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Dual NMDAR signaling in astrocytes: flux-independent pH sensor & flux-dependent mitochondrial regulator through membrane-mitochondria communication

Glutamate N-methyl-D-aspartate (NMDA) receptor (NMDAR) is critical for neurotransmission as a Ca2+ channel. Nonetheless, several reports have also demonstrated flux-independent signaling. Astrocytes express NMDAR distinct from its neuronal counterpart, but cultured astrocytes have no electrophysiological response and controversial findings have questioned NMDAR function. We recently demonstrated that in cultured astrocytes NMDA at pH6 (NMDA/pH&) elicits flux-independent Ca2+ release from the Endoplasmic Reticulum (ER) and depletes mitochondrial membrane potential (mΔψ). Here we show that flux-independent Ca2+ release is mainly due to pH6, whereas mΔψ depletion requires both pH6 and flux-dependent NMDAR signaling. Immunofluorescence exhibited that plasma membrane (PM) NMDAR is apposed to ER and mitochondria or surrounds these organelles. Moreover, NMDA/pH6 treatment generated ER stress, increased endocytosis, mitochondria-ER and -nuclear contacts and strikingly, PM invaginations near mitochondria along with electrodense structures referred here as PM-mitochondrial bridges (PM-m-br). These data and earlier observations strongly suggest PM-mitochondria communication. As a proof of concept of this notion, NMDA/pH6 provoked mitochondria labeling by the PM dye FM-4-64FX. Finally, we analyzed by WB NMDAR subunit GluN1 to explore putative causes of NMDAR dual function, we found fragments with M.W. consistent with previously identified cleavage sites. Accordingly, GluN1 intracellular and extracellular domains presented little colocalization. Our findings demonstrate that NMDAR plays a dual function: a flux-independent pH sensor and a flux-dependent regulator of mΔψ. More importantly, mΔψ depletion seems to be mediated by PM-mitochondria communication. Finally, we found different GluN1 fragments that could be involved in NMDAR dual signaling, although causality awaits demonstration.