Bax deletion further orders the cell death pathway in cerebellar granule cells and suggests a caspase-independent pathway to cell death

Abstract

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 microM 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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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).

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.

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.

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.

Figure 12

Diagrammatic representation of the events associated with programmed cell death in cerebellar granule cells.