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The Rockefeller University Press, 0021-9525/98/09/1583/11 $2.00
The Journal of Cell Biology, Volume 142, Number 6, September 21, 1998 1583–1593
http://www.jcb.org 1583
Blocking Cytochrome c Activity within
Intact Neurons Inhibits Apoptosis
Stephen J. Neame,* Lee L. Rubin,
‡
and Karen L. Philpott
§
*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
Abstract.
Cytochrome c has been shown to play a role
in cell-free models of apoptosis. During NGF with-
drawal–induced apoptosis of intact rat superior cervical
ganglion (SCG) neurons, we observe the redistribution
of cytochrome c from the mitochondria to the cyto-
plasm. 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
9
:5
9
-
cyclic monophosphate (CPT-cAMP) or cycloheximide.
Moreover, microinjection of SCG neurons with anti-
body 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
A
poptosis
is a morphologically and biochemically dis-
tinct form of cell death, activated in multiple tis-
sues 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 ner-
vous system (Oppenheim, 1991; Johnson and Deckwerth,
1993; Burek and Oppenheim, 1996). Many neurons are de-
pendent upon neurotrophic factors, which stimulate sur-
vival 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 accumu-
lating evidence that apoptosis is involved in neurodegen-
erative diseases (Bredesen, 1995; Thompson, 1995; John-
son et al., 1996), such as Parkinson’s disease (Mochizuki
et al., 1996), Alzheimer’s disease (Cotman and Ander-
son, 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
(Hen-
gartner and Horvitz, 1994
a
). 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, 1994
b
), 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., 1996
a
; Srinivasula et al., 1996
a
,
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 responsi-
ble for the chromatin degradation seen in apoptosis (Bort-
ner et al., 1995; Liu et al., 1997; Enari et al., 1998; Sakahira
et al., 1998).
Bcl-2 family members encode proteins that either in-
hibit 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 nu-
clear envelope and endoplasmic reticulum (Krajewski et al.,
1993). They may function as channels to regulate perme-
ability across these membranes (Antonsson et al., 1997;
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@elrl.co.uk
This document was created with FrameMaker 4.0.4
The Journal of Cell Biology, Volume 142, 1998 1584
Minn et al., 1997; Reed, 1997; Schendel et al., 1997). A fur-
ther 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 me-
diated caspase activation when dATP was added to nor-
mal cell extracts (Liu et al., 1996
b
). It has also been re-
ported that cytochrome c partitioned into the cytoplasmic
fraction of cells undergoing apoptosis but partitioned into
the mitochondrial fraction of normal cells (Liu et al.,
1996
b
; Kharbanda et al., 1997; Kluck et al., 1997). This ap-
parent redistribution could be prevented by overexpres-
sion of bcl-2 (Kluck et al., 1997; Yang et al., 1997). Cyto-
chrome 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 (Mur-
phy et al., 1996; F. Li et al., 1997; Reed, 1997). From stud-
ies 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 re-
ports of cytochrome c interacting directly with Bcl-x
L
(Kharbanda et al., 1997).
To examine whether cytochrome c plays a role in neu-
ronal cell death, we have first determined that after re-
moval of NGF, cytochrome c redistributes from the mito-
chondria to the cytoplasm. This event was antagonized by
two different neuroprotective agents, 8-(4-chlorophenyl-
thio)adenosine 3
9
:5
9
-cyclic monophosphate (CPT-cAMP)
and cycloheximide, placing their action upstream of cyto-
chrome c, but not by a caspase inhibitor. We have also
demonstrated that an antibody to cytochrome c, when mi-
croinjected into superior cervical ganglion (SCG) neurons,
was able to prevent apoptosis after NGF withdrawal. Fi-
nally, 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.
Materials and Methods
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
8
C in a 10% CO
2
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
m
g/ml
of the 2G8.B6 anti–cytochrome c antibody (a kind gift from Dr. R. Jem-
merson, University of Minnesota, Minneapolis, MN; Mueller and Jem-
merson, 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
m
g/ml Hoechst 33342 in water before mounting in 0.25%
n-
propyl-
galate 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. (1996
b
). Cells were harvested by centrifugation,
washed in ice-cold PBS, and incubated on ice for 15 min in a fivefold vol-
ume of buffer A (10 mM KCl, 20 mM Hepes, pH 7.4, 1.5 mM MgCl
2
, 1
mM DTT, 1
m
g/ml each of pepstatin and leupeptin, 5
m
g/ml antipain, 10
m
g/ml chymostatin, and 100
m
M 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 N
2
. Caspase 3 activation
reactions consisted of cytosol (50
m
g protein) with 0.002 mg/ml cyto-
chrome C (Sigma Chemical Co.), 0.25 mM dATP (Ultrapure; Pharmacia
Biotech, Piscataway, NJ) made to a final volume of 20
m
l with buffer A.
Reactions were incubated for 1 h at 30
8
C. For fluorescence assay of active
caspase, 25
m
g protein (in 10
m
l) was mixed with 200
m
l of 100 mM Hepes,
pH 7.4, 10% sucrose, 0.1% CHAPS, 10
m
g/ml DEVD-AMC (Enzyme Sys-
tems), of which 100
m
l was placed in duplicate wells of a 96-well plate. The
plates were incubated for 60 min at 37
8
C and measured in a fluorimeter
(model LS 50B; Perkin-Elmer Corp., Norwalk, CT).
Immunoblot Analysis
For immunoblot analysis, 25
m
g protein from the activation reactions (for
caspase 3) or 8–16
3
10
3
cells (for cytochrome c/ERK1/2) were used. Sam-
ples 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; Jemmer-
son 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., Ar-
lington Heights, IL). Filters were washed for up to 1 h with TBST between
each reagent and before development of signal by enhanced chemilumi-
nescence (Amersham Corp.). For analysis of ERK1/2, blots were re-
probed with mAb E16220 or M12320 (Affiniti Research Products, Not-
tingham, 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
3
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
1.
Abbreviations used in this paper
: Apaf, apoptotic protease activating
factor; CPT-cAMP, 8-(4-chlorophenylthio)adenosine 3
9
:5
9
-cyclic mono-
phosphate; DEVD-AMC, Asp-Glu-Val-Asp-amino methyl coumarin;
PKA, protein-dependent kinase A; SCG, superior cervical ganglia;
ZVADfmk, Z-Val-Ala-Asp-fluoromethylketone;
DC
m
, mitochondrial in-
ner membrane potential.
Neame et al.
Cytochrome c and Neuronal Apoptosis
1585
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 in-
jected 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 pos-
sessed all of these characteristics were counted as having survived. Apop-
totic cells possessed a condensed and/or fragmented nucleus and phase
dark, deformed, and shrunken cell body. Approximately 90–95% of in-
jected cells survive the injection process itself.
Results
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, cy-
tochrome c partitions with the mitochondria of normal
cells but with the cytosol of cells induced to undergo apop-
Figure 1. Cytochrome c redistrib-
utes from the mitochondria to the
cytoplasm before nuclear conden-
sation. SCG neurons were cul-
tured on coverslips for 24 h in me-
dium with NGF (A), or medium
lacking NGF supplemented with
carrier alone (B), 100 mM ZVAD-
fmk (C), 1 mg/ml cycloheximide
(D), or 100 mM CPT-cAMP (E).
The cells were then fixed and
stained for cytochrome c (FITC)
and chromatin (Hoechst). Repre-
sentative examples were imaged
using a Xillix Microimager (Im-
provision-Image Processing and
Vision Co. Ltd., Coventry, UK).
Coverslips were mixed in a blind
manner, and 10 random fields
were counted per condition. Cells
were evaluated as being either
normal in cytochrome c distri-
bution and nuclear morphology
(hatched bars), diffuse in cyto-
chrome c distribution and normal
in nuclear morphology (shaded
bars), or diffuse in cytochrome c
and having pyknotic nuclei (open
bars). The graphs show mean per-
centage of cells in each category
and are the result of between four
to seven experiments for each
condition. The error bars repre-
sent SEM. Bar, 10 mm.
The Journal of Cell Biology, Volume 142, 1998 1586
tosis (Liu et al., 1996
b
; Kharbanda et al., 1997; Kluck et al.,
1997). To determine if this differential partitioning indeed
represents a redistribution in intact cells, we have exam-
ined by immunofluorescence the cellular location of cyto-
chrome 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 neu-
rons 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 mitochon-
drial location. However in cells with pyknotic nuclei, the
staining was diffuse and uniform throughout the cyto-
plasm and nuclear space, indicating release from the mito-
chondria. Of note was the small proportion of cells found
in both conditions that displayed a normal nuclear mor-
phology 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 apop-
tosis before nuclear condensation.
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 neu-
rons, as determined by nuclear morphology and plasma
membrane integrity (McCarthy et al., 1997). We therefore
examined the effect of ZVADfmk on cytochrome c redis-
tribution. Addition of 100
m
M ZVADfmk to the medium
at the time of NGF withdrawal had no effect on the num-
ber 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 ef-
fectively inhibiting cellular caspases.
There are a number of other agents, including cyclohex-
imide and CPT-cAMP, known to provide protection
against NGF withdrawal–induced apoptosis in SCG neu-
rons. 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-depen-
dent kinase A (PKA) (Martin et al., 1988, 1992; Rydel and
Greene, 1988; Edwards et al., 1991; Buckmaster and Tol-
kovsky, 1994). We therefore studied the cytochrome c dis-
tribution in SCG neurons deprived of NGF and treated
with these agents. Both cycloheximide and CPT-cAMP
prevented nuclear condensation and cytochrome c redis-
tribution (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 (Mar-
tin et al., 1988), also resulted in blockade of NGF with-
drawal–induced cytochrome c redistribution (data not
shown). These results demonstrate that the redistribution
of cytochrome c is dependent upon transcription/transla-
tion 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 down-
stream 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 through-
out 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 inten-
sity must start soon after release of cytochrome c from the
mitochondria since the number of very bright cells was al-
ways small. To determine whether this was due to a con-
formational 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 cul-
tured 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 reduc-
tion in the amount of cytochrome c present when NGF
was removed, either in the presence or absence of ZVAD-
fmk (Fig. 2). After 24 h, there was only 24–27% of the cy-
tochrome c remaining, while the levels of ERK1 and
Figure 2. Cytochrome c de-
grades after release from the
mitochondria. (A) SCG cul-
tures were maintained in
NGF (1N) or withdrawn
from NGF in the absence
(2N) or presence (1Z) of
100 mM ZVADfmk. Cells
were lysed after 24 h, and
protein from an equivalent
number of cells was analyzed
with an anti–cytochrome c
antibody, 7H8.2C12. (B)
Three representative blots
were scanned on a densito-
meter, and the bars show the
mean 1 SEM of the values
obtained for cells maintained
in NGF (1N) or withdrawn
from NGF without (2N) or
with (1Z) ZVADfmk. (C)
These blots were later reana-
lyzed by immunoblot for
ERK1/2, and the results are
expressed as for the cyto-
chrome c blots in B.
Neame et al.
Cytochrome c and Neuronal Apoptosis
1587
ERK2 changed little as described by Deshmukh et al.
(1996). This therefore suggests that in SCG neurons de-
prived 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.
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 dis-
ruption of the mitochondrial integrity. Mitotracker, a fluo-
rescent dye that is accumulated by functional mitochon-
dria, 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 pat-
terns (Fig. 3
A
). However, in the absence of NGF, cells
with condensed nuclei and diffuse cytochrome c stain fre-
quently had bright Mitotracker staining that was clearly
still arranged in a punctate pattern excluded from the nu-
cleus (Fig. 3
B
,
right-hand cell
), indicating that cytochrome
c release from the mitochondria could occur without dis-
ruption 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 re-
duced and diffuse Mitotracker stain, suggesting that mito-
chondrial inner membrane potential (
DC
m
) had decayed
(Fig. 3
B
,
left-hand cell
). This terminal
DC
m
decay was ap-
parently not inhibited by treatment with ZVADfmk in this
system (data not shown).
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 cyto-
chrome c was essential for neuronal apoptosis. We there-
fore used the 2G8.B6 anti–cytochrome c mAb in an in
vitro system to ascertain whether it could prevent the acti-
vation 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–
Figure 3. Redistribution of
cytochrome c precedes the
loss of mitochondrial inner
membrane potential. SCG
neurons were cultured on
coverslips for 23 h in medium
with NGF (A) or medium
lacking NGF (B). Mito-
tracker was added to the me-
dium at 450 nM and incu-
bated for 30 min, and the
medium was changed. The
cells were incubated for a fur-
ther 30 min and then fixed
and stained as in Fig. 1. Rep-
resentative cells are shown
displaying Mitotracker (MT),
cytochrome c (FITC), and
chromatin (Hoechst) localiza-
tion. Bar, 10 mm.
Figure 4. Anti–cytochrome c
mAb 2G8.B6 inhibits the acti-
vation of caspase 3 in a cell-
free assay. 50 mg Jurkat cy-
tosol was incubated for 1 h
without cytochrome c and
dATP (lane 1) or with cyto-
chrome c and dATP and with
no additional components
(lane 2), with 2 mg 2G8.B6
anti–cytochrome c mAb (lane
3), or with 2 mg mouse Ig
(lane 4). The reaction mix
was split into two and ana-
lyzed by (A) PAGE and im-
munoblot for the proteolytic
processing of caspase 3. p32
indicates the pro-caspase 3
band and the asterisk indi-
cates the added antibody
bands. (B) DEVD-AMC cleavage, for generation of active
caspase. Results are expressed as the percentage fluorescence
generated over an additional 1-h incubation and are the average
of three to six experiments. The error bars represent SEM.
The Journal of Cell Biology, Volume 142, 1998 1588
cytochrome c mAb but not by the control mouse IgG (Fig.
4 A). The extracts were further tested by a fluorescence as-
say based upon hydrolysis of DEVD-AMC, a substrate for
caspase 3. The extracts incubated with no antibody or con-
trol 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–medi-
ated activation of caspase 3 in vitro.
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 injec-
tion 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 mor-
phology after 72 h of NGF deprivation (Fig. 5). This indi-
cates that blocking the action of cytochrome c is sufficient
to halt neuronal apoptosis as defined by morphological
criteria.
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 5
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 cyto-
chrome c was in itself sufficient to induce apoptosis in neu-
rons. We therefore microinjected SCG neurons with a
wide range of cytochrome c concentrations and main-
tained them in the presence of NGF for 48 h. By compari-
son 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 vol-
ume of an SCG neuron is z10–20 pl, and we injected a
maximum of 1/10th volume into each cell. From these esti-
mates, we concluded that to introduce a single cell equiva-
lent of cytochrome c, we should inject a solution in the
range of 70 mg/ml. The range of concentrations of cyto-
chrome c injected cover this value and several log10 con-
centrations higher and lower than this.
Measuring cell survival as described above, we detected
no significant increase in death at any cytochrome c con-
centration (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.
If the cytoplasmic presence of cytochrome c were a lim-
iting factor in neuronal apoptosis, then we might expect its
microinjection to enhance the rate of death in SCG neu-
rons 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.
Figure 5. Microinjection of anti–cyto-
chrome c mAb 2G8.B6 inhibits SCG
apoptosis. SCG neurons were microin-
jected with either 20 mg/ml 2G8.B6
mAb (lane 1) or an equivalent concen-
tration of mouse Ig (lane 2). After 2–4 h,
the injected cells were counted, and the
cultures were withdrawn from NGF. At
48 h, the number of surviving injected
cells were counted and expressed as a
percentage of the cells initially surviv-
ing injection. 150–200 cells were in-
jected per coverslip, and the results shown are the average of five
experiments. The error bars represent SEM.
Figure 6. Microinjection of cytochrome c does not induce or ac-
celerate apoptosis in SCG neurons. SCG neurons were microin-
jected with cytochrome c and counted 2–4 h later. (A) Cells were
maintained in NGF for 48 h before counting the surviving cells.
The amount of cytochrome c injected is shown as log10 multiples
of 1 cell equivalent (70 mg/ml in needle), except for lane TR,
which contained no cytochrome c, and lane Cc, in which 17.5 mg/ml
of cytochrome c was used. (B) Cells were maintained in NGF for
72 h before counting surviving cells. The microinjection mix in
lane TR contained no cytochrome c, 1.45 mM cytochrome c (17.5
mg/ml) in lane Cc, and 1.45 mM microperoxidase in lane Mp. (C)
Cells were withdrawn from NGF for 48 h before counting the sur-
viving cells. The amount of cytochrome c injected is shown as
log10 multiples of 1 cell equivalent (70 mg/ml in needle), except
for lane TR, which contained no cytochrome c, and lane Cc, in
which 17.5 mg/ml of cytochrome c was used. The results are ex-
pressed as a percentage of the cells initially surviving injection.
150–200 cells were injected per coverslip, and the results shown
are the average of three to four experiments. The error bars rep-
resent SEM.
Neame et al. Cytochrome c and Neuronal Apoptosis 1589
Microinjection of Cytochrome c with dATP Does Not
Kill SCG Neurons
In cell-free apoptotic cell extract systems, dATP signifi-
cantly 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 cy-
tochrome c, which we estimated was between 1–103 the
cytochrome c cell content, and coinjected dATP in the
range 100 mM–10 mM (in the needle). This would give an
approximate dATP concentration of 10 mM–1 mM within
the cell (assuming that 10% of the cell volume was in-
jected), which is in a similar range to that used in in vitro
systems. At the lower concentrations of dATP, no apop-
totic effect could be seen (Fig. 7). However, when 10 mM
dATP was used, the cells showed a small decrease in via-
bility 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 con-
junction with additional cytochrome c, does not induce
apoptosis in SCG neurons but may itself have some effect
on survival (Wakade et al., 1995).
Discussion
We have examined the role of cytochrome c during apop-
tosis 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 loca-
tion of cytochrome c during apoptosis by fluorescence mi-
croscopy. We observed that in healthy neurons, cyto-
chrome 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, im-
plying release from the mitochondria. This first observa-
tion is in agreement with the findings of groups who have
compared the partition of cytochrome c between the cyto-
sol 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 condensa-
tion 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 in-
hibitors do not affect the partition of cytochrome c be-
tween 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 neu-
rons 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 immuno-
blotting 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 indi-
rectly mediated by the inactivation of cytochrome c (Kripp-
ner et al., 1996). They detected no degradation of cyto-
chrome c during this process. The cells in their study were
treated for 2 h, so this difference, compared with our re-
sults, 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 en-
tirely different sequence of events from that in SCG neu-
rons 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 re-
lease 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 pro-
teins required for apoptosis (Martin et al., 1988, 1992; Ed-
wards 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 forma-
tion 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 col-
Figure 7. Coinjection of dATP
does not enable cytochrome c
to initiate apoptosis in SCG
neurons. SCG neurons were
microinjected with cyto-
chrome c and dATP, counted
2–4 h later, and maintained in
NGF for a further 72 h. The
microinjection mix in the
first four bars contained 0.7
mg/ml cytochrome c. dATP
was added in the second bar
at 100 mM, in the third bar at 2.5 mM, and in the fourth bar at 10
mM. The fifth bar contained 10 mM dATP alone, and the sixth
bar contained no cytochrome c or dATP. The results are ex-
pressed as a percentage of the cells initially surviving injec-
tion. 150–200 cells were injected per coverslip, and the results
shown are the average of three experiments. The error bars rep-
resent SEM.
The Journal of Cell Biology, Volume 142, 1998 1590
leagues (1996) demonstrated that a form of PKA could
phosphorylate Bad in vitro. In addition, in cells microin-
jected 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 up-
stream of cytochrome c. Thus, we can speculate a survival
pathway activated by the NGF receptor, stimulating phos-
phatidylinositol 3-kinase and Akt kinase, leading to the
phosphorylation of Bad, which in turn antagonizes the for-
mation of an apoptotic Bcl-2 family member complex in
the mitochondrial membrane (Fig. 8). How such a com-
plex 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).
Additional sites of action of cAMP could be put for-
ward. 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 tran-
scriptional level. Indeed modulation of transcription fac-
tors, 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, includ-
ing 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 re-
leased from SCG neurons after NGF withdrawal, we
wished to determine whether this release was associated
with a loss of DCm. This has been reported to be the cause
of cytochrome c release (Susin et al., 1996, 1997) and con-
versely to be the result of caspase activation (Bossy-Wet-
zel et al., 1998). Thus, for some models of apoptotic cell
death it is unclear if DCm loss is crucial to cytochrome c re-
lease from the mitochondria. However, in the case of
NGF-deprived sympathetic neurons, it was clear that com-
plete cytochrome c dispersal and nuclear pyknosis pre-
ceded loss of DCm.
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 mi-
croinjection results show that cytochrome c activity is im-
portant for regulating neuronal apoptosis. This demon-
strates for the first time that cytochrome c is an essential
component of NGF withdrawal–induced apoptosis of sym-
pathetic neurons. Since the anti–cytochrome c mAbs were
injected into the cell cytoplasm, we assume that the inhibi-
tion of cytochrome c occurs post efflux from the mitochon-
dria. We infer that the cytochrome c mode of action is by
activation of a caspase in a similar manner to that previ-
ously 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 part-
ners 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 cyto-
chrome c and dATP, some sources have found that cyto-
chrome 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 cyto-
chrome 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, al-
though 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 mi-
croperoxidase-injected cells appeared normal. Not with-
standing 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 limit-
ing 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 mM (Hunting and Henderson, 1981; Sherman and
Fyfe, 1989), implying that we are supplying sufficient
dATP. Wakade and colleagues (1995) described a 40-fold
Figure 8. A model for the mode of action of NGF, cAMP, and
cycloheximide in neuronal cell death. In this model, NGF is
shown to stimulate phosphatidylinositol 3-kinase, hence Akt ki-
nase activity. Akt kinase can phosphorylate Bad, resulting in its
sequestration in the cytosol and survival. However, in the ab-
sence of NGF, unphosphorylated Bad disrupts Bax:BclxL dimers
in the mitochondrial membrane with the subsequent freeing of
Bax to form Bax homodimers. These might be involved in the re-
lease of cytochrome c from the mitochondria. Cytochrome c
forms a complex with Apaf-1, dATP, and pro-caspase 9, activat-
ing caspase 9. cAMP may act to phosphorylate Bad, and the pos-
sible sites of cycloheximide intervention are indicated by as-
terisks.
Neame et al. Cytochrome c and Neuronal Apoptosis 1591
increase in dATP concentration upon induction of apopto-
sis in chick SCG neurons. However, this death was in-
duced by treatment with 2-deoxyadenosine, a precursor of
dATP, so the increase in its concentration may not be
physiologically relevant to apoptosis. Certainly, dATP lev-
els 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 in-
trinsic 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 re-
cently 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 cyclo-
heximide prevents cytochrome c release, perhaps it also
functions to inhibit the translation of these proteins; how-
ever, the regulation could equally involve other mecha-
nisms. 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 re-
lease of cytochrome c, which appears rapid since we very
rarely see cells displaying intermediate punctate/diffuse
cytochrome c distribution, is differentially regulated com-
pared to its apoptotic partners. It is also possible that the
immediate apoptotic partners of cytochrome c may be un-
regulated, while the downstream mediators are. In this
context, it is interesting that F. Li et al. (1997) report a pa-
rental 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 neu-
rons 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 cyto-
chrome 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 DCm and so must be regulated by
other means. These in turn are dependent upon transcrip-
tion/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 regu-
lated, since the introduction of cytochrome c into the cyto-
plasm is not in itself sufficient in SCG neurons to activate
or accelerate cell death.
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.
Received for publication 16 April 1998 and in revised form 10 July 1998.
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