Saleh Abu-Raya

Neuroprotective and neurotoxic effects of monoamine oxidase-B inhibitors and derived metabolites under ischemia in PC12 cells

Selegiline and rasagiline are selective and irreversible monoamine oxidase-B inhibitors that exert neuroprotective effects in various preclinical models. The aim of the present study was to examine the effect of selegiline and its major metabolite, L-methamphetamine in comparison to rasagiline and its major metabolite, 1-R-aminoindan on oxygen-glucose deprivation induced cell death in nerve growth factor (NGF)-differentiated pheochromocytoma (PC12) cells. Our results show that selegiline reduces oxygen-glucose deprivation induced cell death by 30%. When the cultures were treated with rasagiline at similar concentrations, cell death induced by oxygen-glucose deprivation was reduced by 45-55%. L-methamphetamine, a major selegiline metabolite, but not 1-R-aminoindan, the major rasagiline metabolite, enhanced oxygen-glucose deprivation-induced cell death by 70%. Under normoxic conditions, both metabolites lack neurotoxicity. Concomitant exposure of the cultures under oxygen-glucose deprivation, to a combination of either selegiline and L-methamphetamine or rasagiline and 1-R-aminoindan, indicated that L-methamphetamine, but not 1-R-aminoindan, blocked the neuroprotective effect of the parental drug. These results suggest there may be a neuroprotective advantage of rasagiline over selegiline.

Rasagiline, a monoamine oxidase-B inhibitor, protects NGF-differentiated PC12 cells against oxygen-glucose deprivation

In our in vitro model, rasagiline a selective irreversible monoamine oxidase‐B (MAO‐B) inhibitor, protected nerve growth factor (NGF)‐differentiated PC12 cells from cell death under oxygen and glucose deprivation (OGD). The severity of the OGD insult, as expressed by cell death, was time‐dependent. Exposure of the cells to OGD for 3 hr followed by 18 hr of reoxygenation caused about 30–40% cell death. Under these conditions, the neuroprotective effect of rasagiline was dose‐dependent: rasagiline reducing OGD‐induced cell death by 68% and 80% at 100 nM and 1 μM, respectively. The neuroprotective effect of rasagiline was also observed when added after the OGD insult (55% reduction in cell death). Under rasagiline treatment, there was a lesser decrease in ATP content in cultures exposed to OGD compared with that in untreated cultures. OGD followed by reoxygenation resulted in a several fold increase in PGE2 release into the extracellular medium. Rasagiline (100 nM–1 μM) markedly inhibited OGD‐induced PGE2 release. Clorgyline, a monoamine oxidase‐A (MAO‐A) inhibitor, did not protect NGF‐differentiated PC12 cells against OGD‐induced cell death. As NGF‐differentiated PC12 cells contain exclusively MAO type A, these data suggest that the neuroprotective effect of rasagiline under OGD conditions is independent of MAO inhibition. J. Neurosci. Res. 58:456–463, 1999.

Isolation, characterization and synthesis of a novel pardaxin isoform

We report the isolation of a novel pardaxin isoform from the toxic secretion of the Red Sea Moses sole (Pardachirus marmoratus). Mass spectrometrical analysis of the newly purified peptide revealed a different primary structure compared to the previously known pardaxin isoforms. Sequence analysis disclosed an aspartic acid residue instead of glycine at position 31 of the new isoform. According to the novel sequence, a synthetic Asp‐31‐peptide was compared with the native compound as well as with synthetic Gly‐31‐pardaxin. The isolated Asp‐31‐pardaxin isoform and its synthetic analog exhibited identical elution properties during reverse‐phase HPLC, as well as similar dose‐dependent lytic effects on human erythrocytes at a concentration of 10−6 to 10−5 M. The hemolytic activity of Asp‐31‐pardaxins was lower than that of Gly‐31‐pardaxin and no synergistic effect between these peptides was found. The additional negative charge introduced by Asp‐31 is likely to affect the selectivity of pardaxin pores towards a variety of ions.

Synergism between tumor necrosis factor-α and H2O2 enhances cell damage in rat PC12 cells

Abstract Tumor necrosis factor-α (TNFα) is harmful in the early phase and beneficial in the long-term phase after brain injury. Reactive oxygen species (ROS) are among the most toxic mediators activated by injury. We speculate that part of the TNFα toxicity is mediated by its synergism with ROS. Thus, toxicity of TNFα and ROS, alone or together, were studied in PC12 cells. PC12 cells were exposed for 18 h to TNFα (0–100 ng/ml), to H 2 O 2 (1–300 μM) or to both, each at sub-toxic concentrations. Lactic dehydrogenase release, prostaglandin E 2 accumulation and morphology indicated cell death and stress response. TNFα toxicity was seen at >50 ng/ml, and that of H 2 O 2 at >150 μM, however, when together, sub-lethal levels (25 ng/ml TNFα and 30 μM H 2 O 2 ) induced toxicity. Dexanabinol, an N -methyl- d -aspartate antagonist with antioxidant and anti-TNFα properties, completely rescued the cells. These findings corroborate our hypothesis on the cooperative toxicity exerted by TNFα and ROS after brain injury.

Rasagiline, a novel monoamine oxidase-B inhibitor with neuroprotective effects under ischemic conditions in PC12 cells

Rasagiline (N‐propargyl‐1R‐aminoindan) is a novel, potent, selective, and irreversible inhibitor of monoamine oxidase B (MAO‐B), currently in Phase III trials for the treatment of Parkinson disease (PD). Rasagiline was shown to have neuroprotective properties in various in vitro and in vivo models independently of MAO‐B inhibition. Recently, we developed an in vitro oxygen‐glucose deprivation (OGD) model of time‐course dependent neuronal cell death in nerve growth factor (NGF)‐differentiated PC12 cultures. OGD was accompanied by activation of the arachidonic acid cascade and a decrease in ATP content, changes typical of ischemic conditions. OGD for 3 h followed by reoxygenation for 18 h caused about 30–40% cell death. These conditions are suitable for testing the effect of potential neuroprotective compounds on neuronal cell death. Rasagiline markedly reduced OGD‐induced cell death independently of MAO‐B inhibition, reducing OGD‐induced cell death even when added after the OGD insult. The compound also inhibited OGD‐induced prostaglandin E2 (PGE2) release in a dose‐dependent manner. At present, selegiline remains the only drug approved for PD therapy based on MAO‐B inhibition. However, in contrast to selegiline, rasagiline is not metabolized to amphetamine‐like products, which cause adverse side effects and neuronal cell death. Therefore, rasagiline, whose neuroprotective properties are uncomplicated by the production of neurotoxic metabolites, may have an advantage over selegiline in the treatment of PD. Drug Dev. Res. 50:285–290, 2000.

Pardaxin, a new pharmacological tool to stimulate the arachidonic acid cascade in PC12 cells

The effect of Pardaxin, a neurotoxin that induces neurotransmitter release from neurons, on the arachidonic acid (AA) cascade was studied in PC12 cells. Both native and the synthetic Pardaxin selectively stimulated phospholipase A2 (PLA2) activity (measured by [3H]AA release) in the presence as well as in the absence of extracellular calcium. Pardaxin-stimulated PLA2 activity was also evident in the increased formation of lysophosphatidylcholine. Pardaxin analogs, lacking the alpha-helical structure that is essential for insertion into the plasma membrane, were ineffective in stimulating the AA cascade in PC12 cells. Pardaxin stimulation of PLA2 was markedly inhibited by the nonselective PLA2 inhibitors bromophenacyl bromide and mepacrine, by methyl arachidonyl fluorophosphonate, a dual inhibitor of calcium-dependent cytosolic PLA2 and the calcium-independent PLA2 and by bromoenol lactone[(E)-6-(bromoethylene)tetrahydro-3-(1-naphthalenyl-2H-pyran -2- one], a highly specific inhibitor of calcium-independent PLA2. After Pardaxin treatment, there was increased release of AA metabolites produced by the cyclooxygenase pathway as expressed in an 8-fold increase of PGE2 release. The release of other eicosanoids, such as 6-keto-PGF1alpha and thromboxane B2, was also augmented. Pardaxin-induced PGE2 release was observed in calcium-free medium and in the absence of any increase in cytosolic calcium. Dexamethasone partially inhibited Pardaxin-induced PGE2 release. This effect was reversed by the type II corticosteroid receptor antagonist RU-38486. Our results indicate that Pardaxin stimulates release of AA and eicosanoids, independently of calcium, and suggest that calcium-independent PLA2 plays an important role in Pardaxin stimulation of the AA cascade.