Spreading depolarization activates the cGAS–STING pathway and drives cranial nociception: therapeutic potential of STING modulation

Spreading depolarization (SD) is a transient wave of near-complete neuronal and glial depolarization in the cortex which underlies migraine aura. Beyond its electrophysiological effects, SD has been shown to trigger a cascade of sterile neuroinflammatory responses which may contribute to trigeminal activation and pain sensitization observed in migraine with aura. Recent studies have highlighted the involvement of innate immune system pathways in SD-associated inflammation. The cyclic GMP-AMP synthase (cGAS)-Stimulator of Interferon genes (STING) pathway induces the expression of type I interferons and pro-inflammatory cytokines in response to cellular stress. Although this pathway is increasingly recognized for its role in neuroinflammation and nociception, its specific contribution to SD-induced mechanisms remains incompletely understood. In this study, we investigated whether SD activates the cGAS-STING pathway in the mouse cerebral cortex and evaluated the functional consequences of this pathway activation on SD associated cranial nociception. SD was induced non-invasively by optogenetic stimulation. Animals were subjected to either a single or six SDs. The expression of cGAS-STING pathway proteins in the cortex were assessed by immunofluorescent labeling and capillary-based Western blotting. Sham-treated animals served as controls. The cellular localization of the pathway proteins in the cortex was also determined. Pharmacological modulation of the pathway was achieved via intraperitoneal administration of the STING inhibitor C-176 (20 mg/kg) or intranasal delivery of the STING agonist 2’3’-cGAMP (1 mg/kg). SD threshold was determined with potassium chloride application, and periorbital nociceptive responses were measured using the manual von Frey test. Furthermore, periorbital mechanical allodynia was assessed at 2 and 24 h following optogenetically induced SDs in animals treated with either 2’3’-cGAMP or vehicle. SD induced cGAS-STING signaling and Interferon beta (IFN-β) expression in the mouse cerebral cortex, with prominent neuronal expression and downstream microglial activation. Pathway activation with 2’3’ cGAMP decreased SD susceptibility and significantly alleviated the development of periorbital mechanical allodynia following SD. Our findings suggest that SD activates the cGAS-STING pathway, extending the scope of SD-induced neuroinflammation. These results also highlight the therapeutic potential of modulating STING to mitigate SD-related nociception and neuroinflammatory consequences associated with headache disorders such as migraine with aura.