The pathophysiology of morning migraine - Morning migraine, characterized by attacks that begin between 3-6 AM, presents a unique challenge for treatment due to the patient’s inability to address the onset early, when medications are most effective. During this same 3-6 AM window, the alternative pathway of the complement system undergoes circadian-dependent activation, generating C3a and C5a complement fragments. Concurrently, soluble C3b and C4b fragments bind to complement receptor 1 (CR1) on circulating red blood cells (RBCs), triggering a massive release of ATP. Given the temporal overlap between peak complement activation, RBC-mediated ATP release, and morning migraine onset, we hypothesize that heightened complement activity and ATP release are linked to morning migraine. Specifically, we propose that the peak release of complement fragments C3b, C4b, C3a and C5a cause higher levels of ATP release from RBC in migraine patients that experience morning migraine than in migraine patients that do not experience it because the level of expression of C3a, C5a and of CR1 (all genetically predetermined). Moreover, we postulate that elevated levels of C3a and C5a, along with persistently high extracellular ATP levels, may drive prolonged activation and sensitization of peripheral and central trigeminovascular neurons, thereby contributing to morning migraine. We aim to test the central hypothesis that circadian-driven complement flux and aberrant purinergic responses are key triggers of morning migraine through the following specific aims: Aim 1: Define the quantitative and qualitative profiles of the complement and purinergic receptors and regulators on circulating cells isolated from morning and non-morning migraineurs vs. healthy controls. Circulating nucleated cells will be collected from healthy controls, migraineurs with morning migraine and migraineurs without morning migraine (15 subjects per group) and analyzed as follows: a) Nucleated cells will be analyzed using flow cytometry for complement and purinergic receptor expression levels, and for isoform composition by PacBio analyses. The flow cytometry results will be then confirmed by transcriptomics analyses. b) The functional response of the complement and purinergic receptors on circulating cells will be assessed by flow cytometric measurements of Ca++ influx following C3a/C5a +ATP stimulation. Aim 2: Determine whether and to what extent C5a+ATP activates/sensitizes dura-sensitive trigeminovascular neurons in male and female rats. We propose to study the effects of dural stimulation with C5a+ATP and C3a+ATP on activation and sensitization of peripheral (C- and Ad-meningeal nociceptors) and central (HT and WDR) trigeminovascular neurons. We will also compare these responses to those with systemic administration of these complement fragments and ATP (2a and 2b); and the effects of pre-administration of lidocaine or adenosine on activation of trigeminovascular neurons by C5a/C3a+ATP (2c and 2d). By linking complement and purinergic signaling with circadian physiological events, this research aims to provide novel mechanistic insights into morning migraines.
