thapsigargin and Teratocarcinoma

Amyloid-beta (Abeta) peptide plays a significant role in the pathogenesis of Alzheimer's disease (AD). Previously we found that Abeta induces both mitochondrial and endoplasmic reticulum (ER) dysfunction leading to apoptosis, and now we address the relevance of ER-mitochondria crosstalk in apoptotic cell death triggered by Abeta peptide. Using mitochondrial DNA-depleted rho0 cells derived from the human NT2 teratocarcinoma cell line, characterized by the absence of functional mitochondria, and the parental rho+ cells, we report here that treatment with the synthetic Abeta1-40 peptide, or the classical ER stressors thapsigargin or brefeldin A, increases GRP78 expression levels and caspase activity, two ER stress markers, and also depletes ER calcium stores. Significantly, we show that the presence of functional mitochondria is required for ER stress-mediated apoptotic cell death triggered by toxic insults such as Abeta. We found that the increase in the levels of the pro-apoptotic transcription factor GADD153/CHOP, which mediates ER stress-induced cell death, as well as caspase-9 and -3 activation and increased number of TUNEL-positive cells, occurs in treated parental rho+ cells but is abolished in rho0 cells. Our results strongly support the close communication between ER and mitochondria during apoptotic cell death induced by the Abeta peptide and provide insights into the molecular cascade of cell death in AD.

Topics: Amyloid beta-Peptides; Analysis of Variance; Apoptosis; Brefeldin A; Caspases; Cell Line, Tumor; Endoplasmic Reticulum; Endoplasmic Reticulum Chaperone BiP; Humans; In Situ Nick-End Labeling; Mitochondria; Oxidative Stress; Peptide Fragments; Protein Synthesis Inhibitors; Teratocarcinoma; Thapsigargin; Time Factors

Fluctuations of intracellular Ca2+ ([Ca2+]i) regulate a variety of cellular functions. The classical Ca2+ transport pathways in the endoplasmic reticulum (ER) and plasma membrane are essential to [Ca2+]i oscillations. Although mitochondria have recently been shown to absorb and release Ca2+ during G protein-coupled receptor (GPCR) activation, the role of mitochondria in [Ca2+]i oscillations remains to be elucidated. Using fluo-3-loaded human teratocarcinoma NT2 cells, we investigated the regulation of [Ca2+]i oscillations by mitochondria. Both the muscarinic GPCR agonist carbachol and the ER Ca2+-adenosine triphosphate inhibitor thapsigargin (Tg) induced [Ca2+]i oscillations in NT2 cells. The [Ca2+]i oscillations induced by carbachol were unsynchronized among individual NT2 cells; in contrast, Tg-induced oscillations were synchronized. Inhibition of mitochondrial functions with either mitochondrial blockers or depletion of mitochondrial DNA eliminated carbachol--but not Tg-induced [Ca2+]i oscillations. Furthermore, carbachol-induced [Ca2+]i oscillations were partially restored to mitochondrial DNA-depleted NT2 cells by introduction of exogenous mitochondria. Treatment of NT2 cells with gap junction blockers prevented Tg-induced but not carbachol-induced [Ca2+]i oscillations. These data suggest that the distinct patterns of [Ca2+]i oscillations induced by GPCR and Tg are differentially modulated by mitochondria and gap junctions.

Topics: Calcium; Calcium Signaling; Calcium-Transporting ATPases; Carbachol; Cell Line, Tumor; Endoplasmic Reticulum; Enzyme Inhibitors; Gap Junctions; Humans; Mitochondria; Receptors, G-Protein-Coupled; Teratocarcinoma; Thapsigargin

Chemokines play a key role in the regulation of central nervous system disease. CXCL10 over-expression has been observed in several neurodegenerative diseases, including multiple sclerosis, Alzheimer's disease and HIV-associated dementia. More recent studies by others and us have shown that CXCL10 elicits apoptosis in fetal neurons. The mechanism of CXCL10-mediated neurotoxicity, however, remains unclear. In this study, we provide evidence for the direct role of Ca(2+) dysregulation in CXCL10-mediated apoptosis. We demonstrate that treatment of fetal neuronal cultures with exogenous CXCL10 produced elevations in intracellular Ca(2+) and that this effect was modulated via the binding of CXCL10 to its cognate receptor, CXCR3. We further explored the association of intracellular Ca(2+) elevations with the caspases that are involved in CXC10-induced neuronal apoptosis. Our data showed that increased Ca(2+), which is available for uptake by the mitochondria, is associated with membrane permeabilization and cytochrome c release from this compartment. The released cytochrome c then activates the initiator active caspase-9. This initiator caspase sequentially activates the effector caspase-3, ultimately leading to apoptosis. This study identifies the temporal signaling cascade involved in CXCL10-mediated neuronal apoptosis and provides putative targets for pharmaceutical intervention of neurological disorders associated with CXCL10 up-regulation.

Topics: Aniline Compounds; Blotting, Western; Brain; Calcium; Caspase 3; Caspases; Cell Count; Cell Death; Cells, Cultured; Chemokine CXCL10; Chemokines, CXC; Cytosol; Dose-Response Relationship, Drug; Drug Interactions; Enzyme Inhibitors; Fetus; Gene Expression; Humans; Immunohistochemistry; Mitochondria; Models, Neurological; Neurons; Teratocarcinoma; Thapsigargin; Xanthenes