Wednesday, 11 July 2012

Apoptosis vs. Necroptosis

Cell death is a highly regulated process. Ask any cancer or stroke patient. In the former case, too little cell death is causing problems, in the latter it's too much cell death that's doing the damage. Every day, approximately 100 billion (!) cells die in your body and every day, all those cells are replaced. Every day you die a little and you never even noticed.

Most of those cells die by a process called apoptosis; a programmed form of cell death. Every cell is genetically programmed to undergo apoptosis, a sequence of orchestrated events leading to the cells demise, when the cell has either suffered irreparable internal damage or receives an extrinsic stimulus from its environment. The extrinsic signal to die is given when a cell is, for example, infected with a virus, has become old or redundant or has become dislodged from its usual place in the body. The cell is basically told to quietly commit suicide, dismantle itself and allow its remains to be recycled. Cells are equipped with 'death receptors', members of the TNFa super family, that receive the death signal.

As mentioned above, apoptosis can also be triggered intrinsically. When, for example, a cell's genome or essential organelles have suffered irreparable damage a sequence of events leads to the release of toxic proteins from the mitochondria, such as cytochrome c and SMAC, that induce the cell to undergo apoptosis and remove itself from the population.

Extrinsic versus intrinsic apoptosis (image by author)
Apoptosis is a quiet, dignified form of cell death that does not trigger an inflammatory response. Apoptosis depends on the sequential activation of the caspases; a family of cysteine proteases. At the top of the chain are the initiator caspases (caspase-8 and -10 for death-receptor-induced apoptosis, caspase-9 for intrinsically-triggered apoptosis), at the bottom are the executioner caspases (caspase-3, -6 and -7) that dismantle the cell. Diametrically opposed to apoptosis is necrosis; a messy form of cell death -wherein the cell's contents are spilled into the environment- that does elicit an inflammatory response.

Surprisingly, necrosis can also follow a genetically-encoded program, similar to apoptosis. However, programmed necrosis, now widely known as 'necroptosis', does not depend in caspase activity, but on the activity of a kinase: Receptor Interacting Protein Kinase 1 (RIPK1). RIPK1 functions as the initiator of the pathway, while several downstream kinases (most notable RIPK3) serve as the executioners. We don't know much about necroptosis yet, but new findings on this form of cell death are published almost daily. What we do know for sure is that caspase activity is essential to prevent it. Necroptosis only occurs in the absence of caspase activity (for a free review by some friends of mine, see here).

The initiators of death-receptor-dependent apoptosis, caspase-8 and caspase-10, have both been shown to cleave and inactivate RIPK1 (reviewed by me here). Thus, if those caspases are activated, RIPK1 is inactivated. The opposite is also true: When the gene for caspase-8 is knocked out in mice (mice don't have the gene for caspase-10), the embryo deficient in caspase-8 dies on the eleventh day after gestation (Varfolomeev et al. 1998). This is a crucial day in the development of the murine embryo, since at that time the embryo's own blood circulation kicks in. In the absence of caspase-8 activity, the hearth and blood vessels of the embryonic mice fail to develop. Experiments with conditional knockout mice, mice that are only deficient for caspase-8 in certain tissues, have revealed that caspase-8 activity is also essential for the development of the immune system (see Kang et al.).

This failure of caspase-8 deficient embryos to develop, is entirely due to the activity of RIPK1 and its downstream effector RIPK3. Knock either one of these genes out concomitantly with caspase-8 or the adapter protein FADD (essential for the activation of caspase-8) and the mouse develops just fine (herehere, here and here; only the last one, the one I'm on, is free) . Or at least; it develops past this crucial stage, past day 11, for knockout of RIPK1 is lethal in itself. RIPK1 has pro-life as well as pro-death functions, but knockout of RIPK3 is relatively safe. Mice deficient in RIPK3 as well as caspase-8 develop into relatively healthy individuals. They still have some problems, of course, caspase-8 and RIPK3 are not entirely useless genes you can just dispose of. They have problems dealing with viral infections, for example, and their T cells proliferate unchecked. They may have other problems too, that haven't surfaced yet.

Thus, caspase-8 has a crucial pro-survival role in shutting off RIPK1 and preventing it from inducing necroptosis. But how, then, does a cell wherein caspase-8 is activated not die by apoptosis instead? How does it live to develop into a healthy mouse or human? Caspase-8 activates through dimerization; two molecules of caspase-8 are forcefully brought together to form an active complex. The previously mentioned adapter protein FADD is essential for initiating this process of dimerization, but recent evidence has shown that once a few dimers are formed around clusters of FADD, more caspase-8 dimers can form independent of FADD. An important clue comes from the observation that caspase-8 does not only activate when it dimerises with itself to form a homodimer, but can also when it forms a dimer with its cousin, FLIP (FLICE-like Inhibitory Protein), to form a heterodimer. FLIP is similar to caspase-8 but has no protease activity, it is an inactive caspase homologue. The heterodimer is active, but has a restricetd substrate repertoire; it cleaves the pro-apoptotic substrates of caspase-8 with very low efficiency, while it cleaves the non-apoptotic substrates of caspase-8 just as efficiently as the homodimer. I recently published a very readable review on the proliferative versus the apoptotic functions of caspase-8, find it here.

Apoptosis vs. Necroptosis vs. Survival  (image by author)
Now, this is not the end of it. There is much more to RIPK1 signaling than necroptosis; it plays an important role in immune activation and development too. In addition, recent evidence suggests that direct cleavage of RIPK1 cleavage by caspase-8 may not even be the key to prevention of necroptosis. Instead, caspase-8 may cleave CYLD, a de-ubiquitinating enzyme and an important regulator of RIPK1 activity. As long as CYLD is active, RIPK1 can promote necroptosis but if CYLD is inactivated RIPK1 is more likely to promote cell survival. However, caspase-8 is very bad at cleaving either CYLD or RIPK1. The paracaspase MALT1 can also cleave CYLD, an event that is crucial for the activation of T cells, but it is not so very good at it either. Could there be another caspase, downstream of caspase-8, that cleaves and inactivates RIPK1? Does cleavage of RIPK1 really lead to its inactivation or do the two fragments gain a different function? Does the caspase-8/FLIP heterodimer have other substrates, besides RIPK1 and CYLD? These and other important questions are currently under investigation. We're not even sure yet what the relevance of necroptosis is for either normal human physiology or pathology.

Surely these are exciting times for the fields of cell death and inflammation! I will use this blog to review the latest findings in these fields, both by myself and by others. I hope to attract opinionated readers both inside and outside the fields and get some discussions going.