Paraquat Licences α-Synuclein to Trigger NLRP3-Dependent Pyroptosis in Microglia

Parkinsons disease (PD) is characterized by dopaminergic neurodegeneration and the pathological accumulation of -synuclein (Syn) aggregates. While its etiology is multifactorial, environmental toxicants such as paraquat, a widely used herbicide banned in over 70 countries but still permitted in some regions, including Australia, are strongly implicated. We previously showed that Syn activates the NLRP3 inflammasome in microglia, inducing interleukin-1{beta} (IL-1{beta}) release without triggering pyroptosis. Here, we examine how paraquat modulates microglial NLRP3 activation by Syn. Using primary microglia from wild-type (WT) and Nlrp3 knockout (Nlrp3-/-) mice, alongside human monocyte-derived microglia, we demonstrate that paraquat induces dose-dependent NLRP3 activation in LPS-primed WT microglia via NADPH oxidase-mediated reactive oxygen species (ROS) generation. This results in ASC speck formation and IL-1{beta} release, which is absent in Nlrp3-/- microglia and blocked by the selective NLRP3 inhibitor MCC950. While Syn alone fails to induce pyroptosis, co-treatment with paraquat resulted in caspase-1-dependent lactate dehydrogenase (LDH) release and gasdermin-D cleavage, hallmarks of pyroptotic cell death. This response was also suppressed by NLRP3 inhibition. Our findings reveal that paraquat acts as a critical second hit to license Syn-induced inflammasome-dependent pyroptosis in microglia, highlighting a mechanistic link between environmental toxin exposure and innate immune activation in PD pathogenesis. Targeting this axis with brain-penetrant NLRP3 inhibitors may offer an alternative therapeutic avenue for synucleinopathies associated with environmental toxins.