Joanna B. Strosznajder

Amyloid-beta induced changes in nitric oxide production and mitochondrial activity lead to apoptosis

Increasing evidence suggests an important role of mitochondrial dysfunction in the pathogenesis of Alzheimers disease. Thus, we investigated the effects of acute and chronic exposure to increasing concentrations of amyloid β (Aβ) on mitochondrial function and nitric oxide (NO) production in vitro and in vivo. Our data demonstrate that PC12 cells and human embryonic kidney cells bearing the Swedish double mutation in the amyloid precursor protein gene (APPsw), exhibiting substantial Aβ levels, have increased NO levels and reduced ATP levels. The inhibition of intracellular Aβ production by a functional γ-secretase inhibitor normalizes NO and ATP levels, indicating a direct involvement of Aβ in these processes. Extracellular treatment of PC12 cells with comparable Aβ concentrations only leads to weak changes, demonstrating the important role of intracellular Aβ. In 3-month-old APP transgenic (tg) mice, which exhibit no plaques but already detectable Aβ levels in the brain, reduced ATP levels can also be observed showing the in vivo relevance of our findings. Moreover, we could demonstrate that APP is present in the mitochondria of APPsw PC12 cells. This presence might be directly involved in the impairment of cytochrome c oxidase activity and depletion of ATP levels in APPsw PC12 cells. In addition, APPsw human embryonic kidney cells, which produce 20-fold increased Aβ levels compared with APPsw PC12 cells, and APP tg mice already show a significantly decreased mitochondrial membrane potential under basal conditions. We suggest a hypothetical sequence of pathogenic steps linking mutant APP expression and amyloid production with enhanced NO production and mitochondrial dysfunction finally leading to cell death.

Activation of Ethanolamine Phospholipase A2 in Brain During Ischemia

Abstract: Extracts of acetone‐dried powders from ischemic gerbil brain were examined for phospholipase A1 and A2 activities with phosphatidylethanolamine at pH 7.2. Ischemia was induced by bilateral ligation, and the animals were killed by immersion into liquid nitrogen. Bilateral ligation with ketamine as general anesthetic resulted in a rapid, transient increase in phospholipase A2 activity. The activity increased from 0.46 nmolihimg protein at 0 time to 0.82 nmol/h/mg protein at 1 min of ligation. Phospholipase A1 activity also increased from 0.7 to 1.3 nmol/h/mg protein within the 1st min. When Nembutal was used as anesthetic, the phospholipase activation was earlier, within the first 30 s. Similar results were found for ischemia induced by decapitation of Wistar rats without anesthesia. Bilateral ligation of the carotid arteries of the gerbil is known to increase the concentration of free fatty acids, particularly arachidonate. This increase is, at least in part, due to phospholipase A activation. As ethanolamine phospholipase A2 in brain does not require Ca2+ for activity, these results suggest that phospholipase A2 activation in ischemic brain results from a covalent modification of the enzyme.

Amyloid beta peptide and NMDA induce ROS from NADPH oxidase and AA release from cytosolic phospholipase A2 in cortical neurons.

Increase in oxidative stress has been postulated to play an important role in the pathogenesis of a number of neurodegenerative diseases including Alzheimer’s disease. There is evidence for involvement of amyloid‐β peptide (Aβ) in mediating the oxidative damage to neurons. Despite yet unknown mechanism, Aβ appears to exert action on the ionotropic glutamate receptors, especially the N‐methyl‐D‐aspartic acid (NMDA) receptor subtypes. In this study, we showed that NMDA and oligomeric Aβ1–42 could induce reactive oxygen species (ROS) production from cortical neurons through activation of NADPH oxidase. ROS derived from NADPH oxidase led to activation of extracellular signal‐regulated kinase 1/2, phosphorylation of cytosolic phospholipase A2α (cPLA2α), and arachidonic acid (AA) release. In addition, Aβ1–42‐induced AA release was inhibited by d(−)‐2‐amino‐5‐phosphonopentanoic acid and memantine, two different NMDA receptor antagonists, suggesting action of Aβ through the NMDA receptor. Besides serving as a precursor for eicosanoids, AA is also regarded as a retrograde messenger and plays a role in modulating synaptic plasticity. Other phospholipase A2 products such as lysophospholipids can perturb membrane phospholipids. These results suggest an oxidative‐degradative mechanism for oligomeric Aβ1–42 to induce ROS production and stimulate AA release through the NMDA receptors. This novel mechanism may contribute to the oxidative stress hypothesis and synaptic failure that underline the pathogenesis of Alzheimer’s disease.

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.

P2X7 nucleotide receptor activation enhances IFNγ‐induced type II nitric oxide synthase activity in BV‐2 microglial cells

Under normal and pathological conditions, brain cells release nucleotides that regulate a wide range of cellular responses due to activation of P2 nucleotide receptors. In this study, the effect of extracellular nucleotides on IFNγ‐induced NO release in murine BV‐2 microglial cells was investigated. BV‐2 cells expressed mRNA for metabotropic P2Y and ionotropic P2X receptors. Among the P2 receptor agonists tested, ATP, ADP, 2′,3′‐O‐(4‐benzoylbenzoyl)‐ATP (BzATP), and 2‐methylthio‐ATP (2‐MeSATP), but not UTP, enhanced IFNγ‐induced iNOS expression and NO production, suggesting that the uridine nucleotide receptors P2Y2 and P2Y6 are not involved in this response. U0126, an antagonist for MEK1/2, a kinase that phosphorylates the extracellular signal‐regulated kinases ERK1/2, decreased IFNγ‐induced NO production. BzATP, a potent P2X7 receptor agonist, was more effective than ATP, ADP, or 2‐MeSATP at enhancing IFNγ‐induced ERK1/2 phosphorylation. Consistent with activation of the P2X7 receptor, periodate‐oxidized ATP, a P2X7 receptor antagonist, and suramin, a non‐specific P2 receptor antagonist, inhibited the effect of ATP or BzATP on IFNγ‐induced NO production, whereas pyridoxal‐phosphate‐6‐azophenyl‐2′,4′‐disulfonic acid (PPADS), an antagonist of several P2X receptor subtypes, was ineffective. These results suggest that activation of P2X7 receptors may contribute to inflammatory responses in microglial cells seen in neurodegenerative diseases.

Cyclic GMP and Nitric Oxide Synthase in Aging and Alzheimer's Disease

Cyclic guanosine monophosphate (cGMP) is an important secondary messenger synthesized by the guanylyl cyclases which are found in the soluble (sGC) and particular isoforms. In the central nervous system, the nitric oxide (NO)-sensitive sGC isoform is the major enzyme responsible for cGMP synthesis. Phosphodiesterases (PDEs) are enzymes for hydrolysis of cGMP in the brain, and they are mainly isoforms 2, 5, and 9. The NO/cGMP signaling pathway has been shown to play an important role in the process underlying learning and memory. Aging is associated with an increase in PDE expression and activity and a decrease in cGMP concentration. In addition, aging is also associated with an enhancement of neuronal NO synthase, a lowering of endothelial, and no alteration in inducible activity. The observed changes in NMDA receptor density along with the Ca2+/NO/cGMP pathway underscore the lower synaptic plasticity and cognitive performance during aging. This notion is in agreement with last data indicating that inhibitors of PDE2 and PDE9 improve learning and memory in older rats. In this review, we focus on recent studies supporting the role of Ca2+/NO/cGMP pathway in aging and Alzheimers disease.

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.

Effect of poly(ADP-ribose) polymerase inhibitors on oxidative stress evoked hydroxyl radical level and macromolecules oxidation in cell free system of rat brain cortex

Poly(ADP-ribose) polymerase-1 (PARP-1) is a nuclear enzyme involved in DNA repair, replication and cell cycle. However, its overactivation leads to nicotinamide adenine dinucleotide and ATP depletion and cell death. The inhibitors of PARP-1 were successfully used in the basic studies and in animal models of different diseases. For this reason, it is important to discriminate between specific and non-specific antioxidant properties of PARP-1 inhibitors. The aim of this study was to investigate the effect of PARP-1 inhibitors on the free radical level and oxidation of macromolecules and to compare their properties with the efficacy of antioxidants. Oxidative stress was induced in the brain cortex homogenate by FeCl(2) or CuSO(4) at 25 microM during 15 min incubation at 37 degrees C. PARP-1 inhibitors 3-aminobenzamide (3-AB), 1,5-dihydroxyisoquinoline (DHIQ) and 3,4-dihydro-5-[4-(1-piperidinyl)butoxy]-1(2H)-isoquinolinone (DPQ), and the antioxidants alpha-tocopherol, resveratrol and Tempol were used at 0-5 mM. Free radical contents were estimated by spin-trapping using HPLC. Lipid and protein oxidation were determined by measuring thiobarbituric acid reactive substances and carbonyl groups or using fluorescent probe TyrFluo, respectively. Our data indicate that 3-AB and DHIQ are potent hydroxyl radical scavengers and inhibitors of protein oxidation. DHIQ additionally decreases lipid peroxidation. DPQ has no antioxidant properties and seems to be a specific PARP-1 inhibitor, however, it is a water insoluble compound. Among the investigated antioxidants, the most potent was resveratrol and then alpha-tocopherol and Tempol. These results indicate that 3-A beta, benzamide and DHIQ are potent hydroxyl radical scavengers and antioxidants. These data ought to be taken into consideration when properties of these compounds as PARP inhibitors are evaluated.

Poly(ADP-ribose) Polymerase-1 in Amyloid Beta Toxicity and Alzheimer's Disease

Poly(ADP-ribose) polymerase-1 (PARP-1) is a key enzyme responsible for the maintenance of genome stability, transcriptional regulation, and long-term potentiation in neurons. However, the excessive activation of PARP-1 under pathological conditions may lead to an accumulation of poly(ADP-ribose) (PAR), a novel signaling molecule that induces programmed cell death, or to NAD depletion that induces energy crisis and necrotic cell death. PARP-1 is thought to be primarily a nuclear enzyme, but some data indicate that it can also be localized to the mitochondria where it is responsible for posttranslational modification of electron transport chain complexes and alteration of mitochondria function. The enhancement of PARP-1 activity and the accumulation of PAR were demonstrated in the brain of patients with Alzheimers disease (AD), particularly in neurons of the frontal and temporal lobes and in skin fibroblasts and lymphoblasts. Moreover, it has been reported that PARP-1 gene polymorphisms are highly associated with the development of AD. The activation of PARP-1 by oxidative stress seems to be an early and important event in the pathogenesis of AD. It is now widely accepted that the overproduction and oligomerization of amyloid β (Aβ) are responsible for the activation of a free radical cascade and oxidative stress in AD. Interestingly, the activity of PARP-1 is enhanced in AD and is also increased by Aβ peptides. The activation of PARP-1 by Aβ can lead to the PAR-mediated release of apoptosis-inducing factor from the mitochondria and its translocation to the nucleus, which leads to death of some populations of cells. A role of PARP-1 in the regulation of Aβ precursor protein metabolism processing and Aβ liberation has not been described previously. The study presented in this review indicated the relationship between PARP-1 activation, alteration of mitochondria function, and Aβ toxicity. The presented data should stimulate further studies on the role of PARP-1 in AD pathogenesis and thereby engage a new perspective regarding AD therapy.

Role of nitric oxide in the brain during lipopolysaccharide-evoked systemic inflammation.

Although the inducible isoform of nitric oxide synthase (iNOS) is a well‐established source of nitric oxide (NO•) during inflammation of the central nervous system (CNS), little is known about the involvement of constitutive isoforms of NOS (cNOS) in the inflammatory process. The aim of this study was to compare the responses of the expression and activity of iNOS and the two cNOS isoforms, neuronal and endothelial (nNOS and eNOS, respectively), in the brain to systemic inflammation and their roles in the cascade of events leading to degeneration and apoptosis. A systemic inflammatory response in C57BL/6 mice was induced by intraperitoneal injection of lipopolysaccharide [LPS; 1 mg/kg body weight (b.w.)]. The relative roles of the NOS isoforms were evaluated after injection of NG‐nitro‐L‐arginine (NNLA; 30 mg/kg b.w.), which preferentially inhibits cNOS, or 1400W (5 mg/kg b.w.), an inhibitor of iNOS. Biochemical and morphological alterations were analyzed up to 48 hr after administration of LPS. Systemic LPS administration evoked significant ultrastructural alterations in brain capillary vessels, neuropils, and intracellular organelles of neurons, astrocytes, and microglia. Apoptotic/autophagic processes occurred in many neurons of the substantia nigra (SN), which coincided with exclusive enhancement of iNOS expression and activity in this brain region. Moreover, inhibitors of both iNOS and cNOS prevented LPS‐evoked release of apoptosis‐inducing factor (AIF) from SN mitochondria. Collectively, the results indicate that synthesis of NO• by both the inducible and constitutive NOS isoforms contribute to the activation of apoptotic pathways in the brain during systemic inflammation.