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Activation of the executioner caspases-3 and -7 promotes microglial pyroptosis in models of multiple sclerosis.

ABSTRACT: BACKGROUND:Pyroptosis is a type of proinflammatory regulated cell death (RCD) in which caspase-1 proteolytically cleaves gasdermin D (GSDMD) to yield a cytotoxic pore-forming protein. Recent studies have suggested that additional cell death pathways may interact with GSDMD under certain circumstances to execute pyroptosis. Microglia/macrophages in the central nervous system (CNS) undergo GSDMD-associated pyroptosis in multiple sclerosis (MS) and its animal model experimental autoimmune encephalomyelitis (EAE) but the contribution of other cell death pathways to this phenomenon is unknown. Herein, we tested the hypothesis that multiple RCD pathways underlie microglial pyroptosis in the context of neuroinflammation. METHODS:A siRNA screen of genes with known RCD functions was performed in primary human microglia to evaluate their role in nigericin-induced pyroptosis using supernatant lactate dehydrogenase activity as a read-out of cell lysis. Activation of apoptotic executioner proteins and their contribution to pyroptosis was assessed using semi-quantitative confocal microscopy, high-sensitivity ELISA, immunoblot, cell lysis assays, and activity-based fluorescent probes. Quantification of pyroptosis-related protein expression was performed in CNS lesions from patients with progressive MS and mice with MOG35-55-induced EAE, and in matched controls. RESULTS:Among progressive MS patients, activated caspase-3 was detected in GSDMD immunopositive pyroptotic microglia/macrophages within demyelinating lesions. In the siRNA screen, suppression of caspase-3/7, caspase-1, or GSDMD expression prevented plasma membrane rupture during pyroptosis. Upon exposure to pyroptotic stimuli (ATP or nigericin), human microglia displayed caspase-3/7 activation and cleavage of caspase-3/7-specific substrates (e.g., DFF45, ROCK1, and PARP), with accompanying features of pyroptosis including GSDMD immunopositive pyroptotic bodies, IL-1? release, and membrane rupture. Pyroptosis-associated nuclear condensation and pyroptotic body formation were suppressed by caspase-3/7 inhibition. Pharmacological and siRNA-mediated inhibition of caspase-1 diminished caspase-3/7 activation during pyroptosis. In mice with EAE-associated neurological deficits, activated caspase-3 colocalized with GSDMD immunopositivity in lesion-associated macrophages/microglia. CONCLUSIONS:Activation of executioner caspases-3/7, widely considered key mediators of apoptosis, contributed to GSDMD-associated microglial pyroptosis under neuroinflammatory conditions. Collectively, these observations highlight the convergence of different cell death pathways during neuroinflammation and offer new therapeutic opportunities in neuroinflammatory disease.

SUBMITTER: McKenzie BA

PROVIDER: S-EPMC7456507 | biostudies-literature | 2020 Aug

REPOSITORIES: biostudies-literature

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Activation of the executioner caspases-3 and -7 promotes microglial pyroptosis in models of multiple sclerosis.

McKenzie Brienne A BA Fernandes Jason P JP Doan Matthew A L MAL Schmitt Laura M LM Branton William G WG Power Christopher C

Journal of neuroinflammation 20200829 1

<h4>Background</h4>Pyroptosis is a type of proinflammatory regulated cell death (RCD) in which caspase-1 proteolytically cleaves gasdermin D (GSDMD) to yield a cytotoxic pore-forming protein. Recent studies have suggested that additional cell death pathways may interact with GSDMD under certain circumstances to execute pyroptosis. Microglia/macrophages in the central nervous system (CNS) undergo GSDMD-associated pyroptosis in multiple sclerosis (MS) and its animal model experimental autoimmune e ...[more]

PMID: 32861242

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Project description:BackgroundApoptosis is fundamental in maintaining cell balance in multicellular organisms, and caspases play a crucial role in apoptosis pathways. It is reported that apoptosis plays an important role in tick salivary gland degeneration. Several different caspases have been found in ticks, but the interactions between them are currently unknown. Here, we report three new caspases, isolated from the salivary glands of the tick Rhipicephalus haemaphysaloides.MethodsThe full-length cDNA of the RhCaspases 7, 8 and 9 genes were obtained by transcriptome, and RhCaspases 7, 8 and 9 were expressed in E. coli; after protein purification and immunization in mice, specific polyclonal antibodies (PcAb) were created in response to the recombinant protein. Reverse-transcription quantitative PCR (RT-qPCR) and western blot were used to detect the existence of RhCaspases 7, 8 and 9 in ticks. TUNEL assays were used to determine the apoptosis level in salivary glands at different feeding times after gene silencing. The interaction between RhCaspases 7, 8 and 9 were identified by co-transfection assays.ResultsThe transcription of apoptosis-related genes in R. haemaphysaloides salivary glands increased significantly after tick engorgement. Three caspase-like molecules containing conserved caspase domains were identified and named RhCaspases 7, 8 and 9. RhCaspase8 and RhCaspase9 contain a long pro-domain at their N-terminals. An RT-qPCR assay demonstrated that the transcription of these three caspase genes increased significantly during the engorged periods of the tick developmental stages (engorged larval, nymph, and adult female ticks). Transcriptional levels of RhCaspases 7, 8 and 9 in salivary glands increased more significantly than other tissues post-engorgement. RhCaspase9-RNAi treatment significantly inhibited tick feeding. In contrast, knockdown of RhCaspase7 and RhCaspase8 had no influence on tick feeding. Compared to the control group, apoptosis levels were significantly reduced after interfering with RhCaspase 7, 8 and 9 expressions. Co-transfection assays showed RhCaspase7 was cleaved by RhCaspases 8 and 9, demonstrating that RhCaspases 8 and 9 are initiator caspases and RhCaspase7 is an executioner caspase.ConclusionsTo the best of our knowledge, this is the first study to identify initiator and executioner caspases in ticks, confirm the interaction among them, and associate caspase activation with tick salivary gland degeneration.

Project description:PurposePhotoreceptor (PR) death is the ultimate cause of irreversible vision loss in retinal detachment (RD). Although microglial infiltration in the subretinal space (SRS) was observed after RD, the molecular mechanism underlying microglial activation and the outcomes of infiltrating microglia remain unclear. We aimed to uncover the mechanism of initiation of microglial activation to help explore potential therapy to promote PR survival.MethodsAn RD model was conducted by injecting sodium hyaluronate into SRS of C57BL/6J wild type mice. Adenosine triphosphate (ATP) was measured by a ATP Microplate Assay Kit. Bioinformatics analysis was used to evaluate the upregulated receptor relating to ATP binding in human datasets and mouse transcriptomes of RD. Expression of P2X7, its downstream signaling pathways, and microglial pyroptosis were confirmed by qPCR, WB, and immunofluorescence in vivo and in vitro. The cell viability of PR was measured by cell counting kit-8. Brilliant Blue G, a P2X7 antagonist, was subretinally or intraperitoneally injected to inhibit microglial activation in vivo and was applied for microglia cell line treatment in vitro. The decrease in microglial activation and pyroptosis was detected by immunofluorescence and WB. The protective effect on PR was measured by hematoxylin and eosin staining, TUNEL assay, and electroretinogram analysis.ResultsThe results showed that extracellular ATP released in the SRS after RD triggered P2X7 activation and attracted microglia. The downstream cascade of inflammasome activation induced by P2X7 activation contributed to microglial pyroptosis and then to PR death. ATP-activated microglia led to PR death in vitro. P2X7 blockade rescued PR morphologically and functionally by inhibiting microglial activation and pyroptosis.ConclusionsThese results elucidate that ATP-induced P2X7-mediated microglial activation leads to microglial pyroptosis, contributing to PR death. Appropriate inhibition of microglial pyroptosis might serve as a pharmacotherapeutic strategy for decreasing PR death in RD.

Dataset Information

Activation of the executioner caspases-3 and -7 promotes microglial pyroptosis in models of multiple sclerosis.

Publications

Activation of the executioner caspases-3 and -7 promotes microglial pyroptosis in models of multiple sclerosis.

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