Amyloid-β disrupts unitary calcium entry through endothelial NMDA receptors in mouse cerebral arteries

Abstract

Transient increases in intracellular Ca2+ activate endothelium-dependent vasodilatory pathways. This process is impaired in cerebral amyloid angiopathy, where amyloid- β(1-40) accumulates around blood vessels. In neurons, amyloid- β impairs the Ca2+-permeable N-methyl-D-aspartate receptor (NMDAR), a mediator of endothelium-dependent dilation in arteries. We hypothesized that amyloid- β(1-40) reduces NMDAR-elicited Ca2+ signals in mouse cerebral artery endothelial cells, blunting dilation. Cerebral arteries isolated from 4-5 months-old, male and female cdh5:Gcamp8 mice were used for imaging of unitary Ca2+ influx through NMDAR ( NMDAR sparklets) and intracellular Ca2+ transients. The NMDAR agonist NMDA (10 µmol/L) increased frequency of NMDAR sparklets and intracellular Ca2+ transients in endothelial cells; these effects were prevented by NMDAR antagonists D-AP5 and MK-801. Next, we tested if amyloid- β(1-40) impairs NMDAR-elicited Ca2+ transients. Cerebral arteries incubated with amyloid- β(1-40) (5 µmol/L) exhibited reduced NMDAR sparklets and intracellular Ca2+ transients. Lastly, we observed that NMDA-induced dilation of pial arteries is reduced by acute intraluminal amyloid- β(1-40), as well as in a mouse model of Alzheimer’s disease, the 5x-FAD, linked to downregulation of Grin1 mRNA compared to wild-type littermates. These data suggest that endothelial NMDAR mediate dilation via Ca2+-dependent pathways, a process disrupted by amyloid- β(1-40) and impaired in 5x-FAD mice.

Blood tPA of liver origin contributes to neurovascular coupling involving brain endothelial NMDA receptors

“…All these studies were reported from neuronal investigations. But NMDA receptors are also exhibited by endothelial cells (22,50) and LRP1 too (51,52). On endothelial cells, tPA was reported to mediate NMDAR-dependent signaling, involving differential phosphorylation of proteins associated with tight-junctions, such as the myosin light chain (MLC-1) (50).…”

“…In parallel, Anfray et al have proposed that the circulating tPA could contribute to NVC (18). It is thus interesting to note that, in the central nervous system, the tPA initially reported to be expressed and released by endothelial cells is also expressed and released by neurons; both cells also express NMDA receptors (19)(20)(21)(22).…”

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

“…In agreement with these observations, NMDA induced AP5-sensitive inward currents in the presence of glycine and in the absence of extracellular Mg 2+ in hPASMCs, thereby confirming that NMDARs were functional [ 87 ]. Multiple GluN subunit- and NMDAR-mediated intracellular Ca 2+ signals were also recorded in mouse [ 9 , 88 , 89 ] and human [ 24 , 90 ] cerebrovascular endothelial cells, as more widely discussed in Section 5 .…”

“…Glutamate can induce the opening of the BBB and thereby increase paracellular permeability via NMDAR-mediated intracellular Ca 2+ signals [ 120 , 125 , 126 , 127 , 128 ]. In accord, NMDA-induced elevation in BBB permeability was sensitive to MK-801 [ 125 , 126 , 127 ] or AP5 [ 126 , 128 ] and an NMDA-evoked increase in [Ca 2+ ] i was recorded in both human [ 24 , 115 , 126 ] and mouse [ 129 , 130 ] CMVECs and in mouse cerebral artery endothelial cells [ 88 ]. Intriguingly, NMDA-induced intracellular Ca 2+ signals in hCMEC/D3 cells are sensitive to AP5 and genetic knockdown of GluN1, but not to MK-801 and to removal of extracellular Ca 2+ [ 24 ].…”

“…In accord, NMDARs interact with mGluR1 and mGluR5 to mobilize lysosomal Ca 2+ via TPCs and ER-stored Ca 2+ via InsP 3 receptors and to induce store-operated Ca 2+ entry ( Figure 2 ) [ 24 , 131 ]. Conversely, NMDA-induced intracellular Ca 2+ signals in mouse CMVECs disappear in the absence of extracellular Ca 2+ and in the presence of the pore-blocking antagonist, MK-801 [ 88 ]. Therefore, NMDARs can signal the increase in [Ca 2+ ] i in a flux-dependent manner in mouse CMVECs and in a flux-independent (i.e., metabotropic manner) in human CMVECs.…”