d-jnki-1 and Brain-Ischemia

Neonatal encephalopathy is associated with high mortality and life-long developmental consequences. Therapeutic options are very limited. We assessed the effects of D-JNKi, a small peptide c-Jun N-terminal kinase (JNK) MAP kinase inhibitor, on neuroinflammation, mitochondrial integrity and neuronal damage in a neonatal rat model of ischemic brain damage. Hypoxic-ischemic (HI) brain injury was induced in postnatal-day 7 rats by unilateral carotid artery occlusion and hypoxia, and was followed by intraperitoneal D-JNKi treatment. We demonstrate here for the first time that a single intraperitoneal injection with D-JNKi directly after HI strongly reduces neonatal brain damage by >85% with a therapeutic window of at least 6h. D-JNKi treatment also restored cognitive and motor function as analyzed at 9weeks post-insult. Neuroprotective D-JNKi treatment inhibited phosphorylation of nuclear c-Jun (P-c-Jun), and consequently reduced activity of the AP-1 transcription factor and production of cerebral cytokines/chemokines as determined at 3 and 24h post-HI. Inhibition of P-c-Jun by D-JNKi is thought to be mediated via inhibition of the upstream phosphorylation of cytosolic and nuclear JNK and/or by preventing the direct interaction of phosphorylated (P-)JNK with c-Jun. Surprisingly, however, HI did not induce a detectable increase in P-JNK in cytosol or nucleus. Notably, we show here for the first time that HI induces P-JNK only in the mitochondrial fraction, which was completely prevented by D-JNKi treatment. The hypothesis that mitochondrial JNK activation is key to HI brain injury was supported by data showing that treatment of rat pups with SabKIM1 peptide, a specific mitochondrial JNK inhibitor, is also neuroprotective. Inhibition of HI-induced mitochondrial JNK activation was associated with preservation of mitochondrial integrity as evidenced by prevention of ATP loss and inhibition of lipid peroxidation. The HI-induced increase in apoptotic markers (cytochrome c release and caspase 3 activation) as analyzed at 24h post-HI were also strongly reduced by D-JNKi and the mitochondrial anti-apoptotic proteins Bcl-2 and Bcl-xL were upregulated. Neuroprotection was lost after repeated 0+3h D-JNKi treatment which was associated with complete inhibition of the second peak of AP-1 activity and disability to upregulate mitochondrial Bcl-2 and Bcl-xL. We show here for the first time that D-JNKi treatment efficiently protects the neonatal brain against ischemic brain da

Topics: Animals; Animals, Newborn; Apoptosis; Blotting, Western; Brain Ischemia; Disease Models, Animal; Electrophoretic Mobility Shift Assay; Enzyme Inhibitors; Inflammation; MAP Kinase Kinase 4; Mitochondria; Neuroprotective Agents; Oxidative Stress; Peptides; Phosphorylation; Rats; Rats, Wistar; Real-Time Polymerase Chain Reaction; Reverse Transcriptase Polymerase Chain Reaction; Signal Transduction

Cerebral ischemia is associated with the activation of glial cells, infiltration of leukocytes and an increase in inflammatory mediators in the ischemic brain and systemic circulation. How this inflammatory response influences lesion size and neurological outcome remains unclear. D-JNKI1, an inhibitor of the c-Jun N-terminal kinase pathway, is strongly neuroprotective in animal models of stroke. Intriguingly, the protection mediated by D-JNKI1 is high even with intravenous administration at very low doses with undetectable drug levels in the brain, pointing to a systemic mode of action, perhaps on inflammation.. We evaluated whether D-JNKI1, administered intravenously 3 h after the onset of middle cerebral artery occlusion (MCAO), modulates secretion of the inflammatory mediators interleukin-6 and keratinocyte-derived chemokine in the plasma and from the spleen and brain at several time points after MCAO. We found an early release of both mediators in the systemic circulation followed by an increase in the brain and went on to show a later systemic increase in vehicle-treated mice. Release of interleukin-6 and keratinocyte-derived chemokine from the spleen of mice with MCAO was not significantly different from sham mice. Interestingly, the secretion of these inflammatory mediators was not altered in the systemic circulation or brain after successful neuroprotection with D-JNKI1.. We demonstrate that neuroprotection with D-JNKI1 after experimental cerebral ischemia is independent of systemic and brain release of interleukin-6 and keratinocyte-derived chemokine. Furthermore, our findings suggest that the early systemic release of interleukin-6 and keratinocyte-derived chemokine may not necessarily predict an unfavorable outcome in this model.

Topics: Animals; Brain Ischemia; Chemokine CXCL1; Chemokines; Interleukin-6; JNK Mitogen-Activated Protein Kinases; Male; MAP Kinase Signaling System; Mice; Mice, Inbred ICR; Neuroprotective Agents; Peptides; Predictive Value of Tests; Random Allocation

Both hyperbaric oxygenation (HBO) and inhibition of the c-Jun N-terminal kinases (JNKs) by the peptide inhibitor XG-102 (D-JNKI-1) are efficient protective strategies against ischaemia-induced neurodegeneration. The present study investigated whether the combination of HBO and JNK inhibitor, XG-102, provides additive neuroprotection against cerebral ischaemia.. Rat middle cerebral artery was occluded (MCAO) for 90 min. XG-102 [2 mg/kg, intraperitoneally] or HBO (3 ATA, 60 min) was applied 3 h after the onset of MCAO. For the combination treatment, HBO was started 10 min after the injection of XG-102. Twenty-four hours after MCAO, the infarct area, the neurological score and the immunohistochemistry staining in brain slices for cleaved-PARP, transferase-mediated biotinylated UTP nick end labelling, c-Jun and phosphorylated (activated) c-Jun were observed.. XG-102 or HBO alone reduced the total infarct area by 43% and 63%, respectively. The combination diminished total infarct area by 78%, improved the neurological function and reduced brain oedema. Co-application of HBO and XG-102 also significantly reduced the cleavage of PARP, by 96% and 91% in cortical penumbra and ischaemic core, respectively. Moreover, cotreatment significantly attenuated the number of cells labelled with transferase-mediated biotinylated UTP nick end labelling and phosphorylated c-Jun.. Our study demonstrates that HBO reinforces the efficiency of neuroprotective drugs such as XG-102 and vice versa. Both treatments, physical HBO and pharmacological XG-102, are already in phase I/II studies and promising strategies for clinical use.

Topics: Aging; Animals; Brain; Brain Edema; Brain Ischemia; Enzyme Inhibitors; Hyperbaric Oxygenation; Infarction, Middle Cerebral Artery; JNK Mitogen-Activated Protein Kinases; Male; Neuroprotective Agents; Peptides; Phosphorylation; Proto-Oncogene Proteins c-jun; Random Allocation; Rats; Rats, Sprague-Dawley; Time Factors

The c-Jun-N-terminal kinase signaling pathway (JNK) is highly activated during ischemia and plays an important role in apoptosis and inflammation. We have previously demonstrated that D-JNKI1, a specific JNK inhibitor, is strongly neuroprotective in animal models of stroke. We presently evaluated if D-JNKI1 modulates post-ischemic inflammation such as the activation and accumulation of microglial cells. Outbred CD1 mice were subjected to 45 min middle cerebral artery occlusion (MCAo). D-JNKI1 (0.1 mg/kg) or vehicle (saline) was administered intravenously 3 h after MCAo onset. Lesion size at 48 h was significantly reduced, from 28.2+/-8.5 mm(3) (n=7) to 13.9+/-6.2 mm(3) in the treated group (n=6). Activation of the JNK pathway (phosphorylation of c-Jun) was observed in neurons as well as in Isolectin B4 positive microglia. We quantified activated microglia (CD11b) by measuring the average intensity of CD11b labelling (infra-red emission) within the ischemic tissue. No significant difference was found between groups. Cerebral ischemia was modelled in vitro by subjecting rat organotypic hippocampal slice cultures to oxygen (5%) and glucose deprivation for 30 min. In vitro, D-JNKI1 was found predominantly in NeuN positive neurons of the CA1 region and in few Isolectin B4 positive microglia. Furthermore, 48 h after OGD, microglia were activated whereas resting microglia were found in controls and in D-JNKI1-treated slices. Our study shows that D-JNKI1 reduces the infarct volume 48 h after transient MCAo and does not act on the activation and accumulation of microglia at this time point. In contrast, in vitro data show an indirect effect of D-JNKI1 on the modulation of microglial activation.

Topics: Animals; Brain Ischemia; Hippocampus; Immunohistochemistry; Inflammation; JNK Mitogen-Activated Protein Kinases; Male; Mice; Microglia; Neurons; Organ Culture Techniques; Peptides; Phosphorylation; Rats; Rats, Sprague-Dawley

Inhibition of the c-Jun N-terminal kinase (JNK) pathway by the TAT-coupled peptide XG-102 (formerly D- JNKI1) induces strong neuroprotection in ischemic stroke in rodents. We investigated the effect of JNK inhibition in intracerebral hemorrhage (ICH).. Three hours after induction of ICH by intrastriatal collagenase injection in mice, the animals received an intravenous injection of 100 microg/kg of XG-102. The neurological outcome was assessed daily and the mice were sacrificed at 6 h, 1, 2 or 5 days after ICH.. XG-102 administration significantly improved the neurological outcome at 1 day (p < 0.01). The lesion volume was significantly decreased after 2 days (29 +/- 11 vs. 39 +/- 5 mm(3) in vehicle-treated animals, p < 0.05). There was also a decreased hemispheric swelling (14 +/- 13 vs. 26 +/- 9% in vehicle-treated animals, p < 0.05) correlating with increased aquaporin 4 expression.. XG-102 attenuates cerebral edema in ICH and functional impairment at early time points. The beneficial effects observed with XG-102 in ICH, as well as in ischemic stroke, open the possibility to rapidly treat stroke patients before imaging, thereby saving precious time.

Topics: Animals; Aquaporin 4; Behavior, Animal; Brain; Brain Edema; Brain Ischemia; Cerebral Hemorrhage; Collagenases; Enzyme Activation; Immunohistochemistry; JNK Mitogen-Activated Protein Kinases; Macrophage Activation; Mice; Mice, Inbred ICR; Neuroglia; Peptides; Postural Balance; Protein Kinase Inhibitors; Treatment Outcome

XG-102 (formerly D-JNKI1), a TAT-coupled dextrogyre peptide which selectively inhibits the c-Jun N-terminal kinase, is a powerful neuroprotectant in mouse models of middle cerebral artery occlusion (MCAo) with delayed intracerebroventricular injection. We aimed to determine whether this neuroprotection could also be achieved by intravenous injection of XG-102, which is a more feasible approach for future use in stroke patients. We also tested the compatibility of the compound with recombinant tissue plasminogen activator (rtPA), commonly used for intravenous thrombolysis and known to enhance excitotoxicity.. Male ICR-CD1 mice were subjected to a 30-min-suture MCAo. XG-102 was injected intravenously in a single dose, 6 h after ischemia. Hippocampal slice cultures were subjected to oxygen (5%) and glucose (1 mM) deprivation for 30 min. rtPA was added after ischemia and before XG-102 administration, both in vitro and in vivo.. The lowest intravenous dose achieving neuroprotection was 0.0003 mg/kg, which reduced the infarct volume after 48 h from 62 +/- 19 mm(3) (n = 18) for the vehicle-treated group to 18 +/- 9 mm(3) (n = 5, p < 0.01). The behavioral outcome was also significantly improved at two doses. Addition of rtPA after ischemia enhanced the ischemic damage both in vitro and in vivo, but XG-102 was still able to induce a significant neuroprotection.. A single intravenous administration of XG-102 several hours after ischemia induces a powerful neuroprotection. XG-102 protects from ischemic damage in the presence of rtPA. The feasibility of systemic administration of this promising compound and its compatibility with rtPA are important steps for its development as a drug candidate in ischemic stroke.

Topics: Animals; Brain Ischemia; Drug Therapy, Combination; Fibrinolytic Agents; Hippocampus; Infarction, Middle Cerebral Artery; JNK Mitogen-Activated Protein Kinases; Male; Mice; Mice, Inbred ICR; Organ Culture Techniques; Peptides; Rats; Recombinant Proteins; Tissue Plasminogen Activator

In 2 models of severe ischemic injury, we have evaluated the neuroprotective action of D-JNKI1, a cell-penetrating and protease-resistant peptide selectively inhibiting the c-Jun-N-terminal kinase (JNK).. Hippocampal slices from newborn rats were subjected to oxygen (5%) and glucose (1 mmol/L) deprivation for 30 minutes. Cell death was evaluated with propidium iodide, and the evoked potential responses were recorded in the CA1 region after stimulation in CA3. Male ICR-CD1 mice were subjected to permanent endoluminal "suture" middle cerebral artery occlusion (MCAo). The lesion size was determined after 24 hours by triphenyl-tetrazolium chloride staining, and neurological scores and rotarod treadmill performance were used to evaluate the neurological outcome.. In vitro, D-JNKI administration 6 hours after oxygen glucose deprivation reduced cell death at 24 hours from 21%+/-8% (n=10) to 5%+/-3% (n=7, P<0.01). This protective effect was still seen at 48 hours, paralleled by an improved amplitude of the evoked potential response. In vivo in the mouse, D-JNKI1 administration 3 hours after ischemia significantly reduced the infarct volume from 162+/-27 mm(3) (n=14) to 85+/-27 mm(3) (n=9, P<0.001). The functional outcome was also improved.. JNK inhibition prevents cell death induced by oxygen and glucose deprivation in hippocampal slice cultures in vitro and by permanent suture MCAo in vivo. D-JNKI1 is a powerful neuroprotectant in models of both mild and severe cerebral ischemia, with an extended therapeutic window. Further investigations are needed to identify the relevant JNK target(s) mediating ischemic neuronal death.

Topics: Animals; Animals, Newborn; Brain Ischemia; Cell Death; Culture Media; Glucose; Hippocampus; In Vitro Techniques; Infarction, Middle Cerebral Artery; Male; Mice; Models, Animal; Neuroprotective Agents; Oxygen; Peptides; Psychomotor Performance; Rats