dizocilpine-maleate and benzyloxycarbonylvalyl-alanyl-aspartyl-fluoromethyl-ketone

Unconjugated bilirubin (UCB) induces both apoptosis and necrosis in neurons. To investigate the role of caspases and excitotoxicity in UCB-induced cell death, we exposed NT2-N neurons to 5 microM UCB (a concentration known to induce apoptosis) or 2 microM staurosporine (positive apoptosis control) and investigated the effects of treatments with the specific caspase-3 inhibitor, zDEVD.FMK (20 and 100 microM), or the general caspase inhibitor, zVAD.FMK (20 and 100 microM), and/or the N-methyl-D-aspartate (NMDA) receptor antagonist MK-801 (10 microM) during a 24- or 48-h exposure. UCB increased caspase-3 activity 2.3-fold after 6 h. Despite this, treatment with zDEVD.FMK did not prevent cell death. zVAD.FMK enhanced neuronal survival by reducing apoptotic nuclear fragmentation, while MK-801 enhanced survival by reducing apoptotic nuclear condensation; both without affecting the MTT assays. Combined treatment reduced both apoptotic morphologies (without affecting necrosis), and this effect was also reflected in the MTT assays [corrected] We conclude that NMDA receptor-mediated pathways and caspase-mediated pathways are involved in UCB-induced cell death in human NT2-N neurons. Concomitant inhibition of both pathways results in synergistic protection.

Topics: Amino Acid Chloromethyl Ketones; Apoptosis; Bilirubin; Caspase 3; Caspase Inhibitors; Cysteine Proteinase Inhibitors; Dizocilpine Maleate; DNA Fragmentation; Drug Synergism; Enzyme Activation; Humans; Kernicterus; Neurons; Receptors, N-Methyl-D-Aspartate; Tumor Cells, Cultured

Cell death of neural progenitor cells is the primary problem limiting the value of neural progenitor cell-based therapy for central nervous system disorders. However, little is known about the mechanism of cell death of neural progenitor cells. In this study, we investigated the mechanisms of cell death of a multipotent cell line, MEB5, caused by deprivation of epidermal growth factor (EGF). When EGF was removed from the culture medium, the total number of viable MEB5 cells reduced, and nuclear condensation and elevation of caspase-3-like enzyme activity were observed in MEB5 cells. Treatment with a broad-range caspase inhibitor reduced cell death in a concentration-dependent manner, indicating that MEB5 cells undergo caspase-mediated apoptotic cell death caused by EGF deprivation. We also investigated the effects of glutamate receptor antagonists, antioxidants and nitric oxide synthase inhibitor on EGF deprivation-induced cell death. N-methyl-D-aspartate (NMDA) glutamate receptor antagonists, alpha-amino-3-hydrozy-5-methyl-4-isoxazole propionic acid (AMPA) glutamate receptor antagonist and nitric oxide synthase inhibitor failed to reduce cell death. In contrast, two antioxidants with different chemical structures reduced cell death in a concentration-dependent manner. The production of reactive oxygen species was detected in MEB5 cells after EGF deprivation by monitoring dichlorodihydrofluorescein fluorescence as a marker of reactive oxygen species-related radicals. Our results suggest that oxidative stress triggers caspase-mediated apoptosis of neural progenitor cells by trophic support deprivation.

Topics: 6-Cyano-7-nitroquinoxaline-2,3-dione; Amino Acid Chloromethyl Ketones; Animals; Antioxidants; Apoptosis; Astrocytes; Caspase 3; Caspase Inhibitors; Cell Line; Dizocilpine Maleate; Enzyme Inhibitors; Epidermal Growth Factor; Ethylenediamines; Excitatory Amino Acid Antagonists; L-Lactate Dehydrogenase; Metalloporphyrins; Mice; Multipotent Stem Cells; Neurons; NG-Nitroarginine Methyl Ester; Nitric Oxide Synthase; Organometallic Compounds; Reactive Oxygen Species; Receptors, AMPA; Receptors, N-Methyl-D-Aspartate

Organophosphorus (OP) compounds, used as insecticides and chemical warfare agents, are potent neurotoxins. We examined the neurotoxic effect of paraoxon (O,O-diethyl O-p-nitrophenyl phosphate), an organophosphate compound, and the role of NMDA receptors as a mechanism of action in cultured cerebellar granule cells. Paraoxon is neurotoxic to cultured rat cerebellar granule cells in a time- and concentration-dependent manner. Cerebellar granule cells are less sensitive to the neurotoxic effects of paraoxon on day in vitro (DIV) 4 than neurons treated on DIV 8. Surprisingly, the N-methyl-D-aspartate (NMDA) receptor antagonist, MK-801, enhances paraoxon-mediated neurotoxicity suggesting that NMDA receptors may play a protective role. Pretreatment with a subtoxic concentration of N-methyl-D-aspartate (NMDA) [100 microM] protects about 40% of the vulnerable neurons that would otherwise die from paraoxon-induced neurotoxicity. Moreover, addition of a neuroprotective concentration of NMDA 3 h after treatment with paraoxon provides the same level of protection. Because paraoxon-mediated neuronal cell death is time-dependent, we hypothesized that apoptosis may be involved. Paraoxon increases apoptosis about 10-fold compared to basal levels. The broad-spectrum caspase inhibitor (Boc-D-FMK) and the caspase-9-specific inhibitor (Z-LEHD-FMK) protect against paraoxon-mediated apoptosis, paraoxon-stimulated caspase-3 activity and neuronal cell death. MK-801 increases, whereas NMDA blocks paraoxon-induced apoptosis and paraoxon-stimulated caspase-3 activity. These results suggest that activation of NMDA receptors protect neurons against paraoxon-induced neurotoxicity by blocking apoptosis initiated by paraoxon.

Topics: Amino Acid Chloromethyl Ketones; Animals; Animals, Newborn; Apoptosis; Benzyl Compounds; Bungarotoxins; Caspase 3; Caspase Inhibitors; Caspases; Cell Survival; Cells, Cultured; Cerebellar Cortex; Cholinergic Agonists; Cholinergic Antagonists; Cholinesterase Inhibitors; Cysteine Proteinase Inhibitors; Dizocilpine Maleate; Dose-Response Relationship, Drug; Enzyme Activation; Hippocampus; Hydrocarbons, Fluorinated; N-Methylaspartate; Neurons; Paraoxon; Rats; Rats, Sprague-Dawley; Receptors, N-Methyl-D-Aspartate; Time Factors

The neurotoxic action of the abuse drugs methamphetamine (METH) and 3,4-methylenedioxymethamphetamine (MDMA) on cerebellar granule neurones (CGNs) culture was examined. Treatment for 48 h with METH or MDMA (1-5 mM) induced a higher decrease in viability than 24 h treatment. z.VAD.fmk (100 microM) but not MK-801 nor NBQX recovered control viability values. In both cases, cell death was characterised as apoptotic rather than necrotic by morphology cell observation. Apoptosis measured by flow cytometry indicated an increase in the hypodiploid population after 48 h treatment with METH and MDMA. Apoptosis was reverted by the presence of z.VAD.fmk (100 microM) but not by 10 microM MK-801 or NBQX. Similar results were obtained by analysing nuclear chromatine condensation. These results ruled out excitotoxic participation in amphetamine derivative-induced neurotoxicity in CGNs. Participation of radical oxygen species (ROS) was evaluated using alpha-tocopherol (1-15 microM) and cytometric studies. The co-treatment with 4 mM METH or MDMA for 48 h partially reverted neurotoxic action and apoptotic features, indicating ROS implication in CGNs death by amphetamine derivatives. Alteration of mitochondrial function induced cytochrome C (Cyt C) release after 48-h treatment with METH and MDMA (4 mM). There was also indication of caspase-3-like activation, measured by immunoanalysis and biochemically. Finally, neurodegenerative action caused by amphetamine derivatives may be prevented by using caspase inhibitors.

Topics: alpha-Tocopherol; Amino Acid Chloromethyl Ketones; Animals; Apoptosis; Caspase 3; Caspases; Cerebellum; Cytochromes c; Dizocilpine Maleate; Drug Interactions; Enzyme Activation; Flow Cytometry; Methamphetamine; Microscopy, Fluorescence; Microscopy, Phase-Contrast; Mitochondria; N-Methyl-3,4-methylenedioxyamphetamine; Neurons; Neuroprotective Agents; Quinoxalines; Rats; Reactive Oxygen Species

Characterising the mechanisms of cell death following focal cerebral ischaemia has been hampered by a lack of an in vitro assay emulating both the apoptotic and necrotic features observed in vivo. The present study systematically characterised oxygen-glucose-deprivation (OGD) in primary rat cortical neurones to establish a reproducible model with components of both cell-death endpoints. OGD induced a time-dependent reduction in cell viability, with 80% cell death occurring 24 h after 3 h exposure to 0% O2 and 0.5 mM glucose. Indicative of a necrotic component to OGD-induced cell death, N-methyl-D-aspartate (NMDA) receptor inhibition with MK-801 attenuated neuronal loss by 60%. The lack of protection by the caspase inhibitors DEVD-CHO and z-VAD-fmk suggested that under these conditions neurones did not die by an apoptotic mechanism. Moderating the severity of the insult by decreasing OGD exposure to 60 min did not reduce the amount of necrosis, but did induce a small degree of apoptosis (a slight reduction in cell death was observed in the presence of 10 uM DEVD-CHO). In separate experiments purported to enhance the apoptotic component, cells were gradually deprived of O2, exposed to 4% O2 (as opposed to 0%) during the OGD period, or maintained in serum-containing media throughout. While NMDA receptor antagonism significantly reduced cortical cell death under all conditions, a caspase-inhibitor sensitive component of cell death was not uncovered. These studies suggest that OGD of cultured cortical cells models the excitotoxic, but not the apoptotic component of cell death observed in vivo.

Topics: Amino Acid Chloromethyl Ketones; Animals; Apoptosis; Brain Ischemia; Caspases; Cell Hypoxia; Cell Survival; Cells, Cultured; Cerebral Cortex; Dizocilpine Maleate; Excitatory Amino Acid Antagonists; Glucose; Models, Biological; N-Methylaspartate; Neurons; Oligopeptides; Oxygen; Rats; Rats, Sprague-Dawley; Staurosporine; Time Factors

3-Nitropropionic acid (3NP), an irreversible inhibitor of succinate dehydrogenase, has been used to model features of neurodegenerative disorders including Huntington disease, as well as acute neuronal insults such as cerebral ischemia. 3NP induces rapid necrosis and delayed apoptosis in primary cultures of rat hippocampal neurons. Low levels of extracellular glutamate shift the cell death mechanism to necrosis, whereas antagonism of NMDA receptors results in predominately apoptotic death. In the present study, the involvement of cysteine proteases in the morphologic and biochemical alterations accompanying 3NP-induced neuron death was investigated. Immunoblots of spectrin breakdown products indicated Ca(2+)-dependent cysteine protease (calpain) activation within the 8 hours of 3NP administration, whereas caspase-3 activation was not evident until 16 to 48 hours after treatment. The NMDA receptor antagonist MK-801 (dizocilpine) decreased 3NP-induced calpain activity, but did not alter caspase-3 activity. Similar to MK-801, calpain inhibitors (Z-Val-Phe.H and Z-Leu-Phe-CONHEt) shifted the cell death morphology towards apoptosis and delayed, but did not prevent, the 3NP-induced cell death. Together, the results indicate that following 3NP administration, increased calpain activity precedes caspase-3 activation, contributes to the necrotic morphology, and facilitates and accelerates the cell death.

Topics: Amino Acid Chloromethyl Ketones; Animals; Apoptosis; Calpain; Caspases; Cell Death; Cell Survival; Cells, Cultured; Cysteine Proteinase Inhibitors; Dizocilpine Maleate; Drug Synergism; Embryo, Mammalian; Excitatory Amino Acid Antagonists; Glutamic Acid; Hippocampus; Immunoblotting; Immunohistochemistry; Necrosis; Neurons; Neurotoxins; Nitro Compounds; Oligopeptides; Propionates; Rats; Spectrin; Time Factors

These studies have addressed the role of caspase-3 activation in neuronal death after cerebral ischemia in different animal models. The authors were unable to show activation of procaspase-3 measured as an induction of DEVDase (Asp-Glu-Val-Asp) activity after focal or transient forebrain ischemia in rats. DEVDase activity could not be induced in the cytosolic fraction of the brain tissue obtained from these animals by exogenous cytochrome c/dATP and Ca2+. However, the addition of granzyme B to these cytosolic fractions resulted in a significant activation of DEVDase, confirming that the conditions were permissive to analyze proteolytic cleavage of the DEVD-AMC (7-amino-4-methyl-coumarin) substrate. Consistent with these findings, zVal-Ala-Asp-fluoromethylketone administered after focal ischemia did not have a neuroprotective effect. In contrast to these findings, a large increase in DEVDase activity was detected in a model of hypoxic-ischemia in postnatal-day-7 rats. Furthermore, in postnatal-day-7 animals treated with MK-801, in which it has been suggested that excessive apoptosis is induced, the authors were unable to detect activation of DEVDase activity but were able to induce it in vitro by the addition of cytochrome c/dATP and Ca2+ to the cytosolic fraction. Analysis of cytochrome c distribution did not provide definitive evidence for selective cytochrome c release in the permanent focal ischemia model, whereas in the transient model a small but consistent amount of cytochrome c was found in the cytosolic fraction. However, in both models the majority of cytochrome c remained associated with the mitochondrial fraction. In conclusion, the authors were unable to substantiate a role of mitochondrially derived cytochrome c and procaspase-3 activation in ischemia-induced cell death in adult brain, but did see a clear induction of caspase-3 in neonatal hypoxia.

Topics: Amino Acid Chloromethyl Ketones; Animals; Animals, Newborn; Brain; Brain Ischemia; Caspase 3; Caspases; Cell Death; Cytochrome c Group; Dizocilpine Maleate; Enzyme Activation; Granzymes; Humans; Infarction, Middle Cerebral Artery; Male; Mice; Mice, Inbred C57BL; Neurons; Neuroprotective Agents; Peptide Hydrolases; Protein Precursors; Rats; Rats, Sprague-Dawley; Rats, Wistar; Serine Endopeptidases

Transgenic Huntington's disease (HD) mice, expressing exon 1 of the HD gene with an expanded CAG repeat, are totally resistant to striatal lesion induced by excessive NMDA receptor activation. We now show that striatal lesions induced by the mitochondrial toxin malonate are reduced by 70-80% in transgenic HD mice compared with wild-type littermate controls. This occurred in 6- and 12-week-old HD mice with 150 CAG repeats (line R6/2) and in 18-week-old, but not 6-week-old, HD mice with 115 CAG repeats (line R6/1). Therefore, we show for the first time that the resistance to neurotoxin in transgenic HD mice is dependent on both the CAG repeat length and the age of the mice. Importantly, most HD patients develop symptoms in adulthood and exhibit an inverse relationship between CAG repeat length and age of onset. Transgenic mice expressing a normal CAG repeat (18 CAG) were not resistant to malonate. Although endogenous glutamate release has been implicated in malonate-induced cell death, glutamate release from striatal synaptosomes was not decreased in HD mice. Malonate-induced striatal cell death was reduced by 50-60% in wild-type mice when they were treated with either the NMDA receptor antagonist MK-801 or the caspase inhibitor zVAD-fmk. These two compounds did not reduce lesion size in transgenic R6/1 mice. This might suggest that NMDA receptor- and caspase-mediated cell death pathways are inhibited and that the limited malonate-induced cell death still occurring in HD mice is independent of these pathways. There were no changes in striatal levels of the two anti cell death proteins Bcl-X(L) and X-linked inhibitor of apoptosis protein (XIAP), before or after the lesion in transgenic HD mice. We propose that mutant huntingtin causes a sublethal grade of metabolic stress which is CAG repeat length-dependent and results in up-regulation over time of cellular defense mechanisms against impaired energy metabolism and excitotoxicity.

Topics: Aging; Amino Acid Chloromethyl Ketones; Animals; bcl-X Protein; Blood Glucose; Cell Death; Corpus Striatum; Disease Models, Animal; Dizocilpine Maleate; Excitatory Amino Acid Antagonists; Female; Glutamic Acid; Humans; Huntingtin Protein; Huntington Disease; Immunoblotting; Immunohistochemistry; Male; Malonates; Mice; Mice, Inbred C57BL; Mice, Inbred CBA; Mice, Transgenic; Nerve Tissue Proteins; Neurons; Neuroprotective Agents; Nuclear Proteins; Proteins; Proto-Oncogene Proteins c-bcl-2; Succinate Dehydrogenase; Synaptosomes; Trinucleotide Repeats; X-Linked Inhibitor of Apoptosis Protein

In primary neuronal-astrocyte cultures from mouse brain, ischemic conditions were simulated by combined oxygen-glucose deprival (OGD) for 2 hrs. This treatment resulted in near complete neuronal damage 24 hrs. later and was accompanied by DNA degradation and apoptotic nuclear morphology. Since caspases are key enzymes in the propagation and execution of programmed cell death, we evaluated the effect of the caspase inhibitor z-VAD-fmk. Damage following 2 hrs. OGD could be reduced by up to 56% with z-VAD-fmk (p<0.05). DNA-fragmentation and caspase activation has been also reported in an in vivo model of cerebral ischemia imitating human stroke. In this model the middle cerebral artery (MCA) is permanently occluded resulting in focal cerebral ischemia and subsequent infarction. Since z-VAD.fmk does not penetrate the blood-brain barrier it was applied intraventricularly as a bolus injection given 30 min. before MCA occlusion which was followed by 24 hrs. of infusion. Infarct volume was determined 48 hrs. after MCA occlusion by means of in vivo magnetic resonance imaging. Z-VAD.fmk dose dependently reduced infarct volume reaching a significant decrease of the cortical infarct by 45% when given as a 120 ng bolus followed by 40 ng/hr. infusion (p<0.05). In summary, our study supports the concept that caspase inhibitors are beneficial in brain ischemia.

Topics: Amino Acid Chloromethyl Ketones; Animals; Apoptosis; Astrocytes; Brain; Brain Ischemia; Caspase Inhibitors; Dizocilpine Maleate; Enzyme Inhibitors; Glucose; Immunohistochemistry; In Situ Nick-End Labeling; L-Lactate Dehydrogenase; Magnetic Resonance Imaging; Mice; Neuroprotective Agents; Rats; Rats, Inbred F344; Stroke

Evidence has accumulated that Zn2+ plays a central role in neurodegenerative processes following brain injuries including ischaemia or epilepsy. In the present study, we examined patterns and possible mechanisms of Zn2+ neurotoxicity. Inclusion of 30-300 microM Zn2+ for 30 min caused neuronal necrosis apparent by cell body and mitochondrial swelling in cortical cell cultures. This Zn2+ neurotoxicity was not attenuated by antiapoptosis agents, inhibitors of protein synthesis or caspase. Blockade of glutamate receptors or nitric oxide synthase showed no beneficial effect against Zn2+ neurotoxicity. Interestingly, antioxidants, trolox or SKF38393, attenuated Zn(2+)-induced neuronal necrosis. Pretreatment with insulin or brain-derived neurotrophic factor increased the Zn(2+)-induced free radical injury. Kainate or AMPA facilitated Zn2+ entry and potentiated Zn2+ neurotoxicity in a way sensitive to trolox. Reactive oxygen species and lipid peroxidation were generated in the early phase of Zn2+ neurotoxicity. These findings indicate that entry and accumulation of Zn2+ result in generation of toxic free radicals and then cause necrotic neuronal degeneration under certain pathological conditions in the brain.

Topics: 2,3,4,5-Tetrahydro-7,8-dihydroxy-1-phenyl-1H-3-benzazepine; 6-Cyano-7-nitroquinoxaline-2,3-dione; alpha-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic Acid; Amino Acid Chloromethyl Ketones; Animals; Antioxidants; Apoptosis; Brain-Derived Neurotrophic Factor; Cells, Cultured; Cerebral Cortex; Chromans; Cysteine Proteinase Inhibitors; Dizocilpine Maleate; Dopamine Agonists; Drug Synergism; Excitatory Amino Acid Agonists; Excitatory Amino Acid Antagonists; Female; Free Radicals; Hypoglycemic Agents; Insulin; Kainic Acid; Lipid Peroxidation; Mice; Microscopy, Electron; Mitochondrial Swelling; Necrosis; Nerve Degeneration; Neurons; Neurotoxins; Oligopeptides; Oxidative Stress; Pregnancy; Zinc

Early loss of neurites followed by delayed damage of neuronal somata is a feature of several neurodegenerative diseases. Death by apoptosis would ensure the rapid removal of injured neurons, whereas conditions that prevent apoptosis may facilitate the persistence of damaged cells and favor inflammation and disease progression.. Cultures of cerebellar granule cells (CGC) were treated with microtubule disrupting agents. These compounds induced an early degeneration of neurites followed by apoptotic destruction of neuronal somata. The fate of injured neurons was followed after co-exposure to caspase inhibitors or agents that decrease intracellular ATP (deoxyglucose, S-nitrosoglutathione, 1-methyl-4-phenylpyridinium). We examined the implications of energy loss for caspase activation, exposure of phagocytosis markers, and long-term persistence of damaged cells.. In CGC exposed to colchicine or nocodazole, axodendritic degeneration preceded caspase activation and apoptosis. ATP-depleting agents or protein synthesis inhibition prevented caspase activation, translocation of the phagocytosis marker, phosphatidylserine, and apoptotic death. However, they did not affect the primary neurite loss. Repletion of ATP by enhanced glycolysis restored all apoptotic features. Peptide inhibitors of caspases also prevented the apoptotic changes in the cell bodies, although the axodendritic net was lost. Under this condition cell demise still occurred 48 hr later in a caspase-independent manner and involved plasma membrane lysis at the latest stage.. Inhibition of the apoptotic machinery by drugs, energy deprivation, or endogenous mediators may result in the persistence and subsequent lysis of injured neurons. In vivo, this may favor the onset of inflammatory processes and perpetuate neurodegeneration.

Topics: Adenosine Triphosphate; Amino Acid Chloromethyl Ketones; Animals; Apoptosis; Carrier Proteins; Caspase 3; Caspase Inhibitors; Caspases; Cerebellum; Colchicine; Cyclosporine; Cysteine Proteinase Inhibitors; Deoxyglucose; Dizocilpine Maleate; Energy Metabolism; Enzyme Activation; Enzyme Inhibitors; Glutathione; Mice; Mice, Inbred BALB C; Microfilament Proteins; Microtubules; Neurons; Neuroprotective Agents; Nitroso Compounds; Nocodazole; Oligopeptides; Paclitaxel; Potassium; Protein Biosynthesis; S-Nitrosoglutathione; Tacrolimus; Verapamil

In rats, striatal histotoxic hypoxic lesions produced by the mitochondrial toxin malonate resemble those of focal cerebral ischemia. Intrastriatal injections of malonate induced cleavage of caspase-2 beginning at 6 h, and caspase-3-like activity as identified by DEVD biotin affinity-labeling within 12 h. DEVD affinity-labeling was prevented and lesion volume reduced in transgenic mice overexpressing BCL-2 in neuronal cells. Intrastriatal injection of the tripeptide, N-benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone (zVAD-fmk), a caspase inhibitor, at 3 h, 6 h, or 9 h after malonate injections reduced the lesion volume produced by malonate. A combination of pretreatment with the NMDA antagonist, dizocilpine (MK-801), and delayed treatment with zVAD-fmk provided synergistic protection compared with either treatment alone and extended the therapeutic window for caspase inhibition to 12 h. Treatment with cycloheximide and zVAD-fmk, but not with MK-801, blocked the malonate-induced cleavage of caspase-2. NMDA injections alone resulted in a weak caspase-2 cleavage. These results suggest that malonate toxicity induces neuronal death by more than one pathway. They strongly implicate early excitotoxicity and delayed caspase activation in neuronal loss after focal ischemic lesions and offer a new strategy for the treatment of stroke.

Topics: Amino Acid Chloromethyl Ketones; Animals; Apoptosis; Brain; Caspase 2; Caspase 3; Caspase Inhibitors; Caspases; Corpus Striatum; Cycloheximide; Cysteine Proteinase Inhibitors; Dizocilpine Maleate; Drug Synergism; Genes, bcl-2; Humans; Hypoxia, Brain; In Situ Nick-End Labeling; Male; Malonates; Mice; Mice, Transgenic; Neuroprotective Agents; Proto-Oncogene Proteins c-bcl-2; Rats; Rats, Sprague-Dawley