Apaf-1/cytochrome c apoptosome: an essential initiatorof caspase activation or just a sideshow?

Caspase activation in mammalian cells is mediated via two main routes, often referred to as ‘the intrinsic pathway’ and ‘the extrinsic pathway’.¹ The extrinsic pathway of apoptosis is induced by ligand-mediated activation of the tumour necrosis factor (TNF) family of cell surface receptors. A large protein complex, known as the DISC, forms at the cell membrane and recruits and activates the initiator caspase, caspase-8.^1,2,3,4 Active caspase-8 directly processes and activates effector caspases, which in turn cleave several hundred caspase substrates to induce the characteristic morphological features of apoptosis. Caspase-8 can also induce apoptosis indirectly by cleaving the BH3-only protein, Bid.^1,2,4 Cleavage of Bid triggers its translocation to the mitochondria to mediate membrane permeabilisation by an unknown mechanism and release of cytochrome c. Released cytochrome c then facilitates the formation of the apoptosome-containing adaptor Apaf-1 and another initiator caspase, caspase-9.^1,2,3,4 Like caspase-8, caspase-9 can directly activate the effector caspase, caspase-3. While this serves as an amplification mechanism during the extrinsic pathway of cell death, damage to mitochondria and subsequent apoptosome-mediated caspase-9 activation is widely accepted as the initiating event in the intrinsic pathway of apoptosis.^1,2,3,4 This is perhaps surprising as in Caenorhabditis elegans, there is no involvement of mitochondria in the developmentally programmed cell death (PCD). Recent results indicate that release of cytochrome c is also not required for the activation of the initiator caspase DRONC in Drosophila melanogaster.^5,6

Why then in mammals does the intrinsic pathway of apoptosis proceed solely via the mitochondria? A recent study demonstrates that, in fact, some stress-induced cell death pathways may not require mitochondria after all.⁷ Several other studies also show that during stress signalling caspase-2 activation occurs upstream of mitochondrial damage and the release of cytochrome c, suggesting that caspase-9 activation by the Apaf-1 apoptosome is not an initiator of the caspase cascade.^8,9,10 Using fetal liver cells derived from Apaf-1-deficient and caspase-9-deficient mice to reconstitute the haematopoietic compartments of irradiated donor mice, Marsden et al⁷ demonstrate that apoptosis occurs normally in the absence of either Apaf-1 or caspase-9. While levels of caspase activation are substantially reduced in Apaf-1 or caspase-9-deficient cells than in their wild-type counterparts, the cells show little defect in their ability to undergo apoptosis in response to physiological and pathological stimuli.⁷ Importantly, while Apaf-1 and caspase-9 are dispensable for cell death, BCL-2 does confer a survival advantage to these deficient cells. Thus, in an experimental system where cytochrome c has no involvement, BCL-2 can still protect against cell death, arguing against the widely held view that BCL-2 solely protects cell death by preventing the release of apoptogenic factors from mitochondria and maintaining mitochondrial membrane integrity.⁴ In addition, the authors show that treating cells with several highly specific broad-spectrum caspase inhibitors produced a similar pheno-type as did overexpression of BCL-2, and retarded release of mitochondrial cytochrome c.⁷

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