Bcl2 Proteins Regulators Of Apoptosis

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The continuous presence of death-promoting molecules 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 important 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 mechanism 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 activation remains relatively high. Mitochondrion permeability 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 inactive ced-9 lead to death among cells that would normally 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 mammalian 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 participants 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 prevents the release of cyt c 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 influence 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 studies suggest that there is some redundancy among survival-promoting members of the Bcl-2 family: Targeted 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 mitochondrion 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 obliterated (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 elevation 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

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 contrast 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 permission of Soc of Neuroscience)

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 contrast 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 permission of Soc of Neuroscience)

released from the mitochondria. This result suggests that neurotrophins, at least, prevent cell death by suppressing 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 observation. A member of the IAP family is deleted in humans with a disorder called spinal muscular atrophy, a condition 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 permeable, 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 fact, both safety latches must be removed to kill sympathetic neurons grown in the presence of NGF. Injection of cyt c alone is not able to activate caspases and produce apoptosis, but if Smac is co-injected, then caspases are activated and the neurons die (Du et al., 2000; Verhagen et al., 2000; Deshmukh et al., 2002). In Drosophila, loss of IAP function alone leads to unrestrained 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).

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