MLKL Mediated Necroptosis Accelerates JEV-Induced Neuroinflammation in Mice.
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ABSTRACT:
SUBMITTER: Bian P
PROVIDER: S-EPMC5328978 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Methamphetamine is one of the most prevalent drugs abused in the world. Methamphetamine abusers usually present with hyperpyrexia (39°C), hallucination and other psychiatric symptoms. However, the detailed mechanism underlying its neurotoxic action remains elusive. This study investigated the effects of methamphetamine + 39°C on primary cortical neurons from the cortex of embryonic Sprague-Dawley rats. Primary cortex neurons were exposed to 1 mM methamphetamine + 39°C. Propidium iodide staining and lactate dehydrogenase release detection showed that methamphetamine + 39°C triggered obvious necrosis-like death in cultured primary cortical neurons, which could be partially inhibited by receptor-interacting protein-1 (RIP1) inhibitor Necrostatin-1 partially. Western blot assay results showed that there were increases in the expressions of receptor-interacting protein-3 (RIP3) and mixed lineage kinase domain-like protein (MLKL) in the primary cortical neurons treated with 1 mM methamphetamine + 39°C for 3 hours. After pre-treatment with RIP3 inhibitor GSK'872, propidium iodide staining and lactate dehydrogenase release detection showed that neuronal necrosis rate was significantly decreased; RIP3 and MLKL protein expression significantly decreased. Immunohistochemistry staining results also showed that the expressions of RIP3 and MLKL were up-regulated in brain specimens from humans who had died of methamphetamine abuse. Taken together, the above results suggest that methamphetamine + 39°C can induce RIP3/MLKL regulated necroptosis, thereby resulting in neurotoxicity. The study protocol was approved by the Medical Ethics Committee of the Third Xiangya Hospital of Central South University, China (approval numbers: 2017-S026 and 2017-S033) on March 7, 2017.
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Project description:Mixed lineage kinase domain-like protein (Mlkl) was recently found to interact with receptor interacting protein 3 (Rip3) and to be essential for tumor necrosis factor (TNF)-induced programmed necrosis (necroptosis) in cultured cell lines. We have generated Mlkl-deficient mice by transcription activator-like effector nucleases (TALENs)-mediated gene disruption and found Mlkl to be dispensable for normal mouse development as well as immune cell development. Mlkl-deficient mouse embryonic fibroblasts (MEFs) and macrophages both showed resistance to necrotic but not apoptotic stimuli. Mlkl-deficient MEFs and macrophages were indistinguishable from wild-type cells in their ability to activate NF-κB, ERK, JNK, and p38 in response to TNF and lipopolysaccharides (LPS), respectively. Consistently, Mlkl-deficient macrophages and mice exhibited normal interleukin-1β (IL-1β), IL-6, and TNF production after LPS treatment. Mlkl deficiency protects mice from cerulean-induced acute pancreatitis, a necrosis-related disease, but has no effect on polymicrobial septic shock-induced animal death. Our results provide genetic evidence for the role of Mlkl in necroptosis.
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Project description:The pseudokinase domain of the necroptosis effector mixed lineage kinase domain-like (MLKL) functions as a latch to restrain the unleashing of its N-terminal 4-helix bundle (4HB) domain. Cell death mediated by the 4HB domain relies on membrane association and oligomerization, which can be inhibited by an ATP-mimetic small molecule that binds the pseudokinase domain of MLKL.
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Project description:Crystals cause injury in numerous disorders, and induce inflammation via the NLRP3 inflammasome, however, it remains unclear how crystals induce cell death. Here we report that crystals of calcium oxalate, monosodium urate, calcium pyrophosphate dihydrate and cystine trigger caspase-independent cell death in five different cell types, which is blocked by necrostatin-1. RNA interference for receptor-interacting protein kinase 3 (RIPK3) or mixed lineage kinase domain like (MLKL), two core proteins of the necroptosis pathway, blocks crystal cytotoxicity. Consistent with this, deficiency of RIPK3 or MLKL prevents oxalate crystal-induced acute kidney injury. The related tissue inflammation drives TNF-?-related necroptosis. Also in human oxalate crystal-related acute kidney injury, dying tubular cells stain positive for phosphorylated MLKL. Furthermore, necrostatin-1 and necrosulfonamide, an inhibitor for human MLKL suppress crystal-induced cell death in human renal progenitor cells. Together, TNF-?/TNFR1, RIPK1, RIPK3 and MLKL are molecular targets to limit crystal-induced cytotoxicity, tissue injury and organ failure.
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Project description:Mixed lineage kinase-like protein (MLKL) forms amyloid-like polymers to promote necroptosis; however, the mechanism through which these polymers trigger cell death is not clear. We have determined that activated MLKL translocates to the lysosomal membrane during necroptosis induction. The subsequent polymerization of MLKL induces lysosome clustering and fusion and eventual lysosomal membrane permeabilization (LMP). This LMP leads to the rapid release of lysosomal contents into the cytosol, resulting in a massive surge in cathepsin levels, with Cathepsin B (CTSB) as a significant contributor to the ensuing cell death as it cleaves many proteins essential for cell survival. Importantly, chemical inhibition or knockdown of CTSB protects cells from necroptosis. Furthermore, induced polymerization of the MLKL N-terminal domain (NTD) also triggers LMP, leading to CTSB release and subsequent cell death. These findings clearly establish the critical role of MLKL polymerization induced lysosomal membrane permeabilization (MPI-LMP) in the process of necroptosis.
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Project description:Necroptosis is a programmed cell death pathway triggered by activation of receptor interacting protein kinase 3 (RIPK3), which phosphorylates and activates the mixed lineage kinase-like domain pseudokinase, MLKL, to rupture or permeabilize the plasma membrane. Necroptosis is an inflammatory pathway associated with multiple pathologies including autoimmunity, infectious and cardiovascular diseases, stroke, neurodegeneration, and cancer. Here, we describe protocols that can be used to characterize MLKL as the executioner of plasma membrane rupture in necroptosis. We visualize the process of necroptosis in cells using live-cell imaging with conventional and confocal fluorescence microscopy, and in fixed cells using electron microscopy, which together revealed the redistribution of MLKL from the cytosol to the plasma membrane prior to induction of large holes in the plasma membrane. We present in vitro nuclear magnetic resonance (NMR) analysis using lipids to identify putative modulators of MLKL-mediated necroptosis. Based on this method, we identified quantitative lipid-binding preferences and phosphatidyl-inositol phosphates (PIPs) as critical binders of MLKL that are required for plasma membrane targeting and permeabilization in necroptosis.
| S-EPMC6126679 | biostudies-literature
Project description:Necroptosis is a mechanism by which cells can kill themselves that does not require caspase activity or the presence of the pro-apoptotic Bcl-2 family members Bax or Bak. It has been reported that RIPK3 (receptor interacting protein kinase 3) activates MLKL (mixed lineage kinase domain-like) to cause cell death that requires dynamin-related protein 1 (Drp1), because survival was increased in cells depleted of Drp1 or treated with the Drp1 inhibitor mdivi-1. To analyze necroptosis in a system that does not require addition of tumor necrosis factor (TNF), we used a construct that allows RIPK3 to be induced in cells, and then dimerized via an E. coli gyrase domain fused to its carboxyl-terminus, using the dimeric gyrase binding antibiotic coumermycin. We have previously shown elsewhere that RIPK3 dimerized in this manner not only induces necroptosis but also apoptosis, which can be inhibited by the broad-spectrum caspase inhibitor Q-VD-OPh (QVD). In response to RIPK3 dimerization, wild-type mouse embryonic fibroblasts (MEFs) underwent cell death that was reduced but not completely blocked by QVD. In contrast, death upon dimerization of RIPK3 in Mlkl(-/-) MEFs was completely inhibited with QVD, confirming that MLKL is required for necroptosis. Similar to wild-type MEFs, most Drp1(-/-) MEFs died when RIPK3 was activated, even in the presence of QVD. Furthermore, overexpression of wild-type MLKL or dominant active mutants of MLKL (Q343A or S345E/S347E) caused death of wild-type and Drp1(-/-) MEFs that was not inhibited with QVD. These results indicate that necroptosis caused by RIPK3 requires MLKL but not Drp1.
| S-EPMC3944236 | biostudies-literature
Project description:Defective production of antiviral interferon (IFN)-β is thought to contribute to rhinovirus-induced asthma exacerbations. These exacerbations are associated with elevated lung levels of lactate dehydrogenase (LDH), indicating occurrence of cell necrosis. We thus hypothesized that reduced lung IFN-β could contribute to necrotic cell death in a model of asthma exacerbations. Wild-type and IFN-β-/- mice were given saline or house dust mite (HDM) intranasally for 3 weeks to induce inflammation. Double-stranded RNA (dsRNA) was then given for additional 3 days to induce exacerbation. HDM induced an eosinophilic inflammation, which was not associated with increased expression of cleaved caspase-3, cleaved PARP or elevated bronchoalveolar lavage fluid (BALF) LDH levels in wild-type. However, exacerbation evoked by HDM + dsRNA challenges increased BALF levels of LDH, apoptotic markers and the necroptotic markers receptor-interacting protein (RIP)-3 and phosphorylation of mixed linage kinase domain-like protein (pMLKL), compared to HDM + saline. Absence of IFN-β at exacerbation further increased BALF LDH and protein expression of pMLKL compared to wild-type. We demonstrate that cell death markers are increased at viral stimulus-induced exacerbation in mouse lungs, and that absence of IFN-β augments markers of necroptotic cell death at exacerbation. Our data thus suggest a novel role of deficient IFN-β production at viral-induced exacerbation.
| S-EPMC5844912 | biostudies-literature
Project description:Mixed lineage kinase domain-like (MLKL) is the executioner in the caspase-independent form of programmed cell death called necroptosis. Receptor-interacting serine/threonine protein kinase 3 (RIPK3) phosphorylates MLKL, triggering MLKL oligomerization, membrane translocation and membrane disruption. MLKL also undergoes ubiquitylation during necroptosis, yet neither the mechanism nor the significance of this event has been demonstrated. Here we show that necroptosis-specific multi-mono-ubiquitylation of MLKL occurs following its activation and oligomerization. Ubiquitylated MLKL accumulates in a digitonin-insoluble cell fraction comprising organellar and plasma membranes and protein aggregates. Appearance of this ubiquitylated MLKL form can be reduced by expression of a plasma membrane-located deubiquitylating enzyme. Oligomerization-induced MLKL ubiquitylation occurs on at least four separate lysine residues, and correlates with its proteasome- and lysosome-dependent turnover. Using a MLKL-DUB fusion strategy, we show that constitutive removal of ubiquitin from MLKL licences MLKL auto-activation independent of necroptosis signalling in mouse and human cells. Therefore, in addition to the role of ubiquitylation in the kinetic regulation of MLKL-induced death following an exogenous necroptotic stimulus, it also contributes to restraining basal levels of activated MLKL to avoid unwanted cell death.
| S-SCDT-EMBOJ-2019-103718 | biostudies-other
Project description:Receptor interacting protein kinase 3 (RIPK3)-mediated smooth muscle cell (SMC) necroptosis has been shown to contribute to the pathogenesis of abdominal aortic aneurysms (AAAs). However, the signaling steps downstream from RIPK3 during SMC necroptosis remain unknown. In this study, the roles of mixed lineage kinase domain-like pseudokinase (MLKL) and calcium/calmodulin-dependent protein kinase II (CaMKII) in SMC necroptosis were investigated. We found that both MLKL and CaMKII were phosphorylated in SMCs in a murine CaCl2-driven model of AAA and that Ripk3 deficiency reduced the phosphorylation of MLKL and CaMKII. In vitro, mouse aortic SMCs were treated with tumor necrosis factor α (TNFα) plus Z-VAD-FMK (zVAD) to induce necroptosis. Our data showed that both MLKL and CaMKII were phosphorylated after TNFα plus zVAD treatment in a time-dependent manner. SiRNA silencing of Mlkl-diminished cell death and administration of the CaMKII inhibitor myristoylated autocamtide-2-related inhibitory peptide (Myr-AIP) or siRNAs against Camk2d partially inhibited necroptosis. Moreover, knocking down Mlkl decreased CaMKII phosphorylation, but silencing Camk2d did not affect phosphorylation, oligomerization, or trafficking of MLKL. Together, our results indicate that both MLKL and CaMKII are involved in RIPK3-mediated SMC necroptosis, and that MLKL is likely upstream of CaMKII in this process.
| S-EPMC8471540 | biostudies-literature