Distinct Mechanistic Roles of Calpain and Caspase Activation inNeurodegeneration as Revealed in Mice Overexpressing TheirSpecific Inhibitors*

Received for publication, January 25, 2005, and in revised form, February 4, 2005Published, JBC Papers in Press, February 7, 2005, DOI 10.1074/jbc.M500939200

Makoto Higuchi‡§¶, Masanori Tomioka‡§, Jiro Takano‡�, Keiro Shirotani‡, Nobuhisa Iwata‡,Hajime Masumoto‡, Masatoshi Maki‡�, Shigeyoshi Itohara**, and Takaomi C. Saido‡ ‡‡

From the ‡Laboratory for Proteolytic Neuroscience, RIKEN Brain Science Institute, Wako, Saitama 351-0198, �Laboratoryof Molecular and Cellular Regulation, Department of Applied Biological Sciences, School of Agricultural Sciences, NagoyaUniversity, Furo-cho, Chikusa-ku, Nagoya 464-8601, and **Laboratory for Behavioral Genetics, RIKEN Brain ScienceInstitute, Wako, Saitama 351-0198, Japan

Enzymatic proteolysis has been implicated in diverseneuropathological conditions, including acute/subacuteischemic brain injuries and chronic neurodegenera-tion such as Alzheimer disease and Parkinson disease.Calcium-dependent proteases, calpains, have been in-tensively analyzed in relation to these pathological con-ditions, but in vivo experiments have been hampered bythe lack of appropriate experimental systems for a se-lective regulation of the calpain activity in animals.Here we have generated transgenic (Tg) mice that over-express human calpastatin, a specific and the only nat-ural inhibitor of calpains. In order to clarify the distinctroles of these cell death-associated cysteine proteases,we dissected neurodegenerative changes in these micetogether with Tg mice overexpressing a viral inhibitorof caspases after intrahippocampal injection of kainicacid (KA), an inducer of neuronal excitotoxicity. Immu-nohistochemical analyses using endo-specific antibod-ies against calpain- and caspase-cleaved cytoskeletalcomponents revealed that preclusion of KA-induced cal-pain activation can rescue the hippocampal neuronsfrom disruption of the neuritic cytoskeletons, whereascaspase suppression has no overt effect on the neuriticpathologies. In addition, progressive neuronal loss be-tween the acute and subacute phases of KA-induced in-jury was largely halted only in human calpastatin Tgmice. The animal models and experimental paradigmemployed here unequivocally demonstrate their useful-ness for clarifying the distinct contribution of calpainand caspase systems to molecular mechanisms govern-ing neurodegeneration in adult brains, and our resultsindicate the potentials of specific calpain inhibitors inameliorating excitotoxic neuronal damages.

Diverse proteolytic enzymes have been indicated to mediatemolecular processes of neurodegeneration (1) because axonal,dendritic, and synaptic integrity is targeted by protease activ-ities provoked by different types of insults (2–4). A family of

nonlysosomal calcium-activated neutral cysteine proteases, re-ferred to as calpain family (�- and m-calpain isoforms in thepresent study), has been mechanistically implicated in theregulation of various cellular functions (5, 6). Because multiplelines of evidence indicate that neuronal cytoskeletal constitu-ents, including microtubule-associated proteins (MAPs),1 neu-rofilaments, spectrin, and actin, are preferred substrates forcalpain (7–11), calpain is likely to play essential roles in thepathophysiological derangement of cytoskeletons. Acute andsubacute brain insults such as traumatic injury and hypoxia/ischemia involve prominent calpain activation evoked by cal-cium dysregulation in neurons. Moreover, several independentstudies have highlighted the elevation of calpain activity inchronic neurodegenerative disorders including Alzheimer dis-ease (12, 13) and Parkinson disease (14). However, it remainsunclear whether the relationship between calpain activationand the disorganization of the neuronal cytoskeletons in thesepathologies is causal or coincidental.

To our knowledge, there has been a relatively small amountof in vivo information for the molecular consequences of calpainactivation in neurons. Although attempts to intervene in cal-pain activation in experimental animal models of head trauma(15), ischemia (16), and several other brain pathologies havebeen made, there have been the following limitations in mod-ulating calpain activity (1): knock-out mice lacking the 30-kDasmall regulatory subunit of calpain suffer embryonic lethality(17, 18), indicating difficulty in direct reverse-genetic suppres-sion of calpain expression (2); and all synthetic peptidic, pep-tide-mimetic, and nonpeptidic calpain inhibitors currentlyavailable have problems in terms of specificity, metabolic sta-bility, water-solubility, or penetration through the blood-brainbarrier (19, 20).

Calpastatin (CAST), the only natural inhibitor of both �- andm-calpains (21, 22), is a potentially valuable tool to investigatethe significance of calpain activation in living animals. A recentstudy, utilizing adenovirus-mediated overexpression of CAST forsuppressing the calpain activity in an experimental mouse modelof Parkinson disease, demonstrated protective effects of locallyoverexpressed CAST on striatal neurons (14). However, genera-tion of genetically engineered mice carrying the CAST transgeneis more advantageous in accomplishing a stable and persistent

* This work was supported by a research grant from RIKEN BSI andfrom the Ministry of Education, Culture, Sports, Science, and Technol-ogy. The costs of publication of this article were defrayed in part by thepayment of page charges. This article must therefore be hereby marked“advertisement” in accordance with 18 U.S.C. Section 1734 solely toindicate this fact.

§ Both authors contributed equally to this work.¶ To whom correspondence may be addressed. Tel.: 81-48-462-1111;

Fax: 81-48-467-9716; E-mail: mhiguchi@brain.riken.jp.‡‡ To whom correspondence may be addressed. Tel.: 81-48-467-9715;

Fax: 81-48-467-9716; E-mail: saido@brain.riken.jp.

1 The abbreviations used are: MAPs, microtubule-associated pro-teins; CAST, calpastatin; hCAST, human CAST; KA, kainic acid;mCAST: murine CAST; LSD, least squares difference; MTs, microtu-bules; NF, neurofilament; nTg, nontransgenic; Tg, transgenic; VIC,viral inhibitor of caspases; TUNEL, transferase-mediated dUTP nick-end labeling; ANOVA, analysis of variance.

THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 280, No. 15, Issue of April 15, pp. 15229–15237, 2005© 2005 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in U.S.A.

This paper is available on line at http://www.jbc.org 15229

at NO

UK

AG

AK

U S

OG

O K

EN

KY

U C

TR

on August 2, 2006

ww

w.jbc.org

Dow

nloaded from

overexpression of CAST in broad areas of the mouse brain. Suchtransgenic (Tg) mice could also be cross-bred with numerousmodels of neurodegenerative disorders to examine the contribu-tion of calpain activation to various neuropathologies. We re-cently generated Tg mice overexpressing a viral inhibitor ofcaspases (VIC), and we assessed the effects of caspase inhibitionon neuronal loss induced by a local injection of kainic acid (KA),an agonist for excitatory amino acid receptors (23). A similarexperimental paradigm may be applicable to investigating thepathological role of the calpain-CAST system.

In the present study, we have generated Tg mice overex-pressing human CAST (hCAST) and raised antibodies to cal-pain- and caspase-cleaved cytoskeletal constituents, which canbe employed to immunohistochemically visualize the activitiesof these proteases using both frozen and paraffin sections. ThehCAST and VIC Tg mice and these antibodies permitted us toestablish an experimental paradigm that is ideal for distinc-tively analyzing the mechanistic roles of calpain and caspaseactivation. Whereas both the VIC and hCAST transgenes weredriven by the same neuron-specific calcium/calmodulin-dependent protein kinase-II � subunit promoter (24), only thehCAST Tg mice showed a significant preservation of the neu-ritic cytoskeletons and an attenuation of progressive neuronalloss between the acute and subacute phases following the KAchallenge. In contrast, there was little difference in the patho-logical consequences of KA injection between nontransgenic(nTg) and VIC Tg mice. These findings support the view thatthe calpains-CAST system is critically involved in the molecu-lar events taking place in excitotoxicity-induced neuropathol-ogy in adult brains, and also demonstrate the usefulness of ourexperimental model for obtaining insights into the mechanismsgoverning a wide range of neurodegenerative disorders.

MATERIALS AND METHODS

Tg Mice Overexpressing hCAST and VIC—The hCAST cDNA (25, 26)was cloned into pNN265, from which the NotI fragment was subclonedinto pMM403 containing the calcium/calmodulin-dependent protein ki-nase-II � promoter (27). The SfiI-linearized DNA construct was micro-injected into the pronucleus of C57BL/6Cr zygotes, which were trans-ferred into foster females to create hCAST Tg founders. The genotypewas determined by PCR with primers 5�-CATGAACCACAGACAGCT-TGGTTGAC-3� and 5�-GGAGGATTTGATATTCACCTGGCCCG-3� thatyield a 350-bp product. The VIC Tg mice were described previously (23).We used the line of mice with the highest expression of VIC for thepresent study.

Intrahippocampal Injection of KA—Administration of KA (1 nmol)into the hippocampal CA1 region of 6-month-old nTg, VIC Tg (23), andhCAST Tg mice was performed as described previously (23). Transcar-dial perfusion of the mice for biochemical and neuropathological studieswas performed 4 and 24 h and 7 days after the KA injection. All theanimal experiments were performed in accordance with our institu-tional guidelines.

Western Blot Analysis of hCAST Expression in the Brain—Levels ofTg hCAST protein in different brain regions of untreated nTg and Tgmice were examined by means of immunoblot analysis. The brain tissuesamples taken from the mice were homogenized in 10 ml/g tissueice-cold lysis buffer (50 mM Tris-HCl (pH 8.0) containing 150 mM NaCl,50 mM EDTA, 1% Triton X-100, and protease/phosphatase inhibitormixture) and centrifuged at 14,000 � g for 40 min at 4 °C. Equalamounts (10 �g of protein) of the resultant supernatants were employedfor immunoblot analysis with the monoclonal anti-hCAST antibody.

Histochemical and Immunohistochemical Studies—Mice under deepanesthesia were transcardially perfused with 15 ml of ice-cold PBS,followed by 20 ml of 4% paraformaldehyde in phosphate buffer (23).4-�m-thick paraffin sections and 20-�m-thick frozen sections of thebrains were immunostained based on either a standard protocol forimmunofluorescence staining (23) or the tyramide signal amplificationmethod with a TSA-Direct kit (PerkinElmer Life Sciences) using anti-bodies described below. We employed the staining techniques based onthe method described by Wang et al. (28). for double or triple fluores-cence labeling. Viability of the hippocampal neurons was studied byNeuN immunostaining; cell morphology and DNA fragmentation were

monitored by cresyl violet staining and transferase-mediated dUTPnick-end labeling (TUNEL) assay (Apoptosis Detection System; Pro-mega, Madison, WI), respectively.

Antibodies and Dilutions—Rabbit polyclonal antibody against cal-pain-generated C-terminal fragment of �-spectrin, the 150-kDa frag-ment (1:200 dilution) (29), was used for monitoring the magnitude of thecalpain activity. In addition, we developed rabbit polyclonal antibodiesto the calpain-generated N-terminal fragment of �-spectrin, the 136-kDa fragment (1:2,000 dilution), and the caspase-generated actin frag-ment, clact32 (1:2,000 dilution), using the synthetic peptidesCQQQEVY and CYELPD, respectively (30, 31), as described previously(29). We also raised a polyclonal antibody against mCAST using thesynthetic peptide CKKTEEVSKPKAKEDARHS. We used a mousemonoclonal antibody to hCAST (CSL1–5; 1:1,000 dilution; TakaraBiomed). Mouse monoclonal antibodies to tau used here include T49(1:500 dilution; murine tau-specific, phosphorylation-independent) (32)and AT8 (1:2,000 dilution; phosphorylated tau-specific; Innogenetics)(33). The following antibodies to neurofilament (NF) subunits wereemployed for immunostaining: mouse monoclonal RMdO9 to nonphos-phorylated NFH (1:500 dilution) (34); RMO24 to phosphorylated NFH(1:500 dilution) (34); RMO189 to phosphorylated and nonphosphoryl-ated NFM (1:500 dilution) (34); and rabbit antisera against NFL (aNFL;1:1,000 dilution) (35). Other antibodies used in the present study are asfollows: mouse monoclonal antibodies to MAP2 (AP20; 1:10 dilution;Roche Diagnostics), �-tubulin (DM1A; 1:1,000 dilution; Sigma), synap-tophysin (1:50 dilution; Progen Biotechnik), and NeuN (1:2,000 dilu-tion; Chemicon); and rabbit polyclonal antibodies to SV2A (1:100 dilu-tion; Calbiochem-Novabiochem), GluR1 (1:1,000 dilution; Chemicon),mGluR2/3 (1:1,000 dilution; Chemicon), and Cdk5 activator p25/p35(C19; 1:1,000 dilution; Santa Cruz Biotechnology).

Statistical Analysis—The significance of differences between groupswas examined by means of either t statistics or multiple comparisons byFisher’s LSD test following ANOVA.

RESULTS

The hCAST Tg mice robustly expressed the transgene-de-rived protein in the forebrain regions. One of the founders thatstably expressed hCAST showed the highest copy number oftransgenes and the highest level of hCAST protein expression(Fig. 1A). This line of mice (L7) was used for the followingexperiments. The regional distribution of the overexpressedhCAST was analyzed by immunohistochemistry with anti-hCAST antibody (Fig. 1, B and C). The brain calpastatin activ-ity in biochemical terms, defined as the activity to inhibitpurified bovine m-calpain activity (36), was 3.05 times greaterin the Tg mice than in the nTg controls. The transgene-derivedhCAST was highly expressed in neurons of the hippocampalformation and neocortex (Fig. 1C). Similar to the endogenousmCAST (Fig. 1D), hCAST was present mainly in the neuronalcell bodies, dendrites, and synaptic terminals (Fig. 1E). Doubleimmunolabeling clearly demonstrates the high level expressionof hCAST in the presynaptic terminals (Fig. 1F) and dendrites(Fig. 1G).

None of the hCAST Tg mice exhibited marked motor orbehavioral impairments at any age up to 15 months. In addi-tion, there was no significant difference in body weight orlongevity between the Tg and nTg mice. Immunohistochemicalanalyses for cytoskeletal components and synaptic markers(see “Materials and Methods”) also revealed no pronouncedabnormalities in the amounts and distributions of these mole-cules in the brain of Tg mice at any age.

KA-induced calpain activation was prominently suppressedin the hippocampus of hCAST Tg mice (Fig. 2). Immunohisto-chemistry using antibodies to the 150-kDa fragment (data notshown) and the 136-kDa fragment (Fig. 2, A and B) of calpain-cleaved �-spectrin (fodrin) revealed calpain activation in thehippocampal CA1 neurons 4 h after the KA injection. The136-kDa fragment immunoreactivity in the ipsilateral hip-pocampus of the hCAST mice was significantly decreased by63% in comparison with that of the nTg mice (Fig. 2E).

Caspase activation visualized by means of immunostainingfor a 32-kDa fragment of caspase-cleaved actin, fractin

Calpain and Caspase Activation in Excitotoxicity15230

at NO

UK

AG

AK

U S

OG

O K

EN

KY

U C

TR

on August 2, 2006

ww

w.jbc.org

Dow

nloaded from

(clact32), was observed primarily in the CA3 sector 4 h after theKA injection (Fig. 2B) and spread to the CA1 region by 24 h(Fig. 2, G and H). The clact32 immunoreactivity representsactivation of the caspase cascade (30). The calpain activationpreceded the caspase activation in the CA1 region, and thehCAST Tg mice exhibited diminution of not only the 136-kDafragment signals but also clact32 staining (Fig. 2, F and I),implying a requirement of calpain activation for the caspaseactivation in the CA1 neurons. The VIC Tg mice showed amarked suppression of caspase activity, but not calpain activityat all, in the hippocampus at every time point when comparedwith the nTg mice (Fig. 2, D–F and J).

Overexpression of hCAST also halted excitotoxicity-inducedprogressive loss of hippocampal neurons. The majority of thehippocampal CA1 and CA3 neurons exhibited apparent atrophyand pyknosis as shown by cresyl violet staining 24 h after the KAadministration (Fig. 3, A–C). No significant difference in thenumber of pyknotic neurons was observed among the nTg,hCAST Tg, and VIC Tg mice at this time point, although shrink-age of the neuronal cytoplasm was less severe in the hCAST Tgmice than in the nTg and VIC mice. Additionally, there was nosignificant difference in the number of TUNEL-positive neuronsamong the three groups of mice at any time point after the KAtreatment (data not shown). The CA1 region of the three groupsof mice also showed similar reductions of NeuN immunoreactiv-ity (Fig. 3, D–F and J) 24 h after the KA injection. In contrast,

there was a striking difference in the amount of NeuN-positivenuclei 7 days after the KA treatment (Fig. 3, G–I and J). Notably,the hCAST Tg mice did not show any progression of the NeuNdecline from 24 h to 7 days, unlike the nTg and VIC Tg mice.These results indicate that inhibition of calpains, but notcaspases, protected hippocampal neurons against KA-triggeredsubacute death, whereas the suppression of calpain or caspaseactivities did not modulate acute excitotoxic neuronal degenera-tion in the present experimental paradigm.

Dendrites and axons in hCAST Tg mice retained their integ-rities in the course of KA-induced neurodegeneration in a man-ner much better than those in nTg and VIC Tg mice (Fig. 4).

FIG. 1. Transgenic expression of hCAST in mice. A, Western blotanalyses with anti-hCAST (upper panel) and anti-murine CAST(mCAST) (lower panel) antibodies for the protein samples extractedfrom the brains of nTg and four different lines of hCAST Tg mice. Noremarkable difference in the level of endogenous mCAST was foundamong all the mice analyzed. B and C, immunofluorescence observa-tions of nTg (B) and hCAST Tg (C) mouse brain sections with ananti-hCAST antibody. High levels of hCAST expression were observedin the hippocampus and neocortex of Tg mice. D and E, localization ofendogenous mCAST (D) and transgene-derived hCAST (E) in the den-tate gyrus. Immunofluorescence signals of hCAST were more concen-trated in soma than processes as compared with those of mCAST. ThemCAST immunoreactivity was particularly intense in the middle mo-lecular layer, whereas hCAST immunoreactivity was more prominentin the outer molecular layer. F and G, confocal images of double immu-nofluorescence staining with a combination of antibodies againsthCAST and presynaptic marker SV2 (F) or dendritic marker MAP2 (G)for sections from the CA3 (F) and CA1 (G) sectors of hCAST Tg. Thetransgene-derived hCAST was abundantly present in the presynapticterminals (F) and dendrites (G). Scale bars: 1000 �m (B and C) and 100�m (D–G).

FIG. 2. Calpain- and caspase-catalyzed proteolysis in nTg,hCAST Tg, and VIC Tg mice in early stages after intrahippocam-pal KA injection. A–D, double immunofluorescence staining with theanti-136-kDa fragment (green) and anti-clact32 (red) antibodies forsections of nTg (A and B), hCAST Tg (C), and VIC Tg (D) mousehippocampal formations 4 h after injection of PBS (A) or KA (B–D). TheCA1 sector of nTg mice exhibited marked calpain-catalyzed proteolysisindicated by the 136-kDa fragment immunoreactivity, whereascaspase-catalyzed fractin formation was localized primarily to the CA3region (B). Relative to nTg mice, calpain activation was attenuated inhCAST Tg mice (C); VIC Tg mice showed suppression of caspase acti-vation (D). E, quantification of the intensities of the 136-kDa fragment(green columns) and clact32 (red columns) immunofluorescence signalsin the whole hippocampal formations of nTg, hCAST Tg, and VIC Tgmice 4 h after the KA challenge (n � 3 in each group). Data werenormalized against the mean value given by KA-challenged nTg mice.The quantification was performed within a linear range. F, intensitiesof clact32 immunostaining signals in the CA1 region 4 (pink columns)and 24 h (red columns) after KA injection (n � 3 in each group). Datawere normalized against the mean value given by nTg mice at 4 h. G–J,confocal images of double immunolabeling with the anti-136-kDa frag-ment (green) and anti-clact32 (red) antibodies for the CA1 region of nTg(G and H), hCAST Tg (I), and VIC Tg (J) mice 4 (G) and 24 h (H–J) afterKA injection. Calpain activation preceded caspase activation (G), andthereafter caspase activation emerged, partially colocalizing in thesoma and dendrites in nTg mice (H). Reduced signals of both the136-kDa fragment and clact32 were observed in hCAST Tg mice (I). InVIC Tg mice, calpain was activated in a manner similar to that in nTgmice, whereas caspase activation was prominently attenuated (J). Ver-tical line represents S.E. Scale bars, 300 �m (A–D) and 75 �m (G–J). *,p � 0.05; **, p � 0.01 versus nTg mice by ANOVA/LSD.

Calpain and Caspase Activation in Excitotoxicity 15231

at NO

UK

AG

AK

U S

OG

O K

EN

KY

U C

TR

on August 2, 2006

ww

w.jbc.org

Dow

nloaded from

FIG. 3. Protective effect of overexpressed hCAST on KA-induced subacute death of hippocampal neurons. A–C, cresyl violet stainingof the CA1 neurons in nTg (A and B) and hCAST Tg (C) mice 24 h after PBS (A) or KA (B and C) treatment. The majority of neurons in KA-treatednTg mice exhibited apparent atrophy and pyknosis (B). Although the neurons in hCAST Tg mice also became atrophic, shrinkage of soma was lesssevere than in nTg mice (C). D–I, NeuN immunostaining of the CA1 sector (D and E) and dentate gyros (G–I) in PBS-treated (D and G) andKA-treated (E and H) nTg and KA-treated hCAST Tg (F and I) mice 24 h (D–F) and 7 days (G–I) after injection. J, quantification of NeuNimmunoreactivity in CA1 neurons from nTg, hCAST Tg, and VIC Tg mice 24 h (black columns) and 7 days (gray columns) after KA treatment (n �3 in each group). Vertical bars represent S.E. Scale bars, 50 �m (A–C) and 250 �m (D–I). **, p � 0.01 versus nTg mice by ANOVA/LSD.

Calpain and Caspase Activation in Excitotoxicity15232

at NO

UK

AG

AK

U S

OG

O K

EN

KY

U C

TR

on August 2, 2006

ww

w.jbc.org

Dow

nloaded from

FIG. 4. Attenuation of KA-induced dendritic and axonal degenerations in hCAST Tg mice. A–L, immunofluorescence staining for MAP2(antibody AP12) in the ipsilateral hippocampus (left panels) and for total tau (antibody T49) in the ipsilateral (middle panels) and contralateral(right panels) hippocampi from nTg (A–F), hCAST Tg (G–I), and VIC Tg (J–L) mice 24 h (left and middle panels) and 7 days (right panels) afterinjection of PBS (A–C) or KA (D–L). M–O, quantitative analyses of MAP2 (M) and tau (N and O) immunoreactivities in the ipsilateral (M and N)and contralateral (O) hippocampal formations of nTg, hCAST Tg, and VIC Tg mice 24 h (black columns) and 7 days (gray columns) after KAinjection (n � 5 in each group). Data were normalized against the mean values given by PBS-treated nTg mice. The vertical bars represent S.E.Scale bar, 700 �m (A–L). *, p � 0.05; **, p � 0.01 versus KA-treated nTg mice by ANOVA/LSD.

Calpain and Caspase Activation in Excitotoxicity 15233

at NO

UK

AG

AK

U S

OG

O K

EN

KY

U C

TR

on August 2, 2006

ww

w.jbc.org

Dow

nloaded from

Although the dendritic marker, MAP2, showed a pronouncedreduction in the entire hippocampal formation, except for thedentate gyrus, of the nTg and VIC Tg mice 24 h after the KAinjection, the reduction of MAP2 immunoreactivity in thehCAST Tg mouse hippocampus was significantly smaller (leftpanels in Fig. 4). The retention of the MAP2 immunoreactivityin the hCAST Tg mice was also observed 7 days after thetreatment. Although the tau immunoreactivity in the hip-pocampal axons on the injection side decreased to a lesserextent than the MAP2 signals (middle panels in Fig. 4), theentire hippocampal formation of the nTg and VIC Tg miceexhibited a remarkable and progressive decrease in the tauimmunoreactivity after the KA treatment. In contrast to thesetwo groups of mice, no marked reduction of the tau immuno-staining was found in the hCAST Tg mice. None of the miceshowed marked alterations in the tau immunoreactivity in thecontralateral hippocampus 24 h after the KA administration,whereas the intensity of the tau staining was significantlyattenuated 7 days after the treatment (right panels in Fig. 4).Taking these observations together, suppression of the KA-induced calpain activation is likely to have rescued neurons ofthe entire hippocampal formation from both acute and sub-acute cytoskeletal degeneration of the dendrites and axons.However, subacute loss of axonal tau in the contralateral hip-pocampus, presumably caused by Wallerian degeneration, wasnot overtly inhibited by attenuation of calpain activity.

We next quantified the levels of the cytoskeletal components24 h after the KA injection by Western blotting (Fig. 5A). The

136-kDa fragment immunoreactivity was markedly elevated inthe ipsilateral hippocampal samples from the nTg and hCASTTg mice, and the hCAST Tg mice showed 32% reduction in the136-kDa fragment signal intensity as compared with the nTgmice, consistent with the immunohistochemical observations.The appearance of the 136-kDa fragment signal in the con-tralateral hippocampus was also significantly lower in thehCAST Tg mice than in the nTg mice. In accordance with theimmunohistochemical data, the reduction of the MAP2 signalin the ipsilateral hippocampus was suppressed in the hCASTTg mice. The T49 level, representing total tau, in the ipsilateralhippocampus also remained preserved in the hCAST Tg mice incomparison with the nTg mice. The levels of phosphorylatedtau proteins assessed by AT8 immunostaining were elevated inboth of the nTg and hCAST Tg mice, and the ratio of phospho-rylated tau to total tau (AT8/T49) also showed a marked in-crease in the nTg mice (Fig. 5B). Consistently, the increase inthe AT8/T49 ratio was �30% smaller in the hCAST Tg than inthe nTg mice.

We then investigated proteolytic cleavage of p35, the modu-lator of cyclin-dependent kinase 5 (Cdk5), to p25, since Cdk5 isone of the kinases capable of phosphorylating tau (37, 38). Thep25 signal was increased in the KA-treated mice (Fig. 5A), andthe ratio of p25 to p35 was significantly elevated in the ipsilat-eral hippocampus (Fig. 5C). Notably, the p25/p35 ratio of thenTg mice was �2-fold greater than that in the hCAST Tg mice.Hence, the conversion of p35 to p25 may be promoted by theKA-induced calpain activation, giving rise to the enhanced tau

FIG. 5. Dual impacts of calpain activation on biochemical properties of tau. A, Western blot analyses for the 136-kDa fragment, MAP2a/b(AP20), total tau (T49), phosphorylated tau (AT8), and Cdk5 activator p25/p35 using protein samples extracted from ipsilateral (i) andcontralateral (c) hippocampal formations of nTg and KA-treated hCAST Tg mice 24 h after treatment with PBS or KA. B and C, ratio of AT8 toT49 (B) and ratio of p25 to p35 (C) in the ipsilateral (closed columns) and contralateral (open columns) hippocampi based on densitometricquantification of immunoblot signals (n � 3 in each group). The ratios indicate the relative incidence of phosphorylation and limited proteolysisof tau and p35 proteins per molecule at this time point, respectively. The quantification was performed within a linear range. Vertical barsrepresent S.E. Data for tau phosphorylation are normalized against the mean value given by the PBS-treated mice. D and E, AT8 immunofluo-rescence staining in the CA1 neurons of nTg mice 24 h after PBS (D) or KA (E) treatment. KA-induced calpain activation not only reduced the totalamount of tau but also promoted phosphorylation of remaining tau. Scale bar, 50 �m (D and E). *, p � 0.05; **, p � 0.01 versus PBS-treated nTgmice by ANOVA/LSD.

Calpain and Caspase Activation in Excitotoxicity15234

at NO

UK

AG

AK

U S

OG

O K

EN

KY

U C

TR

on August 2, 2006

ww

w.jbc.org

Dow

nloaded from

phosphorylation by activated Cdk5. Consistently, cytoplasmicAT8 immunoreactivity, representing tau phosphorylation, in-creased in the CA1 and CA3 neurons of the KA-treated mice(Fig. 5, D and E), implying that the phosphorylated tau re-mained unbound to MTs and may consequently accumulate inthe neuronal soma.

Double immunolabeling also revealed apparent dendritic co-localization of calpain activity with MAP2 in contrast to itslimited colocalization with tau only in a small subset of axons(Fig. 6), providing a plausible explanation for the observationthat progression of axonal cytoskeletal disorganization wasmuch slower than that of dendritic disruption (see Fig. 7).Furthermore, the nTg mice exhibited a remarkable KA-inducedreduction of endogenous mCAST in the CA1 region, where adrastic elevation of the calpain activity and substantial loss ofMAP2 took place (Fig. 6). In the hCAST Tg mice, calpainactivation as well as dendritic pathology was largely sup-pressed in the areas where hCAST was abundantly expressed.Hence, the balance between calpain and CAST activities seemsto primarily regulate cytoskeletal derangements during excito-toxic neuronal insults.

DISCUSSION

The central nervous system is highly vulnerable to both non-apoptotic and apoptotic insults mediated by calpains andcaspases, because various protein components of neuronal pro-cesses and synapses are substrates upon which these proteolyticenzymes act (39). Disruption of calcium homeostasis has beenimplicated in neuronal injuries, and thus roles of calpains,strictly regulated by intracellular calcium concentrations, haveattracted particular research interest. In fact, vast arrays ofexperiments have revealed participation of calpain activation inapoptotic (1, 40, 41) as well as nonapoptotic (41–44) neuronaldeath. However, precise molecular mechanisms, primarily gov-erned by calpain activation, in neurodegenerative processes re-main elusive. In this study, the construction of an experimentalparadigm using genetically engineered mice and proteolyticproduct-specific antibodies to monitor calpain and caspase acti-vation enabled us to obtain direct evidence for distinct mechanis-tic roles of these enzymes in excitotoxicity-induced neuronal in-jury in vivo. It is noteworthy that calpain-mediated cytoskeletaldisruption in neurites led to subacute cell death. In addition,stable overexpression of hCAST and VIC in mice without emer-gence of unfavorable phenotypes indicates the potential useful-ness of the hCAST and VIC Tg mice and the endo-specific anti-bodies for unraveling involvement of calpain and caspasesystems in a broad range of pathological circumstances.

In our observations, the cytoskeletal disorganization wasmost prominent in the dendrites, where calpain was activatedmost extensively. Furthermore, this dendritic degenerationwas markedly suppressed by the overexpressed hCAST, whichwas also abundantly present in the dendrites, indicating adirect contribution of the calpain-CAST system to the disrup-tion of the dendritic structural components. The decrease ofMAP2 immunoreactivity exceeded neuronal loss assessed byNeuN staining in the nTg hippocampus at 24 h (96 versus 72%of total CA1 neurons). This finding indicates that the loss ofdendritic cytoskeleton did not arise simply as a consequence ofthe neuronal loss but rather preceded apoptotic events, there-after exerting profound effects on the survival of the cells in asubacute stage. There also was a significant diminution of theaxonal damage by the hCAST overexpression, suggesting thatthe calpain-CAST system also functions as a mediator of theaxonal derangement. Most interestingly, excitotoxic treatmentresulted in both an increase in axonal tau phosphorylation anda decrease in total tau quantity (Fig. 5). Conceivably, both ofthese changes accelerated disorganization of the axonal MTs

because tau phosphorylation lowers its affinity to MTs andthus destabilizes the MT assembly and dynamics (4). Thisphosphorylation is likely to be facilitated by an elevated level ofCdk5 activator p25 that is produced by calpain-mediated cleav-age of p35 (37, 38). Mobilization of the calpain-p35/p25-Cdk5cascade suggested here was implicated previously in the phos-phorylation of tau in excitotoxic neuronal injury (45). As our

FIG. 6. Inverse correlation between KA-induced calpain acti-vation and hCAST overexpression. A–C, immunofluorescence stain-ing showing the 136-kDa fragment (136-kf) (A), MAP2 (B), and merged(C) images in the hippocampus of nTg mouse 24 h after KA treatment.Calpain activation, indicated by the specific limited proteolysis of�-spectrin, was spatially associated with severe loss of MAP2 immuno-reactivity. D–F, confocal images of the 136-kDa fragment/MAP2 (D andE) and 136-kDa fragment/tau (F) double immunofluorescence observa-tions in nTg mice 4 (D and F) and 24 h (E) after the KA challenge. At4 h, the 136-kDa fragment signals (red) colocalized well with MAP2(green in D) but only partially with tau (green in F). Prominent loss ofMAP2 immunostaining was observed 24 h after KA treatment (green inE), whereas the 136-kDa fragment staining remained intense (red in E).G–L, confocal photomicrographs of triple immunolabeling for the 136-kDa fragment (G and J), MAP2 (H and K), and either of mCAST (I) orhCAST (L) in the CA1 neurons of nTg (G–I) and hCAST Tg (J–L) mice24 h after KA injection. Prominent loss of MAP2 signals in nTg mice (H)was closely associated with calpain activation (G) and mCAST reduc-tion (I) as compared with the PBS-treated nTg control (inset in I). Incontrast, hCAST Tg mice exhibited retained hCAST signals (L) closelyassociated with suppressed calpain activation (J) and preserved MAP2immunoreactivity (K). Scale bars, 300 �m (A–C) and 50 �m (D–L).

Calpain and Caspase Activation in Excitotoxicity 15235

at NO

UK

AG

AK

U S

OG

O K

EN

KY

U C

TR

on August 2, 2006

ww

w.jbc.org

Dow

nloaded from

observations indicate that the somato-dendritic areas show thehighest level of calpain activation relative to other cellularcompartments, proteolytic degradation and hyper-phosphoryl-ation of tau may take place primarily in the soma, resulting indepletion of axonal tau that should be constantly supplied fromthe soma. This can explain why the axonal cytoskeletal disrup-tion progressed more slowly than the dendritic degeneration(Fig. 4).

Incidentally, administration of KA at the dosage employed inthe present study (1 nmol) into CAST-knock-out mouse brains(46) resulted in acute death of the mice, whereas the wild-type(nTg) and Tg mice remained alive, allowing the subsequenthistochemical and biochemical analyses of the brain after thetreatment. In the present study, use of this relatively high doseof KA was necessary to induce cytoskeletal disorganizationparticularly in axons. We therefore did not employ CAST-knock-out mice here. The present experimental protocol may inpart mimic the chronic axonal degeneration that arises in anumber of neurological diseases accompanying tauopathy be-cause we were able to observe delayed neurodegeneration andtau hyperphosphorylation (Figs. 3 and 4).

The cytoskeletal disruption arose within 24 h after the KAtreatment and progressed during the next 6 days in the nTgmice, whereas the hCAST Tg mice showed relatively well sus-tained dendritic and axonal integrity. Additionally, we ob-served inexorable subacute loss of the CA1 neurons from 24 hto 7 days after the treatment in the nTg and VIC Tg mice butnot in the hCAST Tg mice. Taken together with the observationthat there was no difference in the number of TUNEL-positiveneurons among the nTg, hCAST Tg, and VIC Tg mice after theKA challenge, these results demonstrate that subacute neuro-nal death involves calpain-mediated cytoskeletal disorganiza-tion independently of DNA fragmentation.

The caspase activation in the soma of CA1 neurons appearedto arise partly as a consequence of the calpain activation be-cause the clact32 immunoreactivity partially colocalizing with

the 136-kDa fragment signals was reduced by the hCAST over-expression. This is indicative of the cross-talk(s) between cal-pain and caspase systems in the Ca2�-induced cellular injuryas demonstrated recently by using primary neurons (45, 47).Notably, the suppression of the caspase activation in the VICTg mice did not rescue the CA1 neurons from subacute death;the pathological significance of the caspase activation in thisregion remains uncertain. Moreover, the cytoskeletal compo-nents in the VIC Tg mice were disrupted in a manner similar tothose in the nTg mice, indicating that caspases do not playmajor roles in the disorganization of cytoskeletal networksfollowing excitotoxic insults despite the previous knowledgethat the dendritic and axonal constituents such as tau havebeen considered as preferred substrates for caspases (48, 49).

In the experimental paradigm employed here, the roles ofcalpain and caspase systems in the machinery of apoptoticneuronal death remain unsettled. The TUNEL-stained cellsseen 24 h after the KA challenge accounted for �45% of thetotal neurons in the hippocampus (Fig. 3). Therefore, the acuteinsult, induced by a relatively high dose of KA, was presumablytoo extensive to be modulated by suppression of calpain orcaspase activity. We may also need to consider the potentialphysiological functions of calpain under excitotoxic conditions;calpain may play a protective role in the early phase of theexcitotoxicity-induced neuronal damage at least in vitro (50,51). Inhibition of calpain activation by the overexpressingCAST may thus not necessarily behave protectively in acuteneuronal injury. This possibility should be further addressedby more intensive investigations using in vivo paradigms.

The present findings also indicate that the balance betweenthe levels of calpain and CAST activities determines the fate ofneurons following excitotoxic challenges. If the amount ofCAST is not sufficient to suppress the KA-induced calpainactivity, CAST may undergo proteolysis as a consequence ofcalpain activation, driving a vicious cycle to promote the im-balance between calpain and CAST activities (Fig. 6). Although

FIG. 7. A proposed scheme for calpain-mediated neurodegenerative processes focusing on cytoskeletal disorganization. Excitotoxicinsults induce calpain and caspase activation, whereas CAST and VIC can suppress activation of these proteases in a specific manner. Reductionof CAST may arise as a consequence of the calpain activation, driving a vicious cycle that further deteriorates the balance of the calpain-calpastatinsystem. Calpain is activated primarily in the somato-dendritic compartments of neurons and consequently degrades dendritic MAP2 andcytoplasmic MT-unbound tau in a preferential manner. Tau in the neuronal soma also undergoes hyper-phosphorylation catalyzed by Cdk5, whichcan be activated by a Cdk5 activator fragment (p25) converted from its intact form (p35) as a consequence of calpain-mediated proteolysis.Dendritic cytoskeletons become deranged as well in these locations by the calpain activation, giving rise to pronounced degeneration of dendritesin an acute phase. In contrast, calpain activation is smaller and slower in the axon, and consequently axonal cytoskeletons degenerate more slowlythan those in the dendrites, resulting from degradation and hyper-phosphorylation of tau in the soma and subsequent depletion of axonal tausupplied from the soma. Finally, cytoskeletal disorganization in the neuritic processes substantially deteriorates neuronal functions, leading toneuronal death in a subacute phase.

Calpain and Caspase Activation in Excitotoxicity15236

at NO

UK

AG

AK

U S

OG

O K

EN

KY

U C

TR

on August 2, 2006

ww

w.jbc.org

Dow

nloaded from

CAST is a possible substrate for calpain (52), caspases may alsoproteolyze CAST during apoptosis (53, 54). However, involve-ment of the caspase system appears to be negligible in the adultbrain due to the apparent absence of caspase-3 (46).

To conclude, the use of Tg mice overexpressing hCAST andVIC has clarified the preeminent role played by the calpain-calpastatin system in the neuron-specific molecular mechanismsunderlying excitotoxicity-induced cytoskeletal degeneration(schematically summarized in Fig. 7). The modulation of thebalance between calpain and CAST activities demonstrated inthe present study also offers a target for potential therapeuticinterventions to treat neuritic pathologies. Exploitation of phar-macological means to solely regulate calpain activity through, forinstance, determining three-dimensional structures of the calci-um-bound forms of calpains may lead to establishment of an idealmedicinal approach to reverse or decelerate neuropathologies indiverse central nervous system disorders.

Acknowledgments—We thank Misaki Sekiguchi, Kaori Watanabeand Yukiko Dohzono for technical assistance. We also thank MarkMayford, the Scripps Research Institute, for kindly providing the plas-mids pNN265 and pMM403. We are grateful to John Q. Trojanowskiand Virginia M.-Y. Lee, University of Pennsylvania School of Medicine,for providing anti-tau antibody T49 and antibodies againstneurofilaments.

REFERENCES

1. Rami, A. (2003) Neurobiol. Dis. 13, 75–882. Chan, S. L., and Mattson, M. P. (1999) J. Neurosci. Res. 58, 167–1903. Coleman, M. P., and Perry, V. H. (2002) Trends Neurosci. 25, 532–5374. Higuchi, M., Lee, V. M.-Y., and Trojanowski, J. Q. (2002) Neuromolecular Med.

2, 131–1505. Melloni, E., and Pontremoli, S. (1989) Trends Neurosci. 12, 438–4446. Goll, D. E., Thompson, V. F., Li, H., Wei, W., and Cong, J. (2003) Physiol. Rev.

83, 731–8017. Johnson, G. V., Jope, R. S., and Binder, L. I. (1989) Biochem. Biophys. Res.

Commun. 163, 1505–15118. Johnson, G. V., Litersky, J. M., and Jope, R. S. (1991) J. Neurochem. 56,

1630–16389. Fischer, I., Romano-Clarke, G., and Grynspan, F. (1991) Neurochem. Res. 16,

891–89810. Banik, N. L., Matzelle, D. C., Gantt-Wilford, G., Osborne, A., and Hogan, E. L.

(1997) Brain Res. 752, 301–30611. Potter, D. A., Tirnauer, J. S., Janssen, R., Croall, D. E., Hughes, C. N., Fiacco,

K. A., Mier, J. W., Maki, M., and Herman, I. M. (1998) J. Cell Biol. 141,647–662

12. Saito, K., Elce, J. S., Hamos, J. E., and Nixon, R. A. (1993) Proc. Natl. Acad.Sci. U. S. A. 90, 2628–2632

13. Nixon, R. A., Saito, K. I., Grynspan, F., Griffin, W. R., Katayama, S., Honda,T., Mohan, P. S., Shea, T. B., and Beermann, M. (1994) Ann. N. Y. Acad. Sci.747, 77–91

14. Crocker, S. J., Smith, P. D., Jackson-Lewis, V., Lamba, W. R., Hayley, S. P.,Grimm, E., Callaghan, S. M., Slack, R. S., Melloni, E., Przedborski, S.,Robertson, G. S., Anisman, H., Merali, Z., and Park, D. S. (2003) J. Neuro-sci. 23, 4081–4091

15. McIntosh, T. K., Saatman, K. E., Raghupathi, R., Graham, D. I., Smith, D. H.,Lee, V. M.-Y., and Trojanowski, J. Q. (1998) Neuropathol. Appl. Neurobiol.24, 251–267

16. Neumar, R. W., Meng, F. H., Mills, A. M., Xu, Y. A., Zhang, C., Welsh, F. A.,and Siman, R. (2001) Exp. Neurol. 170, 27–35

17. Arthur, J. S., Elce, J. S., Hegadorn, C., Williams, K., and Greer, P. A. (2000)Mol. Cell. Biol. 20, 4474–4781

18. Zimmerman, U. J., Boring, L., Pak, J. H., Mukerjee, N., and Wang, K. K. (2000)IUBMB Life 50, 63–68

19. Wang, K. K. W., and Yuen, P. W. (1999) in CALPAIN: Pharmacology andToxicology of Calcium-dependent Protease (Wang, K. K. W., and Yuen,

P. W., eds) pp. 77–101, Taylor & Francis, Philadelphia20. Lubisch, W., Hofmann, H. P., Treiber, H. J., and Moller, A. (2000) Bioorg. Med.

Chem. Lett. 10, 2187–219121. Emori, Y., Kawasaki, H., Imajoh, S., Imahori, K., and Suzuki, K. (1987) Proc.

Natl. Acad. Sci. U. S. A. 84, 3590–359422. Wang, K. K., and Yuen, P. W. (1994) Trends Pharmacol. Sci. 15, 412–41923. Tomioka, M., Shirotani, K., Iwata, N., Lee, H. J., Yang, F., Cole, G. M.,

Seyama, Y., and Saido, T. C. (2002) Brain Res. Mol. Brain Res. 108, 18–3224. Sahyoun, N., LeVine, H., III, Burgess, S. K., Blanchard, S., Chang, K. J., and

Cuatrecasas, P. (1985) Biochem. Biophys. Res. Commun. 132, 878–88425. Asada, K., Ishino, Y., Shimada, M., Shimojo, T., Endo, M., Kimizuka, F., Kato,

I., Maki, M., Hatanaka, M., and Murachi, T. (1989) J. Enzyme Inhib. 3,49–56

26. Hitomi, K., Yokoyama, A., and Maki, M. (1998) Biosci. Biotechnol. Biochem.62, 136–141

27. Mayford, M., Wang, J., Kandel, E. R., and O’Dell, T. J. (1995) Cell 81, 891–90428. Wang, G., Achim, C. L., Hamilton, R. L., Wiley, C. A., and Soontornniyomkij,

V. (1999) Methods 18, 459–46429. Saido, T. C., Yokota, M., Nagao, S., Yamaura, I., Tani, E., Tsuchiya, T., Suzuki,

K., and Kawashima, S. (1993) J. Biol. Chem. 268, 25239–2524330. Yang, F., Sun, X., Beech, W., Teter, B., Wu, S., Sigel, J., Vinters, H. V.,

Frautschy, S. A., and Cole, G. M. (1998) Am. J. Pathol. 152, 379–38931. Manya, H., Inomata, M., Fujimori, T., Dohmae, N., Sato, Y., Takio, K., Na-

beshima, Y., and Endo, T. (2002) J. Biol. Chem. 277, 35503–3550832. Mawal-Dewan, M., Henley, J., Van de Voorde, A., Trojanowski, J. Q., and Lee,

V. M.-Y. (1994) J. Biol. Chem. 269, 30981–3098733. Goedert, M., Jakes, R., Crowther, R. A., Six, J., Luke, U., Vandermeeren, M.,

Cras, P., Trojanowski, J. Q., and Lee, V. M.-Y. (1993) Proc. Natl. Acad. Sci.U. S. A. 90, 5066–5070

34. Carden, M. J., Trojanowski, J. Q., Schlaepfer, W. W., and Lee, V. M.-Y. (1987)J. Neurosci. 7, 3489–3504

35. Tu, P. H., Elder, G., Lazzarini, R. A., Nelson, D., Trojanowski, J. Q., and Lee,V. M.-Y. (1995) J. Cell Biol. 129, 1629–1640

36. Nagao, S., Saido, T. C., Akita, Y., Tsuchiya, T., Suzuki, K., and Kawashima, S.(1994) J. Biochem. (Tokyo) 115, 1178–1184

37. Kusakawa, G., Saito, T., Onuki, R., Ishiguro, K., Kishimoto, T., and Hisanaga,S. (2000) J. Biol. Chem. 275, 17166–17172

38. Lee, M. S., Kwon, Y. T., Li, M., Peng, J., Friedlander, R. M., and Tsai, L. H.(2000) Nature 405, 360–364

39. Saido, T. C., Sorimachi, H., and Suzuki, K. (1994) FASEB J. 8, 814–82240. Mattson, M. P. (2000) Nat. Rev. Mol. Cell. Biol. 1, 120–12941. Yamashima, T. (2000) Prog. Neurobiol. 62, 273–29542. Nath, R., Raser, K. J., Stafford, D., Hajimohammadreza, I., Posner, A., Allen,

H., Talanian, R. V., Yuen, P., Gilbertsen, R. B., and Wang, K. K. (1996)Biochem. J. 319, 683–690

43. Syntichaki, P., Xu, K., Driscoll, M., and Tavernarakis, N. (2002) Nature 419,939–944

44. Pang, Z., Bondada, V., Sengoku, T., Siman, R., and Geddes, J. W. (2003)J. Neuropathol. Exp. Neurol. 62, 633–643

45. Nath, R., Davis, M., Probert, A. W., Kupina, N. C., Ren, X., Schielke, G. P., andWang, K. K. (2000) Biochem. Biophys. Res. Commun. 274, 16–21

46. Takano, J., Tomioka, M., Tsubuki, S., Higuchi, M., Iwata, M., Itohara,S., Maki, M., and Saido, T. C. (February 2, 2005) J. Biol. Chem.10.1074/jbc.M414552200

47. Waterhouse, N. J., Finucane, D. M., Green, D. R., Elce, J. S., Kumar, S.,Alnemri, E. S., Litwack, G., Khanna, K., Lavin, M. F., and Watters, D. J.(1998) Cell Death Differ. 5, 1051–1061

48. Nakagawa, T., and Yuan, J. (2000) J. Cell Biol. 150, 887–89449. Canu, N., Dus, L., Barbato, C., Ciotti, M. T., Brancolini, C., Rinaldi, A. M.,

Novak, M., Cattaneo, A., Bradbury, A., and Calissano, P. (1998) J. Neurosci.18, 7061–7074

50. Gamblin, T. C., Chen, F., Zambrano, A., Abraha, A., Lagalwar, S., Guillozet,A. L., Lu, M., Fu, Y., Garcia-Sierra, F., LaPointe, N., Miller, R., Berry,R. W., Binder, L. I., and Cryns, V. L. (2003) Proc. Natl. Acad. Sci. U. S. A.100, 10032–10037

51. Rami, A., and Krieglstein, J. (1993) Brain Res. 609, 67–7052. Lankiewicz, S., Luetjens, M. C., Truc Bui, N., Krohn, A. J., Poppe, M., Cole,

G. M., Saido, T. C., and Prehn, J. H. (2000) J. Biol. Chem. 275, 17064–1707153. Wang, K. K., Posmantur, R., Nadimpalli, R., Nath, R., Mohan, P., Nixon, R. A.,

Talanian, R. V., Keegan, M., Herzog, L., and Allen, H. (1998) Arch. Biochem.Biophys. 356, 187–196

54. Porn-Ares, M. I., Samali, A., and Orrenius, S. (1998) Cell Death Differ. 5,1028–1033

Calpain and Caspase Activation in Excitotoxicity 15237

at NO

UK

AG

AK

U S

OG

O K

EN

KY

U C

TR

on August 2, 2006

ww

w.jbc.org

Dow

nloaded from