cytochrome-c-t and lactacystin

The proteasome inhibitor model of Parkinson's disease (PD) appears to reproduce many of the important behavioural, imaging, pathological and biochemical features of the human disease. However, the mechanisms involved in the lactacystin-induced, mitochondria-mediated apoptotic pathway remain poorly defined.. We have used lactacystin as a specific inhibitor of the 20S proteasome in the dopaminergic neuroblastoma cell line SH-SY5Y. We over-expressed a green fluorescent protein (GFP)-Bax fusion protein in these cells to study localization of Bax. Free radical scavengers were used to assess the role of reactive oxygen species (ROS) in these pathways.. Lactacystin triggered a concentration-dependent increase in cell death mediated by the mitochondrial apoptotic pathway, and induced a change in mitochondrial membrane permeability accompanied by cytochrome c release. The participation of Bax protein was more critical than the formation of the permeability transition pore in mitochondria. GFP-Bax over-expression demonstrated Bax redistribution from the cytosol to mitochondria after the addition of lactacystin. ROS, but not p38 mitogen-activated protein kinase, participated in lactacystin-induced mitochondrial Bax translocation. Lactacystin disrupted the intracellular redox state by increasing ROS production and depleting endogenous antioxidant systems such as glutathione (GSH). Pharmacological depletion of GSH, using L-buthionine sulphoxide, potentiated lactacystin-induced cell death. Lactacystin sensitized neuroblastoma cells to oxidative damage, induced by subtoxic concentrations of 6-hydroxydopamine.. The lactacystin-induced, mitochondrial-mediated apoptotic pathway involved interactions between ROS, GSH and Bax. Lactacystin could constitute a potential factor in the development of sporadic PD.

Topics: Acetylcysteine; Apoptosis; bcl-2-Associated X Protein; Cell Death; Cell Line, Tumor; Cell Survival; Coloring Agents; Cytochromes c; Cytosol; Dose-Response Relationship, Drug; Enzyme Inhibitors; Green Fluorescent Proteins; Humans; Membrane Potential, Mitochondrial; Mitochondria, Liver; Neuroblastoma; Reactive Oxygen Species; Recombinant Fusion Proteins; Tetrazolium Salts; Thiazoles

Parkinson disease is characterized by the progressive loss of dopaminergic neurons in the substantia nigra pars compacta. It has been proposed that dysfunction of the ubiquitin proteasome system plays an important role in the pathogenesis of Parkinson disease, but the mechanisms underlying ubiquitin proteasome system-related neuron degeneration are unknown. Here, we demonstrate that the proteasome inhibitor lactacystin induces phosphorylation of c-Jun N-terminal kinase (JNK) and c-Jun, the release of cytochrome c, activation of both caspase-9 and caspase-3, and sequential apoptosis of dopaminergic neurons in vitro. Most of these effects can be attenuated by the JNK inhibitor SP600125. Furthermore, infusion of lactacystin in rats in vivo also leads to phosphorylation of JNK before nigral neuron loss; chronic administration of SP600125 also blocks this loss. These results indicate that JNK is involved in proteasome inhibition-induced dopaminergic neuron degeneration through caspase-3-mediated apoptotic pathways, suggesting that this kinase may be a therapeutic target for the prevention of substantia nigra pars compacta degeneration in Parkinson disease patients.

Topics: Acetylcysteine; Animals; Apoptosis; Blotting, Western; Caspase 3; Cell Line; Chromatography, High Pressure Liquid; Cysteine Proteinase Inhibitors; Cytochromes c; Dopamine; Immunohistochemistry; JNK Mitogen-Activated Protein Kinases; Male; Nerve Degeneration; Neurons; Rats; Rats, Sprague-Dawley; Reverse Transcriptase Polymerase Chain Reaction; Substantia Nigra; Tetrazolium Salts; Thiazoles

Loss-of-function mutations of attractin (Atrn) in animals result in age-dependent progressive neurodegeneration including neuronal cell death, hypomyelination and vacuolation. The mechanisms of how age-dependent neurodegeneration occurs in these animals are not clear. In this study, we found that reducing the endogenous expression level of Atrn exacerbated, whereas overexpressing Atrn protected against, the neuronal cell death caused by the neurotoxins, 1-methyl-4-phenylpyridinium (MPP+) and lactacystin. In addition, both MPP+ and lactacystin-induced cytochrome c and apoptosis inducing factor (AIF) release, which was inhibited by overexpressing Atrn and enhanced by knocking down Atrn, indicating that Atrn may be involved in regulating the mitochondrial function. Furthermore, we found that vast majority of the dopaminergic neurons in mice express Atrn and its expression decreases with age. Our findings demonstrated that Atrn may play a protective role against environmental toxins, and implied a potential therapeutic effect of Atrn for neurodegenerative diseases.

Topics: 1-Methyl-4-phenylpyridinium; Acetylcysteine; Analysis of Variance; Apoptosis Inducing Factor; Cell Line, Tumor; Cell Survival; Cytochromes c; Gene Expression Regulation; Humans; In Situ Hybridization; Membrane Proteins; Nerve Degeneration; Neurotoxins; RNA, Small Interfering; Transfection; Tyrosine 3-Monooxygenase

Cells exposed to sustained endoplasmic reticulum (ER) stress undergo programmed cell death and display features typical of apoptosis, such as cysteine aspartyl protease (caspase) activation, cytochrome c release, and DNA fragmentation. Here, we show that the execution of cell death induced by ER stress is mediated via the proteasome. Inhibition of the proteasome by lactacystin prevented ER stress-induced degradation of Bcl-2, release of cytochrome c, processing of effector caspase-3, and exposure of phosphatidylserine. Owing to the ability of lactacystin to inhibit cytochrome c release, we propose that the pro-apoptotic activity of the proteasome lies upstream of mitochondrial activation. Thus, the proteasome serves as a principal mediator of ER stress-induced cell death in this system.

Topics: Acetylcysteine; Animals; Apoptosis; Blotting, Western; Brefeldin A; Caspase 3; Cells, Cultured; Chlorhexidine; Cytochromes c; DNA Fragmentation; Electrophoresis, Polyacrylamide Gel; Endoplasmic Reticulum; Fibroblasts; Mitochondrial Membranes; Models, Biological; Phosphatidylserines; Proteasome Endopeptidase Complex; Proteasome Inhibitors; Proto-Oncogene Proteins c-bcl-2; Rats; Sulfones; Temperature; Ubiquitin-Activating Enzymes

Proteasome inhibitors exhibit antitumor activity against malignancies of different histology. Yet, the mechanisms underlying this effect are poorly understood. Recent evidence indicates that antiapoptotic factors may also accumulate as a consequence of exposure to these drugs, possibly reducing their cytotoxicity. These include the Bcl-2 family member Mcl-1, whose down-regulation has been proposed to initiate apoptosis in response to genotoxic stimuli. In this study, we found that proteasome inhibitors release cyotochrome c and second mitochondria-derived activator of caspase (SMAC)/Diablo and trigger the subsequent apoptotic cascade in spite of concomitant Mcl-1 increase. However, our data indicate that subtraction of Mcl-1 during apoptosis, although not required for early release of proapoptotic factors, is probably relevant in speeding up cell demise, since RNA interference-mediated Mcl-1 silencing is lethal in lymphoma cells. Consistent with this, the cytotoxic effects of proteasome inhibitors are enhanced when Mcl-1 increase is impeded. Thus, this study identifies Mcl-1 accumulation as an unwanted molecular consequence of exposure to proteasome inhibitors, which slows down their proapoptotic effects. Pharmacologic or genetic approaches targeting Mcl-1, including therapeutic RNAi, may increase the effectiveness of these compounds.

Topics: Acetylcysteine; Apoptosis; Apoptosis Regulatory Proteins; Carrier Proteins; Caspases; Cell Survival; Complement Membrane Attack Complex; Complement System Proteins; Cysteine Proteinase Inhibitors; Cytochromes c; Gene Expression; Glycoproteins; Humans; Intracellular Signaling Peptides and Proteins; Jurkat Cells; Leupeptins; Lymphocytes; Mitochondria; Mitochondrial Proteins; Myeloid Cell Leukemia Sequence 1 Protein; Neoplasm Proteins; Oligopeptides; Proteasome Endopeptidase Complex; Proteasome Inhibitors; Proto-Oncogene Proteins c-bcl-2; RNA Interference

The function of the proteasome has been linked to various pathologies, including cancer and neurodegeneration. Proteasomal inhibition can lead to death in a variety of cell types, however the manner in which this occurs is unclear, and may depend on the particular cell type. In this work we have extended previous findings pertaining to the effects of pharmacological proteasomal inhibitors on PC12 cells, by examining in more detail the induced death pathway. We find that cell death is apoptotic by ultrastructural criteria. Caspase 9 and 3 are processed, cytochrome c is released from the mitochondria and a dominant negative form of caspase 9 prevents death. Furthermore, Bax undergoes a conformational change and is translocated to the mitochondria in a caspase-independent fashion. Total cell levels of Bax however do not change, whereas levels of the BH3-only protein Bim increase with proteasomal inhibition. Transient overexpression of bcl-xL or, to a lesser extent, of bcl-2, significantly decreased apoptotic death and prevented Bax conformational change. We conclude that death elicited by proteasomal inhibition of PC12 cells follows a classical "intrinsic" pathway. Significantly, antiapoptotic bcl-2 family members prevent apoptosis by inhibiting Bax conformational change. Increased levels of Bim may contribute to cell death in this model.

Topics: Acetylcysteine; Animals; Apoptosis; Apoptosis Regulatory Proteins; bcl-2-Associated X Protein; Bcl-2-Like Protein 11; bcl-X Protein; Caspase 9; Cytochromes c; Gene Expression; Genes, Dominant; Membrane Proteins; Mitochondria; PC12 Cells; Proteasome Inhibitors; Protein Processing, Post-Translational; Protein Structure, Quaternary; Protein Transport; Proto-Oncogene Proteins; Rats

Proteasomal dysfunction may play a role in a number of neurodegenerative conditions, and in particular Parkinson's disease (PD) and related Lewy body (LB) diseases. Application of proteasomal inhibitors to neuronal cell culture systems is associated with survival-promoting effects or with cell death depending on the model system. We have applied pharmacological proteasomal inhibitors to cultured neonatal mouse sympathetic neurons in order to investigate whether these catecholaminergic neurons, which are affected in PD, are sensitive to proteasomal inhibition and, if so, which cell death pathway is activated. We report here that proteasomal inhibition leads to apoptotic death of mouse sympathetic neurons. This death is accompanied by caspase 3 activation and cytochrome c release from the mitochondria and is abrogated by caspase inhibition. Bax deletion prevented both cytochrome c release and caspase 3 activation, and also provided complete protection against proteasomal inhibition-induced death. Bcl-2 overexpression achieved a similar survival-promoting effect. There was no change in Bax levels following proteasomal inhibition, suggesting that Bax itself is not regulated by the proteasome in this cell culture system, and that a primary increase in Bax is unlikely to account for death. In contrast, levels of the BH3-only protein, Bim, increased with proteasomal inhibition. We conclude that proteasomal inhibition of mouse sympathetic neurons activates the intrinsic apoptotic pathway involving bcl-2 family members and the mitochondria.

Topics: Acetylcysteine; Animals; Animals, Newborn; Apoptosis; Apoptosis Regulatory Proteins; bcl-2-Associated X Protein; Bcl-2-Like Protein 11; Blotting, Western; Carrier Proteins; Caspase 3; Caspases; Cell Survival; Cells, Cultured; Cysteine Endopeptidases; Cysteine Proteinase Inhibitors; Cytochromes c; Dose-Response Relationship, Drug; Drug Interactions; Enzyme Inhibitors; Ganglia, Sympathetic; Genes, bcl-2; Green Fluorescent Proteins; Immunohistochemistry; Luminescent Proteins; Membrane Proteins; Mice; Mice, Knockout; Multienzyme Complexes; Neurons; Oligopeptides; Proteasome Endopeptidase Complex; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-bcl-2; Rhodamines; Signal Transduction; Time Factors; Ubiquitin

Topics: Acetylcysteine; Amino Acid Chloromethyl Ketones; Antioxidants; Apoptosis; bcl-X Protein; Blotting, Western; Caspase 3; Caspase 8; Caspases; Cell Line, Tumor; Cell Survival; Cysteine Endopeptidases; Cytochromes c; Dose-Response Relationship, Drug; Flow Cytometry; Humans; Leupeptins; Membrane Potentials; Microscopy, Fluorescence; Mitochondria; Multienzyme Complexes; Osteosarcoma; Protease Inhibitors; Proteasome Endopeptidase Complex; Proto-Oncogene Proteins c-bcl-2; Reactive Oxygen Species; Retinoblastoma Protein; Time Factors; Transfection; Tumor Suppressor Protein p53