Rotavirus non-structural protein 4 usurps host cellular RIPK1-RIPK3 complex to induce MLKL-dependent necroptotic cell death.

The dynamic interface between invading viral pathogens and programmed cell death (PCD) of the host is a finely regulated process. Host cellular demise at the end of the viral life cycle ensures the release of progeny virions to initiate new infection cycles. Rotavirus (RV), a diarrheagenic virus with double-stranded RNA genome, has been reported to trigger different types of PCD such as apoptosis and pyroptosis in a highly regulated way to successfully disseminate progeny virions. Recently our lab also showed that induction of MLKL-driven programmed necroptosis by RV. However, the host cellular machinery involved in RV-induced necroptosis and the upstream viral trigger responsible for it remained unaddressed. In the present study, the signalling upstream of MLKL-driven necroptosis has been delineated where the involvement of Receptor interacting serine/threonine kinase 3 (RIPK3) and 1 (RIPK1) from the host side and RV non-structural protein 4 (NSP4) as the viral trigger for necroptosis has been shown. Interestingly, RV-NSP4 was found to be an integral component of the necrosome complex by interacting with RIPK1, thereby bypassing the requirement of RIPK1 kinase activity. Subsequently, NSP4-driven elevated cytosolic Ca2+ concentration and Ca2+-binding to NSP4 lead further to RHIM domain-dependent RIPK1-RIPK3 interaction, RIPK3-dependent MLKL phosphorylation, and eventual necroptosis. Overall, this study presents the interplay between RV-NSP4 and the host cellular necrosome complex to induce necroptotic death of host cells.

Atezolizumab Induces Necroptosis and Contributes to Hepatotoxicity of Human Hepatocytes.

Atezolizumab is an immune checkpoint inhibitor (ICI) targeting PD-L1 for treatment of solid malignancies. Immune checkpoints control the immune tolerance, and the adverse events such as hepatotoxicity induced by ICIs are often considered as an immune-related adverse event (irAE). However, PD-L1 is also highly expressed in normal tissues, e.g., hepatocytes. It is still not clear whether, targeting PD-L1 on hepatocytes, the atezolizumab may cause damage to liver cells contributing to hepatotoxicity. Here, we reveal a novel mechanism by which the atezolizumab induces hepatotoxicity in human hepatocytes. We find that the atezolizumab treatment increases a release of LDH in the cell culture medium of human hepatocytes (human primary hepatocytes and THLE-2 cells), decreases cell viability, and inhibits the THLE-2 and THLE-3 cell growth. We demonstrate that both the atezolizumab and the conditioned medium (T-CM) derived from activated T cells can induce necroptosis of the THLE-2 cells, which is underscored by the fact that the atezolizumab and T-CM enhance the phosphorylation of RIP3 and MLKL proteins. Furthermore, we also show that necrostatin-1, a necrosome inhibitor, decreases the amount of phosphorylated RIP3 induced by the atezolizumab, resulting in a reduced LDH release in the culture media of the THLE-2 cells. This finding is further supported by the data that GSK872 (a RIP3 inhibitor) significantly reduced the atezolizumab-induced LDH release. Taken together, our data indicate that the atezolizumab induces PD-L1-mediated necrosome formation, contributing to hepatotoxicity in PD-L1+-human hepatocytes. This study provides the molecular basis of the atezolizumab-induced hepatotoxicity and opens a new avenue for developing a novel therapeutic approach to reducing hepatotoxicity induced by ICIs.

Viral-induced neuronal necroptosis: Detrimental to brain function and regulation by necroptosis inhibitors.

Neuronal necroptosis (programmed necrosis) in the CNS naturally occurs through a caspase-independent way and, especially in neurodegenerative diseases (NDDs) such as Alzheimer's disease (AD), Parknson's disease (PD), Amyotrophic Lateral Sclerosis (ALS) and viral infections. Understanding necroptosis pathways (death receptor-dependent and independent), and its connections with other cell death pathways could lead to new insights into treatment. Receptor-interacting protein kinase (RIPK) mediates necroptosis via mixed-lineage kinase-like (MLKL) proteins. RIPK/MLKL necrosome contains FADD, procaspase-8-cellular FLICE-inhibitory proteins (cFLIPs), RIPK1/RIPK3, and MLKL. The necrotic stimuli cause phosphorylation of MLKL and translocate to the plasma membrane, causing an influx of Ca2+ and Na+ ions and, the immediate opening of mitochondrial permeability transition pore (mPTP) with the release of inflammatory cell damage-associated molecular patterns (DAMPs) like mitochondrial DNA (mtDNA), high-mobility group box1 (HMGB1), and interleukin1 (IL-1). The MLKL translocates to the nucleus to induce transcription of the NLRP3 inflammasome complex elements. MLKL-induced NLRP3 activity causes caspase-1 cleavage and, IL-1 activation which promotes neuroinflammation. RIPK1-dependent transcription increases illness-associated microglial and lysosomal abnormalities to facilitate amyloid plaque (Aß) aggregation in AD. Recent research has linked neuroinflammation and mitochondrial fission with necroptosis. MicroRNAs (miRs) such as miR512-3p, miR874, miR499, miR155, and miR128a regulate neuronal necroptosis by targeting key components of necroptotic pathways. Necroptosis inhibitors act by inhibiting the membrane translocation of MLKL and RIPK1 activity. This review insights into the RIPK/MLKL necrosome-NLRP3 inflammasome interactions during death receptor-dependent and independent neuronal necroptosis, and clinical intervention by miRs to protect the brain from NDDs.

Celastrol inhibits necroptosis by attenuating the RIPK1/RIPK3/MLKL pathway and confers protection against acute pancreatitis in mice.

Necroptosis is a necrotic form of regulated cell death, which is primarily mediated by the receptor-interacting protein kinase 1 (RIPK1), RIPK3, and mixed lineage kinase domain-like (MLKL) pathway in a caspase-independent manner. Necroptosis has been found to occur in virtually all tissues and diseases evaluated, including pancreatitis. Celastrol, a pentacyclic triterpene extracted from the roots of Tripterygium wilfordii (thunder god vine), possesses potent anti-inflammatory and anti-oxidative activities. Yet, it is unclear whether celastrol has any effects on necroptosis and necroptotic-related diseases. Here we showed that celastrol significantly suppressed necroptosis induced by lipopolysaccharide (LPS) plus pan-caspase inhibitor (IDN-6556) or by tumor-necrosis factor-α in combination with LCL-161 (Smac mimetic) and IDN-6556 (TSI). In these in vitro cellular models, celastrol inhibited the phosphorylation of RIPK1, RIPK3, and MLKL and the formation of necrosome during necroptotic induction, suggesting its possible action on upstream signaling of the necroptotic pathway. Consistent with the known role of mitochondrial dysfunction in necroptosis, we found that celastrol significantly rescued TSI-induced loss of mitochondrial membrane potential. TSI-induced intracellular and mitochondrial reactive oxygen species (mtROS), which are involved in the autophosphorylation of RIPK1 and recruitment of RIPK3, were significantly attenuated by celastrol. Moreover, in a mouse model of acute pancreatitis that is associated with necroptosis, celastrol administration significantly reduced the severity of caerulein-induced acute pancreatitis accompanied by decreased phosphorylation of MLKL in pancreatic tissues. Collectively, celastrol can attenuate the activation of RIPK1/RIPK3/MLKL signaling likely by attenuating mtROS production, thereby inhibiting necroptosis and conferring protection against caerulein-induced pancreatitis in mice.

Necrostatin-1S mitigates type-2 diabetes-associated cognitive decrement and lipotoxicity-induced neuro-microglia changes through p-RIPK-RIPK3-p-MLKL axis.

Type-2 diabetes mellitus (T2DM) is associated with neuroinflammation and cognitive decrement. Necroptosis programmed necrosis is emerging as the major contributing factor to central changes. It is best characterized by the upregulation of p-RIPK(Receptor Interacting Kinase), p-RIPK3, and the phosphorylated-MLKL (mixed-lineage kinase domain-like protein). The present study aims to evaluate the neuroprotective effect of Necrostatin (Nec-1S), a p-RIPK inhibitor, on cognitive changes in the experimental T2DM model in C57BL/6 mice and lipotoxicity-induced neuro-microglia changes in neuro2A and BV2 cells. Further, the study also explores whether Nec-1S would restore mitochondrial and autophago-lysosomal function.T2DM was developed in mice by feeding them a high-fat diet (HFD) for 16 weeks and injecting a single dose of streptozotocin (100 mg/kg, i.p) on the 12th week. Nec-1S was administered for 3 weeks at (10 mg/kg, i.p) once every 3 days. Lipotoxicity was induced in neuro2A, and BV2 cells using 200 µM palmitate/bovine serum albumin conjugate. Nec-1S (50 µM), and GSK-872(10 µM) were further used to explore their relative effect. The neurobehavioral performance was assessed using mazes and task-assisted performance tests. To decipher the hypothesis plasma parameters, western blot, immunofluorescence, microscopy, and quantitative reverse transcription-PCR studies were carried out. The Nec-1S treatment restored cognitive performance and reduced the p-RIPK-p-RIPK3-p-MLKL mediated neuro-microglia changes in the brain and in cells as well, under lipotoxic stress. Nec-1S reduced tau, and amyloid oligomer load. Moreover, Nec-1S restored mitochondrial function and autophago-lysosome clearance. The findings highlight the central impact of metabolic syndrome and how Nes-1S, by acting as a multifaceted agent, improved central functioning.

Rubiarbonol B induces RIPK1-dependent necroptosis via NOX1-derived ROS production.

The activation of receptor-interacting protein kinase 1 (RIPK1) by death-inducing signaling complex (DISC) formation is essential for triggering the necroptotic mode of cell death under apoptosis-deficient conditions. Thus, targeting the induction of necroptosis by modulating RIPK1 activity could be an effective strategy to bypass apoptosis resistance in certain types of cancer. In this study, we screened a series of arborinane triterpenoids purified from Rubia philippinesis and identified rubiarbonol B (Ru-B) as a potent caspase-8 activator that induces DISC-mediated apoptosis in multiple types of cancer cells. However, in RIPK3-expressing human colorectal cancer (CRC) cells, the pharmacological or genetic inhibition of caspase-8 shifted the mode of cell death by Ru-B from apoptosis to necroptosis though upregulation of RIPK1 phosphorylation. Conversely, Ru-B-induced cell death was almost completely abrogated by RIPK1 deficiency. The enhanced RIPK1 phosphorylation and necroptosis triggered by Ru-B treatment occurred independently of tumor necrosis factor receptor signaling and was mediated by the production of reactive oxygen species via NADPH oxidase 1 in CRC cells. Thus, we propose Ru-B as a novel anticancer agent that activates RIPK1-dependent cell death via ROS production, and suggest its potential as a novel necroptosis-targeting compound in apoptosis-resistant CRC.

Supramolecular organizing centers at the interface of inflammation and neurodegeneration.

The pathogenesis of neurodegenerative diseases involves the accumulation of misfolded protein aggregates. These deposits are both directly toxic to neurons, invoking loss of cell connectivity and cell death, and recognized by innate sensors that upon activation release neurotoxic cytokines, chemokines, and various reactive species. This neuroinflammation is propagated through signaling cascades where activated sensors/receptors, adaptors, and effectors associate into multiprotein complexes known as supramolecular organizing centers (SMOCs). This review provides a comprehensive overview of the SMOCs, involved in neuroinflammation and neurotoxicity, such as myddosomes, inflammasomes, and necrosomes, their assembly, and evidence for their involvement in common neurodegenerative diseases. We discuss the multifaceted role of neuroinflammation in the progression of neurodegeneration. Recent progress in the understanding of particular SMOC participation in common neurodegenerative diseases such as Alzheimer's disease offers novel therapeutic strategies for currently absent disease-modifying treatments.

Visualizing Endogenous Necrosomes in Necrosomes by In Situ Proximity Ligation Assay.

Necroptosis is a regulated form of necrosis that has been shown to participate in the pathogenesis of major human inflammatory and neurodegenerative diseases. Formation of a necrosome, composed of the RIPK1/RIPK3 complex, drives the execution of necroptosis. Although the co-immunoprecipitation (co-IP) assay has been widely used as a biochemical protocol for studying necrosomes, the technical limitations of co-IP prevent its use for identifying necrosomes in complex tissues and for investigating the subcellular localization of necrosomes. The development of a specific assay for visualizing necrosomes in situ is needed. Here, we developed an in situ proximity ligation assay (PLA), which converts the detection of protein-protein interaction to detection of DNA product by rolling-circle amplification for investigating the endogenous necrosome in situ and in tissues. This protocol describes an in situ PLA that we have developed for visualizing endogenous necrosomes in necroptosis in both human and mouse cells and in mouse embryos with sensitivity and specificity. © 2022 Wiley Periodicals LLC. Basic Protocol 1: Detection of RIPK1/RIPK3 interaction by in situ proximity ligation assay in human and mouse cells.

8-Geranylumbelliferone isolated from Paramignya trimera triggers RIPK1/RIPK3-dependent programmed cell death upon TNFR1 ligation.

In tumor necrosis factor (TNF) signaling, IκB kinase (IKK) complex-mediated activation of NF-κB is a well-known protective mechanism against cell death via transcriptional induction of pro-survival genes occurring as a late checkpoint. However, recent belief holds that IKK functions as an early cell death checkpoint to suppress the death-inducing signaling complex by regulating receptor interacting protein kinase1 (RIPK1) phosphorylation. In this study, we propose that two major gernaylated 7-hydroxy coumarins, 6-geranyl-7-hydroxycoumarin (ostruthin) and 8-geranyl-7-hydroxycoumarin (8-geranylumbelliferone, 8-GU) isolated from Paramignya timera, facilitate RIPK1-dependent dual modes of apoptosis and necroptosis by targeting IKKß upon TNF receptor1 (TNFR1) ligation. Analysis of events upstream of NF-κB revealed that 8-GU and ostruthin drastically inhibited TNF-induced IKK phosphorylation, while having no effect on TAK1 phosphorylation and TNFR1 complex-I formation. Interestingly, 8-GU did not affect the cell death induced by Fas ligand or TNF-related apoptosis-inducing ligand or that induced by DNA-damaging agents, indicating that 8-GU sensitizes TNF-induced cell death exclusively. Moreover, 8-GU accelerated TNF-driven necroptosis by up-regulating necrosome formation in FADD deficient cancer cells harboring RIPK3. Thus, the present study provides new insights into the molecular mechanism underlying geranylated 7-hydroxy coumarin-mediated control of the RIPK1-dependent early cell death checkpoint and suggests that 8-GU is a potential anti-cancer therapeutic via an alternative apoptosis-independent strategy to overcome TNF resistance.

TRIM21, a New Component of the TRAIL-Induced Endogenous Necrosome Complex.

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is a well-known apoptosis inducer and a potential anticancer agent. When caspases and inhibitors of apoptosis proteins (IAPs) are inhibited, TRAIL induces necroptosis. Molecular mechanisms of necroptosis rely on kinase activation, and on the formation of a necrosome complex, bringing together the receptor-interacting protein kinases 1 and 3 (RIPK1, RIPK3), and the mixed lineage kinase domain-like protein (MLKL). In this study, mass spectrometry approach allowed to identify the tripartite motif containing 21 (TRIM21), an E3 ubiquitin-protein ligase as a new partner of the endogenous TRAIL-induced necrosome. Alteration of TRIM21 expression level, obtained by transient transfection of HT29 or HaCat cells with TRIM21-targeted siRNAs or cDNA plasmids coding for TRIM21 demonstrated that TRIM21 is a positive regulator of TRAIL-induced necroptosis. Furthermore, the invalidation of TRIM21 expression in HT29 cells by CRISPR-Cas9 technology also decreased cell sensitivity to TRAIL-induced necroptosis, a shortcoming associated with a reduction in MLKL phosphorylation, the necroptosis executioner. Thus, TRIM21 emerged as a new partner of the TRAIL-induced necrosome that positively regulates the necroptosis process.

RIP1-dependent linear and nonlinear recruitments of caspase-8 and RIP3 respectively to necrosome specify distinct cell death outcomes.

There remains a significant gap in our quantitative understanding of crosstalk between apoptosis and necroptosis pathways. By employing the SWATH-MS technique, we quantified absolute amounts of up to thousands of proteins in dynamic assembling/de-assembling of TNF signaling complexes. Combining SWATH-MS-based network modeling and experimental validation, we found that when RIP1 level is below ~1000 molecules/cell (mpc), the cell solely undergoes TRADD-dependent apoptosis. When RIP1 is above ~1000 mpc, pro-caspase-8 and RIP3 are recruited to necrosome respectively with linear and nonlinear dependence on RIP1 amount, which well explains the co-occurrence of apoptosis and necroptosis and the paradoxical observations that RIP1 is required for necroptosis but its increase down-regulates necroptosis. Higher amount of RIP1 (>~46,000 mpc) suppresses apoptosis, leading to necroptosis alone. The relation between RIP1 level and occurrence of necroptosis or total cell death is biphasic. Our study provides a resource for encoding the complexity of TNF signaling and a quantitative picture how distinct dynamic interplay among proteins function as basis sets in signaling complexes, enabling RIP1 to play diverse roles in governing cell fate decisions.

ICP6 Prevents RIP1 Activation to Hinder Necroptosis Signaling.

Necroptosis is a type of programmed necrosis which depends on the activation of receptor-interacting protein kinase 3 (RIP3). Herpes simplex virus type 1 (HSV-1) is known to block necroptosis by the viral protein ICP6 in human cells, but its specific inhibitory mechanism is not fully understood. Here we reported that ICP6 could promote rather than suppress the formation of necrosome, the necroptosis signaling complex containing RIP3 and upstream regulator receptor-interacting protein kinase 1 (RIP1), but blocked RIP3 activation. Moreover, ICP6 could reduce the necroptosis-specific auto-phosphorylation of RIP1 regardless of the presence of RIP3. These results indicate that ICP6 block necroptosis through preventing RIP1 activation dependent signal transduction in necrosome.

A Non-canonical PDK1-RSK Signal Diminishes Pro-caspase-8-Mediated Necroptosis Blockade.

Necroptosis induction in vitro often requires caspase-8 (Casp8) inhibition by zVAD because pro-Casp8 cleaves RIP1 to disintegrate the necrosome. It has been unclear how the Casp8 blockade of necroptosis is eliminated naturally. Here, we show that pro-Casp8 within the necrosome can be inactivated by phosphorylation at Thr265 (pC8T265). pC8T265 occurs in vitro in various necroptotic cells and in the cecum of TNF-treated mice. p90 RSK is the kinase of pro-Casp8. It is activated by a mechanism that does not need ERK but PDK1, which is recruited to the RIP1-RIP3-MLKL-containing necrosome. Phosphorylation of pro-Casp8 at Thr265 can substitute for zVAD to permit necroptosis in vitro. pC8T265 mimic T265E knockin mice are embryonic lethal due to unconstrained necroptosis, and the pharmaceutical inhibition of RSK-mediated pC8T265 diminishes TNF-induced cecum damage and lethality in mice by halting necroptosis. Thus, phosphorylation of pro-Casp8 at Thr265 by RSK is an intrinsic mechanism for passing the Casp8 checkpoint of necroptosis.

Regulating RIPK1: another way in which ULK1 contributes to survival.

The mammalian ULK1 is the central initiating kinase of bulk and selective macroautophagy/autophagy processes. In the past, both autophagy-relevant and non-autophagy-relevant substrates of this Ser/Thr kinase have been reported. Here, we describe our recent finding that ULK1 also regulates TNF signaling pathways. We find that inhibition of autophagy or specifically ULK1 increases TNF-induced cell death. This autophagy-independent pro-survival function of ULK1 is mediated via the phosphorylation of RIPK1 at Ser357. RIPK1 is the central mediator of pro-inflammatory or pro-death signaling pathways induced by TNF, and ULK1-dependent phosphorylation regulates RIPK1 activation and distribution to different intracellular signaling complexes. Our results indicate that ULK1 exerts a cyto-protective function not only by initiating autophagy, but also by controlling RIPK1-mediated cell death.

The involvement of regulated cell death forms in modulating the bacterial and viral pathogenesis.

Apoptosis, necroptosis and pyroptosis represent three distinct types of regulated cell death forms, which play significant roles in response to viral and bacterial infections. Whereas apoptosis is characterized by cell shrinkage, nuclear condensation, bleb formation and retained membrane integrity, necroptosis and pyroptosis exhibit osmotic imbalance driven cytoplasmic swelling and early membrane damage. These three cell death forms exert distinct immune stimulatory potential. The caspase driven apoptotic cell demise is considered in many circumstances as anti-inflammatory, whereas the two lytic cell death modalities can efficiently trigger immune response by releasing damage associated molecular patterns to the extracellular space. The relevance of these cell death modalities in infections can be best demonstrated by the presence of viral proteins that directly interfere with cell death pathways. Conversely, some pathogens hijack the cell death signaling routes to initiate a targeted attack against the immune cells of the host, and extracellular bacteria can benefit from the destruction of intact extracellular barriers upon cell death induction. The complexity and the crosstalk between these cell death modalities reflect a continuous evolutionary race between pathogens and host. This chapter discusses the current advances in the research of cell death signaling with regard to viral and bacterial infections and describes the network of the cell death initiating molecular mechanisms that selectively recognize pathogen associated molecular patterns.

The Autophagy-Initiating Kinase ULK1 Controls RIPK1-Mediated Cell Death.

Autophagy, apoptosis, and necroptosis are stress responses governing the ultimate fate of a cell. However, the crosstalk between these cellular stress responses is not entirely understood. Especially, it is not clear whether the autophagy-initiating kinase ULK1 and the cell-death-regulating kinase RIPK1 are involved in this potential crosstalk. Here, we identify RIPK1 as a substrate of ULK1. ULK1-dependent phosphorylation of RIPK1 reduces complex IIb/necrosome assembly and tumor necrosis factor (TNF)-induced cell death, whereas deprivation of ULK1 enhances TNF-induced cell death. We observe that ULK1 phosphorylates multiple sites of RIPK1, but it appears that especially phosphorylation of S357 within the intermediate domain of RIPK1 mediates this cell-death-inhibiting effect. We propose that ULK1 is a regulator of RIPK1-mediated cell death.

Tristetraprolin regulates necroptosis during tonic Toll-like receptor 4 (TLR4) signaling in murine macrophages.

The necrosome is a protein complex required for signaling in cells that results in necroptosis, which is also dependent on tumor necrosis factor receptor (TNF-R) signaling. TNFα promotes necroptosis, and its expression is facilitated by mitogen-activated protein (MAP) kinase-activated protein kinase 2 (MK2) but is inhibited by the RNA-binding protein tristetraprolin (TTP, encoded by the Zfp36 gene). We have stimulated murine macrophages from WT, MyD88-/-, Trif-/-, MyD88-/-Trif-/-, MK2-/-, and Zfp36-/- mice with graded doses of lipopolysaccharide (LPS) and various inhibitors to evaluate the role of various genes in Toll-like receptor 4 (TLR4)-induced necroptosis. Necrosome signaling, cytokine production, and cell death were evaluated by immunoblotting, ELISA, and cell death assays, respectively. We observed that during TLR4 signaling, necrosome activation is mediated through the adaptor proteins MyD88 and TRIF, and this is inhibited by MK2. In the absence of MK2-mediated necrosome activation, lipopolysaccharide-induced TNFα expression was drastically reduced, but MK2-deficient cells became highly sensitive to necroptosis even at low TNFα levels. In contrast, during tonic TLR4 signaling, WT cells did not undergo necroptosis, even when MK2 was disabled. Of note, necroptosis occurred only in the absence of TTP and was mediated by the expression of TNFα and activation of JUN N-terminal kinase (JNK). These results reveal that TTP plays an important role in inhibiting TNFα/JNK-induced necrosome signaling and resultant cytotoxicity.

CK1α, CK1δ, and CK1ε are necrosome components which phosphorylate serine 227 of human RIPK3 to activate necroptosis.

Necroptosis is a regulated necrotic cell death pathway, mediated by a supermolecular complex called the necrosome, which contains receptor-interacting protein kinase 1 and 3 (RIPK1, RIPK3) and mixed-lineage kinase domain-like protein (MLKL). Phosphorylation of human RIPK3 at serine 227 (S227) has been shown to be required for downstream MLKL binding and necroptosis progression. Tandem immunoprecipitation of RIPK3 reveals that casein kinase 1 (CK1) family proteins associate with the necrosome upon necroptosis induction, and this interaction depends on the kinase activity of RIPK3. In addition, CK1 proteins colocalize with RIPK3 puncta during necroptosis. Importantly, CK1 proteins directly phosphorylate RIPK3 at S227 in vitro and in vivo. Loss of CK1 proteins abolishes S227 phosphorylation and blocks necroptosis. Furthermore, a RIPK3 mutant with mutations in the CK1 recognition motif fails to be phosphorylated at S227, does not bind or phosphorylate MLKL, and is unable to activate necroptosis. These results strongly suggest that CK1 proteins are necrosome components which are responsible for RIPK3-S227 phosphorylation.

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.

Molecular Insights into the Mechanism of Necroptosis: The Necrosome As a Potential Therapeutic Target.

Necroptosis, or regulated necrosis, is an important type of programmed cell death in addition to apoptosis. Necroptosis induction leads to cell membrane disruption, inflammation and vascularization. It plays important roles in various pathological processes, including neurodegeneration, inflammatory diseases, multiple cancers, and kidney injury. The molecular regulation of necroptotic pathway has been intensively studied in recent years. Necroptosis can be triggered by multiple stimuli and this pathway is regulated through activation of receptor-interacting protein kinase 1 (RIPK1), RIPK3 and pseudokinase mixed lineage kinase domain-like (MLKL). A better understanding of the mechanism of regulation of necroptosis will further aid to the development of novel drugs for necroptosis-associated human diseases. In this review, we focus on new insights in the regulatory machinery of necroptosis. We further discuss the role of necroptosis in different pathologies, its potential as a therapeutic target and the current status of clinical development of drugs interfering in the necroptotic pathway.