Calpain I activation in rat hippocampal neurons in culture is NMDA receptor selective and not essential for excitotoxic cell death

Molecular Brain Research 54 Ž1998. 35–48

Research report

Calpain I activation in rat hippocampal neurons in culture is NMDA receptor selective and not essential for excitotoxic cell death Emil Adamec a

a,b,)

, Mary L. Beermann

a,1

, Ralph A. Nixon

a,b,c

Laboratories for Molecular Neuroscience, Mailman Research Center 104, McLean Hospital, 115 Mill Street, Belmont, MA 02178, USA b Department of Psychiatry, HarÕard Medical School, Boston, MA 02115, USA c Program in Neuroscience, HarÕard Medical School, Boston, MA 02115, USA Accepted 16 September 1997

Abstract Administration of glutamate Ž100 mM. to primary cultures of rat hippocampal neurons for 1 h led to calpain I activation as determined by monitoring the extent of spectrin breakdown with the antibodies designed to specifically recognize the calpain I-mediated spectrin breakdown products. Based on the studies with subtype selective antagonists of glutamate receptors, glutamate caused calpain I activation specifically through the activation of the NMDA receptor. In parallel experiments, the magnitude and the temporal profiles of Ca2q rise were determined by Fura-2 microfluorimetry. Ca2q influx through voltage-sensitive Ca2q channels, even though leading to substantial Ca2q rise, did not by itself activate calpain I. These results indicate that for calpain I activation, the source of Ca2q influx is more important than the magnitude of Ca2q rise. Glutamate-mediated calpain I activation was fully blocked by preincubation Ž30 min. of the cultures with calpain inhibitor I, calpain inhibitor II, or calpeptin Žall 10 mM.. The presence of calpain inhibitors did not, however, in any way ameliorate the massive excitotoxicity resulting from 16 h exposure to glutamate, indicating that calpain I activation and excitotoxicity are not causally related events. Similarly, preincubation with any of the tested calpain inhibitors did not modulate the toxicity resulting from a 10-min exposure to glutamate. Additionally, the presence of calpain inhibitors was detrimental to the clearance of neuritic varicosities resulting from a short-term sublethal exposure to glutamate, suggesting that a physiological level of calpain I activation might actually play an important homeostatic role in the restoration of normal cytoskeletal organization. q 1998 Elsevier Science B.V. Keywords: Hippocampal neuron; Calpain; Spectrin; Excitotoxicity; Imaging; Calcium; Primary culture

1. Introduction Calpains are a family of Ca2q-activated neutral cysteine proteases w16,40,41x. Two extensively studied forms with ubiquitous tissue expression are known as calpain I and calpain II. Calpain I is also sometimes referred to as m-calpain and calpain II as m-calpain, due to their different in vitro Ca2q requirement for activation. Four novel isoforms, known as n-calpains, have tissue-specific expression and await further biochemical characterization w59,65x. In addition to Ca2q, the enzyme activity is regulated also by phospholipids and by a natural inhibitory protein, calpastatin w39x. In the CNS, an increase in the cytoplasmic concentration of free Ca2q ŽwCa2q x i . has an important signaling and )

Corresponding author. Fax: Ž1. Ž617. 855-3198; E-mail: [email protected] 1 Present address: Day Neuromuscular Laboratory, Massachusetts General Hospital, 149 13th Street, Charlestown, MA 02129, USA. 0169-328Xr98r$19.00 q 1998 Elsevier Science B.V. All rights reserved. PII S 0 1 6 9 - 3 2 8 X Ž 9 7 . 0 0 3 0 4 - 5

regulatory function. Ca2q ions serve as important mediators of many physiological processes. Ca2q homeostasis is a tightly regulated process w32,49x. Dysregulation of Ca2q homeostasis is poorly tolerated by nervous cells, leading to cell dysfunction and ultimately death w21x. Indeed, abnormalities in Ca2q homeostasis have been implicated as important contributing factors in the pathophysiology of stroke, CNS trauma, and possibly neurodegenerative disorders. The exact etiopathogenesis of these abnormalities is not known, but increased release of glutamate has been demonstrated both in experimental models of cerebral ischemia w31x, concussive brain injury w30x, and in patients with cerebral infarction w10x. Overstimulation of the receptors for the excitatory amino acids might be, therefore, an important contributing factor in the disruption of Ca2q homeostasis and possibly a crucial mediator of neuronal dysfunction in general w13,15,36x. Upon activation, calpain cleaves biologically important proteins and serves, therefore, as a key regulator of many physiological functions. Abnormalities in Ca2q homeosta-

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sis might, due to the Ca2q dependency of the protease, lead to excessive activation of the enzyme with potential detrimental effects. Excessive activation of calpain has been proposed to serve as a contributing factor in the pathophysiology of acute ischemic neurodegeneration w5x, neurotrauma w29x, and in the chronic neurodegeneration associated with Alzheimer’s disease w45,46,60,62x. The complexity of the CNS makes the investigation of exact regulation of biochemical events very difficult. Nearly pure primary cultures of CNS neurons represent, therefore, a useful alternative to whole-animal studies, adding a distinct advantage of the possibility to address questions directly on a single-cell level. This study, employing rat hippocampal neurons in culture, concentrated on two major objectives. First, since the Ca2q entry through different routes might influence cell function differently w24,68x, the initial goal of this study was to pharmacologically identify the specific, glutamate-activated, Ca2q influx pathway responsible for calpain I activation. Second, a possible association between glutamate-mediated calpain I activation and glutamate-induced cytotoxicity was investigated. 2. Materials and methods 2.1. Materials Neurobasal culture medium ŽNB., B27 medium supplement ŽB27., Ca2q-, Mg 2q-free Hank’s balanced salt solution ŽCMF-HBSS., Hank’s balanced salt solution ŽHBSS., N-Ž2-hydroxyethyl.piperazine-N X-Ž2-ethanesulfonic acid. ŽHEPES., glutamine, and L-glutamic acid were from Life Technologies ŽGaithersburg, MD.. Fetal calf serum ŽFCS. and horse serum ŽHS. were from HyClone Laboratories ŽLogan, UT.. Poly-D-lysine Žmol.wt. 30 000–70 000., aprotinin, cytosine b-D-arabinofuranoside ŽAra-C., ethylene glycol-bisŽb-aminoethyl ether. N, N, N X , N X-tetraacetic acid ŽEGTA., ethylenediaminetetraacetic acid ŽEDTA., trisŽhydroxymethyl.aminomethane ŽTris., dimethyl sulfoxide ŽDMSO., and sodium dodecyl sulfate ŽSDS. were from Sigma ŽSt. Louis, MO.. Fura-2 PE3 acetoxymethylester ŽFura-2 PE3rAM. was from TefLabs ŽAustin, TX.. Pluronic F-127 was from Molecular Probes ŽEugene, OR.. Dizocilpine maleate ŽMK-801. and 6,7-dinitroquinoxaline2,3-dione ŽDNQX. were from Research Biochemicals International ŽNatick, MA.. Calpain inhibitor I Ž N-acetylLeu-Leu-norleucinal., calpain inhibitor II Ž N-acetyl-LeuLeu-methional., leupeptin, and Pefabloc SC were from Boehringer Mannheim ŽIndianapolis, IN.. DNase I was from Worthington Biochemicals ŽFreehold, NJ.. Calpeptin Žbenzyloxycarbonyldipeptidyl aldehyde. was a kind gift of Mitsubishi Kasei Co., Yokohama, Japan. Nitro blue tetrazolium ŽNBT. and 5-bromo-4-chloro-3-indolyl-1-phosphate ŽBCIP. were from Promega ŽMadison, WI.. All other chemicals were purchased from standard commercial sources and were of the highest purity available. Tissue

culture plastiware was from Corning Costar ŽCambridge, MA. and from Greiner Labortechnik ŽIntermountain Scientific Co., Kaysville, UT.. Glass coverslips ŽD type. were from Erie Scientific ŽPorthmouth, NH.. 2.2. Antibodies A mouse monoclonal antibody ŽMAB 1622. to nonerythroid spectrin Žfodrin. was from Chemicon International ŽTemecula, CA.. The antibody specifically recognizes the 240-kDa a-fodrin molecule of all mammalian non-erythroid cells. The rabbit polyclonal antibodies CEP 38 and CEP 39 were a kind gift of Dr. R. Siman ŽCephalon, West Chester, PA.. The preparation of these antibodies has been described previously w55x. Briefly, calpain I cleaves the a-subunit of spectrin approximately in half generating two breakdown products: SBPN and SBPC . For the preparation of the antibodies, rabbits were immunized with peptides corresponding to the sequences of the Nterminal ŽCEP 38. or the C-terminal ŽCEP 39. part of the cleavage site. 2.3. Cell culture Cultures of rat hippocampal neurons were established from 17- to 18-day-old CD1 embryos based on the method of Brewer et al. w8x, with minor modifications. Hippocampi were dissected in cold CMF-HBSS Žsupplemented with 10 mM HEPES and 1 mM pyruvate.. Following trituration 20 = in CMF-HBSS with a fire-polished Pasteur pipet, the cell suspension was centrifuged at 200 = g for 5 min in a table-top centrifuge. The pellet was resuspended in CMFHBSS and triturated again 5 = . The resulting mostly single-cell suspension was added to the NB medium supplemented with B27, 2 mM glutamine, 25 mM glutamate, 1% Žvrv. FCS, 1% Žvrv. HS, and plated at the density of approximately 200–300 cellsrmm2 on poly-D-lysine Ž50 mgrml. precoated glass coverslips, multi-well plates, or 60-mm Petri dishes. The cultures were maintained in a humidified atmosphere of 5% CO 2 –95% air at 378C in a tissue culture incubator. Ara-C Ž10 mM. was added 96 h after plating to prevent the division of non-neuronal cells. The cells were fed at 7 days in vitro and subsequently once a week by the addition of 25% of the starting culture medium volume with fresh NBrB27 mixture. No antibiotics were used. Cultures prepared this way contained less than 10% of glial cells. The protocol for the culture was approved by the IACUC Committee of McLean Hospital. 2.4. Determination of [Ca 2 q ]i wCa2q x i in the somatic regions of individual hippocampal neurons was determined with single-cell digital microfluorimetry as described previously w20x. Cells on glass coverslips ŽNo. 1, diameter 25 mm. were loaded with Fura-2 PE3rAM w69x by incubating with 1 mM of Fura-2 PE3rAM Ž1 mM stock in DMSO. q 0.03% Žvrv. Pluronic

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pixel-to-pixel converted to wCa2q x i based on the equation of Grynkiewicz w25x. K d was taken as 224 nM. Calibration was performed by an in vitro method as described w20x. At the beginning of each experiment, a small rectangle was placed around a cell soma. The wCa2q x i in that area was averaged and the profile of the wCa2q x i change recorded over time. The neurons in a microscope-stage incubator were continuously perfused with a prewarmed ECBS at the rate of 1 mlrmin. Drugs were delivered through a separate in-flow channel with an increased flow rate to 2 mlrmin. During the first 5 min of each experiment, the images were taken every 10 s. The rate of image acquisition was increased to one every 2 s during the initial 2 min of drug

Fig. 1. Glutamate-mediated calpain I activation in rat hippocampal neurons. Cultures were exposed to either standard culture medium ŽC. or glutamate Ž100 mM. for 60 min ŽG. and calpain I activation determined by monitoring the extent of spectrin breakdown by SDS–PAGE Ž15 mg protein per lane. and immunoblotting Ž1:1000. with CEP 38 ŽA., CEP 39 ŽB., and MAB 1622 ŽC. antibody. The immunoblots from an experiment with neurons 17 days in vitro are presented in A, C, and E. Molecular weights standards Žin kDa. are shown on the left. Corresponding densitometric scans are shown in B, D, and F. The numbers adjacent to each peak represent integrated Žbackground subtracted. density=area measurements. The results are representative examples from 2 to 6 determinations on independent cultures.

F-127 Ž20% Žwrv. stock in DMSO. in an extracellular buffer solution ŽECBS, in mM: NaCl, 130; KCl, 5; CaCl 2 , 1.8; MgCl 2 , 1; glucose, 20; HEPES, 20, pH 7.3 with NaOH. for 60 min at 378C in a tissue culture incubator. Following a wash with ECBS, neurons were placed in a thermostatted microscope-stage incubator and maintained at 358C. The cells were visualized with an inverted epifluorescent microscope ŽOlympus IMT-2; 40 = oil immersion objective, 1.3 NA.. Cells were alternatively excited with 340 and 380 nm light through a computer-controlled filter wheel. An intensified CCD camera ŽIonOptix Corporation, Milton, MA. recorded the emitted image at 510 nm. For each image, 5 frames were averaged for each of the two excitation wavelengths. Background was recorded from an area of the coverslip free of any cells. After background subtraction, the ratio of the intensities of emitted light at each of the two excitation wavelengths was calculated and

Fig. 2. The extent of calpain I activation, both under the control condition ŽC. and under glutamate stimulation ŽG., is influenced by the concentration of Ca2q in the extracellular environment ŽwCa2q xe .. Cultures were stimulated for 60 min with glutamate Ž100 mM. and the extent of spectrin breakdown determined by SDS–PAGE Ž15 mg protein per lane. and immunoblotting with CEP 39 antibody Ž1:1000.. Densitometric scans from an experiment with hippocampal neurons 16 days in vitro are shown. wCa2q xe s1.8 mM ŽA., 3.6 mM ŽB., 5.4 mM ŽC.. Data are a representative example from 3 experiments on independent cultures.

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administration and again progressively decreased to one image every 10 or 20 s. For each neuron, the averaged value of wCa2q x i during the first 5 min Žs resting wCa2q x i . was subtracted from the

wCa2q x i at 1 min following the start of the drug administration Ž d 1 . and at 30 min of drug administration Ž d 30 .. The parameters d 1 and d 30 were taken as the measures of a drug-induced wCa2q x i response.

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2.5. Drug treatments All experiments were performed on cultures 12–21 days in vitro. Drugs were prepared as the following stock solutions: glutamate, 100 mM in water; glycine, 10 mM in water; KCl, 3 M in water; MK-801, 10 mM in water; DNQX, 10 mM in 0.1 N NaOH; CI I; CI II; calpeptin, 10 mM in DMSO; and stored at y208C. Administration of glutamate was always supplemented with 10 mM glycine. Drug exposure was carried out by direct addition to the original culture medium. All treatments were performed at 378C in a tissue culture incubator. At the end of each experiment, the culture medium was aspirated and the cells rinsed once with HBSS. Following the wash, the extraction buffer Ž1 mM EGTA, 2 mM EDTA, 1 mM benzamidine, 1 mM dithiothreitol, 25 mgrml leupeptin, 10 mgrml aprotinin, 1 mM Pefabloc SC, 2 mgrml DNase I, and 20 mM Tris-HCl, pH 7.4, in 0.15 M NaCl. was added, the cells were scraped and homogenized on ice. Following homogenization, the samples were incubated on ice for an additional 10 min. Protein concentration was determined by the method of Bradford ŽBio-Rad Laboratories, Hercules, CA.. Samples were either subjected to gel electrophoresis immediately or stored at y708C. 2.6. Gel electrophoresis and immunoblot analysis Protein electrophoresis was performed as described by Laemmli w33x with 0.75 mm Hoefer minigel system ŽPharmacia Biotech Inc., Piscataway, NJ. on 7.5% Žwrv. SDS–polyacrylamide gels ŽSDS–PAGE.. Proteins Ž15 mg. separated by SDS–PAGE were electrophoretically transferred to an Immobilon PVDF membrane ŽMillipore, Bedford, MA. for 1 h at 1 amp in Trisrglycine buffer ŽpH 8.3. with 15% Žvrv. methanol. Non-specific binding sites were blocked by incubating the membrane in TBS Žin mM: NaCl, 150; Tris, 50. containing 5% Žwrv. non-fat dry milk for 1 h at room temperature. Incubation with primary antibodies was carried out overnight at 48C. The next day, after washing with TBS containing 0.05% Žvrv. Tween-20, the membrane was exposed to alkaline phosphatase-conjugated secondary antibodies Ž1:750; Promega, Madison, WI. for 2 h at room temperature. Detection was done with BCIPrNBT.

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Scanned immunoblots ŽApple Color One Scanner. were analyzed on a Macintosh Quadra 650 computer with the public domain NIH Image program Žwritten by Wayne Rasband at the US National Institutes of Health and available from the Internet by anonymous FTP from zippy.nimh.nih.gov.. 2.7. Toxicity determination Two experimental paradigms were employed: Ž1. a continuous exposure to 100 mM glutamate for 16 h; and Ž2. a short-term, 10-min application of 100 mM glutamate. Since hippocampal neurons grown in the absence of glial layer were very sensitive to any medium removal and wash with a buffer solution, drugs were added directly to the original culture medium. For the short-term exposure, the drug action was terminated by the administration of a combination of 10 mM MK-801 and 100 mM DNQX, and the cultures maintained for an additional 16 h. The determination of cytotoxicity was performed on single-cell level w38x. At the end of an experiment, cultures were stained with Trypan blue Ž0.4% Žwrv. solution in phosphatebuffered saline.. Living cells maintained the ability to exclude the dye and, therefore, did not stain. Living cells were counted under phase-contrast microscopy. Cell death led to an increase in membrane permeability to the dye. Dead cells, therefore, appeared blue under bright-field microscopy. In a typical experiment, more than 500 cells were counted and the toxicity estimated as the percentage of dead cells in the total cell number. 2.8. Data analysis Quantitative data are reported as mean " S.E.M. The statistical significance of the differences between individual means was determined, as appropriate, by either an unpaired two-tailed Student’s t-test or by an analysis of variance ŽANOVA. with the JMP software ŽSAS Institute; Cary, NC.. Multiple comparisons were assessed by either Tukey’s or Dunnett’s post-hoc tests. A P-value of - 0.05 was considered as statistically significant. All experimental conditions were repeated on a minimum of two independent cultures.

Fig. 3. Glutamate-induced calpain I activation is caused by Ca2q influx specifically through the NMDA receptor. A: administration of 100 mM glutamate ŽG. for 30 min caused significant calpain activation as determined by monitoring spectrin breakdown with SDS–PAGE Ž15 mg protein per lane. and immunoblotting with CEP 38 antibody Ž1:1000.. C, control. Calpain activation was fully prevented by preincubation Ž30 min. with MK-801 Ž10 mM. q DNQX Ž100 mM., preincubation with DNQX alone was without effect, and preincubation with MK-801 fully blocked the response. A densitometric scan of the 150-kDa band from an experiment with cultures 13 days in vitro is shown. Data are a representative example from 3 experiments on independent cultures. Corresponding profiles of wCa2q x i responses were obtained by Fura-2 digital microfluorimetry as described in Section 2: Materials and methods. Glutamate Ž100 mM. was always administered at time point 5 min and was present for 60 min. Antagonists were preincubated for 30 min before the glutamate administration and were present throughout the recording. B: glutamate. C: preincubation with MK-801q DNQX. D: preincubation with DNQX. E: preincubation with MK-801. F: quantitative evaluation of the drug-induced wCa2q xi responses Ž d wCa2q x i .. The response at 1 min following the drug administration Ž d 1 . is represented by filled bars, the response at 30 min of drug administration Ž d 30 . by open bars. Average resting wCa2q x i s 114.40 " 5.47 nM Ž n s 55.. For d 1 ANOVA, P s 0.0001. ) Significantly different from other groups. For d 30 ANOVA, P s 0.0001. a Significantly different from other groups. The number of neurons monitored is indicated in parentheses.

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3. Results

3.2. The dependence of calpain I actiÕation on [Ca 2 q ]e

3.1. Glutamate-mediated calpain I actiÕation

The next series of experiments addressed the question of the dependence of calpain I activation on the concentration of Ca2q in the extracellular environment ŽwCa2q xe ., both under control conditions and upon glutamate stimula-

The initial series of experiments concentrated on establishing a method of monitoring calpain I activation in primary cultures of rat hippocampal neurons. Spectrin w6,54x, a preferred calpain substrate, is cleaved by calpain I approximately in half generating two breakdown products of an apparent molecular size 150 kDa corresponding to the N-terminal ŽSBPN . and C-terminal ŽSBPC . part of the molecule. Glutamate-mediated calpain I activation was monitored by determining the extent of spectrin breakdown by SDS–PAGE and immunoblotting with antibodies w55x designed to specifically recognize the spectrin breakdown products ŽSBP.. Immunodetection of the SBPs can be considered, therefore, as a measure of calpain I activation w63x. Administration of 100 mM glutamate for 30 min or 1 h reproducibly induced calpain I activation. Representative immunoblots with the corresponding densitometric scans are shown in Fig. 1. Immunoblotting with the antibody CEP 38 ŽSBPN , Fig. 1A,B. led to the appearance of a strong 150-kDa band which was only barely detectable under control conditions. In four experiments on independent cultures, 30 min administration of glutamate increased the intensity of the 150-kDa band 4.2- to 6.7-fold. The antibody CEP 39 ŽSBPC , Fig. 1C,D. recognized both the native spectrin molecule and the SBPC . Upon glutamate stimulation, the intensity of the 150-kDa SBPC band increased Ž2.3- to 3.7-fold in 4 experiments., and the intensity of the band corresponding to the native spectrin molecule decreased. Immunoblotting with a monoclonal antibody towards the a-subunit of spectrin confirmed the SBP nature of the 150-kDa band ŽFig. 1E,F.. Overall, the data presented in Fig. 1 confirm previous reports w3,37,55x about the usefulness and the sensitivity of these antibodies in monitoring drug-induced calpain I activation.

Fig. 4. Ca2q influx through voltage-sensitive Ca2q channels leads to calpain I activation by inducing glutamate release. A: exposure to KCl Ž50 mM. for 30 min induced significant calpain-mediated spectrin breakdown as determined by SDS–PAGE Ž15 mg protein per lane. and immunoblotting with CEP 38 antibody Ž1:1000.. A densitometric scan of the 150-kDa band from an experiment with neurons 12 days in vitro is shown. The data are representative of 3 experiments on independent cultures. Calpain I activation was fully prevented by preadministration Ž30 min. of MK-801 Ž10 mM.qDNQX Ž100 mM.. Corresponding profiles of wCa2q x i responses to the administration Žat time point 5 min. of KCl ŽB. KCl with preincubation of MK-801qDNQX ŽC.. wCa2q x i was monitored in individual neurons with Fura-2 digital microfluorimetry as described in Section 2: Materials and methods. D: quantitative evaluation of the drug-induced wCa2q x i responses Ž d wCa2q xi .. The response at 1 min following the drug administration Ž d 1 . is represented by filled bars, the response at 30 min of drug administration Ž d 30 . by open bars. For d 1 , P s 0.34. For d 30 , P s 0.0031 Žasterisk.. The number of neurons monitored is indicated in the parentheses.

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recognized practically exclusively the 150-kDa SBP and appeared, therefore, the most specific of the three antibodies used in this study. An example of a representative experiment is shown in Fig. 3A. Glutamate-mediated calpain I activation was fully prevented by preincubation Ž30

Fig. 5. Glutamate-mediated calpain I activation: full prevention by calpain inhibitors. Exposure to 100 mM glutamate ŽG. for 30 min leads to significant calpain activation as determined by SDS–PAGE Ž15 mg protein per lane. and immunoblotting with the CEP 38 antibody Ž1:1000.. A densitometric scan of the 150-kDa band from an experiment with hippocampal neurons 13 days in vitro is shown. Data are representative of 3 experiments on independent cultures. Calpain I activation was fully prevented by a 30-min preincubation with calpain inhibitor I ŽCI I., calpain inhibitor II, or calpeptin Žall 10 mM..

tion. The extent of spectrin breakdown was monitored with SDS–PAGE and immunoblotting with the CEP 39 antibody. An example of a representative experiment is presented in Fig. 2. The extent of calpain I activation was assessed by evaluating the ratio of the intensities of the band detecting the native spectrin molecule Ž240 kDa. and the 150-kDa SBPC Žratio 240:150.. Under control condition ŽwCa2q xe s 1.8 mM, Fig. 2A., the ratio 240r150 was ) 1. Increasing wCa2q xe to either 3.6 mM ŽFig. 2B. or 5.4 mM ŽFig. 2C., even without any drug stimulation, led to a decrease in the ratio 240r150 to - 1 indicating calpain I activation. Administration of 100 mM glutamate for 1 h in wCa2q xe s 1.8 mM decreased the 240r150 ratio to - 1. Practically identical results were obtained by glutamate stimulation in either wCa2q xe s 3.6 mM or wCa2q xe s 5.4 mM. The results presented in Fig. 2 show clear dose-dependency of calpain I activation on wCa2q xe even without any drug administration. The activation induced by 100 mM glutamate was, however, already near maximum at wCa2q xe 1.8 mM and increased only minimally at 5.4 mM. 3.3. The dependence of calpain I actiÕation on a particular route of Ca 2 q influx The first major objective of this study was to identify the glutamate-activated Ca2q influx pathwayŽs. specifically responsible for calpain I activation. Glutamate activates both the NMDA and the non-NMDA subtype of glutamate receptors leading to Ca2q influx and increase in wCa2q x i w12x. In addition, glutamate-mediated membrane depolarization might open voltage-sensitive Ca2q channels causing an increase in wCa2q x i and possibly the release of glutamate from glutamatergic nerve terminals. In order to determine if the glutamate-mediated calpain I activation was dependent on Ca2q influx through a specific subtype of glutamate receptors, neurons were stimulated for 30 min with 100 mM glutamate either alone or in the presence of subtype-selective receptor antagonists ŽFig. 3.. The extent of spectrin breakdown was monitored by immunoblotting with the CEP 38 antibody since it

Fig. 6. Cytotoxicity mediated by long-term Ž16 h. exposure to glutamate in rat hippocampal neurons: full protection by preincubation with a combination of an NMDA- and a non NMDA-receptor antagonist. Phase-contrast photomicrographs ŽOlympus CK-2 microscope, 20= objective. from an experiment with cells 13 days in vitro are shown. Data are representative of 4 experiments on independent cultures. A: cells exposed to standard culture conditions. B: exposure to glutamate Ž100 mM. for 16 h leads to extensive degeneration of both cell bodies and neuronal processes. C: preincubation with MK-801 Ž10 mM.qDNQX Ž100 mM. protected practically all neurons. D: administration of MK-801 qDNQX alone for 16 h did not cause any detectable toxicity. Scale bar: A–D, 100 mm.

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min. with either: Ž1. a combination of an NMDA receptor antagonist MK-801 Ž10 mM. and a non-NMDA receptor antagonist DNQX Ž100 mM.; or Ž2. by MK-801 Ž10 mM. alone. Preincubation with DNQX alone had no effect on the extent of glutamate-mediated calpain I activation. In order to directly correlate the drug-induced calpain I activation with the wCa2q x i signal, the magnitude and temporal profiles of the wCa2q x i responses were determined in parallel experiments with Fura-2 single-cell digital microfluorimetry. Administration of glutamate Ž100 mM. led to an immediate wCa2q x i rise to a sharp peak followed by a decline to a plateau ŽFig. 3B.. At different time intervals, all neurons underwent a secondary wCa2q x i rise and established a new plateau which was much higher than the initial response. The wCa2q x i response to glutamate was fully blocked by preincubation Ž30 min. with a combination of MK-801 Ž10 mM. and DNQX Ž100 mM. ŽFig. 3C.. When neurons were preincubated with DNQX alone ŽFig. 3D., the initial response to glutamate was significantly decreased and a stable plateau was maintained throughout the recording period. Only one neuron underwent secondary wCa2q x i destabilization. Administration of glutamate in the presence of MK-801 ŽFig. 3E. led to a sharp peak rise in wCa2q xi followed by a decay to a stable plateau. Since glutamate-induced influx of Ca2q and Naq leads to cell depolarization and opening of voltage-sensitive Ca2q channels, a separate series of experiments addressed specifically the role of Ca2q influx through voltage-sensitive Ca2q channels in calpain I activation ŽFig. 4.. Administration of 50 mM KCl led to calpain I activation. This activation was, however, fully prevented by preincubation Ž30 min. with a combination of MK-801 Ž10 mM. and DNQX Ž100 mM. ŽFig. 4A.. This result indicates that administration of KCl caused calpain I activation through glutamate release and subsequent indirect activation of glutamate receptors. Parallel wCa2q x i imaging of KClmediated responses ŽFig. 4B,C. demonstrated that the presence of glutamate receptor antagonists did not significantly influence the magnitude of the immediate wCa2q x i response Ž d 1 .. The response at 30 min Ž d 30 . was, however, significantly decreased by the presence of the antagonists, indicating small, but significant, contribution of the released glutamate to the overall KCl-induced wCa2q x i rise ŽFig. 4D.. Taken together with the data presented in Fig. 3, these results indicate that glutamate-mediated calpain I

Fig. 7. Cytotoxicity mediated by long-term Ž16 h. administration of glutamate in rat hippocampal neurons: no protection by preincubation with calpain inhibitors. Phase-contrast photomicrographs from an experiment with cultures 12 days in vitro are shown. Data are representative of 3 experiments on independent cultures. A: control culture. B: application of glutamate Ž100 mM. for 16 h caused massive degeneration of both cell soma and neurites. Preincubation Ž30 min. with either 10 mM CI I ŽC., 10 mM CI II ŽD., or 10 mM calpeptin ŽE. did not ameliorate the cytotoxicity. Scale bar: A–E, 100 mm.

activation was caused exclusively by Ca2q influx through the NMDA subtype of glutamate receptors. 3.4. The effect of calpain inhibitors The second major objective of this study was to evaluate the cellular consequences of calpain I activation,

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specifically the association between glutamate-mediated calpain I activation and excitotoxicity. This evaluation depended heavily on the verification that, under the conditions employed in this study, the available calpain inhibitors did actually inhibit calpain I. The following inhibitors were used: calpain inhibitor I; calpain inhibitor II; and calpeptin w67,72x. All three are membrane permeable w2,71x with K i values for calpain I inhibition in the sub-micromolar range w61x. To ensure adequate cell permeation, neurons were preincubated with the inhibitors for 30 min before the administration of glutamate Ž100 mM.. Under these conditions, application of calpain inhibitor I, calpain inhibitor II, or calpeptin Žall 10 mM. did, indeed, fully block any subsequent calpain I activation verifying the pharmacodynamic efficacy of these compounds. An example of a representative experiment is shown in Fig. 5. 3.5. The association between glutamate-mediated calpain I actiÕation and excitotoxic cell death Two experimental paradigms were employed to address the question of the association of glutamate-mediated calpain I activation and excitotoxicity. In the first paradigm, a continuous exposure to glutamate Ž100 mM. for 16 h induced nearly 100% cell death ŽFig. 6B.. The toxic effect was fully prevented by preincubation with a combination of MK-801 Ž10 mM. and DNQX Ž100 mM. ŽFig. 6C.. Since prolonged exposure to glutamate might be possibly toxic also by receptor-independent mechanisms, such as e.g. competitive inhibition of cystine uptake w42x, this result verifies a receptor-mediated mechanism for glutamate-mediated toxicity in mature cultures of hippocampal neurons. Administration of the combination of MK-801 and DNQX alone for 16 h to cultures in a fully developed state Žmore than 10 days in vitro. was not toxic ŽFig. 6D.. The involvement of calpain I activation as an important mediator of excitotoxicity in this paradigm was tested by preincubation Ž30 min. of the cultures with calpain in-

Fig. 8. Lack of cytoprotection by calpain inhibitors against toxicity induced by short-term Ž10 min. exposure to glutamate. Phase-contrast photomicrographs from a representative experiment with hippocampal neurons 19 days in vitro. A: culture exposed for 10 min to glutamate Ž100 mM.. The action of the agonist was terminated by the application of MK-801 Ž10 mM.qDNQX Ž100 mM. which were present for the subsequent 16 h at which time point the picture was taken. The extent of toxicity was determined by staining with 0.4% Trypan blue. Dead cells appear dark due to the uptake of the dye. On the corresponding bright-field image ŽB. dead neurons are clearly stained while living neurons appear only as an outline. Preincubation Ž30 min. with either 10 mM calpain inhibitor I ŽCI I., 10 mM calpain inhibitor II ŽCI II, both not shown., or 10 mM calpeptin ŽC. exhibited no modulatory effect on the glutamatemediated cytotoxicity. Scale bar: A–C, 100 mm. D: quantitative evaluation of the experiment shown above. G, glutamate Ž100 mM.. ) Significantly different from all other groups ŽANOVA, P s 0.0001.. @ Not significantly different from each other ŽANOVA, P s 0.8.. The number of neurons counted is indicated in parentheses. This experiment was repeated on another independent culture with comparable results.

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hibitors Ž10 mM. before the excitotoxin administration. As can be seen on examples of photomicrographs from a representative experiment shown in Fig. 7, the glutamatemediated toxicity was not in any way modified by the presence of calpain inhibitors. This result indicates that calpain I activation does not serve as a crucial mediator of excitotoxicity induced by a long-term exposure to glutamate.

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Glutamate-mediated neuronal death has two distinct components, a Naq-Cly component and a Ca2q component w14,18x. The toxicity resulting from a long-term administration of glutamate might be mediated preferentially by osmotic disturbances caused by Naq and Cly influx and exhibit, therefore, independence on calpain activation. Therefore, in a second paradigm, a short-term exposure, which leads primarily to Ca2q-mediated toxicity, was also tested. In this paradigm, the extent of cell death depended on the age of the cultures. In older, very sensitive, cultures 17–19 days in vitro, a 10-min administration of glutamate Ž100 mM. caused approximately 30% cell death ŽFig. 8.. Preincubation Ž30 min. of calpain inhibitors Ž10 mM. did not significantly influence the extent of cell death. A representative example from an experiment employing

calpeptin is shown in Fig. 8D. Similar results were also obtained with calpain inhibitor I Ž10 mM. and calpain inhibitor II Ž10 mM.. In younger, less sensitive, cultures, 12 days in vitro ŽFig. 9., a 10-min exposure to glutamate Ž100 mM. led to a minimal cell death Ž- 10%.. One striking feature of this short-term exposure of glutamate to the relatively younger cultures was a rapid Žwithin minutes. formation of neuritic varicosities Žnot shown.. These varicosities were, however, nearly completely cleared upon termination of the glutamate action with a combination of MK-801 and DNQX and incubation of the cultures for the subsequent 16 h ŽFig. 9B.. Interestingly, the presence of any of the tested calpain inhibitors inhibited the clearance of these varicosities and was actually detrimental to the restoration of normal smooth neurites. A representative example from an experiment employing calpain inhibitor II Ž10 mM. is shown in Fig. 9C, but identical results were also obtained with calpain inhibitor I Ž10 mM. and calpeptin Ž10 mM.. The presence of calpain inhibitors alone for 16 h did not, by itself, lead to the formation of the varicosities Žnot shown..

4. Discussion

Fig. 9. The presence of calpain inhibitors is detrimental to normal clearance of glutamate-induced neuritic varicosities. Phase-contrast photomicrographs from an experiment with hippocampal neurons 12 days in vitro. A: control neurons had smooth neuritic processes. B: administration of glutamate Ž100 mM. for 10 min Žterminated by 10 mM MK-801q100 mM DNQX. led to a formation of neuritic varicosities within minutes Žnot shown. that were cleared by the time the picture was taken Ž16 h.. Preincubation Ž30 min. with either 10 mM calpain inhibitor I Žnot shown., 10 mM calpain inhibitor II ŽC., or 10 mM calpeptin Žnot shown. inhibited the clearance of the neuritic varicosities. Scale bar: A–C, 50 mm. This experiment was repeated on another independent culture with comparable result.

The first objective of this study was to pharmacologically characterize the specific, glutamate-activated, Ca2q influx pathway responsible for calpain I activation in hippocampal neurons in culture. As demonstrated both in whole-animal models w63,64x and hippocampal slices w3,17x, stimulation of glutamate receptors leads to calpain activation. The type of the Ca2q-permeable receptor or ion channel responsible for this activation has not been yet precisely identified. The presented study clearly demonstrates that activation of the NMDA receptors is necessary for calpain I activation. Activation of the non-NMDA receptors, which leads to depolarization-mediated opening of voltage-sensitive Ca2q channels, or direct activation of voltage-sensitive Ca2q channels, even though both cause substantial Ca2q influx, are by itself not sufficient to activate the enzyme. Calpain I activation, similarly to early calcium neurotoxicity w68x, depends, therefore, in an important, and not yet fully understood, way on the source of Ca2q influx. Also, monitoring wCa2q x i rise with Ca2q microfluorimetry does not, by itself, allow one to predict the biochemical responses arising from the differences in the source of Ca2q rise. It appears that cells handle Ca2q ions entering through different pathways differently. It has been suggested that the total Ca2q accumulation inside a cell might be actually a more relevant parameter for predicting the biochemical consequences of Ca2q uptake than monitoring wCa2q x i rise with microfluorimetry w22x. The second objective of this study was to investigate the association between glutamate-mediated calpain I activation and glutamate-mediated cytotoxicity. This issue has not yet been directly addressed for hippocampal neurons in

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culture. Two previous studies investigated this question in cultured cerebellar granule cells w19,37x. Both studies found no direct association between calpain I activation and glutamate-mediated excitotoxicity. Since both studies employed cerebellar granule cells, the question of the general applicability of this finding to other neuronal types still remained. Also, due to the high sensitivity of the hippocampus to hypoxia–ischemia-induced damage and to the high incidence of hippocampal pathology in Alzheimer’s disease, addressing this question directly in cultured hippocampal neurons was considered important. The lack of association between the two phenomena observed in this study is, however, in full agreement with the previously published finding in cerebellar granule cells, making it likely a more general phenomenon. The association between wCa2q x i rise, calpain I activation, and cytotoxicity resulting from metabolic inhibition was also investigated in cardiomyocytes w2x. In that study, calpain inhibitors also prevented calpain I activation, but did not attenuate cell death, which extends the findings outside the nervous system. Interestingly, two in vitro studies with cerebellar neurons Ždifferent from the cerebellar granule cells mentioned above. demonstrated protective effects of calpain inhibitors on excitotoxic cell death w9,70x. In both studies, the excitotoxin involved a ligand of non-NMDA receptors as opposed to glutamate employed in the presented study. It is therefore possible that either a difference in the cell type or a difference in the stimulus employed might have contributed to the differences in the findings. The study of Brorson et al. w9x also demonstrated a neuroprotective effect of calpain inhibitors against NMDA-mediated toxicity in hippocampal neurons. One possible explanation for the different results might be the type of calpain inhibitors used: MDL-28170 and E-64 in the study of Brorson et al., calpain inhibitor I, calpain inhibitor II, and calpeptin in the present study. The lack of complete cytoprotection of calpain I inhibition on excitotoxic cell death, as reported in some studies employing primary neuronal cultures, is difficult to reconcile with a considerable body of evidence showing protective effects of calpain inhibitors in whole-animal models of cerebral ischemia and brain trauma. Calpain activation has been demonstrated in models of both focal cerebral ischemia w74,75x and global cerebral ischemia w44,73x. In both models, calpain inhibitors have been shown to be neuroprotective w4,26,34,53x. Similarly, calpain activation has been demonstrated in models of traumatic brain injury w28x, and calpain inhibitors have been shown to be neuroprotective w50,57x. Clearly, the in vivo and in vitro models differ in some important aspect. Several points might be considered as a possible explanation for the differences in the findings. First, the nature of the primary pathogenic stimulus might differ. The studies employing glutamate exposure to neurons in culture might not properly reflect the complex

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biochemical events associated with ischemia–hypoxia and brain trauma in whole-animal studies. Second, the intensity of the pathogenic stimulus might differ. It is possible that the exposure of neurons in culture to maximally effective concentrations of glutamate represents a stimulus too strong to appropriately reflect the conditions relevant for most in vivo models. Third, the mechanism of the resultant cell death might be different. In most in vitro and in vivo studies, the mechanism of cell death, necrosis versus apoptosis, has not been adequately characterized. Neurons in culture exposed to maximally effective concentrations of glutamate die by necrosis w7x. For in vivo studies, necrosis probably also plays an important role, at least in the areas of maximal damage, but for the majority of the neurons, apoptosis might actually represent a more important mechanism of cell death. Indeed, the presence of apoptotic cell death has been described in experimental models of focal cerebral ischemia w11,35x, global cerebral ischemia w27x, and closed head injury w51x. Also, intrastriatal administration of NMDA in the rat leads to apoptotic cell death w52x. Calpain activation has been shown to participate in the expression of neuronal apoptosis w43x and in some models of apoptosis, calpain inhibitors have been shown to be protective w66x. Interestingly, the intensity of the pathogenic stimulus and the mechanism of cell death might actually be interconnected. As shown by Ankarcrona et al. w1x, based on the extent of mitochondrial damage, exposure to glutamate might lead to either apoptosis or necrosis. Fourth, the pharmacological effects of calpain inhibitors might differ. Currently available calpain inhibitors are not entirely selective w71x. In addition to calpain, they also inhibit cathepsin B, cathepsin L, proteasome w56,58x, and possibly other enzymes. It is possible that in in vivo models, inhibition of an enzyme other than calpain might have a more decisive contribution to cell survival than it does for neurons in culture. The effect of calpain inhibitors on cathepsins is especially intriguing since a transient brain ischemia in a monkey leads to a rapid disruption of lysosomes with activated m-calpain being localized at the lysosomal membranes w73x. An important, and mostly unresolved, question remains about the biological nature of calpain activation. Prolonged, uncontrolled activation of calpain might certainly have detrimental effects w60x. Relatively short-term activation which would still remain under physiological control might, on the other hand, serve an important homeostatic function. One issue to consider is the role of calpain in the regulation of normal cytoskeletal organization. Sublethal exposure to Ca2q rise causes an early appearance of neuritic varicosities w47,48x. The exact nature of these varicosities will require further characterization, but swelling combined with cytoskeletal alterations are the most likely explanation. As has been demonstrated for mouse cortical neurons by Faddis et al. w23x and for rat hippocampal neurons in the presented study, calpain con-

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tributes in an important way to the clearance of these varicosities. Since cytoskeletal proteins represent an important target of calpain action, calpain-mediated degradation of altered cytoskeletal proteins might be a step necessary for the restoration of normal cytoskeletal organization. In summary, this report presents evidence implicating specifically the Ca2q influx through the NMDA subtype of glutamate receptors as the pathway primarily responsible for calpain I activation following glutamate administration in rat hippocampal neurons in culture. However, no association between glutamate-mediated calpain I activation and glutamate-induced cell death was detected, indicating that calpain I activation does not function as an important mediator of toxic effects of glutamate. Additionally, during sublethal glutamate exposure, calpain I activation appears to serve as an important homeostatic mechanism involved in the restoration of normal cytoskeletal organization disrupted by the action of glutamate.

w10x

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w13x w14x w15x w16x

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Acknowledgements

w18x

We would like to thank Dr. R. Siman ŽCephalon, West Chester, PA. for providing the antibodies CEP 38 and CEP 39. This research was supported by a grant from the National Institute on Aging ŽAG10916..

w19x

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