α-Synuclein Enhances Cadmium Uptake and Neurotoxicity via Oxidative Stress and Caspase Activated Cell Death Mechanisms in a Dopaminergic Cell Model of Parkinson’s Disease

This study examined the role of alpha-synuclein in regulating cadmium (Cd)-induced neurotoxicity using the N27 dopaminergic neuronal model of Parkinson’s disease (PD) that stably expresses wild-type human α-synuclein (α-Syn) or empty vector (Vec) control. We report that α-Syn significantly increased Cd-induced cytotoxicity as compared to Vec control cells upon 24 h exposure. To explore the cellular mechanisms, we examined oxidative stress, caspase activation, and Cd uptake and intracellular accumulation. Expression of α-Syn coupled with Cd-induced cytotoxicity increased oxidative stress. Inductively coupled plasma-mass spectrometry (ICP-MS) revealed an increase in Cd uptake and intracellular accumulation in α-Syn-expressing cells upon Cd exposure. Analysis of the mitochondrial mediated apoptotic pathway showed greater activation of caspase-9 and caspase-3 in α-Syn cells. To functionally evaluate the role of metal transporters in the altered Cd phenotype, we examined Cd toxicity in the presence of nontoxic levels of divalent manganese Mn(II) and iron Fe(II). Co-treatment with Fe(II) or Mn(II) did not significantly attenuate Cd-induced cytotoxicity. We report that Cd exposure decreased the divalent metal transporter 1 and Akt protein levels in the α-Syn-expressing cells without altering native PKCδ protein levels in both Vec control and α-Syn lines. In addition, we show decreased basal metallothionein-3 protein expression in α-Syn-expressing cells. Co-treatment with N-acetyl-l-cysteine was sufficient to attenuate and abolish the α-Syn × Cd-induced cytotoxicity. Collectively, these results demonstrate that α-Syn exhibits neurotoxic properties upon acute Cd exposure to cause cell death by causing oxidative stress, increasing Cd uptake, altering caspase-9 and caspase-3 activation, and diminishing the neuroprotective effect of Akt in a dopaminergic neuronal model of PD.     

Acute exposure to chlorpyrifos caused NADPH oxidase mediated oxidative stress and neurotoxicity in a striatal cell model of Huntington’s disease

We hypothesized that expression of mutant Huntingtin (HTT) would modulate the neurotoxicity of the commonly used organophosphate insecticide, chlorpyrifos (CPF), revealing cellular mechanisms underlying neurodegeneration. Using a mouse striatal cell model of HD, we report that mutant HD cells are more susceptible to CPF-induced cytotoxicity as compared to wild-type. This CPF-induced cytotoxicity caused increased production of reactive oxygen species, reduced glutathione levels, decreased superoxide dismutase activity, and increased malondialdehyde levels in mutant HD cells relative to wild-type. Furthermore, we show that co-treatment with antioxidant agents attenuated the CPF-induced ROS levels and cytotoxicity. Co-treatment with a NADPH oxidase (NOX) inhibitor, apocynin, also attenuated the CPF-induced ROS production and neurotoxicity. CPF caused increased NOX activity in mutant HD lines that was ameliorated following co-treatment with apocynin. Finally, CPF-induced neurotoxicity significantly increased the protein expression of nuclear factor erythroid 2-related factor (Nrf2) in mutant HD cells as compared to wild-type. This study is the first report of CPF-induced toxicity in HD pathophysiology and suggests that mutant HTT and CPF exhibit a disease-toxicant interaction wherein expression of mutant HTT enhances CPF-induced neurotoxicity via a NOX-mediated oxidative stress mechanism to cause neuronal loss in the full length HTT expressing striatal cells.     

The role of D1 dopamine receptors and phospho-ERK in mediating cytotoxicity. Commentary

Striatal neurodegeneration observed in several neurological diseases, occurs through unknown mechanisms. Recent evidence suggests that its pathogenesis may be linked, in part, to high synaptic levels of dopamine (DA), which can then cause neurotoxicity of striatal neurons through mitogen-activated protein kinases (MAPKs). Here we comment on the role of extracellular signal-regulated kinase (ERK) activation in the cytotoxicity mediated upon activation of the D1 DA receptor, and describe a possible mechanism for phospho-ERK (p-ERK) in inducing cytotoxicity.     

Cocaine and human immunodeficiency virus type 1 gp120 mediate neurotoxicity through overlapping signaling pathways

Although it has been well documented that drugs of abuse such as cocaine cause enhanced progression of human immunodeficiency virus (HIV)-associated neuropathological disorders, the underlying mechanisms mediating these effects remain poorly understood. The present study demonstrated that exposure of rat primary neurons to both cocaine and gp120 resulted in increased cell toxicity compared to cells treated with either factor alone. The combinatorial toxicity of cocaine and gp120 was accompanied by an increase in both caspase-3 activity and expression of the proapoptotic protein Bax. Furthermore, increased neurotoxicity in the presence of both the agents was associated with a concomitant increase in the production of intracellular reactive oxygen species and loss of mitochondrial membrane potential. Increased neurotoxicity mediated by cocaine and gp120 was ameliorated by NADPH oxidase inhibitor apocynin, thus underscoring the role of oxidative stress in this cooperation. Signaling pathways including c-jun N-teminal kinase (JNK), p38, extracellular signal-regulated kinase (ERK)/ mitogen-activated protein kinases (MAPK), and nuclear factor (NF)-κB were also identified to be critical in the neurotoxicity induced by cocaine and gp120. These findings thus underscore the role of oxidative stress, mitochondrial and MAPK signal pathways in cocaine and HIV gp120-mediated neurotoxicity.     

Caspase-3 dependent proteolytic activation of protein kinase C delta mediates and regulates 1-methyl-4-phenylpyridinium (MPP+)-induced apoptotic cell death in dopaminergic cells: relevance to oxidative stress in dopaminergic degeneration

1-Methyl-4-phenylpyridinium (MPP+), the neurotoxic metabolite of MPTP (1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine), induces apoptosis in dopaminergic neurons; however, the cellular mechanisms underlying the degenerative process are not well understood. In the present study, we demonstrate that caspase-3 mediated proteolytic activation of protein kinase C delta (PKC delta) is critical in MPP+-induced oxidative stress and apoptosis. MPP+ exposure in rat dopaminergic neuronal cells resulted in time-dependent increases in reactive oxygen species generation, cytochrome c release, and caspase-9 and caspase-3 activation. Interestingly, MPP+ induced proteolytic cleavage of PKC delta (72-74 kDa) into a 41-kDa catalytic and a 38-kDa regulatory subunit, resulting in persistently increased kinase activity. The caspase-3 inhibitor Z-DEVD-fmk effectively blocked MPP+-induced PKC delta cleavage and kinase activity, suggesting that the proteolytic activation is caspase-3 mediated. Similar results were seen in MPP+-treated rat midbrain slices. Z-DEVD-fmk and the PKC delta specific inhibitor rottlerin almost completely blocked MPP+-induced DNA fragmentation. The superoxide dismutase mimetic, MnTBAP also effectively attenuated MPP+-induced caspase-3 activation, PKC delta cleavage, and DNA fragmentation. Furthermore, rottlerin attenuated MPP+-induced caspase-3 activity without affecting basal activity, suggesting positive feedback activation of caspase-3 by PKC delta. Intracellular delivery of catalytically active recombinant PKC delta significantly increased caspase-3 activity, further indicating that PKC delta regulates caspase-3 activity. Finally, over-expression of a kinase inactive PKC delta K376R mutant prevented MPP+-induced caspase activation and DNA fragmentation, confirming the pro-apoptotic function of PKC delta in dopaminergic cell death. Together, we demonstrate for the first time that MPP+-induced oxidative stress proteolytically activates PKC delta in a caspase-3-dependent manner to induce apoptosis and up-regulate the caspase cascade in dopaminergic neuronal cells.     

l-Theanine attenuates cadmium-induced neurotoxicity through the inhibition of oxidative damage and tau hyperphosphorylation

Cadmium (Cd) has long been known to induce neurological degenerative disorders. We studied effects of l-theanine, one of the major amino acid components in green tea, on Cd-induced brain injury in mice. Male ICR mice were intraperitoneally injected with l-theanine (100 or 200 mg/kg/day) or saline and after one hour these mice were orally administrated with CdCl₂ (3.75–6 mg/kg). The treatment was conducted for 8 weeks. l-Theanine significantly reduced Cd level in the mouse brain and plasma. Cd-induced neuronal cell death in the mouse cortex and hippocampus were apparently inhibited by l-theanine treatment. l-Theanine also decreased the levels of malondialdehyde (MDA) and ROS, and obviously elevated the levels of glutathione (GSH) and activities of superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GSH-Px) in the mouse brain. Hyperphosphorylation of tau protein is proposed to be an early event for the evolution of tau pathology, and may play an important role in Cd-induced neurodegeneration. Our results showed that l-theanine significantly suppressed Cd-induced tau protein hyperphosphorylation at Ser199, Ser202, and Ser396. Mechanism study showed that l-theanine inhibited the activation of glycogen synthase kinase-3β (GSK-3β) which contributed to the hyperphosphorylation of tau and Cd-induced cytotoxicity. Furthermore, l-theanine reduced Cd-induced cytotoxicity possibly by interfering with the Akt/mTOR signaling pathway. In conclusion, our study indicated that l-theanine protected mice against Cd-induced neurotoxicity through reducing brain Cd level and relieved oxidative damage and tau hyperphosphorylation. Our foundings provide a novel insight into the potential use of l-theanine as prophylactic and therapeutic agents for Cd-induced neurodegenerative diseases.     

Carboxyl-terminal fragment of amyloid precursor protein and hydrogen peroxide induce neuronal cell death through different pathways

Summary. Carboxyl-terminal fragments (CTs) of the amyloid precursor protein have been shown to be highly neurotoxic and are though to contribute to the neuropathology of Alzheimer’s disease. We compared the effects of expressing CT99 in the human neuroblastoma MC65 with the effects of hydrogen peroxide on the parental SK-N-MC cells. CT99 and hydrogen peroxide generated a different pattern of free radicals and their toxic effects were differentially protected by a battery of antioxidants. Hydrogen peroxide caused a cell cycle arrest at phase S and apoptosis mediated through caspase-3 activation in a pattern similar to that described for amyloid-β neurotoxicity. However, CT99 apoptosis appeared to be mediated through an unidentified mitochondrial pathway. Both oxidative injury types induced heme oxygenase-1 expression as a neuroprotective response. Overall we found a coincidence in the nonespecific stress oxidative effects of CT99 and hydrogen peroxide, but clear differences on their respective potencies and pathways of neurotoxicity.     

Mice overexpressing 70-kDa heat shock protein show increased resistance to malonate and 3-nitropropionic acid

Heat shock proteins (HSPs) are induced in response to oxidative stress, hypoxia-ischemia, and neuronal injury and play a protective role. Malonate and 3-nitropropionic acid (3-NP) are well-characterized animal models of Huntington's Disease (HD). They inhibit succinate dehydrogenase, inducing mitochondrial dysfunction, which triggers the generation of superoxide radicals, secondary excitotoxicity, and apoptosis. In this study, we examined whether the 70-kDa heat shock protein (HSP-70) is protective against neurotoxicity induced by malonate and 3-NP. Homozygous and heterozygous HSP-70 overexpressing mice (HSP-70+/+, HSP-70+/-) and wild-type controls received 3-NP or malonate and striatal lesion sizes were evaluated by stereology. Compared to HSP-70+/+ and HSP-70+/-, wild-type controls showed significantly larger striatal lesions following 3-NP or malonate injections. These findings support the idea that HSP-70 has a neuroprotective role that may be useful in the treatment of neurodegenerative diseases.     

Environmental neurotoxic pesticide dieldrin activates a non receptor tyrosine kinase to promote PKCδ-mediated dopaminergic apoptosis in a dopaminergic neuronal cell model

Oxidative stress and apoptosis are two key pathophysiological mechanisms underlying dopaminergic degeneration in Parkinson's disease (PD). Recently, we identified that proteolytic activation of protein kinase C-delta (PKCδ), a member of the novel PKC family, contributes to oxidative stress-induced dopaminergic degeneration and that phosphorylation of tyrosine residue 311 (tyr311) on PKCδ is a key event preceding the PKCδ proteolytic activation during oxidative damage. Herein, we report that a non-receptor tyrosine kinase Fyn is significantly expressed in a dopaminergic neuronal N27 cell model. Exposure of N27 cells to the dopaminergic toxicant dieldrin (60 μM) rapidly activated Fyn kinase, PKCδ-tyr311 phosphorylation and proteolytic cleavage. Fyn kinase activation precedes the caspase-3-mediated proteolytic activation of PKCδ. Pre-treatment with p60-tyrosine-specific kinase inhibitor (TSKI) almost completely attenuated dieldrin-induced phosphorylation of PKCδ-tyr311 and its proteolytic activation. Additionally, TSKI almost completely blocked dieldrin-induced apoptotic cell death. To further confirm Fyn's role in the pro-apoptotic function of PKCδ, we adopted the RNAi approach. siRNA-mediated knockdown of Fyn kinase also effectively attenuated dieldrin-induced phosphorylation of PKCδ-tyr311, caspase-3-mediated PKCδ proteolytic cleavage, and DNA fragmentation, suggesting that Fyn kinase regulates the pro-apoptotic function of PKCδ. Collectively, these results demonstrate for the first time that Fyn kinase is a pro-apoptotic kinase that regulates upstream signaling of the PKCδ-mediated apoptotic cell death pathway in neurotoxicity models of pesticide exposure.     

Edaravone attenuates cadmium-induced toxicity by inhibiting oxidative stress and inflammation in ICR mice

The neurotoxicity caused by cadmium (Cd) is well known in humans and experimental animals. However, there is no effective treatment for its toxicity. In this study, we established Cd toxicity models in cultured cells or mice to investigate the detoxification effect of edaravone (Eda). We found that Eda protected GL261 cells from Cd toxicity and prevented the loss of cell viability. In Cd-exposed mice, liver, kidney and testicular damage, as well as cognitive dysfunction were observed. Oxidative stress and inflammatory responses, such as decreased SOD and CAT, increased LDH and MDA, and abnormal changes in the inflammatory factors TNF-α, IL-1β, IL-6 and IL-10 were detected in serum and brain tissue. Eda protected mice from Cd-induced toxicity and abrogated oxidative stress and inflammatory responses. Also, Eda prevented inflammatory activation of microglia and astrocytes and was accompanied by restoration of the neuronal marker protein MAP2, indicating restoration of neuronal function. In addition, the BDNF-TrkB/Akt and Notch/HES-1 signaling axes were involved in the response of Eda to the elimination of Cd toxicity. In conclusion, Eda does contribute to the clearance of Cd-induced toxicity.     

Towards a comprehensive understanding of the contributions of mitochondrial dysfunction and oxidative stress in the pathogenesis and pathophysiology of Huntington's disease

Huntington's disease (HD) is a rare autosomal dominant disorder affecting the corticostriatal area of the brain. HD is driven by elongated cytosine–adenine–guanine (CAG) repeat (36 repeats or more) on the short arm of chromosome 4p16.3 in the Huntingtin gene (HTTg) which encode the huntingtin protein (HTT). Although the polyglutamine expansion within HTT is the causative factor in the pathogenesis of HD, the underlying mechanisms that provoke this expansion and the resulting neurodegeneration and clinical symptoms are not fully understood. In this paper, the critical role played by mitochondria dysfunction and oxidative stress in HTT expansion, HD progression, and clinical symptoms are elucidated. Their interactions with the key factors in the disease, as well as treatment strategies, are discussed.     

Activation of Neuronal NMDA Receptors Induces Superoxide-Mediated Oxidative Stress in Neighboring Neurons and Astrocytes

Excitotoxic neuronal death is mediated in part by NMDA receptor-induced activation of NOX2, an enzyme that produces superoxide and resultant oxidative stress. It is not known, however, whether the superoxide is generated in the intracellular space, producing oxidative stress in the neurons responding to NMDA receptor activation, or in the extracellular space, producing oxidative stress in neighboring cells. We evaluated these alternatives by preparing cortical neuron cultures from p47(phox-/-) mice, which are unable to form a functional NOX2 complex, and transfecting the cultures at low density with GFP-tagged p47(phox) to reconstitute NOX2 activity in widely scattered neurons. NMDA exposure did not induce oxidative stress or cell death in the nontransfected, p47-phox(-/-) cultures, but did produce oxidative stress and neuronal death in neurons surrounding the transfected, NOX2-competent neurons. This cell-to-cell spread of NMDA-induced oxidative injury was blocked by coincubation with either superoxide dismutase or the anion channel blocker 4'-diisothiocyanostilbene-2,2'-disulphonate, confirming superoxide anion as the mediating oxidant. In neurons plated on a preexisting astrocyte layer, NMDA induced oxidative stress in both the neurons and the astrocytes, and this was also prevented by superoxide dismutase. These findings show that activation of NMDA receptors on one neuron can lead to oxidative stress and cell death in neighboring neurons and astrocytes by a process involving the extracellular release of superoxide by NOX2.     

Methamphetamine-induced serotonin neurotoxicity is mediated by superoxide radicals

Methamphetamine (METH) causes deleterious effects in brain monoaminergic systems. Evidence has accumulated to suggest that these effects may be mediated via the overproduction of the superoxide radicals. We have recently shown that METH-induced dopamine (DA) depletion is attenuated in copper-zinc superoxide dismutase (CuZnSOD) transgenic (Tg) mice. In the present study, we have used receptor autoradiographic studies of [¹²⁵I]RTI-55 labeled serotonin (5-HT) uptake sites to evaluate the effect of a two dosing schedule (5 mg/kg or 10 mg/kg×4) of METH on striatal 5-HT uptake sites in nontransgenic (Non-Tg), heterozygous (Hetero) and homozygous (Homo) SOD-Tg mice. The low dose caused no significant changes in striatal 5-HT uptake sites in any of the groups. The high dose caused marked decreases (−74%) in striatal 5-HT uptake sites in Non-Tg mice. In contrast, 5-HT uptake sites showed only a 31% decrease in homozygous SOD-Tg mice whereas heterozygous SOD-Tg mice showed 63% depletion. These results show that increased SOD activity can protect against METH-induced neurotoxicity in striatal serotonergic terminals. These data provide further evidence for a role of oxidative stress in the neurotoxic effects of METH.     

Apoptotic death of striatal neurons induced by human immunodeficiency virus-1 Tat and gp120: Differential involvement of caspase-3 and endonuclease G

Human immunodeficiency virus-1 (HIV-1) infection affects the striatum, resulting in gliosis and neuronal losses. To determine whether HIV-1 proteins induce striatal neurotoxicity through an apoptotic mechanism, mouse striatal neurons isolated on embryonic day 15 and the effects of HIV-1 Tat(1-72) and gp120 on survival were assessed in vitro. Mitochondrial release of cytochrome c, caspase-3 activation, and neuron survival, as well as an alternative apoptotic pathway involving endonuclease G (endo G), were assessed at 4 h, 24 h, 48 h, and/or 72 h using enzyme assays and immunoblotting. Both HIV-1 Tat and gp120 significantly increased caspase-3 activation in a concentration-dependent manner in striatal neurons at 4 h following continuous exposure in vitro. Tat(1-72) and gp120 caused significant neuronal losses at 48 h and/or 72 h. Tat(1-72) increased cytochrome c release, and caspase-3 and endo G activation at 4 h, 24 h, and/or 72 h. By contrast, gp120 increased caspase-3 activation, but failed to increase cytochrome c or endo G levels in the cytoplasm at 4 h, 24 h, and/or 72 h. The cell permeant caspase inhibitor Z-DEVD-FMK significantly attenuated gp120-induced, but not Tat(1-72)-induced, neuronal death, suggesting that gp120 acts in large part through the activation of caspase(s), whereas Tat(1-72)-induced neurotoxicity was accompanied by activating an alternative pathway involving endo G. Thus, although Tat(1-72) and gp120 induced significant neurotoxicity, the nature of the apoptotic events preceding death differed. Collectively, our findings suggest that HIV-1 proteins are intrinsically toxic to striatal neurons and the pathogenesis is mediated through separate actions involving both caspase-3 and endo G.     

N-methyl-D-aspartate receptor activation results in regulation of extracellular signal-regulated kinases by protein kinases and phosphatases in glutamate-induced neuronal apototic-like death

Extracellular signal-regulated kinases (ERK1/ERK2) have been shown transiently activated and involved in excitotoxicity. We searched for upstream molecules responsible for the regulation of glutamate-induced ERK1/ERK2 activation and ERK1/ERK2-mediated apototic-like death in cultured rat cortical neurons. ERK1/ERK2 activation (monitored by anti-active ERK1/ERK2 antibody) was almost completely prevented by blockage of NMDA receptor (NMDA-R) or elimination of extracellular Ca(2+), but not any other glutamate receptor or L-type voltage-gated Ca(2+) channel. It was prevented largely by inhibition of protein kinase C (PKC), protein-tyrosine kinases (PTK), respectively, but mildly by that of CaM kinase II. Combined inhibition of CaM kinase II (but not PTK) and PKC had an additive effect. Reversion of ERK1/ERK2 activation was largely prevented by inhibition of protein phosphatase (PP) 1 or protein tyrosine phosphatase (PTP). Combined inhibition of PP 1 and PTP had no additive effect. Glutamate-induced apoptotic-like death (determined by DAPI staining) was largely prevented by inhibition of NMDA-R, PKC, CaM kinase II, PTK and MEK1/MEK2 (ERK1/ERK2 kinase), respectively. Combined inhibition of CaM kinase II (but not PKC or PTK) and MEK1/MEK2 had an additive effect. Glutamate-induced apoptotic-like death was promoted by inhibition of PP1 and PTP, respectively. The above results suggested that in glutamate-induced cortical neurotoxicity ERK1/ERK2 activation be mainly mediated by NMDA-R. Subsequently, a pathway dependent on both PKC and PTK was mainly involved, which was also mainly responsible for ERK1/ERK2-mediated apoptotic-like death, and a CaM kinase II-dependent pathway was relatively mildly involved. Reversion of ERK1/ERK2 activation was mainly mediated by a pathway dependent on both PP1 and PTP, which might be involved in the restrain of glutamate-induced neurotoxicity.     

Rapid activation of ERK by 6-hydroxydopamine promotes survival of dopaminergic cells

Isoforms of the mitogen-activated protein kinase ERK have been implicated in both cell survival and cell death. In the present study we explored their role in cell viability in response to oxidative stress. Using the dopaminergic MN9D cell line, we determined that cell death occurred in a concentration-dependent manner after exposure to 6-hydroxydopamine (6-OHDA). The toxicity of 6-OHDA was mediated through generation of reactive oxygen species and was accompanied by a large increase in phosphorylated ERK1/2 but no significant increase in phosphorylated ERK5. 6-OHDA produced a distinct temporal pattern of ERK1/2 activation, with phosphorylated ERK1/2 peaks occurring after 10-15 min (25-fold increase) and 6-24 hr (13-fold increase). Inhibition of the early phosphorylated ERK1/2 peak with U0126 increased the generation of reactive oxygen species by 6-OHDA as well as 6-OHDA-induced toxicity, whereas inhibition of the late peak did not affect 6-OHDA-induced cell death. The time course of phosphorylation of the prosurvival protein CREB mimicked the temporal profile of ERK1/2 activation after 6-OHDA, and blocking the early phospho-ERK1/2 peak also abolished CREB activation. In contrast, activation of caspase-3 by 6-OHDA was delayed, occurring after about 6 hr, and this activation was increased by inhibition of the first phosphorylated ERK1/2 peak. These results suggest that the rapid activation of ERK1/2 in dopaminergic cells by oxidative stress serves as a self-protective response, reducing the content of reactive oxygen species and caspase-3 activity and increasing downstream ERK1/2 substrates.     

N-Methyl- -aspartate receptor activation results in regulation of extracellular signal-regulated kinases by protein kinases and phosphatases in glutamate-induced neuronal apototic-like death

Extracellular signal-regulated kinases (ERK1/ERK2) have been shown transiently activated and involved in excitotoxicity. We searched for upstream molecules responsible for the regulation of glutamate-induced ERK1/ERK2 activation and ERK1/ERK2-mediated apototic-like death in cultured rat cortical neurons. ERK1/ERK2 activation (monitored by anti-active ERK1/ERK2 antibody) was almost completely prevented by blockage of NMDA receptor (NMDA-R) or elimination of extracellular Ca²⁺, but not any other glutamate receptor or L-type voltage-gated Ca²⁺ channel. It was prevented largely by inhibition of protein kinase C (PKC), protein-tyrosine kinases (PTK), respectively, but mildly by that of CaM kinase II. Combined inhibition of CaM kinase II (but not PTK) and PKC had an additive effect. Reversion of ERK1/ERK2 activation was largely prevented by inhibition of protein phosphatase (PP) 1 or protein tyrosine phosphatase (PTP). Combined inhibition of PP 1 and PTP had no additive effect. Glutamate-induced apoptotic-like death (determined by DAPI staining) was largely prevented by inhibition of NMDA-R, PKC, CaM kinase II, PTK and MEK1/MEK2 (ERK1/ERK2 kinase), respectively. Combined inhibition of CaM kinase II (but not PKC or PTK) and MEK1/MEK2 had an additive effect. Glutamate-induced apoptotic-like death was promoted by inhibition of PP1 and PTP, respectively. The above results suggested that in glutamate-induced cortical neurotoxicity ERK1/ERK2 activation be mainly mediated by NMDA-R. Subsequently, a pathway dependent on both PKC and PTK was mainly involved, which was also mainly responsible for ERK1/ERK2-mediated apoptotic-like death, and a CaM kinase II-dependent pathway was relatively mildly involved. Reversion of ERK1/ERK2 activation was mainly mediated by a pathway dependent on both PP1 and PTP, which might be involved in the restrain of glutamate-induced neurotoxicity.     

P2X7 receptor activation aggravates NADPH oxidase 2-induced oxidative stress after intracerebral hemorrhage

Oxidative stress is a crucial pathological process that contributes to secondary injury following intracerebral hemorrhage. P2X7 receptor(P2X7R), which is activated by the abnormal accumulation of extracellular ATP, plays an important role in the regulation of oxidative stress in the central nervous system, although the effects of activated P2X7R-associated oxidative stress after intracerebral hemorrhage remain unclear. Mouse models of intracerebral hemorrhage were established through the stereotactic injection of 0.075 U VII collagenase into the right basal ganglia. The results revealed that P2X7R expression peaked 24 hours after intracerebral hemorrhage, and P2X7R expressed primarily in neurons. The inhibition of P2X7R, using A438079(100 mg/kg, intraperitoneal), reduced nicotinamide adenine dinucleotide phosphate oxidase 2(NOX2) expression and malondialdehyde generation, increased superoxide dismutase and glutathione/oxidized glutathione levels, and alleviated neurological damage, brain edema, and apoptosis after intracellular hemorrhage. The P2X7R inhibitor A438079(100 mg/kg, intraperitoneal injection) inhibited the activation of extracellular signal-regulated kinase 1/2(ERK1/2) and nuclear factor kappa-B(NF-κB) after intracerebral hemorrhage. Blocking ERK1/2 activation, using the ERK1/2 inhibitor U0126(2 μg, intraventricular injection), reduced the level of NOX2-mediated oxidative stress induced by P2X7R activation after intracellular hemorrhage. Similarly, the inhibition of NF-κB, using the NF-κB inhibitor JSH-23(3.5 μg, intraventricular), reduced the level of NOX2-mediated oxidative stress induced by P2X7R activation. Finally, GSK2795039(100 mg/kg, intraperitoneal), a NOX2 antagonist, attenuated P2X7R-mediated oxidative stress, neurological damage, and brain edema after intracerebral hemorrhage. The results indicated that P2X7R activation aggravated NOX2-induced oxidative stress through the activation of the ERK1/2 and NF-κB pathways following intracerebral hemorrhage in mice. The present study was approved by the Ethics Committee of Huazhong University of Science and Technology, China(approval No. TJ-A20160805) on August 26, 2016.     

Vascular thrombogenicity induced by progressive LDL oxidation: protection by antioxidants

Oxidative modification of LDL, which dysregulates the homeostasis between blood and vascular cells, and alterations of endothelial function are considered among the early events in the pathogenesis of atherosclerosis. This study was designed to evaluate the impact of progressive LDL oxidation on the thrombotic response both in vitro and in vivo, and to address the potential effect of antioxidants. Tissue factor was induced by progressive LDL oxidation in HUVEC, and this event was in parallel to the appearance of the apoptotic phenotype. Both these phenomena were mediated by ERK1/2 activation and were prevented by LDL pre-enrichment with antioxidants. In contrast, antioxidants failed to affect tPA and PAI-1 secretion, which was increased by LDL, either native or oxidized. Tissue factor-pathway inhibitor was also increased upon HUVEC exposure to progressively oxidized LDL. LDL, in the presence of an oxidative agent, trigger a thrombogenic response in vivo, mostly TF-dependent, in an in situ model of platelet deposition. This effect was markedly attenuated when LDL were enriched with antioxidants. It can be concluded that vascular thrombogenicity is induced by progressive LDL oxidation and that alterations of the antioxidant/oxidant balance of the LDL particle in favor of the antioxidant tone are protective against the thrombotic response triggered by oxidative stress. The extrapolation of these data in a clinical setting, even if not easy, offers potential insights for the use of antioxidants in the prevention of thrombotic complications associated with atherothrombosis.     

Selenium reduces the proapoptotic signaling associated to NF-kappaB pathway and stimulates glutathione peroxidase activity during excitotoxic damage produced by quinolinate in rat corpus striatum

Quinolinate (QUIN) neurotoxicity has been attributed to degenerative events in nerve tissue produced by sustained activation of N-methyl-D-aspartate receptor (NMDAr) and oxidative stress. We have recently described the protective effects that selenium (Se), an antioxidant, produces on different markers of QUIN-induced neurotoxicity (Santamaría et al., 2003, J Neurochem 86:479-488.). However, the mechanisms by which Se exerts its protective actions remain unclear. Since some of these events are thought to be related with inhibition of deadly molecular cascades through the activation of antioxidant selenoproteins, in this study we investigated the effects of Se on QUIN-induced cell damage elicited by the nuclear factor kappaB (NF-kappaB) pathway, as well as the time-course response of striatal glutathione peroxidase (GPx) activity. Se (sodium selenite, 0.625 mg/kg/day, i.p.) was administered to rats for 5 days, and 120 min after the last administration, animals received a single striatal injection of QUIN (240 nmol/mul). Twenty-four hours later, their striata were tested for the expression of IkappaB-alpha (the NF-kappaB cytosolic binding protein), the immunohistochemical expression of NF-kappaB (evidenced as nuclear expression of P65), caspase-3-like activation, and DNA fragmentation. Additional groups were killed at 2, 6, and 24 h for measurement of GPx activity. Se reduced the QUIN-induced decrease in IkappaB-alpha expression, evidencing a reduction in its cytosolic degradation. Se also prevented the QUIN-induced increase in P65-immunoreactive cells, suggesting a reduction of NF-kappaB nuclear translocation. Caspase-3-like activation and DNA fragmentation produced by QUIN were also inhibited by Se. Striatal GPx activity was stimulated by Se at 2 and 6 h, but not at 24 h postlesion. Altogether, these data suggest that the protective effects exerted by Se on QUIN-induced neurotoxicity are partially mediated by the inhibition of proapoptotic events underlying IkappaB-alpha degradation, NF-kappaB nuclear translocation, and caspase-3-like activation in the rat striatum, probably involving the early activation of GPx.