The nitric oxide-cGKII system relays death and survival signals during embryonic retinal development via AKT-induced CREB1 activation

During early neurogenesis, retinal neuronal cells display a conserved differentiation program in vertebrates. Previous studies established that nitric oxide (NO) and cGMP accumulation regulate essential events in retinal physiology. Here we used pharmacological and genetic loss-of-function to investigate the effects of NO and its downstream signaling pathway in the survival of developing avian retinal neurons in vitro and in vivo. Six-day-old (E6) chick retinal cells displayed increased calcium influx and produced higher amounts of NO when compared with E8 cells. L-arginine (substrate for NO biosynthesis) and S-nitroso-N-acetyl-D,L-penicillamine (SNAP; a nitrosothiol NO donor) promoted extensive cell death in E6 retinas, whereas in E8 both substances decreased apoptosis. The effect of NO at both periods was mediated by soluble guanylyl cyclase (sGC) and cGMP-dependent kinase (cGK) activation. In addition, shRNA-mediated cGKII knockdown prevented NO-induced cell death (E6) and cell survival (E8). This, NO-induced cell death or cell survival was not correlated with an early inhibition of retinal cell proliferation. E6 cells also responded differentially from E8 neurons regarding cyclic AMP-responsive element-binding protein (CREB) activation in the retina in vivo. NO strongly decreased nuclear phospho-CREB staining in E6 but it robustly enhanced CREB phosphorylation in the nuclei of E8 neurons, an effect that was completely abrogated by cGKII shRNAs at both embryonic stages. The ability of NO in regulating CREB differentially during retinal development relied on the capacity of cGKII in decreasing (E6) or increasing (E8) nuclear AKT (V-Akt murine thymoma viral oncogene) activation. Accordingly, inhibiting AKT prevented both cGKII shRNA-mediated CREB upregulation in E6 and SNAP-induced CREB activation in E8. Furthermore, shRNA-mediated in vivo cGKII or in vitro CREB1 knockdown confirmed that NO/cGKII dualistically regulated the downstream CREB1 pathway and caspase activation in the chick retina to modulate neuronal viability. These data demonstrate that NO-mediated cGKII signaling may function to control the viability of neuronal cells during early retinal development via AKT/CREB1 activity.     

Neuronal nitric oxide synthase (nNOS) modulates the JNK1 activity through redox mechanism: a cGMP independent pathway

Nitric oxide (NO) is a small, uncharged molecule, which is primarily generated by the nitric oxide synthase (NOS) family of proteins, including neuronal nitric oxide synthase (nNOS), inducible NOS (iNOS), and endothelial NOS (eNOS). NO has been implicated in diverse roles in biological systems, such as the regulation of cell death and survival signaling pathways of a variety of cell types, including neuronal cells. In this study, we determined that the NO generated from l-arginine by ectopically overexpressed nNOS in HEK293 cells exerted an inhibitory effect against the activity of c-Jun N-terminal kinase (JNK), an important modulator of neuronal cell death and survival signaling pathways. NO repressed the activation of JNK, but exerted no significant effects on the activities of SEK1/MKK4 and MEKK1, which are the upstream MAPKK and MAPKKK of JNK1, respectively. This NO-mediated inhibition of JNK1 was not affected by the addition of ODQ, a guanylyl cyclase inhibitor, indicating that the effect is independent of the level of cyclic GMP. In an in vitro kinase assay, SNAP, a NO donor, was shown to directly suppress JNK1 activity, thereby indicating that NO is a direct modulator of JNK1. Moreover, the NO-mediated suppression of JNK1 was demonstrated to be redox-sensitive and dependent on the cysteine-116 in JNK1. Finally, according to the results of an immunohistochemical study using rat striatal neurons, we were able to determine that nNOS-expressing neurons evidenced significantly reduced JNK1 activation. Collectively, these data suggest that JNK1 is regulated by nNOS-mediated NO production in neurons, via a thiol-redox-sensitive mechanism.     

Nitric oxide has dual opposite roles during early and late phases of apoptosis in cerebellar granule neurons

The involvement and the role of nitric oxide (NO) as a signaling molecule in the course of neuronal apoptosis, whether unique or modulated during the progression of the apoptotic program, has been investigated in a cellular system consisting of cerebellar granule cells (CGCs) where apoptosis can be induced by lowering extracellular potassium. Several parameters involved in NO signaling pathway, such as NO production, neuronal nitric oxide synthase (nNOS) expression, and cyclic GMP (cGMP) production were examined in the presence or absence of different inhibitors. We provide evidence that nitric oxide has dual and opposite effects depending on time after induction of apoptosis. In an early phase, up to 3 h of apoptosis, nitric oxide supports survival of CGCs through a cGMP-dependent mechanism. After 3 h, nNOS expression and activity decreased resulting in shut down of NO and cGMP production. Residual NO then contributes to the apoptotic process by reacting with rising superoxide anions leading to peroxynitrite production and protein inactivation. We conclude that whilst NO over-production protects neurons from death in the early phase of neuronal damage, its subsequent reduction may contribute to neuronal degeneration and ultimate cell death.     

α-Synuclein induced cell death in mouse hippocampal (HT22) cells is mediated by nitric oxide-dependent activation of caspase-3

Our previous studies indicated that exogenous α-synuclein (ASN) activates neuronal nitric oxide (NO) synthase (nNOS) in rat brain slices. The present study, carried out on immortalized hippocampal neuronal cells (HT22), was designed to extend the previous results by showing the molecular pathway of NO-mediated cell death induced by exogenous ASN. Extracellular ASN (10 μM) was found to stimulate nitric oxide synthase (NOS) and increase caspase-3 activity in HT22 cells, leading to poly(ADP-ribose) polymerase (PARP-1) cleavage. The inhibitor of Ca²⁺-dependent NOS (N-nitro-l-arginine, 100 μM) prevented ASN-evoked caspase-3 activation and PARP-1 degradation. ASN exposure resulted in apoptotic death of HT22 cells and this effect was reversed by inhibition of NO synthesis and caspase-3 activity. Our results demonstrated that extracellular ASN induces neuronal cell death by NO-mediated caspase-3 activation.     

Akt pathway mediates a cGMP-dependent survival role of nitric oxide in cerebellar granule neurones

Apoptotic death results from disrupting the balance between anti-apoptotic and pro-apoptotic cellular signals. The inter- and intracellular messenger nitric oxide is known to mediate either death or survival of neurones. In the present work, cerebellar granule cells were used as a model to assess the survival role of nitric oxide and to find novel signal transduction pathways related to this role. It is reported that sustained inhibition of nitric oxide production induces apoptosis in differentiated cerebellar granule neurones and that compounds that slowly release nitric oxide significantly revert this effect. Neuronal death was also reverted by a caspase-3-like inhibitor and by a cyclic GMP analogue, thus suggesting that nitric oxide-induced activation of guanylate cyclase is essential for the survival of these neurones. We also report that the Akt/GSK-3 kinase system is a transduction pathway related to the survival action of nitric oxide, as apoptosis caused by nitric oxide deprivation is accompanied by down-regulation of this, but not of other, kinase systems. Conversely, treatments able to rescue neurones from apoptosis also counteracted this down-regulation. Furthermore, in transfection experiments, overexpression of the Akt gene significantly decreased nitric oxide deprivation-related apoptosis. These results are the first evidence for a mechanism where endogenous nitric oxide promotes neuronal survival via Akt/GSK-3 pathway.     

Brain-derived neurotrophic factor accelerates nitric oxide donor-induced apoptosis of cultured cortical neurons

Brain-derived neurotrophic factor (BDNF) is known to have important functions in neuronal survival, differentiation, and plasticity. In addition to its role as a survival-promoting factor, BDNF reportedly can enhance neuronal cell death in some cases, for example, the death caused by excitotoxicity or glucose deprivation. The cellular mechanism of the death-enhancing effect of BDNF remains unknown, in contrast to that of its survival-promoting effect. In this work, we found that BDNF markedly accelerated the nitric oxide (NO) donor-induced death of cultured embryonic cortical neurons. BDNF increased the number of cells with nuclear condensation and DNA fragmentation 24 h after treatment with the NO donor, but it did not change the number of those cells 36 h after the treatment. The BDNF-accelerated death of cortical neurons was inhibited by the addition of actinomycin D or cycloheximide. These results suggest that BDNF can accelerate apoptotic cell death elicited by NO donor. TrkB-IgG and K252a blocked the BDNF-induced acceleration of the death, indicating that the death-accelerating effect by BDNF is mediated by TrkB. In addition, the BDNF-accelerated apoptosis was inhibited by the addition of SB202190 and SB203580, specific inhibitors of p38 mitogen-activated protein kinase (MAPK), and U0126, a specific inhibitor of MAPK/ERK kinase 1, indicating that the activation of both p38 MAPK and ERK is involved in the signaling cascade of the BDNF-accelerated, NO donor-induced apoptosis.     

Excitotoxic and apoptotic neuronal death induce different patterns of glial activation in vitro

We have studied glial activation in rat cerebellar neuronal-glial cultures after inducing neuronal death using various stimuli. Cultures were exposed to 100 microm glutamate for 20 min, which induces excitotoxic neuronal death, or to potassium/serum deprivation, which induces apoptosis of granule neurons. We evaluated alterations in several parameters related to glial activation: nuclear factor-kappaB activation, nitric oxide and tumour necrosis factor-alpha production, which are associated with a pro-inflammatory response, glial proliferation and phagocytic activity. Although the two experimental models of neuronal damage resulted in the death of most neuronal cells within 24 h, differences were observed in the response of the various glial parameters evaluated. While nitric oxide production was not detected in any case, tumour necrosis factor-alpha production, nuclear factor-kappaB activation and glial proliferation were only induced in the presence of excitotoxic neuronal death. However, phagocytosis was induced in both cases, although earlier in the case of apoptotic neuronal death. These results show that glial cells respond to excitotoxic neuronal death with an inflammatory response associated with proliferation and phagocytosis. In contrast, whilst glial cells do not produce pro-inflammatory molecules in the presence of apoptotic neuronal death, phagocytic activity is rapidly induced.     

Minocycline inhibits apoptotic cell death via attenuation of TNF-alpha expression following iNOS/NO induction by lipopolysaccharide in neuron/glia co-cultures

We attempted to ascertain the neuroprotective effects and mechanisms of minocycline in inflammatory-mediated neurotoxicity using primary neuron/glia co-cultures treated with lipopolysaccharide (LPS). Neuronal cell death was induced by treatment with LPS for 48 h, and the cell damage was assessed using lactate dehydrogenase (LDH) assays and by counting microtubule-associated protein-2 (MAP-2) positive cells. Through terminal transferase deoxyuridine triphosphate-biotin nick end labeling (TUNEL)-staining and by measuring caspase-3 activity, we found that LPS-induced neuronal cell death was mediated by apoptosis. We determined that pre-treatment with minocycline significantly inhibited LPS-induced neuronal cell death. In addition, LPS induced inducible nitric oxide synthase (iNOS) expression significantly, resulting in nitric oxide (NO) production within glial cells, but not in neurons. Both nitric oxide synthase (NOS) inhibitors (N(G)-monomethyl-L-arginine monoacetate (L-NMMA) and S-methylisothiourea sulfate (SMT)) and minocycline inhibited iNOS expression and NO release, and increased neuronal survival in neuron/glia co-cultures. Pre-treatment with minocycline significantly inhibited the rapid and extensive production of tumor necrosis factor-alpha (TNF-alpha) mediated by LPS in glial cells. We also determined that the signaling cascade of LPS-mediated iNOS induction and NO production was mediated by TNF-alpha by using neutralizing antibodies to TNF-alpha. Consequently, our results show that the neuroprotective effect of minocycline is associated with inhibition of iNOS induction and NO production in glial cells, which is mediated by the LPS-induced production of TNF-alpha.     

Growth factors prevent changes in Bcl-2 and Bax expression and neuronal apoptosis induced by nitric oxide

Recent studies have shown that nitric oxide (NO) donors can trigger apoptosis of neurons, and growth factors such as insulin-like growth factor-1 (IGF-1) and basic fibroblast growth factor (bFGF) can protect against NO-induced neuronal cell death. The purpose of this study was to elucidate the possible mechanisms of NO-mediated neuronal apoptosis and the neuroprotective action of these growth factors. Both IGF-1 and bFGF prevented apoptosis induced by NO donors, sodium nitroprusside (SNP) or 3-morpholinosydnonimin (SIN-1) in hippocampal neuronal cultures. Incubation of neurons with SNP induced caspase-3-like activation following downregulation of Bcl-2 and upregulation of Bax protein levels in cultured neurons. Treatment of neurons with a bax antisense oligonucleotide inhibited the caspase-3-like activation and neuronal death induced by SNP. In addition, treatment of neurons with an inhibitor of caspase-3, Ac-DEVD-CHO, together with SNP did not affect the changes in the protein levels, although it inhibited NO-induced cell death. Pretreatment of cultures with either IGF-1 or bFGF prior to NO exposure inhibited caspase-3-like activation together with the changes in Bcl-2 and Bax protein levels. These results suggest that the changes in Bcl-2 and Bax protein levels followed by caspase-3-like activation are a component in the cascade of NO-induced neuronal apoptosis, and that the neuroprotective actions of IGF-1 and bFGF might be due to inhibition of the changes in the protein levels of the Bcl-2 family.     

Nitric oxide-induced mitochondrial fission is regulated by dynamin-related GTPases in neurons

Mitochondria are present as tubular organelles in neuronal projections. Here, we report that mitochondria undergo profound fission in response to nitric oxide (NO) in cortical neurons of primary cultures. Mitochondrial fission by NO occurs long before neurite injury and neuronal cell death. Furthermore, fission is accompanied by ultrastructural damage of mitochondria, autophagy, ATP decline and generation of free radicals. Fission is occasionally asymmetric and can be reversible. Strikingly, mitochondrial fission is also an early event in ischemic stroke in vivo. Mitofusin 1 (Mfn1) or dominant-negative Dynamin related protein 1 (Drp1(K38A)) inhibits mitochondrial fission induced by NO, rotenone and Amyloid-beta peptide. Conversely, overexpression of Drp1 or Fis1 elicits fission and increases neuronal loss. Importantly, NO-induced neuronal cell death was mitigated by Mfn1 and Drp1(K38A). Thus, persistent mitochondrial fission may play a causal role in NO-mediated neurotoxicity.     

Nitric Oxide Produced During Ischemia Is Toxic but Crucial to Preconditioning-Induced Ischemic Tolerance of Neurons in Culture

The present study investigated the roles of nitric oxide (NO) in preconditioning (PC)-induced neuronal ischemic tolerance in cortical cultures. Ischemia in vitro was simulated by subjecting cultures to both oxygen and glucose deprivation (OGD). A sublethal OGD (PC) significantly increased the survival rate of neurons when cultures were exposed to a lethal OGD 24 h later. Both the inhibition of nitric oxide synthase (NOS) and scavenging of NO during PC significantly attenuated the PC-induced neuronal tolerance. In addition, exposure to an NO donor emulated the PC. In contrast, the inhibition of NOS and the scavenging of NO during lethal OGD tended to increase the survival rate of neurons. This study suggested that NO produced during ischemia was fundamentally toxic, but critical to the development of PC-induced neuronal tolerance.     

Huperzine A attenuates apoptosis and mitochondria-dependent caspase-3 in rat cortical neurons

The neuroprotection of huperzine A against apoptosis was investigated. In cultures of rat primary cortical neurons, neuronal apoptosis was induced by serum deprivation for 24 h, which was accompanied by enhanced caspase-3 activity and the release of cytochrome c into the cytosol from mitochondria. Pretreating the neurons for 2 h with huperzine A (0.1-10 microM) improved neuronal survival. Huperzine A at a concentration of 1 microM significantly attenuated apoptosis by inhibiting the mitochondria-caspase pathway directly and indirectly.     

PKCδ mediates Nrf2-dependent protection of neuronal cells from NO-induced apoptosis

A chemical inhibitor library of 84 compounds was screened to investigate the signaling pathway(s) leading to activation of Nrf2 in response to nitric oxide (NO). We identified the protein kinase C delta (PKCδ) inhibitor rottlerin as the only compound that reduced NO-induced ARE-luciferase reporter activity and diminished NO-induced up-regulation of two Nrf2/ARE-regulated proteins - NAD(P)H:quinone oxidoreductase-1 (NQO1) and hemeoxygenase-1 (HO-1) in SH-Sy5y cells. Rottlerin also sensitized neuroblastoma cells and mouse primary cortical neurons to NO-induced apoptosis. Stable over-expression of PKCδ augmented NO-induced, ARE-dependent gene expression of HO-1 in SH-Sy5y cells, which were more protected from NO killing. Conversely, NO-induced ARE-dependent gene expression was reduced in PKCδ-knockdown SH-EP cells, which displayed greater sensitivity to apoptosis. PKCδ^−/− cortical neurons exhibited increased NO-induced apoptosis and less HO-1 mRNA and protein induction compared with wild type neurons. Hence, PKCδ is an important positive modulator of NO-induced Nrf2/ARE-dependent signaling that counteracts NO-mediated apoptosis in neuronal cells.     

Catalpol attenuates nitric oxide increase via ERK signaling pathways induced by rotenone in mesencephalic neurons

Catalpol has been shown to rescue neurons from kinds of damage in vitro and in vivo in previous reports. However, the effect of catalpol on the nitric oxide (NO) system via MAPKs signaling pathway of mesencephalic neurons largely remains to be verified. The current study examined that whether catalpol modulated NO and iNOS increase by rotenone in primary mesencephalic neurons and investigated its underlying signaling pathways. Present results indicated that catalpol inhibited primary mesencephalic neurons from apoptosis by morphological assay, immunocytochemistry and flow cytometric evaluation. Moreover, the ERK signaling pathway plays an important role in NO-mediated degeneration of neuron. The current results suggest that catalpol is a potential agent for the prevention of neurons apoptosis by regulating NO and iNOS increase in ERK-mediated neurodegenerative disorders.     

Tumor necrosis factor-alpha and phorbol 12-myristate 13-acetate differentially modulate cytotoxic effect of nitric oxide generated by serum deprivation in neuronal PC12 cells

Nitric oxide (NO) is a signaling molecule that mediates several physiological processes in a range of cell and tissue types. Here we investigated the effect of serum deprivation in the absence or presence of phorbol 12-myristate 1 3-acetate (PMA) or tumor necrosis factor-alpha (TNFalpha) on cell viability, NO formation, inducible NO synthase (iNOS) induction, and activation of mitogen-activated protein kinase in neuronal PC12 cells. Within 24 h of serum deprivation, apoptosis occurred in up to 65-70% of the cells, and significant levels of NO were generated. When PMA was added in serum-free medium, NO formation and cell death were decreased. In contrast, addition of TNFalpha in serum-free medium increased the levels of NO formation and apoptosis compared with those in serum-deprived cells. We have demonstrated that differential generation of NO levels by PMA or TNFalpha under conditions of serum deprivation is mediated by the same pattern of iNOS induction. NO formation via iNOS induction resulted in the activation of c-Jun N-terminal kinase (JNK) but not extracellular signal-regulated kinase. From this study it is suggested that the differential formation of cytotoxic NO by serum deprivation plus PMA or TNFalpha is primarily mediated by the induction of iNOS enzymes in neuronal PC12 cells and that its action is mediated by the activation of JNK.     

Nitric oxide can function as either a killer molecule or an antiapoptotic effector in cardiomyocytes.

Caspase enzymes are a family of cysteine proteases that play a central role in apoptosis. Recently, it has been demonstrated that caspases can be S-nitrosylated and inhibited by nitric oxide (NO). The present report shows that in chick embryo heart cells (CEHC), NO donor molecules such as S-nitroso-N-acetylpenicillamine (SNAP), S-nitrosoglutathione, spermine-NO or sodium nitroprusside inhibit caspase activity in both basal and staurosporine-treated cells. However, the inhibitory effect of NO donors on caspase activity is accompanied by a parallel cytotoxic effect, that precludes NO to exert its antiapoptotic capability. N-Acetylcysteine (NAC) at a concentration of 10 mM blocks depletion of cellular glutathione and cell death in SNAP-treated CEHC, but it poorly affects the ability of SNAP to inhibit caspase activity. Consequently, in the presence of NAC, SNAP attenuates not only caspase activity but also cell death of staurosporine-treated CEHC. These data show that changes in the redox environment may inhibit NO-mediated toxicity, without affecting the antiapoptotic capability of NO, mediated by inhibition of caspase enzymes. NO may thus be transformed from a killer molecule into an antiapoptotic agent.