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TrkB pathway in neuroprotecive effect of cyclosporin A in forebrain ischemia
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Neuroscience Vol. 105, No. 3, pp. 571^578, 2001 ß 2001 IBRO. Published by Elsevier Science Ltd Printed in Great Britain. All rights reserved 0306-4522 / 01 $20.00+0.00
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INVOLVEMENT OF THE BRAIN-DERIVED NEUROTROPHIC FACTOR/TrkB PATHWAY IN NEUROPROTECIVE EFFECT OF CYCLOSPORIN A IN FOREBRAIN ISCHEMIA K. MIYATA,a;b * N. OMORI,b H. UCHINO,a;b T. YAMAGUCHI,a;b A. ISSHIKIa and F. SHIBASAKIb a b
Department of Anesthesiology, Tokyo Medical University, Tokyo, Japan
Department of Molecular Cell Physiology, Tokyo Metropolitan Institute of Medical Science, Tokyo, Japan
AbstractöRecent studies have shown that cyclosporin A, a speci¢c antagonist of calcineurin, a phosphatase, ameliorates neuronal cell death in the CA1 sector of the hippocampus after forebrain ischemia in animal models. The mechanism of this neuroprotective e¡ect, however, has not yet been established. Brain-derived neurotrophic factor (BDNF), a member of the neurotrophins, is one of the potent survival and developmental factors whose expression is regulated by cyclic AMP-response element-binding protein (CREB). Activation of CREB is dependent on its phosphorylation at Ser133 , and calcineurin has been reported to dephosphorylate CREB via protein phosphatase 1. Based on these observations, we attempted to investigate how cyclosporin A treatment would a¡ect the changes of phosphorylated CREB (pCREB), BDNF and its receptor tyrosine kinase B (TrkB) after forebrain ischemia in rats. Phosphorylation of CREB was kept augmented throughout the time course examined in cyclosporin A-treated animals, while it ceased without cyclosporin A. Reverse transcription-polymerase chain reaction revealed prolonged maintenance of BDNF mRNA expression in the CA1 sector of cyclosporin A-treated animals. The protein expression of BDNF and TrkB appeared to be up-regulated in cyclosporin A-treated animals, whereas it was transiently up-regulated but decreased to the marginal level of expression without cyclosporin A. From these results we suggest that cyclosporin A induces pCREB by an inhibition of calcineurin, resulting in the induction of BDNF. The mechanisms by which cyclosporin A protects the CA1 region from neuronal cell death in forebrain ischemia may involve the interaction of pCREB, BDNF and TrkB. ß 2001 IBRO. Published by Elsevier Science Ltd. All rights reserved. Key words: forebrain ischemia, cyclosporin A, cyclic AMP-response element-binding protein, brain-derived neurotrophic factor, tyrosine kinase B.
ischemia have not yet been clari¢ed. Several lines of evidence suggest that the damage is somehow coupled to delayed bioenergetic failure and secondary accumulation of calcium in mitochondria, triggering cell death after hours or days of recirculation (Deshpande et al., 1987; Dux et al., 1987; Pulsinelli et al., 1982; Sims and Pulsinelli, 1987; Zaidan and Sims, 1994). The cyclic undecapeptide cyclosporin A (CsA) is an e¡ective immunosuppressant and a speci¢c blocker of calcineurin, calcium/calmodulin-sensitive protein phosphatase. It has been reported that CsA dramatically ameliorates neuronal cell damages in the CA1 sector of the hippocampus during forebrain ischemia (Li et al., 1997; Uchino et al., 1995, 1998). FK506, another blocker of calcineurin, has been reported to reduce the damage due to focal ischemia caused by transient occlusion of the middle cerebral artery (Sharkey and Butcher, 1994). Although little is known about the details of such neuroprotective e¡ects, it seems quite likely that calcineurin is one of the essential factors in regulation of neuronal cell death after ischemia^reperfusion. The cyclic AMP-response element-binding protein (CREB) is one of the family of DNA-binding proteins that mediate the e¡ect of cAMP on the transcriptional regulation of a large number of peptides and proteins
Forebrain or focal ischemia of short to intermediate duration leads to brain injury in the form of neuronal damage, selectively a¡ecting vulnerable regions, for example, the CA1 sector of the hippocampus (Pulsinelli et al., 1982; Smith et al., 1984). This type of damage occurs with a marked delay, suggesting the involvement of events that trigger secondary damage of an in£ammatory or immunological nature. The mechanisms giving rise to delayed neuronal damage following forebrain
*Correspondence to: K. Miyata, 6-7-1 Nishishinjuku, Shinjuku-ku, Tokyo 160-0022, Japan. Tel.: +81-3-3342-6111 (ext. 5811); fax: +81-3-5381-6650. E-mail address: [email protected] (K. Miyata). Abbreviations : ANOVA, analysis of variance; BDNF, brain-derived neurotrophic factor; CREB, cyclic AMP-response element-binding protein; CsA, cyclosporin A; CSA, ischemia^reperfusion rats treated with CsA; DG, dentate gyrus; EDTA, ethylenediaminetetra-acetate; EGTA, ethylene glycol-bis(2-aminoethyl-ether)N,N,NP,NP-tetraacetic acid ; ISC, ischemia^reperfusion only rats treated with vehicle; NGF, nerve growth factor; PBS, phosphatebu¡ered saline ; pCREB, phosphorylated CREB; PKB, protein kinase B; RT-PCR, reverse transcription-polymerase chain reaction; TrkB, tyrosine kinase B; TUNEL, terminal deoxynucleotidyl transferase (TdT)-mediated dUTP-biotin nick end labeling. 571
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(Gonzalez et al., 1989; Yamamoto et al., 1988). The phosphorylation status of CREB is critical for its transactivating activity, and several protein kinases, such as calcium/calmodulin-dependent kinase 1, 2, and 4 and protein kinase C, have been reported to be involved in the activation of CREB by phosphorylation (BeitnerJohnson and Millhorn, 1998; Bito et al., 1996; Matthews et al., 1994; Sheng et al., 1991; Yamamoto et al., 1988). In contrast, little has been elucidated about the dephosphorylation mechanisms; at present, calcineurin is assumed to be responsible for dephosphorylation in hippocampal neurons via the regulation of protein phosphatase 1 (Bito et al., 1996). Phosphorylated CREB (pCREB), the activated form of CREB, regulates many aspects of neuronal function, including excitation of nerve cells (Moore et al., 1996), circadian rhythm (Ginty et al., 1993), pituitary proliferation (Struthers et al., 1991), and long-term memory formation (Struthers et al., 1991). Recently, an increased level of pCREB following hypoxic-ischemia brain injury was shown to be evident in damage-resistant neurons of the dentate gyrus (DG) and neocortex, but not in the vulnerable CA1 neurons that underwent delayed neuronal cell death (Bito et al., 1996; Hu et al., 1999; Walton et al., 1996). In addition, treatment with FK506 has been reported to augment pCREB immunoreactivity in hippocampal neurons and slices of the striatum (Merlio et al., 1993). pCREB mediates a variety of genes, including brain-derived neurotrophic factor (BDNF), nerve growth factor (NGF), c-fos, and Bcl-2 (Beck et al., 1994; Tao et al., 1998; Walton et al., 1996; Wilson et al., 1996). The administration of exogenous factors such as neurotrophic factors and growth factors is known to protect against neuronal cell death in models of brain ischemia (Beck et al., 1994; Miyazaki et al., 1999). In this study, we attempted to investigate the neuroprotective mechanisms of CsA in a model of forebrain ischemia, focusing on the regulation of CREB phosphorylation and on the expression of BDNF and its receptor tyrosine kinase B (TrkB), as a representative of the neurotrophic factor family which is known to be transcriptionally controlled by CREB.
EXPERIMENTAL PROCEDURES
Experimental model Male Wistar rats of the S.P.F. strain (Kyudo, Saga, Japan) weighing 280^330 g were used. Insertion of a needle into the hippocampus of one side was performed 1 week before ischemia. Using a 10-Wl Hamilton syringe (needle outer diameter 450 Wm), four 1-Wl deposits of saline solution were made stereotaxically in the right hippocampus at two cannula penetrations: (a) 2.5 mm caudal to bregma, 1.2 mm lateral to midline with deposits 3.1 mm and 2.7 mm ventral to dura, and (b) 4.5 mm caudal to bregma, 3.6 mm lateral to midline with deposits 2.9 mm and 2.6 mm ventral to dura. The animals were injected daily with CsA (10 mg/kg) or vehicle i.p. for 1 week before ischemia. The CsA was supplied by Novartis (Basel, Switzerland). CsA (250 mg) was dissolved in 65% cremaphol EL. And we used only 65% cremaphol EL without CsA as vehicle for every experiment. Prior to ischemia, the animals were fasted overnight but allowed water ad libitum. Anesthesia was induced with 3% iso-
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£urane in N2 O/O2 (70:30). The animals were intubated and arti¢cially ventilated. The tail artery and tail vein were cannulated for blood sampling, blood pressure recording, and drug infusion. A central venous catheter was inserted into the right jugular vein in order to allow withdrawal of blood during ischemia. The common carotid arteries of both sides were isolated and ¢tted with loose ligatures for later clamping. After the operative procedures, the iso£urane concentration was maintained at 0.3^0.5%. The animals were given heparin (30 U/kg) and paralyzed with vecuronium bromide (2 mg/h). Core and head temperature were monitored and kept close to 37³C by lamp heating. Ventilation and oxygen supply were adjusted to give a PaCO2 of 35^40 mm Hg, PaO2 over 100 mm Hg and pH close to 7.40. Animals were subjected to forebrain ischemia as described elsewhere (Smith et al., 1984). In brief, the procedure involves a 10-min duration of ischemia, which gives dense and maximal damage to the CA1 sector of the hippocampus, by bilateral common carotid artery clamping plus hypotension by exsanguination (mean arterial pressure 40^50 mm Hg). Recirculation was achieved by reinfusing the shed blood and by releasing the clamps placed around the carotid arteries. After reperfusion, bicarbonate (0.6 M, 0.5 ml) was given i.v. to counteract systemic acidosis. Iso£urane and the muscle relaxant were discontinued. When the animals regained spontaneous breathing, they were disconnected from the respirator and extubated. The rats were then housed in cages, given free access to water and pellet food, and monitored for behavioral manifestations or seizures. All animal care and experimentation was performed according to the study guidelines established by the Tokyo Metropolitan Institute of Medical Science subcommittee on laboratory animal care, handling, and termination. All e¡orts were made to minimize the number of animals used and their su¡ering. Tissue preparation After reperfusion, brain samples were collected periodically. At 1, 6, 12, 24, and 72 h after reperfusion, the animals were reanesthetized with 3% iso£urane and killed. The brains were removed and immediately frozen in powdered dry ice. Coronal cryostat sections (30 Wm) were taken through the dorsal hippocampus. Extraction of RNA and RT-PCR To obtain hippocampal CA1 and DG tissues, rats were decapitated and CA1 tissues were dissected out at 1, 6, 12 and 24 h after reperfusion (n = 5 for each interval and experimental group). Using Isogen (Nippon Gene, Tokyo, Japan), total RNAs were extracted followed by DNase I treatment. Reverse transcription-polymerase chain reaction (RT-PCR) was performed with a one-step PCR kit (Takara, Tokyo, Japan) following the manufacturer's instructions. In brief, 0.5 Wg of total RNA was used per reaction and the following program was applied. Reverse transcription: 50³C for 30 min; PCR: 94³CU30 s, 60³CU30 s, 72³CU1 min. Cycle numbers were 25 and 20 for rat BDNF and L-actin, respectively. The oligonucleotide primers used are listed below (Bova et al., 1998). They give rise to 342-bp and 520-bp PCR products for BDNF and L-actin, respectively. Ampli¢cation products were electrophoresed on 1% agarose gel, then stained with ethidium bromide and photographed. Quanti¢cation of signals was performed by NIH Image software. BDNF: sense, 5P-CGTGATCGAGGAGCTGTTGG-3P; antisense, 5P-CTGCTTCAGTTGGCCTTTCG-3P; L-actin: sense, 5P-GGCTGTGTTGTCCCTGTAT-3P; antisense, 5P-CCGCTCATTGCCGATAGTG-3P. Immunohistochemistry Sections were ¢xed with 4% paraformaldehyde in phosphate-
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BDNF/TrkB in neuroprotection by cyclosporin A
bu¡ered saline (PBS) for 30 min and washed three times with PBS for 5 min. Then, blocking was performed with PBS containing 5% bovine serum albumin for 30 min. Rabbit anti-mouse BDNF polyclonal antibody (Chemicon, Temecula, CA, USA), rabbit anti-mouse TrkB polyclonal antibody (Santa Cruz Biotech, Santa Cruz, CA, USA) and rabbit anti-phospho-CREB polyclonal antibody (New England Biolabs, Beverly, MA, USA) were diluted 1:1000 in PBS containing 5% bovine serum albumin and incubated for hybridization overnight at 4³C. After rinsing, secondary antibodies labeled with Alexa 546 (Molecular Probes, Eugene, OR, USA) were hybridized at room temperature for 1 h. After washing several times, sections were mounted with Vectashield (Vector Laboratories, Burlingame, CA, USA) and subjected to immuno£uorescent detection with a Carl Zeiss LSM510 confocal laser scanning microscope. Terminal deoxynucleotidyl transferase (TdT)-mediated dUTP-biotin nick end labelling (TUNEL) staining was performed with a Mebstain Apoptosis Kit (MBL, Nagoya, Japan) following the manufacturer's instructions. Preparation of whole cell extracts Rats were killed and hippocampal CA1 sectors were dissected out at 1, 6, 12 and 24 h after reperfusion (n = 4 for each interval and experimental group). Brain tissues were homogenized with a
Dounce homogenizer (30 strokes) in 10 times volume of homogenization bu¡er containing 15 mM Tris^HCl, pH 7.6, 1 mM dithiothreitol, 0.25 M sucrose, 1 mM MgCl2 , 1.25 Wg/ml pepstatin A, 10 Wg/ml leupeptin, 2.5 Wg/ml aprotinin, 0.5 mM phenylmethylsulfonyl £uoride, 2.5 mM EDTA, 1 mM EGTA, 0.1 M Na3 VO4 , 50 mM NaF, 2 mM sodium pyrophosphate, and 0.1% Triton X-100. The DC protein assay kit (Bio-Rad Laboratories, Hercules, CA, USA) was used to determine the protein concentration. Western blot analysis Western blot analysis was carried out on 8% sodium dodecyl sulfate-polyacrylamide gel electrophoresis. One sample containing 20 Wg of protein was applied to each lane in a slab gel. Following electrophoresis, proteins were transferred to an Immobilon-P membrane. The membranes were incubated with primary polyclonal antibodies as used in immunohistochemistry at a dilution of 1:1000 overnight at 4³C. The membrane was then incubated with horseradish peroxidase-conjugated anti-rabbit secondary antibody for 1 h at room temperature. The enhanced chemiluminescence detection method (ECL; Amersham Pharmacia Biotech, Uppsala, Sweden) was used for the detection of signals, following the manufacturer's instructions. Four animals in each experimental group
Fig. 1. Histopathological ¢ndings in rat hippocampus following 10 min of forebrain ischemia. CsA(+), CsA-treated (10 mg/ kg) animals; CsA(3), vehicle-administered (ISC) animals. (A) The extent of neuronal damage in the CA1 sector in CsA(+) and CsA(3) at 7 days after reperfusion. Scale bar = 200 Wm. (B) Immuno£uorescent images of TUNEL staining of the CA1 sector after 72 h in CsA(+) and CsA(3). Scale bar = 50 Wm.
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Fig. 2. Expression of BDNF and TrkB in the CA1 sector. CsA(+), CsA-treated (10 mg/kg) animals ; CsA(3), vehicle-administered (ISC) animals. (A) Western blot analysis of BDNF and TrkB in the CA1 sector. Samples were collected from either CsA(+) or CsA(3) groups rats at 1, 6, 12 and 24 h. Samples obtained from sham-operated rats were used as control. (B) Immuno£uorescence images of BDNF (upper row) and TrkB (lower row) in the CA1 sector at 24 h. CsA(+) samples are shown in the right column and CsA(3) samples in the left column. Scale bar = 50 Wm.
were used to analyze the level of the proteins by western blotting.
RESULTS
Histopathological ¢ndings
Statistical analysis Evaluation of di¡erences in BDNF mRNA expression among animal groups was performed by one-way analysis of variance (ANOVA) followed by the Bonferroni/Dunn post-hoc test with signi¢cance set at P 6 0.05.
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Hematoxylin^eosin-stained hippocampal slices of rat brain 1 week after ischemia^reperfusion (ISC group) showed massive neuronal cell death in the CA1 sector, whereas cells were completely intact in CsA-treated rats
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(CSA group) (Fig. 1A). CA1 pyramidal cells undergoing cell death were detectable by TUNEL staining at 72 h after ischemia in the ISC group (Fig. 1B). Western blot analysis and immunohistochemical ¢ndings in CA1 sector To investigate involvement of BDNF and TrkB, we examined the expression of BDNF and TrkB by western
blot. In sham-operated animals, both BDNF and TrkB were detectable (Fig. 2A). In the ISC group, BDNF was gradually down-regulated after the transient increase at 1 h, while no marked £uctuation was found in the CSA group throughout the time course. TrkB was, indeed, upregulated in both groups at the early time points, but as seen in BDNF, the expression level was steadily tapered in the ISC group while it was kept at the increased level in the CSA group (Fig. 2A). Fig. 2B clearly demonstrates
Fig. 3. Expression of pCREB. CsA(+), CsA-treated (10 mg/kg) animals; CsA(3), vehicle-administered (ISC) animals. (A) western blot analysis of pCREB and CREB expression in the CA1 sector. Samples were obtained as described in Fig. 2B. (B) Immuno£uorescent images of pCREB in the CA1 sector at 24 h. Scale bar = 50 Wm.
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Fig. 4. Variation of BDNF mRNA levels in the CA1 sector. Each reported value represents the mean of ¢ve independent experiments þ S.D. Each value is represented as a percentage of the ratio between the densitometric scores for the BDNF and L-actin PCR products based on the ratio of sham-operated animals as 100%. *P 6 0.05, one-way ANOVA followed by Bonferroni/Dunn post-hoc test. CsA(+), CsA-treated (10 mg/kg) animals ; CsA(3), vehicle-administered (ISC) animals.
the enhanced immuno£uorescent signals of BDNF and TrkB in the CSA group at 24 h. The speci¢city of the immunohistochemical reaction for the primary antibodies used was con¢rmed by omitting each primary antibody in the procedure. Non-speci¢c signals were very low (data not shown). Next, we monitored the activation of CREB formation of pCREB by immunostaining and western blot (Fig. 3A, B). Even in sham-operated animal, a distinct level of pCREB was observed. An increased level of pCREB was found in both groups immediately after the reperfusion. However, it was transient and became marginally detectable by 24 h without CsA (Fig. 3A). CsA induced more prominent formation of pCREB and it was maintained throughout the course studied, while the whole body of CREB expression was unchanged. As shown in Fig. 3B, pCREB immunoreactivity is considerably maintained only in the CSA group at 24 h. RT-PCR To examine changes in mRNA expression induced by CsA administration, semi-quantitative RT-PCR was performed for BDNF using total RNAs dissected from the CA1 sector (Fig. 4). However, the expression of BDNF was decreased in the ISC group, whereas it was maintained at a normal level by CsA administration. This result indicates that CsA may play a role in maintaining the expression of BDNF, at least at the mRNA level in the CA1 sector.
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DISCUSSION
The novel ¢ndings in this study were: (a) in the CA1 sector, CsA treatment induced an apparent increase of pCREB, and augmented formation of pCREB was maintained throughout the time course examined; (b) concurrent with the induction of pCREB, the expression of BDNF and TrkB remained up-regulated in the CsAtreated animals, while it was transient in the animals without the CsA treatment; (c) the level of BDNF mRNA in the CA1 sector was maintained at a higher level by CsA treatment. Controlling the transcription of various genes, phosphorylation of CREB is one of the most important mechanisms (Gonzalez et al., 1989; Yanamoto et al., 2000). It has been reported that, in the transient ischemic model, the level of pCREB increased in damage-resistant neurons of the DG but not in the vulnerable CA1 neurons (Hu et al., 1999). The cyclic undecapeptide CsA is an e¡ective immunosuppressant that is a speci¢c blocker of the calcium/calmodulin-sensitive phosphatase calcineurin. It has been reported that CsA dramatically ameliorates the neuronal cell damage a¡ecting vulnerable regions, for example, the CA1 sector of the hippocampus after forebrain ischemia (Li et al., 1997; Uchino et al., 1995, 1998). FK506, another blocker of calcineurin, has been reported to reduce the damage due to focal ischemia caused by transient occlusion of the middle cerebral artery (Sharkey and Butcher, 1994). Although little is known about the
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details of such neuroprotective e¡ects, it seems quite likely that calcineurin is one of the essential factors in the regulation of neuronal cell death after ischemia^ reperfusion injury. Our results in CsA-treated rats indicate that formation of pCREB is enhanced in the CA1 sector throughout the time course examined. Calcineurin has been suggested to play a key role through regulation of protein phosphatase 1 activity resulting in dephosphorylation of pCREB (Bito et al., 1996). Since CsA is a speci¢c blocker of calcineurin, we hypothesized that the inhibition of calcineurin by CsA shifted the phosphorylation balance of CREB to be phosphorylated. Therefore, the induction of pCREB by CsA may well be involved in the concurrent induction of BDNF. Administration of exogenous BDNF prevents neuronal death in the CA1 sector after transient forebrain ischemia (Beck et al., 1994), and induces infarct tolerance after focal ischemia (Yanamoto et al., 2000). In the CA1 sector after transient ischemia, BDNF immunoreactivity is reported to increase at early stages after ischemic insult (Yamasaki et al., 1998). Our data of RT-PCR revealed the maintained expression of BDNF mRNA by CsA treatment in the CA1 sector. It is, therefore, conceivable that the augmented expression of BDNF may be involved in prevention of delayed neuronal cell death. Takeda et al. (1993) described that BDNF mRNA gradually decreased in the CA1 sector, and increased in the DG after transient ischemia. Our results are consistent with this report suggesting regulation of BDNF expression at the transcriptional level. BDNF binds and activates a speci¢c high-a¤nity receptor, TrkB, and a low-a¤nity receptor, p75 (Carter and Lewin, 1997). TrkB is a member of the receptor-type tyrosine kinases whose activation potentially leads to several signaling systems, including the phosphatidylinositol 3-kinase pathway which is linked to the phosphorylation of Akt/protein kinase B (PKB) (Atwal et al., 2000; Eves et al., 1998). As CREB is reported to be one of the regulatory targets for Akt/PKB (Du and Montminy, 1998), induction of BDNF by CsA administration may play a part in enhancement of phosphorylation of CREB. Immunohistochemical studies and western blots of TrkB revealed augmented expression in the CA1 sector after CsA treatment. This may be due not only to the de novo synthesis of TrkB (Ferrer et al., 1998; Merlio et al., 1993), but also to the recruit-
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ment of TrkB to the plasma membrane from intracellular stores (Meyer-Franke et al., 1998). Several roles have been proposed for the p75 receptor, including regulation of apoptosis, ligand discrimination, retrograde transport and signal transduction (Carter and Lewin, 1997). Although we could not detect any apparent change of p75 expression by CsA treatment (data not shown), it is possible that a suppressed death signal through the p75 receptor subunit by enhanced signaling through the TrkB subunit may be involved in the rescue of neuronal cells, as reported in the TrkA/p75 (Yoon et al., 1998). It would be intriguing to pursue the details of this possibility in future investigations. The Bcl-2 family proteins, consisting of pro-apoptotic Bad, Bik, Bid, etc. and anti-apoptotic Bcl-2 and Bcl-xl, are important regulators of mammalian apoptosis (Merry and Korsmeyer, 1997). The promoter region of Bcl-2 contains a CRE site. The transcription factor CREB has been identi¢ed as a positive regulator of Bcl-2 expression (Pugazhenthi et al., 1999; Wilson et al., 1996). Up-regulation of Bcl-2 expression has been identi¢ed as a critical mechanism of which growth factors promote cell survival (Pugazhenthi et al., 1999). In a cerebral ischemia model, it has been reported that Bcl-2 is induced in surviving neurons (Chen et al., 1997). Neuronal overexpression of Bcl-2 in transgenic mice ameliorates hippocampal neuronal damage after transient ischemia (Kitagawa et al., 1998). Recent studies have reported that NGF and BDNF induce Bcl-2 in sympathetic or cortical neurons through the activation of CREB (Riccio et al., 1999). In our preliminary experiments, weak but apparent induction of Bcl-2 by CsA was detected, whereas neither sham-operated nor ischemic^ reperfused animals showed a detectable level of immunoreactivity (data not shown). Further studies are required to elucidate the possible involvement of Bcl-2 in the mechanism of neuroprotection. Taken together, our results indicate that the neuroprotective e¡ect of CsA in this model of forebrain ischemia may involve activation of the CREB-BDNF/TrkB pathway through the suppression of calcineurin activity by CsA. AcknowledgementsöWe thank Dr. Yutaka Hosikawa and Dr. Miki Shitasige for scienti¢c advice. This work was supported by a Grant-in-Aid for Scienti¢c Research, Grant 11671524, from the Ministry of Education, Science and Culture of Japan.
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Insulin-like growth factor-I induces bcl-2 promoter through the transcription factor cAMP-response element-binding protein. J. Biol. Chem. 274, 27529^27535. Pulsinelli, W.A., Brierley, J.B., Plum, F., 1982. Temporal pro¢le of neuronal damage in a model of transient forebrain ischemia. Ann. Neurol. 11, 491^498. Riccio, A., Ahn, S., Davenport, C.M., Blendy, J.A., Ginty, D.D., 1999. Mediation by a CREB family transcription factor of NGF-dependent survival of sympathetic neurons. Science 286, 2358^2361. Sharkey, J., Butcher, S.P., 1994. Immunophilins mediate the neuroprotective e¡ects of FK506 in focal cerebral ischaemia. Nature 371, 336^339. Sheng, M., Thompson, M.A., Greenberg, M.E., 1991. CREB: a Ca(2+)-regulated transcription factor phosphorylated by calmodulin-dependent kinases. Science 252, 1427^1430. Sims, N.R., Pulsinelli, W.A., 1987. Altered mitochondrial respiration in selectively vulnerable brain subregions following transient forebrain ischemia in the rat. J. Neurochem. 49, 1367^1374. Smith, M.L., Auer, R.N., Siesjo, B.K., 1984. The density and distribution of ischemic brain injury in the rat following 2^10 min of forebrain ischemia. Acta Neuropathol. 64, 319^332. Struthers, R.S., Vale, W.W., Arias, C., Sawchenko, P.E., Montminy, M.R., 1991. Somatotroph hypoplasia and dwar¢sm in transgenic mice expressing a non-phosphorylatable CREB mutant. Nature 350, 622^624. Takeda, A., Onodera, H., Sugimoto, A., Kogure, K., Obinata, M., Shibahara, S., 1993. Coordinated expression of messenger RNAs for nerve growth factor, brain-derived neurotrophic factor and neurotrophin-3 in the rat hippocampus following transient forebrain ischemia. Neuroscience 55, 23^31. Tao, X., Finkbeiner, S., Arnold, D.B., Shaywitz, A.J., Greenberg, M.E., 1998. Ca2 in£ux regulates BDNF transcription by a CREB family transcription factor-dependent mechanism [published erratum appears in Neuron 20 (1998) 1297]. Neuron 20, 709^726. Uchino, H., Elmer, E., Uchino, K., Li, P.A., He, Q.P., Smith, M.L., Siesjo, B.K., 1998. Amelioration by cyclosporin A of brain damage in transient forebrain ischemia in the rat. Brain Res. 812, 216^226. Uchino, H., Elmer, E., Uchino, K., Lindvall, O., Siesjo, B.K., 1995. Cyclosporin A dramatically ameliorates CA1 hippocampal damage following transient forebrain ischaemia in the rat. Acta Physiol. Scand. 155, 469^471. Walton, M., Sirimanne, E., Williams, C., Gluckman, P., Dragunow, M., 1996. The role of the cyclic AMP-responsive element binding protein (CREB) in hypoxic-ischemic brain damage and repair. Mol. Brain Res. 43, 21^29. Wilson, B.E., Mochon, E., Boxer, L.M., 1996. Induction of bcl-2 expression by phosphorylated CREB proteins during B-cell activation and rescue from apoptosis. Mol. Cell. Biol. 16, 5546^5556. Yamamoto, K.K., Gonzalez, G.A., Biggs, W.H.d., Montminy, M.R., 1988. Phosphorylation-induced binding and transcriptional e¤cacy of nuclear factor CREB. Nature 334, 494^498. Yamasaki, Y., Shigeno, T., Furukawa, Y., Furukawa, S., 1998. Reduction in brain-derived neurotrophic factor protein level in the hippocampal CA1 dendritic ¢eld precedes the delayed neuronal damage in the rat brain. J. Neurosci. Res. 53, 318^329. Yanamoto, H., Nagata, I., Sakata, M., Zhang, Z., Tohnai, N., Sakai, H., Kikuchi, H., 2000. Infarct tolerance induced by intra-cerebral infusion of recombinant brain-derived neurotrophic factor. Brain Res. 859, 240^248. Yoon, S.O., Casaccia-Bonne¢l, P., Carter, B., Chao, M.V., 1998. Competitive signaling between TrkA and p75 nerve growth factor receptors determines cell survival. J. Neurosci. 18, 3273^3281. Zaidan, E., Sims, N.R., 1994. The calcium content of mitochondria from brain subregions following short-term forebrain ischemia and recirculation in the rat. J. Neurochem. 63, 1812^1819. (Accepted 17 April 2001)
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Neuroscience Vol. 105, No. 3, pp. 571^578, 2001 ß 2001 IBRO. Published by Elsevier Science Ltd Printed in Great Britain. All rights reserved 0306-4522 / 01 $20.00+0.00
www.elsevier.com/locate/neuroscience
INVOLVEMENT OF THE BRAIN-DERIVED NEUROTROPHIC FACTOR/TrkB PATHWAY IN NEUROPROTECIVE EFFECT OF CYCLOSPORIN A IN FOREBRAIN ISCHEMIA K. MIYATA,a;b * N. OMORI,b H. UCHINO,a;b T. YAMAGUCHI,a;b A. ISSHIKIa and F. SHIBASAKIb a b
Department of Anesthesiology, Tokyo Medical University, Tokyo, Japan
Department of Molecular Cell Physiology, Tokyo Metropolitan Institute of Medical Science, Tokyo, Japan
AbstractöRecent studies have shown that cyclosporin A, a speci¢c antagonist of calcineurin, a phosphatase, ameliorates neuronal cell death in the CA1 sector of the hippocampus after forebrain ischemia in animal models. The mechanism of this neuroprotective e¡ect, however, has not yet been established. Brain-derived neurotrophic factor (BDNF), a member of the neurotrophins, is one of the potent survival and developmental factors whose expression is regulated by cyclic AMP-response element-binding protein (CREB). Activation of CREB is dependent on its phosphorylation at Ser133 , and calcineurin has been reported to dephosphorylate CREB via protein phosphatase 1. Based on these observations, we attempted to investigate how cyclosporin A treatment would a¡ect the changes of phosphorylated CREB (pCREB), BDNF and its receptor tyrosine kinase B (TrkB) after forebrain ischemia in rats. Phosphorylation of CREB was kept augmented throughout the time course examined in cyclosporin A-treated animals, while it ceased without cyclosporin A. Reverse transcription-polymerase chain reaction revealed prolonged maintenance of BDNF mRNA expression in the CA1 sector of cyclosporin A-treated animals. The protein expression of BDNF and TrkB appeared to be up-regulated in cyclosporin A-treated animals, whereas it was transiently up-regulated but decreased to the marginal level of expression without cyclosporin A. From these results we suggest that cyclosporin A induces pCREB by an inhibition of calcineurin, resulting in the induction of BDNF. The mechanisms by which cyclosporin A protects the CA1 region from neuronal cell death in forebrain ischemia may involve the interaction of pCREB, BDNF and TrkB. ß 2001 IBRO. Published by Elsevier Science Ltd. All rights reserved. Key words: forebrain ischemia, cyclosporin A, cyclic AMP-response element-binding protein, brain-derived neurotrophic factor, tyrosine kinase B.
ischemia have not yet been clari¢ed. Several lines of evidence suggest that the damage is somehow coupled to delayed bioenergetic failure and secondary accumulation of calcium in mitochondria, triggering cell death after hours or days of recirculation (Deshpande et al., 1987; Dux et al., 1987; Pulsinelli et al., 1982; Sims and Pulsinelli, 1987; Zaidan and Sims, 1994). The cyclic undecapeptide cyclosporin A (CsA) is an e¡ective immunosuppressant and a speci¢c blocker of calcineurin, calcium/calmodulin-sensitive protein phosphatase. It has been reported that CsA dramatically ameliorates neuronal cell damages in the CA1 sector of the hippocampus during forebrain ischemia (Li et al., 1997; Uchino et al., 1995, 1998). FK506, another blocker of calcineurin, has been reported to reduce the damage due to focal ischemia caused by transient occlusion of the middle cerebral artery (Sharkey and Butcher, 1994). Although little is known about the details of such neuroprotective e¡ects, it seems quite likely that calcineurin is one of the essential factors in regulation of neuronal cell death after ischemia^reperfusion. The cyclic AMP-response element-binding protein (CREB) is one of the family of DNA-binding proteins that mediate the e¡ect of cAMP on the transcriptional regulation of a large number of peptides and proteins
Forebrain or focal ischemia of short to intermediate duration leads to brain injury in the form of neuronal damage, selectively a¡ecting vulnerable regions, for example, the CA1 sector of the hippocampus (Pulsinelli et al., 1982; Smith et al., 1984). This type of damage occurs with a marked delay, suggesting the involvement of events that trigger secondary damage of an in£ammatory or immunological nature. The mechanisms giving rise to delayed neuronal damage following forebrain
*Correspondence to: K. Miyata, 6-7-1 Nishishinjuku, Shinjuku-ku, Tokyo 160-0022, Japan. Tel.: +81-3-3342-6111 (ext. 5811); fax: +81-3-5381-6650. E-mail address: [email protected] (K. Miyata). Abbreviations : ANOVA, analysis of variance; BDNF, brain-derived neurotrophic factor; CREB, cyclic AMP-response element-binding protein; CsA, cyclosporin A; CSA, ischemia^reperfusion rats treated with CsA; DG, dentate gyrus; EDTA, ethylenediaminetetra-acetate; EGTA, ethylene glycol-bis(2-aminoethyl-ether)N,N,NP,NP-tetraacetic acid ; ISC, ischemia^reperfusion only rats treated with vehicle; NGF, nerve growth factor; PBS, phosphatebu¡ered saline ; pCREB, phosphorylated CREB; PKB, protein kinase B; RT-PCR, reverse transcription-polymerase chain reaction; TrkB, tyrosine kinase B; TUNEL, terminal deoxynucleotidyl transferase (TdT)-mediated dUTP-biotin nick end labeling. 571
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(Gonzalez et al., 1989; Yamamoto et al., 1988). The phosphorylation status of CREB is critical for its transactivating activity, and several protein kinases, such as calcium/calmodulin-dependent kinase 1, 2, and 4 and protein kinase C, have been reported to be involved in the activation of CREB by phosphorylation (BeitnerJohnson and Millhorn, 1998; Bito et al., 1996; Matthews et al., 1994; Sheng et al., 1991; Yamamoto et al., 1988). In contrast, little has been elucidated about the dephosphorylation mechanisms; at present, calcineurin is assumed to be responsible for dephosphorylation in hippocampal neurons via the regulation of protein phosphatase 1 (Bito et al., 1996). Phosphorylated CREB (pCREB), the activated form of CREB, regulates many aspects of neuronal function, including excitation of nerve cells (Moore et al., 1996), circadian rhythm (Ginty et al., 1993), pituitary proliferation (Struthers et al., 1991), and long-term memory formation (Struthers et al., 1991). Recently, an increased level of pCREB following hypoxic-ischemia brain injury was shown to be evident in damage-resistant neurons of the dentate gyrus (DG) and neocortex, but not in the vulnerable CA1 neurons that underwent delayed neuronal cell death (Bito et al., 1996; Hu et al., 1999; Walton et al., 1996). In addition, treatment with FK506 has been reported to augment pCREB immunoreactivity in hippocampal neurons and slices of the striatum (Merlio et al., 1993). pCREB mediates a variety of genes, including brain-derived neurotrophic factor (BDNF), nerve growth factor (NGF), c-fos, and Bcl-2 (Beck et al., 1994; Tao et al., 1998; Walton et al., 1996; Wilson et al., 1996). The administration of exogenous factors such as neurotrophic factors and growth factors is known to protect against neuronal cell death in models of brain ischemia (Beck et al., 1994; Miyazaki et al., 1999). In this study, we attempted to investigate the neuroprotective mechanisms of CsA in a model of forebrain ischemia, focusing on the regulation of CREB phosphorylation and on the expression of BDNF and its receptor tyrosine kinase B (TrkB), as a representative of the neurotrophic factor family which is known to be transcriptionally controlled by CREB.
EXPERIMENTAL PROCEDURES
Experimental model Male Wistar rats of the S.P.F. strain (Kyudo, Saga, Japan) weighing 280^330 g were used. Insertion of a needle into the hippocampus of one side was performed 1 week before ischemia. Using a 10-Wl Hamilton syringe (needle outer diameter 450 Wm), four 1-Wl deposits of saline solution were made stereotaxically in the right hippocampus at two cannula penetrations: (a) 2.5 mm caudal to bregma, 1.2 mm lateral to midline with deposits 3.1 mm and 2.7 mm ventral to dura, and (b) 4.5 mm caudal to bregma, 3.6 mm lateral to midline with deposits 2.9 mm and 2.6 mm ventral to dura. The animals were injected daily with CsA (10 mg/kg) or vehicle i.p. for 1 week before ischemia. The CsA was supplied by Novartis (Basel, Switzerland). CsA (250 mg) was dissolved in 65% cremaphol EL. And we used only 65% cremaphol EL without CsA as vehicle for every experiment. Prior to ischemia, the animals were fasted overnight but allowed water ad libitum. Anesthesia was induced with 3% iso-
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£urane in N2 O/O2 (70:30). The animals were intubated and arti¢cially ventilated. The tail artery and tail vein were cannulated for blood sampling, blood pressure recording, and drug infusion. A central venous catheter was inserted into the right jugular vein in order to allow withdrawal of blood during ischemia. The common carotid arteries of both sides were isolated and ¢tted with loose ligatures for later clamping. After the operative procedures, the iso£urane concentration was maintained at 0.3^0.5%. The animals were given heparin (30 U/kg) and paralyzed with vecuronium bromide (2 mg/h). Core and head temperature were monitored and kept close to 37³C by lamp heating. Ventilation and oxygen supply were adjusted to give a PaCO2 of 35^40 mm Hg, PaO2 over 100 mm Hg and pH close to 7.40. Animals were subjected to forebrain ischemia as described elsewhere (Smith et al., 1984). In brief, the procedure involves a 10-min duration of ischemia, which gives dense and maximal damage to the CA1 sector of the hippocampus, by bilateral common carotid artery clamping plus hypotension by exsanguination (mean arterial pressure 40^50 mm Hg). Recirculation was achieved by reinfusing the shed blood and by releasing the clamps placed around the carotid arteries. After reperfusion, bicarbonate (0.6 M, 0.5 ml) was given i.v. to counteract systemic acidosis. Iso£urane and the muscle relaxant were discontinued. When the animals regained spontaneous breathing, they were disconnected from the respirator and extubated. The rats were then housed in cages, given free access to water and pellet food, and monitored for behavioral manifestations or seizures. All animal care and experimentation was performed according to the study guidelines established by the Tokyo Metropolitan Institute of Medical Science subcommittee on laboratory animal care, handling, and termination. All e¡orts were made to minimize the number of animals used and their su¡ering. Tissue preparation After reperfusion, brain samples were collected periodically. At 1, 6, 12, 24, and 72 h after reperfusion, the animals were reanesthetized with 3% iso£urane and killed. The brains were removed and immediately frozen in powdered dry ice. Coronal cryostat sections (30 Wm) were taken through the dorsal hippocampus. Extraction of RNA and RT-PCR To obtain hippocampal CA1 and DG tissues, rats were decapitated and CA1 tissues were dissected out at 1, 6, 12 and 24 h after reperfusion (n = 5 for each interval and experimental group). Using Isogen (Nippon Gene, Tokyo, Japan), total RNAs were extracted followed by DNase I treatment. Reverse transcription-polymerase chain reaction (RT-PCR) was performed with a one-step PCR kit (Takara, Tokyo, Japan) following the manufacturer's instructions. In brief, 0.5 Wg of total RNA was used per reaction and the following program was applied. Reverse transcription: 50³C for 30 min; PCR: 94³CU30 s, 60³CU30 s, 72³CU1 min. Cycle numbers were 25 and 20 for rat BDNF and L-actin, respectively. The oligonucleotide primers used are listed below (Bova et al., 1998). They give rise to 342-bp and 520-bp PCR products for BDNF and L-actin, respectively. Ampli¢cation products were electrophoresed on 1% agarose gel, then stained with ethidium bromide and photographed. Quanti¢cation of signals was performed by NIH Image software. BDNF: sense, 5P-CGTGATCGAGGAGCTGTTGG-3P; antisense, 5P-CTGCTTCAGTTGGCCTTTCG-3P; L-actin: sense, 5P-GGCTGTGTTGTCCCTGTAT-3P; antisense, 5P-CCGCTCATTGCCGATAGTG-3P. Immunohistochemistry Sections were ¢xed with 4% paraformaldehyde in phosphate-
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bu¡ered saline (PBS) for 30 min and washed three times with PBS for 5 min. Then, blocking was performed with PBS containing 5% bovine serum albumin for 30 min. Rabbit anti-mouse BDNF polyclonal antibody (Chemicon, Temecula, CA, USA), rabbit anti-mouse TrkB polyclonal antibody (Santa Cruz Biotech, Santa Cruz, CA, USA) and rabbit anti-phospho-CREB polyclonal antibody (New England Biolabs, Beverly, MA, USA) were diluted 1:1000 in PBS containing 5% bovine serum albumin and incubated for hybridization overnight at 4³C. After rinsing, secondary antibodies labeled with Alexa 546 (Molecular Probes, Eugene, OR, USA) were hybridized at room temperature for 1 h. After washing several times, sections were mounted with Vectashield (Vector Laboratories, Burlingame, CA, USA) and subjected to immuno£uorescent detection with a Carl Zeiss LSM510 confocal laser scanning microscope. Terminal deoxynucleotidyl transferase (TdT)-mediated dUTP-biotin nick end labelling (TUNEL) staining was performed with a Mebstain Apoptosis Kit (MBL, Nagoya, Japan) following the manufacturer's instructions. Preparation of whole cell extracts Rats were killed and hippocampal CA1 sectors were dissected out at 1, 6, 12 and 24 h after reperfusion (n = 4 for each interval and experimental group). Brain tissues were homogenized with a
Dounce homogenizer (30 strokes) in 10 times volume of homogenization bu¡er containing 15 mM Tris^HCl, pH 7.6, 1 mM dithiothreitol, 0.25 M sucrose, 1 mM MgCl2 , 1.25 Wg/ml pepstatin A, 10 Wg/ml leupeptin, 2.5 Wg/ml aprotinin, 0.5 mM phenylmethylsulfonyl £uoride, 2.5 mM EDTA, 1 mM EGTA, 0.1 M Na3 VO4 , 50 mM NaF, 2 mM sodium pyrophosphate, and 0.1% Triton X-100. The DC protein assay kit (Bio-Rad Laboratories, Hercules, CA, USA) was used to determine the protein concentration. Western blot analysis Western blot analysis was carried out on 8% sodium dodecyl sulfate-polyacrylamide gel electrophoresis. One sample containing 20 Wg of protein was applied to each lane in a slab gel. Following electrophoresis, proteins were transferred to an Immobilon-P membrane. The membranes were incubated with primary polyclonal antibodies as used in immunohistochemistry at a dilution of 1:1000 overnight at 4³C. The membrane was then incubated with horseradish peroxidase-conjugated anti-rabbit secondary antibody for 1 h at room temperature. The enhanced chemiluminescence detection method (ECL; Amersham Pharmacia Biotech, Uppsala, Sweden) was used for the detection of signals, following the manufacturer's instructions. Four animals in each experimental group
Fig. 1. Histopathological ¢ndings in rat hippocampus following 10 min of forebrain ischemia. CsA(+), CsA-treated (10 mg/ kg) animals; CsA(3), vehicle-administered (ISC) animals. (A) The extent of neuronal damage in the CA1 sector in CsA(+) and CsA(3) at 7 days after reperfusion. Scale bar = 200 Wm. (B) Immuno£uorescent images of TUNEL staining of the CA1 sector after 72 h in CsA(+) and CsA(3). Scale bar = 50 Wm.
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Fig. 2. Expression of BDNF and TrkB in the CA1 sector. CsA(+), CsA-treated (10 mg/kg) animals ; CsA(3), vehicle-administered (ISC) animals. (A) Western blot analysis of BDNF and TrkB in the CA1 sector. Samples were collected from either CsA(+) or CsA(3) groups rats at 1, 6, 12 and 24 h. Samples obtained from sham-operated rats were used as control. (B) Immuno£uorescence images of BDNF (upper row) and TrkB (lower row) in the CA1 sector at 24 h. CsA(+) samples are shown in the right column and CsA(3) samples in the left column. Scale bar = 50 Wm.
were used to analyze the level of the proteins by western blotting.
RESULTS
Histopathological ¢ndings
Statistical analysis Evaluation of di¡erences in BDNF mRNA expression among animal groups was performed by one-way analysis of variance (ANOVA) followed by the Bonferroni/Dunn post-hoc test with signi¢cance set at P 6 0.05.
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Hematoxylin^eosin-stained hippocampal slices of rat brain 1 week after ischemia^reperfusion (ISC group) showed massive neuronal cell death in the CA1 sector, whereas cells were completely intact in CsA-treated rats
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(CSA group) (Fig. 1A). CA1 pyramidal cells undergoing cell death were detectable by TUNEL staining at 72 h after ischemia in the ISC group (Fig. 1B). Western blot analysis and immunohistochemical ¢ndings in CA1 sector To investigate involvement of BDNF and TrkB, we examined the expression of BDNF and TrkB by western
blot. In sham-operated animals, both BDNF and TrkB were detectable (Fig. 2A). In the ISC group, BDNF was gradually down-regulated after the transient increase at 1 h, while no marked £uctuation was found in the CSA group throughout the time course. TrkB was, indeed, upregulated in both groups at the early time points, but as seen in BDNF, the expression level was steadily tapered in the ISC group while it was kept at the increased level in the CSA group (Fig. 2A). Fig. 2B clearly demonstrates
Fig. 3. Expression of pCREB. CsA(+), CsA-treated (10 mg/kg) animals; CsA(3), vehicle-administered (ISC) animals. (A) western blot analysis of pCREB and CREB expression in the CA1 sector. Samples were obtained as described in Fig. 2B. (B) Immuno£uorescent images of pCREB in the CA1 sector at 24 h. Scale bar = 50 Wm.
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Fig. 4. Variation of BDNF mRNA levels in the CA1 sector. Each reported value represents the mean of ¢ve independent experiments þ S.D. Each value is represented as a percentage of the ratio between the densitometric scores for the BDNF and L-actin PCR products based on the ratio of sham-operated animals as 100%. *P 6 0.05, one-way ANOVA followed by Bonferroni/Dunn post-hoc test. CsA(+), CsA-treated (10 mg/kg) animals ; CsA(3), vehicle-administered (ISC) animals.
the enhanced immuno£uorescent signals of BDNF and TrkB in the CSA group at 24 h. The speci¢city of the immunohistochemical reaction for the primary antibodies used was con¢rmed by omitting each primary antibody in the procedure. Non-speci¢c signals were very low (data not shown). Next, we monitored the activation of CREB formation of pCREB by immunostaining and western blot (Fig. 3A, B). Even in sham-operated animal, a distinct level of pCREB was observed. An increased level of pCREB was found in both groups immediately after the reperfusion. However, it was transient and became marginally detectable by 24 h without CsA (Fig. 3A). CsA induced more prominent formation of pCREB and it was maintained throughout the course studied, while the whole body of CREB expression was unchanged. As shown in Fig. 3B, pCREB immunoreactivity is considerably maintained only in the CSA group at 24 h. RT-PCR To examine changes in mRNA expression induced by CsA administration, semi-quantitative RT-PCR was performed for BDNF using total RNAs dissected from the CA1 sector (Fig. 4). However, the expression of BDNF was decreased in the ISC group, whereas it was maintained at a normal level by CsA administration. This result indicates that CsA may play a role in maintaining the expression of BDNF, at least at the mRNA level in the CA1 sector.
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DISCUSSION
The novel ¢ndings in this study were: (a) in the CA1 sector, CsA treatment induced an apparent increase of pCREB, and augmented formation of pCREB was maintained throughout the time course examined; (b) concurrent with the induction of pCREB, the expression of BDNF and TrkB remained up-regulated in the CsAtreated animals, while it was transient in the animals without the CsA treatment; (c) the level of BDNF mRNA in the CA1 sector was maintained at a higher level by CsA treatment. Controlling the transcription of various genes, phosphorylation of CREB is one of the most important mechanisms (Gonzalez et al., 1989; Yanamoto et al., 2000). It has been reported that, in the transient ischemic model, the level of pCREB increased in damage-resistant neurons of the DG but not in the vulnerable CA1 neurons (Hu et al., 1999). The cyclic undecapeptide CsA is an e¡ective immunosuppressant that is a speci¢c blocker of the calcium/calmodulin-sensitive phosphatase calcineurin. It has been reported that CsA dramatically ameliorates the neuronal cell damage a¡ecting vulnerable regions, for example, the CA1 sector of the hippocampus after forebrain ischemia (Li et al., 1997; Uchino et al., 1995, 1998). FK506, another blocker of calcineurin, has been reported to reduce the damage due to focal ischemia caused by transient occlusion of the middle cerebral artery (Sharkey and Butcher, 1994). Although little is known about the
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details of such neuroprotective e¡ects, it seems quite likely that calcineurin is one of the essential factors in the regulation of neuronal cell death after ischemia^ reperfusion injury. Our results in CsA-treated rats indicate that formation of pCREB is enhanced in the CA1 sector throughout the time course examined. Calcineurin has been suggested to play a key role through regulation of protein phosphatase 1 activity resulting in dephosphorylation of pCREB (Bito et al., 1996). Since CsA is a speci¢c blocker of calcineurin, we hypothesized that the inhibition of calcineurin by CsA shifted the phosphorylation balance of CREB to be phosphorylated. Therefore, the induction of pCREB by CsA may well be involved in the concurrent induction of BDNF. Administration of exogenous BDNF prevents neuronal death in the CA1 sector after transient forebrain ischemia (Beck et al., 1994), and induces infarct tolerance after focal ischemia (Yanamoto et al., 2000). In the CA1 sector after transient ischemia, BDNF immunoreactivity is reported to increase at early stages after ischemic insult (Yamasaki et al., 1998). Our data of RT-PCR revealed the maintained expression of BDNF mRNA by CsA treatment in the CA1 sector. It is, therefore, conceivable that the augmented expression of BDNF may be involved in prevention of delayed neuronal cell death. Takeda et al. (1993) described that BDNF mRNA gradually decreased in the CA1 sector, and increased in the DG after transient ischemia. Our results are consistent with this report suggesting regulation of BDNF expression at the transcriptional level. BDNF binds and activates a speci¢c high-a¤nity receptor, TrkB, and a low-a¤nity receptor, p75 (Carter and Lewin, 1997). TrkB is a member of the receptor-type tyrosine kinases whose activation potentially leads to several signaling systems, including the phosphatidylinositol 3-kinase pathway which is linked to the phosphorylation of Akt/protein kinase B (PKB) (Atwal et al., 2000; Eves et al., 1998). As CREB is reported to be one of the regulatory targets for Akt/PKB (Du and Montminy, 1998), induction of BDNF by CsA administration may play a part in enhancement of phosphorylation of CREB. Immunohistochemical studies and western blots of TrkB revealed augmented expression in the CA1 sector after CsA treatment. This may be due not only to the de novo synthesis of TrkB (Ferrer et al., 1998; Merlio et al., 1993), but also to the recruit-
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ment of TrkB to the plasma membrane from intracellular stores (Meyer-Franke et al., 1998). Several roles have been proposed for the p75 receptor, including regulation of apoptosis, ligand discrimination, retrograde transport and signal transduction (Carter and Lewin, 1997). Although we could not detect any apparent change of p75 expression by CsA treatment (data not shown), it is possible that a suppressed death signal through the p75 receptor subunit by enhanced signaling through the TrkB subunit may be involved in the rescue of neuronal cells, as reported in the TrkA/p75 (Yoon et al., 1998). It would be intriguing to pursue the details of this possibility in future investigations. The Bcl-2 family proteins, consisting of pro-apoptotic Bad, Bik, Bid, etc. and anti-apoptotic Bcl-2 and Bcl-xl, are important regulators of mammalian apoptosis (Merry and Korsmeyer, 1997). The promoter region of Bcl-2 contains a CRE site. The transcription factor CREB has been identi¢ed as a positive regulator of Bcl-2 expression (Pugazhenthi et al., 1999; Wilson et al., 1996). Up-regulation of Bcl-2 expression has been identi¢ed as a critical mechanism of which growth factors promote cell survival (Pugazhenthi et al., 1999). In a cerebral ischemia model, it has been reported that Bcl-2 is induced in surviving neurons (Chen et al., 1997). Neuronal overexpression of Bcl-2 in transgenic mice ameliorates hippocampal neuronal damage after transient ischemia (Kitagawa et al., 1998). Recent studies have reported that NGF and BDNF induce Bcl-2 in sympathetic or cortical neurons through the activation of CREB (Riccio et al., 1999). In our preliminary experiments, weak but apparent induction of Bcl-2 by CsA was detected, whereas neither sham-operated nor ischemic^ reperfused animals showed a detectable level of immunoreactivity (data not shown). Further studies are required to elucidate the possible involvement of Bcl-2 in the mechanism of neuroprotection. Taken together, our results indicate that the neuroprotective e¡ect of CsA in this model of forebrain ischemia may involve activation of the CREB-BDNF/TrkB pathway through the suppression of calcineurin activity by CsA. AcknowledgementsöWe thank Dr. Yutaka Hosikawa and Dr. Miki Shitasige for scienti¢c advice. This work was supported by a Grant-in-Aid for Scienti¢c Research, Grant 11671524, from the Ministry of Education, Science and Culture of Japan.
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Cyaan Magenta Geel Zwart