Dynamin-Related Protein 1 Inhibitors Protect against Ischemic Toxicity through Attenuating Mitochondrial Ca2+ Uptake from Endoplasmic Reticulum Store in PC12 Cells

Ye Tian,Ge-Juan Zhang,Yong Liu,Bin Li,Ming-Ze Chang,Zheng-Li Di,Wen-Zhen Shi

doi:10.3390/ijms15023172

Abstract

Intracellular calcium homeostasis disorder and mitochondrial dysfunction are involved in many acute and chronic brain diseases, including ischemic brain injury. An imbalance in mitochondrial fission and fusion is one of the most important structural abnormalities found in a large number of mitochondrial dysfunction related diseases. Here, we investigated the effects of mitochondrial division inhibitor A (mdivi A) and mdivi B, two small molecule inhibitors of mitochondrial fission protein dunamin-related protein 1 (Drp-1), in neuronal injury induced by oxygen-glucose deprivation (OGD) in PC12 cells. We found that mdivi A and mdivi B inhibited OGD-induced neuronal injury through attenuating apoptotic cell death. These two inhibitors also preserved mitochondrial function, as evidenced by reduced reactive oxygen species (ROS) generation and cytochrome c release, as well as prevented loss of mitochondrial membrane potential (MMP). Moreover, mdivi A and mdivi B significantly suppressed mitochondrial Ca2+ uptake, but had no effect on cytoplasmic Ca2+ after OGD injury. The results of calcium imaging and immunofluorescence staining showed that Drp-1 inhibitors attenuated endoplasmic reticulum (ER) Ca2+ release and prevented ER morphological changes induced by OGD. These results demonstrate that Drp-1 inhibitors protect against ischemic neuronal injury through inhibiting mitochondrial Ca2+ uptake from the ER store and attenuating mitochondrial dysfunction.

Highlights

Stroke is the second most common cause of death and a significant cause of long-term disability worldwide
Cultured PC12 cells were treated with mitochondrial division inhibitor A (mdivi A) or mdivi B in different concentrations
Both mdivi A and mdivi B significantly increased the expression of optic atrophy type 1 (Opa1) and mitofusin 1 (Mfn1), two fusion related mitochondrial dynamic proteins, and decreased the expression of dynamin-related protein 1 (Drp-1) (Figure 1D)

Summary

Introduction

Stroke is the second most common cause of death and a significant cause of long-term disability worldwide. The mechanisms of ischemic stroke are considered to be related to hyper-activation of ionotropic and metabotropic glutamate receptors, massive influx of calcium ions, over-production of reactive oxygen species (ROS) and over-activation of Ca2+-dependent lethal enzymes, but the exact molecular mechanisms are not fully understood [2,3]. Brain ischemic injury results in O2 and ATP depletion and toxic metabolites buildup, which is followed by re-establishment of blood flow through increased generation of reactive oxygen and nitrogen species [2,4]. Mitochondria play essential roles in energy metabolism, and increasing evidence suggests that structural and functional abnormalities in mitochondria are involved in the regulation of the cell death pathways in response to ischemic brain injury [5]. Small molecule inhibitors were used to investigate Drp-1 dependent mitochondrial death pathways in oxygen-glucose deprivation (OGD) in PC12 cells.

Results

Discussion

Conclusion