title: Mitochondrial JNK phosphorylation as a novel therapeutic target to inhibit neuroinflammation and apoptosis after neonatal ischemic brain damage.
abstract: 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 damage and subsequent cognitive and motor impairment. We propose that inhibition of phosphorylation of mitochondrial JNK is a pivotal step in preventing early loss of mitochondrial integrity leading to reduced neuroinflammation and inhibition of apoptotic neuronal loss. Moreover we show the crucial role of upregulation of mitochondrial anti-apoptotic proteins to maintain neuroprotection.
authors: Laboratory of Neuroimmunology and Developmental Origins of Disease (NIDOD), University Medical Center Utrecht, Utrecht, The Netherlands. C.Nijboer@umcutrecht.nl
authors: Cora H
authors: CH
authors: Nijboer
authors: Hilde J C
authors: HJ
authors: Bonestroo
authors: Jitske
authors: J
authors: Zijlstra
authors: Annemieke
authors: A
authors: Kavelaars
authors: Cobi J
authors: CJ
authors: Heijnen
mesh_terms: D000818:Animals; D000831:Animals, Newborn; D017209:Apoptosis / Q000187:drug effects; D015153:Blotting, Western; D002545:Brain Ischemia / Q000150:complications / Q000378:metabolism* / Q000503:physiopathology*; D004195:Disease Models, Animal; D024202:Electrophoretic Mobility Shift Assay; D004791:Enzyme Inhibitors / Q000494:pharmacology; D007249:Inflammation / Q000209:etiology / Q000378:metabolism; D048670:MAP Kinase Kinase 4 / Q000037:antagonists & inhibitors*; D008928:Mitochondria / Q000187:drug effects / Q000378:metabolism*; D018696:Neuroprotective Agents / Q000494:pharmacology*; D018384:Oxidative Stress / Q000187:drug effects; D010455:Peptides / Q000494:pharmacology*; D010766:Phosphorylation; D051381:Rats; D017208:Rats, Wistar; D060888:Real-Time Polymerase Chain Reaction; D020133:Reverse Transcriptase Polymerase Chain Reaction; D015398:Signal Transduction / Q000187:drug effects
chemical_list: D004791:Enzyme Inhibitors; D018696:Neuroprotective Agents; D010455:Peptides; D048670:MAP Kinase Kinase 4; C478085:D-JNKI-1