Mitochondrial fragmentation and neuronal cell death in response to the Bcl-2/Bcl-x(L)/Bcl-w antagonist ABT-737.

Inhibition of pro-survival Bcl-2 family proteins by BH3-only proteins is a key initial step leading to apoptotic cell death. In neurons, investigating cell death pathways is often hampered by the multi-factorial nature of the stress stimuli employed. Here we investigate the action of ABT-737, a small molecule inhibitor which specifically targets the BH3-protein binding domain of pro-survival Bcl-2, Bcl-X(L) and Bcl-w. ABT-737 produced a time- and concentration-dependent neuronal cell death which displayed the classical hallmarks of apoptosis. Cell death was maximal by around 4 h ABT-737 treatment, and the effect of ABT-737 could be delayed by the broad spectrum caspase inhibitor zVADfmk. Examining, using real-time confocal microscopy, the molecular basis for the onset of response demonstrated recruitment of pro-apoptotic Bax to specific mitochondrial foci, followed by mitochondrial fragmentation. Treatment of neurons with ABT-737 also produced cleavage of Bid, a BH3-only protein known to be a caspase substrate. Interestingly, cleaved Bid translocated to mitochondria but did not colocalise with Bax foci. zVADfmk inhibited Bid cleavage and slowed the rate of fragmentation, suggesting a role for cleaved Bid in the amplification of the apoptotic response. siRNA-mediated knockdown of Bax significantly inhibited ABT-737 induced cell death, whereas knockdown of the BH3-only proteins Bid or Bim had no effect. ABT-737 therefore appears to be a useful tool with which to examine neuronal apoptotic pathways. Our data suggests that caspase-dependent cleavage of Bid may be a downstream amplification event which enhances the rate of mitochondrial fragmentation.

Caspase-9 cleavage, do you need it?

Caspase-9, which is activated by association with the Apaf-1 (apoptotic protease-activating factor-1) apoptosome complex, cleaves and activates the downstream effector caspases-3 and -7, thereby executing the caspase-cascade and cell-death programme. Although caspase-9 does not need to be cleaved to be active, apoptotic cell death is always accompanied by autocatalytic cleavage and by further downstream effector caspase-dependent cleavage of caspase-9. In this issue of the Biochemical Journal, Denault and co-workers evaluate the role of caspase-3-dependent cleavage of caspase-9 and conclude that this mechanism mainly serves to enhance apoptosis by alleviating XIAP (X-linked inhibitor of apoptosis) inhibition of the apical caspase.

Caspase-7 is directly activated by the approximately 700-kDa apoptosome complex and is released as a stable XIAP-caspase-7 approximately 200-kDa complex.

MCF-7 cells lack caspase-3 but undergo mitochondrial-dependent apoptosis via caspase-7 activation. It is assumed that the Apaf-1-caspase-9 apoptosome processes caspase-7 in an analogous manner to that described for caspase-3. However, this has not been validated experimentally, and we have now characterized the caspase-7 activating apoptosome complex in MCF-7 cell lysates activated with dATP/cytochrome c. Apaf-1 oligomerizes to produce approximately 1.4-MDa and approximately 700-kDa apoptosome complexes, and the latter complex directly cleaves/activates procaspase-7. This approximately 700-kDa apoptosome complex, which is also formed in apoptotic MCF-7 cells, is assembled by rapid oligomerization of Apaf-1 and followed by a slower process of procaspase-9 recruitment and cleavage to form the p35/34 forms. However, procaspase-9 recruitment and processing are accelerated in lysates supplemented with caspase-3. In lysates containing very low levels of Smac and Omi/HtrA2, XIAP (X-linked inhibitor of apoptosis) binds tightly to caspase-9 in the apoptosome complex, and as a result caspase-7 processing is abrogated. In contrast, in MCF-7 lysates containing Smac and Omi/HtrA2, active caspase-7 is released from the apoptosome and forms a stable approximately 200-kDa XIAP-caspase-7 complex, which apparently does not contain cIAP1 or cIAP2. Thus, in comparison to caspase-3-containing cells, XIAP appears to have a more significant antiapoptotic role in MCF-7 cells because it directly inhibits caspase-7 activation by the apoptosome and also forms a stable approximately 200-kDa complex with active caspase-7.

Pro-apoptotic proteins released from the mitochondria regulate the protein composition and caspase-processing activity of the native Apaf-1/caspase-9 apoptosome complex.

The apoptosome is a large caspase-activating ( approximately 700-1400 kDa) complex, which is assembled from Apaf-1 and caspase-9 when cytochrome c is released during mitochondrial-dependent apoptotic cell death. Apaf-1 the core scaffold protein is approximately 135 kDa and contains CARD (caspase recruitment domain), CED-4, and multiple (13) WD40 repeat domains, which can potentially interact with a variety of unknown regulatory proteins. To identify such proteins we activated THP.1 lysates with dATP/cytochrome c and used sucrose density centrifugation and affinity-based methods to purify the apoptosome for analysis by MALDI-TOF mass spectrometry. First, we used a glutathione S-transferase (GST) fusion protein (GST-casp9(1-130)) containing the CARD domain of caspase-9-(1-130), which binds to the CARD domain of Apaf-1 when it is in the apoptosome and blocks recruitment/activation of caspase-9. This affinity-purified apoptosome complex contained only Apaf-1XL and GST-casp9(1-130), demonstrating that the WD40 and CED-4 domains of Apaf-1 do not stably bind other cytosolic proteins. Next we used a monoclonal antibody to caspase-9 to immunopurify the native active apoptosome complex from cell lysates, containing negligible levels of cytochrome c, second mitochondria-derived activator of caspase (Smac), or Omi/HtrA2. This apoptosome complex exhibited low caspase-processing activity and contained four stably associated proteins, namely Apaf-1, pro-p35/34 forms of caspase-9, pro-p20 forms of caspase-3, X-linked inhibitor of apoptosis (XIAP), and cytochrome c, which was only bound transiently to the complex. However, in lysates containing Smac and Omi/HtrA2, the caspase-processing activity of the purified apoptosome complex increased 6-8-fold and contained only Apaf-1 and the p35/p34-processed subunits of caspase-9. During apoptosis, Smac, Omi/HtrA2, and cytochrome c are released simultaneously from mitochondria, and thus it is likely that the functional apoptosome complex in apoptotic cells consists primarily of Apaf-1 and processed caspase-9.