sq-23377 and Infarction--Middle-Cerebral-Artery

Calpains are a family of calcium-dependent cysteine proteases that are ubiquitously expressed in mammals and play critical roles in neuronal death by catalyzing substrate proteolysis. Here, we developed two-dimensional gel electrophoresis-based protease proteomics to identify putative calpain substrates. To accomplish this, cellular lysates from neuronal cells were first separated by pI, and the immobilized sample on a gel strip was incubated with a recombinant calpain and separated by molecular weight. Among 25 altered protein spots that were differentially expressed by at least 2-fold, we confirmed that arsenical pump-driving ATPase, optineurin, and peripherin were cleaved by calpain using in vitro and in vivo cleavage assays. Furthermore, we found that all of these substrates were cleaved in MN9D cells treated with either ionomycin or 1-methyl-4-phenylpyridinium, both of which cause a calcium-mediated calpain activation. Their cleavage was blocked by calcium chelator or calpain inhibitors. In addition, calpain-mediated cleavage of these substrates and its inhibition by calpeptin were confirmed in a middle cerebral artery occlusion model of cerebral ischemia, as well as a stereotaxic brain injection model of Parkinson disease. Transient overexpression of each protein was shown to attenuate 1-methyl-4-phenylpyridinium-induced cell death, indicating that these substrates may confer protection of varying magnitudes against dopaminergic injury. Taken together, the data indicate that our protease proteomic method has the potential to be applicable for identifying proteolytic substrates affected by diverse proteases. Moreover, the results described here will help us decipher the molecular mechanisms underlying the progression of neurodegenerative disorders where protease activation is critically involved.

Topics: 1-Methyl-4-phenylpyridinium; Animals; Arsenite Transporting ATPases; Calpain; Cell Death; Cell Line; Dipeptides; Dopaminergic Neurons; Electrophoresis, Gel, Two-Dimensional; Glycine; Infarction, Middle Cerebral Artery; Ionomycin; Peripherins; Proteome; Proteomics; Rats; Rats, Sprague-Dawley

In the brain, the expression of the pleiotropic cytokine interleukin-6 (IL-6) is enhanced in various chronic or acute central nervous system disorders. However, the significance of IL-6 production in such neuropathologic states remains controversial. The present study investigated the role of IL-6 after cerebral ischemia. First, the authors showed that focal cerebral ischemia in rats early up-regulated the expression of IL-6 mRNA, without affecting the transcription of its receptors (IL-6Ralpha and gp130). Similarly, the striatal injection of N-methyl-D-aspartate (NMDA) in rats, a paradigm of excitotoxic injury, activated the expression of IL-6 mRNA. The involvement of glutamatergic receptor activation was further investigated by incubating cortical neurons with NMDA or alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA). NMDA and ionomycin (a calcium ionophore) up-regulated IL-6 mRNA, suggesting that neurons may produce IL-6 in response to the calcium influx mediated through NMDA receptors. The potential role of IL-6 during ischemic/excitotoxic insults was then studied by testing the effect of IL-6 against apoptotic or excitotoxic challenges in cortical cultures. IL-6 did not prevent serum deprivation- or staurosporine-induced apoptotic neuronal death, or AMPA/kainate-mediated excitotoxicity. However, in both mixed and pure neuronal cultures, IL-6 dose-dependently protected neurons against NMDA toxicity. This effect was blocked by a competitive inhibitor of IL-6. Overall, the results suggest that the up-regulation of IL-6 induced by cerebral ischemia could represent an endogenous neuroprotective mechanism against NMDA receptor-mediated injury.

Topics: Animals; Apoptosis; Astrocytes; Brain Chemistry; Cells, Cultured; Cerebral Cortex; Excitatory Amino Acid Agonists; Gene Expression; Infarction, Middle Cerebral Artery; Interleukin-6; Ionomycin; Ionophores; Ischemic Attack, Transient; Male; N-Methylaspartate; Neurons; Neuroprotective Agents; Neurotoxins; Polymerase Chain Reaction; Rats; Rats, Sprague-Dawley; Receptors, AMPA; Receptors, Interleukin-6; Receptors, Kainic Acid; Receptors, N-Methyl-D-Aspartate; RNA, Messenger; Transcription, Genetic