Brain Research, 541 (1991) 171-174 Elsevier

171

BRES 24535

Reduction of protein kinase C activity in the adult rat brain following transient forebrain ischemia Jean-Claude Louis 1'2'*, Ella Magal 2'* , Alain Brixi 3, Regis Steinberg 3, Ephraim Yavin 2 and Guy Vincendon 1 1Centre de Neurochimie du CNRS, Strasbourg (France), 2Department of Neurobiology, The Weizmann Institute of Science, Rehovot (Israel) and 3Laboratoire de Neurobiochimie, Centre de Recherche SANOFI, Montpellier (France)

(Accepted 6 November 1990) Key words: Protein kinase C; Ischemia; Striatum; Cortex; Hippocampus; Neurodegeneration

Acute forebrain ischemia reduced protein kinase C (PKC) activity in the adult rat cortex, striatum and hippocampus by 60-70% after 20 min ischemia episodes, followed by 48 h of recirculation. Ischemia of 1 min, followed by recirculation, produced a less pronounced but significant decrease in PKC activity. The ischemia-induced decrease of PKC affected both the soluble and the membrane-bound kinase. Alterations of PKC predate neuronal death following ischemia.

Ischemia leads to a complex sequence of events culminating in the loss of functional integrity of the nervous system and, ultimately, in neuronal cell death 18' 19 Substantial evidence supports the hypothesis that intracellular accumulation of calcium ions and activation of phospholipases A 2 and C, leading to release of free fatty acids 2'5 and diacylglycerols1'4, resp., from membrane phospholipids trigger the cascade of ischemic neuronal cell damage (reviewed in ref. 19). Sustained increased intracellular Ca 2÷ levels, excess formation of free arachidonic acid and diacylglycerols may alter the activity and function of the Ca2+/diacylglycerol-dependent protein kinase C (PKC) 1233. As a result, protein phosphorylation and normal signal transduction may be impaired, and neuronal death may follow. We have recently reported that short ischemic episodes (5 min) cause a near complete and reversible translocation of PKC from the cytosolic to the membrane compartment in the fetal rat. Prolonged ischemic episodes (30 min), in contrast, are characterized by a significant decrease in PKC activity, concomitant with the appearance of a Ca2÷/diacylglycerol-independent kinase activity 8. These observations led us to extend these studies and to investigate the consequences of ischemia on the activity of PKC in the adult rat brain. The 4-vessel-occlusion model of the adult rat, which produces a uniform and reversible cessation of blood supply to the forebrain and results in a reproducible pattern of delayed

neuronal cell damage was employed. Herein is demonstrated that PKC activity of the cortex, striatum and hippocampus is decreased after 24 h following ischemic episodes of duration as short as 1 min. Male Wistar rats weighing 250-300 g (Jackson Laboratories, U.K.) were subjected to bilateral forebrain ischemia, by a modification of the model described by PulsineUi and Brierley 16. Briefly, the rats were anesthetized with chloral hydrate (300 mg/kg b.wt. administered i.p.) and both common carotid arteries were exposed and encircled with sterile thread (0.3 mm diameter) that was passed through plastic Tygon tubes. The alar foramina of the first cervical vertebra were then exposed, and both vertebral arteries were occluded by electrocauterization. After 24 h, the rats were restrained and cerebral ischemia was induced by pulling out the threads, which were held in position with a metallic clamp, in order to occlude the carotid arteries. After designated periods of ischemia, the clamps were removed and the animals were either sacrificed for immediate sample processing, or recirculation was permitted for 24-48 h. The control groups were: (i) sham-operated animals, in which carotid arteries were exposed and C 1 n e r v e s protruding out of the alar foramina divided, but vertebral arteries were not electrocauterized, and (ii) a two-vessel-occlusion group, in which vertebral arteries were occluded, but carotid arteries remained untouched. On the day of the experiment, these animals were restrained, but the threads

* Present address: Department of Biology M-001, University of California at San Diego, La Jolla, CA 92093, U.S.A. Correspondence: J.C. Louis, Department of Biology M-001, University of California at San Diego, La Joila, CA 92093, U.S.A. 0006-8993/91/$03.50 (~) 1991 Elsevier Science Publishers B.V. (Biomedical Division)

172 around the carotids were not pulled out. After decapitation, the rat brains were quickly removed and the cortical (Cx), hippocampal (Hc) and striatal (St) structures were each dissected and separated from coronal sections and immediately frozen in liquid nitrogen. The collected structures were individually homogenized at 0-4 °C in 1.2 ml of homogenizing buffer consisting of 20 mM Tris-HCl buffer pH 7.5, 2 mM EDTA, 0.5 mM EGTA, 2 mM dithiotreitol, 2 mM phenylmethylsulfonylfluoride and other protease inhibitors (pepstatin A, leupeptin, aprotinin, 10 #g/ml, each). The resulting homogenates were then centrifuged at 100,000 g for 1 h at 4 °C to separate soluble and membrane fractions. The resulting pellets were resuspended in 1.2 ml of homogenizing buffer containing 0.3% Triton X-100, incubated for 1 h at 4 °C and centrifuged at 100,000 g for 1 h, all at 4 °C. The cytosolic and Triton X-100-solubilized membrane fractions were subjected to DEAE-cellulose chromatography, in order to partially purify PKC, the activity of which was measured as described previously8. Bilateral forebrain ischemia produced by occlusion of the 4 major arteries for 20 min, resulted in a decrease of PKC activity in several brain areas after 48 h of recirculation. Total PKC activity was reduced by 40-50%

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in Cx, St and Hc (Fig. 1). However, no significant changes were seen in the hypothalamus and the cerebellum (data not shown). Membrane-bound PKC activities, particularly, decreased in the Cx, St and Hc by 52%, 58% and 55%, respectively, but also in the soluble fraction. Decrease of PKC activity was not detected when the rats were sacrificed immediately after the ischemia without recirculation (Fig. 2). Furthermore, no significant change in total (soluble plus particulate) PKC activity of the examined brain structures occurred following ischemia sessions of 1 min, 20 min or four consecutive episodes each for 5 min (with 1 h of recirculation between each session). The decrease in PKC activity was dependent on the duration of ischemia. When PKC activity was measured after a 24 h recirculation period following ischemia, maximal loss was observed after a 20 min ischemia session, in the Cx, St and Hc (70%, 60% and 72%, respectively) (Fig. 2). The decrease observed after 1 min of ischemia (followed by 24 h of recirculation) was less pronounced, although significant (48%, 55% and 22% for Cx, St and Hc, respectively). Four consecutive CORTEX

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Fig. 1. Reduction of protein kinase C (PKC) activity in ischemic cortex, striatum and hippocampus. Male Wistar rats were subjected to bilateral forebrain ischemia for 20 min followed by 48 h of recirculation. Cytosolic and Triton X-100-solubilized membrane fractions were prepared from cortex, hippocampus and striatum, and aliquots (corresponding to 0.5 mg protein) were transferred to assay tubes containing 0.5 ml of DEAE-cellulose (Whatman), previously equilibrated in 20 mM Tris-HCl buffer pH 7.5, containing 2 mM EDTA, 0.5 mM EGTA and 2 mM dithiotreitol (buffer A). After 15 min, the cellulose was washed with 5 ml of buffer A containing 30 mM NaCI and protein kinase C was then eluted with 1 ml of buffer A containing 0.2 M NaCI. The eluates were diluted (1:5) with buffer A and PKC activity was determined on 40 ml aliquots by measuring the incorporation of [32p] from [y-32p]ATP (0.3 x 106 d.p.m./100 ml, Amersham) into lysine-rich histone (type III-S, Sigma) in the presence of 1.5 mM CaCI2, 50/tg/ml phosphatidylserine and 0.3 #g/ml 1,2-sn-dioctanoylglycerol, as previously described s. PKC activity is calculated from the difference in 32p incorporated into histone in the presence and absence of added phosphatidylserine and dioctanoyglycerol and expressed as U/mg of total brain protein (1 U = 1 nmol of [32p] incorporated into histone III-S/min). Basal activity, measured in the absence of the activators, represented 5-10% of the total activity and was not significantly affected by ischemia. Each value is the mean + S.D. (n = 4-5). Significant differences from sham-operated controls at *P < 0.001, ** P < 0.02, or ***P < 0.05 (Student's t-test).

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Fig. 2. Influence of the duration of ischemia on protein kinase C (PKC) activity reduction. Total PKC activity (soluble plus particulate) was determined in cortex, hippocampus and striatum of rats, subjected to forebrain ischemia for 1 min, 20 min or 4 episodes of ischemia each for 5 min and 1 h reperfusion between each session, either immediately after the ischemia session (black bars) or after 24 h of recirculation (white bars). 2-V.O. = animals in which the vertebral arteries were occluded, but the carotids remained untouched. Values are expressed as percentage (+ S.D.) of the sham-operated controls. Significant differences from controls at *P < 0.001, **P < 0.02, or ***P < 0.05 (Student's t-test).

173 sessions of 5 min of ischemia, each separated by 1 h of recirculation, resulted in a lesser decrease of P K C activity than a 20 min session of continuous ischemia in all the tested regions, particularly in the striatum. Occlusion of the two vertebral arteries by electrocauterization without carotid ligation did not affect the level of P K C activity (Fig. 2). The principal finding of the present study is the decrease of P K C activity upon recirculation after bilateral forebrain ischemia in the adult rat brain. The reduction in P K C activity (of Cx, St and Hc upon recirculation), although maximal after an initial episode of 20 min, was already observed after 1 min of ischemia. We have reported previously ischemia-induced alterations of PKC, depending on the length of blood flow obstruction s. A specific reduction of the phosphorylation of endogenous proteins by endogenous PKC has recently been reported in a rabbit spinal cord ischemia model 7. Bilateral forebrain ischemia in the adult rat also caused an increase of phorbol ester binding in the CA1 layer in the Hc, followed by a delayed decrease during the late postischemic period TM. In addition, Taft et al. 21 reported that 5 rain of bilateral forebrain ischemia in adult gerbils provoked a significant and long-lasting decrease in Ca 2+calmodulin-dependent protein kinase activity in regions showing histological neuronal loss after ischemia. Thus, it appears that impaired Ca2+-dependent phosphorylation systems, such as P K C and Ca2+-calmodulin kinases, may possibly contribute to irreversible neuronal damage and subsequent death. Loss of PKC activity, as shown in this study, may be the result of enhanced proteolytic degradation by CaE+-activated neutral proteases, which display high levels of activity in brain 9. Neutral proteases were shown to act on purified PKC in vitro 7 and in

cultured cells exposed to long-term phorbol ester treatment 1°'11. Alternatively, P K C may be inhibited by endogenous inhibitors produced during ischemia 7. The decrease of P K C activity was observed at 24 and 48 h postischemia, a time when neuronal death in the 3 brain structures is not yet evident histologically 15"22. The selection of these structures for this study was based on their higher vulnerability to ischemia 22. Indeed, alterations of PKC precede morphological damage and persist during the period where neuronal death occurs. However, the marked decrease of P K C activity in these areas (60-80%), appears too pronounced to be attributed only to discrete groups of neurons and, therefore, must reflect changes in a larger number of neurons and, possibly, glial cells in these structures. A question posed by these studies is whether changes in PKC activity and content can indicate the state of reversible or irreversible ischemia. P K C is believed to lower the receptor-mediated elevated intracellular Ca 2÷ levels and, by doing so, to provide a negative feedback mechanism 12'13. Losses of PKC in sensitive brain structures during the course of ischemia, would distort this negative control and, as a result, amplify damage. Whether discrete populations of neurons in the hippocampus CA1 subfield, the middle layers of the cerebral cortex, the caudate nucleus and the amygdala, which receive glutamatic inputs and possess N-methyl-Daspartate receptors 3'22, may be more prone to PKC alterations, remains to be seen.

1 Abe. K., Kogure, K., Yamamoto, H., Imazawa, M. and Miyamoto, K., Mechanism of arachidonic acid liberation during ischemia in gerbil cerebral cortex, J. Neurochem., 48 (1987) 503-509. 2 Bazan, N., Free arachidonic acid and other lipids in the nervous system during early ischemia and after electroshock, Adv. Exp. Med. Biol., 72 (1976) 317-335. 3 Foster, A.C. and Fagg, G.E., Acidic amino acid binding sites in mammalian neuronal membranes: their characteristics and relationship to synaptic receptors, Brain Res. Rev., 7 (1984) 103-164. 4 Huang, S.E-L. and Sun, G.-Y., Cerebral ischemia induced quantitative changes in rat brain membrane lipids involved in phosphoinositide metabolism, Neurochem. Int., 9 (1986) 185190. 5 Ikeda, M., Yoshida, S., Busto, R., Santiso, M. and Ginsberg, M.D., Polyphosphoinositides as a probable source of brain free fatty acid accumulated at the onset of isehemia, J. Neurochem., 47 (1986) 123-132. 6 Kishimoto, A., Kajikawa, N., Shiota, M. and Nishizuka, Y., Proteolytic activation of calcium-activated phospholipid-dependent protein kinase by calcium-dependent neutral protease, J. Biol. Chem., 258 (1983) 1156-1164.

7 Kochhar, A., Saitoh, T. and Zivin, J., Reduced protein kinase C activity in ischemic spinal cord, J. Neurochem., 53 (1989) 946-952. 8 Louis, J.-C., Magal, E. and Yavin, E., Protein kinase C alterations in the fetal brain after global ischemia, J. Biol. Chem., 263 (1988) 19282-19285. 9 Lynch, G. and Baudry, M., The biochemistry of memory: a new and specific hypothesis, Science, 224 (1984) 1057-1063. 10 Melloni, E. and Pontremoli, S., The Calpains, Trends Neurosci., 12 (1989) 438-444. 11 Murray, A.W., Fournier, A. and Hardy, S.J., Proteolytic activation of protein kinase C: a physiological reaction?, Trends Biochem. Sci. 12 (1987) 53-54. 12 Nishizuka, Y., Studies and perspectives of protein kinase C, Science, 233 (1986) 305-312. 13 Nishizuka, Y., The molecular heterogeneity of protein kinase C and its implications for cellular regulation, Nature, 334 (1988) 661-665. 14 Onodera, H., Araki, T. and Kogure, K., Protein kinase C activity in the rat hippocampus after forebrain ischemia: autoradiographic analysis by [3H]phorbol 12,13-dibutyrate, Brain Research, 481 (1989) 1-7. 15 Plum, F., What causes infarction in ischemic brain?, Neurology,

We wish to thank Mr. Amnon Kohen for technical help during the course of this study. Part of these studies were supported by a grant made available by the Revson Foundation of the Israel Academy of Sciences and Humanities, Jerusalem.

174 33 (1983) 222-233. 16 Pulsinelli, W.A. and Brierley, J.B., A new model of bilateral hemispheric ischemia in the unanesthetized rat, Stroke, 10 (1979) 267-272. 17 Pulsinelli, W.A., Brierley, J.B. and Plum, E, Temporal profile of neuronal damage in a model of transient forebrain ischemia, Ann. Neurol., 11 (1982) 491-498. 18 Raichle, M.E., The pathophysiology of brain ischemia, Ann. Neurol., 13 (1983) 2-10. 19 Siesj6, B.K., Historical overview: calcium, ischemia and death of brain cells, Ann. N.Y. Acad. Sci., 522 (1988) 638-661.

20 Steinberg, R., Gueniau, C., Scarna, H., Keller, A., Worcel, M. and Pujol, J.F., Enolase level in cerebrospinal fluid as an index of neuronal damage, J. Neurochem., 43 (1984) 19-24. 21 Taft, W.C., Tennes-Rees, K.A., Blair, R.E., Clipton, G.C. and DeLorenzo, R.J., Cerebral ischemia decreases endogenous calcium-dependent protein phosphorylation in gerbil brain, Brain Research, 447 (1988) 159-163. 22 Wieloch, T., Neurochemical correlates to selective neuronal vulnerability. In: K. Kogure, A. Hossmann, B.K. Siesj6 and F.A. Welsh (Eds.), Progress in Brain Research Vol. 63, 1985, pp. 69-85.