Abstract
Lysosomal membrane permeabilization caused either via phagocytosis of particulates or the uptake of protein aggregates can trigger the activation of NLRP3 inflammasome- an intense inflammatory response that drives the release of the pro-inflammatory cytokine IL-1β by regulating the activity of CASPASE 1. The maintenance of lysosomal homeostasis and lysosomal membrane integrity is facilitated by the AAA+ ATPase, VCP/p97 (VCP). However, the relationship between VCP and NLRP3 inflammasome activity remains unexplored. Here, we demonstrate that the VCP inhibitors, DBeQ and ML240 elicit the activation of NLRP3 inflammasome in bone marrow-derived macrophages (BMDMs) when used as activation stimuli. Moreover, genetic inhibition of VCP or VCP chemical inhibition enhances lysosomal membrane damage and augments LLoME-associated NLRP3 inflammasome activation in BMDMs. Similarly, VCP inactivation also augments NLRP3 inflammasome activation mediated by aggregated alpha-synuclein fibrils and lysosomal damage. These data suggest that VCP is a participant in the complex regulation of NLRP3 inflammasome activation.
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No datasets were generated or analysed during the current study.
Abbreviations
- AAA+ ATPase:
-
ATPase associated with different cellular activities adenosine triphosphatase
- BMDMs:
-
Bone-marrow-derived macrophages
- DAMPs:
-
Damage-associated molecular patterns
- NLRP3:
-
Nucleotide-binding domain, leucine-rich–containing family, pyrin domain–containing-3
- IL-1β:
-
Interleukin 1β
- VCP:
-
Valosin-containing protein
- iPSCS:
-
Induced pluripotent stem cells
- LLoMe:
-
L-Leucyl-L-Leucine methyl ester
- ERAD:
-
Endoplasmic reticulum-associated degradation
- NOD:
-
Nucleotide-binding oligomerization domain
- LRR:
-
Leucine-rich repeats
- IBMPFD:
-
Inclusion body myopathy with Paget disease of bone and frontotemporal dementia
- MSP:
-
Multisystem proteinopathy
- RNA:
-
Ribonucleic acid
- SQSTM1:
-
Sequestosome-1
- αS PFF:
-
α-Synuclein preformed fibrils
- TDP-43:
-
Transactive response DNA binding protein-43
- ALS:
-
Amyotrophic lateral sclerosis
- NFκB:
-
Nuclear factor kappa-light-chain-enhancer of activated B cells
- PD:
-
Parkinson's disease
- TNF:
-
Tumor necrosis factor
- PBST:
-
Phosphate Buffer Saline, 0.1% Tween 20
- RT:
-
Room Temperature
- BSA:
-
Bovine Serum Albumin
- EGF:
-
Epidermal growth factor
- DAMPS:
-
Damage-associated molecular patterns
- ASC:
-
Apoptosis-associated speck-like protein containing a caspase recruitment domain
- ATP:
-
Adenosine triphosphate
- LPS:
-
Lipopolysaccharide
- GAL-3:
-
GALECTIN-3
- VIN:
-
VINCULIN
- FTD:
-
Frontotemporal dementia
References
Place, D.E., and T.-D. Kanneganti. 2018. Recent advances in inflammasome biology. Current Opinion in Immunology 50: 32–38. https://doi.org/10.1016/j.coi.2017.10.011.
Baroja-Mazo, A., F. Martín-Sánchez, A.I. Gomez, C.M. Martínez, J. Amores-Iniesta, V. Compan, M. Barberà-Cremades, et al. 2014. The NLRP3 inflammasome is released as a particulate danger signal that amplifies the inflammatory response. Nature Immunology 15: 738–748. https://doi.org/10.1038/ni.2919.
Fu, J., and W. Hao. 2023. Structural Mechanisms of NLRP3 Inflammasome Assembly and Activation. Annual Review of Immunology 41: 301–316. https://doi.org/10.1146/annurev-immunol-081022-021207.
Dinarello, Charles A. 2007. A signal for the caspase-1 inflammasome free of TLR. Immunity 26: 383–385. https://doi.org/10.1016/j.immuni.2007.04.005.
Mendiola, A. S., and A. E. Cardona. 2018. The IL-1β phenomena in neuroinflammatory diseases Journal of Neural Transmission (Vienna Austria : 1996) 125: 781–795. https://doi.org/10.1007/s00702-017-1732-9.
Hornung, V., F. Bauernfeind, A. Halle, E.O. Samstad, H. Kono, K.L. Rock, K.A. Fitzgerald, and E. Latz. 2008. Silica crystals and aluminum salts activate the NALP3 inflammasome through phagosomal destabilization. Nature Immunology 9: 847–856. https://doi.org/10.1038/ni.1631.
Franchi, L., R. Muñoz-Planillo, and G. Núñez. 2012. Sensing and reacting to microbes through the inflammasomes. Nature Immunology 13: 325–332. https://doi.org/10.1038/ni.2231.
Lima, Heriberto Jr, Lee S. Jacobson, Michael F. Goldberg, Kartik Chandran, Felipe Diaz-Griffero, Michael P. Lisanti, and Jürgen Brojatsch. 2013. Role of lysosome rupture in controlling Nlrp3 signaling and necrotic cell death. Cell cycle (Georgetown, Tex.) 12: 1868–1878. https://doi.org/10.4161/cc.24903.
Gross, Olaf, Amir S. Yazdi, Christina J. Thomas, Mark Masin, Leonhard X. Heinz, Greta Guarda, Manfredo Quadroni, Stefan K. Drexler, and Jurg Tschopp. 2012. Inflammasome activators induce interleukin-1α secretion via distinct pathways with differential requirement for the protease function of caspase-1. Immunity 36: 388–400. https://doi.org/10.1016/j.immuni.2012.01.018.
Amores-Iniesta, J., M. Barberà-Cremades, C.M. Martínez, J.A. Pons, B. Revilla-Nuin, L. Martínez-Alarcón, F. Di Virgilio, P. Parrilla, A. Baroja-Mazo, and P. Pelegrín. 2017. Extracellular ATP Activates the NLRP3 Inflammasome and Is an Early Danger Signal of Skin Allograft Rejection. Cell Reports 21: 3414–3426. https://doi.org/10.1016/j.celrep.2017.11.079.
Gurcel, L., L. Abrami, S. Girardin, J. Tschopp, and F. Gisou van der Goot. 2006. Caspase-1 activation of lipid metabolic pathways in response to bacterial pore-forming toxins promotes cell survival. Cell 126: 1135–1145. https://doi.org/10.1016/j.cell.2006.07.033.
Perregaux, D., and C.A. Gabel. 1994. Interleukin-1 beta maturation and release in response to ATP and nigericin. Evidence that potassium depletion mediated by these agents is a necessary and common feature of their activity. The Journal of Biological Chemistry 269: 15195–15203.
Tang, T., X. Lang, X. Congfei, X. Wang, T. Gong, Y. Yang, J. Cui, et al. 2017. CLICs-dependent chloride efflux is an essential and proximal upstream event for NLRP3 inflammasome activation. Nature Communications 8: 202. https://doi.org/10.1038/s41467-017-00227-x.
Liu, Q., D. Zhang, D. Hu, X. Zhou, and Y. Zhou. 2018. The role of mitochondria in NLRP3 inflammasome activation. Molecular Immunology 103: 115–124. https://doi.org/10.1016/j.molimm.2018.09.010.
Sanman, LE., Y. Qian, N.A. Eisele, T.M. Ng, W.A. van der Linden, D.M. Monack, E. Weerapana, and M. Bogyo. 2016. Disruption of glycolytic flux is a signal for inflammasome signaling and pyroptotic cell death. eLife 5: e13663. https://doi.org/10.7554/eLife.13663.
Muñoz-Planillo, R., P. Kuffa, G. Martínez-Colón, B.L. Smith, T.M. Rajendiran, and G. Núñez. 2013. K+ efflux is the common trigger of NLRP3 inflammasome activation by bacterial toxins and particulate matter. Immunity 38: 1142–1153. https://doi.org/10.1016/j.immuni.2013.05.016.
Hou, Y., H. He, M. Ma, and R. Zhou. 2023. Apilimod activates the NLRP3 inflammasome through lysosome-mediated mitochondrial damage. Frontiers in Immunology 14: 1128700. https://doi.org/10.3389/fimmu.2023.1128700.
Zhou, Y., L. Ming, D. Ren-Hong, C. Qiao, C.-Y. Jiang, K.-Z. Zhang, J.-H. Ding, and H. Gang. 2016. MicroRNA-7 targets Nod-like receptor protein 3 inflammasome to modulate neuroinflammation in the pathogenesis of Parkinson’s disease. Molecular Neurodegeneration 11: 28. https://doi.org/10.1186/s13024-016-0094-3.
Gordon, R., E.A. Albornoz, D.C. Christie, M.R. Langley, V. Kumar, S. Mantovani, A.A.B. Robertson, et al. 2018. Inflammasome inhibition prevents α-synuclein pathology and dopaminergic neurodegeneration in mice. Science Translational Medicine. https://doi.org/10.1126/scitranslmed.aah4066.
Tresse, E., F.A. Salomons, J. Vesa, L.C. Bott, V. Kimonis, T.-P. Yao, N.P. Dantuma, and J.P. Taylor. 2010. VCP/p97 is essential for maturation of ubiquitin-containing autophagosomes and this function is impaired by mutations that cause IBMPFD. Autophagy 6: 217–227. https://doi.org/10.4161/auto.6.2.11014.
Papadopoulos, C., P. Kirchner, M. Bug, D. Grum, L. Koerver, N. Schulze, R. Poehler, et al. 2017. VCP/p97 cooperates with YOD1, UBXD1 and PLAA to drive clearance of ruptured lysosomes by autophagy. The EMBO Journal 36: 135–150. https://doi.org/10.15252/embj.201695148.
Seguin, S.J., F.F. Morelli, J. Vinet, D. Amore, S. De Biasi, A. Poletti, D.C. Rubinsztein, and S. Carra. 2014. Inhibition of autophagy, lysosome and VCP function impairs stress granule assembly. Cell Death and Differentiation 21: 1838–1851. https://doi.org/10.1038/cdd.2014.103.
Magnaghi, P., R. D’Alessio, B. Valsasina, N. Avanzi, S. Rizzi, D. Asa, F. Gasparri, et al. 2013. Covalent and allosteric inhibitors of the ATPase VCP/p97 induce cancer cell death. Nature Chemical Biology 9: 548–556. https://doi.org/10.1038/nchembio.1313.
Chou, Tsui-Fen., Steve J. Brown, Dmitriy Minond, B.E. Nordin, K. Li, A.C. Jones, P. Chase, et al. 2011. Reversible inhibitor of p97, DBeQ, impairs both ubiquitin-dependent and autophagic protein clearance pathways. Proceedings of the National Academy of Sciences of the United States of America 108: 4834–4839. https://doi.org/10.1073/pnas.1015312108.
Fang, C.-J., L. Gui, X. Zhang, D.R. Moen, K. Li, K.J. Frankowski, H.J. Lin, F.J. Schoenen, and T.-F. Chou. 2015. Evaluating p97 inhibitor analogues for their domain selectivity and potency against the p97–p47 complex. ChemMedChem 10: 52–56. https://doi.org/10.1002/cmdc.201402420.
Anderson, D.J., R. Le Moigne, S. Djakovic, B. Kumar, J. Rice, S. Wong, J. Wang, et al. 2015. Targeting the AAA ATPase p97 as an Approach to Treat Cancer through Disruption of Protein Homeostasis. Cancer Cell 28: 653–665. https://doi.org/10.1016/j.ccell.2015.10.002.
Al-Obeidi, E., S. Al-Tahan, A. Surampalli, N. Goyal, A.K. Wang, A. Hermann, M. Omizo, C. Smith, T. Mozaffar, and V. Kimonis. 2018. Genotype-phenotype study in patients with valosin-containing protein mutations associated with multisystem proteinopathy. Clinical Genetics 93: 119–125. https://doi.org/10.1111/cge.13095.
Arhzaouy, K., C. Papadopoulos, N. Schulze, S.K. Pittman, H. Meyer, and C.C. Weihl. 2019. VCP maintains lysosomal homeostasis and TFEB activity in differentiated skeletal muscle. Autophagy 15: 1082–1099. https://doi.org/10.1080/15548627.2019.1569933.
Asai, T., Y. Tomita, S.-I. Nakatsuka, Y. Hoshida, A. Myoui, H. Yoshikawa, and K. Aozasa. 2002. VCP (p97) regulates NFkappaB signaling pathway, which is important for metastasis of osteosarcoma cell line. Japanese Journal of Cancer Research 93: 296–304. https://doi.org/10.1111/j.1349-7006.2002.tb02172.x.
Dec, E., P. Rana, V. Katheria, R. Dec, M. Khare, A. Nalbandian, S.-Y. Leu, et al. 2014. Cytokine profiling in patients with VCP-associated disease. Clinical and Translational Science 7: 29–32. https://doi.org/10.1111/cts.12117.
Nalbandian, A., A.A. Khan, R. Srivastava, K.J. Llewellyn, B. Tan, N. Shukr, Y. Fazli, V.E. Kimonis, and L. BenMohamed. 2017. Activation of the NLRP3 Inflammasome Is Associated with Valosin-Containing Protein Myopathy. Inflammation 40: 21–41. https://doi.org/10.1007/s10753-016-0449-5.
Dhavale, D.D., C. Tsai, D.P. Bagchi, L.A. Engel, J. Sarezky, and P.T. Kotzbauer. 2017. A sensitive assay reveals structural requirements for α-synuclein fibril growth. The Journal of Biological Chemistry 292: 9034–9050. https://doi.org/10.1074/jbc.M116.767053.
Heneka, M.T., M.P. Kummer, A. Stutz, A. Delekate, S. Schwartz, A. Vieira-Saecker, A. Griep, et al. 2013. NLRP3 is activated in Alzheimer’s disease and contributes to pathology in APP/PS1 mice. Nature 493: 674–678. https://doi.org/10.1038/nature11729.
Codolo, G., N. Plotegher, T. Pozzobon, M. Brucale, I. Tessari, L. Bubacco, and M. de Bernard. 2013. Triggering of inflammasome by aggregated α-synuclein, an inflammatory response in synucleinopathies. PloS One 8: e55375. https://doi.org/10.1371/journal.pone.0055375.
Debye, B., L. Schmülling, L. Zhou, G. Rune, C. Beyer, and S. Johann. 2018. Neurodegeneration and NLRP3 inflammasome expression in the anterior thalamus of SOD1(G93A) ALS mice. Brain Pathology 28: 14–27. https://doi.org/10.1111/bpa.12467.
Rawat, R., T.V. Cohen, B. Ampong, D. Francia, A. Henriques-Pons, E.P. Hoffman, and K. Nagaraju. 2010. Inflammasome up-regulation and activation in dysferlin-deficient skeletal muscle. The American Journal of Pathology 176: 2891–2900. https://doi.org/10.2353/ajpath.2010.090058.
Root, J., P. Merino, A. Nuckols, M. Johnson, and T. Kukar. 2021. Lysosome dysfunction as a cause of neurodegenerative diseases: Lessons from frontotemporal dementia and amyotrophic lateral sclerosis. Neurobiology of Disease 154.
Kimonis, V.E., S.G. Mehta, E.C. Fulchiero, D. Thomasova, M. Pasquali, K. Boycott, E.G. Neilan, et al. 2008. Clinical studies in familial VCP myopathy associated with Paget disease of bone and frontotemporal dementia. American Journal of Medical Genetics Part A 146A: 745–757. https://doi.org/10.1002/ajmg.a.31862.
Chauhan, S., S. Kumar, A. Jain, M. Ponpuak, M.H. Mudd, T. Kimura, S.W. Choi, et al. 2016. TRIMs and Galectins Globally Cooperate and TRIM16 and Galectin-3 Co-direct Autophagy in Endomembrane Damage Homeostasis. Developmental Cell 39: 13–27. https://doi.org/10.1016/j.devcel.2016.08.003.
Jia, J., Y.P. Abudu, A. Claude-Taupin, G. Yuexi, S. Kumar, S.W. Choi, R. Peters, et al. 2019. Galectins control MTOR and AMPK in response to lysosomal damage to induce autophagy. Autophagy 15: 169–171. https://doi.org/10.1080/15548627.2018.1505155.
Chou, T.-F., K. Li, K.J. Frankowski, F.J. Schoenen, and R.J. Deshaies. 2013. Structure-activity relationship study reveals ML240 and ML241 as potent and selective inhibitors of p97 ATPase. ChemMedChem 8: 297–312. https://doi.org/10.1002/cmdc.201200520.
Zhang, X., L. Jiang, Y. Li, Q. Feng, X. Sun, Y. Wang, and M. Zhao. 2023. Discovery of novel benzylquinazoline molecules as p97/VCP inhibitors. Frontiers in Pharmacology 14: 1209060. https://doi.org/10.3389/fphar.2023.1209060.
Zhu, J., S. Pittman, D. Dhavale, R. French, J.N. Patterson, M.S. Kaleelurrrahuman, Y. Sun, et al. 2022. VCP suppresses proteopathic seeding in neurons. Molecular Neurodegeneration 17: 30. https://doi.org/10.1186/s13024-022-00532-0.
Ye, Y., W.K. Tang, T. Zhang, and D. Xia. 2017. A Mighty, “Protein Extractor” of the Cell: Structure and Function of the p97/CDC48 ATPase. Frontiers in Molecular Biosciences 4: 39. https://doi.org/10.3389/fmolb.2017.00039.
Meyer, H., M. Bug, and S. Bremer. 2012. Emerging functions of the VCP/p97 AAA-ATPase in the ubiquitin system. Nature Cell Biology 14: 117–123. https://doi.org/10.1038/ncb2407.
Ahlstedt, B.A., R. Ganji, and M. Raman. 2022. The functional importance of VCP to maintaining cellular protein homeostasis. Biochemical Society Transactions 50: 1457–1469. https://doi.org/10.1042/BST20220648.
Ghalandary, M., Y. Li, T. Fröhlich, T. Magg, Y. Liu, M. Rohlfs, S. Hollizeck, et al. 2022. Valosin-containing protein-regulated endoplasmic reticulum stress causes NOD2-dependent inflammatory responses. Scientific Reports 12: 3906. https://doi.org/10.1038/s41598-022-07804-1.
Li, J.-M., W. Hongyu, W. Zhang, M.R. Blackburn, and J. Jin. 2014. The p97-UFD1L-NPL4 protein complex mediates cytokine-induced IκBα proteolysis. Molecular and Cellular Biology 34: 335–347. https://doi.org/10.1128/MCB.01190-13.
Custer, S.K., M. Neumann, L. Hongbo, A.C. Wright, and J.P. Taylor. 2010. Transgenic mice expressing mutant forms VCP/p97 recapitulate the full spectrum of IBMPFD including degeneration in muscle, brain and bone. Human Molecular Genetics 19: 1741–1755. https://doi.org/10.1093/hmg/ddq050.
Bright, F., E.L. Werry, C. Dobson-Stone, O. Piguet, L.M. Ittner, G.M. Halliday, J.R. Hodges, et al. 2019. Neuroinflammation in frontotemporal dementia. Nature Reviews Neurology 15: 540–555. https://doi.org/10.1038/s41582-019-0231-z.
Blevins, H.M., X. Yiming, S. Biby, and S. Zhang. 2022. The NLRP3 Inflammasome Pathway: A Review of Mechanisms and Inhibitors for the Treatment of Inflammatory Diseases. Frontiers in Aging Neuroscience 14.
Yue, Y., N.R. Nabar, C. S. Shi, O. Kamenyeva, X. Xiao, and II-Y Hwang, M Wan, and JH Kehrl. 2018. SARS-Coronavirus Open Reading Frame-3a drives multimodal necrotic cell death. Cell Death & Disease 9: 904. https://doi.org/10.1038/s41419-018-0917-y.
Funding
This work was supported by National Institute on Aging (Grant number: R01AG031867), National Institute of Arthritis and Musculoskeletal and Skin Disease (Grant number: K24AR073317), National Institutes of Health(Grant number: NS110436) and National Institutes of Health (Grant number: NS097799).
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AS designed and performed the experiments, did the formal analysis, and wrote the original draft, DD, and PK provided Recombinant Alpha-synuclein fibrils used in Figs. 3 and 4 and CW conceptualized, guided the experiments, and reviewed and edited the manuscript. All authors read and approved the manuscript.
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Sharma, A., Dhavale, D.D., Kotzbauer, P.T. et al. VCP Inhibition Augments NLRP3 Inflammasome Activation. Inflammation (2024). https://doi.org/10.1007/s10753-024-02013-6
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DOI: https://doi.org/10.1007/s10753-024-02013-6