The gaseous signalling molecule nitric oxide (NO) is among a number of molecules that have both autocrine and paracrine activities. During the past 20 years, thou‑sands of papers have appeared in the literature unravel‑ling the biological functions of NO, thus contributing to NO being named Molecule of the Year in 1992. In 1998, the importance of NO in the life sciences was finally underscored when the Nobel Prize for Physiology and Medicine was awarded to Robert Furchgott, Louis Ignarro and Ferid Murad for their significant contributions to this field.

NO is produced from the amino acid l‑arginine by the members of the NO synthase (NOS) family of proteins, and is involved in several cellular functions, including neurotransmission, regulation of blood‑vessel tone and the immune response. Different members of the NOS family are known to regulate different func‑tions. In the CNS, NO production is associated with cognitive function, its role spanning from the induction and maintenance of synaptic plasticity to the control of sleep, appetite, body temperature and neurosecretion1–3 (FIG. 1). In the PNS, NO regulates the non‑adrenergic, non‑cholinergic relaxation of smooth muscle cells. This has consequences for a number of tissues: smooth‑muscle relaxation in the corpora cavernosa promotes penile erection4 (FIG. 1); NO also allows the stomach to accommodate a large volume of ingested food with‑out any significant increase in intraluminal pressure; it regulates the muscle tone of intestinal sphincters;

and it has an important role in the peristalsis of the gastrointestinal tract5,6.

NO is able to interact with many intracellular targets to trigger an array of signal transduction pathways, resulting in stimulatory or inhibitory output signals. Apart from the above mentioned physiological functions, NO becomes noxious if it is produced in excess7; furthermore, if a cell is in a pro‑oxidant state, NO can undergo oxidative– reductive reactions to form toxic compounds (these belong to a family known as ‘reactive nitrogen species’ , or RNS), which cause cellular damage1,7. Recently, the term ‘nitrosative stress’ has been used to indicate the cellular damage that is elicited by excess NO and RNS (mainly peroxynitrite and nitrogen (III) oxide)8,9, and NO and RNS have been implicated in the pathogenesis of neurodegenerative disorders1,10,11. In fact, some of the initial studies carried out on NO led to the hypothesis that peroxynitrite, formed by the reaction between NO and a superoxide anion, might be responsible for the cel‑lular damage in neurodegenerative disorders; this con‑cept brings together oxidative stress and nitrosative stress and is a widely accepted explanation for the contribution of nitrosative stress to Alzheimer’s disease1,7.

Given the broad range of functions of NO, we con‑centrate in this Review on both the physiological and pathological implications of NO activity in the regula‑tion of the CNS. In particular, we focus our attention on the multifaceted functions of NO as a neuromodulator, a neuroprotective and a neurotoxic agent.

*Department of Chemistry, Biochemistry and Molecular Biology Section, Faculty of Medicine, University of Catania, Catania, Italy. ‡Institute of Pharmacology and §Department of Internal Medicine, Catholic University School of Medicine, Roma, Italy. ||Department of Chemistry, Sanders-Brown Center on Aging and Center of Membrane Sciences, University of Kentucky, Lexington, Kentucky, USA. Correspondence to V.C. e-mail: calabres@unict.itdoi:10.1038/nrn2214

Nitric oxide in the central nervous system: neuroprotection versus neurotoxicityVittorio Calabrese*, Cesare Mancuso‡, Menotti Calvani§, Enrico Rizzarelli*, D. Allan Butterfield|| and Anna Maria Giuffrida Stella*

Abstract | At the end of the 1980s, it was clearly demonstrated that cells produce nitric oxide and that this gaseous molecule is involved in the regulation of the cardiovascular, immune and nervous systems, rather than simply being a toxic pollutant. In the CNS, nitric oxide has an array of functions, such as the regulation of synaptic plasticity, the sleep–wake cycle and hormone secretion. Particularly interesting is the role of nitric oxide as a Janus molecule in the cell death or survival mechanisms in brain cells. In fact, physiological amounts of this gas are neuroprotective, whereas higher concentrations are clearly neurotoxic.

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The nitric oxide synthase familyThe NOS family of enzymes is responsible for the synthesis of NO; in the presence of oxygen, these enzymes catalyse the conversion of l‑arginine to l‑citrulline plus NO (FIG. 2). Recently, many tools, including NOS inhibitors, NO donors and NO scavengers, have been discovered and developed (TABLE 1); their appropriate use allows researchers to specifically manipulate the NOS–NO system.

Isoform expression. The NOS family consists of three isoforms: neuronal NOS (nNOS, type I); endothelial NOS (eNOS, type III); and inducible NOS (iNOS, type II)1,12,13. Neuronal NOS and eNOS are constitutively expressed and require the formation of Ca2+–calmodulin complexes for their activation, whereas iNOS exerts its activity in a Ca2+‑independent manner. All three NOS isoforms need co‑factors, such as haem, tetrahydrobi‑opterin, flavin adenine dinucleotide, flavin mononucle‑otide and reduced nicotinamide‑adenine dinucleotide phosphate, for catalytic activity1,12 (FIG. 2).

Isoform localization. Several studies carried out in rodents and primates have shown nNOS to be abundant in brain areas such as the cerebral cortex, the ventral endopiriform nucleus, the claustrum, the olfactory bulb, the olfac‑tory nuclei, the nucleus accumbens, the striatum, the amygdala, the hippocampus (in particular the CA1 region and the dentate gyrus), the hypothalamus (the supraoptic and paraventricular nuclei), the thalamus, the lateral dorsal and pedunculopontine tegmental nuclei, the trapezoid body, the raphe magnus, the nucleus of sol‑itary tract and the cerebellum13–16. In the CNS, nNOS has also been found in astrocytes and cerebral blood vessels1. In addition to this central localization, nNOS has been found in peripheral non‑adrenergic, non‑cholinergic neurons, which innervate the smooth muscle in the gastrointestinal tract17, as well as in the penile corpora cavernosa, the urethra and the prostate4,18.

In the brain, eNOS is expressed in cerebral endothe‑lial cells, where it regulates cerebral blood flow, by a small population of pyramidal neurons of the CA1, CA2 and CA3 subfields in the hippocampus, and by granule cells of the dentate gyrus19. endothelial NOS has been also found in rat astrocyte cultures20. In the periphery, eNOS has been found in vascular/sinusoi‑dal endothelium and in the smooth muscle of human corpora cavernosa19,21.

Levels of iNOS in the CNS are low, but iNOS can be induced in astrocytes or microglial cells following events such as inflammation, viral infection or trauma12,19.

Nitric oxide signallingInitial studies into NO‑mediated signalling indicated that this gas interacts with soluble guanylyl cyclase (sGC) and stimulates its activity (FIG. 3). The consequent increase in intracellular levels of cyclic GMP can influence synaptic plasticity, smooth‑muscle relaxation, neurosecretion and neurotransmission22–25. NO has subsequently been shown to interact with members of the haemoprotein family, such as cyclooxygenase26 and haem oxygenase 1 (REF. 27). This family of proteins is involved in metabolic, inflammatory and cellular stress responses.

NO also regulates the Akt kinase pathway and the transcription factor cyclic‑AMP‑responsive‑element‑binding protein (CReb), two pathways that promote cell survival and neuroprotection28,29. Finally, NO has been shown to regulate cell signalling events by S‑nitrosylation of pathway components, in which it binds covalently to thiol groups of proteins and non‑protein molecules30. Through this reaction, NO exerts both neuroprotective and neurotoxic effects (see below).

Nitric oxide and neurotransmissionThe first evidence of a role for NO as a neurotrans‑mitter was reported by Garthwaite et al., who dem‑onstrated that stimulation of cerebellar NMDA (N‑methyl‑d‑aspartate) receptors by glutamate caused the release of a diffusible molecule with strong similarities to endothelial‑derived relaxation factor (eDRF)31. Shortly before this study was published, NO had been identified as the eDRF molecule32,33. Subsequently, it was shown that NO acts as a neuro‑transmitter in both the CNS and PNS by mechanisms that are dependent on cyclic GMP34,35 (FIG. 3).

before discussing the direct effect of NO in neuro‑transmission in the next section, it is interesting to note that this gaseous compound regulates the release of ‘classical’ neurotransmitters in many brain areas. In fact, NO has been shown to indirectly stimulate the release of acetylcholine in the nucleus accumbens by stimulating

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Central effects

Peripheral effects

NO in the nervous system

NeurotransmissionRegulation of food intakeControl of the sleep–wake cycleModulation of hormone releaseThermal regulationNeuroprotectionNeurotoxicity

Control of smooth-muscle relaxation• Gastrointestinal tract• Urogenital tract

Figure 1 | Nitric oxide in the CNS and PNS. The gaseous signalling molecule nitric oxide (NO) is able to mediate several processes in the CNS and PNS.

Nature Reviews | Neuroscience

NH

H2N NH2

COO–H3N+

+

H3N+

NH

H2N O

COO–

Haem, BH4, Flavin

NADPH O2

L-arginine L-citrulline

+ NONOS

Figure 2 | The metabolic pathway that leads to nitric oxide formation. In the presence of oxygen, NADPH and co-factors such as flavin mononucleotide (FMN), flavin adenine dinucleotide (FAD), haem and tetrahydrobiopterin (BH4), nitric oxide synthase (NOS) catalyses the oxidation of the terminal guanidinyl nitrogen of the amino acid l-arginine to form l-citrulline and nitric oxide (NO)12,19. Once formed, NO easily diffuses within the cell or across the cell membrane, and is involved in both autocrine and paracrine actions.

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adjacent glutamatergic neurons36. basal NO concentra‑tions have been shown to reduce the release of GAbA (γ‑aminobutyric acid) in a Ca2+‑ and Na+‑dependent manner37,38, whereas high levels of NO increase GAbA release. NO donors stimulated the release of noradrena‑line and glutamate in the hippocampus39 , whereas haemoglobin, an endogenous NO scavenger, inhibited the release of these molecules. In the rat medial preoptic area, NO increased the release of both dopamine and serotonin40 in an sGC–cGMP‑dependent way41.

In the telencephalon and the cerebellum, NO has an important role in the regulation of the synaptic plasticity that is involved in cognitive processes, such as memory. Long‑term potentiation (LTP) and long‑term depres‑sion (LTD) of synaptic transmission are well‑established components of synaptic plasticity. Several lines of evi‑dence have shown that NO, produced presynaptically or in interneurons, acts postsynaptically during cerebellar and striatal LTD, whereas the postsynaptic generation of this gaseous molecule and its action at presynaptic sites characterize NO as a retrograde diffusible messenger in hippocampal and cortical LTP 42. NO‑dependent LTP in rat hippocampal and amygdala slices is inhibited by the sGC inhibitor 1H‑[1,2,4]oxadiazolo[4,3‑a]quinoxalin‑1‑one (ODQ) (TABLE 1), but enhanced by the sGC activator 3‑(5‑hydroxymethyl‑2‑furyl)‑1‑benzyl‑indazole (YC‑1) (TABLE 1), demonstrating that the NO‑mediated modula‑tion of synaptic plasticity is an sGC–cGMP‑dependent mechanism43,44.

In the diencephalon, NO is a major regulator of the neurosecretory activity of the hypothalamus (see below for further information). In the mesencephalon, NO is involved in the regulation of many functions, including the sleep–wake cycle. l‑arginine, the precursor of NO (FIG. 2; TABLE 1), caused an increase in slow‑wave sleep in rats when it was administered during the light phase into the pedunculopontine tegmentum (a brain area

assigned to sleep control)45. Similarly, the microinjection of the NO donor S‑nitroso‑acetyl‑penicillamine into cat pedunculopontine tegmentum during wakefulness increased both slow‑wave sleep and rapid‑eye move‑ment sleep46. Interestingly, 3‑bromo‑7‑nitroindazole, a specific inhibitor of nNOS, was used to show that NO produced specifically by this NOS isoform regulates the sleep process in rats47.

Nitric oxide and neurosecretionNeuronal NOS is localized in the hypothalamic supraoptic nucleus and the paraventricular nucleus, both of which are mainly involved in the neurosecre‑tory activity of this brain area48. In fact, the hypotha‑lamic paraventricular nucleus (parvicellular and magnocellular portions) and the supraoptic nucleus (magnocellular portion) contain the cell bodies of neurons that release corticotropin‑releasing hormone (CRH), arginine vasopressin (AvP) and oxytocin49 — hormones that are implemented in stress and sleep regulation, respectively.

The stress axis. CRH and AvP are the major neuropep‑tides that control the stress axis. when activated in response to stress, neurons in the paraventricular nucleus release both CRH and AvP in the median eminence; these neuropeptides then travel to the anterior hypophysis through the portal vessel system49. Once in the pituitary, CRH and AvP activate corticotroph cells, which release adrenocorticotropin‑releasing hormone (ACTH) into the general circulation. ACTH, in turn, stimulates the adrenal glands to release glucocorticoids50. AvP and oxy‑tocin can also be released from hypothalamic neurons in the posterior pituitary gland (the neurohypophysis), and from there directly into the systemic circulation, where AvP regulates water reabsorption by the kidney and oxy‑tocin is involved in the contraction of uterine smooth

Table 1 | Pharmacological tools used in nitric oxide research

Substance effect refs

l-arginine Substrate for NOS; increases NO production 1

NG-nitro-l-arginine methyl ester (L-NAME) Non-selective NOS inhibitor 118

Nω-propyl-l-arginine Highly selective and potent inhibitor of nNOS 118

l-argNO2-l-Dbu-NH2 The most selective inhibitor of nNOS 118

N5-(1-iminoethyl)-l-ornithine (L-NIO) Potent inhibitor of eNOS 118

N-[[3-(aminomethyl)phenyl]methyl] ethanimidamide (1400W)

Potent, highly selective human iNOS inhibitor 118

1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ) Selective inhibitor of sGC 23,43

3-(5-hydroxymethyl-2-furyl)-1-benzyl-indazole (YC-1) Activator of sGC 23,44

S-nitrosothiols Endogenous NO donors 30

2-phenyl-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (PTIO)

A scavenger of NO 119

Haemoglobin and its derivatives An endogenous scavenger of NO 39,75,120

Bilirubin A novel endogenous scavenger of NO and RNS in reconstituted systems

75,121

NO, nitric oxide; NOS, nitric oxide synthase (e, endothelial; i, inducible; n, neuronal); RNS, reactive nitrogen species; sGC, soluble guanylyl cyclase.

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muscle at term. During lactation, oxytocin also regu‑lates the contraction of myoepithelial cells surrounding alveoli in the mammary gland49,51.

NO-mediated regulation of stress. The contribution of NO to the regulation of stress has long been debated. early studies showed that NO did not affect basal CRH and AvP release but did inhibit the release of these neuropetides from rat hypothalamic explants that had been stimulated with potassium chloride (which depo‑larizes the explants and increases their intracellular Ca2+ concentration52: the main physiological stimulus leading to neurotransmitter release into the synaptic cleft) or the cytokine interleukin‑1β53,54 (which is a well known mediator of immuno‑inflammatory stress55). by contrast, other studies showed that NO stimulated the release of CRH from the rat mediobasal hypothalamus in vitro3,56. On the basis of in vivo studies, the current view reconciles these contradictory findings and proposes that NO has opposing effects on the different components of the stress axis. The NOS inhibitor L‑NAMe (NG‑nitro‑l‑arginine methyl ester) has been shown to decrease the ACTH response in response to shock and to decrease the upreg‑ulation of CRH and AvP expression in the hypothalamic

paraventricular nucleus following exposure to neuro‑genic stressors; accordingly, the intracerebroventricular administration of NO increased the number of CRH and AvP transcripts in the rat paraventricular nucleus2. Taken together, these results strengthen the hypothesis that NO has a stimulatory role in the hypothalamus. by contrast, L‑NAMe augmented the release of ACTH from the pituitary in response to AvP and circulating pro‑ inflammatory cytokines, which implies an inhibitory role for NO at the level of the median eminence or the pitui‑tary2,57. The ultimate effect of NO‑mediated regulation of the stress axis is the fine balance between the opposing effects of this gas on the paraventricular nucleus and the pituitary gland — an effect that also depends on the type of stress stimulus.

The effect of NO on fluid balance and reproduction. In addition to this ‘central’ effect, NO has been shown to exert a tonic inhibition on ‘circulating’ AvP levels under physiological iso‑osmotic conditions because of its inhibitory activity on hypothalamic magnocellular neurons58. In the case of osmotic stress (as occurs during hypovolaemia or haemorrhage), however, NO‑mediated inhibition of AvP neurons is absent. The net effect of this regulatory loop is to specifically increase AvP release in situations that require the correction of fluid imbalance58.

NO has been shown to affect reproductive proc‑esses, mainly through the central regulation of the hypothalamic release of gonadotropin‑releasing hor‑mone (GnRH)3,59. However, NO has also been shown to facilitate reproductive processes through the activation of oxytocinergic neurons located in the hypothalamic paraventricular nucleus, which ultimately leads to penile erection60. NO also has a stimulatory effect on the hypothalamic release of growth‑hormone‑releasing hormone3. It is worth noting that NO shares control of the stress axis and the reproductive processes with another gaseous neuromodulator, carbon monoxide, the product of the enzymatic activity of haem oxygenase61. In vitro and in vivo studies support the idea that carbon monoxide, like NO, inhibits at the hypothalamic level the increase in both CRH and AvP that is elicited by depo‑larizing and inflammatory stimuli62–64. Furthermore, in concert with NO, carbon monoxide contributes to the regulation of reproduction mainly by stimulat‑ing the release of GnRH from mediobasal hypothalami incubated in vitro59.

Nitric oxide as a neuroprotectantNO confers a neuroprotective effect through multiple mechanisms. The following sections summarize findings from several experimental models.

Akt and CREB. In primary rat cerebellar granule cells that had been cultured for 7 days, inhibition of NO synthesis resulted in a significant increase in apop‑totic cell death through the activation of caspase 3. Apoptosis following NO deprivation in these cells was mimicked by the sGC inhibitor ODQ and reversed by treatment with NO donors or cGMP analogues28. using

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NO

sGC

GTP cGMP

PDE

PKG

• Smooth-muscle tone • Neurotransmission

Cyclic nucleotide-gated channels

Figure 3 | Nitric oxide activates soluble guanylyl cyclase. Soluble guanylyl cyclase (sGC) is a cytosolic haem-containing enzyme that catalyses the transformation of guanosine triphosphate (GTP) into 3′,5′-cyclic guanosine monophosphate (cGMP)23. sGC is activated by the binding of nitric oxide (NO) to its haem moiety, and the intracellular concentration of cGMP is subsequently increased23. cGMP has several downstream effectors, the most important being protein kinase G (PKG) and the cyclic nucleotide-gated channels 23. Through these pathways, NO exerts its effects on smooth-muscle motility and neurotransmission23. Phosphodiesterase (PDE) hydrolyses cGMP and therefore acts to avoid excessive accumulation of this molecule. By reducing cGMP degradation, PDE inhibitors such as sildenafil ameliorate smooth-muscle relaxation. These inhibitors are currently used to treat impotence and pulmonary hypertension23,94,97.

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this experimental system, the intracellular pathways through which NO exerts neuroprotective effects have been delineated. The kinase Akt and the transcription factor CReb have been shown to be involved in the survival pathway that is elicited by NO in cerebellar granule cells28; notably, both proteins are important as signal transducers of neurotrophin‑mediated survival and for protection against various neurodegenerative challenges, and this similarity contributes to the neu‑roprotective role of NO28,29 (FIG. 4). The effect of NO on both Akt and CReb seems to be mediated by cGMP and the sequential stimulation of protein kinase G, which is a crucial intermediate in the NO‑mediated activation of both Akt and CReb28.

Neuroprotection through S-nitrosylation. NO also confers neuroprotection in the NMDA‑mediated neurotoxicity model, in which prolonged stimulation of NMDA receptors causes excitotoxic cell death1. NO protects against such excitotoxicity by S‑nitrosylating the NR1 and NR2 subunits of the NMDA receptor65,66 (FIG. 4), reducing the intracellular Ca2+ influx that is responsible for neuronal death67. Prolonged nNOS stimulation, which occurs in response to sustained NMDA receptor activation, generates superoxide radicals68, and these, in turn, react with NO to form peroxynitrite7. Consequently, it is conceivable that NO formed during excessive NMDA activation S‑nitrosylates the NMDA subunits, and thereby dimin‑ishes either the formation of peroxynitrite or Ca2+ influx that is to promote neuronal survival (FIG. 4).

NO can also confer cytoprotection through the inhibi‑tion of caspase activity by S‑nitrosylating cysteines of the catalytic site69,70 (FIG. 4). S‑nitrosylation has been shown to reduce the activity of caspases in several cell lines, including neurons70–72. Recent studies demonstrated that cortical neurons that had been treated with several NO

donors, including S‑nitrosothiols, exhibited a signifi‑cant reduction in staurosporin‑induced caspase 3 and caspase 9 activation, possibly due to the NO‑mediated S‑nitrosylation of the cysteine residue in the catalytic site of these caspases; moreover, NO treatment inhibited the appearance of the classical apoptotic nuclear morphol‑ogy73. Surprisingly, caspase 3 and caspase 9 inhibition by NO was not paralleled by a significant increase in neuronal cell viability, which implies the occurrence of an alternative, caspase‑independent form of cell death in neurons exposed to NO, in accordance with previous findings69,73.

Neuroprotection through the overexpression of haem oxygenase. The induction of haem oxygenase 1 is con‑sidered to be an early event in the cellular response to oxidative stress, and it has a neuroprotective function61. Moreover, under pro‑oxidant conditions, the upregulation of iNOS and the following formation of excess NO and RNS occur1. In the brain, NO has been shown to induce haem oxygenase 1 in rat astrocytes and microglia as well as in the hippocampus74. The upregulation of haem oxy‑genase 1 protein and the following increase in biliverdin, which is further reduced by biliverdin reductase into the antioxidant and antinitrosative molecule bilirubin, can be considered a secondary mechanism through which NO can exert neuroprotective effects19,61,75.

Nitric oxide in neurodegenerationThe involvement of nitrosative stress in the development of neurodegenerative disorders is no longer a matter of question. In these diseases, NO is produced in excess by iNOS induction owing to the pro‑inflammatory response, which is a common feature of neurodegen‑erative disorders. Moreover, NO is much more harmful under pathological conditions that involve the produc‑tion of reactive oxygen species (ROS), such as superoxide anions, and the formation of peroxynitrite1,7 (FIG. 5). The formation of nitrotyrosine, a marker of nitrosative stress, has been documented in patients with Alzheimer’s dis‑ease and Parkinson’s disease1,10,11,76. Furthermore, NO has been shown to activate both the constitutive and the inducible isoforms of cyclooxygenase, which are upregulated in brain cells under pro‑inflammatory con‑ditions26,77. During the catalytic cycle of cyclooxygenase, the release of free radicals and the formation of pros‑taglandins occur, two events that are closely related to the development of neuroinflammation77. Interestingly, inducible cyclooxygenase is upregulated in the brain of patients affected by Alzheimer’s disease and is consid‑ered a marker of the progression of dementia in this dis‑ease26,77. Keeping this in mind, the activation of inducible cyclooxygenase can be considered as an indirect way for NO to exert neurotoxicity (FIG. 5).

Alzheimer’s disease. Redox proteomics techniques78 have been used to identify ten proteins that show increased specific nitrotyrosine immunoreactivity in the brains of patients with Alzheimer’s disease (BOX 1): α‑enolase, triosophosphate isomerase, neuropolypeptide h3, β‑actin, l‑lactate dehydrogenase, carbonic anhydrase II,

Nature Reviews | Neuroscience

NO

NMDARNR1NR2 Caspase-3

S-NO

CREB

sGC–cGMP–PKG

Akt HO-1

Ca2+

Neuroprotection

Figure 4 | Neuroprotective effects of nitric oxide. Nitric oxide (NO) confers neuroprotection by several mechanisms. NO S-nitrosylates caspase 3 and the NR1 and NR2 subunits of the N-methyl-d-aspartate receptor (NMDAR); as a consequence of these reactions, Ca2+ influx through NMDARs and caspase 3 activity are both inhibited, leading to a decrease in cell death65–67,69–72. Through the stimulation of the soluble guanylate cyclase (sGC)–cyclic GMP (cGMP)–protein kinase G (PKG) pathway, NO activates cyclic-AMP-responsive-element-binding protein (CREB) and Akt, two proteins that are mainly involved in neuroprotection28,29. In addition to these pathways, NO induces the activity of haem oxygenase 1 (HO-1), which generates biliverdin, the precursor of the powerful antioxidant and antinitrosative molecule bilirubin27,61,74,75,77. nNOS, neuronal nitrogen oxide synthase; S-NO, S-nitrosylation.

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glyceraldehyde 3‑phosphate dehydrogenase, ATP syn‑thase α‑chain, voltage‑dependent anion channel protein 1 and γ‑enolase10,11. Of these proteins, α‑enolase has prev‑iously been identified as being specifically oxidized in the brains of people with Alzheimer’s disease79. It is one of the subunits of enolase, which catalyses the reversible conversion of 2‑phosphoglycerate to phos‑phoenolpyruvate in glycolysis. Taken together with the increased nitration of triosephosphate isomerase — which interconverts dihydroxyacetone phosphate and 3‑phosphoglyceraldehyde in glycolysis — that is also seen in those with the condition, these results indi‑cate a possible mechanism to explain the altered glucose tolerance and metabolism that is exhibited in patients with Alzheimer’s disease80,81. Neuropolypeptide h3, also known as phosphatidylethanolamine‑binding protein, hippocampal cholinergic neurostimulating peptide and Raf‑kinase inhibitor protein, has various functions in the brain. In vitro, it has been shown to upregulate levels of choline acetyltransferase in cholinergic neu‑rons after NMDA‑receptor activation82. Choline acetyl‑transferase activity is known to be decreased in patients with Alzheimer’s disease, and cholinergic deficits are prominent in the brains of such patients83,84. Nitration of neuropolypeptide h3, and the consequent lack of neurotrophic action on cholinergic neurons of the hip‑pocampus and basal forebrain, might help to explain the decline in cognitive function.

Carbonic anhydrase II is crucial for the maintenance of pH and control of carbon dioxide levels; its activity is also altered in Alzheimer’s disease85. Glyceraldehyde 3‑phosphate dehydrogenase (GAPDH) is not only important in ATP production, but also has a role as a nitrosative stress sensor86. In fact, the active site Cys149 can undergo several modifications on reaction with NO (and RNS) that result in a reversible or irreversible inhi‑bition of GAPDH enzymatic activity (depending on the severity of the pro‑oxidant stimulus)86. As a consequence of this inhibition, the glucose metabolism is shifted towards the pentose phosphate shunt, which produces NADPH that is required for the activity of glutathione reductase and uncouples glucose metabolism from the production of ATP and oxidative intermediates86. Moreover, GAPDH interacts with a key protein to func‑tion as a transcription factor (see below). ATP synthase α‑chain is clearly important in energy metabolism, and voltage‑dependent anion channel protein 1 is involved in the mitochondrial permeability transition pore, which has consequent apoptotic considerations, as well as in mitochondrial Ca2+ homeostasis10.

Huntington’s and Parkinson’s diseases. In Huntington’s disease, another age‑related neurodegenerative disorder that often gives rise to dementia, there is evidence of oxidative and nitrosative damage in the basal ganglia (for a review, see REF. 87).

In 2002, it was shown that matrix metalloprotei‑nase 9 (MMP9), which causes neuronal apoptosis, is S‑nitrosylated by NO that is derived from the endog‑enous nitrosothiol S‑nitrosocysteine88. Matrix metal‑loproteinases are involved in the pathogenesis of acute and chronic neurodegenerative disorders, such as stroke, Alzheimer’s disease, HIv‑associated dementia and multi‑ple sclerosis88,89. A similar mechanism has been proposed for Parkinson’s disease. Yao et al.90 and Chung et al.91 independently demonstrated that S‑nitrosocysteine‑derived NO is able to nitrosylate parkin, an e3 ubiquitin ligase. Mutations in parkin are known to cause auto‑somal recessive‑juvenile parkinsonism. Nitrosylation of cysteine residues in parkin initially increases but later decreases the e3 ubiquitin ligase activity of this protein, and thereby reduces its protective function. NO has also been shown to S‑nitrosylate GAPDH, thereby reducing its activity and allowing GAPDH to bind to another e3 ubiquitin ligase, SIAH1. The GAPDH–SIAH1 complex then translocates into the nucleus to induce apoptosis92. A direct consequence of this study was the identification of a new mechanism of action for selegiline, a drug that is already used to treat patients with Parkinson’s disease because of its ability to inhibit monoamine oxidase type b. At nanomolar concentrations, selegiline prevented S‑nitrosylation of GAPDH, thereby blocking its inter‑action with SIAH1 and any further induction of apop‑tosis. Selegiline shared this neuroprotective effect with TCH346, a derivative that has no monoamine oxidase type b inhibitory activity92.

Another target for NO‑induced neurotoxicity is protein‑disulphide isomerase (PDI), which catalyses thiol‑disulphide exchange, thereby promoting the

Nature Reviews | Neuroscience

Oxidative stress

NMDA

iNOS

nNOS

L-arginine

O2–. NO

ONOO–

Protein nitration

Parkin–S-NOMMP9–S-NOGAPDH–S-NOPDI–S-NO

COX

FRs PGs

Neurotoxicity

Figure 5 | Neurotoxic effects of nitric oxide. If it is produced in excess, or if a cell is in a pro-oxidant state, nitric oxide (NO) has cytotoxic effects. It is well established that NO can react with superoxide anions (O2–·; produced by inducible nitric oxide synthase (iNOS) under inflammatory conditions or neuronal nitric oxide synthase (nNOS), as in the case of excitotoxicity) to form peroxynitrite (ONOO–), an anion with strong oxidant properties1,7,68. As a consequence of the interaction between peroxynitrite and cellular components, protein nitration takes place, resulting in damage to cellular components1,7. The NO-mediated S-nitrosylation (S-NO) of certain substrates, such as matrix metalloproteinase 9 (MMP9)88, parkin90,91, GAPDH92 and protein-disulphide isomerase (PDI)93, has been proposed to be a novel mechanism through which NO becomes neurotoxic. NO also activates the haemoprotein cyclooxygenase (COX). During its catalytic cycle, COX generates free radicals (FRs) and prostaglandins (PGs), both of which have strong pro-inflammatory features77. NMDA, N- methyl-d-aspartate.

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formation of disulphide bonds and protein rearrange‑ment93. During the course of neurodegenerative dis‑eases and cerebral ischaemia, immature and denatured proteins accumulate, the latter of which are toxic to neurons. under normal circumstances, PDI upregula‑tion would reduce the abnormal accumulation of mis‑folded proteins to protect neurons. However, in brains from patients with Alzheimer’s disease or Parkinson’s disease, this protective role has been lost owing to the S‑nitrosylation of crucial cysteines and the inhibition of PDI enzymatic activity93.

Therapeutic potentialKnowledge of the endogenous physiological actions of NO in the nervous system highlighted here raises the possibility of manipulating the NO system for therapeutic benefit. Although NO, or other molecules acting on the NO–cGMP pathway, are used to treat important disorders such as impotence94, respiratory diseases95,96 and pulmo‑nary hypertension97, current data indicate that NO‑based therapies would not be appropriate for the treatment of CNS diseases. This pessimism arises from the intrinsic difficulty of delivering NO into the CNS without any side effects, as well as from the dual role — neuroprotective or neurotoxic — of NO in the brain.

Heat shock proteins. More fascinating is the possibil‑ity of counteracting the neurotoxic effects of NO or RNS by modulating members of the vitagene family, which include heat shock proteins. The evidence that these endogenous proteins can be manipulated using

nutritional products or pharmacological compounds represents an innovative approach to therapeutic intervention in diseases characterized by both oxida‑tive and nitrosative stress, such as neurodegenerative disorders19,77.

Curcumin. The polyphenolic molecule curcumin is among a number of natural substances that show promise in reducing nitrosative brain injury and delaying the onset of neurodegenerative disorders. It is a strong antioxidant that can inhibit lipid peroxidation, effectively intercept and neutralize ROS and RNS98, and significantly increase haem oxygenase 1 expression in astrocytes and neurons99. Dietary curcumin suppressed indicators of inflammation and oxidative damage in the brains of a transgenic mouse model of Alzheimer’s disease77,98.

Ferulic acid. Ferulic acid, which is found in fruit and vegetables, is another phenolic compound with strong antioxidant and anti‑inflammatory properties. It also protects synaptosomal membrane systems and neuro‑nal cell culture systems against hydroxyl and peroxyl radical oxidation100 and has been shown to protect mice against amyloid‑β‑peptide‑induced microglial activation101. Ferulic acid ethyl ester protected cortical neurons in vitro and brain tissue in vivo from amyloid‑β toxicity by inducing the expression of haem oxygenase 1 and other members of the heat shock protein family, as well as by decreasing neuronal 3‑nitrotyrosine levels and, therefore, NOS activity102.

Acetyl-l-carnitine. Acetyl‑l‑carnitine might be of thera‑peutic benefit for Alzheimer’s disease, multiple sclerosis, chronic fatigue syndrome, depression in the elderly, HIv infection, diabetic neuropathies, ischaemia and reperfusion of the brain, cognitive impairment resulting from alcoholism, and ageing103,104. It is involved in cel‑lular energy production and in maintenance and repair processes in neurons103, and has been shown to induce the expression of haem oxygenase 1 and heat‑shock pro‑tein 72 in rat neurons105. Other studies have shown that acetyl‑l‑carnitine protects neurons from oxidative dam‑age and neurotoxicity induced by amyloid‑β peptide105.

Carnosine. Recently, carnosine, a natural di‑peptide, received great attention owing to its neuroprotective prop‑erties, some of which relate to its close interaction with the NO system. In the brain, carnosine is found in glial cells and in some types of neurons106, and has been shown to induce neuroprotective pathways that counteract both oxidative and nitrosative stress107. Importantly, carnosine has been shown to prevent amyloid‑β aggregation and toxicity108, perhaps through its ability to inhibit protein misfolding and prevent the formation of advanced‑ glycation end products109. Furthermore, carnosine has been shown to counteract peroxynitrite‑dependent protein alterations, such as tyrosine nitration110. Recent evidence demonstrates that carnosine prevents the upregulation of iNOS and the induction of both haem oxygenase 1 and heat‑shock protein 70 that occurs after exposure to strong nitrosative conditions107.

Box 1 | Redox proteomics and the identification of nitrated proteins

A redox proteomics approach was used to identify proteins that were modified specifically by nitration in the brains of patients with Alzheimer’s disease and mild cognitive impairment10,11,116. This approach has provided new insight into potential mechanisms of onset and progression of Alzheimer’s disease. Redox proteomics has the potential to detect disease markers and identify potential targets for drug therapy in neurodegenerative disorders. This technique78 involves the separation of brain proteins by two-dimensional (2D) SDS–PAGE, followed by the detection, usually immunochemically, of nitrated proteins (either from a 2D Western blot followed by spot excision from a 2D gel, or from column eluates). Subsequent mass spectrometric analysis of tryptic digests, combined with database searches, is used for protein identification78 (see figure). Almost uniformly, proteins that are identified as being oxidatively modified by redox proteomics are dysfunctional117.

Nature Reviews | Neuroscience

Sample(protein mixture)

2D-PAGE

2D blot

2D gel map Image analysis

In-gel trypsin digestion

Mass spectrometryDatabase searching

Proteinidentification

Spot excision

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Chemotherapy-induced somnolence. NO is involved in the brain effects that are induced by peripheral adminis‑tration of the chemotherapeutic agent adriamycin111–113. butterfield and co‑workers demonstrated that intraperi‑toneal administration of adriamycin leads to oxidative and nitrosative damage in the brain111. This treatment enhances the levels of the cytokine tumour necrosis fac‑tor (TNF) in the bloodstream, which in turn causes TNF to cross the blood–brain barrier, resulting in oxidative damage to neurons112 and the translocation of the pro‑apoptotic protein p53 to brain mitochondria. These effects lead to neuronal cell death112. when these stud‑ies were repeated in mice lacking the gene that encodes iNOS, no brain nitrosative stress, no mitochondrial respiratory dysfunction and no nitration of manganese superoxide dismutase were found, although brain TNF levels were still elevated113. As patients that have been treated with cancer chemotherapeutics often complain of somnolence for years after the cessation of chemother‑apy, and as the brains of such patients show changes in metabolism when examined by positron emission tom‑ography (PeT) imaging114, the results in mice described above, if translatable to humans, would suggest that NO contributes significantly to somnolence.

Conclusions and perspectivesNitric oxide presents both challenges and opportuni‑ties to intervene in and promote human health. This Review highlights the many effects that NO has in the nervous system and discusses its roles in neuroprotec‑tion and neurodegeneration, as well as its therapeutic potential for neurodegenerative disorders. However, as outlined above, the use of drugs such as NO donors, NOS inhibitors or PDe inhibitors in humans can not be considered safe for such disorders because of the complex effects of this gas in the nervous system. The potential use of natural antioxidants, such as polyphenols, in the prevention of neurodegenerative disorders has been proposed98, owing to their abil‑ity to enhance cellular survival pathways such as the heat‑shock response77,98. However, although there is an impressive amount of in vitro data to support the neuroprotective action of these substances, there are important limitations on their use in humans, mainly owing to the pharmacokinetics of these sub‑stances115. Naturally occurring antioxidants could be chemically modified to render them more effective for therapeutic use in disorders of the CNS, including neurodegenerative disorders.

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AcknowledgementsThis work was supported by grants from Ministero dell’Università e della Ricerca Cofin 2000, Progetti di Ricerca di Interesse Nazionale 2005, Fondo per gli Investimenti della Ricerca di Base RBNE01ZK8F and by National Institutes of Health grant AG-10836; AG-05119.

Competing interests statementThe authors declare no competing financial interests.

DATABASESEntrez Gene: http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=geneACTH | Akt | AVP | CREB | CRH | eNOS | iNOS | nNOS | oxytocin | PDI | SIAH1OMIM: http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=OMIMAlzheimer’s disease | Huntington’s disease | Parkinson’s disease

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