Nitric Oxide in Health and Disease of the Nervous System H-Y Yun1,2, VL Dawson1,3,4 and TM Dawson1,3

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PROGRESS Nitric oxide in health and disease of the nervous system H-Y Yun1,2, VL Dawson1,3,4 and TM Dawson1,3

Departments of 1Neurology; 3Neuroscience; 4Physiology, Johns Hopkins University School of Medicine, Baltimore, MD, USA

Nitric oxide (NO) is a widespread and multifunctional biological messenger molecule. It mediates vasodilation of blood vessels, host defence against infectious agents and tumors, and neurotransmission of the central and peripheral nervous systems. In the nervous system, NO is generated by three nitric oxide synthase (NOS) isoforms (neuronal, endothelial and immunologic NOS). Endothelial NOS and neuronal NOS are constitutively expressed and activated by elevated intracellular calcium, whereas immunologic NOS is inducible with new RNA and protein synthesis upon immune stimulation. Neuronal NOS can be transcriptionally induced under conditions such as neuronal development and injury. NO may play a role not only in physiologic neuronal functions such as neurotransmitter release, neural development, regeneration, synaptic plasticity and regulation of gene expression but also in a variety of neurological disorders in which excessive production of NO leads to neural injury. Keywords: nitric oxide synthase; endothelium-derived relaxing factor; neurotransmission; neurotoxicity; neurological diseases

Nitric oxide is probably the smallest and most versatile NO synthases isoforms and regulation of NO bioactive molecule identified. Convergence of multi- generation disciplinary efforts in the field of immunology, cardio- vascular pharmacology, chemistry, toxicology and neu- NO is formed by the enzymatic conversion of the guan- robiology led to the revolutionary novel concept of NO idino nitrogen of l-arginine by NO synthase (NOS). as an unorthodox messenger.1–3 The discovery of NO Three NOS isoforms, neuronal NOS (nNOS; type I), as a messenger molecule in the nervous system4 also immunologic NOS (iNOS; type II) and endothelial NOS revised conventional concepts of neurotransmitters. (eNOS; type III) have been isolated and cloned5–8 NO is freely diffusible across membranes and is not (Table 1). All three isoforms are expressed in the cen- stored in synaptic vesicles and released by exocytosis tral nervous system (CNS). NO can also be generated upon membrane depolarization. NO seems to be ter- nonenzymatically by the direct reduction of nitrite to minated primarily by reaction with its targets. Thus, NO under acidic and highly reduced conditions that control of NO synthesis, by NOS, is the primary way may occur under ischemic conditions.9–10 nNOS and to regulate NO actions. Rapid progress is being made eNOS are calcium/calmodulin-dependent and under in understanding the regulation of NOS activity and most conditions are constitutively expressed. However, the cellular and molecular targets of NO under physiol- recent studies indicate that both enzymes can be ogic and pathologic conditions. Some of the emerging induced (ie new protein synthesis) under conditions roles of NO in the nervous system include regulation or of stress, injury and differentiation.11 iNOS is calcium- control of neuronal morphogenesis, short-term or long- independent and its expression is primarily regulated term synaptic plasticity, regulation of gene expression at the level of transcription. Interestingly, in the and modification of sexual and aggressive behavior. absence of the substrate, l-arginine, nNOS is able to − Deregulated formation of NO from all three NOS iso- produce superoxide anion (O2· ) and hydrogen per- forms plays a role in neural injury in a host of neuro- oxide.12,13 Under conditions of reduced arginine con- logical disorders. centrations substantial amounts of superoxide may be 14 − generated by NOS. NOS generation of O2· and NO in arginine-depleted cells can lead to peroxynitrite- mediated cellular injury.15 Since NO is freely diffusible, highly reactive, and synthesized on demand without being stored, its gener- Correspondence: Dr TM Dawson, Department of Neurology, Johns ation is the only way by which it is regulated. Thus, Hopkins University School of Medicine, 600 N Wolfe St there is a multiplicity of regulatory sites on the Pathology 2-210, Baltimore, MD 21287, USA. E-mail: enzymes that synthesize NO. Consensus sites for phos- ted dawsonȰqmail.bs.jhu.edu 2Present address; Department of Biochemistry, Chung-Ang Uni- phorylation are evident in all NOS isoforms. Although versity College of Medicine, Seoul, Korea there are some discrepancies, it appears likely that Received and accepted 27 February 1997 nNOS catalytic activity is decreased by phosphoryl- Nitric oxide in the nervous system H-Y Yun et al 301 Table 1 NOS isoforms

Type 1 nNOS Type II iNOS Type III eNOS

Ca2+ dependency Activity depends on elevated Activity is independent of Activity depends on elevated Ca2+ elevated Ca2+ Ca2+ First identified in Neurons Macrophages Endothelial cells Expression Constitutive Inducible Constitutive Inducible under appropriate Inducible under appropriate conditions conditions Putative AP-2, TEF-1/MCBF IFN-␥, TNF-␣, NF-␬B AP-1, AP-2, NF-1 transcription CREB/ATF/cFos A-activator-binding Heavy metal acute phase regulatory sites NRF-1, Ets, NF-1 response, Shear stress and NF-␬B sterol regulatory elements Subcellular Soluble or membrane-bound Soluble or membrane-bound Largely membrane-bound localization Sarcoplasmic membrane localization in skeletal muscle Phenotypes of null Pyloric stenosis Increased susceptibility to Deficient acetylcholine mutant mice Resistant to vascular stroke Listeria and Leishmania vasodilation Resistant to MPTP neurotoxicity infection and lymphoma cell Elevated mean blood pressure Excessive sexual and aggressive proliferation l-nitroarginine-induced behavior Resistant to endotoxin hypotension Urinary bladder hypertrophy hypotension and carrageenan Increased infarct volume and loss of urethral and inflammation following MCA occlusion bladder muscle relaxation Reduced infarct volume by NO Normal hippocampal LTP and inhibitor in MCA occlusion cerebellar LTD Reduced hippocampal LTP in nNOS and eNOS double knockouts

ation by protein kinase A (PKA), protein kinase C tochemistry has localized nNOS to the Golgi apparatus, (PKC), cyclic GMP-dependent protein kinase (PKG) endoplasmic reticulum, spines, dendritic shafts and and calcium-calmodulin protein kinase (CAM-k).16,17 axon terminals.24,25 In addition, nNOS and the NMDA- Calcineurin, a calcium-calmodulin dependent protein receptor subunits are colocalized in nNOS neurons.26 phosphatase, is able to dephosphorylate NOS and sub- The presence of a PDZ domain (DHR domain or GLGF sequently increase its catalytic activity.18 The immuno- repeat) in nNOS27 and its role in the membrane associ- suppressant and calcineurin inhibitor, FK506 ation of nNOS in skeletal muscle28 may provide insight (Tacrolimus), protects cultured cortical neurons into regulation of nNOS. PDZ domain interactions may against glutamate neurotoxicity by maintaining the mediate binding of nNOS to synaptic junctions through phosphorylated, catalytically inactive state of nNOS.18 interactions with postsynaptic density-95 protein NO formation is also tightly regulated by controlling (PSD-95) and PSD-93.29 Skeletal muscle membrane the levels of various necessary cofactors within the from patients with Duschene Muscular Dystrophy cell.19 Recent studies identiﬁed a novel protein, PIN, which lack dystrophin also lack nNOS.28,30 nNOS is which interacts with and potently inhibits the activity also signiﬁcantly reduced in the skeletal muscle from of nNOS.20 PIN destabilizes the nNOS dimer, a confor- mdx mice, which do not express full-length dystro- mation necessary for activity. The highly conserved phin. nNOS mRNA, protein and enzymatic activity are sequence of PIN suggests it plays a role in regulating all markedly reduced in mdx mouse skeletal muscle,31 numerous NO-related biological processes. Regulation suggesting a role of NO in maintaining integrity and of iNOS primarily occurs at the level of gene transcrip- functional regulation of skeletal muscle membranes. tion and analysis of the promoter region indicates that iNOS has a complex pattern of regulation.21,22 iNOS Biochemistry of NO expression is induced in response to cytokines, lipopolysaccharide (LPS) and a host of other agents.19 In NO is considered a highly reactive free radical due to the nervous system, NO generated by nNOS in glial its short half-life (3–6 s) in comparison to traditional cells negatively regulates iNOS expression through neuronal messenger molecules. NO, once produced, inhibition of NF␬B.23 does not persist in its native form for very long, in that NOS may also be regulated by changes in its intra- it is either rapidly oxidized, reduced, or complexed to cellular localization. Electron microscopic immunohis- various biomolecules (Table 2). The biological outcome Nitric oxide in the nervous system H-Y Yun et al 302 Table 2 Cellular targets of NO

Molecules Actions Cellular consequences

Guanylyl cyclase Activation Increase cGMP and possible neuroprotection Ras Activation Activation of extracellular signal-activated protein kinase and possible neuroprotection NADPH-ubiquinone oxidoreductase Inhibition Decreased oxidative phosphorylation NADH-succinate oxidoreductase Inhibition Decreased oxidative phosphorylation cis-Acotinase Inhibition Decreased glycolysis Cytochrome oxidase Inhibition Decreased oxidative phosphorylation Creatine kinase Inhibition Decreased availability of ATP DNA DNA strand break Depletes NAD and ATP which activate PARS − − − O2 Forms ONOO ONOO is a potent oxidant which is a major endogenous neurotoxin

− of NO is critically dependent upon the local micro- O2· reacts with NO to produce the potent oxidant environment in which it is generated. peroxynitrite.40–42 This reaction is much faster than the Thiols are commonly assumed to be major targets for reaction of the enzyme, superoxide dismutase (SOD) NO. Nitrosothiols with biological relevance have been that catalyzes the dismutation of the superoxide anion isolated and characterized including S-nitrosogluta- to hydrogen peroxide. Thus, NO can effectively out- 32 − thione and the nitrosothiol of serum albumin. Vari- compete SOD for the O2· . Peroxynitrite can readily ous oxidation states of NO exist, and, thus probably react with sulfhydryls and with zinc thiolate moieties, influence its biologic function.33 In addition to the free directly nitrate and hydroxylate aromatic amino acid radical form, there are oxidized and reduced forms, residues, and it can also oxidize lipids, proteins and NO+ (nitrosonium ion) and NO− (nitroxide ion), DNA. Thus, peroxynitrite is thought to be a key respectively, of NO. All three oxidation states of NO mediator of NO-mediated cytotoxicity. may exist in the brain and, in part, may be responsible Nitrosylation of many proteins appears to be a poten- for some of the conflicting activities attributed to NO. tially important regulatory system accounting for some The NO free radical (NO·) is probably the form that of NO’s physiologic actions. Protein-associated targets exists in solution and is thought to be most likely the of NO include heme, cysteine (Cys) and tyrosine (Tyr) form of NO that is produced on stimulation of NOS. residues. At physiological pH, Cys residues are NO binds to the heme moiety of guanylyl cyclase to efficiently nitrosylated, while other reactions such as induce a conformational change which activates the further oxidation of nitrosothiol to sulfonic acid and enzyme and results in cGMP formation. NO increases nitration of Tyr occur at a much slower rate.43 NO may prostaglandin production by activating another heme- physiologically modulate glutamatergic neuro- containing enzyme, cyclooxygenase.34 NO reacts with transmission through nitrosylation of the NMDA recep- non-heme iron particularly with iron sulfur clusters in tor. 33 However, other studies suggest that NO inhibits numerous enzymes. Peroxynitrite, a reaction product NMDA-induced currents through an interaction with − of NO and O2· , rather than NO, may react with iron cations rather than through the redox modulatory site sulfur clusters.35 These reactions readily occur after of the NMDA receptor channel.44 NO reacts with an macrophage activation. However, in contrast with the active site cysteine of glyceraldehyde-3-phosphate reversible reaction of NO with heme, the reaction of dehydrogenase (GAPDH) resulting in the direct bind- NO with iron sulfur clusters results in the dissolution ing of NAD to cysteine and inhibition of GAPDH cata- of the cluster.36 Through these interactions NO may lytic activity and depression of glycolysis.45,46 NO also inhibit oxidative respiration and cause cytotoxicity. In reacts with cysteine residue of hemoglobin in the lung the presence of NO cytosolic aconitase functions as the forming an S-nitrosyl hemoglobin.47 NO is released iron-responsive element binding protein; and in its from S-nitrosyl hemoglobin during arterial-venous absence it functions as a cytosolic aconitase.37,38 NO transit and appears to control blood pressure, which disrupts aconitase activity and exposes its RNA bind- may facilitate efficient oxygen delivery to tissues. ing site permitting binding of the iron-responsive NO reversibly inhibits the growth of rat dorsal root element binding protein to the iron-responsive ganglion neurites.48 This may be due to inhibition by element.39 Production of NO, through stimulation of NO of thioester-linked long-chained fatty acylation of NMDA receptors in rat brain slices, stimulates the neuronal proteins probably through a direct modifi- RNA-binding function of the iron-responsive element cation of substrate cysteine thiols in the neuronal binding protein while diminishing its aconitase growth cone. The role of calcineurin and its inhibitor, activity. The iron-responsive element binding protein immunosuppressant FK506 in neurite outgrowth49,50 has a discreet neuronal localization, thus it may be an further implicates NO’s participation in growth cone important molecular target for NO action in the brain.39 dynamics since calcineurin enhances NOS activity by Nitric oxide in the nervous system H-Y Yun et al 303 maintaining it in a dephosphorylated, catalytically ive postures by wild-type males nor do they elicit sub- active state.18 missive postures in response to attack by another mouse.63 These behaviors may be a selective conse- quence of the loss of nNOS, since nNOS null mice have NO-mediated neural function no detectable abnormalities in brain structure53 and Studies on the peripheral nervous system have pro- have normal synaptic plasticity in the hippocampus64 vided the best evidence for a transmitter role of NO. and cerebellum.65 Furthermore, plasma testosterone NOS inhibitors selectively block non-adrenergic and levels, which could account for both aggression and non-cholinergic (NANC) mediated relaxation of the sexual behavior, do not differ between wild-type and gastrointestinal tract.51 These data coupled with the nNOS null mice.63 In newt, brain NOS activity selective localization of nNOS to the myenteric plexus increases transiently during males’ courtship behavior, indicate that NO functions as the NANC neuro- suggesting that NO’s role in reproductive behavior may transmitter.52 Studies of gastrointestinal function in be conserved throughout vertebrate evolution.66 mice with targeted disruption of the nNOS gene53 Long-term potentiation (LTP) in the hippocampus further support a role of nNOS in gastrointestinal func- and long-term depression (LTD) in the cerebellum are tion. These mice have marked enlargement of the stom- forms of synaptic plasticity that are thought to be ach and hypertrophy of the inner circular muscle layer. involved with learning and memory.67,68 Induction of NO is thought to mediate relaxation of the pyloric LTP in the CA1 region of hippocampal slices usually sphincter in response to a food bolus. The grossly dis- requires calcium influx through post-synaptic NMDA tended stomach and hypertrophy of the inner circular receptor channels and enhancement of glutamate muscle layer is felt to be a compensatory response due release by Schaeffer collaterals and is thought to to the inability of the pyloric sphincter to properly require release of a retrograde messenger from postsyn- relax in response to a food bolus. The pathology aptic CA1 pyramidal cells. Support for the role of NO resembles hypertrophic pyloric stenosis, in which the as a retrograde messenger includes studies in hippo- lack of NO may play a role since a recent study shows campal neurons in which NO produces an increase in an absence of NADPH diaphorase staining in the the frequency of spontaneous miniature excitatory myenteric neurons in human male infants with pyloric post-synaptic potentials and observations that direct stenosis.54 Furthermore, the nNOS gene is a suscepti- application of NO elicits LTP. Furthermore, NOS bility locus for the infantile pyloric stenosis.55 In circu- inhibitors interfere with the establishment of LTP as lar smooth muscle of gastric fundus, NO acts as an does hemoglobin which binds NO and some NOS inhibitory neurotransmitter that mediates the slow inhibitors block LTP when directly injected into CA1 component of inhibitory junction potentials.56 pyramidal neurons.69–72 Intracellular application of an nNOS is highly expressed in the pelvic plexus in NO scavenger or use of a UV-sensitive NO donor also axonal processes including the cavernosal nerve and supports the contention that postsynaptically gener- plexus. nNOS is also present in the adventitia of the ated NO acts directly on the postsynaptic neuron to deep cavernosal arteries and sinusoids in the periphery produce LTP.73 Similar evidence exists for a role of NO of the corpus cavernosa. NO functions as the NANC in LTD.74,75 Genetic knockout of nNOS or eNOS failed neurotransmitter that mediates penile erections. Penile to support NO’s role in LTP. LTP in nNOS null or erections elicited by electrical stimulation of the eNOS null mice appeared normal and is still blockable cavernous nerve are blocked by low doses of NOS by NOS inhibition.64,76 The role of NO in LTP has been inhibitors in intact rats and NOS inhibitors block clarified by Kandel and colleagues who recently gener- NANC-stimulated relaxation of isolated cavernosal ated double knockout mice lacking both nNOS and strips.57,58 nNOS null mice display hypertrophied uri- eNOS expression and found that hippocampal LTP in nary bladders and loss of neurally mediated relaxation the stratum radiatum of CA1 was markedly reduced.76 of urethral and bladder muscle providing a model for These mutant mice showed normal LTP in the stratum idiopathic voiding disorder in humans.59 Additionally oriens of hippocampal CA1. Thus, there is both NO- NO may regulate both sympathetic and parasympa- dependent and NO-independent of LTP. eNOS may thetic outflow.60 play a primary role in NO-mediated LTP in wild-type NO may play a role in modulating behavior. Inhi- mice as adenovirus-mediated disruption of eNOS bition of NOS attenuates alcohol consumption in alco- activity in wild-type hippocampal slices eliminates hol-preferring rats and prevents the acquisition of tol- NO-dependent LTP.77 In honeybee, inhibition of NOS erance to the ataxic effects of alcohol.61 NO may be impairs a specific form of memory formed by olfactory involved in light phase shifts of circadian rhythm in conditioning carried out by multiple learning trials.78 that NOS inhibitors block light-induced resetting of Thus, involvement of NO in learning and memory may behavioral rhythms.62 Studies in nNOS null mice pro- be conserved throughout the animal kingdom. vide further insight into the behavioral roles of NO. NO influences neurotransmitter release presumably Male nNOS null mice exhibit inappropriate sexual through cGMP activation of cGMP-dependent protein behavior, mounting females indiscriminately regard- kinases to augment the phosphorylation of synaptic less of whether the females are in estrus or not. Fur- vesicle proteins associated with neurotransmitter thermore, male mice when housed together, fight release. The role of NO production in NMDA receptor- incessantly and do not respond to appropriate submiss- mediated neurotransmitter release is evident from the Nitric oxide in the nervous system H-Y Yun et al 304 studies in which inhibitors of NOS block NMDA recep- hypertrophy and ectopic NOS expression in larvae has tor-mediated neurotransmitter release from the cerebral the opposite effects, implicating NO as an antiprolifer- cortex or striatal synaptosomes.78,80 NO is involved in ative signal during development.90 The molecular glutamate or norepinephrine-stimulation and GABA- mechanism for NO’s role in neural development is not inhibition of hypothalamic luteinizing hormone-releas- known. One intriguing hypothesis is that activation of ing hormone (LHRH) release.81 In synaptosomes neuro- mitogen-activated protein kinase (MAPK) pathway by transmitter release evoked by stimulation of NMDA NO may be a key to how NO regulates neuronal growth, receptors is blocked by NOS inhibitors whereas release differentiation, survival and death (Figure 1).91,92 Our by potassium depolarization is unaffected. Presumably, recent studies indicate that stimulation of NMDA potassium depolarizes all terminals so that the effects receptor in cultured cortical neurons activates Ras-ERK of NO are diminished due to the limited population of pathway via calcium-dependent activation of nNOS NOS-containing terminals. The mechanism of how NO and NO generation.93 NMDA-stimulated phosphoryl- influences neurotransmitter release is unclear. How- ation of cyclic AMP-response element binding protein ever, NO may act on both calcium-sensitive and cal- (CREB), a downstream effector of ERK, is also NO- cium-insensitive pools of synaptic vesicles.82 NO’s dependent.92 Activation of Ras/ERK pathway by NO actions on neurotransmitter release could involve cyc- may be mediated by direct activation of Ras GTPase lic GMP activation of cyclic GMP-dependent protein activity presumably by nitrosylation of cysteine kinase to augment phosphorylation of synaptic vesicle through a redox-sensitive interaction.93,94 Since cal- proteins or through activation of cyclic GMP depen- cium-dependent activation of Ras-MAPK pathway is dent-cation channels.83 Recent studies indicate that NO thought to be a major pathway of neural activity- may facilitate vesicle docking/fusion events by promot- dependent long-term changes in nervous system,95 NO ing VAMP/SNAP-25/syntaxin 1a complex formation may be a key mediator linking activity to gene and by inhibiting n-sec1 binding to syntaxin 1a.84 This expression and long-term plasticity. NO donors acti- effect of NO may be mediated by nitrosylation of vate all three types of MAPK (ERK, JNK/SAPK, p38 sulfhydryl groups of protein(s) involved. Thus, there MAPK) with different extent and time courses in T cell may be multiple molecular targets of NO acting in con- leukemic cell lines.91 ERK and JNK/p38 MAPK path- cert to modulate neurosecretion. ways are implicated in neuronal survival and death, NO may play a key role in nervous system morpho- respectively.96 Since the MAPK signaling pathway genesis and synaptic plasticity. nNOS is transiently expressed in the cerebral cortical plate neurons from embryonic day E15–E19 of rats.85 These cells extend their processes through to the corpus striatum and thalamus. This intense labeling of the cortical plate neurons and their processes decreases rapidly and van- ishes by the 15th postnatal day. Embryonic sensory ganglia are nNOS positive and this decreases to less than 1% of the cells at birth. Thus, the transient expression of NOS may reflect a role in developmental processes. Molecular maturation of adult spinal cord motor neurons may involve NO.86 NO may also play a role in the development of proper patterns of connections in the retinotectal system as NOS expression peaks at the time when refinement of the initial pattern of connections is occurring.87 Neurons of the developing olfactory epithelium during migration and the establishment of primary synapses in the olfactory bulb of rats express nNOS during embryonic development.88 Olfactory nNOS expression rapidly declines after birth and is undetectable by postnatal day 7. In addition, nNOS expression is rapidly induced in the regenerating olfactory receptor neuron after bulbec- tomy and is particularly enriched in their outgrowing axons. Thus, NO may play a role in activity-dependent establishment of connections in both developing and regenerating olfactory neurons. During neuronal differentiation nitric oxide may trigger a switch to growth arrest during differentiation of neuronal cells; and thus, NOS serves as a growth arrest gene initiating the 89 switch to cytostasis during differentiation. In Droso- Figure 1 A schematic diagram showing the role of NO in phila, NOS is highly expressed in developing imaginal coupling NMDA receptor-mediated calcium influx to Ras/Erk discs and inhibition of NOS in larvae causes organ pathway. (Reproduced with permission).92 Nitric oxide in the nervous system H-Y Yun et al 305 plays a central role in growth factor response (ERK) or generative diseases such as Huntingdon’s disease, Alz- stress response (JNK, p38 MAPK) in the nervous sys- heimer’s disease and vascular stroke.111–114 nNOS neu- tem, NO-MAPK signaling may underlie NO’s role in rons may possess protective factors that render them neuronal survival, differentiation and apoptotic cell resistant to the toxic effects of NO. To address this puz- death during development. zling phenomenon, we developed an NO-resistant PC12 cell line and determined using sequential analysis of gene expression that MnSOD accounts for the NO in neurological disorders resistance to NO. We then showed that nNOS neurons Deregulated excess generation of NO can initiate a neu- in the cortex are enriched in manganese SOD (MnSOD) rotoxic cascade.97 Excess glutamate acting via NMDA which may prevent the local formation of toxic peroxy- receptors mediates cell death in focal cerebral nitrite, rendering nNOS neurons resistant to NO’s toxic ischemia.98 Glutamate neurotoxicity may also play a actions.115 Overexpression of MnSOD by recombinant part in neurodegenerative diseases such as Hunt- adenovirus in cultured cortical neurons markedly ington’s disease and Alzheimer’s disease.99 NMDA increased resistance to NMDA-mediated cell death.115 applied only for a short (5 min) period of time is able Furthermore, antisense knockdown of MnSOD renders to elicit cell death in cortical cultures assessed 24 h only nNOS neurons more susceptible to NO and later. This type of toxicity has been called ‘delayed or NMDA toxicity. Thus, MnSOD is the major protective rapidly triggered neurotoxicity’ in which irreversible protein in nNOS neurons. Other putative protective processes are set in motion by the 5-min application of factors may exist in nNOS neurons and may represent NMDA.100 This type of toxicity is exquisitely depen- specifically expressed gene products in these cells. dent upon calcium. Furthermore, calcium influx via NO presumably kills neurons via peroxynitrite.116 − NMDA receptors elicits more potent toxicity than other NO interacts with the O2· to form the toxic radical per- modes of calcium entry. This type of toxicity involves oxynitrite. This powerful oxidant is thought to mediate NO since treatment with NOS inhibitors, removal of l- most NO neurotoxicity. Peroxynitrite may further arginine or reduced hemoglobin which scavenges NO, decompose to hydroxyl and nitrogen dioxide radicals blocks this form of toxicity.101 Further evidence of the in vivo117 which are also highly reactive and biologi- role of NO in NMDA neurotoxicity comes from the cally destructive. studies in nNOS null mice which are relatively resist- NOS inhibitors block ischemic damage following ant to NMDA neurotoxicity as well as combined oxy- middle cerebral artery (MCA) ligation in several animal gen and glucose deprivation.102 Despite the critical evi- models.118 The notion that neuronally derived NO dence for a role of NO in glutamate neurotoxicity, plays a role in neural damage following stroke is sup- others have failed to confirm that NO is necessary for ported by the studies in nNOS null mice. These mice glutamate neurotoxicity.103–105 These discrepancies have reduced infarct volumes compared to age- may be due to the finding that NO may possess either matched wild-type controls following permanent neurodestructive and neuroprotective properties, middle cerebral artery occlusion.119 Inhibition of eNOS depending on its oxidation-reduction status, with NO· may account for the deleterious effects of NOS inhibi- being neurodestructive and NO+ being neuroprotec- tors. MCA occlusion of eNOS null mice leads to larger tive.33 Furthermore, recent studies indicate that the infarcts than in wild-type mice.120 This effect is asso- conditions under which neurons are cultured can pro- ciated with reduction in cerebral blood flow in the foundly influence the expression of nNOS and sub- ischemic penumbra region. The selective nNOS inhibi- sequent involvement of NO in excitotoxicity.106 The tor, 7-nitroindazole, which has a minimal effect on cer- expression of nNOS in primary cultures at levels equi- ebral blood flow is neuroprotective in animal models valent to in vivo expression (1–2% of the neuronal of focal ischemia.121 Furthermore, when the nNOS null population) is critical for the ability to observe NO- mice are treated with the NOS inhibitor, nitro-l-argi- mediated neurotoxicity. In primary cultures the nine methyl-ester, there are adverse effects on cerebral expression of nNOS is dependent on both the method blood flow and stroke volume is increased. Conversely, of culture and the age of the cultures.106,107 Neurotro- when eNOS null mice are treated with NOS inhibitors phins, despite their general role in attenuating excito- during MCA occlusion, infarct volume is reduced.122 toxic neuronal injury, were recently shown to increase Thus, neuronally derived NO plays an important role nNOS neurons in cortical culture grown on glial feeder in mediating neuronal cell death following focal layers and render neurons more sensitive to NMDA.106 ischemia and endothelial-derived NO plays an This finding is consistent with a previous report by important protective role by regulating and main- Choi and colleagues on the enhancement of excitotoxic taining proper cerebral blood flow.121–123 In the late neuronal injury by neurotrophins under certain con- stage of cerebral ischemia (Ͼ6 h), post-ischemic ditions.108 However, when neurons are grown on a inflammation induces iNOS expression and sustained poly-ornithine matrix, neurotrophins failed to enhance generation of large amounts of NO leads to delayed the expression of nNOS and are neuroprotective, con- neural injury.124 Supporting this idea is the observation sistent with the neuroprotective role of neurotrophins that iNOS null mice have smaller infarcts after cer- in neurotoxicity studies in intact animals.109,110 ebral ischemia.125 nNOS neurons are resistant to a variety of toxic NO may mediate other forms of neurotoxicity. NOS insults including NMDA neurotoxicity and neurode- inhibitors protect against CNS oxygen toxicity.126 Nitric oxide in the nervous system H-Y Yun et al 306 Selective nNOS inhibitors protect against the dopami- teins to inhibit the calcium-calmodulin dependent pro- nergic neurotoxin, MPTP, which is a model of Parkin- tein phosphatase, calcineurin, and inhibit its son’s disease.127,128 Further evidence of a role for neu- activation. Calcineurin dephosphorylates nNOS and ronally derived NO in Parkinson’s disease is the recent contributes to its activation. Since phosphorylation observation that mice lacking the nNOS gene129 and 7- renders nNOS catalytically inactive, FK506 and cyclos- nitroindazole-injected baboon130 are markedly resistant porin A maintain NOS in a catalytically inactive state to MPTP neurotoxicity. NO may also play a role in the and thus serve as NOS inhibitors. Both FK506 and pathogenesis of AIDS dementia. Both neuronally and cyclosporin A are neuroprotective against NMDA- immunologically derived NO may contribute to AIDS induced neurotoxicity in cortical cultures.18 The physi- dementia. The HIV coat protein, gp120, which is shed ologic relevance of this observation was recently con- by the virus, kills neurons in primary cortical cultures, firmed by the report that FK506 is profoundly neuro- in part, via the activation of nNOS.131 Additionally, the protective in a rat model of focal ischemia.135 coat proteins gp160, gp120 and gp41 induce iNOS in Gangliosides, which are neuroprotective in a variety of microglial and astrocyte cultures.132,133 The role of animal models, may be neuroprotective through inhi- iNOS in AIDS dementia has been established by recent bition of NOS as gangliosides inhibit NOS activity, and studies which show that iNOS is markedly induced in the protective potency of gangliosides closely parallels patients with severe AIDS dementia.134 their affinities for calmodulin and their potencies for Since NO may be involved in stroke and other neuro- inhibiting NOS.136 Other compounds such as 7-nitroin- degenerative disorders, strategies are currently being dazole and ARL17477, relatively selective nNOS developed to find selective NOS inhibitors. NOS can inhibitors, and lamotrigine, a voltage-sensitive sodium be inhibited indirectly, thus providing alternative stra- channel blocker,137 also have therapeutic potential in tegies for protection against NO-mediated cell death. NO-mediated neurotoxicity.125,137 The immunosuppressants FK506 and cyclosporin A Understanding the neurotoxic mechanisms of NO which bind small soluble receptor proteins, designated requires uncovering how NO acts on numerous poten- FK506-binding proteins and cyclophilins, respectively, tial targets to which it can initiate neurotoxic cascades inhibit NOS and are neuroprotective.18,135 Immunosup- (Figure 2). NO probably mediates most of its toxic − pressants interact with their respective binding pro- effects through interactions with O2· to form peroxy-

Figure 2 A schematic diagram of the mechanisms of NO-mediated neuronal cell death in excitotoxic neural injury. Abbrevi- ations: NMDA-R, N-methyl d-aspartate receptor; CaMK II, calcium/calmodulin-dependent protein kinase II; ICE, interleukin- 1␤ converting enzyme. Nitric oxide in the nervous system H-Y Yun et al 307 nitrite. Of the multiple mechanisms of toxic effects of tive therapeutic strategies for pathologic neuronal func- NO or peroxynitrite, the best established candidate for tions caused by malfunction of the NO system. the target of NO and peroxynitrite is poly(ADP-ribose) synthetase [PARS; also called poly(ADP-ribose) poly- Acknowledgements merase or NAD+:protein(ADP-ribosyl)transferase]. PARS is a nuclear enzyme that is involved with the VLD is supported by US PHS Grants NS 33142, NS DNA repair process.138 PARS is activated by damaged 26643, the American Foundations for AIDS Research, DNA and adds ADP-ribose units to nuclear proteins the National Alliance for Research on Schizophrenia, such as histone and PARS itself.139 PARS can add sev- the American Heart Association and the Alzheimer’s eral hundred ADP-ribose groups onto nuclear proteins Association. TMD is an Established Investigator of the and by doing so is thought to spread DNA strands American Heart Association and is supported by US apart, facilitating access for the DNA repair enzymes. PHS Grants NS 22643, NS 33277, NS 01578, and the During the process of ADP-ribose attachment, one mole Paul Beeson Faculty Scholar Award in Aging Research. of NAD is consumed and four free energy equivalents Under an agreement between the Johns Hopkins Uni- of ATP are consumed to regenerate NAD. Thus, acti- versity and Guilford Pharmaceuticals, TMD and VLD vation of PARS can rapidly deplete energy stores. are entitled to a share of sales royalty received by the PARS inhibitors are neuroprotective in several model University from Guilford. TMD and the University also systems including cortical cell cultures,140 cerebellar own Guilford stock, which is subject to certain restric- granule cell cultures141 and hippocampal slices.142 Tar- tions under University policy. The terms of this geted disruption of PARS provided compelling evi- arrangement have been reviewed and approved by the dence for participation of PARS in NO-mediated tox- University in accordance with its conflict of interest icity.143 Mutant mouse islet cells lacking PARS do not policies. show DNA damage-induced NAD depletion and are more resistant to NO toxicity. 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