Selpercatinib mitigates microglia-mediated neuroinflammation by inhibiting RET signaling and NLRP3 inflammasome activation

Abstract

Selpercatinib is a highly selective rearranged during transfection (RET) receptor tyrosine kinase inhibitor approved for RET fusion-positive cancers and known to penetrate the blood–brain barrier. Although RET signaling has been implicated in neural development and cell survival, its role in microglia-mediated neuroinflammation and NOD-like receptor family pyrin domain-containing 3 (NLRP3) inflammasome activation remains poorly understood. This study investigated whether Selpercatinib attenuates neuroinflammatory signaling in microglia by modulating RET-dependent kinase cascades. Neuroinflammation was induced in BV2 microglial cells using lipopolysaccharide (LPS; 200 ng/mL), followed by post-treatment with Selpercatinib (5 μM). For in vivo validation, male C57BL/6N mice received systemic LPS administration (10 mg/kg, i.p.) followed by Selpercatinib treatment (10 mg/kg, i.p.), and hippocampal tissues were collected for analysis. Proinflammatory gene expression, RET-associated signaling pathways, microglial phenotypic markers, and NLRP3 inflammasome components were assessed using quantitative real-time polymerase chain reaction, western blotting, enzyme-linked immunosorbent assay, immunocytochemistry, and flow cytometry. RET-targeting siRNA was used to genetically validate the involvement of RET in LPS-induced inflammatory signaling. Selpercatinib reduced RET phosphorylation at Tyr1062 in LPS-stimulated BV2 microglia and reduced the mRNA expression of proinflammatory mediators, including il-1β, il-6, tnf-α, cox-2 , inos, ccl-2 , and ccl-7 in BV2 microglia. Mechanistically, Selpercatinib attenuated AKT and ERK phosphorylation, inhibited NF-κB nuclear translocation, and reduced CREB phosphorylation. Similarly, RET knockdown reduced LPS-induced proinflammatory gene expression and attenuated AKT/ERK–NF-κB/CREB signaling, providing genetic evidence for the involvement of RET in these inflammatory responses. Selpercatinib also modulated LPS-induced microglial activation by decreasing the expression of reactive microglial markers, including CD16/32, disease-associated microglia-associated Clec7a, and microglial neurodegenerative phenotype-associated Galectin-3, while increasing CD206, the marker associated with tissue repair. In addition, Selpercatinib significantly reduced LPS-induced intracellular ROS generation. Furthermore, Selpercatinib inhibited NLRP3 inflammasome activation, as evidenced by reduced levels of NLRP3, ASC, pro-caspase-1, cleaved caspase-1, GSDMD, and IL-1β in BV2 microglia. Consistent with the in vitro findings, Selpercatinib attenuated LPS-induced hippocampal il-1β gene expression in vivo and reduced NLRP3 inflammasome-related proteins, including ASC, pro-caspase-1, cleaved caspase-1 and GSDMD-F and N in the hippocampus of mice. Selpercatinib attenuated the development of microglia-mediated neuroinflammatory responses through modulation of RET-associated AKT/ERK–NF-κB/CREB signaling and suppression of NLRP3 inflammasome activation. These findings provide a mechanistic basis for further investigating Selpercatinib as an early intervention agent for neuroinflammatory responses.