c-Jun N-terminal kinase (JNK) is an important stress-responsive kinase that is activated by various forms of brain insults. In this study, we have examined the role of JNK activation in neuronal cell death in a murine model of focal ischemia and reperfusion; furthermore, we investigated the mechanism of JNK in apoptosis signaling, focusing on the mitochondrial-signaling pathway. We show here that JNK activity was induced in the brain 0.5 to 24 h after ischemia. Systemic administration of SP600125, a small molecule JNK-specific inhibitor, diminished JNK activity after ischemia and dose-dependently reduced infarct volume. c-Jun N-terminal kinase inhibition also attenuated ischemia-induced expression of Bim, Hrk/DP5, and Fas, but not the expression of Bcl-2 or FasL. In strong support of a role for JNK in promoting the mitochondrial apoptosis-signaling pathway, JNK inhibition prevented ischemia-induced mitochondrial translocation of Bax and Bim, release of cytochrome c and Smac, and activation of caspase-9 and caspase-3. The potential mechanism by which JNK promoted Bax translocation after ischemia was further studied using coimmunoprecipitation, and the results revealed that JNK activation caused serine phosphorylation of 14-3-3, a cytoplasmic sequestration protein of Bax, leading to Bax disassociation from 14-3-3 and subsequent translocation to mitochondria. These results confirm the role of JNK as a critical cell death mediator in ischemic brain injury, and suggest that one of the mechanisms by which JNK triggers the mitochondrial apoptosis-signaling pathway is via promoting Bax and Bim translocation.

Background and Purpose-Mitochondrial biogenesis is regulated through the coordinated actions of both nuclear and mitochondrial genomes to ensure that the organelles are replenished on a regular basis. This highly regulated process has been well defined in skeletal and heart muscle, but its role in neuronal cells, particularly when under stress or injury, is not well understood. In this study, we report for the first time rapidly increased mitochondrial biogenesis in a rat model of neonatal hypoxic/ischemic brain injury (H-I). Methods-Postnatal day 7 rats were subjected to H-I induced by unilateral carotid artery ligation followed by 2.5 hours of hypoxia. The relative amount of brain mitochondrial DNA (mtDNA) was measured semiquantitatively using long fragment PCR at various time points after H-I. HSP60 and COXIV proteins were detected by Western blot. Expression of three genes critical for the transcriptional regulation of mitochondrial biogenesis, peroxisome proliferator-activated receptor coactivator-1 (PGC-1), nuclear respiratory factor-1 (NRF-1), and mitochondrial transcription factor A (TFAM), were examined by Western blot and RT-PCR. Results-Brain mtDNA content was markedly increased 6 hours after H-I, and continued to increase up to 24 hours after H-I. Paralleling the temporal change in mtDNA content, mitochondrial number and proteins HSP60 and COXIV, and citrate synthase activity were increased in neurons in the cortical infarct border zone after H-I. Moreover, cortical expression of NRF-1 and TFAM were increased 6 to 24 hours after H-I, whereas PGC-1 was not changed. Conclusions-Neonatal

Peroxisome proliferator-activated receptor gamma (PPAR-c) is a nuclear membrane-associated transcription factor that governs the expression of various inflammatory genes. PPAR-c agonists protect peripheral organs from ischemic injury. In the present study, we investigated whether the PPAR-c agonist rosiglitazone is neuroprotective against focal ischemic brain injury. C57/B6 mice underwent 1.5-h middle cerebral artery occlusion, and received either vehicle or rosiglitazone treatment of 0.75, 1.5, 3, 6 or 12 mg/kg (n ¼ 9 per group). Cerebral infarct volume, neurological function, expression of proinflammatory proteins and neutrophil accumulation were assessed after ischemia and reperfusion. At 48 h after ischemia, infarct volume was significantly decreased with 3-12 mg/kg of rosiglitazone, with a time window of efficacy of 2 h after ischemia at the optimal dose (6 mg/kg). Neutrophil accumulation was significantly decreased in the brain parenchyma of rosiglitazone-treated mice. Ischemia-induced expression of several inflammatory cytokines and chemokines was markedly reduced in rosiglitazone-treated brains, as determined using proteomic-array analysis. Rosiglitazone treatment improved neurological function at 7 days after ischemia. Moreover, in cultured cortical primary microglia, rosiglitazone attenuated inflammatory responses by decreasing lipopolysaccharide-induced release of tumor necrosis factor-a, interleukin (IL)-1b and IL-6. These results suggest that the PPAR-c agonist rosiglitazone has neuroprotective properties that are at least partially mediated via anti-inflammatory actions, and is thus a potential novel therapeutic agent for stroke.

Background and Purpose-Erythropoietin (EPO) has been well characterized and shown to improve functional outcomes after ischemic injury, but EPO may also have unexplored effects on neurovascular remodeling and neuronal replacement in the neonatal ischemic brain. The current study investigates the effects of exogenous administration of EPO on revascularization and neurogenesis, 2 major events thought to contribute to neuronal replacement, in the neonatal brain after hypoxia/ischemia (H/I). Methods-Seven-day-old rat pups were treated with recombinant human EPO or vehicle 20 minutes after H/I and again on postischemic days 2, 4, and 6. Rats were euthanized 7 or 28 days after H/I for evaluation of infarct volume, revascularization, neurogenesis, and neuronal replacement using bromodeoxyuridine incorporation, immunohistochemistry, and lectin labeling. Neurological function was assessed progressively for 28 days after H/I by gait testing, righting reflex and foot fault testing. Results-We demonstrate that exogenous EPO-enhanced revascularization in the ischemic hemisphere correlated with decreased infarct volume and improved neurological outcomes after H/I. In addition to vascular effects, EPO increased both neurogenesis in the subventricular zone and migration of neuronal progenitors into the ischemic cortex and striatum. A significant number of newly synthesized cells in the ischemic boundary expressed neuronal nuclei after EPO treatment, indicating that exogenous EPO led to neuronal replacement. Conclusions-Our data suggest that treatment with EPO contributes to neurovascular remodeling after H/I by promoting tissue protection, revascularization, and neurogenesis in neonatal H/I-injured brain, leading to improved neurobehavioral outcomes.