The effect of selective inhibition of cyclic GMP hydrolyzing phosphodiesterases 2 and 5 on learning and memory processes and nitric oxide synthase activity in brain during aging.

Our previous studies have shown that there is a lower cGMP concentration in the aged brain as well as an alteration in the activity of cGMP-hydrolyzing phosphodiesterases (PDEs) and nitric oxide synthase (NOS). The aim of this study was to investigate the effect of specific inhibitors of selected PDEs on object recognition memory and locomotor activity during aging, and to correlate their action with NOS activity in the following brain regions: hippocampus, striatum, and cerebral cortex. The study was carried out using 3, 12, and 24 month-old rats. Inhibitors of PDE2 and PDE5 (Bayer 60-7550 and zaprinast, respectively) were used. Evaluation of memory and locomotor activity was carried out using an object recognition task and the open field test. NOS activity was determined using a radiochemical method after behavioral analysis in the cytosolic fraction from all brain areas investigated. We have found that the inhibitor of PDE2, Bay60-7550, improves object recognition memory in all age groups investigated and increases basal constitutive NOS activity in the hippocampus and striatum. Moreover, in 3 month-old rats, additional inhibition of PDE5 by zaprinast improves object memory and elevates NOS activity in all brain regions studied. Specific inhibition of nNOS eliminates the effect of Bay60-7550 on memory function and on NOS activity in 24 month-old rats. In summary, our results indicate that inhibition of PDE2 is able to improve cognition and memory function in 3, 12, and 24 month-old rats through the enhancement of nNOS activity in the brain, whereas inhibition of PDE5 is effective only in 3 month-old animals.

Alpha-synuclein alters differently gene expression of Sirts, PARPs and other stress response proteins: implications for neurodegenerative disorders.

Alpha-synuclein (ASN) is a presynaptic protein that can easily change its conformation under different types of stress. It's assumed that ASN plays an important role in the pathogenesis of Parkinson's and Alzheimer's disease. However, the molecular mechanism of ASN toxicity has not been elucidated. This study focused on the role of extracellular ASN (eASN) in regulation of transcription of sirtuins (Sirts) and DNA-bound poly(ADP-ribose) polymerases (PARPs) - proteins crucial for cells' survival/death. Our results indicate that eASN enhanced the free radicals level, decreased mitochondria membrane potential, cells viability and activated cells' death. Concomitantly eASN activated expression of antioxidative proteins (Sod2, Gpx4, Gadd45b) and DNA-bound Parp2 and Parp3. Moreover, eASN upregulated expression of Sirt3 and Sirt5, but downregulated of Sirt1, which plays an important role in cell metabolism including Aß precursor protein (APP) processing. eASN downregulated gene expression of APP alpha secretase (Adam10) and metalloproteinases Mmp2, Mmp10 but upregulated Mmp11. Additionally, expression and activity of pro-survival sphingosine kinase 1 (Sphk1), Akt kinase and anti-apoptotic protein Bcl2 were inhibited. Moreover, higher expression of pro-apoptotic protein Bax and enhancement of apoptotic cells' death were observed. Summarizing, eASN significantly modulates transcription of Sirts and enzymes involved in APP/Aß metabolism and through these mechanisms eASN toxicity may be enhanced. The inhibition of Sphk1 and Akt by eASN may lead to disturbances of survival pathways. These results suggest that eASN through alteration of transcription and by inhibition of pro-survival kinases may play important pathogenic role in neurodegenerative disorders.

Nitric oxide alters arachidonic acid turnover in brain cortex synaptoneurosomes.

Nitric oxide (NO) and arachidonic acid (AA) and also its metabolites are very important inter- and intracellular second messengers. They are involved in mechanisms of learning and memory. However, liberated in excessive amount in brain ischemia, Parkinson and Alzheimer diseases they are responsible for cell degeneration and death. Previously, we could show that Alzheimer disease's amyloid-beta protein enhanced nitric oxide liberation. The role of NO in AA metabolism is till now not well understood. Therefore, the aim of the present study was to investigate the mechanisms of NO-evoked activation of AA release and inhibition of AA incorporation into phospholipids of cortical rat brain synaptoneurosomes. The studies were carried out using NO donors, butyryl-cGMP (b-cGMP) and H2O2. All these compounds enhanced AA liberation from phosphatydilinositol (PI) and phosphatidylcholine (PC). Protein kinase ERK1/2, protein kinase C (PKC), cGMP-dependent protein kinase G (PKG) were involved in basal and NO-induced cytosolic phospholipase A2 (cPLA2) activation. Moreover, NO donors, b-cGMP and hydrogen peroxide (H2O2) exerted inhibitory effect on AA incorporation into PI and PC influencing arachidonyl-CoA transferase (AA-CoA-T) activity. AA-CoA synthase (AA-CoA-S) activity did not change. Specific inhibitors of protein kinase ERK1/2 (UO126), PKC (GF109203X), PKG (KT5823) had no effect on NO-mediated lowering of AA incorporation into PI and PC but inhibited the basal AA-CoA-S activity. Our data indicated that AA (10 microM) itself markedly decreased AA incorporation by about 50% into phospholipids of synaptoneurosomes membranes. Increasing release of AA and its metabolites causes the lowering of AA incorporation evoked by NO, b-cGMP and H2O2. Antioxidant, Resveratrol (100 microM) prevented NO- and cGMP-evoked inhibition of AA incorporation. These results suggest that NO affects the intracellular level of AA through alteration of cPLA2 and AA-CoA acyltransferase activities and may have an important implication in alterations of nerve endings properties and function.

Expression of alpha-synuclein in different brain parts of adult and aged rats.

The synucleins are a family of presynaptic proteins that are abundant in neurons and include alpha-, beta, and gamma-synuclein. Alpha-synuclein (ASN) is involved in several neurodegenerative age-related disorders but its relevance in physiological aging is unknown. In the present study we investigated the expression of ASN mRNA and protein in the different brain parts of the adult (4-month-old) and aged (24-month-old) rats by using RT-PCR technique and Western blot, respectively. Our results indicated that mRNA expression and immunoreactivity of ASN is similar in brain cortex, hippocampus and striatum but markedly lower in cerebellum comparing to the other brain parts. Aging lowers ASN mRNA expression in striatum and cerebellum by about 40%. The immunoreactivity of ASN in synaptic plasma membranes (SPM) from aged brain cortex, hippocampus and cerebellum is significantly lower comparing to adult by 39%, 24% and 65%, respectively. Beta-synuclein (BSN) was not changed in aged brain comparing to adult. Age-related alteration of ASN may affect the nerve terminals structure and function.

Differential effect on inositol-phospholipid hydrolysis, cytosolic-free Ca2+ concentration, protein kinase C activity and protein phosphorylation of 1-oleoyl-2-acetyl-sn-glycerol growth-stimulated ascites tumor cells.

This study shows that the membrane-permeable stereospecific 1-oleoyl-2-acetyl-sn-glycerol (OAG), which is the analog of the natural 1,2-diacylglycerol (DAG), can stimulate the growth of ascites tumor cells. OAG can fully replace high serum concentrations in the culture medium and stimulates DNA synthesis in a dose-dependent manner. Investigation of the protein kinase C (PKC) isolated from a Triton extract of a 100,000g membrane pellet revealed that OAG can directly activate this enzyme. Concomitantly the phosphorylation of several cytosolic proteins with the molecular weights of 26, 33, 49, 55, 64, and 90 kDa is observed which is also found in serum-stimulated cells. Since DAG as a second messenger molecule originates from the hydrolysis of phosphoinositides we have investigated the metabolism of these lipids after labeling the cells with [3H]inositol. In detail, we have measured the amount of radioactive inositol trisphosphate (IP3) and the phosphodiesterase hydrolyzing phosphatidylinositol-4,5-bisphosphate (PIP2). The decreased radioactivity level of IP3 in OAG-stimulated cells as compared to non-growing cells (1-2% serum) indicates a feedback regulation of PIP2 hydrolysis which is substantiated by a profound reduction of PIP2-specific phospholipase C activity. The reduced IP3 formation has apparently no inhibitory effect on the cytoplasmic free Ca2+ concentration of OAG-stimulated cells, suggesting that the Ca2+ release is not directly correlated to the amount of IP3, which is also demonstrated for the non-growing cells. These data indicate that OAG apparently has a duel effect on the inositol phospholipid-mediated signal transfer system.(ABSTRACT TRUNCATED AT 250 WORDS)

Amyloid beta peptide 25-35 modulates hydrolysis of phosphoinositides by membrane phospholipase(s) C of adult brain cortex.

Phosphoinositide-specific phospholipase C (PLC) is a key enzyme in signal transduction. A subset of muscarinic cholinergic receptors are linked to G-proteins that activate phospholipase C. Cholinergic pathways are important in learning and memory, and deficits in cholinergic transmission have been implicated in Alzheimer's disease (AD). AD is also associated with increased beta-amyloid plaques. In the present study, we have investigated the effect of the amyloid beta (A beta) synthetic peptide homologous to residue 25-35 of A beta in nonaggregated and aggregated forms on the degradation of inositol phospholipids. Synaptic plasma membranes (SPM) and the cytosolic fraction from rat brain cortex served as a source of enzymes. The studies were carried out with radioactive inositol phospholipids in the presence of endogenous and 2 mM CaCl2. The enzyme(s) activity was evaluated by determination of the product formation of [3H]inositol-1-phosphate (IP1) or [3H]inositol-1,4,5-trisphosphate (IP3). Results show that the PI-PLC activity was significantly higher in cytosol compared to SPM, and this enzyme was stimulated by 2 mM CaCl2, but not by GTPgammaS or carbachol, a cholinergic receptor agonist. Activity of the SPM-bound PIP2-PLC was similar to that in cytosol and was not activated by 2 mM CaCl2. The SPM PIP2-PLC was significantly stimulated by GTPgammaS together with the cholinergic agonist, carbachol. Fresh-water-soluble A beta 25-35 activated PI-PLC in SPM markedly by two- to threefold, but this effect was absent in the presence of 2 mM CaCl2. Moreover, A beta 25-35 had no effect on basal PIP2-PLC activity and cytosolic PI-PLC and PIP2-PLC. The aggregated form of A beta 25-35 significantly inhibited PIP2-PLC only in the presence of endogenous CaCl2. It also inhibited the carbachol and GTP(gamma)S-stimulated PIP2-PLC. Our findings show that depending on the aggregation state and Ca2+ concentration, A beta modulates phosphoinositide degradation differently and exclusively in brain synaptic plasma membranes. Our data suggested that aggregated A beta peptide may be responsible for the significant impairment of phosphoinositide signaling found in brain membranes during AD.

Effect of amyloid beta peptide on poly(ADP-ribose) polymerase activity in adult and aged rat hippocampus.

It is suggested that the fibrillar amyloid beta peptide (A beta) in brain plays a direct role in neurodegeneration in Alzheimer's disease, probably through activation of reactive oxygen species formation. Free radicals and numerous neurotoxins elicit DNA damage that subsequently activates poly(ADP-ribose) polymerase (PARP, EC 2.4.2.30). In this study the effect of neurotoxic fragment (25-35) of full length A beta peptide on PARP activity in adult and aged rat hippocampus was investigated. In adult (4 month old) rat hippocampus the A beta 25-35 peptide significantly enhanced PARP activity by about 80% but had no effect on PARP activity in cerebral cortex and in hippocampus from aged (24-27 month old) rats. The effect of A beta peptide was reduced by half by the nitric oxide synthase inhibitor N-nitro-L-arginine. Stimulation of glutamate receptor(s) itself enhanced PARP activity by about 80% in adult hippocampus. However, A beta 25-35 did not exert any additional stimulatory effect. These results indicate that A beta, through NO and probably other free radicals, induces activation of DNA bound PARP activity exclusively in adult but not in aged hippocampus.

Age-related alteration of poly(ADP-ribose) polymerase activity in different parts of the brain.

Poly(ADP-ribose) polymerase (PARP) is a conserved enzyme involved in the regulation of DNA repair and genome stability. The role of PARP during aging is not well known. In this study PARP activity was investigated in nuclear fractions from hippocampus, cerebellum, and cerebral cortex of adult (4 months), old adult (14 months) and aged (24-27 months) rats. Concomitantly, the free radical evoked lipid peroxidation was estimated as thiobarbituric acid reactive substances (TBARS). The specific activity of PARP in adult brain was about 25, 21 and 16 pmol/mg protein per min in hippocampus, cerebellum and cerebral cortex, respectively. The enzyme activity was higher in all investigated parts of the brain of old adults. In aged animals PARP activity was lower in hippocampus by about 50%, and was unchanged in cerebral cortex and in cerebellum comparing to adult rats. The concentration of TBARS was the same in all parts of the brain and remained unchanged during aging. There is no direct correlation between PARP activity and free radical evoked lipid peroxidation during brain aging. The lowered enzyme activity in aged hippocampus may decrease DNA repair capacity which subsequently may be responsible for the higher vulnerability of hippocampal neurons to different toxic insults.

Pergolide mesylate, a dopaminergic receptor agonist, applied with L-DOPA enhances serum antioxidant enzyme activity in Parkinson disease.

The aim of this study was to compare patients with Parkinson disease (PD) patients treated with pergolide mesylate (PM), a dopaminergic receptor agonist, together with L-DOPA and those these treated with L-DOPA alone on the concentration of free radicals (FR), glutathione, and the activity of superoxide dismutase (SOD) and catalase in the serum. The study was carried out using 16 age-matched control subjects, 16 PD patients treated with L-DOPA at a dose of 1 to 1.5 g daily, and 16 PD patients treated with L-DOPA 1 to 1.5 g daily with PM 0.75 to 1.25 mg daily. The mean duration of treatment of PD was 6 years (range, 2-8 years) with l-DOPA, and 2 years with PM + L-DOPA or L-DOPA alone. Although there was no significant difference in lipid peroxidation products among the 3 groups, patients treated with L-DOPA showed high levels of FR as determined by dichlorofluorescein. Although catalase and SOD activities were elevated in both groups of PD patients, additional treatment with PM further enhanced catalase activity compared with those treated with l-DOPA alone. Interestingly, patients treated with PM + L-DOPA showed a significantly increased level of glutathione compared with those treated with L-DOPA alone. Collectively, these data suggest that PM + L-DOPA is a more efficient therapy in maintaining an antioxidative defense in PD patients compared with treatment with L-DOPA alone.

Cyclic GMP metabolism and its role in brain physiology.

Cyclic GMP (cGMP) is synthesized by guanylyl cyclase (GC) in response to nitric oxide (NO) and carbon monoxide (CO) or natiuretic peptides (NPs); atrial, brain and C-type (ANP, BNP and CNP). cGMP is degraded by several cGMP-specific phosphodiesterases (PDEs). Guanylate cyclases (GC) are differentiated into: membrane-bound/particulate (pGC) and cytosolic/soluble (sGC). In recent years evidence has accumulated that NO is the main activator of sGC and NO/cGMP plays important role in glutaminergic, cholinergic and dopaminergic signaling pathways. cGMP in the nervous system is involved in long term potentiation and depression (LTP, LTD) suggesting its participation in learning and memory mechanism. cGMP regulates calcium homeostasis and phototransduction. Its level is regulated by PDEs and their specific inhibitors protect cGMP level in cells and are very important from clinical point of view.

Effect of 1-oleoyl-2-acetyl-sn-glycerol on inositol lipid metabolism of ascites tumor cells in culture.

1-Oleoyl-2-acetyl-sn-glycerol (OAG), the membrane-permeable analogue of 1,2-diacylglycerol (DAG), which stimulates ascites tumor cell proliferation, was used to study its effect on phosphoinositide metabolism. Culturing of ascites cells labeled with [3H]inositol at low serum concentration in the presence of OAG suppressed the radioactivity level of the inositol phosphates, particularly IP3. Membrane-bound, Ca(2+)- and GTP gamma S-sensitive PI- and PIP2-specific phosphodiesterase (phospholipase C) showed much lower activities in OAG-stimulated cells, which could be enhanced by GTP gamma S in these but not in the unstimulated cells. A high susceptibility to Ca2+ of the PI- and PIP2-specific phospholipase C of non-stimulated cells was observed. The PIP-kinase activity was similarly reduced by about 85% in OAG-stimulated cells. These data indicate a negative feedback regulation of the phosphoinositide metabolism mediated by OAG. Reduction in synthesis and degradation of PIP2, which furnishes the two second messengers, DAG and IP3, provides a means of controlling the intracellular level of these molecules, which is important for a balanced proliferation rate.

Metabolism of [14C]arachidonic acid-labeled lipids in quiescent and OAG-stimulated ascites tumor cells.

1. A rapid uptake and esterification of [14C]arachidonic acid during the first 4 hr of cultivation of ascites cells in serum-deprived medium was observed followed by a fast turnover of the fatty acid. 2. Labeling and turnover of esterified arachidonate in individual phospholipid classes was in the order: phosphatidylcholine (PC) greater than phosphatidylinositol (PI) much greater than phosphatidylinositol-4-phosphate (PIP) and -4,5-bisphosphate (PIP2) greater than phosphatidylethanolamine (PE) greater than PE-plasmalogens. 3. In cells stimulated with 1-oleoyl-2-acetyl-sn-glycerol a transient course of arachidonic acid incorporation into PC, PI, PIP and PIP2 was determined peaking 30 min after stimulation, indicating both esterification and release under these conditions. 4. The release of arachidonate was blocked by quinacrine which is a specific inhibitor of phospholipase A2.

Aging modulates nitric oxide synthesis and cGMP levels in hippocampus and cerebellum. Effects of amyloid beta peptide.

The biological roles of nitric oxide (NO) and cGMP as inter- and intracellular messengers have been intensively investigated during the last decade. NO and cGMP both mediate physiological effects in the cardiovascular, endocrinological, and immunological systems as well as in central nervous system (CNS). In the CNS, activation of the N-methyl-D-aspartic acid (NMDA) type of glutamatergic receptor induces Ca(2+)-dependent NOS and NO release, which then activates soluble guanylate cyclase for the synthesis of cGMP. Both compounds appear to be important mediators in long-term potentiation and long-term depression, and thus may play important roles in the mechanisms of learning and memory. Aging and the accumulation of amyloid beta (A beta) peptides are important risk factors for the impairment of memory and development of dementia. In these studies, the mechanism of basal- and NMDA receptor-mediated cGMP formation in different parts of adult and aged brains was evaluated. The relative activity of the NO cascade was determined by assay of NOS and guanylate cyclase activities. In addition, the effect of the neurotoxic fragment 25-35 of A beta (A beta) peptide on basal and NMDA receptor-mediated NOS activity was investigated. The studies were carried out using slices of hippocampus, brain cortex, and cerebellum from 3- and 28-mo-old rats. Aging coincided with a decrease in the basal level of cGMP as a consequence of a more active degradation of cGMP by a phosphodiesterase in the aged brain as compared to the adult brain. Moreover, a loss of the NMDA receptor-stimulated enhancement of the cGMP level determined in the presence of cGMP-phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine (IBMX) was observed in hippocampus and cerebellum of aged rats. However, this NMDA receptor response was preserved in aged brain cerebral cortex. A significant enhancement of the basal activity of NOS by about 175 and 160% in hippocampus and cerebellum, respectively, of aged brain may be involved in the alteration of the NMDA receptor response. The neurotoxic fragment of A beta, peptide 25-35, decreased significantly the NMDA receptor-mediated calcium, and calmodulim-dependent NO synthesis that may then be responsible for disturbances of the NO and cGMP signaling pathway. We concluded that cGMP-dependent signal transduction in hippocampus and cerebellum may become insufficient in senescent brain and may have functional consequences in disturbances of learning and memory processes. A beta peptide accumulated during brain aging and in Alzheimer disease may be an important factor in decreasing the NO-dependent signal transduction mediated by NMDA receptors.

Aggregated beta amyloid peptide 1-40 decreases Ca2+- and cholinergic receptor-mediated phosphoinositide degradation by alteration of membrane and cytosolic phospholipase C in brain cortex.

The effects of full-length amyloid beta protein, A(beta) (1-40), on phosphoinositide-specific phospholipase C (PLC) were investigated in synaptic plasma membranes (SPM) and cytosol prepared from the cerebral cortex of adult rats. Moreover, the role of A(beta) (1-40) on the activation of lipid peroxidation was evaluated. The activity of phospholipase C (PLC) acting on phosphatidylinositol (PI) and phosphatidylinositol-4,5-bisphosphate (PIP2) was determined using exogenous labeled substrates. The subcellular fractions were the source of enzyme(s). The radioactivity of lipid messengers derived from degradation of [14C- arachidonoyl] PI was also determined. The stable aggregated form of beta-amyloid peptide (1-40) at 25 microM concentration exerted reproducible effects. The aggregated form of A(beta) (1-40) inhibited Ca(2+)-regulated PI and PIP2 degradation by SPM and cytosolic enzymes. Aggregated A(beta) also decreased significantly the level of diacylglycerol, the product of PLC. This additionally supports the inhibitory effect of A(beta) on membrane-bound and cytosolic PLC. Moreover, A(beta) (1-40) significantly decreased the basal activity of the PIP2-PLC in SPM and the enzyme activity regulated through cholinergic receptors. However, in spite of the lower enzyme activity, the percentage distribution of inositol (1,4,5) P3 radioactivity (IP3) in the total pool of inositol metabolites was not significantly changed. The aggregated neurotoxic fragment, A(beta) (25-35), mimicked the effect of full-length A(beta) (1-40). A(beta) (1-40) enhanced the level of malondialdehyde indicating an activation of free radical stimulated membrane lipid peroxidation that may be involved in alteration of phospholipase(s) activity. Our results indicated that aggregated A(beta) (1-40) alters Ca(2+)-dependent phosphoinositide degradation affecting synaptic plasma membrane and cytosolic phospholipase(s) activity. Moreover, this peptide significantly decreased the phosphoinositide-dependent signal transduction mediated by cholinergic receptors. The effect of aggregated A(beta) (1-40) is more pronounced than that of the neurotoxic fragment A(beta) (25-35). Our study suggests that the deposition of aggregated A(beta) may alter phosphoinositide signaling in brain.

Inhibition of nitric oxide synthase prevents energy failure and oxidative damage evoked in the brain by lipopolysaccharide.

The inducible nitric oxide synthase (iNOS) plays an important role in endotoxic shock. However,little is known about the involvment of constitutive isoform(s) of NOS (cNOS). The aim of this study was to determine the role of cNOS in the mouse brain after lipopolysaccharide (LPS) injection. Concentrations of nicotinamide adenine dinucleotide (NAD(+)), carbonyl group and thiobarbituric acid reactive substances were determined spectrophotometrically, cNOS mRNA was evaluated by RT-PCR. Our data showed that LPS significantly decreased NAD(+) level, and enhanced protein and lipid oxidation, but had no effect on cNOS mRNA expression. Inhibitors of cNOS protected the cells against alterations evoked by LPS, suggesting involvement of cNOS isoforms in pathology.

Detection of protein oxidation in endothelial cells by fluorescently labelled tyramine.

BACKGROUND: Endothelial cell injury mediated by activated polymorphonuclear leucocytes (PMN) occurs during inflammation or reperfusion after brain ischemia. Protein oxidation caused by activated PMN may lead to functional disturbances, degeneration and death of the endothelial cells. The aim of this study was to detect protein oxidation in endothelial cells induced by activated neutrophils by using a novel fluorescent probe. MATERIAL AND METHODS: Protein oxidation of Human Umbilical Vein Endothelial Cells (HUVEC) in culture was investigated by a 15-min incubation with human neutrophils activated by phorbol myristate acetate (PMA) in the presence of tyramine coupled to the succinimidyl ester of (fluorescein -5 (and-6)-carboxamido) hexanoic acid. Dityrosine bond formation as reflected by the linkage of the fluorescent tyramine to proteins was determined by Western-blotting. RESULTS: The oxidative burst generated by activated neutrophils induced dityrosine formation in the extracellular proteins (ECP) of HUVEC. Similar results were obtained, when horseradish peroxidase (HRP) was used for the induction of oxidative stress. However, when hydrogen peroxide (0.1 mM) was used, dityrosine formation was not detected. CONCLUSIONS: Fluorescently labelled tyramine is a powerful tool for the detection of ECP oxidation in endothelial cells. As long as the oxidation by the activated neutrophils is limited to ECP, the endothelial cells may be protected by antioxidants.

Elevated nitric oxide production mediates beta-amyloid-induced mitochondria failure.

Mitochondrial dysfunction has been identified in a large proportion of neurodegenerative disorders including Alzheimer's disease (AD). In addition, the involvement of nitric oxide (NO) has been implicated in the pathogenesis of AD. Thus, we investigated the effects of the Swedish double mutation (K670M/N671L) in the beta-amyloid precursor protein (APPsw) on NO levels and mitochondrial function in PC12 cells. Interestingly, APPsw PC12 cells showed increased NO levels, decreased cytochrome C oxidase activity and reduced ATP levels compared to wild-type APP bearing cells and empty vector transfected cells. On the basis of our data, we propose a hypothetical sequence of events linking amyloid beta-peptide and NO production with mitochondria failure.