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Targeting receptor‐interacting protein kinase 1 (RIPK1) has emerged as a promising therapeutic stratagem for neurodegenerative disorders, particularly Alzheimer's disease (AD). A positron emission tomography (PET) probe enabling brain RIPK1 imaging can provide a powerful tool to unveil the neuropathology associated with RIPK1. Herein, the development of a new PET radioligand, [11C]CNY‐10 is reported, which may enable brain RIPK1 imaging. [11C]CNY‐10 is radiosynthesized with a high radiochemical yield (41.8%) and molar activity (305 GBq/µmol). [11C]CNY‐10 is characterized by PET imaging in rodents and a non‐human primate, demonstrating good brain penetration, binding specificity, and a suitable clearance kinetic profile. It is performed autoradiography of [11C]CNY‐10 in human AD and healthy control postmortem brain tissues, which shows strong radiosignal in AD brains higher than healthy controls. Subsequently, it is conducted further characterization of RIPK1 in AD using [11C]CNY‐10‐based PET studies in combination with immunohistochemistry leveraging the 5xFAD mouse model. It is found that AD mice revealed RIPK1 brain signal significantly higher than WT control mice and that RIPK1 is closely related to amyloid plaques in the brain. The studies enable further translational studies of [11C]CNY‐10 for AD and potentially other RIPK1‐related human studies. [ABSTRACT FROM AUTHOR]

Aim: To explore the regulatory mechanisms of microglia‐mediated cytotoxic CD8+ T‐cell infiltration in the white matter injury of perioperative stroke (PIS). Methods: Adult male C57BL/6 mice were subjected to ileocolic bowel resection (ICR) 24 h prior to permanent distant middle cerebral artery occlusion (dMCAO) to establish model PIS. White matter injury, functional outcomes, peripheral immune cell infiltration, and microglia phenotype were assessed up to 28 days after dMCAO using behavioral phenotyping, immunofluorescence staining, transmission electron microscopy, western blot, and FACS analysis. Results: We found surgery aggravated white matter injury and deteriorated sensorimotor deficits up to 28 days following PIS. The PIS mice exhibited significantly increased activation of peripheral and central CD8+ T cells, while significantly reduced numbers of mature oligodendrocytes compared to IS mice. Neutralizing CD8+ T cells partly reversed the aggravated demyelination following PIS. Pharmacological blockage or genetic deletion of receptor‐interacting protein kinase 1 (RIPK1) activity could alleviate CD8+ T‐cell infiltration and demyelination in PIS mice. Conclusion: Surgery exacerbates demyelination and worsens neurological function by promoting infiltration of CD8+ T cells and microglia necroptosis, suggesting that modulating interactions of CD8+ T cells and microglia could be a novel therapeutic target of long‐term neurological deficits of PIS. [ABSTRACT FROM AUTHOR]

Necroptosis, a potent host defense mechanism, limits viral replication and pathogenesis through three distinct initiation pathways. Toll‐like receptor 3 (TLR3) via TIR‐domain‐containing adapter‐inducing interferon‐β (TRIF), Z‐DNA‐binding protein 1 (ZBP1) and tumor necrosis factor (TNF)α mediate necroptosis, with ZBP1 and TNF playing pivotal roles in controlling viral infections, with the role of TLR3–TRIF being less clear. ZBP1‐mediated necroptosis is initiated when host ZBP1 senses viral Z‐form double stranded RNA and recruits receptor‐interacting serine/threonine‐protein kinase 3 (RIPK3), driving a mixed lineage kinase domain‐like pseudokinase (MLKL)‐dependent necroptosis pathway, whereas TNF‐mediated necroptosis is initiated by TNF signaling, which drives a RIPK1–RIPK3–MLKL pathway, resulting in necroptosis. Certain viruses (cytomegalovirus, herpes simplex virus and vaccinia) have evolved to produce proteins that compete with host defense systems, preventing programmed cell death pathways from being initiated. Two engineered viruses deficient of active forms of these proteins, murine cytomegalovirus M45mutRHIM and vaccinia virus E3∆Zα, trigger ZBP1‐dependent necroptosis in mouse embryonic fibroblasts. By contrast, when bone‐marrow‐derived macrophages are infected with the viruses, necroptosis is initiated predominantly through the TNF‐mediated pathway. However, when the TNF pathway is blocked by RIPK1 inhibitors or a TNF blockade, ZBP1‐mediated necroptosis becomes the prominent pathway in bone‐marrow‐derived macrophages. Overall, these data implicate a cell‐type preference for either TNF‐mediated or ZBP1‐mediated necroptosis pathways in host responses to viral infections. These preferences are important to consider when evaluating disease models that incorporate necroptosis because they may contribute to tissue‐specific reactions that could alter the balance of inflammation versus control of virus, impacting the organism as a whole. [ABSTRACT FROM AUTHOR]

Aims: Chronic hyperglycemia‐induced inflammation of the hippocampus is an important cause of cognitive deficits in diabetic patients. The receptor for advanced glycation end products (RAGE), which is widely expressed in the hippocampus, is a crucial factor in this inflammation and the associated cognitive deficits. We aimed to reveal the underlying mechanism by which RAGE regulates neuroinflammation in the pathogenesis of diabetes‐induced cognitive impairment. Methods: We used db/db mice as a model for type 2 diabetes to investigate whether receptor‐interacting serine/threonine protein kinase 1 (RIPK1), which is expressed in microglia in the hippocampal region, is a key protein partner for RAGE. GST pull‐down assays and AutoDock Vina simulations were performed to identify the key structural domain in RAGE that binds to RIPK1. Western blotting, co‐immunoprecipitation (Co‐IP), and immunofluorescence (IF) were used to detect the levels of key proteins or interaction between RAGE and RIPK1. Cognitive deficits in the mice were assessed with the Morris water maze (MWM) and new object recognition (NOR) and fear‐conditioning tests. Results: RAGE binds directly to RIPK1 via the amino acid sequence (AAs) 362–367, thereby upregulating phosphorylation of RIPK1, which results in activation of the NLRP3 inflammasome in microglia and ultimately leads to cognitive impairments in db/db mice. We mutated RAGE AAs 362–367 to reverse neuroinflammation in the hippocampus and improve cognitive function, suggesting that RAGE AAs 362–367 is a key structural domain that binds directly to RIPK1. These results also indicate that hyperglycemia‐induced inflammation in the hippocampus is dependent on direct binding of RAGE and RIPK1. Conclusion: Direct interaction of RAGE and RIPK1 via AAs 362–367 is an important mechanism for enhanced neuroinflammation in the hyperglycemic environment and is a key node in the development of cognitive deficits in diabetes. [ABSTRACT FROM AUTHOR]

Temporomandibular joint osteoarthritis (TMJOA), prevalent in adolescents and the elderly, has serious physical and psychological consequences. TMJOA is a degenerative disease of the cartilage and bone, mostly driven by inflammation, and synoviocytes are the first and most important inflammatory factor releasers. Receptor‐interacting serine/threonine‐protein kinase (RIPK1) promotes inflammatory response and cell death during an array of illnesses. This research aimed to explore the impacts of RIPK1 inhibitor therapy in TMJOA and the mechanism of RIPK1 in inducing inflammation during TMJOA. Herein, inhibition of RIPK1 suppressed the elevated levels of inflammatory factors, nuclear factor kappa B (NF‐κB), along with markers of apoptosis and necroptosis after tumour necrosis factor (TNF)‐α/cycloheximide (CHX) treatment in synoviocytes. Moreover, inflammation models were constructed in vivo through complete Freund's adjuvant (CFA) induction and disc perforation, and the findings supported that RIPK1 inhibition protected TMJ articular cartilage against progressive degradation. RIPK1 regulates NF‐κB activation via cellular inhibitor of apoptosis proteins (cIAP), apoptosis via caspase‐8, and necroptosis via RIPK3/mixed lineage kinase domain‐like (MLKL) in synoviocytes, which in turn facilitates TMJOA inflammation progression. [ABSTRACT FROM AUTHOR]

The clinical syndrome caused by cleavage‐resistant RIPK1 is known as CRIA (Cleavage‐resistant RIPK1‐induced autoinflammatory) syndrome. We present a family with three generations affected by CRIA syndrome. Our index patient (P1), a boy born of a non‐consanguineous marriage, developed recurrent episodes of fever after 5 months of age, with variable periodicity. His father (P2) and paternal grandmother also had periodic fever. At 23 months of age, P1 was diagnosed with renal biopsy‐proven steroid‐responsive nephrotic syndrome. His first visit to our center was at 2 years of age. At presentation, he had failure to thrive, microcytic hypochromic anemia, and elevated inflammatory markers and interleukin‐6 levels. Amyloid A protein was elevated, serum creatinine was normal, and proteinuria resolved after addition of steroids. Next‐generation sequencing showed heterozygous mutation (c.970G>A, p.Asp324His) in RIPK1. This mutation has been reported to cause CRIA syndrome. P2 and P1's asymptomatic younger brother had the same mutation. All the affected members showed variability with respect to frequency and duration of periodic fever as well as the age of onset. Both P1 and P2 had elevated amyloid A, with no evidence of renal dysfunction. P1 and P2 showed improvement in the intensity of fever spikes with colchicine treatment; however, both continue to have periodic fever. [ABSTRACT FROM AUTHOR]

Thiosemicarbazide derivatives have been the focus of scientists owing to their broad biological activities such as anticancer, antimicrobial, and anti‐inflammatory. Herein, we designed and synthesized a new thiosemicarbazide derivative (TS‐1) and evaluated its antiproliferative potential against the human hepatocellular carcinoma cell line (HEPG2) and human umbilical vein endothelial cell line (ECV‐304). Also, it was aimed to investigate the necroptotic and apoptotic cell death effects of TS‐1 in HEPG2 cells, and these effects were supported by molecular docking. The new synthesized compound structure was characterized using various spectroscopic methods such as FT‐IR, 1H‐NMR, 13C‐NMR, and elemental analysis. The cytotoxic activity of the tested compound was measured by the MTT assay. Apoptotic and necroptotic properties of the TS‐1 were evaluated by indirect immunoperoxidase method using antibodies against Ki‐67, Bax, Bcl‐2, caspase‐3, caspase‐8, caspase‐9, RIP3, and RIPK1. Apoptotic and necroptotic effects of TS‐1 were supported by molecular docking. Compound TS‐1 was synthesized as a pure compound with a high yield. The effective value of TS‐1 was 10 μM in HEPG2 cells. TS‐1 did not show any cytotoxic effect on ECV‐304. Caspase‐3 and RIPK1 immunoreactivities were significantly increased in HEPG2 cells after being treated with TS‐1. As the results of the molecular docking studies, the molecular docking showed that the TS‐1 exhibits H‐bond interaction with various significant amino acid residues in the active site of both RIPK1. It could be concluded that TS‐1 could be a promising novel therapeutic agent by inducing apoptosis rather than necroptosis in HEPG2 cells. [ABSTRACT FROM AUTHOR]

Cellular inhibitor of apoptosis proteins (cIAPs) are RING‐containing E3 ubiquitin ligases that ubiquitylate receptor‐interacting protein kinase 1 (RIPK1) to regulate TNF signalling. Here, we established mice simultaneously expressing enzymatically inactive cIAP1/2 variants, bearing mutations in the RING domains of cIAP1/2 (cIAP1/2 mutant RING, cIAP1/2MutR). cIap1/2MutR/MutR mice died during embryonic development due to RIPK1‐mediated apoptosis. While expression of kinase‐inactive RIPK1D138N rescued embryonic development, Ripk1D138N/D138N/cIap1/2MutR/MutR mice developed systemic inflammation and died postweaning. Cells expressing cIAP1/2MutR and RIPK1D138N were still susceptible to TNF‐induced apoptosis and necroptosis, implying additional kinase‐independent RIPK1 activities in regulating TNF signalling. Although further ablation of Ripk3 did not lead to any phenotypic improvement, Tnfr1 gene knock‐out prevented early onset of systemic inflammation and premature mortality, indicating that cIAPs control TNFR1‐mediated toxicity independent of RIPK1 and RIPK3. Beyond providing novel molecular insights into TNF‐signalling, the mouse model established in this study can serve as a useful tool to further evaluate ongoing therapeutic protocols using inhibitors of TNF, cIAPs and RIPK1. Synopsis: cIAPs control RIPK1‐dependent and ‐independent TNF toxicity in mouse. This study finds ubiquitylation and inhibition of RIPK1 by cIAPs to be required for mouse embryonic development, while RIPK1‐independent cIAP inhibition of TNF‐induced cell death regulates tissue inflammation in adult mice. cIAP1/2 E3 ubiquitin ligase activity is required for mouse embryonic development.Catalytic activity of cIAPs regulates RIPK1 kinase activity to promote embryonic survival.TNF induces apoptosis and necroptosis in cells lacking the cIAPs‐RIPK1 regulatory axis.cIAPs control non‐overlapping signalling processes downstream of RIPK1 and TNFR1. [ABSTRACT FROM AUTHOR]

Dysregulation of the adaptor protein Abelson interactor 1 (ABI1) is linked to malignant transformation. To interrogate the role of ABI1 in cancer development, we mapped the ABI1 interactome using proximity‐dependent labeling (PDL) with biotin followed by mass spectrometry. Using a novel PDL data filtering strategy, considering both peptide spectral matches and peak areas of detected peptides, we identified 212 ABI1 proximal interactors. These included WAVE2 complex components such as CYFIP1, NCKAP1, or WASF1, confirming the known role of ABI1 in the regulation of actin‐polymerization‐dependent processes. We also identified proteins associated with the TAK1‐IKK pathway, including TAK1, TAB2, and RIPK1, denoting a newly identified function of ABI1 in TAK1‐NF‐κB inflammatory signaling. Functional assays using TNFα‐stimulated, ABI1‐overexpressing or ABI1‐deficient cells showed effects on the TAK1‐NF‐kB pathway‐dependent signaling to RIPK1, with ABI1‐knockout cells being less susceptible to TNFα‐induced, RIPK1‐mediated, TAK1‐dependent apoptosis. In sum, our PDL‐based strategy enabled mapping of the ABI1 proximal interactome, thus revealing a previously unknown role of this adaptor protein in TAK1/RIPK1‐based regulation of cell death and survival. [ABSTRACT FROM AUTHOR]

Necroptosis is a highly regulated cell death (RCD) form in various inflammatory diseases. Receptor‐interacting protein kinase 1 (RIPK1) and RIPK3 are involved in the pathway. Targeting the kinase domains of RIPK1 and/or 3 is a drug design strategy for related diseases. It is generally accepted that essential reoccurring features are observed across the human kinase domains, including RIPK1 and RIPK3. They present common N‐ and C‐terminal domains that are built up mostly by α‐helices and β‐sheets, respectively. The current RIPK1/3 kinase inhibitors mainly interact with the kinase catalytic cleft. This article aims to present an in‐depth profiling for ligand–kinase interactions in the crucial cleft areas by carefully aligning the kinase‐ligand cocrystal complexes or molecular docking models. The similarity and differential structural segments of ligands are systematically evaluated. New insights on the adaption of the conserved and selective kinase domains to the diversity of chemical scaffolds are also provided. In a word, our analysis can provide a better structural requirement for RIPK1 and RIPK3 inhibition and a guide for inhibitor discovery and optimization of their potency and selectivity. [ABSTRACT FROM AUTHOR]

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. Synopsis: ZBP1 is a nucleic acid sensor that induces cell death via RIPK3. This study describes a ZBP1‐induced inflammatory signaling pathway that is independent of cell death and is mediated by non‐degradative ubiquitin chains, and RIPK1 and RIPK3 as scaffolds. ZBP1 stimulates signaling and cytokine production independently of cell death.ZBP1 signaling relies on the scaffolding functions of RIPK3 and RIPK1, but not their kinase activities.ZBP1 signaling is mediated by K63‐ and M1‐linked ubiquitin chains generated by cIAPs and LUBAC.ZBP1 contributes to cytokine production after SARS‐CoV‐2 infection. [ABSTRACT FROM AUTHOR]

Necroptosis is defined as a novel programmed cell necrosis that is mediated by receptor interacting serine–threonine protein kinase 1 (RIPK1) and other related signals. Necrosis, apoptosis and inflammation are commonly considered as the leading mechanism in acute kidney injury (AKI) induced by gentamicin (GEN), which is a useful antibiotic for treating the infection of Gram‐negative bacterial. However, the necroptosis in the pathogenesis of GEN‐induced AKI is unknown. In this study, to investigate the process and function of necroptosis in GEN‐induced AKI, NRK‐52E and HK‐2 cells and SD rats were used as the models. The necroptosis‐related proteins, including RIPK1, RIPK3, mixed lineage kinase domain‐like (MLKL) and phosphorylated MLKL (p‐MLKL), were all increasing time‐dependently when GEN was continuously given. By using the RIPK1 inhibitor necrostatin‐1 (NEC‐1) and RIPK3 inhibitor (CPD42), the GEN‐induced toxicity of tubular cells was alleviated. Moreover, it was validated that GEN‐induced cell apoptosis and inflammation were attenuated after treating with NEC‐1 or CPD42, both in vivo and in vitro. When MLKL was knocked down by siRNA, NEC‐1 and CPD42 can not further protect the damage of tubular cells by GEN. Although the using of pan‐caspase inhibitor Z‐VAD significantly decreased GEN‐induced apoptosis, it enhanced necroptosis and slightly promoted the decreased cell viability in GEN‐treated cells, with the protective effects weaker than NEC‐1 or CPD42. Finally, in vitro minimum inhibitory concentration (MIC) tests and bacteriostatic ring studies showed that NEC‐1 did not interfere with the antibiotic effects of GEN. Thus, suppressing necroptosis can serve as a promising strategy for the prevention of GEN‐induced nephrotoxicity. [ABSTRACT FROM AUTHOR]

The recently defined necroptosis process participates in the pathophysiology of several tissue injuries. Targeting the necroptosis mediator receptor‐interacting protein kinase (RIPK1) by necrostatin‐1 in different phases of ischaemia–reperfusion injury (IRI) may provide new insight into the protection against renal IRI. The rat groups included (n = 8 in each group): 1) Sham; 2) Renal IRI; 3) Necrostatin‐1 treatment 20 min before ischaemia induction in a dose of 1.65 mg/kg/intravenous; 4) Necrostatin‐1 injection just before reperfusion; 5) Necrostatin‐1 injection 20 min after reperfusion establishment; and 6) drug injection at both the pre‐ischaemia and at reperfusion time in the same dose. Timing dependent, necrostatin‐1 diminished RIPK1 (p < 0.001), and aborted the necroptosis‐induced renal cell injury. Necrostatin‐1 decreased the renal chemokine (CXCL1), interleukin‐6, intercellular adhesion molecule (ICAM‐1), myeloperoxidase, and the nuclear factor (NFκB), concomitant with reduced inducible nitric oxide synthase (iNOS), inflammatory cell infiltration, and diminished cell death represented by apoptotic cell count and the BAX/Bcl2 protein ratio. In group 6, the cell injury was minimal and the renal functions (creatinine, BUN and creatinine clearance) were almost normalised. The inflammatory markers were diminished (p < 0.001) compared to the IRI group. The results were confirmed by histopathological examination. In conclusion, RIPK1 inhibition ameliorates the inflammatory immune response induced by renal IRI. The use of two doses was more beneficial as the pathophysiology of cell injury is characterised. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. SYNOPSIS: Promyelocytic leukemia protein promotes TNF‐induced necroptosis via two distinct pathways, firstly by inhibiting MAPK activated kinase 2 activation, and secondly by enhancing autocrine TNF generation. Promyelocytic leukemia protein (PML) promotes necroptosis.PML targets MAPK activated kinase 2 (MK2) and inhibits MK2 activation to enhance necroptosis.PML also increases autocrine production of tumor necrosis factor (TNF) and associated necroptosis induction. [ABSTRACT FROM AUTHOR]

Objectives: Critically ill coronavirus disease 2019 (COVID‐19) patients are characterised by a severely dysregulated cytokine profile and elevated neutrophil counts, impacting disease severity. However, it remains unclear how neutrophils contribute to pathophysiology during COVID‐19. Here, we assessed the impact of the dysregulated cytokine profile on the regulated cell death (RCD) programme of neutrophils. Methods: Regulated cell death phenotype of neutrophils isolated from critically ill COVID‐19 patients or healthy donors and stimulated with COVID‐19 or healthy plasma ex vivo was assessed by flow cytometry, time‐lapse microscopy and cytokine multiplex analysis. Immunohistochemistry of COVID‐19 patients and control biopsies were performed to assess the in situ neutrophil RCD phenotype. Plasma cytokine levels of COVID‐19 patients and healthy donors were measured by multiplex analysis. Clinical parameters were correlated to cytokine levels of COVID‐19 patients. Results: COVID‐19 plasma induced a necroptosis‐sensitive neutrophil phenotype, characterised by cell lysis, elevated release of damage‐associated molecular patterns (DAMPs), increased receptor‐interacting serine/threonine‐protein kinase (RIPK) 1 levels and mixed lineage kinase domain‐like pseudokinase (MLKL) involvement. The occurrence of neutrophil necroptosis MLKL axis was further confirmed in COVID‐19 thrombus and lung biopsies. Necroptosis was induced by the tumor necrosis factor receptor 1 (TNFRI)/TNF‐α axis. Moreover, reduction of soluble Fas ligand (sFasL) levels in COVID‐19 patients and hence decreased signalling to Fas directly increased RIPK1 levels, exacerbated TNF‐driven necroptosis and correlated with disease severity, which was abolished in patients treated with glucocorticoids. Conclusion: Our results suggest a novel role for sFasL signalling in the TNF‐α‐induced RCD programme in neutrophils during COVID‐19 and a potential therapeutic target to curb inflammation and thus influence disease severity and outcome. [ABSTRACT FROM AUTHOR]

Necroptosis, a distinctive type of programmed cell death different from apoptosis or necrosis, triggered by a series of death receptors such as tumor necrosis factor receptor 1 (TNFR1), TNFR2, and Fas. In case that apoptosis process is blocked, necroptosis pathway is initiated with the activation of three key downstream mediatorswhich are receptor-interacting serine/threonine protein kinase 1 (RIPK1), RIPK3, and mixed lineage kinase domain-like protein (MLKL). The whole process eventually leads to destruction of the cell membrane integrity, swelling of organelles, and severe inflammation. Over the past decade, necroptosis has been found widely involved in life process of human beings and animals. In this review, we attempt to explore the therapeutic prospects of necroptosis regulators by describing its molecular mechanism and the role it played in pathological condition and tissue homeostasis, and to summarize the research and clinical applications of corresponding regulators including small molecule inhibitors, chemicals, Chinese herbal extracts, and biological agents in the treatment of various diseases. [ABSTRACT FROM AUTHOR]

Aim: To explore the regulatory mechanisms of microglia-mediated cytotoxic CD8 + T-cell infiltration in the white matter injury of perioperative stroke (PIS)., Methods: Adult male C57BL/6 mice were subjected to ileocolic bowel resection (ICR) 24 h prior to permanent distant middle cerebral artery occlusion (dMCAO) to establish model PIS. White matter injury, functional outcomes, peripheral immune cell infiltration, and microglia phenotype were assessed up to 28 days after dMCAO using behavioral phenotyping, immunofluorescence staining, transmission electron microscopy, western blot, and FACS analysis., Results: We found surgery aggravated white matter injury and deteriorated sensorimotor deficits up to 28 days following PIS. The PIS mice exhibited significantly increased activation of peripheral and central CD8 + T cells, while significantly reduced numbers of mature oligodendrocytes compared to IS mice. Neutralizing CD8 + T cells partly reversed the aggravated demyelination following PIS. Pharmacological blockage or genetic deletion of receptor-interacting protein kinase 1 (RIPK1) activity could alleviate CD8 + T-cell infiltration and demyelination in PIS mice., Conclusion: Surgery exacerbates demyelination and worsens neurological function by promoting infiltration of CD8 + T cells and microglia necroptosis, suggesting that modulating interactions of CD8 + T cells and microglia could be a novel therapeutic target of long-term neurological deficits of PIS., (© 2024 The Author(s). CNS Neuroscience & Therapeutics published by John Wiley & Sons Ltd.)

Various forms of cell death have been identified over the last decades with each relying on a different subset of proteins for the activation and execution of their respective pathway(s). In addition to the three best characterized pathways—apoptosis, necroptosis, and pyroptosis—other forms of regulated cell death including autophagy‐dependent cell death (ADCD), mitochondrial permeability transition pore (MPTP)‐mediated necrosis, parthanatos, NETosis and ferroptosis, and their relevance for organismal homeostasis are becoming better understood. Importantly, it is increasingly clear that none of these pathways operate alone. Instead, a more complex picture is emerging with many pathways sharing components and signaling principles. Finally, a number of cell death regulators are implicated in human diseases and represent attractive therapeutic targets. Therefore, better understanding of physiological and mechanistic aspects of cell death signaling should yield improved reagents for addressing unmet medical needs. [ABSTRACT FROM AUTHOR]

Dynamic control of ubiquitination by deubiquitinating enzymes is essential for almost all biological processes. Ubiquitin‐specific peptidase 22 (USP22) is part of the SAGA complex and catalyzes the removal of mono‐ubiquitination from histones H2A and H2B, thereby regulating gene transcription. However, novel roles for USP22 have emerged recently, such as tumor development and cell death. Apart from apoptosis, the relevance of USP22 in other programmed cell death pathways still remains unclear. Here, we describe a novel role for USP22 in controlling necroptotic cell death in human tumor cell lines. Loss of USP22 expression significantly delays TNFα/Smac mimetic/zVAD.fmk (TBZ)‐induced necroptosis, without affecting TNFα‐mediated NF‐κB activation or extrinsic apoptosis. Ubiquitin remnant profiling identified receptor‐interacting protein kinase 3 (RIPK3) lysines 42, 351, and 518 as novel, USP22‐regulated ubiquitination sites during necroptosis. Importantly, mutation of RIPK3 K518 reduced necroptosis‐associated RIPK3 ubiquitination and amplified necrosome formation and necroptotic cell death. In conclusion, we identify a novel role of USP22 in necroptosis and further elucidate the relevance of RIPK3 ubiquitination as crucial regulator of necroptotic cell death. SYNOPSIS: Ubiquitin‐specific protease 22 (USP22) controls necroptotic cell death by regulating RIPK3 phosphorylation and RIPK3 K518 ubiquitination in human tumor cell lines. USP22 affects RIPK3 phosphorylation and ubiquitination.Identification of novel, USP22‐regulated RIPK3 ubiquitin sites at K42, K351 and K518 upon necroptosis progression.USP22‐regulated RIPK3 K518 ubiquitination controls necroptosis. [ABSTRACT FROM AUTHOR]

Microglia are rapidly activated following ischaemic stroke and participate in the induction of neuroinflammation, which exacerbates the injury of ischaemic stroke. However, the mechanisms regulating ischaemic microglia remain unclear. In the present study, middle cerebral artery occlusion and oxygen and glucose deprivation models were established for in vivo and vitro monitoring of experimental stroke. We applied recombinant human thioredoxin‐1 (rhTrx‐1) and Necrostatin‐1 (Nec‐1, inhibitor of RIPK1) to examine the role of receptor‐interacting protein kinase 1 (RIPK1) in the development of inflammation in ischaemic microglia via explored the inflammatory responses and the associated mechanisms. Molecular docking results indicated that rhTrx‐1 could directly bind to RIPK1. In vivo and vitro data revealed that rhTrx‐1 reduced necroptosis, mitochondrial membrane potential damage, reactive oxygen species accumulation and NLR Family, pyrin domain‐containing 3 protein (NLRP3) inflammasome activation and regulated the microglial M1/M2 phenotypic changes by inhibiting RIPK1 expression in ischaemic microglia. Consistent with these findings, further in vivo experiments revealed that rhTrx‐1 treatment attenuated cerebral ischaemic injury by inhibiting the inflammatory response. Our data demonstrated the role of RIPK1 in microglia‐induced neuroinflammation following cerebral ischaemia. Administration of rhTrx‐1 provides neuroprotection in ischaemic stroke‐induced microglial neuroinflammation by inhibiting RIPK1 expression. [ABSTRACT FROM AUTHOR]

Efficient brain drug delivery has been a challenge in the treatment of Alzheimer's disease (AD) and other brain disorders as blood‐brain barrier (BBB) impedes most drugs to reach brain. To overcome this obstacle, a novel poly(lactic‐co‐glycolic acid) (PLGA) nanoparticle conjugated with CD47 extracellular domain via reactive oxygen species (ROS)‐responsive phenylborate ester bond exhibiting "do not eat me" signal and BBB penetrating peptide CRTIGPSVC (CRT) and microglia modulation agent Nec‐1s encapsulated in it is developed. The experimental results show that the designed nanoparticle efficiently increases its half‐life in blood circulation by preventing engulfment via phagocytes, and enhances its brain distribution by synergistic effect of CD47 and CRT. The high level of ROS in mouse brain releases CD47 from the nanoparticles and the resultant particles are effectively phagocytized by resident microglia. The engulfed Nec‐1s modulates pathological microglia to a beneficial state, which reduces Aβ burden, microgliosis and astrocytosis, decreases cytokine production and oxidative stress in the brains of AD mice, and finally attenuates cognition deficits and synapse loss. The results first demonstrate that the conditionally releasable "do not eat me" CD47 signal remarkably facilitates microglia‐targeted drug delivery and warrants further study to develop therapeutic agent for AD treatment. [ABSTRACT FROM AUTHOR]

Drugs that mobilise the immune system against cancer are dramatically improving care for many people. Dying cancer cells play an active role in inducing anti‐tumour immunity but not every form of death can elicit an immune response. Moreover, resistance to apoptosis is a major problem in cancer treatment and disease control. While the term "immunogenic cell death" is not fully defined, activation of receptor‐interacting serine/threonine‐protein kinase 1 (RIPK1) can induce a type of death that mobilises the immune system against cancer. However, no clinical treatment protocols have yet been established that would harness the immunogenic potential of RIPK1. Here, we report the first pre‐clinical application of an in vivo treatment protocol for soft‐tissue sarcoma that directly engages RIPK1‐mediated immunogenic cell death. We find that RIPK1‐mediated cell death significantly improves local disease control, increases activation of CD8+ T cells as well as NK cells, and enhances the survival benefit of immune checkpoint blockade. Our findings warrant a clinical trial to assess the survival benefit of RIPK1‐induced cell death in patients with advanced disease at limb extremities. Synopsis: The pathways that initiate and execute immunogenic cell death are complex, genetically encoded, and subject to significant regulation. This study focusses on the possibility to engage RIPK1‐mediated immunogenic cell death for the treatment of soft tissue sarcomas that occur at limb extremities. Inhibition of IAPs via small‐molecule SMAC mimetics potently sensitised extremity malignancies to the standard‐of‐care treatment TNF/Melphalan.Co‐treatment with SMAC mimetics converted non‐immunogenic intrinsic apoptosis to Ripk1‐assisted immunogenic cell death.Ripk1‐induced cell death augmented tumour immune infiltration, delayed tumour growth and prolonged survival.Combination therapy with SMAC mimetics and checkpoint inhibitors further prolonged recurrence‐free survival following isolated limb perfusion‐mediated TNF/Mel treatment.SMAC mimetics improved anti‐tumour immunity of standard‐of‐care treatment for soft tissue sarcomas that occur at limb extremities. [ABSTRACT FROM AUTHOR]

In tumor necrosis factor (TNF) signaling, phosphorylation and activation of receptor interacting protein kinase 1 (RIPK1) by upstream kinases is an essential checkpoint in the suppression of TNF‐induced cell death. Thus, discovery of pharmacological agents targeting RIPK1 may provide new strategies for improving the therapeutic efficacy of TNF. In this study, we found that 3‐O‐acetylrubianol C (3AR‐C), an arborinane triterpenoid isolated from Rubia philippinesis, promoted TNF‐induced apoptotic and necroptotic cell death. To identify the molecular mechanism, we found that in mouse embryonic fibroblasts, 3AR‐C drastically upregulated RIPK1 kinase activity by selectively inhibiting IKKβ. Notably, 3AR‐C did not interfere with IKKα or affect the formation of the TNF receptor1 (TNFR1) complex‐I. Moreover, in human cancer cells, 3AR‐C was only sufficient to sensitize TNF‐induced cell death when c‐FLIPL expression was downregulated to facilitate the formation of TNFR1 complex‐II and necrosome. Taken together, our study identified a novel arborinane triterpenoid 3AR‐C as a potent activator of TNF‐induced cell death via inhibition of IKKβ phosphorylation and promotion of the cytotoxic potential of RIPK1, thus providing a rationale for further development of 3AR‐C as a selective IKKβ inhibitor to overcome TNF resistance in cancer therpay. [ABSTRACT FROM AUTHOR]

Mutations of fms‐like tyrosine kinase 3 (FLT3) are the most frequent mutations in acute myeloid leukemia (AML). Furthermore, the internal tandem duplication (ITD) represents the most common mutation of FLT3 in AML. To explore therapeutic strategies for AML patients carrying FLT3‐ITD, we analyzed death receptor (DR) signaling networks in AML cells comprising FLT3‐ITD. We have started with murine myeloid progenitor 32D cells that ectopically express human FLT3‐ITD (32D‐ FLT3‐ITD) and found that RIPK1 is strongly upregulated in these cells. Subsequently, we have shown that combinatorial treatment of 32D‐FLT3‐ITD cells with the SMAC mimetic BV6 and CD95L sensitizes these cells toward apoptosis and necroptosis. Moreover, combinatorial treatment with death ligands (DLs), for example, CD95L or tumor necrosis factor‐related apoptosis‐inducing ligand (TRAIL) and BV6 enhanced cell death in primary AML blasts from patients carrying FLT3‐ITD mutation. Finally, pharmacological and genetic targeting of RIPK1 inhibited DL/BV6‐mediated cell death in cells with FLT3‐ITD mutations. Taken together, our study suggests a promising therapeutic opportunity for AML cancer cells harboring FLT3‐ITD mutation via targeting RIPK1 pathways. What's new? Mutations in fms‐like tyrosine kinase 3 (FLT3), which influences the termination of hematopoietic progenitor cell growth and survival, occur at relatively high frequency in acute myeloid leukemia (AML). Here, the authors analyzed death receptor (DR) signaling networks in AML cells carrying FLT3 internal tandem duplication (ITD) mutations. Cells with FLT3‐ITD mutation were found to have increased levels of receptor interacting protein kinase 1 (RIPK1). RIPK1 targeting via pharmacological or genetic disruption inhibited cell death mediated by death ligand and BV6. The findings suggest that RIPK1 is a promising therapeutic target in AML cells harboring FLT3‐ITD mutations. [ABSTRACT FROM AUTHOR]

MUC1 (mucin 1), a membrane‐tethered mucin glycoprotein, is highly expressed on the surface of respiratory epithelial cells and plays a key role in anti‐inflammatory and antiapoptotic responses against infections. However, little is known about the link between MUC1 and necroptosis in asthma. This study aimed to investigate the effects of MUC1 on TNF‐α‐induced necroptosis in human bronchial epithelial (16HBE) cells and the underlying molecular mechanism. Negative control and MUC1‐siRNA cells were treated with TNF‐α in the presence or absence of necrostatin‐1 (Nec‐1). Necroptosis was investigated using flow cytometry analyses, and the protein expression levels of MUC1, receptor‐interacting protein kinase‐1 (RIPK1), RIPK3, and phosphorylated RIPK1 were detected by western blot analysis. In addition, the interactions between RIPK and MUC1 were analyzed by coimmunoprecipitation. The results demonstrated that TNF‐α could induce necroptosis of 16HBE cells, and MUC1 expression was increased upon treatment with TNF‐α. The coimmunoprecipitation outcomes showed that MUC1 interacted with RIPK1 but not with RIPK3 in 16HBE cells, and the interaction was augmented by TNF‐α. Furthermore, MUC1 downregulation obviously increased the TNF‐α‐induced necroptosis of 16HBE cells and enhanced the expression of p‐RIPK1‐Ser166 and RIPK3, whereas these phenomena were partially attenuated by Nec‐1. These results may provide a new insight into the mechanism of severe asthma‐related necroptosis and lay a foundation for the future development of new anti‐inflammatory drugs for asthma. [ABSTRACT FROM AUTHOR]

MLKL is a central mediator for necroptosis. Its knockout significantly relieves acute kidney injury (AKI). However, its upstream regulatory mechanism in AKI has not been fully elucidated. We recently reviewed how microRNAs (miRNAs), a type of well-studied epigenetic regulator, play critical roles in AKI. Here, we evaluated miRNAs that potentially target MLKL and evaluated their function in human tubular epithelial cells in response to toxic and ischemic insults. TargetScan analysis showed that miR-194-5P, miR-338-3P, miR-500a-3P, and miR-577 had MLKL binding sites. Although all 4 miRNAs are reduced in AKI, our data show that only hsa-miR-500a-3P was significantly suppressed in cisplatin-treated human tubular epithelial (HK2) cells. We further found that hsa-miR-500a-3P alleviated cisplatin-induced HK2 cell death, which was confirmed by transmission electron microscopy and flow cytometry. In addition, overexpression of hsa-miR-500a-3P decreased kidney injury molecule-1 mRNA and protein levels. Real-time PCR, ELISA, and immunofluorescence data show that hsa-miR-500a-3P protected against inflammatory response, evidenced by decreased monocyte chemotactic protein-1 and proinflammatory cytokines TNF-α and IL-8. Further, hsa-miR-500a-3P attenuated P65 NF-κB phosphorylation and promoter activity. Mechanistically, luciferase reporter assay showed that hsa-miR-500a-3P bound the 3′UTR of MLKL, thereby suppressing phosphorylation and membrane translocation of MLKL. In agreement with these findings, we identified that overexpression of hsa-miR-500a-3P attenuated cell injury and the inflammatory response in response to sodium azide treatment in an in vitro model. Results show that circulating exosomes from patients with AKI down-regulated miR-500a-3P, which suppressed cell injury and inflammation in HK2 cells. hsa-miR-500a-3P alleviated toxic and ischemic insults that were triggered by cell necroptosis and the inflammatory response in human HK2 cells by targeting MLKL. This may serve as a novel therapeutic target for treatment of AKI. [ABSTRACT FROM AUTHOR]

Therapies that suppress RIPK1 kinase activity are emerging as promising therapeutic agents for the treatment of multiple inflammatory disorders. The ability to directly measure drug binding of a RIPK1 inhibitor to its target is critical for providing insight into pharmacokinetics, pharmacodynamics, safety and clinical efficacy, especially for a first-in-class small-molecule inhibitor where the mechanism has yet to be explored. Here, we report a novel method for measuring drug binding to RIPK1 protein in cells and tissues. This TEAR1 (Target Engagement Assessment for RIPK1) assay is a pair of immunoassays developed on the principle of competition, whereby a first molecule (ie, drug) prevents the binding of a second molecule (ie, antibody) to the target protein. Using the TEAR1 assay, we have validated the direct binding of specific RIPK1 inhibitors in cells, blood and tissues following treatment with benzoxazepinone (BOAz) RIPK1 inhibitors. The TEAR1 assay is a valuable tool for facilitating the clinical development of the lead RIPK1 clinical candidate compound, GSK2982772, as a first-in-class RIPK1 inhibitor for the treatment of inflammatory disease. [ABSTRACT FROM AUTHOR]

GSK2982772 is a highly selective inhibitor of receptor-interacting protein kinase 1 (RIPK1) being developed to treat chronic inflammatory diseases. This first-in-human study evaluated safety, tolerability, pharmacokinetics (PK), and exploratory pharmacodynamics (PD) of GSK2982772 administered orally to healthy male volunteers. This was a Phase I, randomized, placebo-controlled, double-blind study. In Part A, subjects received single ascending doses of GSK2982772 (0.1-120 mg) or placebo in a crossover design during each of 4 treatment periods. In Part B, subjects received repeat doses of GSK2982772 (20 mg once daily [QD] to up to 120 mg twice daily [BID]) or placebo for 14 days. Part C was an open-label relative bioavailability study comparing 20-mg tablets vs capsules. Safety, tolerability, pharmacokinetics (PK), RIPK1 target engagement (TE), and pharmacodynamics (PD) were assessed. The most common adverse events (AEs) were contact dermatitis and headache. Most AEs were mild in intensity, and there were no deaths or serious AEs. The PK of GSK2982772 was approximately linear over the dose range studied (up to 120 mg BID). There was no evidence of drug accumulation upon repeat dosing. Greater than 90% RIPK1 TE was achieved over a 24-hour period for the 60-mg and 120-mg BID dosing regimens. Single and repeat doses of GSK2982772 were safe and well tolerated. PK profiles showed dose linearity. The high levels of RIPK1 TE support progression into Phase II clinical trials for further clinical development. [ABSTRACT FROM AUTHOR]

Regulated cell death pathways have important functions in host defense and tissue homeostasis. Studies in genetic mouse models provided evidence that cell death could cause inflammation in different tissues. Inhibition of RIPK3- MLKL-dependent necroptosis by FADD and caspase-8 was identified as a key mechanism preventing inflammation in epithelial barriers. Moreover, the interplay between IKK/ NF-κB and RIPK1 signaling was recognized as a critical determinant of tissue homeostasis and inflammation. NEMO was shown to regulate RIPK1 kinase activity-mediated apoptosis by NF-κB-dependent and -independent functions, which are critical for averting chronic tissue injury and inflammation in the intestine and the liver. In addition, RIPK1 was shown to exhibit kinase activity-independent functions that are essential for preventing cell death, maintaining tissue architecture and inhibiting inflammation. In the intestine, RIPK1 acts as a scaffold to prevent epithelial cell apoptosis and preserve tissue integrity. In the skin, RIPK1 functions via its RHIM to counteract ZBP1/ DAI-dependent activation of RIPK3- MLKL-dependent necroptosis and inflammation. Collectively, these studies provided evidence that the regulation of cell death signaling plays an important role in the maintenance of tissue homeostasis, and suggested that cell death could be causally involved in the pathogenesis of inflammatory diseases. [ABSTRACT FROM AUTHOR]

Necrosis is a hallmark of several widespread diseases or their direct complications. In the past decade, we learned that necrosis can be a regulated process that is potentially druggable. RIPK3- and MLKL-mediated necroptosis represents by far the best studied pathway of regulated necrosis. During necroptosis, the release of damage-associated molecular patterns ( DAMPs) drives a phenomenon referred to as necroinflammation, a common consequence of necrosis. However, most studies of regulated necrosis investigated cell lines in vitro in a cell autonomous manner, which represents a non-physiological situation. Conclusions based on such work might not necessarily be transferrable to disease states in which synchronized, non-cell autonomous effects occur. Here, we summarize the current knowledge of the pathophysiological relevance of necroptosis in vivo, and in light of this understanding, we reassess the morphological classification of necrosis that is generally used by pathologists. Along these lines, we discuss the paucity of data implicating necroptosis in human disease. Finally, the in vivo relevance of non-necroptotic forms of necrosis, such as ferroptosis, is addressed. [ABSTRACT FROM AUTHOR]

The programmed self-destruction of infected cells is a powerful antimicrobial strategy in metazoans. For decades, apoptosis represented the dominant mechanism by which the virus-infected cell was thought to undergo programmed cell death. More recently, however, new mechanisms of cell death have been described that are also key to host defense. One such mechanism in vertebrates is programmed necrosis, or 'necroptosis', driven by receptor-interacting protein kinase 3 ( RIPK3). Once activated by innate immune stimuli, including virus infections, RIPK3 phosphorylates the mixed lineage kinase domain-like protein ( MLKL), which then disrupts cellular membranes to effect necroptosis. Emerging evidence demonstrates that RIPK3 can also mediate apoptosis and regulate inflammasomes. Here, we review studies on the mechanisms by which viruses activate RIPK3 and the pathways engaged by RIPK3 that drive cell death. [ABSTRACT FROM AUTHOR]

Necroptosis is a form of programmed cell death that is both mechanistically and morphologically distinct from apoptosis, the canonical mechanism of cell suicide. Although early descriptions of necroptosis date back decades, the last 5 years have seen a proliferation of studies of this process. This surge in interest has included the recent publication of several excellent, in-depth reviews of the literature [Chan FK-M et al. (2014) Annu Rev Immunol 33, 141210135520002; Weinlich R & Green DR (2014) Mol Cell 56, 469-480; Silke J et al. (2015) Nat Immunol 16, 689-697; Linkermann A & Green DR (2014) N Engl J Med 370, 455-465]. Rather than contribute another summary to this well-summarized field, in this Minireview I will briefly discuss key recent findings, then touch on some of the major outstanding questions - the known unknowns - that remain. [ABSTRACT FROM AUTHOR]

In the past decade, studies have shown how instrumental programmed cell death ( PCD) can be in innate and adaptive immune responses. PCD can be a means to maintain homeostasis, prevent or promote microbial pathogenesis, and drive autoimmune disease and inflammation. The molecular machinery regulating these cell death programs has been examined in detail, although there is still much to be explored. A master regulator of programmed cell death and innate immunity is receptor-interacting protein kinase 1 ( RIPK1), which has been implicated in orchestrating various pathologies via the induction of apoptosis, necroptosis, and nuclear factor-κB-driven inflammation. These and other roles for RIPK1 have been reviewed elsewhere. In a reflection of the ability of tumor necrosis factor ( TNF) to induce either survival or death response, this molecule in the TNF pathway can transduce either a survival or a death signal. The intrinsic killing capacity of RIPK1 is usually kept in check by the chains of ubiquitin, enabling it to serve in a prosurvival capacity. In this review, the intricate regulatory mechanisms responsible for restraining RIPK1 from killing are discussed primarily in the context of the TNF signaling pathway and how, when these mechanisms are disrupted, RIPK1 is free to unveil its program of cellular demise. [ABSTRACT FROM AUTHOR]

Background Neuron apoptosis is a pivotal process for brain damage in cerebral ischemia. Dot1L (disruptor of telomeric silencing 1-like) is only known histone H3K79 methyltransferase. It is not clear whether the role and mechanism of Dot1L on cerebral ischemia is related to regulate neuron apoptosis. Methods and Results We use a combination of mice middle cerebral artery occlusion stroke and neurons exposed to oxygen-glucose deprivation followed by reoxygenation to investigate the role and mechanism of Dot1L on cerebral ischemia. We find knockdown or inhibition of Dot1L reversed ischemia-induced neuronal apoptosis and attenuated the neurons injury treated by oxygen-glucose deprivation followed by reoxygenation. Further, blockade of Dot1L prevents RIPK1 (receptor-interacting protein kinase 1)-dependent apoptosis through increased RIPK1 K63-ubiquitylation and decreased formation of RIPK1/Caspase 8 complexes. In line with this, H3K79me3 enrichment in the promoter region of deubiquitin-modifying enzyme A20 and deubiquitinase cylindromatosis gene promotes the increasing expression in oxygen-glucose deprivation followed by reoxygenation -induced neuronal cells, on the contrary, oxygen-glucose deprivation followed by reoxygenation decreases H3K79me3 level in the promoter region of ubiquitin-modifying enzyme cIAP1 (cellular inhibitors of apoptosis proteins), and both these factors ultimately cause K63-deubiquitination of RIPK1. Importantly, knockdown or inhibition of Dot1L in vivo attenuates apoptosis in middle cerebral artery occlusion mice and reduces the extent of middle cerebral artery occlusion -induced brain injury. Conclusions These data support for the first time, to our knowledge, that Dot1L regulating RIPK1 to the apoptotic death trigger contributes to cerebral ischemia injury. Therefore, targeting Dot1L serves as a new therapeutic strategy for ischemia stroke.