PANoptosis-like cell death in ischemia/reperfusion injury of retinal neurons

PANoptosis is a newly identified type of regulated cell death that consists of pyroptosis,apoptosis,and nec roptosis,which simultaneously occur during the pathophysiological process of infectious and inflammatory diseases.Although our previous lite rature mining study suggested that PANoptosis might occur in neuronal ischemia/repe rfusion injury,little experimental research has been reported on the existence of PANoptosis.In this study,we used in vivo and in vitro retinal neuronal models of ischemia/repe rfusion injury to investigate whether PAN optosis-like cell death(simultaneous occurrence of pyroptosis,apo ptosis,and necroptosis)exists in retinal neuronal ischemia/repe rfusion injury.Our results showed that ischemia/repe rfusion injury induced changes in morphological features and protein levels that indicate PANoptosis-like cell death in retinal neurons both in vitro and in vivo.Ischemia/repe rfusion inju ry also significantly upregulated caspase-1,caspase-8,and NLRP3 expression,which are important components of the PANoptosome.These results indicate the existence of PANoptosis-like cell death in ischemia/reperfusion injury of retinal neurons and provide preliminary experimental evidence for future study of this new type of regulated cell death.

Ligustroflavone reduces necroptosis in rat brain after ischemic stroke through targeting RIPK1/RIPK3/MLKL

OBJECTIVE To explore the effect of ligustroflavone on ischemic brain injury in stroke rat and the under⁃lying mechanisms.METHODS A rat model of ischemic stroke was established by middle cerebral artery occlusion(MCAO).Administration of ligustroflavone(10,30,60 mg·kg-1,ig)15 min before ischemia,after which neurological deficit score and infarct volume were detected by longa score and TTC stain.The cell viability and necrosis rate of hypoxia-cultured PC12 cells(O2/N2/CO2,1:94:5,8 h)were evaluated by MTS and LDH release rate.Flow cytometry further verified the mortality rate of PC12 cells.Necroptosis-associated proteins(RIPK1,RIPK3 and MLKL/p-MLKL)were detected by Western blotting.The interaction between RIPK3 and RIPK1 or MLKL were confirmed by immunoprecipitation.Operating Environ⁃ment(MOE)program demonstrated the possible combination of ligustroflavone with RIPK1,RIPK3 and MLKL.RESULTS Ischemic injury(increase in neurological deficit score and infarct volume)and upregulation of necroptosis-associated proteins were showed in rat MCAO model.Administration of ligustroflavone(30 mg·kg^-1,ig)evidently improved neurological func⁃tion,reduced infarct volume,and decreased the levels of necroptosis-associated proteins except the RIPK1.Consistently,hypoxia-cultured PC12 cells caused cellular injury(LDH release and necroposis)concomitant with up-regulation of necroptosis-associated proteins,and these phenomena were blocked in the presence of ligustroflavone(25μmol·L^-1)except the elevated RIPK1 levels.Using the Molecular Operating Environment(MOE)program,we identified RIPK1,RIPK3,and MLKL as potential targets of ligustroflavone.Further studies showed that the interaction between RIPK3 and RIPK1 or MLKL was significantly enhanced,which was blocked in the presence of ligustroflavone.CONCLUSION Ligus⁃troflavone protects rat brain from ischemic injury,and its beneficial effect is related to the prevention of necroptosis through a mechanism involving targeting RIPK1,RIPK3,and/or MLKL.

TRAF2-MLK3 interaction is essential for TNF-α-induced MLK3 activation

Mixed lineage kinase 3 （MLK3） is a mitogen-activated protein kinase kinase kinase that is activated by tumor necrosis factor-α （TNF-α） and specifically activates c-Jun N-terminal kinase （JNK） on TNF-a stimulation. The mecha- nism by which TNF-α activates MLK3 is still not known. TNF receptor-associated factors （TRAFs） are adapter molecules that are recruited to cytoplasmic end of TNF receptor and mediate the downstream signaling, including activation of JNK. Here, we report that MLK3 associates with TRAF2, TRAF5 and TRAF6; however only TRAF2 can significantly induce the kinase activity of MLK3. The interaction domain of TRAF2 maps to the TRAF domain and for MLK3 to its C-terminal half （amino acids 511-847）. Endogenous TRAF2 and MLK3 associate with each other in response to TNF-α treatment in a time-dependent manner. The association between MLK3 and TRAF2 mediates MLK3 activation and competition with the TRAF2 deletion mutant that binds to MLK3 attenuates MLK3 kinase activity in a dose-dependent manner, on TNF-α treatment. Furthermore the downstream target of MLK3, JNK was activated by TNF-α in a TRAF2-dependent manner. Hence, our data show that the direct interaction between TRAF2 and MLK3 is required for TNF-α-induced activation of MLK3 and its downstream target, JNK.

MLKL forms cation channels

The mixed lineage kinase domain-like(MLKL)protein is a key factor in tumor necrosis factor-induced necroptosis.Recent studies on necroptosis execution revealed a commitment role of MLKL in membrane disruption.However,our knowledge of how MLKL functions on membrane remains very limited.Here we demonstrate that MLKL forms cation channels that are permeable preferential y to Mg2+rather than Ca2+in the presence of Na+and K+.Moreover,the N-terminal domain containing six helices(H1-H6)is sufficient to form channels.Using the substituted cysteine accessibility method,we further determine that helix H1,H2,H3,H5 and H6 are transmembrane segments,while H4 is located in the cytoplasm.Finally,MLKL-induced membrane depolarization and cell death exhibit a positive correlation to its channel activity.The Mg2+-preferred permeability and five transmembrane segment topology distinguish MLKL from previously identified Mg2+-permeable channels and thus establish MLKL as a novel class of cation channels.

Complex roles of necroptosis in cancer

Necroptosis is a tightly regulated form of necrosis that requires the activation of receptor-interacting protein(RIP)kinases RIPK1 and RIPK3,as well as the RIPK3 substrate mixed lineage kinase domain-like protein(MLKL).Because of membrane rupture,necroptotic cells release damage-associated molecular patterns(DAMPs)that evoke immune responses.Necroptosis is emerging as an important cellular response in the modulation of cancer initiation,progression,and metastasis.Necroptosis of cancer cells is considered to be an immunogenic cell death capable of activating anti-tumor immunity.Necroptosis also participates in the promotion of myeloid cell-induced adaptive immune suppression and thus contributes to oncogenesis.In addition,necroptosis of endothelial cells and tumor cells is conducive to tumor metastasis.In this review,we summarize the current knowledge of the complex role of necroptosis in cancer and discuss the potential of targeting necroptosis components for cancer therapies.