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Activation and Measurement of NLRP3 Inflammasome Activity Using IL-1β in Human Monocyte-derived Dendritic Cells
Published on: May 22, 2014
The microglial NLRP3 inflammasome is activated by amyotrophic lateral sclerosis proteins
Vandana Deora1, John D Lee1,2, Eduardo A Albornoz1,3
1School of Biomedical Sciences, Faculty of Medicine, The University of Queensland, St. Lucia, Queensland, Australia.
Abstract:
Microglial NLRP3 inflammasome activation is emerging as a key contributor to neuroinflammation during neurodegeneration. Pathogenic protein aggregates such as β-amyloid and α-synuclein trigger microglial NLRP3 activation, leading to caspase-1 activation and IL-1β secretion. Both caspase-1 and IL-1β contribute to disease progression in the mouse SOD1G93A model of amyotrophic lateral sclerosis (ALS), suggesting a role for microglial NLRP3. Prior studies, however, suggested SOD1G93A mice microglia do not express NLRP3, and SOD1G93A protein generated IL-1β in microglia independent to NLRP3. Here, we demonstrate using Nlrp3-GFP gene knock-in mice that microglia express NLRP3 in SOD1G93A mice. We show that both aggregated and soluble SOD1G93A activates inflammasome in primary mouse microglia leading caspase-1 and IL-1β cleavage, ASC speck formation, and the secretion of IL-1β in a dose- and time-dependent manner. Importantly, SOD1G93A was unable to induce IL-1β secretion from microglia deficient for Nlrp3, or pretreated with the specific NLRP3 inhibitor MCC950, confirming NLRP3 as the key inflammasome complex mediating SOD1-induced microglial IL-1β secretion. Microglial NLRP3 upregulation was also observed in the TDP-43Q331K ALS mouse model, and TDP-43 wild-type and mutant proteins could also activate microglial inflammasomes in a NLRP3-dependent manner. Mechanistically, we identified the generation of reactive oxygen species and ATP as key events required for SOD1G93A -mediated NLRP3 activation. Taken together, our data demonstrate that ALS microglia express NLRP3, and that pathological ALS proteins activate the microglial NLRP3 inflammasome. NLRP3 inhibition may therefore be a potential therapeutic approach to arrest microglial neuroinflammation and ALS disease progression.
Insights
Amyotrophic lateral sclerosis (ALS) proteins activate microglial NLRP3 inflammasomes, driving neuroinflammation. Inhibiting NLRP3 may halt ALS progression and microglial activation.
Area of Science:
- Neuroscience
- Immunology
- Molecular Biology
Background:
- Microglial NLRP3 inflammasome activation contributes to neuroinflammation in neurodegenerative diseases.
- Pathogenic protein aggregates like beta-amyloid and alpha-synuclein trigger NLRP3 activation, leading to IL-1β secretion.
- Previous studies suggested conflicting roles for NLRP3 in the SOD1G93A mouse model of ALS.
Purpose of the Study:
- To investigate the expression and role of microglial NLRP3 inflammasome in ALS.
- To determine if ALS-associated proteins SOD1 and TDP-43 activate the NLRP3 inflammasome in microglia.
- To explore potential therapeutic strategies targeting microglial NLRP3 in ALS.
Main Methods:
- Utilized Nlrp3-GFP gene knock-in mice to track NLRP3 expression in microglia.
- Assessed inflammasome activation (caspase-1, IL-1β cleavage, ASC speck formation) in primary microglia exposed to SOD1G93A and TDP-43 proteins.
- Employed Nlrp3-deficient microglia and the NLRP3 inhibitor MCC950 to confirm NLRP3 dependency.
- Investigated upstream signaling events including reactive oxygen species and ATP generation.
Main Results:
- Confirmed microglial NLRP3 expression in SOD1G93A mice.
- Demonstrated that both aggregated and soluble SOD1G93A activate the NLRP3 inflammasome in a dose- and time-dependent manner.
- Showed that SOD1G93A-induced IL-1β secretion is critically dependent on NLRP3.
- Observed NLRP3 upregulation in a TDP-43Q331K ALS model, with TDP-43 proteins also activating NLRP3.
- Identified reactive oxygen species and ATP as key mediators of SOD1G93A-induced NLRP3 activation.
Conclusions:
- ALS-associated proteins, including SOD1 and TDP-43, activate the microglial NLRP3 inflammasome.
- Microglial NLRP3 inflammasome plays a significant role in ALS pathogenesis.
- Targeting microglial NLRP3 represents a promising therapeutic avenue for ALS treatment by reducing neuroinflammation.
