Inhibition of Calpain Prevents N-Methyl-D-aspartate-InducedDegeneration of the Nucleus Basalis and AssociatedBehavioral Dysfunction□S

Volker Nimmrich, Robert Szabo, Csaba Nyakas, Ivica Granic, Klaus G. Reymann,Ulrich H. Schroder, Gerhard Gross, Hans Schoemaker, Karsten Wicke,Achim Moller, and Paul LuitenNeuroscience Research, GPRD, Abbott, Ludwigshafen, Germany (V.N., G.G., H.S., K.W., A.M.); Departments of MolecularNeurobiology (C.N., I.G., P.L.) and Biological Psychiatry, University of Groningen (P.L.), Haren, The Netherlands;Neuropsychopharmacology Research Unit of Semmelweis University and Hungarian Academy of Sciences, Budapest, Hungary(C.N., R.S.); and Projektgruppe Neuropharmakologie, Leibniz Institute for Neurobiology, Magdeburg, Germany (K.G.R., U.H.S.)

Received June 25, 2008; accepted August 12, 2008

ABSTRACTN-Methyl-D-aspartate (NMDA) receptor-mediated excitotoxicityis thought to underlie a variety of neurological disorders, andinhibition of either the NMDA receptor itself, or molecules of theintracellular cascade, may attenuate neurodegeneration inthese diseases. Calpain, a calcium-dependent cysteine pro-tease, has been identified as part of such an NMDA receptor-induced excitotoxic signaling pathway. The present study ad-dressed the question of whether inhibition of calpain canprevent neuronal cell death and associated behavioral deficitsin a disease-relevant animal model, which is based on excito-toxic lesions of the cholinergic nucleus basalis magnocellularisof Meynert. Excitotoxic lesions of the nucleus basalis withNMDA induced a markedly impaired performance in the novelobject recognition test. Treatment with the calpain inhibitor,N-(1-benzyl-2-carbamoyl-2-oxoethyl)-2-[E-2-(4-diethlyamino-methylphenyl) ethen-1-yl]benzamide (A-705253), dose-depen-dently prevented the behavioral deficit. Subsequent analysis of

choline acetyltransferase in the cortical mantle of the lesionedanimals revealed that application of A-705253 dose-depen-dently and significantly attenuated cholinergic neurodegen-eration. Calpain inhibition also significantly diminished the ac-companying gliosis, as determined by immunohistochemicalanalysis of microglia activation. Finally, inhibition of calpain byA-705253 and the peptidic calpain inhibitor N-acetyl-Leu-Leu-Nle-CHO did not impair long-term potentiation in hippocampalslices, indicating that calpain inhibition interrupts NMDA exci-totoxicity pathways without interfering with NMDA receptor-mediated signaling involved in cognition. We conclude thatinhibition of calpains may represent a valuable strategy for theprevention of excitotoxicity-induced neuronal decline withoutinterfering with the physiological neuronal functions associatedwith learning and memory processes. Thus, calpain inhibitionmay be a promising and novel approach for the treatment ofvarious neurodegenerative disorders.

Calpains in the central nervous system are Ca2�-depen-dent cysteine proteases, which can modulate the conforma-tion, localization, and activity of various proteins. Calpain-induced modifications have a profound affect on biologicalprocesses as different as cell cycle progression and vesiculartrafficking. They are an integrative part of several signalingcascades, but they can also contribute to apoptotic and ne-

crotic cell death in pathological conditions. For this reason,calpains have been discussed extensively as target for ther-apeutic interventions in a number of neurodegenerative dis-ease that are associated with neuronal loss (for review, seeHuang and Wang, 2001; Goll et al., 2003; Zatz and Starling,2005).

In that respect, it has recently been shown that inhibitionof calpains prevents excitotoxic neuronal cell death in vitro(Caba et al., 2002; Ray et al., 2006) and in vivo (Chiu et al.,2005; Takano et al., 2005). Excitotoxicity is caused by anoverstimulation of N-methyl-D-aspartate (NMDA) receptors,and there is now growing evidence that calpain cleaves sev-

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doi:10.1124/jpet.108.142679.□S The online version of this article (available at http://jpet.aspetjournals.org)

contains supplemental material.

ABBREVIATIONS: NMDA, N-methyl-D-aspartate; A-705253, N-(1-benzyl-2-carbamoyl-2-oxoethyl)-2-[E-2-(4-diethlyaminomethylphenyl) ethen-1-yl]benzamide; NBM, nucleus basalis magnocellularis; LTP, long-term potentiation; PBS, phosphate-buffered saline; MK-801, 5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10imine; ALLN, N-acetyl-Leu-Leu-Nle-CHO; ChAT, choline acetyltransferase; fEPSP, field excitatorypostsynaptic potential; SBDP, spectrin breakdown product; APV, (2R)-amino-5-phosphonovaleric acid.

0022-3565/08/3272-343–352$20.00THE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS Vol. 327, No. 2Copyright © 2008 by The American Society for Pharmacology and Experimental Therapeutics 142679/3397325JPET 327:343–352, 2008 Printed in U.S.A.

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eral downstream targets that are critical for the progressionof excitotoxic neuronal degenerative processes (Wu et al.,2004; Hou et al., 2006).

We have recently introduced a highly specific low-molecu-lar-weight inhibitor of calpain, A-705253, which does notaffect the multifunctional proteasome complex (Lubisch etal., 2003). Selectivity against the proteasome is necessary toevaluate the role of calpain, because the ubiquitin-protea-some system also contributes to some neurodegenerative dis-eases (Rubinsztein, 2006; Pan et al., 2008). By using such aspecific calpain inhibitor, we addressed the questions ofwhether inhibition of calpain-mediated cascades would1) prevent neurodegeneration in a model relevant for neuro-degenerative diseases, 2) maintain the behavioral function ofthe affected brain area, and 3) affect normal NMDA receptor-dependent physiology. To analyze the neuroprotective effectof calpain inhibition, we used an established model of exci-totoxicity-induced nucleus basalis magnocellularis (NBM)degeneration in rats (Horvath et al., 2000; Harkany et al.,2001, 2002). The NBM in rats is analogous to the nucleusbasalis of Meynert in humans. Cholinergic fibers originatingfrom the NBM extensively project to the neocortical mantleand play an essential role in the attention phase of informa-tion storage processing (Blokland 1995; Van der Zee andLuiten 1999). Lesioning of the NBM in rodents causes adegeneration of cholinergic cortical projections, which inevi-tably leads to severe cognitive deficits in affected rats andmimics the characteristic loss of forebrain cholinergic inner-vation in Alzheimer’s disease (Bartus et al., 1982; Gaykemaet al., 1992). Compounds that interfere with downstreamcalcium-associated intracellular pathways initiated by gluta-mate-induced calcium influx may well affect signaling cas-cades that are crucial for memory processes. To assesswhether calpain inhibition would affect normal NMDA-de-pendent physiology, we also examined long-term potentiation(LTP) in hippocampal slices.

In this study, we present data showing that calpain inhi-bition prevents NMDA-induced neurodegeneration of NBMand associated decrease of cortical cholinergic innervation.Furthermore, calpain inhibition attenuates cognitive deficitsthat occur as a result of such neurodegeneration. NormalNMDA receptor-dependent physiology is not affected, indi-cating that only those downstream cascades are interrupted,which contribute to cellular pathology.

Materials and MethodsMaterials

A-705253 (Lubisch et al., 2003) was solubilized in 0.9% sodiumchloride with a pH between 5 and 5.5, and injected intraperitoneallyin doses of 1, 3, and 10 mg/kg b.wt. NMDA (Sigma-Aldrich, St. Louis,MO) solution was prepared in phosphate-buffered saline (PBS) solu-tion (pH7.4) in a concentration of 60 nmol per 1 �l. The selectivenoncompetitive NMDA receptor antagonist (�)-MK-801 hydrogenmaleate (Sigma-Aldrich) was dissolved in 0.9% sodium chloride forintraperitoneal injection in a dose of 2.5 mg/kg. N-Acetyl-Leu-Leu-Nle-CHO (ALLN; Calbiochem, San Diego, CA) stock solutions wereprepared in dimethyl sulfoxide.

Animals and Surgical Procedure

Eight-month-old male Wistar rats (8–11 per group; Harlan,Borchen, Germany) were anesthetized with an intraperitoneal injec-tion of sodium pentobarbital (60 mg/kg b.wt.). All care and treat-

ments were carried out in accordance with the European Communi-ties Council Directive on the use of experimental animals. Onemicroliter of the freshly prepared solution of NMDA (60 nmol) in PBSwas applied by a Hamilton microsyringe into the right nucleusbasalis (anterior-posterior coordinate, �1.5 mm; lateral, 3.2 mm) attwo dorsoventral positions, 6.2 and 7.0 mm ventral to the dura.Sham controls followed the same surgical procedures, but they wereinjected with the same quantity of the PBS alone. For more meth-odological details, we can refer to our earlier report (Luiten et al.,1995).

Drug Administration

A-705253, MK-801, or vehicle were applied 1 h before NMDAinjection, which was followed by a second application 12 h aftersurgery and 2 daily injections for the next 2 days. MK-801 was alsoinjected 1 h before and 12 h after surgery, but it was given only onceper day for the two postsurgery days (at 2.5 mg/kg).

At postoperative day 12, the animals were transcardially perfusedunder deep pentobarbital anesthesia with a fixative solution of 4%paraformaldehyde in 0.1 M phosphate buffer, pH 7.4, after a shortprerinse with heparinized saline. The brains were removed, post-fixed for 2 days in the same fixative, and stored in phosphate bufferat 0°C, then cryoprotected by 30% sucrose for 4 days, and sectionedon a cryostat microtome at a thickness of 20 �m. Free-floating brainsections were processed for immunocytochemical staining.

ChAT Staining of Cholinergic Fibers

An immunostaining procedure was applied to visualize cholineacetyltransferase (ChAT)-positive cholinergic neurons in NBM andtheir projection axon ramifications in the target brain areas, i.e., inthe parietal neocortex. Every eighth section was stained for ChATand CD11b as a microglia marker. Goat anti-ChAT primary antibody(Chemicon, Temecula, CA) was used in a 1:500 dilution. Biotinylatedrabbit anti-goat antibody and Vectastain ACB kit was obtained fromVector Laboratories (Burlingame, CA). The staining was completedwith nickel-enhanced diaminobenzidine reaction in the presenceof H2O2.

Visualization of Microglial Activation by CD11b Antibody

Mouse anti-rat integrin �M [CD11b] monoclonal antibody (Chemi-con) was used in a dilution rate of 1:1500 to recognize activatedmicroglia. The biotinylated secondary antibody and the ABC kit wereobtained from Vector Laboratories (see above). Diaminobenzidinereaction was enhanced by nickel ammonium sulfate, as describedabove.

Quantification of Cholinergic Fiber Density

The quantification procedure for cholinergic fiber density is estab-lished in our laboratory and was published several times in the past(Horvath et al., 2000; Harkany et al., 2001). In brief, the parietalneocortex, topographically corresponding to the anterior-posteriorsite of the NMDA lesion in the NBM and with the highest level ofcholinergic fiber loss, was analyzed for fiber density with the Quan-timet 600HR (Leica, Wetzlar, Germany) computer program. Theexact measurement took place in the superficial sublayer of the layerV cortical area, which receives the highest density of cholinergicinnervation. Three brain sections were analyzed per animal, and theresults were averaged. Fiber density in percentage surface area ofpositively stained fibers was computed in both sides of the brainsection. The ChAT-positive fiber density ipsilateral to the lesion wascompared with the intact contralateral side, and the percentageloss was calculated as an indicator of cholinergic neuron and fiberdegeneration.

Magnitude of Microglial Reaction

Microglial activation around the lesion was remarkably localizedand could be quantified with a computerized technique that mea-

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sured the size of the brain region infiltrated by the activated micro-glia (Quantimet; Leica). Area of microglial activation was manuallydelineated and measured at the level of NBM injection indicated bythe overstained injection channel. Here again, three sections wereselected from a total of 10 to 12 for measurements. The selection ofsections and measurements were unbiased and carried out blindly.

Behavioral Testing

Small Open-Field Behavior. Seventy-two hours postsurgery,the behavior of the animals was assessed in a light-independentsmall open-field paradigm (Nyakas et al., 1991). Animals wereplaced into individual Perspex arenas, 24 � 24 cm in diameter with30-cm high transparent walls, for 25 min. Six rats were observedsimultaneously, and every 10 s the following behaviors were scoredby a sampling technique: 1) rearing; 2) sniffing with head turning; 3)walking; 4) grooming; and 5) immobility (resting). Ambulation wasalso expressed by a combined score of rearing, sniffing, and walking(ambulation � 3 � rearing � 1 � sniffing � 2 � walking scores). Therepresentative scores of each behavioral component were summed upin 5-min blocks and analyzed statistically.

Novel Object Recognition. Novel object recognition was as-sessed 5 days postsurgery. A day before testing novel object recog-nition ability, the rats were placed into a cylindrical open-field arenaof 80-cm diameter surrounded by a 60-cm high reflective aluminumwall for 3 min. During this period, the rats were allowed to habituateto the experimental surroundings. The next day, during the 1stsession of the novel object recognition test, the rats were allowed toexplore the same open-field apparatus for 5 min while two identicalobjects (A � A) were placed into the arena. During the 2nd session,carried out 3 h after the 1st session, one of the two objects wasreplaced by a novel one (B � A). The ratio of visiting the novel versusknown objects illustrated the object recognition ability, i.e., the at-tention behavior. The total period spent with exploration of theobjects during the 1st and 2nd sessions were recorded. The recogni-tion ability of the novel object at the 2nd session was calculated inthe following way: duration of exploration of the novel (B) object wasdivided by the duration of exploration of both novel (B) and familiar(A) objects and expressed in percentages.

Electrophysiology

Seven to 8-week-old male Wistar rats (Harlan) were decapitated,and 400-�m transverse hippocampal slices were prepared with aself-constructed tissue chopper. The slices were transferred to asubmerged recording chamber and adapted for at least 1 h at 33°C inartificial cerebrospinal fluid (124 mM NaCl, 4.9 mM KCl, 1.3 mMMgSO4, 2.5 mM CaCl, 1.2 mM KH2PO4, 25.6 mM NaHCO3, and 10mM glucose 10, pH7.4).

The Schaffer collateral was locally stimulated by a monopolarstimulation electrode (impedance 20 � 150 �; Sprint Metall Work,Reichshof, Germany) using a constant current, biphasic stimulusgenerator (voltage, 1–5 V; impulse duration, one polarity 0.1 ms andwhole impulse 0.2 ms) (2100; AM-Systems Isolated Pulse Stimulator;AM-Systems, Carlsborg, WA). Field excitatory postsynaptic poten-tials (fEPSPs) were recorded from the stratum radiatum with glasselectrodes (borosilicate glass with filament, 1–5 MOhm, diameter: l.5mm, tip diameter: 3–20 �m) filled with experimental solution. Thefield potentials were filtered by a low-pass filter (5 kHz). For thestatistical analysis of the experiments, the slope of the fEPSP wasdetermined. Recording, analysis, and supervision of the experimentswere performed by means of a software program (PWIN), which wasdeveloped at the Leibniz Institute for Neurobiology (Department ofNeurophysiology) in Magdeburg, Germany. LTP was induced using atheta burst stimulation protocol (four times two paired pulses wereapplied in intervals of 200 ms, the interval between the paired pulseswas 10 ms, and the width of a single pulse was 0.2 ms).

Effect of A-705253 on Spectrin Cleavage in HippocampalSlice Cultures

Hippocampal slices were prepared from 9 to 11-day-old Wistarrats. Slices, 350-�m thick, were placed on Millicell single-well PCFinserts (Millipore, Schwalbach, Germany) in 6-well cell cultureplates (Greiner Bio-One GmbH, Frickenhausen, Germany) and keptin an incubator for 8 days at 34°C with 5% CO2 air. The slices werecultured in medium A [25 mM HEPES, pH 7.3, 40% basal mediumEagle with Earle’s salt, 25% horse serum, 25% Earle’s balanced saltsolution, 1 mM GlutaMAX (Invitrogen GmbH, Karlsruhe, Germany),and 28 mM glucose] for the first 3 days. Thereafter, medium A wasreplaced with Neurobasal A medium (Neurobasal A medium, 1 mMGlutaMAX, and 25 mM glucose; Invitrogen GmbH), and the sliceswere kept in culture for an additional 5 days. Neurobasal A mediumwas replaced twice a week.

After cultivation of the hippocampal slices for 8 days, the mediumwas aspirated and replaced with 1 ml of Neurobasal A medium. Atotal of 0.001 ml of different A-705253 inhibitor solutions at 10, 1,and 0.1 mM was added, respectively. After a 30-min incubation at34°C with 5% CO2 air, NMDA was added (10 �M final concentration)and incubated for 5 h at 34°C with 5% CO2 air. After this time, theslices were collected in 1.5-ml Eppendorf tubes and solubilized by theaddition of 0.025 ml of lysis buffer containing 50 mM Tris, pH 7.4,150 mM NaCl, 5 mM EDTA, 10% sucrose, 0.1% CHAPS, 1 mMdithiothreitol, 1:25 CompletePlus (Roche Diagnostics GmbH, Mann-heim, Germany), and 1:100 phosphatase inhibitor cocktail set II(Calbiochem-Merck Biosciences GmbH, Darmstadt, Germany).Slices were homogenized with a plastic pistill and kept on ice for 5min. The insoluble material was removed by centrifugation for 5 minat 13,000 rpm and 4°C, and the supernatant was collected. Super-natant protein concentrations were determined with MicroBCA(Pierce Inc., Rockford, IL). Samples (equal total protein of 20 �g/lane) were mixed with NuPAGE LDS sample buffer 4� (InvitrogenGmbH), heated for 5 min at 95°C, and thereafter resolved by 4 to 12%SDS � polyacrylamide gel electrophoresis (Invitrogen GmbH). Aftertransfer of proteins to nitrocellulose membranes, the proteins wereanalyzed by immunoblotting using a murine monoclonal antibody(mAB 1622; Chemicon) specific for spectrin and calpain-mediatedspectrin breakdown products (SBDPs) followed by a peroxidase-con-jugated secondary sheep anti-mouse antibody (A6782; Sigma-Al-drich) and a peroxidase substrate. To determine the percentageinhibition, the integrated optical density of the SBDPs in the pres-ence and absence of inhibitor was determined using the VersaDocImaging System (Bio-Rad Laboratories GmbH, Munchen, Germany).

ResultsCalpain Inhibition Markedly Attenuates Degenera-

tion of Cholinergic Fibers after Excitotoxic Lesion ofthe Nucleus Basalis Magnocellularis. We analyzed theeffect of calpain inhibition on the cholinergic innervation ofthe parietal neocortex after induction of an excitotoxic insultto the nucleus basalis magnocellularis in rats. Immunostain-ing visualized ChAT-positive cholinergic innervation fibersin the parietal neocortex (Fig. 1, 2), and fiber density wasmeasured in the superficial sublayer of the layer V as de-scribed in Horvath et al. (2000). The ChAT-positive fiberdensity ipsilaterally to the lesion was compared with theintact contralateral side and the percentage of fiber loss inthe various experimental groups summarized in Fig. 3. In-jection of NMDA to the NBM decreased the density of theChAT-positive fibers in the parietal cortex by 50%. Applica-tion of 2.5 mg/kg MK-801 nearly completely prevented thisdecrease. The calpain inhibitor A-705253 dose-dependentlyprotected the cholinergic neurons and their projection fibers

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against the excitotoxic injury and reduced the fiber degener-ation with 44 and 49% with 3 and 10 mg/kg, respectively.Application of 10 mg/kg A-705253 did not affect the densityof the cholinergic innervation, when given to sham-treatedanimals.

Calpain Inhibition Decreased Microglia Activationafter Excitotoxic Lesion of the Nucleus Basalis Magno-cellularis. We further sought to examine whether calpaininhibition would prevent microglial activation that normallyaccompanies neurodegenerative processes. Therefore, wemeasured the area of microglial activation at the level of the

NBM injection by staining microglia for integrin. Injection ofNMDA strongly increased this area, which corresponds to thespread of the neurotoxin in the injection locus (Harkany etal., 2000) (Figs. 4 and 5). When rats were treated with cal-

Fig. 1. Anatomical description of studying the cholinergicsystem after NBM lesion with NMDA. A, the posterior partof the NBM was lesioned, and most of the magnocellularcholinergic neurons are absent after lesion (lower leftpanel, which is the magnification of the nucleus basalisarea outlined by dashed lines at A). The parietal neocortexof lesioned rat represents a marked loss of ChAT-positivefibers (middle left panel). The precise anatomical regionbetween parietal cortex area 1 (Par1; primary somatosen-sory cortex) and parietal cortex area 2 (Par2; supplemen-tary somatosensory cortex) (Zilles 1985), where most of theareas were sampled for quantitative measurements of fiberdensity, is outlined by dashed lines. B and the right panelsrepresent the nonlesioned control side of the same rat. Thebar in B is 1 mm.

Fig. 2. Photomicrographs of cholinergic innervation of the parietal neo-cortex in 5 groups of animals: sham-operated and vehicle-treated,NMDA-treated and vehicle-injected, NMDA-treated and A-705253-in-jected in three different doses of 1, 3, and 10 mg/kg (labeled 1, 3, and 10,respectively). Quantitative measurement of fiber density was performedbetween the two arrows in the superficial part of layer V. The bar at theright represents 100 �m.

Fig. 3. Attenuation of cholinergic neurodegeneration by calpain inhibitorA-705253. Groups are sham-lesioned and NMDA-treated, and they re-ceived injections with either the vehicle or the different doses of A-705253(1, 3, and 10 mg/kg) or MK-801 (MK). ��, p 0.01 versus sham-lesionedand saline-treated groups; not significant from sham-0; ##, p 0.01versus NMDA-injected and vehicle-treated groups.

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pain inhibitor A-705253, the area of activation was dose-dependently decreased, and it reached significance at a doseof 3 mg/kg. The NMDA receptor antagonist MK-801 alsostrongly reduced the area size of microglia activation.

Calpain Inhibition As Well As NMDA Lesioning DoesNot Affect Behavior in the Small Open-Field Test. Wefurther examined whether NMDA lesioning of the NBM orthe drug treatment itself would influence the rats ability toperform spontaneous behavior in an open-field test (experi-ments were performed blindly). No significant effects of theNMDA lesion and of the treatment with A-705253 were re-corded at day 4 after the lesion. The different components of

ambulatory behavior, i.e., rearing, walking, and sniffing withhead movements, were not different from sham-treated con-trol levels (data are not shown). Time spent with groomingand immobility was also unaltered (Fig. 6). Ambulation ingeneral was not influenced by the different experimentalmanipulation. Therefore, the general condition to performspontaneous behavior was not significantly altered by thelesion and by drug treatment.

Calpain Inhibition Attenuated Cognitive DeficitsOccurring after Excitotoxic Lesion of the NucleusBasalis Magnocellularis. After habituation to the open-field box, attention was tested at day 6 (5 days after surgery;experiments were performed blindly). The results obtainedare shown in Fig. 7A. Spontaneous exploration of differentobjects placed in the open-field arena was not influenced bythe lesion or by the treatments. MK-801-treated rats ex-plored somewhat less, but this change was not significant.The objects were placed on the floor of the open field, andthus they could be approached by horizontal ambulation.This was not different among the groups.

Recognizing the novel object in contrast to the familiar onewas markedly decreased by the NMDA lesion (Fig. 7B).Treatment with A-705253 dose-dependently prevented thedeficit. ANOVA revealed a significant difference betweensham control and lesioned and A-705253-treated groups (p 0.005). The smallest dose of A-705253 was ineffective. Thedose of 3 mg/kg attenuated the effect of the NMDA lesion, butthe interference just approached statistical significance (p �0.074). However, performance of this group was closer to thevalues of sham controls (p � 0.104). The highest dose of 10mg/kg completely prevented the effect of lesion (p 0.005).The NMDA-lesioned group that was treated with MK-801also displayed an attention level to novel stimuli comparablewith the sham-lesioned controls.

Calpain Inhibition Did Not Affect NMDA Receptor-Dependent LTP. To examine whether general NMDA re-ceptor-mediated physiological functions and related intracel-

Fig. 4. Photomicrographs show the extent of activated microglia reactionat the level of brain injection indicated by the stained injection channelin representative rats of five different experimental groups: sham-oper-ated and vehicle-treated (sham-0), NMDA-treated and vehicle-injected(NMDA-0), NMDA-treated and A-705253-injected in three different dosesof 1, 3, and 10 mg/kg (NMDA-1, NMDA-3, and NMDA-10, respectively).For quantitative measurement of the extent of microglia activation, thetotal surface area was quantified that was infiltrated by activated micro-glia. In the right upper panel, individual activated microglia are shown tobe immunoreactive for CD11b with higher magnification (bar represents200 �m). The bar at the right lowest panel shows 1 mm.

Fig. 5. Attenuation of microglia activation by calpain inhibitor A-705253.Groups are sham-lesioned and NMDA-treated, and they received injec-tions with either the vehicle (0 mg) or the different doses of A-705253(1, 3, and 10 mg/kg) or MK-801 (MK). �, p 0.05 and ��, p 0.01 versussham-lesioned and saline-treated group; ns, not significant from thesham group; #, p 0.05 and ##, p 0.01 versus NMDA-injected andvehicle-treated groups.

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lular signaling cascades were affected by calpain inhibition,we analyzed LTP during administration of A-705253 in hip-pocampal slices (Fig. 8, A and B). Calpain inhibition by

A-705253 did not affect LTP at 100 nM, which is comparablewith brain levels of A-705253 at the dose of 10 mg/kg (Sup-plemental Fig. 1). We also tested the peptidic inhibitor ALLNfor possible effects on LTP (Fig. 9, A and B), but we did notdetect any LTP suppression. Basic synaptic transmissionwas also unaltered, as assessed by the input/output relation(Figs. 8C and 9C). To determine whether the LTP protocolused in this study was indeed NMDA receptor-dependent, weused the same LTP induction paradigm during treatmentwith the NMDA receptor blocker APV (Fig. 9D). LTP wascompletely blocked under this condition, indicating that theLTP induction with our tetanization paradigms in CA1 wasindeed NMDA receptor-dependent.

Calpain Was Activated by NMDA in HippocampalSlice Cultures and Dose-Dependently Inhibited byA-705253. To examine whether application of NMDA acti-vates calpain in neurons, and whether A-705253 sufficientlyinhibits the activated calpain, we cultured rat hippocampalslices and exposed them to NMDA in the presence or absenceof different concentrations of A-705253. Spectrin was cleavedunder 10 �M NMDA treatment into its spectrin degradationproducts of 150/145 kDa (Fig. 10A), indicating that calpainwas indeed activated in neurons treated with NMDA. Appli-cation of A-705253 dose-dependently prevented calpain acti-vation, as indicated by a decrease of SBDPs. The calculatedIC50 is 408 nM � 29 nM for free calpain inhibitor (consider-ing 93% protein binding for A-705253; Fig. 10B).

Fig. 6. Components of behavioral activity in small open field are shown.Ambulation expressed as a combined score of rearing, walking, andsniffing is depicted in A, grooming is depicted in B, and immobility isdepicted in C. Scores collected by behavioral sampling every 10 s for 25min are shown in blocks of 5 min. No statistically significant differencecould be obtained between groups.

Fig. 7. Exploratory behavior in the novel object recognition test. A, theduration of exploration of objects irrespective of their nature; B, therelative preference of novel object exploration compared with the familiarobject. The different treatments did not influence object exploration ingeneral. The NMDA lesion decreased attention toward the novel object,and A-705253 treatment interfered with the lesion effect. The dotted lineat 50% represents the chance level. xx, p 0.01 versus sham-operatedcontrol; �, p 0.05 and ��, p 0.01 versus NMDA-lesioned group.

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DiscussionExcitotoxic cell death has been discussed as a common

principle for several progressive neurological disorders (Lip-ton and Rosenberg, 1994; Harkany et al., 2000; Mattson,2004). Inadequate control over NMDA receptor stimulationcauses excessive calcium influx into neurons, activating asubset of intracellular Ca2�-dependent signaling cascades.As a result, affected cells perish, can eventually die, andconsequently the functionality of affected brain areas is im-paired. During this process, Ca2�-activated calpain cleavesseveral proteins that are involved in cell death signaling. Forexample, Wu et al. (2004) provide data that cleavage of cal-cineurin by calpain contributes to neuronal degeneration. Xuet al. (2007) could show that truncation of mGluR1� bycalpain is essential to excitotoxicity. Furthermore, collapsinresponse mediator-protein 3 triggers neuronal death aftercleavage by calpain (Hou et al., 2006). In a more chronicmodel, Kim et al. (2007) could show that degeneration ofcortical neurons in vitro depends on calpain cleavage of p35.If calpain is part of such degenerative cascades, then inhibi-tion of calpain should attenuate neuronal decline. Indeed,Caba et al. (2002) could show that inhibition of calpain wasneuroprotective in hippocampal slice cultures exposed toNMDA receptor overstimulation. In vivo, Chiu et al. (2005)revealed that calpain inhibition should reduce neurodegen-eration in the rat retina. For these reasons, calpain has been

discussed as a target for neurodegenerative disorders (Huangand Wang, 2001; Zatz and Starling, 2005; Saez et al., 2006).

The data presented here strongly suggest calpain thatinhibition by A-705253 is neuroprotective in a model ofNMDA-induced cholinergic cell lesion. After such cholinergicbasal forebrain damage by NMDA and subsequent cholin-ergic cortical denervation, learning behavior was compro-mised as assessed in a novel object recognition test, in whicha dose-dependent protective effect of A-705253 on behavioraldecline was demonstrated. Anatomical findings further con-firmed the neuroprotective effect of calpain inhibition. Boththe degree of cholinergic denervation in the parietal cortexand the extent of microglia activation around the injection inthe NBM were markedly and dose-dependently attenuatedby the inhibitor. The effective dose was shown to inhibitcalpain in vivo, as determined by experiments on tau phos-phorylation in the rat brain (data not shown). The presentfindings support the conclusion that inhibition of calpain notonly prevents neuronal decline in an animal model of excito-toxicity, but it also attenuates accompanying behavioral def-icits and neuropathology, including gliosis.

Most of the previous studies on calpain inhibition usedpeptidic inhibitors that are not specific for calpain. Further-more, due to low solubility, these compounds had to be ap-plied at high concentrations, which again may have resultedin unspecific side effects. The compound used in the present

Fig. 8. Effect of A-705253.13 (100 nM) on thepotentiation of the fEPSPs after a weak tetanus.A, averaged LTP time course. When applied 45min before until 10 min after a weak tetanus,A-705253.13 (100 nM) did not affect LTP elicitedby weak tetanization (control: n � 10 slices, 5animals; A-705253.13: n � 10 slices, 5 animals;p � 0.742, repeated measures ANOVA). B, origi-nal traces taken before (solid line) and after thetetanus (dotted line). C, input/output relation.Open circles, control; filled circles, 100 nMA-705253.

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study was recently introduced by Lubisch et al. (2003), and ithas a high inhibitory potency for calpain (Ki � 27 nM) with-out affecting the proteasome (Ki 10,000 nM) and caspases(Ki 10,000 nM). Therefore, the observed neuroprotectiveeffects are probably mediated by inhibition of calpain and notby reducing proteasome or caspase activity. This finding isimportant in light of the fact that the proteasome complexcontributes to a variety of neurodegenerative disorders (Ru-binsztein, 2006; Pan et al., 2008). A-705253 has previouslybeen shown to rapidly and effectively penetrate myocardialtissues, and it exerts its effects on cellular targets withinminutes after perfusion (Neuhof et al., 2003, 2004), making itan ideal compound for studying calpain in vivo.

Excitotoxicity-induced neurodegeneration is caused by anoveractivation of the NMDA receptor (Liu et al., 2007; vonEngelhardt et al., 2007). Consequently, blockade of theNMDA receptor has been widely discussed and tested as apharmacological target for excitotoxicity-induced neurode-generation (Harkany et al., 1999; Lipton 2004; Gardoni andDi Luca, 2006). However, one of the caveats of this approachis that a functional NMDA receptor is indispensable for avariety of physiological and behavioral brain functions, in-cluding synaptic plasticity, long-term potentiation, and mem-ory performance (Lynch, 2004; Fedulov et al., 2007), and it iswell established that blockade of the NMDA receptor impairscognition (Morris, 1989; Fedulov et al., 2007). In the study

Fig. 9. Effect ALLN and AP-V on the potentiation of the fEPSPs after a weak tetanus. A, averaged LTP time course under ALLN (left panel). WhenALLN (6 �M) was applied 60 min before until 120 min after tetanization (6 �M), LTP was not affected (dimethyl sulfoxide control: n � 10 slices, 5animals; ALLN: n � 10 slices, 5 animals; p � 0.230, repeated measures ANOVA). Right panel, input/output-relation. B, original traces taken before(solid line) and after the tetanus (dotted line). C, averaged LTP time course under APV. When APV (AP-V) (25 �M) was applied 20 min before until10 min after tetanization, LTP was completely suppressed (control: n � 5 slices, 3 animals; APV: n � 5 slices, 3 animals). Open circles, control; filledcircles, compound.

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presented here, calpain inhibition by A-705253 was similarlyeffective as MK-801 against the excitotoxic insult. This indi-cates that blocking the calpain-mediated downstream path-way in specific neurotoxic conditions can be as effective insuppressing the NMDA receptor. Therefore, we investigatedwhether calpain inhibition would also prevent or affect nor-mal physiological function of the NMDA receptor, as doesMK-801. To address this question, we examined LTP in thehippocampal CA1 region under exposure to A-705253. LTP atthe Schaffer collateral requires functional NMDA receptors(Harris et al., 1984; Reymann et al., 1989). We did not detectany LTP impairment during calpain inhibition, allowing theconclusion that normal NMDA receptor function and down-stream calcium-dependent mechanisms of synaptic plasticityare not affected by A-705253 treatment. The concentrationused effectively inhibited calpain in hippocampal slices aswell as cultured neurons, as determined by tau and dynamindegradation as well as tau hyperphosphorylation (data notshown). LTP during application of the peptidic calpain inhib-itor ALLN also yielded LTP equal to controls. To ensure thatour LTP protocol indeed induces NMDA receptor-dependentLTP, the NMDA blocker (2R)-amino-5-phosphonovaleric acid(APV) was applied under the same protocol. Under this con-dition, LTP was completely prevented. Therefore, it is un-likely that calpain is involved with all of the cascades inducedby NMDA receptor activation, and it may thus be a moreappropriate target for neuroprotection than the NMDA re-ceptor itself.

With the exception of traumatic brain injury and stroke,most neurodegenerative diseases progress slowly over a pe-riod of many years. The excitotoxic insult used in this modelinduced a more rapid decline, and behavioral deficits werealready measurable after days. At this point, it is not knownwhether calpain inhibition would also prevent a more chronicneurodegeneration of the nucleus basalis, as observed inAlzheimer’s disease. Further experiments are needed to eval-uate whether calpain inhibition can also counteract a chronicloss of cholinergic cells.

Taken together, this study shows that inhibition of calpain1) is sufficient to prevent NMDA-induced degeneration ofNBM and its associated cholinergic fiber projections, 2) at-tenuates behavioral deficits associated with such lesion, and3) does not interfere with the physiological role of the synap-tic NMDA receptor. These findings indicate that inhibitingcalpain could be an attractive neuroprotective strategy, with-out interfering with physiologically relevant NMDA receptor-related processes and its behavioral functions.

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Fig. 10. Spectrin cleavage in rat hippocampal slice cultures for thedetermination of calpain activity. A, immunoblot of slice cultures treatedwith 10 �M NMDA in the absence and presence of calpain inhibitorA-705253 (CI) using an antibody directed against spectrin and spectrindegradation products. The 150/145-kDa spectrin degradation bands areshown in the image. Control lanes are untreated cultures. B, IC50 forcalpain inhibition by A-705253 determined by graphical analysis of the150/145-kDa band signal intensities at different inhibitor concentrations.The NMDA band was used as 100%.

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Address correspondence to: Dr. Volker Nimmrich, Neuroscience Research,GPRD, Abbott, Knollstrasse, D-67061 Ludwigshafen, Germany. E-mail:volker.nimmrich@abbott.com

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