thapsigargin and Brain-Ischemia

Background Cornu ammonis 3 (CA3) hippocampal neurons are resistant to global ischemia, whereas cornu ammonis (CA1) 1 neurons are vulnerable. Hamartin expression in CA3 neurons mediates this endogenous resistance via productive autophagy. Neurons lacking hamartin demonstrate exacerbated endoplasmic reticulum stress and increased cell death. We investigated endoplasmic reticulum stress responses in CA1 and CA3 regions following global cerebral ischemia, and whether pharmacological modulation of endoplasmic reticulum stress or autophagy altered neuronal viability . Methods In vivo: male Wistar rats underwent sham or 10 min of transient global cerebral ischemia. CA1 and CA3 areas were microdissected and endoplasmic reticulum stress protein expression quantified at 3 h and 12 h of reperfusion. In vitro: primary neuronal cultures (E18 Wistar rat embryos) were exposed to 2 h of oxygen and glucose deprivation or normoxia in the presence of an endoplasmic reticulum stress inducer (thapsigargin or tunicamycin), an endoplasmic reticulum stress inhibitor (salubrinal or 4-phenylbutyric acid), an autophagy inducer ([4'-(N-diethylamino) butyl]-2-chlorophenoxazine (10-NCP)) or autophagy inhibitor (3-methyladenine). Results In vivo, decreased endoplasmic reticulum stress protein expression (phospho-eIF2α and ATF4) was observed at 3 h of reperfusion in CA3 neurons following ischemia, and increased in CA1 neurons at 12 h of reperfusion. In vitro, endoplasmic reticulum stress inducers and high doses of the endoplasmic reticulum stress inhibitors also increased cell death. Both induction and inhibition of autophagy also increased cell death. Conclusion Endoplasmic reticulum stress is associated with neuronal cell death following ischemia. Neither reduction of endoplasmic reticulum stress nor induction of autophagy demonstrated neuroprotection in vitro, highlighting their complex role in neuronal biology following ischemia.

Topics: Animals; Brain Ischemia; CA1 Region, Hippocampal; CA3 Region, Hippocampal; Cell Death; Cells, Cultured; Disease Models, Animal; Endoplasmic Reticulum Stress; Enzyme Inhibitors; Hypoglycemia; Hypoxia; Male; Neurons; Neuroprotective Agents; Rats, Wistar; Thapsigargin; Tuberous Sclerosis Complex 1 Protein; Tunicamycin

The present study aims to investigate the effect of noscapine (0.5-2.5 μM), an alkaloid from the opium poppy, on primary murine fetal cortical neurons exposed to oxygen-glucose deprivation (OGD), an in vitro model of ischemia.. Cells were transferred to glucose-free DMEM and were exposed to hypoxia in a small anaerobic chamber. Cell viability and nitric oxide production were evaluated by MTT assay and the Griess method, respectively.. The neurotoxicities produced by all three hypoxia durations tested were significantly inhibited by 0.5 μM noscapine. Increasing noscapine concentration up to 2.5 μM produced a concentration-dependent inhibition of neurotoxicity. Pretreatment of cells with MK-801 (10 μM), a non-competitive NMDA antagonist, and nimodipine (10nM), an L-type Ca(2+) channel blockers, increased cell viability after 30 min OGD, while the application of NBQX (30 μM), a selective AMPA-kainate receptor antagonist partially attenuated cell injury. Subsequently, cells treated with noscapine in the presence of thapsigargin (1 μM), an inhibitor of endoplasmic reticulum Ca(2+) ATPases. After 60 min OGD, noscapine could inhibit the cell damage induced by thapsigargin. However, noscapine could not reduce cell damage induced by 240 min OGD in the presence of thapsigargin. Noscapine attenuated nitric oxide (NO) production in cortical neurons after 30 min OGD.. We concluded that noscapine had a neuroprotective effect, which could be due to its interference with multiple targets in the excitotoxicity process. These effects could be mediated partially by a decrease in NO production and the modulation of intracellular calcium levels.

Topics: Animals; Brain Ischemia; Cell Survival; Dizocilpine Maleate; Dose-Response Relationship, Drug; Glucose; Hypoxia; Mice; Neurons; Neuroprotective Agents; NG-Nitroarginine Methyl Ester; Nimodipine; Nitric Oxide; Noscapine; Primary Cell Culture; Quinoxalines; Thapsigargin

Transient cerebral ischemia leads to endoplasmic reticulum (ER) stress. However, the contributions of ER stress to cerebral ischemia are not clear. To address this issue, the ER stress activators tunicamycin (TM) and thapsigargin (TG) were administered to transient middle cerebral artery occluded (tMCAO) mice and oxygen-glucose deprivation-reperfusion (OGD-Rep.)-treated neurons. Both TM and TG showed significant protection against ischemia-induced brain injury, as revealed by reduced brain infarct volume and increased glucose uptake rate in ischemic tissue. In OGD-Rep.-treated neurons, 4-PBA, the ER stress releasing mechanism, counteracted the neuronal protection of TM and TG, which also supports a protective role of ER stress in transient brain ischemia. Knocking down the ER stress sensor Eif2s1, which is further activated by TM and TG, reduced the OGD-Rep.-induced neuronal cell death. In addition, both TM and TG prevented PARK2 loss, promoted its recruitment to mitochondria, and activated mitophagy during reperfusion after ischemia. The neuroprotection of TM and TG was reversed by autophagy inhibition (3-methyladenine and Atg7 knockdown) as well as Park2 silencing. The neuroprotection was also diminished in Park2(+/-) mice. Moreover, Eif2s1 and downstream Atf4 silencing reduced PARK2 expression, impaired mitophagy induction, and counteracted the neuroprotection. Taken together, the present investigation demonstrates that the ER stress induced by TM and TG protects against the transient ischemic brain injury. The PARK2-mediated mitophagy may be underlying the protection of ER stress. These findings may provide a new strategy to rescue ischemic brains by inducing mitophagy through ER stress activation.

Topics: Animals; Apoptosis; Brain Ischemia; Endoplasmic Reticulum Stress; Glucose; Mice; Mitophagy; Neuroprotective Agents; Oxygen; Reperfusion Injury; Signal Transduction; Thapsigargin; Tunicamycin; Ubiquitin-Protein Ligases

Astrocytes are essential cells for maintaining brain integrity. We have recently shown that the transcription factor C/EBP homologous protein (CHOP), associated with endoplasmic reticulum (ER) stress, plays a key role in the astrocyte death induced by ischemia. Meanwhile, mediators of apoptosis downstream of CHOP in the ER stress-dependent pathway remain to be elucidated. Our aim in this work was to determine whether caspase-11, able to activate apoptotic and proinflammatory pathways, is implicated in ER stress-dependent astrocyte death in ischemic conditions. According to our results, caspase-11 is up-regulated in primary astrocyte cultures following either oxygen and glucose deprivation (OGD) or treatment with the ER-stress inducers thapsigargin and tunicamycin. Moreover, these same stimuli increased caspase-11 mRNA levels and luciferase activity driven by a caspase-11 promoter, indicating that caspase-11 is regulated at the transcriptional level. Our data also illustrate the involvement of ER stress-associated CHOP in caspase-11 regulation, insofar as CHOP overexpression by means of an adenoviral vector caused a significant raise in caspase-11. In turn, caspase-11 suppression with siRNA rescued astrocytes from OGD- and ER stress-induced death, supporting the idea that caspase-11 is responsible for the deleterious effects of ischemia on astrocytes. Finally, inhibition of caspase-1 and caspase-3 significantly reduced astrocyte death, which indicates that these proteases act as death effectors of caspase-11. In conclusion, our work contributes to clarifying the pathways leading to astrocyte death in response to ischemia by defining caspase-11 as a key mediator of the ER stress response acting downstream of CHOP.

Topics: Animals; Apoptosis; Astrocytes; Brain Ischemia; Caspase Inhibitors; Caspases; Cells, Cultured; Disease Models, Animal; Endoplasmic Reticulum; Genetic Vectors; Rats; Rats, Sprague-Dawley; RNA Interference; RNA, Messenger; Signal Transduction; Stress, Physiological; Thapsigargin; Transcription Factor CHOP; Transfection; Tunicamycin

The mechanisms of myocardial dysfunction and calcium handling disturbance underlying cerebral ischemia remain obscure. Here we for the first time report that acute cerebral ischemia significantly increased left ventricular end diastolic pressure (LVEDP), but decreased +dP/dt, -dP/dt, and left ventricular systolic pressure (LVSP). Significant increase in either the resting or KCl-induced [Ca2+](i)in ventricular myocytes was also detected by scanning confocal microscopy at 2 and 24 hours after cerebral ischemia. Verapamil as a blocker of I(Ca,L), ryanodine as a specific inhibitor of RyR, thapsigargin as a highly specific inhibitor of sarco(endo)plasmic reticulum Ca(2+)-ATPase 2a (SERCA2a) and SEA0400 as a selective NCX inhibitor changed the area under the curve of averaged ratio of fluorescence (FI/F(0)I) induced by KCl. Cardiac expression of Ca(v)1.2 was significantly up-regulated at 2 and 24 hours after cerebral ischemia, whereas cardiac expression of SERCA2a and Na(+)-Ca(2+) exchanger (NCX) was significantly down-regulated at the same time period after cerebral ischemia. Cardiac expression of phospholamban (PLB) was significantly elevated at 2 hours after cerebral ischemia but was restored to about normal level at 24 hours after injury. These data suggest that acute cerebral ischemia may specifically disturb cardiac function and calcium homeostasis, which are related to increase of Ca(v)1.2 and decrease of through up-regulating Ca(v)1.2 and PLB, down-regulating SERCA2a and NCX, subsequently leading to Ca2+ overload by the enhancement of Ca2+ influx and inhibition of intracellular Ca2+ extrusion and cerebral ischemia-induced myocardial dysfunction.

Topics: Aniline Compounds; Animals; Brain Ischemia; Calcium; Calcium Channel Blockers; Calcium Channels, L-Type; Enzyme Inhibitors; Heart Ventricles; Male; Muscle Contraction; Myocytes, Cardiac; Phenyl Ethers; Potassium Chloride; Rats; Rats, Wistar; Ryanodine; Ryanodine Receptor Calcium Release Channel; Sarcoplasmic Reticulum Calcium-Transporting ATPases; Sodium-Calcium Exchanger; Thapsigargin; Time Factors; Verapamil

Accumulation of misfolded proteins in the endoplasmic reticulum (ER) induces a highly conserved homeostatic response in all eukaryotic cells, termed the unfolded-protein response (UPR). Here we describe the characterization of stanniocalcin 2 (STC2), a mammalian homologue of a calcium- and phosphate-regulating hormone first identified in fish, as a novel target of the UPR. Expression of STC2 gene is rapidly upregulated in cultured cells after exposure to tunicamycin and thapsigargin, by ATF4 after activation of the ER-resident kinase PERK. In addition, STC2 expression is also activated in neuronal cells by oxidative stress and hypoxia but not by several cellular stresses unrelated to the UPR. In contrast, expression of another homologue, STC1, is only upregulated by hypoxia independent of PERK or ATF4 expression. In vivo studies revealed that rat cortical neurons rapidly upregulate STC2 after transient middle cerebral artery occlusion. Finally, siRNA-mediated inhibition of STC2 expression renders N2a neuroblastoma cells and HeLa cells significantly more vulnerable to apoptotic cell death after treatment with thapsigargin, and overexpression of STC2 attenuated thapsigargin-induced cell death. Consequently, induced STC2 expression is an essential feature of survival component of the UPR.

Topics: Activating Transcription Factors; Animals; Apoptosis; Blood Proteins; Brain Ischemia; Cell Line; Cell Survival; Cricetinae; Cytoprotection; eIF-2 Kinase; Endoplasmic Reticulum; Gene Expression Profiling; Gene Expression Regulation, Neoplastic; Glycoproteins; Golgi Apparatus; Humans; Hydrogen Peroxide; Intercellular Signaling Peptides and Proteins; Intracellular Signaling Peptides and Proteins; Mice; Oligonucleotide Array Sequence Analysis; Oxidative Stress; Protein Folding; Protein Transport; Rats; Signal Transduction; Thapsigargin; Transcription Factors; Up-Regulation

Inhibiting Ca(2+) uptake by the sarcoendoplasmic reticular Ca(2+)-ATPase pump (SERCA) causes release of Ca(2+) from the endoplasmic reticulum (ER), increased cytosolic Ca(2+) ([Ca(2+)](cyt)) and depletion of ER Ca(2+) stores. These studies were designed to test the effects of SERCA inhibition on neuronal viability, using as a model the human neuroblastoma cell line, SH-SY5Y. Continuous exposure to the SERCA inhibitor thapsigargin (TG) decreased SH-SY5Y viability to <30% after 48 h exposure, and produced DNA laddering. Two other SERCA inhibitors, BHQ and cyclopiazonic acid CPA, were similarly toxic, although at 1000-fold higher concentrations. BHQ and CPA toxicity was prevented by removing drug within several hours, whereas TG toxicity was essentially irreversible. All three SERCA inhibitors caused an increase in [Ca(2+)](cyt) that was partially blocked by the ryanodine receptor inhibitors, dantrolene and DHBP. Pretreatment with 40 microM dantrolene gave substantial protection against TG- or BHQ-induced cell death but it did not inhibit death from staurosporine, which does not cause release of ER Ca(2+). DHBP (20-100 microM) also gave partial protection against TG toxicity, as did ruthenium red (2 microM), but not ryanodine (10 microM). Inhibition of capacitative Ca(2+) entry with EGTA or LaCl(3) or low extracellular Ca(2+), or chelation of [Ca(2+)](cyt) with BAPTA-AM, failed to inhibit TG toxicity, although they prevented increases in [Ca(2+)](cyt) caused by TG. Taken together, these data suggest that toxicity caused by SERCA inhibition in SH-SY5Y cells is caused by ER Ca(2+) depletion, which triggers an apparent apoptotic pathway.

Topics: Apoptosis; Brain Ischemia; Calcium; Calcium-Transporting ATPases; Cytosol; Dantrolene; Egtazic Acid; Endoplasmic Reticulum; Enzyme Inhibitors; Humans; Hydroquinones; Indoles; Muscle Relaxants, Central; Neuroblastoma; Neurons; Sarcoplasmic Reticulum Calcium-Transporting ATPases; Sesquiterpenes; Staurosporine; Thapsigargin; Tumor Cells, Cultured

Ischemia is associated with a loss of cytosolic calcium homeostasis. Intracellular stores, particularly in endoplasmic reticulum, are critical for the maintenance of calcium homeostasis. Recent studies have shown that ischemia significantly inhibited microsomal calcium uptake mediated by Mg(2+)/Ca(2+) ATPase, the major mechanism of endoplasmic reticulum calcium sequestration. This study was initiated to determine whether the decreased calcium uptake caused by ischemia was the result of inhibition of Mg(2+)/Ca(2+) ATPase activity or an uncoupling of calcium uptake from ATP hydrolysis. The microsomal Mg(2+)/Ca(2+) ATPase specific inhibitor thapsigargin partially inhibited ATPase activity and completely inhibited calcium uptake. ATPase inhibited by thapsigargin was considered microsomal Mg(2+)/Ca(2+) ATPase. Ischemia from 5 to 60 min had no significant effect on thapsigargin sensitive ATPase activity. However, under identical conditions, increasing ischemia from 5 to 60 min significantly inhibited microsomal calcium uptake. Comparing calcium uptake to ATP hydrolysis as ischemia increased from 5 to 60 min revealed that the coupling ratio of calcium molecules sequestered to ATP molecules hydrolyzed became significantly decreased. The results demonstrated that the effect of ischemia on microsomal calcium uptake was mediated by an uncoupling of calcium transport from Mg(2+)/Ca(2+) ATPase activity.

Topics: Adenosine Triphosphate; Animals; Brain; Brain Ischemia; Ca(2+) Mg(2+)-ATPase; Calcium; Calcium-Transporting ATPases; Enzyme Inhibitors; Female; Hydrolysis; Microsomes; Rats; Rats, Sprague-Dawley; Thapsigargin; Time Factors

Gap junctions are highly conductive channels that allow the direct transfer of intracellular messengers such as Ca2+ and inositol triphosphate (IP3) between interconnected cells. In brain, astrocytes are coupled extensively by gap junctions. We found here that gap junctions among astrocytes in acutely prepared brain slices as well as in culture remained open during ischemic conditions. Uncoupling first occurred after the terminal loss of plasma membrane integrity. Gap junctions therefore may link ischemic astrocytes in an evolving infarct with the surroundings. The free exchange of intracellular messengers between dying and potentially viable astrocytes might contribute to secondary expansion of ischemic lesions.

Topics: Animals; Apoptosis; Astrocytes; Brain Ischemia; Calcium; Cell Membrane; Cell Survival; Cells, Cultured; Cerebral Cortex; Cerebral Infarction; Enzyme Inhibitors; Female; Fluorescent Dyes; Gap Junctions; Hippocampus; Hydrogen-Ion Concentration; Ionophores; Isoquinolines; Male; Organ Culture Techniques; Phosphorylation; Protons; Rats; Rats, Sprague-Dawley; Second Messenger Systems; Thapsigargin

Ca2+-activated K+ currents in rat locus coeruleus neurons induced by experimental ischemia, anoxia, and hypoglycemia. J. Neurophysiol. 78: 2674-2681, 1997. The effects of metabolic inhibition on membrane currents and N-methyl--aspartic acid (NMDA)-induced currents were investigated in dissociated rat locus coeruleus (LC) neurons by using the nystatin perforated patch recording mode under voltage-clamp conditions. Changes in the intracellular Ca2+ concentration ([Ca2+]i) during the metabolic inhibition were also investigated by using the microfluometry with a fluorescent probe, Indo-1. Removal of both the oxygen and glucose (experimental ischemia), deprivation of glucose (hypoglycemia), and a blockade of electron transport by sodium cyanide (NaCN) or a reduction of the mitochondrial membrane potential with carbonyl cyanide-p-trifluoromethoxyphenyl-hydrazone(FCCP) as experimental anoxia all induced a slowly developing outward current (IOUT) at a holding potential of -40 mV. The application of 10(-4) M NMDA induced a rapid transient peak and a successive steady state inward current and a transient outward current immediately after washout. All treatments related to metabolic inhibition increased the NMDA-induced outward current(INMDA-OUT) and prolonged the one-half recovery time of INMDA-OUT. The reversal potentials of both IOUT and INMDA-OUT were close to the K+ equilibrium potential (EK) of -82 mV. Either charybdotoxin or tolbutamide inhibited the IOUT and INMDA-OUT, suggesting the contribution of Ca2+-activated and ATP-sensitive K+ channels, even though the inhibitory effect of tolbutamide gradually diminished with time. Under the metabolic inhibition, the basal level of [Ca2+]i was increased and the one-half recovery time of the NMDA-induced increase in [Ca2+]i was prolonged. The IOUT induced by NaCN was inhibited by a continuous treatment of thapsigargin but not by ryanodine, indicating the involvement of inositol 1,4, 5-trisphosphate (IP3)-induced Ca2+ release (IICR) store. These findings suggest that energy deficiency causes Ca2+ release from the IICR store and activates continuous Ca2+-activated K+ channels and transient ATP-sensitive K+ channels in acutely dissociated rat LC neurons.

Topics: Animals; Brain Ischemia; Calcium; Calcium Channels; Carbonyl Cyanide p-Trifluoromethoxyphenylhydrazone; Charybdotoxin; Glucose; Glyburide; Hypoglycemia; Hypoxia, Brain; In Vitro Techniques; Locus Coeruleus; Membrane Potentials; N-Methylaspartate; Neurons; Oxygen; Partial Pressure; Rats; Rats, Wistar; Ryanodine; Sodium Cyanide; Thapsigargin; Tolbutamide

The neuroprotective effects of dantrolene, an inhibitor of calcium release from intracellular stores, were investigated in a model of cell death induced by calcium release from endoplasmic reticulum in vitro. Thapsigargin (50 nM), a selective inhibitor of endoplasmic reticular Ca(2+)-ATPase, significantly increased the cytosolic Ca2+ concentration to 230% over basal levels, induced DNA fragmentation, and reduced cell viability from 94% in control cells to 41% after a 24-h treatment in GT1-7 hypothalamic neurosecretory cells. Pretreatment with dantrolene for 30 min significantly inhibited elevation of cytosolic Ca2+ levels, DNA fragmentation, and GT1-7 cell death induced by thapsigargin in a dose-dependent manner. To determine if dantrolene would also be protective in an in vivo model of neurodegeneration, it was administered intravenously immediately following a 5-min global cerebral ischemia in gerbils, and the number of intact hippocampal CA1 pyramidal neurons was counted 7 days later. The effects of dantrolene on brain and rectal temperature were monitored in a separate experiment. Dantrolene significantly increased the number of intact CA1 pyramidal neurons from 40% (untreated ischemic animals) to 67 (10 mg/kg), 78 (25 mg/kg), or 83% (50 mg/kg) of values in sham controls (all p < 0.001). No significant changes in brain or rectal temperature were detected for 4 h following 50 mg/kg dantrolene. These results suggest that abnormal Ca2+ release from intracellular stores can induce neuronal death and that such a mechanism may contribute to delayed hippocampal neuronal death after cerebral ischemia. Dantrolene may be a potentially useful drug for neuroprotection after cerebral ischemia.

Topics: Animals; Brain Ischemia; Calcium; Cell Count; Cell Death; Cells, Cultured; Dantrolene; Enzyme Inhibitors; Gerbillinae; Hippocampus; Muscle Relaxants, Central; Neuroprotective Agents; Pyramidal Cells; Thapsigargin

The increase in cytoplasmatic calcium concentration during cerebral ischemia has been proposed as a key event leading to neuronal death. In order to investigate a possible role of calcium-release from intracellular stores in ischemic neuronal injury, intracellular calcium pools were depleted prior to ischemia by the use of thapsigargin. Evoked activity (population spike) in rat hippocampal slices was monitored during a 30 min control period, 9 min of energy deprivation and 60 min of recovery. The population spike recovered to 27% (17-33) (median and 95% confidence interval) following energy deprivation in normal calcium, to 56% (50-58) in calcium-free incubation fluid and to 83% (75-88) in slices pretreated with 1 microM thapsigargin. Combining calcium removal and thapsigargin pretreatment did not improve recovery further. Both removal of extracellular calcium and emptying intracellular calcium stores prior to energy deprivation thus improved functional recovery following energy deprivation, however the latter was more effective. These results suggest that calcium release from intracellular stores may be of major importance in calcium-related neuronal injury during cerebral ischemia.

Topics: Action Potentials; Adenosine Triphosphate; Animals; Brain Ischemia; Calcium; Cytotoxins; Endoplasmic Reticulum; Energy Metabolism; Enzyme Inhibitors; Hippocampus; Organ Culture Techniques; Rats; Rats, Wistar; Thapsigargin