 7 are the CA1 region, and lanes 4 and 8 are the CA3 region. Note the stronger inductions of mRNA are observed especially in the vulnerable regions. Co, cortex; St, striatum; Con, control; Pos, postischemia.
frozen with liquid nitrogen. Under a surgical microscope, the specimens of parietal cortex, the striatum, and the CA1 and CA3 regions of hippocampus were removed on crushed ice. The above procedure was carried out without the induction of ischemic insults to provide a control group. Northern blotting analysis was carried out according to the method of Sasaki et al. [17]. Briefly, after total RNA was prepared by the single-step total RNA isolation method, 10 mg samples of total RNAs were electrophoresed and transferred to a membrane, and hybridized with 32P-labeled PP1a and 1g1 probes. The Western blotting analysis was performed according to the method of Shima et al. [18]. Briefly, the cytosolic fraction was harvested and each 10 mg of sample was electrophoresed and transferred to membranes. The membranes were incubated with rabbit polyclonal antibodies against the C-terminal of the two rat PP1 isotypes, and with peroxidase conjugated anti-rabbit IgG. Each of the bands, which were visualized with DuPont NEN(r) chemiluminescence, was analyzed with a computer-assisted image analyzer (Luzex-F). In this method, the amount of each band was expressed as the value obtained by multiplying the area by the mean density. The results of Northern blotting analysis revealed the increases in PP1a and 1g1 mRNA 24 h after transient fore-
brain ischemia (Figs. 1 and 2). It was particularly noteworthy that induction of these isotypes was stronger in the CA1 area and striatum than in the CA3 area and cortex. Western blotting analysis revealed that there is no remarkable change in expression of PP1a 24 h after ischemia, in contrast, the expression of PP1g1 tended to decrease in all regions (Figs. 3 and 4). This is the first study that examined the relationship
Fig. 2. The results of semiquantitative analysis of Fig. 1. The values of the area multiplied by the mean density of each band (designated as Area*Dens) were calculated with a computerassisted image analyzer.
T. Horiguchi et al. / Neuroscience Letters 325 (2002) 115–118
117
Fig. 3. The effect of transient forebrain ischemia on the protein expression of each PP in the cortex, striatum, and CA1 and CA3 regions in the hippocampus of Mongolian gerbil brains. The specific bands of PP1a (A) and 1g1 (B) are obtained. Lanes 1–4 are control brains and lanes 5–8 are postischemic brains. Lanes 1 and 5 are the cortex, lanes 2 and 6 are the striatum, lanes 3 and 7 are the CA1 region, and lanes 4 and 8 are the CA3 region. Note that PP1g1 has a tendency to decrease in all subfields contrary to the results of Northern blotting analysis 24 h after ischemia. On the other hand, PP1a has no remarkable change of expression in all regions 24 h after ischemia. Co, cortex; St, striatum; Hippo, hippocampus; Con, control; Pos, postischemia.
between PP1 and DND. We interpret the discrepancy of the expression between PP1 mRNA and protein in the vulnerable regions as possibly being associated with selective neuronal damage. The precise role of PP1a and PP1g1 in the brain has not been clarified. Hashikawa et al. reported localization of PP1g1 in the distal dendrites of cerebellar Purkinje cells [3]. The pharmacological studies revealed that the long-term depression (LTD) in the CA1 and cerebellum were inhibited by a PP1 inhibitor [6,11]. For sustaining LTD, the activation of alpha-amino-3-hydroxy-5methyl-4-isoxazolepropionic acid (AMPA) receptors plays a key role [9]. According to these studies, we believe the essential role of PP1g1 in the signal transduction through AMPA receptors in not only normal but also ischemic brain. The underlying mechanisms involved in alterations in PP1 mRNA and protein expression are unclear. The close relationship between selective vulnerability to ischemia and the persistent depression of protein synthesis is well known [1]. Transient global ischemia induced accumulation of mRNA without protein synthesis in vulnerable areas has been obtained in not only PPs but also with other gene products [12]. Therefore, the depressed protein synthesis may be the one possible explanation of the increases in PP1 mRNA. However, we speculate that the increases of PP1g1 mRNA, especially in the more vulnerable regions, may be a reaction to the hyperphosphorylated state.
Although the reduction of PP1g1 expression in protein levels was observed in the cortex and CA3 region, the expression in mRNA level does not change in the same region after ischemia. Another equally important mechanism to explain our data is the translocation of PP1 after transient ischemia. A similar change of protein kinases (PKs) has been reported in hippocampal cells after ischemia [4,14]. Hu et al. observed translocation of Ca 21/calmodulin-
Fig. 4. The results of semiquantitative analysis of Fig. 3. The values of the area multiplied by the mean density of each band (designated as Area*Dens) were calculated with a computerassisted image analyzer.
118
T. Horiguchi et al. / Neuroscience Letters 325 (2002) 115–118
dependent protein kinase II (CaM kinase II) to the synaptic junction after cerebral ischemia and concluded that persistent translocation of CaM kinase II in the vulnerable ischemic CA1 area is related to DND [4]. They emphasized events occurring in the nerve terminal and synaptic junctions after ischemia. Recent studies have revealed that changes in the activity of ser/thr PKs, e.g. PKC, or CAM kinase II, may contribute to neuronal death after ischemic insults [4,14]. However, little is known about ser/thr PPs in the ischemic brain. Morioka et al. demonstrated the preservation of immunoreactivity for PP2B (Calcineurin) in CA1 region. Since the activity of CAM kinase II was depressed in their study, they concluded that the difference between those enzymes might play a key role in DND [10]. However, the activities of each PP are regulated by a combination of targeting and regulatory subunits and by specific inhibitors [5]. We believe that the function of PP1 in a phosphorylation equilibrium is different from PP2B. In conclusion, our results suggest that PP1s may play a key role in DND. This suggestion is largely in agreement with pharmacological data demonstrating the crucial role of PPs in cell survival [13,16]. Further investigation to elucidate the cardinal role of PP1s may serve the clue to clarify the molecular mechanisms in ischemic neuronal injury. [1] Bodsch, W., Takahashi, K., Barbier, A., Grosse, O.B. and Hossmann, K.A., Cerebral protein synthesis and ischemia, Prog. Brain Res., 63 (1985) 197–210. [2] Hara, A., Niwa, M., Nakashima, M., Iwai, T., Uematsu, T., Yoshimi, N. and Mori, H., Protective effect of apoptosisinhibitory agent, N-tosyl-l-phenylalanyl chloromethyl ketone against ischemia-induced hippocampal neuronal damage, J. Cereb. Blood Flow Metab., 18 (1998) 819–823. [3] Hashikawa, T., Nakazawa, K., Mikawa, S., Shima, H. and Nagao, M., Immunohistochemical localization of protein phosphatase isoforms in the rat cerebellum, Neurosci. Res., 22 (1995) 133–136. [4] Hu, B.R. and Wieloch, T., Persistent translocation of Ca 21/ Calmodulin-dependent protein kinase II to synaptic junctions in the vulnerable hippocampal CA1 region following transient ischemia, J. Neurochem., 6 (1995) 277–284. [5] Hunter, T., Protein kinases and phosphatases: the yin and yang of protein phosphorylation and signaling, Cell, 80 (1995) 225–236. [6] Ito, M. and Karachot, L., Protein kinases and phosphatase inhibitors mediating long-term desensitization of glutamate receptors in cerebellar Purkinje cells, Neurosci. Res., 14 (1992) 27–38.
[7] Kindy, M.S., Inhibition of tyrosine phosphorylation prevents delayed neuronal death following cerebral ischemia, J. Cereb. Blood Flow Metab., 13 (1993) 372–377. [8] Kirino, T., Delayed neuronal death in the gerbil hippocampus following ischemia, Brain Res., 239 (1982) 57–69. [9] Linden, D.J., Dickinson, M.H., Smeyne, M. and Connor, J.A., A long-term depression of AMPA currents in cultured cerebellar Purkinje neurons, Neuron, 7 (1991) 81–89. [10] Morioka, M., Fukunaga, K., Yasugawa, S., Nagahiro, S., Ushio, Y. and Miyamoto, E., Regional and temporal alterations in Ca 21/calmodulin-dependent protein kinase II and calcineurin in the hippocampus of rat brain after transient forebrain ischemia, J. Neurochem., 58 (1992) 1798–1809. [11] Mulkey, R.M., Herron, C.E. and Malenka, R.C., An essential role for protein phosphatases in hippocampal long-term depression, Science, 261 (1993) 1051–1055. [12] Nowak Jr., T.S., Localization of 70 kDa stress protein mRNA induction in gerbil brain after ischemia, J. Cereb. Blood Flow Metab., 11 (1991) 432–439. [13] Nuydens, R., de Jong, M., Van Den Kieboom, G., Heers, C., Dispersyn, G., Cornelissen, F., Nuyens, R., Borgers, M. and Geerts, H., Okadaic acid-induced apoptosis in neuronal cells: evidence for an abortive mitotic attempt, J. Neurochem., 70 (1998) 1124–1133. [14] Olah, Z., Ikeda, J., Anderson, W.B. and Joo, F., Altered protein kinase C activity in different subfields of hippocampus following cerebral ischemia, Neurochem. Res., 15 (1990) 515–518. [15] Pulsinelli, W.A., Brierley, J.B. and Plum, F., Temporal profile of neuronal damage in a model of transient forebrain ischemia, Ann. Neurol., 11 (1982) 491–498. [16] Runden, E., Seglen, P.O., Haug, F.M., Ottersen, O.P., Wieloch, T., Shamloo, M. and Laake, J.H., Regional selective neuronal degeneration after protein phosphatase inhibition in hippocampal slice cultures: evidence for a MAP kinase-dependent mechanism, J. Neurosci., 18 (1998) 7296–7305. [17] Sasaki, K., Shima, H., Kitagawa, Y., Irino, S., Sugimura, T. and Nagao, M., Identification of members of the protein phosphatase 1 gene family in the rat and enhanced expression of protein phosphatase 1a gene in rat hepatocellular carcinomas, Jpn. J. Cancer Res., 81 (1990) 1272–1280. [18] Shima, H., Hatano, Y., Chun, Y.S., Sugimura, T., Zhang, Z., Lee Ernest, Y.C. and Nagao, M., Identification of PP1 catalytic subunit isotypes PP1g1, PP1d, and PP1a in various rat tissues, Biochem. Biophys. Res. Commun., 192 (1993) 1289–1296. [19] Ward, Y., Gupta, S., Jensen, P., Wartmann, M., Davis, R.J. and Kelly, K., Control of MAO kinase activation by the mitogen-induced threonine/tyrosine phosphatase PAC1, Nature, 367 (1994) 651–654. [20] Wiessner, C., The dural specific phosphatase PAC-1 is transcriptionally induced in the rat brain following transient forebrain ischemia, Brain Res. Mol. Brain Res., 28 (1995) 353–356.