Abstract
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.
Highlights
Excitotoxicity triggers complex signal transduction events that induce the neuronal death program
The minimal essential region of growth arrest and DNA damageinducible 45β (GADD45β) that interacts with MKK7 is at residues 60–86, but another region seems to have a more marginal role to stabilize the interaction between GADD45β and MKK7 (Papa et al.15) (Figure 1a)
GADD45β interacts with MKK7 in proximity of its ATPbinding site[9] and this may justify the presence of acidic residues able to establish electrostatic interactions with the basic residues in this site.[9]
Summary
Excitotoxicity triggers complex signal transduction events that induce the neuronal death program. We linked the effector peptide to a TATcargo in order to penetrate neuronal plasma membrane.[17] The selected cell-permeable MKK7 inhibitor peptide (GADD45β-Ι) confers neuroprotection in vitro against NMDA and oxygen– glucose deprivation (OGD) toxicity, as well as in vivo in two models of MCAo with a clinically relevant post-ischemic temporal window (6 h) at both 24 h and 1 week after lesion. These data shed light on a new approach for the treatment of ischemia
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