Interrogating the relevance of mitochondrial apoptosis for vertebrate development and postnatal tissue homeostasis

Abstract

"Programmed cell death or 'apoptosis' is critical for organogenesis during embryonic development and tissue homeostasis in the adult. Its deregulation can contribute to a broad range of human pathologies, including neurodegeneration, cancer, or autoimmunity…" These or similar phrases have become generic opening statements in many reviews and textbooks describing the physiological relevance of apoptotic cell death. However, while the role in disease has been documented beyond doubt, facilitating innovative drug discovery, we wonder whether the former is really true. What goes wrong in vertebrate development or in adult tissue when the main route to apoptotic cell death, controlled by the BCL2 family, is impaired? Such scenarios have been mimicked by deletion of one or more prodeath genes within the BCL2 family, and gene targeting studies in mice exploring the consequences have been manifold. Many of these studies were geared toward understanding the role of BCL2 family proteins and mitochondrial apoptosis in disease, whereas fewer focused in detail on their role during normal development or tissue homeostasis, perhaps also due to an irritating lack of phenotype. Looking at these studies, the relevance of classical programmed cell death by apoptosis for development appears rather limited. Together, these many studies suggest either highly selective and context-dependent contributions of mitochondrial apoptosis or significant redundancy with alternative cell death mechanisms, as summarized and discussed here.

Keywords: BCL2 family; BH3-only proteins; cell death; development; tissue homeostasis.

Figure 1.

Signaling cascades implicated in developmental cell death and tissue homeostasis. Prevention of classic apoptosis pathways by genetic deletion of Bax and Bak (intrinsic) or impaired death receptor (DR) signaling (extrinsic) does not lead to major developmental abnormalities during embryogenesis or in surviving adults. Hence, additional cell death pathways have been implicated in the removal of excess cells during development. These pathways may involve caspases (green boxes) or not (blue boxes). Caspase-dependent cell death modalities involve apoptosis, ultimately engaging effector caspases 3, 6, and 7 and immunologically silent cell death that ultimately leads to corpse removal by phagocytes. Pyroptosis—a pathway activated by Caspases 1 and 11 in innate immune cells, mainly macrophages—leads to the cleavage of Gasdermin D and possibly other members of this family that ultimately triggers plasma membrane rupture. Lysosomal cell death involves lysosomal membrane permeabilization (LMP) and can be induced by several triggers, such as Ca²⁺ overload, H₂O₂, extensive DNA damage, or possibly even DR signaling, leading to the release of cathepsins. These enzymes were reported to trigger either a necrotic, apoptotic, or apoptosis-like response, e.g., by involving the BCL2 family protein BID. Accordingly, caspase-dependent as well as caspase-independent forms of lysosomal cell death have been reported. Entosis is a cell death that is triggered by signals from neighboring cells expressing E-cadherins or α-catenin, and those cells are engulfed, followed by phagosome-to-lysosome fusion, leading to cell degradation. Necroptosis is characterized by the activation of RIPK3 and phosphorylation of MLKL. It requires engagement of cell surface receptors, such as toll-like receptor 4 (TLR4), TNFR family members, or the interferon receptor (IFN). Engagement of these or other intracellular receptors, including DAI or TLR3, under conditions where Caspase 8 is inhibited triggers necroptosis. Caspase 8 can cleave and thereby inhibit RIPK1/RIPK3, which are needed to activate MLKL's pore-forming potential, leading to plasma membrane rupture during necroptosis. Programmed necrosis is induced by excess of Ca²⁺, increased reactive oxygen species (ROS) levels, or heavy metals, causing persistent mitochondrial membrane permeabilization through the mitochondrial permeability transition (MPT) pore that spans both the inner and outer mitochondrial membrane. Finally, ferroptosis is triggered by deprivation of cysteine, leading to glutathione (GSH) depletion and inhibition of the detoxifying enzyme GPX4, thereby causing lipid peroxidation and plasma membrane rupture by so-far undefined mechanisms.

Figure 2.

Models for the activation of BAX/BAK and apoptotic pore formation at the MOM. (A) The neutralization model proposes that BAX and BAK are sequestered in their active form by BCL2 prosurvival proteins and that apoptotic stimuli lead to the recruitment of BH3-only proteins by either post-translational stabilization or transcriptional induction. These BH3-only proteins then bind to BCL2-like proteins, thereby releasing BAX/BAK, which are intrinsically active. (B) The “direct activation” model implies that BAX or BAK needs to be directly engaged by “activator BH3-only” proteins in order to impose a conformational change needed for dimerization and subsequent oligomerization. In order to inhibit the initiation of apoptosis, BCL2 proteins sequester activator BH3-only proteins to prevent BAX/BAK activation. Binding of BCL2 to sensitizer BH3-only proteins (e.g., BAD, HRK, BMF) is needed to free activator BH3-only proteins (BIM, BID, and PUMA) to activate BAX or BAK. (C) The embedded together model implies that BCL2-like proteins sequester BH3-only proteins at the MOM, thereby inhibiting their interaction with and direct activation of BAK/BAX. In the MOM, BCL2-like proteins themselves also prevent dimerization and pore formation of BH3-only protein-activated/embedded BAX/BAK monomers. Additionally, the retrotranslocation concept proposes that BAX (and also BAK with slower kinetics) is in an equilibrium and is extracted from the MOM in a BCL2/BCLX-dependent manner and shuttled back into the cytoplasm, where it oscillates between an autoinhibitory dimer and a monomeric state. Only the latter is able to translocate to the mitochondria, where it needs to accumulate in order to form an active dimer that can then form the apoptotic pore upon oligomerization.