thapsigargin and thiazolyl-blue

Previously we reported that endoplasmic reticulum (ER)-mitochondria crosstalk is involved in amyloid-β (Aβ)-induced apoptosis. Now we show that mitochondrial dysfunction affects the ER stress response triggered by Aβ using cybrids that recreate the defect in mitochondrial cytochrome c oxidase (COX) activity detected in platelets from Alzheimer's disease (AD) patients. AD and control cybrids were treated with Aβ or classical ER stressors and the ER stress-mediated apoptotic cell death pathway was accessed. Upon treatment, we found increased glucose-regulated protein 78 (GRP78) levels and caspase-4 activation (ER stress markers) which were more pronounced in AD cybrids. Treated AD cybrids also exhibited decreased cell survival as well as increased caspase-3-like activity, poli-ADP-ribose-polymerase (PARP) levels and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL)-positive apoptotic cells. Finally, we showed that Aβ-induced caspase-3 activation in both cybrid cell lines was prevented by dantrolene, thus implicating ER Ca(2+) release in ER stress-mediated apoptosis. Our results demonstrate that mitochondrial dysfunction occurring in AD patients due to COX inhibition potentiates cell susceptibility to Aβ-induced ER stress. This study further supports the close communication between ER and mitochondria during apoptosis in AD.

Topics: Aged; Alzheimer Disease; Amyloid beta-Peptides; Analysis of Variance; Blood Platelets; Brefeldin A; Caspase 3; Cell Death; Cell Fusion; Cell Line, Tumor; Endoplasmic Reticulum; Endoplasmic Reticulum Chaperone BiP; Female; Gene Expression Regulation; Heat-Shock Proteins; Humans; Hybrid Cells; In Situ Nick-End Labeling; Male; Middle Aged; Mitochondria; Peptide Fragments; Poly(ADP-ribose) Polymerases; Protein Synthesis Inhibitors; Tetrazolium Salts; Thapsigargin; Thiazoles

The endoplasmic reticulum (ER) plays a critical role in the maintenance of intracellular homeostasis and its dysfunction is thought to lead to neuronal death, which results in neurodegenerative disorders. Since phospholipase C (PLC) isozymes are involved in maintenance of the intracellular Ca2+ concentration by regulating Ca2+ release from the ER, their expression might be affected by ER stress. Of these isozymes, PLC-beta 1 and -gamma 1, in particular, are known to protect cells from oxidative stress and thus alteration of their expression profile under ER stress-loaded conditions is interesting. Using primary cultured rat cortical neurons, we here examined whether expression of PLC-beta 1 and -gamma 1 was altered in ER stress-loaded neurons induced by tunicamycin (Tm). In ER stress-loaded neurons treated with Tm in the range of 0.03-3 microg/ml for 20 h, the viability of the neurons was decreased dose-dependently, the decrease being significant with 0.3 or more microg/ml, and expression of the representative ER stress markers, GRP78/BiP, and cleaved caspase-3 and -12, was increased after 24 h postincubation, confirming the induction of ER stress in the neurons. In the ER stress-loaded neurons obtained on Tm treatment, the expression level of PLC-beta 1 decreased dose-dependently. On the other hand, there was no difference in the PLC-gamma 1 protein expression level between control and ER stress-loaded neurons. Overall, we demonstrated that ER stress decreases the expression of PLC-beta 1, but not -gamma 1, in neurons.

Topics: Animals; Blotting, Western; Calcimycin; Calcium; Caspase 12; Caspase 3; Cell Survival; Cells, Cultured; Endoplasmic Reticulum; Enzyme Inhibitors; Neurons; Phospholipase C beta; Phospholipase C gamma; Rats; Stress, Physiological; Tetrazolium Salts; Thapsigargin; Thiazoles; Tunicamycin

Endoplasmic reticulum (ER) stress has been implicated in the pathogenesis of many diseases and in cancer therapy. Although the unfolded protein response is known to alleviate ER stress by reducing the accumulation of misfolded proteins, the exact survival elements and their downstream signaling pathways that directly counteract ER stress-stimulated apoptotic signaling remain elusive. Here, we have shown that endogenous Akt and ERK are rapidly activated and act as downstream effectors of phosphatidylinositol 3-kinase in thapsigargin- or tunicamycin-induced ER stress. Introduction of either dominant-negative Akt or MEK1 or the inhibitors LY294002 and U0126 sensitized cells to ER stress-induced cell death in different cell types. Reverse transcription-PCR analysis of gene expression during ER stress revealed that cIAP-2 and XIAP, members of the IAP family of potent caspase suppressors, were strongly induced. Transcription of cIAP-2 and XIAP was up-regulated by the phosphatidylinositol 3-kinase/Akt pathway as shown by its reversal by dominant-negative Akt or LY294002. Ablation of these IAPs by RNA interference sensitized cells to ER stress-induced death, which was reversed by the caspase inhibitor benzyloxycarbonyl-VAD-fluoromethyl ketone. The protective role of IAPs in ER stress coincided with Smac release from mitochondria to the cytosol. Furthermore, it was shown that mTOR was not required for Akt-mediated survival. These results represent the first demonstration that activation of endogenous Akt/IAPs and MEK/ERK plays a critical role in controlling cell survival by resisting ER stress-induced cell death signaling.

Topics: Amino Acid Chloromethyl Ketones; Apoptosis; Blotting, Western; Cell Line, Tumor; Cell Survival; Cytosol; Endoplasmic Reticulum; Enzyme Inhibitors; Gene Expression Regulation, Enzymologic; Genes, Dominant; Humans; In Situ Nick-End Labeling; MAP Kinase Kinase 1; Microscopy, Fluorescence; Mitochondria; Mitogen-Activated Protein Kinase 3; Models, Biological; Phosphatidylinositol 3-Kinases; Plasmids; Protein Denaturation; Protein Folding; Protein Kinases; Protein Serine-Threonine Kinases; Proteins; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-akt; Reverse Transcriptase Polymerase Chain Reaction; RNA Interference; Signal Transduction; Subcellular Fractions; Tetrazolium Salts; Thapsigargin; Thiazoles; Time Factors; TOR Serine-Threonine Kinases; Transcription, Genetic; Tunicamycin; Up-Regulation; X-Linked Inhibitor of Apoptosis Protein