Mitochondrial impairment in microglia amplifies NLRP3 inflammasome proinflammatory signaling in cell culture and animal models of Parkinson’s disease

Rotenone and tebufenpyrad activate the NLRP3 inflammasome in primary murine microglia. a MTS assay of cell viability after treating lipopolysaccharide (LPS)-primed primary microglia with the mitochondrial complex-1 inhibiting pesticides rotenone (ROT) or tebufenpyrad (Tebu) (both 1？μM) for 24？h. b Luminex assay showing an increased IL-1β release from LPS-primed microglia after pesticide treatments for 24？h. Treatment with rotenone or tebufenpyrad did not alter TNFα release. c Western blot analysis of pesticide-treated, primed microglial cells showing the cleavage of pro-caspase-1 to its active form caspase-1 p20. d Duolink proximal ligation assay reveals ASC and NLRP3 interaction in pesticide-exposed, primed microglial cells but not in unprimed cells following 3？h of pesticide exposure. Scale bar, 15？μm. e ICC showing ASC speck formation in pesticide-treated primed microglial cells for 2？h. Scale bar, 20？μm. f q-RT-PCR analysis of vehicle- and rotenone-gavaged (n？=？4 each group, 30？mg/kg for 28 days) mice for striatal NLRP3 gene expression. g Western blot analysis for vehicle- and rotenone-gavaged (30？mg/kg for 28 days) mice for NLRP3, caspase-1 and IL-1β. (H-K) Densitometric analysis of Western blot for NLRP3. h, caspase-1 i, caspase-1 p20 j, pro-IL-1β k, and cleaved IL-1β l. For all Western blots, samples derive from the same experiment and were processed in parallel. Data analyzed via Student’s t test, or via two-way ANOVA with Bonferroni adjustment, *p？<？0.05, **p？<？0.01, ***p？<？0.001 and are represented as Mean？±？SEM with n？=？3–