Temporal Analysis of Changes in Neuronal c-fos mRNA Levels Induced by Depletion of Endoplasmic Reticulum Calcium Stores: Effect of Clamping Cytoplasmic Calcium Activity at Resting Levels

Abstract: Activation of immediate early gene expression is a key event in stress-induced neuronal cell injury. To study whether changes in cytoplasmic calcium activity are necessary to activate neuronal immediate early gene expression, endoplasmic reticulum (ER) calcium stores of primary neurons were depleted by exposing cells to thapsigargin (Tg), an irreversible inhibitor of ER Ca²⁺-ATPase. Tg-induced rise in [Ca²⁺]_i and the effect of loading neurons with the cell-permeable calcium chelator BAPTA-AM on this increase in [Ca²⁺]_i were measured in fura-2-loaded cells by fluorescence microscopy. Changes in c- fos mRNA levels were evaluated by quantitative PCR. Tg treatment of neurons produced a pronounced rise in c- fos mRNA levels (∼10-fold more than DMSO) which peaked at 1 h after exposure. The Tg-induced rise in c- fos mRNA content was unchanged (hippocampal neurons) or even increased further (cortical neurons) by preloading cells with BAPTA before incubation with Tg. It is concluded that in neuronal cells an increase in cytoplasmic calcium activity is not a prerequisite for a rise in mRNA levels of c- fos . Thus, stress-induced changes in mRNA levels of immediate early genes of neurons may also result from disturbances in ER calcium homeostasis and not necessarily by an overload of cells with calcium ions. The results of the present series of experiments cast further doubt on the widely accepted hypothesis that the stress-induced cytoplasmic overload of neurons with calcium ions is the primary event triggering cell injury.     

Unfolded protein response in a Drosophila model for retinal degeneration

Stress in the endoplasmic reticulum (ER stress) and its cellular response, the unfolded protein response (UPR), are implicated in a wide variety of diseases, but its significance in many disorders remains to be validated in vivo. Here, we analyzed a branch of the UPR mediated by xbp1 in Drosophila to establish its role in neurodegenerative diseases. The Drosophila xbp1 mRNA undergoes ire-1-mediated unconventional splicing in response to ER stress, and this property was used to develop a specific UPR marker, xbp1-EGFP, in which EGFP is expressed in frame only after ER stress. xbp1-EGFP responds specifically to ER stress, but not to proteins that form cytoplasmic aggregates. The ire-1/xbp1 pathway regulates heat shock cognate protein 3 (hsc3), an ER chaperone. xbp1 splicing and hsc3 induction occur in the retina of ninaE(G69D)-/+, a Drosophila model for autosomal dominant retinitis pigmentosa (ADRP), and reduction of xbp1 gene dosage accelerates retinal degeneration of these animals. These results demonstrate the role of the UPR in the Drosophila ADRP model and open new opportunities for examining the UPR in other Drosophila disease models.     

Brain trauma induces X-box protein 1 processing indicative of activation of the endoplasmic reticulum unfolded protein response

Brain trauma was induced in mice using a closed head injury (CHI) model. At 1, 6 or 24 h after trauma, brains were dissected into the cortex, striatum and hippocampus. Changes in levels of processed X-box protein 1 (xbp1), glucose-regulated protein 78 (grp78), growth arrest and DNA damage-inducible gene 153 (gadd153) and heat-shock protein 70 (hsp70) mRNA, indicating impaired endoplasmic reticulum (ER) and cytoplasmic functioning, were evaluated by quantitative PCR. In the cortex, processed xbp1 mRNA levels rose to 2000% of control 1 h after CHI, and stayed high throughout the experiments. In the hippocampus and striatum, processed xbp1 mRNA levels rose in a delayed fashion, peaking at 6 h (1000% of control) and 24 h after CHI (1500% of control) respectively. Levels of grp78 mRNA were only slightly increased in the cortex 24 h after CHI (150% of control), and were unchanged or transiently decreased in the hippocampus and striatum. Levels of gadd153 mRNA did not change significantly after trauma. A transient rise in hsp70 mRNA levels was observed only in the cortex, peaking at 1 h after CHI (600% of control). Processing of xbp1 mRNA is a sign of activation of the unfolded protein response indicative of ER dysfunction. The results suggest that brain trauma induces ER dysfunction, which spreads from the ipsilateral cortex to the hippocampus and striatum. These observations may have clinical implications and should therefore be considered for future investigations on therapeutic intervention of brain injury caused by contusion-induced neurotrauma.     

Peroxidative stress selectively down-regulates the neuronal stress response activated under conditions of endoplasmic reticulum dysfunction

Oxidative stress has been implicated in mechanisms leading to neuronal cell injury in various pathological states of the brain. Here, we investigated the effect of peroxide exposure on the expression of genes coding for cytoplasmic and endoplasmic reticulum (ER) stress proteins. Primary neuronal cell cultures were exposed to H(2)O(2) for 6 h and mRNA levels of hsp70, grp78, grp94, gadd153 were evaluated by quantitative PCR. In addition, peroxide-induced changes in protein synthesis and cell viability were investigated. Peroxide treatment of cells triggered an almost 12-fold increase in hsp70 mRNA levels, but a significant decrease in grp78, grp94 and gadd153 mRNA levels. To establish whether peroxide exposure blocks the ER-resident stress response, cells were also exposed to thapsigargin (Tg, a specific inhibitor of ER Ca(2+)-ATPase) which has been shown to elicit the ER stress response. Tg exposure induced 7.2-fold, 3.6-fold and 8.8-fold increase in grp78, grp94 and gadd153 mRNA levels, respectively. However, after peroxide pre-exposure, the Tg-induced effect on grp78, grp94 and gadd153 mRNA levels was completely blocked. The results indicate that oxidative damage causes a selective down-regulation of the neuronal stress response activated under conditions of ER dysfunction. This down-regulation was only observed in cultures exposed to peroxide levels which induced severe suppression of protein synthesis and cell injury, implying a causative link between peroxide-induced down-regulation of ER stress response system and development of neuronal cell injury. These observations could have implications for our understanding of the mechanisms underlying neuronal cell injury in pathological states of the brain associated with oxidative damage, including Alzheimer's disease where the neuronal stress response activated under conditions of ER dysfunction has been shown to be down-regulated. Down-regulation of ER stress response may increase the sensitivity of neurones to an otherwise nonlethal form of stress.     

1,2-bis(2-Aminophenoxy)ethane-N,N,N',N'-tetraacetic acid induces caspase-mediated apoptosis and reactive oxygen species-mediated necrosis in cultured cortical neurons.

Sustained alteration in [Ca(2+)]i triggers neuronal death. We examined morphological and signaling events of Ca(2+)-deficiency-induced neuronal death. Cortical cell cultures exposed to 20 microM 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA-AM), an intracellular calcium chelator, underwent neuronal apoptosis within 12 h that was evident by shriveled cell bodies, aggregated and condensed nuclear chromatin, and disrupted nuclear membrane. Thereafter, surviving neurons revealed typical necrosis, accompanied by swelling of cell body and mitochondria, over 24 h. Both apoptosis and necrosis were prevented by inclusion of 1 microg/mL cycloheximide, a protein synthesis inhibitor. Treatment with BAPTA-AM induced translocation of Bax into mitochondria within 4 h and release of cytochrome c from mitochondria over 4-12 h. An active fragment of caspase-3, a downstream mediator of cytochrome c, was observed within 8 h and cleaved PHF-1-positive tau. Administration of zVAD-fmk, a broad inhibitor of caspases, or DEVD-amc, a selective inhibitor of caspase-3, selectively prevented the apoptosis component of BAPTA-AM neurotoxicity. In contrast, BAPTA-AM-induced necrosis was propagated through sequential production of superoxide, mitochondrial and cytoplasmic reactive oxygen species. Combined treatment with caspase inhibitors and antioxidants blocked BAPTA-AM neurotoxicity. The present study suggests that neurons deficient in [Ca(2+)]i undergo caspase-3-mediated apoptosis and reactive oxygen species (ROS)-mediated necrosis.     

Deficiency of presenilin-1 increases calcium-dependent vulnerability of neurons to oxidative stress in vitro

We examined the function of presenilin-1 (PS1) on neuronal resistance to oxidative stress. CNS neurons cultured from PS1-deficient mice exhibited increased vulnerability to H2O2 treatment compared with those from wild-type mice. Antioxidants protected the cultured neurons against the oxidative stress. An intracellular calcium chelator, BAPTA AM, as well as an L-type voltage-dependent calcium channel blocker, nifedipine, rescued the neurons from H2O2-induced death, while an N-type voltage-dependent calcium channel blocker, omega-conotoxin, or calcium release blockers from ER stores, dantrolene and xestospongin C, failed to rescue them. Wild-type and PS1-deficient neurons showed comparable increases of cytoplasmic free calcium levels after exposure to H2O2. Taken together with the data that PS1-deficient neurons exhibited increased vulnerability to glutamate, these findings imply that PS1 confers resistance to oxidative stress on neurons in calcium-dependent manners.     

Involvement of endoplasmic reticulum stress and activation of MAP kinases in beta-lapachone-induced human prostate cancer cell apoptosis

Beta-lapachone, an o-naphthoquinone, induces various carcinoma cells to undergo apoptosis, but the mechanism is poorly understood. In the present study, we found that the beta-lapachone-induced apoptosis of DU145 human prostate carcinoma cells was associated with endoplasmic reticulum (ER) stress, as shown by increased intracellular calcium levels and induction of GRP-78 and GADD-153 proteins, suggesting that the endoplasmic reticulum is a target of beta-lapachone. Beta-Lapachone-induced DU145 cell apoptosis was dose-dependent and accompanied by cleavage of procaspase-12 and phosphorylation of p38, ERK, and JNK, followed by activation of the executioner caspases, caspase-7 and calpain. However, pretreatment with the general caspase inhibitor, z-VAD-FMK, or calpain inhibitors, including ALLM or ALLN, failed to prevent beta-lapachone-induced apoptotic cell death. Blocking the enzyme activity of NQO1 with dicoumarol, a known NQO1 inhibitor, or preventing an increase in intracellular calcium levels using BAPTA-AM, an intracellular calcium chelator, substantially inhibited MAPK phosphorylation, abolished the activation of calpain, caspase-12 and caspase-7, and provided significant protection of beta-lapachone-treated cells. These findings show that beta-lapachone-induced ER stress and MAP kinase phosphorylation is a novel signaling pathway underlying the molecular mechanism of the anticancer effect of beta-lapachone.     

亚硝酸钠诱导的草鱼肝细胞凋亡中内质网应激IRE1通路的作用研究

为探究亚硝酸钠诱导草鱼肝细凋亡中内质网应激IRE1通路的作用, 草鱼(Ctenopharyngodon idellus)肝细胞L8824分别置于亚硝酸钠浓度为0、5、20和50 mg/L暴露12h和24h。同时采用三磷酸肌醇受体拮抗剂2-APB和IRE1α抑制剂STF-083010分别和20 mg/L亚硝酸钠共同孵育L8824细胞24h。检测细胞凋亡以及c-Jun氨基末端激酶(c-Jun N-terminal kinase, jnk)、B淋巴细胞瘤-2(B-cell lymphoma-2, bcl-2)、bcl-2相关X蛋白(bcl-2 associated X protein, bax)、caspase9、caspase3、肌醇酶1α (inositol requiring enzyme 1α, ire1α)、X盒结合蛋白 1s (X-box binding protein 1s, xbp1s)和葡萄糖调节蛋白78 (glucose-regulated protein78, grp78)基因的表达和细胞质内钙离子浓度。结果表明: 与对照组相比, 亚硝酸钠处理组细胞凋亡率显著上升, jnk、bax、caspase9、caspase3、ire1α、xbp1s和grp78 mRNA的表达显著升高, bcl-2 mRNA的表达量显著下降, 细胞质内钙离子浓度显著上升。与亚硝酸钠单处理组相比, STF-083010处理组细胞凋亡率显著下降, ire1α、xbp1s、grp78、jnk、bax、caspase3 mRNA表达量显著下降, bcl-2 mRNA表达量显著上升, 2-APB和STF-083010两个处理组细胞质内钙离子浓度均显著下降。结果显示, 高浓度的亚硝酸钠可诱导草鱼肝细胞凋亡和钙离子紊乱, 内质网应激IRE1通路发挥了作用。     

Ge-Jee-Bok-Ryung-Hwan induces apoptosis in human cervical carcinoma HeLa cells--an endoplasmic reticulum stress pathway--

Ge-Jee-Bok-Ryung-Hwan (GJBRH), a commonly used herb formulation in Korea, Japan and China, caused a decrease of viability in HeLa human cervical carcinoma cells. The treatment of GJBRH resulted in genomic DNA fragmentation as well as the increase of Sub-G1 portion in cell cycle analysis. In this study, GFP-Bax over-expression system showed that Bax, pro-apoptotic Bcl-2 family protein, was translocated to mitochondria by the presence of GJBRH. The treatment of BAPTA-AM, permeable endogenous calcium chelator, inhibited GJBRH-induced caspase-3 and -9 activations, the release of cytochrome c and Smac/DIABLO into cytoplasm and the resultant cell death in HeLa human cervical carcinoma cells. The treatment of BAPTA-AM increased the expression of XIAP, which mediates binding to and inhibiting caspases and showed protective effect, in GJBRH-treated cells. GJBRH induced the expression of Glucose Response Protein 78 (GRP 78), a positive ER stress marker protein. However, BAPTA-AM did not interfere with the ER-stress response pathway that triggers the expression of GRP 78. This study showed that GJBRH induces cell death, which occurs downstream of or parallel to this point in the ER-stress pathway linked to apoptosis. In conclusion, GJBRH induces apoptosis in HeLa cells via ER stress-pathway associated mitochondria-dependent apoptosis mechansim.     

Mesenchymal stromal cells synchronize the rhythm of protein synthesis under the effect of an exogenous signal

A comparative study was performed of dense 5-hour cultures of rat hepatocytes and equal-density cultures of mesenchymal stromal cells (MSC) isolated from human adipose tissue of rat bone marrow. The cells were grown on collagen-coated class slides in serum-free medium. Unlike in hepatocytes, no rhythm of protein synthesis was initially revealed in MSC, but such a rhythm manifested itself when the culture medium was supplemented with melatonin (2 nM, 5 min). The results of experiments with cytoplasmic calcium chelator BAPTA-AM and protein kinase inhibitor H7 indicate that the mechanism of protein synthesis synchronization in MSC consists in calcium-dependent phosphorylation of cell proteins.     

Oxygen-regulated protein-150 prevents calcium homeostasis deregulation and apoptosis induced by oxidized LDL in vascular cells

Oxidized LDLs (oxLDLs) induce apoptosis, which contributes to the pathogenesis of atherosclerosis. The 150 kDa oxygen-regulated protein (ORP150), an endoplasmic reticulum (ER)-resident chaperone, is upregulated by hypoxia and prevents ischemia-induced cell death. The aim of this work was to investigate whether and how ORP150 can prevent apoptosis induced by oxLDLs in vascular cells. OxLDLs induced ORP150 expression in the ER of human microvascular endothelial cell line (HMEC-1). ORP150 expression was blocked by antioxidants, by the permeant calcium chelator BAPTA-AM, and by inhibitors of the inositol-1,4,5 trisphosphate (IP3) receptors, 2-aminoethyl diphenylborinate (2-APB) and xestospongin C. ORP150 silencing by siRNA-enhanced oxLDL-induced apoptosis, while forced ORP150 expression increased the resistance of cells via an inhibition of the oxLDL-induced calcium rise, and of subsequent calpain activation, cytochrome c release, caspase 3 activation and apoptosis. A similar protective effect was achieved by BAPTA-AM, 2-APB and xestospongin C. Altogether, these data indicate that (i)ORP150 inhibits oxLDL-induced apoptosis by blocking calcium signaling and subsequent apoptosis, (ii)calcium released from ER stores through IP3 channels is involved in the oxLDL-induced calcium rise and apoptosis, and is inhibited by ORP150. Finally, ORP150 is expressed in advanced atherosclerotic lesions, where it may locally participate to reduce the apoptotic effect of oxLDLs and the subsequent risk of plaque rupture.     

Regulating cytoplasmic calcium homeostasis can reduce aluminum toxicity in yeast

Our previous study suggested that increased cytoplasmic calcium (Ca) signals may mediate aluminum (Al) toxicity in yeast (Saccharomyces cerevisiae). In this report, we found that a yeast mutant, pmc1, lacking the vacuolar calcium ion (Ca(2+)) pump Ca(2+)-ATPase (Pmc1p), was more sensitive to Al treatment than the wild-type strain. Overexpression of either PMC1 or an anti-apoptotic factor, such as Bcl-2, Ced-9 or PpBI-1, decreased cytoplasmic Ca(2+) levels and rescued yeast from Al sensitivity in both the wild-type and pmc1 mutant. Moreover, pretreatment with the Ca(2+) chelator BAPTA-AM sustained cytoplasmic Ca(2+) at low levels in the presence of Al, effectively making the cells more tolerant to Al exposure. Quantitative RT-PCR revealed that the expression of calmodulin (CaM) and phospholipase C (PLC), which are in the Ca(2+) signaling pathway, was down-regulated under Al stress. This effect was largely counteracted when cells overexpressed anti-apoptotic Ced-9 or were pretreated with BAPTA-AM. Taken together, our results suggest that the negative regulation of Al-induced cytoplasmic Ca signaling is a novel mechanism underlying internal resistance to Al toxicity.     

The role of endoplasmic reticulum in cadmium-induced mesangial cell apoptosis

Cd is an industrial and environmental pollutant that affects many organs in humans and other mammals. However, the molecular mechanisms of Cd-induced nephrotoxicity are unclear. In this study, we show that endoplasmic reticula (ER) played a pivotal role in Cd-induced apoptosis in mesangial cells. Using Fluo-3 AM, the intracellular concentration of calcium ([Ca²⁺]_i) was detected as being elevated as time elapsed after Cd treatment. Co-treatment with BAPTA-AM, a calcium chelator, was able to significantly suppress Cd-induced apoptosis. Calcineurin is a cytosolic phosphatase, which was able to dephosphorylate the inositol-1,4,5-triphosphate receptor (IP₃R) calcium channel to prevent the release of calcium from ER. Cyclosporine A, a calcineurin inhibitor, increased both [Ca²⁺]_i and the percentage of Cd-induced apoptosis. However, EGTA and the IP₃R inhibitor, 2-APB, were able to partially modulate Cd cytotoxicity. These results led us to suggest that the extracellular and ER-released calcium plays a crucial role in Cd-induced apoptosis in mesangial cells. Following this line, we further detected the ER stress after Cd treatment since ER is one of the major calcium storage organelles. After Cd exposure, GADD153, a hallmark of ER stress, was upregulated (at 4 h of exposure), followed by activation of ER-specific caspase-12 and its downstream molecule caspase-3 (at 16 h of exposure). The pan caspase inhibitor, Z-VAD, and BAPTA-AM were able to reverse the Cd-induced cell death and ER stress, respectively. Furthermore, the mitochondrial membrane potential (ΔΨ_m) was depolarized significantly and cytochrome c was released after 24 h of exposure to Cd and followed by mild activation of caspase-9 at the 36-h time point, indicating that mitochondria stress is a late event. Therefore, we concluded that ER is the major killer organelle in Cd-induced mesangial cell apoptosis and that calcium oscillation plays a pivotal role.     

Induction of ER stress in response to oxygen-glucose deprivation of cortical cultures involves the activation of the PERK and IRE-1 pathways and of caspase-12

Disturbance of calcium homeostasis and accumulation of misfolded proteins in the endoplasmic reticulum (ER) are considered contributory components of cell death after ischemia. However, the signal-transducing events that are activated by ER stress after cerebral ischemia are incompletely understood. In this study, we show that caspase-12 and the PERK and IRE pathways are activated following oxygen-glucose deprivation (OGD) of mixed cortical cultures or neonatal hypoxia–ischemia (HI). Activation of PERK led to a transient phosphorylation of eIF2α, an increase in ATF4 levels and the induction of gadd34 (a subunit of an eIF2α-directed phosphatase). Interestingly, the upregulation of ATF4 did not lead to an increase in the levels of CHOP. Additionally, IRE1 activation was mediated by the increase in the processed form of xbp1, which would be responsible for the observed expression of edem2 and the increased levels of the chaperones GRP78 and GRP94. We were also able to detect caspase-12 proteolysis after HI or OGD. Processing of procaspase-12 was mediated by NMDA receptor and calpain activation. Moreover, our data suggest that caspase-12 activation is independent of the unfolded protein response activated by ER stress.