Selective translocation of diacylglycerol kinase ζ in hippocampal neurons under transient forebrain ischemia

Neuroscience Letters 372 (2004) 190–195

Selective translocation of diacylglycerol kinase ␨ in hippocampal neurons under transient forebrain ischemia Hasmat Alia,b , Tomoyuki Nakanoa , Sachiko Saino-Saitoa , Yasukazu Hozumia , Yuji Katagiria , Hideyuki Kamiib , Shinya Satob , Takamasa Kayamab , Hisatake Kondoc , Kaoru Gotoa,∗ a

c

Department of Anatomy and Cell Biology, Yamagata University School of Medicine, Iida-nishi 2-2-2, Yamagata 990-9585, Japan b Department of Neurosurgery, Yamagata University School of Medicine, Iida-nishi 2-2-2, Yamagata 990-9585, Japan Division of Histology, Department of Cell Biology, Tohoku University Graduate School of Medicine, Seiryo-cho 2-1, Sendai 980-8575, Japan Received 6 August 2004; received in revised form 10 September 2004; accepted 11 September 2004

Abstract The molecular mechanisms responsible for differential neuronal vulnerability to ischemic injury are incompletely understood. Previous studies have reported that the expression and activity of protein kinase C (PKC), some subtypes of which are activated by Ca2+ and diacylglycerol (DG), are altered after ischemic insults. Therefore, DG kinase (DGK), which is responsible for controlling PKC activity through DG metabolism, may also be involved in this process. DGK␨, which is abundantly expressed in the brain, contains a nuclear localization signal (NLS), suggesting its involvement in some nuclear processes in neuronal cells. To elucidate the functional implications of DGK␨ in ischemia, we examined detailed localization of DGK␨ in rat brain after ischemic insults. We used an ischemic model of global cerebral ischemia for 20 min by bilateral common carotid artery occlusion combined with hypotension and followed time-points of reperfusion. DGK␨ expression was evaluated by immunohistochemistry using affinity-purified anti-DGK␨ antibody. In sham-operated rats, a strong DGK␨-immunoreactivity was observed in the nucleus of neurons in various parts of the brain. In the global ischemic model DGK␨-immunoreactivity was reduced in intensity in the hippocampal formation and detected in the cytoplasm of CA1 pyramidal neurons throughout reperfusion time courses. Change in the subcellular localization was restricted to the pyramidal cells in CA1 and later in CA3, but not observed in other areas of hippocampus. No change was observed in the cerebral and cerebellar cortices. The present study suggests that DGK␨ might be involved in the process of selective vulnerability of hippocampal pyramidal neurons in postischemic brain. © 2004 Elsevier Ireland Ltd. All rights reserved. Keywords: Diacylglycerol kinase; Rat brain; Ischemia; Hippocampus; Subcellular localization

Neurons in certain parts of the brain are especially susceptible to ischemia and anoxia, which are caused by arterial occlusion or heart attack. Understanding the molecular mechanisms responsible for differential neuronal vulnerability to ischemic injury is of considerable clinical importance because one of the most common causes of neuronal injury in humans is ischemia or anoxia. A well-known example of selective vulnerability is the case of the hippocampal neurons following ischemia [1]. The mechanism underlying this selective

∗

Corresponding author. Tel.: +81 23 628 5207; fax: +81 23 628 5210. E-mail address: [email protected] (K. Goto).

0304-3940/$ – see front matter © 2004 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.neulet.2004.09.052

vulnerability is incompletely understood, but pharmacological studies showing that glutamate receptor antagonists block neuronal degeneration suggest that glutamate-mediated excitotoxicity is involved [5,27]. Subtypes of glutamate receptor are linked to both ion channels and G proteins that activate phospholipase C. Activation of Ca2+ - and diacylglycerol (DG)-dependent signalling pathways by glutamate may, therefore, contribute to the development of ischemic neuronal injury. In support of this, ischemia-induced increases in intracellular messengers, such as DG, Ca2+ , and arachidonic acid have been described previously [31,34]. In addition several studies have reported that in rat brain the activity of protein kinase C (PKC), some subtypes of which are activated

H. Ali et al. / Neuroscience Letters 372 (2004) 190–195

by Ca2+ and DG, is initially increased and subsequently decreased after global cerebral ischemia [3,6,33] and that PKC is redistributed from the cytosolic fraction to cell membranes in the ischemic rat brain [4,19,33]. Furthermore, it is also demonstrated that topical administration of staurosporine, a relatively specific and potent PKC inhibitor, prevented postischemic neuronal damage in the CA1 field of rat and gerbil, whereas inhibitors of cAMP-dependent, cGMP-dependent, and calmodulin-dependent kinases had no neuroprotective effect [18,35]. Taken together, these studies strongly suggest that activation of PKC isozymes by ischemic injury could play a central role in the pathogenesis of neuronal damage. DG kinase (DGK) is an enzyme responsible for attenuation of DG signal by phosphorylation of DG to produce phosphatidic acid (PA). Because DG serves as an activator of PKC, DGK is thought to regulate PKC activity through DG metabolism. Others, and we have isolated several DGK isozymes from mammalian cDNA libraries [16,28,32]. We also examined their mRNA expression in rat brain by in situ hybridization histochemistry [12–18]. Interestingly, it is revealed that the mRNA for each isozyme is expressed in a distinct pattern in the brain. Molecular diversity and distinct expression patterns may reflect functional importance of DGK isozymes in the brain and suggest that each isozyme plays a unique role in physiologic and pathologic brain functions at the right place in response to various signalling pathway under distinct regulatory mechanisms, although very little is known about the details. Among these, DGK␨ (previously also termed DGK-IV for rat clone), which is abundantly expressed in the brain including the olfactory bulb, cerebral and cerebellar cortices, and hippocampus, is characterized by the presence of a nuclear localization signal (NLS) together with C-terminal four tandem of ankyrin-like repeats [2,8,14,21], suggesting a possible involvement of DGK␨ in some nuclear processes in neuronal cells. To elucidate functional implications of DGK␨ in ischemia, we examined detailed immunohistochemical localization of DGK␨ in rat brain after ischemic insults. We used an ischemic model of global cerebral ischemia for 20 min by bilateral common carotid artery occlusion combined with hypotension and followed time-points of reperfusion. Here we show the altered expression and subcellular localization of DGK␨ in the hippocampal neurons of ischemic brain. The Animal Research Committee of the Yamagata University School of Medicine approved all animal experimental protocols. Adult Wistar rats (250–300 g) were given free access to food and water until the day of experimentation. They were anesthetized initially with 2% sevoflurane, 70% N2 O, and 30% O2 by an induction chamber, and then were mechanically ventilated. The sevoflurane was reduced to 1.0–1.5% for the remainder operation. Rectal temperature of the animal was maintained at 37 ◦ C with a heating pad. The femoral arteries were cannulated with polyethylene catheters with 50 U/ml heparinized saline solution for continuous blood pressure monitoring, blood sampling for arterial blood gas determinations, and induction of hemorrhagic hypotension

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during ischemia. The common carotid arteries were exposed by a longitudinal midline ventral incision and dissected free of surrounding nerve fibers. Global cerebral ischemia was initiated by the combination of bilateral common carotid artery occlusion with hypovolumic hypotension to mean arterial blood pressure (MABP) of 45–50 mmHg with some modification [25,29]. Hypovolumic hypotension was achieved by rapidly withdrawing blood from femoral arterial catheter into a heparinized syringe. Once a MABP of 45 mmHg was achieved, both common carotid arteries were then occluded for 20 min by the surgical aneurysm clips. Blood pressure was maintained at 45–50 mmHg throughout the occlusion period, and blood gas was adjusted as needed. The shed blood was maintained at 37 ◦ C during the ischemic period. In the group without reperfusion the shed blood was discarded, while in the reperfused ischemic group the clips were removed and the shed blood was reinfused to allow recirculation. The animals were sacrificed at 0 (without reperfusion), 3, 24, and 72-h reperfusion after 20 min global ischemia (n = 4 each). Sham-operated animals were also examined, which were subjected to anesthesia and surgical preparation without bilateral carotid occlusion and hypovolumic hypotension. Sham-operated and ischemic rats were anesthetized with ether and fixed with a transcardiac infusion of periodatelysine-paraformaldehyde (PLP) fixative for immunohistochemistry. The brain was removed, immersed in the same solution for a further 2 h at 4 ◦ C, and kept in 30% sucrose in 0.1 M phosphate buffer (pH 7.0) until use. For immunoblot analysis, the hippocampus was freshly removed, homogenized and subjected to SDS/10% PAGE. Immunoreactive bands for anti-rat DGK␨ antibody were visualized using the chemiluminescent ECL+Plus Western blotting detection system (Amersham Pharmacia Biotech UK Limited, Buckinghamshire, UK) as described [21]. For immunohistochemistry, coronal sections (30 ␮m) were cut on a cryostat at the level of the hippocampus for the global ischemia. Free-floating sections were soaked with 0.3% Triton X-100 in phosphate buffered saline (PBS) for 2 h at room temperature (RT) to facilitate antibody penetration. Endogenous peroxidase activity was inactivated with 3% H2 O2 for 10 min at RT, and non-specific binding sites were blocked with 5% normal goat serum (NGS) in PBS for 1 h at RT. The sections were then incubated at 4 ◦ C for overnight with affinity purified rabbit anti-rat DGK␨ antibody (0.1 ␮g/ml) in PBS containing 5% NGS and 0.1% Tween-20 [21]. After washing in PBS several times, they were incubated with a biotinylated anti-rabbit IgG antibody in the same solution for 30 min at RT followed by avidin-biotin-peroxidase complex (ABC) method using a kit (Vector Laboratories, Burlingame, CA) for 30 min at RT. After rinsing, immunolabel was visualized with 0.03% diaminobenzidine tetrahydrocloride (DAB) with 0.002% H2 O2 in 50 mM Tris–HCl buffer (pH 7.5). For immunofluorescent examination, the sections were incubated with streptavidin conjugated-Alexa 488 (similar to FITC)(Molecular Probes, Inc., OR) in PBS for 30 min at RT, instead of ABC. To stain nuclei, some sections were also incubated with propidium

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iodide (15 ␮g/ml), which gives red fluorescence similar to Rhodamine [22]. The images were taken under the confocal laser scanning microscope (PASCAL, Carl Zeiss, Germany) and processed using Adobe Photoshop. Expression of DGK␨ was evaluated by immunohistochemistry on rat brains at various time-points of reperfusion following global cerebral ischemia in comparison with the sham-operated control brains. In sham-operated brains, DGK␨-immunoreactivity was observed throughout gray matter regions, as suggested by our previous study showing wide expression of the mRNA signals in rat brain [14,16,21]. Intense immunoreactivity was detected in the cerebral and cerebellar cortices, hippocampal formation, and amygdaloid nuclei, and moderately in the striatum, thalamus, and hypothalamus. In the cerebral cortex, most neurons were immunoreactive for DGK␨ (Fig. 1). The degree of staining varied according to cell type and seemed to be directly proportional to the size of neurons: large pyramidal cells were more intensely stained for DGK␨ than other, smaller neurons. DGK␨immunoreactivity was typically detected in nuclei of these neurons. In the hippocampus, most of the neurons were immunoreactive for DGK␨, including pyramidal cells in CA1-3, dentate granule cells, and scattered interneurons (Fig. 1). In the cerebellum, Purkinje and granule cells were stained, although both the nucleus and the cytoplasm were immunoreactive in Purkinje cells (data not shown). No immunoreactiv-

ity was observed in the white matter, such as corpus callosum, striatal fiber bundles, and cerebellar medulla. These data show that the immunoreactivity of DGK␨ in sham-operated brain is compatible with our previous report in normal rat brain [21]. In global ischemic brain total DGK␨-immunoreactivity was reduced in intensity in all areas of the hippocampal formation. At 0-h reperfusion, i.e., immediately after ischemia, the most prominent change was observed in CA1 pyramidal neurons. The immunoreactivity was greatly reduced in the nucleus, but rather detected in the perikaryal cytoplasm, suggesting that DGK␨ is translocated from the nucleus to the cytoplasm in these cells under ischemic insult (Fig. 1, arrows). This change in the subcellular localization was selectively observed in CA1 pyramidal neurons, but not in other areas of the hippocampal formation including CA3 and dentate gyrus. On closer examination, the nuclear immunoreactivity still remained in large interneurons scattered in the stratum pyramidale of CA1 (Fig. 1, arrowheads). This further supports the idea that the nucleo-cytoplasmic translocation of DGK␨ occurs in a cell-specific manner, but not layer-specific. At 24-h reperfusion the immunoreactivity was observed in the hippocampus in a fashion similar to that detected at 0-h reperfusion, though at reduced intensity. In addition, about half of the CA3 pyramidal neurons also exhibited perikaryal cytoplasmic staining (Fig. 1 and Table 1). At 72-h reperfusion more than 90% of the pyramidal neurons showed cytoplasmic staining in the CA3 region. In contrast to the hippocampal formation, no change was observed in neurons of the cerebral cortex and dentate gyrus in terms of the subcellular distribution throughout the experiment. To further examine the subcellular localization of DGK␨ in higher resolution, we used confocal laser scanning microscopy. As shown in Fig. 2, the immunoreactivity in ischemic CA1 pyramidal neurons was observed in the cytoplasm as puncta of various sizes (arrows). The intensity decreased gradually during the time course of reperfusion. Table 1 Quantitative anlysis of subcellular localization of DGK␨ in rat brain after ischemic insult

Fig. 1. DGK␨-immunoreactivity in control and 20-min global ischemia rat brains. Sections of the cerebral cortex, hippocampus (CA1 and CA3) and dentate gyrus (DG) in sham-operated control and ischemic brains at 0, 24, and 72 h after reperfusion are shown. The immunoreactivity is observed in the perikaryal cytoplasm of hippocampal CA1 pyramidal neurons (thick arrows) immediately after ischemic insult and is never detected in the nucleus during reperfusion. Note that the nuclear immunoreactivity remains in scattered large interneurons (arrowheads). In the CA3 region, neurons gradually show cytoplasmic staining (thin arrows) after 24-h reperfusion. No change is observed in neurons of the cerebral cortex and detate gyrus during the experiment. Scale bar: 50 ␮m.

Sections of the cerebral cortex, hippocampus (CA1 and CA3), and dentate gyrus (DG) were immuno-stained and more than 100 neurons in shamoperated control (c) and ischemic brains at 0, 24, 72 h after reperfusion were classified into two categories: nuclear dominant staining (open bar); cytoplasmic dominant staining (solid bar).

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Fig. 3. Immunoblot analysis of control and ischemic rat hippocampus with anti-DGK␨ antibody. Hippocampal homogenates (total protein, 20 ␮g) in control (cont) and ischemic (ische) brains (at 0-h reperfusion) were subjected to SDS/10% PAGE and analyzed by immunoblot using anti-DGK␨ antibody. Size markers are indicated on the left in kiloDalton.

Fig. 2. Confocal images of DGK␨-immunoreactivity in neurons of the CA1 and dentate gyrus (DG) of control and ischemic rat brains. (A) The immunoreactivity is detected in a punctate pattern in the cytoplasm of CA1 pyramidal neurons after ischemia and is decreasing in intensity during reperfusion. No change is observed in neurons of the dentate gyrus, and (B) double labeling of DGK␨ (green) and nucleus (red) in the ischemic CA1 pyramidal neurons (at 0-h reperfurion). Scale bar: 10 ␮m.

No immunoreactivity was detected in the nucleus stained for propidium iodide (Fig. 2B). In contrast, neurons in other areas, such as the dentate gyrus, still showed a nuclear staining of speckled appearance similar to that of the control, though at reduced level. In immunoblot analysis using anti-DGK␨ antibody, a single band was detected in the hippocampus of ischemic brain (at 0-h reperfusion) at the same position as in the control,

but less intensely, suggesting no apparent proteolysis of this molecule at an early phase of ischemia (Fig. 3). It also shows that the protein detected with this antibody in ischemic brain is the same species as in normal brain, but not the longer species of alternative spliced form expressed in the muscle [7]. The present study reveals that DGK␨-immunoreactivity shows remarkable changes in its expression and localization in the hippocampal neurons of rat brain insulted by the experimental ischemia, suggesting that DGK␨ might be involved in the process of ischemic injury in these neurons. One of the major findings in the present study of global ischemia is that DGK␨ might be translocated from the nucleus to the perikaryal cytoplasm in CA1 pyramidal cells at a very early phase of ischemic insult. With regard to the functional implication of this phenomenon, the cause and effect relationship between this translocation and subsequent neuronal death remains to be determined. However, it should be noted that in the ischemic CA1 neurons DGK␨-immunoreactivity remains cytoplasmic at decreasing intensity during the time course of reperfusion and has never been relocated to the nucleus. This suggests that the translocation of DGK␨ is not just due to passive diffusion, which might be a reversible response to hypoxic condition, but is caused by an active transport mechanism leading to subsequent processes. Previous studies have shown that the increase in second messengers, such as DG and Ca2+ , is induced by ischemia [31,34] and that the activity of PKC, some subtypes of which are activated by these messengers, is upregulated initially after global cerebral ischemia [3,33]. The nucleo-cytoplasmic translocation of DGK␨ in CA1 neurons at an early phase of ischemic insult may result in a greatly reduced DGK activity in the nucleus of these neurons. As DGK plays a major role of attenuating DG, this might lead to a sustained increase in DG level and subsequent activation of PKC in the nucleus. Therefore, it is plausible that the ischemia-induced increase

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in DG and upregulation of PKC activity reported previously might, to some extent, reflect a nuclear process. It is also suggested that delayed neuronal death in CA1 neurons occurs after 48–72-h reperfusion in the global ischemia model [24]. At the time point immediately after 20 min of ischemia, when the nucleo-cytoplasmic translocation of DGK␨ is observed, there is no apparent morphological change in CA1 neurons under the light microscope. Furthermore, the delayed neuronal death of these neurons after transient ischemia is not necrotic but apoptotic by the fact that those cells show chromatin condensation, TUNEL reaction, and DNA laddering [26]. In apoptotic cell death, nuclear changes are thought to play a key role, in which DNA instability, such as fragmentation, is linked to irreversible alterations of the neurons. Taken the previous findings and the present data together, we suggest a hypothesis that transient ischemia induces removal of DGK␨ from the nucleus, which leads to the reduced DGK activity and sustained increase in DG level in the nucleus. As a result, PKC activity would be upregulated in the nucleus, which might be one of the triggering signals in the nuclear process of delayed neuronal death. Further studies are needed to clarify these points. With regard to the punctate pattern of the immunoreactivity in the perikaryal cytoplasm in CA1 neurons of ischemic brain revealed by confocal microscopic examination, it remains to be elucidated at present whether this punctate immunoreactive pattern of DGK␨ corresponds to lysosomes or cytoplasmic aggregates. In either case, the decreasing immunoreactivity during the time course of reperfusion suggests that degradation process is operated on the translocated molecule. Protein shuttling between the nucleus and the cytoplasm is mediated, if not completely controlled, by members of a nuclear localization signal family, which interact with the ␣ subunit of importin, the NLS receptor [9]. In this regard we identified for the first time a bipartite type of NLS in the regulatory domain of rat DGK␨ [14]. Previous study suggests that phosphorylation of the MARCKS (myristoylated alanine-rich C-kinase substrate) domain overlapping the NLS by PKC might regulate nuclear localization of DGK␨ [30]. On the other hand, it is suggested that interaction of the Cterminal PDZ-binding motif of DGK␨ with the PDZ domain of ␥1-syntrophin may control nuclear localization of this isozyme [20]. It should be also mentioned that nuclear export may be mediated by a family of leucine-rich nuclear export signal (NES) peptides, such as the LxxLxxLxL motif. The NES mediates signal-dependent transport of proteins from the nucleus back into the cytoplasm, depending on the nuclear exporter Crm1/exportin 1 [10,11]. Interestingly, the leucinerich residues are also included in the amino acid sequence of DGK␨ (362-LSTLDQLRL-370) in rat sequence [14], which is compatible with the consensus motif of NES. Further studies are required to elucidate precise molecular machinery exerted on the nuclear export of DGK␨ in ischemia. Collectively, the present study reveals that the subcellular localization of DGK␨ is changed from the nucleus to

the cytoplasm in hippocampal CA1 neurons at a very early phase of ischemic insult. This strongly suggests that DGK␨ might be involved in the process of selective vulnerability of those neurons in postischemic brain. Although functional significance of DGK␨ in those processes remains to be elucidated, it is of particular interest to note that immunoreactivity for the immunophilin FK506-binding protein (FKBP12) exhibits close resemblance to that for DGK␨ in the global ischemia model [23]. This model includes reduction of FKBP12-immunoreactivity and its partial translocation from the nucleus to the cytoplasm in the CA1 neurons following global ischemia. Considering the physiologic roles of FKBP12 in the modulation of Ca2+ influx via interaction with ryanodine receptor and inositol trisphosphate (IP3 ) receptor and the augmentation of nerve regeneration, DGK␨ may also participate in those processes in a still unknown manner. The findings of this study warrant further investigation.

References [1] J.B. Brierley, Cerebral hypoxia, in: W. Blackwood, J.A.N. Corsellis (Eds.), Greenfield’s Neuropathology, third ed., Arnold, London, pp. 41–85. [2] M. Bunting, W. Tang, G.A. Zimmerman, T.M. McIntyre, S.M. Prescott, Molecular cloning and characterization of a novel human diacylglycerol kinase ␨, J. Biol. Chem. 271 (1996) 10230–10236. [3] R. Busto, M.Y. Globus, J.T. Neary, M.D. Ginsberg, Regional alterations of protein kinase C activity following transient cerebral ischemia: effects of intraischemic brain temperature modulation, J. Neurochem. 63 (1994) 1095–1103. [4] M. Cardell, H. Bingren, T. Wieloch, J. Zivin, T. Saitoh, Protein kinase C is translocated to cell membranes during cerebral ischemia, Neurosci. Lett. 119 (1990) 228–232. [5] D.W. Choi, Glutamate neurotoxicity and diseases of the nervous system, Neuron 1 (1988) 623–634. [6] R.C. Crumrine, G. Dubyak, J.C. LaManna, Decreased protein kinase C activity during cerebral ischemia and reperfusion in the adult rat, J. Neurochem. 55 (1990) 2001–2007. [7] L. Ding, M. Bunting, M.K. Topham, T.M. McIntyre, G.A. Zimmerman, S.M. Prescott, Alternative splicing of the human diacylglycerol kinase zeta gene in muscle, Proc. Natl. Acad. Sci. U.S.A. 94 (1997) 5519–5524. [8] L. Ding, T.M. McIntyre, G.A. Zimmerman, S.M. Prescott, The cloning and developmental regulation of murine diacylglycerol kinase ␨, FEBS Lett. 429 (1998) 109–114. [9] C. Dingwall, R.A. Laskey, Nuclear targeting sequences-a consensus? Trends Biochem. Sci. 16 (1991) 478–481. [10] M. Fornerod, M. Ohno, M. Yoshida, I.W. Mattaj, CRM1 is an export receptor for leucine-rich nuclear export signals rich nuclear export signals, Cell 90 (1997) 1051–1060. [11] M. Fukuda, S. Asano, T. Nakamura, M. Adachi, M. Yoshida, M. Yanagida, E. Nishida, CRM1is responsible for intracellular transport mediated by the nuclear export signal, Nature 390 (1997) 308–311. [12] K. Goto, M. Funayama, H. Kondo, Cloning and expression of a cytoskeleton-associated diacylglycerol kinase that is dominantly expressed in cerebellum, Proc. Natl. Acad. Sci. U.S.A. 91 (1994) 13042–13046. [13] K. Goto, H. Kondo, Molecular cloning and expression of a 90-kDa diacylglycerol kinase that predominantly localizes in neurons, Proc. Natl. Acad. Sci. U.S.A. 90 (1993) 7598–7602.

H. Ali et al. / Neuroscience Letters 372 (2004) 190–195 [14] K. Goto, H. Kondo, A 104-kDa diacylglycerol kinase containing ankyrin-like repeats localizes in the cell nucleus, Proc. Natl. Acad. Sci. U.S.A. 93 (1996) 11196–111201. [15] K. Goto, H. Kondo, Heterogeneity of diacylglycerol kinase in terms of molecular structure, biochemical characteristics and gene expression localization in the brain, J. Lipid Mediat. Cell Signal. 14 (1996) 251–257. [16] K. Goto, H. Kondo, Diacylglycerol kinase in the central nervous system—molecular heterogeneity and gene expression, Chem. Phys. Lipids. 98 (1999) 109–117. [17] K. Goto, M. Watanabe, H. Kondo, H. Yuasa, F. Sakane, H. Kanoh, Gene cloning, sequence, expression and in situ localization of 80 kDa diacylglycerol kinase specific to oligodendrocyte of rat brain, Brain Res. Mol. Brain Res. 16 (1992) 75–87. [18] H. Hara, H. Onodera, M. Yoshidomi, Y. Matsuda, K. Kogure, Staurosporine, a novel protein kinase C inhibitor, prevents postischemic neuronal damage in the gerbil and rat, J. Cereb. Blood Flow Metab. 10 (1990) 646–653. [19] K. Harada, T. Maekawa, K.M. Abu Shama, T. Yamashima, K. Yoshida, Translocation and down-regulation of protein kinase C␣, -␤, and -␥ isoforms during ischemi-reperfusion in rat brain, J. Neurochem. 72 (1999) 2556–2564. [20] A. Hogan, L. Shepherd, J. Chabot, S. Quenneville, S.M. Prescott, M.K. Topham, S.H. Gee, Interaction of ␥ 1-syntrophin with diacylglycerol kinase-␨. Regulation of nuclear localization by PDZ interactions, J. Biol. Chem. 276 (2001) 26526–26533. [21] Y. Hozumi, T. Ito, T. Nakano, T. Nakagawa, M. Aoyagi, H. Kondo, K. Goto, Nuclear localization of diacylglycerol kinase ␨ in neurons, Eur. J. Neurosci. 18 (2003) 1448–1457. [22] B.R. Hu, S. Janelidze, M.D. Ginsberg, R. Busto, M. Perez-Pinzon, T.J. Sick, B.K. Siesjoe, C.L. Liu, Protein aggregation after focal brain ischemia and reperfusion, J. Cereb. Blood Flow Metab. 21 (2001) 865–875. [23] H. Kato, T. Oikawa, K. Otsuka, A. Takahashi, Y. Itoyama, Postischemic changes in the immunophilin FKBP12 in the rat brain, Brain Res. Mol. Brain Res. 84 (2000) 58–66. [24] T. Kirino, K. Sano, Selective vulnerability in the gerbil hippocampus following transient ischemia, Acta Neuropathol. 62 (1984) 201–208.

195

[25] A.N. Miles, B.T. Majda, B.P. Meloni, N.W. Knucky, Postischemic intravenous administration of magnesium sulfate inhibits hippocampal CA1 neuronal death after transient global ischemia in rats, Neurosurgery 49 (2001) 1443–1450. [26] T. Nitatori, N. Sato, S. Waguri, Y. Karasawa, H. Araki, K. Shibanai, E. Kominami, Y. Uchiyama, Delayed neuronal death in the CA1 pyramidal cell layer of the gerbil hippocampus following transient ischemia is apoptosis, J. Neurosci. (1995) 15. [27] S.M. Rothman, J.W. Olney, Glutamate and the pathophysiology of hypoxic-ischemic brain damage, Ann. Neurol. 19 (1986) 105– 111. [28] F. Sakane, H. Kanoh, Molecules in focus: diacylglycerol kinase, Int. J. Biochem. Cell Biol. 29 (1997) 1139–1143. [29] M.L. Smith, G. Bendek, N. Dahlgren, I. Rosen, T. Wieloch, B.K. Siesjo, Models for studying long term recovery following forebrain ischemia in the rat. 2. A 2-vessel occlusion model, Acta Neurol. Scand. 69 (1984) 385–401. [30] M.K. Topham, M. Bunting, G.A. Zimmerman, T.M. McIntyre, P.J. Blackshear, S.M. Prescott, Protein kinase C regulates the nuclear localization of diacylglycerol kinase-␨, Nature 394 (1998) 697– 700. [31] D. Uematsu, J.H. Greenberg, M. Reivich, A. Karp, In vivo measurement of cytosolic free calcium during cerebral ischemia and reperfusion, Ann. Neurol. 24 (1988) 420–428. [32] W.J. van Blitterswijk, B. Houssa, Properties and functions of diacylglycerol kinases, Cell Signal. 12 (2000) 595–605. [33] T. Wieloch, M. Cardell, H. Bingren, J. Zivin, T. Saitoh, Changes in the activity of protein kinase C and the differential subcellular redistribution of its isozymes in the rat striatum during and following transient forebrain ischemia, J. Neurochem. 56 (1991) 1227– 1235. [34] S. Yoshida, M. Ikeda, R. Busto, M. Santiso, E. Martinez, M.D. Ginsberg, Cerebral phosphoinositide, triacylglycerol, and energy metabolism in reversible ischemia: origin and fate of free fatty acids, J. Neurochem. 47 (1986) 744–757. [35] M. Yoshidomi, K. Kogure, H. Hara, K. Abe, Y. Matsuda, Inhibition of protein kinase activities ameliorates post ischemic brain cell damage, J. Cereb. Blood Flow Metab. 9 (1989) S186.