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fact, both safety latches must be removed to kill sym-
pathetic neurons grown in the presence of NGF. Injec-
tion of cyt c alone is not able to activate caspases and
produce apoptosis, but if Smac is co-injected, then cas-
pases are activated and the neurons die (Du et al., 2000;
Ve r hagen et al., 2000; Deshmukh et al., 2002). In
Drosophila , loss of IAP function alone leads to unre-
strained activation of caspases and death. Conversely,
the loss of gene products that inhibit IAP ( grim , reaper ,
and hid ) lead to almost no apoptosis (White et al., 1994;
Hay et al., 1995).
Some of the earliest experimental manipulations of
target size suggested that functional synaptic contacts
were correlated with survival. Removal of the nasal
placodes in salamander embryos did not, at first,
decrease the size of the innervating forebrain region.
However, after the system became functional, loss of
the target did produce a hypoplasia (Burr, 1916).
Observations from the NMJ also suggest that func-
tional synapses are involved in motor neuron survival.
There is a very tight correlation between the onset of
neuromuscular activity in chicks and the onset of
normal motor neuron cell death.
If synapse activity at the target is necessary for sur-
vival, then one would predict that more neurons
would die in its absence. To test this hypothesis, chick
embryos were treated with an acetylcholine receptor
(AChR) antagonist (curare or bungarotoxin) during a
four- day period that overlapped the normal period of
motor neuron death. Curare was quite effective at
blocking neuromuscular transmission, as spontaneous
movements were virtually eliminated for much of the
treatment period. Rather than increasing cell death, the
surprising result was that synapse blockade saved
motor neurons (Figure 7.29). Over 90% of the motor
neurons that would have died were still alive after the
period of normal cell death ended and the curare had
been removed. This effect is due to the interaction at
the neuromuscular junction because agents that block
AChRs in the CNS do not prevent cell death (Pittman
and Oppenheim, 1979; Oppenheim et al., 2000). Fur-
thermore, curare produced a threefold increase in the
number of motor axon branches and synapses during
the period when normal cell death occurs (Dahm and
Landmesser, 1991). A similar decrease in normal cell
death is found in the isthmo-optic nucleus when activ-
ity is blocked in its target, the eye, by injecting TTX
during development (PĂ©quignot and Clarke, 1992).
FIGURE 7.28 In vivo regulation of neuron survival by pro- and
anti-apoptotic regulatory proteins. A. A spinal cord tissue section
from an E12 bcl-x-/- mouse shows many pyknotic nuclei, indicative
of massive cell death (left). TUNEL-positive cells (red) are common
in the E12 bcl-x-/- spinal cord (middle). A nuclear stain (bright blue)
indicates condensed chromatin (right). B. A spinal cord tissue section
from an E12 bcl-x-/-/bax-/- double mutant mouse shows few
pyknotic nuclei, indicating that cell death is curtailed (left). In con-
trast to the bcl-x-/- cord, there are few TUNEL positive cells in bcl-
x-/-/bax-/- mice (middle). In contrast to the bcl-x-/- cord, the
nuclear stain reveals few cases of condensed chromatin in bcl-x-/-
/bax-/- mice (right). (Adopted from Shindler et al., 1997, by per-
mission of Soc of Neuroscience)
released from the mitochondria. This result suggests
that neurotrophins, at least, prevent cell death by sup-
pressing the pathway that leads to pro-apoptitic protein
expression (Whitfield et al., 2001).
The second major point of regulation is located at the
caspases themselves. One family of regulators, called
inhibitors of apoptosis proteins (IAPs), act directly on
the caspases (Figure 7.26). IAPs have been identified
that bind directly to caspase-3 or caspase-9, and block
their proteinase activity. One striking indication that
IAPs keep neurons alive comes from a clinical observa-
tion. A member of the IAP family is deleted in humans
with a disorder called spinal muscular atrophy, a condi-
tion in which spinal cord motor neurons gradually die
(Roy et al., 1995). There are also antagonists to the IAPs.
A mitochondrial protein called Smac/DIABLO, which
is also released when the outer membrane becomes per-
meable, can bind to the apoptosome and inhibit IAP.
This promotes caspase activation and apoptosis.
Therefore, there are two safety latches on caspase
activation. The first is control of cyt c release from
mitochondria, and the second is IAP (Figure 7.27). In
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