calpastatin and Brain-Ischemia

Calpains, calcium-activated cysteine proteases with a neutral pH optimum, lead to degration of cystoskeletion and structural protein, and delayed neuronal death. The activation of calpains contribute to apoptosis. Calpain inhibitors provide a novel and potential treatment for cerebral ischemia due to improvement of cerebral infarct and ischemia.

Topics: Animals; Apoptosis; Brain Ischemia; Calcium-Binding Proteins; Calpain; Humans

Topics: Alzheimer Disease; Aminopeptidases; Amyloid beta-Peptides; Brain Ischemia; Calcium-Binding Proteins; Calpain; Caspase 1; Cathepsins; Cysteine Endopeptidases; Humans; Signal Transduction

Taurine as an endogenous substance possesses a number of cytoprotective properties. In the study, we have evaluated the neuroprotective effect of taurine and investigated whether taurine exerted neuroprotection through affecting calpain/calpastatin or caspase-3 actions during focal cerebral ischemia, since calpain and caspase-3 play central roles in ischemic neuronal death.. Male Sprague-Dawley rats were subjected to 2 h of middle cerebral artery occlusion (MCAo), and 22 h of reperfusion. Taurine was administrated intravenously 1 h after MCAo. The dose-responses of taurine to MCAo were determined. Next, the effects of taurine on the activities of calpain, calpastatin and caspase-3, the levels of calpastatin, microtubule-associated protein-2 (MAP-2) and alphaII-spectrin, and the apoptotic cell death in penumbra were evaluated.. Taurine reduced neurological deficits and decreased the infarct volume 24 h after MCAo in a dose-dependent manner. Treatment with 50 mg/kg of taurine significantly increased the calpastatin protein levels and activities, and markedly reduced the m-calpain and caspase-3 activities in penumbra 24 h after MCAo, however, it had no significant effect on mu-calpain activity. Moreover, taurine significantly increased the MAP-2 and alphaII-spectrin protein levels, and markedly reduced the ischemia-induced TUNEL staining positive score within penumbra 24 h after MCAo.. Our data demonstrate the dose-dependent neuroprotection of taurine against transient focal cerebral ischemia, and suggest that one of protective mechanisms of taurine against ischemia may be blocking the m-calpain and caspase-3-mediated apoptotic cell death pathways.

Topics: Animals; Apoptosis; Brain Ischemia; Calcium-Binding Proteins; Calpain; Caspase 3; Dose-Response Relationship, Drug; Down-Regulation; Enzyme Activation; Male; Microfilament Proteins; Microtubule-Associated Proteins; Models, Biological; Neuroprotective Agents; Rats; Rats, Sprague-Dawley; Taurine; Up-Regulation; Vesicular Transport Proteins

Some data have shown the functional connection between calpain and caspase-3. Here, we investigated the cross-talk between calpain and caspase-3 in penumbra and core during focal cerebral ischemia-reperfusion.. The activities of calpain and the levels of calpastatin, microtubule-associated protein-2 (MAP-2), and spectrin in penumbra and core at 3 or 23 h of reperfusion (R 3 h or R 23 h) after 1-h focal cerebral ischemia in rats were determined in sham- or caspase-3 inhibitor z-DEVD-CHO-treated rats.On the other hand, the determination of the activities of caspase-3 and the levels of MAP-2 and spectrin was done in sham- or calpain-inhibitor I-treated rats.. z-DEVD-CHO (600 ng/rat, i.c.v.) markedly reduced the mu- and m-calpain activities in penumbra and the m-calpain activities in core at R 3 h and R 23 h, and enhanced the calpastatin levels in penumbra at R 3 h and in core at R 3 h and R 23 h significantly; however, it had no significant effects on the mu-calpain activities in core and the calpastatin levels in penumbra at R 23 h. Calpain inhibitor I (0.8 mg/rat, i.c.v.) markedly reduced the caspase-3 activities in core at R 3 h and R 23 h, but not in penumbra. Both calpain and caspase-3 inhibitors increased the levels of MAP-2 and spectrin in penumbra and core significantly after focal cerebral ischemia-reperfusion.. Our data provide direct evidence to demonstrate the cross-talk between calpain and caspase-3 in penumbra and core during focal cerebral ischemia-reperfusion.

Topics: Animals; Brain Ischemia; Calcium-Binding Proteins; Calpain; Caspase 3; Caspase Inhibitors; Glycoproteins; Male; Microtubule-Associated Proteins; Models, Biological; Protease Inhibitors; Rats; Rats, Sprague-Dawley; Reperfusion; Spectrin

Microsphere embolism (ME)-induced cerebral ischemia can elicit various pathological events leading to neuronal death. Western blotting and immunohistochemical studies revealed that expression of calpastatin, an endogenous calpain inhibitor, decreased after ME induction. Calpain activation after ME was apparently due to, in part, a decrease in calpastatin in a late phase of neuronal injury. The time course of that decrease also paralleled caspase-3 activation. In vitro studies demonstrated that calpastatin was degraded by caspase-3 in a Ca(2+)/calmodulin (CaM)-dependent manner. Because CaM binds directly to calpastatin, we asked whether a novel CaM antagonist, 3-[2-[4-(3-chloro-2-methylphenylmethyl)-1-piperazinyl]ethyl]-5,6-dimethoxy-1-(4-imidazolylmethyl)-1H-indazole dihydro-chloride 3.5 hydrate (DY-9760e), inhibits caspase-3-induced calpastatin degradation during ME-induced neuronal damage. We also tested the effect of DY-9760e on degradation of fodrin, a calpain substrate. Consistent with our hypothesis, DY-9760e (25 or 50 mg/kg i.p.) treatment inhibited degradation of calpastatin and fodrin in a dose-dependent manner. Because DY-9760e showed powerful neuroprotective activity with concomitant inhibition of calpastatin degradation, cross-talk between calpain and caspase-3 through calpastatin possibly accounts for ME-induced neuronal injury. Taken together, both inhibition of caspase-3-induced calpastatin degradation and calpain-induced fodrin breakdown by DY-9760e in part mediate its neuroprotective action.

Topics: Animals; Brain Ischemia; Calcium-Binding Proteins; Calmodulin; Calpain; Caspase 3; Caspases; Disease Models, Animal; Dose-Response Relationship, Drug; Enzyme Activation; Indazoles; Intracranial Embolism; Male; Microspheres; Neurons; Neuroprotective Agents; Protein Binding; Rats; Rats, Wistar

In brain ischemia, gating of postsynaptic glutamate receptors and other membrane channels triggers intracellular Ca2+ overload and cell death. In excitotoxic settings, the initial Ca2+ influx through glutamate receptors is followed by a second uncontrolled Ca2+ increase that leads to neuronal demise. Here we report that the major plasma membrane Ca2+ extruding system, the Na+/Ca2+ exchanger (NCX), is cleaved during brain ischemia and in neurons undergoing excitotoxicity. Inhibition of Ca2+-activated proteases (calpains) by overexpressing their endogenous inhibitor protein, calpastatin or the expression of an NCX isoform not cleaved by calpains, prevented Ca2+ overload and rescued neurons from excitotoxic death. Conversely, down-regulation of NCX by siRNA compromised neuronal Ca2+ handling, transforming the Ca2+ transient elicited by non-excitotoxic glutamate concentrations into a lethal Ca2+overload. Thus, proteolytic inactivation of NCX-driven neuronal Ca2+ extrusion is responsible for the delayed excitotoxic Ca2+ deregulation and neuronal death.

Topics: Amino Acid Sequence; Animals; Brain Ischemia; Calcium; Calcium-Binding Proteins; Calpain; Cell Death; Cell Membrane; Cells, Cultured; Cysteine Proteinase Inhibitors; Ion Channel Gating; Membrane Transport Proteins; Molecular Sequence Data; Neurons; Rats; Rats, Wistar; Receptors, Glutamate; Sodium-Calcium Exchanger

Inappropriate imbalances between proteases and protease inhibitors are known to occur under cerebral ischemia and neurodegenerative processes, and could be contributors to various diseases that are characterized by excessive (ischemia, AIDS) or inadequate (cancer, autoimmunity) cell death. For instance, calpain is activated in various necrotic and apoptotic conditions, whereas caspase-3 is only activated in neuronal apoptosis. Caspases and calpains are cysteine proteases that require proteolytic cleavage for activation. The substrates cleaved by caspases include cytoskeletal and associated proteins, kinases, members of the Bcl-2 family of apoptosis-related proteins, presenilins, and DNA-modulating enzymes. Calpain substrates include cytoskeletal and associated proteins, kinases and phosphatases, membrane receptors and transporters, and steroid receptors. This article provides a review of the properties of caspases and calpains, their roles in cell death pathways following cerebral ischemia, and the substrates upon which they act. Because calpain inhibitors and caspase inhibitors appear to protect brain tissue by distinct mechanisms in cerebral ischemia, the possible therapeutic interactions between these drugs in a well-defined rodent model of global ischemia are briefly discussed and documented.

Topics: Animals; Apoptosis; Brain Ischemia; Calcium-Binding Proteins; Calpain; Caspases; Hippocampus; Necrosis; Neurons; Rats

In the CNS, where Ca(2+) overload has been established as a mechanism contributing to neuronal damage associated with excitotoxicity, stroke and ischemia, there is interest in understanding the role of calpain inhibition in rescuing neurons from death. In these settings, the activation of large stores of latent calpain may rapidly lead to the demise of the neuron within hours. The activity of calpain is strictly regulated by calcium concentrations and interactions with calpastatin (endogenous calpain inhibitor). The interaction between calpains and calpastatin is calcium dependent, and little is known about the regulation of the neuronal calpain-calpastatin system in vivo. It has been postulated that calpastatin can be modulated by nerve growth factors (NGFs). We have demonstrated in vitro as well as in vivo a neuroprotective effect of the beta(2)-adrenoceptor agonist clenbuterol (CLN) mediated through an increased NGF expression. In this study we attempt to find out whether CLN is capable (1) of modulating proteolysis regulated by the calpain-calpastatin system and (2) of attenuating DNA-fragmentation induced by cerebral ischemia. Rats received CLN daily for 1 week, were then subjected to ischemia and finally perfused at different times post-ischemia. The proteolytic activity of calpain was measured by the immunolocalisation of calpastatin and spectrin-breakdown products (SBP). The time course of apoptosis was assessed by terminal dUTP nick end-labeling (TUNEL)-staining. CLN reduced CA1-hippocampal cell damage by 23%, attenuated DNA-laddering and decreased proteolysis of spectrin by enhancing calpastatin activity. These results provide evidence that CLN is a potent neuroprotective substance, which through the enhancement of calpastatin synthesis attenuates the apoptotic machinery and modulates proteolysis.

Topics: Animals; Apoptosis; Blotting, Western; Brain Ischemia; Calcium-Binding Proteins; Calpain; Clenbuterol; Electrophoresis, Polyacrylamide Gel; Enzyme-Linked Immunosorbent Assay; Hippocampus; Immunohistochemistry; In Situ Nick-End Labeling; Male; Neurons; Neuroprotective Agents; Rats; Rats, Wistar; Receptors, Adrenergic, beta-3

In a model of cerebral hypoxia-ischemia in the immature rat, widespread brain injury is produced in the ipsilateral hemisphere, whereas the contralateral hemisphere is left undamaged. Previously, we found that calpains were equally translocated to cellular membranes (a prerequisite for protease activation) in the ipsilateral and contralateral hemispheres. However, activation, as judged by degradation of fodrin, occurred only in the ipsilateral hemisphere. In this study we demonstrate that calpastatin, the specific, endogenous inhibitor protein to calpain, is up-regulated in response to hypoxia and may be responsible for the halted calpain activation in the contralateral hemisphere. Concomitantly, extensive degradation of calpastatin occurred in the ipsilateral hemisphere, as demonstrated by the appearance of a membrane-bound 50-kDa calpastatin breakdown product. The calpastatin breakdown product accumulated in the synaptosomal fraction, displaying a peak 24 h post-insult, but was not detectable in the cytosolic fraction. The degradation of calpastatin was blocked by administration of CX295, a calpain inhibitor, indicating that calpastatin acts as a suicide substrate to calpain during hypoxia-ischemia. In summary, calpastatin was up-regulated in areas that remain undamaged and degraded in areas where excessive activation of calpains and infarction occurs.

Topics: Animals; Animals, Newborn; Brain; Brain Ischemia; Calcium-Binding Proteins; Calpain; Cell Membrane; Cysteine Proteinase Inhibitors; Dipeptides; Enzyme Activation; Female; Functional Laterality; Hypoxia; Male; Rats; Rats, Wistar; Up-Regulation

Calpains, Ca(2+)-dependent neutral proteinases (microM and mM Ca(2+)-sensitive), and their endogenous inhibitor calpastatin were examined in rat brain. Specific activity of m-calpain exceeded almost 10 times that of mu-calpain, and the both isoforms of calpain together with calpastatin were mainly located in the soluble fraction of homogenate. Acute postdecapitative ischemia of 15 min duration resulted in a gradual, time-dependent decrease of total mu-calpain activity (to 60% of control values) and in the moderate elevation of calpastatin activity (by 28%). The decrease of total mu-calpain activity coincided with its remarkable increase (above 300% of control values) in particulate fraction. In the case of m-calpain, the only observed effect of ischemia was its redistribution and, as a consequence, the elevation of activity in particulate fraction. The accumulation of breakdown products, resulting from calpain-catalyzed proteolysis of fodrin (as revealed by Western blotting) indicated activation of calpain under ischemia. The findings suggest that this rapid activation involves partial enzyme translocation toward membranes, and is followed (at least in acute phase) by mu-calpain downregulation and increased calpastatin activity.

Topics: Animals; Blotting, Western; Brain; Brain Ischemia; Calcium-Binding Proteins; Calpain; Carrier Proteins; Down-Regulation; Male; Membrane Proteins; Microfilament Proteins; Rats; Rats, Wistar

Based on our previous observation that transient forebrain ischemia induces calpain-catalyzed proteolysis in gerbil hippocampus in a region-specific manner, we examined the effect of ischemia on the quantity and localization of the endogenous calpain-specific inhibitor protein, calpastatin, in the tissue. Brief (5 min) forebrain ischemia followed by reperfusion induced an overall increase of calpastatin immunoreactivity in hippocampus, particularly in pyramidal cells, in 4 h as analyzed by Western blotting and immunohistochemistry. The amount of calpastatin, however, decreased to the preischemic level and lower in 24 h to 7 days due to proteolysis except in CA2 showing continuously elevated calpastatin immunoreactivity. Because calpastatin is not only a potent inhibitor but also a preferred substrate for calpain and because CA2 neurons are less vulnerable to ischemic stress than the adjacent CA1 neurons, these observations imply involvement of calpastatin in calpain regulation as a bait substrate and, possibly, in neuroprotection under ischemic conditions. Calpastatin may participate in the stress responses together with the previously known ischemia-induced stress proteins such as heat shock proteins.

Topics: Animals; Brain Ischemia; Calcium-Binding Proteins; Cysteine Proteinase Inhibitors; Down-Regulation; Female; Gerbillinae; Hippocampus; Immunohistochemistry; Up-Regulation

Calpains and calpastatin in the brain of the rabbit were examined in experimental situations that could mimic some features of brain ischemia. Incubations of bisected brains in saline at 39 degrees C for 0.5, 1, or 1.5 h resulted in a decreased calpain I activity in the cytosol and in an increased hydrophobicity of cytosolic calpain II activity. Incubation of brain homogenates at different pH levels demonstrated an almost-complete transfer of calpains from the cytoplasmic compartment to the membranes when pH was lowered from 6 to 5. At pH values lower than 5, the total calpain activity (soluble plus membrane-bound) markedly decreased. No significant changes of calpastatin activity or its subcellular distribution was found following incubation of the homogenates at different pH levels.

Topics: Animals; Brain; Brain Ischemia; Calcium-Binding Proteins; Calpain; Chromatography, High Pressure Liquid; Cytosol; Dithiothreitol; Electrophoresis, Polyacrylamide Gel; Hydrogen-Ion Concentration; In Vitro Techniques; Perfusion; Rabbits; Subcellular Fractions