Roles of receptor‐interacting protein kinase 1 in SH‐SY5Y cells with beta amyloid‐induced neurotoxicity

Alzheimer''s disease (AD), the major cause of dementia, affects the elderly population worldwide. Previous studies have shown that depletion of receptor‐interacting protein kinase 1 (RIPK1) expression reverted the AD phenotype in murine AD models. Necroptosis, executed by mixed lineage kinase domain‐like (MLKL) protein and activated by RIPK1 and RIPK3, has been shown to be involved in AD. However, the role of RIPK1 in beta‐amyloid (Aβ)‐induced necroptosis is not yet fully understood. In this study, we explored the role of RIPK1 in the SH‐SY5Y human neuroblastoma cells treated with Aβ 1–40 or Aβ 1–42. We showed that Aβ‐induced neuronal cell death was independent of apoptosis and autophagy pathways. Further analyses depicted that activation of RIPK1/MLKL‐dependant necroptosis pathway was observed in vitro. We demonstrated that inhibition of RIPK1 expression rescued the cells from Aβ‐induced neuronal cell death and ectopic expression of RIPK1 was found to enhance the stability of the endogenous APP. In summary, our findings demonstrated that Aβ can potentially drive necroptosis in an RIPK1‐MLKL‐dependent manner, proposing that RIPK1 plays an important role in the pathogenesis of AD.     

Promyelocytic leukemia protein targets MK2 to promote cytotoxicity

Promyelocytic leukemia protein (PML) is a tumor suppressor possessing multiple modes of action, including induction of apoptosis. We unexpectedly find that PML promotes necroptosis in addition to apoptosis, with Pml ^−/− macrophages being more resistant to TNF‐mediated necroptosis than wild‐type counterparts and PML‐deficient mice displaying resistance to TNF‐induced systemic inflammatory response syndrome. Reduced necroptosis in PML‐deficient cells is associated with attenuated receptor‐interacting protein kinase 1 (RIPK1) activation, as revealed by reduced RIPK1[S166] phosphorylation, and attenuated RIPK1‐RIPK3‐MLKL necrosome complex formation. We show that PML deficiency leads to enhanced TNF‐induced MAPK‐activated kinase 2 (MK2) activation and elevated RIPK1[S321] phosphorylation, which suppresses necrosome formation. MK2 inhibitor treatment or MK2 knockout abrogates resistance to cell death induction in PML‐null cells and mice. PML binds MK2 and p38 MAPK, thereby inhibiting p38‐MK2 interaction and MK2 activation. Moreover, PML participates in autocrine production of TNF induced by cellular inhibitors of apoptosis 1 (cIAP1)/cIAP2 degradation, since PML‐knockout attenuates autocrine TNF. Thus, by targeting MK2 activation and autocrine TNF, PML promotes necroptosis and apoptosis, representing a novel tumor‐suppressive activity for PML.     

Necroptosis contributes to chronic inflammation and fibrosis in aging liver

Inflammaging, characterized by an increase in low‐grade chronic inflammation with age, is a hallmark of aging and is strongly associated with various age‐related diseases, including chronic liver disease (CLD) and hepatocellular carcinoma (HCC). Because necroptosis is a cell death pathway that induces inflammation through the release of DAMPs, we tested the hypothesis that age‐associated increase in necroptosis contributes to chronic inflammation in aging liver. Phosphorylation of MLKL and MLKL oligomers, markers of necroptosis, as well as phosphorylation of RIPK3 and RIPK1 were significantly upregulated in the livers of old mice relative to young mice and this increase occurred in the later half of life (i.e., after 18 months of age). Markers of M1 macrophages, expression of pro‐inflammatory cytokines (TNFα, IL6 and IL1β), and markers of fibrosis were all significantly upregulated in the liver with age and the change in necroptosis paralleled the changes in inflammation and fibrosis. Hepatocytes and liver macrophages isolated from old mice showed elevated levels of necroptosis markers as well as increased expression of pro‐inflammatory cytokines relative to young mice. Short‐term treatment with the necroptosis inhibitor, necrostatin‐1s (Nec‐1s), reduced necroptosis, markers of M1 macrophages, fibrosis, and cell senescence as well as reducing the expression of pro‐inflammatory cytokines in the livers of old mice. Thus, our data show for the first time that liver aging is associated with increased necroptosis and necroptosis contributes to chronic inflammation in the liver, which in turn appears to contribute to liver fibrosis and possibly CLD.     

Inhibition of Pseudomonas aeruginosa LPS‐Induced airway inflammation by RIPK3 in human airway

Although the physiological function of receptor‐interacting protein kinase (RIPK) 3 has emerged as a critical mediator of programmed necrosis/necroptosis, the intracellular role it plays as an attenuator in human lungs and human bronchial epithelia remains unclear. Here, we show that the expression of RIPK3 dramatically decreased in the inflamed tissues of human lungs, and moved from the nucleus to the cytoplasm. The overexpression of RIPK3 dramatically increased F‐actin formation and decreased the expression of genes for pro‐inflammatory cytokines (IL‐6 and IL‐1β), but not siRNA‐RIPK3. Interestingly, whereas RIPK3 was bound to histone 1b without LPS stimulation, the interaction between them was disrupted after 15 min of LPS treatment. Histone methylation could not maintain the binding of RIPK3 and activated movement towards the cytoplasm. In the cytoplasm, overexpressed RIPK3 continuously attenuated pro‐inflammatory cytokine gene expression by inhibiting NF‐κB activation, preventing the progression of inflammation during Pseudomonas aeruginosa infection. Our data indicated that RIPK3 is critical for the regulation of the LPS‐induced inflammatory microenvironment. Therefore, we suggest that RIPK3 is a potential therapeutic candidate for bacterial infection‐induced pulmonary inflammation.     

An incoherent feedforward loop interprets NFκB/RelA dynamics to determine TNF‐induced necroptosis decisions

Balancing cell death is essential to maintain healthy tissue homeostasis and prevent disease. Tumor necrosis factor (TNF) not only activates nuclear factor κB (NFκB), which coordinates the cellular response to inflammation, but may also trigger necroptosis, a pro‐inflammatory form of cell death. Whether TNF‐induced NFκB affects the fate decision to undergo TNF‐induced necroptosis is unclear. Live‐cell microscopy and model‐aided analysis of death kinetics identified a molecular circuit that interprets TNF‐induced NFκB/RelA dynamics to control necroptosis decisions. Inducible expression of TNFAIP3/A20 forms an incoherent feedforward loop to interfere with the RIPK3‐containing necrosome complex and protect a fraction of cells from transient, but not long‐term TNF exposure. Furthermore, dysregulated NFκB dynamics often associated with disease diminish TNF‐induced necroptosis. Our results suggest that TNF''s dual roles in either coordinating cellular responses to inflammation, or further amplifying inflammation are determined by a dynamic NFκB‐A20‐RIPK3 circuit, that could be targeted to treat inflammation and cancer.     

Human ZBP1 induces cell death‐independent inflammatory signaling via RIPK3 and RIPK1

ZBP1 is an interferon‐induced cytosolic nucleic acid sensor that facilitates antiviral responses via RIPK3. Although ZBP1‐mediated programmed cell death is widely described, whether and how it promotes inflammatory signaling is unclear. Here, we report a ZBP1‐induced inflammatory signaling pathway mediated by K63‐ and M1‐linked ubiquitin chains, which depends on RIPK1 and RIPK3 as scaffolds independently of cell death. In human HT29 cells, ZBP1 associated with RIPK1 and RIPK3 as well as ubiquitin ligases cIAP1 and LUBAC. ZBP1‐induced K63‐ and M1‐linked ubiquitination of RIPK1 and ZBP1 to promote TAK1‐ and IKK‐mediated inflammatory signaling and cytokine production. Inhibition of caspase activity suppressed ZBP1‐induced cell death but enhanced cytokine production in a RIPK1‐ and RIPK3 kinase activity‐dependent manner. Lastly, we provide evidence that ZBP1 signaling contributes to SARS‐CoV‐2‐induced cytokine production. Taken together, we describe a ZBP1‐RIPK3‐RIPK1‐mediated inflammatory signaling pathway relayed by the scaffolding role of RIPKs and regulated by caspases, which may induce inflammation when ZBP1 is activated below the threshold needed to trigger a cell death response.     

The Lck inhibitor,AMG-47a,blocks necroptosis and implicates RIPK1 in signalling downstream of MLKL

Necroptosis is a form of caspase-independent programmed cell death that arises from disruption of cell membranes by the mixed lineage kinase domain-like (MLKL) pseudokinase after its activation by the upstream kinases, receptor interacting protein kinase (RIPK)-1 and RIPK3, within a complex known as the necrosome. Dysregulated necroptosis has been implicated in numerous inflammatory pathologies. As such, new small molecule necroptosis inhibitors are of great interest, particularly ones that operate downstream of MLKL activation, where the pathway is less well defined. To better understand the mechanisms involved in necroptosis downstream of MLKL activation, and potentially uncover new targets for inhibition, we screened known kinase inhibitors against an activated mouse MLKL mutant, leading us to identify the lymphocyte-specific protein tyrosine kinase (Lck) inhibitor AMG-47a as an inhibitor of necroptosis. We show that AMG-47a interacts with both RIPK1 and RIPK3, that its ability to protect from cell death is dependent on the strength of the necroptotic stimulus, and that it blocks necroptosis most effectively in human cells. Moreover, in human cell lines, we demonstrate that AMG-47a can protect against cell death caused by forced dimerisation of MLKL truncation mutants in the absence of any upstream signalling, validating that it targets a process downstream of MLKL activation. Surprisingly, however, we also found that the cell death driven by activated MLKL in this model was completely dependent on the presence of RIPK1, and to a lesser extent RIPK3, although it was not affected by known inhibitors of these kinases. Together, these results suggest an additional role for RIPK1, or the necrosome, in mediating human necroptosis after MLKL is phosphorylated by RIPK3 and provide further insight into reported differences in the progression of necroptosis between mouse and human cells.Subject terms: Kinases, Necroptosis     

The role of RIPK1 mediated cell death in acute on chronic liver failure

Acute-on-chronic liver failure (ACLF) is characterized predominantly by non-apoptotic forms of hepatocyte cell death. Necroptosis is a form of programmed lytic cell death in which receptor interacting protein kinase (RIPK) 1, RIPK3 and phosphorylated mixed lineage kinase domain-like (pMLKL) are key components. This study was performed to determine the role of RIPK1 mediated cell death in ACLF. RIPK3 plasma levels and hepatic expression of RIPK1, RIPK3, and pMLKL were measured in healthy volunteers, stable patients with cirrhosis, and in hospitalized cirrhotic patients with acutely decompensated cirrhosis, with and without ACLF (AD). The role of necroptosis in ACLF was studied in two animal models of ACLF using inhibitors of RIPK1, necrostatin-1 (NEC-1) and SML2100 (RIPA56). Plasma RIPK3 levels predicted the risk of 28- and 90-day mortality (AUROC, 0.653 (95%CI 0.530–0.776), 0.696 (95%CI 0.593–0.799)] and also the progression of patients from no ACLF to ACLF [0.744 (95%CI 0.593–0.895)] and the results were validated in a 2^nd patient cohort. This pattern was replicated in a rodent model of ACLF that was induced by administration of lipopolysaccharide (LPS) to bile-duct ligated rats and carbon tetrachloride-induced fibrosis mice administered galactosamine (CCL₄/GalN). Suppression of caspase-8 activity in ACLF rodent model was observed suggesting a switch from caspase-dependent cell death to necroptosis. NEC-1 treatment prior to administration of LPS significantly reduced the severity of ACLF manifested by reduced liver, kidney, and brain injury mirrored by reduced hepatic and renal cell death. Similar hepato-protective effects were observed with RIPA56 in a murine model of ACLF induced by CCL₄/GalN. These data demonstrate for the first time the importance of RIPK1 mediated cell death in human and rodent ACLF. Inhibition of RIPK1 is a potential novel therapeutic approach to prevent progression of susceptible patients from no ACLF to ACLF.Subject terms: Hepatitis, Liver diseases     

The role of MLKL in Hepatic Ischemia-Reperfusion Injury of Alcoholic Steatotic Livers

Alcohol-related liver disease (ALD) and non-alcoholic fatty liver disease (NAFLD) are the primary causes of chronic liver disease in western countries. Liver transplantation is currently one of the most efficient approaches to save patients with liver failure, which is often associated with hepatic ischemia-reperfusion (IR) injury. IR injury is exacerbated by hepatic steatosis, yet the mechanism remains elusive. Necroptosis is a form of regulated cell death mediated by receptor-interacting protein kinase 1 (RIP1), RIP3 and mixed lineage kinase domain-like (MLKL) protein, which has been implicated in the pathogenesis of ALD and NAFLD. Though necroptosis plays an important role in IR injury of high fat diet - induced steatotic livers, the role of necroptosis in IR injury of ethanol - induced steototic livers has not been investigated. In the present study, we used chronic plus binge alcohol (Gao-binge) feeding followed by IR surgery to investigate IR liver injury with ethanol-associated steatosis. We found that the levels of key necroptotic proteins MLKL and RIP3 increased in alcohol-fed mouse livers. Moreover, we observed increased liver injury after IR in control diet-fed mice, which was further exacerbated by alcohol feeding based on serum alanine aminotransferase (ALT) levels and TUNEL staining of necrotic cells. Hepatic neutrophil infiltration also increased in alcohol-fed mice after IR surgery. However, deletion of Mlkl did not protect against IR liver injury in alcohol-fed mice compared with matched wild-type mice. In conclusion, alcoholic steatosis promotes IR injury, which seems to be independent of MLKL-mediated necroptosis.     

Fueling the Flames: Mammalian Programmed Necrosis in Inflammatory Diseases

Programmed necrosis or necroptosis is an inflammatory form of cell death driven by TNF-like death cytokines, toll-like receptors, and antigen receptors. Unlike necrosis induced by physical trauma, a dedicated pathway is involved in programmed necrosis. In particular, a kinase complex composed of the receptor interacting protein kinase 1 (RIPK1) and RIPK3 is a central step in necrotic cell death. Assembly and activation of this RIPK1–RIPK3 “necrosome” is critically controlled by protein ubiquitination, phosphorylation, and caspase-mediated cleavage events. The molecular signals cumulate in formation of intracellular vacuoles, organelle swelling, internal membrane leakage, and eventually plasma membrane rupture. These morphological changes can result in spillage of intracellular adjuvants to promote inflammation and further exacerbate tissue injury. Because of the inflammatory nature of necrosis, it is an attractive pathway for therapeutic intervention in acute inflammatory diseases.Necrosis and tissue inflammation are two tightly linked phenomena. Cell injury induced by excessive trauma such as heat shock and osmotic shock can result in cell death with “necrotic morphology.” These relatively nonspecific means to trigger necrosis have contributed to the notion that necrosis is caused by excessive insults and does not involve elaborate intracellular signaling pathways. In contrast to the notion that necrosis is associated with harmful pathologies, recent work indicates that necrosis can have beneficial roles in certain biological responses. Proteomic approaches and RNA interference screens have identified several crucial regulators of necrosis induced by TNF-like death cytokines. Because a dedicated molecular circuitry is involved, the terms “programmed necrosis” and “necroptosis” have been used to distinguish these types of necrotic cell death from necrosis induced by physical trauma or insults. Here I will discuss the molecular pathway that regulates programmed necrosis/necroptosis. For the sake of simplicity, we will use the term necrosis to refer to programmed necrosis induced by defined death cytokines.     

PLEKHO2 inhibits TNFα-induced cell death by suppressing RIPK1 activation

Receptor interaction protein kinase 1 (RIPK1) plays a diverse role in tumor necrosis factor α (TNFα) signalings. The ubiquitination of RIPK1 is essential for NF-κB activation, whereas its kinase activity promotes apoptosis and necroptosis. However, the mechanisms underlying have not been fully illuminated. Here we report that PH domain-containing family O member 2 (PLEKHO2) inhibits RIPK1-dependent cell death and is necessary for NF-κB activation in response to TNFα. Cells of PLKEHO2 deficiency are more susceptible to TNF-α induced apoptosis and necroptosis with increased RIPK1 activation, which is consistent with the observation that the susceptibility of PLEKHO2−/− cells is effectively prevented by treatment of RIPK1 kinase inhibitor. Moreover, PLEKHO2 deficient cells exhibit compromised RIPK1 ubiquitination and NF-κB activation in response to TNFα. Ultimately, PLEKHO2-deficient mice display greatly increased hepatotoxicity and lethality after TNFα-induced hepatitis. In summary, our study revealed that PLEKHO2 is a novel inhibitor of apoptosis and necroptosis, which plays a key role in regulating RIPK1 ubiquitination and activationSubject terms: Apoptosis, Ubiquitylation     

RIPK3 contributes to TNFR1-mediated RIPK1 kinase-dependent apoptosis in conditions of cIAP1/2 depletion or TAK1 kinase inhibition

Receptor-interacting protein kinase (RIPK) 1 and RIPK3 have emerged as essential kinases mediating a regulated form of necrosis, known as necroptosis, that can be induced by tumor necrosis factor (TNF) signaling. As a consequence, inhibiting RIPK1 kinase activity and repressing RIPK3 expression levels have become commonly used approaches to estimate the contribution of necroptosis to specific phenotypes. Here, we report that RIPK1 kinase activity and RIPK3 also contribute to TNF-induced apoptosis in conditions of cellular inhibitor of apoptosis 1 and 2 (cIAP1/2) depletion or TGF-β-activated kinase 1 (TAK1) kinase inhibition, implying that inhibition of RIPK1 kinase activity or depletion of RIPK3 under cell death conditions is not always a prerequisite to conclude on the involvement of necroptosis. Moreover, we found that, contrary to cIAP1/2 depletion, TAK1 kinase inhibition induces assembly of the cytosolic RIPK1/Fas-associated protein with death domain/caspase-8 apoptotic TNF receptor 1 (TNFR1) complex IIb without affecting the RIPK1 ubiquitylation status at the level of TNFR1 complex I. These results indicate that the recruitment of TAK1 to the ubiquitin (Ub) chains, and not the Ub chains per se, regulates the contribution of RIPK1 to the apoptotic death trigger. In line with this, we found that cylindromatosis repression only provided protection to TNF-mediated RIPK1-dependent apoptosis in condition of reduced RIPK1 ubiquitylation obtained by cIAP1/2 depletion but not upon TAK1 kinase inhibition, again arguing for a role of TAK1 in preventing RIPK1-dependent apoptosis downstream of RIPK1 ubiquitylation. Importantly, we found that this function of TAK1 was independent of its known role in canonical nuclear factor-κB (NF-κB) activation. Our study therefore reports a new function of TAK1 in regulating an early NF-κB-independent cell death checkpoint in the TNFR1 apoptotic pathway. In both TNF-induced RIPK1 kinase-dependent apoptotic models, we found that RIPK3 contributes to full caspase-8 activation independently of its kinase activity or intact RHIM domain. In contrast, RIPK3 participates in caspase-8 activation by acting downstream of the cytosolic death complex assembly, possibly via reactive oxygen species generation.     

Staurosporine Induces Necroptotic Cell Death under Caspase-Compromised Conditions in U937 Cells

For a long time necrosis was thought to be an uncontrolled process but evidences recently have revealed that necrosis can also occur in a regulated manner. Necroptosis, a type of programmed necrosis is defined as a death receptor-initiated process under caspase-compromised conditions. The process requires the kinase activity of receptor-interacting protein kinase 1 and 3 (RIPK1 and RIPK3) and mixed lineage kinase domain-like protein (MLKL), as a substrate of RIPK3. The further downstream events remain elusive. We applied known inhibitors to characterize the contributing enzymes in necroptosis and their effect on cell viability and different cellular functions were detected mainly by flow cytometry. Here we report that staurosporine, the classical inducer of intrinsic apoptotic pathway can induce necroptosis under caspase-compromised conditions in U937 cell line. This process could be hampered at least partially by the RIPK1 inhibitor necrotstin-1 and by the heat shock protein 90 kDa inhibitor geldanamycin. Moreover both the staurosporine-triggered and the classical death ligand-induced necroptotic pathway can be effectively arrested by a lysosomal enzyme inhibitor CA-074-OMe and the recently discovered MLKL inhibitor necrosulfonamide. We also confirmed that the enzymatic role of poly(ADP-ribose)polymerase (PARP) is dispensable in necroptosis but it contributes to membrane disruption in secondary necrosis. In conclusion, we identified a novel way of necroptosis induction that can facilitate our understanding of the molecular mechanisms of necroptosis. Our results shed light on alternative application of staurosporine, as a possible anticancer therapeutic agent. Furthermore, we showed that the CA-074-OMe has a target in the signaling pathway leading to necroptosis. Finally, we could differentiate necroptotic and secondary necrotic processes based on participation of PARP enzyme.     

Viral dosing of influenza A infection reveals involvement of RIPK3 and FADD,but not MLKL

RIPK3 was reported to play an important role in the protection against influenza A virus (IAV) in vivo. Here we show that the requirement of RIPK3 for protection against IAV infection in vivo is only apparent within a limited dose range of IAV challenge. We found that this protective outcome is independent from RIPK3 kinase activity and from MLKL. This shows that platform function of RIPK3 rather than its kinase activity is required for protection, suggesting that a RIPK3 function independent of necroptosis is implicated. In line with this finding, we show that FADD-dependent apoptosis has a crucial additional effect in protection against IAV infection. Altogether, we show that RIPK3 contributes to protection against IAV in a narrow challenge dose range by a mechanism that is independent of its kinase activity and its capacity to induce necroptosis.Subject terms: Cell death, Cell death and immune response, Infection     

The expression of RIPK3 is associated with cell turnover of gastric mucosa in the mouse and human stomach

Necroptosis is a novel manner of programmed cell death and important for tissue development, homeostasis, damage, and repair. Activation of receptor-interacting protein kinase 3 (RIPK3), a key member of receptor-interacting protein family in contributing significantly to necroptosis, in tissues is a hallmark of cells dying by necroptosis. However, there are few studies that examine the expression of RIPK3 in the glandular cells of stomachs under physiological condition. We have therefore conducted this study to immunohistochemically characterize the key element of necroptosis, RIPK3, in the mouse and human stomach. Results showed that RIPK3 positive cells could be observed in the surface mucosal cells, granular cells, and lamina propria cells in both mouse and human stomach tissues. Ratios of PCNA/RIPK3 positive cells in the glandular cells were?~?2.1 in mouse and?~?4.15 in human sections respectively. Morphological and double immunofluorescence analysis confirmed that these RIPK3 positive cells were mucous, parietal and lamina propria cells. Our results indicate that the expression of RIPK3 in different cell types might contribute to cell turnover of gastric mucosa in the mouse and human stomach under physiological condition.

     

Human RIPK3 C-lobe phosphorylation is essential for necroptotic signaling

Necroptosis is a caspase-independent, pro-inflammatory mode of programmed cell death which relies on the activation of the terminal effector, MLKL, by the upstream protein kinase RIPK3. To mediate necroptosis, RIPK3 must stably interact with, and phosphorylate the pseudokinase domain of MLKL, although the precise molecular cues that provoke RIPK3 necroptotic signaling are incompletely understood. The recent finding that RIPK3 S227 phosphorylation and the occurrence of a stable RIPK3:MLKL complex in human cells prior to exposure to a necroptosis stimulus raises the possibility that additional, as-yet-unidentified phosphorylation events activate RIPK3 upon initiation of necroptosis signaling. Here, we sought to identify phosphorylation sites of RIPK3 and dissect their regulatory functions. Phosphoproteomics identified 21 phosphorylation sites in HT29 cells overexpressing human RIPK3. By comparing cells expressing wild-type and kinase-inactive D142N RIPK3, autophosphorylation sites and substrates of other cellular kinases were distinguished. Of these 21 phosphosites, mutational analyses identified only pT224 and pS227 as crucial, synergistic sites for stable interaction with MLKL to promote necroptosis, while the recently reported activation loop phosphorylation at S164/T165 negatively regulate the kinase activity of RIPK3. Despite being able to phosphorylate MLKL to a similar or higher extent than wild-type RIPK3, mutation of T224, S227, or the RHIM in RIPK3 attenuated necroptosis. This finding highlights the stable recruitment of human MLKL by RIPK3 to the necrosome as an essential checkpoint in necroptosis signaling, which is independent from and precedes the phosphorylation of MLKL.Subject terms: Kinases, Necroptosis     

Akt and mTOR mediate programmed necrosis in neurons

Necroptosis is a newly described form of regulated necrosis that contributes to neuronal death in experimental models of stroke and brain trauma. Although much work has been done elucidating initiating mechanisms, signaling events governing necroptosis remain largely unexplored. Akt is known to inhibit apoptotic neuronal cell death. Mechanistic target of rapamycin (mTOR) is a downstream effector of Akt that controls protein synthesis. We previously reported that dual inhibition of Akt and mTOR reduced acute cell death and improved long term cognitive deficits after controlled-cortical impact in mice. These findings raised the possibility that Akt/mTOR might regulate necroptosis. To test this hypothesis, we induced necroptosis in the hippocampal neuronal cell line HT22 using concomitant treatment with tumor necrosis factor α (TNFα) and the pan-caspase inhibitor N-benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone. TNFα/zVAD treatment induced cell death within 4 h. Cell death was preceded by RIPK1–RIPK3–pAkt assembly, and phosphorylation of Thr-308 and Thr473 of AKT and its direct substrate glycogen synthase kinase-3β, as well as mTOR and its direct substrate S6 ribosomal protein (S6), suggesting activation of Akt/mTOR pathways. Pretreatment with Akt inhibitor viii and rapamycin inhibited Akt and S6 phosphorylation events, mitochondrial reactive oxygen species production, and necroptosis by over 50% without affecting RIPK1–RIPK3 complex assembly. These data were confirmed using small inhibitory ribonucleic acid-mediated knockdown of AKT1/2 and mTOR. All of the aforementioned biochemical events were inhibited by necrostatin-1, including Akt and mTOR phosphorylation, generation of oxidative stress, and RIPK1–RIPK3–pAkt complex assembly. The data suggest a novel, heretofore unexpected role for Akt and mTOR downstream of RIPK1 activation in neuronal cell death.     

Depletion of RIPK3 or MLKL blocks TNF-driven necroptosis and switches towards a delayed RIPK1 kinase-dependent apoptosis

In human cells, the RIPK1–RIPK3–MLKL–PGAM5–Drp1 axis drives tumor necrosis factor (TNF)-induced necroptosis through mitochondrial fission, but whether this pathway is conserved among mammals is not known. To answer this question, we analyzed the presence and functionality of the reported necroptotic axis in mice. As in humans, knockdown of receptor-interacting kinase-3 (RIPK3) or mixed lineage kinase domain like (MLKL) blocks TNF-induced necroptosis in L929 fibrosarcoma cells. However, repression of either of these proteins did not protect the cells from death, but instead induced a switch from TNF-induced necroptosis to receptor-interacting kinase-1 (RIPK1) kinase-dependent apoptosis. In addition, although mitochondrial fission also occurs during TNF-induced necroptosis in L929 cells, we found that knockdown of phosphoglycerate mutase 5 (PGAM5) and dynamin 1 like protein (Drp1) did not markedly protect the cells from TNF-induced necroptosis. Depletion of Pink1, a reported interactor of both PGAM5 and Drp1, did not affect TNF-induced necroptosis. These results indicate that in these murine cells mitochondrial fission and Pink1 dependent processes, including Pink-Parkin dependent mitophagy, apparently do not promote necroptosis. Our data demonstrate that the core components of the necrosome (RIPK1, RIPK3 and MLKL) are crucial to induce TNF-dependent necroptosis both in human and in mouse cells, but the associated mechanisms may differ between the two species or cell types.