Putting the STING in the Arresting: Novel Immune Mechanism Linking Migraine Aura and Headache

Spreading depolarization (SD) is considered to be the underlying cause of migraine aura and implicated in headache initiation through neuroinflammation. This study reveals a novel immune system pathway, that is involved in SD associated neuroinflammation and affects nociceptive behaviours.
Putting the STING in the Arresting: Novel Immune Mechanism Linking Migraine Aura and Headache
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Spreading depolarization activates the cGAS–STING pathway and drives cranial nociception: therapeutic potential of STING modulation - The Journal of Headache and Pain

Background 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. Methods 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. Results 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. Conclusions 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. Graphical Abstract

Spreading depolarization (SD) is a massive wave of cellular depolarization that propagates through the brain's gray matter at a velocity of 2–5 mm/min. Because it spreads across the cortex akin to a tsunami wave over the ocean, it is colloquially termed as a “brain tsunami”. SD can be triggered by the collective depolarization of cells within a certain brain volume. Once initiated, it wreaks havoc on the affected tissue by causing near-complete depolarization of neurons and glial cells, which leads to a severe disruption of ionic and neurotransmitter homeostasis. Consequently, this causes transient suppression in the brain activity and demands a significant amount of metabolic energy for recovery.

SD is heavily implicated in a range of neurological disorders, such as ischemic stroke and traumatic brain injury, where it plays a critical role in secondary tissue damage and lesion expansion. It is also tied to migraine pathophysiology, where it is considered the underlying cause of the migraine aura. Furthermore, experimental studies show that SD can evoke behaviors associated with head pain in rodents, suggesting it is involved in the initiation of migraine headache. One potential mechanism linking SD to headache initiation is neuroinflammation. SD has been shown to trigger a robust, sterile inflammatory response in both the brain parenchyma and the overlying meninges. This inflammatory activation is initiated and maintained by a complex interplay among neurons, astrocytes, and microglia within the parenchyma, which subsequently drives inflammation in the meninges. The resulting release of inflammatory substances in the meninges activates trigeminal nerve endings, which transmit pain signals that ultimately generate the sensation of a headache.

Our study aimed to investigate SD-associated neuroinflammation and its role in headache initiation through a previously unrecognized immune pathway: the cyclic GMP-AMP synthase (cGAS)-Stimulator of Interferon Genes (STING) pathway. The cGAS-STING pathway is a component of the innate immune system with established roles in neuroinflammation across a wide range of neurological disorders. Because this pathway is closely linked to key drivers of SD-associated neuroinflammation and has demonstrated robust modulatory effects on nociception in both acute and chronic pain conditions, we questioned whether it is also involved in SD-associated neuroinflammation and migraine-like nociceptive behaviors in rodents.

To investigate this, we employed optogenetic stimulation to non-invasively induce SD in the cerebral cortex of mice. We then assessed cGAS-STING pathway activation in the cortex and evaluated its cell-type-specific effects on neurons, microglia, and astrocytes using immunofluorescent staining and capillary Western blotting. Additionally, we pharmacologically targeted the cGAS-STING pathway to determine its effects on SD characteristics and associated nociceptive behaviors by evaluating SD susceptibility and behavioural testing with von Frey test to assess periorbital pain sensitivity.

Our results demonstrate that a single SD wave activates the cGAS-STING pathway in the cerebral cortex for up to 24 hours, with predominantly strong activation observed in neurons. This pathway activation also contributed to microglial activation, as pharmacological inhibition of the pathway prevented microglial activation following SD. Interestingly, activating the pathway with specific agonists decreased both SD susceptibility and periorbital pain sensitivity. Importantly, pathway activation prior to SD induction prevented the development of periorbital allodynia (pain response to a normally non-painful stimulus) typically observed after multiple SD waves.

In conclusion, our findings reveal the cGAS-STING pathway as a key driver of SD-associated neuroinflammation. Its robust activation in neurons and clear role in microglial activation underscore its significance in SD induced neuroinflammatory processes. Moreover, the acute modulatory effects of pathway activation on SD susceptibility and headache-like behaviors position this pathway as a promising therapeutic target for migraine and other SD-related neurological disorders. This study marks the beginning of identifying a novel neuroimmune mechanism in migraine pathophysiology. Future studies investigating the specific role of the cGAS-STING pathway in trigeminovascular system activation and employing other clinically relevant migraine models will be essential next steps to fully unraveling its role in headache mechanisms.

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Migraine
Life Sciences > Biological Sciences > Neuroscience > Neurological Disorders > Headache > Migraine
Translational Research
Life Sciences > Health Sciences > Biomedical Research > Translational Research
Neuroimmunology
Life Sciences > Biological Sciences > Neuroscience > Neuroimmunology
Optogenetics
Life Sciences > Biological Sciences > Biological Techniques > Biological Sensors and Probes > Optogenetics

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