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Research Report

Calpeptin provides functional neuroprotection to rat retinalganglion cells following Ca2+ influx

Arabinda Dasa, Dena P. Garnera, Angelo M. Del Rea, John J. Woodwarda,D. Maneesh Kumarb, Neeraj Agarwalb, Naren L. Banika, Swapan K. Raya,⁎aDepartment of Neurosciences, Medical University of South Carolina (MUSC), 96 Jonathan Lucas Street, Suite 323K, P.O. Box 250606,Charleston, SC 29425, USAbDepartment of Cell Biology and Genetics, UNT Health Science Center, 3500 Camp Bowie Boulevard Fort Worth, TX 76107, USA

A R T I C L E I N F O

⁎ Corresponding author. Fax: +1 843 792 8626.E-mail address: raysk@musc.edu (S.K. Ray

0006-8993/$ – see front matter © 2006 Elsevidoi:10.1016/j.brainres.2006.02.051

A B S T R A C T

Article history:Accepted 7 February 2006Available online 5 April 2006

Apoptosis of retinal ganglion cells (RGCs) impairs vision in glaucoma patients. RGCs are alsodegenerated in multiple sclerosis (MS), resulting in loss of visual perception in MS patients.We examined the involvement of calpain and caspase cascades in apoptosis of the ratretinal ganglion cell line RGC-5 following 24 h of exposure to 250 nM ionomycin (IMN) or300 units/ml interferon-gamma (IFN-γ) and then evaluated functional neuroprotection with2 μM calpeptin (CP, a calpain-specific inhibitor). Morphological and biochemical features ofapoptosis were detected in RGC-5 cells following exposure to IMN or IFN-γ. Fura-2 assaydetermined significant increases in intracellular free [Ca2+] following exposure to IMN orIFN-γ. Pretreatment with CP for 1 h prevented Ca2+ influx, proteolytic activities, andapoptosis in RGC-5 cells. Western blot analyses showed an increase in activities of calpainand caspase-12, upregulation of Bax:Bcl-2 ratio, release of cytochrome c frommitochondria,and increase in caspase-9 and caspase-3 activities during apoptosis. Increased caspase-3activity was also confirmed by a colorimetric assay. Activation of caspase-8 and cleavage ofBid to tBid in RGC-5 cells following exposure to IFN-γ indicated co-operation betweenextrinsic and intrinsic pathways of apoptosis. Patch-clamp recordings showed thatpretreatment with CP attenuated apoptosis and maintained normal whole-cell membranepotential, indicating functional neuroprotection. Taken together, our results demonstratedthat Ca2+ overload could be responsible for activation of calpain and caspase cascadesleading to apoptotic death of RGC-5 cells and CP provided functional neuroprotection.

© 2006 Elsevier B.V. All rights reserved.

Keywords:ApoptosisCa2+ influxCalpain and caspasesIonomycin (IMN)Interferon-gamma (IFN-γ)Retinal ganglion cell line

1. Introduction

Glaucoma is a group of eye diseases that gradually cause lossof vision due to progressive loss of retinal ganglion cells(RGCs), the neurons that encode and transmit informationfrom the eye to the brain. In addition, the degeneration ofRGCs has also been implicated in loss of visual perception in

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er B.V. All rights reserved

multiple sclerosis (MS) patients (Sattler et al., 2004, 2005).Although it is known that RGCs undergo apoptotic death in theoptic nerve head resulting in progressive loss of vision inglaucoma patients (Kermer et al., 1998) as well as in themonkey model of glaucoma (Quigley et al., 1995), the specificmechanisms involved in apoptosis of RGCs remain mostlyundetermined.

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Fig. 1 – Determination of morphological features ofapoptosis. Treatments (24 h): CTL; 250 nM IMN; 300 units/mlIFN-γ; 2 μM CP; pretreatment with 2 μM CP for 1 h + 250 nMIMN; and pretreatment with 2 μM CP for 1 h + 300 units/mlIFN-γ. (A) Photomicrographs show cells from each treatment,and arrows indicate apoptotic cells. (B) Bar graph shows thepercentage of apoptotic cells counted from each group.

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In some forms of apoptosis, the extrinsic apoptoticpathway is initiated by activation of the apical caspase-8 following death receptor ligation. Caspase-8 processes Bidto truncated Bid (tBid), the C-terminal part of Bid, whichthen translocates to the mitochondrial membrane andtriggers cytochrome c release (Buki et al., 2000; Li et al.,1998; Luo et al., 1998). In other forms, cellular stress leadsto activation of the intrinsic apoptotic pathway initiated bythe apical caspase-9. These pathways converge withactivation of the executioner caspase-3. Superimposed onthis scheme is an “integration model” in which bothupstream and downstream caspases as well as otherproteases cooperate for mediating cell-specific apoptosis(Leist and Jaattela, 2001). Caspase-12, which is specificallylocalized on the outer membrane of the endoplasmicreticulum (ER), is processed by calpain following ER stress(Nakagawa and Yuan, 2000; Nakagawa et al., 2000). ERstress-mediated apoptosis is partly suppressed by caspase-12 deficiency (Nakagawa and Yuan, 2000), suggesting thatcaspase-12 is involved in ER stress-mediated apoptosis.

Regarding apoptosis of RGCs, activation of caspase-9, anupstream player of mitochondria mediated caspase cas-cade, has been reported in axotomized rat retinal ganglioncells in vivo (Kermer et al., 2000) and in a rat model ofexperimental glaucoma (Hansen et al., 1990). Activation ofcaspase-3, the common final effector of caspase cascades,has also been demonstrated in axotomized rat retinalganglion cells in vivo (Chaudhary et al., 1999; Kerrigan etal., 1997).

Previous studies showed induction of apoptosis incultured cells exposed to IMN (Vanags et al., 1997)induced apoptosis with activation of calpain via anextrinsic pathway due to potentiation of pro-apoptoticfeatures of the Bcl-2 family members (Gil-Parrado et al.,2002). It has also been reported that IFN-γ-mediated glialcell apoptosis is associated with calpain upregulation (Rayet al., 1999).

Our current studies indicate that RGC-5 cells (Krishna-moorthy et al., 2001) exposed to IMN and IFN-γ commitapoptotic death via an increase in Bax:Bcl-2 ratio, release ofcytochrome c from mitochondria, and increase in the expres-sion and activity of calpain and caspases. Activation ofcaspase-8 indicated involvement of receptor-mediated path-way of apoptosis in RGC-5 cells exposed to IFN-γ. Calpeptin(CP) prevented apoptosis and provided functionality to RGC-5cells.

2. Results

2.1. Morphological and biochemical features of apoptosisin RGC-5 cells

Wright staining was used to determine the amount ofapoptotic cell death based on morphological featuresunder the light microscope (Fig. 1). Apoptotic RGC-5 cellsshowed characteristic morphological features such ascondensation of the nucleus and cytoplasm, cytoplasmicblebbing, and formation of apoptotic bodies (Fig. 1A).Compared to control (CTL), treatment of cells with IMN

and IFN-γ showed significant increase (P = 0.002) in thepercentage of apoptotic cells (Fig. 1B). CP alone did nothave any effect on cells. Compared to IMN and IFN-γexposure alone, pretreatment with CP decreased thenumber of apoptotic cells by 3-fold following IMN andIFN-γ exposure.

Results of Wright staining were further supported by theApopTag assay (Fig. 2). Qualitatively, both CTL cells andcells treated with CP alone showed no brown color,indicating absence of apoptosis. In contrast, cells treatedwith IMN and IFN-γ alone demonstrated a substantialincrease in brown labeling, indicating occurrence of apo-ptosis (Fig. 2A). Quantitatively, pretreatment of the cellswith CP prevented IMN- and IFN-γ-induced apoptosis asevidenced by a significant decrease in ApopTag-positivecells (Fig. 2B).

Fig. 2 – ApopTag assay for labeling of DNA fragmentation in RGC-5 cells. Treatments (24 h): CTL; 250 nM IMN; 300 units/mlIFN-γ; 2μMCP; pretreatmentwith 2μMCP for 1 h + 250 nM IMN; and pretreatmentwith 2μMCP for 1 h + 300 units/ml IFN-γ. (A)Photomicrographs show cells from each treatment and arrows indicate apoptotic cells. (B) Bar graph shows the percentage ofapoptotic cells counted from each group.

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2.2. Increased intracellular free [Ca2+] following exposureto IMN and IFN-γ

Intracellular free [Ca2+] was determined in all treatmentgroups using fura-2 fluorescence method (Fig. 3). Cells treatedwith IMN and IFN-γ alone for 24 h had a significant increase(P = 0.002) in intracellular free [Ca2+], compared to CTL. Therewas no significant difference (P = 0.999) in intracellular free[Ca2+] measurements between CTL cells and cells treated with

Fig. 3 – Determination of percent of increase of intracellularfree [Ca2+] using fura-2. Data were from RGC-5 cells grown inphenol-red free medium, treated for 24 h, and then exposedto fura-2. Treatments (24 h): CTL; 250 nM IMN; 300 units/mlIFN-γ; 2 μM CP; pretreatment with 2 μM CP for 1 h + 250 nMIMN; and pretreatment with 2 μM CP for 1 h + 300 units/mlIFN-γ.

CP prior to exposure to IMN or IFN-γ, indicating an efficacy ofCP in preventing increase in intracellular free [Ca2+]. Further-more, no significant difference (P = 0.999) was seen betweenCTL cells and cells treated with CP alone.

2.3. Activation of caspase-8 and cleavage of Bid to tBidfollowing exposure to IFN-γ

We examined activation of extrinsic pathway of apoptosis bymonitoring caspase-8 activation and Bid cleavage to tBid (Fig.4). The extrinsic apoptotic pathway can be initiated at the cellsurface through cytokine-induced death-receptor-mediatedsequential activation of caspase-8 and caspase-3 (Hengartner,2000). It may or may not be amplified by additional activationof caspase-9 and caspase-3 via the intrinsic pathway (Kram-mer, 2000). Our results showed a connection between extrinsicand intrinsic apoptotic pathways because activation ofcaspase-8 (Fig. 4A) caused proteolytic cleavage of Bid to tBid(Fig. 4B), which could translocate from cytosol to mitochon-drial membrane to stimulatemore efficient oligomerization ofBax for activation of the intrinsic pathway (Desagher et al.,1999; Eskes et al., 2000; Li et al., 1998). Here, we examined themitochondrial fraction for the measuring amount of tBidtranslocated to the mitochondria (Fig. 4A). The activation ofcaspase-8 by IFN-γ treatment induced cleavage of Bid to tBid.We found that significant increase (P = 0.001) in 18-kDacaspase-8 active band (Fig. 4B) and proteolytic cleavage of Bidto tBid for translocation to mitochondrial membrane (Fig. 4C)

Fig. 4 – Measurement of caspase-8 activation and activity inBid cleavage to tBid using Western blot analysis. Treatments(24 h): CTL; 250 nM IMN; 300 units/ml IFN-γ; 2 μM CP;pretreatment with 2 μM CP for 1 h + 250 nM IMN; andpretreatment with 2 μM CP for 1 h + 300 units/ml IFN-γ.Representative blots showing (A) 18-kDa caspase-8 activeband, 15-kDa tBid band, and 42-kDa β-actin band.Densitometry showed percent changes in (B) the 18-kDacaspase-8 active band and (C) the tBid 15-kDa band.

Fig. 5 – The ratio of Bax to Bcl-2 measured by Western blotanalysis. Treatments (24 h): CTL; 250 nM IMN; 300 units/mlIFN-γ; 2 μM CP; pretreatment with 2 μM CP for 1 h + 250 nMIMN; and pretreatment with 2 μM CP for 1 h + 300 units/mlIFN-γ. The representative gel pictures showing (A) a 21-kDaBax protein, 26-kDa Bcl-2 protein, and 42-kDa β-actinprotein. Densitometry showed (B) the Bax:Bcl-2 ratio.

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in IFN-γ-treated RGC-5 cells, and the proteolysis of Bid wasinhibited by a pretreatment with CP. Furthermore, there issome possibility of calpain-mediated Bid cleavage to tBid(Chen et al., 2001). However, we could not detect anysignificant production of tBid in IMN-treated RGC-5 cells.

2.4. Apoptosis with an increase in Bax:Bcl-2 ratio

A commitment to apoptosis was measured by examiningany changes in Bax and Bcl-2 expression resulting in anincrease in Bax:Bcl-2 ratio (Fig. 5). The Western blotexperiments showed expressions of Bax and Bcl-2 (Fig.5A) in all treatment groups. Exposure of cells to IMN andIFN-γ caused significant increase (P = 0.005) in the Bax:Bcl-2 ratio (Fig. 5B), compared to CTL. The rise in Bax:Bcl-2ratio in cells exposed to IMN and IFN-γ was influenced

more by a change in Bax than a change in Bcl-2expression. There was a significant difference (P = 0.005)in Bax:Bcl-2 ratio between cells treated with IMN or IFN-γalone and those with CP alone. But, there was nosignificant difference (P = 0.986) between CTL cells andcells pretreated with CP and then exposed to IMN or IFN-γ.CP treatment alone did not significantly (P = 0.999) alterthe Bax:Bcl-2 ratio.

2.5. Mitochondrial cytochrome c release and caspase-9activation

Cytosolic and mitochondrial fractions were prepared andanalyzed for the amounts of cytochrome c by Westernblottings (Fig. 6) in order to assess the involvement ofmitochondrial cytochrome c release in IMN- and IFN-γ-induced apoptosis via caspase-9 activation. Treatment ofRGC-5 cells with IMN and IFN-γ caused an appearance ofcytochrome c in the cytosolic fraction and some disappear-ance of it from the mitochondrial fraction (Fig. 6A) of thetreated cells, indicating that IMN and IFN-γ induced mito-chondrial cytochrome c release. Our results also showed anincrease in appearance of 37-kDa caspase-9 active bandfollowing exposure of cells to IMN and IFN-γ (Fig. 6A). Almostuniform levels of β-actin in all lanes indicated that equalamounts of protein were loaded in all lanes. The release ofcytochrome c from mitochondria to cytosol was higher (Fig.6B) in IMN-treated cells (P = 0.001) than in IFN-γ-treated cells(P = 0.002). Cytosolic cytochrome c interacts with pro-caspase-9 to cause caspase-9 activation (Scarlett et al., 2000). Ourresults showed a significant increase (P = 0.001) in a 37-kDa

Fig. 6 – Analysis of cytochrome c release from mitochondriaand activation of caspase-9 by Western blot analysis.Treatments (24 h): CTL; 250 nM IMN; 300 units/ml IFN-γ;2 μM CP; pretreatment with 2 μM CP for 1 h + 250 nM IMN;and pretreatment with 2 μM CP for 1 h + 300 units/mlIFN-γ. Representative blots show (A) 15 kDa cytochrome cfrom cytosolic fraction, 15 kDa cytochrome c frommitochondrial fraction, 37-kDa caspase-9 active band, and42 kDaβ-actin protein. Densitometry shows percent changesin (B) cytochrome c from mitochondrial fraction and (C)caspase-9 active band.

Fig. 7 – Determination of calpain and caspase-3 activitiesusing Western blot analysis of α-spectrin breakdownproducts (SBDPs). Treatments (24 h): CTL; 250 nM IMN;300 units/ml IFN-γ; 2 μM CP; pretreatment with 2 μM CP for1 h + 250 nM IMN; and pretreatment with 2 μM CP for1 h + 300 units/ml IFN-γ. Representative blots showing (A)SBDPs and β-actin. Densitometry showed percent changesin (B) the calpain-specific 145-kDa SBDP and (C) thecaspase-3-specific 120-kDa SBDP. (D) Determination ofcaspase-3 activity using a colorimetric assay.

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caspase-9 active band (Fig. 6C) in cells exposed to IMNand IFN-γ. Treatment of the cells with CP prior to IMN and IFN-γexposures appeared to block the cytochrome c release frommitochondria (Fig. 6B) as well as activation of caspase-9 (Fig.6C). Treatment of cells with CP alone caused non-significant(P = 0.634) release of cytochrome c over CTL.

2.6. Calpain inhibitor blocked α-spectrin breakdown

The degradation of 270-kDa α-spectrin to a 145-kDa spectrinbreakdown product (SBDP) (Nath et al., 1996) and 120-kDaSBDP (Wang et al., 1998) has been attributed to activation ofcalpain and caspase-3, respectively. We determined calpainand caspase-3 activities indirectly by measuring the calpain-specific 145-kDa SBDP and caspase-3-specific 120-kDa SBDP,respectively, on the Western blots (Fig. 7). Almost uniformlevels of β-actin indicated that equal amounts of protein wereloaded in all lanes.

Compared to CTL, the levels of 145-kDa SBDP in cellsexposed to IMN and IFN-γ (Fig. 7B) were 2-fold more intense(P = 0.001), indicating that the extent of calpain activity wasgreater in cells treated with IMN and IFN-γ. Formation of the145-kDa SBDP was significantly prevented in cells pretreatedwith CP. Moreover, the difference in amounts of 145-kDa SBDPbetween CTL cells and cells pretreated with CP and thenexposed to IMN or IFN-γwas not significant. Cells treated withCP alone demonstrated no significant increase (P = 0.537) in145-kDa SBDP over CTL.

The generation of 120-kDa SBDP, an indication ofcaspase-3 activity, in cells exposed to IMN and IFN-γ (Fig.7C) was almost twice (P = 0.001) the amount seen in CTLcells. Treatment of cells with CP prior to IMN and IFN-γexposures decreased the upregulation of caspase-3 activity.

Fig. 9 – Measurement of membrane potential. Treatments(24 h): CTL; 250 nM IMN; 300 units/ml IFN-γ; 2 μM CP;pretreatment with 2 μM CP for 1 h + 250 nM IMN; andpretreatment with 2 μM CP for 1 h + 300 units/ml IFN-γ.

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Furthermore, there was no significant difference (P = 0.900)between CTL cells and cells pretreated with CP and thenexposed to IMN and IFN-γ. Compared to CTL, treatmentwith CP alone did not cause a significant change (P = 0.999)in caspase-3 activity.

2.7. Confirmation of caspase-3 activity directly bycolorimetric method

Caspase-3 activity was also determined directly in alltreatment groups using a colorimetric method (Fig. 7D).Cells exposed to IMN and IFN-γ showed a significantincrease (P = 0.001) in caspase-3 activity, compared toCTL. However, there was no significant difference(P = 0.900) in caspase-3 activity between CTL cells andcells treated with CP prior to exposure to IMN and IFN-γ.Furthermore, no significant difference (P = 0.413) was seenin caspase-3 activity between CTL cells and cells treatedwith CP alone. These results further confirmed thatpretreatment with CP decreased caspase-3 activity in thecells exposed to IMN and IFN-γ.

2.8. Caspase-12 activation in apoptosis of RGC-5 cells

The ER is an important organelle that participates incellular Ca2+ homeostasis by regulating Ca2+ signaling andprotein folding (Ermak and Davies, 2002). However, dysre-gulation of intracellular free Ca2+ homeostasis (Paschen andFrandsen, 2001) and oxidative stress (McCullough et al.,2001) can cause ER stress and apoptosis (Chen et al., 2001).It was reported earlier that caspase-12 was cleaved andactivated by calpain during ER stress (Nakagawa and Yuan,2000; Nakagawa et al., 2000). Here, we examined whetherER stress events might participate in cellular demisefollowing IMN and IFN-γ exposures. We used Westernblottings to measure activation of caspase-12 (Fig. 8A). β-

Fig. 8 – Determination of caspase-12 activation usingWestern blot analysis. Treatments (24 h): CTL; 250 nM IMN;300 units/ml IFN-γ; 2 μM CP; pretreatment with 2 μM CP for1 h + 250 nM IMN; and pretreatment with 2 μM CP for1 h + 300 units/ml IFN-γ. Representative blots showing (A)caspase-12 and β-actin. Densitometry showed percentchanges in (B) the 40-kDa caspase-12 band.

actin expression was used to ensure that equal amounts ofprotein were loaded in all lanes (Fig. 8A). Analysis of datashowed an increase (P = 0.001) in 40-kDa caspase-12 activeband (Fig. 8B), indicating caspase-12 activation. Treatmentwith CP alone did not cause any significant change(P = 0.399) in caspase-12 activation over CTL. Thus, ourdata provided a correlation of the events showing a rise inintracellular free [Ca2+] (Fig. 3) and activation of bothcalpain (Figs. 7A and B) and caspase-12 (Fig. 8). Pretreat-ment with CP decreased the formation of 40-kDa caspase-12 active band, indicating that calpain activity was requiredfor caspase-12 activation.

2.9. Electrophysiological recordings for measuringwhole-cell membrane potential

Patch-clamp recordings were performed to measure thewhole-cell membrane potential (Fig. 9). The majority of thecells exposed to IMN and IFN-γ committed apoptotic death,and therefore nomembranepotentialwas recorded. Therewasno significant difference (P = 0.998) in whole-cell membrane

Fig. 10 – A schematic presentation of molecular eventsleading to activation of calpain and caspase cascades inapoptosis of RGC-5 cells due to IMN and IFN-γ exposure.Increase in intracellular free [Ca2+] and activation of calpainand caspase cascades caused apoptosis in RGC-5 cells.

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potential between CTL cells and cells pretreated with CP andthen exposed to IMN and IFN-γ, thereby suggesting that CPprovided functional neuroprotection. The concentration of CPalone used in the experiments also hadno adverse effect in thewhole-cellmembrane potential. To assesswhether RGC-5 cellsshowed spontaneous activity, cells were held at −60 mV in thepresence of 10 μM glycine to allow for activation of any N-methyl-D-aspartic acid (NMDA) receptor-dependent currents.Cells were also stimulated with depolarizing pulses (+100 mV)to activate any voltage-gated ion channels. In the presence orabsence of glycine, there was no evidence of any spontaneousactivity. In addition, only small (20–30 pA) currents wereobserved following the voltage steps, suggesting that thesecells did not show expression of the voltage-dependent Na+ orCa2+ channels.

3. Discussion

Apoptosis of the RGCs in the optic nerve head results inprogressive loss of vision in glaucoma and MS patients. UsingRGC-5 as a cell culture model, we studied the mechanism ofapoptosis of RGCs following exposure to IMN and IFN-γ andneuroprotective effect of CP, an inhibitor of calpain. Our datashowed an increase in apoptosis in RGC-5 cells exposed to IMNand IFN-γ, and pretreatmentwith CP attenuated apoptosis. Anincrease in intracellular free [Ca2+] has been observed incourse of this apoptotic process. Only IFN-γ increasedcaspase-8-mediated cleavage of Bid to tBid, indicating aninvolvement of the extrinsic pathway of apoptosis. Incontrast, exposure of RGC-5 cells to either IMN or IFN-γincreased in Bax:Bcl-2 ratio, released cytochrome c frommitochondria, and activated caspase-9, calpain, and cas-pase-3, suggesting participation of the intrinsic pathway ofapoptosis. Our data also showed an increase in caspase-12activity in RGC-5 cells treated with IMN and IFN-γ. Impor-tantly, pretreatment of RGC-5 cells with CP preventedapoptotic features and maintained normal whole-cell mem-brane potential, indicating functional neuroprotection. Fig. 10schematically summarizes the mechanism of apoptosiselucidated by the present study.

Calpain inhibitors can inhibit calpain-mediated proteolysisof talin and actin-binding protein (Wiedmer et al., 1990) aswellas protein–tyrosine phosphatases (Schumacher et al., 1999).Selective inhibitors of calpain also block calpain-mediatedproteolysis in animal models of traumatic brain injury (TBI)(Hall et al., 1999; Kupina et al., 2001; Saatman et al., 1996),spinal cord injury (SCI) (Horrocks et al., 1985; Ray et al., 2000c,2001), brain ischemia (Bartus et al., 1994), and retinal ischemia(Sakamoto et al., 2000), improving functional outcome in somecases (Kupina et al., 2001; Saatman et al., 1996; Schumacher etal., 2000; Smith et al., 1998). Consistent with these studies, CPprovided functional neuroprotection in RGC-5 cells.

The increases in intracellular free [Ca2+] that predisposedthe cells to damage via numerous pathways have beendocumented inmany neurodegenerative conditions includingTBI (Chen et al., 2001; Kupina et al., 2001) and SCI (Happel et al.,1981; Ray et al., 2001). Upregulation of calpain pathway hasstrongly been implicated in the pathophysiology of many CNSinjuries and diseases (Ray and Banik, 2003; Ray et al., 2000a).

Calpain induction by elevated intracellular free [Ca2+]may leadto degradation of cytoskeletal proteins (Yanagisawa et al.,1983) and cell death (Ray et al., 2001). Our findings support adirect relationship between an increase in intracellular free[Ca2+] and cell death due to elevated calpain activity followingexposure of RGC-5 cells to IMN or IFN-γ. This is confirmed bythe protective effect of CP, which decreased intracellular free[Ca2+] and calpain activity elicited by IMN or IFN-γ. These datasuggest that CP may have changed a pathway upstream of theCa2+ influx in RGC-5 cells following IMN or IFN-γ exposure.Calpainmaymediate Ca2+ influx during cell injury and also actsubsequent to Ca2+ influx (Waters et al., 1997). Both peptideand non-peptide inhibitors of calpain are capable of blockingCa2+ influx (Liu et al., 2002). Neuroprotective effect of CPmay bedue to direct blocking of Ca2+ influx to some degree as wereported recently (Das et al., 2005). Because our current resultsshowed that CP pretreatment significantly attenuated celldeath by inhibiting activation of calpain and caspase-3, weinferred that the efficacy of CP was not dependent onprevention of Ca2+ influx but rather on downstream events ofCa2+ influx. These data support the report that IMN treatmentcan cause unpreventable increases in intracellular free [Ca2+](Gil-Parrado et al., 2002). Our data also showed an associationamong increased intracellular free [Ca2+], activation of calpain,and activation of caspase-12. It is known that activation ofcaspase-12 is related to ER stress and requires calpain-mediated cleavage (Nakagawa and Yuan, 2000; Nakagawa etal., 2000). Pretreatment with CP reduced the Ca2+-dependentactivation of both calpain and caspase-12 in RGC-5 cells. Theintensity of the caspase-12 active band was 3-fold higher incells treated with IMN than in cells treated with IFN-γ,indicating that only IMN treatment caused strong ER stressand activated calpain and caspase-12. Taken together, thesefindings suggest that the neuroprotective effect of CP in RGC-5cells is directly related to the prevention of calpain activationfollowing IMN or IFN-γ exposure.

Another mechanism of apoptosis is known to bemitochondrial damage due to intra- and extra-mitochon-drial calpain activation (Buki et al., 2000; Varghese et al.,2001). Pro-apoptotic Bax resides in mitochondria and isactivated by calpain (Olson and Kornbluth, 2001). Consis-tent with the previous studies (Choi et al., 2001; Gil-Parradoet al., 2002; Ray et al., 2000b), we found that alterations inlevels of Bax and Bcl-2 correlated well with increasedcalpain expression and cell death, indicating the participa-tion of pro-apoptotic Bax and altered mitochondrial per-meability in RGC-5 cell death. A consequence of alteredmitochondrial permeability is the release of cytochrome c.Members of the Bcl-2 family have been implicated inregulation of cytochrome c release from the mitochondrialintermembrane space into the cytosol (Scarlett et al., 2000).Cytosolic cytochrome c then interacts with pro-caspase-9and Apaf-1 to activate caspase-9 and then caspase-3leading to apoptosis (Scarlett et al., 2000). The pro-apoptoticproteins (Bax and Bak) of the Bcl-2 family can promoteopening of the voltage-dependent anion channels in theouter mitochondrial membrane followed by release ofcytochrome c for induction of apoptosis (Scarlett et al.,2000; Wong and Cortopassi, 1997). Pretreatment of RGC-5cells with CP blocked cytochrome c release and caspase-9

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activation, supporting the notion that neuroprotection is, inpart, due to inhibition of the mitochondrial apoptoticpathway that involves activities of calpain and caspases.

Previous studies have suggested involvement of cyto-kines both in normal CNS development (Zhao andSchwartz, 1988) and in neurodegenerative diseases (Merrilland Jonakait, 1995). Many cytokines and their receptors areexpressed in CNS cells (Eddleston and Mucke, 1993; Eng etal., 1996). Some cytokines act as neuroimmune messengers(Rothwell, 1997). This concept is further supported by thefindings of neurotransmitter receptors on lymphocytes andcytokine receptors on neurons and glial cells (Sawada etal., 1993). IFN-γ induces expression of calpain at the mRNAand protein levels in U937 and THP-1 cells (Deshpande etal., 1995) and also in C6 cells (Ray et al., 2003). In thecurrent study, RGC-5 cells treated with IFN-γ showed anincrease in caspase-8 active band and proteolytic cleavageof Bid to tBid leading to apoptosis, which was inhibited bypretreatment with CP. An earlier report has shown thatcalpain inhibitor I is capable of blocking the TNF-α-mediated apoptotic signal in human T-cells (Diaz andBourguignon, 2000) and also inhibiting fenretinide-inducedactivation of caspase-8 and apoptosis in hepatoma cells(You et al., 2001). Calpain inhibitors also protected matureoligodendrocytes from cell death due to inhibition ofactivation of caspases-3, -8, and -9 following exposure tostaurosporine, kainate, and thapsigargin (Benjamins et al.,2003). The present study showed that pretreatment of RGC-5 cells with CP partially blocked caspase-8 activity. Takentogether, these findings indicate that the neuroprotectiveeffect of a calpain inhibitor is in part due to its action onextrinsic pathway of apoptosis.

In conclusion, our results with the use of RGC-5 cellsprovide evidence that the calpain inhibitor CP can suppressapoptosis-inducing enzymes in RGCs and thereby inhibitapoptosis. This RGC-5 cell model may partially reflect howRGCs would react to Ca2+ influx in course of glaucoma.Further in vivo studies may prove therapeutic efficacy of CPfor the functional protection of RGCs in glaucoma.

4. Experimental procedures

4.1. Cell culture

The rat retinal ganglion cell line RGC-5 was grown inmonolayer to sub-confluency in 75-cm2 flasks containing10 ml of 1× Dulbecco's modification of Eagle's medium with1 gm/L glucose and L-glutamine, supplemented with 1%penicillin and streptomycin (GIBCO-Invitrogen Corporation,Grand Island, NY, USA), and 10% fetal bovine serum in afully humidified incubator containing 5% CO2 at 37 °C. Dose–response studies were conducted to determine the appro-priate doses of IMN, IFN-γ, and CP. Cells were treated withIMN, IFN-γ, or CP alone in the medium as stated above for24 h to examine the morphological and biochemical featuresof cell death. In some experiments, cells were pretreatedwith 2 μM CP for 1 h and subsequently treated with IMN orIFN-γ for 24 h. For all experiments, cells were always allowedto grow in the fully humidified incubator containing 5% CO2

at 37 °C. After all treatments, cells were used for variousexperiments as stated below.

4.2. Wright staining for morphological analysis ofapoptosis

The RGC-5 cells from each treatment were detached with acell scraper to harvest the attached and detached cellstogether. Cells were washed twice in phosphate-bufferedsaline (PBS, pH 7.4) and sedimented onto the microscopicslides using an Eppendorf 5804R centrifuge (BrinkmannInstruments, Westbury, NY, USA) at 106 × g for 5 min.Cells were fixed and stained with Wright stain as wereported (Das et al., 2004). Cellular morphology was exam-ined by optical microscopy to assess apoptosis. Cells wereconsidered apoptotic if they showed reduction in cell volumeand condensation of the chromatin and/or the presence ofcell membrane blebbing (Geng et al., 1996). At least 600 cellswere counted in each treatment, and the percentage ofapoptotic cells was calculated.

4.3. ApopTag peroxidase assay for biochemical evidence ofapoptosis

Apoptotic cells were detected using the ApopTag peroxidaseassay kit (Intergen Company, Purchase, NY, USA) according tothe supplier's instructions. The cells were also counterstainedwith methyl green. Methyl green stained normal nuclei a paleto medium green. The nuclei that contain DNA fragments orcondensation were positively stained dark brown (by theApopTag detection procedure) and were not stained with themethyl green. Apoptosis was quantified by counting ApopTag-stained cells on the grid of the microscope field (using 40×objective). Experiments were performed in triplicate, and thebrown-colored ApopTag-positive cells were counted under thelight microscope to determine the percentage of apoptosis.

4.4. Determination of intracellular free [Ca2+] using fura-2assay

Level of intracellular free [Ca2+] in RGC-5 cells was measuredusing the fluorescence Ca2+ indicator fura-2/AM, as wedescribed recently (Das et al., 2004, 2005) with modification ofa previously reported method (Grynkiewicz et al., 1985). Theintracellular free [Ca2+] was calculated spectrofluorometricallywith the use of the following formula: [Ca2+] = Kdβ(R − Rmin) /(Rmax − R), where β is the ratio of F380max (the fluorescenceintensity exciting at 380 nm for zero level of free Ca2+) to F380min

(the fluorescence intensity at saturating level of free Ca2+), asreported previously (Hanninen et al., 2002). The determinationof fluorescence ratio (R) was performed using an SLM 8000spectrofluorimeter (Thermospectronic, Rochester, NY, USA)at 340 and 380 nm wavelengths. The maximal fluorescenceratio (Rmax) was determined by adding 200 μl of 250 μMdigitonin (Fisher Scientific, Pittsburgh, PA) to 1 × 106 cells/ml, and subsequently minimal fluorescence ratio (Rmin) wasdetermined by adding 200 μl of 500 mM EGTA (Sigma) tothe same cell suspension. The value of Kd, a cell-specificconstant, was determined experimentally to be 0.387 μM forthe RGC-5 cells using standards of the Calcium Calibration

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Buffer Kit with Magnesium (Molecular Probes, Eugene, OR,USA).

4.5. Antibodies

Monoclonal Bax and Bcl-2 primary IgG antibodies (Santa CruzBiotechnology, Santa Cruz, CA, USA) were used to assessapoptotic threshold by determining the Bax:Bcl-2 ratio.Monoclonal α-spectrin primary IgG antibody (Affiniti, Exeter,UK) was used to measure SBDPs produced by calpain andcaspase-3 activities. Monoclonal caspase-9, polyclonal cas-pase-8 and caspase-12 primary IgG antibodies (Santa CruzBiotech, Santa Cruz, CA, USA) were used to assess theinvolvement of the caspase cascades in apoptosis. PolyclonalBid primary IgG antibody (Santa Cruz Biotechnology, SantaCruz, CA, USA) was used to assess the Bid cleavage.Monoclonal β-actin primary IgG antibody (clone AC-15,Sigma Chemical Co., St. Louis, MO, USA) was used tostandardize protein loading on the SDS-PAGE gels. Thehorseradish-peroxidase (HRP)-conjugated goat anti-mousesecondary IgG antibody (ICN Biomedicals, aurora, OH, USA)was used to identify all the monoclonal primary antibodies,whereas HRP-conjugated goat anti-rabbit secondary IgGantibody (ICN Biomedicals, Aurora, OH, USA) was used todetect the polyclonal primary antibodies.

4.6. Western blot analyses of specific proteins

Western blottings were performed following the procedureas we described previously (Das et al., 2004, 2005). After SDS-PAGE runs, resolved total proteins from the gels weretransferred to nylon membranes (Millipore, Bedford, MA,USA) in the electroblotting apparatus Genie (Idea Scientific,Minneapolis, MN, USA). The membrane was then blocked in5% powdered non-fat milk in a Tris/Tween solution (20 mMTris–HCl, pH 7.6, 0.1% Tween-20 in saline) for 1 h. Theprimary antibodies were diluted (1:100 for Bax, Bcl-2, Bid,caspase-3, caspase-8, caspase-9, caspase-12; and 1:500 forcalpain; 1:2000 for α-spectrin; and 1:15,000 for β-actin) inblocking solution and then added to the blots for 1 h. Theblots were then covered with an alkaline HRP-conjugatedsecondary IgG antibody (goat anti-rabbit for Bid, calpain,caspase-8, caspase-12, and goat anti-mouse for all others) ata 1:2000 dilution for 1 h. Between steps, blots were washedthree times in Tris/Tween solution. Thereafter, blots wereincubated with enhanced chemiluminescence (ECL) reagents(Amersham Pharmacia, Buckinghamshire, UK) and exposedto X-OMAT AR films (Eastman Kodak, Rochester, NY, USA).The ECL autoradiograms were scanned on a UMAX Power-Look Scanner (UMAX Technologies, Fremont, CA, USA) usingPhotoshop software (Adobe Systems, Seattle, WA, USA), andoptical density of each band was determined using QuantityOne software (Bio-Rad Laboratories, Hercules, CA, USA).

4.7. Analysis of cytochrome c release from mitochondria tocytosol

The release of cytochrome c from mitochondria to cytosolwas assessed by Western blottings after collection of cellularfractions containing mitochondria and cytosol (Pique et al.,

2000). Cells (107) from each treatment were harvested,washed once with ice-cold PBS, and gently lysed for 1 minin 100 μl ice-cold lysis buffer (250 mM sucrose, 1 mM EDTA,0.05% digitonin, 25 mM Tris–HCl, pH 6.8, 1 mM dithiothreitol,1 μg/ml leupeptin, 1 μg/ml pepstatin, 1 μg/ml aprotinin,1 mM benzamidine, and 0.1 mM phenylmethylsulfonylfluoride). Lysates were centrifuged at 12,000 × g at 4 °C for3 min to obtain the pellet (fraction containing mitochondria)and the supernatant (cytosolic extract without mitochon-dria). Pellets and supernatants were analyzed by Westernblottings using cytochrome c antibody (BD Biosciences, SanJose, CA, USA).

4.8. Colorimetric assay for direct assessment of caspase-3activity

Measurement of caspase-3 activity directly was done withthe commercially available Caspase-3 Assay kit (Sigma, St.Louis, MO, USA). The caspase-3 colorimetric assay is basedon the hydrolysis of the peptide substrate acetyl-Asp-Glu-Val-Asp-p-nitroanilide (Ac-DEVD-pNA) by caspase-3, result-ing in the release of the p-nitroaniline (pNA) moiety. ThepNA has a high absorbance at 405 nm (∈mM = 10.5).Proteolytic reactions were carried out in extraction buffercontaining 20 μg of cytosolic protein extract and 40 μM Ac-DEVD-pNA. The reaction mixtures were incubated at roomtemperature for 2 h, and the formation of pNA wasmeasured at 405 nm using a colorimeter. The concentrationof the pNA released from the substrate was calculated fromthe absorbance values at 405 nm. Experiments wereperformed in triplicate.

4.9. Electrophysiology for recording of whole-cellmembrane potentials

For electrophysiology, RGC-5 cells were grown on 35 mmculture dishes. Standard patch-clamp techniques wereemployed for recording whole-cell membrane potential(Hamill et al., 1981). Recordings were made with an Axopatch200B amplifier in conjunction with Axograph software (AxonInstruments, Union City, CA, USA). RGC-5 cells grown on 35-mm culture dishes were perused at room temperature withextracellular recording solution containing 135 mM NaCl,5 mM KCl, 1.8 mM CaCl2, 10 mM glucose, and 5 mM HEPES(pH adjusted to 7.2 with NaOH and osmolarity adjusted to325 mosM with sucrose). Patch electrodes (2.5–4.0 mΩ) werefilled with an internal solution containing 150mMKCl, 2.5mMNaCl, 4 mMMg-ATP, 2 mMNa-ATP, 0.3 mMNa-GTP, 5 mMNa-phosphocreatine, and 10 mM HEPES (pH adjusted to 7.4 withNaOH and osmolarity adjusted to 310 mosM with sucrose).The liquid junction potential was 4.1mV andwas corrected forin all recordings. Following seal formation and breakthroughin voltage-clamp (holding potential −60mV), the amplifier wasswitched to current-clamp mode with zero holding currentand the resulting membrane potential was recorded.

4.10. Statistical analysis

Results were assessed using StatView software (AbacusConcepts, Berkeley, CA, USA) and compared using one-way

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analysis of variance (ANOVA) with Fisher's protected leastsignificant difference (PLSD) post hoc test at a 95% confidenceinterval. Data were presented as mean ± standard error ofmean (SEM, n ≥ 3). Difference between two treatments wasconsidered significant at P ≤ 0.01. Significant difference CTLand IMN (or IFN-γ) was indicated by *. Significant differencebetween IMN (or IFN-γ) treatment and CP pretreatment + IMN(or IFN-γ) was indicated by #.

Acknowledgments

This work was supported in part by the R01 grants from theNCI (CA-91460) and NINDS (NS-31622, NS-38146, NS-41088,and NS-45967) and also by the Spinal Cord Injury ResearchFund (SCIRF-0803) from the State of South Carolina.

R E F E R E N C E S

Bartus, R.T., Hayward, N.J., Elliott, P.J., Sawyer, S.D., Baker, K.L.,Dean, R.L., Akiyama, A., Straub, J.A., Harbeson, S.L., Li, Z.,Powers, J., 1994. Calpain inhibitor AK295 protects neurons fromfocal brain ischemia. Effects of postocclusion intra-arterialadministration. Stroke 25, 2265–2270.

Benjamins, J.A., Nedelkoska, L., George, E.B., 2003. Protection ofmature oligodendrocytes by inhibitors of caspases andcalpains. Neurochem. Res. 28, 143–152.

Buki, A., Okonkwo, D.O., Wang, K.K., Povlishock, J.T., 2000.Cytochrome c release and caspase activation in traumaticaxonal injury. J. Neurosci. 20, 2825–2834.

Chaudhary, P., Ahmed, F., Quebada, P., Sharma, S.C., 1999. Caspaseinhibitors block the retinal ganglion cell death following opticnerve transection. Mol. Brain Res. 67, 36–45.

Chen, M., He, H., Zhan, S., Krajewski, S., Reed, J.C., Gottlieb, R.A.,2001. Bid is cleaved by calpain to an active fragment in vitroand during myocardial ischemia/reperfusion. J. Biol. Chem.276, 30724–30728.

Choi, W.S., Lee, E.H., Chung, C.W., Jung, Y.K., Jin, B.K., Kim, S.U.,Oh, T.H., Saido, T.C., Oh, Y.J., 2001. Cleavage of Bax ismediated by caspase-dependent or -independent calpainactivation in dopaminergic neuronal cells: protective role ofBcl-2. J. Neurochem. 77, 1531–1541.

Das, A., Banik, N.L., Patel, S.J., Ray, S.K., 2004. Dexamethasoneprotected human glioblastoma U87MG cells fromtemozolomide induced apoptosis by maintaining Bax:Bcl-2ratio and preventing proteolytic activities. Mol. Cancer 3, 1–10.

Das, A., Sribnick, E.A., Wingrave, J.M., Del Re, A.M., Woodward, J.J.,Appel, S.H., Banik, N.L., Ray, S.K., 2005. Calpain activation inapoptosis of ventral spinal cord 4.1 (VSC4.1) motoneuronsexposed to glutamate: Calpain inhibition provided functionalneuroprotection. J. Neurosci. Res. 81, 551–562.

Desagher, S., Osen-Sand, A., Nichols, A., Eskes, R., Montessuit, S.,Lauper, S., Maundrell, K., Antonsson, B., Martinou, J.C., 1999.Bid-induced conformational change of Bax is responsible formitochondrial cytochrome c release during apoptosis. J. CellBiol. 144, 891–901.

Deshpande, R.V., Goust, J.M., Chakrabarti, A.K., Barbosa, E., Hogan,E.L., Banik, N.L., 1995. Calpain expression in lymphoid cells.Increased mRNA and protein levels after cell activation. J. Biol.Chem. 270, 2497–2505.

Diaz, F., Bourguignon, L.Y., 2000. Selective down-regulation ofIP(3) receptor subtypes by caspases and calpain duringTNF-α-induced apoptosis of human T-lymphoma cells. CellCalcium 27, 315–328.

Eddleston, M., Mucke, L., 1993. Molecular profile of reactiveastrocytes implications for their role in neurologic disease.Neuroscience 54, 15–36.

Eng, L.F., Ghirnikar, R.S., Lee, Y.L., 1996. Inflammation in EAE: roleof chemokine/cytokine expression by resident and infiltratingcells. Neurochem. Res. 21, 511–525.

Ermak, G., Davies, K.J., 2002. Calcium and oxidative stress: fromcell signaling to cell death. Mol. Immunol. 38, 713–721.

Eskes, R., Desagher, S., Antonsson, B., Martinou, J.C., 2000. Bidinduces the oligomerization and insertion of Bax into the outermitochondrial membrane. Mol. Cell. Biol. 20, 929–935.

Geng, Y.J., Hellstrand, K., Wennmalm, A., Hansson, G.K., 1996.Apoptotic death of human leukemic cells induced by vascularcells expressing nitric oxide synthase in response togamma-interferon and tumor necrosis factor-alpha. CancerRes. 56, 866–874.

Gil-Parrado, S., Fernandez-Montalvan, A., Assfalg-Machleidt, I.,Popp, O., Bestvater, F., Holloschi, A., Knoch, T.A., Auerswald,E.A., Welsh, K., Reed, J.C., Fritz, H., Fuentes-Prior, P., Spiess,E., Salvesen, G.S., Machleidt, W., 2002. Ionomycin-activatedcalpain triggers apoptosis: a probable role for Bcl-2 familymembers. J. Biol. Chem. 277, 27217–27226.

Grynkiewicz, G., Poenie, M., Tsien, R.Y., 1985. A new generation ofCa2+ indicators with greatly improved fluorescence properties.J. Biol. Chem. 260, 3440–3450.

Hall, E.D., Kupina, N.C., Althaus, J.S., 1999. Peroxynitritescavengers for the acute treatment of traumatic brain injury.Ann. N. Y. Acad. Sci. 890, 462–468.

Hamill, O.P., Marty, A., Neher, E., Sakmann, B., Sigworth, F.J., 1981.Improved patch-clamp techniques for high-resolution currentrecording from cells and cell-free membrane patches. PflugersArch. 391, 85–100.

Hanninen, V.A., Pantcheva, M.B., Freeman, E.E., Poulin, N.R.,Grosskreutz, C.L., 2002. Activation of caspase-9 in a rat modelof experimental glaucoma. Curr. Eye Res. 25, 389–395.

Hansen, C.A., Monck, J.R., Williamson, J.R., 1990. Measurement ofintracellular free calcium to investigate receptor-mediatedcalcium signaling. Methods Enzymol. 191, 691–706.

Happel, R.D., Smith, K.P., Banik, N.L., Powers, J.M., Hogan, E.L.,Balentine, J.D., 1981. Ca2+-accumulation in experimental spinalcord trauma. Brain Res. 211, 476–479.

Hengartner, M.O., 2000. The biochemistry of apoptosis. Nature 407,770–776.

Horrocks, L.A., Demediuk, P., Saunders, R.D., Dugan, L., Clendenon,N.R., Means, E.D., Anderson, D.K., 1985. The degradation ofphospholipids, formation of metabolites of arachidonic acid,and demyelination following experimental spinal cord injury.Cent. Nerv. Syst. Trauma 2, 115–120.

Kermer, P., Klocker, N., Labes, M., Bahr, M., 1998. Inhibitionof CPP32-like proteases rescues axotomized retinal ganglioncells from secondary cell death in vivo. J. Neurosci. 18,4656–4662.

Kermer, P., Ankerhold, R., Klocker, N., Krajewski, S., Reed, J.C.,Bahr, M., 2000. Caspase-9: involvement in secondary death ofaxotomized rat retinal ganglion cells in vivo. Mol. Brain Res. 85,144–150.

Kerrigan, L.A., Zack, D.J., Quigley, H.A., Smith, S.D., Pease, M.E.,1997. TUNEL-positive ganglion cells in human primaryopen-angle glaucoma. Arch. Ophthalmol. 115, 1031–1035.

Krammer, P.H., 2000. CD95's deadly mission in the immunesystem. Nature 407, 789–795.

Krishnamoorthy, R.R., Agarwal, P., Prasanna, G., Vopat, K.,Lambert, W., Sheedlo, H.J., Pang, I.H., Shade, D., Wordinger, R.J.,Yorio, T., Clark, A.F., Agarwal, N., 2001. Characterization of atransformed rat retinal ganglion cell line. Mol. Brain Res. 86,1–12.

Kupina, N.C., Nath, R., Bernath, E.E., Inoue, J., Mitsuyoshi, A., Yuen,P.W., Wang, K.K., Hall, E.D., 2001. The novel calpain inhibitorSJA6017 improves functional outcome after delayed

156 B R A I N R E S E A R C H 1 0 8 4 ( 2 0 0 6 ) 1 4 6 – 1 5 7

administration in a mouse model of diffuse brain injury.J. Neurotrauma 18, 1229–1240.

Leist, M., Jaattela, M., 2001. Four deaths and a funeral: fromcaspases to alternativemechanisms. Nat. Rev., Mol. Cell Biol. 2,589–598.

Li, H., Zhu, H., Xu, C.J., Yuan, J., 1998. Cleavage of BID bycaspase-8 mediates the mitochondrial damage in the Faspathway of apoptosis. Cell 94, 491–501.

Liu, X., Harriman, J.F., Schnellmann, R.G., 2002. Cytoprotectiveproperties of novel nonpeptide calpain inhibitors in renal cells.J. Pharmacol. Exp. 302, 88–94.

Luo, X., Budihardjo, I., Zou, H., Slaughter, C., Wang, X., 1998. Bid, aBcl-2 interacting protein, mediates cytochrome c release frommitochondria in response to activation of cell surface deathreceptors. Cell 94, 481–490.

McCullough, K.D., Martindale, J.L., Klotz, L.O., Aw, T.Y., Holbrook,N.J., 2001. Gadd153 sensitizes cells to endoplasmic reticulumstress by down-regulating Bcl-2 and perturbing the cellularredox state. Mol. Cell. Biol. 21, 1249–1259.

Merrill, J.E., Jonakait, G.M., 1995. Interactions of the nervous andimmune systems in development, normal brain homeostasis,and disease. FASEB J. 9, 611–618.

Nakagawa, T., Yuan, J., 2000. Cross talk between two cysteineprotease families. Activation of caspase-12 by calpain inapoptosis. J. Cell Biol. 150, 887–894.

Nakagawa, T., Zhu, H., Morishima, N., Li, E., Xu, J., Yankner, B.A.,Yuan, J., 2000. Caspase-12 mediatesendoplasmic-reticulum-specific apoptosis and cytotoxicity byamyloid-β. Science 403, 98–103.

Nath, R., Raser, K.J., Stafford, D., Hajimohammadreza, I., Posner,A., Allen, H., Talanian, R.V., Yuen, P., Gilbertsen, R.B., Wang,K.K., 1996. Non-erythroid α-spectrin breakdown by calpainand interleukin-1β-converting-enzyme-like protease(s) inapoptotic cells: contributory roles of both protease families inneuronal apoptosis. Biochem. J. 319, 683–690.

Olson, M., Kornbluth, S., 2001. Mitochondria in apoptosis andhuman disease. Curr. Mol. Med. 1, 91–122.

Paschen, W., Frandsen, A., 2001. Endoplasmic reticulumdysfunction—A common denominator for cell injury in acuteand degenerative diseases of the brain? J. Neurochem. 79,719–725.

Pique, M., Barragan, M., Dalmau, M., Bellosillo, B., Pons, G., Gil, J.,2000. Aspirin induces apoptosis through mitochondrialcytochrome c release. FEBS Lett. 480, 193–196.

Quigley, H.A., Nickells, R.W., Kerrigan, L.A., Pease, M.E., Thibault,D.J., Zack, D.J., 1995. Retinal ganglion cell death in experimentalglaucoma and after axotomy occurs by apoptosis. Invest.Ophthalmol. Visual Sci. 36, 774–786.

Ray, S.K., Banik, N.L., 2003. Calpain and its involvement in thepathophysiology of CNS injuries and diseases: therapeuticpotential of calpain inhibitors for prevention ofneurodegeneration. Curr. Drug Targets CNS Neurol. Disord. 2,173–189.

Ray, S.K., Wilford, G.G., Crosby, C.V., Hogan, E.L., Banik, N.L., 1999.Diverse stimuli induce calpain overexpression and apoptosisin C6 glioma cells. Brain Res. 829, 18–27.

Ray, S.K., Fidan, M., Nowak, M.W., Wilford, G.G., Hogan, E.L., Banik,N.L., 2000a. Oxidative stress and Ca2+ influx upregulatecalpain and induce apoptosis in PC12 cells. Brain Res. 852,326–334.

Ray, S.K., Matzelle, D.C., Wilford, G.G., Hogan, E.L., Banik, N.L.,2000b. E-64-d prevents both calpain up-regulation andapoptosis in the lesion and penumbra following spinal cordinjury in rats. Brain Res. 867, 80–89.

Ray, S.K., Matzelle, D.D., Wilford, G.G., Hogan, E.L., Banik, N.L.,2000c. Increased calpain expression is associated withapoptosis in rat spinal cord injury: calpain inhibitor providesneuroprotection. Neurochem. Res. 25, 1191–1198.

Ray, S.K., Matzelle, D.D., Wilford, G.G., Hogan, E.L., Banik, N.L.,

2001. Cell death in spinal cord injury (SCI) requires de novoprotein synthesis. Calpain inhibitor E-64-d providesneuroprotection in SCI lesion and penumbra. Ann. N. Y. Acad.Sci. 939, 436–449.

Ray, S.K., Hogan, E.L., Banik, N.L., 2003. Calpain in thepathophysiology of spinal cord injury: neuroprotection withcalpain inhibitors. Brain Res. Rev. 42, 169–185.

Rothwell, N.J., 1997. Neuroimmune interactions: the role ofcytokines. Br. J. Pharmacol. 121, 841–847.

Saatman, K.E., Murai, H., Bartus, R.T., Smith, D.H., Hayward, N.J.,Perri, B.R., McIntosh, T.K., 1996. Calpain inhibitor AK295attenuates motor and cognitive deficits followingexperimental brain injury in the rat. Proc. Natl. Acad. Sci. U. S. A.93, 3428–3433.

Sakamoto, Y.R., Nakajima, T.R., Fukiage, C.R., Sakai, O.R., Yoshida,Y.R., Azuma, M.R., Shearer, T.R., 2000. Involvement of calpainisoforms in ischemia–reperfusion injury in rat retina. Curr. EyeRes. 21, 571–580.

Sattler, M.B., Merkler, D., Maier, K., Stadelmann, C., Ehrenreich, H.,Bahr, M., Diem, R., 2004. Neuroprotective effects andintracellular signaling pathways of erythropoietin ina rat model of multiple sclerosis. Cell Death Differ. 11,S181–S192.

Sattler, M.B., Diem, R., Merkler, D., Demmer, I., Boger, I.,Stadelmann, C., Bahr, M., 2005. Simvastatin treatment does notprotect retinal ganglion cells from degeneration in a rat modelof autoimmune optic neuritis. Exp. Neurol. 193, 163–171.

Sawada, M., Itoh, Y., Suzumura, A., Marunouchi, T., 1993.Expression of cytokine receptors in cultured neuronal and glialcells. Neurosci. Lett. 160, 131–134.

Scarlett, J.L., Sheard, P.W., Hughes, G., Ledgerwood, E.C., Ku, H.H.,Murphy, M.P., 2000. Changes in mitochondrial membranepotential during staurosporine-induced apoptosis in Jurkatcells. FEBS Lett. 475, 267–272.

Schumacher, P.A., Eubanks, J.H., Fehlings, M.G., 1999. Increasedcalpain I-mediated proteolysis, and preferential loss ofdephosphorylated NF200, following traumatic spinal cordinjury. Neuroscience 91, 733–744.

Schumacher, P.A., Siman, R.G., Fehlings, M.G., 2000.Pretreatment with calpain inhibitor CEP-4143 inhibitscalpain I activation and cytoskeletal degradation,improves neurological function, and enhances axonalsurvival after traumatic spinal cord injury.J. Neurochem. 74, 646–1655.

Smith, M.E., van der Maesen, K., Somera, F.P., 1998. Macrophageand microglial responses to cytokines in vitro: phagocyticactivity, proteolytic enzyme release, and free radicalmproduction. J. Neurosci. Res. 54, 68–78.

Vanags, D.M., Orrenius, S., Aguilar-Santelises, M., 1997. Alterationsin Bcl-2/Bax protein levels in platelets form part of anionomycin-induced process that resembles apoptosis. Br. J.Haematol. 99, 824–831.

Varghese, J., Radhika, G., Sarin, A., 2001. The role of calpain incaspase activation during etoposide induced apoptosis in Tcells. Eur. J. Immunol. 31, 2035–2041.

Wang, K.K., Posmantur, R., Nath, R., McGinnis, K., Whitton, M.,Talanian, R.V., Glantz, S.B., Morrow, J.S., 1998. Simultaneousdegradation of αII- and βII-spectrin by caspase-3 (CPP32) inapoptotic cells. J. Biol. Chem. 273, 22490–22497.

Waters, S.L., Sarang, S.S., Wang, K.K., Schnellmann, R.G., 1997.Calpains mediate calcium and chloride influx during thelate phase of cell injury. J. Pharmacol. Exp. Ther. 283,1177–1184.

Wiedmer, T., Shattil, S.J., Cunningham, M., Sims, P.J., 1990.Role of calcium and calpain in complement-inducedvesiculation of the platelet plasma membrane and in theexposure of the platelet factor Va receptor. Biochemistry29, 623–632.

Wong, A., Cortopassi, G., 1997. mtDNA mutations confer cellular

157B R A I N R E S E A R C H 1 0 8 4 ( 2 0 0 6 ) 1 4 6 – 1 5 7

sensitivity to oxidant stress that is partially rescued by calciumdepletion and cyclosporin A. Biochem. Biophys. Res. Commun.239, 139–145.

Yanagisawa, K., Sato, S., Amaya, N., Miyatake, T., 1983.Degradation of myelin basic protein by calcium-activatedneutral protease in human brain and inhibition by E-64analogue. Neurochem. Res. 8, 1285–1293.

You, K.R., Shin, M.N., Park, R.K., Lee, S.O., Kim, D.G., 2001. Activationof caspase-8 during N-(4-hydroxyphenyl)retinamide-inducedapoptosis in Fas-defective hepatoma cells. Hepatology 34,1119–1127.

Zhao, B., Schwartz, J.P., 1988. Involvement of cytokines in normalCNS development and neurological diseases: recent progressand perspectives. J. Neurosci. Res. 52, 7–16.