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.

Heat shock protein 27 (Hsp27), a recently discovered member of the heat shock protein family, is markedly induced in the brain after cerebral ischemia and other injury states. In non-neuronal systems, Hsp27 has potent cell death-suppressing functions. However, the mechanism of Hsp27-mediated neuroprotection has not yet been elucidated. Using transgenic and viral overexpression of Hsp27, we investigated the molecular mechanism by which Hsp27 exerts its neuroprotective effect. Overexpression of Hsp27 conferred long-lasting tissue preservation and neurobehavioral recovery, as measured by infarct volume, sensorimotor function, and cognitive tasks up to 3 weeks following focal cerebral ischemia. Examination of signaling pathways critical to neuronal death demonstrated that Hsp27 overexpression led to the suppression of the MKK4/JNK kinase cascade. While Hsp27 overexpression did not suppress activation of an upstream regulatory kinase of the MKK/JNK cascade, ASK1, Hsp27 effectively inhibited ASK1 activity via a physical association through its N-terminal domain and the kinase domain of ASK1. The N-terminal region of Hsp27 was required for neuroprotective function against in vitro ischemia. Moreover, knockdown of ASK1 or inhibition of the ASK1/MKK4 cascade effectively inhibited cell death following neuronal ischemia. This underscores the importance of this kinase cascade in the progression of ischemic neuronal death. Inhibition of PI3K had no effect on Hsp27-mediated neuroprotection, suggesting that Hsp27 does not promote cell survival via activation of PI3K/Akt. Based on these findings, we conclude that overexpression of Hsp27 confers long-lasting neuroprotection against ischemic brain injury via a previously unexplored association and inhibition of ASK1 kinase signaling.

Background and Purpose-Leptin is the major adipose hormone that regulates body weight and energy expenditure by activating leptin receptors in the hypothalamus. Leptin receptors are also present in other cell types, and a potent antiapoptotic effect for leptin has recently been reported. We investigated whether leptin was neuroprotective against ischemic brain injury. Methods-In vitro ischemic injury was induced in rat primary neuronal culture by oxygen-glucose deprivation for 90 minutes. In vivo ischemic brain injury was induced by middle cerebral artery occlusion in mice for 60 minutes. Results-Leptin receptors were detected in cultured rat cortical neurons, as well as in the mouse cortex, striatum, and hippocampus. In vitro results showed that leptin, 50 to 100 g/mL, protected primary cortical neurons against death induced by oxygen-glucose deprivation in a concentration-dependent manner. In vivo studies in the mouse brain demonstrated that the intraperitoneal administration of leptin, 2 to 8 mg/kg, dose-dependently reduced infarct volume induced by middle cerebral artery occlusion. Leptin was effective when injected 5 minutes before or 30 to 90 minutes after reperfusion, but not 2 hours after reperfusion. Leptin improved animal body weight recovery and behavioral parameters after cerebral ischemia. Leptin enhanced the phosphorylation of extracellular signal-related kinase 1/2. Both extracellular signal-related kinase 1/2 activation and neuroprotection were abolished by the administration of PD98059 in vitro and in vivo. Conclusions-Leptin is neuroprotective against ischemic neuronal injury. Our findings suggest that leptin is a legitimate candidate for the treatment of ischemic stroke.

The death of midbrain dopaminergic neurons in sporadic Parkinson disease is of unknown etiology but may involve altered growth factor signaling. The present study showed that leptin, a centrally acting hormone secreted by adipocytes, rescued dopaminergic neurons, reversed behavioral asymmetry, and restored striatal catecholamine levels in the unilateral 6-hydroxydopamine (6-OHDA) mouse model of dopaminergic cell death. In vitro studies using the murine dopaminergic cell line MN9D showed that leptin attenuated 6-OHDA-induced apoptotic markers, including caspase-9 and caspase-3 activation, internucleosomal DNA fragmentation, and cytochrome c release. ERK1/2 phosphorylation (pERK1/2) was found to be critical for mediating leptin-induced neuroprotection, because inhibition of the MEK pathway blocked both the pERK1/2 response and the pro-survival effect of leptin in cultures. Knockdown of the downstream messengers JAK2 or GRB2 precluded leptin-induced pERK1/2 activation and neuroprotection. Leptin/pERK1/2 signaling involved phosphorylation and nuclear localization of CREB (pCREB), a well known survival factor for dopaminergic neurons. Leptin induced a marked MEK-dependent increase in pCREB that was essential for neuroprotection following 6-OHDA toxicity. Transfection of a dominant negative MEK protein abolished leptin-enhanced pCREB formation, whereas a dominant negative CREB or decoy oligonucleotide diminished both pCREB binding to its target DNA sequence and MN9D survival against 6-OHDA toxicity. Moreover, in the substantia nigra of mice, leptin treatment increased the levels of pERK1/2, pCREB, and the downstream gene product BDNF, which were reversed by the MEK inhibitor PD98059. Collectively, these data provide evidence that leptin prevents the degeneration of dopaminergic neurons by 6-OHDA and may prove useful in the treatment of Parkinson disease.

6-Hydroxydopamine (6-OHDA)-induced loss of dopamine (DA) neurons has served to produce an animal model of DA neuron loss in Parkinson's disease. We report here the use of 6-OHDA to produce an in vitro model of this phenomena using dissociated cultures prepared from neonatal rat mesencephalon. Cultures were exposed to 6-OHDA (40-100 lM, 15 min) in an antioxidant medium, and DA and GABA neurons evaluated by immunocytochemistry. 6-OHDA induced morphological and biochemical signs of cell death in DA neurons within 3 h, followed by loss of tyrosine hydroxylase immunoreactive neurons within 2 days. In substantia nigra (SN) cultures, DA neurons were much more affected by 6-OHDA than were GABA neurons. In contrast, DA neurons from the ventral tegmental area were only lost at higher, non-specific concentrations of 6-OHDA. The effects of 6-OHDA on nigral DA neurons were blocked by inhibitors of high affinity DA transport and by z-DEVD-fmk (150 lM), a caspase inhibitor. Glial cell line-derived neurotrophic factor (GDNF) treatment reduced TUNEL labeling 3 h after 6-OHDA exposure, but did not prevent loss of DA neurons at 48 h. Thus, 6-OHDA can selectively destroy DA neurons in post-natal cultures of SN, acting at least in part by initiating caspase-dependent apoptosis, and this effect can be attenuated early but not late by GDNF.

Background and Purpose-NADϩ is an essential cofactor for cellular energy production and participates in various signaling pathways that have an impact on cell survival. After cerebral ischemia, oxidative DNA lesions accumulate in neurons because of increased attacks by ROS and diminished DNA repair activity, leading to PARP-1 activation, NAD