Aberrant Protein S-Nitrosylation
in Neurodegenerative Diseases
Tomohiro Nakamura,1,* Shichun Tu,1Mohd Waseem Akhtar,1Carmen R. Sunico,1Shu-ichi Okamoto,1
and Stuart A. Lipton1,*
1Del E. Web Center for Neuroscience, Aging, and Stem Cell Research, Sanford-Burnham Medical Research Institute, 10901 North Torrey
Pines Road, La Jolla, CA 92037, USA
*Correspondence: firstname.lastname@example.org (T.N.), email@example.com (S.A.L.)
S-Nitrosylation is a redox-mediated posttranslational modification that regulates protein function via cova-
lent reaction of nitric oxide(NO)-related species with acysteine thiol group on the target protein. Under phys-
iological conditions, S-nitrosylation can be an important modulator of signal transduction pathways, akin to
phosphorylation. However, with aging or environmental toxins that generate excessive NO, aberrant S-nitro-
sylation reactions can occur and affect protein misfolding, mitochondrial fragmentation, synaptic function,
apoptosis or autophagy. Here, we discuss how aberrantly S-nitrosylated proteins (SNO-proteins) play a
crucial role in the pathogenesis of neurodegenerative diseases, including Alzheimer’s and Parkinson’s dis-
eases. Insight into the pathophysiological role of aberrant S-nitrosylation pathways will enhance our under-
standing of molecular mechanisms leading to neurodegenerative diseases and point to potential therapeutic
The global prevalence of neurodegenerative disorders such as
Parkinson’s disease (PD), Alzheimer’s disease (AD), and amyo-
trophic lateral sclerosis (ALS),isincreasing with extendedlife ex-
pectancy. Although precise cellular and molecular mechanisms
underlying neurodegeneration still remain enigmatic, key fea-
tures of these devastating disorders have been identified,
including elevated oxidative/nitrosative stress, mitochondrial
dysfunction, protein misfolding/aggregation, synapse loss, and
decreased neuronal survival. Notably, oxidative/nitrosative
stress appears to influence the manifestation of other patholog-
ical features, including synaptic loss and neuronal cell death,
suggesting that these pathways may be a common determinant
In most mammalian cells, reactive oxygen/nitrogen species
(ROS/RNS) are normally produced at low levels and act
as important physiological messengers of intracellular sig-
naling pathways (Finkel, 2011). However, exposure to environ-
mental toxins or even the normal process of brain aging can
trigger an imbalance between the production of ROS/RNS
and the availability of cell defense systems, including anti-
oxidant enzymes, glutathione and molecular chaperones,
resulting in an overabundance of ROS/RNS that causes oxida-
tive and nitrosative stress. Neurons are particularly vulner-
able to oxidative/nitrosative stress due to their high demand
for energy from ROS/RNS-generating mitochondrial meta-
bolism and the fact that they contain lower levels of certain
antioxidants compared to other cells (Mattson et al., 2002).
Additionally, in some cases genetic mutations that are
pathogenic for inherited neurodegenerative diseases can lead
to increases in basal ROS/RNS production, thus rendering
neurons more vulnerable to additional oxidative/nitrosative
ROS/RNS include reactive free radical groups that exert their
biological effects, at least in part, via reaction with cellular mac-
romolecules. NO is a small, highly diffusible molecule generated
by a family of NO synthases (NOS) that convert L-arginine to
L-citrulline using molecular oxygen and NADPH (Bredt et al.,
1991). This family includes three members: neuronal NOS
NOS (eNOS or NOS3) (Fo ¨rstermann et al., 1991). nNOS and
eNOS have been named after cell types in which they are consti-
tutively and predominantly expressed, while expression of iNOS
is typically induced by acute inflammatory stimuli. All three NOS
leagues have shown that production of NO-related species or
related compounds, possibly including dinitrogen trioxide
(N2O3) or the reaction intermediate nitrosonium cation (NO+),
can lead to S-nitrosylation, a reversible, covalent chemical reac-
tion involving the addition of an NO moiety to a critical cysteine
thiol(-SH) group(or moreproperly thiolate anion,-S?)ona target
protein to regulate its function. This nitrosation reaction forms an
S-nitrosothiol (-SNO), and an S-nitrosylated protein is thus
referred to as a SNO-protein (Lei et al., 1992; Stamler et al.,
1992; Lipton et al., 1993; Stamler et al., 2001). It is important to
note that S-nitrosylation of cysteine thiol is a chemically distinct
redox reaction from nitration of tyrosine residues, representing
another NO-dependent posttranslational modification gener-
ated, for example, via reaction of tyrosine with peroxynitrite
(ONOO?) (Ischiropoulos et al., 1992).
Under normal physiological conditions, S-nitrosylation modu-
lates the function of substrate proteins, thus playing a dynamic
role in a variety of biological processes (Figure 1). Like other
conformational changes, activate or inhibit protein activity, alter
protein-protein interactions, affect protein aggregation, or influ-
ence protein localization. These alterations affect cell signal
Neuron 78, May 22, 2013 ª2013 Elsevier Inc.
transduction pathways and neuronal function (Choi et al., 2000;
Qu et al., 2011; Shi et al., 2013; Uehara et al., 2006). Under path-
ological conditions, aberrant S-nitrosylation of specific proteins
stimulates cell destructive processes, contributing to neurode-
generation. Insults mediated by S-nitrosylation include protein
misfolding, endoplasmic reticulum (ER) stress, mitochondrial
dysfunction, synaptic degeneration, and apoptosis (Nakamura
and Lipton, 2007). Nitrosative stress-associated excitotoxicity
is implicated in a number of neurological disorders, ranging
from acute hypoxia-ischemia to chronic neurodegenerative dis-
eases. Manipulating S-nitrosylation can affect pathology; for
example, pharmacological inhibition of nNOS or knockout of
the nNOS gene provides neuroprotection against ischemia
(Huang et al., 1994).
In this review, we summarize progress made in the identifica-
tion and characterization of key S-nitrosylated proteins that have
been found to play a role in the pathogenesis of AD, PD, and
other neurodegenerative diseases. SNO-proteins discussed in
this review are summarized in Table 1. We will also discuss
how S-nitrosylation affects the function of specific target pro-
teins and influences the onset or development of neurodegener-
ation. Further, we develop a concept for disease pathogenesis
concerning the nearly ubiquitous nature of aberrant S-nitrosyla-
tion reactions in sporadic neurodegenerative disorders. This
contrasts with the rarity of familial forms of these diseases due
to genetic mutations. We propose that excessive nitrosative/
oxidative stress might cause the more common ‘‘sporadic’’
form of neurodegenerative diseases by lowering the threshold
for, or even mimicking, the effect of rare genetic mutations. In
support of this hypothesis, a number of studies have found aber-
rantly S-nitrosylated proteins contributing to disease pathogen-
esis, and theseproteins are encoded bygenesthat manifest rare
mutations causing the same disease phenotype.
Generation of NO in the CNS
A well-established route for NO production in several neurode-
generative disorders involves activation of NMDA receptors
(NMDARs). In brain, nNOS is predominantly expressed in neu-
rons and physically tethered to NMDARs due to mutual interac-
tions with PSD-95 at the postsynaptic density (Brenman et al.,
1996). Activated NMDARs are permeable to Ca2+, which in turn
activates nNOS to produce NO (Bredt et al., 1991). Additionally,
activation of NMDARs also generates ROS (Lafon-Cazal et al.,
Neuroinflammatory stimuli or other toxins (e.g., Ab oligomers
or 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine [MPTP]) can
induce the expression of iNOS in the brain, predominantly in as-
trocytes, macrophages, and microglia cells. iNOS can produce
high concentrations of NO to contribute to neurotoxicity, and
tance against MPTP-induced neurotoxicity in ananimal model of
Figure 1. NO and SNO Signaling in the CNS
(A) S-Nitrosylation plays a dynamic role in both normal and aberrant neuronal
signal transduction pathways. Initially, activation of soluble guanylate cyclase,
with consequent increase in production of cGMP, was identified as an NO-
mediated signal transduction pathway. Additionally, peroxynitrite (ONOO?),
derived from reaction of NO$ and superoxide anion (O2?$), can mediate
neurotoxicity in part via a protein posttranslational modification of tyrosine
residues termed nitration. However, emerging evidence suggests that NO
species mediate signal transduction predominantly via protein S-nitrosylation,
a posttranslational redox modification of critical cysteine residues that affects
protein activity and function. Under physiological conditions, NO is produced
in neurons predominantly by nNOS, which is activated by calcium influx
through NMDAR-associated ion channels. Extrasynaptic in addition to syn-
apticNMDARs canleadtoNOproduction,particularly duringexcitotoxic injury
when excessive NO is generated. This can lead to aberrant protein S-nitro-
sylation whereby cysteine residues, which would not ordinarily be S-nitro-
sylated by physiological levels of NO, undergo S-nitrosylation because of the
high levels of NO generated in disease states.
(normal) levels of NO can mediate neuroprotective effects, at least in part,
by S-nitrosylating caspase and HDAC2. Additionally, during periods of mod-
erate stress, NO can still facilitate protection of neurons, for instance, via
S-nitrosylation of NMDARs to downregulate excessive activity, representing a
negative feedback mechanism. However, persistent hyperactivation of
stimulation of nNOS and thus increased production of NO that contributes to
synaptic injury and cell death. Glial cells (astrocytes and microglia) can also
generate high levels of NO via iNOS activity. We and others have reported
evidence that overproduction of NO can be neurotoxic via aberrant S-nitro-
sylation of parkin, PDI, GAPDH, MMP-2/9, Cdk5, Drp1, and other proteins that
contribute to accumulation of misfolded proteins, mitochondrial dysfunction,
synaptic damage, and neuronal cell death.
Neuron 78, May 22, 2013 ª2013 Elsevier Inc.
Table 1. Summary of SNO Proteins Discussed in this Review
CategoryTarget of S-Nitrosylation Effect of S-Nitrosylation
Synapse/membrane-related proteinsNMDA receptor Downregulation of excessive receptor activity
PSD-95 Decrease in its synaptic expression
NSF Increase in surface expression of AMPA receptors
Stargazin Increase in surface expression of AMPA receptors
AMPAR (GluA1) Increase in its phosphorylation, and enhanced endocytosis
Serine racemase Decrease in generation of D-serine
Syntaxin 1 Possibly prevention of excessive release of glutamate
SNAP-25 Decrease in evoked neurotransmitter release, while increasing
RyR Increased channel opening
GRK2 Prevention of receptor internalization
b-arrestin Acceleration of receptor internalization
Quality control machineriesParkin Dysregulation of its ubiquitin E3 ligase activity
PDIInhibition of its chaperone and isomerase activities
(1) Inhibition of its kinase activity
(2) Inhibition of autophagy flux
HSP90 Inhibition of its chaperone activity
Proteasome Inhibition of 26S proteasome activity
N-end-rule Regulation of UPS-dependent N-end rule
Cell death/survival or other signaling pathways CaspasesInhibition of its protease activity
XIAP (1) Suppression of its anti-apoptotic activity
(2) S-Nitrosylation of caspases (via transnitrosylation)
FLIP Inhibition of its proteasomal degradation
Bcl-2Inhibition of its proteasomal degradation
GAPDH (1) Activation of cell death pathway
(2) S-Nitrosylation of nuclear proteins (via transnitrosylation)
GOSPEL Inhibition of SNO-GAPDH-mediated cell death
MMPs Activation of its metalloproteinase activity
DJ-1 Possible regulation of its anti-cell death activity
Akt Inhibition of its kinase activity
PTEN Inhibition of its phosphatase activity
SHP-2Inhibition of its phosphatase activity
MAP1BIncrease of its microtubule binding affinity
Cdk5 (1) Increase in its kinase activity
(2) S-Nitrosylation of Drp1 (via transnitrosylation)
Prx2 Inhibition of its antioxidant activity
COX-2 Activation of its prostaglandin synthesis activity
IDE Inhibition of its metalloprotease activity
ApoEPossibly lowering its binding affinity to LDL receptors
Mitochondrial function Drp1 Upregulation of its GTPase activity, excessive mitochondrial
fission, bioenergetic failure, synaptic dysfunction and loss
Complex I Inhibition of mitochondrial respiration activity
Complex IVInhibition of mitochondrial respiration activity
F1 ATPaseInhibition of its ATPase activity
Aconitase Suppression of the citric acid cycle
ALDH2 Suppression of alcohol metabolism
Iron homeostasisIRP2Increase in its proteasome degradation
DexrasAugmentation of iron uptake
Neuron 78, May 22, 2013 ª2013 Elsevier Inc.
in an animal model of AD (amyloid precursor protein-presenilin 1
[APP-PS1] double transgenic) ameliorated AD-like symptoms
such as premature mortality, cerebral plaque formation,
increased b-amyloid (Ab) levels, and astrocytosis/microgliosis
(Nathan et al., 2005). However, in a study utilizing a different
mouse AD model (Tg2576 APP transgenic), deletion of iNOS
worsened spatial memory, learning, and tau pathology, suggest-
ing that NO manifested a neuroprotective effect in this case (Wil-
cock et al., 2008).
Protein S-Nitrosylation, Denitrosylation, and
Hundreds, if not thousands, of proteins with potential S-nitrosy-
lation sites have been identified (Seth and Stamler, 2011).
Although the majority of cellular proteins possess multiple
cysteine residues, only specific cysteine residues are S-nitrosy-
lated. One well-characterized determinant of S-nitrosylation
depends on proximity. For example, formation of a complex
with nNOS regulates the S-nitrosylation of NMDARs and PSD-
95 (Lei et al., 1992; Lipton et al., 1993; Ho et al., 2011). Similarly,
the brain-enriched small GTPase Dexras1 is linked to nNOS via
an adaptor protein CAPON, and Dexras1 is activated by S-nitro-
of S-nitrosylation entails the presence of a signature SNO motif
of amino acid residues adjacent to the target cysteine. Acidic
and/or basic amino acids typically exist within 6 to 8 A˚of the
hydryl to facilitate S-nitrosylation (Stamler et al., 1997; Hess
et al., 2005; Doulias et al., 2010; Marino and Gladyshev, 2010).
In addition, local hydrophobicity may also promote the speci-
ficity of S-nitrosylation via increased stability of the S-nitroso-
thiol. Accordingly, S-nitrosylation can occur in a facile manner
within or near biological membranes.
The process of SNO-protein formation is counterbalanced by
denitrosylation enzymes, such as S-nitrosoglutathione reduc-
tase, the thioredoxin system, and PDI (see Benhar et al., 2009
for a detailed review of protein denitrosylation). These denitrosy-
lases are involved in the removal of NO from S-nitrosylated
Figure 2. Protein-Protein
Transnitrosylation as an Enzymatic
Nitrosation Mechanism to Produce SNO-
Increasing evidence suggests that a prominent
S-nitrosylating reaction mechanism involves pro-
tein-protein transnitrosylation reactions (repre-
senting transfer of an NO group from one protein
thiol to another). Transnitrosylation reactions re-
ported to date include the following:
(A) hemoglobin-to-anion exchanger 1 (AE1).
(B) Thioredoxin 1 (Trx1)-to-Caspase-3 (Casp-3).
(C) Caspase-3 (Casp-3)-to-X-linked inhibitor of
(E) Cdk5-to-dynamin related protein 1 (Drp1).
cysteine residues and thus can poten-
tially ameliorate nitrosative stress under
Transnitrosylation may be the major enzymatic mechanism to
generate S-nitrosylated proteins in biological systems. Recent
studies have identified S-nitrosylating enzymes, acting via pro-
tein-protein transnitrosylation, as a primary source of S-nitrosy-
lase activity (Kornberg et al., 2010; Mitchell and Marletta, 2005;
Nakamura et al., 2010). Transnitrosylation reactions catalyze
the transfer of an NO group from a donor protein to the reactive
cysteine residue on an acceptor protein, producing both a deni-
trosylated protein and a SNO-protein. Thus, the protein accept-
ing the NO group in a transnitrosylation reaction may also serve
to its protein thiol from another S-nitrosylated protein. Examples
of transnitrosylases (representing the NO donor protein) demon-
strated to date include SNO-hemoglobin, SNO-thioredoxin (Trx),
SNO-GAPDH, SNO-Caspase-3, and SNO-cyclin-dependent ki-
nase 5 (Cdk5) (Figure 2).
Oxidation Reactions Downstream of S-Nitrosylation
S-Nitrosylation can result in conformational changes in protein
structure, which may facilitate further oxidation reactions with
less active ROS, resulting in sulfenic acid (-SOH), sulfinic acid
(-SO2H), or sulfonic acid (-SO3H) derivatization of the cysteine
thiol group. Sulfonation (-SO3H) reactions cannot be reversed
by known enzymes and can therefore result in permanent/path-
ological changes to protein structure and activity (Gu et al.,
2002). Interestingly, sulfination (-SO2H), unlike sulfonation
firedoxin is transcriptionally induced and that this can occur in
neurons via increased synaptic NMDAR activity (Papadia et al.,
In addition, S-nitrosylation has been reported to influence
additional posttranslational modifications of cysteine residues.
For instance, when there are two neighboring cysteine residues,
tion between them (Lipton et al.,2002; Stamler and Toone, 2002;
Cho et al., 2009). In contrast, if both cysteine residues are S-
nitrosylated, e.g., under severe nitrosative conditions, S-nitrosy-
lation inhibits disulfide formation (Hess et al., 2005; Uehara et al.,
2006). Moreover, S-nitrosylation of a cysteine residue can
Neuron 78, May 22, 2013 ª2013 Elsevier Inc.
precede and thus inhibit palmitoylation (Ho et al., 2011). Since
palmitoylation increases the association of a protein to the cell
membrane, S-nitrosylation can decrease membrane targeting.
Furthermore, S-nitrosylated as well as sulfenated cysteine resi-
dues may react with glutathione (Martı ´nez-Ruiz and Lamas,
2007), but it is unclear whether SNO-proteins are intermediates
for protein glutathionylation in vivo. Future studies are likely to
reveal novel relationships between S-nitrosylation and other
types of posttranslational modifications.
Oxidative and Nitrosative Stress in Alzheimer’s and
The increase in free radicals and decrease in antioxidant poten-
tial that occurduring aging may contribute to the development of
neurodegenerative conditions, such as AD and PD. Consistent
with this idea, postmortem human AD brains exhibit increased
oxidative and nitrosative stress with elevated levels of free radi-
cals (Sayre et al., 2008). While relatively rare genetic mutations,
for example to the genes encoding APP or PS1/PS2 can cause
AD, it appears that this same phenotype can be mimicked by
of proteins critical to synaptic function and neuronal survival are
aberrantly S-nitrosylated in AD, contributing to synaptic loss and
neurodegeneration (Figure 3).
PD is the second most common neurodegenerative disease
and most common motor disorder. It is characterized byspecific
loss of dopaminergic neurons in the substantia nigra pars com-
pacta and is often accompanied by protein inclusions known as
Lewy bodies (LBs). The major proteinaceous component of LBs
is a-synuclein (a-syn), whose encoding gene is mutated in an
autosomal dominant familial form of PD with LB dementia (Poly-
meropoulos et al., 1997). Mutations in additional genes, for
example, encoding parkin, PINK1, and DJ-1, have also been
identified as causal factors for autosomal recessive forms of fa-
milial PD in humans (Bonifati, 2012). In animal models, mutation
of these genes can cause abnormal responses to oxidative
ER stress, synaptic injury, and ultimately cell death (Dawson
et al., 2010). However, similar to AD, the majority of PD cases
Figure 3. Representative Protein
Substrates for Aberrant S-Nitrosylation
Aberrant protein S-nitrosylation plays a patholog-
ical role in many neurodegenerative conditions.
Production of excessive NO can S-nitrosylate
parkin, PDI, Drp1, Cdk5, Prx2, MMP-9, COX-2,
GAPDH, PTEN/Akt, JNK/IKKb, MAP1B, and XIAP.
Subsequently, these aberrant S-nitrosylation re-
actions trigger neurotoxic signaling pathways
leading to ER stress, protein misfolding, mito-
chondrial fragmentation, bioenergetic compro-
mise, and consequent synaptic/neuronal damage.
These processes can contribute to the patho-
genesis of PD, AD, HD, ALS, stroke, and poten-
tially other neurodegenerative disorders.
(>90%–95%) are sporadic (although mu-
tations in the LRRK2 gene may in fact be
present in a number of these cases).
Studies suggest that sporadic PD can
be affected by both genetic factors, usually causing early-onset
disease, and environmental factors, possibly including agricul-
tural pesticides, herbicides, fungicides, or other neurotoxins
that are known to act as mitochondrial toxins to generate oxida-
tive and nitrosative stress (Betarbet et al., 2000; Yao et al., 2004;
Chung et al., 2004; Uehara et al., 2006). Thus, PD may represent
another example of a disease in which a rare genetic mutation
can be phenocopied by more common nitrosative/oxidative
posttranslational modifications of critical proteins.
Dopaminergic neurons are especially susceptible to oxidative
damage (Miller et al., 2009). Compelling evidence suggests that
S-nitrosylation induced by nitrosative stress is a major contrib-
uting factor in the development of PD. As discussed below,
S-nitrosylationofproteins,including parkin,DJ-1,X-linked inhib-
itor of apoptosis protein (XIAP or IAP3), peroxiredoxin (Prx) 2,
and glyceraldehyde-3-phosphate dehydrogenase (GAPDH), is
involved in the pathological process, and can contribute to ubiq-
uitin-proteasome impairment, ER stress, mitochondrial dysfunc-
tion, and apoptosis (Figure 3).
S-Nitrosylation of Parkin
Parkin is an E3 ubiquitin ligase that participates in the ubiquitin-
proteasome system (UPS) responsible for targeting specific pro-
teins for degradation (Shimura et al., 2000). Parkin is also
involved in protein degradation during ER stress via its interac-
tion with the chaperones, heat shock protein (Hsp)70 and
carboxyl-terminus of Hsp70 interacting protein (CHIP), and
thus participates in ER-associated degradation (ERAD) (Wang
and Takahashi, 2007). Disruption of parkin activity causes
dysfunction in protein degradation, leading to accumulation/
aggregation ofneurotoxic proteinsandresultingERstress(Daw-
son and Dawson, 2003; Lindholm et al., 2006).
In addition to its E3 ligase activity, parkin also suppresses the
transcription of the oncogene p53 and serves a neuroprotective
function against PD-associated apoptosis of dopaminergic
neurons (da Costa et al., 2009). Mutations in parkin (PARK2)
have been identified as causal for autosomal recessive juvenile
PD and early onset PD (Kitada et al., 1998). Emerging evidence
points to the possibility that parkin, together with PINK1, whose
gene can also be mutated in familial PD, participates in
Neuron 78, May 22, 2013 ª2013 Elsevier Inc.
mitophagy whereby damaged mitochondria are removed by
quality control machinery such as autophagy (Youle and van
der Bliek, 2012). In this proposed model, PINK1 is initially trans-
located to impaired mitochondria, which in turn recruit parkin
from the cytosol to the damaged mitochondrial membrane.
Recent evidence suggests that PINK1-phosphorylated mitofu-
sin 2 is a parkin receptor on the mitochondrial membrane
(Chen and Dorn, 2013). Parkin then ubiquitinates mitochondrial
outer membrane proteins to enhance autophagic removal of
the unhealthy mitochondria. Parkin’s neuroprotective activity
and role in protein ubiquitination/degradation can also be
compromised by environmental neurotoxic factors, such as
pesticides and herbicides, probably because these toxins
generate excessive amounts of NO and ROS. Several studies
have shown that high levels of NO and ROS can precipitate
S-nitrosylation or further oxidation/sulfonation of parkin (Chung
et al., 2004; Yao et al., 2004; Meng et al., 2011). Parkin has
multiple cysteine residues that can react with NO to form
SNO-parkin. For instance, exposure to the pesticide rotenone
or the neurotoxin MPTP leads to S-nitrosylation of parkin
(Chung et al., 2004; Yao et al., 2004). Intriguingly, upon S-nitro-
sylation, parkin’s E3 ligase activity initially increases but is sub-
sequently inhibited, possibly because of autoubiquitination.
Consequently, the downregulated E3 ligase activity impairs
ubiquitination and degradation of substrate proteins, potentially
contributing to LB formation and neuronal cell injury or death
(Chung et al., 2004; Yao et al., 2004; Lipton et al., 2005;
in the brains of human cases of sporadic PD as well as in animal
models of PD, further supporting a role for S-nitrosylated parkin
in disease pathogenesis (Chung et al., 2004; Yao et al., 2004).
These findings are consistent with our hypothesis that aberrant
S-nitrosylation can contribute to more common forms of neuro-
degenerative diseases by mimicking the effects of rare genetic
S-Nitrosylation of PDI
In neurodegenerative conditions, accumulation of unfolded or
misfolded proteins can induce ER stress. In response, cellular
defense proteins, such as PDI, can be upregulated to increase
chaperone and isomerase activity (Conn et al., 2004; Tanaka
et al., 2000). However, this neuroprotective effect can be in-
hibited by S-nitrosylation, as formation of SNO-PDI compro-
mises its ability to correct protein misfolding (Uehara et al.,
2006; Figure 4). Mitochondrial complex I inhibitors, including
pesticides thought to possibly contribute to the pathogenesis
of PD, also increase SNO-PDI formation in cell-based experi-
ments (Uehara et al., 2006). Additionally, in animal models of
ALS and stroke, iNOS-dependent formation of SNO-PDI
increased the aggregation of ubiquitinated proteins (Chen
et al., 2012; Chen et al., 2013). Consistent with a pathological
role, substantial levels of SNO-PDI are present in human brains
manifesting sporadic PD, AD, and ALS (Uehara et al., 2006;
Walker et al., 2010). These results suggest that PDI is aberrantly
lational modification may contribute to the progression of the
disease, as it compromises chaperone/isomerase activity of
PDI and thus aggravates protein misfolding and ER stress. In
addition to PDI, S-nitrosylation of another ER chaperone folding
protein, glucose-related protein (GRP), may also occur in these
diseases (Dall’Agnol et al., 2006). Moreover, rare mutations in
genes encoding PDI-family proteins have been suggested to
contribute to neurodegenerative conditions, consistent with the
hypothesis that rare mutations may be mimicked by environ-
mental factors that induce S-nitrosylation.
S-Nitrosylation of Cdk5
Cdk5 isa serine/threonine kinase that isimportant in brain devel-
opment, regulating neuronal differentiation and migration, axon
Figure 4. Neurodegenerative Signaling
Pathways Triggered by Aberrant S-
Genetic mutations associated with neurodegener-
ative diseases, environmental
as certain pesticides, and misfolded proteins
including Ab oligomers can all lead to excessive
nitrosative stress. The resulting aberrant S-nitro-
in the pathogenesis of several neurodegenerative
disorders. For example, S-nitrosylation of specific
proteins can cause ‘‘loss-of-function’’ by impairing
E3 ligase ubiquitin ligase activity (parkin and XIAP)
or isomerase/molecular chaperone activity (PDI).
neurotoxic proteins and activation of apoptotic
pathways. Additionally, oligomeric Ab peptide can
result in increased generation of NO and S-nitro-
sylation of Drp1, resulting in excessive mitochon-
drial fragmentation, bioenergetic compromise, and
activated when Ab increases calpain activity to
cleave the Cdk5 regulatory subunit p35top25. The
resulting neurotoxic kinase activity of Cdk5 is
further enhanced by S-nitrosylation. Formation of
SNO-Cdk5 may also contribute to Ab-induced
spine loss by transnitrosylating Drp1, with the
resultant SNO-Drp1 participating in mitochondrial
fragmentation and synaptic loss.
Neuron 78, May 22, 2013 ª2013 Elsevier Inc.
guidance, and synaptic plasticity (Kim et al., 2006; Ohshima
et al., 1996). In addition, excessive Cdk5 activity has been impli-
cated in the development of several neurological disorders,
including AD (Cheung and Ip, 2012). In the adult brain, Cdk5 is
in close proximity to a membrane-bound protein complex con-
taining nNOS/PSD-95/NMDAR. Cdk5 can form a complex with
nNOS and become a target of S-nitrosylation under pathological
conditions (Zhang et al., 2010; Qu et al., 2011, 2012). Indeed,
SNO-Cdk5 is highly expressed in human AD postmortem brains
but not in normal control brains (Qu et al., 2011).
NO was found to S-nitrosylate Cdk5 on residues Cys83 and
Cys157 and thus activate Cdk5 (Figure 4). This activation leads
to increased phosphorylation of substrates such as ataxia telan-
giectasia mutated (ATM), a proapoptotic protein kinase (Qu
et al., 2011). However, S-nitrosylation induced by high (nonphy-
siological) concentrations of NO can exert the opposite effect on
Cdk5 kinase activity, i.e., inhibiting the enzyme (Zhang et al.,
2010). Nonetheless, when adding high exogenous levels of NO
donors, the pH of the solution can be dramatically lowered,
tivity after adding high concentrations of exogenous NO donors
the observed discordant effects of NO on Cdk5 activity. SNO-
Cdk5 has also been shown to contribute to Ab-induced synaptic
degeneration; Ab-induced dendritic spine loss in cultured
cortical neurons was at least partially inhibited by nonnitrosylat-
able mutant-Cdk5, and the NOS inhibitor, N-nitro-L-arginine
(NNA) also blocked synaptic spine loss (Qu et al., 2011). These
results suggest that NO production by NOS and subsequent
S-nitrosylation of Cdk5 are important steps in Ab-induced syn-
aptic loss. Interestingly, Ab-induced synaptic loss is also in
part dependent on NMDAR activity (Shankar et al., 2007; Snyder
et al., 2005). NMDAR activity activates nNOS, and NMDARs are
aberrantly phosphorylated and activated by Cdk5 under patho-
logical conditions (Wang et al., 2003), apparently representing
a positive-feedback loop. Thus, NMDAR-mediated activity may
play an important role in SNO-Cdk5-mediated synaptic loss.
Consistent with this postulate, NMDA-induced spine loss in
cultured cortical neurons was blocked by pretreatment with
NNA and partially blocked by the Cdk5 inhibitor, Roscovitine
(Qu et al., 2011). Taken together, these results describe a unique
regulatory mechanism whereby SNO-Cdk5 mediates, at least in
part, Ab-induced spine loss in AD.
S-Nitrosylation of Drp1
Mitochondria provide energy and play a crucial role in normal
neuronal synaptic activity and maintenance of synapses (Li
et al., 2004). Ab-induced oxidative stress can contribute to mito-
chondrial dysfunction, including excessive fragmentation, which
in turn leads to synaptic dysfunction, neuronal injury, and ulti-
mately cell death (Knott et al., 2008; Reddy and Beal, 2008).
Drp1 is a GTPase involved in normal mitochondrial fission. In
cultured primary cortical neurons, exposure to oligomeric Ab re-
sults in S-nitrosylation of Drp1 at Cys644. Such formation of
SNO-Drp1 results in excessive mitochondrial fission, energy
compromise, and eventually dendritic spine loss (Cho et al.,
2009; Figure 4). S-Nitrosylation also increases the GTPase activ-
ity of both dynamin 1 and 2, close homologs of Drp1 (Kang-
Decker et al., 2007; Wang et al., 2006). Importantly, preventing
Drp1(C644A) abrogated Ab-induced synaptic damage (Cho
et al., 2009). In support of a pathophysiological role for SNO-
Drp1, Drp1 is apparently S-nitrosylated aberrantly because it is
found at high levels in postmortem human AD brains but not in
control brains (Cho et al., 2009; Wang et al., 2009), as well as
in peripheral blood lymphocytes of AD but not control patients
(Wang et al., 2012). Taken together, these findings also suggest
that SNO-Drp1 may represent a potential therapeutic target for
protecting neurons and their synapses in AD.
Interestingly, S-nitrosylation of Drp1 is likely mediated at least
in part by transnitrosylation from SNO-Cdk5, which is present at
high levels in human postmortem AD brains. SNO-Cdk5 can
function as a nitrosylase enzyme and NO donor by transferring
its NO group to Drp1 (Qu et al., 2011). Importantly, these findings
indicate that Cdk5 can regulate Ab-induced spine loss by a
mechanism independent of its kinase activity, suggesting that
both phosphorylase and nitrosylase activity can be important
for the pathological consequences of this dual-function enzyme
(Qu et al., 2011) (Figures 2 and 4).
S-Nitrosylation of XIAP and Caspases
Another transnitrosylase system is represented by the XIAP/cas-
pase pair of enzymes. Caspases belong to a cysteine protease
family that is important for the execution of apoptotic cell death.
NO has been reported to S-nitrosylate caspases-3, -8, and -9 at
the active site cysteine, thus inhibiting enzymatic activity and af-
fording neuroprotection (Dimmeler et al., 1997; Melino et al.,
1997; Tenneti et al., 1997; Mannick et al., 1999). Interestingly, af-
ter exposure to cell death stimuli, mitochondrial Trx2 selectively
denitrosylates SNO-caspase-3 (Benhar et al., 2008), relieving in-
hibition of the active site cysteine from -SNO. This form of deni-
trosylation of caspase-3 requires the oxidoreductase activity of
Trx. In contrast, however, after Trx1 is S-nitrosylated at Cys73
(one of its nonactive site cysteines), the resulting SNO-Trx1
has been reported to transnitrosylate caspase-3 (Mitchell and
Marletta, 2005; Figure 2). Since S-nitrosylation of Cys73 occurs
only when the active site cysteines of Trx1 have been oxidized,
Trx1 acts as a nitrosylase only under conditions of oxidative/
nitrosative stress (Barglow et al., 2011).
Additionally, S-nitrosylation of the E3 ubiquitin ligase XIAP can
occur under pathological conditions (Nakamura et al., 2010;
Tsang et al., 2009). Normally, XIAP targets active caspases-3,
-7, and -9 for ubiquitination and degradation, thus suppressing
caspase-mediated apoptosis and promoting cell survival
(Deveraux et al., 1999). XIAP is the most potent endogenous
teins (IAPs) (Eckelman et al., 2006). However, this neuroprotec-
tive activity can be abrogated during nitrosative stress via
S-nitrosylation of XIAP (Nakamura et al., 2010; Tsang et al.,
2009). This nitrosylation inhibits the E3 ubiquitin ligase activity
of XIAP, which would otherwise lead to the degradation and
hence inactivation of caspases (Figure 4). The level of SNO-
XIAP is significantly increased in human brains from AD, PD,
and Huntington’s disease (HD) patients compared to controls
(Nakamura et al., 2010; Tsang et al., 2009), consistent with the
notion that SNO-XIAP plays a pathological role in these
diseases. A significant increase in SNO-XIAP is also found in
both cell-based and animal models of PD (Nakamura et al.,
formation withthe nonnitrosylatablemutant
Neuron 78, May 22, 2013 ª2013 Elsevier Inc.
2010; Tsang et al., 2009). Additionally, XIAP can be S-nitrosy-
lated via transnitrosylation from SNO-caspase-3. Since S-nitro-
lation from SNO-caspase to XIAP acts as an apoptotic switch,
both relieving direct inhibition of caspases by S-nitrosylation
and inhibiting caspase degradation by XIAP via SNO-XIAP for-
mation (Nakamura et al., 2010).
In addition, NO can indirectly influence the apoptotic caspase
cascade via S-nitrosylation of FLICE inhibitory protein (FLIP) and
Bcl-2 (Azad et al., 2006; Chanvorachote et al., 2005). FLIP is an
antiapoptotic protein that inhibits binding of caspase-8 to the
Fas-associated death domain (FADD). Bcl-2 can inhibit mito-
chondrial-mediated apoptosis via binding to and inhibiting pro-
apoptotic proteins such as Bax and Bak. S-Nitrosylation of
both of these antiapoptotic proteins to enhance their prosurvival
function. Thus, under physiological conditions or during the pre-
symptomatic stage of neurodegenerative diseases, NO may act
as an anti-apoptotic messenger via formation of SNO-FLIP and
S-Nitrosylation of GAPDH
GAPDH is an important glycolytic enzyme, but S-nitrosylation of
GAPDH at Cys150 initiates an apoptotic cell death cascade, for
example, in the MPTP mouse model of PD (Hara et al., 2005,
2006). S-Nitrosylation enhances the binding of GAPDH to
Siah1, an E3 ubiquitin ligase, and the SNO-GAPDH/Siah1 pro-
tein complex is translocated to the nucleus, where it mediates
apoptosis (Figure 5; Hara et al., 2005). The GAPDH-Siah1
tingtin protein (mtHtt), mediating, at least in part, neurotoxicity in
HD (Bae et al., 2006). In the nucleus, GAPDH stimulates the ac-
tivity of p300/CBP and activates downstream targets including
Figure 5. Schematic Representation of
Formation of SNO-GAPDH can trigger multiple
signaling pathways leading to neurodegeneration.
Pathway 1: NO generated from nNOS, iNOS, or
eNOS can S-nitrosylate GAPDH in the cytosol.
to translocate into the nucleus. Pathway 3: in the
nucleus, Siah promotes degradation of nuclear
proteins such as nuclear corepressor (NcoR).
Pathway 4: SNO-GAPDH also increases p300/
CBP activity and induces downstream gene
expression. Pathway 5: SNO-GAPDH serves as a
nuclear nitrosylase, producing SNO-SIRT1 and
mutant huntingtin (mtHtt) protein. Pathway 7:
physiological levels of NO can S-nitrosylate GOS-
PEL, to form a SNO-GOSPEL/GAPDH complex,
preventing the association of GAPDH and Siah 1.
GAPDH regulates iNOS activity as well as ribo-
somal protein L13a degradation.
p53 to precipitate neuronal cell death
(Sen et al., 2008). Additionally, Siah1,
translocated to the nucleus along with
SNO-GAPDH, promotes degradation of
SUV39H1, facilitating acetylation of histone H3 and enhanced
CREB-mediated neurite outgrowth (Sen and Snyder, 2011).
Intriguingly, SNO-GAPDH can also transnitrosylate other nu-
clear proteins, including the deacetylating enzyme sirtuin-1
(SIRT1), histone deacetylase-2 (HDAC2), and DNA-activated
protein kinase (DNA-PK) (Kornberg et al., 2010). A similar
pathway involving S-nitrosylation of GAPDH and Siah1-medi-
ated nuclear translocation has been reported to occur in a rat
modelof cerebralischemiaduringtheearlystages ofreperfusion
injury (Li et al., 2012). A negative regulator of the SNO-GAPDH
pathway involves S-nitrosylation of the protein GOSPEL, which
has been shown to protect neurons from NMDAR-mediated ex-
citotoxicity. SNO-GOSPEL binds to GAPDH, thereby inhibiting
the SNO-GAPDH-mediated apoptosis cascade (Sen et al.,
2009). Another inhibitory mechanism of the pro-apoptotic
SNO-GAPDH pathway involves transnitrosylation of B23/nucle-
ophosmin by SNO-GAPDH. This reaction results in decreased
GAPDH-Siah binding and enhanced B23-Siah interaction, abro-
gating the ubiquitin E3 ligase activity of Siah1 (Lee et al., 2012).
In addition to its effect in the nucleus, recent studies have sug-
gested that SNO-GAPDH also influences cell function in the
cytosol. For instance, GAPDH binds to iNOS in a SNO-depen-
dent manner: GAPDH enhances heme insertion into iNOS to
form active iNOS (Chakravarti et al., 2010); S-nitrosylation of
GAPDH inhibits heme insertion into iNOS since SNO-GAPDH
feedback loop to suppress iNOS-dependent NO production in
glial cells under neurodegenerative conditions. Moreover, in
free ribosomal protein L13a from ubiquitin-proteasome degra-
dation. The resulting degradation of L13a causes defective
translational control (Jia et al., 2012).
Neuron 78, May 22, 2013 ª2013 Elsevier Inc.
Taken together, these studies support the existence of
multiple pathways to cell injury and dysfunction mediated by
SNO-GAPDH. In the canonical pathway, formation of SNO-
GAPDH results in Siah1-dependent nuclear translocation and
neuronal apoptosis, and this cascade may contribute to the
pathogenesis of PD, HD, stroke, and other disorders. However,
SNO-GAPDH also appears to mediate physiological functions
of NO, e.g., via degradation of SUV39H1 or transnitrosylation
of HDAC2 (Kornberg et al., 2010; Sen and Snyder, 2011). Thus,
additional studies are warranted to elucidate the mechanism
determining whether SNO-GAPDH contributes to physiological
or pathological pathways involving NO.
S-Nitrosylation of Prx2
The Prx family of antioxidant proteins belongs to a class of en-
zymes that catalyze reduction of intracellular peroxides by redox
reactions (Rhee et al., 2005). Prx2 is the most abundant in
mammalian neurons (Sarafian et al., 1999), and the level of
Prx2 is increased in a number of neurodegenerative diseases,
probably in an attempt to counteract oxidative stress (Krapfen-
bauer et al., 2003). The antioxidant activity of Prx2 is inhibited
by S-nitrosylation of the two critical cysteine residues that are
involved in its antioxidant activity (Cys51 and Cys172) (Fang
et al., 2007; Romero-Puertas et al., 2007). The result is compro-
mise of the normal redox cycle to detoxify peroxides, involving
coordinated action of Prx with the thioredoxin pathway (thiore-
doxin reductase, thioredoxin and NADPH to reduce -SOH to
-SH) and with the sulfiredoxin pathway (reducing -SO2H to -SH).
Consistent with the pathophysiological relevance of this nitro-
sylation reaction, the level of SNO-Prx2 is significantly elevated
in both animal models and human postmortem PD brains
(Fang etal., 2007). Since formation of SNO-Prx2 prevents reduc-
tivity of Prx2 is compromised by S-nitrosylation, apparently
contributing to the pathogenesis of PD.
S-Nitrosylation of DJ-1
The DJ-1 (PARK7) gene has been shown to be deleted or
mutated in patients with early-onset autosomal recessive PD
and also in rare cases of adult PD (Bonifati et al., 2003). The
DJ-1 protein can be S-nitrosylated under pathological condi-
tions, in some sense mimicking the effect of these rare muta-
tions. DJ-1 colocalizes with Hsp70 and CHIP, and may thus
functionasaredox-sensitive chaperoneinvolved intheoxidative
stress response (Batelli et al., 2008). In fact, consistent with an
anti-oxidant mechanism of defense, DJ-1 expression increases
in response to the nitrosative/oxidative stress induced by the
herbicide paraquat (Mitsumoto et al., 2001). Moreover, exoge-
nous expression of DJ-1 protects neurons from the toxicity
induced by A53T mutant or overexpression of WT a-syn in cell-
lation of DJ-1 enhances cell death induced by oxidative stress
not only in cell-based models but also in vivo in flies and mice
together, these results suggest that DJ-1 plays an important
neuroprotective role in response to oxidative stress. DJ-1 pos-
sesses three potentially redox-active cysteine residues (Cys46,
Cys53, and Cys106). Cys106 is sulfinated (forming a cysteine
sulfinic acid, -SO2H) in response to oxidative stress and may
thus detoxify reactive oxygen species to offer neuroprotection
(Blackinton et al., 2009), while Cys46 and Cys53 have been re-
of nitrosylation remain contentious and further work will be
needed to determine the exact site(s) and effect of S-nitrosyla-
tion of DJ-1. Importantly, whether S-nitrosylation regulates the
neuroprotective activity of DJ-1 remains an open question.
S-Nitrosylation of Matrix Metalloproteinases
In focal cerebral ischemia, specific subtypes of metalloprotei-
nases (MMPs), including MMP-2 and -9, are acutely activated.
These MMPs degrade components of the extracellular matrix,
leading to deleterious consequences in the affected region of
the brain. As a mechanism for MMP involvement, NO, generated
by the excitotoxic and neuroinflammatory consequences of a
stroke, was shown to directly activate MMP-9 by S-nitrosylation
of the so-called ‘‘cysteine switch,’’ whose oxidation leads to
enzyme activation. The resulting acute, excessive activity of
MMPs early on in the evolution of ischemia/reperfusion contrib-
aration of cells from the extracellular matrix triggers an apoptotic
form of cell death (Gu et al., 2002; Manabe et al., 2005).
Mechanistically, the catalytic site of the latent (or proform) of
MMP-9 contains a Zn2+atom that is coordinated by three His
and one Cys residues (constituting the cysteine switch), which
prevent substrates in the extracellular matrix from reaching the
active site. Gu et al. (2002) discovered that one mechanism of
activation of MMPs involves an NO group reacting with the crit-
ical cysteine residue in this region to disrupt the Cys-Zn2+inter-
action, exposing the catalytic Zn2+in the active site to substrate.
Consistent with a role for NO in MMP activation, MMP-9 coloc-
alizes with nNOS during cerebral ischemia and with iNOS in
migrating cells (Gu et al., 2002; Harris et al., 2008). Following
S-nitrosylation, the critical Cys residue in the cysteine switch of
(-SO2H) or sulfonic(-SO3H) acid derivatives,the latter resultingin
apparently irreversible activation of the enzyme. Thus, S-nitrosy-
lation and subsequent oxidation can result in pathological acti-
vation of MMPs, contributing to neuronal injury and death during
stroke and possibly other neurological disorders.
S-Nitrosylation of Akt, PTEN, and Other Protein Tyrosine
Phosphatase and tensin homolog (PTEN) was initially identified
tidylinositol-30-kinase (PI3K)/protein kinase B (PKB or Akt)
pathway by dephosphorylating phosphatidylinositol (3,4,5)-
trisphosphate (PIP3). Accordingly, in the brain, inactivation or
deletion of PTEN may contribute to the pathogenesis of glioblas-
toma. However, recent evidence suggests that downregulation
of PTEN can also play a neuroprotective role in neurodevelop-
erozygous for PTEN manifest resistance to oxidative stress
compared to WT cells (Li et al., 2002). Additionally, S-nitrosyla-
tion of PTEN at Cys83 inhibits PTEN activity and hence exerts
a neuroprotective effect via the Akt pathway (Kwak et al.,
2010; Numajiri et al., 2011). Moreover, S-nitrosylated PTEN
can be selectively ubiquitinated by NEDD4-1 and thus degraded
via the ubiquitin-proteasome pathway (Kwak et al., 2010).
nificance, the formation of SNO-PTEN was discovered in
Neuron 78, May 22, 2013 ª2013 Elsevier Inc.
postmortem human AD brain as well as in ischemic mouse brain
(Kwak et al., 2010; Numajiri et al., 2011; Pei et al., 2009). These
nitrosylation events may represent a negative feedback mecha-
nism during nitrosative stress whereby NO can curb excessive
cell death under specific circumstances by activating the Akt
neuroprotective pathway. However, S-nitrosylation of Akt can
directly inhibit its kinase activity to effectively ‘‘trump’’ the effect
of SNO-PTEN formation (Yasukawa et al., 2005; Numajiri et al.,
2011; Banerjee et al., 2012). Recently, these effects of S-nitrosy-
lation, which seemingly produce opposite effects on the PTEN/
PI3K/Akt pathway, were clarified by the demonstration that low
(and thus neuroprotective) concentrations of NO preferentially
induce SNO-PTEN formation, while higher (neurotoxic) concen-
trations of NO also S-nitrosylate Akt to directly inhibit its neuro-
protective activity (Numajiri et al., 2011).
In addition to PTEN, another member of the protein tyrosine
phosphatase (PTP) family, Src homology region 2-containing
protein tyrosine phosphatase-2 (SHP-2) was identified as an
important substrate for S-nitrosylation during ischemic stroke
(Shi et al., 2013). S-Nitrosylation of SHP-2 occurs at the catalytic
cysteine and thus inhibits its phosphatase activity. Formation of
SNO-SHP-2 therefore blocks downstream activation of the neu-
roprotective extracellular signal-regulated kinase 1/2 (ERK1/2)
pathway, thereby enhancing susceptibility to excitotoxicity.
This finding is in agreement with recent studies demonstrating
an inhibitory effect of S-nitrosylation on other PTPs in nonneuro-
nal cells (Barrett et al., 2005; Chen et al., 2008; Mikkelsen and
Wardman, 2003). Additionally, ROS can induce sulfination
(-SO2H) or sulfonation (-SO3H) of the active site cysteine of
PTPs, thus inhibiting their activity (Tonks, 2006). Since S-nitrosy-
lation of other proteins can facilitate further oxidation of their
redox-active cysteine residues to sulfinic or sulfonic acid deriva-
tives (Gu et al., 2002; Hara et al., 2005; Uehara et al., 2006), it is
conceivable that the S-nitrosylated cysteine residue of SHP-2 or
other PTPs can also initiate further oxidation in this manner.
Nonetheless, for PTEN, S-nitrosylation by NO, unlike oxidation
by H2O2, occurs at an allosteric cysteine residue rather than at
the active site cysteine (Lee et al., 2002; Numajiri et al., 2011).
S-Nitrosylation of Microtubule-Associated Protein 1B
Axonal retraction and degeneration frequently occur in neurode-
generative diseases, including AD, PD, and multiple sclerosis.
NO has been suggested to participate in this process by S-nitro-
sylating microtubule-associated protein 1B (MAP1B) (Stroiss-
nigg et al., 2007). MAP1B is highly expressed in neurons and
bindstomicrotubulesto activelyextend axonlength. Stroissnigg
et al. (2007) demonstrated that MAP1B interacts with nNOS,
facilitating S-nitrosylation of human MAP1B at Cys2464 in the
protein’s light chain. S-Nitrosylation of MAP1B causes a confor-
mational change in the protein that increases its microtubule
binding affinity. Enhanced binding affinity results in inhibition of
the molecular motor dynein, thus contributing to axonal retrac-
tion in response to NO. Recently, S-nitrosylation of MAP1B in
mitochondria was also shown to promote its own degradation,
mediated by the mitochondrial ubiquitin E3 ligase MITOL (Yona-
shiro et al., 2012). In that study, MITOL-dependent degradation
of mitochondrial SNO-MAP1B protected neurons from NO-
induced mitochondrial dysfunction and subsequent cell death.
Taken together, these results suggest that SNO-MAP1B can
mediate axonal retraction, but, as a negative-feedback mecha-
by MITOL; the latter effect results in protection of neurons from
mitochondrially mediated cell death induced by NO. Further
work will be needed to determine how these effects may be
involved in specific neurodegenerative disorders.
S-Nitrosylation of COX-2
Cyclooxygenase (COX-2) converts arachidonic acid (AA) to
prostaglandin (PG)H2, a precursor of many other biologically
active PGs. During inflammation, iNOS has been shown to
interact with, S-nitrosylate, and thus enhance the activity of
COX-2 (Kim et al., 2005b). Similarly, iNOS appears to bind to,
S-nitrosylate, and increase the activity of phospholipase A2
(PLA2), an AA-generating enzyme (Xu et al., 2008). Thus, NO
can promote PG generation via S-nitrosylation of the two key en-
zymes(PLA2and COX-2)involved in PG biosynthesis. Moreover,
COX-2 also binds to nNOS, and S-nitrosylation via nNOS acti-
vates COX-2-mediated NMDAR neurotoxicity (Tian et al.,
2008). Thus, S-nitrosylation of COX-2 and PLA2may contribute
to the neuroinflammatory component of neurodegeneration.
S-Nitrosylation of Insulin-Degrading Enzyme (IDE)
IDE is a zinc metalloprotease that cleaves a number of biologi-
cally active peptides, including both insulin and Ab. Exposure
to NO donors such as S-nitrosoglutathione (GSNO) can
decrease IDE activity via S-nitrosylation of multiple cysteine res-
idues (Cordes et al., 2009; Malito et al., 2008). Hence, S-nitrosy-
lation of IDE may result in increased levels of both insulin and Ab,
as observed in metabolic syndrome/type 2diabetes mellitus and
AD, respectively. Mechanistically, the NO group initially reacts
with IDE at Cys819, located near the catalytic site of the enzyme.
This reaction perturbs local structure, facilitating S-nitrosylation
of a second cysteine residue (Cys110), which is found near the
zinc-binding center (Ralat et al., 2009). The resulting S-nitrosyla-
tion of both Cys819 and Cys110 results in complete inactivation
of IDE, at least in vitro. Alternatively, S-nitrosylation at Cys789
and Cys966 has been reported to trigger not only inhibition of
IDE but also its oligomerization. Finally, S-nitrosylation of
Cys178 reverses the inhibition of IDE caused by S-nitrosylation
of the aforementioned four cysteine residues (Ralat et al.,
2009). Although further work is needed to elucidate the effects
of these reactions in vivo, it appears that S-nitrosylation of IDE
could contribute to AD pathophysiology by decreasing degrada-
tion of Ab.
S-Nitrosylation of JNK1 and IKKb in Autophagy
Autophagic/lysosomal degradation of misfolded proteins or
damaged organelles is an important cellular response to stress
that affects the pathogenesis of neurodegenerative diseases.
Well-characterized cell signaling pathways regulating the auto-
phagic process include the JNK/Bcl-2/Beclin 1 and IKKb/
AMPK/mTORC 1 cascades. Aberrant S-nitrosylation of JNK1
and IKKb inhibits autophagy in models of HD (Sarkar et al.,
2011). Specifically, S-nitrosylation of JNK1 inhibits its activity,
disrupting Beclin 1 complex formation, which is required for
the development of the autophagosome. Additionally, inhibition
of IKKb activity by S-nitrosylation leads to decreased phosphor-
ylation of AMPK, resulting in activation of the autophagy inhibi-
tor, mTORC1. Conversely, and consistent with these findings,
Neuron 78, May 22, 2013 ª2013 Elsevier Inc.
downregulation of endogenous NO production enhances auto-
phagy flux. Additional SNO-proteins have also been implicated
in the control of autophagy, and further work will be necessary
to determine these pathways.
S-Nitrosylation of ApoE
The apolipoprotein E (ApoE) gene encodes a class of lipopro-
teins that plays a critical role in CNS cholesterol homeostasis.
ApoE4 represents a major genetic risk factor for developing
late-onset AD (Bertram et al., 2010). The ApoE isoforms can
bind to nNOS and both ApoE2/E3 can be S-nitrosylated in hu-
man hippocampal lysates (Abrams et al., 2011). In silico analysis
of the ApoE 3D structure further suggested that S-nitrosylation
may result in conformational alteration of the protein, lowering
its binding affinity for low-density lipoprotein (LDL) receptors
(Abrams et al., 2011), raising the interesting question of whether
S-nitrosylation of ApoE might contribute to the onset or progres-
sion of AD by disrupting normal lipid metabolism.
Protein S-Nitrosylation Regulates Diverse Aspects of
Although less well characterized, a large number of additional
SNO-proteins have also been linked to modulation of normal or
pathological brain function. During the neurodegenerative pro-
cess, these SNO-proteins may impact a wide variety of cellular
mechanisms (Figure 6). Below, we discuss possible roles for
several of these SNO-proteins under both physiological and
pathophysiological conditions in the brain.
S-Nitrosylation of Synaptic Proteins
In neurons, NO is typically generated by nNOS coupled to
NMDARs (Bredt et al., 1991; Sattler et al., 1999). This process
in turn leads to S-nitrosylation of various proteins in the cell.
One such protein is N-ethylmaleimide sensitive factor (NSF); its
S-nitrosylation leads to increased surface expression of AMPA
receptors (AMPARs) (Huang et al., 2005; Matsushita et al.,
2003). Surface expression of AMPARs can also be upregulated
by S-nitrosylation of the regulatory subunit stargazin (Selvaku-
mar et al., 2009). In addition, S-nitrosylation of the AMPAR sub-
unit GluA1 facilitates its phosphorylation, producing an increase
in single-channel conductance, and enhancing endocytosis
(Selvakumar et al., 2013). Additionally, PSD-95 can be S-nitrosy-
lated, which decreases its synaptic expression as well as the
expression of proteins that are associated with it, including
ditions, NMDAR-mediated excitotoxicity (which appears to be
mediated predominantly by extrasynaptic NMDARs) can cause
massive Ca2+influx that subsequently leads to the production
of excessive amounts of NO (Dawson et al., 1991; Lipton et al.,
1993; Bonfoco et al., 1995; Hardingham and Bading, 2010). As
a negative-feedback mechanism, NMDARs are S-nitrosylated
by NO, and this S-nitrosylation can downregulate excessive re-
ceptor activity (Lei et al., 1992; Lipton et al., 1993; Choi et al.,
Additionally, NO may act as a retrograde messenger to pre-
synaptic sites, leading, for example, to S-nitrosylation of serine
racemase (SR); this nitrosylation reaction decreases the genera-
tion of D-serine, a co-agonist at NMDARs (Mustafa et al., 2007).
Furthermore, S-nitrosylation of syntaxin 1 can possibly prevent
excessiverelease ofglutamate (Palmer etal.,2008);thiscoupled
with the finding that SNAP-25 can also be S-nitrosylated (Hess
et al., 1993) may account for the observation that NO can
decrease evoked neurotransmitter release while increasing
spontaneous release (Pan et al., 1996). Effects on these or other
members of the core complex of synaptic proteins involved in
docking and fusion could contribute to a phenotype of
decreased evoked and increased spontaneous release. There-
fore, S-nitrosylation of synaptic proteins potentially mediates
both physiological and neuroprotective aspects of NO signaling.
S-Nitrosylation of Other Membrane Proteins
NO can affect many signaling pathways by S-nitrosylating mem-
brane receptors and ion channels. In addition to NMDARs, a
number of SNO-regulated membrane proteins that potentially
affect brain function have been discovered, including the ryano-
dine receptor (RyR) (Eu et al., 2000), transient receptor potential
(TRP) channels (Yoshida et al., 2006), Na+channels (Rengana-
than et al., 2002), K+channels (Nu ´n ˜ez et al., 2006; Asada et al.,
2009), and voltage-gated Ca2+channels (Chen et al., 2002).
The effects of S-nitrosylation on NMDARs and RyRs have been
particularly well characterized. There are five sites of nitrosyla-
tion on NMDARs (Choi et al., 2000, 2001; Lipton et al., 2002).
The relative hypoxia of the brain enhances S-nitrosylation of
the NMDAR by a unique mechanism involving an ‘‘NO-reactive
oxygen sensor motif’’ whose determinants include Cys744 and
Cys798 of the GluN1 (NR1) subunit (Takahashi et al., 2007).
Free thiols at these two cysteine residues are involved in redox
reactions that sensitize other NMDAR cysteines to S-nitrosyla-
tion and result in receptor inhibition. Interestingly, however,
S-nitrosylation of Cys744/Cys798 themselves has little effect
on NMDAR activity (Figure 7). Solving the crystal structure of
the ligand-binding domain of NR1 under oxidizing conditions re-
vealed a flexible disulfide bond (Cys744-Cys798), which may ac-
count for its susceptibility to reduction and subsequent reaction
with NO that is observed with biochemical techniques in the
presence of NO. In accord with these findings, when crystals
of NR1 were formed under relatively hypoxic conditions in the
presence of NO, electron density consistent with S-nitrosylation
Figure 6. Physiological and Pathophysiological Protein
S-Nitrosylation Plays an Important Role in Diverse Aspects of Cell
Function in the CNS
Production of both normal and pathological levels of NO results in S-nitro-
sylation of a number of proteins implicated in synaptic transmission, re-
ceptors/ion channels, transcription factors, iron homeostasis, protein quality
control, and mitochondrial function.
Neuron 78, May 22, 2013 ª2013 Elsevier Inc.
in the region of Cys744/Cys798 was observed (Takahashi et al.,
increasing (pathological) hypoxia, thus preventing excessive ac-
tivity associated with cytotoxicity while avoiding blockade of
physiologically active NMDARs.
The RyR1-type ryanodine receptor mediates Ca2+release
from the ER (or sarcoplasmic reticulum in skeletal muscle cells)
into the cytosol, and S-nitrosylation of RyR1 at Cys3635 has
been shown to increase channel opening in muscle cells.
Although RyR1 is expressed in brain cells, the role of SNO-
RyR1 in neuronal cells has remained enigmatic until recently
when, in cortical neurons, S-nitrosylation of RyR1 was shown
to contribute to NO-induced Ca2+release and neuronal cell
death (Kakizawa et al., 2012).
tor (GPCR) signaling machinery via S-nitrosylation of GPCR-
associated proteins such as G protein receptor kinase 2
(GRK2) (Whalen et al., 2007) and b-arrestin (Ozawa et al.,
2008). As exemplified for b-adrenergic receptors, S-nitrosylation
of GRK2 prevents receptor internalization, while SNO-b-arrestin
displays increased binding to clathrin heavy chain/b-adaptin to
accelerate receptor internalization. As GPCR signaling is impli-
cated in many aspects of neuronal function, these studies sug-
gest that SNO-dependent regulation of these proteins may
play a role in neurodegenerative conditions, although its exact
involvement is as yet to be determined.
S-Nitrosylation of Transcriptional Regulators
S-Nitrosylation can also regulate gene transcription by targeting
transcription factors and their regulatory proteins, including nu-
clear factor-kB (NF-kB)/IKKb (Marshall and Stamler, 2001), hyp-
oxia-inducible factor (HIF) (Li et al., 2007; Sumbayev et al., 2003;
Yasinska and Sumbayev, 2003), c-Jun N-terminal kinase (JNK)/
c-Jun (Park et al., 2000), human homolog of mouse double min-
ute-2 (HDM2)/p53 (Schonhoffetal.,2002),Kelch-like ECH-asso-
ciated protein 1 (Keap1)/nuclear factor erythroid 2-related factor
2 (Nrf2) (Fourquet et al., 2010; Um et al., 2011), nuclear hormone
receptors such as the estrogen receptor (Garba ´n et al., 2005),
and histone deacetylase 2 (HDAC2) (Nott et al., 2008). Addition-
ally, NO can indirectly regulate the activity of transcriptional fac-
tors via S-nitrosylation of a number of upstream effectors,
including Ras, Src, and ASK1 (Akhand et al., 1999; Lander
et al., 1995; Park et al., 2004; Hess et al., 2005). Despite the
well-characterized effects of S-nitrosylation on many transcrip-
tional activities, the pathophysiological significance of these
redox modifications on neurodegenerative diseases is only
now being elucidated.
S-Nitrosylation of Iron Homeostasis Regulatory Proteins
Increasing evidence suggests that progressive accumulation of
iron in the aged brain may underlie a number of neurodegenera-
tive disorders due to induction of oxidative stress (Zecca et al.,
2004). Iron homeostasis is tightly regulated by a series of car-
riers, enzymes, and associated proteins, including transferrin,
transferrin receptor (TfR), ferritin, and iron-regulatory proteins
(IRPs). IRPs interact with the mRNA of the TfR (which is involved
mRNA and blocking ferritin mRNA translation. Accordingly, IRP2
knockout mice manifest significant accumulation of iron de-
posits as well as profound movement disorders and neurode-
generation (LaVaute et al., 2001). Interestingly, S-nitrosylation
of IRP2 at Cys178 reportedly promotes UPS-dependent degra-
dation of IRP2, leading to significant accumulation of iron with
the iron storage protein, ferritin (Kim et al., 2004). These findings
iron homeostasis pathway, and is implicated in nitrosative
Additionally, Dexras1, a small GTPase that is activated by
S-nitrosylation, affects NO-regulated iron homeostasis. NMDAR
Figure 7. Hypoxia Sensitizes Thiol to S-Nitrosylation-Mediated
Inhibition of the NMDAR
(A) Ambient O2levels are significantly higher than levels in living brain tissue or
during hypoxic insults, favoring disulfide formation between cysteine residues
Cys744 and Cys798 on the GluN1 (NR1) subunit of the NMDAR (red line).
(B) S-Nitrosylation can occur on various NMDAR subunits (designated GluNs)
at two or three different sites on each subunit. However, in the presence of
disulfide, NO cannot be transferred to cysteine residues since no free thiol
exists for reaction. S-Nitrosylation can occur at free thiols on the GluN2A
(NR2A) subunit at Cys399 (cysteine residue at left) as well as on NR2A Cys87
and Cys320 (and homologous cysteine residues on GluN2B [NR2B, not
shown]), but only in the presence of high concentrations of NO donor.
(C) Under physiological or even more hypoxic (pathological) conditions, the
relatively reducing state favors free thiol groups on the NMDAR over disulfide
(D) Under relatively hypoxic conditions, the free thiol groups are more readily
available to react with NO to form S-nitrosothiol, and even low concentrations
of an NO donor can effect this reaction. The exact reaction route that leads
from S-nitrosylation to disulfide formation of vicinal thiols (NR1[C744,C798])
remains unknown, although it is likely that in this scenario, only one of these
thiols is S-nitrosylated. During relative hypoxia, our hypothesis is that NO is
more likely to react with both Cys744 and Cys798 to S-nitrosylate their thiol
groups, and under these conditions, disulfide formation would be blocked.
Such dual S-nitrosylation of Cys744/Cys798 would then lead to the increased
NO sensitivity that we have observed experimentally at other NMDAR sites,
such as Cys399, Cys87, and Cys320 on NR2A. Therefore, NO itself may not be
regulatory at NR1(C744,C798), but the redox status of these cysteine residues
would exert an allosteric influence on S-nitrosylation of other thiol groups on
the NMDAR (i.e., Cys399 in the loose linker region of NR2A, and Cys87 and
Cys320 on the amino terminal domain of NR2A (or the homologous cysteine
residues of NR2B, not shown). Adapted from Takahashi et al. (2007).
Neuron 78, May 22, 2013 ª2013 Elsevier Inc.
stimulation of nNOS can produce S-nitrosylation of Dexras on
Cys11 (Fang et al., 2000; Jaffrey et al., 2002). Dexras1 forms a
complex with the divalent metal transporter 1 (DMT1), and
SNO activation of Dexras1 augments iron uptake via DMT1
(Cheah et al., 2006). Importantly, selective iron chelation amelio-
rates NMDA-nNOS neurotoxicity, implying that iron uptake in
response to NMDAR-nNOS activation can play an important
S-Nitrosylation Protein Quality Control Machinery
Abnormal accumulation of toxic misfolded proteins, such as
oligomeric Ab and a-synuclein, represents a characteristic
feature of many neurodegenerative diseases. It is generally
to the appearance of misfolded proteins. The quality control
system encompasses multiple cellular elements, including mo-
lecular chaperones, the UPS, and the autophagy/lysosomal
pathway. As discussed above, S-nitrosylation disrupts both au-
tophagic degradation of misfolded proteins and the protein re-
folding/chaperone activity of PDI and GRP in the ER. In addition,
S-nitrosylation of HSP90 inhibits its ATPase activity and chap-
erone function in the cytoplasm (Martı ´nez-Ruiz et al., 2005).
Concerning components of the UPS, S-nitrosylation has been
shown to cause dysfunctional ubiquitin E3 ligase activity in par-
kin, XIAP, and pVHL (Yao et al., 2004; Chung et al., 2004; Naka-
mura et al., 2010; Tsui et al., 2011). Additionally, S-nitrosylation
can directly inhibit 26S proteasome activity via targeting 10
cysteine residues in the 20S catalytic core (Kapadia et al.,
2009). Moreover, S-nitrosylation and further oxidation of an
N-terminally located cysteine residue appear to control the
UPS-dependent N-end rule (Hu et al., 2005). The N-end rule de-
of its N-terminal amino acid residue(s); in this manner, Asn, Gln,
or Cys at the N terminus destabilizes the protein in mammalian
cells (Sriram et al., 2011). Hu et al. (2005) demonstrated that
NO targets the N-terminal cysteine when a basic residue is
located next to the cysteine (reminiscent of the prior report of
an S-nitrosylation motif in peptides [Stamler et al., 1997]), and
subsequent oxidation of the cysteine residue to a sulfonic acid
the protein. Thus, nitrosative/oxidative stress can cause specific
degradation of proteins bearing an N-terminal ‘‘cysteine-basic
residue motif’’ via S-nitrosylation and further oxidation.
S-Nitrosylation and Mitochondrial Dysfunction
Aberrant redox reactions triggered by excessive amounts of NO
can result in mitochondrial dysfunction, although basal levels of
NO serve as a physiological regulator of mitochondrial activity.
As described above, arguably the best-characterized SNO-
protein that affects mitochondrial function during neurodegener-
ation is SNO-Drp1 (Cho et al., 2009). Additionally, ubiquitination
of mitochondrial proteins by parkin is implicated in mitophagy
(Youle and van der Bliek, 2012). As S-nitrosylation of parkin
esize that SNO-parkin might affect mitophagy during the degen-
The main function of mitochondria entails ATP synthesis via
oxidative phosphorylation (OX/PHOS). NO has been reported
to negatively affect mitochondrial respiratory activity in part
through cysteine S-nitrosylation of complexes I and IV (Cleeter
et al., 1994; Clementi et al., 1998; Zhang et al., 2005; Dahm
et al., 2006; Burwell et al., 2006). It should also be noted that
NO directly reacts with iron in the iron-sulfur center of mitochon-
drialcomplexes IandII,decreasing boththetransferofelectrons
and ATP production (Drapier and Hibbs, 1988). Additionally, S-
nitrosylation may decrease F1 ATPase activity to further disrupt
OX/PHOS, as demonstrated in cardiomyocytes (Sun et al.,
2007). However, direct evidence demonstrating the link between
SNO-mediated inhibition of mitochondrial complexes and neu-
rodegeneration is still lacking, although this mechanism has
been suggested for diseases such as PD (Brown and Borutaite,
2004). In fact, in animal models of PD, administration of complex
I inhibitors, such as MPTP or rotenone, recapitulates many fea-
tures of sporadic PD, including degeneration of dopaminergic
neurons, overproduction and aggregation of a-syn, accumula-
tion of Lewy body-like intraneuronal inclusions, and impairment
of behavioral function (Beal, 2001; Betarbet et al., 2000). Inhibi-
tion of mitochondrial complexes by S-nitrosylation may poten-
tially generate excessive ROS/RNS, providing a possible posi-
tive feedback loop to accelerate neuronal injury (Beal, 2001;
Betarbet et al., 2000; Chung et al., 2004; Uehara et al., 2006;
Yao et al., 2004). Further studies are needed to determine
whether regulation of other mitochondrial metabolic pathways
by S-nitrosylation contributes to neurodegenerative conditions.
Conclusions and Perspectives
Protein S-nitrosylation plays an important role in the pathogen-
esis of a number of neurodegenerative disorders, including AD,
PD, HD, and ALS. Aberrant protein S-nitrosylation reactions
can occur in response to excessive nitrosative/oxidative stress
and may contribute to neurodegeneration via disruption of a
number of pathways. To date, aberrant protein S-nitrosylation
has been identified on an increasing number of targets, and we
expect that many more S-nitrosylated proteins will be found to
play a role in neurodegenerative diseases. There is the potential
for hundreds of such proteins to be identified by improved SNO-
proteome technology coupled with bioinformatics (Forrester
et al., 2009; Hao et al., 2006; Paige et al., 2008; Xue et al.,
2010). Elucidation of the aberrant SNO-proteome in the brain
should expedite our understanding of the pathological role of
S-nitrosylation in these neurodegenerative diseases. Better
characterization of the aberrant SNO-proteome will also facili-
tate identification of potential therapeutic targets for drug dis-
appear to be present in each neurodegenerative condition, inter-
vention that affects a single nitrosylation event can ameliorate
the condition, at least in animal models (Gu et al., 2002; Uehara
et al., 2006; Hara et al., 2006; Cho et al., 2009). This finding sug-
gests that there may be redundancy in redox-mediated path-
ways to injury, but interfering with even a single pathway can
offer therapeutic benefit—raising hope for the development of
The authors’ work related to this research area was supported in part by fund-
ing from the Alzheimer’s Association (to T.N.), grants from the Michael J. Fox
Neuron 78, May 22, 2013 ª2013 Elsevier Inc.
Foundation(toT.N.and S.A.L.),and NIHgrantsR21NS080799,P01 HD29587,
P01 ES01673, and P30 NS076411 (to S.A.L.).
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