The microglial NLRP3 inflammasome is activated by amyotrophic lateral sclerosis proteins.

Microglial NLRP3 inflammasome activation is emerging as a key contributor to neuroinflammation during neurodegeneration. Pathogenic protein aggregates such as ß-amyloid and α-synuclein trigger microglial NLRP3 activation, leading to caspase-1 activation and IL-1ß secretion. Both caspase-1 and IL-1ß 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-1ß 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-1ß cleavage, ASC speck formation, and the secretion of IL-1ß in a dose- and time-dependent manner. Importantly, SOD1G93A was unable to induce IL-1ß 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-1ß 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.

The Ketone Body ß-Hydroxybutyrate Does Not Inhibit Synuclein Mediated Inflammasome Activation in Microglia.

Parkinson's disease (PD) is recognized as the most common neurodegenerative movement disorder and results in debilitating motor deficits. The accumulation and spread of neurotoxic synuclein aggregates in the form of Lewy bodies is a key pathological feature of PD. Chronic activation of the NLRP3 inflammasome by protein aggregates is emerging as a major pathogenic mechanism in progressive neurodegenerative disorders and is considered an important therapeutic target. Recently the ketone body, ß-hydroxy butyrate (BHB), was shown to efficiently inhibit the NLRP3 inflammasome in macrophages, and in vivo models of inflammatory disease. Furthermore, BHB can readily cross the blood brain barrier suggesting that it could have therapeutic benefits for the management of PD. In this study, we evaluated if BHB could inhibit chronic microglial inflammasome activation induced by pathological fibrillar synuclein aggregates. Interestingly, we found that BHB treatment almost completely blocked all aspects of inflammasome activation and pyroptosis induced by ATP and monosodium urate (MSU) crystals, consistent with previously published reports in macrophages. Surprisingly however, BHB did not inhibit inflammasome activation and release of IL-1ß or caspase-1 induced by synuclein fibrils. Our results demonstrate that BHB does not block the upstream pathways regulating inflammasome activation by synuclein fibrils and suggest that synuclein mediated inflammasome activation proceeds via distinct mechanisms compared to traditional NLRP3 activators such as ATP and MSU.

Activation of µ-opioid receptor and Toll-like receptor 4 by plasma from morphine-treated mice.

In this study, we quantified the ability of opioids present in biological samples to activate the µ-opioid receptor and TLR4 using cell-based assays. Each assay was standardised, in the presence of plasma, using morphine, its µ receptor-active metabolite morphine-6 glucuronide (M6G) and its µ receptor-inactive, but TLR4-active metabolite morphine-3 glucuronide (M3G). Specificity was verified using antagonists. Morphine- and M6G-spiked plasma samples exhibited µ receptor activation, which M3G-spiked plasma lacked. In contrast, M3G showed moderate but consistent activation of TLR-4. Plasma samples were collected at a number of time points from mice administered morphine (1 or 10mg/kg every 12h for 3days) or saline. Morphine administration led to intermittent µ receptor activation, reversed by µ receptor antagonists, and to TRL4 activation at time points where M3G is measured in plasma. Interestingly, this protocol of morphine administration also led to TLR4-independent NF-κB activation, at time points where M3G was not detected, presumably via elevation of circulating cytokines including, but not limited to, TNFα. Circulating TNFα was increased after three days of morphine administration, and TNFα mRNA elevated in the spleen of morphine-treated mice.