Glucose-regulated protein 75 determines ER\u2013mitochondrial coupling and sensitivity to oxidative stress in neuronal cells

Birgit Honrath,Amalia M Dolga,Jennifer Rieusset,Isabell Metz,Carsten Culmsee,Nadia Bendridi

doi:10.1038/cddiscovery.2017.76

Abstract

The crosstalk between different organelles allows for the exchange of proteins, lipids and ions. Endoplasmic reticulum (ER) and mitochondria are physically linked and signal through the mitochondria-associated membrane (MAM) to regulate the transfer of Ca2+ from ER stores into the mitochondrial matrix, thereby affecting mitochondrial function and intracellular Ca2+ homeostasis. The chaperone glucose-regulated protein 75 (GRP75) is a key protein expressed at the MAM interface which regulates ER–mitochondrial Ca2+ transfer. Previous studies revealed that modulation of GRP75 expression largely affected mitochondrial integrity and vulnerability to cell death. In the present study, we show that genetic ablation of GRP75, by weakening ER–mitochondrial junctions, provided protection against mitochondrial dysfunction and cell death in a model of glutamate-induced oxidative stress. Interestingly, GRP75 silencing attenuated both cytosolic and mitochondrial Ca2+ overload in conditions of oxidative stress, blocked the formation of reactive oxygen species and preserved mitochondrial respiration. These data revealed a major role for GRP75 in regulating mitochondrial function, Ca2+ and redox homeostasis. In line, GRP75 overexpression enhanced oxidative cell death induced by glutamate. Overall, our findings suggest weakening ER–mitochondrial connectivity by GRP75 inhibition as a novel protective approach in paradigms of oxidative stress in neuronal cells.

Highlights

In order to investigate whether impaired Endoplasmic reticulum (ER)–mitochondrial contact formation could alter cell death signaling induced by oxidative stress, we downregulated glucose-regulated protein 75 (GRP75) expression by two different small interfering RNA (siRNA) sequences followed by initiation of cell death with glutamate
GRP75 is suggested to mediate organelle communication between the ER and mitochondria through interacting with both the IP3R and voltagedependent anion channel 1 (VDAC1).10 In this study, we performed an in situ proximity ligation assay between IP3R1 and VDAC1, and confirmed that GRP75 is a component of the complex that forms the mitochondria-associated membrane (MAM)
We showed that GRP75 knockdown and pharmacological inhibition reduced the number of interaction sites between IP3R1 and VDAC1, reduced ER–mitochondrial coupling

Summary

Introduction

Maintaining intracellular Ca2+ ([Ca2+]i) homeostasis is of major importance to preserve cell survival in neuronal tissues, as for instance oxidative stress induces massive Ca2+ influx through different receptor-operated or voltage-dependent Ca2+ channels.[1,2] Enhanced Ca2+ influx together with Ca2+ release from internal stores such as the endoplasmic reticulum (ER) leads to mitochondrial Ca2+ overload and cell death.[3,4,5,6]Small Ca2+ microdomains are frequently transferred from the ER to the mitochondria as part of homeostatic organelle communication.[7,8,9] The propagation of these Ca2+ microdomains is regulated by a multiprotein complex formed by voltagedependent anion channel 1 (VDAC1) located at the outer mitochondrial membrane, the inositol-1,4,5-trisphosphate receptor (IP3R) on the ER membrane and glucose-regulated protein 75 (GRP75), a member of the heat shock protein 70 family.[10,11,12,13,14]Proper integration of this multiprotein complex into the mitochondria-associated membrane (MAM) is critical for Ca2+transfer into the mitochondrial matrix via the tightly regulated mitochondrial Ca2+ uniporter which drives mitochondrial metabolism.[15,16,17,18,19]By establishing local contact points between ER and mitochondria, GRP75 has a major role in maintaining crosstalk between these organelles through coordinating the exchange and transfer of Ca2+, and to drive subsequent signaling cascades.[11,20,21,22,23] GRP75 has been extensively studied in various cancer cells where its expression increased susceptibility to cell death.[24,25] the consequences of an alteration in GRP75 expression to neuronal cell survival are not entirely clear. Small Ca2+ microdomains are frequently transferred from the ER to the mitochondria as part of homeostatic organelle communication.[7,8,9] The propagation of these Ca2+ microdomains is regulated by a multiprotein complex formed by voltagedependent anion channel 1 (VDAC1) located at the outer mitochondrial membrane, the inositol-1,4,5-trisphosphate receptor (IP3R) on the ER membrane and glucose-regulated protein 75 (GRP75), a member of the heat shock protein 70 family.[10,11,12,13,14]. Further studies are required to clarify the function of GRP75 in paradigms of cell death relevant to neurodegenerative diseases

Methods

Results

Conclusion