Nam Sang Cheung

The novel neuromodulator hydrogen sulfide: an endogenous peroxynitrite ‘scavenger’?

Hydrogen sulfide (H2S) is a well‐known cytotoxic gas. Recently it has been shown to stimulate N‐methyl‐d‐aspartate (NMDA) receptors to enhance long‐term potentiation suggesting a novel neuromodulatory role in vivo. Endogenous levels of H2S in the brain are reported to range between 10 and 160 µm. Considerably lower H2S levels are reported in the brains of Alzheimers disease (AD) patients, where levels of brain protein nitration (probably mediated by peroxynitrite) are markedly increased. Activation of NMDA receptors leads to intracellular tyrosine nitration by peroxynitrite. Because H2S and peroxynitrite are important mediators in brain function and disease, we investigated the effects of the H2S ‘donor’, sodium hydrogen sulfide (NaSH) on peroxynitrite‐mediated damage to biomolecules and to cultured human SH‐SY5Y cells. H2S significantly inhibited peroxynitrite‐mediated tyrosine nitration and inactivation of α1‐antiproteinase to a similar extent to reduced glutathione at each concentration tested (30–250 µm). H2S also inhibited peroxynitrite‐induced cytotoxicity, intracellular protein nitration and protein oxidation in human neuroblastoma SH‐SY5Y cells. These data suggest that H2S has the potential to act as an inhibitor of peroxynitrite‐mediated processes in vivo and that the potential antioxidant action of H2S deserves further study, given that extracellular GSH levels in the brain are very low.

Mice with a Homozygous Null Mutation for the Most Abundant Glutathione Peroxidase, Gpx1, Show Increased Susceptibility to the Oxidative Stress-inducing Agents Paraquat and Hydrogen Peroxide*

Glutathione peroxidases have been thought to function in cellular antioxidant defense. However, some recent studies on Gpx1 knockout (−/−) mice have failed to show a role for Gpx1 under conditions of oxidative stress such as hyperbaric oxygen and the exposure of eye lenses to high levels of H2O2. These findings have, unexpectedly, raised the issue of the role of Gpx1, especially under conditions of oxidative stress. Here we demonstrate a role for Gpx1 in protection against oxidative stress by showing that Gpx1 (−/−) mice are highly sensitive to the oxidant paraquat. Lethality was already detected within 24 h in mice exposed to paraquat at 10 mg·kg−1 (approximately 1 7 the LD50of wild-type controls). The effects of paraquat were dose-related. In the 30 mg·kg−1-treated group, 100% of mice died within 5 h, whereas the controls showed no evidence of toxicity. We further demonstrate that paraquat transcriptionally up-regulatesGpx1 in normal cells, reinforcing a role forGpx1 in protection against paraquat toxicity. Finally, we show that cortical neurons from Gpx1 (−/−) mice are more susceptible to H2O2; 30% of neurons fromGpx1 (−/−) mice were killed when exposed to 65 μm H2O2, whereas the wild-type controls were unaffected. These data establish a function for Gpx1 in protection against some oxidative stressors and in protection of neurons against H2O2. Further, they emphasize the need to elucidate the role of Gpx1 in protection against different oxidative stressors and in different disease states and suggest thatGpx1 (−/−) mice may be valuable for studying the role of H2O2 in neurodegenerative disorders.

Micromolar L-glutamate induces extensive apoptosis in an apoptotic-necrotic continuum of insult-dependent, excitotoxic injury in cultured cortical neurones

Excitotoxicity induced by L-glutamate (Glu), when examined in a pure neuronal cortical culture, involved widespread apoptosis at concentrations of 1-10 microM as part of a continuum of injury, which at its most servere was purely necrotic. Cells, maintained in chemically defined neurobasal/B27 medium, were exposed at d7 for 2 h to Glu (1-500 microM), and cellular injury was analysed 2 and 24 h after insult using morphology (phase-contrast microscopy), a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) viability assay, nuclear staining with 4,6-diamidino-2-phenylindole (DAPI), terminal transferase-mediated dUTP nick end-labelling (TUNEL) and DNA fragmentation by gel electrophoresis. Glu-mediated neurotoxicity was prevented by MK-801 (5 microM), whilst CNQX (20 microM) attenuated injury by 20%. Exposure to intensive insults (100 and 500 microM Glu) induced necrosis characterized by rapid cell swelling (< 2 h) and lack of chromatin condensation, confirmed by DAPI nuclear staining. In contrast, mild insults (< 20 microM Glu) failed to produce acute neuronal swelling at < 2 h, but 24 h after injury resulted in a large number of apoptotic nuclei as confirmed by TUNEL and electrophoretic evidence of DNA fragmentation, which was attenuated by cycloheximide (0.1 microg/ml). Our findings indicate for the first time that physiological concentrations of Glu produce neuronal injury across a continuum involving apoptosis (< 20 microM) and increasingly necrosis(> 20 microM), dependent on the severity of the initial insult.

Oxidative Stress: Emerging Mitochondrial and Cellular Themes and Variations in Neuronal Injury

Oxidative stress plays a central role in neuronal injury and cell death in acute and chronic pathological conditions. The cellular responses to oxidative stress embrace changes in mitochondria and other organelles, notably endoplasmic reticulum, and can lead to a number of cell death paradigms, which cover a spectrum from apoptosis to necrosis and include autophagy. In Alzheimers disease, and other pathologies including Parkinsons disease, protein aggregation provides further cellular stresses that can initiate or feed into the pathways to cell death engendered by oxidative stress. Specific attention is paid here to mitochondrial dysfunction and programmed cell death, and the diverse modes of cell death mediated by mitochondria under oxidative stress. Novel insights into cellular responses to neuronal oxidative stress from a range of different stressors can be gained by detailed transcriptomics analyses. Such studies at the cellular level provide the key for understanding the molecular and cellular pathways whereby neurons respond to oxidative stress and undergo injury and death. These considerations underpin the development of detailed knowledge in more complex integrated systems, up to the intact human bearing the neuropathology, facilitating therapeutic advances.

Proteasome inhibition by lactacystin in primary neuronal cells induces both potentially neuroprotective and pro-apoptotic transcriptional responses: A microarray analysis

Although inhibition of the ubiquitin proteasome system has been postulated to play a key role in the pathogenesis of neurodegenerative diseases, studies have also shown that proteasome inhibition can induce increased expression of neuroprotective heat‐shock proteins (HSPs). The global gene expression of primary neurons in response to treatment with the proteasome inhibitor lactacystin was studied to identify the widest range of possible pathways affected. Our results showed changes in mRNA abundance, both at different time points after lactacystin treatment and at different lactacystin concentrations. Genes that were differentially up‐regulated at the early time point but not when most cells were undergoing apoptosis might be involved in an attempt to reverse proteasome inhibitor‐mediated apoptosis and include HSP70, HSP22 and cell cycle inhibitors. The up‐regulation of HSP70 and HSP22 appeared specific towards proteasome inhibitor‐mediated cell death. Overexpression of HSP22 was found to protect against proteasome inhibitor‐mediated loss of viability by up to 25%. Genes involved in oxidative stress and the inflammatory response were also up‐regulated. These data suggest an initial neuroprotective pathway involving HSPs, antioxidants and cell cycle inhibitors, followed by a pro‐apoptotic response possibly mediated by inflammation, oxidative stress and aberrant activation of cell cycle proteins.

Peroxynitrite mediates calcium-dependent mitochondrial dysfunction and cell death via activation of calpains

Chondrocyte cell death is a hallmark of inflammatory and degenerative joint diseases such as rheumatoid arthritis (RA) and osteoarthritis (OA), but the molecular and cellular mechanisms involved have yet to be elucidated. Because 3‐nitrotyrosine, a marker for reactive nitrogen species such as peroxynitrite, has been observed in OA and RA cartilage and has been associated with chondrocyte cell death, we investigated the mechanisms by which peroxynitrite induces cell death in human articular chondrocytes. The earliest biochemical event observed, subsequent to treatment with either peroxynitrite or the peroxynitrite generator SIN‐1, was a rapid rise in intracellular calcium that lead to mitochondrial dysfunction and cell death. Although, chondrocyte death exhibited several classical hallmarks of apoptosis, including annexin V labeling, increased fraction of cells with subG1 DNA content and DNA condensation, we did not find evidence for caspase involvement either by Western blotting, fluorimetric assays, or caspase inhibition. Additionally, peroxynitrite did not inhibit cellular caspase activity. Furthermore, using other established assays of cell viability, including the MTT assay and release of lactate dehydrogenase, we found that the predominant mode of cell death involved calcium‐dependent cysteine proteases, otherwise known as calpains. Our data show, for the first time, that peroxynitrite induces mitochondrial dysfunction in cells via a calcium‐dependent process that leads to caspase‐independent apoptosis mediated by calpains.

Hydrogen sulfide induced neuronal death occurs via glutamate receptor and is associated with calpain activation and lysosomal rupture in mouse primary cortical neurons.

Hydrogen sulfide (H(2)S) is a cytotoxic gas recently proposed as a novel neuromodulator. Endogenous levels of H(2)S in the brain range between 50 and 160 microM and perturbed H(2)S synthesis has been reported in the brains from stroke, Alzheimers disease and Down syndrome patients. Recently, in immature non-glutamate receptor expressing mouse cortical neurons H(2)S was shown to inhibit cell death exhibited by high concentrations of glutamate whereas H(2)S was not cytotoxic. Due to the reported role of H(2)S in facilitating LTP through NMDA receptors we examined the effects of H(2)S on glutamate receptor functioning using mature cortical neurons expressing functional glutamate receptor subtypes. Addition of 100 microM glutamate exhibited extensive cell death which was exacerbated by co-incubation with < or = 200 microM of the H(2)S donor sodium hydrosulfide (NaHS). At <200 microM NaHS induced apoptosis whereas >200 microM NaHS induced necrosis. Cell death was inhibited by pharmacological glutamate receptor antagonists MK801 and APV (NMDA receptor antagonists), and CNQX (kainate and AMPA receptor antagonist) but not kynurenate (broad spectrum glutamate receptor antagonist), GYKI52466 (more selective AMPA receptor antagonist) and CYZ (AMPA receptor potentiator). Although markers of apoptosis were observed, we did not detect caspase activation either by Western blotting or fluorescence assays and caspase inhibitors did not prevent cell death. Rather, H(2)S induced calpain activation and lysosomal membrane destabilization; processes inhibited by preferential antagonists of NMDA and kainate receptors. These data suggest that H(2)S induced neuronal death through ionotropic glutamate receptors, which recruits apoptosis to ensure cellular demise and employs calpains and lysosomal rupture. This study provides novel insights into cell death observed in neurodegenerative diseases involving glutamate receptor activation and perturbed H(2)S synthesis.

Hypochlorous acid induces apoptosis of cultured cortical neurons through activation of calpains and rupture of lysosomes

3‐Chlorotyrosine, a bio‐marker of hypochlorous acid (HOCl) in vivo, was reported to be substantially elevated in the Alzheimers disease (AD) brains. Thus, HOCl might be implicated in the development of AD. However, its effect and mechanism on neuronal cell death have not been investigated. Here, we report for the first time that HOCl treatment induces an apoptotic‐necrotic continuum of concentration‐dependent cell death in cultured cortical neurons. Neurotoxicity caused by an intermediate concentration of HOCl (250 µm) exhibited several biochemical markers of apoptosis in the absence of caspase activation. However, the involvement of calpains was demonstrated by data showing that calpain inhibitors protect cortical neurons from apoptosis and the formation of 145/150 kDa α‐fodrin fragments. Moreover, an increase in cytosolic Ca2+ concentration was associated with HOCl neurotoxicity and Ca2+ channel antagonists, and Ca2+ chelators prevented cleavage of α‐fodrin and the induction of apoptosis. Finally, we found that calpain activation ruptured lysosomes. Stabilization of lysosomes by calpain inhibitors or imidazoline drugs, as well as inhibition of cathepsin protease activities, rescued cells from HOCl‐induced neurotoxicity. Our results showed for the first time that HOCl induces apoptosis in cortical neurons, and that the cell death process involves calpain activation and rupture of lysosomes.

Apoptosis Induced via AMPA‐Selective Glutamate Receptors in Cultured Murine Cortical Neurons

Abstract: We have investigated the mechanisms of cell death induced by long‐term exposure to the glutamate receptor agonist (S)‐α‐amino‐3‐hydroxy‐5‐methyl‐4‐isoxazolepropionate [(S)‐AMPA]. Using primary cultures of pure neurons (95%) grown in serum‐free conditions, we found that 24‐h exposure to (S)‐AMPA (0.01–1,000 µM) induced concentration‐dependent neuronal cell death (EC50 = 3 ± 0.5 µM) with cellular changes including neurite blebbing, chromatin condensation, and DNA fragmentation, indicative of apoptosis. (S)‐AMPA induced a delayed cell death with DNA fragmentation occurring in ∼50% of cells at concentrations between 100 and 300 µM detected using terminal transferase‐mediated dUTP nick end‐labeling (TUNEL) and agarose gel electrophoresis. Apoptotic chromatin condensation was detected using 4,6‐diamidino‐2‐phenylindole, a fluorescent DNA binding dye. Cell death induced by (S)‐AMPA was attenuated by the AMPA receptor‐selective antagonist LY293558 (10 µM) and the non‐NMDA receptor antagonist 6‐cyano‐7‐nitroquinoxaline‐2,3‐dione (CNQX; 50 µM), yielding EC50 values of 73 ± 5 and 265 ± 8 µM, respectively, and was unaffected by the NMDA receptor antagonist MK‐801 (10 µM). The number of apoptotic nuclei induced by 300 µM (S)‐AMPA (57%) was also reduced substantially by the antagonists LY293558 and CNQX, with only 20% and 18% of neurons, respectively, staining TUNEL‐positive at 24 h. In addition, cycloheximide (0.5 µg/ml) also inhibited (S)‐AMPA‐induced DNA fragmentation and cell death. Our results show that long‐term exposure to AMPA can induce substantial neuronal death involving apoptosis in cultured cortical neurons, suggesting a wide involvement of AMPA‐sensitive glutamate receptors in excitotoxic injury and neurodegenerative pathologies.

Neurotoxin domoic acid produces cytotoxicity via kainate- and ampa-sensitive receptors in cultured cortical neurones

Domoic acid, a naturally occurring kainoid, has been responsible for several outbreaks of fatal poisoning after shellfish ingestion, and we examined its neurotoxic mechanism in cultured murine cortical neurones. Using observations of neuronal viability and morphology, exposure to domoic acid for 24 h was found to induce substantial concentration-dependent neuronal cell death. Domoic acid-mediated neuronal death was attenuated by the non-N-methyl-D-aspartate receptor antagonist 6-cyano-7-nitro-quinoxaline-2,3-dione and the alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate (AMPA) receptor-selective antagonist LY293558 ((3S,4aR,6R,8aR)-6-[2-(1H-tetrazol-5-yl)-ethyl]-1,2,3, 4,4a,5,6,7,8,8a-decahydroisoquinoline-3-carboxylic acid), but unaffected by NS-102 (5-nitro-6,7,8,9-tetrahydrobenzo[g]indole-2, 3-dione-3-oxime)--a low-affinity kainate receptor antagonist. Domoic acid was equipotent with (S)-AMPA (EC50 values 3.8 and 3.4 microM respectively); however, (S)-AMPA induced only 50% cell death compared to > 80% cell death induced by domoic acid. Kainate also killed > 80% of cortical neurones; however, domoic acid was about 19 times more potent than kainate (EC50 75 microM). We show the potent neurotoxicity of domoic acid for the first time in a pure neuronal model and indicate that domoic acid acts via high-affinity AMPA- and kainate-sensitive glutamate receptors to produce excitotoxic cell death.