Magdalena Cakala

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.

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.

Poly(ADP-ribose) polymerase-1 inhibition protects the brain against systemic inflammation.

Poly(ADP-ribose) polymerase-1 (PARP-1) is involved in DNA repair, but its overactivation can induce cell death. Our aim was to investigate the role of PARP-1 in activation of programmed cell death processes in the brain during systemic inflammation. Our data indicated that lipopolysaccharide (1mg/kgb.w., i.p.)-evoked systemic inflammation enhanced PARP-1 activity in the mouse brain, leading to the lowering of beta-NAD(+) concentration, to translocation of apoptosis inducing factor from mitochondria to the nucleus, and to enhanced lipid peroxidation. Inhibitor of PARP-1, 3-aminobenzamide (30 mg/kgb.w., i.p.), protected the brain against prooxidative and cell death processes, suggesting involvement of PARP-1 in systemic inflammation-related processes in the brain.

AIF and PARP-1, the molecular targets for Amyloid beta-toxicity: Alzheimer's disease genetic mutation increases cells susceptibility to nitrosative stress

Background: Moderate to severe Alzheimer’s disease (AD) is characterized by a high degree of cognitive, functional, and behavioral dysfunction. There are relatively few treatments available for this stage of illness, and these appear to provide symptomatic benefit without altering the course of the disease. The most widespread symptomatic treatments for AD are acetylcholinesterase inhibitors, including donepezil, which provide temporary cognitive benefits. In addition to acetylcholinesterase inhibitors, several clinical studies have shown support for the use of the NMDA receptor antagonist memantine, either as monotherapy or in combination with a cholinesterase inhibitor for moderate to severe AD. Methods: In this study, a battery of cognitive tasks was used to test the effects of donepezil, memantine, and combined treatment in 3xTg-AD mice (PS1M146V, APPSwe and tauP301L). These transgenic mice develop both plaques and tangles in a region-specific and age-progressive manner, and also exhibit progressive learning deficits. Donepezil (1mg/kg/day), memantine (30mg/kg/day), donepezil-memantine combination, or vehicle (saline) were administered orally via drinking water to cohorts of adult (15 months of age; severe pathology) and young mice (6 months of age; mild pathology), for three months. Results: In both adult and young mice, memantine and donepezil-memantine combination treatment significantly improved spatial memory (both acquisition and retention), compared to the vehicle-treated animals, whereas donepezil improved the retention, but not acquisition, of spatial memory. Conclusions: These results suggest that memantine treatment, or combination therapy using donepezil and memantine, is more effective than donepezil monotherapy at improving cognitive performance in both young and old mice demonstrating cognitive deficits and AD-like pathology.