* Eisai London Research Laboratories, Bernard Katz Building, University College London, London WC1E 6BT, United
Kingdom; Ontogeny Inc., Cambridge, Massachusetts 02138; and § SmithKline Beecham, New Frontiers Science Park, Harlow,
Essex CM19 5AW, United Kingdom
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Abstract |
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Cytochrome c has been shown to play a role in cell-free models of apoptosis. During NGF withdrawal-induced apoptosis of intact rat superior cervical ganglion (SCG) neurons, we observe the redistribution of cytochrome c from the mitochondria to the cytoplasm. This redistribution is not inhibited by the caspase inhibitor Z-Val-Ala-Asp-fluoromethylketone (ZVADfmk) but is blocked by either of the neuronal survival agents 8-(4-chlorophenylthio)adenosine 3':5'-cyclic monophosphate (CPT-cAMP) or cycloheximide. Moreover, microinjection of SCG neurons with antibody to cytochrome c blocks NGF withdrawal-induced apoptosis. However, microinjection of SCG neurons with cytochrome c does not alter the rate of apoptosis in either the presence or absence of NGF. These data suggest that cytochrome c is an intrinsic but not limiting component of the neuronal apoptotic pathway.
Key words: cytochrome c; mitochondria; neuron; apoptosis; cAMP ![]() |
Introduction |
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APOPTOSIS is a morphologically and biochemically distinct form of cell death, activated in multiple tissues during development and homeostasis (Ellis
et al., 1991; Jacobson et al., 1997
). It is thought to play an
important role in the development and shaping of the nervous system (Oppenheim, 1991
; Johnson and Deckwerth, 1993
; Burek and Oppenheim, 1996
). Many neurons are dependent upon neurotrophic factors, which stimulate survival pathways within the cell, and their removal can lead
to apoptosis (Raff et al., 1993
; Silos-Santiago et al., 1995
;
Segal and Greenberg, 1996
). In addition, there is accumulating evidence that apoptosis is involved in neurodegenerative diseases (Bredesen, 1995
; Thompson, 1995
; Johnson et al., 1996
), such as Parkinson's disease (Mochizuki et al., 1996
), Alzheimer's disease (Cotman and Anderson, 1995
), amylotrophic lateral sclerosis (Yoshiyama et
al., 1994
; Ghadge et al., 1997
), and stroke (Linnik et al.,
1993
; Choi, 1996
).
Many of the genes involved in apoptosis have first been
identified in the nematode Caenorhabditis elegans (Hengartner and Horvitz, 1994a). These include the prototype
gene for the caspase family of proteases, ced-3 (Yuan et al.,
1993
), ced-9, which is homologous to the bcl-2 gene family
(Hengartner and Horvitz, 1994b
), and most recently Apaf-1,
which is homologous to both ced-4 and ced-3 (Zou et al.,
1997
). Caspases are activated by cleavage after aspartate
residues, and it has been shown in vitro that they can be
activated by other active caspases (Fernandes-Alnemri
et al., 1996
; Liu et al., 1996a
; Srinivasula et al., 1996a
,b).
Caspases are now thought to form a proteolytic network
within the cell, resulting in the breakdown of key enzymes
and cellular structures (Nicholson and Thornberry, 1997
;
Salvesen and Dixit, 1997
). Their target proteins include
poly(ADP-ribose) polymerase (Nicholson et al., 1995
;
Tewari et al., 1995
), nuclear lamins (Lazebnik et al., 1995
;
Takahashi et al., 1996
), retinoblastoma protein (Janicke et
al., 1996
), DNA-dependent protein kinase (Song et al.,
1996
), and Bcl-2 family members (Cheng et al., 1997
; Clem
et al., 1998
). In addition, caspase 3 has been shown to
cause activation of DNase activity thought to be responsible for the chromatin degradation seen in apoptosis (Bortner et al., 1995
; Liu et al., 1997
; Enari et al., 1998
; Sakahira
et al., 1998
).
Bcl-2 family members encode proteins that either inhibit or accelerate apoptosis in response to a wide range of
death stimuli (Nunez and Clarke, 1994; Kroemer, 1997
).
The mechanism for this is unclear, but they are known to
form dimers, which, while located predominantly in the
outer mitochondrial membrane, are also found in the nuclear envelope and endoplasmic reticulum (Krajewski et al.,
1993
). They may function as channels to regulate permeability across these membranes (Antonsson et al., 1997
;
Minn et al., 1997
; Reed, 1997
; Schendel et al., 1997
). A further insight into the function of Bcl-2-like proteins has
come about after the surprising discovery that cytochrome
c plays a role in apoptosis. Cytochrome c, originally
termed Apaf-2,1 was isolated as one of the factors that mediated caspase activation when dATP was added to normal cell extracts (Liu et al., 1996b
). It has also been reported that cytochrome c partitioned into the cytoplasmic
fraction of cells undergoing apoptosis but partitioned into
the mitochondrial fraction of normal cells (Liu et al.,
1996b
; Kharbanda et al., 1997
; Kluck et al., 1997
). This apparent redistribution could be prevented by overexpression of bcl-2 (Kluck et al., 1997
; Yang et al., 1997
). Cytochrome c was found to require other cytoplasmic partners
to activate caspases. Recently, Apaf-1 (Zou et al., 1997
) and caspase 9 (Apaf-3) have been isolated and shown
to be sufficient, with cytochrome c and dATP, to activate
caspase 3 (P. Li et al., 1997
). These experiments suggest
that Bcl-2-like proteins may act by preventing the release
of cytochrome c from the mitochondria, but the possibility
that they have additional functions is not excluded (Murphy et al., 1996
; F. Li et al., 1997
; Reed, 1997
). From studies in C. elegans, we know that ced-4 lies downstream of
ced-9 but upstream of ced-3 (Shaham and Horvitz, 1996
).
In addition, CED-4 has been shown to directly interact
with CED-9, CED-3, and Bcl-2 (Chinnaiyan et al., 1997
;
Wu et al., 1997
; Huang et al., 1998
). Caspase 9 and Apaf-1
association has been demonstrated in vitro (P. Li et al.,
1997
), so by analogy with C. elegans, a bcl-2-like protein
may interact directly with Apaf-1. There have also been reports of cytochrome c interacting directly with Bcl-xL
(Kharbanda et al., 1997
).
To examine whether cytochrome c plays a role in neuronal cell death, we have first determined that after removal of NGF, cytochrome c redistributes from the mitochondria to the cytoplasm. This event was antagonized by two different neuroprotective agents, 8-(4-chlorophenylthio)adenosine 3':5'-cyclic monophosphate (CPT-cAMP) and cycloheximide, placing their action upstream of cytochrome c, but not by a caspase inhibitor. We have also demonstrated that an antibody to cytochrome c, when microinjected into superior cervical ganglion (SCG) neurons, was able to prevent apoptosis after NGF withdrawal. Finally, cytochrome c injected into neurons did not increase the amount of death either in the presence or absence of NGF, suggesting that redistribution of cytochrome c is not the only regulated step during neuronal cell death.
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Materials and Methods |
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Cell Culture and Apoptosis Induction
Rat SCG neurons were isolated and maintained as described previously
(Philpott et al., 1996). Cells were routinely maintained in 100 ng/ml NGF
(Promega Corp., Madison, WI). 5-7-d-old cells were deprived of NGF by
rinsing twice in media without NGF and incubating the cells with 100 ng/ml
anti-NGF antibody (GIBCO BRL; Life Technologies, Rockville, MD).
CPT-cAMP, cycloheximide, and actinomycin D were obtained from
Sigma Chemical Co. (St. Louis, MO). Z-Val-Ala-Asp-fluoromethylketone
(ZVADfmk) was obtained from Enzyme Systems Products (Dublin, CA).
Stock solutions of CPT-cAMP were in water, and the others were in
DMSO.
Jurkat cells were grown in DME (4.5 mg/ml glucose)/10% FCS and were cultured at 37°C in a 10% CO2 atmosphere.
Immunofluorescence
Cells were fixed with 3% paraformaldehyde in PBS for 15 min, blocked
with 10 mM glycine in PBS for 10 min, and then rinsed in PBS. The cells
were permeabilized in binding buffer (0.5% Triton X-100, 0.2% gelatine,
0.5% BSA, PBS) for 5 min before incubation in this solution with 20 µg/ml
of the 2G8.B6 anti-cytochrome c antibody (a kind gift from Dr. R. Jemmerson, University of Minnesota, Minneapolis, MN; Mueller and Jemmerson, 1996) for 1-2 h. After a 20-min wash in fresh binding buffer, the
cells were incubated in 1:100 FITC-conjugated anti-mouse antibody
(Jackson Laboratories, Bar Harbor, ME) for an additional 1 h. The cells
were finally washed in fresh binding buffer for up to 1 h and costained
with 1 µg/ml Hoechst 33342 in water before mounting in 0.25% n-propylgalate and 90% glycerol in PBS.
For labeling of functional mitochondria, cells were incubated in the presence of 450 nM Mitotracker (Molecular Probes, Eugene, OR) for 30- 40 min followed by a 30-60-min incubation in fresh medium and fixation as described above.
Fluorescence Assay of Caspase Activation
Jurkat cytosol preparation and caspase activation were according to a
modification of Liu et al. (1996b). Cells were harvested by centrifugation,
washed in ice-cold PBS, and incubated on ice for 15 min in a fivefold volume of buffer A (10 mM KCl, 20 mM Hepes, pH 7.4, 1.5 mM MgCl2, 1 mM DTT, 1 µg/ml each of pepstatin and leupeptin, 5 µg/ml antipain, 10 µg/ml chymostatin, and 100 µM PMSF). The cells were disrupted by 20 strokes of a glass/Teflon Dounce homogenizer, and the nuclei and debris
were removed by centrifugation for 15 min at 1,000 g, followed by 1 h at
100,000 g. Cytosol was frozen in aliquots in liquid N2. Caspase 3 activation reactions consisted of cytosol (50 µg protein) with 0.002 mg/ml cytochrome C (Sigma Chemical Co.), 0.25 mM dATP (Ultrapure; Pharmacia Biotech, Piscataway, NJ) made to a final volume of 20 µl with buffer A. Reactions were incubated for 1 h at 30°C. For fluorescence assay of active
caspase, 25 µg protein (in 10 µl) was mixed with 200 µl of 100 mM Hepes,
pH 7.4, 10% sucrose, 0.1% CHAPS, 10 µg/ml DEVD-AMC (Enzyme Systems), of which 100 µl was placed in duplicate wells of a 96-well plate. The
plates were incubated for 60 min at 37°C and measured in a fluorimeter
(model LS 50B; Perkin-Elmer Corp., Norwalk, CT).
Immunoblot Analysis
For immunoblot analysis, 25 µg protein from the activation reactions (for
caspase 3) or 8-16 × 103 cells (for cytochrome c/ERK1/2) were used. Samples were run under reducing conditions on a 15% SDS-polyacrylamide
gel and electroblotted onto nitrocellulose membrane. Membranes were
blocked in TBST (10 mM Tris-HCl, pH 8, 150 mM NaCl, 0.1% Tween 20)
supplemented with 1% Tween 20 and 5% dried low-fat milk for 20 min
and incubated in the same buffer with 250 ng/ml anti-caspase 3 mAb
(Transduction Laboratories, Lexington, KY) or 1:1,000 anti-cytochrome c
antibody, 7H8.2C12, ascites (a kind gift from Dr. R. Jemmerson; Jemmerson et al., 1991). This was followed by up to 1 h each with 1:1,000 dilution
of biotin anti-mouse IgG and HRP-streptavidin (Amersham Corp., Arlington Heights, IL). Filters were washed for up to 1 h with TBST between
each reagent and before development of signal by enhanced chemiluminescence (Amersham Corp.). For analysis of ERK1/2, blots were reprobed with mAb E16220 or M12320 (Affiniti Research Products, Nottingham, UK) at 1:500 and treated as above. Blots were quantified using a
scanner (model GS670; Bio-Rad Labs, Hercules, CA) and NIH Image
software.
Microinjection of Neurons
Neurons were microinjected with 0.5× PBS, 5 mg/ml 70-kD Texas red dextran (Molecular Probes), plus either bovine cytochrome c (Sigma Chemical Co.), microperoxidase (Sigma Chemical Co.), 2G8.B6, or 6H2.B4 (PharMingen, San Diego, CA) anti-cytochrome c antibody or mouse (Pierce and Warriner, Rockford, IL) IgG at 20 mg/ml. Cells were counted 2-4 h after injection (time zero) and again 48 or 72 h later. Cells were evaluated by microscopic assessment of several parameters. Initially, only cells that were stained with Texas red, indicating that these were injected cells and that they possessed an uncompromised plasma membrane, were counted. These cells were further assessed under phase illumination for normal nuclear morphology and phase-bright cell body. Cells that possessed all of these characteristics were counted as having survived. Apoptotic cells possessed a condensed and/or fragmented nucleus and phase dark, deformed, and shrunken cell body. Approximately 90-95% of injected cells survive the injection process itself.
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Results |
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Cytochrome c Redistributes from the Mitochondria to the Cytoplasm after NGF Withdrawal
Previous studies in numerous model systems have shown
that upon physical disruption and fractionation of cells, cytochrome c partitions with the mitochondria of normal
cells but with the cytosol of cells induced to undergo apoptosis (Liu et al., 1996b; Kharbanda et al., 1997
; Kluck et al.,
1997
). To determine if this differential partitioning indeed
represents a redistribution in intact cells, we have examined by immunofluorescence the cellular location of cytochrome c in SCG neurons. SCG neurons cultured in the
presence of NGF display a normal morphology with
plump nuclei (Fig. 1 A). Approximately 45% of these neurons cultured for 24 h in the absence of NGF had shrunken
cytoplasm (data not shown) and pyknotic nuclei (Fig. 1 B),
which is consistent with apoptosis. Immunofluorescent
staining showed that healthy cells had a bright, punctate
cytoplasmic cytochrome c distribution, clearly excluded
from the nuclear space and consistent with a mitochondrial location. However in cells with pyknotic nuclei, the
staining was diffuse and uniform throughout the cytoplasm and nuclear space, indicating release from the mitochondria. Of note was the small proportion of cells found
in both conditions that displayed a normal nuclear morphology but the diffuse cytochrome c pattern (Fig. 1, A
and B). This suggests that cytochrome c is released from
the mitochondria during NGF withdrawal-induced apoptosis before nuclear condensation.
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Cytochrome c Release from the Mitochondria Is Inhibited by the Neuroprotective Agents CPT-cAMP and Cycloheximide but Not by the Caspase Inhibitor ZVADfmk
We have previously shown that the cell-permeable caspase
inhibitor ZVADfmk can inhibit apoptosis in SCG neurons, as determined by nuclear morphology and plasma
membrane integrity (McCarthy et al., 1997). We therefore
examined the effect of ZVADfmk on cytochrome c redistribution. Addition of 100 µM ZVADfmk to the medium
at the time of NGF withdrawal had no effect on the number of cells in which cytochrome c was released from the
mitochondria (Fig. 1 C). The addition of ZVADfmk did,
however, inhibit nuclear pyknosis, indicating that it was effectively inhibiting cellular caspases.
There are a number of other agents, including cycloheximide and CPT-cAMP, known to provide protection
against NGF withdrawal-induced apoptosis in SCG neurons. Cycloheximide may act by inhibiting translation of
proteins necessary for apoptosis (Martin et al., 1988; Rydel
and Greene, 1988
; Edwards et al., 1991
; Buckmaster and
Tolkovsky, 1994
). Alternatively, the reduction in protein synthesis may result in additional cysteine being available
for the formation of the antioxidant glutathione (Ratan et al.,
1994
). cAMP might function via activation of protein-dependent kinase A (PKA) (Martin et al., 1988
, 1992
; Rydel and
Greene, 1988
; Edwards et al., 1991
; Buckmaster and Tolkovsky, 1994
). We therefore studied the cytochrome c distribution in SCG neurons deprived of NGF and treated
with these agents. Both cycloheximide and CPT-cAMP
prevented nuclear condensation and cytochrome c redistribution (Fig. 1, D and E). Similar experiments with 100 ng/ml actinomycin D, another neuronal survival agent
thought to act through its inhibition of transcription (Martin et al., 1988
), also resulted in blockade of NGF withdrawal-induced cytochrome c redistribution (data not shown). These results demonstrate that the redistribution
of cytochrome c is dependent upon transcription/translation and is regulated by systems that are influenced by
CPT-cAMP. In addition, they suggest that the activation
of caspases, leading to nuclear condensation, occurs downstream or parallel to cytochrome c redistribution.
Cytochrome c Is Degraded after Release from the Mitochondria
We observed in our immunofluorescence experiments that
there was some variation in the intensity of cytochrome c
stain in cells that had lost the normal punctate pattern. A
small number of bright, diffusely stained cells were always
observed, but the majority of the cells were fainter. While
the stain in all of these cells was uniformly spread throughout the cytoplasm and nucleus, there was a wide range of
intensity from cell to cell, with some appearing barely
above background. It seemed that this reduction of intensity must start soon after release of cytochrome c from the
mitochondria since the number of very bright cells was always small. To determine whether this was due to a conformational change or veiling of the epitope recognized by
the 2G8.B6, or more simply due to protein degradation,
we analyzed cell lysates by immunoblot. Cells were cultured in NGF or withdrawn from NGF in the presence or
absence of ZVADfmk for 24 h. The cells were harvested
and analyzed by immunoblotting using an alternative anti-
cytochrome c antibody, 7H8.2C12. We saw a clear reduction in the amount of cytochrome c present when NGF
was removed, either in the presence or absence of ZVADfmk (Fig. 2). After 24 h, there was only 24-27% of the cytochrome c remaining, while the levels of ERK1 and
ERK2 changed little as described by Deshmukh et al. (1996). This therefore suggests that in SCG neurons deprived of NGF, cytochrome c is initially released from the
mitochondria and is then rapidly degraded. Furthermore,
ZVADfmk was unable to prevent this decay, suggesting
that caspases are not involved in the degradation.
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Cytochrome c Release from the Mitochondria Occurs before Breakdown of Mitochondrial Inner Membrane Potential
We were interested to discover if the observed apoptotic
release of cytochrome c from the mitochondria occurred
in conjunction with or independently of a generalized disruption of the mitochondrial integrity. Mitotracker, a fluorescent dye that is accumulated by functional mitochondria, was used to stain SCG neurons that were costained
for cytochrome c and chromatin. Cells in the presence of
NGF with normal nuclei displayed bright, punctate, and
overlapping cytochrome c and Mitotracker staining patterns (Fig. 3 A). However, in the absence of NGF, cells
with condensed nuclei and diffuse cytochrome c stain frequently had bright Mitotracker staining that was clearly
still arranged in a punctate pattern excluded from the nucleus (Fig. 3 B, right-hand cell), indicating that cytochrome
c release from the mitochondria could occur without disruption of the inner membrane. Cells in which apoptosis
was well advanced (as indicated by low or complete loss of
cytochrome c and chromatin stain) did, however, show reduced and diffuse Mitotracker stain, suggesting that mitochondrial inner membrane potential (m) had decayed
(Fig. 3 B, left-hand cell). This terminal
m decay was apparently not inhibited by treatment with ZVADfmk in this
system (data not shown).
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An Anti-cytochrome c Antibody Blocks Caspase Activation in Extracts and Apoptosis in Neurons
The above data show a correlation between cytochrome c distribution and survival in intact neurons. However, we wanted to determine whether mitochondrial loss of cytochrome c was essential for neuronal apoptosis. We therefore used the 2G8.B6 anti-cytochrome c mAb in an in vitro system to ascertain whether it could prevent the activation of caspases. Normal Jurkat cytosol was incubated either in the absence of cytochrome c and dATP or in the presence of cytochrome c and dATP, with or without the anti-cytochrome c or control antibody. The extracts were then analyzed for activation of caspase 3 by immunoblot. This demonstrated that the activation of caspase 3 leading to the loss of the p32 precursor, induced by the addition of cytochrome c and dATP, was clearly inhibited by the anti- cytochrome c mAb but not by the control mouse IgG (Fig. 4 A). The extracts were further tested by a fluorescence assay based upon hydrolysis of DEVD-AMC, a substrate for caspase 3. The extracts incubated with no antibody or control antibody generated a significant amount of free AMC, whereas the extract incubated with the 2G8.B6 mAb did not (Fig. 4 B). These experiments therefore indicate that the 2G8.B6 mAb is able to prevent the cytochrome c-mediated activation of caspase 3 in vitro.
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The effect of this antibody was then examined in intact neurons. 2G8.B6 mAb or control mouse IgG was injected into the cytoplasm of SCG neurons, and the cells were withdrawn from NGF 2-4 h later. Cells that survived injection were counted at this time and 72 h later. On average, 86% of cells injected with 2G8.B6 mAb, but only 16% of the control IgG-injected cells, displayed a normal morphology after 72 h of NGF deprivation (Fig. 5). This indicates that blocking the action of cytochrome c is sufficient to halt neuronal apoptosis as defined by morphological criteria.
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We have recently obtained another anti-cytochrome c mAb, 6H2.B4, which also inhibits caspase 3 activation in extracts. Microinjection of this mAb into SCG neurons (three experiments) leads to survival of 65% (SEM 19%) of injected cells (control Ig injection: mean survival = 15%, SEM 5%).
Microinjection of Cytochrome c Does Not Kill SCG Neurons
Since the presence of cytochrome c in the cytoplasm did appear to play a crucial role in neuronal apoptosis, we wanted to determine whether microinjection of cytochrome c was in itself sufficient to induce apoptosis in neurons. We therefore microinjected SCG neurons with a wide range of cytochrome c concentrations and maintained them in the presence of NGF for 48 h. By comparison with known amounts of cytochrome c on Western blots, we estimate that SCG neurons contain between 100- 500 fg of cytochrome c per cell (data not shown). The volume of an SCG neuron is ~10-20 pl, and we injected a maximum of 1/10th volume into each cell. From these estimates, we concluded that to introduce a single cell equivalent of cytochrome c, we should inject a solution in the range of 70 µg/ml. The range of concentrations of cytochrome c injected cover this value and several log10 concentrations higher and lower than this.
Measuring cell survival as described above, we detected no significant increase in death at any cytochrome c concentration (Fig. 6 A). The small fall in viability at the greatest cytochrome c concentrations was also seen with similar molar concentrations of microperoxidase, a control heme containing fragment of cytochrome c (Fig. 6 B). Thus, cytochrome c alone is not able to induce apoptosis in these cells.
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If the cytoplasmic presence of cytochrome c were a limiting factor in neuronal apoptosis, then we might expect its microinjection to enhance the rate of death in SCG neurons deprived of NGF. We therefore repeated the above experiment but withdrew the cells from NGF for 48 h after microinjection (Fig. 6 C). Again, no clear enhancement of death was detected under these conditions, suggesting that cytoplasmic cytochrome c is not a rate-limiting factor in neuronal apoptosis.
Microinjection of Cytochrome c with dATP Does Not Kill SCG Neurons
In cell-free apoptotic cell extract systems, dATP significantly increased the rate of cytochrome c-induced caspase
activation (Liu et al., 1996b). We therefore examined
whether dATP was a limiting factor in neuronal apoptosis
induced by cytochrome c. We chose a concentration of cytochrome c, which we estimated was between 1-10× the
cytochrome c cell content, and coinjected dATP in the
range 100 µM-10 mM (in the needle). This would give an approximate dATP concentration of 10 µM-1 mM within
the cell (assuming that 10% of the cell volume was injected), which is in a similar range to that used in in vitro
systems. At the lower concentrations of dATP, no apoptotic effect could be seen (Fig. 7). However, when 10 mM
dATP was used, the cells showed a small decrease in viability in the presence or absence of coinjected cytochrome c. No further decrease in viability was detected when
higher concentrations of dATP were used (data not
shown). Hence, we conclude that dATP, alone or in conjunction with additional cytochrome c, does not induce
apoptosis in SCG neurons but may itself have some effect on survival (Wakade et al., 1995
).
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Discussion |
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We have examined the role of cytochrome c during apoptosis in a model of physiological neuronal cell death, NGF
withdrawal-induced death of SCG neurons (Martin et al.,
1988; Edwards et al., 1991
). Initially, we studied the location of cytochrome c during apoptosis by fluorescence microscopy. We observed that in healthy neurons, cytochrome c was found in a punctate pattern, in keeping with its normal mitochondrial location, but that in neurons with
pyknotic nuclei it had assumed a diffuse distribution, implying release from the mitochondria. This first observation is in agreement with the findings of groups who have
compared the partition of cytochrome c between the cytosol and mitochondria of normal and apoptotic populations
of cells using disruption and fractionation techniques (Liu
et al., 1996b
; Ellerby et al., 1997
; Kharbanda et al., 1997
;
Kim et al., 1997
; Kluck et al., 1997
; Yang et al., 1997
) and
more recent observations in intact nonneuronal cells (Bossy-Wetzel et al., 1998
).
After NGF withdrawal, we consistently observed a small
number of cells, which although displaying normal nuclear
morphology, had a diffuse cytochrome c staining pattern.
This would suggest that the process of nuclear condensation occurred after the release of cytochrome c from the
mitochondria. The number of cells with this intermediate
morphology was greatly increased by treatment with the
broad range caspase inhibitor ZVADfmk. Hence caspase activation, thought to be necessary to induce pyknosis (Liu
et al., 1997), is downstream or independent of cytochrome
c release from the mitochondria. These data are also in
keeping with groups who have reported that caspase inhibitors do not affect the partition of cytochrome c between the cytosolic and mitochondrial cellular fractions
during apoptosis (Kharbanda et al., 1997
; Kluck et al., 1997
; Bossy-Wetzel et al., 1998
). We found that many neurons deprived of NGF showed weak cytochrome c staining
and that the level of staining was not maintained by the
addition of ZVADfmk. This, together with our immunoblotting data, suggests that cytochrome c is degraded after
release from the mitochondria and that this is a caspase-independent proteolysis. A previous report has shown that
anti-Fas induction of apoptosis in Jurkat cells involves a
down regulation of cytochrome c oxidase activity indirectly mediated by the inactivation of cytochrome c (Krippner et al., 1996
). They detected no degradation of cytochrome c during this process. The cells in their study were
treated for 2 h, so this difference, compared with our results, may be simply due to cytochrome c degradation only
being detectable over a longer period. However, in their
model ZVADfmk inhibited both the nuclear condensation and the cytochrome oxidase inactivation, suggesting an entirely different sequence of events from that in SCG neurons undergoing NGF withdrawal.
The neuroprotective agents cycloheximide and CPT-cAMP were also examined for their effect on cytochrome
c redistribution. Both agents prevented cytochrome c release from the mitochondria, suggesting they act upstream
of cytochrome c. The points of action of these compounds
are thought to be distinct (Edwards et al., 1991); NGF and
cAMP can rescue SCG neurons from NGF withdrawal at a
later time than cycloheximide. This suggests that while
cells can be rescued by preventing the production of proteins required for apoptosis (Martin et al., 1988
, 1992
; Edwards et al., 1991
), there is a period in which cells can
be rescued by posttranslational mechanisms by NGF or
cAMP (Edwards et al., 1991
; Deckwerth and Johnson,
1993
). How might these agents be inhibiting cytochrome c
release? One possible explanation is that NGF and cAMP
addition may result in the phosphorylation of Bad, a
proapoptotic member of the Bcl-2 family (Yang et al.,
1995
). Phosphorylated Bad is unable to displace Bax from
Bax:BclxL heterodimers, which would result in the formation of Bax homodimers and apoptosis (Yang et al., 1995
;
Zha et al., 1996
). One kinase that phosphorylates Bad in
cerebellar granule neurons is Akt kinase (Datta et al., 1997
). We, and others, have previously demonstrated that
Akt kinase can inhibit apoptosis in primary neurons
(Dudek et al., 1997
; Philpott et al., 1997
), and Zha and colleagues (1996) demonstrated that a form of PKA could
phosphorylate Bad in vitro. In addition, in cells microinjected with Akt kinase and withdrawn from NGF, we find
that the cytochrome c distribution is normal in surviving cells (data not shown), indicating that Akt kinase is upstream of cytochrome c. Thus, we can speculate a survival
pathway activated by the NGF receptor, stimulating phosphatidylinositol 3-kinase and Akt kinase, leading to the
phosphorylation of Bad, which in turn antagonizes the formation of an apoptotic Bcl-2 family member complex in
the mitochondrial membrane (Fig. 8). How such a complex would lead to cytochrome c release is not clear, but
Bcl-2-like proteins have a structure resembling pore-forming proteins and could function in this manner (Reed,
1997
).
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Additional sites of action of cAMP could be put forward. cAMP is known to modulate gene expression via the
phosphorylation of transcription factors by PKA (Lalli
and Sassone-Corsi, 1994) and may be acting at the transcriptional level. Indeed modulation of transcription factors, such as c-Jun, can prevent apoptosis in SCG neurons
(Ham et al., 1995
). Cycloheximide could act at multiple
sites by inhibiting the translation of many proteins, including proapoptotic members of the bcl-2 family and proteins necessary for the activation of caspase 9 (Fig. 8). We are
currently examining some of these issues.
Having demonstrated that cytochrome c was indeed released from SCG neurons after NGF withdrawal, we
wished to determine whether this release was associated
with a loss of m. This has been reported to be the cause
of cytochrome c release (Susin et al., 1996
, 1997
) and conversely to be the result of caspase activation (Bossy-Wetzel et al., 1998
). Thus, for some models of apoptotic cell
death it is unclear if
m loss is crucial to cytochrome c release from the mitochondria. However, in the case of
NGF-deprived sympathetic neurons, it was clear that complete cytochrome c dispersal and nuclear pyknosis preceded loss of
m.
In vitro experiments by several groups have shown that
addition of cytochrome c to normal cytosolic extracts
causes activation of endogenous caspase 3 (Liu et al.,
1996b; Ellerby et al., 1997
; Kharbanda et al., 1997
; Kluck
et al., 1997
; Neame, S.J., unpublished data). Our mAb microinjection results show that cytochrome c activity is important for regulating neuronal apoptosis. This demonstrates for the first time that cytochrome c is an essential
component of NGF withdrawal-induced apoptosis of sympathetic neurons. Since the anti-cytochrome c mAbs were
injected into the cell cytoplasm, we assume that the inhibition of cytochrome c occurs post efflux from the mitochondria. We infer that the cytochrome c mode of action is by
activation of a caspase in a similar manner to that previously described in vitro (Liu et al., 1996b
; P. Li et al., 1997
;
Zou et al., 1997
), involving a CED-4 homologous protein and a caspase with a CED-3 homologous prodomain. We
are presently investigating the identity of the protein partners of cytochrome c in SCG neurons.
While in several reports the optimal in vitro activation
of caspase 3 was achieved upon the addition of both cytochrome c and dATP, some sources have found that cytochrome c alone is sufficient (Ellerby et al., 1997; Kluck et al.,
1997
; Hampton et al., 1998
; Neame, S.J., unpublished
data). This suggests that all the necessary partners of cytochrome c are already present in the cells from which the
cytoplasmic extracts are made. If this was true in SCG neurons, microinjection of cytochrome c would induce
apoptosis, as has been found in human kidney 293 cells (F. Li et al., 1997
) and NRK cells (Zhivotovsky et al., 1998
).
To ensure that the cytochrome c we were using was, although clearly active in extracts (Fig. 4), not defective in
intact cells, we injected Rat-1 cells with cytochrome c at
5 mg/ml. We found that >90% of injected cells displayed
apoptotic morphology within 2 h of injection, while microperoxidase-injected cells appeared normal. Not withstanding the efficacy of the cytochrome c, we observed no
apoptosis in SCG neurons injected with a wide range of
cytochrome c concentrations, suggesting some other limiting factor. Coinjection of dATP with cytochrome c did not
induce apoptosis if introduced at below 1 mM. While there
was a small induction of death at 10 mM dATP (with or
without cytochrome c), this was not the rapid death seen in other cell types upon injection of cytochrome c (F. Li et
al., 1997
; Zhivotovsky et al., 1998
; data not shown) and so
may be caused by nonapoptotic processes. Estimates of
normal cellular dATP concentration suggest a range of
5-10 µM (Hunting and Henderson, 1981
; Sherman and
Fyfe, 1989
), implying that we are supplying sufficient
dATP. Wakade and colleagues (1995) described a 40-fold increase in dATP concentration upon induction of apoptosis in chick SCG neurons. However, this death was induced by treatment with 2-deoxyadenosine, a precursor of
dATP, so the increase in its concentration may not be
physiologically relevant to apoptosis. Certainly, dATP levels are not increased in all cases of apoptosis and actually
fall during apoptosis induced by IL-3 withdrawal from
BAF3 cells (Oliver et al., 1996
). The primary physiological enzyme responsible for dATP synthesis is ribonucleotide
reductase, an enzyme that has a free radical at its active
site, obligatory for function. At low O2 concentrations, this
radical is abolished (Brischwein et al., 1997
), yet cells can
undergo apoptosis under similar conditions (Jacobson and
Raff, 1995
), implying that increases in dATP are not intrinsic to apoptosis.
That we see no induction of apoptosis upon cytochrome
c injection suggests that some partner to cytochrome c in
the apoptotic process may be regulated. Clearly, the recently identified Apaf-1 (Zou et al., 1997) and Apaf-3
(caspase 9; P. Li et al., 1997
) are possible candidates for
this regulation. While we have demonstrated that cycloheximide prevents cytochrome c release, perhaps it also functions to inhibit the translation of these proteins; however, the regulation could equally involve other mechanisms. We find that the accumulation of cytochrome c in
the cytoplasm does not appear to be the rate-limiting step
in induction of nuclear pyknosis. This implies that the release of cytochrome c, which appears rapid since we very
rarely see cells displaying intermediate punctate/diffuse cytochrome c distribution, is differentially regulated compared to its apoptotic partners. It is also possible that the
immediate apoptotic partners of cytochrome c may be unregulated, while the downstream mediators are. In this
context, it is interesting that F. Li et al. (1997)
report a parental MCF7 cell line that was not induced to undergo
apoptosis upon injection of cytochrome c, while a sibling
cell line in which pro-caspase 3 was expressed, could be. In
some neuronal cells, caspase 3 mRNA has been shown to
increase in level after an apoptotic insult (Miller et al., 1997
). However, while caspase 3 is present in SCG neurons at detectable levels by Western blotting (Deshmukh
et al., 1996
; McCarthy et al., 1997
), it does not appear to
increase in level after NGF withdrawal (Deshmukh et al.,
1996
; data not shown). Thus, it is unclear to date which
component is limiting apoptosis in SCG neurons and
whether this can vary with cell type.
In conclusion, we have shown that the release of cytochrome c from the mitochondria is of crucial importance
in the NGF withdrawal-induced apoptosis of sympathetic
neurons. We have also shown that cytochrome c release is
independent of loss of m and so must be regulated by
other means. These in turn are dependent upon transcription/translation and can be modulated by Akt kinase or a
cAMP-mediated kinase. We have also implied that the
partners in cytochrome c activation of caspases are regulated, since the introduction of cytochrome c into the cytoplasm is not in itself sufficient in SCG neurons to activate
or accelerate cell death.
![]() |
Footnotes |
---|
Received for publication 16 April 1998 and in revised form 10 July 1998.
Address all correspondence to Stephen J. Neame, Eisai London Research
Laboratories, Bernard Katz Building, University College London, Gower
Street, London WC1E 6BT, UK. Tel.: 0171 413 1130. Fax: 0171 413 1121. E-mail: sjneame{at}elrl.co.uk
We would like to thank Dr. C. Gatchalian and Dr. J. Taylor for critical reading of this manuscript, Ms. K. Ferguson for editorial assistance, and Dr. R. Jemmerson for generously supplying anti-cytochrome c antibodies.
![]() |
Abbreviations used in this paper |
---|
Apaf, apoptotic protease activating
factor;
CPT-cAMP, 8-(4-chlorophenylthio)adenosine 3':5'-cyclic monophosphate;
DEVD-AMC, Asp-Glu-Val-Asp-amino methyl coumarin;
PKA, protein-dependent kinase A;
SCG, superior cervical ganglia;
ZVADfmk, Z-Val-Ala-Asp-fluoromethylketone;
m, mitochondrial inner membrane potential.
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