Non-decameric NLRP3 follows an MTOC-independent inflammasome activation pathway

Abstract

Die Dissertation ist gesperrt bis zum 30. November 2026 !

NLRP3 is a pivotal molecular initiator of the innate immune response, playing a significant role in the pathogenesis of various non-genetic neurodegenerative diseases such as Alzheimer's and Parkinson's, as well as genetic autoinflammatory disorders like cryopyrin-associated periodic syndrome (CAPS). Despite its critical involvement in these conditions, the precise cell biological mechanisms governing NLRP3 activation remain incompletely understood. Recent advancements in cellular and structural biology have elucidated that the inactive form of NLRP3 assembles into an oligomeric 'cage' structure, specifically a decamer in humans, which exhibits a high affinity for negatively charged membranes like those of the trans-Golgi network (TGN). Under basal conditions, NLRP3 exists in a dynamic equilibrium between its monomeric form in the cytosol and the decameric cage associated with the TGN. Upon activation by various stimuli, the TGN reorganizes into endosomal-like vesicles that transport the NLRP3 cage toward the microtubule organizing center (MTOC) via the microtubule network, where it forms a functional inflammasome. However, whether non-decameric forms of NLRP3 can also achieve full activation remains unclear. In this thesis, we systematically investigate the activation mechanisms of decameric versus non-decameric NLRP3 in monocytic THP-1 cells by engineering a deletion mutant (NLRP3 Δexon3) that cannot form decamers, resulting in predominantly monomeric and dimeric species. Using biochemical assays, confocal and live-cell microscopy, and inflammasome activity measurements, we demonstrate that smaller NLRP3 assemblies can form functional inflammasomes without reliance on the TGN or MTOC, defining an MTOC-independent pathway. Importantly, this pathway remains unaffected by microtubule disruption with the FDA-approved drug colchicine, highlighting its independence from the traditional microtubule-mediated transport system. Furthermore, the MTOC-independent pathway exhibits different kinetics compared to its MTOC-dependent counterpart and selectively responds to potassium-dependent stimuli while not responding to the potassium-independent stimuli imiquimod. Collectively, our findings reveal two parallel and biologically distinct NLRP3 activation pathways, enhancing the understanding of inflammasome regulation.