Calpain inhibitor entrapped in liposome rescues ischemic neuronal damage

Brain Research 819 Ž1999. 8–14

Research report

Calpain inhibitor entrapped in liposome rescues ischemic neuronal damage Masayuki Yokota a

a, )

, Eiichi Tani a , Satoshi Tsubuki d , Ikuya Yamaura a , Ikuko Nakagaki b , Seiki Hori b , Takaomi C. Saido c

Department of Neurosurgery, Hyogo College of Medicine, Mukogawacho 1-1, Nishinomiya, Hyogo 663, Japan Department of Physiology, Hyogo College of Medicine, Mukogawacho 1-1, Nishinomiya, Hyogo 663, Japan c Laboratory for Proteolytic Neuroscience, Riken Brain Science Institute, Japan d Department of Molecular Biology, Tokyo Metropolitan Institute of Medical Science, Japan

b

Accepted 8 December 1998

Abstract Transient forebrain ischemia induces activation of calpain and proteolysis of a neuronal cytoskeleton, fodrin, in gerbil hippocampus. This phenomenon precedes delayed neuronal death in hippocampal CA1 neurons. We examined effects of a calpain inhibitor on delayed neuronal death after transient forebrain ischemia. In gerbils, a selective calpain inhibitor entrapped in liposome was given transvenously and 30 min later, 5-min forebrain ischemia was produced by occlusion of both common carotid arteries. On day 7, CA1 neuronal damage was examined in the hippocampal slices stained with cresyl violet. Calpain-induced proteolysis of fodrin was also examined by immunohistochemistry and immunoblot. Additionally, to assure entrapment of the inhibitor by CA1 neurons, the inhibitor-liposome complex was labeled with FITC and given to gerbils. Fluorescence in the hippocampal slices was examined by confocal laser scanning microscope. Selective CA1 neuronal damage induced by forebrain ischemia was prevented by administration of the inhibitor in a dose-dependent manner. Calpain-induced proteolysis of fodrin was also extinguished by the calpain inhibitor in a dose-dependent manner. Bright fluorescence of the FITC-labeled inhibitor was observed in the CA1 neurons. The data show an important role of calpain in the development of the ischemic delayed neuronal death. Calpain seems to produce neuronal damage by degrading neuronal cytoskeleton. Our data also show a palliative effect of the calpain inhibitor on the neurotoxic damage, which offers a new and potent treatment of transient forebrain cerebral ischemia. q 1999 Elsevier Science B.V. All rights reserved. Keywords: Calpain; Transient forebrain cerebral ischemia; Calpain inhibitor; Liposome; Hippocampus

1. Introduction Transient global forebrain ischemia in rodents induces selective degeneration affecting hippocampal CA1 and CA4 neurons with relative sparing of CA3 neurons and dentate granule cells. Although the degeneration of CA4 neurons begins within hours after the ischemic insult, CA1 neurons remain intact for up to 2 days and then degenerate Ždelayed neuronal death. w10x. This neuronal damage in postischemic brain has been implied to involve activation of calpain, calcium-dependent cysteine protease w3,8, 15,17,19,31x. Calpain proteolyzes the neuronal cytoskeleton, fodrin. We developed the polyclonal antibody against the proteolyzed form of fodrin. The immunohistochemical analysis using this antibody can visualize the

) Corresponding author. [email protected]

Fax:

q 81-798-456457;

E-m ail:

activation of calpain. In the previous study, we demonstrated that global forebrain ischemia induces three phases of calpain-induced proteolysis of fodrin in gerbil hippocampus w22x. Degree of the proteolysis closely associated with the duration of ischemic insult. On the basis of these results, we postulated that inhibition of calpain in ischemic brain may prevent the ischemic neuronal damage. In preliminary study, we administrated a calpain inhibitor to gerbils to rescue ischemic neuronal damage but got a little beneficial effect. Brain has a strict barrier for substances to pass through the brain capillary, which is named blood–brain barrier ŽBBB.. Long-term ischemia disrupts the barrier but less than 5-min forebrain ischemia does not affect it until 4 days after the ischemic insult w12x. This barrier may have prevented transportation of the calpain inhibitor into neurons. By entrapping in liposome, drugs may pass BBB w9x. Therefore, we designed liposomeentrapped calpain inhibitor to be able to deliver it through BBB.

0006-8993r99r$ - see front matter q 1999 Elsevier Science B.V. All rights reserved. PII: S 0 0 0 6 - 8 9 9 3 Ž 9 8 . 0 1 3 3 4 - 1

M. Yokota et al.r Brain Research 819 (1999) 8–14

2. Materials and methods 2.1. Preparation of calpain inhibitor N-acetyl-leucyl-leucyl-norleucine amide ŽALLNal. is a selective and potent inhibitor of calpain w29x. It was entrapped in liposome as follows. Powder of ALLNal Ž3.2–9.6 mg. was dissolved in 1.7 ml of redistilled chloroformrmethanol Ž3:1; vrv. solution containing L-a-phosphatidylcholine Ž5.9 mgrml. from bovine brain, cholesterol Ž1.19 mgrml., and sulfatides Ž0.89 mgrml. from bovine brain. The solution in a glass tube was dried under nitrogen gas and then under near vacuum for 1 h. The resultant lipid film was suspended in 0.8 ml of phosphatebuffered saline by vigorous vortexing followed by sonication ŽBrabnson bath type Sonifier 185, power 7 for 2 min.. A total of 250 ml of the suspension containing 1–3 mg of the inhibitor was used per gerbil. To examine the effect of the vehicle, 250 ml of the vehicle without ALLNal was administrated to another group of gerbils. 2.2. EÕaluation of the specificity of N-acetyl-leucyl-leucylnorleucine amide A typical peptide aldehyde calpain inhibitor like ALLNal also inhibits the trypsin-like activity of proteasome. Therefore, we measured the inhibition of purified 20S proteasome and m-calpain by ALLNal in vitro to evaluate the specificity of the inhibitor. For the m-calpain inhibitory assay, the 0.5 ml reaction mixture contained 0.1 molrl Tris–HCl ŽpH 7.5., 6 mmolrl CaCl 2 , 28 mmolrl 2-mercaptoethanol, 0.94 unit of m-calpain, ALLNal, 0.24% alkali-denatured casein. The reaction was started by the addition of m-calpain solution and stopped by the addition of 0.5 ml of 10% trichloroacetic acid after incubation at 308C for 15 min. After centrifugation at 1300 = g for 10 min, the absorbency of the supernatant at 280 nm was measured. For the 20S proteasome inhibitory assay, the 1 ml reaction mixture contained 0.1 molrl Tris–acetate, pH 7.0, 2.65 mgrml 20S proteasome, ALLNal, and 25 mmolrl each substrate dissolved in dimethyl sulfoxide. The substrates were succinyl-leucyl-leucyl-valyl-tyrosine-4-methylcoumaryl-7-amide ŽSucLLVY-MCA. for measuring the chymotrypsin-like activity of proteasome, tertiary-butyloxycarbonyl-valyl-leucyl-lysine-4-methylcoumaryl-7-amide ŽBoc-VLK-MCA. for measuring the trypsin-like activity of proteasome, and benzyloxycarbonyl-leucyl-leucyl-glutamic acid-b-naphthylamide ŽZLLE-NA. for measuring the peptidylglutamyl-peptide hydrolyzing activities of proteasome. After incubation at 378C for 15 min, the reaction was stopped by addition of 0.1 ml of 10% SDS and 0.9 ml of 0.1 molrl Tris–acetate, pH 9.0. The fluorescence of the reaction products was measured. To determine the IC 50 against m-calpain and 20S proteasome, various concentrations of ALLNal were included in the assay mixture. While IC 50 of ALLNal against the alkalin-denatured ca-

9

sein-degrading activity of m-calpain is 0.22 mmolrl, IC 50 of ALLNal against 20S proteasome are 17, 38, 54 mmolrl Žeach substrate is SucLLVY-MCA, BocVLK-MCA, and ZLLE-NA.. Thus, ALLNal is around 100 times more effective against m-calpain than against proteasome. 2.3. Animal model Using forebrain ischemia model in gerbil, we documented the effects of the calpain inhibitor on ischemic neuronal damage. In 10 animals, sham operation was performed with only exposure of bilateral CCA Žnonischemic group.. Sixty animals were divided into three groups and loaded ischemic insult; Group 1: 12 animals not treated with the vehicle or ALLNal–liposome complex Žnon-treated group. Group 2: 12 animals treated with only the vehicle of ALLNal–liposome complex Žvehicle group.; Group 3: each 12 animals treated with 20 mgrkg, 40 mgrkg or 60 mgrkg of ALLNal–liposome complex Žtreated group.. The animals of Groups 2 and 3 were injected with the vehicle or ALLNal–liposome complex from right femoral vein 30 min before the ischemia. Forebrain ischemia was produced as previously reported w31x. Briefly, after anesthesia using diethyl ether, bilateral common carotid arteries were exposed under the microscope. Then anesthesia was discontinued and bilateral common carotid arteries were occluded for 5 min by aneurysmal clips ŽSugita temporary mini clip.. Body temperature in temporalis muscle and rectum were monitored and maintained at 36–378C with a homeothermic heating blanket ŽCMA, Stockholm, Sweden. and warming lamp during the surgical procedure and ischemia. Four hours Žeach two animals of Group 1–3. and 7 days Žall 10 animals of nonischemic group and each 10 animals of Group 1–3. after recirculation, the animals were anesthetized with an overdose of sodium pentobarbital and then perfused with 20 mmolrl Tris–HCl buffer ŽpH 7.6. containing 5 mmolrl EDTA, 5 mmolrl b-mercaptoethanol, 250 mmolrl sucrose, and 0.1 molrl leupeptin followed by 2% paraformaldehyde in 0.1 molrl phosphate buffer ŽpH 7.4.. The brain was removed and coronally sectioned alternately at 5 mm and 20 mm intervals on a cryostat. ŽIn animals sacrificed 4 h after ischemia, only 20-mm thick sections were made and served for immunohistochemical study.. The 5-mm thick sections were stained with cresyl violet to assess tissue architecture and count the damaged neurons in CA1 sector, according to the method of Kirino et al. w11x. The 20-mm thick sections were processed for immunohistochemistry of calpain-proteolyzed 150-kD form of fodrin. Immunohistochemical study was performed as described previously. Briefly, each 20-mm thick section was preincubated with 3% normal goat serum and 0.3% hydrogen peroxide in PBS for 30 min at room temperature. Then the sections were incubated with the antibody to the proteolyzed form of fodrin at room temperature for one night. After being rinsed, sections were incubated with

10

M. Yokota et al.r Brain Research 819 (1999) 8–14

biotinylated goat anti-rabbit IgG for 2 h and then with ABC peroxidase complex for 2 h at room temperature. After rinsing of the sections, immunolabel was visualized with 0.015% diaminobenzidine tetrahydrochloride and 0.003% hydrogen peroxide in 50 mmolrl Tris–HCl buffer ŽpH 7.6.. Sections were counterstained with methylgreen solution. To assure the immunohistochemical study, immunoblot analysis for calpain-proteolyzed 150-kD form of fodrin was performed. Six animals loaded with a 5-min ischemia and another six animals treated with 60 mgrkg of ALLNal–liposome complex before a 5-min ischemia were perfused with Tris–HCl buffer ŽpH 7.6. containing 5 mmolrl EDTA, 5 mmolrl b-mercaptoethanol, 250 mmolrl sucrose, and 0.1 mmolrl leupeptin at intervals of 4 h and 7 days Žthree animals at each interval.. Furthermore, six sham-operated animals were perfused in the same fashion 7 days after operation. The hippocampus was dissected under microscope and immersed in liquid nitrogen, and remained frozen at y858C until further processing. Western blotting of the hippocampus was performed by use the antibody against calpain-proteolyzed 150-kD form of fodrin as described in the previous study w31x. Each immunolabeled band was scanned with a HP Scan Jet llcx scanner

interfaced to a Macintosh Performa 6210 16MB RAMHD500r4= CDrMonitor and the scans were imported into NIH Image Žversion 1.61. for quantisation. To demonstrate that ALLNal entrapped in liposome can pass through the BBB, ALLNal was labeled by fluorescein isothiocyanate ŽFITC. and entrapped with liposome as described below. A fluorescence-labeled peptidase inhibitor analogue, FITC-LLNamide Žfluorescein isothiocyanate-conjugated leucyl-leucyl-norleucine amide., was produced as previously described for producing FITClabeled calpastatin peptide w6x. First, a tripeptide, LLNamide, was synthesized on a rink resin using an ACT396 peptide synthesizer ŽAdvanced Chemtech. as described w21x. After deprotection of the Fmoc group, the resin carrying 60 peptide was incubated with 360 mmol FITC ŽSigma. in 1 ml of dimethylformaminde at room temperature overnight. The resin was then extensively washed with dimethylformaminde, methanol, and diethyl ether on a glass filter and dried under near vacuum. The product was subjected to cleavage and purification as described w6x. The FITC-labeled ALLNal–liposome complex was injected intravenously in each three animals 30 min before the ischemia or sham operation. As a control group, FITC-labeled ALLNal without liposome was injected

Fig. 1. High power photomicrograph of CA1 sector 7 days after ischemia or sham operation Župper-left.. Five-minute ischemia induced degeneration of almost all CA1 neurons Župper right.. The calpain inhibitor, ALLNal, rescued CA1 neurons from degeneration in a dose-dependent manner Žlower-left, middle, right.. Cresyl violet staining. Bar s 100 mm.

M. Yokota et al.r Brain Research 819 (1999) 8–14 Table 1 Neuronal cell density of the CA1 sector 7 days after ischemia Dose of ALLNal

Neuronal density

0 mgrkg Žnon-treated. Vehicle 20 mgrkg 40 mgrkg 60 mgrkg Control Žnon-ischemic.

37.2"19.8 42.2"37.1 71.8"35.9) 94.0"36.6)) 114.8"48.4)) 212.2"17.8

The number of neurons in the linear length Ž1 mm. of the CA1 pyramidal layer was counted in dorsal hippocampus of each specimen. The data of treated groups are compared with non-treated group and analyzed by Wilcoxon’s rank sum test. The drug effect is statistically significant Ž) P - 0.05, )) P - 0.01.. The values are expressed as mean "S.D. ns10 for all measurements.

intravenously in three animals 30 min before the ischemia. The animals were euthanized with an overdose of sodium pentobarbital Ž60 mgrkg, i.p.. and then perfused with PBS 30 min after the ischemic insult or sham operation. The

11

brain was immediately removed and frozen by immersion in liquid nitrogen. The brain was coronally sectioned in 20 mm thickness on a cryostat. Under room temperature, fluorescence of the sections was observed by confocal laser scanning microscope ŽLeica, Heidelberg, Germany.. The intensity of fluorescence in CA1 neurons was measured by the same method as the immunoblot experiment and the data were compared between two groups. The numbers mentioned above indicate the numbers of animals with no epileptic movement during the experiment. Four animals with epileptic movement were excluded from the experiments. Two non-treated, ischemic animals and two animals treated with vehicle showed epileptic movement but none of the animals treated with the inhibitor did. It may reflect that seizure induces calpain activation in rat brain w2x. Additionally, three animals were dead during the experiments and they were also exclude from the experiments. One of non-treated, ischemic animals was dead during the ischemia likely because of respiratory distress caused by injury of vagal nerve. Immediately after injection of the inhibitor Ž60 mgrkg., two

Fig. 2. Immunohistochemical distribution of calpain-proteolyzed fodrin in hippocampus. In sham-operated animal, the immunoreactivity was not observed ŽA.. Four hours after ischemia, heavy immunoreactivity was observed in stratum lacunosum moleculare, stratum oriens of CA3 and the stratum oriens of CA1 ŽB.. Seven days after ischemia, the immunoreactivity was observed in whole CA1 sector, especially in pyramidal neurons ŽC.. The calpain inhibitor Ž60 mgrkg. attenuated the immunoreactivity in the hippocampus both 4 h ŽD. and 7 days ŽE. after the ischemia. Bar s 500 mm.

M. Yokota et al.r Brain Research 819 (1999) 8–14

12

Table 2 Density of immunolabeled band on Western blotting Sham-operated

5.5"4.6

Ischemia

IschemiaqALLNal

4h

7 days

4h

7 days

100

22.5"11.9

31.4"14.0))

13.5"6.2)

Data are given as percentile of value for 4 h of ischemia and expressed as mean"S.D. The data of treated groups are compared with each nontreated group and analyzed by the two-tailed Student’s t-test for paired variates Ž) P - 0.05, )) P - 0.01.. ns6 for all measurements.

animals were dead. It may reflect the adverse effect of the inhibitor because the vehicle and low dose of the inhibitor did not cause death of the animals. 2.4. Statistical methods Statistical analysis to examine the effect of calpain inhibitor was done by comparing the neuronal density of hippocampus in each group. Statistical significance was determined by Wilcoxon’s rank sum test. Statistical analysis for immunoblot study between untreated and treated group was done by using the two-tailed Student’s t-test for paired variates. The animals were carefully handled in a humane fashion and our experimental protocols met the U.S. Public Health Service standards described in the 1985 ‘Guide for the Care and Use of Laboratory Animals’ of the National Institutes of Health.

3. Results Consistent neuronal degeneration in CA1 pyramidal neurons of hippocampus was observed in non-treated group ŽFig. 1, upper right. and vehicle group but not at all in non-ischemic group ŽFig. 1, upper left. 7 days after the ischemic insult. In treated group, the CA1 neuronal degeneration was significantly decreased depending on the dose of ALLNal–liposome complex ŽTable 1, Fig. 1, lower-left, -middle and -right.. In immmunohistochemical study, non-ischemic group ŽFig. 2A. showed no or very little proteolysis. On the other hand, non-treated ischemic group ŽFig. 2B. and vehicle

group showed obvious calpain-induced proteolysis of fodrin in the stratum lacunosum moleculare, stratum oriens of CA3 sector and the stratum oriens of CA1 sector 4 h after the ischemia. Then, 7 days later, the proteolysis was observed in entire CA1 sector ŽFig. 2C. as described previously w31x. The heavy immunoreactivity was observed in the soma and dendrites of the CA1 neurons associated with moderate immunoreactivity in neuropils of stratum oriens and stratum radiatum. The heavy, varicose immunoreactivity was observed in the stratum lacunosum moleculare of CA1 sector. CA3 sector and dentate gyrus showed little or no immunoreactivity. We could not find any significant difference between non treated group and vehicle group. In treated group, the immunoreactivity was generally weak in whole hippocampus both 4 h ŽFig. 2D. and 7 days ŽFig. 2E. after the ischemia. The intensity of the immunoreactivity was inversely proportional to the dose of ALLNal–liposome complex. Immunoblot analysis confirmed the result of immunohistochemical study ŽTable 2.. Sham-operated animals showed no or very faint immunolabeled band of proteolyzed fodrin Ž150 kD. ŽFig. 3, lane 1.. Non-treated ischemic animals showed dense immunolabeled band 4 h after ischemia ŽFig. 3, lane 2. and moderately dense immunolabeled band 7 days later ŽFig. 3, lane 3., while treated animals showed significantly weak density ŽFig. 3, lane 4; 4 h and 5, 7 days after ischemia.. After administration of FITC-labeled ALLNal–liposome complex, both sham-operated and ischemic groups showed obvious fluorescence in the cytoplasm of the hippocampal neurons in CA1 sector Žintensity of fluorescence was 63.9 " 35.8, 74.6 " 20.0; mean " S.D., respectively, no significant difference. ŽFig. 4B and C.. These findings demonstrated that ALLNal passed through the BBB and was entrapped in the neuronal cytoplasm. It was difficult to observe fluorescence in fine structures such as dendrites and axons because of no fixation of the slices. Intensity of fluorescence showed a small trend to increase in the hippocampal neurons of the ischemic group rather than the sham-operated group. In the animals injected with FITC-labeled ALLNal without liposome, weak fluorescence was observed in CA1 neurons Žintensity of fluorescence was 5.8 " 4.2; mean " S.D.. ŽFig. 4A.. No signifi-

Fig. 3. Photograph of Western blot for fodrin proteolysis in the ischemic hippocampus with or without the calpain inhibitor. The blots are stained with the antibody against the proteolyzed fodrin Ž150 kD.. Lanes are as follows: lane 1, sham-operated animals; lane 2, 4 h and lane 3, 7 days after the ischemia; lane 4, 4 h and lane 5, 7 days after the ischemia treated with the calpain inhibitor entrapped in liposome.

M. Yokota et al.r Brain Research 819 (1999) 8–14

Fig. 4. Photomicrograph of CA1 sector after administration of the FITClabeled calpain inhibitor with or without liposome by confocal laser scanning microscope. The drug was injected through femoral vein. Fluorescence in CA1 sector was detected by confocal laser scanning microscope. CA1 neurons showed weak fluorescence in without-liposome group ŽA. 30 min after ischemia but bright fluorescence in with-liposome group 30 min after sham operation ŽB. or ischemia ŽC..

cant difference of the intensity was observed among animals in each group.

4. Discussion Up to date, the mechanism of the delayed neuronal death after global forebrain ischemia has been partly clarified w5,24x. As the first step, ischemic insult induces release of excessive amount of excitatory amino acid from pre-synaptic vesicles and post-synaptic neurons, which leads activation of glutamate receptors w27x. Glutamate receptors are categorized into two groups, ionotropic and metabotropic w14x. Activation of the former group induces influx of extracellular calcium ion into the neurons w16x. Activation of the latter group produces inositol 1,4,5-triphosphate ŽIP3. which releases Ca2q from intracellular endoplasmic reticulum w1x. Increase of intracellular Ca2q causes many signal transduction such as activation of protein kinase C, calpain, phospholipase A, xanthine oxi-

13

dase and nitric oxide synthetase w7,25,28x. However, the final and definitive pathway to the neuronal death has not been elucidated. In the previous study, we demonstrated that calpain is activated in the ischemic brain and in the present study, demonstrated that the selective calpain inhibitor can prevent ischemic neuronal damage in a dosedependent manner. The evaluation of the specificity of ALLNal demonstrated that ALLNal inhibits calpain activation potently and selectively. Therefore, activation of the protease must play an important role in development of the ischemic neuronal damage, at least triggering a key step to the catastrophe. Although calpain activation seems nearly complete as reflected by immunoblot study, the protection of hippocampal neurons is not complete but partial. This may suggest that other mechanisms concomitantly or independently play a crucial role in the development of neuronal damage, such as oxidative stress w5x. Caspase may also explain the reason why calpain inhibitor cannot completely block the ischemic neuronal damage. In the recent study, caspase-3 may mediate neuronal damage in the hippocampus after transient forebrain cerebral ischemia w4x. Although we did not examine the effect of the inhibitor on caspase-3, Stefanelli et al. w26x reported that ALLNal increases spontaneous caspase activity. Therefore, in our experiment, treatment with ALLNal may increase caspase activity, which could result in the adverse effect. The present study also suggests how calpain induces neuronal degeneration. Fodrin, a main subplasmalemmal cytoskeleton of neuron, is degraded by calpain after ischemic insult as other studies showed w3,13x. This phenomenon precedes the neuronal death w31x. Fodrin is a family of spectrin which regulates cell morphology by sustaining the integrity of the membrane w30x. Therefore, degraded fodrin could not sustain the neuronal framework after ischemia. As another possibility, further degraded 20-kD fragment of fodrin by other protease such as caspase might have neurotoxic effect Žunpublished data.. Calpain proteolyzes a variety of substrates, not only cytoskeletal proteins but also growth factors, adhesion molecules and kinases which are enriched in neurons w23x. However, we could not yet find other pivotal substrates which are degraded in ischemic insult. For example, proteolysis of integrin by calpain was observed in normal hippocampus and not altered after the ischemic insult Ždata not shown.. While calpain-induced proteolysis of protein kinase C, the other important substrate, is undergoing an experimental checkup, the degradation of fodrin in neuron may be the main cause of the neuronal death. Our data suggest that the calpain inhibitor-liposome complex might be a potent drug for transient forebrain cerebral ischemia. The lack of a significant beneficial effect of ALLNal alone despite its small molecular weight Žm.w. 383. and lipid-soluble character might be related to two factors. First, its molecular weight is near the upper limit Žm.w. 400–600. to penetrate BBB. Second, by conjugating ALLNal with liposome, ALLNal can maintain

14

M. Yokota et al.r Brain Research 819 (1999) 8–14

higher concentration in brain than ALLNal alone w18x. By conjugating with liposome, the calpain inhibitor can be administered via venous route, instead of complicated transventricular or transcisternal route w13,20x. This means that clinical application of the inhibitor could become possible.

w13x

w14x

w15x

Acknowledgements These studies were supported by research grant from the Ministry of Education, Science and Culture of Japan ŽM.Y. and T.C.S... We are grateful to Dr. S. Sasaki for valuable advice.

w16x

w17x

w18x

References w1x M.J. Berridge, Inositol triphosphate and calcium signaling, Nature 361 Ž1993. 315–325. w2x X. Bi, V. Chang, R. Siman, G. Tocco, M. Baudry, Regional distribution and time-course of calpain activation following kainateinduced seizure activity in adult rat brain, Brain Res. 726 Ž1996. 98–108. w3x K. Blomgren, M. Amanda, E. Bona, T.C. Saido, J.O. Karlsson, H. Hagberg, Degradation of fodrin and MAP2 after neonatal cerebral hypoxic-ischemia, Brain Res. 684 Ž1995. 136–142. w4x J. Chen, T. Nagayama, K. Jin, R.A. Stetler, R.L. Zhu, S.T. Graham, R.P. Simon, Induction of caspase-3-like protease may mediate delayed neuronal death in the hippocampus after transient cerebral ischemia, J. Neurosci. 18 Ž1998. 4914–4928. w5x J.T. Coyle, P. Puttfarcken, Oxidative stress, glutamate, and neurodegenerative disorders, Science 262 Ž1993. 670–689. w6x A. Eto, Y. Akita, T.C. Saido, K. Suzuki, S. Kawashima, The role of the calpain–calpastatin system in thyrotropin-releasing hormone-induced selective down-regulation of a protein kinase C isozymes, nPKC ´ , in rat pituitary GH 4 C 1 cells, J. Biol. Chem. 270 Ž1995. 25115–25120. w7x A. Ghosh, M.E. Greenberg, Calcium signaling in neurons: molecular mechanisms and cellular consequences, Science 268 Ž1995. 239–247. w8x S.C. Hong, Y. Goto, G. Lanzino, S. Soleau, N.F. Kassel, K.S. Lee, Neuroprotection with a calpain inhibitor in a model of focal transient forebrain cerebral ischemia, Stroke 25 Ž1994. 663–669. w9x S. Imaizumi, V. Woolworth, R.A. Fishman, P.H. Chan, Liposomeentrapped superoxide dismutase reduces cerebral infarction in transient forebrain cerebral ischemia in rats, Stroke 21 Ž1990. 1312– 1317. w10x T. Kirino, Delayed neuronal death in the gerbil hippocampus following ischemia, Brain Res. 239 Ž1982. 57–69. w11x T. Kirino, A. Tamura, K. Sano, A reversible type of neuronal injury following ischemia in the gerbil hippocampus, Stroke 17 Ž1986. 455–459. w12x K. Kitagawa, M. Matsumoto, T. Ohtsuki, M. Tagaya, T. Okabe, R. Hata, H. Ueda, N. Handa, K. Sobue, T. Kamada, The characteristics of blood–brain barrier in three different conditions—infarction, selective neuronal death and selective loss of presynaptic terminals

w19x

w20x

w21x

w22x

w23x

w24x w25x w26x

w27x

w28x w29x w30x

w31x

—following transient forebrain cerebral ischemia, Acta Neuropathol. 84 Ž1992. 378–386. K.S. Lee, S. Frank, P. Vanderklish, A. Arai, G. Lynch, Inhibition of proteolysis protects hippocampal neurons from ischemia, Proc. Natl. Acad. Sci. USA 88 Ž1991. 7233–7237. S.A. Lipton, P.A. Rosenberg, Excitatory amino acids as a final common pathway for neurogenic disorders, N. Engl. J. Med. 330 Ž1994. 613–622. C.G. Markgraf, N.L. Velayo, M.P. Johnson, B.S. McCarty, S. Medhi, J.R. Koehl, P.A. Chmielewski, M.D. Linnik, Six-hour window of opportunity for calpain inhibition in focal ischemia in rats, Stroke 29 Ž1998. 152–158. A. Mitani, H. Yanase, K. Sakai, Y. Wake, K. Kataoka, Origin of intracellular Ca2q elevation induced by in vitro ischemia-like condition in hippocampal slices, Brain Res. 601 Ž1993. 103–110. R.W. Neumar, S.M. Hagle, D.J. DeGracia, G.S. Krause, B.C. White, Brain m-calpain autolysis during global transient forebrain cerebral ischemia, J. Neurochem. 66 Ž1996. 421–424. W.M. Pardridge, Drug delivery to the brain, J. Cereb. Blood Flow Metab. 17 Ž1997. 713–731. L.C. Pettigrew, M.L. Holtz, S.D. Craddock, S.L. Minger, N. Hall, J.W. Geddes, Microtubular proteolysis in focal transient forebrain cerebral ischemia, J. Cereb. Blood Flow Metab. 16 Ž1996. 1189– 1202. A. Rami, J. Krieglstein, Protective effects of calpain inhibitors against neuronal damage caused by cytotoxic hypoxia in vitro and ischemia in vivo, Brain Res. 609 Ž1993. 67–70. T.C. Saido, S. Nagao, M. Shiramine, M. Tsukaguchi, H. Sorimachi, H. Murofushi, T. Tsuchiya, H. Ito, K. Suzuki, Autolytic transition of m-calpain upon activation as resolved by antibodies distinguishing between the pre- and post-autolysis forms, J. Biochem. 111 Ž1992. 81–86. T.C. Saido, M. Yokota, S. Nagao, I. Yamaura, E. Tani, T. Tsuchiya, K. Suzuki, S. Kawashima, Spatial resolution of fodrin proteolysis in postischemic brain, J. Biol. Chem. 268 Ž1993. 25239–25243. T.C. Saido, H. Sorimachi, K. Suzuki, Calpain: new perspectives in molecular diversity and physiological–pathological involvement, FASEB J. 8 Ž1994. 814–822. R. Schmidt-Kastner, T.F. Freund, Selective vulnerability of the hippocampus in brain ischemia, Neuroscience 40 Ž1991. 599–636. P.B. Simpson, R.A.J. Challiss, S.R. Nahorski, Neuronal Ca2q stores: activation and function, Trends Neurosci. 18 Ž1995. 299–306. C. Stefanelli, F. Bonavita, I. Stanic, C. Pignatti, G. Farruggia, L. Masotti, C. Guarnieri, C.M. Caldarera, Inhibition of etoposide-induced apoptosis with peptide aldehyde inhibitors of proteasome, Biochem. J. 332 Ž1998. 661–665. M. Szatkowski, D. Attwell, Triggering and execution of neuronal death in brain ischemia: two phases of glutamate release by different mechanisms, Trends Neurosci. 17 Ž1994. 359–365. B.F. Trump, I.K. Berezesky, Calcium-mediated cell injury and cell death, FASEB J. 9 Ž1995. 219–228. K.K.W. Wang, Developing selective inhibitors of calpain, Trends Pharmacol. Sci. 11 Ž1990. 139–142. Y. Yan, E. Winograd, A. Viel, T. Cronin, S.C. Harrison, D. Branton, Crystal structures of the repetitive segment of spectrin, Science 262 Ž1993. 2027–2030. M. Yokota, T.C. Saido, E. Tani, S. Kawashima, K. Suzuki, Three distinct phases of fodrin proteolysis induced in postischemic hippocampus; involvement of calpain and unidentified protease, Stroke 26 Ž1995. 1901–1907.