Nitric Oxide

The NOS family of enzymes is responsible for NO synthesis, which catalyzes the conversion of arginine to citrulline and NO. NO synthase (NOS), localized in the central nervous system (CNS) and in the periphery, is present in three isoforms named (a) neuronal NOS (nNOS, type I), (b) endothelial NOS (eNOS, type III) and (c) inducible NOS (iNOS, type II) (3,4). Recently, a fourth isoform has been discovered and called mitochondrial NOS (mtNOS); indeed mtNOS is an isoform of nNOS present in the inner mitochondrial membrane (18). Activation of different isoforms of NOS requires various factors and cofactors. Formation of calcium-calmodulin complexes is a prerequisite before the functionally active dimer exhibits NOS activity, which depends also on cofactors such as tetrahydrobiopterin (BH4), flavins (FAD), FMN and NADPH (19). In contrast to nNOS and eNOS, iNOS can bind to calmodulin even at very low concentration of intracellular calcium, thus iNOS can exert its activity in a calcium-independent manner. The levels of iNOS in the CNS are generally fairly low. However, an increased expression of iNOS in astrocytes and microglia occurs following viral infection and trauma (20). Activation of iNOS requires gene transcription, and the induction can be influenced by endotoxin and cytokines (interleukin-1, interleukin-2, lipopolysaccharide (LPS), interferon-y, tumor necrosis factor). This activation can be blocked by anti-inflammatory drugs (dexametha-sone), inhibitory cytokines (interleukin-4, interleukin-10) prostaglandins (PGA2), tissue growth factors or inhibitors of protein synthesis, e.g. cycloheximide (4,21).

From a chemical point of view, NO is a free radical because of the unpaired electron in its outer orbital. This allows NO to exist in different redox-related forms. In fact, the removal of this electron generates nitrosonium (NO+), whereas the addition of another electron to this orbital forms nitroxyl anion (NO-) (22). The real importance of NO+ and NO- in the regulation of biological systems is still under debate, mainly by virtue of their very short half-lives. In fact, NO+ is rapidly hydrolyzed in aqueous solution to give nitrous acid with a life time of 3 x 10-10 s whereas NO- has a life time in the order of milliseconds (23). These data, along with the high reactivity of NO itself, half-life in the order of 0.1 s (24), raise the problem of the biological activity of this gas. In fact, first studies demonstrated that endothelium-derived relaxing factor had a relatively longer half-life, in the order of seconds, than these redox forms. This discrepancy, led to the hypothesis that NO, once formed by NOS activity, reacts with carrier molecules that stabilize it and prolong its halflife. S-Nitrosothiols (RSNOs) are a group of substances formed by the attachment of the NO and its congeners to the sulfhydryl centers (S-nitrosylation) of proteins and non-protein molecules (24). Notably, low-molecular-weight RSNO, i.e. S-nitrosocysteine (SNOC) and S-nitroso-glutathione (GSNO), are the main non-protein RSNO in cells and extracellular fluids (25,26), whereas albumin-SNO is the main circulating protein RSNO (27). The much greater half-life of RSNO with respect to free NO and their ability to release the gas in response to many stimuli, makes RSNO as important intermediates in the cellular metabolism and bioactivity of NO (27). The biological role for NO in the S-nitrosylation of many proteins is emerging as an important physiological regulatory system (28). The NMDA receptor is inactivated by nitrosylation, hence NO may modulate glutamatergic neurotransmission by this mechanism (29); NO has been demonstrated to stimulate the auto-ADP ribosylation of glyceraldehyde-3-phosphate dehydrogenase (GADPH) by its reaction with a critical cysteine with resulting binding of NAD to the catalytic cysteine, inhibition of GADPH activity and depression of glycolysis (30). Through formation of GSNO, NO can cause GSH depletion and hence trigger redox-dependent changes in cellular signaling (31) as well as modification of key intracellular enzymes, such as chain respiratory complex activities (32). Recently, the S-nitrosylation of cyclooxygenase-2 (COX-2) by iNOS and the increase in the catalytic activity of COX-2 has been documented; this evidence is potentially very useful because it allows the development of new drugs which can behave as anti-inflammatory by inhibiting the direct interaction of iNOS and COX-2 (33). Finally, NO has been shown to S-nitrosylate dynamin, an enzyme endowed with GTPase activity and involved in the vesciculation and intracellular vescicle trafficking. The S-nitrosylation of this enzyme is important in the downstream effect of adrenergic beta2-receptor as well as in the internalization of pathogens such as uropathogenic Escherichia coli (34).

One of the most significant biological reactions of NO is with transition metals resulting in NO-metal complexes, which occurs with iron in the heme moiety of guanylate cyclase (35). This interaction through the induction of conformational changes in the heme moiety results in the activation of the enzyme with rise in cGMP levels. Other heme protein targets for NO are heme oxygenase, catalase, cytochrome c, hemoglobin and peroxidase. NO also reacts with non-heme iron, such as iron-sulfur cluster present in numerous enzymes including, NADH-ubiquinone oxidoreductase, cis-aconitase and NADH: succinate oxidoreductase (36). In contrast with the reversible reaction of NO with heme, binding of NO to non-heme iron results in irreversible enzyme inactivation. Through this mechanism, NO (a) irreversibly inactivates the enzyme ribonucleotide reductase (thereby inhibiting DNA synthesis), (b) moves iron from iron-storage proteins such as ferritin and (c) mobilizes Cu+ from ceruloplasmin and metallothionein. NO can also influence iron metabolism at the post-transcriptional level by interacting with cytosolic aconitase, which after binding NO, functions as iron-responsive-binding protein diminishing its aconitase activity (37).

The reaction of NO with ROS is very important from a pathological point of view. NO reacts with the superoxide anion (O2-) to produce the potent oxidant,

(OONO-) (38). The rate of this reaction is three times faster than the rate of superoxide dismutase (SOD) in catalyzing the dismutation of the superoxide anion to hydrogen peroxide. Therefore when present at appropriate concentrations, NO effectively competes with SOD for O^-. Peroxynitrite is a strong oxidant capable of reacting with sulfhydryl groups, such as those of proteins, or directly nitrate aromatic aminoacids and possibly affect their participation in signal transduction mechanisms (39). In addition, peroxynitrite oxidizes lipids (39), proteins (40) and DNA (41).

NO and brain aging

Several lines of evidence have shown a "physiologic" impairment of the NO system during aging. A decrease in NADPH diaphorase positive neurons (i.e. containing nNOS) has been described in cerebral cortex and striatum of aged rats (42). Furthermore, a dramatic decrease in NO-responsive cGMP-synthesizing cholinergic neurons has been found in septum, diagonal band of Broca and caudate-putamen in aged rats (43). Finally, iNOS was not detected under normal conditions in aged rats (44), whereas eNOS was significantly reduced only in cerebellum (45). All these data demonstrate that during aging NO production in brain is mainly sustained by constitutive NOS and is reduced with respect to young. This reduction in NO content could explain some of the pathological changes occurring during age. The relationship between NO and cognitive functions is well established (4,46) and NO reduction during age could be a plausible explanation of this impairment. In support of this theory, the administration of NO donors to old rats ameliorated cognitive functions in many behavioral tasks (46). Interestingly, the reduction of cerebral blood flow and increase in cerebral vascular resistance reported in old men with respect to young volunteers was related to the NO impairment (47).

A particular aspect of the brain-NO interaction during aging is the dysregulation of the neuroendocrine hypothalamo-pituitary axes. McCann et al. recently revisited the so-called "nitric oxide theory of aging" and proposed that during old age, bacterial or viral infections (very common in old people) can up-regulate iNOS and cause protein nitration and cell damage both in hypothalamic nuclei and in pituitary cell groups (48). This dysfunction affects primarily the stress axis because the cell damage is particularly evident in the hypothalamic paraventricular and arcuate nuclei, the first containing the cell bodies of the corticotropin-releasing-hormone (CRH)-secreting neurons (14). This reduction in the stress axis activity along with the thermoregulatory impairment can alter the physiologic response to infections and contribute to the acceleration of brain damage.

Nitrosative stress and neurodegenerative disorders

The involvement of nitrosative stress in the development of neurodegenerative disorders is no longer a matter of question. In these disease states, NO is produced in excess by the iNOS induction due to the pro-inflammatory response, the latter being a common feature of neurodegenerative disorders. Moreover, NO is much more harmful under pathological conditions involving the production of ROS such as superoxide anion and the final formation of the peroxynitrite (3,4,11). Nitrotyrosine formation, as a marker of nitrosative stress, has been documented in AD, PD and ALS (9,49-52). The role played by the cytokine system is crucial in triggering nitrosative stress. Cytokines (IFN-y) which are present in normal brain are elevated in numerous pathological states, including PD, AD, multiple sclerosis (MS), ischemia, encephalitis and central viral infections (53). Accordingly, as cytokines promote the induction of iNOS in brain, a possible role for a glial-derived NO in the pathogenesis of these diseases has been suggested (53). Excessive formation of NO from glial origin has been evidenced in some study in which NADPH diaforase (a cytochem-ical marker of NOS activity)-positive glial cells have been identified in the substantia nigra of postmortem brains obtained from individuals with PD. Loss of nigral GSH is considered an early and crucial event in the pathogenesis of PD and as a consequence, decreased peroxynitrite scavenging may also occur. Therefore, such perturbations in thiol homeostasis may constitute the starting point for a vicious cycle leading to excessive per-oxynitrite generation in PD (53). Moreover, in support of this, it has been reported that the selective inhibition of nNOS prevents 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced parkinsonism in experimental animals (54).

Proteomic techniques have been used in our laboratory to determine which proteins are post-translationally modified to 3-NT in AD brain. Six proteins were identified which exhibited increased specific 3-NT immunoreactivity: a-enolase, triosephosphate isomerase, neuropolypeptide h3, P-actin, L-lactate dehydrogenase and y-enolase (3). Three of the proteins, a-enolase, triosephosphate isomerase and neuropolypeptide h3 were significantly increased in 3-NT immunoreactivity (55). a-Enolase had been previously identified as specifically oxidized in AD brain (56), and is one of the subunits comprising the enzyme enolase. Enolase catalyzes the reversible conversion of 2-phosphoglycerate to phosphoenolpyruvate in glycolysis. Taken together with the increased nitration of triosephosphate isomerase, which interconverts dihydroxyacetone phosphate and 3-phosphoglyceraldehyde in glycolysis, these results indicate a possible mechanism to explain the altered glucose tolerance and metabolism exhibited in AD (57,58). Neuropolypeptide h3, also known as phosphatidylethanolamine-binding protein (PEBP), hippocampal cholinergic neurostimulating peptide (HCNP) and raf-kinase inhibitor protein (RKIP), has a variety of functions in the brain. Among them is in vitro up-regulation of the production of choline acetyltransferase in cholinergic neurons following NMDA receptor activation (59). Choline acetyltransferase activity is known to be decreased in AD (60), and cholinergic deficits are prominent in AD brain (61,62). Nitration of neuropolypeptide h3 may help to explain the decline in cognitive function due to lack of neurotrophic action on cholinergic neurons of the hippocampus and basal forebrain.

A novel mechanism by which NO can trigger neurodegenerative disorders has been recently proposed. In 2002, Gu et al. demonstrated that NO derived from the endogenous SNOC S-nitrosylates metalloproteinase-9 (MMP-9) (9). MMPs are a group of proteins involved in the pathogenesis of acute and chronic neurodegenerative disorders such as stroke, AD, HIV-associated dementia and MS (9,63-65). The S-nitrosylation reaction activated MMP-9 and caused neuronal apoptosis. A similar mechanism has been proposed for PD. Yao et al. (10) and Chung et al. (8), in two elegant articles, independently demonstrated that SNOC-derived NO is able to nitrosylate parkin, an E3 ubiquitin ligase protein, with chaperone activity, which plays a fundamental role in the proteasome-mediated destruction of misfolded proteins. Mutation in parkin activity results in autosomal recessive-juvenile parkinsonism. The nitrosylation of cysteine residues of parkin can initially increase and later decrease the E3 ubiquitin ligase activity of this protein, thus reducing its protective function. Finally, NO has been shown to S-nitrosylate GAPDH thus reducing its activity and allowing the enzyme to bind to Siahl, the latter being an E3-ubiquitin ligase like parkin. Once formed, the GAPDH-Siahl complex translocates into the nucleus where it begins to induce apoptosis (66). A direct consequence of this study was the unravelling of a new mechanism of action for selegiline, a drug used to treat PD patients by virtue of its mono-amine oxidase-B (MAO-B) inhibitory activity. Hara et al. demonstrated that selegiline at nanomolar concentrations is able to prevent S-nitrosylation of GAPDH thus blocking its binding to Siahl and further induction of apoptosis. Selegiline shares this neuroprotective effect with TCH346, a derivative without any MAO-B inhibitory activity (66).

Taken together, these findings provide new evidence about a novel approach to neurodegenerative disorders. In fact, drugs which can modify the NO-mediated activation of MMP, parkin or GAPDH as well as activate the ubiquitin-proteasome system could be very useful in the treatment of neurodegenerative disorders such as AD and PD.

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