NMDAR in cultured astrocytes: Flux‐independent pH sensor and flux‐dependent regulator of mitochondria and plasma membrane‐mitochondria bridging

Abstract

Glutamate N-methyl-D-aspartate (NMDA) receptor (NMDAR) is critical for neurotransmission as a Ca 2+ channel. Nonetheless, flux-independent signaling has also been demonstrated. Astrocytes express NMDAR distinct from its neuronal counterpart, but cultured astrocytes have no electrophysiological response to NMDA. We recently demonstrated that in cultured astrocytes, NMDA at pH6 (NMDA/pH6) acting through the NMDAR elicits flux-independent Ca 2+ release from the Endoplasmic Reticulum (ER) and depletes mitochondrial membrane potential (mΔΨ). Here we show that Ca 2+ release is due to pH6 sensing by NMDAR, whereas mΔΨ depletion requires both: pH6 and flux-dependent NMDAR signaling. Plasma membrane (PM) NMDAR guard a non-random distribution relative to the ER and mitochondria. Also, NMDA/pH6 induces ER stress, endocytosis, PM electrical capacitance reduction, mitochondria-ER, and-nuclear contacts. Strikingly, it also produces the formation of PM invaginations near mitochondria along with structures referred to here as PM-mitochondrial bridges (PM-m-br). These and earlier data strongly suggest PM-mitochondria communication. As proof of the concept of mass transfer, we found that NMDA/pH6 provoked mitochondria labeling by the PM dye FM-4-64FX. NMDA/pH6 caused PM depolarization, cell acidification, and Ca 2+ release from most mitochondria. Finally, the MCU and microtubules were not involved in mΔΨ depletion, while actin cytoskeleton was partially involved. These findings demonstrate that NMDAR has concomitant flux-independent and flux-dependent actions in cultured astrocytes. K E Y W O R D S astrocyte, calcium, flux-independent, mitochondria, NMDAR, organelle communication | 16623 MONTES dE OCA BALdERAS ET AL.

Mitochondria–plasma membrane interactions and communication

“…In addition, MACs have not been observed associated with tubular invaginations of the PM, as mitochondria in yeast, although in cardiac myocytes, where apparently no MACs are present, similar tubular invaginations radiating from caveolae towards mitochondria have been shown (Fig.3A-5)(18). Moreover, caveolae have been found proximal to subplasmalemmal mitochondria in epithelial cells and astrocytes under different J o u r n a l P r e -p r o o f conditions and could be the means to achieve direct contact between mitochondria and PM domains, as our findings support (Figs.3A-4and 3B-6)(17). More research is required to disentangle the role of PM invaginations for these interactions.…”

“…This transfer was mediated by Ca 2+ , CD38, and ADP ribose signaling and resulted in enhanced neuronal survival. Despite cellular details for such transfer were not described, it is possible that observations by Kolikova et al (49) and ours, show the early steps before mitochondria exit the cell (17,49). In this regard, a striking mechanism of mitochondria transfer between cells through TNT has been reported…”

“…to the PM that contacts dendrites ( 44), similar to cultured astrocytes (17). In these same cells, a different group observed vesicles that could be caveolae between mitochondria and PM.…”

“…B6 , Fig. 6 B in ( 17 ) showing the PM–mitochondria bridges in cultured rat astrocytes. This structure is related to endocytic vesicles, most probably, caveolae for their size ( arrow ).…”

“…However, and although the interaction of mitochondria with the PM could have enormous implications for cell and mitochondria physiology, these interactions have not been deeply investigated. Only in the last decade, the attachment of mitochondria to the PM that occurs during cell budding in yeast has been studied at the molecular level, and to my knowledge, only a few reports have demonstrated its functional induction by stressors in mammalian cells, but no molecular players mediating this interaction have been identified ( 10 , 17 , 18 ). In this section, the literature that has studied or suggested the interaction between mitochondria and the PM is reviewed.…”