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© The Rockefeller University Press, 0021-9525/1997//205 $5.00
The Journal of Cell Biology, Volume 139, Number 1, , 1997 205-217

Bax Deletion Further Orders the Cell Death Pathway in Cerebellar Granule Cells and Suggests a Caspase-independent Pathway to Cell Death

Timothy M. Miller^*, Krista L. Moulder^*, C. Michael Knudson, Douglas J. Creedon^*, Mohanish Deshmukh^*, Stanley J. Korsmeyer, and Eugene M. Johnson, Jr.^*

^* Department of Neurology and Department of Molecular Biology and Pharmacology, Department of Medicine and Department of Pathology, Howard Hughes Medical Institute, Washington University School of Medicine, St. Louis, Missouri 63110

Dissociated cerebellar granule cells maintained in medium containing 25 mM potassium undergo an apoptotic death when switched to medium with 5 mM potassium. Granule cells from mice in which Bax, a proapoptotic Bcl-2 family member, had been deleted, did not undergo apoptosis in 5 mM potassium, yet did undergo an excitotoxic cell death in response to stimulation with 30 or 100 µM NMDA. Within 2 h after switching to 5 mM K⁺, both wild-type and Bax-deficient granule cells decreased glucose uptake to <20% of control. Protein synthesis also decreased rapidly in both wild-type and Bax-deficient granule cells to 50% of control within 12 h after switching to 5 mM potassium. Both wild-type and Bax –/– neurons increased mRNA levels of c-jun, and caspase 3 (CPP32) and increased phosphorylation of the transactivation domain of c-Jun after K⁺ deprivation. Wild-type granule cells in 5 mM K⁺ increased cleavage of DEVD–aminomethylcoumarin (DEVD-AMC), a fluorogenic substrate for caspases 2, 3, and 7; in contrast, Bax-deficient granule cells did not cleave DEVD-AMC. These results place BAX downstream of metabolic changes, changes in mRNA levels, and increased phosphorylation of c-Jun, yet upstream of the activation of caspases and indicate that BAX is required for apoptotic, but not excitotoxic, cell death. In wild-type cells, Boc-Asp-FMK and ZVAD-FMK, general inhibitors of caspases, blocked cleavage of DEVD-AMC and blocked the increase in TdT-mediated dUTP nick end labeling (TUNEL) positivity. However, these inhibitors had only a marginal effect on preventing cell death, suggesting a caspase-independent death pathway downstream of BAX in cerebellar granule cells.

Abbreviations used in this paper: BAF, Boc-aspartyl(OMe)-fluoromethylketone; NMDA, N-methyl-D-aspartic acid; PI-3-K, phosphatidylinositol-3 kinase; PCD, programmed cell death; TUNEL, TdT-mediated dUTP nick end labeling.

EXTENSIVE cell death is an important part of the development and ongoing maintenance of tissues in multicellular organisms (Glucksman, 1951; Clarke and Clarke, 1996). The demise of many cells is modulated by members of the BCL-2 family of proteins (Korsmeyer, 1996; White, 1996). BCL-2 was first recognized as a modulator of cell death in studies of follicular B cell lymphoma in which overexpression of BCL-2 promotes transformation by rendering cells more resistant to apoptosis. The antiapoptotic activity of BCL-2 has been borne out in numerous cell types in response to a variety of stimuli (for review see Korsmeyer, 1996; White, 1996).

Several homologues of BCL-2 have been isolated, including some members of this family that are antiapoptotic, like BCL-2, and others that are proapoptotic (for review see Korsmeyer, 1996; White, 1996). BCL-2 family members that inhibit apoptosis include CED-9, MCL-1, A1, and E1B-19K. Family members that promote apoptosis include BAX, BAK, and BAD. BCL-X, another BCL-2 homologue, has both an antiapoptotic, long-splice variant, BCL-X_L, and a proapoptotic, short-splice variant, BCL-X_S. While the essential biochemical function of BCL-2 family members remains under investigation, their activity is regulated, in part, by dimerization among the various family members (Korsmeyer, 1996; Oltvai et al., 1993). In this rheostat model, the ratio of proapoptotic versus antiapoptotic molecules determines the fate of a cell. Dimerization among family members is mediated by three highly conserved domains, BH1, BH2, and BH3. BH1 and BH2 appear critical for antiapoptotic members (BCL-2 and BCL-X_L) to heterodimerize with the proapoptotic BCL-2 family member, BAX (Yin et al., 1994), while the BH3 domain of BAX is essential for its heterodimerization and activity (Chittenden et al., 1995) .

Studies of mice deficient in BCL-X or BAX have highlighted these two family members as particularly important for the nervous system. Apoptosis in developing sensory and central nervous system neurons is greatly increased in mice deficient in Bcl-X (Motoyama et al., 1995). Dissociated sympathetic neurons from Bax-deficient animals are remarkably resistant to trophic factor deprivation–induced death, and motor neurons from the facial nucleus do not degenerate in response to axotomy (Deckwerth et al., 1996). In contrast, isolated thymocytes from Bax-deficient mice are not resistant to apoptosis (Knudson et al., 1995). In this study, we determined whether BAX was critical for cerebellar granule cell apoptosis.

Dissociated cerebellar granule cells from early postnatal rats can be maintained in serum-containing medium by elevating extracellular potassium levels (25 mM) (Gallo et al., 1987), or by adding low concentrations of N-methyl-D-aspartic acid (NMDA)¹ to the culture medium (Balazs et al., 1988). Both low concentrations of NMDA and depolarization are presumed to mimic endogenous excitatory activity (Burgoyne et al., 1993); the survival promotion is mediated by increases in intracellular calcium (Gallo et al., 1987). Overstimulation of glutamate receptors on granule cells leads to an excitotoxic death (Schramm et al., 1990; Dessi et al., 1993; Lafon-Cazal et al., 1993). Dissociated cerebellar granule cells develop characteristics of mature cerebellar granule cells in vivo including an extensive neuritic network, expression of excitatory amino acid receptors, and production and release of L-glutamate (Burgoyne et al., 1993). Removal of both potassium and serum from dissociated cerebellar granule cells triggers a cell death that is morphologically apoptotic, accompanied by DNA fragmentation, and dependent on macromolecular synthesis (D'Mello et al., 1993; Nardi et al., 1997). This apoptotic cell death presumably mimics the naturally occurring death of 20–30% of granule cells (Caddy and Biscoe, 1979), which is important for matching the number of granule cells with Purkinje cells, that occur during the third through fifth postnatal weeks (Wetts and Herrup, 1983; Williams and Herrup, 1988).

In the current study, we demonstrate that BAX is required for programmed cell death (PCD), but not excitotoxic cell death, of cerebellar granule cells. We used this homogeneous population of BAX-dependent neurons to assess when BAX was acting in the program that commits a cell to die. We found that while Bax-deficient granule cells did not die, they did progress through several early changes associated with neuronal PCD (Deckwerth and Johnson, 1993; Estus et al., 1994; Freeman et al., 1994; Miller and Johnson, 1996) including decreases in protein synthesis and glucose uptake, and an increase in the mRNA level of the AP-1 transcription factor c-jun. In contrast to these events that do occur in both Bax +/+ and Bax –/– cells, we found that Bax –/– lysates did not cleave Ac-DEVD-aminomethylcoumarin (DEVD-AMC), a fluorogenic substrate for caspases 2, 3, and 7 that was cleaved by lysates from wild-type cells undergoing apoptosis. These results identify a central nervous system cell type that requires BAX to undergo PCD and clearly place BAX downstream of changes in glucose uptake, protein synthesis, and certain mRNA levels and upstream of the activation of a DEVD-selective caspase. We further investigated the role of the caspases in PCD of granule cells by using peptide inhibitors of caspases. Although these compounds blocked cleavage of DEVD-AMC completely and blocked the increase in TdT-mediated dUTP nick end labeling (TUNEL)–positivity, they only had a marginal effect on cell survival, suggesting the presence of caspase-independent cell death effectors downstream of BAX.

	MATERIALS AND METHODS

Top Abstract MATERIALS AND METHODS RESULTS BAX –/– NEURONS ARE... DISCUSSION REFERENCES

 
Breeding and Genotyping of Mice with Different Gene Dosages of Bax
Mice heterozygous for Bax (Knudson et al., 1995) were mated to yield F1 offspring with Bax –/–, Bax +/–, and wild-type genotypes. At postnatal day 4–5, tail DNA was prepared and screened by PCR as described (Deckwerth et al., 1996).

Cell Culture Media
All culture media were based on Basal Medium Eagle (Life Technologies, Grand Island, NY) containing 100 U/ml penicillin and 100 µg/ml streptomycin. The following additions were made: K5+S medium, 10% dialyzed FBS 10,000 mol wt cutoff (Sigma Chemical Co., St. Louis MO); K25+S medium, 10% dialyzed FBS, 20 mM KCl; K25–S medium, 20 mM KCl; K5–S medium, no additions. Dialyzed serum was used because adding fresh medium containing nondialyzed serum to cerebellar granule cells is toxic. This sensitivity to nondialyzed serum develops after several days in culture because of the glutamate in the serum (Schramm et al., 1990).

Neuronal Culture
This cell culture protocol is a modification of previous protocols (Levi et al., 1984) and is extensively detailed in Miller and Johnson (1996). The only difference was that cerebella from Bax +/+, Bax +/–, Bax –/– were treated as separate, parallel dissections. In brief, cerebella were dissected from postnatal day seven (P7) mice, sliced into 1-mm pieces, and incubated at 37°C for 15 min in 0.30 mg/ml trypsin (Worthington Biochemical Corp., Freehold, NJ). The tissue was then triturated in K25+S medium with 0.5 mg/ml trypsin inhibitor (Sigma Chemical Co.) by using a flame-polished Pasteur pipette. The resulting cell suspension was spun at 500 g for 6 min. The pellet was gently triturated in fresh K25+S medium and filtered through a nitex filter (size 3-20/14; Tetko Inc., Elmsford, NY). Trypan blue exclusion was used to determine the number of the living neurons before plating: 2–2.5 x 10⁵ cells/cm² in either four-well (Nunc, Naperville, IL) or 35-mm dishes (Corning Inc., Corning, NY). Before plating, dishes were coated with 0.1 mg/ml poly-L-lysine (P2636; Sigma Chemical Co.). The granule cells were kept at 35°C in a humidified incubator with 5% CO₂/95% air for 7 d. Fresh K25+S medium was added after 4 d in vitro. To reduce the number of nonneuronal cells, aphidicolin (3.3 µg/ ml, Sigma Chemical Co.) was added to the medium 24–36 h after initial plating. The culture conditions do not support the survival of other neuronal cell types (Thangnipon et al., 1983; Kingsbury et al., 1985); the nonneuronal contamination was 1–2% (Miller and Johnson, 1996).

Treatment of Cultures
For studies of potassium deprivation, phosphatidylinositol-3 kinase (PI-3-K) inhibition, or inhibition of caspases, at 7 d in vitro, culture medium was replaced with either K5–S or K5+S medium after washing cells once with the respective medium. Control cultures were treated identically with K25+S medium. Special care was taken to assure that all media had been preincubated at 35°C, 5% CO₂/95% air for 18–24 h. 30 µM LY 294002 (Biomol Research Laboratories, Inc., Plymouth, PA) was added to K25+S medium to inhibit PI-3-K. Boc-aspartyl(OMe)-fluoromethylketone (BAF) and Z-VAD-fluoromethylketone were obtained from Enzyme Systems Products (Dublin, CA).

For NMDA stimulation, cells were treated at 10 d in vitro as described in Lafon-Cazal et al. (1993). Culture medium was removed and placed at 35°C, 5% CO₂/95% air. Cultures were washed twice for 15 min at 35°C in Mg²⁺-free Locke's solution (154 mM NaCl, 5.6 mM KCl, 3.6 mM NaHCO₃, 2.7 mM CaCl₂, 5.6 mM D-glucose, 5 mM Hepes, pH 7.4) and then incubated at 35°C for 30 min in Mg²⁺-free Locke's solution with 3 µM glycine (Sigma Chemical Co.) and 10, 30, or 100 µM NMDA (Sigma Chemical Co.), or 100 µM NMDA + 150 nM MK-801 (Research Biochemicals International, Natick, MA). Cells were washed twice for 15 min with Mg²⁺-free Locke's solution at 35°C. The original culture medium was replaced and the cells were incubated at 35°C, 5% CO₂/95% air for 24 h after which cell viability was assessed.

Determination of Cell Viability
Cell viability was quantified from photomicrographs of representative fields of cells labeled with calcein AM (Molecular Probes Inc., Eugene, OR). Calcein AM is an acetoxymethyl ester fluorescein derivative, which is cleaved and trapped inside viable cells that have nonspecific esterase activity (Bozyczko et al., 1993). The photomicrographs were both taken and scored by an observer naive to the experimental condition and to the genotype of the animal from which the culture was derived. Six photomicrographs at 200 magnification were taken for each condition and the number of calcein AM–positive cells was counted. This method of quantifying cell viability is detailed and validated in Miller and Johnson (1996).

Metabolic Parameters
Experiments were performed in four-well dishes (Nunc) with 400,000 cells per well. After 7 d in vitro, culture medium was replaced with K5+S or K25+S medium (as described above). Detailed description of the following methods may be found in Deckwerth and Johnson (1993) and Miller and Johnson (1996).

Rate of Protein Synthesis.
Neuronal cultures were labeled for 1 h at 35°C with 10 µCi/ml L-[4,5-³H]leucine (159 Ci/mmol; Amersham Life Science, Inc., Arlington Heights, IL) in K25+S or K5+S medium containing 10 µM unlabeled L-leucine. Cultures were lysed, precipitated with 10% trichloroacetic acid (TCA), filtered, and counted in a liquid scintillation counter (Beckman Instrs., Fullerton, CA).

Rate of 2-deoxyglucose Uptake.
Neuronal cultures were labeled for 30 min at 35°C with 2.5 µCi/ml [1,2-³H]2-deoxy-D-glucose (30 Ci/mmol; ICN Biomedicals, Inc., Irvine,CA) in K25+S or K5+S medium containing 500 µM D-glucose. Cultures were washed three times, lysed, and added directly to liquid scintillation fluid and counted. Assays were linear with respect to time during the indicated measuring period (data not shown).

RT-PCR
Semiquantitative reverse transcription-PCR (RT-PCR) assays are based on those used for sympathetic neurons described in Freeman et al. (1994) and Estus et al. (1994), and extensively detailed by Estus (1997). Briefly, granule cells were switched to K5+S for the indicated times. Polyadenylated (poly-A) RNA was isolated from 400,000 cerebellar granule cells by using an oligo-dT-cellulose mRNA purification kit as directed by the manufacturer (QuickPrep Micro Kit; Pharmacia LKB Biotechnology Inc., Piscataway, NJ). Half of the poly-A RNA was converted to cDNA by reverse transcription (RT) with Moloney murine leukemia virus reverse transcriptase with random hexamers (16 µM) as primers. cDNA from 4,000 cells was used in a 50 µl PCR reaction. After amplification, the PCR products were separated by electrophoresis on 10% polyacrylamide gels, visualized by autoradiography of the dried gels, and quantified with a PhosphorImager (Molecular Dynamics, Inc., Sunnyvale, CA). Preliminary experiments with cerebellar granule cell cultures validated that the RT-PCR technique was linear with respect to the amount of input RNA used for RT and with respect to the amount of cDNA used for PCR within the ranges used in these experiments. No product was amplified when purified RNA was used as input for a PCR reaction. Results were repeated in at least two independent RNA preparations. The sequences of the PCR products were confirmed (Estus et al., 1994; Freeman et al., 1994; data not shown). Primer sequences for cyclophilin and cyclin D1 are reported in Freeman et al. (1994). Sequences for c-fos and c-jun are reported in Miller and Johnson (1996). Bax primer sequences are detailed in Greenlund et al. (1995). Primer sequences for CPP32 (EMBL/GenBank/DDBJ accession number D86352), ICH-1 (U13022), and GFAP (X02801) are as follows (5' to 3'): CPP32 (–569) AGA GTA AGC ATA CAG GAA GTC GGC; CPP32(+351) GAT TCT AAG TCA TGG AGA TGA AGG; ICH1 (fwd) GGT TGA GAT GGC AAA CTG CT; ICH1 (rev) CCA GCA TCA CTC CC TCA CA; GFAP (+2092) CAA TGG AGT TGG AAG TTG TAG GC; and GFAP (–2245) GAT AGA CCT TCA CAA CTG AGA CG.

DEVD-AMC Cleavage Assay
DEVD-AMC cleavage was measured essentially as described (Armstrong et al., 1997). After 0, 4, 8, 12, 24, or 48 h in K5+S medium, 400,000 granule cells were washed once with PBS and lysed in 100 µl of buffer A (10 mM Hepes, pH 7.4, 42 mM KCl, 5 mM MgCl₂, 1 mM DTT, 0.5% CHAPS, 1 mM PMSF, 1 µg/ml leupeptin). In a 96-well plate, 25 µl of lysate was combined with 75 µl of buffer B (25 mM Hepes, 1 mM EDTA, 0.1% CHAPS, 10% sucrose, 3 mM DTT, pH 7.5) containing 30 µM Ac-DEVD-AMC (Biomol Research Laboratories, Plymouth, PA) and incubated for 20 min at room temperature in the dark. Fluorescence was measured at excitation 360 nm and emission 460 nm in a fluorescent plate reader (Titertek Fluoroskan II; Flow Laboratories, Inc., McLean, VA). 20 min and 25 µl were both in the linear range of the assay with respect to time and amount of lysate added.

Although cultures were extensively washed before lysing cells, we were concerned that residual BAF or ZVAD from the culture medium might account for the block in DEVD-AMC cleavage in our enzyme assay. To test this possibility, we combined 8-h K⁺ deprivation lysates from BAF- or ZVAD-treated cultures with 8-h K⁺ deprivation lysates from untreated cultures and determined DEVD-AMC cleavage. We found that the 8-h lysates from the BAF- or ZVAD-treated cultures did not themselves contain any ability to inhibit the DEVD-AMC cleavage activity in our assay, demonstrating that the BAF and ZVAD in the culture medium had been adequately washed out before lysis (data not shown).

Transfections of Cerebellar Granule Cells
All constructs were under the cytomegalovirus (CMV) promoter. pGreen Lantern-1 (green fluorescent protein) was obtained from GIBCO BRL (Gaithersburg, MD). The BCL-2 construct is detailed in Greenlund et al. (1995a). The p35 construct was a generous gift from Dr. Lois K. Miller. pBluescript II was used as control DNA (Short et al., 1988). Granule cells were transfected essentially as described in Xia et al. (1995). In brief, after 5 d in vitro, cultures were switched to K25–S medium for 1 h after which DNA/CaCl₂ precipitates were prepared as follows: An equal volume of solution containing 0.25 M CaCl₂, 67 µg/ml pGreen Lantern-1 DNA, and 67 µg/ml of either Bax, BCL-2, or p35 DNA was added to 2x Hepes-buffered saline (274 mM NaCl, 10 mM KCl, 1.4 mM Na₂HPO₄-7H₂0, 15 mM dextrose, 42 mM Hepes-free acid, pH 7.07) and incubated in the dark at room temperature for 25 min. 30 µl of the CaCl₂ precipitate (1 µg of DNA) was added dropwise to each well of a Nunc four-well plate containing 400,000 granule cells and incubated for 1 h at 37°C. Cells were washed twice with K25–S medium and returned to K25+S medium for 2 d after which the number of transfected cells was assessed by counting the number of pGreen Lantern-1 (fluorescent) cells (200) in a defined area for two separate wells of a Nunc four-well dish. Transfection efficiency was typically 0.2%. Cells were then switched to K5+S or K25+S medium, and the number of fluorescent cells was again assessed for the same defined area after 24 and 48 h and compared to the original number for a particular well. All cultures were counted by a naive observer. pGreen Lantern-1 positivity correlated well with a healthy, viable cell as assessed by phase-contrast microscopy, nuclear morphology, and exclusion of trypan blue. Preliminary experiments using both pGreen Lantern-1 and LacZ as markers of transfection demonstrated that all cells that were pGreen Lantern-1–positive were also LacZ-positive and vice versa. In addition, from the earliest time that pGreen Lantern-1 could reliably be detected (24 h), expression of the constructs was not toxic to the cells up to 9 d after transfection.

Phospho-Jun Staining
Neuronal cultures were washed once with PBS, pH 7.4, before a 30-min fixation with 4% paraformaldehyde in PBS at 4°C. Neuronal cultures were washed three times with TBS (100 mM Tris, 0.9% NaCl, pH 7.6), blocked for 30 min in TBS containing 5% goat serum (Sigma Chemical Co.) and 0.3% Triton X-100 at room temperature, and incubated overnight at 4°C with anti–phospho–c-jun antibody (Ser 63; New England Biolabs Inc., Beverly, MA) diluted 1:200 in TBS containing 1% goat serum and 0.3% Triton X-100. Cultures were washed three times with TBS then incubated overnight at 4°C in a 1:400 dilution of 1.5 mg/ml Cy3-donkey anti–rabbit antibody (Jackson Immunoresearch Laboratories, Inc., Westgrove, PA) in the same buffer as the primary antibody. Cultures were washed twice with TBS, stained with 1 µg/ml bisbenzimide (Hoechst 33258; Molecular Probes Inc., Eugene, OR) for 20 min to visualize nuclei, then washed two additional times with TBS. The phospho-jun antibody does not react with the nonphosphorylated form of c-Jun and does not appreciably cross-react with the phosphorylated form of JunD or JunB (New England Biolabs Inc.).

TUNEL Staining
At 7 d in vitro, culture medium from cerebellar granule cells was replaced with either K5+S, K5+S plus 100 µM BAF, or fresh K25+S after washing the cells once with the appropriate medium. At 24 h after this treatment, cells were examined for DNA fragmentation using the In Situ Cell Death Detection Kit (Fluorescein; Boehringer Mannheim Biochemicals, Indianapolis, IN) according to the manufacturer's instructions. In brief, cells were fixed for 30 min in 4% paraformaldehyde/PBS at room temperature, washed once with PBS, and permeabilized in 0.1% Triton X-100, 0.1% sodium citrate for 20 min at 4°C. After two more washes with PBS, the cells were incubated with 50 µl of the TUNEL reagent for 1 h at 37°C in the dark. Cells were then washed twice with PBS, stained with 1 µg/ml Hoechst 33258 (Molecular Probes Inc.), and washed twice more with PBS. The percentage of TUNEL-positive cells was calculated as the number of TUNEL-positive cells divided by the number of Hoechst-stained cells. For each condition, a naive observer counted two to five fields, each containing over 200 bisbenzimide-stained neurons. Results represent mean ± range for two independent experiments.

	RESULTS

Top Abstract MATERIALS AND METHODS RESULTS BAX –/– NEURONS ARE... DISCUSSION REFERENCES

 
Bax –/– Neurons are Resistant to Apoptosis
Dissociated cerebellar granule cells maintained in 25 mM K⁺ and serum undergo a PCD that is apoptotic if they are deprived of both potassium and serum (K5–S medium) (D'Mello et al., 1993). To determine whether BAX, a proapoptotic BCL-2 family member, was important in cerebellar granule cell apoptosis, we used cultures of dissociated cerebellar granule cells from P7 wild-type and Bax-deficient mice (Knudson et al., 1995). After 7 d in vitro, cultures of Bax +/+, and Bax –/– cerebellar granule cells were switched to medium containing low potassium and serum (K5+S) or maintained in high potassium and serum (K25+S). After 72 h, cultures were stained with calcein AM, which stains living cells. Phase-contrast images and the corresponding calcein AM photomicrographs are presented in Fig. 1. While virtually all of the Bax +/+ cells died by 72 h (compare Fig. 1, a and b with e and f ), granule cells from Bax –/– mice did not undergo apoptosis (compare Fig. 1, c and d with g and h). To determine the number of surviving cells, we counted the number of neurons on photomicrographs of cultures stained with calcein AM after 0, 12, 24, 48, or 72 h in K5+S medium (Fig. 2). All the granule cells from Bax –/– mice were protected from cell death (Fig. 2, open triangles) while more than 90% of the wild-type granule cells died by 72 h (Fig. 2, open circles). Granule cells from heterozygous animals died completely by 72 h (Fig. 2, open squares), though the time course of death was slightly slower. Nuclei from Bax –/– cultures deprived of K⁺ for 72 h were indistinguishable from control cultures maintained in K25+S medium (data not shown), while Bax +/+ and Bax +/– cultures displayed characteristic apoptotic nuclear changes 6 h after switching to K5+S medium (data not shown).

View larger version (29K): [in this window] [in a new window] [Download PPT slide]   Figure 1 Bax –/– cerebellar granule cells do not undergo apoptosis in response to K+ deprivation. Cerebellar granule cells from Bax +/+ (a, b, e, and f) and Bax –/– (c, d, g, and h) were maintained for 7 d in vitro and then switched to K25+S medium (a–d) or K5+S medium (e–h). Photomicrographs of phase contrast (a, c, e, and g) and the corresponding calcein AM–stained images (b, d, f, and h) were taken 72 h after the media were switched. Bar, 20 µm.

View larger version (16K): [in this window] [in a new window] [Download PPT slide]   Figure 2 Bax-deficient granule cells are fully protected from K+ or K+/serum deprivation-induced death. After 7 d in vitro, granule cells were switched to either K5+S (open circle, square, and triangle) or K5-S medium (closed circle, square, and triangle) for 12, 24, 48, or 72 h. Survival was assessed by counting neurons in photomicrographs of calcein AM-stained cultures and compared to cultures maintained in K25+S. Circles, squares, and triangles represent Bax +/+, Bax +/–, and Bax –/– cultures, respectively. Data represent mean ± range for two independent experiments.

We have previously shown that PI-3-K activity is increased by K⁺ depolarization (Miller et al., 1997). This increase appears critical to the survival of depolarized neurons as shown by the ability of two inhibitors of PI-3-K to produce death indistinguishable from that caused by removal of high K⁺ (Miller et al., 1997). Because BCL-2 family members have been recently implicated in signaling pathways (Gajewski and Thompson, 1996; Wang et al., 1996; Zha et al., 1996), we directly tested the ability of Bax –/– cells to survive in the absence of PI-3-K activity. Neurons maintained for 7 d in vitro were switched to K25+S with 30 µM LY 294002, an inhibitor of PI-3-K activity. Viability was assessed after 48 h by calcein AM staining (Fig. 3). Similar to previous results (Miller et al., 1997), LY 294002 blocked the survival-promoting activity of K⁺ in Bax +/+ cells. Results from cultures of animals heterozygous for Bax were similar to those from wild type. In contrast, neurons from Bax –/– cultures survived in the presence of LY 294002, implying that Bax deficiency blocked apoptosis downstream of PI-3-K.

View larger version (10K): [in this window] [in a new window] [Download PPT slide]   Figure 3 Inhibition of PI-3-K does not induce apoptosis in Bax –/– granule cells. After 7 d in vitro, granule cells were switched to K25+S medium in the presence of 30 µM LY 294002, an inhibitor of PI-3-K. Control cells were switched to K25+S medium. Survival was determined by counting neurons in photomicrographs of calcein AM–stained cultures 48 h after treatment. Data represent mean ± range for two independent experiments.

	BAX –/– NEURONS ARE NOT PROTECTED FROM EXCITOTOXIC DEATH

Top Abstract MATERIALS AND METHODS RESULTS BAX –/– NEURONS ARE... DISCUSSION REFERENCES

View larger version (15K): [in this window] [in a new window] [Download PPT slide]   Figure 4 Bax-deficient granule cells are not protected from NMDA-induced excitotoxic cell death. After 10 d in vitro, granule cells were stimulated with 0, 10, 30, and 100 µM NMDA, or 100 µM NMDA with 150 nM MK-801 for 30 min at 37°C. Survival was assessed after 24 h by counting neurons in photomicrographs of calcein AM–stained cultures. Data represent mean ± range for two independent experiments.

View larger version (12K): [in this window] [in a new window] [Download PPT slide]   Figure 5 BAX deletion does not alter changes in metabolic parameters associated with PCD. (A) Glucose uptake was determined 2, 6, 12, 24, or 48 h after switching granule cell cultures to K5+S medium by measuring the incorporation of [1, 2-3H]2-deoxy-D-glucose and compared to control cultures that were switched to K25+S medium. (B) Protein synthesis was determined after 6, 12, 24, or 48 h by measuring the incorporation of L-[4, 5-3H]leucine and compared to control cultures that were switched to K25+S medium. Data represent mean ± range for two independent experiments.

Whether the increase in c-jun mRNA and presumably the action of c-jun is downstream or upstream of BAX is unknown. To address this issue and examine other message levels, cultures from Bax +/+ and Bax –/– animals were switched to K5+S for 1, 3, 6, 9, 12, 18, 24, 48, or 72 h and mRNA was isolated, reverse transcribed, and selectively amplified by PCR. mRNA levels of cyclophilin declined rapidly in wild-type granule cells (Fig. 6, A and B); actin and neuron-specific enolase were similar to cyclophilin (data not shown). mRNA levels of these same messages in Bax –/– cultures declined far less than in wild-type cultures reflecting the lack of death of Bax –/– cells (Fig. 6, A and C). Similar to results seen in rat cerebellar granule cells (Miller and Johnson, 1996), mRNA levels for c-jun increased approximately fourfold in K⁺-deprived wild-type granule cells (Fig. 6 B). c-jun mRNA also increased in K⁺-deprived, Bax-deficient granule cells (Fig. 6 C), demonstrating that increases in c-jun were upstream of Bax.

View larger version (44K): [in this window] [in a new window] [Download PPT slide]   Figure 6 mRNA levels of c-jun and CPP32 increase in both Bax +/+ and Bax –/– granule cells after K+ deprivation. Cultures were switched to K5+S and cDNA was prepared from granule cells after 1, 3, 6, 9, 12, 18, 24, 48, or 72 h. Control cultures were maintained in K25+S medium. cDNA from 4,000 cells was used in a 50-µl PCR reaction as described in Materials and Methods. Identical results were obtained in an independent neuronal preparation. (A) PCR products. (B) PhosphorImager quantitation of Bax +/+ PCR products. (C) PhosphorImager quantitation of Bax –/– PCR products. Time 0 was used as control level for B and C.

mRNA levels of caspase 3 (CPP32) increased in both wild-type and Bax-deficient cultures deprived of K⁺ (Fig. 6). Caspase 3 has been clearly implicated in several cell death paradigms (for review see Henkart, 1996) and in developmental neuronal cell death (Kuida et al., 1996), although whether increases in mRNA levels of caspases have any significance for cell death is unclear. The mRNA level of at least one other caspase, caspase 2 (Ich-1), was not increased after K⁺ deprivation (data not shown).

c-Jun Is Phosphorylated on Ser 63 in Both Bax +/+ and Bax –/– Cultures
In addition to increases in mRNA levels, another indication of an increase in c-Jun activity is the phosphorylation of c-Jun on serines 63 and 73 of the transactivation domain (Binetruy et al., 1991; Smeal et al., 1991, 1992). Therefore, we determined the phosphorylation status of the c-Jun transactivation domain during PCD by immunostaining with an antibody that specifically recognizes the serine 63– phosphorylated form of c-Jun (Fig. 7). Cells maintained in K25+S medium (Fig. 7, a and e) had almost no staining. After 6 h of K⁺ deprivation, wild-type granule cells showed significant phosphorylation of c-Jun on serine 63 (Fig. 7 c). Similar to the parallel increases in c-jun mRNA levels in Bax +/+ and Bax –/– cultures, phospho-Jun staining also increased in Bax –/– granule cells deprived of K⁺ for 6 h (Fig. 7 g). Staining was not increased in the few nonneuronal cells in these cultures (data not shown). In nuclei counterstained with bisbenzimide (Fig. 7, b, d, f, and h), some K⁺-deprived, wild-type cells showed nuclear margination and chromatin condensation at 6 h (Fig. 7 d). The increase in c-Jun phosphorylation and c-jun mRNA in both wild-type and Bax –/– cultures indicates that BAX functions downstream of the increase in c-Jun activity that is associated with PCD.

View larger version (22K): [in this window] [in a new window] [Download PPT slide]   Figure 7 c-Jun phosphorylation increases in both Bax +/+ and Bax –/– granule cells after K+ deprivation. Bax +/+ (a–d) and Bax –/– (e–h) cultures were switched to K25+S (a, b, e, and f) or K5+S (c, d, g, and h) medium for 6 h and immunostained with an antibody that specifically recognizes the ser-63 phosphorylated form of c-Jun (a, c, e, and g) and stained with the nuclear dye bisbenzimide (b, d, f, and h). Bar, 5 µm.

View larger version (13K): [in this window] [in a new window] [Download PPT slide]   Figure 8 BAX is required for increases in caspase activity. Cultures were switched to K5+S medium, lysed after 4, 8, 12, 24, 48, or 72 h, and cleavage of DEVD-AMC was determined. Control cultures were switched to K25+S medium and treated identically. Data represent mean ± SD for triplicate measurements from one experiment and are representative of two additional independent experiments.

View larger version (15K): [in this window] [in a new window] [Download PPT slide]   Figure 9 Inhibitors of caspases do not block death, but do block DEVD-AMC cleavage. Cultures were switched to K5+S, K5+S plus 100 µM BAF, or K5+S plus 100 µM ZVAD-FMK. Control cultures were switched to K25+S medium. (A) After 12, 24, or 48 h neuronal survival was determined by calcein AM staining. (mean ± SD, N = 3 experiments) (B) After 8, 12, 18, 24, or 48 h cultures were lysed and assayed for DEVD-AMC cleavage. Fluorescence was measured after 20 min at room temperature (mean ± range, N = 2 experiments).

View larger version (44K): [in this window] [in a new window] [Download PPT slide]   Figure 10 Cell death in the presence of BAF is TUNEL-negative. Cultures were switched to K5+S (b and e) or K5+S plus 100 µM BAF (c and f). Control cultures were switched to K25+S medium (a and d). After 24 h, cultures were fixed, stained with the TUNEL reagent (d–f), and stained with bisbenzimide (a–c) as described in Materials and Methods. The percent TUNEL-positive cells is the number of TUNEL-positive cells divided by the number of bisbenzimide-stained cells (g). Data represent the mean ± range for two independent experiments.

View larger version (14K): [in this window] [in a new window] [Download PPT slide]   Figure 11 p35 does not block cell death. After 5 d in vitro, granule cells were transfected, as described in Materials and Methods, with pGreen Lantern-1 and either pBluescriptII, a BCL-2 construct, or a p35 construct. At 7 d in vitro, the number of pGreen Lantern-1–positive (fluorescent) cells was determined before switching cells to K25+S or K5+S medium. The number of pGreen Lantern-1–positive cells was again determined after 24 and 48 h and compared to the original number of pGreen Lantern-1–positive cells before the medium was switched. Data represent mean ± SEM for four independent experiments. *indicates significance at P < 0.05 in a paired t test in which the data passed a test for normality.

	DISCUSSION

Top Abstract MATERIALS AND METHODS RESULTS BAX –/– NEURONS ARE... DISCUSSION REFERENCES

View larger version (9K): [in this window] [in a new window] [Download PPT slide]   Figure 12 Diagrammatic representation of the events associated with programmed cell death in cerebellar granule cells.

BAX Is Not Required for Changes in Metabolic Parameters or mRNA Levels
The early, dramatic fall in metabolic parameters that we saw in Bax –/– cultures is part of the cell death program in cerebellar granule cells (Miller and Johnson, 1996; Nardi et al., 1997), sympathetic neurons (Deckwerth and Johnson, 1993), and thymocytes (Makman et al., 1966; Munck, 1968; Cidlowski, 1982). Thus, we have hypothesized that these events may represent an early trigger for PCD. The early decreases in glucose uptake and protein synthesis rate were not affected by the Bax genotype. This suggests that the actions of BAX are not related to early signal transduction pathways mediating neuronal survival by trophic signals. This conclusion is further supported by the inability of PI-3-K inhibitors to induce cell death in Bax –/– cells. It is also consistent with previous observations that overexpression of BCL-2, while greatly retarding sympathetic neuronal death after NGF deprivation, has no effect on the decrease in protein synthesis that presages that death (Greenlund et al., 1995b).

The deletion of BAX also failed to block changes in mRNA levels, another step in the cell death program; c-jun mRNA increased in both Bax +/+ and Bax –/– cultures. Several reports have suggested that c-Jun may be important for cell death. Microinjection of neutralizing antibodies to c-Jun or a dominant negative c-jun construct delays the death of NGF-deprived sympathetic neurons (Estus et al., 1994; Ham et al., 1995). Dominant negative c-jun constructs also block cell death in human monocytic leukemia cells (U937) treated with ceramide (Verheij et al., 1996). An increase in c-Jun is evidently part of cell death in vivo. c-Jun increases in granule cells undergoing PCD in the weaver mouse (Gillardon et al., 1995) and in irradiated animals (Ferrer et al., 1996). c-Jun amino-terminal kinase (JNK) (Hibi et al., 1993) may be the kinase responsible for the increased phosphorylation on serine 63 of c-Jun in both Bax +/+ and Bax –/– cells (Fig. 7). Several reports have implicated this pathway during cell death (Xia et al., 1995; Chen et al., 1996; Latinis and Koretzky, 1996; Park et al., 1996; Verheij et al., 1996; Wilson et al., 1996). The increase in c-Jun both in vitro and in vivo, the fact that blocking c-Jun delays cell death, and increases in c-Jun phosphorylation during PCD imply that an increase in c-Jun is an important early occurrence during PCD. In PC12 cells, overexpression of Bcl-2 is able to block the increase in JNK activity associated with PCD, suggesting that the BCL-2 family functions upstream of c-Jun (Park et al., 1996). Our loss of function study with BAX is in apparent disagreement with these data. In cerebellar granule cells, the increase in c-jun message level and the phosphorylation of c-Jun are clearly upstream of BAX (Figs. 6 and 7).

Caspase Activity
We examined the role of caspases in PCD in granule cells by determining changes in mRNA levels of caspase 3 (CPP32) and increases in the activity of DEVD selective caspases. K⁺ deprivation increased caspase 3 mRNA levels in Bax +/+ and Bax –/– cultures. An increase in the message level of caspase 3 also occurs in response to focal ischemic injury (Asahi et al., 1997). Similarly, loss of expression of caspase 1 (ICE) in mammary epithelial cells correlates with the suppression of apoptosis by extracellular matrix (Boudreau et al., 1995). However, whether these changes in mRNA levels are important for cell death or lead to increases in activity of caspases is unclear.

We directly measured the activity of caspases 2, 3, and 7 and found that wild-type granule cells increased DEVD-specific substrate cleavage after K⁺deprivation (Fig. 8). Other groups have similarly reported that DEVD-AMC is cleaved in granule cells after K⁺/serum deprivation (Armstrong et al., 1996; Nath et al., 1996). The increase in the activity of DEVD-selective caspases is an event that clearly occurs downstream of BAX in cerebellar granule cells (Fig. 8) since Bax –/– cultures showed no increase in DEVD-AMC cleavage after K⁺ deprivation. Similarly, overexpression of BCL-2 prevents activation of caspases in Jurkat cells (Armstrong et al., 1996; Chinnaiyan et al., 1996), in GT1-7 neural cells (Srinivasan et al., 1996), and in PC12 cells (Stefanis et al., 1996).

Caspase Inhibitors Do Not Block Granule Cell Death
We were surprised by the failure of the baculoviral protein p35 or pharmacological caspase inhibitors to block PCD in cerebellar granule cells. Although inhibiting caspases does delay cell death in granule cells up to 24 h (Nath et al., 1996; Schulz et al., 1996; Armstrong et al., 1997), our data indicate that these compounds did not offer any substantial long-term protection (Fig. 9). This contrasts with sympathetic neurons in which PCD is prevented completely for at least 7 d by BAF (Deshmukh et al., 1996). These results suggest that granule cells have, in addition to a caspase-dependent pathway, a caspase-independent pathway downstream of BAX that insures cell death.

Our suggestion that at least two pathways exist downstream of BAX presumes that all potential caspases were effectively blocked in these studies. We demonstrated that DEVD-AMC cleavage was blocked completely in these cells, showing that the compounds used were able to penetrate the cells and function as caspase inhibitors. BAF, ZVAD-FMK (Armstrong et al., 1996; Cain et al., 1996; Deshmukh et al., 1996), and p35 (Bump et al., 1995; Xue and Horvitz, 1995) are nonspecific inhibitors with reasonable IC₅₀s that block the activity of a wide spectrum of caspases. We cannot formally exclude the possibility that an atypical caspase might have been activated in granule cells in the presence of these inhibitors. Similarly, cell death in the presence of p35 might be accounted for by inadequate levels of protein expression.

The fact that cell death in the presence of caspase inhibitors was TUNEL-negative, while K⁺ deprivation induced was TUNEL-positive, provided further evidence for the ability of caspase inhibitors to function inside of cells. The nuclear morphology of dying BAF- and ZVAD-treated granule cells may also be an indication that these compounds blocked caspases. While BAF- and ZVAD-treated nuclei did condense, we found no evidence for margination or clumping of the chromatin typically observed in apoptotic cells, though these changes were apparent in untreated, K⁺-deprived cells (Miller, T.M., personal observation). The ability of caspase inhibitors to block lamin proteolysis (Lazebnik et al., 1995) may account for the lack of margination and chromatin clumping (Rao et al., 1996). In addition to demonstrating that these caspase inhibitors did function within cells, these data raise the possibility that the modestly slower, caspase-independent cell death in granule cells differs fundamentally from the cell death that normally occurs after K⁺ deprivation when caspases are unimpeded. Further studies of this caspase-independent, TUNEL-negative cell death may elucidate important differences between these two cell death pathways.

Evidence in a different system using an alternative approach has also recently suggested a BAX-dependent, protease-independent mechanism of cell death. BAX overexpression in Jurkat cells leads to an apoptotic-like death that is not blocked by caspase inhibitors, calpain inhibitors, serine protease inhibitors, granzyme B inhibitors, or proteasome inhibitors (Xiang et al., 1996). In these overexpressing Jurkat cells, as observed here, caspase inhibitors do block DNA fragmentation and cleavage of substrates for caspases; however, these cells still die. In Jurkat cells, a fall in mitochondrial membrane potential and an increase in ROS occurs (Xiang et al., 1996). These mitochondrial changes and the membrane localization of BAX suggest that a mitochondria-dependent cell death predominates in BAX gain of function experiments (Xiang et al., 1996). Data here in the experimental setting of BAX loss-of-function provide support for the possibility that a non–caspase-dependent, TUNEL-negative, cell death pathway resides downstream of BAX.

In summary (Fig. 12), K⁺ and IGF-I block the activation of the cell death program in granule cells. When these agents are removed and the program is activated, MAP kinase activity, PI-3-K activity, metabolic parameters, and most mRNA levels decrease, while some mRNA levels, e.g., c-jun, increase. Our data from the Bax –/– mice place BAX downstream of these changes. The PCD events subsequent to BAX include increased caspase activity and some other change(s) that promote DNA fragmentation, chromatin condensation, and cell death.

Bax –/– granule cell cultures offer an informative perspective on cell death because these cells clearly progress through part of the apoptotic program and then are completely halted. These cultures should be a valuable tool for further ordering the molecular pathways leading to cell death, for defining the role of the BCL-2 family in apoptosis, and, potentially, for identifying a caspase-independent cell-death pathway. Finally, Bax –/– animals bred to mice harboring various cerebellar mutations will determine whether BAX deletion can also rescue "diseased" granule cells in vivo. Assessing both the extent of neuroprotection and the recovery of cerebellar function may offer important insights into how modulation of the BCL-2 family may affect neurological disease.

	Acknowledgments

 
We thank our colleagues at Washington University School of Medicine (St. Louis, MO) B. Klocke, G. Brown, and J. Harding for care of Bax-deficient mice, and R. Easton and P. Osborne for critically evaluating this manuscript.

Supported by National Institutes of Health Grants NS 24679 and AG12947 to E. Johnson, and by CA 49712 to S. Korsmeyer. K. Moulder is supported as a Lucille P. Markey predoctoral fellow, C. Knudson by a Pfizer Postdoctoral Fellowship, and M. Deshmukh by a Paralyzed Veterans of America Spinal Cord Research Foundation Grant 1786.

Submitted: 21 March 1997

Revised: 29 July 1997

Address all correspondence to Eugene M. Johnson, Jr., Washington University School of Medicine, Department of Molecular Biology and Pharmacology, 660 South Euclid Avenue, Box 8103, St. Louis, MO 63110. Tel.: (314) 362-3926. Fax: (314) 362-7058. E-mail: ejohnson{at}pharmdec.wustl.edu

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