Suping Wang

Neuroprotection against focal ischemic brain injury by the peroxisome proliferator-activated receptor-γ agonist rosiglitazone

Peroxisome proliferator‐activated receptor gamma (PPAR‐γ) is a nuclear membrane‐associated transcription factor that governs the expression of various inflammatory genes. PPAR‐γ agonists protect peripheral organs from ischemic injury. In the present study, we investigated whether the PPAR‐γ 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 = 9 per group). Cerebral infarct volume, neurological function, expression of pro‐inflammatory 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‐α, interleukin (IL)‐1β and IL‐6. These results suggest that the PPAR‐γ 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.

Neuroprotective Effects of Leptin Against Ischemic Injury Induced by Oxygen-Glucose Deprivation and Transient Cerebral Ischemia

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 &mgr;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.

Leptin Protects against 6-Hydroxydopamine-induced Dopaminergic Cell Death via Mitogen-activated Protein Kinase Signaling

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.

Protective effects and mechanisms of sirtuins in the nervous system

Silent information regulator two proteins (sirtuins or SIRTs) are a group of histone deacetylases whose activities are dependent on and regulated by nicotinamide adenine dinucleotide (NAD(+)). They suppress genome-wide transcription, yet upregulate a select set of proteins related to energy metabolism and pro-survival mechanisms, and therefore play a key role in the longevity effects elicited by calorie restriction. Recently, a neuroprotective effect of sirtuins has been reported for both acute and chronic neurological diseases. The focus of this review is to summarize the latest progress regarding the protective effects of sirtuins, with a focus on SIRT1. We first introduce the distribution of sirtuins in the brain and how their expression and activity are regulated. We then highlight their protective effects against common neurological disorders, such as cerebral ischemia, axonal injury, Alzheimers disease, Parkinsons disease, amyotrophic lateral sclerosis, and multiple sclerosis. Finally, we analyze the mechanisms underlying sirtuin-mediated neuroprotection, centering on their non-histone substrates such as DNA repair enzymes, protein kinases, transcription factors, and coactivators. Collectively, the information compiled here will serve as a comprehensive reference for the actions of sirtuins in the nervous system to date, and will hopefully help to design further experimental research and expand sirtuins as therapeutic targets in the future.

Erythropoietin protects CA1 neurons against global cerebral ischemia in rat: Potential signaling mechanisms

Erythropoietin (EPO) is a hormone that is neuroprotective in models of neurodegenerative diseases. This study examined whether EPO can protect against neuronal death in the CA1 region of the rat hippocampus following global cerebral ischemia. Recombinant human EPO was infused into the intracerebral ventricle either before or after the induction of ischemia produced by using the four‐vessel‐occlusion model in rat. Hippocampal CA1 neuron damage was ameliorated by infusion of 50 U EPO. Administration of EPO was neuroprotective if given 20 hr before or 20 min after ischemia, but not 1 hr following ischemia. Coinjection of the phosphoinositide 3 kinase inhibitor LY294002 with EPO inhibited the protective effects of EPO. Treatment with EPO induced phosphorylation of both AKT and its substrate, glycogen synthase kinase‐3β, in the CA1 region. EPO also enhanced the CA1 level of brain‐derived neurotrophic factor. Finally, we determined that ERK activation played minor roles in EPO‐mediated neuroprotection. These studies demonstrate that a single injection of EPO ICV up to 20 min after global ischemia is an effective neuroprotective agent and suggest that EPO is a viable candidate for treating global ischemic brain injury.

Hsp27 Protects against Ischemic Brain Injury via Attenuation of a Novel Stress-Response Cascade Upstream of Mitochondrial Cell Death Signaling

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.

Apoptosis inducing factor mediates caspase-independent 1-methyl-4-phenylpyridinium toxicity in dopaminergic cells.

Parkinsons disease is a debilitating neurodegenerative disease characterized by loss of midbrain dopaminergic neurons. These neurons are particularly sensitive to the neurotoxin 1‐methyl‐4‐phenylpyridinium (MPP+), the active metabolite of 1‐methyl‐4‐phenyl‐1,2,3,6‐tetrahydropyridine (MPTP), which causes parkinsonian syndromes in humans, monkeys and rodents. Although apoptotic cell death has been implicated in MPTP/MPP+ toxicity, several recent studies have challenged the role of caspase‐dependent apoptosis in dopaminergic neurons. Using the midbrain‐derived MN9D dopaminergic cell line, we found that MPP+ treatment resulted in an active form of cell death that could not be prevented by caspase inhibitors or over‐expression of a dominant negative inhibitor of apoptotic protease activating factor 1/caspase‐9. Apoptosis inducing factor (AIF) is a mitochondrial protein that may mediate caspase‐independent forms of regulated cell death following its translocation to the nucleus. We found that MPP+ treatment elicited nuclear translocation of AIF accompanied by large‐scale DNA fragmentation. To establish the role of AIF in MPP+ toxicity, we constructed a DNA vector encoding a short hairpin sequence targeted against AIF. Reduction of AIF expression by RNA interference inhibited large‐scale DNA fragmentation and conferred significant protection against MPP+ toxicity. Studies of primary mouse midbrain cultures further supported a role for AIF in caspase‐independent cell death in MPP+‐treated dopaminergic neurons.

Calcium dysregulation induces apoptosis-inducing factor release: cross-talk between PARP-1- and calpain-signaling pathways.

Recent discoveries show that caspase-independent cell death pathways are a pervasive mechanism in neurodegenerative diseases, and apoptosis-inducing factor (AIF) is an important effector of this mode of neuronal death. There are currently two known mechanisms underlying AIF release following excitotoxic stress, PARP-1 and calpain. To test whether there is an interaction between PARP-1 and calpain in triggering AIF release, we used the NMDA toxicity model in rat primary cortical neurons. Exposure to NMDA resulted in AIF truncation and nuclear translocation, and shRNA-mediated knockdown of AIF resulted in neuroprotection. Both calpain and PARP-1 are involved with AIF processing as AIF truncation, nuclear translocation and neuronal death were attenuated by calpain inhibition using adeno-associated virus-mediated overexpression of the endogenous calpain inhibitor, calpastatin, or treatment with the PARP-1 inhibitor 3-ABA. Activation of PARP-1 is necessary for calpain activation as PARP-1 inhibition blocked mitochondrial calpain activation. Finally, NMDA toxicity induces mitochondrial Ca(2+) dysregulation in a PARP-1 dependent manner. Thus, PARP-1 and mitochondrial calpain activation are linked via PARP-1-induced alterations in mitochondrial Ca(2+) homeostasis. Collectively, these findings link the two seemingly independent mechanisms triggering AIF-induced neuronal death.

Cellular NAD Replenishment Confers Marked Neuroprotection Against Ischemic Cell Death Role of Enhanced DNA Repair

Background and Purpose— NAD+ 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+ depletion, and cell death. The objective of this study was to determine the neuroprotective effects of NAD+ repletion against ischemic injury and the underlying mechanism. Methods— In vitro ischemic injury was induced in rat primary neuronal cultures by oxygen-glucose deprivation (OGD) for 1 to 2 hours. NAD+ was replenished by adding NAD+ directly to the culture medium before or after OGD. Cell viability, oxidative DNA damage, and DNA base-excision repair (BER) activity were measured quantitatively up to 72 hours after OGD with or without NAD+ repletion. Knockdown of BER enzymes was achieved in cultures using AAV-mediated transfection of shRNA. Results— Direct NAD+ repletion in neurons either before or after OGD markedly reduced cell death and OGD-induced accumulation of DNA damage (AP sites, single and double strand breaks) in a concentration- and time-dependent manner. NAD+ repletion restored nDNA repair activity by inhibiting serine-specific phosphorylation of the essential BER enzymes AP endonuclease and DNA polymerase-β. Knocking down AP endonuclease expression significantly reduced the prosurvival effect of NAD+ repletion. Conclusion— Cellular NAD+ replenishment is a novel and potent approach to reduce ischemic injury in neuronal cultures. Restoration of DNA repair activity via the BER pathway is a key signaling event mediating the neuroprotective effect of NAD+ replenishment.

Omega-3 Fatty Acids Protect the Brain against Ischemic Injury by Activating Nrf2 and Upregulating Heme Oxygenase 1

Ischemic stroke is a debilitating clinical disorder that affects millions of people, yet lacks effective neuroprotective treatments. Fish oil is known to exert beneficial effects against cerebral ischemia. However, the underlying protective mechanisms are not fully understood. The present study tests the hypothesis that omega-3 polyunsaturated fatty acids (n-3 PUFAs) attenuate ischemic neuronal injury by activating nuclear factor E2-related factor 2 (Nrf2) and upregulating heme oxygenase-1 (HO-1) in both in vitro and in vivo models. We observed that pretreatment of rat primary neurons with docosahexaenoic acid (DHA) significantly reduced neuronal death following oxygen-glucose deprivation. This protection was associated with increased Nrf2 activation and HO-1 upregulation. Inhibition of HO-1 activity with tin protoporphyrin IX attenuated the protective effects of DHA. Further studies showed that 4-hydroxy-2E-hexenal (4-HHE), an end-product of peroxidation of n-3 PUFAs, was a more potent Nrf2 inducer than 4-hydroxy-2E-nonenal derived from n-6 PUFAs. In an in vivo setting, transgenic mice overexpressing fatty acid metabolism-1, an enzyme that converts n-6 PUFAs to n-3 PUFAs, were remarkably resistant to focal cerebral ischemia compared with their wild-type littermates. Regular mice fed with a fish oil-enhanced diet also demonstrated significant resistance to ischemia compared with mice fed with a regular diet. As expected, the protection was associated with HO-1 upregulation, Nrf2 activation, and 4-HHE generation. Together, our data demonstrate that n-3 PUFAs are highly effective in protecting the brain, and that the protective mechanisms involve Nrf2 activation and HO-1 upregulation by 4-HHE. Further investigation of n-3 PUFA neuroprotective mechanisms may accelerate the development of stroke therapies.