Time-Dependent Increases in ProteaseActivities for Neuronal Apoptosis inSpinal Cords of Lewis Rats DuringDevelopment of Acute ExperimentalAutoimmune Encephalomyelitis

Arabinda Das,1 M. Kelly Guyton,2 Denise D. Matzelle,1 Swapan K. Ray,3

and Naren L. Banik1*1Department of Neurosciences, Medical University of South Carolina, Charleston, South Carolina2Department of Microbiology and Immunology, Medical University of South Carolina, Charleston, South Carolina3Department of Pathology, Microbiology and Immunology, University of South Carolina School ofMedicine, Columbia, South Carolina

Multiple sclerosis (MS) is characterized by axonal demy-elination and neurodegeneration, the latter having beeninadequately explored in the MS animal model experi-mental autoimmune encephalomyelitis (EAE). The pur-pose of this study was to examine the time-dependentcorrelation between increased calpain and caspaseactivities and neurodegeneration in spinal cord tissuesfrom Lewis rats with acute EAE. An increase in TUNEL-positive neurons and internucleosomal DNA fragmenta-tion in EAE spinal cords suggested that neuronal deathwas a result of apoptosis on days 8–10 following induc-tion of EAE. Increases in calpain expression in EAE cor-related with activation of pro-apoptotic proteases, leadingto apoptotic cell death beginning on day 8 of EAE, whichoccurred before the appearance of visible clinical symp-toms. Increases in calcineurin expression and decreasesin phospho-Bad (p-Bad) suggested Bad activation in ap-optosis during acute EAE. Increases in the Bax:Bcl-2 ratioand activation of caspase-9 showed the involvement ofmitochondria in apoptosis. Further, caspase-8 activationsuggested induction of the death receptor–mediatedpathway for apoptosis. Endoplasmic reticulum stressleading to caspase-3 activation was also observed, indi-cating that multiple apoptotic pathways were activatedfollowing EAE induction. In contrast, cell death wasmostly a result of necrosis on the later day (day 11), whenEAE entered a severe stage. From these findings, weconclude that increases in calpain and caspase activitiesplay crucial roles in neuronal apoptosis during the devel-opment of acute EAE. VVC 2008 Wiley-Liss, Inc.

Key words: acute EAE; calpain; neuronal apoptosis;spinal cord

Multiple sclerosis (MS) is a T-cell-mediated auto-immune demyelinating disease of the central nervoussystem (CNS) with neurodegenerative components

including axonal damage and neuronal and glial celldeath. MS is the major disabling disease of young indi-viduals in the United States (Martin and McFarland,1995; Bauer et al., 1999) and can lead to fatigue, dizzi-ness, impaired vision, pain, and paralysis of one or morelimbs (Keegan and Noseworthy, 2002). Despite numer-ous studies, the etiology of MS remains unknown. Ex-perimental autoimmune encephalomyelitis (EAE), ananimal model of MS, has been extensively used to studythe pathogenesis of MS and its treatment options (Bech-told et al., 2004). In MS, an abnormal immune responseto myelin antigens causes mononuclear cell infiltration,demyelination, oligodendrocyte loss, and axonal degen-eration. Currently, the consensus on the pathogenesis ofMS is that autoreactive T cells activated in the periphery(Mokhtarian et al., 1994) cross the blood–brain barrier(BBB) where they encounter the CNS antigen presentedto them by antigen-presenting cells (APCs; Sospedra andMartin, 2005). This process causes secondary influx ofadditional lymphocytes and blood-borne macrophages tothe inflammatory site. The inflammatory cells and resi-dent glial cells produce cytokines and proteases, includ-ing calpain, that individually or together contribute tothe destruction of myelin and eventually cause damage

Contract grant sponsor: NINDS; Contract grant numbers: NS-45967,

NS-57811; Contract grant sponsor: NCI; Contract grant number: CA-

91460; Contract grant sponsor: NIH; Contract grant number: C06

RRO15455.

*Correspondence to: Naren L. Banik, PhD, Department of Neuroscien-

ces, Medical University of South Carolina, 96 Jonathan Lucas Street, 310

CSB, Charleston, SC 29425. E-mail: baniknl@musc.edu

Received 12 December 2007; Revised 6 February and 19 February

2008; Accepted 21 February 2008

Published online 2 June 2008 in Wiley InterScience (www.

interscience.wiley.com). DOI: 10.1002/jnr.21737

Journal of Neuroscience Research 86:2992–3001 (2008)

' 2008 Wiley-Liss, Inc.

to axons (Benveniste, 1997). In fact, increased calpainexpression and activity in the spinal cord and optic nerveof Lewis rats with an acute form of EAE (Guyton et al.,2005a, 2005b) and in MS plaques (Shields et al., 1999a,1999b) have been previously demonstrated. Up-regulationof calpain and caspase activity has also been implicated inapoptotic cell death in other neurodegenerative disorders,including spinal cord injury (Ray et al., 1999) and ische-mia (Yamashima, 2000) and in the aging brain (Sloaneet al., 2003; Hinman et al., 2004). From these and otherfindings, increases in calpain expression and activity havebeen suggested to play a crucial role in axonal damageand neuronal death in the spinal cord with the onset ofclinical symptoms in EAE rats (Guyton et al., 2005a).

Although neuronal apoptosis during EAE has beenpreviously demonstrated (Guyton et al., 2005a), themechanisms involved in this cell death process have notbeen clearly elucidated. To this end, several intracellularsignal transduction cascades involving proteases arethought to contribute early in this process. Elevated cal-pain expression and activity in the white matter of MSpatients (Shields et al., 1999b) and in inflammatory cellsin EAE (Shields and Banik, 1998) have implicated animportant role for calpain in the pathogenesis of demye-linating disease. Although calpain inhibition has beenshown to prevent cell death and reduce degradation ofmyelin protein in vitro (Deshpande et al., 1995; Rayet al., 1999; Das et al., 2005), it is equally important toexamine the role of other proteases in the apoptosis ofneurons and glial cells and axonal damage. Caspases, afamily of cysteine proteases mainly involved in apoptoticpathways, are essential for the control of cell populationsnot only during development but also in pathology. Cas-pase-3, one member of the caspase family, appears to bethe main effector caspase involved in neuronal apoptosis,and activation of caspase-3 has been shown to be a criti-cal event in neuronal apoptosis; however, other caspasesand signaling proteins such as calcineurin, Bax, and poly(ADP-ribose) polymerase-1 (PARP-1) may also be im-portant.

Thus, the goal of this investigation was to charac-terize time-dependent alterations in apoptotic proteins asthey correlate with neuron death following EAE induc-tion in Lewis rats. Our results indicated that increases incalpain and caspase activities contributed to neuronal ap-optosis with the onset EAE.

MATERIALS AND METHODS

EAE Induction

Adult male Lewis rats (180–200 g) were purchased fromCharles River Breeding Laboratories (Wilmington, MA), pro-vided with water and food ad libitum, and maintained andused in the proposed experiments in accordance with the Lab-oratory Welfare Act and the Guide for the Care and Use of Lab-oratory Animals of the National Institutes of Health (Bethesda,MD). To induce EAE, rats were immunized subcutaneouslywith a 0.2-mL emulsion (1:1) of complete Freund’s adjuvant(CFA) containing Mycobacterium tuberculosis H37Ra (10 mg/mL;

Difco, Detroit, MI) and phosphate-buffered saline (PBS) con-taining guinea pig spinal cord (GPSC) homogenate (200 mg/mL) and myelin basic protein (MBP; 200 lg/mL). Controlanimals received PBS/CFA without GPSC/MBP. Two hourslater, all rats received an intraperitoneal injection of Pertussistoxin (1.25 lg/rat). After induction of EAE, the rats weremonitored daily for weight loss and appearance of clinicalsymptoms (usually occurring 8–11 days postinoculation). Theclinical scores for severity of EAE symptoms were based onthe following grades: 0, no change; 1, limp tail; 2, hind limbweakness with difficulty in righting; 3, one limb plegic; 4,two limbs plegic; 5, quadriplegic or moribund.

Isolation and Sectioning of Spinal Cord

Animals were sacrificed under anesthesia (ketamine, 95mg/kg; xylazine, 5 mg/kg) on days 7, 8, 9, and 11. Lumbarspinal cord regions were surgically removed. Tissue sampleswere dissected on ice and immediately immersed in tissue-freezing medium (Histo Prep; Fisher Scientific, Fair Lawn,NJ) for immunofluorescent studies or fresh-frozen for Westernblot analyses. Tissue samples were stored at 2708C until theexperiments were performed.

Combined TUNEL and DoubleImmunofluorescent Labeling

Before processing, the samples were warmed to 2168C,and thin sections (5 lm) were cut using a Reichart-Jung cryo-stat (Cryocut 1800, Leica, Wetzlar, Germany). Sections werefixed at room temperature first in 95% ethanol for 10 min andthen in 4% methanol-free formaldehyde (Polysciences, War-rington, PA) for 15 min and finally were stored in PBS (pH7.4). The slides were saturated for 5 min with terminal deoxy-nucleotidyl transferase (TdT) buffer (50 lL/slide; Promega,Madison, WI) and then replaced with the TUNEL reactionmixture (50 lL/slide) containing digoxigenin (DIG) labelingmixture with DIG-11-dUTP (2.5 lL; Roche Diagnostics,Mannheim, Germany) and TdT (25 U; Promega) in TdTbuffer. The slides were covered with Hybrislip hybridizationcoverslips (Research Products Int., Mount Prospect, IL) andincubated at 378C in an OmniSlide thermal cycler (HybaidInt.) for 1 hr. The TUNEL reaction was terminated by sub-mersing the slides in a NaCl and sodium citrate solution(SSC; Promega). The slides were then washed three timeswith PBS to remove any unincorporated DIG-11-dUTP. Thetissue was blocked for 1 hr with blocking buffer containing2% horse serum and 2% sheep serum in PBS. Then the sec-tions were incubated for 1 hr in blocking buffer with mouseanti-NeuN antibody (1:100). The slides were washed twicewith PBS for 5 min each and then incubated for 30 min inthe dark with sheep anti-DIG conjugated to FITC (1:100;Roche) and horse antimouse IgG conjugated to Texas red(1:100). The slides were washed twice with PBS and oncewith double-distilled water and then mounted with 1 drop ofVectashield Mounting Medium (Vector Laboratories) andcoverslip. The slides were viewed under a fluorescence micro-scope at 4003 magnification.

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Analysis of DNA Fragmentation

Genomic DNA fragmentation was analyzed by agarosegel electrophoresis of DNA isolated from spinal cord tissue, asreported previously. (Ray et al., 2000). Briefly, rat spinal cordsegments were homogenized in a buffer [10 mM Tris-HCl (pH8.0), 150 mM NaCl, 50 mM EDTA] using a battery-operatedhomogenizer (Kontes Instruments, Vineland, NJ). The homog-enates were digested in a solution (10 mM Tris-HCl, pH 8.0,50 mM NaCl, 10 mM EDTA, 0.5% SDS, 250 ng/lL protein-ase K) at 378C for 24 hr. The digests were extracted twice witha 1:1 (v/v) mixture of phenol and chloroform and once withchloroform alone. Total genomic DNA was precipitated, driedin air, and dissolved in TE buffer [10 mM Tris-HCl (pH 8.0), 1mM EDTA containing RNase A (50 ng/mL)] for 1 hr at 378C.Equal amounts of the samples were loaded onto 1.6% agarosegels and electrophoresed in a TAE [40 mM Tris-acetate (pH8.3), 1 mM EDTA] buffer. Gels were stained with ethidium bro-mide (1 lg/mL) and photographed on a UV (303 nm) transillu-minator using Alpha Innotech (San Leandro, CA).

Protein Extraction and Western Blot Analysis

Western blotting was performed as we described previ-ously (Das et al., 2006). The blots were washed three times (10min each), and protein bands were detected by alkaline horse-radish peroxidase (HRP)-catalyzed oxidation of luminol in thepresence of H2O2 using an enhanced chemiluminescence(ECL) system (Amersham Life Science, Buckinghamshire, UK).The blots were exposed immediately to X-OMAT XAR-2film (Eastman Kodak Co., Rochester, NY) for autoradiogra-phy. The ECL autoradiograms were scanned on a PowerLookscanner (UMAX, Fremont, CA), then imaged and digitizedusing Adobe Photoshop (Adobe Systems, San Jose, CA). Bandswere quantitated using the NIH Image software.

Antibodies

Monoclonal antibody against a-spectrin (Affiniti, Exeter,UK) was used to measure calpain as well as caspase-3 activity.Rabbit polyclonal 80-kDa calpain and 76-kDa calpain anti-bodies, which were raised in our laboratory (Guyton et al.,2005b), were used to determine calpain expression and calpainactivation, respectively. Monoclonal antibody against a-spec-trin (Affiniti, Exeter, United Kingdom) was used to measurethe activity of calpain as well as of caspase-3. Bax and Bcl-2monoclonal antibodies (Santa Cruz Biotech, Santa Cruz, CA)were used to assess apoptotic threshold by determining theBax:Bcl-2 ratio. All other antibodies were purchased eitherfrom Calbiochem (San Diego, CA) or Santa Cruz Biotechnol-ogy (Santa Cruz, CA). Monoclonal antibody against b-actin(clone AC-15, Sigma Chemical Co., St. Louis, MO) was usedto standardize protein loading in SDS-PAGE. The secondaryantibody used was HRP–conjugated goat antimouse IgG(ICN Biomedicals, Aurora, OH) except for calpain and a-spectrin, for which we used HRP-conjugated goat antirabbitIgG (ICN Biomedicals, Aurora, OH).

Statistical Analysis

Data from Western blots were analyzed for detectionof significant differences between two groups by one-way

analysis of variance followed by Fisher’s post hoc tests usingStatview software (Abacus Concepts, Berkeley, CA). Resultswere expressed as the mean 6 the standard error of themean (SEM) of three or more independent experiments.Differences from the control were considered significant at P� 0.05.

RESULTS

Time-Dependent Increases in Neuron-Specific CellDeath and Apoptosis in Spinal Cord of EAE Rats

One goal of the study was to determine when neu-ron cell death occurred in acute EAE following chal-lenge with myelin antigens. We determined the occur-rence of neuronal apoptosis with internucleosomal DNAfragmentation during the development of acute EAE inLewis rats (Fig. 1). The studies showed a substantialincrease in neuron death 11 days after challenge (grades3–4 EAE) compared with day 9 EAE, when few cellswere costained with TUNEL and NeuN (Fig. 1A).These data indicated that neuronal death occurred dur-ing the late phase of disease progression. Because neuro-nal death was increased in EAE spinal cord (Fig. 1A),we expected that many of these were dying via apopto-tic pathways. Therefore, to confirm the occurrence ofapoptotic death, we examined formation of internucleo-somal DNA fragmentation, a hallmark of apoptosis, incontrol and EAE spinal cord samples that were collectedon different days (Fig. 1B). Like the control, no DNAladdering was found in day 7 EAE spinal control. Incontrast, there was increased DNA laddering in EAE spi-nal cord from days 8 to 11 (Fig. 1B), compared with thecontrol. We found that apoptosis started on day 8 inEAE spinal cord (Fig. 1B), before the appearance of clin-ical symptoms. Our results showed a dramatic increasein internucleosomal DNA fragmentation on day 10(grade 4 EAE; Fig 1B). In addition, a substantial numberof cells were found to be necrotic on day 11 (grades 3–4), as evidenced by massive random DNA fragmentation(Fig. 1B).

Increased Calpain Expression and Activityduring Acute EAE

Previous studies have demonstrated calpain expres-sion to be up-regulated in both glial and infiltratingimmune cells in the spinal cord of Lewis rats with EAEafter the onset of characteristic clinical symptoms of dis-ease (Schaecher et al., 2002). In the present study, cal-pain expression and activity were examined via Westernblot analysis of control and EAE spinal cord tissues (Fig.2). Calpain expression was examined using antibodiesagainst 80-kDa inactive calpain (Fig. 2A) and 76-kDaactive calpain (Fig. 2B), and calpain activity was meas-ured by determining formation of 145-kDa SpectrinBreak Down Product (SBDP; Nath et al., 1996, Wanget al., 1998a) in lumbar spinal cord tissues from controland EAE animals (grades 2–4) on different days follow-ing challenge (Fig. 2C). Calpain expression and activitywere significantly (P � 0.05) increased with the onset

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and severity of disease. On day 8 post–EAE induction,very little calpain expression and activity were seen incontrol and EAE tissue. In contrast, significant increasesin 80-kDa inactive calpain and 76-kDa active calpainexpression were observed on days 9–11 (P � 0.05) inEAE spinal cord at or after onset of disease (Fig. 2A,B).There was also a gradual loss of calpastatin, the endoge-nous inhibitor of calpain, at the onset of disease on day8, but this loss was greater with the severity of disease

Fig. 1. Neuronal death and DNA fragmentation in acute EAE withtime. A: Immunofluorescence staining of neuron-specific cell deathon days 8–11 post–EAE induction using the antibodies to NeuN(red), a neuron marker, and TUNEL staining (green), which identi-fied cell death with anti-DIG antibody. Arrows indicate TUNEL-positive neurons (4003 magnification). B: Agarose gel electrophore-sis of genomic DNA for detection of DNA fragmentation (C, con-trol; E, EAE; D7–D11, days 7–11).

Fig. 2. Calpain and calpastatin levels during EAE. Time-dependentchanges in the expression of 80-kDa (A) and 76-kDa (B) calpain,145-kDa SBDP (C), and 110-kDa calpastatin (D) during acute EAE.Upper panels (A–D) show representative Western blot, and lowerpanels show the corresponding bar graphs (n � 3 animals per group).b-Actin was used as a control for equal protein loading. Data arepresented as percent increase compared with the control set at 100%(C, control; E, EAE; D7–D11, days 7–11).

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on days 9–11 compared with that in the control (Fig.2D).

Up-regulated Calcineurin and DephosphorylatedBad during Acute EAE

Calcineurin is a Ca21/calmodulin-dependent pro-tein phosphatase that is highly expressed in the CNS.Calcineurin is cleaved by calpain to form the activated60-kDa calcineurin A subunit, which contains a catalyticdomain, the binding site for calmodulin, and a C-termi-nal autoinhibitory domain (Wang et al., 1999). On acti-vation by calpain or other signaling proteins, calcineurindephosphorylates and activates the proapoptotic proteinBad. Based on this knowledge, we surmised that calci-neurin activation caused Bad dephosphorylation to con-tribute to apoptosis in EAE. Compared with the control,indeed, a significant increase (P � 0.05) in the 60-kDacalcineurin subunit and a decrease in phospho-Bad (p-

Bad) on day 10 (grade 4 EAE) were observed (Fig. 3),suggesting that up-regulation of calcineurin dephospho-rylated Bad for apoptosis during acute EAE (Fig. 3A,B).Thus, these results confirmed a role for calcineurin inactivating Bad, thereby linking calcineurin to the apo-ptosis machinery in EAE.

Caspase-8 Activation and Proteolytic Cleavage ofBid in the Course of EAE

Caspase-8 plays a central role in the cytokine-medi-ated caspase cascade. Current in vivo observations indi-cated involvement of caspase-8 in cytokine-induced celldeath in EAE (Fig. 4). There was a significantly (P 50.009) greater level (1.5-fold to 2.5-fold) of caspase-8cleaved (active) product (18-kDa fragment) generated inEAE with the onset of disease after 8 days to severe gradeafter 10–11 days, compared with the control (Fig. 4A).The present results showed a connection between extrin-sic and intrinsic apoptotic pathways because activationof caspase-8 caused proteolytic cleavage of Bid to tBid

Fig. 3. Up-regulated calcineurin and Bad dephosphorylation duringEAE. Time-dependent changes in 61-kDa calcineurin (A), 22-kDaBad, and 24-kDa p-Bad (B) expression during acute EAE. Upperpanels (A–D) show representative Western blots and lower panelsshow the corresponding bar graphs (n � 3 animals per group). b-Actin was used as a control for equal protein loading. Data are pre-sented as percent increase compared with the control set at 100%(C, control; E, EAE; D7–D11, days 7–11).

Fig. 4. Caspase-8 activity during EAE. Time-dependent changes in42-kDa and 18-kDa caspase-8 (A) and 15-kDa tBid (B) expressionduring acute EAE. Upper panels (A–D) show representative Westernblots, and lower panels show the corresponding bar graphs (n � 3animals per group). b-Actin was used as a control for equal proteinloading. Data are presented as percent increase compared with thecontrol set at 100% (C, control; E, EAE;. D7–D11, day 7 to day 11).

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(Fig. 4B). The level of 15-kDa tBid was also very signifi-cantly (P 5 0.008) increased with the severity of diseaseon days 9–11, compared with the conrol (Fig 4B). Thus,the Bcl-2 family member Bid can induce apoptosis whenit is activated following proteolytic cleavage by caspase-8during induction of EAE.

Increase in Bax:Bcl-2 Ratio and Activation ofCaspase-9 during Acute EAE

Proteins of the Bcl-2 family, including the proapop-totic protein Bax and the antiapoptotic protein Bcl-2, col-lectively regulate the predisposition of a cell to apoptosisthrough sequestration of critical activation factors or altera-tions of mitochondrial permeability (Das et al., 2007). Weexamined involvement of the mitochondrial pathway ofapoptosis in acute EAE (Fig. 5). Our studies showed thatBax was significantly increased (P 5 0.02) in EAE and thatthe elevation correlated with the development of disease(Fig. 5A). The observation of higher Bax and lower Bcl-2expression among EAE animals suggested that mitochon-drial pathway of apoptosis might play a predominant role intriggering cell death (Fig. 5A). There was a significant dif-ference (P 5 0.021) in the Bax:Bcl-2 ratio between lumbarspinal cord tissues from control and EAE (grades 2–4) ondifferent days following challenge. Also, in mitochodria-de-pendent apoptosis, cytochrome c was released from mito-chondria into cytosol, which could interact with Apaf-1,thereby triggering autoproteolysis of 46-kDa pro-caspase-9to form the 37-kDa caspase-9 active fragment (Fig. 5B). Asignificantly (P 5 0.029) greater level of caspase-9 activa-tion was seen in the acute phase of EAE, reaching its peaklevel on days 9–10 post-challenge, compared with that inthe control (Fig. 5B), suggesting the importance of mito-chondrial regulation of the mechanism of apoptosis. Thus,the increase in the Bax:Bcl-2 ratio correlated with theincrease in the caspase-9 active fragment in EAE (Fig.5A,B).

Activation of Caspase-12 and Caspase-3 andPARP-1 Cleavage in the Course of EAE

Because both calpain and caspase-12 are activated byelevation of intracellular free [Ca21] and both may playimportant roles in mediating neuronal degeneration, weexamined the possible interaction between these two cys-teine proteases. Our experiments demonstrated the degra-dation of the inactive 55-kDa pro-caspase-12 form to the40-kDa caspase-12 active form during EAE development(Fig. 6). This activation could be carried out by theincreased calpain activity in EAE (Fig. 2). The signifi-cantly increased (P 5 0.011) production of the 40-kDacaspase-12 active fragment was evident at the onset of thesevere stage of the disease (Fig. 6A). Also, Western blotanalysis previously showed a significant decrease (P 50.031) in calpastatin, the endogenous inhibitor of calpain(Fig. 2). Caspase-3 activation was assessed by examiningthe generation of the 20-kDa caspase-3 active fragmentand the formation of the120-kDa SBDP on the Westernblots (Fig. 6B,C). The increased level of the 20-kDa cas-

pase-3 active band was correlated with the severity of thedisease, compared with the control (Fig. 6C). Activationof caspase-3 subsequently leads to apoptotic cell deaththrough cleavage of a broad spectrum of central cellulartarget proteins in the cell nucleus, including PARP-1.Western blot analysis also showed an increase (1.5–3.5times) in the formation of the 85-kDa PARP-1 fragmentin acute EAE (Fig. 6D). These results correlated withendoplasmic reticulum stress and activation of calpain andcaspase-3, suggesting the requirement of different levels ofactivity of these proteases for mediation of neuronal apo-ptosis in acute EAE.

DISCUSSION

The goal of this study was to determine whethertime-dependent elevation of protease activity (calpain andcaspases) was involved in apoptosis signaling and neurode-generation during the development of acute EAE. Thepresent investigation indicated that expression and activa-tion of caspases in acute EAE spinal cord tissues were sig-

Fig. 5. Apoptotic protein alteration during EAE. Time-dependentchanges in 24-kDa and 21-kDa Bax, 26-kDa Bcl-2 (A) and 37-kDacaspase-9 (B) expression during acute EAE. Upper panels (A–D)show representative Western blots and lower panels show corre-sponding the bar graphs (n � 3 animals per group). b-Actin was usedas a control for equal protein loading. Data are presented as percentincrease compared with the control set at 100% (C, control; E, EAE;D7–D11, days 7–11).

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nificantly up-regulated during days 9–11 post-EAE induc-tion, which correlated with a significant increase in cal-pain expression, neuronal apoptosis, and axonal damage atthese times (Shields et al., 1999a; Schaecher et al., 2002;Guyton et al., 2005a, 2005b; Hassen et al., 2006).

Calpain expression and activity were increased insplenic cells before the onset of the disease (Shields et al.,1999a), and increased activity was evident in infiltratingimmune cells and glial cells of the spinal cord white mat-ter and optic nerve (Shields and Banik, 1998) after theappearance of clinical symptoms. The current study dem-onstrated (Fig. 1) and confirmed our previous findings(Schaecher et al., 2002; Guyton et al., 2005b) that calpainexpression was increased in EAE spinal cord in a time-dependent manner. This finding also suggested that cal-pain was involved in the neurodegenerative process inEAE and perhaps in MS. Although no significant numberof TUNEL-positive neurons was observed on day 8 fol-lowing challenge (Guyton et al., 2005b), increased DNAladdering, the hallmark of apoptosis, indicates occurrenceof non-neuronal apoptosis in day 8 EAE spinal cord, sug-gesting that the apoptotic process was initiated early in theinflammatory phase in EAE development, even before theappearance of clinical symptoms. Apoptotic cell death wasmore evident on day 9 following challenge and was at amaximum on day 10 (Fig. 1). This finding also suggeststhat in the inflammatory phase, cells expressing the deathmediators on day 8 are destined to die with progression ofthe disease. Thus, axons and neurons may be degeneratedin the early phase of disease development. Such axonaldegeneration has been found in EAE (Guyton, et al.,2005b, Kim et al., 2006; Budde et al., 2007) as well as inpatients with early, chronic, and progressive phases of MS(Bitsch et al., 2000; Kornek et al., 2000; Kuhlmann et al.,2002). Although up-regulation of calpain occurred on day9 post-EAE induction (Fig. 2), significant neuronal deathdid not occur until day 10, suggesting that calpain expres-sion preceded neuronal loss in the acute model of EAE.Although DNA fragmentation was increased in EAE spinalcord on day 11 (grades 3–4), the severe phase of the disease,most of the cells went into the necrotic phase, as seen byrandom DNA fragmentation or DNA smearing. Nonethe-less, these findings suggest that the neuronal death identifiedin EAE spinal cord was at least partially a result of an apo-ptotic mechanism. The current study also supports previousfindings that greater neuronal death and axonal degenera-tion occur during the demyelinating phase of EAE and MS(Kornek et al., 2000; Guyton et al., 2005a).

Fig. 6. Involvement of endoplasmic reticulum and caspase-3 activityin apoptosis during EAE. Time-dependent changes in the 55- and40-kDa caspase-12 (A), 120-kDa SBDP (B), 20-kDa caspase-3 (C),and 85-kDa PARP-1 (D) expression during acute EAE. Upper pan-els (A–D) show representative Western blots and lower panels showthe corresponding bar graphs (n � 3 animals per group). b-Actin wasused as a control for equal protein loading. Data are presented as per-cent increase compared with the control set at 100% (C, control; E,EAE; D7–D11, days 7–11).

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This observation is supported by earlier investiga-tions demonstrating the role of calpain in other neuro-degenerative disorders, including ischemia, spinal cordinjury, brain trauma, Alzheimer’s disease, Parkinson’sdisease, and epilepsy (Ray and Banik, 2003). Theseresults also suggested that calpain may be a potentialtherapeutic target for the amelioration of neurodegenera-tive diseases such as MS. Studies in our and other labo-ratories have demonstrated that treatment with calpaininhibitors decreased loss of neurofilament protein and cal-pain-specific spectrin breakdown and protected neuronsand glial cells in spinal cord injury as well as in EAE,indicating the importance of calpain inhibition as a prom-ising therapeutic option (Ray and Banik, 2003; Zhanget al., 2003; Guyton et al., 2005b; Hassen, et al., 2006).

In the development and progression of diseases,elevation of intracellular free [Ca21] and calpain activa-tion can induce apoptotic cell death through multiplemechanisms (Das et al., 2007). The finding that redis-tribution of Bad and apoptosis is suppressible by an in-hibitory mutant of calcineurin suggests that calcineurin isa significant mediator of apoptotic death signals. Thus,the possibility that Bad activation by calcineurin activitymight be involved in cell death in EAE was explored.The results suggested that calcineurin induced apoptosisby dephosphorylating Bad in EAE (Fig. 3). The interac-tion of calcineurin with Bad provides a Ca21-mediatedmechanism for regulating the phosphorylation state ofBad and thus making Bad a proapoptotic protein, whichfurther confirms the participation of calcineurin in theapoptosis machinery.

Ischemic and hypoxic injuries to the nervous sys-tem have been shown to involve the release of celldeath–inducing cytokines and the activation of deathreceptors. It is likely that these events involve caspase-8-mediated cleavage of the BH3-only protein Bid. Thispathway provides evidence that activation of caspase-8cleaves Bid to tBid. Once Bid is cleaved by caspase-8,tBid translocates from the cytosol to the mitochondria tocause mitochondrial damage, release of cytochrome c,increase in caspase activity nuclear condensation, and ap-optotic cell shrinkage (Li et al., 1998; Das et al., 2006).In addition, tBid is able to activate Bax to form Baxmultimers in the mitochondria, thereby inducing therelease of cytochrome c. In the present study, caspase-8activation correlated with Bid cleavage in acute EAE(Fig. 4), again demonstrating induction of cell death sig-nals early in disease development.

Calpain has been reported to play several roles inactivating factors that may be involved in apoptosis andhas a variety of possible interactions with the Bcl-2 fam-ily of proteins. Calpain may activate proapoptotic Baxdirectly through cleavage of Bax (Choi et al., 2001).Thus, activation of Bax is an essential component of thecell death mechanism. An increased level of cleaved cas-pase-9 early in the apoptotic process in EAE was alsodemonstrated (Fig. 5). Active caspase-9 further processesother caspase members, including caspase-3, to initiate acaspase cascade, which ultimately leads to apoptosis. In-

hibition of calpain has been shown to inhibit mitochon-drial transition pore formation in renal cells (Gores et al.,1998). Similar mechanisms of neuronal death may alsoexist in EAE. Calpain interacts and modifies caspasepathways. Specifically, calpain has been shown to acti-vate caspase-3 (Blomgren et al., 2001; Varghese et al.,2001) and caspase-12 (Nakagawa and Yuan, 2000). Cas-pase also has been shown to cleave calpastatin in vitro,indicating that caspase activity may also enhance calpainactivity by removing calpastatin, the regulator of calpain(Wang et al., 1998b). However, the interactionsbetween these two protease families are complex, andinhibition of calpain has also been shown to modulatecaspase-3 activity (Lankiewicz et al., 2000; Neumaret al., 2003). Activation of caspase-3, the final execu-tioner in apoptosis, which degrades the cellular targetprotein PARP-1, is elevated early in EAE (Fig. 6).Nevertheless, these results in particular indicate a pivotalcooperative role of calpain and caspases, generating cru-cial cell death signals early in disease development inEAE. This hypothesis is further supported by both invitro and in vivo studies, in which calpain inhibition hasbeen found to inhibit caspase-3 and to block glial andneuronal apoptosis (Das et al., 2005). For example, treat-ment with the calpain inhibitors E-64-d and calpeptindecreased apoptotic indicators, including internucleoso-mal DNA fragmentation and proapoptotic proteins in ratmodel of spinal cord injury (Ray et al., 1999).

The present study used the processing of pro-cas-pase-12 as an indirect biochemical marker for activationof the endoplasmic reticulum (ER)–mediated apoptoticpathway (Fig. 6). ER stress-induced apoptosis is depend-ent on this ER-resident protease (Nakagawa and Yuan,2000), activation of which likely involves proteolyticprocessing and oligomeric assembly in a manner similarto all other caspases (Nakagawa and Yuan, 2000). Thecurrent study confirms that pro-caspase-12, which isenriched in ER, is processed by the family of cytosolicCa21-dependent cysteine proteases such as calpain dur-ing EAE (Fig. 6). These results support the concept thata rise in cytosolic free [Ca21] and activation of calpaintrigger caspase-12 activation and ER apoptotic signaling,although they do not exclude the possibility that otherproteases may also be involved in activation of caspase-12 (Das et al., 2006). We propose a novel pathway ofcalpain-mediated caspase-12 activation induced by dis-turbance in intracellular Ca21 homeostasis. These resultssuggest the involvement of ER stress in the apoptoticmechanism of neuronal death in EAE (Fig. 7).

In conclusion, the finding of up-regulation of cal-pain and caspases on day 8 post-EAE induction suggeststhat calpain and caspase expression and activity play im-portant and cooperative roles in apoptotic neuronaldeath signaling early in the development of acute EAEin Lewis rats. Later, in the severe phase of the disease,on day 11, cells die mostly as a result of necrosis. Theresults of this investigation also imply that inhibition ofthese proteases may reduce the inflammatory cascade becausecalpain is involved in T-cell activation (Schaecher et al.,

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2002; Imam et al., 2007). Also, the inhibition of theseproteases may preserve and protect axons, neurons, andglia. Therefore, these proteases may be potential thera-peutic targets for treatment of EAE and MS.

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