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Evidence that NF-κB and MAPK Signaling Promotes NLRP Inflammasome Activation in Neurons Following Ischemic Stroke

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Abstract

Multi-protein complexes, termed “inflammasomes,” are known to contribute to neuronal cell death and brain injury following ischemic stroke. Ischemic stroke increases the expression and activation of nucleotide-binding oligomerization domain (NOD)-like receptor (NLR) Pyrin domain containing 1 and 3 (NLRP1 and NLRP3) inflammasome proteins and both interleukin (IL)-1β and IL-18 in neurons. In this study, we provide evidence that activation of either the NF-κB and MAPK signaling pathways was partly responsible for inducing the expression and activation of NLRP1 and NLRP3 inflammasome proteins and that these effects can be attenuated using pharmacological inhibitors of these two pathways in neurons and brain tissue under in vitro and in vivo ischemic conditions, respectively. Moreover, these findings provided supporting evidence that treatment with intravenous immunoglobulin (IVIg) preparation can reduce activation of the NF-κB and MAPK signaling pathways resulting in decreased expression and activation of NLRP1 and NLRP3 inflammasomes, as well as increasing expression of anti-apoptotic proteins, Bcl-2 and Bcl-xL, in primary cortical neurons and/or cerebral tissue under in vitro and in vivo ischemic conditions. In summary, these results provide compelling evidence that both the NF-κB and MAPK signaling pathways play a pivotal role in regulating the expression and activation of NLRP1 and NLRP3 inflammasomes in primary cortical neurons and brain tissue under ischemic conditions. In addition, treatment with IVIg preparation decreased the activation of the NF-κB and MAPK signaling pathways, and thus attenuated the expression and activation of NLRP1 and NLRP3 inflammasomes in primary cortical neurons under ischemic conditions. Hence, these findings suggest that therapeutic interventions that target inflammasome activation in neurons may provide new opportunities in the future treatment of ischemic stroke.

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Acknowledgements

This work was supported by the National Medical Research Council Research Grants (NMRC/CG/013/2013 and NMRC-CBRG-0102/2016), NUHS Seed Fund for Basic Science Research [R-185-000-255-112] and Singapore Ministry of Education Tier 1 grants [R-185-000-285-112].

Author Contributions

DYF, PC, GRD, CGS, DGJ, CLC, and TVA conceived and designed the experiments. DYF, YLC, KZL, SHB performed experiments. DYF, STD, CGY, CLC, YAL, DGJ, STD, YAL, CLC, and TVA were involved in drafting and editing the manuscript, and interpreted primary data. YAL, STD, and CLC contributed reagents. All authors read and approved the final manuscript.

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Authors and Affiliations

  1. Department of Physiology, Yong Loo Lin School of Medicine, National University of Singapore, MD9, Singapore, Singapore

    David Yang-Wei Fann, Yi-Lin Cheng, Ker-Zhing Lok & Thiruma V. Arumugam

  2. Department of Pharmacology, National University of Singapore, Singapore, Singapore

    Yun-An Lim & Christopher Li-Hsian Chen

  3. School of Biomedical Sciences, The University of Queensland, St Lucia, QLD, 4072, Australia

    Prasad Chunduri

  4. School of Pharmacy, Sungkyunkwan University, Suwon, South Korea

    Sang-Ha Baik, Dong-Gyu Jo & Thiruma V. Arumugam

  5. Department of Pharmacology, Monash University, Clayton, VIC, Australia

    Grant R. Drummond & Christopher G. Sobey

  6. Department of Anatomy, The Yong Loo Lin School of Medicine, National University of Singapore, Singapore, Singapore

    S. Thameem Dheen

  7. Memory Aging and Cognition Centre, National University Health System, Singapore, Singapore

    Christopher Li-Hsian Chen

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  1. David Yang-Wei Fann
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Supplementary Figure 1

Inhibition of the NF-κB and MAPK pathways and cell death in primary cortical neurons following simulated ischemic conditions. (A and B). Representative immunoblots and quantification illustrating the effect of increasing concentrations (μM) of a NF-κB inhibitor (Bay-11-7082) on levels of p-P65 NF-κB, total NF-κB and cleaved caspase-3 proteins in primary cortical neurons subjected to oxygen and glucose deprivation for 6 h (OGD6hr). (C and D). Representative immunoblots and quantification illustrating the effect of increasing concentrations (μM) of a JNK MAPK inhibitor (SP600125) on levels of p-JNK MAPK, total JNK MAPK and cleaved caspase-3 proteins in primary cortical neurons subjected to oxygen and glucose deprivation for 6 h (OGD6hr). (E and F). Representative immunoblots and quantification illustrating the effect of increasing concentrations (μM) of a P38 MAPK inhibitor (SB203580) on levels of p-P38 MAPK, total P38 MAPK and cleaved caspase-3 proteins in primary cortical neurons subjected to oxygen and glucose deprivation for 6 h (OGD6hr). (G and H). Representative immunoblots and quantification illustrating the effect of increasing concentrations (μM) of an ERK MAPK inhibitor (U-0126) on levels of p-ERK MAPK, total ERK MAPK and cleaved caspase-3 proteins in primary cortical neurons subjected to oxygen and glucose deprivation for 6 h (OGD6hr). β-actin was used as a loading control. Data are represented as mean ± S.E.M. n = 4 cultures. *** P < 0.001 compared to control; **P < 0.01 compared to OGD6 + VehicleI; ### P < 0.001 compared to OGD6 + VehicleI (JPEG 1266 kb)

Supplementary Figure 2

Inhibition of the NF-κB and MAPK pathways and cell death in primary cortical neurons following simulated ischemic/reperfusion (I/R) conditions. (A and B). Representative immunoblots and quantification illustrating the effect of increasing concentrations (μM) of a NF-κB inhibitor (Bay-11-7082) on levels of p-P65 NF-κB, total P65 NF-κB and cleaved caspase-3 proteins in primary cortical neurons subjected to oxygen and glucose deprivation (OGD3hr) followed by neurobasal reperfusion and re-oxygenation (24 h). (C and D). Representative immunoblots and quantification illustrating the effect of increasing concentrations (μM) of a JNK MAPK inhibitor (SP600125) on levels of p-JNK MAPK, total JNK MAPK and cleaved caspase-3 proteins in primary cortical neurons subjected to oxygen and glucose deprivation (OGD3hr) followed by neurobasal reperfusion and re-oxygenation (24 h). (E and F). Representative immunoblots and quantification illustrating the effect of increasing concentrations (μM) of a P38 MAPK inhibitor (SB203580) on levels of p-P38 MAPK, total P38 MAPK and cleaved caspase-3 proteins in primary cortical neurons subjected to oxygen and glucose deprivation (OGD3hr) followed by neurobasal reperfusion and re-oxygenation (24 h). (G and H). Representative immunoblots and quantification illustrating the effect of increasing concentrations (μM) of an ERK MAPK inhibitor (U-0126) on levels of p-ERK MAPK, total ERK MAPK and cleaved caspase-3 proteins in primary cortical neurons subjected to oxygen and glucose deprivation (OGD3hr) followed by neurobasal reperfusion and re-oxygenation (24 h). β-actin was used as a loading control. Data are represented as mean ± S.E.M. n = 4 cultures. *** P < 0.001 compared to control; **P < 0.01 compared to OGD3 + R24 + VehicleI; ### P < 0.001 compared to OGD3 + R24 + VehicleI. (JPEG 1296 kb)

Supplementary Figure 3

NF-κB and MAPKs inhibitors attenuate NF-κB and MAPKs signaling in the brain following focal ischemic stroke. (A and B). Representative immunoblots and quantification illustrating increases in the activation levels of NF-κB (p-P65) and MAPKs such as p-P38, p-JNK, p-ERK and p-c-Jun in ipsilateral brain tissues following middle cerebral artery occlusion (1 h) and reperfusion (24 h). The administration of NF-κB (10 mg/kg) and MAPK inhibitors (P38 inhibitor, 10 mg/kg; JNK inhibitor, 10 mg/kg; ERK inhibitor, 10 mg/kg) significantly reduced the activation levels of NF-κB (p-P65) and MAPKs such as p-P38, p-JNK, p-ERK and p-c-Jun. β-actin was used as a loading control. Data are represented as mean ± S.E.M. n = 5–6 animals in each experimental group. *** P < 0.001 compared with SHAM; $$$ P < 0.001 compared with I/R24Hr + VehicleI. (JPEG 724 kb)

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Fann, D.YW., Lim, YA., Cheng, YL. et al. Evidence that NF-κB and MAPK Signaling Promotes NLRP Inflammasome Activation in Neurons Following Ischemic Stroke. Mol Neurobiol 55, 1082–1096 (2018). https://doi.org/10.1007/s12035-017-0394-9

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