Relieving ER-mitochondrial crosstalk protects neuronal HT22 cells from oxidative glutamate toxicity

The crosstalk between the endoplasmic reticulum (ER) and mitochondria facilitates the transfer of proteins, lipids and calcium between these organelles. ER-mitochondrial coupling maintains the driving force for calcium that is released from inositol-1,4,5-triphosphate receptors (IP3R) into the mitochondrial matrix where it determines mitochondrial respiration. ER-mitochondrial contact points are built by ER-bound IP3 receptors and voltage dependent anion channel 1 (VDAC1) on the outer mitochondrial membrane, that are connected by the chaperone glucose-regulated protein 75 (GRP75/mortalin). Enhanced mitochondrial calcium [Ca2+]m uptake, thus [Ca2+]m overload, and mitochondrial integrity are critical parameters during oxidative glutamate toxicity in neuronal HT22 cells. In our study, we investigated the role of ER-mitochondrial coupling in immortalized mouse hippocampal HT22 cells in response to glutamate-induced cell death. In these cells, we confirm that GRP75 determines ER-mitochondrial contact formation as shown by an in situ proximity ligation assay. Using siRNA-mediated knockdown, CRISPR/Cas9-mediated knockout and pharmacological inhibition of GRP75, we show that relieving ER-mitochondrial crosstalk protects against glutamate-induced cell death. In response to the glutamate challenge, we observe preservation of mitochondrial morphology, reduction of mitochondrial ROS formation and attenuation of both [Ca2+]c and [Ca2+]m levels. We provide for the first time evidence that disrupting ER-mitochondrial coupling, thus [Ca2+]m uptake through [Ca2+]ER release, through silencing GRP75 conferred protection against glutamate toxicity in neuronal HT22 cells.