pH Changes Drive Blood Brain Barrier Opening and Anti-Migraine Agent Blood to Brain Uptake

Abstract

Deciphering the precise pathophysiologic mechanisms underlying migraine has proven to be quite elusive. Cortical spreading depression (CSD) has long been a postulated culprit, yet a cogent mechanistic link remains undiscovered. Pathologies of the blood brain barrier (BBB) have been observed in multiple disease states of the CNS, however a discrete role of BBB pathology in migraine remains uninvestigated. CSD events trigger release of several substances into the cellular microenvironment of the CNS, a major constituent being H+ ions, which acidify the regions they are released and are regulated by the NHE1 Na+/H+ antiporter. Previous studies reported sex hormone divergence in protective abilities of endothelial barriers, and migraine incidence shows a similar trend. Sex hormone modulation of endothelial barrier integrity via NHE1 regulation and control of pH flux was investigated to assess effects of testosterone, estradiol, and progesterone in the context of endothelial barrier integrity. Testosterone was found to tighten the endothelial barrier, while estradiol reduced cell surface NHE1 localization without impacting barrier integrity. Progesterone was found to increase surface expression of NHE1, implicating NHE1 activity in progesterone responsive barrier permeability. Quantitative proteomics demonstrated protein enrichment divergence due to hormone treatment. This suggests sex hormone dependent modulation of pH homeostasis via divergent regulation of NHE1 functional expression and modifications to the endothelial proteome. NHE1 functions as a primary regulator of pH within the CNS. Extracellular K+ flux following CSD events is a known inducer of BBB permeability, and it was hypothesized to act indirectly through change in NHE1 function to induce loss of barrier integrity while also increasing triptan uptake through an NHE1 dependent mechanism. With the use of NHE1 knockdowns and pharmacological inhibition, downregulation of NHE1 mitigated paracellular leak of an endothelial barrier, while this same loss of expression induced sumatriptan uptake in the presence of elevated K+ ions, suggesting alteration to pH homeostasis via K+ induced NHE1 relocalization contributes to BBB functional integrity. Endothelial cells form extremely restrictive tight junctions in the CNS, modulated by several critical proteins forming the physical portion of the BBB. Permeability and imaging assays demonstrated CSD induced dynamic opening of the endothelial barrier in vitro specifically due to relocalization and loss of functional expression of several tight junction and cytoskeletal proteins. Global proteome analysis of endothelial cells demonstrated significant alterations to expression of these proteins following acidic and K+ insult, while phospho-proteomic data demonstrated a potential mechanism based on post translational modifications. Specifically, ZO-1 phosphorylation states following insult allowed for PTM “signatures” following discrete CSD modelled insults. This data implies that increase of K+ and H+ ions due to CSD induces breach at the BBB due to dynamic relocalization of critical tight junction proteins through alterations to post translational modifications. In total these studies demonstrate a clear link between CSD, BBB pathology, and sex hormones through the common element of pH fluctuation. This could provide a foundation into the observed divergence in migraine incidence between the sexes, as well as provide a physiologic foundation for CSD induction of migraine, as well as illuminating new potential therapeutic and biomarker targets in the form of ZO-1 PTM states.