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Friday, March 16, 2018 by Sali

Introduction

Most age-related degenerative disorders are characterized by neuronal damage. PD is one such progressive debilitating disorder which affects over one million people all over the world and manifests as degeneration of movement. PD is characterized by the progressive degeneration of dopaminergic neurons in the substantia nigra and striatal regions of the brain. Clinical symptoms are manifested when more than 80% of neuronal loss has occurred and currently, the available therapeutic options only aim at alleviating the symptoms of the disease. Since neuronal damage is irreversible and the degeneration is progressive, a better understanding of the underlying mechanisms of the neurodegeneration in PD holds the key to development of better treatment strategies.

Findings over the last decade have implicated nitric oxide (NO) in the pathogenesis of Parkinson's disease (PD). NO is gaseous and short lived, yet has profound effects on several physiological conditions and is also found up-regulated in many pathological conditions. Reactive nitrogen species are generated by the biochemical reactions of NO or by enzymatic catalysis of NO metabolism. NO reacts with the superoxide anion generated through dopamine metabolism and mitochondrial aerobic respiration to form peroxynitrate ion (ONOO-). In the presence of cupric ions, ONOO- is heterolytically cleaved to form hydroxyl radical and nitronium cation. The formation of ONOO- is governed by the concentration of nitric oxide, which is found in excess amounts in conditions of neuronal damage. During mitochondrial dysfunction and glutamate excitotoxicity, the excessive intracellular calcium accumulation leads to over-activation of calcium dependant NO synthesizing enzymes. Then again, the excess NO so produced reacts with superoxide to form ONOO-. It can nitrate tyrosine residues to 3-Nitrotyrosine (Coyle and Puttfarcken, 1993, Torreilles et al., 1999). Via nitration, ONOO- may inhibit tyrosine phosphorylation and alter protein formation and function by inserting negative charges and tagging the protein for proteolysis. Increased 3-Nitrotyrosine immunostaining in Lewy bodies was observed in PD patients and in 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) treated mice (Good et al., 1998). An enzymatic distribution of nitric oxide synthase (NOS) predominant near the dopaminergic neuronal tracts, points at the potential interaction of NO with the reactive intermediates of dopamine metabolism, giving rise to peroxynitrate like reactive ions (Egberongbe et al., 1994).

In addition to causing cytotoxicity through free radical damage, NO itself is involved directly. It inhibits complexes I, II and IV of the mitochondrial respiratory chain, aconitase of citric acid cycle, the rate limiting DNA replication enzyme ribonucleotide reductase etc. Complex I activity is found decreased in the pars compacta region of substantia nigra of PD brain (Ebadi and Sharma, 2003). Nitric oxide further exacerbates oxidative damage by depleting the antioxidant system. Neuromelanin, an important neuroprotective material is found depleted in the dopaminergic neurons in PD brain. It was also demonstrated that Glutathione reductase, an enzyme which regenerates glutathione from its oxidized form, is susceptible to peroxynitrate (Barker et al., 1996). Therefore NO and peroxynitrate could contribute by depleting the cells of major antioxidant systems and rendering them more vulnerable to oxidative stress. Involvement of NO in neurodegeneration can be inferred from the increased levels of iron in the substantia nigra of PD brains, increased lipid per oxidation levels and DNA damage in the region.

Post mortem analysis of PD brains have revealed higher than normal levels of NO producing enzyme, nitric oxide synthase (NOS) in the nigrostriatal regions (Hunot et al., 1996). The involvement of NO in the pathogenesis of PD is also confirmed by various experimental models using neurotoxins like MPTF, 6-hydroxydopamine (6-OHDA) lipopolysaccharide (LPS), where an upregulation of NO producing enzymes and their genes has been observed and NO inhibitors, where neuroprotection was demonstrated (Schulz et al., 1995, Przedborski et al., 1996, Possel et al., 2000, Barthwal et al., 2001, Iravani et al., 2002Singh et al., 2005). An understanding about the biosynthesis and regulatory mechanisms of NO is pivotal in addressing the role of NO in PD and design of therapeutic agents. This review recapitulates the findings about the NOS, the NO producing enzyme, their regulation and involvement in PD.

NOS – structure and chemistry

NO is synthesized during the redox conversion of L-Arginine (L-Arg) to L-Citrulline (L-Cit), catalyzed by NOS. At least three major isoforms of NOS have been identified and based on their localization, classified as neuronal NOS (nNOS), endothelial NOS (eNOS) and inducible or macrophage NOS (iNOS). The NOS enzyme is made up of two catalytic units – a C terminal reductase domain and an N terminal oxygenase domain. Six important cofactors participate in the NOS catalyzed reaction. Flavin adenine dinucleotide (FAD) and flavin mononucleotide (FMN) of the reductase domain mediate the electron transfer from nicotinamide adenine dinucleotide phosphate (NADPH) to haem in the oxygenase domain. This electron transfer is dependant on the intracellular calcium ions.


Calmodulin, a calcium binding protein, binds to the region between the two domains and facilitates the electron transfer process and results in the production of NO via oxidation of L-arginine to L-citrulline. The binding of calmodulin to NOS determines the catalytic property of the NOS. In a recent report, a novel calmodulin dependant regulation of constitutive NOS activity has been described. Auto inhibitory loops found in the FMN binding site, together with NADPH, induces a conformational lock in the enzyme which is relieved upon calmodulin binding (Daff, 2003).

Dimerization of NOS proteins is essential for their activity. The interaction of two oxygenase domains creates an interface where tetrahydrobiopterin (H4B) interacts with residues in both subunits of the dimer and also hydrogen bonds to the active site haem. Dimerization activates NOS in at least three ways: it sequesters haem from solvent, creates high affinity binding sites for substrate L-Arg and cofactor, H4B, and enables transfer of electrons between the two domains. Recently, it has been reported that the strengths of the dimers formed by the oxygenase domain of NOS was highest for eNOS, followed by nNOS and iNOS (Venema et al., 1997). H4B is not required for eNOS dimerization, whereas bound L-arginine and H4B increased the stability of the NOS dimmers, particularly of nNOS and iNOS (Panda et al., 2002).

Isoforms of NOS – Localization and genetic make-up

At least three isoforms of NOS have been isolated so far. Based on their localizations, they are denoted as neuronal NOS (nNOS), endothelial NOS (eNOS) and inducible NOS (iNOS). nNOS and eNOS are constitutive isoforms. Their activity is dependant on intracellular calcium and calmodulin levels. iNOS is the inducible isoform. It produces sustained and high concentrations of NO. iNOS is able to bind tightly to calmodulin even at very low cellular concentration of calcium. Consequently its activity doesn't respond to changes in calcium levels in the cell and as a result the production of NO by it lasts much longer than from the other isoforms of NOS.

nNOS: It is found mainly in the neuronal cells (Bredt et al., 1990), astrocytes (Murphy et al., 1993) and polymorphonuclear cells (Gatto et al., 2000). Various tissues and organs harbor other variants of nNOS, which will be discussed later. The human nNOS gene is localized on chromosome 12 (Marsden et al., 1994). It comprises of 29 exons and 28 introns in its genome which encodes 1434 amino acids in its open reading frame (Hall et al., 1994). The transcription initiation and termination sites are present in exon 2 and exon 29 respectively. Exon 2 contains a post synaptic density Zo-1 domain (PDZ) which is important for docking of nNOS protein to NMDA (N-methyl D-aspartate) receptors. Sequence analysis of the 5′-flanking region in the nNOS genome reveals sites for binding of several transcription regulation factors like nuclear factor NF-kB etc. In neurons harboring nNOS, it is present both in particulate as well as soluble form and found localized in the cytoplasm as well as in the membrane.

nNOS variants: nNOS is one of the most structurally diverse human gene described. It is expressed in diverse cell types and tissues apart from the neurons. The molecular diversity in mRNA is generated by selective alternative splicing, results in tissue-specific transcriptional regulation of nNOS, but their functional significance have not been fully understood. The natural full-length nNOS (160 kDa), denoted as nNOS-1 or nNOS-a has 29 exons and expresses 1434 amino acids in its primary structure. Targeted disruption of exon-2 of the nNOS-1 gene in mice by homologous recombination has reported a minimal NOS enzyme activity in the brains of the mutant mice (Huang et al., 1993). Two alternatively spliced forms of nNOS, b and g lacked exon-2 and have a regional distribution (Brenman et al., 1996). nNOS b is approximately 136 kDa in size and is prominent in several brain regions of wild type animals viz. ventral cochlear nuclei and lateral tegmental nuclei. It contains exon 2 deletion and splicing with exon la and has some catalytic activity. nNOS-g (125 kDa) contains exon-2 deletion and splicing with exon-lb.. Since PDZ domian is absent in these two variants, they are found in soluble form in the cytosol. A natural alternatively spliced variant with a deletion of 105 amino acids between the haem and calmodulin binding site in its primary sequence, denoted as nNOS-2 (144 kDa) has also been described (Iwasaki et al., 1999). It lacks catalytic function and is expressed specifically and either constitutively or stage and tissue specifically in mouse, rat, and human central nervous system and Drosophila head cells (Ogura et al., 1993, Regulski and Tully 1995). Another catalytically active tissue isoform of nNOS, nNOS-m (165 kDa) has been located in skeletal muscle. It contains 102-base pair (34-amino acid) alternatively spliced segment between the two FMN binding sites in exons 16 and 17 (Silvagno et al., 1996). Similarly, a novel, testis specific nNOS, TnNOS mRNA encoding an N-terminal truncation with 1098 amino acids has been isolated. TnNOS has been found distributed in both cytosol and particulate fractions in cell. It also possesses the catalytic activity of nNOS-1. Detailed studies on other alternative spliced nNOS forms and their expressional regulation may lend more insight; to derive significance from these findings and aid in better approaches to inhibit nNOS.

iNOS: It is found in various cells like alveolar macrophages, hepatocytes, microglia, astrocytes and several other tissues. Recently iNOS has also been localized to neuronal cells (Minc-Golomb et al., 1994, Heneka and Feinsfen 2001). iNOS gene is present on chromosome 17 and encodes a protein of 131 kDa. It contains 26 exons and 25 introns; with the transcription initiation and termination sites in exons 2 and 26 respectively (Chartrain et al., 1994). Binding sites for calmodulin, FMN, FAD and NADPH are encoded within distinct exons. The human iNOS gene also has consensus sequences for several transcription factors including NF-kB binding. Several sites of alternative splicing have been identified in iNOS gene (Eissa et al., 1996). Exon 5 deletion results in a truncated protein and the mRNA transcript is found abundant in cerebellum. Alternate spliced variant with cassette deletion of exons 8 and 9, denoted as iNOS 8–9-results in an in-frame deletion of 242–335 amino acids in the haem domain (Eissa et al., 1998). Cassette deletions of exons 9, 10 and 11; and exons 15 and 16 have also identified. Exon 14 deletions have also been reported in lymphocytic leukemia ? cells (Tiscomia et al., 2004). These result in iNOS proteins with in-frame deletions, but their functional relevance is to be studied.

iNOS and high NO levels – altered regulation in PD

Post mortem samples of PD showed cells in substantia nigra pars compacta expressed considerable amounts of iNOS (Hunot et al., 1996). Intense reactive gliosis expressing iNOS was also seen in the substantia nigra of PD brain and in experimental models (Chao et al., 1992). The cellular conditions contributing to the production of high levels of NO by iNOS are poorly understood.

iNOS activation requires increased cellular uptake of L-Arginine. L-Arginine is transported into brain cells by cationic amino acid transporters (CAT), predominantly by System y + which is encoded by CAT genes, cat-1 and cat-2. Their cDNA clones are denoted as CAT-1, CAT-2, CAT-2A and CAT-2B. In a related experiment, it has been shown that rat hepatocytes stimulated with LPS expressed decreased CAT-2 and CAT-2B mRNA and increased iNOS mRNA (Yang et al., 2005), and no change in CAT-2A levels. In cultured astroglial cells, LPS/IFN-g induced the expression of transporter, System y + and CAT-2 mRNA (Stevens et al., 1996). Further, in rat alveolar macrophages, it has been shown that NF-kB is essential for induction of iNOS and up-regulation of CAT-2B transporter (Hammermann et al., 2000). Production of NO requires availability of arginine. Citrulline which is formed as a by-product in the biosynthesis of NOS is involved in an argininosuccinate pathway to regenerate arginine. Argininosuccinate synthetase (AS) and argininosuccinate lyase (AL) are the two enzymes associated with it. Along with iNOS and CAT-2, AS mRNA is also reported to be induced by LPS/IFN-g stimulation of murine microglial cells (Kawahara et al., 2001). Similarly, Arginase was found to reduce NO production by depleting intracellular arginine concentrations (Mori and Gotoh, 2000).

NF-kB is a transcription factor which is present as a heterotrimer composed of p50, p65 (Rel A) and a It has been shown to be activated by elimination of the regulatory subunit IkB-a from the heterotrimer. This occurs due to phosphorylation of serines 32 and 36 of IkB-a, followed by polyubiquitination and 26S proteasomal degradation IkB-a. This activation of NF-kB is essential for iNOS gene expression and NO production (Nomura, 2001). While NF-kB inhibitors have shown to decrease iNOS expression, they also stabilized CAT transporter mRNAs in rat hepatocytes (Yang et al., 2005). Recent studies have suggested a novel mechanism of inhibition of nNOS by LPS and cytokines via arachidonic acid, which is extensively released during inflammation, promotes tyrosine phosphorylation of nNOS by tyrosine kinase and activation of NF-kB, which leads to iNOS expression (Palomba et al., 2004, Won et al, 2005). Interestingly, it was also demonstrated that LPS/IFN-g were potent activators of nNOS. This presents a specific pathway for the induction and suppression of the NOS isoforms in glial cells. Cytokines and endotoxins probably activate both iNOS and nNOS expression. But through activation of NF- kB and arachidonic acid, they induce iNOS while inhibiting nNOS. NO from nNOS also inhibits NF- kB and regulates the expression of iNOS.

NOS Polymorphisms and implication in PD

Various genetic polymorphisms have been reported in the nNOS gene and iNOS. The functional significance of these polymorphisms is yet to be fully understood. Three intragenic microsatellites have been found in the 5′ flanking region, intron 2 and exon 29 (Hall et al., 1994). Allelic diversity of nNOS gene have also been reported. Frequencies of aCAdinucleotide repeat in exon 29 and an intronic AAT repeat was significantly different between two different ethnic populations (Grasemann et al., 1999). A short tandem repeat located in the promoter region of iNOS gene has been reported to exhibit polymorphisms and allelic differences (Lu et al., 2002). Since alleles of a gene that differs in number of dinucleotide repeats may express differences in promoter activity or manifest as a disease, association of such polymorphisms with diseases have been investigated. Significantly small allelic size distribution was reported in Chinese PD patients as compared to normal subjects in the polymorphic regions of nNOS gene (Lo et al., 2002). A repeat polymorphism in the promoter region of iNOS gene has been associated with Dementia with Lewy bodies (Xu et al., 2000). Hence, such a possibility in PD could exist. Polymorphisms in exons 29 and 18 of nNOS gene and exon 22 of iNOS gene have been genotyped in a community based case-control study of PD (Levecque et al., 2003). Association between PD and the polymorphisms in iNOS exon 22 and nNOS exon 29 was found to be significant. Since exon 29 is located on the 5′ untranslated region of nNOS which regulates transcription and mRNA stability, such polymorphisms can possibly affect the protein expression. Thus, nNOS and iNOS appear to play an important role in susceptibility to PD.

NOS Inhibitors and therapeutic potential

Experimental and clinical findings have indicated an association of iNOS induction with neurodegeneration, thus suggesting iNOS inhibition to be a possible therapeutic target for PD treatment. ). L-Arginine analogues, NG-monomethyl-L-arginine (L-NMMA), NG-nitro-L-arginine (L-NA), NG-nitro-L-arginine methyl ester (L-NAME) are potent NOS inhibitors. L-NMMA acts as a substrate for NOS and gets metabolized, causing irreversible inactivation of the enzyme (Feldman et al, 1993). These compounds do not differentiate between the NOS isoforms and inhibited all of them (Olken et al., 1994). L-NAME and L-NA have been used in PD models and shown protection against the neurodegeneration (Barthwal et al., 2001, Singh et al., 2005). 7-Nitroindazole (7-N1), an equipotent inhibitor of all three NOS isoforms, has also been effective against l-methyl-4-phenyl-l,2,3,6-tetrahydropyridine (MPTP) induced nigral cell loss in mice and baboons (Babbedge et al., 1993, Mayer et al., 1994, Schulz et al., 1995, Hantraye et al., 1996). Other agents capable of reducing NO production are H4B synthesis inhibitors, agents interfering with the haem moiety, calmodulin inhibitors, NO scavengers etc (Hobbs et al., 1999). Since iNOS is upregulated in PD and appears to mediate the neuronal damage, specificity to the particular isoform is necessary in designing iNOS inhibitors. Non-specific NOS inhibitors can cause side effects because they inhibit both the pathogenic NOS (usually the inducible, high output iNOS) as well as the basal production from other NOS isoforms. Inhibitors to be used for PD, must reduce excessive NO production in the nigral region, but allow maintenance of the physiological basal levels to maintain cardiovascular homeostasis. Selective iNOS inhibitors might prove a potential avenue for developing neuroprotective agents, though their action on location specific isoforms and pharmacological effectiveness need to be evaluated.

Conclusion

NO is implicated in PD and NOS has a definite role in the generating high NO concentrations which exacerbate the process of neurodegenerátion. Understanding the regulatory mechanisms that govern NOS expression and activity will help answer many of the questions surrounding the neuronal death seen in PD and other degenerative disorders. By elucidating the role of each NOS isoform in the pathogenesis of PD, NOS inhibitors could be developed as therapeutic agents .targeting at the prevention of neuronal death. However, specificity to a particular isoform of NOS is essential, so that the normal physiological function of NOS is not disrupted.

http://annalsofneurosciences.org/journal/index.php/annal/article/viewArticle/117/487