in vivo 18: 367-376 (2004)

Review Regulation of Programmed Cell in Neuronal Cells by Nitric Oxide

YUN-CHUL KANG1, PETER K. KIM2, BYOUNG-MIN CHOI3, HUN-TAEG CHUNG3, KWON-SOO HA1, YOUNG-GUEN KWON4 and YOUNG-MYEONG KIM1

1Vascular System Research Center and Department of Molecular and Cellular Biochemistry, College of Medicine, Kangwon National University, Chunchon, Kangwon-do, Korea; 2Department of Surgery, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania, U.S.A; 3Department of Microbiology and Immunology, College of Medicine, Wonkwang University, Iksan, Chonbuk; 4Department of Biochemistry, College of Science, Yonsei University, Seoul 120-749, Korea

Abstract. Nitric oxide (NO), produced from L-arginine and (nNOS), inducible NOS (iNOS) and endothelial NOS molecular oxygen in a reaction catalyzed by one of three NO (eNOS) (1, 2). The functional role of NO can vary by cell synthase isoenzymes, can prevent or induce neuronal type and enzyme isotype. The major function of eNOS is depending on its concentration and cellular redox state. This vasodilation by regulating vascular smooth muscle relaxation, molecule affords neuroprotection by post-translational S- while the main role for nNOS may involve neurotransmission nitrosylation of NMDA receptor, and p21ras, and by creating retrograde signaling between synapses. An increases the expression of cytoprotective genes such as HSP70, inducible form of NOS (iNOS) can be up-regulated heme oxygenase and Bcl-2. Moreover, the NO/cGMP pathway considerably by the bacterial component activates the anti-apoptotic serine/threonine kinase Akt by lipopolysaccharide and/or cytokines which play a role in G-dependent activation of phosphatidylinositol regulation of immune cells. At normal intracellular calcium 3-kinase. A high concentration of NO and peroxynitrite, a levels, NO production by iNOS is limited only by the amount reaction product of NO with superoxide anion, can promote of enzyme, substrate, or cofactor present (3, 4) and iNOS apoptotic pathways in neuronal cells through the indirect at the transcriptional level via the activation activation of caspases. We review the molecular mechanism by of nuclear transcription factor NF-Î B. In contrast, eNOS and which NO exerts both pro- and anti-apoptotic actions in nNOS are constitutively expressed and both are inactive at neuronal cells and the clinical implications for regulating normal calcium concentration. These isoenzymes of NOS neuronal apoptosis. produce puffs of NO in response to transient increases in cytosolic calcium level, but chronic calcium elevation will Function and mode of action of NO in biological system cause persistent NO production (5, 6). Also, the catalytic activity of eNOS can be increased by post-translational Nitric oxide (NO) is a pleiotropic, ubiquitous modulator of modification of palmitoylation and phosphorylation. many cellular functions. NO is synthesized from L-arginine NO interacts directly with several cellular target and molecular oxygen in a reaction catalyzed by one of three molecules, such as thiol, transition metals, molecular oxygen isotypes of NO synthases (NOS) such as neuronal NOS and superoxide, before diffusing out of NO-producing cells. Since NO is small and hydrophobic, it can pass easily through membranes and, as it persists in vivo for a few seconds, NO can diffuse several cell diameters from its site Correspondence to: Young-Myeong Kim, Vascular System of synthesis (7). When immune-activated hepatocytes were Research Center and Department of Molecular and Cellular co-cultured with red blood cells, the biological scavengers of Biochemistry, College of Medicine, Kangwon National University, Chunchon, Kangwon-Do 200-701, Republic of Korea. Tel: +82-33- NO, the nitrosyl iron complexes were mostly formed in red 250-8831, Fax: +82-33-244-3286, e-mail: [email protected] blood cells, but few within NO-producing hepatocytes (8). This result indicates that the transcellular diffusion of NO is Key Words: Nitric oxide, neuronal cells, apoptosis, review. more rapid than the rate of its intracellular reaction in NO-

0258-851X/2004 $2.00+.40 367 in vivo 18: 367-376 (2004) generating cells. Consequently the steady-state NO formation of dinitrosyl iron complexes may be involved in concentration experienced by a cell is determined by the transnitrosylation to the redox-sensitive sulfhydryl group of number of NO-producing cells nearby (7). NO produced several biomolecules responsible for apoptotic , within cells, therefore, interacts with biomolecules through resulting in the modulation of neuronal apoptotic cell death. both autocrine and paracrine action modes. Types of cell death Biochemical reactivity with biological molecules (PCD) is a normal physiological NO can interact both with oxygen and a great number of other process that occurs during embryonic development as well as in biological molecules such as transition metals, redox-sensitive the maintenance of tissue homeostasis. Eukaryotic cells that thiol and tyrosine residues. Although NO reacts with O2 in die by PCD undergo an astonishingly stereotypical series of aqueous solution to produce nitrite, this reaction is very slow at biochemical and morphological changes. PCD is believed to physiological concentrations of O2. NO reacts rapidly with occur by -dependent and -independent mechanisms - superoxide anion (O2 ) and forms the strong oxidant (17, 18). These two mechanisms have distinct histological and peroxynitrite (ONOO-), a cytotoxic modulator in neuronal cells. biochemical signatures. Caspase-dependent PCD, or apoptosis, NO also interacts with many iron-containing proteins, such as involves the activation of a cascade of events that orchestrate hemoproteins and nonheme-iron-containing proteins. NO the destruction of the cell and thus apoptosis is blocked by reacts with the heme iron of hemoglobin and myoglobin to inhibition of caspase activation and activity. Apoptosis is a form nitrate. NO binds the heme moiety of soluble guanylate strictly regulated device responsible for the ordered removal cyclase and increases its catalytic activity to generate more of superfluous, aged and damaged cells (19, 20). cGMP from GTP. The augmentation of this enzymatic activity Morphologically, in cells undergoing apoptosis there is ruffling, plays a key role in regulation of vascular tone and blebbing and condensation of the plasma and nuclear neurotransmission (9). Binding of NO to heme or the iron- membranes and, subsequently, aggregation of nuclear sulfur cluster of enzymes inhibits their catalytic activity such as chromatin. Mitochondria and ribosomes retain their gross cytochrome oxidase, , cytochrome P-450, ferrochelatase structure and at least partial function. There is disruption of and aconitase (10). The reaction of NO with biological the cytoskeletal architecture; the cell shrinks and then - components such as metals, thiols, O2 and O2 produces a fragments into a cluster of membrane-enclosed "apoptotic variety of secondary products ranging from the innocuous bodies" that are rapidly ingested by adjacent macrophages or - - oxidized components (NO2 and NO3 ) to the reactive nitrogen other neighboring phagocytic cells. Apoptotic cells display a intermediates such as nitrosonium equivalent (NO+), ONOO, characteristic pattern of DNA involving fragmentation into - S-nitrosothiol, nitroxyl ion (NO ), dinitrogen trioxide (N2O3) distinct segments that can be visualized as a ladder of bands by and nitrogen dioxide (NO2). These reactive nitrogen species, gel electrophoresis. Other features of apoptosis are a reduction including NO, interact with other biomolecules to cause lipid in the membrane potential of mitochondria, intracellular oxidation, protein modification (e.g. cysteine and tyrosine acidification, generation of free radicals and externalization of residue), DNA damage or base modification and regulation of phosphatidylserine residues (21, 22). enzyme activity. Contrary to earlier expectations, the inhibition of caspase Because NO is a radical molecule and does not effectively activation does not necessarily protect against cell death nitrosylate thiol groups, a NO reaction product is implicated stimuli. Instead it might reveal, or even enhance, underlying in the post-translational S-nitrosylation of cysteine residue. caspase-independent cell death programs. Although these NO+ equivalent can nitrosylate thiols (11). Free endogenous programs might resemble apoptosis-like PCD, the caspase- NO+ exists only at low pH, such as that of the stomach (12). independent mode includes two patterns of cell death, such N2O3 formed by the reaction of NO with molecular oxygen as "apoptosis-like PCD" and "-like PCD". In has a strong propensity to nitrosate both amine and thiol apoptosis-like PCD, chromatin may condense, but not to the moieties at physiological pH (12). Reactive nitrogen oxide characteristic geometric shape associated with classic species, including NO+ and its equivalents, can be generated apoptosis, and -recognition molecules including by the reaction of NO with O2 (13), iron-sulfur clusters (13), phosphatidylserine are displayed before cell lysis of the or heme iron (15). We have demonstrated that chemically plasma membrane. In necrosis-like PCD, chromatin may not synthesized dinitrosyl iron complexes induce post- condense at all, but other apoptosis-like features, including translational S-nitrosylation of caspase proteins in vitro (16). externalization of phosphatidylserine, might occur before the Furthermore, dinitrosyl iron complexes have been identified lysis (23). Furthermore, necrosis-like cell death that is in NO-producing cells (10, 16) and could provide a programmatic requires gene expression, because inhibition mechanism for protein modification, in particular, trans-S- of gene expression by treatment either with actinomycin D nitrosation (16). These results indicate that the endogenous or cyclohexamide blocks this necrosis-like cell death (24).

368 Kang et al: Regulation of Neuronal Cell Apoptosis by Nitric Oxide

Caspase-independent PCD includes , paraptosis, that these apogenic conditions can trigger the activation of zeiosis and dark cell death. Autophagy is a major intracellular caspases by two different mechanisms. Non-receptor degradation/recycling system ubiquitous in eukaryotic cells. apoptotic stimuli cause release of mitochondrial proteins to It contributes to the turnover of cellular components by activate the "intrinsic pathway" (28). Mitochondrial protein delivering portions of the and organelles to release often occurs after BH-3-only members of the Bcl-2 , where they are digested. Autophagy is mediated family bind to and neutralize anti-apoptotic members of the by membrane trafficking of unique double-membrane Bcl-2 family. Pro-apoptotic Bcl-2 family proteins (Bax, Bid structures, the so-called autophagosomes, which are formed and Bad) promote release of mitochondrial cytochrome c, transiently. Moreover, autophagy is dramatically induced which interacts with Apaf-1, procaspase-9 and dATP to under starvation conditions to maintain an amino acid pool form a complex called the apoptosome. This complex so that essential proteins may be synthesized. Recent studies dimerizes and activates caspase-9, which then activates have given insights into the molecular basis of membrane effector caspases including caspase-3 to induce apoptosis. dynamics and the regulation of autophagy, which had Mitochondria also release other apoptosis-promoting remained cryptic for a long time. Paraptosis (paraptosis, from proteins such as apoptosis-inducing factor (AIF), para = next to or related to and apoptosis) is a form of Smac/Diablo, endonuclease G and Omi/HtrA2. Recent programmed cell death that does not involve DNA work showed that these mitochondrial events can occur by fragmentation and traditional apoptotic body formation (24). activation of caspase-2. On the other hand, activation of the This killing resists various caspase inhibitors and Bcl-xL cytotoxic receptor activates an "extrinsic pathway". The treatment, which distinguishes it from apoptosis. Paraptotic death receptors contain an intracellular globular protein cell death is characterized by cytoplasmic vacuolization, which interaction domain called a death domain. Upon ligand begins with progressive swelling of the mitochondria and binding to death receptors probably in the form of pre- . Zeiosis, which comes from the Greek associated receptor trimers, activated death receptors word ZÂȈ meaning "to boil over" (25), is characterized by recruit an adaptor protein called Fas-associated death violent cytokinesis with continuous bleb extension and domain (FADD). FADD consists of two protein interaction retraction. Dark cell death occurs through a process that is domains: a death domain and a death effector domain morphologically and biochemically distinct from apoptosis (DED). The current model is that FADD binds (directly or and is associated with the presence of intracellular via another adaptor such as TNF receptor-associated death aggregation of damaged or mutant protein. domain (TRADD), which binds to TNF receptor-1) to the Accidental necrosis or cell lysis is sudden cell death receptor through interactions between death domains and without caspase activation or gene expression and it is the to procaspase-8 through DED interactions to form a conceptual counterpart to PCD. The fate of necrotic death complex at the receptor called the death-inducing signaling of cells appears to depend on the presence of serious complex (DISC). Recruitment of caspase-8 through FADD pathophysiological circumstances in their surroundings, such leads to its auto-cleavage and activation. Active caspase-8 as high concentrations of detergents, oxidants, ionophores in turn activates effector caspases such as caspase-3, causing and pathologic stimuli (26). Intracellular energy levels and the cell to undergo apoptosis by digesting terminal death mitochondrial function are other critical factors to determine substrates leading to the systematic destruction of the cell. whether cells are driven towards either apoptosis or necrosis Active caspase-3 preferentially cleaves the inhibitor of (27). Conditions of intracellular ATP depletion, obtained by caspase-activated DNase (ICAD) and allows the blocking mitochondrial and/or glycolytic ATP generation, translocation of the activated CAD into the nucleus, switches the type of demise caused by the two classic resulting in DNA degradation. An endogenous inhibitor, c- apoptotic triggers (staurosporine and CD95 stimulation) from FLIP, which is related to caspase-8 but has no protease apoptosis to necrosis. Necrosis is often associated with activity, is thought to function by competing with caspase-8 cellular edema (organelle swelling) and is due to postmortem for binding to the DISC. events that occur after plasma membrane lysis. Previously, it was reported that there are two types of cells that differ with respect to their requirement for Caspase activation is a key process of apoptosis mitochondria during death receptor-mediated apoptosis (29). In type I cells, caspase-8 is activated without Apoptosis is biologically initiated by ligation of cytotoxic involvement of mitochondria to a level sufficient to process receptors under unfavorable environmental conditions. Cell the effector caspase-3, and the inhibition of the release of death receptors include the receptors of tumor necrosis apoptogenic factors (i.e. cytochrome c and apoptosis- factor alpha (TNF-·), CD95/Fas/Apo-1 and TRAIL, as well inducing factor) from mitochondria by heterologous as non-physiological environments such as nutrient expression of Bcl-2 or Bcl-XL had no effect on caspase-8 or deprivation, pH change and oxidative stress. This indicates caspase-3 cleavage or on cytotoxic sensitivity of these cells

369 in vivo 18: 367-376 (2004)

(29). In contrast, in type II cells a mitochondria-dependent endogenous regulator of apoptosis induced by trophic factor amplification loop is required to fully activate caspase-8 and deprivation or 6-hydroxydopamine in primary neurons and transduce an apoptotic signal (29). In these cells, cytotoxic PC12 cells. The different effects of NO could be attributed, at cytokine-induced apoptotic signaling is transmitted by least in part, to the different redox-related species produced sequential reactions, activation of caspase-8 in the DISC by the molecule and their different chemical properties. The and cleavage of the cytosolic BH3-only protein Bid (p22) to neuroprotective properties of NO have been ascribed to its a p15 fragment. This fragment translocates to mitochondria oxidized form nitrosonium ion (NO+), which can down- to activate the intrinsic pathway (i.e. cytochrome c release), regulate NMDA-receptor activity by reaction with thiol thus connecting the two caspase activation pathways and groups in the redox modulatory site of the receptor (11, 41). amplifying the death receptor apoptotic signal. NO+ modifies caspase activity by redox-based modification of the enzyme. NO is known to activate soluble guanylate Roles of NO in PCD of neuronal cells cyclase, resulting in the production of intracellular cGMP. In addition, the protective effect of NO against apoptosis is also Neuronal PCD is required for normal development of the dependent on production of the second messenger cGMP in nervous system but also occurs during pathological states. embryonic motor neurons and PC12 cells. Extensive neuronal cell death is observed after acute brain injury, including stroke and trauma and is thought to Pro-apoptotic mechanism of NO contribute to neurodegenerative diseases, such as Parkinson's disease and Alzheimer's disease. Caspase-3- and caspase-9- It has been shown that stimulation of the NMDA receptor deficient mice exhibit increased brain size associated with in brain neurons leads to an increase in NO production via decreased apoptosis. Caspase-3-like protease inhibitors block the activation of nNOS (42). Stimulation of the NMDA apoptotic cell death of cultured neurons in response to receptor by agonists (glutamate and glycine) allows the several stimuli, including trophic factor deprivation, serum movement of sodium and calcium ions into the cell (43). deprivation, 6-hydroxydopamine, N-methyl-D-aspartic acid The increase in intracellular calcium leads to activation of (NMDA) and 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine. nNOS via calmodulin binding (44). The neurotoxicity of These observations indicate that neuronal apoptosis is primary cultured cortical neurons by treatment with NMDA directly linked to activation of caspase proteases. was attenuated by NOS inhibitor, the removal of L-arginine NO synthesized by nNOS was identified originally as a from the medium, or the NO scavenger hemoglobin (45). neurotransmitter (30) and later was found to be an These results indicate that NO is a neuronal messenger that endogenous regulator of neuronal cell death (31, 32). plays a critical role in the nervous system in both normal Although NO can lead to neuronal cell death when produced and pathological states and appears to be involved in certain in excess (33, 34), the physiological level of NO produced aspects of cytotoxicity. from NOS or NO donors can function as an endogenous Some mechanisms regarding NO- and/or peroxynitrite- regulator of apoptosis in primary cultured neurons (31, 35), induced cytotoxicity in neurons have been proposed. It has motor neurons and PC12 pheochromocytoma cells (32). In been reported that NO can enhance ADP-ribosylation or S- several studies, it has been observed that NO is involved in nitrosylation of an increasing number of proteins and two of neurotoxic events (36). In particular, in the presence of these proteins have been identified as NO-target proteins. factors such as excessive formation, generation of reactive One is glyceraldehyde 3-phosphate dehydrogenase (GAPDH), oxygen intermediates and deficient antioxidant system, NO which is S-nitrosylated by NO, thereby inhibiting enzyme can induce PCD in motor neurons (37), PC12 cells (38) and activity. GAPDH is known to be a key enzyme in the glycolytic cortical neurons (39), probably by the formation of conversion of glucose to pyruvic acid and represents an peroxynitrite (36). The neurotoxicity of peroxynitrite may be important pathway for carbohydrate metabolism in most triggered by a dual activity. In fact, peroxynitrite inhibits organisms. Hence, inhibition of GAPDH activity by NO mitochondrial proteins with consequent decrease in ATP would diminish the amount of ATP formed from glycolysis synthesis and can modify and break DNA strands; DNA and decrease the flow of substrates to the electron transport breakage then activates the nuclear enzyme poly(ADP- chain. NO and/or peroxynitrite also activates the nuclear ribose) polymerase (PARP) to participate in DNA repair enzyme PARP in association with damage to DNA. (40). PARP catalyzes the attachment of ADP-ribose units Consequently, it is believed that ATP is also depleted, with from NAD to nuclear proteins such as histone and PARP cell death occurring due to energy depletion. itself. ADP-ribosylation reduces the cellular level of NAD, The cytotoxic effects of NO and peroxynitrite have also been resulting in a rapid drop in energy stores and consequently to shown to result from DNA damage and the concomitant up- cell death. These observations contrast with the results regulation of the tumor suppressor gene . NO can interfere showing that the physiological level of NO can function as an with the proteolytic activity of the 20S , which leads

370 Kang et al: Regulation of Neuronal Cell Apoptosis by Nitric Oxide to the accumulation of p53 (46). The inhibition of proteasome in PC12 cells (32, 35), motor and sympathetic neurons (31, 35) activity by NO should block IÎ B degradation and subsequently and cultured hippocampal neurons (53). This protective inhibit NF-Î B activation, which is a well-known transcription mechanism has been shown to proceed via either cGMP- factor of anti-apoptotic gene expression (47). NO-mediated dependent or -independent pathways. NO can either induce or increases in p53 induce apoptosis by increasing the ratio of prevent apoptosis, depending on the conditions studied (31, 35, pro-apoptotic Bax to anti-apoptotic Bcl-XL, expression of 54). The anti-apoptotic mechanisms of NO in neuronal cells several target genes (Apaf-1, DR5, p53AIP, PUMA and Noxa) have been examined in some detail. Concurrent NO (48) and cytochrome c release. Cytosolic cytochrome c production can prevent apoptosis by direct inhibition of the interacts with Apaf-1 and procaspase-9 in the presence of activity of caspases (55) and glutamate-mediated NMDA dATP to form the apoptosome complex that results in cleavage receptor (41), mitochondrial cytochrome c release (56), and activation of caspase-9. The active caspase-9 then activates activation of p21ras (57) and the up-regulation of cytoprotective downstream effector caspases including caspase-3, which is genes such as Bcl-2 family protein (58), HSP70 (59) and heme thought to assure the cleavage of target proteins that are oxygenase (60). The molecular mechanism underlying required to complete the terminal events of apoptosis. NO/cGMP-mediated anti-apoptotic effects may depend on NO can trigger apoptotic neuronal death via caspase apogenic stimuli or cellular redox properties with multiple activation by cytochrome c release following direct loss of signal pathways. mitochondrial membrane potential (49) or intracellular signal cascade (50). Low concentrations of NO reversibly inhibit Anti-apoptosis-related gene expression. NO interacts with the cytochrome oxidase in competition with oxygen. Higher heme moiety of soluble guanylate cyclase and produces concentrations of NO can react with peroxide and other cGMP, which is an important second messenger in neuronal oxygen radicals to form peroxynitrite, that irreversibly inhibits and vascular systems. Furthermore, the NO/cGMP pathway mitochondrial respiration by oxidizing and damaging can prevent apoptosis in neuronal cells, such as PC12 cells mitochondrial complexes I, II, IV and V. Peroxynitrite can and motor neuron and other cells, such as hepatocytes, B cause mitochondrial damage including mitochondrial lymphocytes, eosinophils and ovarian follicles. Recent swelling, oxidation of mitochondrial membrane lipid and studies provide new biological and pharmacological evidence depolarization with alterations in the permeability transition, suggesting that physiological concentrations of NO prevent and thus result in cytochrome c release and caspase human brain-derived SH-SY5Y cells from serum activation. These results suggest that both the cytochrome c deprivation-induced apoptosis by increasing cytoprotective release and consequent activation of caspase are heavily genes such as thioredoxin and thioredoxin peroxidase (61, involved in NO-induced neuronal apoptosis. 62). This protective effect was reduced by the cGMP- NO can also modulate multiple pathways that lead to cell dependent protein kinase (PKG) inhibitor, Rp-8-pCPT- death, including those mediated by ceramide and stress cGMPS. This result indicates that cGMP may play a key role kinases. By enhancement of neutral sphingomyelinase activity in expression of these cytoprotective genes. Indeed, PKG and inhibition of acid ceramidase activity, NO donors also activation promotes the elevation of c-Jun, phosphorylated increase cellular ceramide levels that can induce several MAPK/ERK1/2 and c-Myc, consistent with the notion that death pathways including release of cytochrome c from PKG enhances the expression of thioredoxin through its mitochondria, activation of caspases-9 and -3 and activation c-Myc-, AP-1- and PEA3-binding motifs. Furthermore, of stress-related protein kinases (51). NO, generated by elevation of thioredoxin expression can induce the neuronal PC12 cells treated with PMA or TNF-· under biosynthesis of Bcl-2 (61) and Mn(II)-superoxide dismutase conditions of serum deprivation, induced apoptotic cell death (63), known to inhibit mitochondria-mediated apoptosis and through the activation of c-Jun N-terminal kinase/stress- to protect cells from peroxynitrite-mediated cellular toxicity. activated protein kinases (JNK/SAPK) (52). These findings In addition, we showed first that NO potentially induces the suggest that NO can modulate the intracellular signal expression of several cytoprotective genes including HSP70 transduction responsible for the initiation and amplification (59) and HSP32 (heme oxygenase) (10, 64), which prevent of neuronal apoptotic signal pathway. neuronal cell death in an ischemic animal model (65) and neuronal cell cultures (66). The molecular mechanism Anti-apoptotic mechanisms of NO in neuronal cells underlying the anti-apoptotic effect of NO-induced HSP70 may be associated with two possibilities: the first is that Although NO induces neuronal apoptosis under some HSP70 inhibits oligomerization of Apaf-1 by associating with pathological conditions, it becomes apparent that a low level the caspase-recruitment domain of Apaf-1, resulting in the of NO prevents neuronal cells from apoptosis induced by suppression of apoptosome formation (67). The other is that different types of stimuli such as trophic factor deprivation and HSP70 involves the chaperone-mediated import of precursor 6-hydroxydopamine. NO has been shown to prevent apoptosis proteins into the mitochondria, thus inhibiting cytochrome c

371 in vivo 18: 367-376 (2004) release (68). Heme oxygenase is the rate-limiting enzyme in nitrosative modification of caspase-3 and -1 has also been heme catabolism, which leads to the generation of biliverdin, identified in vivo (64). By suppressing caspase activity free iron and carbon monoxide (CO). Bilirubin has proven through redox-based post-translational modification, NO to be a potent antioxidant (66). This property is ascribed to produced either by NO donor or nNOS transfection inhibits a powerful redox cycle based on the reduction of biliverdin apoptotic cell death in PC12 cells (32) and primary cultured to bilirubin by biliverdin reductase (69). This redox cycle may cerebrocortical neurons (81). These findings have important constitute the principal cytoprotective function of bilirubin implications for the regulation of neuronal apoptosis by against oxidative brain injury. Free iron, the second catalytic post-translational nitrosative modification of caspases. by-product, can also be considered cytotoxic secondary to its A recognized hallmark of cytotoxic neuronal death seems pro-oxidant properties based on Fenton chemistry. However, to be massive influx of Ca2+ by NMDA receptor activation release of iron from heme stores via heme oxygenase (82, 83). Glutamate neurotoxicity has been implicated in the induction up-regulates ferritin biosynthesis and subsequently underlying neuronal damage found in cerebral ischemia, as induces the net effect of decreasing intracellular free iron, well as in the pathogenesis of different neurodegenerative thus acting as an antioxidant (70). The third catalytic gaseous diseases (83, 84). The NMDA receptor contains redox- product, carbon monoxide, can also activate soluble modulatory cysteine residues, which can be post- guanylate cyclase by binding its heme moiety and translationally modified by NO (85, 86). S-nitrocysteine, subsequently increases the intracellular level of cGMP. The which generates NO+ equivalents, inhibited NMDA receptor sGC/cGMP pathway mediates the effects of CO on vascular activity as demonstrated by whole-cell recording (86) or by relaxation, neurotransmission and the inhibition of platelet digital calcium imaging (11). During single-channel recording, aggregation, and smooth muscle proliferation. CO S-nitrosocysteine decreased the opening frequency of NMDA potentially influences other intracellular signal transduction receptor-operated channels in outside-out patches from pathways. It has been demonstrated that CO can protect cerebrocortical neurons (87). These observations indicate that apoptotic cell death by the selective activation of the post-translational modification of NMDA receptor by NO MKK3/p38‚ protein MAP kinase pathway (71), the can prevent neuronal PCD by suppressing Ca2+ influx. activation of NF-Î B (72) and increase in the ratio of Bcl-2 The protooncogene p21ras, a monomeric G protein family to Bax (73). Modulation of the p38 MAPK pathway can member, is a guanine nucleotide-binding protein that cycles modulate neuronal cell apoptosis, probably by activating the between inactive GDP-bound and active GTP-bound states to downstream signal mediator of protein kinase-myocyte regulate a diverse array of cellular processes, including cell enhancer factor 2 transcription factor (74). growth, apoptosis and differentiation. The guanine nucleotide- bound state of Ras is tightly maintained by regulatory factors Post-translational modification of caspases. It has been to promote regulated growth control. p21ras possesses a clearly shown that NO can covalently modify redox-sensitive critical site of redox regulation at cys118, which can be S- cysteine residues and transition metals in proteins through nitrosylated (88-90), and triggers guanine nucleotide exchange three chemical reactions: nitrosylation, oxidation (RS-SR) and downstream signaling including phosphatidylinositol 3- and ADP ribosylation (75). These post-translational kinase (PI3K)/Akt (91). When recombinant wild-type p21ras modifications serve in the regulation of cellular responses and mutant p21rasC118S were incubated with NO donor in (75, 76). Particularly, redox-based S-nitrosylation of proteins vitro, S-nitrosylated modification was identified in wild-type or non-protein thiols can occur both in vitro and in vivo (64, protein, but not in the mutated p21 (92). A physiological level 77). S-nitrosylation regulates the gene expression and of NO produced by nNOS or NO donor prevented neuronal cellular homeostasis through an alteration in protein cell apoptosis by post-translational S-nitrosylation of p21ras function (64, 75, 78). (90) and the protective effect was reduced by Ras inhibitor, Caspases, a family of cysteine proteases consisting of 14 Erk blocker or PI3K inhibitor. This suggests that Erk and isoforms, play an essential role in the apoptotic signal PI3K/Akt are involved in the downstream signal cascade of cascade. All caspases contain a single redox-sensitive p21ras (93). Furthermore, NO could not prevent apoptotic cell cysteine at the enzyme catalytic site. This thiol is susceptible death of neuronal PC12 cells, which were transfected with to redox modification in the presence of NO or related mutant p21ras C118S (90). Thus, post-translational S- molecules with NO+-like characteristics. We have shown nitrosylation of Ras may be an important event of NO- that seven members of the recombinant caspases are mediated anti-apoptosis of neuronal cells. susceptible to reversible inhibition by NO through this The post-translational S-nitrosylation of NMDA receptor redox-based post-translational modification (79). The and caspases inhibits their biological activities and stoichiometric ratio of the enzyme to S-nitrosylation is contributes to NO-mediated neuroprotection. On the other found to be 1: 1 in caspase-3 and -8 following treatment hand, S-nitrosylation of p21ras increases its enzymatic with NO donor (16, 80). Evidence for post-translational activity, in some way akin to phosphorylation, resulting in

372 Kang et al: Regulation of Neuronal Cell Apoptosis by Nitric Oxide anti-apoptotic signal pathways. Thus, this post-translational exogenous NO through a slow-releasing NO donor (101). modification is gaining acceptance as a newly-recognized These results indicate that CREB phosphorylation is a molecular switch to control protein function via reactive downstream signal mediator of the NO/cGMP pathway. It thiol groups. In addition, pharmacological intervention with was demonstrated that PKG can phosphorylate CREB NO produced by NOS gene transfection or chemical NO directly in vitro and as a consequence of NO-dependent donors could be implemented in the treatment of cGMP activity in vivo (102). Thus, in neuronal cells, CREB neurological disorders associated with unwanted cell death. phosphorylation by the NO/cGMP pathway may play an essential role in NO-mediated survival in neuronal cells. Anti-apoptotic signal cascade by NO. Although NO and its related molecules prevent apoptotic cell death via changes in Conclusion cellular redox potential and post-translational S-nitrosylation of proteins, NO-mediated cGMP production has been shown NO and its reaction product cGMP can regulate cellular to prevent apoptosis in PC12 cells (32, 35), motor and homeostasis in order to maintain the balance between the sympathetic neurons (31, 35) and cultured hippocampal induction and prevention of PCD in neuronal cells. neurons (53). The anti-apoptotic effect of cGMP may be Abnormal homeostasis of neuronal cells is linked to many associated with PKG activation in trophic factor-deprived human diseases, such as formation of brain tumors, which PC12 cells (32). Consistent with these previous reports, we may be initiated by the suppression of apoptosis. On the found that the concentration of NO adequate to activate other hand, hyperapoptosis of neuronal cells may influence soluble guanylate cyclase protects PC12 cells from apoptotic such neuronal diseases as ischemic neuronal disease, death via a cGMP-dependent mechanism (32). Although NO traumatic brain injury, Parkinson's disease and Alzheimer's can suppress apoptosis by post-translational modification of disease. NO and/or cGMP induces anti-apoptotic gene caspases, NMDA receptor and p21ras, this appears, in part, to expression, post-translational S-nitrosylation of caspases, occur in PC12 cells. Instead, we clearly showed that the NMDA receptor and p21ras, and activation of anti-apoptotic NO/cGMP/protein kinase G pathway inhibits 6- signal pathways, which are central mediators of the PCD hydroxydopamine (6-OHDA)-induced undifferentiated and signal cascade in multiple biological and pathological differentiated PC12 cell apoptosis by a PKG-dependent processes. These events result in the suppression of activation of the PI3K/Akt pathway, resulting in Bad apoptosis of neuronal cells. In addition, NO induces phosphorylation and subsequent suppression of cytochrome c nitrosative stress, which activates mitochondria-dependent release and caspase-3 activation (94). This mechanism was apoptotic signaling pathways that involve the activation of further supported by the observations that the anti-apoptotic caspases and release of AIF, Smac and endonuclease G. effect of the membrane permeable cGMP analog 8-Br-cGMP These data indicate that NO, produced either by was blocked both by PKG inhibitor and PI3K inhibitors. Thus, endogenous biological enzymes or exogenous NO donors, it demonstrated that the NO/cGMP pathway suppresses PC12 can serve as an in vivo regulatory mechanism of neuronal cell apoptosis by Bad phosphorylation and inhibition of PCD. Thus, regulation of NO production may prove to be cytochrome c release and caspase-3 activation through a PKG- a pertinent therapeutic target for various neuronal diseases dependent activation of the PI3K/Akt pathway. where unwanted PCD is involved. The transcription factor cAMP-response element-binding protein (CREB) activation through phosphorylation has Acknowledgements been implicated in neuronal survival (95-97) and it has recently been demonstrated that disruption of CREB This work was support by the Vascular System Research Center function in the brain leads to (98). grant from KOSEF. Death of cerebellar granule neurons due to NO deprivation has also been shown to be accompanied by down-regulation References of CREB activity (99). Interestingly long-lasting exposure to the NOS inhibitor L-NAME caused apoptosis in cultured 1 Ignarro LJ, Buga GM, Wood KS, Byrns RE and Chaudhuri G: cerebellar granule cells (100) and neuroblastoma cells (99) Endothelium-derived relaxing factor produced and released by down-regulating the serine/threonine kinase (Akt) from artery and vein is nitric oxide. Proc Natl Acad Sci USA survival pathway and CREB transcription factor activity, 84: 9265-9269, 1987. respectively. Indeed, CREB phosphorylation in nNOS- 2 Nathan C: Nitric oxide as a secretory product of mammalian cells. FASEB J 6: 3051-3064, 1992. overexpressing neuroblastoma cells was strongly reduced by 3 Mayer B, John M and Bohme E: Purification of a concomitant exposure to the NOS inhibitor L-NAME or the Ca2+/calmodulin-dependent nitric oxide synthase from porcine specific inhibitor of soluble guanylate cyclase ODQ, and the cerebellum. Cofactor-role of tetrahydrobiopterin. 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