NO homeostasis_Hannibal_CAR 2016 (1)

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Current Alzheimer Research, 2016, 13, 135-149 135
Nitric Oxide Homeostasis in Neurodegenerative Diseases
Luciana Hannibala,b,c,*
a
Department of Pathobiology, Lerner Research Institute, Cleveland Clinic, 9500 Euclid Ave., Cleve-
land 44195, USA; b
Lehrstuhl für Bioanorganische Chemie, Department Chemie und Pharmazie, Uni-
versität Erlangen-Nürnberg, Egerlandstraße 1, D-91058 Erlangen, Germany; c
Departamento de Bio-
química and Centro de Investigaciones Biomédicas (CEINBIO), Facultad de Medicina, Universidad
de la República, Avda. General Flores 2125, 11800 Montevideo, Uruguay
Abstract: The role of nitric oxide in the pathogenesis and progression of neurodegenerative illnesses
such as Parkinson’s and Alzheimer’s diseases has become prominent over the years. Increased activity
of the enzymes that produce reactive oxygen species, decreased activity of antioxidant enzymes and
imbalances in glutathione pools mediate and mark the neurodegenerative process. Much of the oxida-
tive damage of proteins is brought about by the overproduction of nitric oxide by nitric oxide synthases (NOS) and its
subsequent reactivity with reactive oxygen species. Proteomic methods have advanced the field tremendously, by facilitat-
ing the quantitative assessment of differential expression patterns and oxidative modifications of proteins and alongside,
mapping their non-canonical functions. As a signaling molecule involved in multiple biochemical pathways, the level of
nitric oxide is subject to tight regulation. All three NOS isoforms display aberrant patterns of expression in Alzheimer’s
disease, altering intracellular signaling and routing oxidative stress in directions that are uncompounded. This review dis-
cusses the prime factors that control nitric oxide biosynthesis, reactivity footprints and ensuing effects in the development
of neurodegenerative diseases.
Keywords: Alzheimer’s disease, interactome, metal homeostasis, neurodegenerative disease, nitric oxide, NOS, oxidative
stress, proteomics.
INTRODUCTION
Alzheimer’s disease is a progressive neurodegenerative
illness that manifests primarily in the elderly and leads to
various degrees of dementia. Extracellular deposition of neu-
ritic plaques containing amyloid-β and intracellular neurofi-
brillary tangles enriched in phosphorylated tau protein are
the best characterized markers of the disease [1]. The pres-
ence of vascular comorbidity in approximately 60% of Alz-
heimer’s disease patients [2], has led to the distinction be-
tween vascular and the Alzheimer’s disease-type dementias.
Escalating evidence suggests that the vascular endothelium
partakes heavily in promoting or preventing neuronal dete-
rioration (reviewed in [3]). Indeed, many of the risk factors
associated with cardiovascular disease are commonly identi-
fied in neurodegenerative processes [4]. Endothelial dys-
function has been documented as a major contributor to
Parkinson’s and Alzheimer’s disease and amyotrophic lateral
sclerosis. Increased levels of inflammation markers such as
C-reactive protein, interleukins 6, 8 and 1b [5, 6] as well as
dysfunctional mitochondria [7, 8] have been reported in
brain vascular cells of patients with Alzheimer’s disease.
*Address correspondence to this author at the Department of Pathobiology,
Lerner Research Institute, Cleveland Clinic. 9500 Euclid Ave., Cleveland
44195, USA; Lehrstuhl für Bioanorganische Chemie, Department Chemie
und Pharmazie, Universität Erlangen-Nürnberg. Egerlandstraße 1, D-91058
Erlangen, Germany; Departamento de Bioquímica and Centro de Investiga-
ciones Biomédicas (CEINBIO), Facultad de Medicina, Universidad de la
República, Avda. General Flores 2125, 11800 Montevideo, Uruguay;
E-mail: mhanniba@kent.edu
Because endothelial function is exquisitely reliant on nitric
oxide homeostasis and negatively affected by oxidative
stress, understanding the cellular sources, reactivity and fate
of reactive oxygen species is essential to comprehend the
molecular mechanisms underlying neurodegenerative dis-
eases.
NITRIC OXIDE BIOSYNTHESIS
Nitric oxide is synthesized by a group of enzymes known
as nitric oxide synthases (EC 1.14.13.39). Nitric oxide syn-
thases are homodimeric dual flavoenzymes containing
NADPH, FAD, FMN, tetrahydrobiopterin and heme. NOS
catalyze the conversion of L-arginine (L-Arg) into citrulline
and nitric oxide via a process that involves oxygen activation
to generate N-hydroxyarginine, the first stable intermediate
in the biosynthesis of nitric oxide [9, 10] (Fig. 1). Three iso-
forms of NOS exist in humans, namely inducible (iNOS),
endothelial (eNOS) and neuronal (nNOS), which are classi-
fied according to their predominant site of expression and
susceptibility to undergo induction under conditions of in-
flammation [9, 10]. The enzymatic activity of eNOS and
nNOS responds to calcium levels through the interaction
with calmodulin [11, 12], whereas iNOS activity is inde-
pendent of calcium and largely inducible by cytokines [9,
10]. Each NOS isoform displays distinct catalytic behavior
in spite of significant sequence and tridimensional structure
homology [13-15]. The flavoenzyme domain of NOS con-
trols the overall reaction as electron transfer from the flavins
to the heme is the rate-limiting step in NO biosynthesis [16].
1 - /16 $58.00+.00 © 2016 Bentham Science Publishers

136 Current Alzheimer Research, 2016, Vol. 13, No. 2 Luciana Hannibal
Fig. (1). Biosynthesis of nitric oxide. A. Structure of the oxygenase domain of murine inducible NOS (accession number 1NOD). The sub-
strate L-Arg, tetrahydrobiopterin and the heme moiety are depicted as sticks. The structure was created using Pymol software. B. The biosyn-
thesis of NO occurs through two consecutive reactions that convert L-Arg into the stable intermediate N-hydroxyarginine (a), and then the
latter into citrulline and NO (b). Electrons provided by NADPH in the reductase domain reduce the heme center present in the oxygenase
domain to activate oxygen. A highly reactive compound I species (or the alike) enables the hydroxylation of the substrate L-Arg. Tetrahydro-
biopterin serves as electron donor to generate the highly reactive heme-centered species. The resulting tetrahydrobiopterin radical is reduced
back by the reductase domain of NOS. A stable FeIII
-NO enzyme complex is formed, and the timely release of NO from the heme center en-
sures maximum NO synthesis yield by minimizing the unwanted reduction of the FeIII
-NO complex. Uncoupled NOS diverts oxygen in reac-
tion (a) into forming superoxide rather than channeling electron transfer toward L-Arg hydroxylation.
The expression of each NOS isoform responds to different
signals and stressors. The traditional notion of tissue-
specificity and constitutive versus inducible expression has
been recently challenged by experimental observations dem-
onstrating non-canonical expression patterns for all three
NOS isoforms (reviewed in [17, 18]). Astrocytes, the major
cell type in the central nervous system, have been shown to
release NO under basal conditions and upon stimulation by
trauma and pathological insult [19]. Since all three isoforms
of NOS are active in astrocytes [19-21], the output of NO
release under stress conditions would be conceivably high.
Redox imbalance by an altered biosynthesis of NO leads to
proteome instability by oxidative post-translational modifi-
cation of proteins and the concomitant upregulation of mo-
lecular chaperones involved in cellular stress [22]. Protein
misfolding has been recognized as a hallmark of Alzheimer’s
disease along with other neurological disorders. Upregula-
tion of cellular stress chaperones may be one means to re-
move excess amyloid-β and tau proteins from the neuron
[23-27]. A fundamental mechanism that leads to a decrease
in NOS activity is through impairments in heme insertion.
Importantly, one of the heat shock proteins, Hsp90, is in-
volved in the maturation of NOS and the NO receptor, solu-
ble guanylate cyclase (sGC), by controlling heme insertion
[28-32]. Nitric oxide biosynthesis by NOS and signaling via
soluble guanylate cyclase take place at their respective
bound heme moieties, hence protein maturation and cofactor
insertion is essential for proper function. Thus, the assembly
of both NOS and sGC to form the fully mature, heme-
containing enzymes requires a) that heme is available and b)
the assistance of Hsp90 [28-32]. These findings suggest that
metal homeostasis might be essential to support adequate

Nitric Oxide Homeostasis in Neurodegenerative Diseases Current Alzheimer Research, 2016, Vol. 13, No. 2 137
NO synthesis in Alzheimer’s and other neurodegenerative
diseases [33, 34]. An enhancement of the cellular stress re-
sponse may function as a compensatory mechanism to sup-
port heme insertion and therefore to sustain nitric oxide bio-
synthesis and signaling. Nutritional and functional deficien-
cies of heme may have detrimental effects on the homeosta-
sis of NO. Both NO and the chemically related small gas
messenger CO, are important factors in the regulation of
cellular stress response proteins in neurodegenerative proc-
esses and aging [35].
NOS INHIBITION BY ENDOGENOUS SUBSTRATE
ANALOGUES
Some naturally occurring analogs of the substrate L-Arg
inhibit NOS, resulting in a decrease in gasotransmitter avail-
ability. Such is the case of asymmetric dimethylarginine
(ADMA) and NG
-monomethyl-arginine (L-MMA), both of
which are otherwise degraded to citrulline and dimethy-
lamines by dimethylarginine dimethylaminohydrolases
(DDAHs) [36, 37]. These substrate analogues are a product
of the degradation of proteins harboring methylated arginine
residues, a post-translational modification exerted by methyl
transferases (PRMT) 1 and 2 [38]. Cytosolic ADMA can be
exported out into circulation reaching all cells in the body.
The erythrocyte has been proposed as the main reservoir and
source of free ADMA [39]. Independent groups have re-
ported elevated plasma ADMA [40, 41] in patients with Alz-
heimer’s disease and low to normal levels of the inhibitor in
cerebrospinal fluid [41]. The levels of ADMA in cerebrospi-
nal fluid correlated well with the presence of phosphorylated
protein tau, but not with amyloid-β in Alzheimer’s disease
[42]. These findings provide a direct link between the enzy-
matic activity of NOS, NO availability and the deposition of
phosphorylated tau. However, a study performed with a
small cohort of patients (N=20) showed that during the early
stages of Alzheimer’s disease, ADMA levels did not differ
significantly from control patients, and therefore, no altera-
tions are expected in NOS activity [43]. Additional studies
are thus essential to reveal the exact time frame of the regu-
lation of nitric oxide homeostasis in the development of Alz-
heimer’s disease and other dementias.
NOS UNCOUPLING AND TETRAHYDROBIOP-
TERIN HOMEOSTASIS
Uncoupling of NOS diverts the biosynthesis of NO to-
ward the production of superoxide and hydrogen peroxide.
Uncoupling of NO biosynthesis occurs under deficiency of
tetrahydrobiopterin (H4B) [44]. Tetrahydrobiopterin is essen-
tial for the electron transfer reaction required for oxygen
activation during NO biosynthesis, the dimerization of NOS
enzymes and for preserving the integrity of the heme elec-
tronic environment of NOS [45-80]. Tetrahydrobiopterin
distribution has been shown to be tissue-specific, which pro-
vides a means to modulate NO synthesis depending on site-
specific needs [44, 81]. The intracellular levels of tetrahy-
drobiopterin and its oxidized form, dihydrobiopterin, are
controlled by both de novo and salvage pathways [82-85].
Several reports indicate lower levels of H4B in the brain tis-
sue and cerebrospinal fluid of patients with Parkinson’s and
Alzheimer’s disease as well as in other unrelated dementias
[86-91]. Likewise, an increased level of serum neopterin,
which would result from impairments in the regeneration of
H4B from dehydroneopterin triphosphate, has been noted in a
small cohort of patients with advanced stage Alzheimer’s
disease [92]. An imbalance of cellular and serum H4B has
direct repercussions in the activity of all NOS isoforms,
which compromises downstream NO-dependent signaling.
Besides the direct impact on the NO pathway, a deficiency
of H4B has been associated with impaired neurotransmitter
biosynthesis [82, 86]. Tetrahydrobiopterin is the cofactor of
tyrosine hydroxylase, thus serving an essential role in the
biosynthesis of dopamine and related neurotransmitters [88,
90]. Evidence that alterations in dopamine metabolism con-
tribute to Alzheimer’s disease pathogenesis and progression
is mounting [93-95]. In light of this development, an under-
lying deficiency of H4B would not only disrupt nitric oxide
homeostasis but also the major neurotransmitter pathways
involved in cognitive deterioration. In practice, H4B pools
can be effectively refurnished through the folate pathway.
Supplementation of N5
-methyltetrahydrofolate and vitamin
B12 has been shown to correct an underlying H4B deficiency,
a process mediated by the enzymatic activity of dihydrofo-
late reductase [96]. This is an important consideration for the
treatment of Alzheimer’s disease, which is often accompa-
nied by a deficiency of vitamin B12 and/or folate [97-104].
From a therapeutic perspective, direct supplementation with
H4B may be dangerous, since excess H4B has been shown to
cause mitochondrial dysfunction in a model of Parkinson’s
disease by disrupting the function of respiratory chain com-
plexes and inducing cytochrome c release [105].
NITRIC OXIDE REACTIVITY: SUPEROXIDE AND
PEROXYNITRITE
Nitric oxide is a double-edged sword chemical: too much
and too little of it has been associated with cardiovascular,
neurological and inflammatory disorders, yet, its presence is
indispensable for cell survival and proliferation [106, 107].
The cytotoxic actions of NO are mainly driven by its reactiv-
ity with superoxide to form the powerful oxidant peroxyni-
trite [108-111]. Peroxynitrite formation occurs under basal
metabolic conditions and it is notoriously increased under
oxidative stress, where buildup of precursors nitric oxide and
superoxide exceed the antioxidant capacity of the cells [112].
The basal level of peroxynitrite formation in non-stressed
mitochondria of endothelial cells has been estimated to be
0.2-0.4 µM/s (2-3 nM peroxynitrite, considering competing
reactions) [112, 113], and studies predict that this could be
augmented 2 to 3 orders of magnitude in phagosomes and in
dysfunctional mitochondria [112]. Detection of peroxynitrite
in biological systems has been challenging due to: a) Its ex-
tremely short half-life of 10 ms that hampers isolation and
characterization and b) The footprints of its oxidative dam-
age are indicative of its existence but are not entirely specific
[113]. Experimental evidence from cultured cells and brain
tissue of patients with degenerative diseases such as Alz-
heimer’s and Parkinson’s indicates that oxidative stress is a
major contributor to the alteration of signaling pathways in
neuronal cells [1, 114-116]. Lipid peroxidation, DNA oxida-
tion, protein oxidation, advanced glycation end-products and
reactive nitrogen species are among the most consistently
characterized markers of oxidative stress in brains of patients

with Alzheimer’s disease [89, 117, 118]. Fingerprints of oxi-
dative stress in neurodegenerative diseases have been identi-
fied by several research groups worldwide via the analysis of
oxidative post-translational modifications of proteins (Table
1). Nitration of tyrosine residues and S-nitrosylation of cys-
teine residues have been identified by independent groups
(Table 1) and represent an undeniable mark of an altered
nitric oxide homeostasis. The large number of protein targets
identified through redox proteomics impetrates for follow up
studies, to understand the molecular mechanism by which
these oxidative modifications aggravate or protect neurons
from the ongoing disease.
NITRIC OXIDE AND HOMOCYSTEINE METABO-
LISM
Several groups have reported elevated levels of serum
homocysteine in patients with Alzheimer’s disease compared
to age-matched controls (Table 2) [119-129]. A comprehen-
sive imaging study showed that elevated levels of homocys-
teine was associated with lower gray matter thickness in
bilateral, frontal, parietal, occipital, and right temporal re-
gions as well as lower gray matter volumes in left frontal,
parietal, temporal, and occipital regions of the brain of pa-
tients with Alzheimer’s disease [130]. A study reported that
elevated plasma homocysteine in patients with Alzheimer’s
disease was associated with worsening of behavioral and
psychological symptoms [131]. The relationship between
plasma homocysteine and nitric oxide levels has yielded con-
flicting results [123, 132, 133]. A study showed that hyper-
homocysteinemia disrupts the pools of tetrahydrobiopterin
and dihydrobiopterin leading to NOS uncoupling and oxida-
tive stress [134]. Another group identified a direct inhibition
of DDAHs by homocysteine, which leads to the buildup of
the endogenous NOS inhibitor ADMA, and the concomitant
inactivation of NOS [135, 136]. An independent group found
that homocysteine inactivates NOS via activation of protein
kinase C, which phosphorylates Thr495
of eNOS in human
aortic endothelial cells and lowers its expression, without
altering tetrahydrobiopterin pools [137]. While the exact
mechanism by which elevated homocysteine inactivates
NOS begs for further research, consensus exists that reduc-
ing levels of homocysteine would be beneficial to prevent
secondary complications in neurodegenerative and vascular
disorders. Homocysteine is the substrate for the cytosolic
enzyme methionine synthase, a key point in one-carbon me-
tabolism. Methionine synthase catalyzes the conversion of
homocysteine into methionine with 5-methyl-
tetrahydrofolate serving as a methyl donor and methyl-
cobalamin as a cofactor [138]. Co-administration of folate
and vitamin B12 is the first course of action to reduce ele-
vated homocysteine and this therapeutic approach has been
utilized with success to normalize plasma levels of homocys-
teine in patients with Alzheimer’s disease and other forms of
dementia [139, 140]. It should be noted that reduction of
homocysteine not always results in improved cognitive per-
formance [139, 141, 142]. This implies that homocysteine
may exert its oxidative effect via alternative mechanisms, for
example, via N- and S-homocysteinylation of proteins [143-
150]. An emerging aspect of nitric oxide homeostasis in the
nervous system concerns the biochemistry of the smallest
thiol, hydrogen sulfide [151, 152], and the role of the trans-
sulfuration pathway in the brain [153]. Understanding the
exact pathways involved in the actions of this gasotransmit-
ter awaits further investigation.
POST-TRANSLATIONAL MODIFICATIONS
The evidence that neurodegenerative processes are ac-
companied by the post-translational modification of proteins
is profuse (Table 1) [154, 155]. The oxidative modification
of proteins can result in gain and loss of function by means
of electronic and conformational changes. This in turn could
influence the way oxidized proteins interact with other pro-
teins in the complex cellular milieu. In some cases, post-
translational modifications can lead to protein aggregation
and misfolding and act as a trigger of cell death [156, 157].
S-nitrosation of proteins has been recognized as a marker of
aging and Alzheimer’s disease [158, 159]. Redox proteomics
and metabolomic studies have been critical to elucidate the
biochemical elements and pathways involved in neurodegen-
eration, especially those involving nitric oxide and its de-
rived oxidizing partners [160, 161]. Table 1 presents a
summary of selected post-translational modifications re-
ported to date. Widespread oxidative stress manifests in Alz-
heimer’s, Parkinson’s, Down syndrome and unrelated forms
of dementia and mild cognitive impairment through the in-
creased levels of protein oxidation post-translation. Major
changes in post-translational modifications involve proteins
of carbon and energy metabolism, cellular stress response,
pterin metabolism, oxidative stress and protein degradation.
A number of protein targets display expression levels and
oxidative modifications that are common to unrelated forms
of neurodegeneration. This points to the highly conserved
routes involved in the progression of neurodegenerative
processes and suggest that these disorders may be precipi-
tated by similar triggers.
ANTIOXIDANT DEFENSE: GLUTATHIONE AND
DETOXYFYING ENZYMES
Glutathione imbalance has been widely recognized as a
marker of both the onset and progression of several neurode-
generative disorders [162]. Reduced glutathione pools have
been detected in both blood and brain tissue of patients with
neurodegenerative diseases [162]. Since reduced glutathione
constitutes the most readily available barrier against oxida-
tive damage, even transient insufficiency of the reduced thiol
is guaranteed to contribute to cellular stress. Reduced glu-
tathione is abundant (1-10 mM) and its homeostasis involves
several proteins and enzymes (GPx, GR, GST, and GCL)
whose expression and activity are also impaired in neurode-
generative disorders [162]. Notably, greater expression and
lower activity of superoxide dismutase (SOD) has been ob-
served in Alzheimer’s disease [163]. This loss of function
could be the result of post-translational modifications, as
observed with mitochondrial SOD (Table 1). Likewise, the
activities of glutathione peroxidase and catalase are also re-
duced in Alzheimer’s disease [163]. The expression of per-
oxiredoxin isoforms has been found to be abnormal in brain
tissue of patients with Alzheimer’s disease and Down syn-
drome [164-166] . Further, oxidized peroxiredoxins 2 and 6
in plasma have been proposed as biomarkers of Alzheimer’s
disease [167]. Peroxiredoxins are essential for the removal

Table 1. Selected post-translational modifications identified in neurodegenerative disorders, protein targets and the associated
disorders or model animals.
Protein Modification Disease References
creatine kinase BB, glutamine synthase, and ubiquitin carboxy-terminal hydrolase L-1 Carbonylation Alzheimer’s disease [198]
dihydropyrimidinase-related protein 2, alpha-enolase and heat shock cognate 71 Carbonylation Alzheimer’s disease [199]
Ubiquitin carboxyl-terminal hydrolase L1 (UCH-L1), gamma-enolase, actin, and
dimethylarginine dimethylaminohydrolase 1 (DMDMAH-1)
Carbonylation Alzheimer’s disease [200]
enolase, glyceraldehyde-3-phosphate dehydrogenase, ATP synthase alpha chain, car-
bonic anhydrase-II, and voltage-dependent anion channel-protein
Nitration Alzheimer’s disease [201]
peptidyl prolyl cis-trans isomerase, phosphoglycerate mutase 1, ubiquitin carboxyl ter-
minal hydrolase 1, dihydropyrimidinase related protein-2 (DRP-2), carbonic anhydrase
II, triose phosphate isomerase, alpha-enolase, and gamma-SNAP
Carbonylation Alzheimer’s disease [202]
Pin1 Carbonylation Alzheimer’s disease [203]
beta-actin (ACTB), glutamine synthase (GS), and neurofilament 66 (NF-66) Carbonylation Healthy old mice [204]
Alpha-enolase, Glucose regulated protein precursor, Aldolase, Malate dehydrogenase,
GSTM3, MRP3 protein, Peroxiredoxin, Heat shock protein 70 (HSPA8), Structural
dysfunction Dihydropyrminidase like-2, Fascin 1, 14-3-3 protein-gamma
Nitration
Amnestic mild cognitive
impairment
[205]
glutamate dehydrogenase [NAD (P)], glyceraldehyde-3-phosphate dehydrogenase
(GAPDH), alpha-enolase, neurofilament triplet L protein, glutathione-S-transferase
(GST) and fascin actin bundling protein
Carbonylation
Canine model of human
aging
[206]
Neuropolypeptide h3, carbonyl reductase (NADPH), alpha-enolase, lactate dehydro-
genase B, phosphoglycerate kinase, heat shock protein 70, ATP synthase alpha chain,
pyruvate kinase, actin, elongation factor Tu, and translation initiation factor alpha
4-hydroxy-2-
nonenal (HNE)
Amnestic mild cognitive
impairment
[207]
peroxiredoxin 2, triose phosphate isomerase, glutamate dehydrogenase, neuropolypep-
tide h3, phosphoglycerate mutase1, H(+)- transporting ATPase, alpha-enolase and fruc-
tose-1,6-bisphosphate aldolase
Nitration Early Alzheimer’s disease [208]
α-enolase, aldolase, Prx6, aconitase, and α-tubulin HNE
Alzheimer’s disease
(hippocampus)
[209]
ATP synthase a chain, glutamine synthase, DRP-2, and MnSOD HNE
(inferior parietal lobule)
[209]
Synapsin 1, Gamma-enolase, Guanosine diphosphate dissociation inhibitor 1 (GDP),
Phosphoglycerate mutase (PGM), Heat shock protein 70 (Hsp70), ATP synthase, Alpha-
spectrin
Nitration
Traumatic brain-injured
rats
[210]
carbonic anhydrase II (CA II), heat shock protein 70 (Hsp70), mitogen-activated protein
kinase I (MAPKI), and syntaxin binding protein I (SBP1)
Carbonylation
Mild cognitive impair-
ment and early
Alzheimer's disease
[211]
Alpha enolase, Gamma enolase, Glyceraldehyde-3-phosphate dehydrogenase, Creatine
kinase B-type, NAD-dependent deacetylase, sirtuin-2, Fructose-bisphosphate
aldolase C, NADH dehydrogenase, [ubiquinone] iron-sulfur protein 3, mitochondrial, 6-
phosphogluconate dehydrogenase, decarboxylating, Glyoxylate reductase/
hydroxypyruvate reductase, Dihydropteridine reductase, Glial fibrillary acidic protein P,
Mitochondrial inner membrane protein, Transitional endoplasmic reticulum ATPase,
Dihydropyrimidine related protein, Dual specificity mitogen activated protein kinase
kinase 1, Guanine nucleotide-binding protein G(o) subunit alpha, Rab GDP dissociation
inhibitor beta
Phosphorylation Alzheimer's disease [212]
phosphatidylethanolamine-binding protein
1 and Pin-1
Nitration
Transgenic mouse, model
of Alzheimer’s disease
[213]

(Table 1) contd….
Protein Modification Disease References
Haptoglobin b chain, Serotransferrin,
a2-Macroglobulin, Complement factor B
Carbonylation
(plasma)
[214]
RP78, UCH-L1, V0-ATPase, cathepsin D and GFAP Carbonylation
Down syndrome prior to
the development of Alz-
heimer's disease neuropa-
thology
[180]
Glutamate dehydrogenase 1, mitochondrial, Syntaxin-binding protein 1, Dihydro-
pyrimidinase-related protein 2, Dihydropyrimidinase-related protein 1, 78-kDa glucose-
regulated protein, Superoxide dismutase 1 (Cu,Zn), Glial fibrillary acidic protein, Cyto-
chrome b–c1 complex subunit Rieske, mitochondrial, T-complex protein 1 subunit β,
Pyruvate kinase isozymes M1/M2, Heat shock cognate 71-kDa protein, Neurofilament
medium polypeptide, Glyceraldehyde-3-phosphate dehydrogenase, α-Enolase, Malate
dehydrogenase, cytoplasmic, Septin 11
HNE
Down syndrome brain.
Proteins that are specific
for Alzheimer's disease.
[215]
Superoxide dismutase [Mn], mitochondrial, Voltage-dependent anion selective channel
protein 2, Fructose-bisphosphate aldolase C, Actin, cytoplasmic 1, Alpha-crystallin B
chain, Alpha-enolase Alpha-internexin, Aspartate aminotransferase, cytoplasmic ATP
synthase subunit beta, mitochondrial, Carbonyl reductase [NADPH] 1, Carbonic anhy-
drase 2, Cofilin 1, Dihydropteridine reductase, Dihydropyrimidinase-related protein 2,
Fructose-bisphosphate aldolase A, Fructose-bisphosphate aldolase C,
Glial fibrillary acidic protein, Glutamine synthetase, Heat shock cognate 71 kDa protein,
Hemoglobin subunit alpha, Hemoglobin subunit beta, Ig gamma-1 chain C region, l-
lactate dehydrogenase B chain, l-lactate dehydrogenase A chain, Malate dehydro-
genase, cytoplasmic, Neurofilament light polypeptide, Peroxiredoxin-1, Peroxiredoxin-6,
Peptidyl-prolyl cis–trans isomerase A, Pyruvate kinase isozymes M1/M2 Phosphoglyc-
erate kinase, Serum albumin, Superoxide dismutase [Cu–Zn], Superoxide dismutase
[Mn], mitocondrial, Triosephosphate isomerase, Tubulin alpha-1A chain, Tubulin beta-
2C chain, Tubulin alpha-1B chain, 14-3-3 protein epsilon, 14-3-3 protein zeta/delta, 14-
3-3 protein theta, Phosphatidylethanolamine-binding protein 1, Glyceraldehyde-3-
phosphate dehydrogenase, Guanine nucleotide-binding protein G(I)/G(S)/G(T) subunit
beta-1, Glutamate dehydrogenase 1,mitochondrial, NADP-regulated thyroid-hormone-
binding protein, Voltage-dependent anion-selective channel protein 1, Voltage-
dependent anion-selective channel protein 2
S-nitrosylation
Alzheimer’s Disease
hippocampus, substantia
nigra and cortex
[216]
Cdk5 S-nitrosylation Alzheimer’s disease [217, 218]
Protein disulfide isomerase (PDI) P5 S-nitrosylation Alzheimer’s disease [219-221]
ApoE S-nitrosylation Alzheimer’s disease [222]
Drp1 S-nitrosylation
Neurodegenerative disor-
ders; the role of S-
nitrosylation of Drp1
remains controversial
[223-225]
Parkin S-nitrosylation Parkinson’s disease [226-228]
DJ-1 to PTEN Transnitrosylation Parkinson’s disease [229]
Mitochondrial complex I
S-nitrosylation
Nitration
Parkinson’s disease [230]
Heme oxygenase 1 (HO-1)
Carbonylation
HNE
Alzheimer’s disease [231]
Biliverdin reductase (BLVR)
Phosphorylation
Nitration
Alzheimer’s disease [177, 178]

Table 2. Selected metabolites strongly associated with the onset and progression of neurodegenerative diseases, their site of detec-
tion and the affected metabolic pathways.
Marker or mediator Level compared to control Compartment Metabolic pathway References
Neopterin High Plasma
Folate and tetrahydrobiopterin biosyn-
thesis
[92, 232-236]
Tetrahydrobiopterin Low
Brain
CSF
Folate and tetrahydrobiopterin biosyn-
thesis
[74-79]
Folates Low Plasma One-carbon metabolism [120, 131, 237]
Vitamin B12 Low Plasma One-carbon metabolism [131, 238]
Nitric oxide Low Plasma
Signaling, vascular tone, cell prolifera-
tion
[123]
High Plasma Endogenous NOS inhibitor [36, 37, 41, 42, 89]ADMA
Low CSF Endogenous NOS inhibitor [41, 239]
L-MMA High Plasma Endogenous NOS inhibitor [36, 37]
Iron uptake High Neuroblastoma Iron metabolism [184]
Heme
Functional deficiency caused by binding to
excess Amyloid-β
Neuroblastoma
Tetrapyrrole metabolism, Iron metabo-
lism
[182-184]
Glutathione, reduced Low Brain
Glutathione metabolism, transsulfura-
tion
[240, 241]
High Plasma
One-carbon metabolism
Marker of folate and/or vitamin B12
deficiency
[41, 42, 89, 119,
131]
Homocysteine
Normal CSF
Marker of folate and/or vitamin B12
deficiency
[242]
High Plasma
Marker of vitamin B12 deficiency
[119, 238]
Methylmalonic acid
Normal CSF
Marker of vitamin B12 deficiency
[242]
Phospholipids
Low Plasma
Carbon metabolism
Lipid Metabolism
Membrane integrity
Signaling
[196]
of hydrogen peroxide and organic hydroperoxides to water
and alcohol, respectively. Their inactivation by oxidative
modification can contribute to the mismanagement of oxida-
tive stress in the degenerating brain [168]. Excessive pro-
duction of ROS and glutathione depletion induce the upregu-
lation of heme oxygenase 1 (HO-1) and biliverdin reductase
A (BVR-A) [169]. Heme oxygenases catalyze the decompo-
sition of heme to the linear tetrapyrrole biliverdin, carbon
monoxide and ferrous iron. Biliverdin reductase catalyzes
the conversion of biliverdin into bilirubin, the latter possess-
ing enhanced antioxidant properties. The induction of HO-
1/BVR-A affords antioxidant protective effects during the
early stages of neurodegeneration by reducing the pools of
toxic, free heme [169]. Apart from heme detoxification, the
other two products of the enzymatic reactions of HO and
BLVR participate in cell proliferation and apoptosis, thus
contributing to cellular life and death, respectively [170-
172]. For instance, while elevated CO is toxic, low concen-
trations of CO have been shown to be beneficial by antago-
nizing apoptosis and stimulating cell proliferation [173-175].
Likewise, apart from its intrinsic antioxidant property, bili-
rubin stimulates neuronal NOS expression and NO biosyn-
thesis [176], hence supporting the benign roles of NO in the
brain. Increased ROS upregulate the expression of BVR-A,

Fig. (2). Nitric oxide homeostasis in neurodegenerative diseases. Nitric oxide biosynthesis is stimulated by calcium in the case of eNOS
and nNOS and cytokines in the case of iNOS. Under normal conditions, nitric oxide supports cellular proliferation and vascular functions via
signaling events. Endogenous L-arginine analogues (1), PKC-mediated phosphorylation of NOS (2), imbalance of biopterins (3), defective
heme insertion (4) and oxidative damage (5) inhibit NOS or lead to its uncoupling. Elevated homocysteine caused by deficiency of folate or
vitamin B12 contributes to NOS inactivation via pathways 1, 2 and 3. Uncoupled NOS produces superoxide and hydrogen peroxide, which
oxidize DNA, lipids and proteins (carbonylation, HNE-adduct formation) altering their functions. Overproduction of ROS reduces NO
bioavailability via the formation of additional reactive species such as peroxynitrite. This favors the occurrence of NO-derived post-
translational modification of proteins (Tyr nitration, S-nitrosation) with the subsequent gain or loss of function. Amyloid-β protein can bind
heme leading to functional heme deficiency. A local shortage of heme could impair NOS maturation thereby reducing NO synthesis in the
brain. The HO-1/BLVR system protects against oxidative damage during the early stages of Alzheimer’s disease and conceivably in other
dementias by limiting the amount of toxic free heme in the cells. However, persisting conditions of oxidative stress inhibit the HO-1/BLVR
pair via post-translational modifications. Low bioavailability of NO due to uncoupling or inhibition of NOS along with increased ROS con-
tributes to endothelial and mitochondrial dysfunction. Imbalances in glutathione metabolism, impairments in antioxidants enzymes and down-
regulation of proteins of the cellular stress response accompany the onset and progression of neurodegenerative disorders.
however, this is accompanied by a reduction in enzyme ac-
tivity [177, 178]. At a first glance, this finding challenged
the proposed role of the HO-1/BVR-A pair in the protection
against oxidative stress in neurodegeneration. A detailed
analysis of the timeframe for the observed changes in protein
expression and activity led to the reconciling paradigm that
the role of HO-1/BVR-A in Alzheimer’s disease is biphasic
in nature [169]. During the early stages of neurodegenerative
disease the HO-1/BVR-A system proves efficient in the de-
toxification of unbound heme and in stimulating cell prolif-
eration and anti-apoptotic pathways. As the disease pro-
gresses and sustained oxidative stress prevails, the HO-
1/BVR-A system undergoes oxidative post-translational
modification, reducing its capacity to protect the cell against
further damage. This biphasic response of the HO-1/BVR-A
system is not unprecedented, but rather a vivid illustration of
the role of proteostasis in neurodegenerative diseases [179].
CONCLUSION
Neurodegenerative diseases are illnesses of elusive ori-
gin. Oxidative stress and impairments in cofactor metabo-
lism are common features observed in the pathogenesis and
progression of various neurodegenerative disorders. Proteo-
mic (Table 1) and metabolomic (Table 2) footprints of a dis-
rupted nitric oxide homeostasis are commonly seen in Alz-
heimer’s, Parkinson’s, Down syndrome and unrelated de-
mentias [154, 180, 181] (Fig. 2). An outstanding and perhaps
underappreciated characteristic of neurological impairments
is the derangement of metal metabolism. Functional heme
deficiency caused by amyloid-β binding to heme has been

reported in Alzheimer’s disease [182-184]. Downregulation
of DJ-1, a copper chaperone that furnishes the metal needs of
SOD, has been noted in Parkinson’s disease [185] and in
MMACHC disease, a functional deficiency of vitamin B12, a
cobalt-containing macrocycle [186, 187]. Vitamin B12 defi-
ciency, whether nutritional or functional unravels in neuro-
logical deterioration with various degrees of dementia and
hematological abnormalities [188]. Importantly, the pro-
teome of MMACHC disease displays alterations in protein
expression levels that are typically observed in neurological
disorders [186, 187]. Functional cobalamin deficiency, as
observed in MMACHC disease, is also characterized by oxi-
dative stress [189-191] and low glutathione levels [192]. The
trafficking of metals and its derived cofactors, namely, heme
[193] and cobalamin [194, 195], is complex and involves
several cellular compartments. It is possible that alterations
in metal homeostasis and oxidative damage to metal centers
in proteins contributes significantly to the neurological dete-
rioration observed in these seemingly unrelated neurological
disorders. Prompted by the substantial overlap of common-
alities among neurological disorders of unrelated origin, the
search for biomarkers took a new direction with the study of
Mapstone and colleagues, who investigated the lipidome of
Alzheimer’s disease [196]. The authors identified 10 phos-
pholipids present in plasma that could predict the onset of
neurocognitive impairment 3 years prior to the emergence of
symptoms, with 90% accuracy [196]. While full validation in
large-scale clinical studies is crucial, this is the first study to
identify biomarkers that show specificity for Alzheimer’s
disease and that are easily assessed in plasma samples. At
the cellular level, model studies with C. elegans, a nematode
with a well-characterized neuronal network, promise to ad-
vance our knowledge on the role of oxidative stress in neu-
rodegenerative diseases due to its easily manageable genetic
modification and inexpensive growth conditions [34, 197].
The complexity of neurodegenerative disorders calls for the
deciphering of the interactome for the integrative analysis of
the cellular and plasma components that determine disease
onset and progression.
CONFLICT OF INTEREST
The author(s) confirm that this article content has no con-
flict of interest.
ACKNOWLEDGEMENTS
The author thanks the DAAD (German Academic Ex-
change Service) for financial support through the Visiting
Professorship Program. The author is grateful to Prof. Dr.
Ivana Ivanovic-Burmazovic for serving as a host of the
DAAD-sponsored program.
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Received: June 16, 2015 Revised: August 12, 2015 Accepted: August 18, 2015

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