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a decrease in cell death in vivo (Li et al., 1997), similar to
the effects of ced-4 inactivation.
A caspase begins with the release of a mitochon-
drial flavoprotein, called apoptosis-inducing factor
(AIF). In this case, the AIF enters the nucleus and ini-
titates DNA cleavage (Figure 7.27).
Several caspases have now been implicated in
neuron cell death. There are 14 members in mam-
malian genome, 6 in flies, and only 3 in worms. There
are many lines of evidence that caspase activity is
required for neuronal apoptosis. When a caspase
inhibitor (a cytokine response modifier, crmA) is
microinjected into chick DRG neurons in vitro, they
can survive the withdrawal of NGF (Gagliardini et al.,
1994). Members of the caspase family may also
mediate the death-promoting effect of “death domain”
containing proteins, such as p75 NTR , and the Drosophila
protein caller Reaper (discussed above). For example,
Reaper overexpression in the Drosophila eye causes all
the cells to die, but a caspase inhibitor is able to block
this effect (White et al., 1996; Vernooy et al., 2000).
To determine whether caspases are involved in the
normal period of cell death, chick embryos were treated
with a synthetic peptide inhibitor of caspase on embry-
onic day 8, the peak of motor neuron death. After 24
hours, the number of pyknotic cell bodies was cut in
half compared to animals treated with a less selective
protease inhibitor (Milligan et al., 1995). However, it
remains possible that the synthetic peptide inhibitor
blocked more than one member of the caspase family.
To examine the effect of a single protease, transgenic
mice were produced lacking caspase-3, the closest
mammalian homolog of CED-3. The brains of these
animals are disorganized, and there are few signs of the
pyknotic cell clusters that accompany nervous system
morphogenesis, suggesting a decrease in normal cell
death. This is most apparent in the proliferative zone
or immature populations in the forebrain (Kuida et al.,
1996, Pompeiano et al., 2000). In contrast, cell death of
motor, sensory, and sympathetic neurons proceeds
unchecked when caspase-3 activity is genetically elimi-
nated. Interestingly, electron micrographs of the dying
neurons suggest that they die in a somewhat different
manner than those in control mice; there is little sign of
chromatin condensation or fragmentation into apop-
totic bodies. Together, these results suggest that other
caspases, or caspase-independent pathways, mediate
cell death in many developing neuronal populations.
They also suggest that TUNEL labeling alone does not
characterize cell death. It is possible that cell death is
occurring, but without DNA fragmentation, in cells
that are not stained with TUNEL (Oppenheim et al.,
2001). It is not too surprising to learn that nerve cells
commit suicide by destroying their own proteins. This
ced-3 expressing cells die
ced-3 -/- cells live
CED-3 (nematode)
ICE (mammal)
DCP-1 (fly)
FIGURE 7.25 The role of caspases in cell death. A. In C. elegans ,
CED-3-expressing cells (yellow cytoplasm) die during development,
but almost all cell death is prevented when the ced-3 gene is mutated.
B. CED-3 is a member of the caspase family, enzymes that specifi-
cally cleave proteins after an aspartate residue. There are homologs
of CED-3 in mammals (ICE) and fruit flies (DCP-1). (Panel A adapted
from Yuan and Horvitz, 1990)
inactive form of CED-3 and leads to its activation (Figure
7.26). In mammals and flies, the pathway involves ced-3
and ced-4 homologs, but the mechanism leading to
caspase activation begins at the mitochondrion. Various
stimuli, such as the withdrawal of growth factor, lead to
increased permeability of the outer mitochondrial mem-
brane. When this occurs, a member of the electron trans-
port chain, called cytochrome c (cyt c ), leaks out of the
mitochondrion. As it enters the cytoplasm, cyt c binds
tightly to an adaptor protein called apoptosis protease
activating factors-1 (Apaf-1). This is the mammalian
homolog of ced-4 . Procaspase-9 is then recruited to this
complex, which is called an apoptosome. This is the site
where caspase-9 becomes activated autoproteolytically.
The activated caspase-9, in turn, cleaves pro-caspase-3,
resulting in its activation. Mutations of this Apaf lead to
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