Exploring the role of MKK7 in excitotoxicity and cerebral ischemia: a novel pharmacological strategy against brain injury.
Excitotoxicity following cerebral ischemia elicits a molecular cascade, which leads to neuronal death. c-Jun-N-terminal kinase (JNK) has a key role in excitotoxic cell death. We have previously shown that JNK inhibition by a specific cell-permeable peptide significantly reduces infarct size and neuronal death in an in vivo model of cerebral ischemia. However, systemic inhibition of JNK may have detrimental side effects, owing to blockade of its physiological function. Here we designed a new inhibitor peptide (growth arrest and DNA damage-inducible 45β (GADD45β-I)) targeting mitogen-activated protein kinase kinase 7 (MKK7), an upstream activator of JNK, which exclusively mediates JNK's pathological activation. GADD45β-I was engineered by optimizing the domain of the GADD45β, able to bind to MKK7, and by linking it to the TAT peptide sequence, to allow penetration of biological membranes. Our data clearly indicate that GADD45β-I significantly reduces neuronal death in excitotoxicity induced by either N-methyl-D-aspartate exposure or by oxygen-glucose deprivation in vitro. Moreover, GADD45β-I exerted neuroprotection in vivo in two models of ischemia, obtained by electrocoagulation and by thromboembolic occlusion of the middle cerebral artery (MCAo). Indeed, GADD45β-I reduced the infarct size when injected 30 min before the lesion in both models. The peptide was also effective when administrated 6 h after lesion, as demonstrated in the electrocoagulation model. The neuroprotective effect of GADD45β-I is long lasting; in fact, 1 week after MCAo the infarct volume was still reduced by 49%. Targeting MKK7 could represent a new therapeutic strategy for the treatment of ischemia and other pathologies involving MKK7/JNK activation. Moreover, this new inhibitor can be useful to further dissect the physiological and pathological role of the JNK pathway in the brain.
1] Department of Neuroscience, NICO - Neuroscience Institute Cavalieri Ottolenghi, Neuroscience Institute of Turin (NIT), University of Turin, Turin I-10125, Italy [2] National Institute of Neuroscience, Corso Raffaello 30, Turin, Italy.
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Vercelli
Neuronal Death and Neuroprotection Laboratory, IRCCS - Istituto di Ricerche Farmacologiche, 'Mario Negri', Via La Masa 19, Milan 20156 Italy.
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Biggi
Neuronal Death and Neuroprotection Laboratory, IRCCS - Istituto di Ricerche Farmacologiche, 'Mario Negri', Via La Masa 19, Milan 20156 Italy.
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Sclip
Department of Neuroscience, NICO - Neuroscience Institute Cavalieri Ottolenghi, Neuroscience Institute of Turin (NIT), University of Turin, Turin I-10125, Italy.
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Repetto
Neuronal Death and Neuroprotection Laboratory, IRCCS - Istituto di Ricerche Farmacologiche, 'Mario Negri', Via La Masa 19, Milan 20156 Italy.
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Cimini
Neuronal Death and Neuroprotection Laboratory, IRCCS - Istituto di Ricerche Farmacologiche, 'Mario Negri', Via La Masa 19, Milan 20156 Italy.
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Falleroni
Department of Neuroscience, NICO - Neuroscience Institute Cavalieri Ottolenghi, Neuroscience Institute of Turin (NIT), University of Turin, Turin I-10125, Italy.
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Tomasi
Department of Neuroscience, NICO - Neuroscience Institute Cavalieri Ottolenghi, Neuroscience Institute of Turin (NIT), University of Turin, Turin I-10125, Italy.
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Monti
Sanipedia, Via Ariosto 21, Bresso (MI) 20091, Italy.
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Tonna
Neuronal Death and Neuroprotection Laboratory, IRCCS - Istituto di Ricerche Farmacologiche, 'Mario Negri', Via La Masa 19, Milan 20156 Italy.
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Morelli
1] Department of Neuroscience, NICO - Neuroscience Institute Cavalieri Ottolenghi, Neuroscience Institute of Turin (NIT), University of Turin, Turin I-10125, Italy [2] Neuronal Death and Neuroprotection Laboratory, IRCCS - Istituto di Ricerche Farmacologiche, 'Mario Negri', Via La Masa 19, Milan 20156 Italy.
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Grande
Neuronal Death and Neuroprotection Laboratory, IRCCS - Istituto di Ricerche Farmacologiche, 'Mario Negri', Via La Masa 19, Milan 20156 Italy.
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Stravalaci
Neuronal Death and Neuroprotection Laboratory, IRCCS - Istituto di Ricerche Farmacologiche, 'Mario Negri', Via La Masa 19, Milan 20156 Italy.
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Biasini
Department of Biomedical Sciences, University of Padova, Padova 35121, Italy.
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Marin
1] Sanipedia, Via Ariosto 21, Bresso (MI) 20091, Italy [2] Fondazione Fernando Santarelli, Neuroinflammation Lab, Corso Venezia 18, Milan, Italy.
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Bianco
1] Department of Physics, Sapienza University of Rome, Rome, Italy [2] Department of Chemistry, Brooklyn College, City University of New York, Brooklyn, New York, USA.
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di Marino
1] Neuronal Death and Neuroprotection Laboratory, IRCCS - Istituto di Ricerche Farmacologiche, 'Mario Negri', Via La Masa 19, Milan 20156 Italy [2] Department of Pharmacological and Biomolecular Sciences, University of Milan, Milan, Italy.
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Borsello
D000595:Amino Acid Sequence; D000818:Animals; D000831:Animals, Newborn; D000943:Antigens, Differentiation / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D015687:Cell Hypoxia; D002540:Cerebral Cortex / Q000187:drug effects / Q000378:metabolism / Q000473:pathology; D004564:Electrocoagulation; D005786:Gene Expression Regulation; D005947:Glucose / Q000633:toxicity; D020244:Infarction, Middle Cerebral Artery / Q000188:drug therapy* / Q000235:genetics / Q000378:metabolism / Q000473:pathology; D048031:JNK Mitogen-Activated Protein Kinases / Q000235:genetics / Q000378:metabolism; D048688:MAP Kinase Kinase 7 / Q000037:antagonists & inhibitors* / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D008297:Male; D062105:Molecular Docking Simulation; D008969:Molecular Sequence Data; D016202:N-Methylaspartate / Q000633:toxicity; D009474:Neurons / Q000187:drug effects* / Q000378:metabolism / Q000473:pathology; D018696:Neuroprotective Agents / Q000138:chemical synthesis / Q000494:pharmacology*; D010455:Peptides / Q000138:chemical synthesis / Q000494:pharmacology*; D061251:Primary Cell Culture; D015202:Protein Engineering; D051381:Rats; D017207:Rats, Sprague-Dawley; D015398:Signal Transduction; D013923:Thromboembolism; D046509:Tissue Culture Techniques; D000097642:GADD45 Proteins
D000943:Antigens, Differentiation; D005947:Glucose; D048031:JNK Mitogen-Activated Protein Kinases; D048688:MAP Kinase Kinase 7; D016202:N-Methylaspartate; D018696:Neuroprotective Agents; D010455:Peptides; C490125:Gadd45b protein, mouse; D000097642:GADD45 Proteins; C482570:Map2k7 protein, mouse