A brief period of hypoxia causes proteolysis of cytoskeletal proteins in hippocampal slices

Brain Research, 555 (1991) 276-280 © 1991 Elsevier Science Publishers B.V. 0006-8993/91/$03.50 ADONIS 0006899391168476

276

BRES 16847

A brief period of hypoxia causes proteolysis of cytoskeletal proteins in hippocampal slices A m y Arai 1, Peter Vanderklish 1, Markus Kessler 1, Kevin Lee 2 and Gary Lynch 1 t Center for the Neurobiology of Learning and Memory, University of California, lrvine, CA 92717 (U. S.A.) and 2Department of Neurological Surgery, University of Virginia, Health Sciences Center, Charlottesville, VA 22908 (U.S.A.)

(Accepted 5 March 1991) Key words: Ischemia; Calpain; Protease, Hippocampus; Calpain inhibitor I; Spectrin

Breakdown products (BDPs) resulting from the partial proteolysis of spectrin were examined in hippocampal slices after periods of hypoxia lasting for 5 or 10 min. The concentration of a -155 kDa BDP increased nearly twofold after 5 min of hypoxia; further increases were not seen with 10 rain episodes or 10 min of hypoxia followed by reoxygenation. The hypoxia-induced proteolysis was blocked by prior infusion of a newly introduced inhibitor of caipain (calpain inhibitor I, 200/aM). Together with previously published data showing improved recovery of hippocampal slices from hypoxia in the presence of calpain inhibitors, these data suggest that activation of calpain may contribute significantly to the pathophysiology of ischemia.

INTRODUCTION Transient ( - 1 0 min) periods of ischemia produced by carotid artery occlusion cause a massive loss of cells in field CA1 of the rodent hippocampus that begins to appear 2-4 days after the event. The sequence of pathogenic events occurring between the ischemic episode and the disappearance of neurons is still largely unknown. Increased excitability, changes in binding to adenosine A1 and muscarinic cholinergic receptors, changes in inositol phosphate and forskolin binding and in protein kinase C activity have all been observed during the first two days after the ischemic episode 14A6'17. However, it is not clear how or to what degree these processes contribute to the ensuing cell degeneration. Considerable evidence suggests that one of the earliest factors in the chain of reactions leading to cell death is an increase in intracellular free calcium 24. Of the various enzyme systems activated by calcium, calcium-activated proteases (calpains) are prominent candidates for mediating deleterious effects. These enzymes have a logical relationship to pathology since they degrade several cytoskeletal proteins thought to be critical to the maintenance of cell shape and intracellular transport. In support of a role for calpains in neuronal degeneration, a stable breakdown product resulting from the cleavage

of spectrin by calpain has been found to increase dramatically in several pathogenic conditions including toxin administration 22, denervation 1°'19, exposure to excitotoxin z°'25 and the brindled mouse mutation 23. In certain of these instances, it was possible to show that spectrin degradation precedes overt signs of cell degeneration, suggesting that calpain activation is a cause rather than a consequence of cell degeneration. There is also evidence that ischemia triggers calpain activation. Concentrations of two cytoskeletal substrates of the protease (spectrin and MAP-2) are significantly reduced within 1 h of ischemia in gerbils s'12'13 while the above noted spectrin breakdown product is increased 5 to 10-fold within 15 min in the same experimental paradigm 21. Recently, we reported that either of two drugs (leupeptin and calpain inhibitor I) that suppress calpain activity, reduce the pathophysiological effects resulting from brief periods of hypoxia in slices of hippocampus 2. This provided indirect evidence, (i) linking one of the central events of ischemia (hypoxia) to calpain activation and (ii) implicating the protease in the development of physiological disturbances. In the present experiments, we tested the idea that short episodes of hypoxia cause a rapid increase in calpain mediated proteolysis. Certain of these results have been reported in a preliminary form ~5.

Correspondence: A. Arai, CNLM, University of California, Irvine, CA 92717, U.S.A.

277 MATERIALS AND METHODS Hippocampal slices (400/~m) were prepared from male SpragueDawley rats (6-7 weeks) using conventional methods 6 and maintained in an interface chamber at 35 °C. The lower surface of the slices was in contact with an artificial cerebrospinal fluid (ACSF), while the upper surface was exposed to a humidified atmosphere of O2/CO~ (95:5). ACSF contained (in raM): 124, NaCI; 3, KC1; 1.2, KH2PO4; 2, MgSO4; 2, CaCI2; 26, NaHCO3; 10, glucose and 100 /~M antipaln. Preparing hippocampal slices itself can increase the background level of spectrin breakdown products (BDPs), presumably because the intracellular compartments of damaged cells are exposed to extracellular calcium. In order to reduce the high background, we used a slightly modified version of a previously described method 2°. Immediately after cutting, the slices were placed in cold medium containing low calcium (1 mM) and high magnesium (3 mM) as well as 1 mM of antipain and 13/~M of B A P T A / A M . Antipain penetrates intact membranes only very poorly; BAFFA/AM is a calcium chelator which becomes active when cleaved by a cytoplasmic esterase. These two conditions were used in an effort to reduce calpain activation in broken cells. Following a minute of exposure to this condition, slices were transferred to the recording chamber with normal ACSF containing 100 jaM antipain. Air flow rate was kept at 1.5 I/rain. After 1 h incubation in normal ACSF, slices were exposed to hypoxia by substituting N 2 for 0 2 in the gas phase for a duration of 5 or 10 min. Samples consisted of 3 or 4 slices collected immediately before, at the end of, and 20 min after termination of hypoxia. Non-hypoxia control slices were collected in separate experiments at the same time points as in nypoxia experiments (i.e. 60, 65, 70 and 90 min after preparation). In a subset of the hypoxia experiments, slices were randomly distributed in a dual well chamber where the medium in one of the wells contained 200/zM calpain inhibitor I and 0.1% DMSO, while the control well contained ACSF with 0.1% DMSO alone. Since DMSO by itself had no effect on the degree of spectrin proteolysis during hypoxia (or on the recovery of electrical responses from hypoxia2), data for hypoxia treatments with or without DMSO were pooled. The concentration of spectrin breakdown products (BDP) in each sample was determined on Western blots as previously described 19. Groups of 4 hippocampal slices were homogenized by sonication in an ice-cold buffer containing 0.32 M sucrose, 10 mM Tris-HCl, 2 mM EDTA, 1 mM EGTA, 100/zM leupeptin and 20/zg/ml TPCK (pH 7.4). An aliquot of the homogenate was added to 1/3volume of 3x concentrated SDS-PAGE sample buffer (150 mM Tris-HC1, 6% SDS, 30% glycerol, 3.75 mM EDTA and 3% fl-mercaptoethanol, pH 6.8). This mixture was heated in a boiling water bath for 5 min. The protein concentration in the homogenate was determined by the method of Bradford 4 and used to adjust the concentration in the boiled samples to 0.33 mg/ml. An aliquot containing 12 ~g of protein was then subjected to SDS-PAGE on a 3-10% gradient gel. Proteins resolved by SDS-PAGE were transferred in a Bio-Rad Transblot cell onto nitrocellulose membranes over a 12-15 h period at 45 V (+ 2 h at 60 V) in a buffer containing 25 mM "Iris, 190 mM glycine, 0.01% SDS and 20% methanol. Immunodetection of spectrin and its proteolytic subspecies utilized an affinity-purified polyclonal antibody to brain spectrin 9. The procedures for blocking of nitrocellulose sheets, primary and secondary antibody incubation, and color development were as recommended by Bio-Rad using an anti-rabbit IgG alkaline phosphatase conjugate with the BCIP/NBT substrate system of detection. The percentage of spectrin immunoreactivity present as 150 kDa and 155 kDa BDP was quantified by reflective scanning laser densitometry. Protein load and development time were such that the amount of spectrin staining was proportional to the amount of protein loaded. Chemicals: calpain inhibitor 1 (Calbiochem) was dissolved in DMSO to prepare a 200 mM stock solution, which was then diluted 1000 times with ACSF for every experiment (final concentration of DMSO was 0.1%). Antipain (Sigma) was dissolved in ACSE

BAPTA/AM (Calbiochem) was dissolved in DMSO to a concentration of 13 mM and diluted 1000 times with ACSF before each experiment.

RESULTS Fig. 1 shows t h e t i m e c o u r s e o f c h a n g e s in B D P f o r m a t i o n e x p r e s s e d as a p e r c e n t a g e o f t o t a l i m m u n o r e activity f o l l o w i n g t h e p r e p a r a t i o n o f slices. B D P f o r m a tion was 0.84 + 0 . 1 4 %

(n

=

11) i m m e d i a t e l y after

cutting and t h e n i n c r e a s e d a l m o s t linearly to 6.0 + 0 . 7 % (n = 37) within 60 min. It is n o t e w o r t h y t h a t t h e levels o f B D P are m u c h g r e a t e r in slices p r e p a r e d by c o n v e n tional t e c h n i q u e s 2°. A f t e r 60 m i n i n c u b a t i o n , t h e conc e n t r a t i o n s of b r e a k d o w n p r o d u c t a p p e a r e d to stabilize and did n o t c h a n g e f u r t h e r with t i m e . T h e r e f o r e , h y p o x i a e p i s o d e s w e r e s t a r t e d at that t i m e point. T h r e e h y p o x i a g r o u p s w e r e tested: 5 m i n (n = 6 e x p e r i m e n t s ) , 10 m i n (n = 22), a n d 10 m i n plus 20 m i n o f r e - o x y g e n a t i o n (n = 16). F o r e a c h e x p e r i m e n t , o n e g r o u p o f slices was r e m o v e d i m m e d i a t e l y b e f o r e t h e hypoxia episode (-60

m i n after cutting) and a s e c o n d

g r o u p was c o l l e c t e d after t h e e p i s o d e . C o m p a r i s o n s w e r e t h e n m a d e for e a c h e x p e r i m e n t o f t h e a m o u n t o f B D P in t h e p o s t - h y p o x i a g r o u p vs. t h e p r e - h y p o x i a g r o u p . In a s e p a r a t e e x p e r i m e n t c o n d u c t e d o n t h e s a m e day, c o n t r o l slices w e r e run for e a c h t i m e p o i n t , i.e. g r o u p s o f slices w i t h o u t h y p o x i a w e r e r e m o v e d at 60 (n = 18), 65 (n = 4), 70 (n = 15), o r 90 (n = 12) m i n after b e i n g p l a c e d in t h e c h a m b e r . T h e a m o u n t o f s p e c t r i n B D P p r e s e n t at t h e l a t t e r t h r e e t i m e p o i n t s for e a c h e x p e r i m e n t was again expressed

as a d i f f e r e n c e f r o m that

c o l l e c t e d at 60 min.

Fig.

found

2 summarizes

in slices

the

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e x p r e s s e d as a c h a n g e in t h e p e r c e n t o f t o t a l i m m u n o -

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0

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10 20 30 40 50 60 70 80 Time (rain)

90

Fig. 1. Time course of spectrin breakdown product (SBDP) formation after preparation of hippocampal slices. The amount of spectrin breakdown product is expressed as a percentage of total spectrin immunoreactivity on Western blots. Each point represents the mean + S.E.M., for the number of experiments indicated in parenthesis.

278

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Fig. 2. Change in spectrin breakdown product (SBDP) levels in hippocampal slices during and after hypoxia. Sixty minutes after preparation, slices were exposed to 5 or 10 min hypoxia or to 10 min hypoxia followed by 20 min reoxygenation. Left side: Western blot showing the aand fl-spectrin doublet around 240 kDa and the predominant spectrin breakdown product (SBDP) at 155 kDa. The two lines of numbers at the bottom of the blot give the time (in min) after cutting the slices and after initiation of hypoxia. Right side: averaged data for hypoxia treated slices (0) and for control slices which were held in the chamber for the equivalent amount of time without hypoxia (O). The duration of the hypoxia episode is indicated by the horizontal bar at the bottom. For each experiment one group of slices was removed at 60 min (immediately before the hypoxia period) and a second group was taken out at one of the later time points. The amount of breakdown product present at 60 rain (expressed as percent of total speetrin immunoreactivity) was then subtracted from that present at the later time point to determine the increase in SBDP for that particular experiment. The figure shows the averaged data, each point representing the mean + S.E.M. obtained from 16--23 experiments. *P < 0.05; ***P < 0.005. reactivity represented by the 150-155 k D a spectrin breakdown products. As can be seen, 5 and 10 min of hypoxia resulted in a conversion of nearly 6% of the total immunoreactivity into the BDP, i.e. spectrin breakdown product increased from 6.0% to 10.7 and 12.1% of total immunoreactivity in comparisons of slices removed immediately before hypoxia vs. those removed after 5 and 10 min of hypoxia, respectively ( P < 0.005, paired t-test). Effects of similar magnitude were obtained after 10 min of hypoxia plus re-oxygenation (Fig. 2). No such increase was observed in the control slices not exposed to hypoxia (i.e. removed at 65 to 90 min vs. 60 min after cutting). The differences in the a m o u n t of B D P between the hypoxia and control groups were significant at P < 0.005 (65 and 70 min) or at P < 0.05 (90 min) according to Student's t-test. The above results indicate that hypoxia causes a rapid and substantial proteolysis of spectrin. To test if this was due to activation of calpain, slices were incubated in the presence of a newly introduced inhibitor of the protease that is both more potent and selective than previously used drugs 18'27. Slices perfused for 1 h with calpain inhibitor ! at 200/zM before the hypoxia episode had lower levels of spectrin B D P (4.5 + 1.1%, n = 21) than those assayed at the same time point in the first study.

10' [] Hypoxia • Hypoxia + Calpain inhibitor I

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10 min Hypoxia

20 min Reoxygenation

Fig. 3. Effect of calpain inhibitor I on hypoxia-induced increase in SBDP levels. Slices were incubated for 1 h with ACSF containing 200/~M calpain inhibitor I plus 0.1% DMSO or 0.1% DMSO alone. Following the same protocol as in Fig. 2, groups of slices were removed from the chamber immediately before the initiation of hypoxia and after either 5 or 10 min hypoxia or after 10 min hypoxia followed by 20 min re-oxygenation. The left and right columns in each group show the pooled data for hypoxia treated slices without and with inhibitor, respectively. Each column represents the mean + S.E.M. of 6 (5 min), 22 (10 min, no inhibitor), 11 (10 min, + inhibitor), 16 (re-oxygenation, no inhibitor) and 4 (re-oxygenation, + inhibitor) experiments. *P < 0.05; ***P < 0.005

279 Hypoxia had little if any effect on spectrin proteolysis in slices treated with calpain inhibitor I. Fig. 3 summarizes the data for drug treated slices given 5 or 10 min of hypoxia or 10 min plus 20 min of re-oxygenation. As can be seen, the increase in spectrin BDP found in the absence of the drug (data from the first experiment are shown) did not occur in the presence of calpain inhibitor I. DISCUSSION The present findings indicate that even relatively brief episodes of hypoxia increase the breakdown of spectrin apparently via the activation of calpain. This effect is comparable in magnitude to that previously observed in the hippocampus of gerbils after transient ischemia 21. An interesting and unexpected result was that the proteolytic response did not increase further after 5 min of hypoxia or with re-oxygenation. This could reflect any of several possibilities acting alone or in combination. (i) The proteolytic response is localized. Calpain activity is much higher in the soluble component of 'synaptosomes' than in other subcellular fractions 3 and this may indicate that only a restricted fraction of spectrin in intact cells is exposed to the protease during hypoxia. (ii) Spectrin breakdown products are rapidly degraded. Biochemical experiments indicate that the 150 kDa spectrin BDP is only very slowly degraded by calpain but the possibility exists that some other protease in situ digests it. Nonetheless, continued breakdown of spectrin should have increased the percent total immunoreactivity values for the BDP even if the absolute concentrations of the latter had reached an equilibrium value. (iii) Autolysis of calpain. An initial autolysis step involves cleavage of a small terminal sequence 7'25'26 and is thought to lead to full activation but in the continued presence of calcium autolysis may produce inactivation. Possibly then, prolonged hypoxia begins to result in reduced levels of active protease, thereby reducing the level of spectrin breakREFERENCES 1 Aitken, EG. and Schiff, S.J., Selective neuronal vulnerability to hypoxia in vitro, Neurosci. Lett., 67 (1986) 92-96. 2 Arai, A., Kessler, M., Lee, K. and Lynch, G., Calpain inhibitors improve the recovery of synaptic transmission from hypoxia in hippocampal slices, Brain Research, 532 (1990) 63-68. 3 Baudry, M., DuBrin, R. and Lynch, G., Subcellular compartmentalization of calcium-dependent and calcium-independent neutral proteases in brain, Synapse, 1 (1987) 506-511. 4 Bradford, M.M., A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principles of protein-dye binding, Anal. Biochem., 72 (1976) 248-254. 5 Del Cerro, S., Larson, J., Oliver, M.W. and Lynch, G., Development of hippocampal long-term potentiation is suppressed by calpain inhibitors, Brain Research, 530 (1990) 91-95. 6 Dunwiddie, T. and Lynch, G., Long-term potentiation and

down. It will be of interest to assay levels of calpain in slices at various intervals after the onset of hypoxia. The finding that calpain inhibitor I blocks hypoxiainduced spectrin cleavage provides evidence that calpain is the responsible protease. It also indicates that drugs of this type will be useful in assessing the effects of calpain-mediated proteolysis on cell functioning. In a previous study, we found that infusion of calpain inhibitor I at the concentrations used in the present experiments substantially increased the degree to which synaptic potentials in field CA1 of slices recovered after hypoxia 2. Those experiments also established that the inhibitor does not affect baseline synaptic potentials (although it does block long-term potentiation; see also ref. 5) or the physiological response to hypoxia. Thus, the suppression of proteolysis, as well as the enhanced recovery of field excitatory postsynaptic potentials, produced by calpain inhibitor I are not likely to be due to a secondary effect on the initial reactions to hypoxia. Finally, the present results represent a first step towards defining the events occurring during hypoxia that trigger calpain. Intra- and extra-cellular recording studies have shown that a marked reduction in the membrane potentials and a loss of fiber volleys usually occurs 3-4 min after the onset of hypoxia; if hypoxia continues for about 2 min beyond this point, then full recovery of synaptic potentials does not occur upon re-oxygenation 2. Possibly then the depolarized cell admits calcium, an effect that would be augmented by intense stimulation of N M D A receptors by glutamate released from depolarized afferents. Comparisons of the physiological TM and proteolytic responses to hypoxia across subfields of hippocampus provide an opportunity for further testing of the mechanisms responsible for calpain activation. Acknowledgements. This research was supported by grants from NIA (AG-00538) to G.L. and grants from NIH to M.K. (NS-21860) and to K.L. (NS-24782). We thank Jackie Porter for secretarial assistance.

depression of synaptic responses in the rat hippocampus: localization and frequency dependency, J. Physiol., 276 (1978) 353-367. 7 Inomata, M., Kasai, Y., Nakamura, M. and Kawashima, S., Activation mechanism of calcium-activated neutral protease, J. Biol. Chem., 263 (1988) 19783-19787. 8 Inuzuka, T., Tamura, A., Sato, S., Kirino, T., Toyoshima, I. and Miyatake, T., Suppressive effect of E-64c on ischemic degradation of cerebral proteins following occlusion of the middle cerebral artery in rats, Brain Research, 526 (1990) 177-179. 9 Ivy, G.O., Seuhert, P., Baudry, M. and Lynch, G., Presence of brain spectrin in dendrites of mammalian brain: technical factors involved in immunocytochemical detection, Synapse, 2 (1988) 329-333. 10 Ivy, G., Seubert, P., Lynch, G. and Baudry, M., Lesions of entorhinal cortex produce a calpain-mediated degradation of brain spectrin in dentate gyrus. II. Anatomical studies, Brain Research, 459 (1988) 233-240.

280 11 Kawasaki, K., Traynelis, S.E and Dingledine, R., Different responses of CA1 and CA3 regions to hypoxia in rat hippocampal slice, J. Neurophysiol., 63 (1990) 385-394. 12 Kitagawa, K., Matsumoto, M., Niinobe, M., Mikoshiba, K., Hata, R., Ueda, H., Handa, N., Fukunaga, R., Isaka, Y., Kimura, K. and Kamada, T., Microtubule-associated protein 2 as a sensitive marker for cerebral ischemic damage - immunohistochemical investigation of dendritic damage, Neuroscience, 31 (1989) 401-411. 13 Kuwaki, T., Satoh, H., Ono, T., Shibayama, F., Yamashita, T. and Nishimura, T., Nilvadipine attenuates ischemic degradation of gerbil brain cytoskeletal proteins, Stroke, 20 (1989) 78-83. 14 Lee, K.S., Tetzlaff, W. and Kreutzberg, G.W., Rapid down regulation of hippocampal adenosine receptors following brief anoxia, Brain Research, 380 (1986) 155-158. 15 Lee, K.S., Frank, S., Vanderklish, P., Arai, A. and Lynch, G., Inhibition of proteolysis protects hippocampal neurons from ischemia, Proc. Natl. Acad. Sci., U.S.A., in press. 16 Onodera, H. and Kogure, K., Mapping second messenger systems in the rat hippocampus after transient forebrain ischemia: in vitro 3H-forskolin and 3H-inositol 1,4,5-triphosphate binding, Brain Research, 487 (1989) 343-349. 17 Onodera, H., Sato, G. and Kogure, K., Quantitative autoradiographic analysis of muscarinic cholinergic and adenosine A1 binding sites after transient forebrain ischemia in the gerbil, Brain Research, 415 (1987) 309-322. 18 Saito, M., Kawaguchi, N., Hashimoto, M., Komada, T., Higuchi, N., Tanaka, T., Nomoto, K. and Murachi, T., Purification and structure of novel cysteine proteinase inhibitors, staccopins P1 and P2, from Staphylococcus tanabeensis, Agric.

Biol. Chem., 51 (1987) 861-868. 19 Seubert, P., Ivy, G., Larson, J., Lee, J., Shahi, K., Baudry, M. and Lynch, G., Lesions of entorhinal cortex produce a calpainmediated degradation of brain spectrin in dentate gyrus. I. Biochemical studies, Brain Research, 459 (1988) 226-232. 20 Seubert, P., Larson, J., Oliver, M., Jung, M.W., Baudry, M. and Lynch, G., Stimulation of NMDA receptors induces proteolysis of spectrin in hippocampus, Brain Research, 460 (1988) 189-194. 21 Seubert, P., Lee, K. and Lynch, G., Ischemia triggers NMDA receptor-linked cytoskeletal proteolysis in hippocampus, Brain Research, 492 (1989) 366-370. 22 Seubert, P., Nakagawa, Y., Ivy, G., Vanderklish, P., Baudry, M. and Lynch, G., Intrahippocampal colchicine injection results in spectrin proteolysis, Neuroscience, 31 (1989) 195-202. 23 Seubert, P., Peterson, C., Vanderklish, P., Cotman, C. and Lynch, G., Increased spectrin proteolysis in brindled mouse brain, Neurosci. Lett., 108 (1990) 303-308. 24 Siesj6, B.K., Bengtsson, E, Grampp, W. and Theander, S., Calcium, excitotoxins, and neuronal death in the brain, Ann. N.Y. Acad. Sci., 568 (1989) 234-251. 25 Siman, R. and Noszek, J.C., Excitatory amino acids activate caipain I and induce structural protein breakdown in vivo, Neuron, 1 (1988) 279-287. 26 Suzuki, K., Imajoh, S., Emori, Y., Kawasaki, H., Minami, Y. and Ohno, S., Calcium-activated neutral protease and its endogenous inhibitor: activation at the cell membrane and biological function, FEBS Lett., 220 (1987) 271-277. 27 Wang, K.K.W., Developing selective inhibitors of calpain, Trends Pharmacol. Sci., 11 (1990) 139-142.