M Experimental Figure 2229

Different classes of sensory neurons are lost in knockout mice lacking different trophic factors or their receptors. In animals lacking nerve growth factor (NGF) or its receptor TrkA, small nociceptive (pain-sensing) neurons (blue) that innervate the skin are missing. These neurons express TrkA receptor and innervate NGF-producing targets. In animals lacking either neurotrophin-3 (NT-3) or its receptor TrkC, large propioceptive neurons (red) innervating muscle spindles are missing. Muscle produces NT-3 and the propioceptive neurons express TrkC. Mechanoreceptors (brown), another class of sensory neurons in the dorsal root ganglion, are unaffected in these mutants. [Adapted from W. D. Snider, 1994, Cell 77:627.]

A Cascade of Caspase Proteins Functions in One Apoptotic Pathway

Key insights into the molecular mechanisms regulating cell death came from genetic studies in C. elegans. As we noted in Section 22.1, scientists have traced the lineage of all the somatic cells in C. elegans from the fertilized egg to the mature worm (see Figure 22-9). Of the 947 nongonadal cells generated during development of the adult hermaphrodite form, 131 cells undergo programmed cell death.

Specific mutations have identified a variety of genes whose encoded proteins play an essential role in controlling programmed cell death during C. elegans development. For instance, programmed cell death does not occur in worms carrying loss-of-function mutations in the ced-3 gene or the ced-4 gene; as a result, the 131 "doomed" cells survive (Figure 22-30). CED-4 is a protease-activating factor that causes autocleavage of the CED-3 precursor protein, creating an active CED-3 protease that initiates cell death (Figure 22-31). In contrast, in ced-9 mutants, all cells die during embryonic life, so the adult form never develops. These genetic studies indicate that the CED-3 and CED-4 proteins are required for cell death, that CED-9 suppresses apoptosis, and that the apoptotic pathway can be activated in all cells. Moreover, the finding that cell death does not occur in ced-9/ced-3 double mutants suggests that CED-9 acts "upstream" of CED-3 to suppress the apoptotic pathway.

The confluence of genetic studies in worms and studies on human cancer cells first suggested that an evolutionarily conserved pathway mediates apoptosis. The first apoptotic gene to be cloned, bcl-2, was isolated from human follicular lymphomas. A mutant form of this gene, created in lymphoma cells by a chromosomal rearrangement, was shown to act as an oncogene that promoted cell survival rather than cell death (Chapter 23). The human Bcl-2 protein and worm CED-9 protein are homologous, and a bcl-2 transgene can

▲ EXPERIMENTAL FIGURE 22-30 Mutations in the ced-3 gene block programmed cell death in C. elegans.

(a) Newly hatched larva carrying a mutation in the ced-1 gene. Because mutations in this gene prevent engulfment of dead cells, highly refractile dead cells accumulate (arrows), facilitating their visualization. (b) Newly hatched larva with mutations in both the ced-1 and ced-3 genes. The absence of refractile dead cells in these double mutants indicates that no cell deaths occurred. Thus CED-3 protein is required for programmed cell death. [From H. M. Ellis and H. R. Horvitz, 1986, Cell 91:818; courtesy of Hilary Ellis.]


▲ FIGURE 22-31 Overview of the evolutionarily conserved apoptotis pathway in C. elegans and vertebrates. Three general types of proteins are critical in this conserved pathway. Regulators either promote or suppress apoptosis; two orthologous regulators shown here, CED-9 and Bcl-2, suppress apoptosis in the presence of trophic factors. Adapters interact with both regulators and effectors; in the absence of trophic factors, they promote activation of effectors. Proteases called caspases serve as effector proteins; their activation leads to degradation of various intracellular substrates and eventually cell death. [Adapted from D. L. Vaux and S. J. Korsemeyer, 1999, Cell 96:245.]

block the extensive cell death found in ced-9 mutant worms. Thus both proteins act as regulators that suppress the apop-totic pathway see (Figure 22-31). In addition, both proteins contain a single transmembrane domain and are localized to the outer mitochondrial, nuclear, and endoplasmic reticulum membranes, where they serve as sensors that control the apoptotic pathway in response to external stimuli. As we discuss below, other regulators promote apoptosis.

The effector proteins in the apoptotic pathway are enzymes called caspases in vertebrates, so named because they contain a key cysteine residue in the catalytic site and selectively cleave proteins at sites just C-terminal to aspartate residues. The principal effector protease in C. elegans is the CED-3 caspase. Humans have 15 different caspases, all of which are initially made as procaspases that must be cleaved to become active. Such proteolytic processing of proproteins is used repeatedly in blood clotting, generation of digestive enzymes, and generation of hormones (Chapter 17). Initiator caspases (e.g., caspase-9) are activated by autoproteolysis induced by other types of proteins (CED-4 in worms, for example), which help the initiators to aggregate. Activated initiator caspases cleave effector caspases (e.g., caspase-3) and thus quickly amplify the total caspase activity level in the dying cell. The various effector caspases recognize and cleave short amino acid sequences in many different target proteins. They differ in their preferred target sequences. Their specific intracellular targets include proteins of the nuclear lamina and cytoskeleton whose cleavage leads to the demise of a cell. Seven lines of knockout mice have been made, each lacking a particular caspase function. Some homozygotes die as embryos; others are viable but with subtle defects. Strikingly, some exhibit an excessive number of neurons (i.e., neuronal hyperplasia), reaffirming the importance of cell death in development of the nervous system; other mutants have cells that are resistant to apoptosis.

While caspases are critical in many apoptosis events, caspase-independent apoptosis also occurs. Apoptosis-

inducing factor (AIF; a flavoprotein) and endonuclease G are two proteins that kill cells on their release from mitochondria.

Cell-culture studies have yielded important insights about a group of adapter proteins that coordinate the action of regulators and effectors to control apoptosis (see Figure 22-31). For instance, expression of C. elegans CED-4 in a human kidney cell line induces rapid apoptosis. This can be blocked by co-expression of the negative regulator CED-9 (or mammalian Bcl-2), indicating that CED-9 opposes CED-4 action. In addition, CED-9 has been shown to directly bind to CED-4 and move it from the cytosol to intracellular membranes. Thus the pro-apoptotic function of CED-4 is directly suppressed by the anti-apoptotic function of CED-9. CED-4 also binds directly to the CED-3 caspase (and related mammalian caspases) and promotes activation of its protease activity, as noted previously. Biochemical studies have shown that CED-4 can simultaneously bind to both CED-9 and CED-3. These results fit with the genetics, which shows that the absence of CED-9 has no effect if CED-3 is also missing (ced-3/ced-9 double mutants have no cell death, like ced-3 mutants).

Pro-Apoptotic Regulators Permit Caspase Activation in the Absence of Trophic Factors

Having introduced the major participants in the apoptotic pathway, we now take a closer look at how the effector cas-pases are activated in mammalian cells. Although the normal function of CED-9 or Bcl-2 is to suppress the cell-death pathway, other intracellular regulatory proteins promote apopto-sis. The first pro-apoptotic regulator to be identified, named Bax, was found associated with Bcl-2 in extracts of cells expressing high levels of Bcl-2. Sequence analysis demonstrated that Bax is related in sequence to CED-9 and Bcl-2, but overproduction of Bax induces cell death rather than protecting cells from apoptosis, as CED-9 and Bcl-2 do. Thus this family of regulatory proteins comprises both anti-apoptotic members (e.g., CED-9, Bcl-2) and pro-apoptotic members (e.g., Bax). All members of this family, which we refer to as the Bcl-2 family, are single-pass transmembrane proteins and can participate in oligomeric interactions. In mammals, six Bcl-2 family members prevent apoptosis and nine promote it. Thus the fate of a given cell—survival or death—may reflect the particular spectrum of Bcl-2 family members made by that cell and the intracellular signaling pathways regulating them.

Some Bcl-2 family members preserve or disrupt the integrity of mitochondria, thereby controlling release of mito-chondrial proteins such as cytochrome c. In normal healthy cells, cytochrome c is localized between the inner and outer mitochondrial membrane, but in cells undergoing apoptosis, cytochrome c is released into the cytosol. This release can be blocked by overproduction of Bcl-2; conversely, overproduction of Bax promotes release of cytochrome c into the cytosol and apoptosis. Moreover, injection of cytochrome c into the cytosol of cells induces apoptosis. A variety of death-inducing stimuli cause Bax monomers to move from the cytosol to the outer mitochondrial membrane where they oligomerize. Bax homodimers, but not Bcl-2 homodimers or Bcl-2/Bax het-erodimers, permit influx of ions through the mitochondrial membrane. It remains unclear how this ion influx triggers the release of cytochrome c. Once cytochrome c is released into the cytosol, it binds to the adapter protein Apaf-1 (the mammalian homolog of CED-4) and promotes activation of a cas-pase cascade leading to cell death (Figure 22-32a). Bax can cause cell death by a second pathway that triggers mitochon-drial dysfunction independently of any caspase action, at least in part by stimulating mitochondrial depolarization.

Gene knockout experiments have dramatically confirmed the importance of both pro-apoptotic and anti-apoptotic Bcl-2 family members in neuronal development. Mice lacking the bcl-xl gene, which encodes an anti-apoptotic protein, have massive defects in nervous system development with widespread cell death in the spinal cord, dorsal root ganglion, and brain of developing embryos. In contrast, bax knockouts exhibit a marked increase in neurons in some regions of the nervous system. These knockouts also demonstrate that the effects of Bcl-2 family members generally are tissue specific.

Some Trophic Factors Induce Inactivation of a Pro-Apoptotic Regulator

We saw earlier that neurotrophins such as nerve growth factor (NGF) protect neurons from cell death. The intracellular signaling pathways linking such survival factors to inactiva-tion of the cell-death machinery are quite elaborate. The finding that trophic factors appear to work largely independent of protein synthesis suggested that these external signals lead to changes in the activities of preexisting proteins rather than to activation of gene expression. Scientists demonstrated that in the absence of trophic factors, the nonphosphorylated form of Bad is associated with Bcl-2/Bcl-xl at the mitochondrial membrane (see Figure 22-32a). Binding of Bad inhibits the anti-apoptotic function of Bcl-2/Bcl-xl, thereby promoting

(a) Absence of trophic factor: Caspase activation

- Trophic factor receptor

(b) Presence of trophic factor: Inhibition of caspase activation

Trophic factor-

Plasma membrane

Death /

Cleavage of substrates

Caspase 3

Caspase 9

Procaspase 9

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