RIP1 kinase activity-dependent roles in embryonic development of Fadd-deficient mice

Abstract

RIP1 is an essential regulator of TNF-induced signaling complexes mediating NF-κB activation, apoptosis and necroptosis. Loss of Rip1 rescues the embryonic lethality of Fadd or Caspase-8-deficient mice, even though the double knockout mice die shortly after birth like Rip1-deficient mice. Recent studies demonstrated that mice expressing RIP1 kinase-dead mutants developed normally and resisted necroptotic stimuli in vitro and in vivo. However, the impact of RIP1 kinase activity on Fadd embryonic development remains unknown. Here, we engineered two RIP1 kinase inactive mutant mouse lines, a Rip1 mouse line as previously reported and a novel Rip1 mouse line with an altered P-loop in the kinase domain. While RIP1 could not rescue the embryonic lethality of Fadd-deficient mice at E11.5, RIP1 rescued lethality of Fadd mice at E11.5 and FaddRip1 mice eventually died at E16.5 due to excessive death of fetal liver cells and unregulated inflammation. Under necropotosis-inducing conditions, comparing to Rip1 cells, Rip1cells displayed reduced phosphorylation and oligomerization of RIP3 and MLKL, which lead to increased cell viability. Thus, our study provides genetic evidence that different kinase inactive mutations have distinct impacts on the embryogenesis of Fadd-deficient mice, which might attribute to their extents of protection on necroptosis signaling.

Catalytically inactive RIP1 and RIP3 deficiency protect against acute ischemic stroke by inhibiting necroptosis and neuroinflammation

“…Previous study reported by Liu et al showed that Rip1 Δ/Δ mice is more effective than Rip1 K45A/K45A mice in restoring embryonic lethality caused by FADD deficiency, which might attribute to their extents of inhibition on necroptosis 32 . In our study, we found that both RIP1 kinase-dead mutant mice(Rip1 K45A/K45A or Rip1 Δ/Δ ) had protective effects against cerebral injury after ischemic stroke, which were reflected in the reduction of infarct area and neurobehavioral score.…”

“…To investigate the role of RIP1 kinase activity in acute ischemic stroke, two types of RIP1 kinase-dead mice ( Rip1 K45A/K45A and Rip1 Δ/Δ mice 32 ) were investigated by the MCAO/R model. In comparison with WT (wild-type) mice, infarct volumes in both Rip1 K45A/K45A mice and Rip1 Δ/Δ mice were significantly reduced (Fig.…”

Molecular mechanisms of cell death: recommendations of the Nomenclature Committee on Cell Death 2018

“…As for the first condition, compelling experimental findings demonstrate that the concerted activity of CASP8, FADD, and c-FLIP L tonically inhibits necroptosis [ 432 , 466 , 478 , 479 482 – 484 ]. Thus, the embryonic lethality imposed on mice by the loss of Casp8 or Fadd can be rescued by concurrent ablation of Ripk3 or Mlkl , even though these double knockout animals generally display lymphoproliferative and/or systemic autoimmune disorders as adults [ 432 , 466 , 484 , 485 ]. Of note, Cflar −/− mice require the concomitant knockout of Ripk3 and Fadd to develop into adulthood, which underscores the inhibitory role of c-FLIP in both necroptosis and extrinsic apoptosis reported above [ 483 , 486 ].…”

Necroptosis and RIPK1-mediated neuroinflammation in CNS diseases

“…Receptor-interacting protein kinase 1 (RIPK1) is an important therapeutic target for the treatment of neurodegenerative diseases. Inactivation of RIPK1 by various genetic mutations, including D138N, K45A and ΔG26F27, leads to strong resistance to TNF and no other gross phenotype in mouse models 151–154 . Furthermore, RIPK1 provides a pharmacologically ‘druggable’ target, as its kinase domain presents a hydrophobic pocket that is highly amenable for specific small-molecule inhibitors 155,156 .…”