Necroptosis - RIP3-dependent Programmed Necrosis

Necroptosis is best defined as RIP3-dependent programmed necrosis. RIP3-deficient animals are protected in various disease models, like ischemia/reperfusion injury (IRI, shown for brain, heart and liver), TNF-mediated shock and TNF/zVAD-mediated hyperacute shock, cisplatin-induced acute kidney injury (AKI) and in some models of chronic inflammatory bowel disease, pancreatitis and a model for sepsis. The clinical relevance of necroptosis is therefore securely identified.

Signalling Pathways of Necroptosis

In untreated cells, death receptor ligation results in activation of the apoptosis cascade and the assembly of the caspase 8/cFLIPlong heterodimer that inactivates key players of necroptosis like CYLD, and presumably leads to the cleavage of RIP1 and RIP3, maybe by caspase-6 that is activated by the active caspase 8 homodimer (see apoptosis). If CYLD is inactivated, the cIAP1/2 complex remains polyubiquitinylated and in turn k63-polyubiquitinylates RIP1. In parallel, the linear ubiquitinylation complex LUBAC assures the linear polyubiquitinylation of RIP1. These two events go hand-in-hand to prevent the exposition of the RIP1-assembly region that is required for the so-called complex II to be formed. If the heterodimer or LUBAC are inactivated (by viral proteins, caspase-inhibitors,etc.), RIP1 becomes deubiquitinylated and exerts its pro cell-death functions. In parallel, RIP1 controlls a critical initiation step of autophagy by directly interating with p62/Sequestosome-1. RIP1 therefore is the master switch to determine life and death decisition of the cell.

If RIP1 is deubiquitinylated and caspases and/or LUBAC are blocked/inactivated, RIP1 will bind to RIP3 (in RIP3-positive cells) and activate the downstream cascade of necroptosis, a classical example for immunogenic cell death as well as programmed necrosis. The RIP1-RIP3-interaction is mediated by the so-called RHIM-doamin and results in a series of phosphorylation events, probably involving an unknown kinase. This leads to the recruitment of MLKL and the assembly of the supramolecular structure called the necroptosome. This assembly is required for further transduction of the necroptosis signal into mitochondria via PGAM5L/S and subsequent Drp1-stimulation, but the downstream partners of RIP3 and the overall dependence on mitochondria are currently heavily debated. However, mitochondria do undergo fragmentation processes, but these are hardly specific for necroptosis because they are also seen in MPT-dependent PN and PARP-1-dependent PN. The subsequent ATP-depletion leads to plasma membrane rupture  (see picture above) and release of cellular content into the interstitium due to an entirely unidentified signalling network. This immunogenic cell death presumably defines the basis on which a well-defined inflammatory infiltration of ischemic organs, like the kidney after transplantation or after cardiac surgery, is built on and is therapeutically targeted. To prevent the primary event of cell death might prevent the subsequent inflammation, which certainly amplifies organ damage.

Application of etoposide or SMAC-mimetics leads to the receptor-independent assembly of a 2 MDa complex that consists of RIP1, FADD, FLIP and caspase 8 (and presumably many other, yet undefined components). This complex initiates both apoptotic and necroptotic signals at the same time and is called the ripoptosome. It si possible that such complexes might form in some AKI models, but the precise pathways of ripoptosome assembly and the in vivo relevance of the ripoptosome (unlike the necroptosome) are still lacking.

AKI = acute kidney injury, IRI = ischemia/reperfusion injury, zVAD = pan-caspase inhibitor, poly-ub = polyubiquitinylation, LUBAC = linear ubiquitin chain assembly complex, RIP1: receptor-interacting protein kinase 1, PGAM5 = Phosphoglycerade mutase 5, MLKL = Mixed-lineage kinase domain like, CYLD = cylindromatosis, cIAP = cellular inhibitor of apoptosis protein, FADD = Fas associated death domain. From Linkermann et al., NDT 2012