Necrosome complex detected in granulovacuolar degeneration is associated with neuronal loss in Alzheimer's disease.

Alzheimer's disease (AD) is characterized by a specific pattern of neuropathological changes, including extracellular amyloid ß (Aß) deposits, intracellular neurofibrillary tangles (NFTs), granulovacuolar degeneration (GVD) representing cytoplasmic vacuolar lesions, synapse dysfunction and neuronal loss. Necroptosis, a programmed form of necrosis characterized by the assembly of the necrosome complex composed of phosphorylated proteins, i.e. receptor-interacting serine/threonine-protein kinase 1 and 3 (pRIPK1 and pRIPK3) and mixed lineage kinase domain-like protein (pMLKL), has recently been shown to be involved in AD. However, it is not yet clear whether necrosome assembly takes place in brain regions showing AD-related neuronal loss and whether it is associated with AD-related neuropathological changes. Here, we analyzed brains of AD, pathologically defined preclinical AD (p-preAD) and non-AD control cases to determine the neuropathological characteristics and distribution pattern of the necrosome components. We demonstrated that all three activated necrosome components can be detected in GVD lesions (GVDn+, i.e. GVD with activated necrosome) in neurons, that they colocalize with classical GVD markers, such as pTDP-43 and CK1δ, and similarly to these markers detect GVD lesions. GVDn + neurons inversely correlated with neuronal density in the early affected CA1 region of the hippocampus and in the late affected frontal cortex layer III. Additionally, AD-related GVD lesions were associated with AD-defining parameters, showing the strongest correlation and partial colocalization with NFT pathology. Therefore, we conclude that the presence of the necrosome in GVD plays a role in AD, possibly by representing an AD-specific form of necroptosis-related neuron death. Hence, necroptosis-related neuron loss could be an interesting therapeutic target for treating AD.

Post-ischemic salubrinal administration reduces necroptosis in a rat model of global cerebral ischemia.

Ischemic stroke is one of the most important causes of death and disability worldwide. Subroutines underlying cell death after stroke are largely unknown despite their importance in the design of novel therapies for this pathology. Necroptosis, a recently described form of regulated cell death, has been related with inflammation and, in some models, with endoplasmic reticulum (ER) stress. We hypothesize that alleviation of ER stress following a salubrinal treatment will reduce the ischemic-dependent necroptosis. To probe the hypothesis, we measured, at 48 and 72 h after transient global cerebral ischemia in rat, in cerebral cortex and cornu ammonis 1, the main hallmarks of necroptosis: mRNA levels and phosphorylation of mixed lineage kinase domain like pseudokinase as well as receptor interacting serine/threonine protein kinase 3, along the years 2017-2018. Selective neuronal loss after 7 days of the ischemic insult, and other markers related with the inflammatory response were also measured. This study shows that necroptosis in cerebral cortex can be detected after 72 h of the insult and seems to be elicited before 48 h of reperfusion. The type of necroptosis here observed seems to be tumor necrosis factor receptor 1 independent. Necroptotic response is less evident in the cornu ammonis 1 hippocampal area than in cerebral cortex. The treatment with salubrinal administered 1 and 24 h after the ischemia, decreased the necroptotic marker levels and reduced the areas of selective neuronal loss, supporting the presence of ischemic-dependent necroptosis, and the notion that ER stress is involved in the necroptotic response. Open Science: This manuscript was awarded with the Open Materials Badge For more information see: https://cos.io/our-services/open-science-badges/.

Sodium sulfite triggered hepatic apoptosis, necroptosis, and pyroptosis by inducing mitochondrial damage in mice and AML-12 cells.

Sodium sulfite (SS) is a biological derivative of the air pollutant sulfur dioxide, and is often used as a food and pharmaceutical additive. Improper or excessive SS exposure in liver cell death. The phenomenon of simultaneous regulation of apoptosis, necroptosis, and pyroptosis is defined as PANoptosis. However, the specific types of programmed cell death (PCD) caused by SS and their interconnections remain unclear. In the present study, C57BL/6 mice were orally administered SS for 30 d, consecutively, to establish an in vivo mouse exposure model. AML-12 cells were treated with SS for 24 h to establish an in vitro exposure model. The results showed that SS-induced mitochondrial reactive oxygen species (mtROS) accumulation activated the BAX/Bcl-2/caspase 3 pathway to trigger apoptosis and RIPK1/RIPK3/p-MLKL to trigger necroptosis. Interestingly, ROS-activated p-MLKL perforated not the cell membrane as well as the lysosomal membrane. We determined that p-MLKL mediates lysosomal membrane permeabilization (LMP), resulting in cathepsin B (CTSB) release. Furthermore, knockdown of MLKL, a CTSB inhibitor (CA074-ME) and an NLRP3 inhibitor (MCC950) alleviated SS-induced pyroptosis. In summary, our study showed that SS induced apoptosis and necroptosis though mtROS accumulation, whereas the activation of p-MLKL mediated NLRP3-dependent pyroptosis by causing CTSB leakage through LMP. This study comprehensively explored the mechanism unerlying SS-induced PCD and provided an experimental basis for p-MLKL as a potential regulatory protein in PANoptosis.

Hypoxic Preconditioning Attenuates Neuroinflammation via Inhibiting NF-κB/NLRP3 Axis Mediated by p-MLKL after Transient Global Cerebral Ischemia.

Hypoxic preconditioning (HPC) has been reported to alleviate neuronal damage and microglial activation in hippocampal CA1 after transient global cerebral ischemia (tGCI). However, the molecular mechanism is unclear. Recent studies identified that nuclear factor-kappa-B (NF-κB)/oligomerization domain-like receptors protein (NLRP) 3 inflammasome pathway is mainly involved in the activation of microglia and that phosphorylated (p)-mixed lineage kinase domain-like (MLKL) is related to the regulation of NF-κB/NLRP3 axis. Hence, in this study, we set out to investigate whether HPC attenuates neuronal damage and microglial activation through inhibiting NF-κB/NLRP3 axis mediated by p-MLKL after tGCI in CA1 of male rats. We found that HPC decreased NLRP3 inflammasome in microglia and inhibited M1 polarization of microglia in CA1 after tGCI. Mechanistically, HPC inhibited the activation of NF-κB signaling pathway and reduced the mRNA and protein levels of NLRP3 inflammasome after tGCI. Additionally, the knockdown of p-MLKL by short hairpin RNA (shRNA) administration inhibited the activation of the NF-κB signaling pathway and reduced the formation of NLRP3 inflammasome, thus attenuating M1 polarization of microglia and decreasing the release of interleukin 1 beta (IL-1ß) and necrosis factor alpha (TNF-α) in CA1 post ischemia. We consider that p-MLKL in microglia may be derived from necroptotic neurons after tGCI. In conclusion, the new finding in this study is that HPC-induced neuroprotection against tGCI through inhibiting NF-κB/NLRP3 pathway mediated by p-MLKL.

RIPK1 and RIPK3 are positive prognosticators for cervical cancer patients and C2 ceramide can inhibit tumor cell proliferation in vitro.

Introduction: The enzymes Receptor-interacting serine/threonine-protein kinase 1 (RIPK1) und 3 (RIPK3) as well as the protein Mixed lineage kinase domain like pseudokinase (pMLKL) play a role in the signaling cascade of necroptosis. This is a form of programmed cell death which is caspase-independent. High-risk human papilloma virus infection can inhibit necroptosis. Thereby, a persistent infection and consequently the development of cervical cancer can be triggered. Aim of this study was the analysis of the expression of RIPK1, RIPK3 and pMLKL in cervical cancer tissue and the evaluation of its prognostic value on overall survival, progression-free survival and additional clinical parameters. Methods: The expression of RIPK1, RIPK3, and pMLKL in cervical cancer tissue microarrays of n = 250 patients was analyzed immunohistochemically. Further, the effect of C2 ceramide on several cervical cancer cell lines (CaSki, HeLa, SiHa) was examined. C2 ceramide is a biologically active short-chain ceramide that induces necroptosis in human luteal granulosa cells. Results: Significantly longer overall survival and progression-free survival rates could be detected in cervical cancer patients expressing nuclear RIPK1 or RIPK3 alone or simultaneously (RIPK1 and RIPK3). Cell viability and proliferation was reduced through C2 ceramide stimulation of cervical cancer cells. Simultaneous stimulation of C2 ceramide and the pan-caspase inhibitor Z-VAD-fmk, or the RIPK1-inhibitor necrostatin-1, partly reversed the negative effect of C2 ceramide on cell viability. This observation could imply that caspase-dependent and -independent forms of cell death, including necroptosis, can occur. AnnexinV-FITC apoptosis staining induced a significant increase in apoptotic cells in CaSki and SiHa cells. The stimulation of CaSki cells with C2 ceramide led to a significant percentual increase in necrotic/intermediate (dying) cells after stimulation with C2 ceramide. In addition, after stimulation with C2 ceramide, CaSki and HeLa cells live cell imaging showed morphological changes which are common for necroptosis. Discussion: In conclusion, RIPK1 and RIPK3 are independent positive predictors for overall survival and progression-free survival in cervical cancer patients. C2 ceramide can reduce cell viability and proliferation in cervical cancer cells by inducing most likely both apoptosis and necroptosis.

Baicalin inhibits necroptosis by decreasing oligomerization of phosphorylated MLKL and mitigates caerulein-induced acute pancreatitis in mice.

Necroptosis is a form of regulated necrosis mainly controlled by receptor-interacting protein kinases 3 (RIPK3) and mixed lineage kinase domain-like protein (MLKL). Necroptosis has important roles in defensing against pathogenic infections, but it is also implicated in various inflammatory diseases including pancreatitis. Baicalin, a flavonoid from Scutellaria baicalensis Georgi, has been shown to possess anti-inflammatory and anti-pyroptosis properties, yet it is unclear whether baicalin can inhibit necroptosis and confer protection against necroptosis-related diseases. Here we reported that baicalin significantly inhibited necroptosis in macrophages induced by lipopolysaccharide plus pan-caspase inhibitor (IDN-6556), or by tumor-necrosis factor-α in combination with LCL-161 (Smac mimetic) and IDN-6556 (TSI). Mechanistically, baicalin did not inhibit the phosphorylation of RIPK1, RIPK3 and MLKL, nor membrane translocation of p-MLKL, during necroptotic induction, but instead inhibited p-MLKL oligomerization that is required for executing necroptosis. As intracellular reactive oxygen species (ROS) has been reported to be involved in p-MLKL oligomerization, we assessed the effects of N-acetyl-L-cysteine (NAC), an ROS scavenger, on necroptosis and found that NAC significantly attenuated TSI-induced necroptosis and intracellular ROS production concomitantly with reduced levels of oligomerized p-MLKL, mirroring the effect of baicalin. Indeed, inhibitory effect of baicalin was associated with reduced TSI-induced superoxide (indicating mitochondrial ROS) production and increased mitochondrial membrane potential within cells during necroptosis. Besides, oral administration of baicalin significantly reduced the severity of caerulein-induced acute pancreatitis in mice, an animal model of necroptosis-related disease. Collectively, baicalin can inhibit necroptosis through attenuating p-MLKL oligomerization and confers protection against caerulein-induced pancreatitis in mice.

LATE-NC aggravates GVD-mediated necroptosis in Alzheimer's disease.

It has become evident that Alzheimer's Disease (AD) is not only linked to its hallmark lesions-amyloid plaques and neurofibrillary tangles (NFTs)-but also to other co-occurring pathologies. This may lead to synergistic effects of the respective cellular and molecular players, resulting in neuronal death. One of these co-pathologies is the accumulation of phosphorylated transactive-response DNA binding protein 43 (pTDP-43) as neuronal cytoplasmic inclusions, currently considered to represent limbic-predominant age-related TDP-43 encephalopathy neuropathological changes (LATE-NC), in up to 70% of symptomatic AD cases. Granulovacuolar degeneration (GVD) is another AD co-pathology, which also contains TDP-43 and other AD-related proteins. Recently, we found that all proteins required for necroptosis execution, a previously defined programmed form of neuronal cell death, are present in GVD, such as the phosphorylated necroptosis executioner mixed-lineage kinase domain-like protein (pMLKL). Accordingly, this protein is a reliable marker for GVD lesions, similar to other known GVD proteins. Importantly, it is not yet known whether the presence of LATE-NC in symptomatic AD cases is associated with necroptosis pathway activation, presumably contributing to neuron loss by cell death execution. In this study, we investigated the impact of LATE-NC on the severity of necroptosis-associated GVD lesions, phosphorylated tau (pTau) pathology and neuronal density. First, we used 230 human post-mortem cases, including 82 controls without AD neuropathological changes (non-ADNC), 81 non-demented cases with ADNC, i.e.: pathologically-defined preclinical AD (p-preAD) and 67 demented cases with ADNC. We found that Braak NFT stage and LATE-NC stage were good predictors for GVD expansion and neuronal loss in the hippocampal CA1 region. Further, we compared the impact of TDP-43 accumulation on hippocampal expression of pMLKL-positive GVD, pTau as well as on neuronal density in a subset of nine non-ADNC controls, ten symptomatic AD cases with (ADTDP+) and eight without LATE-NC (ADTDP-). Here, we observed increased levels of pMLKL-positive, GVD-exhibiting neurons in ADTDP+ cases, compared to ADTDP- and controls, which was accompanied by augmented pTau pathology. Neuronal loss in the CA1 region was increased in ADTDP+ compared to ADTDP- cases. These data suggest that co-morbid LATE-NC in AD impacts not only pTau pathology but also GVD-mediated necroptosis pathway activation, which results in an accelerated neuronal demise. This further highlights the cumulative and synergistic effects of comorbid pathologies leading to neuronal loss in AD. Accordingly, protection against necroptotic neuronal death appears to be a promising therapeutic option for AD and LATE.

Boosting mitochondria activity by silencing MCJ overcomes cholestasis-induced liver injury.

BACKGROUND & AIMS: Mitochondria are the major organelles for the formation of reactive oxygen species (ROS) in the cell, and mitochondrial dysfunction has been described as a key factor in the pathogenesis of cholestatic liver disease. The methylation-controlled J-protein (MCJ) is a mitochondrial protein that interacts with and represses the function of complex I of the electron transport chain. The relevance of MCJ in the pathology of cholestasis has not yet been explored. METHODS: We studied the relationship between MCJ and cholestasis-induced liver injury in liver biopsies from patients with chronic cholestatic liver diseases, and in livers and primary hepatocytes obtained from WT and MCJ-KO mice. Bile duct ligation (BDL) was used as an animal model of cholestasis, and primary hepatocytes were treated with toxic doses of bile acids. We evaluated the effect of MCJ silencing for the treatment of cholestasis-induced liver injury. RESULTS: Elevated levels of MCJ were detected in the liver tissue of patients with chronic cholestatic liver disease when compared with normal liver tissue. Likewise, in mouse models, the hepatic levels of MCJ were increased. After BDL, MCJ-KO animals showed significantly decreased inflammation and apoptosis. In an in vitro model of bile-acid induced toxicity, we observed that the loss of MCJ protected mouse primary hepatocytes from bile acid-induced mitochondrial ROS overproduction and ATP depletion, enabling higher cell viability. Finally, the in vivo inhibition of the MCJ expression, following BDL, showed reduced liver injury and a mitigation of the main cholestatic characteristics. CONCLUSIONS: We demonstrated that MCJ is involved in the progression of cholestatic liver injury, and our results identified MCJ as a potential therapeutic target to mitigate the liver injury caused by cholestasis. LAY SUMMARY: In this study, we examine the effect of mitochondrial respiratory chain inhibition by MCJ on bile acid-induced liver toxicity. The loss of MCJ protects hepatocytes against apoptosis, mitochondrial ROS overproduction, and ATP depletion as a result of bile acid toxicity. Our results identify MCJ as a potential therapeutic target to mitigate liver injury in cholestatic liver diseases.