calpain and thiazolyl-blue

The characteristic pathological change of Alzheimer's disease (AD) include deposits of β-amyloid protein (Aβ) in brain, neurofibrillary tangles (NFTs), as well as a few neuronal loss. Evidence shows that Aβ causes calcium influx and induces the cleavage of p35 into p25. Furthermore, the binding of p25 to cyclin-dependent kinase 5 (Cdk5) constitutively activates Cdk5. The p25/Cdk5 complex then hyperphosphorylates tau. Tanshinone IIA (tanIIA), a natural product extracted from Chinese herbal medicine Salvia miltiorrhiza BUNGE, has been reported to exert antioxidative activity. However, its neuroprotective activity remains unclear. The present study determined whether tanIIA protects neurons against Aβ(25-35)-induced cytotoxicity and detected the association of this protective effect with calpain and the p35/Cdk5 pathway. The results showed that tanIIA protected neurons against the neurotoxicity of Aβ(25-35), increased the viability of neurons, decreased expression of phosphorylated tau in neurons induced by Aβ(25-35), improved the impairment of the cell ultrastructure (such as nuclear condensation and fragmentation, and neurofibril collapse). Further more, we found that tanIIA maintained the normal expression of p35 on peripheral membranes, and decreased p25 expression in the cytoplasm. TanIIA also inhibited the translocation of Cdk5 from the nucleus into the cytoplasm of primary neurons induced by Aβ(25-35). These data suggested that tanIIA possessed neuroprotective action and the protection may involve in calpain and the p35/Cdk5 pathway.

Topics: Abietanes; Amyloid beta-Peptides; Animals; Blotting, Western; Calpain; Cell Nucleus; Cell Survival; Cells, Cultured; Cerebral Cortex; Cytoplasm; Female; Immunohistochemistry; Mice; Microscopy, Electron, Transmission; Neurons; Neuroprotective Agents; Peptide Fragments; Phosphorylation; Phosphotransferases; Pregnancy; Protein Transport; Signal Transduction; tau Proteins; Tetrazolium Salts; Thiazoles

Cyclin-dependent kinase (Cdk) 5 and p38 activities are significantly increased in Alzheimer's Disease (AD). Both p38 and Cdk5 promote neurodegeneration upon deregulation. However, to date the mechanistic link between Cdk5 and p38 remains unclear. This study presents the first mechanism showing Cdk5 as a major regulator of p38 cascade in neurons and in transgenic mouse model of AD. Using beta-amyloid and glutamate as the neurotoxic stimuli, our results show that deregulated Cdk5 induces p38 activation by increasing reactive oxygen species (ROS) in neuronal cells and in primary cortical neurons. Elimination of ROS inhibits p38 activation, revealing ROS as major stimuli of the p38 cascade. Importantly, Cdk5-mediated p38 activation increases c-Jun expression, thereby revealing a mechanistic link between deregulated Cdk5 and c-Jun level in AD brains. c-Jun is over-expressed in AD, and is believed to contribute significantly to neurodegeneration. Based on the proposed mechanism, Cdk5 inhibition is more neuroprotective relative to p38 and c-Jun, suggesting that Cdk5 is an upstream regulator of neurodegenerative pathways triggered by p38 and a preferable therapeutic target for AD.

Topics: Alzheimer Disease; Amyloid beta-Peptides; Animals; Blotting, Western; Calpain; Coloring Agents; Cyclin-Dependent Kinase 5; Glutamic Acid; Humans; Immunohistochemistry; MAP Kinase Kinase 6; Mice; Mice, Transgenic; Neurons; p38 Mitogen-Activated Protein Kinases; Phosphorylation; Rats; Rats, Sprague-Dawley; Reactive Oxygen Species; Signal Transduction; Tetrazolium Salts; Thiazoles

The invariant characteristic features associated with Alzheimer's disease (AD) brain include the presence of extracellular neuritic plaques composed of amyloid beta (Abeta) peptide, intracellular neurofibrillary tangles containing hyper-phosphorylated tau protein and the loss of basal forebrain cholinergic neurons. Studies of the pathological changes that characterize AD and several other lines of evidence indicate that in vivo accumulation of Abeta(1-42) may initiate the process of neurodegeneration observed in AD brains. However, the cause of degeneration of the basal forebrain cholinergic neurons and their association to Abeta peptides or phosphorylated tau protein have not been clearly established. In the present study, using rat primary septal cultures, we have shown that Abeta(1-42), in a time (1-48 h) and concentration (0.01-20 microM)-dependent manner, induce toxicity in cultured neurons. Subsequently, we have demonstrated that Abeta toxicity is mediated via activation of cysteine proteases, i.e., calpain and caspase, and proteolytic breakdown of their downstream substrates tau, microtubule-associated protein-2 and alpha II-spectrin. Additionally, Abeta-treatment was found to induce phosphorylation of tau protein along with decreased levels of phospho-Akt and phospho-Ser(9)glycogen synthase kinase-3beta. Exposure to specific inhibitors of caspase or calpain can partially protect cultured neurons against Abeta-induced toxicity but their effects are not found to be additive. These results, taken together, suggest that Abeta peptide can induce toxicity in rat septal cultured neurons by activating multiple intracellular signaling molecules. Additionally, evidence that inhibitors of caspase and calpains can partially protect the cultured basal forebrain neurons raised the possibility that their inhibitors could be of therapeutic relevance in the treatment of AD pathology.

Topics: Amyloid beta-Peptides; Animals; Calcium-Binding Proteins; Calpain; Caspases; Cell Death; Cells, Cultured; Dose-Response Relationship, Drug; Embryo, Mammalian; Enzyme Inhibitors; Female; Glycogen Synthase Kinase 3; Glycogen Synthase Kinase 3 beta; Microscopy, Electron, Scanning; Microtubule-Associated Proteins; Neurofibrillary Tangles; Neurons; Oligopeptides; Peptide Fragments; Pregnancy; Rats; Septum of Brain; Tetrazolium Salts; Thiazoles; Time Factors

Zinc and copper are trace elements essential for proper folding, stabilization and catalytic activity of many metalloenzymes in living organisms. However, disturbed zinc and copper homeostasis is reported in many types of cancer. We have previously demonstrated that copper complexes induced proteasome inhibition and apoptosis in cultured human cancer cells. In the current study we hypothesized that zinc complexes could also inhibit the proteasomal chymotrypsin-like activity responsible for subsequent apoptosis induction. We first showed that zinc(II) chloride was able to inhibit the chymotrypsin-like activity of a purified 20S proteasome with an IC(50) value of 13.8 microM, which was less potent than copper(II) chloride (IC(50) 5.3 microM). We then compared the potencies of a pyrrolidine dithiocarbamate (PyDT)-zinc(II) complex and a PyDT-copper(II) complex to inhibit cellular proteasomal activity, suppress proliferation and induce apoptosis in various human breast and prostate cancer cell lines. Consistently, zinc complex was less potent than copper complex in inhibiting the proteasome and inducing apoptosis. Additionally, zinc and copper complexes appear to use somewhat different mechanisms to kill tumor cells. Zinc complexes were able to activate calpain-, but not caspase-3-dependent pathway, while copper complexes were able to induce activation of both proteases. Furthermore, the potencies of these PyDT-metal complexes depend on the nature of metals and also on the ratio of PyDT to the metal ion within the complex, which probably affects their stability and availability for interacting with and inhibiting the proteasome in tumor cells.

Topics: Animals; Apoptosis; Blotting, Western; Calcium-Binding Proteins; Calpain; Caspase 3; Cell Line, Tumor; Chymotrypsin; Copper; Cysteine Proteinase Inhibitors; Dose-Response Relationship, Drug; Female; Humans; In Situ Nick-End Labeling; Kinetics; Proteasome Inhibitors; Pyrrolidines; Rabbits; Tetrazolium Salts; Thiazoles; Thiocarbamates; Time Factors; Zinc

Evidence has implicated apoptosis as a mechanism underlying cell death in diverse neurodegenerative diseases including Parkinson's disease (PD). Endogenous agents such as TNF-alpha, INF-gamma, IL-1beta and others stress signals activate the sphingomyelin pathway increasing ceramide levels. Ceramide triggers apoptotic pathways while inhibiting survival signalling, and is involved in the regulation of intracellular Ca(2+) homeostasis and compartmentalisation. The contribution of caspases in neuronal apoptosis has been highlighted by the increased survival exerted by caspase inhibition, but the involvement of calpains during neuronal apoptosis and the potential benefit of their inhibition is still controversial. In the present paper, we have analysed the contribution of caspases and calpains to cell death of CAD cells, a catecholaminergic cell line of mesencephalic origin, following C2-ceramide exposure. Ceramide caused CAD cell death by a dose and time dependant mechanism. 25microM of C2-ceramide caused apoptosis. Analysis of activation of caspases and calpains by differential cleavage of alpha-fodrin showed that although calpains are activated before caspases following C2-ceramide exposure, only caspase inhibition increased cell survival. These results demonstrate the activation of caspases and calpains in C2-ceramide-induced cell death, and support the role of caspase inhibition as a neuroprotective strategy and a plausible therapeutic approach to decrease catecholaminergic cell death.

Topics: Analysis of Variance; Animals; Calpain; Caspases; Cell Death; Cell Line; Dipeptides; Dose-Response Relationship, Drug; Drug Interactions; Enzyme Inhibitors; Mice; Neurons; Neuroprotective Agents; Sphingosine; Tetrazolium Salts; Thiazoles; Time Factors

The goal of this study was to assess if neurons exposed to amyloid-beta peptide (Abeta) exclusively in distal axons, undergo apoptosis. This is relevant to the loss of cholinergic neurons in Alzheimer's disease. Using a three-compartmented culture system for rat sympathetic neurons, we demonstrate that exposure of axons to Abeta1-42 activates an independent destruction program in axons, which leads to nuclear apoptosis. Abeta-induced axonal degeneration does not involve local caspase activation, but causes caspase activation in cell bodies. Accordingly, inhibition of caspase activation blocks Abeta-induced apoptosis but not axonal degeneration. In agreement with previous suggestions that disruption of nerve growth factor (NGF)-mediated signaling might contribute to the loss of cholinergic neurons, we found that provision of NGF to cell bodies protects sympathetic neurons from Abeta-induced apoptosis. However, our data indicate that Abeta-induced axonal degeneration follows a mechanism different than that activated by NGF withdrawal. Only Abeta-induced axonal degeneration is prevented by the calpain inhibitor calpastatin and is insensitive to the inhibitor of the ubiquitin-proteasome system MG132. Importantly, inhibition of Abeta-induced axonal degeneration by calpastatin prevents nuclear apoptosis.

Topics: Amyloid beta-Peptides; Animals; Animals, Newborn; Antidotes; Apoptosis; Axons; Blotting, Western; Calcium-Binding Proteins; Calpain; Cells, Cultured; Cysteine Proteinase Inhibitors; Dose-Response Relationship, Drug; Drug Interactions; Egtazic Acid; Nerve Degeneration; Nerve Growth Factor; Neurons; Peptide Fragments; Prosencephalon; Rats; Rats, Sprague-Dawley; Superior Cervical Ganglion; Tetrazolium Salts; Thiazoles

It is well known that traumatic injury in the central nervous system can be viewed as a primary injury and a secondary injury. Increases in oxidative stress lead to breakdown of membrane lipids (lipid peroxidation) during secondary injury. Acrolein, an alpha,beta-unsaturated aldehyde, together with other aldehydes, increases as a result of self-propagating lipid peroxidation. Historically, most research on the pathology of secondary injury has focused on reactive oxygen species (ROS) rather than lipid peroxidation products. Little is known about the toxicology and cell death mediated by these aldehydes. In this study, we investigated and characterized certain features of cell death induced by acrolein on PC12 cells as well as cells from dorsal root ganglion (DRG) and sympathetic ganglion in vitro. In the companion paper, we evaluated a possible means to interfere with this toxicity by application of a compound that can bind to and inactivate acrolein. Here we use both light and atomic force microscopy to study cell morphology after exposure to acrolein. Administration of 100 microM acrolein caused a dramatic change in cell morphology as early as 4 hr. Cytoskeletal structures significantly deteriorated after exposure to 100 microM acrolein as demonstrated by fluorescence microscopy, whereas calpain activity increased significantly at this concentration. Cell viability assays indicated significant cell death with 100 microM acrolein by 4 hr. Caspase 3 activity and DNA fragmentation assays were performed and supported the notion that 100 microM acrolein induced PC12 cell death by the mechanism of necrosis, not apoptosis.

Topics: Acrolein; Animals; Calpain; Caspase 3; Caspases; Cell Death; Cell Differentiation; Cytokines; DNA Fragmentation; Dose-Response Relationship, Drug; Immunohistochemistry; Microscopy, Atomic Force; Microtubules; Mitochondria; Nerve Growth Factor; Neurons; PC12 Cells; Rats; Tetrazolium Salts; Thiazoles; Time Factors

3-Chlorotyrosine, a bio-marker of hypochlorous acid (HOCl) in vivo, was reported to be substantially elevated in the Alzheimer's disease (AD) brains. Thus, HOCl might be implicated in the development of AD. However, its effect and mechanism on neuronal cell death have not been investigated. Here, we report for the first time that HOCl treatment induces an apoptotic-necrotic continuum of concentration-dependent cell death in cultured cortical neurons. Neurotoxicity caused by an intermediate concentration of HOCl (250 microm) exhibited several biochemical markers of apoptosis in the absence of caspase activation. However, the involvement of calpains was demonstrated by data showing that calpain inhibitors protect cortical neurons from apoptosis and the formation of 145/150 kDa alpha-fodrin fragments. Moreover, an increase in cytosolic Ca2+ concentration was associated with HOCl neurotoxicity and Ca2+ channel antagonists, and Ca2+ chelators prevented cleavage of alpha-fodrin and the induction of apoptosis. Finally, we found that calpain activation ruptured lysosomes. Stabilization of lysosomes by calpain inhibitors or imidazoline drugs, as well as inhibition of cathepsin protease activities, rescued cells from HOCl-induced neurotoxicity. Our results showed for the first time that HOCl induces apoptosis in cortical neurons, and that the cell death process involves calpain activation and rupture of lysosomes.

Topics: Adenosine Triphosphate; Animals; Apoptosis; Calcium; Calcium Channels; Calpain; Caspase 3; Caspases; Cell Survival; Cells, Cultured; Dipeptides; Dose-Response Relationship, Drug; Drug Interactions; Enzyme Inhibitors; Hypochlorous Acid; Lysosomes; Mice; Microscopy, Electron, Transmission; Neocortex; Neurons; Nifedipine; Oxidants; Tetrazolium Salts; Thiazoles; Time Factors

We examined the mechanism of 17beta-estradiol (estrogen)-mediated inhibition of apoptosis in C6 (rat glioma) cells following exposure to hydrogen peroxide (H(2)O(2)). Cells were preincubated with 4 microM estrogen for 2 h and then exposed to 100 microM H(2)O(2) for 24 h. Exposure to H(2)O(2) caused significant increases in intracellular calcium (Ca(2+)), as determined by fura-2, which was attenuated by preincubation with estrogen. H(2)O(2) and ionomycin caused cell death in a dose-dependent manner, as measured by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay. Preincubation with estrogen restored viability in cells exposed to H(2)O(2) but not in cells exposed to ionomycin. Western blot analysis showed an increase in Bax/Bcl-2 ratio, calpain activity, and caspase-3 activity following treatment with H(2)O(2), and estrogen pretreatment decreased levels of all three. Cell morphology, as evaluated by Wright staining, indicated apoptosis in cells treated with H(2)O(2), and pretreatment with estrogen reduced apoptosis. Results from MTT and Wright staining were further supported by the terminal deoxyribonucleotidyl transferase (TdT)-mediated dUTP Nick End Labeling (TUNEL) assay. These results indicate a role for estrogen in preventing apoptosis in C6 glial cells exposed to H(2)O(2). Our results suggest that estrogen may have a protective role in minimizing glial cell apoptosis in neurological diseases such as demyelinating disease or central nervous system trauma.

Topics: Animals; Apoptosis; bcl-2-Associated X Protein; Blotting, Western; Calcium; Calpain; Carrier Proteins; Caspase 3; Caspases; Cell Line; Cell Survival; Dose-Response Relationship, Drug; Estrogens; Fura-2; Glioma; Hydrogen Peroxide; In Situ Nick-End Labeling; Ionomycin; Ionophores; Oxidative Stress; Peptide Fragments; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-bcl-2; Rats; Receptors, Estrogen; Spectrin; Tetrazolium Salts; Thiazoles; Time Factors