Drug-Resistant Temporal Lobe Epilepsy Alters the Expression and Functional Coupling to Gαi/o Proteins of CB1 and CB2 Receptors in the Microvasculature of the Human Brain.

Vicente Sánchez-Valle,Fruzsina Walter,María De Los Ángeles Nuñez-Lumbreras,Luisa Rocha,Rosalinda Guevara-Guzmán,María Guadalupe Valle-Dorado,Maria A Deli,José Luis Castañeda-Cabral,Iris Martínez-Juárez,Francia Carmona-Cruz,Mario Alonso-Vanegas,Sandra Orozco-Suárez

doi:10.3389/fnbeh.2020.611780

Abstract

Cannabinoid receptors 1 and 2 (CB1 and CB2, respectively) play an important role in maintaining the integrity of the blood–brain barrier (BBB). On the other hand, BBB dysfunction is a common feature in drug-resistant epilepsy. The focus of the present study was to characterize protein expression levels and Gαi/o protein-induced activation by CB1 and CB2 receptors in the microvascular endothelial cells (MECs) isolated from the brain of patients with drug-resistant mesial temporal lobe epilepsy (DR-MTLE). MECs were isolated from the hippocampus and temporal neocortex of 12 patients with DR-MTLE and 12 non-epileptic autopsies. Immunofluorescence experiments were carried out to determine the localization of CB1 and CB2 receptors in the different cell elements of MECs. Protein expression levels of CB1 and CB2 receptors were determined by Western blot experiments. [35S]-GTPγS binding assay was used to evaluate the Gαi/o protein activation induced by specific agonists. Immunofluorescent double-labeling showed that CB1 and CB2 receptors colocalize with tight junction proteins (claudin-5, occludin, and zonula occludens-1), glial fibrillary acidic protein and platelet-derived growth factor receptor-β. These results support that CB1 and CB2 receptors are expressed in the human isolated microvessels fragments consisting of MECs, astrocyte end feet, and pericytes. The hippocampal microvasculature of patients with DR-MTLE presented lower protein expression of CB1 and CB2 receptors (66 and 43%, respectively; p < 0.001). However, its Gαi/o protein activation was with high efficiency (CB1, 251%, p < 0.0008; CB2, 255%, p < 0.0001). Microvasculature of temporal neocortex presented protein overexpression of CB1 and CB2 receptors (35 and 41%, respectively; p < 0.01). Their coupled Gαi/o protein activation was with higher efficiency for CB1 receptors (103%, p < 0.006), but lower potency (p < 0.004) for CB2 receptors. The present study revealed opposite changes in the protein expression of CB1 and CB2 receptors when hippocampus (diminished expression of CB1 and CB2) and temporal neocortex (increased expression of CB1 and CB2) were compared. However, the exposure to specific CB1 and CB2 agonists results in high efficiency for activation of coupled Gαi/o proteins in the brain microvasculature of patients with DR-MTLE. CB1 and CB2 receptors with high efficiency could represent a therapeutic target to maintain the integrity of the BBB in patients with DR-MTLE.

Highlights

The blood–brain barrier (BBB) regulates the access of molecules, drugs, neurotoxins, and pathogens from the bloodstream into the cerebral parenchyma
The immunofluorescence images and their Z-stack analysis confirmed that receptors CB1 and CB2 proteins were expressed in the brain microvessels obtained from patients with drug-resistant mesial temporal lobe epilepsy (DR-MTLE) and non-epileptic autopsies (Figure 1; Supplementary Videos 1–4)
Immunofluorescent doublelabeling showed that CB1 and CB2 receptors colocalize with claudin-5, occludin, and ZO-1 proteins (Figures 2–4)

Summary

Introduction

The blood–brain barrier (BBB) regulates the access of molecules, drugs, neurotoxins, and pathogens from the bloodstream into the cerebral parenchyma. Tight junctions involve the interaction of transmembrane (occludin, claudins, and junctional adhesion molecules), as well as cytoplasmic [zonula occludens (ZO)] proteins (Hawkins and Davis, 2005). Changes in the expression and interaction of these proteins facilitate BBB disruption (Kook et al, 2012). The activation of CB1 receptors prevents downregulation of ZO-1, claudin-5, and junctional adhesion molecules-1 proteins and the consequent BBB breakdown in cocultures of human brain MECs and astrocytes (Lu et al, 2008). The activation of CB2 receptors increases the transendothelial electrical resistance and the expression of tight junction proteins in MECs and reduces neuroinflammation effects (Ramirez et al, 2012). In autopsy brain samples from patients with multiple sclerosis, the BBB overexpressed CB2 receptors in chronic inactive plaques (Zhang et al, 2011), a finding that supports their protective role

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