REVIEWS

Nitric oxide in the central nervous

system: neuroprotection versus
neurotoxicity
Vittorio 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
sleepwake 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.

*Department of Chemistry,
Biochemistry and Molecular
Biology Section,
Faculty of Medicine,
University of Catania,
Catania, Italy.
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 larginine
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
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

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.it
doi:10.1038/nrn2214
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 neurosecretion13
(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;
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 Alzheimers 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.

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2007 Nature Publishing Group

Neurotransmission
Regulation of food intake
Control of the sleepwake cycle
Central effects Modulation of hormone release
Thermal regulation
NO in the Neuroprotection
nervous system Neurotoxicity
Control of smooth-muscle relaxation
Peripheral effects 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
The nitric oxide synthase family
The NOS family of enzymes is responsible for the synthesis
of NO; in the presence of oxygen, these enzymes catalyse
the conversion of larginine to lcitrulline plus NO
(FIG.2). Recently, many tools, including NOS inhibitors,
NO donors and NO scavengers, have been discovered and
developed (TABLE1); their appropriate use allows researchers
to specifically manipulate the NOSNO system.
Isoform expression. The NOS family consists of three
isoforms: neuronal NOS (nNOS, typeI); endothelial
NOS (eNOS, typeIII); and inducible NOS (iNOS,
typeII)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 cerebellum1316. 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.
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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 signalling
Initial studies into NOmediated 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 neurotransmission2225. 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 Snitrosylation
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 neurotransmission
The first evidence of a role for NO as a neurotrans
mitter was reported by Garthwaite etal., who dem
onstrated that stimulation of cerebellar NMDA
(Nmethyld-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
H2N + NH2 H2N O
Haem, BH4, Flavin
NH NH
NADPH O2
+ NO
NOS
H3N+ COO
H3 N+ COO
L-arginine L-citrulline
Figure 2 | The metabolic pathwayNaturethat leads
Reviews to nitric
| Neuroscience
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
larginine to form lcitrulline 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.
REVIEWS
Table 1 | Pharmacological tools used in nitric oxide research
Substance
l-arginine
NG-nitrol-arginine methyl ester (L-NAME)
N-propyll-arginine
l-argNO2l-Dbu-NH2
N (1-iminoethyl)l-ornithine (L-NIO)
5
N[[3-(aminomethyl)phenyl]methyl] ethanimidamide
(1400W)
1H[1,2,4]oxadiazolo[4,3a]quinoxalin1-one (ODQ)
3(5-hydroxymethyl2-furyl)1-benzyl-indazole (YC1)
S-nitrosothiols
2-phenyl4,4,5,5-tetramethylimidazoline1-oxyl3-oxide
(PTIO)
Haemoglobin and its derivatives
Bilirubin
NO, nitric oxide; NOS, nitric oxide synthase (e, endothelial; i, inducible; n, neuronal); RNS, reactive nitrogen species; sGC, soluble
guanylyl cyclase.
adjacent glutamatergic neurons36. Basal NO concentra
tions have been shown to reduce the release of GABA
(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 sGCcGMP-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 LTP42. NOdependent LTP in
rat hippocampal and amygdala slices is inhibited by the
sGC inhibitor 1H[1,2,4]oxadiazolo[4,3a]quinoxalin1-
one (ODQ) (TABLE1), but enhanced by the sGC activator
3(5-hydroxymethyl2-furyl)1-benzyl-indazole (YC1)
(TABLE1), demonstrating that the NOmediated modula
tion of synaptic plasticity is an sGCcGMP-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 sleepwake cycle. larginine, the precursor of NO
(FIG.2; TABLE1), caused an increase in slow-wave sleep
in rats when it was administered during the light phase
into the pedunculopontine tegmentum (a brain area
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Effect Refs
Substrate for NOS; increases NO production 1
Non-selective NOS inhibitor 118
Highly selective and potent inhibitor of nNOS 118
The most selective inhibitor of nNOS 118
Potent inhibitor of eNOS 118
Potent, highly selective human iNOS inhibitor 118
Selective inhibitor of sGC 23,43
Activator of sGC 23,44
Endogenous NO donors 30
A scavenger of NO 119
An endogenous scavenger of NO 39,75,120
A novel endogenous scavenger of NO and RNS 75,121
in reconstituted systems
assigned to sleep control)45. Similarly, the microinjection
of the NO donor Snitroso-acetyl-penicillamine into
cat pedunculopontine tegmentum during wakefulness
increased both slow-wave sleep and rapid-eye move
ment sleep46. Interestingly, 3bromo7-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 neurosecretion
Neuronal 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

NO
sGC PDE
GTP cGMP
Cyclic nucleotide-
PKG
gated channels
Smooth-muscle tone
Neurotransmission
Figure 3 | Nitric oxide activates soluble guanylyl
Nature
cyclase. Soluble guanylyl cyclase Reviews
(sGC) | Neuroscience
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.
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 interleukin153,54 (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 invitro3,56.
On the basis of invivo 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 LNAME (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
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REVIEWS
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, LNAME 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 NOmediated 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, NOmediated
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.
Invitro and invivo 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 stimuli6264. 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 invitro59.
Nitric oxide as a neuroprotectant
NO 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
REVIEWS
NO
sGCcGMPPKG
NR1
NMDAR NR2 Caspase-3 CREB Akt HO-1
S-NO
Ca2+
Neuroprotection
Figure 4 | Neuroprotective effects of nitric oxide. Nitric oxide (NO) confers
neuroprotection by several mechanisms. NO Snitrosylates caspase 3 and the NR1 and
NR2 subunits of the Nmethyld-aspartate receptor (NMDAR);Nature as a consequence of these
Reviews | Neuroscience
reactions, Ca2+ influx through NMDARs and caspase 3 activity are both inhibited, leading
to a decrease in cell death6567,6972. 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 (HO1), 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.
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 NOmediated activation of
both Akt and CREB28.
Neuroprotection through Snitrosylation. 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 Snitrosylating
the NR1 and NR2 subunits of the NMDA receptor65,66
(FIG.4) , reducing the intracellular Ca 2+ influx that is
responsible for neuronal death 67. 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
Snitrosylates 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 Snitrosylating cysteines of the
catalytic site69,70 (FIG.4). Snitrosylation has been shown
to reduce the activity of caspases in several cell lines,
including neurons7072. Recent studies demonstrated that
cortical neurons that had been treated with several NO
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donors, including Snitrosothiols, exhibited a signifi
cant reduction in staurosporin-induced caspase 3 and
caspase 9 activation, possibly due to the NOmediated
Snitrosylation 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 neurodegeneration
The 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 Alzheimers dis
ease and Parkinsons 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 Alzheimers 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).
Alzheimers disease. Redox proteomics techniques78 have
been used to identify ten proteins that show increased
specific nitrotyrosine immunoreactivity in the brains
of patients with Alzheimers disease (BOX1): enolase,
triosophosphate isomerase, neuropolypeptide h3,
actin, llactate dehydrogenase, carbonic anhydrase II,

L-arginine
Oxidative stress iNOS
NMDA nNOS
O2. NO
ParkinS-NO COX
MMP9S-NO
GAPDHS-NO
ONOO PDIS-NO
FRs PGs
Protein nitration
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 nitricReviews
Nature oxide synthase
| Neuroscience
(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 NOmediated Snitrosylation (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- methyld-aspartate.
glyceraldehyde 3phosphate dehydrogenase, ATP syn
thase -chain, voltage-dependent anion channel protein 1
and enolase10,11. Of these proteins, enolase has prev
iously been identified as being specifically oxidized
in the brains of people with Alzheimers disease 79. It
is one of the subunits of enolase, which catalyses the
reversible conversion of 2phosphoglycerate to phos
phoenolpyruvate in glycolysis. Taken together with
the increased nitration of triosephosphate isomerase
which interconverts dihydroxyacetone phosphate
and 3phosphoglyceraldehyde 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 Alzheimers 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. Invitro, 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 Alzheimers 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.
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Carbonic anhydrase II is crucial for the maintenance
of pH and control of carbon dioxide levels; its activity
is also altered in Alzheimers disease85. Glyceraldehyde
3phosphate 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
-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.
Huntingtons and Parkinsons diseases. In Huntingtons
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
Snitrosylated by NO that is derived from the endog
enous nitrosothiol Snitrosocysteine88. Matrix metal
loproteinases are involved in the pathogenesis of acute
and chronic neurodegenerative disorders, such as stroke,
Alzheimers disease, HIV-associated dementia and multi
ple sclerosis88,89. A similar mechanism has been proposed
for Parkinsons disease. Yao etal.90 and Chung etal.91
independently demonstrated that Snitrosocysteine-
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 Snitrosylate GAPDH, thereby reducing
its activity and allowing GAPDH to bind to another E3
ubiquitin ligase, SIAH1. The GAPDHSIAH1 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 Parkinsons disease
because of its ability to inhibit monoamine oxidase type B.
At nanomolar concentrations, selegiline prevented
Snitrosylation 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 NOinduced neurotoxicity is
protein-disulphide isomerase (PDI), which catalyses
thiol-disulphide exchange, thereby promoting the
REVIEWS
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 Alzheimers disease and mild
cognitive impairment10,11,116. This approach has provided new insight into potential
mechanisms of onset and progression of Alzheimers 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) SDSPAGE, 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.
2D blot
Sample 2D-PAGE
(protein mixture) Image analysis
2D gel map
In-gel trypsin
digestion
Spot excision
Protein Database
Mass spectrometry
identification searching
Natureand
formation of disulphide bonds Reviews | Neuroscience
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 Alzheimers disease or Parkinsons
disease, this protective role has been lost owing to the
Snitrosylation of crucial cysteines and the inhibition of
PDI enzymatic activity93.
Therapeutic potential
Knowledge 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
NOcGMP pathway, are used to treat important disorders
such as impotence94, respiratory diseases95,96 and pulmo
nary hypertension97, current data indicate that NObased
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
772 | october 2007 | volume 8 www.nature.com/reviews/neuro
2007 Nature Publishing Group
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 Alzheimers 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--peptide-induced microglial
activation101. Ferulic acid ethyl ester protected cortical
neurons invitro and brain tissue invivo from amyloid-
toxicity by inducing the expression of haem oxygenase 1
and other members of the heat shock protein family,
as well as by decreasing neuronal 3nitrotyrosine levels
and, therefore, NOS activity102.
Acetyll-carnitine. Acetyll-carnitine might be of thera
peutic benefit for Alzheimers 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
acetyll-carnitine protects neurons from oxidative dam
age and neurotoxicity induced by amyloid- 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- 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.
 REVIEWS

Chemotherapy-induced somnolence. NO is involved in Conclusions and perspectives

the brain effects that are induced by peripheral adminis
tration of the chemotherapeutic agent adriamycin111113.
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 bloodbrain 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.
Nitric 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 proposed 98, owing to their abil
ity to enhance cellular survival pathways such as the
heat-shock response 77,98. However, although there
is an impressive amount of invitro 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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This was the first paper to describe proteomics- di Interesse Nazionale 2005, Fondo per gli Investimenti della
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