C2-Ceramide Mediates CerebellarGranule Cells Apoptosis by Activationof Caspases-2, -9, and -3

Alfredo Minano,1,2 Andrea Caballero-Benıtez,1,3 Monica Lluch,1

Julio Moran,3 and Jose Rodrıguez-Alvarez1,2*1Institut de Neurociencies and Department de Bioquımica i Biologıa Molecular,Universitat Autonoma de Barcelona, Barcelona, Spain2Centro de Investigacion Biomedica en Red sobre EnfermedadesNeurodegenerativas (CIBERNED), Spain3Departmento de Neurociencias, Instituto de Fisiologıa Celular, Universidad NacionalAutonoma de Mexico, D.F., Mexico

Ceramide is able to induce the apoptotic death of cere-bellar granule cells (CGC) in culture. However, previousreports did not agree on whether ceramide-induced ap-optosis of CGC requires caspase activation. Here wehave shown that addition of C2-ceramide is able to pro-duce extensive death of cultured CGC, which is associ-ated with chromatin condensation, ladder-like DNA frag-mentation, and activation of caspases. Our results showthat C2-ceramide activates caspases-3, -9, and -2 butnot caspases-1 and -8. Caspase-9 activation was asso-ciated with cytochrome c release from mitochondria to-ward the cytosol and was followed by activation of cas-pases-2 and -3. PARP proteolysis was also observed af-ter caspase-3 and -2 activation. The involvement ofcaspases-9, -3, and -2 in ceramide-mediated apoptoticdeath of CGC was further supported by the use of spe-cific inhibitors. VVC 2008 Wiley-Liss, Inc.

Key words: neurons; neuronal death; cerebellum; rat

Apoptosis is a normal event during the developmentof the nervous system, and it has also been associatedwith several pathologic processes (Thompson, 1995; Matt-son, 2000). A family of aspartate-specific cystein proteasesknown as caspases plays a key role in the execution of ap-optosis (Lazebnik et al., 1994; Alnemri, 1997). Typicalmorphological changes of apoptotic death, such as nuclearcondensation, chromatin fragmentation, and DNA degra-dation, are controlled by the effector caspases-3 or -7,which disassemble subcellular structures and are activatedby initiator caspases, such as caspases-8 or -9.

Ceramide is a sphingosine-based lipid signallingmolecule that is involved in the regulation of differentcellular processes, including proliferation, senescence,differentiation, and cell cycle arrest (Kolesnick, 1991;Hannun, 1994). In addition, during the past years, sev-eral pieces of evidence have indicated that ceramidecould be a major regulator of apoptotic death (Obeidet al., 1993; Kolesnick and Hannun, 1999). In the nerv-

ous system, the role of ceramide in apoptotic cell deathhas been more controversial. For example, as in periph-eral tissues, several reports have suggested that ceramideis a potent inductor of apoptosis in PC12 cells (Hartfieldet al., 1997), mesencephalic cortical neurons (Brugget al., 1996; Stoica et al., 2003), and embryonic chickneurons (Wiesner and Dawson, 1996). However, otherstudies indicate that ceramide could have a neuroprotec-tive effect against certain proapoptotic stimuli. The cell-permeabIe analogue C2-ceramide protected culturedsympathetic neurons from apoptosis triggered by nervegrowth factor deprivation (Ito and Horigome, 1995).

Cultured cerebellar granule cells (CGC) have beenshown to be a useful model for characterizing and study-ing the apoptotic death induced by a large variety of ex-perimental conditions. CGC die by apoptosis duringtheir migration toward the internal granular layer if theydo not receive excitatory inputs from the mossy fibers(Williams and Herrup, 1988; Wood et al., 1993). Thisprocess could be mimicked in vitro, because CGC die

The first two authors contributed equally to this work.

Supplementary Material for this article is available online at http://

www.mrw.interscience.wiley.com/suppmat/0360-4012/suppmat/ (www.

interscience.wiley.com).

Contract grant sponsor: Ministerio de Ciencia y Tecnologia; Contract

grant number: SAF2001-1941; Contract grant number: SAF2005-5106;

Contract grant sponsor: Ministerio de Sanidad y Consumo; Contract

grant number: Red G03/167 (to J.R.-A.); Contract grant sponsor: Cona-

cyt-Mexico; Contract grant number: 47158-Q; (to J.M.); Contract grant

sponsor: Ministerio de Educacion y Ciencia (to A.M.).

*Correspondence to: Jose Rodrıguez-Alvarez, Institut de Neurociencies,

Edifici M, Universitat Autonoma de Barcelona, Campus de Bellaterra,

08193 Cerdanyola del Valles, Barcelona, Spain.

E-mail: jose.rodriguez@uab.es

Received 20 September 2007; Revised 22 November 2007; Accepted 24

November 2007

Published online 29 February 2008 in Wiley InterScience (www.

interscience.wiley.com). DOI: 10.1002/jnr.21633

Journal of Neuroscience Research 86:1734–1747 (2008)

' 2008 Wiley-Liss, Inc.

by apoptosis in the presence of physiological concentra-tions of KCl (5 mM or K5), but they survive in thepresence of depolarizing KCl concentrations (25 mM orK25; Gallo et al., 1987). Ceramide seems to be involvedin CGC apoptosis induced by KCl deprivation. Anincrease in the intracellular concentration of ceramidehas been described when CGC are switched to K5 me-dium (Toman et al., 2002). Accordingly, it has beenshown that CGC die apoptotically in a concentration-and age-dependent manner when exposed to C2-cer-amide (Manev and Cagnoli, 1997; Centeno et al., 1998).However, although the role of caspases induced by dif-ferent stimuli has been evaluated with detail in CGC(Bellacosa et al., 1998; D’Mello et al., 1998; Moranet al., 1999; Caballero-Benitez and Moran, 2003), fewdata exist on the activation of caspases by ceramide inCGC. A recent report has shown that caspase-3 activa-tion is involved in C2-ceramide-mediated apoptosis ofCGC (Vaudry et al., 2003). By contrast, other reportssuggest that C2-ceramide-mediated apoptosis is caspase-3independent (Monti et al., 2001), in accordance withrecent data obtained in jurkat cells (Thon et al., 2005).

In the present study, we further characterized theeffect of C2-ceramide on the activation of several cas-pases in cultured CGC. We found an activation of cas-pases-9, -3, and -2 after different periods (3–24 hr) ofceramide treatment. The caspase activation was precededby the translocation of cytochrome c to the cytosol andwas related to the hydrolysis of PARP. Moreover, CGCapoptotic death induced by ceramide was reduced bycaspase inhibitors.

MATERIALS AND METHODS

Materials

Fetal calf serum and penicillin/streptomycin wereobtained from Gibco (Grand Island, NY). Poly-L-lysine (MW< 300,000), trypsin, soybean trypsin inhibitor, DNAase, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT),and reagents for polyacrylamide gel electrophoresis (PAGE)were obtained from Sigma (St. Louis, MO). C2-ceramide (N-acetyl-D-sphingosine) was obtained by Tocris (Ellisville, MO).Enhanced chemiluminiscence detecting agent for phosphatasealkaline-conjugated antibodies and molecular weight markers(kaleidoscope) were purchased from Bio-Rad Laboratories(Hercules, CA), and polyvinylidene difluoride (PVDF) mem-branes were from Millipore (Bedford, MA). Antibody againstcaspase-1 was purchased from Thermo Scientific (ref. rb1608).Antibody against caspase-2 was obtained from Abcam (Cam-bridge, MA; ref. ab2251). Antibody against caspase-8 wasfrom Santa Cruz (Santa Cruz, CA; ref. sc-5263). Poly (ADP-ribose) polymerase (PARP) and cytochrome-c antibodies werepurchased from Roche (ref. 135238) and BD Biosciences (ref.65981A), respectively. Hsp60 antibody was obtained fromStressgen (ref. SPA-806), and GAPDH antibody was pur-chased from Ambion (Austin, TX; ref. 4300). Antibodiesagainst cleaved caspases-3 and -9 were purchased from CellSignalling (Beverly, MA; ref. 9661 and 9507). Caspase sub-strates and inhibitors were from Peptides International. All

others chemicals were of the purest grade available from regu-lar commercial sources.

Cell Cultures

CGC were obtained as previously described (Moran andPatel, 1989). Briefly, the dissociated cell suspensions from thecerebella of 8-days-old rats were plated at a density of 2.65 3105 cell/cm2 in plastic dishes previously coated with poly-L-lysine (5 lg/ml). The culture medium contained basal Eagle’smedium supplemented with 10% heat-inactivated fetal calf se-rum, 2 mM glutamine, 25 mM KCl, 50 lg/ml streptomycin,and 50 U/ml penicillin. The culture dishes were incubated at378C in a humidified 5% CO2/95% air atmosphere, and cyto-sine arabinoside (10 lM) was added after 20 hr. After 7–8DIV, CGC were treated with C2-ceramide (3, 10, 30, 40, and60 lM) for different times. In some experiments, cells wereincubated with 10 or 100 lM of the broad-spectrum caspaseinhibitor ZVAD or the preferring inhibitors of caspase-1, -3, -2, -8, and -9 YVAD-CHO, DEVD-CHO, VDVAD-CHO,IETD-CHO, and LEHD-CHO, respectively. Inhibitors wereadded simultaneously to the treatment with ceramide, and theywere present until the end of the experiment.

MTT Metabolism

Cell viability was estimated by MTT assay (Mosmann,1983). Yellow MTT is converted to the formazan blue productby metabolically active mitochondria, and the absorbance isdirectly proportional to the number of the viable cells. MTT(0.2 mg/ml) was added to the cultures, and the cells were incu-bated 1 hr at 378C. After aspiration of the medium containingthe remaining MTT, dimethyl sulfoxide was used to extract theformazan blue produced. Samples were read at a wavelength of570 nm and a reference wavelength of 630 nm. The data werecalculated as the absorbance at 570 nm minus the absorbance at630 nm and expressed as percentage of control groups.

Hoechst Staining

Nuclear morphology was assessed after Hoechst staining.Briefly, cells were harvested and fixed with 4% paraformalde-hyde in phosphate-buffered saline (PBS) for 10 min andstained with Hoechst 33258 (1lg/ml) for 5 min. Nuclearstaining was observed with a fluorescence microscope (Leica).Condensed and/or fragmented nuclei were considered as apo-ptotic nuclei.

DNA Laddering

CGC were grown on 6-cm culture dishes for DNA lad-dering determination. Cultures were washed twice with PBS,lysed in Tris-EDTA buffer (5 mM Tris-HCl, 200 mMEDTA, and 0.5% Triton X-100), and maintained for 20 minat 48C. Lysates were then centrifuged at 20,000g for 30 sec at48C, and the supernatant was treated with proteinase K(200 lg/ml) and the pellet added to lysis buffer and proteinaseK (200 lg/ml), with incubation for 30 min at 608C and over-night at 378C. DNA was purified by using phenol/chloroform(1:1) extraction and precipitated with ethanol at –208C over-night. DNA was resuspended in Tris-EDTA buffer containingDnase-free RNase (20 lg/ml) for 1 hr at 378C. DNA (1 lg)

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was loaded onto 1% agarose gels and stained with ethidiumbromide (0.5 lg/ml).

Immunohistochemistry Assay

Cultures were rinsed twice with phosphate-buffered sa-line (PBS) at 378C and fixed in 4% paraformaldehyde in PBS0.1 M, pH 7.2, for 1 hr at 48C. The plates were washed twicewith Tris-buffered saline/Tween 20 (TTBS) buffer (100 mMTris-HCl, 150 mM NaCl, and 0.1% Tween, pH 7.5) at roomtemperature and blocked with TTBS buffer containing 0.05%FCS and 0.03% BSA, for 45–60 min at room temperature.Cells were incubated with the primary antibody directedagainst the active fragment of caspase-3 or caspase-9 (1:100) inTTBS/5% BSA for 24 hr at 48C, washed twice with TTBS,and incubated with FITC-conjugated secondary antibody(Jackson Immunoresearch, Wst Grove, PA; 1:500) in TTBS/5% BSA for 1 hr. After washing, the preparations were proc-essed for visualization. Cultures were examined in an epifluo-rescence microscope (Leica) using a wavelength of 490 nm.

Subcellular Fractionation

CGC cultured in 100-mm-diameter dishes were rinsedtwice with PBS at 378C harvested in 350 ll of homogeniza-tion buffer containing 10 mM Tris-HCl, 0.3 mM ethylenebi-s(oxyethylenenitrilo)-tetra-acetic acid (EGTA), and 0.25 Msucrose, pH 7.4, in the presence of 1 lM phenylmethylsulfo-nyl fluoride (PMSF), 2 lg/ml aprotinin, 1 lg/ml pepstatin,and 5 lg/ml leupeptin. Cells were homogenized with an all-glass homogenizer and centrifuged at 5,000g for 10 min, andthe supernatant was then centrifuged at 14,500g for 30 min at48C. The pellet was resuspended in 50 ml of homogenizationbuffer and used as mitochondrial fraction. Supernatant wasfurther centrifuged at 100,000g for 60 min at 48C, and theresulting supernatant was used as cytosolic fraction. Mitochon-drial and cytosolic fractions were processed for Western blot-ting as indicated below.

Immunoblotting

Cells were rinsed twice with PBS at 378C, homoge-nized, sonicated in a lysis buffer (25 mM Tris-HCl, 50 mMNaCl, 2% Nonodet P-40, 0.2% SDS, 1lM PMSF, 2 lg/mlaprotinin, 1lg/ml pepstatin, and 5 lg/ml leupeptin, pH 7.4)and centrifuged at 3,400g for 20 min at 48C. The proteinconcentrations of the cell homogenates were determined bythe method of Bradford. Homogenates (50 lg of protein perlane) were run under nonreducing conditions in a one-dimen-sional SDS-PAGE and then electroblotted onto PVDF mem-branes at 100 V for 1 hr. Blots were blocked with TTBS/5%nonfat dry milk for 3 hr at 48C, washed three times withTTBS, and incubated overnight with the primary antibody at48C. After further washing, the blots were incubated withalkaline phosphatase-conjugated secondary antibody for 1 hrat room temperature. After washing, the blots were processedfor visualization using the enhanced chemiluminescense systemaccording to the manufacturer’s recommendations (Bio-RadLaboratories) and exposed to Kodak XAR-5 film.

Caspase Activity Measurement

CGC were washed with PBS and then homogenized inlysis buffer. The homogenates were diluted 1:1 (vol/vol) withglycerol and stored at –708C. Caspase activities were assayedby a fluorimetric method in a luminescence spectrometer(Luminescense Spectrometer AMINCO Bowman Series 2)using the tetrapeptides Ac-YVAD-AMC, Ac-VDVAD-AMC,Ac-DEVD-AMC, IETD-AMC, and LEHD-AMC as prefer-ring substrates to detect the activity of caspases-1, -2, -3, -8,and -9, respectively. The reactions were followed for 15 minafter addition of substrate (25 lM) and cell homogenate(30 mg/ml) in a standard solution [100 mM HEPES, 10%(wt/vol) sucrose, 0.1% (wt/vol) 3-[(3-cholamidopropyl) dime-thylammonio]-1-propane sulfonate (CHAPS), 10 mM dithio-threitol, 1 mM EDTA, 1 mM PMSF, 2 lg/ml aprotinin,1 lg/ml pepstatin, and 5 lg/ml leupeptin]. Results areexpressed as change of fluorescence intensity/hr/mg prot.

Statistical Analysis

Statistical significance was determined by one-way anal-ysis of variance (ANOVA), followed by Tukey’s multiple-comparisons test. A value of P < 0.05 was accepted as denot-ing statistical significance.

RESULTS

C2-Ceramide Induced CGC Apoptotic Death

In previous studies, it was observed that the death-promoting effect of C2-ceramide in CGC was depend-ent on the developmental state of the neurons in culturescontaining foetal calf serum (Taniwaki et al., 1999). Bycontrast, others studies (Monti et al., 2001; Vaudryet al., 2003) have shown that C2-ceramide induces apo-ptosis independently of the cell maturation conditionwhen grown in medium without fetal calf serum. In thisstudy, we confirmed the proapoptotic effect of C2-cer-amide on mature CGC cultures in serum-containing(Fig. 1) and serum-free (data not shown) medium bytesting the effect of different concentration of C2-cer-amide (3, 10, 30, 40, and 60 lM) on the viability ofCGC at 24 and 48 hr. In CGC grown in serum-con-taining media, treatment for 24 hr with C2-ceramidehad a significant effect only above 30 lM (Fig. 1A). Weobserved that CGC viability was reduced by 30% and60% at 40 and 60 lM of C2-ceramide, respectively. Af-ter 48 hr, C2-ceramide-mediated cell death was observedfrom 20lM. When CGC were treated for 48 hr with30, 40, or 60 lM of C2-ceramide, cell viability was sig-nificantly reduced up to 65%, 85%, or 95%, respectively(Fig. 1A). When CGC were cultured in a serum-freemedium, C2-ceramide produced a larger decrease inMTT metabolism than in a serum-containing media.For example, 30 lM of C2-ceramide produced a 40%and a 75% decrease in CGC metabolism at 24 and 48hr, respectively (data not shown).

The decrease in cell metabolism induced by C2-ceramide was in agreement with the observed morpho-logical appearance of CGC treated with this lipid andobserved by phase-contrast microscopy (Fig. 1B). For

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Fig. 1. Effect of C2-ceramide on the viability and morphology ofCGC. At 7 DIV, the cultures of CGC grown in K25 were treatedwith 3, 10, 30, 40, and 60 lM of C2-ceramide. A: Cell viability ofCGC was evaluated as MTT transformation (for details see Materialsand Methods). MTT transformation was measured 24 and 48 hr after

application of different concentrations of C2-ceramide. Values aremean 6 SEM of five independent experiments performed in tripli-cate. *Significantly different from control (P < 0.01). B: Cultureswere observed under phase contrast after 24 hr or 48 hr. See insetsfor more detailed morphology of cultured CGC. Scale bar 5 50 lm.

example, in serum-containing media, the morphologicalappearance of the cultures treated with 30 lM C2-cer-amide for 24 hr was similar to that of control cells(K25); however, after 48 hr, when the MTT assay indi-cated an important decrease in cell viability (65%; Fig.1A), CGC showed marked morphological alterations,characterized by a decreased cell volume and an appreci-able degeneration of CGC soma and neuritic processes,as well as an evident number of dead cells (Fig. 1B).

Oligonucleosomal fragmentation of DNA is con-sidered a characteristic feature of apoptotic cell death. Asshown in Figure 2A, C2-ceramide (40lM) producedDNA laddering after 24 or 48 hr. A similar DNA lad-dering pattern was observed in K5 cultured CGC,whereas no DNA laddering was observed in control(K25) CGC. To confirm further the apoptotic nature ofC2-ceramide-mediated death of CGC, we observed thenuclear morphology by Hoechst staining. Figure 2Bshows a significant increase in the condensed or frag-mented nuclei in C2-ceramide (40 lM)-treated CGCcompared with control (K25) cultures.

Caspase Activation by C2-Ceramide

Caspase activation is considered to be a key factorleading to cell apoptosis. Previous results have shown

that caspase-3 activation is induced in KCl-deprived orC2-ceramide-treated CGC (D’Mello et al., 1998; Vaudryet al., 2003). Supporting the involvement of caspase acti-vation in C2-ceramide-mediated CGC apoptotic death,we observed that a pan-inhibitor of caspases, zVAD-CHO (10 lM), was able to abrogate the morphologicalchanges induced by C2-ceramide (Fig. 1B).

To identify the caspases activated by C2-ceramide,we decided to evaluate the activity of caspases-1, -2, -3,-8, and -9 by using fluorogenic substrates that have beendemonstrated to be preferentially cleaved by the differentcaspases (Thornberry et al., 1997). DEVD-AMC wasused to determine the activation of caspase-3. WhenCGC were treated with 40 lM C2-ceramide, a signifi-cant increase in DEVD cleavage was detected at 6 hr,and a maximal increase (250% vs. control) was observedat 12 hr. DEVD cleavage remained increased until 36 hrafter treatment (Fig. 3A). No significant DEVD cleavagewas observed before 6 hr. Staurosporine (0.5 lM) alsoproduced an increase of DEVD cleavage (400% vs. con-trol) after 8–12 hr (Fig. 3A), as previously shown (Cabal-lero-Benitez and Moran, 2003). To confirm these resultsobtained by fluorimetric analysis, we also evaluated byWestern blot analysis the generation of the active frag-ment of caspase-3 in homogenates obtained from cells

Fig. 2. Oligo-nucleosomal cleavage of genomic DNA and nuclearcondensation in C2-ceramide-treated CGC. A: Cerebellar granule neu-rons were cultured in medium alone (K25 control), low-potassium me-dium (K5), or K25 plus 40 lM C2-ceramide for 24 hr or 48 hr. Onlygenomic DNA from K5- and ceramide-treated CGC revealed the typi-cal ladder-like pattern. In parallel, the amount of high-molecular-weight genomic DNA decreased with ceramide treatment. B: CGC

were cultured in K25 until 8 DIV, when the medium was changed to aK5-based medium or K25 with C2-ceramide 40 lM. Chromatin con-densation was determined at 9 DIV after Hoechst staining. Note theappearance of several condensed or fragmented nuclei in K5 or C2-cer-amide vs. K25. Cells were counted from three independent experi-ments. In each experiment, >500 cells were examined from at leastthree culture wells for each condition. Results are mean 6 SEM.

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Fig. 3. Effect of C2-ceramide on the activation (DEVD cleavage)and proteolysis of caspase-3 in CGC. Cells maintained in K25 (C)for 7 DIV were treated with 40 lM of C2-ceramide or 10 lM stau-rosporine (STS) for the indicated periods of time. A: Time course ofcaspase-3 activity measured as the cleavage of Ac-DEVD-AMC.Results are expressed as delta of fluorescence per hour per milligramprotein. Values are mean 6 SD of four independent experimentsperformed in triplicate. *Significantly differences vs. control (K25)

CGC (P < 0.05). B: Time course of C2-ceramide-mediated procas-pase-3 proteolysis. CGC were treated as in A, and the appearance ofthe 17-kDa band was revealed by Western blotting. A representativeblot from four independent experiments is shown. C: Time courseof PARP fragmentation induced by C2-ceramide in CGC revealedby Western blot. Arrows indicate the intact (116 kDa) and the 85-kDa fragment of PARP, generated by caspase-3 after different timesof C2-ceramide application.

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treated with C2-ceramide at different times. Figure 3Bshows the appearance of the active fragment of caspase-3(17 kDa) from 6 hr to 48 hr after C2-ceramide treat-ment. To confirm further the activation of caspase-3 byC2-ceramide, we examined the 85-kDa PARP fragmentthat results from the specific activity of caspase-3 (Fig.3C). The PARP processing in homogenates of CGCtreated with C2-ceramide was similar to that observed inother studies (Eldadah et al., 1997). We found that theapplication of C2-ceramide for 16–28 hr leads to genera-

tion of the 85-kDa fragment, concomitantly with adecrease in the 116-kDa band. The activation of cas-pase-3 by C2-ceramide was also studied via immunohis-tochemistry by using an antibody directed against theactive fragment of caspase-3. Figure 4 shows that cellswith activated caspase-3 were clearly observed after 6–8hr of C2-ceramide treatment, whereas the maximalnumber of cells with active caspase-3 was seen at 24 hr.Afterward, the number of cells immunoreactive foractive caspase-3 declined, and almost no positive cells

Fig. 4. Determination of cleaved caspase-3 in C2-ceramide-treatedCGC. DIV 7 cultures grown in K25 were treated with 40 lM of C2-ceramide for 3, 6, 8, 12, 24, 36, and 48 hr and immunostained withan antibody against cleaved caspase-3. Immunoreactivity to active cas-

pase-3 (green) was observed in cultures exposed to C2-ceramide. Totalcells were stained with Hoechst (blue). No activation of caspase-3 wasevident in control (K25) cultures. Inset shows the cytosolic and/ornuclear localization of active caspase-3. Scale bar 5 10 lm.

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were observed at 48 hr. As expected, CGC culturestreated with the proapoptotic agent staurosporine alsoshowed cells with activated caspase-3 (data not shown).However, activation was observed earlier than with C2-ceramide, insofar as numerous positive cells were alreadyobserved at 8 hr of staurosporine treatment.

The caspase responsible for the activation of cas-pase-3 in the intrinsic pathway to apoptosis is caspase-9.In this study, the activity of caspase-9 was measured asAc-LEHD-AMC cleavage (Thornberry et al., 1997) aswell as by the detection of the active fragments byWestern blot analysis and immunohistochemistry. When

Fig. 5. Effect of C2-ceramide on the activation (LEHD cleavage) andproteolysis of caspase-9 in CGC. Cells maintained in K25 (C) for 7DIV were treated with 40 lM of C2-ceramide or 10 lM of stauro-sporine (STS) for the indicated periods of time. A: Time course ofcaspase-9 activity measured as the cleavage of Ac-LEHD-AMC.Results are expressed as delta of fluorescence per hour per milligram

protein. Values are mean 6 SEM of four independent experimentsperformed in triplicate. *P < 0.05 vs. control (K25). B: Time courseof C2-ceramide-mediated procaspase-9 proteolysis. Cells were treatedas described for A, and the presence of the active fragment (10 kDa)of caspase-9 was revealed by Western blotting. A representative blotfrom three independent experiments is shown.

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CGC were treated with 40 lM C2-ceramide, wedetected a significant LEHD cleavage at 6 hr (110% overcontrol), reaching a maximum at 32 hr after treatment(400% over control; Fig. 5A). In line with previous stud-ies (Caballero-Benitez and Moran, 2003), we found thatstaurosporine (0.5 lM) also induced the fragmentationof LEDH. The presence of the active fragment of cas-pase-9 (10 kDa) by immunoblotting was alreadyobserved at 6 hr (Fig. 5B), peaked at 36 hr, and persisteduntil 48 hr after C2-ceramide addition. Similar results

were observed when activation of caspase-9 was moni-tored by immunohistochemistry. As shown in Figure 6,cells immunoreactive for active caspase-9 were observedat 6 hr after C2-ceramide treatment, reaching a maxi-mum at about 24–36 hr. Similar results were observedwith staurosporine-treated CGC cultures (data notshown).

It has been suggested that caspase-9 is activated bythe release of cytochrome-c from mitochondria to cyto-sol (Krajewski et al., 1999). Figure 7 shows an increase

Fig. 6. Determination of cleaved caspase-9 in C2-ceramide-treatedCGC. DIV 7 cultures grown in K25 were treated with 40 lM of C2-ceramide for 3, 6, 8, 12, 24, 36, and 48 hr and immunostained withan antibody against cleaved caspase-9. Immunoreactivity to active cas-

pase-9 was observed in cultures exposed to C2-ceramide. Total cellswere stained with Hoechst (blue). No activation of caspase-9 was evi-dent in control (K25) cultures. Inset shows the cytosolic and/or nu-clear localization of active caspase-9. Scale bar 5 10 lm.

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of cytosolic cytochrome-c, detected as a 17-kDa band,from 3 hr of C2-ceramide treatment, which is accompa-nied by a decrease in the mitochondrial fraction.

Activation of effector caspases in the extrinsic apo-ptotic pathway involves the activation of plasma mem-brane receptors from the tumor necrosis factor-a (TNF-a) receptor superfamily and of initiator caspases such ascaspase-8 or -2 (Juo et al., 1998). In this study, we havemeasured caspase-8 activity by using the preferring sub-strate Ac-IETD-AMC (Thornberry et al., 1997). WhenCGC were incubated with 40 lM C2-ceramide, we didnot observe any significant increase in IETD cleavageduring the first 48 hr compared with control cultures(Fig. 8A). Similar results were observed by Western blotanalysis; no differences in the amount of cleaved procas-pase-8 was seen in C2-ceramide-treated cells vs. controlcultures (Fig. 8B). By contrast, when caspase-2 activitywas monitored by VDVAD-AMC cleavage, an increasein the activity was observed from 8 hr until 48 hr afterC2-ceramide addition compared with control cultures(Fig. 9A). Similar results were observed when proteolysisof caspase-2 was determined by immunoblotting (Fig.9B). On the other hand, the presence of caspase-2 in-hibitor did not prevent caspase-9 activation by C2-cer-amide. Also, the specific caspases-9 inhibitor did notblock the activation of caspase-2 by the lipid (see Suppl.Fig. 1). These results indicate that C2-ceramide in-dependently activates caspases-2 and -9. Staurosporinewas also able to activate caspase-2, as determined byboth measurement of activity (Fig. 9A) and immuno-blotting (Fig. 9B). Finally, no activation of caspase-8was observed in staurosporine-treated cultures (data notshown).

We have also monitored the activity of caspase-1in C2-ceramide-treated CGC cultures. Caspase-1 belongsto the subfamily of caspases involved in proinflammatoryprocesses. As expected, we did not detect caspase-1 acti-vation; no cleavage of Ac-YVAD-AMC was observed in

ceramide (40 lM)-treated cells at the studied times (3–48 hr; data not shown).

Effect of Caspase Inhibitors on CellViability of CGC

Our results indicate that C2-ceramide activates cas-pases-2, -3, and -9 but not caspase-8 in CGC. To con-firm these results further, cell viability was measured inCGC treated with 40 lM C2-ceramide in the presenceof specific caspases inhibitors (Fig. 10A). We found thatthe broad-spectrum caspase inhibitor ZVAD-CHO, thecaspase-3 inhibitor DEVD-CHO, and the caspase-9 in-hibitor LEHD-CHO completely prevented the effect of40 lM C2-ceramide after 48 hr. Inhibition of caspase-2with VDVAD-CHO reduced by 55% C2-ceramide-mediated CGC death. By contrast, no decrease in C2-ceramide-mediated CGC death was observed withIETD-CHO, an inhibitor of caspase-8 (data not shown).When the morphological appearance of CGC treatedwith C2-ceramide was evaluated in the presence of thesecaspase inhibitors, we found that all caspase inhibitorstested, except for IETD-CHO, showed a protectiveeffect of the CGC morphology (Fig. 10).

DISCUSSION

Apoptosis has a key role during CNS developmentby maintaining cell homeostasis. Moreover, apoptosis hasbeen involved in the neuronal death observed in severalneurodegenerative disease and in cerebral ischemia(Chen et al., 1998). The molecular processes and themechanisms involved in this type of death have receivedparticular attention during the past decade. A set of mol-ecules participates actively in the regulation and imple-mentation of the death process. Among them, caspaseshave been demonstrated to be one of the main compo-nents of the apoptotic machinery (Cohen, 1997; Nichol-son and Thornberry, 1997).

Fig. 7. C2-ceramide induces the release of cytochrome-c form mito-chondria in CGC. Cells maintained in K25 (C) for 7 DIV weretreated with 40 lM of C2-ceramide for the indicated periods of time.Cells were fractionated to yield mitochondrial and cytosolic fractions as

described in Materials and Methods. Representative Western blots (ofthree independent experiments) of cytochrome-c in the mitochondrialand cytosolic fractions of CGC are shown. Hsp60 was used to monitorthe absence of mitochondrial contamination in the cytosolic fraction.

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It has been shown that deprivation of excitatoryinputs triggers massive apoptotic death of CGC duringtheir postnatal migration toward the internal granule celllayer. Also, CGC apoptosis can be observed in culturesthat are switched from a medium with depolarizing po-tassium concentrations (25 mM; K25) to a medium withnormal potassium (5 mM; K5). Several studies haveshown that apoptosis triggered by deprivation of depola-rizing inputs is mediated by caspase-9 and caspase-3(Eldadah et al., 1997; D’Mello et al., 1998; Moran et al.,1999; Gerhardt et al., 2001). On the other hand, anincrease has been reported in the intracellular levels ofceramide and the disialoganglioside GD3 under theseconditions (Melchiorri et al., 2002; Toman et al., 2002;Castiglione et al., 2004), It is still not clear which is the

pathway linking ceramide synthesis and the deprivationof excitatory inputs in CGC, although it has beenreported that it could be mediated by the activation ofFas receptor (Castiglione et al., 2004).

Short-chain analogues of ceramide, in particular,C2-ceramide and C6-ceramide, may mimic some of thebiological effects of endogenous ceramide, so they havebeen used to study ceramide-mediated apoptosis in a va-riety of cell types. Several studies have shown that C2-ceramide elicits CGC apoptosis (Manev and Cagnoli,1997; Centeno et al., 1998; Monti et al., 2001; Vaudryet al., 2003). The effect of ceramide seems to depend onthe concentration of exogenous ceramide and the matu-ration state of the cell. In this respect, it has beendescribed (Taniwaki et al., 1999) that a cell-permeableanalogue, C2-ceramide, induces CGC apoptotic deathonly in immature and not in mature cultures. By con-trast, in this study, we found that C2-ceramide inducedcell death in mature CGC (8 DIV).

Fig. 8. Effect of C2-ceramide on activation (IETD cleavage) andproteolysis of caspase-8 in CGC. Cells maintained in K25 (C) for 7DIV were treated with 40 lM of C2-ceramide for the indicated peri-ods of time. A: Time course of caspase-8 activation measured ascleavage of Ac-IETD-AMC. Results are expressed as delta of fluores-cence per hour per milligram protein. Values are mean 6 SEM offour independent experiments performed in triplicate. *P < 0.05 vs.control (K25). B: Time course of procaspase-8 proteolysis by 40 lMof C2-ceramide was analyzed by Western blot. Bands correspond toprocaspase-8 (p55) and the cleavage products (p10 and p20). A repre-sentative blot from six independent experiments is shown.

Fig. 9. Effect of C2-ceramide on the activation (VDVAD cleavage)and poteolysis of caspase-2 in CGC. Cells maintained in K25 (C) for 7DIV were treated with 40 lM of C2-ceramide for the indicated peri-ods of time. A: Time course of caspase-2 activation measured by thecleavage of Ac-VDVAD-AMC. Results are expressed as delta of fluo-rescence per hour per milligram protein. Values are mean 6 SEM offour independent experiments performed in triplicate. *P < 0.05 vs.control (K25). B: Time course of procaspase-2 proteolysis by 40 lMof C2-ceramide was analyzed by Western blot. Bands correspond tothe cleavage products of caspase-2 proenzyme (p12 and p19). A repre-sentative blot from six independent experiments is shown.

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Apoptosis is usually mediated by caspase activation,and several reports have shown the activation of caspase-3 in ceramide-mediated apoptosis (Keane et al., 1997;Movsesyan et al., 2002; Vaudry et al., 2003). However,the role of caspases in C2-ceramide-mediated apoptotic

death of CGC is controversial. It has been reported that,when CGC are cultured in serum-free medium, C2-cer-amide-mediated cell death is independent of caspase acti-vation (Monti et al., 2001). By contrast, Vaudry andcoworkers (2003) have reported that caspase-3 activationis involved in C2-ceramide-mediated apoptosis in CGCgrown in serum-containing medium.

In the present study, we found that cell death ofCGC induced by C2-ceramide shows internucleosomalcleavage of DNA and the condensation and fragmentationof chromatin, suggesting that C2-ceramide induces apo-ptotic death in this model. To confirm this suggestion,we further explored the involvement of different caspasesby using different technical approaches, such as fluorimet-ric assays, Western blot determination of caspase proteoly-sis, and immunohistochemical detection of caspase activefragments. Our results clearly show that C2-ceramide pro-duces an increase in caspase-3 activity. Activation of cas-pase-3 begins 6 hr after C2-ceramide administration toCGC. Maximal activation is observed between 12 and 36hr. The time course of caspase-3 activation agrees withthe observed proteolysis of the caspase-3 substrate PARP.Western blot analysis showed that PARP proteolysis isfirst observed after 16 hr of C2-ceramide treatment, 4 hrafter the detection of maximal DEVD cleavage. Ourresults agree with previous observations (Vaudry et al.,2003) that C2-ceramide activates caspase-3 in CGC. Acti-vation of caspase-3 is related to C2-ceramide-mediatedCGC death, in that we were able markedly to reduce celldeath with an inhibitor of caspase-3.

Caspase-3 is an effector caspase that triggers theproteolysis of several cellular substrates, leading to apo-ptotic death. Activation of caspase-3 is usually mediatedby upstream caspases (or initiator caspases) such as cas-pases-2, -8, or -9. Caspase-9 activation, observed in theso-called intrinsic apoptotic pathway, requires formationof the apoptosome by interaction of caspase-9, apoptosisactivating factor-1, ATP, and cytochrome-c releasedfrom the mitochondria (Cecconi et al., 1998; Yoshidaet al., 1998; Krajewski et al., 1999). Caspase-2 and -8activation is considered to be involved in the extrinsicapoptotic pathways usually triggered by activation ofplasma membrane receptors (Juo et al., 1998; Wagneret al., 2004). With our experimental conditions, wehave found that C2-ceramide activates caspases-2 and -9but not -8. Accordingly, inhibition of caspases-2 and -9with VDVAD-CHO and LEDH-CHO, respectively,blocked C2-ceramide-mediated CGC death, but inhibi-tion of caspase-8 did not protect CGC from C2-cer-amide-mediated apoptosis. We have detected that activa-tion of caspase-2 starts 8 hr after C2-ceramide additionand that it remains activated after 48 hr. A similar timecourse was observed when proteolysis of caspase-2 wasdetermined by Western blot analysis.

On the other hand, we observed an earlier activa-tion of caspase-9 by immunodetection of proteolysedcaspase-9 by and immunohistochemistry after 6 hr ofC2-ceramide addition. Similarly, caspase-9 activity wasnot detected earlier than 6 hr after treatment of CGC

Fig. 10. Effect of caspase inhibitors on CGC viability in culturestreated with C2-ceramide. A: Cells were grown for 7 DIV and thentreated with 40 lM of C2-ceramide for 48 hr in the presence or ab-sence of caspase inhibitors (10 lM). Cell viability was determined bythe MTT assay. Results are mean 6 SEM of three experiments per-formed in triplicate. *P < 0.05 vs. C2-ceramide treated cultures. B:Effect of caspase inhibitors on the morphology of C2-ceramide-treated CGC. Cells were cultured for 7 DIV and then treated with40 lM of C2-ceramide for 48 hr in the presence or absence of cas-pase inhibitors (10 lM). Control (a), C2-ceramide (b), C2-ceramide1 DEVD (c), C2-ceramide 1 LEDH (d), C2-ceramide 1 VDVAD(e), and C2-ceramide 1 IETD (f). Scale bar 5 50 lm.

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cultures with the lipid. As expected, the release from mi-tochondria of cytochrome-c started before caspase-9activation. The fact that caspase-9 was activated beforecaspase-2 does not mean that caspase-2 is activated bycaspase-9 as described previously in the human neuro-blastoma cell line SK-N-MC (Ito and Horigome, 1995),because the inhibition of caspase-9 with LEDH-CHOdid not affect the activation of caspase-2 by C2-cer-amide. Also, the inhibition of caspase-2 with VDVAD-CHO did not modify the activation of caspase-9 by thelipid (data not shown), suggesting that C2-ceramide acti-vates both caspases by different pathways. It has beendescribed that C2-ceramide is able to activate caspase-9in cortical neurons by a pathway that mediates the inac-tivation of Akt activity and the dephosphorylation ofBad (Zhou et al., 1998; Stoica et al., 2003). Dephospho-rylated BAD is believed to translocate to the mitochon-dria, where it promotes cytochrome c release and subse-quent caspase-9 activation (Datta et al., 1997). Caspase-2has a CARD domain that can interact with several adap-tor proteins implicated in the pathways associated withcell surface receptors of the TNF-a receptor superfamily(Wagner et al., 2004). Also, it is believed that dimeriza-tion triggers initial caspase-2 activation (Butt et al.,1998). However, the mechanism involved in caspase-2activation by ceramide is uncertain, and further experi-ments are necessary to address this issue fully.

In summary, ceramide triggers the apoptotic deathof CGC by a caspase-mediated mechanism. We havefound that ceramide activates caspases-2, -3, and -9. Theinitiator caspases, caspase-9 and caspase-2, were activatedby different pathways, because inhibition of one caspasedid not prevent ceramide-mediated activation of theother caspases. However, the blockade of caspase-9 orcaspase-2 was sufficient to reverse ceramide-mediatedapoptosis, showing that both caspases should be active inceramide-mediated apoptosis of cerebellar granule neu-rons. No increase in caspase-1 or caspase-8 activity wasobserved in cultures treated with ceramide.

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