* 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.
EXTENSIVE cell death is an important part of the development and ongoing maintenance of tissues in
multicellular organisms (Glucksman, 1951 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 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 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 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 Breeding and Genotyping of Mice with Different Gene
Dosages of Bax
Mice heterozygous for Bax (Knudson et al., 1995 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 Neuronal Culture
This cell culture protocol is a modification of previous protocols (Levi et
al., 1984 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 For NMDA stimulation, cells were treated at 10 d in vitro as described
in Lafon-Cazal et al. (1993) 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 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) 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) DEVD-AMC Cleavage Assay
DEVD-AMC cleavage was measured essentially as described (Armstrong
et al., 1997 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) 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.
Bax 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
In a more rigorous test of the ability of Bax We have previously shown that PI-3-K activity is increased by K+ depolarization (Miller et al., 1997
Overexpression of BCL-2 is neuroprotective in some
models of stroke and excitotoxic injury (Linnik, 1996
BAX Is Not Required for the Early Decrease in
Metabolic Parameters
The experiments described above indicate that BAX is required for mediating apoptosis in cerebellar granule cells.
We were able to use this granule cell culture model to gain
insight into where BAX functions in the cell death pathway. One of the earliest changes in cerebellar granule cells
undergoing PCD is a dramatic decrease in metabolic parameters such as glucose uptake and protein synthesis
(Miller and Johnson, 1996
BAX Is Not Required for Changes in mRNA Levels
Associated with PCD
PCD of many cell types (Freeman et al., 1993 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
c-fos mRNA levels increase in dying sympathetic neurons and the Fos family of proteins may be important for
cell death in sympathetic neurons (Estus et al., 1994 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 c-Jun Is Phosphorylated on Ser 63 in Both Bax +/+
and Bax 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
BAX Is Required for Increases in Caspase Activity
Caspases have been implicated in several cell death models (for review see Henkart, 1996
Blocking Caspases Delays but Does Not
Block Cell Death
Inhibiting caspases with peptide inhibitors blocks PCD in
several models (Henkart, 1996
Further evidence that BAF was able to function inside
cells was found in a TUNEL assay of K+-deprived granule
cells. TUNEL serves as an in situ marker of DNA fragmentation (Gavrieli et al., 1992
A second method we used to inhibit caspase activity was
expression of p35, a baculoviral protein. Recombinant p35
blocks the enzymatic activity of purified ICE, Ich-1, Ich-2,
and CPP32 (caspases 1, 2, 4, and 3, respectively) (Bump et
al., 1995
In this study, we demonstrated that PCD in cerebellar
granule cells induced by K+ deprivation, K+/serum deprivation, or inhibition of PI-3-K requires BAX. In contrast, BAX was not required for excitotoxic cell death in granule
cells since we found that Bax
Bax Deletion Does Not Appear to Result in Activation
of Upstream Signal Transduction Pathways
Survival-promoting agents, such as IGF-I and K+, are
likely to block the initial activation of the cell death program by activating intracellular signaling pathways. We
have previously shown that both K+ and IGF-I activate
two intracellular signaling pathways, PI-3-K and mitogen-activated protein (MAP) kinase in these cells (Miller et al.,
1997 BAX Is Not Required for Changes in Metabolic
Parameters or mRNA Levels
The early, dramatic fall in metabolic parameters that we
saw in Bax 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 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 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 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 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 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 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 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 Bax ; 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
).
; 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-XL, and a proapoptotic, short-splice variant, BCL-XS.
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-XL) 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
) .
). 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.
), 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
).
; 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
) 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
).
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
).
) 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 × 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
).
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).
. 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.
). 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)
.
and
Miller and Johnson (1996)
.
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.
). 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.
. 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 2× 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.
RESULTS
/
Neurons are Resistant to Apoptosis
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).
Fig. 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 of this Image (59K GIF file)]
Fig. 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.
[View Larger Version of this Image (24K GIF file)]
/
granule
cells to resist apoptosis, we also determined the time
course of death after both K+ and serum were removed
(K5
S). Again, Bax
/
granule cells did not die while
Bax +/+ and Bax +/
cells died completely by 72 h (Fig.
2); the death in Bax +/+ and Bax +/
was slightly faster when both serum and potassium were removed. Because
removing K+ alone was an inherently simpler model in
which only one source of trophic support was removed,
this paradigm was used for all subsequent experiments.
). 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.
Fig. 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.
[View Larger Version of this Image (14K GIF file)]
BAX
/
NEURONS ARE NOT PROTECTED FROM
EXCITOTOXIC DEATH
).
Therefore, we tested whether the absence of BAX would
be protective in an NMDA model of excitotoxic death in
cerebellar granule cells. After 10 d in vitro, cultures from
Bax +/+ and Bax
/
were stimulated with 10, 30, or 100 µM NMDA for 30 min and viability was assessed 24 h
later by calcein AM staining (Fig. 4). In contrast to K+
deprivation or treatment with an inhibitor of PI-3-K, the
absence of Bax was not neuroprotective in this excitotoxic
paradigm. The lack of death in cultures simultaneously exposed to 100 µM NMDA and 150 nM MK-801, a highly
potent noncompetitive NMDA receptor antagonist (Wong
et al., 1986
), demonstrated that the death was specific to
stimulation of NMDA receptors.
Fig. 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 of this Image (21K GIF file)]
; Nardi et al., 1997
). We measured glucose uptake and protein synthesis in K+-deprived
granule cells from Bax
/
animals to determine at what point Bax
/
cells are arrested in the cell death program
and to provide an indication of the metabolic status of
these cells. Cultures were switched to K5+S for 2, 6, 12, 24, or 48 h, and metabolic parameters were measured. The
dramatic early fall in glucose uptake was seen in cells from
Bax
/
animals; 2-deoxyglucose uptake fell to <20% of
control within 2 h of K+ deprivation (Fig. 5 A). The early
fall in protein synthesis, as measured by uptake of L-[4,5-
3H]leucine, was also observed in Bax
/
cultures (Fig. 5
B). However, Bax
/
cells did maintain a low basal level
of protein synthesis even after 48 h of K+ deprivation.
Therefore, BAX was not required for the early fall in metabolic parameters. Consistent with a decrease in metabolic parameters, granule cells from K+-deprived Bax
/
cultures, while alive, ceased to increase in diameter compared
to those maintained in 25 mM potassium which continued
to grow (Miller, T.M., personal observation). A similar relative decrease in soma diameter is seen in Bax
/
sympathetic neurons deprived of NGF (Deckwerth et al.,
1996
).
Fig. 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.
[View Larger Version of this Image (18K GIF file)]
), including
cerebellar granule cells (D'Mello et al., 1993
), is markedly
attenuated by inhibitors of macromolecular synthesis, implying that transcription and translation of new gene products are important during cell death in these cells. Though
most mRNA levels decline rapidly as cells die, we have
previously identified increases in the mRNA levels of several genes during programmed cell death in granule cells
and sympathetic neurons (Estus et al., 1994
; Freeman et al., 1994
; Miller and Johnson, 1996
). The increase in the
mRNA level of the AP-1 transcription factor c-Jun may be
particularly important for cell death because microinjection of a dominant negative c-jun construct (Ham et al.,
1995
) or neutralizing antibodies directed against c-Jun
(Estus et al., 1994
) significantly delays PCD of sympathetic neurons.
/
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.
Fig. 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.
[View Larger Versions of these Images (16 + 16 + 47K GIF file)]
). Examination of c-fos mRNA levels in K+-maintained granule
cells was uninformative since K+ depolarization itself
drives the expression of c-fos (Ghosh et al., 1994
). cyclin
D1, a cell cycle regulator that increases in dying sympathetic neurons (Freeman et al., 1994
), remained relatively constant in both wild-type and Bax-deficient animals (Fig.
6 A). Since this message level did not decrease in the wild-type cultures even after 120 h of K+ deprivation (data not
shown) when very few, if any, granule cells remained viable, we concluded that the majority of the cyclin D1 message level is in the few contaminating nonneuronal cells; therefore, we could not determine whether cyclin D1 increased in the neurons. Glial fibrillary acidic protein
(GFAP) is expressed in the astrocytes, which are the main
contaminating nonneuronal cell. GFAP, as expected, remained relatively constant as the neurons died. Bax
mRNA levels did not increase in wild-type granule cells undergoing apoptosis and, as expected, no Bax mRNA
was expressed in Bax
/
cells (Fig. 6 A).
) 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).
/
Cultures
; 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.
Fig. 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 of this Image (54K GIF file)]
; Schwartz and Milligan,
1996
). We examined whether caspases 2, 3, and 7 were activated after K+ deprivation. The activity of this subset of
proteases can be measured by monitoring the cleavage of
the fluorogenic substrate DEVD-AMC. In Bax +/+ and
Bax +/
cells, DEVD-selective caspase activity increased
to 18 times control levels by 8 h after K+ deprivation and
then decreased as the cells died over the next 64 h. In stark
contrast, granule cells from Bax
/
animals did not increase DEVD-specific caspase activity at any time after K+ deprivation (Fig. 8). These results indicate that BAX is
required for activation of this subset of caspases in response to removal of potassium.
Fig. 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 of this Image (18K GIF file)]
; Schwartz and Milligan,
1996
). For example, BAF or ZVAD-FMK, two inhibitors
of caspases, have dramatic effects on saving sympathetic
neurons from PCD induced by trophic factor deprivation
(Deshmukh et al., 1996
). We examined the effect of these
compounds on K+ deprivation-induced cell death in granule cells. Neither compound was toxic to granule cells. After 3 d exposure to either 100 µM ZVAD or 100 µM BAF
in K25+S medium, survival was 96 ± 1 and 103 ± 3%, respectively. In K5+S medium, while both BAF and ZVAD
were able to delay PCD in granule cells at 12 h, neither afforded significant protection at later time points (Fig. 9 A).
One possible explanation for the lack of protection was
that BAF and ZVAD did not function as caspase inhibitors in these cells, perhaps because they did not effectively
cross the plasma membrane. To rule out this possibility, we deprived cells of K+ in the presence of 100 µM BAF or
100 µM ZVAD and then measured caspase activity. Cells
treated with either compound showed no increase in
DEVD-AMC cleavage after K+ deprivation (Fig. 9 B),
demonstrating that BAF and ZVAD did indeed penetrate
granule cells and function as caspase inhibitors in these
cells.
Fig. 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 of this Image (22K GIF file)]
), one of the hallmarks of
apoptosis. The number of TUNEL-positive cells was determined in cells switched to K25+S, K5+S, or K5+S with
100 µM BAF (Fig. 10, d-f, respectively). To facilitate visualization of the total number of cells, the nuclei were
stained with bisbenzimide (Fig. 10, a-c). As expected, the
percent of TUNEL-positive cells increased markedly after
switching cells from K25+S to K5+S (Fig. 10, d and e)
from 14 to 84% (Fig. 10 g). Although BAF did not block
cell death (Fig. 9 A), BAF blocked the increase in TUNEL
positivity associated with depriving granule cells of potassium (Fig. 10, f and g).
Fig. 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 Versions of these Images (25 + 62K GIF file)]
; Xue and Horvitz, 1995
). Expression of p35 delays
PCD in NGF-deprived sympathetic neurons (Martinou et
al., 1995
) and a serum-deprived neuronal cell line (Rabizadeh et al., 1993
), and also blocks developmental cell death
in Caenorhabditis elegans (Sugimoto et al., 1994
; Xue and
Horvitz, 1995
) and Drosophila (Hay et al., 1994
). After 5 d
in vitro, we transfected pGreen Lantern-1 in combination
with constructs encoding p35, BCL-2, or the pBluescriptII
plasmid into cerebellar granule cells (see Materials and
Methods). At 7 d, the number of transfected cells was determined by counting pGreen Lantern-1-transfected (fluorescent) cells. Cultures were then switched to K5+S medium or maintained in K25+S medium. The number of
surviving transfected cells was determined after 24 and 48 h
and compared to the number of transfected cells before
switching medium (Fig. 11). Although expression of BCL-2
significantly retarded cell death in K5+S medium, expression of p35 had no effect on cell death (Fig. 11). These
data further suggest a caspase-independent cell death
pathway downstream of BAX.
Fig. 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.
[View Larger Version of this Image (20K GIF file)]
DISCUSSION
/
granule cells were not
protected from NMDA-induced cell death. Because Bax
/
granule cells did not die in response to K+ deprivation, we were able to use this model to define where BAX is acting in the pathway leading to apoptosis. We analyzed
several metabolic and genetic events associated with PCD
in granule cells and found that Bax deficiency did not affect these early events. On the other hand, we found that
BAX was required for the activation of caspases since Bax
+/+, but not Bax
/
cells cleaved a fluorogenic DEVD
substrate in response to K+ deprivation. Our data suggest
that while likely to be involved in cell death, caspases may
not be solely responsible for causing cell death in granule
cells since blocking these proteases with peptide inhibitors
delayed, but did not significantly block, the loss of cells.
These studies of Bax-knockout cells are consistent with
BAX or BAK induction studies in which cell death also
proceeded in the presence of caspase inhibitors (Xiang et
al., 1996
; McCarthy et al., 1997
). From these results and
previous studies, we are now able to define a sequence of
events associated with PCD in granule cells (Fig. 12).
Fig. 12.
Diagrammatic representation of the events associated
with programmed cell death in cerebellar granule cells.
[View Larger Version of this Image (12K GIF file)]
). MAP kinase is not likely to be regulating survival directly since inhibition of this pathway does not lead to
cell death in granule cells (Miller et al., 1997
) or sympathetic neurons (Creedon et al., 1996
; Virdee and Tolkovsky, 1996
). In contrast, inhibition of PI-3-K in granule cells
in the presence of IGF-I or K+ induces cell death that is indistinguishable from potassium deprivation, including a
similar time course of death, dependence on macromolecular synthesis, and characteristic morphology of apoptotic
nuclei (Miller et al., 1997
). Further support for the importance of the PI-3-K pathway in granule cells is the survival-promoting effect of overexpressing AKT (Dudek et al.,
1997
), a downstream target of PI-3-K (Bos, 1995
; Franke
et al., 1995
). The ability of Bax
/
granule cells to survive without PI-3-K activity and the fact that metabolic changes and changes in mRNA levels still occur in Bax
/
cells implies that BAX lies downstream of both.
/
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
).
/
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).
/
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.
; 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
).
; 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.
; 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.
) 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.
). 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.
/
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.
/
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.
Received for publication 21 March 1997 and in revised form 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.eduWe 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.
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.
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