Investigating ASC-αSynuclein Complex-triggered Inflammatory Responses in Murine Microglia

Abstract

Neuroinflammation is a fundamental hallmark of several chronic neurodegenerative diseases, including Parkinson’s disease (PD), and is closely associated with microglial activation. PD is characterized by the progressive loss of dopaminergic neurons in the substantia nigra and the accumulation of misfolded α-synuclein (αSyn) protein aggregates in surviving neurons. A key signaling mechanism that activates chronic inflammation is based on NLRP3 inflammasome activation, which can occur via aggregated forms of amyloid-β (Aβ) and αSyn. The inflammasome adaptor protein, ASC, has an intrinsic ability to oligomerize and, upon cell death, is released as specks into the extracellular space. These ASC specks are recognized by microglia as ‘danger signals’, thereby driving inflammation and neurodegeneration. Recent evidence has suggested that extracellular ASC specks can bind Aβ, and that the internalization of ASC-Aβ complexes by microglia results in the amplification and perpetuation of pro-inflammatory responses. Moreover, studies on human samples and preclinical animal models of PD have established a link between αSyn aggregation, microglial NLRP3 inflammasome activation and neuronal cell death. However, the underlying molecular mechanisms that perpetuate chronic inflammatory responses are incompletely understood. In this study, I produced ASC-αSyn complexes and evaluated their effects on NLRP3 inflammasome activation using a mouse SIM-A9 microglial cell culture model. Examination of electron microscopic images revealed that αSyn was found in close proximity and clustered around ASC fibrils. I further demonstrated that ASC-αSyn complexes were capable of activating the NLRP3 inflammasome to a greater extent than ASC or αSyn alone. This was determined by an increased expression of the NLRP3 sensor at the protein level, augmentation of ASC speck formation and activation of caspase-1. In addition, ASC-αSyn complexes triggered caspase-1-dependent interleukin (IL)-1β/IL-18 processing by SIM-A9 cells, resulting in increased release of IL-1β and IL-18 into their conditioned media. This study links ASC and αSyn to mechanisms that amplify pro-inflammatory responses, thereby promoting a state of chronic inflammation. Overall, these results reveal a role for ASC specks in perpetuating inflammation and point at the possible importance of ASC-αSyn complexes, as triggers, in the propagation and exacerbation of αSyn-linked pathology.