title: Neuroprotective effects of MAPK/ERK1/2 and calpain inhibitors on lactacystin-induced cell damage in primary cortical neurons.
abstract: The dysfunction of the proteasome system is implicated in the pathomechanism of several chronic neurodegenerative diseases. Lactacystin (LC), an irreversible proteasome inhibitor, induces cell death in primary cortical neurons, however, the molecular mechanisms of its neurotoxic action has been only partially unraveled. In this study we aimed to elucidate an involvement of the key enzymatic pathways responsible for LC-induced neuronal cell death. Incubation of primary cortical neurons with LC (0.25-50 μg/ml) evoked neuronal cell death in concentration- and time-dependent manner. Lactacystin (2.5 μg/ml; 6.6μM) enhanced caspase-3 activity, but caspase-3 inhibitor, Ac-DEVD-CHO did not attenuate the LC-evoked cell damage. Western blot analysis showed a time-dependent, prolonged activation of MAPK/ERK1/2 pathway after LC exposure. Moreover, inhibitors of MAPK/ERK1/2 signaling, U0126 and PD98052 attenuated the LC-evoked cell death. We also found that LC-treatment resulted in the induction of calpains and calpain inhibitors (MDL28170 and calpeptin) protected neurons against the LC-induced cell damage. Neuroprotective action of MAPK/ERK1/2 and calpain inhibitors were connected with attenuation of LC-induced DNA fragmentation measured by Hoechst 33342 staining and TUNEL assay. However, only MAPK/ERK1/2 but not calpain inhibitors, attenuated the LC-induced AIF (apoptosis inducing factor) release. Further studies showed no synergy between neuroprotective effects of MAPK/ERK1/2 and calpain inhibitors given in combination when compared to their effects alone. The obtained data provided evidence for neuroprotective potency of MAPK/ERK1/2 and calpain, but not caspase-3 inhibition against the neurotoxic effects of LC in primary cortical neurons and give rationale for using these inhibitors in the treatment of neurodegenerative diseases connected with proteasome dysfunction.
authors.affiliation: Department of Experimental Neuroendocrinology, Institute of Pharmacology, Polish Academy of Sciences, Smetna 12, PL 31-343 Krakow, Poland. jantas@if-pan.krakow.pl
authors.forename: D
authors.initials: D
authors.lastname: Jantas
authors.forename: E
authors.initials: E
authors.lastname: Lorenc-Koci
authors.forename: M
authors.initials: M
authors.lastname: Kubera
authors.forename: W
authors.initials: W
authors.lastname: Lason
mesh_terms: D000111:Acetylcysteine / Q000031:analogs & derivatives* / Q000633:toxicity; D000818:Animals; D017209:Apoptosis / Q000187:drug effects; D015153:Blotting, Western; D002070:Butadienes / Q000494:pharmacology; D002154:Calpain / Q000037:antagonists & inhibitors* / Q000378:metabolism; D053148:Caspase 3 / Q000378:metabolism; D061945:Caspase Inhibitors; D002478:Cells, Cultured; D002540:Cerebral Cortex / Q000187:drug effects* / Q000196:embryology / Q000201:enzymology / Q000473:pathology; D015853:Cysteine Proteinase Inhibitors / Q000633:toxicity*; D019610:Cytoprotection; D004151:Dipeptides / Q000494:pharmacology; D004305:Dose-Response Relationship, Drug; D004789:Enzyme Activation; D005865:Gestational Age; D020287:In Situ Nick-End Labeling; D051379:Mice; D019950:Mitogen-Activated Protein Kinase 1 / Q000037:antagonists & inhibitors* / Q000378:metabolism; D048052:Mitogen-Activated Protein Kinase 3 / Q000037:antagonists & inhibitors* / Q000378:metabolism; D009474:Neurons / Q000187:drug effects* / Q000201:enzymology / Q000473:pathology; D018696:Neuroprotective Agents / Q000494:pharmacology*; D009570:Nitriles / Q000494:pharmacology; D046988:Proteasome Endopeptidase Complex / Q000378:metabolism; D061988:Proteasome Inhibitors; D047428:Protein Kinase Inhibitors / Q000494:pharmacology*; D015398:Signal Transduction / Q000187:drug effects; D013049:Spectrin / Q000378:metabolism; D013997:Time Factors
chemical_list: D002070:Butadienes; D061945:Caspase Inhibitors; D015853:Cysteine Proteinase Inhibitors; D004151:Dipeptides; D018696:Neuroprotective Agents; D009570:Nitriles; D061988:Proteasome Inhibitors; D047428:Protein Kinase Inhibitors; C113580:U 0126; D013049:Spectrin; C067713:lactacystin; C071482:calpeptin; D019950:Mitogen-Activated Protein Kinase 1; D048052:Mitogen-Activated Protein Kinase 3; D002154:Calpain; C505409:Casp3 protein, mouse; D053148:Caspase 3; D046988:Proteasome Endopeptidase Complex; C058076:calpain inhibitor III; D000111:Acetylcysteine