Exploring the Role of the 2-AG Endocannabinoid System in Migraine Prevalence, Physiology, and Treatment

Abstract

Migraine headache is a debilitating disorder of unknown etiology that has been documented in medical records dating back thousands of years. While advancements have been made in understanding the physiology that underlies migraine, medical scientists still struggle to fully explain the symptomology that corresponds with migraine. Trigeminal nociception is heavily implicated in migraine development, as is the role of vasodilation and alterations to blood-brain barrier integrity, yet the more that is elucidated about migraine, the more it becomes clear that migraine represents an incredibly complex neurovascular event. Current therapeutics rely primarily on inducing meningeal vasoconstriction with 5HT1B/D agonists and CGRP antagonism, but symptom relief is rarely absolute. Other options for migraine treatment include anti-inflammatory therapies, such as NSAIDs, however there is always the possibility of developing tolerance or even secondary overuse headache. Within the past two decades, the role of the endocannabinoid system has been increasingly investigated in migraine physiology. Evidence has shown that migraineurs have decreased central levels of the endocannabinoids, 2-AG and AEA, and that exogenous cannabinoid agonists can often help to alleviate migraine pain. The theory of Clinical Endocannabinoid Deficiency has emerged based off this evidence, touting that migraine, and other functional pain disorders, may stem from the observed decrease in endocannabinoid tone. Prior work in animal models has demonstrated that inhibition of AEA and 2-AG hydrolysis can ameliorate induced headache pain; however, clinical trials of AEA hydrolysis inhibitors were forced to halt due to fatal adverse effects and lack of efficacy. Therefore, investigation into endocannabinoid-based therapeutics has shifted to studies of 2-AG. In this thesis, we seek to validate reduced endocannabinoid tone during headache and to better understand the role that 2-AG plays during induced headache. As clinical prevalence of migraine shows a sex difference of roughly 3:1 for females: males, we began by investigating whether sex differences exist within the endocannabinoid system between female and male rodents. Females were shown to have reduced levels of 2-AG as compared to males within the periaqueductal gray (PAG), an important region for descending pain modulation. Furthermore, immunohistochemistry and proteomic analysis revealed that females have greater expression of the 2-AG hydrolyzing enzymes, MAGL and ABHD6, within the PAG. These results indicate that the increased prevalence of functional pain disorders in the female population may indeed arise from decreased 2-AG signaling within the PAG. We next sought to recapitulate reductions of 2-AG during migraine by utilizing three models of headache: KCl administration, sumatriptan overuse, and morphine overuse. In all three models, 2-AG was reduced within the PAG as compared to controls. Within the KCl model, we observed increased expression of MAGL and ABHD6 following headache induction, as well as increases in PGE2 and glial activation markers (GFAP). This demonstrates that currently utilized models of headache lead to reductions in endocannabinoid tone and increases in neuroinflammation in pertinent nociceptive regions. Following evidence that endocannabinoid tone is reduced during headache, and that females display reduced levels of 2-AG as compared to males, we investigated whether we could induce headache phenotypes by exogenously depleting 2-AG. DAGL synthesizes 2-AG from DAG, with DAGLα representing the primary 2-AG synthesizing enzyme within the central nervous system (CNS). We found that DAGL and specific DAGLα inhibition led to reductions of 2-AG within the PAG that corresponded to induction of periorbital allodynia without hind paw allodynia. Female and male rodents were utilized, and it was observed that females displayed greater allodynia for longer periods of time than males. DAGLα inhibition also led to increased photophobia and anxiety behaviors within animals. Taken together, these results indicate that depletion of 2-AG is sufficient to trigger headache, that females are more sensitive than males to 2-AG depletion, and that 2-AG depletion primarily induces cephalic allodynia. This validates 2-AG signaling as a player in headache development and DAGLα inhibition as a potential novel means of modeling episodic migraine phenotypes in rodents. To explore the potential of endocannabinoid-based therapeutics, we built off evidence that 2-AG is reduced during headache due to increased degradation. MAGL and ABHD6 inhibitors were employed before and after KCl induced headache to test for their abilities to prevent and reverse induced headache. We also examined for the receptor dependency of these effects by co-administering hydrolysis inhibitors with antagonists of the CB1R and CB2R. We observed that both MAGL and ABHD6 inhibition can prevent and reverse KCl induced headache. Furthermore, ABHD6 was shown to be independent of the cannabinoid receptors as a preventative treatment, while its reversal was primarily mediated via the CB1R. MAGL, on the other hand, showed dependence on the CB2R for both prevention and reversal. These findings indicate that 2-AG hydrolysis inhibitors may represent novel headache therapeutics. As the CB2R primarily signals at microglia to reduce neuroinflammation, MAGL inhibition is particularly appealing in its ability to avoid the psychoactive effects associated with CB1R agonism. Alterations to the blood-brain barrier are well documented during headache, so our final objective was to investigate the contributions of 2-AG signaling to barrier integrity. In vivo analysis of barrier permeability via carotid perfusion of 14C-sucrose demonstrated increased extravasation of 14C-sucrose into PAG tissue following both DAGLα inhibition and medication overuse, correlating with previous findings within a KCl model of headache. In vitro analysis of bEnd.3 cells following DAGLα inhibition demonstrated reduced trans-endothelial electrical resistance, increased trans-endothelial leak of 14C-sucrose, and changes in cell morphology that correlated with reductions of the tight junction protein VE-Cadherin. Further investigation of the tight junction effects elucidated that VE-Cadherin underwent cleavage following DAGLα inhibition. VE-Cadherin is primarily cleaved via a calcium dependent kinase, therefore we investigated whether DAGLα inhibition induced greater levels of intracellular calcium, which was confirmed with the calcium imaging dye Fura-2, AM. Finally, we confirmed that DAGLα inhibition was reducing endothelial cell levels of 2-AG via LC-MS. Based on this data, we conclude that DAGLα inhibition reduces integrity of the blood-brain barrier by altering the function of tight junction proteins. The results of this thesis demonstrate that 2-AG signaling plays a major role during headache development. Female rodents displayed reduced 2-AG levels in the PAG that correspond with the female prevalence of clinical migraine, currently utilized models of headache likewise reduced PAG 2-AG signaling, exogenous inhibition of 2-AG synthesis induced headache pain in a sex-dependent manner, inhibition of 2-AG hydrolysis alleviated induced headache, and depletion of 2-AG led to loss of integrity of the blood-brain barrier. This work translates well to clinical research, as the induced headache phenotypes following DAGLα inhibition correlate directly with episodic migraine. Further investigation of the effects of 2-AG depletion on the blood-brain barrier is warranted, as well as research into the potential of reversible 2-AG hydrolysis inhibitors, namely MAGL inhibitors, for clinical treatment of migraine.