Luminal Ca2+ depletion during the unfolded protein response in Xenopus oocytes: Cause and consequence

The endoplasmic reticulum (ER) is a Ca2+ storing organelle that plays a critical role in the synthesis, folding and post-translational modifications of many proteins. The ER enters into a condition of stress when the load of newly synthesized proteins exceeds its folding and processing capacity. This activates a signal transduction pathway called the unfolded protein response (UPR) that attempts to restore homeostasis. The precise role of ER Ca2+ in the initiation of the UPR has not been defined. Specifically, it has not been established whether ER Ca2+ dysregulation is a cause or consequence of ER stress. Here, we report that partial depletion of ER Ca2+ stores induces a significant induction of the UPR, and leads to the retention of a normally secreted protein Carboxypeptidase Y. Moreover, inhibition of protein glycosylation by tunicamycin rapidly induced an ER Ca2+ leak into the cytosol. However, blockade of the translocon with emetine inhibited the tunicamycin-induced Ca2+ release. Furthermore, emetine treatment blocked elF2α phosphorylation and reduced expression of the chaperone BiP. These findings suggest that Ca2+ may be both a cause and a consequence of ER protein misfolding. Thus, it appears that ER Ca2+ leak is a significant co-factor for the initiation of the UPR.Fil: Paredes, R. Madelaine. University of Texas; Estados UnidosFil: Bollo, Mariana Ines. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Instituto de Investigación Médica Mercedes y Martín Ferreyra. Universidad Nacional de Córdoba. Instituto de Investigación Médica Mercedes y Martín Ferreyra; ArgentinaFil: Holstein, Deborah. University of Texas; Estados UnidosFil: Lechleiter, James D.. University of Texas; Estados Unido

Calcineurin Interacts with PERK and Dephosphorylates Calnexin to Relieve ER Stress in Mammals and Frogs

Background: The accumulation of misfolded proteins within the endoplasmic reticulum (ER) triggers a cellular process known as the Unfolded Protein Response (UPR). One of the earliest responses is the attenuation of protein translation. Little is known about the role that Ca 2+ mobilization plays in the early UPR. Work from our group has shown that cytosolic phosphorylation of calnexin (CLNX) controls Ca 2+ uptake into the ER via the sarco-endoplasmic reticulum Ca 2+-ATPase (SERCA) 2b. Methodology/Principal Findings: Here, we demonstrate that calcineurin (CN), a Ca 2+ dependent phosphatase, associates with the (PKR)-like ER kinase (PERK), and promotes PERK auto-phosphorylation. This association, in turn, increases the phosphorylation level of eukaryotic initiation factor-2 a (eIF2-a) and attenuates protein translation. Data supporting these conclusions were obtained from co-immunoprecipitations, pull-down assays, in-vitro kinase assays, siRNA treatments and [ 35 S]-methionine incorporation measurements. The interaction of CN with PERK was facilitated at elevated cytosolic Ca 2+ concentrations and involved the cytosolic domain of PERK. CN levels were rapidly increased by ER stressors, which could be blocked by siRNA treatments for CN-Aa in cultured astrocytes. Downregulation of CN blocked subsequent ER-stress-induced increases in phosphorylated elF2-a. CN knockdown in Xenopus oocytes predisposed them to induction of apoptosis. We also found that CLNX was dephosphorylated by CN when Ca 2+ increased. These data were obtained from [c 32 P]-CLN

Luminal Ca2+ depletion during the unfolded protein response in Xenopus oocytes: Cause and consequence

The endoplasmic reticulum (ER) is a Ca2+ storing organelle that plays a critical role in the synthesis, folding and post-translational modifications of many proteins. The ER enters into a condition of stress when the load of newly synthesized proteins exceeds its folding and processing capacity. This activates a signal transduction pathway called the unfolded protein response (UPR) that attempts to restore homeostasis. The precise role of ER Ca2+ in the initiation of the UPR has not been defined. Specifically, it has not been established whether ER Ca2+ dysregulation is a cause or consequence of ER stress. Here, we report that partial depletion of ER Ca2+ stores induces a significant induction of the UPR, and leads to the retention of a normally secreted protein Carboxypeptidase Y. Moreover, inhibition of protein glycosylation by tunicamycin rapidly induced an ER Ca2+ leak into the cytosol. However, blockade of the translocon with emetine inhibited the tunicamycin-induced Ca2+ release. Furthermore, emetine treatment blocked elF2α phosphorylation and reduced expression of the chaperone BiP. These findings suggest that Ca2+ may be both a cause and a consequence of ER protein misfolding. Thus, it appears that ER Ca2+ leak is a significant co-factor for the initiation of the UPR.Fil: Paredes, R. Madelaine. University of Texas; Estados UnidosFil: Bollo, Mariana Ines. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Instituto de Investigación Médica Mercedes y Martín Ferreyra. Universidad Nacional de Córdoba. Instituto de Investigación Médica Mercedes y Martín Ferreyra; ArgentinaFil: Holstein, Deborah. University of Texas; Estados UnidosFil: Lechleiter, James D.. University of Texas; Estados Unido

Clozapine-Induced Mitochondria Alterations and Inflammation in Brain and Insulin-Responsive Cells

<div><p>Background</p><p>Metabolic syndrome (MetS) is a constellation of factors including abdominal obesity, hyperglycemia, dyslipidemias, and hypertension that increase morbidity and mortality from diabetes and cardiovascular diseases and affects more than a third of the population in the US. Clozapine, an atypical antipsychotic used for the treatment of schizophrenia, has been found to cause drug-induced metabolic syndrome (DIMS) and may be a useful tool for studying cellular and molecular changes associated with MetS and DIMS. Mitochondria dysfunction, oxidative stress and inflammation are mechanisms proposed for the development of clozapine-related DIMS. In this study, the effects of clozapine on mitochondrial function and inflammation in insulin responsive and obesity-associated cultured cell lines were examined.</p> <p>Methodology/Principal Findings</p><p>Cultured mouse myoblasts (C2C12), adipocytes (3T3-L1), hepatocytes (FL-83B), and monocytes (RAW 264.7) were treated with 0, 25, 50 and 75 µM clozapine for 24 hours. The mitochondrial selective probe TMRM was used to assess membrane potential and morphology. ATP levels from cell lysates were determined by bioluminescence assay. Cytokine levels in cell supernatants were assessed using a multiplex array. Clozapine was found to alter mitochondria morphology, membrane potential, and volume, and reduce ATP levels in all cell lines. Clozapine also significantly induced the production of proinflammatory cytokines IL-6, GM-CSF and IL12-p70, and this response was particularly robust in the monocyte cell line.</p> <p>Conclusions/Significance</p><p>Clozapine damages mitochondria and promotes inflammation in insulin responsive cells and obesity-associated cell types. These phenomena are closely associated with changes observed in human and animal studies of MetS, obesity, insulin resistance, and diabetes. Therefore, the use of clozapine in DIMS may be an important and relevant tool for investigating cellular and molecular changes associated with the development of these diseases in the general population.</p> </div

The effect of clozapine treatment on mitochondria morphology in 3T3-L1, C2C12, FL83B and RAW 264.7 cells.

<p>Cells were treated with 0, 25, 50 or 75 µM clozapine for 24 hours and then incubated with TMRM for 20 minutes at 37°C with 5% CO₂. Mitochondria morphology was observed by fluorescent confocal microscopy. All images were taken using a 63x/1.4 Oil objective; images of RAW 264.7 cells are digitally enlarged 3× since these cells are smaller and appear to shrink with clozapine treatment.</p

The effect of clozapine treatment on ATP production in 3T3-L1, C2C12, FL83B, and RAW264.7 cells.

<p>ATP levels were assessed using a bioluminescence assay in cells treated with 0, 25, 50 and 75 µM clozapine for 24 hours. Luminescence intensity corresponds to relative levels of ATP. Luminescence was corrected by using lysate A260 values to correct for the number of viable cells contributing to ATP levels. P values shown are based on a Bonferroni post-hoc test.</p

The effect of clozapine treatment on mitochondria.

<p>A) membrane potential, B) morphology, and C) volume in SKNSH neuroblastoma cells. SKNSH cells were treated with 0, 10, 20 or 50 µM clozapine for 24 hours and then incubated with TMRM for 20 minutes at 37°C with 5% CO₂. Confocal micrographs analyzed for mitochondrial membrane potential and volume using the Nernst Potential MulPro2D plug-in for Image J software.</p

The effect of clozapine treatment on production of inflammatory cytokines in 3T3-L1, C2C12 and RAW 264.7 cells.

<p>Cells were treated with 0, 25, 50 and 75 µM clozapine for 24 hours. Cytokine levels were measured using the MILLIPLEX MAP Mouse Cytokine/Chemokine-Premixed 13 Plex array. Data shown are baseline corrected to show the relative changes in cytokine levels (in pg of cytokine per mg of protein lysate) with increasing clozapine compared to the untreated control cells. Only cytokines that are significantly altered by clozapine treatment in each cell line are shown. P values shown are based on a Bonferroni post-hoc test.</p