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.     

A pathway of neuronal apoptosis induced by hypoxia/reoxygenation: roles of nuclear factor-kappaB and Bcl-2

As a model of the reperfusion injury found in stroke, we have exposed neurons to hypoxia followed by reoxygenation. Neurons treated with hypoxia/reoxygenation (H/R) respond by activating nuclear factor-kappaB (NFkappaB), releasing cytochrome c from their mitochondria, and ultimately dying. Further supporting an apoptotic mechanism, expression of the antiapoptotic Bcl-2 and Bcl-x proteins was increased following H/R. In this model, adenoviral-mediated transduction of lkappaB expression inhibited NFkappaB activation and significantly accelerated cytochrome c release and caspase-dependent neuronal death. At the same time, expression of mutated lkappaB prevented the increased expression of endogenous Bcl-2 and Bcl-x. In the presence of mutated lkappaB, singular overexpression of only Bcl-2 by adenoviral-mediated transduction significantly inhibited cytochrome c release, caspase-3-like activation, and cell death in response to H/R. These findings suggest a pathway where NFkappaB activation induces overexpression of Bcl-2 and Bcl-x, which function to prevent apoptotic cell death following H/R treatments.     

Activation of Akt Kinase Inhibits Apoptosis and Changes in Bcl-2 and Bax Expression Induced by Nitric Oxide in Primary Hippocampal Neurons

Emerging data indicate that growth factors such as insulin-like growth factor-1 (IGF-1) prevent neuronal death due to nitric oxide (NO) toxicity. On the other hand, growth factors can promote cell survival by acting on phosphatidylinositol 3-kinase (PI3-kinase) and its downstream target, serine-threonine kinase Akt, in various types of cells. Here, we examined the mechanism by which IGF-1 inhibits neuronal apoptosis induced by NO in primary hippocampal neurons. IGF-1 was capable of preventing apoptosis and caspase-3-like activation induced by a NO donor, sodium nitroprusside or 3-morpholin-osydnonimine. Incubation of neurons with a P13-kinase inhibitor, wortmannin or LY294002, blocked the effects of IGF-1 on NO-induced neurotoxicity and caspase-3-like activation. In addition, the P13-kinase inhibitors blocked the effect of IGF-1 on down-regulation in Bcl-2 and upregulation in Bax expression induced by NO. Adenovirus-mediated overexpression of the activated form of Akt significantly inhibited NO-induced cell death, caspase-3-like activation, and changes in Bcl-2 and Bax expression. Moreover, expression of the kinase-defective form of Akt almost completely blocked the effects of IGF-1. These findings suggest that activation of Akt is necessary and sufficient for the effect of IGF-1 and is capable of preventing NO-induced apoptosis by modulating the NO-induced changes in Bcl-2 and Bax expression.     

Inflammatory neurodegeneration mediated by nitric oxide,glutamate, and mitochondria

In inflammatory, infectious, ischemic, and neurodegenerative pathologies of th central nervous system (CNS) glia become “activated” by inflammatory mediators, and express new proteins such as the inducible isoform of nitric oxide synthase (iNOS). Although these activated glia have beneficial roles, in vitro they potently kill cocultured neurons, and there is increasing evidence that they contribute to pathology in vivo. Nitric oxide (NO) from iNOS appears to be a key mediator of such glial-induced neuronal death. The high sensitivity of neurons to NO is partly due to NO causing inhibition of respiration, rapid glutamate release from both astrocytes and neurons, and subsequent excitotoxic death of the neurons. NO is a potent inhibitor of mitochondrial respiration, due to reversible binding of NO to cytochrome oxidase in competition with oxygen, resulting in inhibition of energy production and sensitization to hypoxia. Activated astrocytes or microglia cause a potent inhibition of respiration in cocultured neurons due to glial NO inhibiting cytochrome oxidase within the neurons, resulting in ATP depletion and glutamate release. In some conditions, glutamate-induced neuronal death can itself be mediated by N-methyl-d-aspartate (NMDA)-receptor activation of the neuronal isoform of NO synthase (nNOS) causing mitochondrial damage. In addition NO can be converted to a number of reactive derivatives such as peroxynitrite, NO₂, N₂O₃, and S-nitrosothiols that can kill cells in part by inhibiting mitochondrial respiration or activation of mitochondrial permeability transition, triggering neuronal apoptosis or necrosis.     

Prevention of nuclear localization of activated caspases correlates with inhibition of apoptosis

The caspase family proteases are principal components of the apoptotic pathway. In this study we demonstrate that caspase-1-like proteases and interleukin-1 are important for death induced by various stimuli in cell lines, primary fibroblasts and primary sensory neurons. Furthermore, we show by immunohistochemistry that during the cell death process endogenous caspase-1-like proteases translocate into the nucleus. This translocation is stimulated by interleukin-1 receptor activation. Translocation of caspase-1-like proteases and cell death can be partially prevented by blocking the interleukin-1 receptor with the interleukin-1 receptor antagonist. This finding offers for the first time a mechanistic explanation for the protective effect of the interleukin-1 receptor antagonist against cell death. Furthermore, our data suggest that caspase-1-like proteases have a function in the nucleus which is necessary for completion of the cell death program.In cultured DRG neurons from embryonic mice the combined inhibition of caspases and the interleukin-1 receptor have an additive effect and fully prevent semaphorin III-induced neuronal death. This shows that endogenous caspases work together with IL-1 in Semaphorin III-induced neuronal death. We hypothetize that the cell death process involves a double activation step, probably including an interleukin-1 autocrine loop. This model can explain our finding that combined inhibition of caspases and interleukin-1 receptor is necessary to strongly inhibit the cell death process.     

Metabotropic glutamate receptor 3 activation prevents nitric oxide‐induced death in cultured rat astrocytes

Altered glial function may contribute to the initiation or progression of neuronal death in neurodegenerative diseases. Thus, modulation of astrocyte death may be essential for preventing pathological processes in the CNS. In recent years, metabotropic glutamate receptor (mGluR) activation has emerged as a key target for neuroprotection. We investigated the effect of subtype 3 mGluR (mGluR3) activation on nitric oxide (NO)‐induced astroglial death. A mGluR3 selective agonist, LY379268, reduced inducible NO synthase expression and NO release induced by bacterial lipopolysaccharide and interferon‐γ in cultured rat astrocytes. In turn, a NO donor (diethylenetriamine/NO) induced apoptotic‐like death in cultured astrocytes, which showed apoptotic morphology and DNA fragmentation, but no caspase 3 activation. LY379268 prevented astrocyte death induced by NO exposure, which correlates with a reduction in: phosphatidylserine externalization, p53 and Bax activation and mitochondrial permeability. The reported effects of LY379268 were prevented by the mGluR3 antagonist (s)‐α‐ethylglutamic acid. All together, these findings show the protective effect of mGluR3 activation on astroglial death and provide further evidence of a role of these receptors in preventing CNS injury triggered by several inflammatory processes associated with dysregulated NO production.     

S100β Induces Neuronal Cell Death Through Nitric Oxide Release from Astrocytes

Abstract: The glial-derived neurotrophic protein S100β has been implicated in the development and maintenance of the nervous system. S100β has also been postulated to play a role in mechanisms of neuropathology because of its specific localization and selective overexpression in Alzheimer's disease. However, the exact relationship between S100β overexpression and neurodegeneration is unclear. Recent data have demonstrated that treatment of cultured rat astrocytes with high concentrations of S100β results in a potent activation of inducible nitric oxide synthase (iNOS) and a subsequent generation of nitric oxide (NO), which can lead to astrocytic cell death. To investigate whether S100β-induced NO release from astrocytes might influence neurons, we studied S100β effects on neuroblastoma B104 cells or primary hippocampal neurons co-cultured with astrocytes. We found that S100β treatment of astrocyte-neuron co-cultures resulted in neuronal cell death by both necrosis and apoptosis. Neuronal cell death induced by S100β required the presence of astrocytes and depended on activation of iNOS. Cell death correlated with the levels of NO and was blocked by a specific NOS inhibitor. Our data support the idea that overexpression of S100β may be an exacerbating factor in the neurodegeneration of Alzheimer's disease.     

Apocynum venetum leaf extract protects rat cortical neurons from injury induced by oxygen and glucose deprivation in vitro

This study aimed to investigate the protective effect of Apocynum venetum leaf extract (AVLE) on an in vitro model of ischemia-reperfusion induced by oxygen and glucose deprivation (OGD) and further explored the possible mechanisms underlying protection. Cell injury was assessed by morphological examination using phase-contrast microscopy and quantified by measuring the amount of lactate dehydrogenase (LDH) leakage; cell viability was measured by XTT reduction. Neuronal apoptosis was determined by flow cytometry, and electron microscopy was used to study morphological changes of neurons. Caspase-3,?-8, and?-9 activation and Bcl-2/Bax protein expression were determined by Western blot analysis. We report that treatment with AVLE (5 and 50?μg/mL) effectively reduced neuronal cell death and relieved cell injury induced by OGD. Moreover, AVLE decreased the percentage of apoptotic neurons, relieved neuronal morphological damage, suppressed overexpression of active caspase-3 and?-8 and Bax, and inhibited the reduction of Bcl-2 expression. These findings indicate that AVLE protects against OGD-induced injury by inhibiting apoptosis in rat cortical neurons by down-regulating caspase-3 activation and modulating the Bcl-2/Bax ratio.     

Specific protein nitration in nitric oxide-induced apoptosis of human monocytes

The sustained overproduction of nitric oxide (NO) observed in inflammatory conditions can contribute to cell demise by affecting apoptosis. Nitration of tyrosine residues occurs in a range of diseases involving macrophage activation. Since NO induces apoptosis in monocytes/macrophages, we tested the hypothesis that nitration of specific proteins could result in apoptotic cell death. The peroxynitrite generator SIN-1 promoted apoptosis in monocytes based on oligonucleosomal DNA fragmentation, caspase-3 and -9 activation, Bcl-2 depletion and accumulation of Bax and p53 proteins. We also found that the signaling pathway triggered by SIN-1 was initiated through tyrosine kinase and Rac activation and resulted in increased JNK and p38 activities. Among the tyrosine-nitrated proteins, Rac and Lyn were identified. Using specific inhibitors for different signaling and effector molecules involved in the apoptotic process we demonstrate that NO, via protein-nitration, could play an important role in controlling the inflammatory response by regulation of monocyte homeostasis.     

Mechanism of nitric oxide-induced apoptosis in human neuroblastoma SH-SY5Y cells

We have attempted to elucidate the precise mechanism of nitric oxide (NO)-induced apoptotic neuronal cell death. Enzymatic cleavages of DEVD-AFC, VDVAD-AFC, and LEHD-AFC (specific substrates for caspase-3-like protease (caspase-3 and -7), caspase-2, and caspase-9, respectively) were observed by treatment with NO. Western blot analysis showed that pro-forms of caspase-2, -3, -6, and -7 are decreased during apoptosis. Interestingly, Ac-DEVD-CHO, a caspase-3-like protease inhibitor, blocked not only the decreases in caspase-2 and -7, but also the formation of p17 from p20 in caspase-3 induced by NO, suggesting that caspase-3 exists upstream of caspase-2 and -7. Bongkrekic acid, a potent inhibitor of mitochondrial permeability transition, specifically blocked both the loss of mitochondrial membrane potential and subsequent DNA fragmentation in response to NO. Thus, NO results in neuronal apoptosis through the sequential loss of mitochondrial membrane potential, caspase activation, and degradation of inhibitor of caspase-activated DNase (CAD) (CAD activation).     

Essential role of caspase-11 in activation-induced cell death of rat astrocytes

We have previously shown that rat astrocytes undergo apoptosis upon inflammatory activation. Nitric oxide (NO) produced by activated astrocytes was the major cytotoxic mediator in this type of autoregulatory apoptosis. However, an inhibitor of nitric oxide synthase did not completely block the apoptosis of activated astrocytes, suggesting the presence of other apoptotic pathways. Here, we present evidence that caspase-11 is an essential molecule in NO-independent apoptotic pathway of activated astrocytes. Inflammatory activation (lipopolysaccharide, interferon-gamma, and tumor necrosis factor-alpha treatment) of rat astrocyte cultures and C6 glioma cells led to the induction of caspase-11 followed by activation of caspases-11, -1, and -3. In contrast, NO donors induced activation of caspase-3 only. Inactivation of caspase-11 by the transfection of dominant negative mutant or treatment with the caspase inhibitors rendered the astrocytes partially resistant to the apoptosis following inflammatory activation, but not NO donor exposure. These results indicate that inflammatory stimuli not only induce the production of cytotoxic NO, but also initiate NO-independent apoptotic pathway through the induction of caspase-11 expression.     

Neuroprotection by BDNF against glutamate-induced apoptotic cell death is mediated by ERK and PI3-kinase pathways

Neurotrophins protect neurons against glutamate excitotoxicity, but the signaling mechanisms have not been fully elucidated. We studied the role of the phosphatidylinositol 3-kinase (PI3-K) and Ras/mitogen-activated protein kinase (MAPK) pathways in the protection of cultured hippocampal neurons from glutamate induced apoptotic cell death, characterized by nuclear condensation and activation of caspase-3-like enzymes. Pre-incubation with the neurotrophin brain-derived neurotrophic factor (BDNF), for 24 h, reduced glutamate-evoked apoptotic morphology and caspase-3-like activity, and transiently increased the activity of the PI3-K and of the Ras/MAPK pathways. Inhibition of the PI3-K and of the Ras/MAPK signaling pathways abrogated the protective effect of BDNF against glutamate-induced neuronal death and similar effects were observed upon inhibition of protein synthesis. Moreover, incubation of hippocampal neurons with BDNF, for 24 h, increased Bcl-2 protein levels. The results indicate that the protective effect of BDNF in hippocampal neurons against glutamate toxicity is mediated by the PI3-K and the Ras/MAPK signaling pathways, and involves a long-term change in protein synthesis.     

Nitric oxide and oxygen radicals induced apoptosis via bcl-2 and p53 pathway in hypoxia-reoxygenated cardiomyocytes

Neonatal rat cardiomyocytes were subjected to 24 h of hypoxia 95%N2/5%CO2 and 24 h of hypoxia plus 4 h of reoxygenation 95%O2/5%CO2. 24 h of hypoxia increased the levels of NO, TBARS and LDH. 24 h of hypoxia plus 4 h of reoxygenation decreased the levels of NO, but further increased TBARS and LDH. The hypoxia up-regulated the expression of bcl-2, p53 and p21/waf1/cip1 but the reoxygenation down-regulated the expression of bcl-2, and further up-regulated p53 and p21/waf1/cip1. The hypoxia increased cell apoptosis and reoxygenation further increased both apoptotic and necrotic cell death. NO, TBARS, DNA fragmentation and cell apoptosis were enhanced by SNP and inhibited by L-NAME respectively. In addition, SOD/catalase down-regulated the expression of p53, p21/wafl/cipl and TBARS but up-regulated bcl-2 and increased indirectly the level of NO, and inhibited DNA fragmentation. The results suggest that hypoxia-induced cell death is associated with the activation of NO, bcl-2 and p53 pathway, while hypoxia-reoxygenation induced cell death via the generation of reactive oxygen species and activation of p53 pathway. The present study clarified that NO may be an initiative signal to apoptotic cell death and the activation of bcl-2, p53 and p21/waf1/cip1 pathway in hypoxic and hypoxia-reoxygenated cardiomyocytes.     

Hypoxia/Reoxygenation induces nitric oxide and TNF-alpha release from cultured microglia but not astrocytes of the rat

Hypoxia/reoxygenation (H/R) elicits neuronal cell injury and glial cell activation within the central nervous system (CNS). Neuroinflammation is a process that primarily results from the acute or chronic activation of glial cells. This overactive state of glial cells results in the increased release of nitric oxide (NO) and/or tumor necrosis factor alpha (TNF-alpha), a process which can lead to neuronal damage or death. In this study, we found that hypoxia for eight or twelve hours (h) followed by 24 h reoxygenation (H8/ R24 or H12/R24) induced NO production and TNF-alpha release from cultures of enriched microglial or mixed glial cells. However, microglial cells could not survive longer periods of hypoxia (> or = 12 h) in microglia-enriched culture. While astrocytes retained a 95% viability following longer periods of H/R in astrocyte-enriched cultures, they did not produce any significant quantities of NO and TNF-alpha. Reoxygenation for prolonged periods (three and five days) following H24 resulted in progressively greater increases in NO production (about two-fold greater level in hypoxia as compared to normoxic conditions) accompanied by relatively less increases in TNF-alpha release in mixed glial cell cultures. Our data indicate that inflammatory mediators such as NO and TNF-alpha are released from glia-enriched mix culture in response to H/R. While microglial cells are more vulnerable than astrocytes during H/R, they survive longer in the presence of astrocyte and are the major cell type producing NO and TNF-alpha. Furthermore, the TNF-alpha release precedes NO production in response to a prolonged duration of reoxygenation following hypoxia for 24 h.     

FLIP protects against hypoxia/reoxygenation-induced endothelial cell apoptosis by inhibiting Bax activation

Hypoxia/reoxygenation causes cell death, yet the underlying regulatory mechanisms remain partially understood. Recent studies demonstrate that hypoxia/reoxygenation can activate death receptor and mitochondria-dependent apoptotic pathways, involving Bid and Bax mitochondrial translocation and cytochrome c release. Using mouse lung endothelial cells (MLEC), we examined the role of FLIP, an inhibitor of caspase 8, in hypoxia/reoxygenation-induced cell death. FLIP protected MLEC against hypoxia/reoxygenation by blocking both caspase 8/Bid and Bax/mitochondrial apoptotic pathways. FLIP inhibited Bax activation in wild-type and Bid(-/-) MLEC, indicating independence from the caspase 8/Bid pathway. FLIP also inhibited the expression and activation of protein kinase C (PKC) (alpha, zeta) during hypoxia/reoxygenation and promoted an association of inactive forms of PKC with Bax. Surprisingly, FLIP expression also inhibited death-inducing signal complex (DISC) formation in the plasma membrane and promoted the accumulation of the DISC in the Golgi apparatus. FLIP expression also upregulated Bcl-X(L), an antiapoptotic protein. In conclusion, FLIP decreased DISC formation in the plasma membrane by blocking its translocation from the Golgi apparatus and inhibited Bax activation through a novel PKC-dependent mechanism. The inhibitory effects of FLIP on Bax activation and plasma membrane DISC formation may play significant roles in protecting endothelial cells from the lethal effects of hypoxia/reoxygenation.     

ATP and glutamate released via astroglial connexin 43 hemichannels mediate neuronal death through activation of pannexin 1 hemichannels

Inflammation contributes to neurodegeneration in post-ischemic brain, diabetes, and Alzheimer's disease. Participants in this inflammatory response include activation of microglia and astrocytes. We studied the role of microglia treated with amyloid-β peptide (Aβ) on hemichannel activity of astrocytes subjected to hypoxia in high glucose. Reoxygenation after 3?h hypoxia in high glucose induced transient astroglial permeabilization via Cx43 hemichannels and reduction in intercellular communication via Cx43 cell-cell channels. Both responses were greater and longer lasting in astrocytes previously exposed for 24 h to conditioned medium from Aβ-treated microglia (CM-Aβ). The effects of CM-Aβ were mimicked by TNF-α and IL-1β and were abrogated by neutralizing TNF-α with soluble receptor and IL-1β with a receptor antagonist. Astrocytes under basal conditions protected neurons against hypoxia, but exposure to CM-Aβ made them toxic to neurons subjected to a sub-lethal hypoxia/reoxygenation episode, revealing the additive nature of the insults. Astrocytes exposed to CM-Aβ induced permeabilization of cortical neurons through activation of neuronal pannexin 1 (Panx1) hemichannels by ATP and glutamate released through astroglial Cx43 hemichannels. In agreement, inhibition of NMDA or P2X receptors only partially reduced the activation of neuronal Panx1 hemichannels and neuronal mortality, but simultaneous inhibition of both receptors completely prevented the neurotoxic response. Therefore, we suggest that responses to ATP and glutamate converge in activation of neuronal Panx1 hemichannels. Thus, we propose that blocking hemichannels expressed by astrocytes and/or neurons in the inflamed nervous system could represent a novel and alternative strategy to reduce neuronal loss in various pathological states including Alzheimer's disease, diabetes and ischemia.     

Phosphorylation of p38 MAPK induced by oxidative stress is linked to activation of both caspase-8- and -9-mediated apoptotic pathways in dopaminergic neurons

We evaluated the contribution of p38 mitogen-activated protein kinase and the events upstream/downstream of p38 leading to dopaminergic neuronal death. We utilized MN9D cells and primary cultures of mesencephalic neurons treated with 6-hydroxydopamine. Phosphorylation of p38 preceded apoptosis and was sustained in 6-hydroxydopamine-treated MN9D cells. Co-treatment with PD169316 (an inhibitor of p38) or expression of a dominant negative p38 was neuroprotective in death induced by 6-hydroxydopamine. The superoxide dismutase mimetic and the nitric oxide chelator blocked 6-hydroxydopamine-induced phosphorylation of p38, suggesting a role for superoxide anion and nitric oxide in eliciting a neurotoxic signal by activating p38. Following 6-hydroxydopamine treatment, inhibition of p38 prevented both caspase-8- and -9-mediated apoptotic pathways as well as generation of truncated Bid. Consequently, 6-hydroxydopamine-induced cell death was rescued by blockading activation of caspase-8 and -9. In primary cultures of mesencephalic neurons, the phosphorylation of p38 similarly appeared in tyrosine hydroxylase-positive, dopaminergic neurons after 6-hydroxydopamine treatment. This neurotoxin-induced phosphorylation of p38 was inhibited in the presence of superoxide dismutase mimetic or nitric oxide chelator. Co-treatment with PD169316 deterred 6-hydroxydopamine-induced loss of dopaminergic neurons and activation of caspase-3 in these neurons. Furthermore, inhibition of caspase-8 and -9 significantly rescued 6-hydroxydopamine-induced loss of dopaminergic neurons. Taken together, our data suggest that superoxide anion and nitric oxide induced by 6-hydroxydopamine initiate the p38 signal pathway leading to activation of both mitochondrial and extramitochondrial apoptotic pathways in our culture models of Parkinson's disease.     

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.