Activation of non-classical NMDA receptors by glycine impairs barrier function of brain endothelial cells

Abstract

Blood–brain barrier (BBB) integrity is necessary to maintain homeostasis of the central nervous system (CNS). NMDA receptor (NMDAR) function and expression have been implicated in BBB integrity. However, as evidenced in neuroinflammatory conditions, BBB disruption contributes to immune cell infiltration and propagation of inflammatory pathways. Currently, our understanding of the pathophysiological role of NMDAR signaling on endothelial cells remains incomplete. Thus, we investigated NMDAR function on primary mouse brain microvascular endothelial cells (MBMECs). We detected glycine-responsive NMDAR channels, composed of functional GluN1, GluN2A and GluN3A subunits. Importantly, application of glycine alone, but not glutamate, was sufficient to induce NMDAR-mediated currents and an increase in intracellular Ca2+ concentrations. Functionally, glycine-mediated NMDAR activation leads to loss of BBB integrity and changes in actin distribution. Treatment of oocytes that express NMDARs composed of different subunits, with GluN1 and GluN3A binding site inhibitors, resulted in abrogation of NMDAR signaling as measured by two-electrode voltage clamp (TEVC). This effect was only detected in the presence of the GluN2A subunits, suggesting the latter as prerequisite for pharmacological modulation of NMDARs on brain endothelial cells. Taken together, our findings argue for a novel role of glycine as NMDAR ligand on endothelial cells shaping BBB integrity.

GluK2 Is a Target for Gene Therapy in Drug‐Resistant Temporal Lobe Epilepsy

“…Ten μg were reverse‐transcribed into cDNA by Maxima First Strand cDNA Synthesis Kit (Thermofischer). Real‐time qPCR was performed in a LightCycler480 real‐time PCR system (Roche) using TaqMan Gene Expression Assays (Thermo Fisher Scientific) for mouse grik1 (Mm00446882_m1), grik2 (Mm01181234‐m1), grik3 (Mm01179722_m1), grik4 (Mm00615472_m1), grik5 (Mm00433774_m1), gria1 (Mm00433753_m1), gria2 (Mm01220174_m1), grin2a (Mm00433802_m1), grabrb2 (Mm00549788_s1), gabrb3 (Mm00433473_m1), and gapdh (Mm99999915‐g1) as a housekeeping gene 29,30 …”

The Emerging Role of N-Methyl-D-Aspartate (NMDA) Receptors in the Cardiovascular System: Physiological Implications, Pathological Consequences, and Therapeutic Perspectives

“…The molecular architecture of endothelial NMDARs in human CNS microvessels has been investigated by exploiting primary CMVECs, which express GluN1 [ 115 , 117 ], and in the hCMEC/D3 cell line, which has been established from the human temporal lobe microvessels [ 118 ] and express GluN1, GluN2C, and GluN3B subunits ( Table 3 ) [ 24 ]. Therefore, the molecular configuration of endothelial NMDARs in cortical vessels is enriched with GluN2C and GluN3 subunits, which are likely to yield a low sensitivity to extracellular Mg 2+ , low single-channel conductance, and sensitivity to glycine [ 10 , 24 , 119 ].…”

Ferroptosis: Underlying mechanism and the crosstalk with other modes of neuronal death after intracerebral hemorrhage

“…Glutamate and glutamine are also important regulators of ferroptosis ( Gao et al, 2015 ). Researchers have found in mice, rabbits, and patients with ICH that glutamate levels in brain tissue surrounding the hematoma were elevated ( Li et al, 2017b ; Epping et al, 2022 ). The addition of human glutamate to the culture medium of HT22 hippocampal neurons resulted in a significant increase in cell death ( Chen et al, 2022a ).…”