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ß-Amyloid inhibits NOS activity by subtracting NADPH availability¹

GIORGIO VENTURINI, MARCO COLASANTI, TIZIANA PERSICHINI, EMANUELA FIORAVANTI, PAOLO ASCENZI, LETIZIA PALOMBA^*, ORAZIO CANTONI^* and GIOVANNI MUSCI²

Department of Biology, University "Roma Tre", Rome, Italy;
^* Institute of Pharmacology and Pharmacognosy, University of Urbino, Urbino, Italy; and
Department of Microbiological, Genetic and Molecular Sciences, University of Messina, Messina, Italy

²Correspondence: Department of Microbiological, Genetic and Molecular Sciences, University of Messina, Salita Sperone, 31, 98166 Messina, Italy. E-mail: musci{at}unime.it

SPECIFIC AIM

The experiments reported here aimed to define the effect of ß-amyloid peptides (Aß) on the enzymatic activity of nitric oxide synthase (NOS), with emphasis on constitutive (i.e., endothelial and neuronal) NOS (cNOS) in both cell-free and cellular model systems. Another goal was to distinguish between the effect of the soluble form of Aß vs. the aggregated derivative, since the two forms may play different roles in the onset and maintenance of Alzheimer’s disease (AD).

PRINCIPAL FINDINGS

1. Aß1–42 and Aß25–35 inhibit the activity of cNOS
We first analyzed the effect of either Aß1–42 or Aß25–35 on the catalytic activity of neuronal NOS-I and endothelial NOS-III by measuring the conversion of [³H]arginine to [³H]citrulline in vitro. The activity of NOS-I and NOS-III was significantly inhibited by 50 µM Aß1–42 or Aß25–35. Inhibition of cNOS by Aß was studied in greater detail with the shorter Aß25–35 peptide. The inhibitory action was peculiar to the soluble form of Aß25–35, whereas the aggregated form of the peptide at the same concentration was totally ineffective (Fig. 1 A). The inverted Aß35–25 peptide was ineffective (Fig. 1A ), proving that the inhibition of NOS activity was specifically elicited by the Aß25–35 amino acid sequence derived from the amyloid precursor protein.

View larger version (21K): [in this window] [in a new window]   Figure 1. Effect of Aß25–35 on enzymatic activity of cNOS. A) The effect of 200 µM soluble, aggregated, or inverted Aß on the activity of NOS-I and NOS-III. Effect of NADPH (10–1000 µM) on the extent of inhibition of NOS-I (B) and NOS-III (C) activity by increasing concentrations (0–600 µM) of Aß25–35. Bars represent the mean ± SE from 3 separate experiments. One-way ANOVA followed by Bonferroni’s test was used to determine significant differences: *P < 0.001 vs. relative control.

2. Inhibition of NOS by Aß is NADPH dependent
The inhibitory effect of the peptides could be relieved by increasing the concentration of NADPH in the assay mixture, suggesting an action for Aß either in directly recruiting NADPH or interacting with the NADPH-binding site on NOS. On the other hand, addition of excess arginine, FAD, FMN, or Ca²⁺-calmodulin could not reverse the inhibition by Aß. The compensatory effect of NADPH was better specified with Aß25–35, as shown in Fig. 1B, C for NOS-I and NOS-III, respectively. The inhibition of NOS by Aß25–35 was progressively relieved by increasing concentrations of NADPH. As expected, the effect of Aß25–35 was not limited to constitutive NOSs, inducible NOS-II being inhibited in a similar pattern of NADPH dependence.

3. Aß inhibits other NADPH-dependent enzymes
A further enzymatic assay helped to define the simple mechanism of NADPH recruitment by Aß. This was achieved by testing another NADPH-dependent enzyme, namely, microsomal cytochrome c reductase. In line with the results obtained on NOS, Aß25–35 significantly inhibited the enzymatic reduction of cytochrome c, the inhibition being dependent on NADPH concentration.

4. Spectroscopic evidence of the interaction of Aß with NADPH
Absorption and fluorescence spectroscopies were used to find evidence of a molecular interaction of NADPH with either Aß1–42 or Aß25–35. The optical and fluorescence spectra of NADPH were both greatly affected by the soluble amyloids. Interaction of Aß25–35 with NADPH was further proved by ¹H-NMR spectroscopy, which disclosed significant changes of the proton NMR spectrum line shape of NADPH in the presence of Aß25–35. The signal at -0.199 ppm (relative to dioxane) shifted to -0.123 ppm and -0.108 ppm after addition of 0.5 and 0.75 equivalents of Aß25–35, respectively.

5. Intracellular accumulation of Aß25–35 leads to impaired cNOS activity
The physiopathological relevance of the findings reported above was assessed by testing whether Aß25–35 could impair the enzymatic activity of cNOS directly in the cells. First, we sought to verify whether Aß25–35, which is known to enter different cell types, did so under our experimental conditions. Neuronal-like PC12 and glial-derived C6 cell lines were chosen as models of possible cellular targets of Aß peptides in the central nervous system (CNS). Aß25–35 dose-dependently accumulated in both cell lines, the uptake being statistically significant (P<0.01 vs. untreated cells) at Aß25–35 concentration in the medium as low as 5 µM. An optimal uptake was attained at 15 µM Aß25–35 (P<0.001 vs. untreated cells). Second, the effect of intracellularly accumulated soluble Aß25–35 on NO production from cNOS was measured in PC12 and C6 cells, using the DAF-2DA detection system. As shown in Fig. 2 A, a basal level of DAF fluorescence was observed in unstimulated cells, which was not abolished by the NOS inhibitor L-NAME (1 mM), suggesting that it derived from arginine-independent metabolism(s). This background level (15 and 17 arbitrary units for PC12 and C6, respectively) can somewhat hide the small amount of NO produced by the cNOS enzyme in control cells. To enhance cNOS activity, cells were therefore treated with either the calcium ionophore A23187, which induces calcium influx prevalently from the extracellular milieu, or Tg, which specifically promotes calcium mobilization from intracellular stores. When cells were treated with 2.5 µM A23187 or 100 nM Tg, DAF fluorescence was strongly enhanced above the background level in PC12 and C6 cells (Fig. 2A ). As expected, the increase was abolished by treating cells for 5 min with 1 mM L-NAME before the addition of A23187 or Tg (Fig. 2A ), indicating that the Ca²⁺-dependent increase of DAF fluorescence was consequent to activation of the L-arginine-NO pathway. Figure 2B shows that Aß25–35 (5÷50 µM) dose-dependently inhibited the ionophore-induced activation of cNOS in PC12 and C6 cells. The effect was already statistically significant (P<0.01 vs. untreated cells) at 5 µM Aß25–35 and reached its maximum at 15–25 µM (P<0.001 vs. untreated cells). Consistently, 15 µM Aß25–35 inhibited Tg-dependent cNOS activation (Fig. 2B , inset), proving that the inhibitory effect of the peptide did not depend on the mechanism of intracellular calcium rise.

View larger version (31K): [in this window] [in a new window]   Figure 2. Effect of Aß25–35 on the A23187- or Tg-induced DAF-fluorescence response in PC12 or C6 glioma cells. A) DAF fluorescence of control cells and of cells treated with 2.5 µM A23187, or 100 nM Tg. Also shown is the effect of 1 mM L-NAME preincubated with cells for 5 min. B) DAF fluorescence of PC12 and C6 cells treated with 2.5 µM A23187 in the presence of increasing concentrations of Aß25–35. The inset shows the effect of 15 µM Aß25–35 on the DAF-fluorescence response evoked by 100 nM Tg. Data are expressed as percent of maximum DAF fluorescence. Bars represent the mean ± SE from 3–5 separate experiments, each performed in duplicate. °P < 0.001 vs. control (A); *P < 0.01 and #P < 0.001 vs. cNOS agonist-treated cells (B).

6. Soluble Aß25–35 modulates NOS-II mRNA expression in glial cells
To address whether depression of constitutive levels of NO by Aß25–35 resulted in transcriptional hyperactivation of inducible NOS-II, rat C6 glioma cells were used as a model of cells expressing constitutive and inducible NOS isoforms. Soluble Aß25–35 (40 µM) was found to enhance NOS-II mRNA expression in C6 cells suboptimally induced for 4 h with a mixture of LPS (1 µg/mL) plus IFN- (100 U/mL). Restoring the basal levels of NO by pretreating cells for 30 min with 10 µM sodium nitroprusside (SNP), an established NO donor, prevented NOS-II hyperinduction by soluble Aß25–35.

CONCLUSIONS AND SIGNIFICANCE

The amyloid peptides Aß1–42 and Aß25–35 (collectively referred to as Aß) are inhibitors of NOS activity, an effect observed in vitro and in vivo systems. Intracellular accumulation of soluble Aß25–35 has been found to impair cNOS functioning in PC12 and C6 lines, two cell types representing possible models of cellular targets for Aß in the CNS.

The phenomena reflect the strong inhibition of cNOS activity by soluble, but not aggregated, Aß observed in cell-free enzymatic assays. The primary mechanism seems to be the efficient recruitment of NADPH by the soluble (but not the aggregated) peptide, as shown by spectroscopic and competition kinetics data.

Results of the in vitro experiments match those obtained in in vivo systems. The discovery that soluble, intracellular Aß strongly inhibits cNOS activity in vivo may shed some light on the possible mechanisms underlying the onset of AD when considering the following. 1) Basal NO levels produced by cNOS have been hypothesized to be involved in neuro- and vascular protection. Evidence suggests that soluble Aß can induce dysfunction in perfused rat cerebral vessels and cultured endothelial cells through inhibition of NO release from endothelia, a finding in line with the hypothesis that AD may have a vascular (rather than neuronal) origin. 2) At physiological basal levels (e.g., either derived from NO donors or produced by cNOS), NO may have a protective role in that it can prevent NOS-II-dependent NO synthesis through suppression of the transcriptional factor NF-B and consequent inhibition of NOS-II mRNA expression. Our findings are compatible with the concept that NOS-II gene overexpression induced by Aß25–35 in C6 cells may be attributable to a sharp decline in constitutive NO, this effect reflecting the removal of the inhibitory action of physiological NO levels produced by NOS-I on NOS-II transcription. The reversibility of Aß25–35-induced NOS-II overexpression by the NO donor SNP confirms this picture. 3) Intraneuronal Aß accumulation has been shown to precede the appearance of typical hallmarks of AD (i.e., Aß secretion, neurofibrillary tangle, and Aß plaque deposition), suggesting that intracellular buildup of Aß represents an early event in the pathogenesis of AD. Neuritic plaques in AD are surrounded by reactive astrocytes and microglia, in which an early intracellular accumulation of Aß can occur. Aß accumulation in reactive astrocytes has been found to depend on the distribution of APP into intracellular compartments, the cell surface APP being only accessible for nonamyloidogenic cleavage. Note that internalization of extracellular amyloid peptides can take place. Our results consistently show that PC12 and C6 cells are able to internalize soluble Aß25–35, as reported elsewhere.

It is tempting to speculate that the inhibition of cNOS activity taking place in the presence of a soluble amyloid fragment may reflect an early event in the onset of AD, since APP can be proteolytically processed in some cell types, such as glia and endothelia, beside neurons. It could be envisaged that the accumulation of intracellular amyloid peptides in cells expressing cNOS isoforms (e.g., glia, endothelia, and neurons) decreases basal NO level beneath a putative threshold value that maintains an inhibitory state. The lack of an adequate basal NO would then facilitate an activation state, thus contributing very early to promote AD.

View larger version (25K): [in this window] [in a new window]   Figure 3. Schematic diagram of the putative role of Aß in the onset of AD. Under normal conditions (left), physiological levels of NO produced by constitutive endothelial and neuronal forms of NOS (NOS-I and NOS-III) elicit a positive role in neurovascular protection. A chronic drop in basal NO levels induced by soluble Aß would lead to activation of proinflammatory pathways and consequent neurovascular damage (right).

FOOTNOTES

¹ To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.02-0186fje; to cite this article, use FASEB J. (October 18, 2002) 10.1096/fj.02-0186fje

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