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studies suggest that there is some redundancy among
survival-promoting members of the Bcl-2 family: Tar-
geted disruption of the bcl-2 gene does not have an
effect on neuron survival, despite its high expression
in the developing nervous system (Veis et al., 1993). In
contrast, disruption of a different anti-apoptotic gene,
bcl-x , does produce a clear increase of apoptosis in the
nervous system (Motoyama et al., 1995).
In dying neurons, the pro-apoptotic members of the
Bcl-2 family bind to members that maintain survival
(i.e., Bcl-2 and Bcl-x), and then mount an assault on the
mitochondria (Figure 7.27). One subset of pro-apop-
totic proteins (those containing only a single Bcl-2
homology domain, such as Bad, Bid, and Bim) recruit
a second subset into play (Bax subfamilymembers
such as Bax and Bak). This latter set of pro-apoptotic
proteins oligomerizes and becomes associated with the
mitochondrial membrane. At this point, Bax and Bak
apparently interact with a voltage-dependent anion
channel within the mitochondrial wall and facilitate its
opening (Shimizu et al., 1999). This increases mito-
chondrion permeability and permits cyt c to be
released. At the same time that the mitochondria are
under attack, pro-apoptotic proteins are dimerizing
with anti-apoptotic members and inactivating them.
Bad binds to Bcl-x, and Bax binds to Bcl-2. As the cas-
pases become activated, anti-apoptotic members of the
Bcl-2 family are themselves a substrate. When Bcl-2 is
cleaved by a caspase, its protective influence is oblit-
erated (Cheng et al., 1997).
The pro-apoptotic members have a clear influence
on neuron cell death in vivo. Apoptosis in ganglia and
motor neurons is virtually eliminated in bax knockout
mice, and it is significantly reduced in many areas of
the CNS (White et al., 1998). The functional interaction
between pro- and anti-apoptotic members is illustrated
in a mouse with deletions of one or both members
(Figure 7.28). Mice deficient for a bcl-x exhibit increased
apoptosis, suggesting that pro-apoptotic proteins are
no longer being suppressed. To test this idea, a double
knockout mouse deficient for both bcl-x and the pro-
apoptotic member, bax , was examined. When apoptosis
was examined in the spinal cord of bcl-x -/- / bax -/- mice,
the level had returned to normal (Shindler et al., 1997).
One recent experiment has tied together elements of
the neurotrophin withdrawal response, including ele-
vation of pro-apoptotic Bcl-2 family members. When
sympathetic neurons were transfected with virus that
expresses a dominant negative form for c-Jun, the cells
could survive NGF withdrawal. Without active c-Jun,
there were two basic changes to the cells physiology
when NGf was withdrawn. First, the expression of a
pro-apoptotic Bcl-2 family member, Bim, did not
increase as it normally does. Second, cyt c was not
The continuous presence of death-promoting mole-
cules in the cytoplasm is rather like keeping several
loaded guns scattered about the house; safety locks of
some sort are essential. In fact, there are several impor-
tant checks and balances to ensure that only the correct
neurons die. Once again, the molecular acronyms will
fly, so there is some appeal in grasping the basic mech-
anism first. There are two key points of regulation.
First, the mitochondrion outer membrane must remain
relatively impermeable so that cytochrome c does not
leak into the cytoplasm. Second, inactive pro-caspases
must be constrained so that their threshold for activa-
tion remains relatively high. Mitochondrion perme-
ability is controlled by a large family of proteins that
interact with the outer mitochondrial membrane as
well as with one another. The activation of pro-cas-
pases is regulated by two interacting molecules, one of
which is released from mitochondria.
In C. elegans , activation of the ced-9 gene can prevent
apoptosis in all cells. Conversely, mutations that inac-
tive ced-9 lead to death among cells that would nor-
mally survive through development (Hengartner et
al., 1992). CED-9 apparently blocks death by complex-
ing with CED-4 and interfering with its ability to
activate the protease, CED-3 (Figure 7.26). The mam-
malian homolog of CED-9 is a membrane-associated
protein called Bcl-2 (named for its discovery in B Cell
Lymphoma cells) that was originally discovered in
studies of tumor formation. The Bcl-2 family has since
been found to include members that promote survival
(anti-apoptotic), as well as those that promote apopto-
sis (pro-apoptotic). To date, 12 pro-apoptotic and 7
anti-apoptotic members have been described in
mammals, although few have been evaluated as par-
ticipants in naturally occurring neuron cell death.
In healthy neurons, anti-apoptotic members of the
Bcl-2 family (Bcl-2 and Bcl-x) are closely associated
with mitochondria (Figure 7.27). Bcl-x apparently pre-
vents the release of cy tc by interacting with a voltage-
dependent anion channel in the mitochondrial outer
wall, causing it to remain closed (Shimizu et al., 1999).
Bcl-x also associates with two other proteins to keep
them inactive: a pro-apoptotic member of the Bcl-2
family (Bax) and the caspase activator (Apaf-1).
One of the more impressive displays of Bcl-2 influ-
ence on neuron survival comes from transgenic mice
that overexpress this protein. These mice have much
bigger brains than controls, and cell counts in the facial
nucleus and retina reveal a 40% increase in neuron
number (Martinou et al., 1994). However, genetic
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