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Immunosupressants and calcineurin inhibitors, cyclosporin A and FK506, reversibly inhibit epileptogenesis in amygdaloid kindled rat
BRAIN RESEARCH ELSEVIER
Brain Research 648 (1994) 337-341
Short Communication
Immunosuppressants and calcineurin inhibitors, cyclosporin A and FK506, reversibly inhibit epileptogenesis in amygdaloid kindled rat Lizomar J.M.P. Moia, Hideki Matsui, Guilherme A.M. de Barros, Kazuhito Tomizawa, Kazuhiro Miyamoto, Yoshihiro Kuwata, Masaaki Tokuda, Toshifumi Itano, Osamu Hatase * Department of Physiology, The Kagawa Medical School, 1750-1 lkenobe, Miki, Kagawa 761-07,Japan (Accepted 29 March 1994)
Abstract
Calcineurin (CAN) immunoreactivity and content increased markedly in kindled rat brain, and this increment was due to CaN in the membrane fraction. Investigation of the effects of cyclosporin A and FK506 (immunosuppressants which inhibit CaN activity in T lymphocytes) in the kindling phenomena showed that the kindling stage progression was reversibly blocked by these drugs. These findings suggest that calcineurin may play an essential role in acquiring epileptogenesis in kindling. Key words: Calcineurin; Kindling; Rat brain; Hippocampus; Neocortex; Cyclosporin A; FK506; Immunosuppressant
Kindling is a model of intractable human temporal lobe epilepsy in which progressive changes in electroencephalographic and behavioral seizures are evoked by repeated subthreshold electrical stimulations [9]. Remodeling of neuronal networks and axonal sprouting have been implicated as an important mechanism in kindling progression [5] and thus, it also provides a useful model of neuronal plasticity. Calcineurin (CAN), a calcium-calmodulin dependent protein phosphatase has been identified as a key signalling enzyme in T cell activation [6]. The enzyme is also identified as an intra-cellular target for two immunosuppressive drugs, namely cyclosporin A (CysA) and FK506 [17,16]. Although calcineurin is widely distributed in the whole body, the content of the enzyme, is 10-100 times higher in the brain than in other organs including lymphoid tissues [2]. The immunophilins, cyclophilin and FK506 binding proteins (FKBP), which are essential for cyclosporin A and FK506, respectively, to interact with calcineurin are also extraordinarily abundant in the brain. These immunophilins are also colocalized with calcineurin in the brain [14,18,27]. However, the physiological function and regulatory mechanisms of calcineurin and
* Corresponding author. Fax: (81) (878) 98-7107. 0006-8993/94/$07.00 © 1994 Elsevier Science B.V. All rights reserved SSDI 0 0 0 6 - 8 9 9 3 ( 9 4 ) 0 0 3 9 3 - Q
immunophilins in the central nervous system are not fully elucidated in contrast to their role in the immune system. In the present study we report that calcineurin content is markedly increased in kindled rat hippocampus and neocortex. We further show that cyclosporin A and FK506 inhibit the progression of kindling stage in reversible manner. Male Sprague-Dawley (SD) rats were bought from Japan Shizuoka Laboratory Center (SLC), Inc., Japan, weighing around 300 g. The rats were anesthetized with pentobarbital sodium solution 50 m g / k g body weight (Abbott Lab., North Chicago, USA), and were stereotaxically implanted with stainless steel bipolar electrode for stimulation and recording. The electrode was inserted in the left amygdala (4.5 mm lateral, 0.2 mm posterior and 8.3 mm ventral from bregma). The rats had access to food and water ad libitum and were housed in individual cages under diurnal lighting conditions, with lights on from 08.00 to 20.00 h. One week after operation, rats were divided in two groups: (a) electrode implanted but non-stimulated (sham operated rats); and (b) electrically stimulated twice a day at 10.00 and 16.00 h, with a biphasic square wave pulse (100-200/zA, 60 Hz, for one second) using two electric stimulators (NEC San-Ei Co., Tokyo, Japan). From this point we observed progressive changes in behaviour, which were classified according to Racine [25]. When 3 - 6 consecutive generalized motor seizures (stage 5)
338
L.J.M.P. Moia et al./ Brain Research 648 (1994) 337-341
occurred, stimulations were suspended. The normal and epileptic rats were perfused transcardiacally with 0.9% NaC1 solution followed by perfusion with 10% formaldehyde in phosphate buffer. The brains were removed and fixed in Bouin's fluid (1 g picric acid, 25 ml of 37% formaldehyde solution and 5 ml Acetic acid for 80 ml distilled water) overnight in cold-room. Vibratome sections of 20 mm thickness were incubated either with V J6 antibody (a monoclonal antibody specific to CaN catalytic subunit A) [22] or non-specific mouse IgG (Jackson I m m u n o R e s e a r c h Lab. Inc., West Grove, USA) overnight in cold-room. The subsequent steps were done according to the immunostaining method of Vectastain-ABC System monoclonal Kit (Vector Lab. Inc., Burlingame, USA). Normal, sham and kindled rats forebrain were ho-
mogenized each in 5 vols. of 20 mM Tris-HC1 buffer (pH 7.4), containing 100 /.tM of CaCI 2 and protease inhibitors. The homogenates were centrifuged at 15,000 × g for 5 min. The pellet was discarded and the supernatant was further centrifuged at 105,000 × g for 70 min. The second supernatant (cytosolic fractions) was saved. The second pellet was further resuspended and homogenized with 20 mM Tris-HCl buffer (pH 7.4), containing 1 mM E G T A ( e t h y l e n e g l y c o l - N , N , N ' N'-tetraacetic acid, Dojindo) and 0.1% Tween 20 (a non-ionic detergent from Bio-Rad Lab., Richmond, USA), to obtain the membrane bound proteins. After centrifugation at 105,000 × g for 90 min, the supernatant was saved as the membrane fraction. From membrane and cytosolic fractions 150 /xg sample each was resolved on 12.5% sodium dodecyl sulfate (SDS)-
Fig. 1. Immunohistochemical analysis of brain coronal sections from kindled and control rats, using a monoclonal antibody against CaN A subunit, VJ6. Kindled (a) and control (b) rat brain stained with VJ6 (× 8). These data are representatives of three independent experiments showing essentially the same results.
L.J.M.P. Moia et al. / Brain Research 648 (1994) 337-341
polyacrylamide gel electrophoresis (PAGE) [15], and transferred to the nitrocellulose membrane as described by Towbin et al. [29]. The nitrocellulose membranes were incubated with V J6 antibody overnight, followed by 2 h incubation with peroxidase conjugated goat anti-mouse IgG (Organon Teknika Corp., Wester Chester, USA), and developed with DAB (Diaminobenzidine tetrahydrochloride from Dojindo, Kumamoto, Japan). The effects of CysA and FK506 (supplied by Sandoz Pharmaceutical Co. Ltd., Tokyo, Japan and Fujisawa Pharmaceutical Co. Ltd., Osaka, Japan, respectively) in kindling were also investigated. Electrode implantation, analysis of changes in clinical behaviour and stimulation protocols were carried out as described above. CysA (10 m g / k g b.wt.) and FK506 (0.5 m g / k g b.wt.) were injected intraperitoneally, once every day, 30 min before the morning electrical stimulation. Two types of control were used: (1) only vehicle (99% ethanol) injeca
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Fig. 2. Analysis of CaN content by Western blotting and densitometric scanning, a: membrane fraction prepared from normal (lane 2), kindled (lane 3) and sham operated (lane 4) rat brain, b: cytosolic fraction from normal (lane 2), kindled (lane 3) and sham operated (lane 4) rat brain. The purified bovine brain CaN (lane 1, a and b). Arrows in Fig. 2, a and b indicate the position of CaN A subunit, c: densitometry of CaN A bands from normal, sham and kindled rat brain: cytosol (solid bars) and membrane (pattern bars). The ordinate shows the percent of control of CaN A content taking the normal rat brain CaN A content as 100%. The bars represent the mean values and standard deviations from three independent experiments (,1= 3 of each normal, sham operated and kindled rats). • P < 0.05 as compared to the respective controls, normal membrane and kindled cytosol fractions (Student's t statistic).
339
tion; and (2) neither any drug nor ethanol injection. The stage progression of kindling for both controls was the same. We performed immunohistochemical study, using a monoclonal antibody against calcineurin catalytic subunit A (VJ6), on stage 5 (fully kindled) and control rat brains. In normal rat brain (Fig. lb), CaN showed wide distribution including in hippocampus, neocortex, striatum and thalamus. These observations are in good agreement with the previous reports using monoclonal and polyclonal antibodies [10,20,21]. On the other hand, kindled rat brain (Fig. la) showed distinctively stronger CaN immunoreactive staining than control rat brain (Fig. lb). In kindled brain, the most striking increment was observed on the ipsilateral side of the hippocampus (left) and in the ipsilateral and contralateral sides of the neocortex. In the ipsilateral hippocampus, the CaN increment was seen in CA1, CA2, CA3 and CA4 and in dentate gyrus. In contralateral hippocampus, CaN immunoreactive staining level was essentially the same as in normal rat (Fig. la,b). No specific immunostaining was observed either in control, or in kindled rat brain sections stained with non-specific mouse IgG (data not shown). In order to confirm the increment of CaN content in kindled rat brain, we performed immunoblotting analysis. Membrane and cytosol fractions were prepared from kindled, sham operated and control rat forebrains by the method indicated above. The monoclonal antibody VJ6 detected CaN A subunit (61 kDa) and some degradative products in purified bovine brain CaN (Fig. 2a,b, lane 1) and in rat brain samples (Fig. 2 a,b, lanes 2-4). In membrane fraction of kindled rat brain CaN A subunit was significantly increased compared to corresponding fractions from normal and sham operated brains (Fig. 2a, lanes 2-4). The amount of CaN in cytosol fractions did not show any difference (Fig. 2b, lanes 2-4). CaN content was quantified by densitometric scanning (Fig. 2c). The content in membrane fraction from kindled rat brain was 2.2-fold higher than that in fractions from controls and sham operated rat brains. However, the content of CaN in cytosol fractions did not show any difference (Fig. 2c). These data confirmed that CaN content was increased in kindled rat brain, and further showed that this increment was due to CaN in the membrane fraction. Two possible mechanisms can be postulated to explain the mechanism of increase in the membrane bound CaN: (1) CaN may translocate from cytosol to membrane fraction in calcium dependent manner; and (2) membrane binding subtype of CaN may specifically increase in kindled brain. The former mechanism is more probable because we have detected the calcium dependent translocation of CaN from cytosol to membrane [28]. Although CaN has multiple subtypes, mem-
340
L.J.M.P. Moia et al ./ Br ai n Research 648 (1994) 337-341
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Fig. 3. Cyclosporin A inhibits kindling stage progression between stage 2 and stage 3, and the inhibition is reversible, a: normal control rat without CysA injection (e). Three individual rats (©, A, D) were injected with CysA. Control rat (e) reached stage 5 (full kindling) by 11 stimulations. On the other hand, CysA injected rats (©, A, [] ) stopped developing kindling stage at stage 2 and did not reach stage 3 even after 18 stimulations, b: control rat (e) reached stage 5 by 9 stimulations. Rat 1 ([]) and Rat 2 (A) did not develop kindling while receiving CysA. However, when CysA administration was stopped after 9 stimulations (Rat 1) and 14 stimulations (Rat 2) as indicated by arrows, both rats started to develop kindling. Both rats 1 and 2 reached stage 5 by 9 stimulations after stopping the drug administration.
brane binding subtypes have not been reported. Further investigation of the characteristics of the increased CaN in kindled brain will be required. Our findings that CaN is increased in kindled rat brain, in addition to the knowledge of inhibitory effect of immunosuppressants, CysA and FK506, on the CaN activity, led us to investigate the effect of these immunosuppressants on the progression of kindling. Control rats without immunosuppressant injection reached kindling stage 5 (generalized motor seizures) after 9 to 11 electrical stimulations (Fig. 3a and Fig. 3b, respectively). Rats with intraperitoneally administered CysA once everyday did not develop kindling stages beyond stage 2 even when 14 electrical stimulations or more had been applied (Fig. 3a). However, when we stopped the drug injection, the same rats reached stage 5 seizures with 9 electrical stimulations which was essentially the same as in normal control rats (Fig 3b). On the other hand, if CysA was administered at stage 3, 4 or 5, the drug did not inhibit the stage progression (data not shown). FK506 also showed essentially the same inhibitory effect on kindling stage progression as CysA. FK506 blocked stage progression between stage 2 and stage 3, and the blockade was reversible (data not shown). These data indicated that CysA and FK506 have a very similar, if not, the same mechanism, in inhibiting the progression of kindling. The reversibility indicates that the inhibitory effects of CysA and FK506 on kindling are not due to the result of non-specific neuronal damage but due to the result of specific pharmacological inhibitory effects of these drugs, most likely on CaN. The general clinical conditions of the animals injected with the immunosuppressants were similar to that of controls, except that the
formers presented slight diarrhea and 5% body weight loss. We did not observed any epileptic activity induced by CysA or FKS06, on the contrary these drugs inhibited the progression of seizure stage. The pharmacological effects of CysA and FK506 are well studied in T lymphocytes activation [24,26]. These i m m u n o s u p p r e s s a n t s form complexes with immunophilins and inhibit calcineurin activity, and thus suppress the dephosphorylation and translocation of N F - A T (nuclear factor of activated T cell) which leads to the inhibition of interleukin 2 (IL-2) production [19]. IL-2 and 1L-2 receptor are both proven to be present in rat brain hippocampus, striatum and neocortex [1]. However, it has not been examined whether IL-2 production increases in kindled rat brain. Other physiological events which are necessary for the production of IL-2 in T cell activation are also detected in kindled brain, such as increment of protein kinase C activity, induction of c-fos, increase in phosphatidyl inositol turn over and increase in the level of intracellular calcium [4,11,12,13,23]. These observations, in concert with the data presented in this paper lead us to postulate that a mechanism very similar to T cell activation is also involved in the epileptogenesis acquiring step in kindling. If so, it seems very reasonable to assume that CysA and FKS06 which are known as immunosuppressants and CaN inhibitors, block the stage progression of kindling. Since CysA and FK506 are very hydrophobic, they can penetrate through b l o o d - b r a i n barrier and reach neuronal cells. The pharmacological effects of clinical doses of C~sA on central nervous system including tremor, ataxia, seizures, quadriparesis and coma are reported [3,7]. As for FK506, tremor is reported in significant number of cases [8]. We are now
L.J.M.P. Moia et al. /Brain Research 648 (1994) 337-341
investigating to see if C3,sA and FK506 injected intraperitoneally can inhibit CaN activity in brain. We will also confirm that the increment of CaN will be long-lasting. We would like to express our special thanks to Dr. T.H. Murakami in the Department of Biology and Dr. T. Negi in the Department of Sports Medicine in The Kagawa Medical School for their technical instruction and support to prepare kindling rats. We also thank to Dr. Najma Janjua for the review of the English of this manuscript. This work was supported by a Grant-in-aid for Scientific Research from the Ministry of Education, Science and Culture of Japan. [1] Araujo, D.M., Lapchak, P.A. Collier, B. and Quirion R., Localization of interleukin-2 immunoreactivity and interleukin-2 receptors in the rat brain: interaction with the cholinergic system, Brain Res., 498 (1989) 257-266. [2] Asami, M., Kuno, T., Mukai, H. and Tanaka, C., Detection of the FICS06-FKBP-calcineurin complex by a simple binding assay, Biochem. Biophys. Res. Commun., 192 (1993) 1388-1394. [3] Atkinson, K., Biggs, J., Darveniza, P., Boland, J., Concannon, A. and Dodds, A., Cyclosporine-associated central nervous system toxicity after allogeneic bone marrow transplantation, Transplantation, 38 (1984) 34-37. [4] Buzsaki, G., Hsu, M., Horvath, Z. Horsburgh, K., Sundsmo, M., Masliah, E. and Saitoh, T., Kindling-induced changes of protein kinase C levels in hippocampus and neocortex, Mol. Neurobiol. Epilepsy, Epilepsy Res., Suppl. 9 (1992) 279-284. [5] Cavazos, J.E., Golarai, G. and Sutula, T.P., Mossy fiber synaptic reorganization induced by kindling: time course of development, progression, and permanence, Z Neurosci., 11 (1991)2795-2803. [6] Clipstone, N.A. and Crabtree, G.R., Identification of calcineurin as a key signalling enzyme in T-lymphocyte activation, Nature, 357 (1992) 695-697. [7] Deierhoi, M.H., Kalayoglu, M., Sollinger, H.W. and Belzer, F.O., Cyclosporin neurotoxicity in liver transplant recipients: report of three cases, Transplant. Proc., XX (1988) 116-118. [8] Eidelman, B.H., Abu-Elmagd, K., Wilson, J., Fung, J.J., Aleissiani, M., Jain, A., Takaya, S., Todo, S.N., Tzakis, A., Thiel, D.V., Shannon, W. and Starzl, T.E., Transplant. Proc., 23 (1991) 3175-3178. [9] Goddard, G.V., Mclntyre, D.C. and Leech, C.K., A permanent change in brain function resulting from daily electrical stimulation, Exp. Neurol., 25 (1969) 295-330. [10] Goto, S., Matsukado, Y., Mihara, Y., Inoue, N. and Miyamoto, E., The distribution of calcineurin in rat brain by light and electron microscopic immunohistochemistry and enzyme-immunoassay, Brain Res., 397 (1986) 161-172. [11] Iadorola, M.J., Nicolleti, F., Naranjo, J.R., Putnam, F. and Costa, E., Kindling enhances the stimulation of inositol phospholipid hydrolysis elicited by ibotenic acid in rat hippocampal slices, Brain Res., 374 (1986) 174-178. [12] Kohira, I., Akiyama, K., Daigen, A. and Otsuki, S., Enduring increase in membrane-associated protein kinase C activity in the hippocampal-kindled rat, Brain Res., 593 (1992) 82-88. [13] Labiner, D.M., Butler, L.S., Cao, Z., Hosford, D.A., Shin, C.
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and Mcnamara, J.O., Induction of c-los mRNA by kindled seizures: complex relationship with neuronal burst firing, J. Neurosci., 13 (1993) 744-751. [14] Lad, R.P., Smith, M.A. and Hilt, D.C., Molecular cloning and regional distribution of rat brain cyclophilin, Mol. Brain Res., 9 (1991) 239-244. [15] Laemmli, U.K., Cleavage of structural proteins during the assembly of the head of bacteriophage T4, Nature, 227 (1970) 680-685. [16] Liu, J., Albers, M.W., Wandless, T.J., Luan, S., Alberg, D.G., Belshaw, P.J., Cohen, P., MacKintosh, C., Klee, C.B. and Schreiber, S.L., Inhibition of T cell signaling by immunophilinligand complexes ccorrelates with loss of calcineurin phosphatase activity, Biochemistry, 31 (1992) 3896-3901. [17] Liu, J., Farmer Jr., J.D., Lane, W.S., Friedman, J., Weissman, I. and Schreiber, S.L., Calcineurin is a common target of cyclophilin-cyclosporin A and FKBP-FK506 complexes, Cell, 66 (1991) 807-815. [18] Maki, N., Sekiguchi, F., Nishimaki, J., Miwa, K., Hayano, T., Takahashi, N. and Suzuki, M., Complementary DNA encoding the human T-cell FK506-binding protein, a peptidylprolyl cistrans isomerase distinct from cyclophilin, Proc. NatL Acad. Sci. USA, 87 (1990) 5440-5443. [19] McCaffrey, P.G., Perrino, B.A., Soderling, T.R. and Rao, A., NF-ATp, a T lymphocyte DNA-binding protein that is a target for calcineurin and immunosuppressive drugs, Z Biol. Chem., 268 (1993) 3747-3752. [20] Matsui, H., Doi, A., Itano, T., Shimada, M., Wang, J.H. and Hatase O., Immunohistochemical localization of calcineurin, calmodulin-stimulated phosphatase, in the rat hippocampus using a monoclonal antibody, Brain Res., 402 (1987) 193-196. [21] Matsui, H., Itano, T., Etoh, S., Tokuda, M., Wang, J.H. and Hatase, O., Demonstration of different regional distributions of calcineurin subunits using monoclonal antibodies, AdL,. Exp. Med. BioL, 255 (1989) 369-375. [22] Matsui, H., Pallen, C.J., Adachi, A.-M., Wang, J.H. and Lam, P.H.-Y., Demonstration of different metal ion-induced calcineurin conformations using a monoclonal antibody, J. Biol. Chem., 260 (1985) 4174-4179. [23] Mody, I. and Heinemann, U., NMDA receptors of dentate gyrus granule cells participate in synaptic transmission following kindling, Nature, 326 (1987) 701-704. [24] O'Keefe, S.J., Tamura, J., Kincaid, R.L., Tocci, M.J. and O'Neill, E.A,, FK-506- and CsA-sensitive activation of the interleukin-2 promoter by calcineurin, Nature, 357 (1992) 692-694. [25] Racine, R.J., Modification of seizure activity by electrical stimulation: II. Motor seizure, Electroencephalogr. Clin. Neurophysiol., 32 (1972) 281-294. [26] Sigal, N.H. and Dumont, F.J., Cyclosporin A, FK-506, and rapamycin: pharmacologic probes of lymphocyte signal transduction, Annu. Ret,. lmmunol., 10 (1992) 519-560. [27] Steiner, J.P., Dawson, T.M., Fotuhi, M., Glatt, C.E., Snowman, A.M., Cohen, N. and Snyder, S.H., High brain densities of the immunophilin FKBP colocalized with calcineurin, Nature, 358 (1992) 584-586. [28] Tokuda, M., Matsui, H., Itano, T., Masaki, T. and Hatase, O., Possible mechanism of translocation of calcineurin between membrane and cytosol fractions, in submission. [29] Towbin, H., Stadhelin, T. and Gordon, J., Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications, Proc. Natl. Acad. Sci. USA, 76 (1979) 350-4354.
Brain Research 648 (1994) 337-341
Short Communication
Immunosuppressants and calcineurin inhibitors, cyclosporin A and FK506, reversibly inhibit epileptogenesis in amygdaloid kindled rat Lizomar J.M.P. Moia, Hideki Matsui, Guilherme A.M. de Barros, Kazuhito Tomizawa, Kazuhiro Miyamoto, Yoshihiro Kuwata, Masaaki Tokuda, Toshifumi Itano, Osamu Hatase * Department of Physiology, The Kagawa Medical School, 1750-1 lkenobe, Miki, Kagawa 761-07,Japan (Accepted 29 March 1994)
Abstract
Calcineurin (CAN) immunoreactivity and content increased markedly in kindled rat brain, and this increment was due to CaN in the membrane fraction. Investigation of the effects of cyclosporin A and FK506 (immunosuppressants which inhibit CaN activity in T lymphocytes) in the kindling phenomena showed that the kindling stage progression was reversibly blocked by these drugs. These findings suggest that calcineurin may play an essential role in acquiring epileptogenesis in kindling. Key words: Calcineurin; Kindling; Rat brain; Hippocampus; Neocortex; Cyclosporin A; FK506; Immunosuppressant
Kindling is a model of intractable human temporal lobe epilepsy in which progressive changes in electroencephalographic and behavioral seizures are evoked by repeated subthreshold electrical stimulations [9]. Remodeling of neuronal networks and axonal sprouting have been implicated as an important mechanism in kindling progression [5] and thus, it also provides a useful model of neuronal plasticity. Calcineurin (CAN), a calcium-calmodulin dependent protein phosphatase has been identified as a key signalling enzyme in T cell activation [6]. The enzyme is also identified as an intra-cellular target for two immunosuppressive drugs, namely cyclosporin A (CysA) and FK506 [17,16]. Although calcineurin is widely distributed in the whole body, the content of the enzyme, is 10-100 times higher in the brain than in other organs including lymphoid tissues [2]. The immunophilins, cyclophilin and FK506 binding proteins (FKBP), which are essential for cyclosporin A and FK506, respectively, to interact with calcineurin are also extraordinarily abundant in the brain. These immunophilins are also colocalized with calcineurin in the brain [14,18,27]. However, the physiological function and regulatory mechanisms of calcineurin and
* Corresponding author. Fax: (81) (878) 98-7107. 0006-8993/94/$07.00 © 1994 Elsevier Science B.V. All rights reserved SSDI 0 0 0 6 - 8 9 9 3 ( 9 4 ) 0 0 3 9 3 - Q
immunophilins in the central nervous system are not fully elucidated in contrast to their role in the immune system. In the present study we report that calcineurin content is markedly increased in kindled rat hippocampus and neocortex. We further show that cyclosporin A and FK506 inhibit the progression of kindling stage in reversible manner. Male Sprague-Dawley (SD) rats were bought from Japan Shizuoka Laboratory Center (SLC), Inc., Japan, weighing around 300 g. The rats were anesthetized with pentobarbital sodium solution 50 m g / k g body weight (Abbott Lab., North Chicago, USA), and were stereotaxically implanted with stainless steel bipolar electrode for stimulation and recording. The electrode was inserted in the left amygdala (4.5 mm lateral, 0.2 mm posterior and 8.3 mm ventral from bregma). The rats had access to food and water ad libitum and were housed in individual cages under diurnal lighting conditions, with lights on from 08.00 to 20.00 h. One week after operation, rats were divided in two groups: (a) electrode implanted but non-stimulated (sham operated rats); and (b) electrically stimulated twice a day at 10.00 and 16.00 h, with a biphasic square wave pulse (100-200/zA, 60 Hz, for one second) using two electric stimulators (NEC San-Ei Co., Tokyo, Japan). From this point we observed progressive changes in behaviour, which were classified according to Racine [25]. When 3 - 6 consecutive generalized motor seizures (stage 5)
338
L.J.M.P. Moia et al./ Brain Research 648 (1994) 337-341
occurred, stimulations were suspended. The normal and epileptic rats were perfused transcardiacally with 0.9% NaC1 solution followed by perfusion with 10% formaldehyde in phosphate buffer. The brains were removed and fixed in Bouin's fluid (1 g picric acid, 25 ml of 37% formaldehyde solution and 5 ml Acetic acid for 80 ml distilled water) overnight in cold-room. Vibratome sections of 20 mm thickness were incubated either with V J6 antibody (a monoclonal antibody specific to CaN catalytic subunit A) [22] or non-specific mouse IgG (Jackson I m m u n o R e s e a r c h Lab. Inc., West Grove, USA) overnight in cold-room. The subsequent steps were done according to the immunostaining method of Vectastain-ABC System monoclonal Kit (Vector Lab. Inc., Burlingame, USA). Normal, sham and kindled rats forebrain were ho-
mogenized each in 5 vols. of 20 mM Tris-HC1 buffer (pH 7.4), containing 100 /.tM of CaCI 2 and protease inhibitors. The homogenates were centrifuged at 15,000 × g for 5 min. The pellet was discarded and the supernatant was further centrifuged at 105,000 × g for 70 min. The second supernatant (cytosolic fractions) was saved. The second pellet was further resuspended and homogenized with 20 mM Tris-HCl buffer (pH 7.4), containing 1 mM E G T A ( e t h y l e n e g l y c o l - N , N , N ' N'-tetraacetic acid, Dojindo) and 0.1% Tween 20 (a non-ionic detergent from Bio-Rad Lab., Richmond, USA), to obtain the membrane bound proteins. After centrifugation at 105,000 × g for 90 min, the supernatant was saved as the membrane fraction. From membrane and cytosolic fractions 150 /xg sample each was resolved on 12.5% sodium dodecyl sulfate (SDS)-
Fig. 1. Immunohistochemical analysis of brain coronal sections from kindled and control rats, using a monoclonal antibody against CaN A subunit, VJ6. Kindled (a) and control (b) rat brain stained with VJ6 (× 8). These data are representatives of three independent experiments showing essentially the same results.
L.J.M.P. Moia et al. / Brain Research 648 (1994) 337-341
polyacrylamide gel electrophoresis (PAGE) [15], and transferred to the nitrocellulose membrane as described by Towbin et al. [29]. The nitrocellulose membranes were incubated with V J6 antibody overnight, followed by 2 h incubation with peroxidase conjugated goat anti-mouse IgG (Organon Teknika Corp., Wester Chester, USA), and developed with DAB (Diaminobenzidine tetrahydrochloride from Dojindo, Kumamoto, Japan). The effects of CysA and FK506 (supplied by Sandoz Pharmaceutical Co. Ltd., Tokyo, Japan and Fujisawa Pharmaceutical Co. Ltd., Osaka, Japan, respectively) in kindling were also investigated. Electrode implantation, analysis of changes in clinical behaviour and stimulation protocols were carried out as described above. CysA (10 m g / k g b.wt.) and FK506 (0.5 m g / k g b.wt.) were injected intraperitoneally, once every day, 30 min before the morning electrical stimulation. Two types of control were used: (1) only vehicle (99% ethanol) injeca
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Fig. 2. Analysis of CaN content by Western blotting and densitometric scanning, a: membrane fraction prepared from normal (lane 2), kindled (lane 3) and sham operated (lane 4) rat brain, b: cytosolic fraction from normal (lane 2), kindled (lane 3) and sham operated (lane 4) rat brain. The purified bovine brain CaN (lane 1, a and b). Arrows in Fig. 2, a and b indicate the position of CaN A subunit, c: densitometry of CaN A bands from normal, sham and kindled rat brain: cytosol (solid bars) and membrane (pattern bars). The ordinate shows the percent of control of CaN A content taking the normal rat brain CaN A content as 100%. The bars represent the mean values and standard deviations from three independent experiments (,1= 3 of each normal, sham operated and kindled rats). • P < 0.05 as compared to the respective controls, normal membrane and kindled cytosol fractions (Student's t statistic).
339
tion; and (2) neither any drug nor ethanol injection. The stage progression of kindling for both controls was the same. We performed immunohistochemical study, using a monoclonal antibody against calcineurin catalytic subunit A (VJ6), on stage 5 (fully kindled) and control rat brains. In normal rat brain (Fig. lb), CaN showed wide distribution including in hippocampus, neocortex, striatum and thalamus. These observations are in good agreement with the previous reports using monoclonal and polyclonal antibodies [10,20,21]. On the other hand, kindled rat brain (Fig. la) showed distinctively stronger CaN immunoreactive staining than control rat brain (Fig. lb). In kindled brain, the most striking increment was observed on the ipsilateral side of the hippocampus (left) and in the ipsilateral and contralateral sides of the neocortex. In the ipsilateral hippocampus, the CaN increment was seen in CA1, CA2, CA3 and CA4 and in dentate gyrus. In contralateral hippocampus, CaN immunoreactive staining level was essentially the same as in normal rat (Fig. la,b). No specific immunostaining was observed either in control, or in kindled rat brain sections stained with non-specific mouse IgG (data not shown). In order to confirm the increment of CaN content in kindled rat brain, we performed immunoblotting analysis. Membrane and cytosol fractions were prepared from kindled, sham operated and control rat forebrains by the method indicated above. The monoclonal antibody VJ6 detected CaN A subunit (61 kDa) and some degradative products in purified bovine brain CaN (Fig. 2a,b, lane 1) and in rat brain samples (Fig. 2 a,b, lanes 2-4). In membrane fraction of kindled rat brain CaN A subunit was significantly increased compared to corresponding fractions from normal and sham operated brains (Fig. 2a, lanes 2-4). The amount of CaN in cytosol fractions did not show any difference (Fig. 2b, lanes 2-4). CaN content was quantified by densitometric scanning (Fig. 2c). The content in membrane fraction from kindled rat brain was 2.2-fold higher than that in fractions from controls and sham operated rat brains. However, the content of CaN in cytosol fractions did not show any difference (Fig. 2c). These data confirmed that CaN content was increased in kindled rat brain, and further showed that this increment was due to CaN in the membrane fraction. Two possible mechanisms can be postulated to explain the mechanism of increase in the membrane bound CaN: (1) CaN may translocate from cytosol to membrane fraction in calcium dependent manner; and (2) membrane binding subtype of CaN may specifically increase in kindled brain. The former mechanism is more probable because we have detected the calcium dependent translocation of CaN from cytosol to membrane [28]. Although CaN has multiple subtypes, mem-
340
L.J.M.P. Moia et al ./ Br ai n Research 648 (1994) 337-341
a
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.
.
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10
i
15
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i
0
5
10
15
20
25
30
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Fig. 3. Cyclosporin A inhibits kindling stage progression between stage 2 and stage 3, and the inhibition is reversible, a: normal control rat without CysA injection (e). Three individual rats (©, A, D) were injected with CysA. Control rat (e) reached stage 5 (full kindling) by 11 stimulations. On the other hand, CysA injected rats (©, A, [] ) stopped developing kindling stage at stage 2 and did not reach stage 3 even after 18 stimulations, b: control rat (e) reached stage 5 by 9 stimulations. Rat 1 ([]) and Rat 2 (A) did not develop kindling while receiving CysA. However, when CysA administration was stopped after 9 stimulations (Rat 1) and 14 stimulations (Rat 2) as indicated by arrows, both rats started to develop kindling. Both rats 1 and 2 reached stage 5 by 9 stimulations after stopping the drug administration.
brane binding subtypes have not been reported. Further investigation of the characteristics of the increased CaN in kindled brain will be required. Our findings that CaN is increased in kindled rat brain, in addition to the knowledge of inhibitory effect of immunosuppressants, CysA and FK506, on the CaN activity, led us to investigate the effect of these immunosuppressants on the progression of kindling. Control rats without immunosuppressant injection reached kindling stage 5 (generalized motor seizures) after 9 to 11 electrical stimulations (Fig. 3a and Fig. 3b, respectively). Rats with intraperitoneally administered CysA once everyday did not develop kindling stages beyond stage 2 even when 14 electrical stimulations or more had been applied (Fig. 3a). However, when we stopped the drug injection, the same rats reached stage 5 seizures with 9 electrical stimulations which was essentially the same as in normal control rats (Fig 3b). On the other hand, if CysA was administered at stage 3, 4 or 5, the drug did not inhibit the stage progression (data not shown). FK506 also showed essentially the same inhibitory effect on kindling stage progression as CysA. FK506 blocked stage progression between stage 2 and stage 3, and the blockade was reversible (data not shown). These data indicated that CysA and FK506 have a very similar, if not, the same mechanism, in inhibiting the progression of kindling. The reversibility indicates that the inhibitory effects of CysA and FK506 on kindling are not due to the result of non-specific neuronal damage but due to the result of specific pharmacological inhibitory effects of these drugs, most likely on CaN. The general clinical conditions of the animals injected with the immunosuppressants were similar to that of controls, except that the
formers presented slight diarrhea and 5% body weight loss. We did not observed any epileptic activity induced by CysA or FKS06, on the contrary these drugs inhibited the progression of seizure stage. The pharmacological effects of CysA and FK506 are well studied in T lymphocytes activation [24,26]. These i m m u n o s u p p r e s s a n t s form complexes with immunophilins and inhibit calcineurin activity, and thus suppress the dephosphorylation and translocation of N F - A T (nuclear factor of activated T cell) which leads to the inhibition of interleukin 2 (IL-2) production [19]. IL-2 and 1L-2 receptor are both proven to be present in rat brain hippocampus, striatum and neocortex [1]. However, it has not been examined whether IL-2 production increases in kindled rat brain. Other physiological events which are necessary for the production of IL-2 in T cell activation are also detected in kindled brain, such as increment of protein kinase C activity, induction of c-fos, increase in phosphatidyl inositol turn over and increase in the level of intracellular calcium [4,11,12,13,23]. These observations, in concert with the data presented in this paper lead us to postulate that a mechanism very similar to T cell activation is also involved in the epileptogenesis acquiring step in kindling. If so, it seems very reasonable to assume that CysA and FKS06 which are known as immunosuppressants and CaN inhibitors, block the stage progression of kindling. Since CysA and FK506 are very hydrophobic, they can penetrate through b l o o d - b r a i n barrier and reach neuronal cells. The pharmacological effects of clinical doses of C~sA on central nervous system including tremor, ataxia, seizures, quadriparesis and coma are reported [3,7]. As for FK506, tremor is reported in significant number of cases [8]. We are now
L.J.M.P. Moia et al. /Brain Research 648 (1994) 337-341
investigating to see if C3,sA and FK506 injected intraperitoneally can inhibit CaN activity in brain. We will also confirm that the increment of CaN will be long-lasting. We would like to express our special thanks to Dr. T.H. Murakami in the Department of Biology and Dr. T. Negi in the Department of Sports Medicine in The Kagawa Medical School for their technical instruction and support to prepare kindling rats. We also thank to Dr. Najma Janjua for the review of the English of this manuscript. This work was supported by a Grant-in-aid for Scientific Research from the Ministry of Education, Science and Culture of Japan. [1] Araujo, D.M., Lapchak, P.A. Collier, B. and Quirion R., Localization of interleukin-2 immunoreactivity and interleukin-2 receptors in the rat brain: interaction with the cholinergic system, Brain Res., 498 (1989) 257-266. [2] Asami, M., Kuno, T., Mukai, H. and Tanaka, C., Detection of the FICS06-FKBP-calcineurin complex by a simple binding assay, Biochem. Biophys. Res. Commun., 192 (1993) 1388-1394. [3] Atkinson, K., Biggs, J., Darveniza, P., Boland, J., Concannon, A. and Dodds, A., Cyclosporine-associated central nervous system toxicity after allogeneic bone marrow transplantation, Transplantation, 38 (1984) 34-37. [4] Buzsaki, G., Hsu, M., Horvath, Z. Horsburgh, K., Sundsmo, M., Masliah, E. and Saitoh, T., Kindling-induced changes of protein kinase C levels in hippocampus and neocortex, Mol. Neurobiol. Epilepsy, Epilepsy Res., Suppl. 9 (1992) 279-284. [5] Cavazos, J.E., Golarai, G. and Sutula, T.P., Mossy fiber synaptic reorganization induced by kindling: time course of development, progression, and permanence, Z Neurosci., 11 (1991)2795-2803. [6] Clipstone, N.A. and Crabtree, G.R., Identification of calcineurin as a key signalling enzyme in T-lymphocyte activation, Nature, 357 (1992) 695-697. [7] Deierhoi, M.H., Kalayoglu, M., Sollinger, H.W. and Belzer, F.O., Cyclosporin neurotoxicity in liver transplant recipients: report of three cases, Transplant. Proc., XX (1988) 116-118. [8] Eidelman, B.H., Abu-Elmagd, K., Wilson, J., Fung, J.J., Aleissiani, M., Jain, A., Takaya, S., Todo, S.N., Tzakis, A., Thiel, D.V., Shannon, W. and Starzl, T.E., Transplant. Proc., 23 (1991) 3175-3178. [9] Goddard, G.V., Mclntyre, D.C. and Leech, C.K., A permanent change in brain function resulting from daily electrical stimulation, Exp. Neurol., 25 (1969) 295-330. [10] Goto, S., Matsukado, Y., Mihara, Y., Inoue, N. and Miyamoto, E., The distribution of calcineurin in rat brain by light and electron microscopic immunohistochemistry and enzyme-immunoassay, Brain Res., 397 (1986) 161-172. [11] Iadorola, M.J., Nicolleti, F., Naranjo, J.R., Putnam, F. and Costa, E., Kindling enhances the stimulation of inositol phospholipid hydrolysis elicited by ibotenic acid in rat hippocampal slices, Brain Res., 374 (1986) 174-178. [12] Kohira, I., Akiyama, K., Daigen, A. and Otsuki, S., Enduring increase in membrane-associated protein kinase C activity in the hippocampal-kindled rat, Brain Res., 593 (1992) 82-88. [13] Labiner, D.M., Butler, L.S., Cao, Z., Hosford, D.A., Shin, C.
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