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Endoplasmic reticulum Ca2+ signaling and mitochondrial Cyt c release in astrocytes following oxygen and glucose deprivation.

ABSTRACT: In the present study, we investigated changes of cytosolic Ca2+([Ca2+](cyt)), endoplasmic reticulum Ca2+([Ca2+](ER)) and mitochondrial Ca2+(Ca2+(m)) in astrocytes following oxygen/glucose deprivation and reoxygenation (OGD/REOX). Two hours OGD did not cause changes in [Ca2+](cyt), but led to a significant increase in [Ca2+](ER). The elevation in [Ca2+](ER) continued and reached a peak level (130 +/- 2 microM) by 90 min REOX. An abrupt release of Ca2+(ER) occurred during 1.5-2.5 h REOX, which was accompanied with a delayed and sustained rise in [Ca2+](cyt). Moreover, Ca2+(m) content was increased significantly within 15 min REOX followed by a secondary rise (approximately 4.5-fold) and a release of mitochondrial cytochrome c (Cyt c). Astrocytes exhibited translocation of Cyt c from mitochondria to endoplasmic reticulum (ER) and up regulation of ER stress protein p-eIF2alpha. Blocking Na+-K+-Cl(-) cotransporter isoform 1 activity, either by its potent inhibitor bumetanide or genetic ablation, abolished release of ER Ca2+, delayed rise in [Ca2+](cyt) and Ca2+(m). Inhibition of the reverse mode operation of the Na+/Ca2+ exchanger significantly attenuated OGD/REOX-mediated Cyt c release. In summary, this study illustrates that OGD/REOX triggers a time-dependent loss of Ca2+ homeostasis in cytosol and organelles (ER and mitochondria) in astrocytes. Collective stimulation of Na+-K+-Cl(-) cotransporter isoform 1 and reverse mode function of Na+/Ca2+ exchanger contributes to these changes.

SUBMITTER: Liu Y

PROVIDER: S-EPMC2923237 | biostudies-literature | 2010 Sep

REPOSITORIES: biostudies-literature

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Endoplasmic reticulum Ca2+ signaling and mitochondrial Cyt c release in astrocytes following oxygen and glucose deprivation.

Liu Yan Y Kintner Douglas B DB Begum Gulnaz G Algharabli Jehad J Cengiz Pelin P Shull Gary E GE Liu Xiang-Jun XJ Sun Dandan D

Journal of neurochemistry 20100614 5

In the present study, we investigated changes of cytosolic Ca2+([Ca2+](cyt)), endoplasmic reticulum Ca2+([Ca2+](ER)) and mitochondrial Ca2+(Ca2+(m)) in astrocytes following oxygen/glucose deprivation and reoxygenation (OGD/REOX). Two hours OGD did not cause changes in [Ca2+](cyt), but led to a significant increase in [Ca2+](ER). The elevation in [Ca2+](ER) continued and reached a peak level (130 +/- 2 microM) by 90 min REOX. An abrupt release of Ca2+(ER) occurred during 1.5-2.5 h REOX, which was ...[more]

PMID: 20557423

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Project description:Phototransduction in Drosophila is mediated by phospholipase C (PLC) and Ca2+-permeable TRP channels, but the function of endoplasmic reticulum (ER) Ca2+ stores in this important model for Ca2+ signaling remains obscure. We therefore expressed a low affinity Ca2+ indicator (ER-GCaMP6-150) in the ER, and measured its fluorescence both in dissociated ommatidia and in vivo from intact flies of both sexes. Blue excitation light induced a rapid (tau ∼0.8 s), PLC-dependent decrease in fluorescence, representing depletion of ER Ca2+ stores, followed by a slower decay, typically reaching ∼50% of initial dark-adapted levels, with significant depletion occurring under natural levels of illumination. The ER stores refilled in the dark within 100-200 s. Both rapid and slow store depletion were largely unaffected in InsP3 receptor mutants, but were much reduced in trp mutants. Strikingly, rapid (but not slow) depletion of ER stores was blocked by removing external Na+ and in mutants of the Na+/Ca2+ exchanger, CalX, which we immuno-localized to ER membranes in addition to its established localization in the plasma membrane. Conversely, overexpression of calx greatly enhanced rapid depletion. These results indicate that rapid store depletion is mediated by Na+/Ca2+ exchange across the ER membrane induced by Na+ influx via the light-sensitive channels. Although too slow to be involved in channel activation, this Na+/Ca2+ exchange-dependent release explains the decades-old observation of a light-induced rise in cytosolic Ca2+ in photoreceptors exposed to Ca2+-free solutions.SIGNIFICANCE STATEMENT Phototransduction in Drosophila is mediated by phospholipase C, which activates TRP cation channels by an unknown mechanism. Despite much speculation, it is unknown whether endoplasmic reticulum (ER) Ca2+ stores play any role. We therefore engineered flies expressing a genetically encoded Ca2+ indicator in the photoreceptor ER. Although NCX Na+/Ca2+ exchangers are classically believed to operate only at the plasma membrane, we demonstrate a rapid light-induced depletion of ER Ca2+ stores mediated by Na+/Ca2+ exchange across the ER membrane. This NCX-dependent release was too slow to be involved in channel activation, but explains the decades-old observation of a light-induced rise in cytosolic Ca2+ in photoreceptors bathed in Ca2+-free solutions.

Project description:BackgroundProlonged cellular activity may overload cell function, leading to high rates of protein synthesis and accumulation of misfolded or unassembled proteins, which cause endoplasmic reticulum (ER) stress and activate the unfolded protein response (UPR) to re-establish normal protein homeostasis. Previous molecular work has demonstrated that sleep deprivation (SD) leads to ER stress in neurons, with a number of ER-specific proteins being upregulated to maintain optimal cellular proteostasis. It is still not clear which cellular processes activated by sleep deprivation lead to ER- stress, but increased cellular metabolism, higher request for protein synthesis, and over production of oxygen radicals have been proposed as potential contributing factors. Here, we investigate the transcriptional and ultrastructural ER and mitochondrial modifications induced by sleep loss.ResultsWe used gene expression analysis in mouse forebrains to show that SD was associated with significant transcriptional modifications of genes involved in ER stress but also in ER-mitochondria interaction, calcium homeostasis, and mitochondrial respiratory activity. Using electron microscopy, we also showed that SD was associated with a general increase in the density of ER cisternae in pyramidal neurons of the motor cortex. Moreover, ER cisternae established new contact sites with mitochondria, the so-called mitochondria associated membranes (MAMs), important hubs for molecule shuttling, such as calcium and lipids, and for the modulation of ATP production and redox state. Finally, we demonstrated that Drosophila male mutant flies (elav > linker), in which the number of MAMs had been genetically increased, showed a reduction in the amount and consolidation of sleep without alterations in the homeostatic sleep response to SD.ConclusionsWe provide evidence that sleep loss induces ER stress characterized by increased crosstalk between ER and mitochondria. MAMs formation associated with SD could represent a key phenomenon for the modulation of multiple cellular processes that ensure appropriate responses to increased cell metabolism. In addition, MAMs establishment may play a role in the regulation of sleep under baseline conditions.

Project description:Inhibition of phosphodiesterase 4 (PDE4) produces neuroprotective effects against cerebral ischemia. However, the involved mechanism remains unclear. Augmentation of endoplasmic reticulum (ER) stress promotes neuronal apoptosis, and excessive oxidative stress is an inducer of ER stress. The present study aimed to determine whether suppression of ER stress is involved in the protective effects of PDE4 inhibition against cerebral ischemia. We found that exposing HT-22?cells to oxygen-glucose deprivation (OGD) significantly activated ER stress, as evidenced by increased expression of the 78-kDa glucose-regulated protein (GRP78), phosphorylated eukaryotic translation-initiation factor 2? (eIF2?), and C/EBP-homologous protein (CHOP). Overexpression of PDE4B increased ER stress, while knocking down PDE4B or treatment with the PDE4 inhibitor, FCPR03, prevented OGD-induced ER stress in HT-22?cells. Furthermore, FCPR03 promoted the translocation of nuclear factor erythroid 2-related factor 2 (Nrf-2) from the cytoplasm to the nucleus. Importantly, the Nrf-2 inhibitor, ML385, blocked the inhibitory role of FCPR03 on OGD-induced ER stress. ML385 also abolished the protective role of FCPR03 in HT-22?cells subjected to OGD. Knocking down heme oxygenase-1 (HO-1), which is a target of Nrf-2, also blocked the protective role of FCPR03, enhanced the level of reactive oxygen species (ROS), and increased ER stress and cell death. We then found that FCPR03 or the antioxidant, N-Acetyl-l-cysteine, reduced oxidative stress in cells exposed to OGD. This effect was accompanied by increased cell viability and decreased ER stress. In primary cultured neurons, we found that FCPR03 reduced OGD-induced production of ROS and phosphorylation of eIF2?. The neuroprotective effect of FCPR03 against OGD in neurons was blocked by ML385. These results demonstrate that inhibition of PDE4 activates Nrf-2/HO-1, attenuates the production of ROS, and thereby attenuates ER stress in neurons exposed to OGD. Additionally, we conclude that FCPR03 may represent a promising therapeutic agent for the treatment of ER stress-related disorders.

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Endoplasmic reticulum Ca2+ signaling and mitochondrial Cyt c release in astrocytes following oxygen and glucose deprivation.

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Endoplasmic reticulum Ca2+ signaling and mitochondrial Cyt c release in astrocytes following oxygen and glucose deprivation.

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