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Although alterations in mitochondrial dynamics are associated with cellular responses to injury, the functional role of these dynamic changes in ischemic neurons is underexplored. One of these dynamic responses to injury includes mitochondrial biogenesis. Various sublethal pre-conditioning stimuli that induce an ischemic-tolerant state [e.g., lipopolysaccharide ( LPS)] may also induce mitochondrial biogenesis. Using neuron-enriched cultures, we found that sublethal LPS pre-conditioning induced both ischemic tolerance and markers of mitochondrial biogenesis with overlapping dose-response temporal kinetics. Sublethal LPS transiently increased the expression of critical components of the mitochondrial transcriptional machinery, including nuclear respiratory factor 1 ( NRF1) and mitochondrial transcription factor A ( TFAM), as well as mt DNA copy number, mitochondrial protein levels, and markers of functional mitochondria, such as increased cellular ATP content, citrate synthase activity, and maximal respiration capacity. Importantly, knockdown of TFAM abrogated both the induction of mitochondrial biogenesis and the neuroprotective pre-conditioning effects of LPS. Several signaling pathways coordinated these events. AMPK inhibition suppressed NRF1 and TFAM expression by LPS, whereas PI3K/Akt signaling was necessary for the nuclear translocation of NRF1 and subsequent induction of TFAM. This is the first demonstration that LPS pre-conditioning initiates multiple signaling pathways leading to mitochondrial biogenesis in neurons and that these dynamic changes contribute to ischemic tolerance. [ABSTRACT FROM AUTHOR]

J. Neurochem. (2010) 114, 1039–1048. Our previous studies show that group III secreted phospholipases A2 (sPLA2s III) induces extensive neuronal apoptosis in brain cortical cultures. However, the molecular mechanisms underlying sPLA2 III-induced neuronal injury/death are still unknown. Also it is not clear whether hypoxic pre-conditioning (HPC) is able to protect neurons from the sPLA2 III insult. In this report, we demonstrate that sPLA2 III significantly decreased production of Bcl-xl and the ratio of Bcl-xl/Bax, and increased expression of Bax, cleaved caspase 3, and cleaved α-Fodrin in primary neuronal culture. HPC prevented the sPLA2 III-induced decreases in production of Bcl-xl and the ratio of Bcl-xl/Bax, and increases in expression of Bax, cleaved caspase 3, and α-Fodrin. However, the HPC-produced neuronal protection was eliminated or attenuated by AG490, rapamycin, and STAT3 shRNA. Our results suggest that sPLA2 III-induced neuronal apoptosis is likely because of its alterations in expression and activity of Bcl-xl, Bax, caspase 3, and its target gene fodrin; and that HPC-produced neuroprotection against the sPLA2 III toxicity is mediated via JAK-STAT signal pathways that regulate the expression of Bcl-xl, Bax, and cleaved caspase 3 in cultured cortical neurons. [ABSTRACT FROM AUTHOR]

J. Neurochem. (2010) 114, 843–852. The aim of the present study was to investigate whether late pre-conditioning using 3-nitropropionic acid (3NP) prevents the 5-hydroxytryptamine (5-HT) deficits caused by the amphetamine derivative 3,4-methylenedioxymethamphetamine (MDMA) in the rat. For this purpose we administered 3NP 24 h before MDMA (3 × 5 mg/kg i.p., every 2 h) and rats were killed 7 days later. Pre-treatment of 3NP afforded complete protection against MDMA-induced 5-HT deficits independent of any effect on MDMA-induced hyperthermia or 5-HT transporter activity. To identify the transductional mechanisms responsible for the neuroprotective effect of 3NP, we first examined the involvement of nitric oxide (NO) by using selective inhibitors of all three nitric oxide synthase isoforms. Inhibition of endothelial and neuronal nitric oxide synthase, but not inducible nitric oxide synthase, reversed 3NP-induced pre-conditioning. The NO donor S-Nitroso- N-acetylpenicilamine mimicked 3NP effects further suggesting the involvement of NO in mediating 3NP protection. To investigate the involvement of NOS/soluble guanylate cyclase (sGC)/protein kinase G/mitochondrial ATP-sensitive potassium channels (mitoKATP) signaling pathway we examined the effect of 5-hydroxydecanoate (5-HD), a selective mitoKATP blocker, and 1 H-(1,2,4)oxadiazolo[4,3- a]quinoxaline-1-one, a potent inhibitor of sGC, on 3NP-induced tolerance. 5-hydroxydecanoate, but not 1 H-(1,2,4)oxadiazolo[4,3- a]quinoxaline-1-one, suppressed 3NP-mediated protection suggesting that mitoKATP opening, but not NO-mediated activation of sGC, participates in the mechanism underlying tolerance to MDMA. Our data also showed that the protective effect of 3NP was abolished by cycloheximide, supporting the involvement of de novo protein synthesis. In conclusion, 3NP-induced delayed tolerance against 5-HT deficits caused by MDMA occurs via NO production. [ABSTRACT FROM AUTHOR]

Carbon monoxide (CO) is an endogenous product of mammalian cells generated by heme-oxygenase, presenting anti-apoptotic properties in several tissues. The present work demonstrates the ability of small amounts of exogenous CO to prevent neuronal apoptosis induced by excitotoxicity and oxidative stress in mice primary culture of cerebellar granule cells. Additionally, our data show that endogenous CO is a heme-oxygenase product critical for its anti-apoptotic activity. Despite being neuroprotective, CO also induces reactive oxygen species generation in neurons. These two phenomena suggest that CO induces pre-conditioning (PC) to prevent cell death. The role of several PC mediators, namely soluble guanylyl cyclase, nitric oxide (NO) synthase, and ATP-dependent mitochondrial K channel (mitoKATP) was addressed. Inhibition of soluble guanylyl cyclase or NO synthase activity, or closing of mitoKATP abolishes the protective effect conferred by CO. In addition, CO treatment triggers cGMP and NO production in neurons. Opening of mitoKATP, which appears to be critical for CO prevention of apoptosis, might be a later event. We also demonstrated that reactive oxygen species generation and de novo protein synthesis are necessary for CO PC effect and neuroprotection. In conclusion, CO induces PC and prevents neuronal apoptosis, therefore constituting a novel and promising candidate for neuroprotective therapies. [ABSTRACT FROM AUTHOR]

Basic fibroblast growth factor (bFGF) has proven neuroprotective efficacy in the rodent retina against a diverse array of injurious stimuli. However, there is no consensus to date as to the molecular mechanisms underlying this neuroprotection. The study presented herein demonstrates increased expression of endogenous bFGF in the albino mouse retina in response to acute exposure to sublethal levels of light stress. The increased expression correlates with significant photoreceptor protection from light damage. The neuroprotection is likely to be mediated by bFGF as we demonstrate that a shorter exposure to bright light stress that does not up-regulate bFGF fails to protect photoreceptors from light damage. Furthermore, intravitreal bFGF injection into the retina of mice 3 h prior to light damage affords almost complete photoreceptor protection from light-induced degeneration. In addition, injected bFGF induces the activation of protein kinase B and extracellular signal-regulated kinase 1/2 signalling which correlate directly with the pathways we find to be activated in response to light stress and up-regulated bFGF. Moreover, we demonstrate that both bright light pre-conditioning and intravitreal bFGF injection result in dramatic increases in levels of inactive glycogen synthase kinase 3β and cyclic AMP response element binding protein phosphorylation indicating a potential mechanism by which bFGF promotes survival of photoreceptors in vivo. [ABSTRACT FROM AUTHOR]

The protective effect of pituitary adenylate cyclase-activating polypeptide (PACAP) in stroke models is poorly understood. We studied patterns of PACAP, vasoactive intestinal peptide, and the PACAP-selective receptor PAC1 after middle cerebral artery occlusion and neuroprotection by PACAP in cortical cultures exposed to oxygen/glucose deprivation (OGD). Within hours, focal ischemia caused a massive, NMDA receptor (NMDAR)-dependent up-regulation of PACAP in cortical pyramidal cells. PACAP expression dropped below the control level after 2 days and was normalized after 4 days. Vasoactive intestinal peptide expression was regulated oppositely to that of PACAP. PAC1 mRNA showed ubiquitous expression in neurons and astrocytes with minor changes after ischemia. In cultured cortical neurons PACAP27 strongly activated Erk1/2 at low and p38 MAP kinase at higher nanomolar concentrations via PAC1. In astrocyte cultures, effects of PACAP27 on Erk1/2 and p38 were weak. During OGD, neurons showed severely reduced Erk1/2 activity and dephosphorylation of Erk1/2-regulated Ser112 of pro-apoptotic Bad. PACAP27 stimulation counteracted Erk1/2 inactivation and Bad dephosphorylation during short-term OGD but was ineffective after expanded OGD. Consistently, PACAP27 caused MEK-dependent neuroprotection during mild but not severe hypoxic/ischemic stress. While PACAP27 protected neurons at 1–5 nmol/L, full PAC1 activation by 100 nmol/L PACAP exaggerated hypoxic/ischemic damage. PACAP27 stimulation of astrocytes increased the production of Akt-activating factors and conferred ischemic tolerance to neurons. Thus, ischemia-induced PACAP may act via neuronal and astroglial PAC1. PACAP confers protection to ischemic neurons by maintaining Erk1/2 signaling via neuronal PAC1 and by increasing neuroprotective factor production via astroglial PAC1. [ABSTRACT FROM AUTHOR]

Abstract We measured and manipulated intracellular potassium (K+ ) fluxes in cultured hippocampal neurons in an effort to understand the involvement of K+ in neuronal death under conditions of ischemia and exposure to apoptotic stimuli. Measurements of the intracellular K+ concentration using the fluorescent probe 1,3-benzenedicarboxylic acid, 4,4′-[1,4,10,13-tetraoxa-7,16-diazacyclooctadecane-7,16-diylbis(5-methoxy-6,2-benzofurandiyl)]bis-, tetrakis [(acetyloxy) methyl] ester (PBFI) revealed that exposure of neurons to cyanide (chemical hypoxia), glutamate (excitotoxic insult) or staurosporine (apoptotic stimulus) results in efflux of K+ and cell death. Treatment of neurons with 5-hydroxydecanoate (5HD), an inhibitor of mitochondrial K+ channels, reduced K+ fluxes in neurons exposed to each insult and increased the resistance of the cells to death. K+ efflux was attenuated, levels of oxyradicals were decreased, mitochondrial membrane potential was stabilized and release of cytochrome c from mitochondria was attenuated in neurons treated with 5HD. K+ was rapidly released into the cytosol from mitochondria when neurons were exposed to the K+ channel opener, diazoxide, or to the mitochondrial uncoupler, carbonyl cyanide 4(trifluoromethoxy)phenylhydrazone (FCCP), demonstrating that the intramitochondrial K+ concentration is greater than the cytosolic K+ concentration. The release of K+ from mitochondria was followed by efflux through plasma membrane K+ channels. In vivo studies showed that 5HD reduces ischemic brain damage without affecting cerebral blood flow in a mouse model of focal ischemic stroke. These findings suggest that intracellular K+ fluxes play a key role in modulating neuronal oxyradical production and cell survival under ischemic conditions, and that agents that modify K+ fluxes... [ABSTRACT FROM AUTHOR]