Tiziana Persichini

Induction of nitric oxide synthase mRNA expression. Suppression by exogenous nitric oxide.

The reactive nitrogen species, nitric oxide (NO), plays an important role in the pathogenesis of neurodegenerative diseases. The suppression of NO production may be fundamental for survival of neurons. Here, we report that pretreatment of human ramified microglial cells with nearly physiological levels of exogenous NO prevents lipopolysaccharide (LPS)/tumor necrosis factor α (TNFα)-inducible NO synthesis, because by affecting NF-κB activation it inhibits inducible Ca-independent NO synthase isoform (iNOS) mRNA expression. Using reverse transcriptase polymerase chain reaction, we have found that both NO donor sodium nitroprusside (SNP) and authentic NO solution are able to inhibit LPS/TNFα-inducible iNOS gene expression; this effect was reversed by reduced hemoglobin, a trapping agent for NO. The early presence of SNP during LPS/TNFα induction is essential for inhibition of iNOS mRNA expression. Furthermore, SNP is capable of inhibiting LPS/TNFα-inducible nitrite release, as determined by Griess reaction. Finally, using electrophoretic mobility shift assay, we have shown that SNP inhibits LPS/TNFα-elicited NF-κB activation. This suggests that inhibition of iNOS gene expression by exogenous NO may be ascribed to a decreased NF-κB availability.

Nitric oxide mediates anti‐inflammatory action of extracorporeal shock waves

Here, we show that extracorporeal shock waves (ESW), at a low energy density value, quickly increase neuronal nitric oxide synthase (nNOS) activity and basal nitric oxide (NO) production in the rat glioma cell line C6. In addition, the treatment of C6 cells with ESW reverts the decrease of nNOS activity and NO production induced by a mixture of lipopolysaccharides (LPS), interferon‐γ (IFN‐γ) plus tumour necrosis factor‐α (TNF‐α). Finally, ESW treatment efficiently downregulates NF‐κB activation and NF‐κB‐dependent gene expression, including inducible NOS and TNF‐α. The present report suggests a possible molecular mechanism of the anti‐inflammatory action of ESW treatment.

Nitric oxide: an inhibitor of NF-κB/Rel system in glial cells

Abstract Nitric oxide (NO) has been reported to regulate NF-κB, one of the best-characterized transcription factors playing important roles in many cellular responses to a large variety of stimuli. NO has been suggested to induce or inhibit the activation of NF-κB, its effect depending, among others, on the cell type considered. In this review, the inhibitory effect of NO on NF-κB (and subsequent suppression of NF-κB-dependent gene expression) in glial cells is reported. In particular, exogenous and endogenous NO has been observed to keep NF-κB suppressed, thus preventing the expression of NF-κB-induced genes, such as inducible NO synthase itself or HIV-1 long terminal repeat. Furthermore, the possible molecular mechanisms of NO-mediated NF-κB inhibition are discussed. More specifically, NO has been reported to suppress NF-κB activation inducing and stabilizing the NF-κB inhibitor, IκB-α. On the other hand, NO may inhibit NF-κB DNA binding through S-nitrosylation of cysteine residue (i.e., Cys62) of the p50 subunit. As a whole, a novel concept that the balance of intracellular NO levels may control the induction of NF-κB in glial cells has been hypothesized.

Mitochondrial type I nitric oxide synthase physically interacts with cytochrome c oxidase

Nitric oxide (NO) regulates key aspects of cell metabolism through reversible inhibition of cytochrome c oxidase (CcOX), the terminal electron acceptor (complex IV) of the mitochondrial respiratory chain, in competition with oxygen. Recently, a constitutive mitochondrial NOS corresponding to a neuronal NOS-I isoform (mtNOS-I) has been identified in several tissues. The role of this enzyme might be to generate NO close enough to its target without a significant overall increase in cellular NO concentrations. An effective, selective, and specific NO action might be guaranteed further by a physical interaction between mtNOS-I and CcOX. This possibility has never been investigated. Here we demonstrate that mtNOS-I is associated with CcOX, as proven by electron microscopic immunolocalization and co-immunoprecipitation studies. By affinity chromatography, we found that association is due to physical interaction of mtNOS-I with the C-terminal peptide of the Va subunit of CcOX, which displays a consensus sequence for binding to the PDZ domain of mtNOS-I previously unreported for CcOX. The molecular details of the interaction have been analyzed by means of molecular modeling and molecular dynamics simulations. This is the first evidence of a protein-protein interaction mediated by PDZ domains involving CcOX.

Human ramified microglial cells produce nitric oxide upon Escherichia coli lipopolysaccharide and tumor necrosis factor α stimulation

This study shows that human ramified microglial cells derived from fetal brain primary cultures, are able to produce nitric oxide (NO). In fact, stimulation with Escherichia coli lipopolysaccharide (LPS) (1 microgram ml-1) or tumor necrosis factor alpha (TNF alpha) (500 U ml-1) enhances nitrite release in cell supernatants, as determined by the Griess reaction. A synergistic effect is achieved following treatment with LPS plus TNF alpha, this effect being inhibited by pretreating cells with NOS inhibitor N omega-nitro-L-arginine methyl ester (L-NAME). Using reverse transcriptase-polymerase chain reaction (RT-PCR) and Southern blot analysis, we also found that LPS/TNF alpha produce an increase of inducible NO synthase (iNOS) mRNA expression.

S-nitrosylation of viral proteins: Molecular bases for antiviral effect of nitric oxide

Nitric oxide (NO) is considered an important signaling molecule implied in various different physiological processes, including nervous transmission, vascular regulation, and immune defence, as well as the pathogenesis of several diseases. NO reportedly also has an antiviral effect on several DNA and RNA virus families. The NO‐mediated S‐nitrosylation of viral and host (macro)molecules appears to be an intriguing general mechanism for the control of the virus life cycle. In this respect, NO is able to nitrosylate cysteine‐containing enzymes (e.g., proteases, reverse transcriptase, and ribonucleotide reductase). Moreover, zinc‐fingers and related domains present in enzymes (e.g., HIV‐1 encoded integrase or herpes simplex virus type‐1 heterotrimeric helicase primase complex) or nucleocapsid proteins may beconsidered as NO targets. Also, NO may regulate both host (e.g., nuclear factor‐kappaB) and viral‐encoded (e.g., HIV‐1 tat protein or Epstein‐Barr virus Zta) transcriptional factors that are involved in virus replication. Finally, NO‐mediated S‐nitrosylation of cysteine‐containing glycoproteins and hemagglutinin may also occur. Here, NO targets are summarised, and the molecular bases for the antiviral effect of NO are discussed.

Bacterial Lipopolysaccharide Plus Interferon-γ Elicit a Very Fast Inhibition of a Ca2+-dependent Nitric-oxide Synthase Activity in Human Astrocytoma Cells

Previous results indicate that induction of inducible nitric-oxide synthase (iNOS) expression may be kept suppressed by the endogenous NO level as produced by a constitutive NOS (cNOS) enzyme. In cell types possessing both cNOS and iNOS, this may represent an evident paradox. Here, we report that lipopolysaccharide and interferon-γ, which are able to strongly induce iNOS in astrocytoma cells, can rapidly inhibit the NO production generated by the constitutive NOS isoform, thus obtaining the best conditions for iNOS induction and resolving the apparent paradox. In fact, a 30-min treatment of T67 cells with the combination of lipopolysaccharide plus interferon-γ (MIX) strongly inhibits the cNOS activity, as determined by measuring [3H]citrulline production. In addition, the effect of MIX is also observed by measuring nitrite, the stable breakdown product of NO: a 30-min pretreatment of T67 cells with MIX is able to reduce significantly the N-methyl-D-aspartate-induced nitrite production. Finally, using reverse transcriptase-polymerase chain reaction, we have observed that a 30-min treatment of T67 cells with MIX does not affect expression of mRNA coding for the neuronal NOS-I isoform. These results suggest the novel concept of a possible role of a cNOS isoform in astrocytes as a control function on iNOS induction.

Beta-amyloid inhibits NOS activity by subtracting NADPH availability.

The amyloid peptides Aβ1–42 and Aβ25–35 strongly inhibited the activity of constitutive neuronal and endothelial nitric oxide synthases (i.e., NOS‐I and NOS‐III, respectively) in cell‐free assays. The molecular mechanism of NOS inhibition by Aβ fragments was studied in detail with Aβ25–35. The inhibitory ability was mostly NADPH‐dependent and specific for the soluble form of Aβ25–35. Optical, fluorescence, and NMR spectroscopy showed that the soluble, but not aggregated, Aβ25–35 interacted with NADPH, thus suggesting that a direct recruitment of NADPH may result in diminished availability of the redox cofactor for NOS functioning. To assess the physiological relevance of our findings, rat neuronal‐like PC12 and glioma C6 cell lines were used as cellular models. After Aβ25–35 internalization into cells was verified, the activity of constitutive NOS was measured using the DAF‐2DA detection system and found to be severely impaired upon Aβ25–35 uptake. Consistent with previous results on the molecular cross‐talk between NOS isoforms, repression of constitutive NOS by Aβ25–35 resulted in enhanced expression of inducible NOS (NOS‐II) mRNA in C6 cells. Our results represent the first evidence that amyloid fragments impair constitutive NOS activity in cell‐free and cellular systems, providing a possible molecular mechanism for the onset and/or maintenance of Alzheimers disease.

Interleukin-1β up-regulates iron efflux in rat C6 glioma cells through modulation of ceruloplasmin and ferroportin-1 synthesis

A number of pathologies, including neurodegeneration and inflammation, have been associated with iron dysmetabolism in the brain. Hence, systems involved in iron homeostasis at the cellular level have aroused considerable interest in recent years. The iron exporter ferroportin-1 (FP) and the multicopper oxidase ceruloplasmin (CP) are essential for iron efflux from cells. By using RT-PCR, we demonstrate that FP and CP gene expression is up-regulated by treatment with the pro-inflammatory cytokine IL-1beta in rat C6 cells, taken as a glial cellular model. Following stimulation with IL-1beta, a higher expression level of CP and FP was also confirmed by Western blotting. Moreover, IL-1beta has been found to increase iron efflux from C6 cells, suggesting that both proteins may play a crucial role in iron homeostasis in pathological brain conditions, such as inflammatory and/or neurodegenerative diseases.

Dominant Mutants of Ceruloplasmin Impair the Copper Loading Machinery in Aceruloplasminemia

The multicopper oxidase ceruloplasmin plays a key role in iron homeostasis, and its ferroxidase activity is required to stabilize cell surface ferroportin, the only known mammalian iron exporter. Missense mutations causing the rare autosomal neurodegenerative disease aceruloplasminemia were investigated by testing their ability to prevent ferroportin degradation in rat glioma C6 cells silenced for endogenous ceruloplasmin. Most of the mutants did not complement (i.e. did not stabilize ferroportin) because of the irreversible loss of copper binding ability. Mutant R701W, which was found in a heterozygous very young patient with severe neurological problems, was unable to complement per se but did so in the presence of copper-glutathione or when the yeast copper ATPase Ccc2p was co-expressed, indicating that the protein was structurally able to bind copper but that metal loading involving the mammalian copper ATPase ATP7B was impaired. Notably, R701W exerted a dominant negative effect on wild type, and it induced the subcellular relocalization of ATP7B. Our results constitute the first evidence of “functional silencing” of ATP7B as a novel molecular defect in aceruloplasminemia. The possibility to reverse the deleterious effects of some aceruloplasminemia mutations may disclose new possible therapeutic strategies.