Maura Floreani

Neurotrophins rescue cerebellar granule neurons from oxidative stress-mediated apoptotic death: selective involvement of phosphatidylinositol 3-kinase and the mitogen-activated protein kinase pathway.

Abstract: Cerebellar granule neurons maintained in medium containing serum and 25 mM K+ reliably undergo an apoptotic death when switched to serum‐free medium with 5 mM K+. New mRNA and protein synthesis and formation of reactive oxygen intermediates are required steps in K+ deprivation‐induced apoptosis of these neurons. Here we show that neurotrophins, members of the nerve growth factor gene family, protect from K+/serum deprivation‐induced apoptotic death of cerebellar granule neurons in a temporally distinct manner. Switching granule neurons, on day in vitro (DIV) 4, 10, 20, 30, or 40, from high‐K+ to low‐K+/serum‐free medium decreased viability by >50% when measured after 30 h. Treatment of low‐K+ granule neurons at DIV 4 with nerve growth factor, brain‐derived neurotrophic factor (BDNF), neurotrophin‐3, or neurotrophin‐4/5 (NT‐4/5) demonstrated concentration‐dependent (1–100 ng/ml) protective effects only for BDNF and NT‐4/5. Between DIV 10 and 20, K+‐deprived granule neurons showed decreasing sensitivity to BDNF and no response to NT‐4/5. Cerebellar granule neuron death induced by K+ withdrawal at DIV 30 and 40 was blocked only by neurotrophin‐3. BDNF and NT‐4/5 also circumvented glutamate‐induced oxidative death in DIV 1–2 granule neurons. Granule neuron death caused by K+ withdrawal or glutamate‐triggered oxidative stress was, moreover, limited by free radical scavengers like melatonin. Neurotrophin‐protective effects, but not those of antioxidants, were blocked by selective inhibitors of phosphatidylinositol 3‐kinase or the mitogen‐activated protein kinase pathway, depending on the nature of the oxidant stress. These observations indicate that the survival‐promoting effects of neurotrophins for central neurons, whose cellular antioxidant defenses are challenged, require activation of distinct signal transduction pathways.

Neuroprotection by melatonin from kainate-induced excitotoxicity in rats.

In this study, we injected 10 mg/kg kainate i.p. into rats. This resulted in a brain injury, which we quantified in the hippocampus, the amygdala, and the pyriform cortex. Neuronal damage was preceded by a set of typical behavioral signs and by biochemical changes (noradrenaline decrease and 5‐hydroxyindoleacetic acid increase) in the af‐fected brain areas. Melatopin (2.5 mg/kg) was injected i.p. four times: 20 min before kainate, immediately after, and 1 and 2 h after the kainate. The cumulative dose of 10 mg/kg melatonin prevented kainate‐induced neuronal death as well as behavioral and biochemical disturbances. A possible mechanism of melatonin‐provided neuroprotection lies in its antioxidant action. Our results suggest that melatonin holds potential for the treatment of pathologies such as epilepsy‐associated brain damage, stroke, and brain trauma.—Giusti, P., Lipartiti, M., Franceschini, D., Schiavo, N., Floreani, M., Manev, H. Neuroprotection by melatonin from kainate‐induced excitotoxicity in rats. FASEB J. 10, 891‐896 (1996)

Excitotoxicity, Oxidative Stress, and the Neuroprotective Potential of Melatonin

The Brain Consumes Large Quantities of Oxygen Relative to its Contribution to total body mass. This, together with its paucity of oxidative defense mechanisms, places this organ at risk for damage mediated by reactive oxygen species. The pineal secretory product melatonin possesses broad‐spectrum free radical scavenging and antioxidant activities, and prevents kainic acid‐induced neuronal lesions, glutathione depletion, and reactive oxygen species‐mediated apoptotic nerve cell death. Melatonins action is thought to involve electron donation to directly detoxify free radicals such as the highly toxic hydroxyl radical, which is a probable end‐product of the reaction between NO· and peroxynitrite. Moreover, melatonin limits NO·‐induced lipid peroxidation, inhibits cerebellar NO· synthase, scavenges peroxynitrite, and alters the activities of enzymes that improve the total antioxidative defense capacity of the organism. Melatonin function as a free radical scavenger and antioxidant is likely facilitated by the ease with which it crosses morphophysiological barriers, e.g., the blood‐brain barrier, and enters cells and subcellular compartments. Pinealectomy, which eliminates the nighttime rise in circulating and tissue melatonin levels, worsens both reactive oxygen species‐mediated tissue damage and brain damage after focal cerebral ischemia and excitotoxic seizures. That melatonin protects against hippocampal neurodegeneration linked to excitatory synaptic transmission is fully consistent with the last study. Conceivably, the decreased melatonin secretion that is documented to accompany the aging process may be exaggerated in populations with dementia.

Melatonin maintains glutathione homeostasis in kainic acid-exposed rat brain tissues.

Reduced glutathione (GSH) is a key component of the cellular defense cascade against injury caused by reactive oxygen species. Because kainic acid (KA) neurotoxicity is probably mediated at least in part by oxidative stress, we examined the influence of KA treatment on GSH content and GSH‐related enzyme activities in adult rats. A single injection of KA (10 mg/kg i.p.) time‐dependently decreased forebrain GSH (maximal reduction at 48 h). KA also markedly lowered GSH levels in amygdala and hippocampus, but not in the corpus striatum, which is resistant to KA injury. The pineal secretory product melatonin has been shown to exert neuroprotective effects against KA‐induced excitotoxicity in rats. Melatonin (2.5 mg/kg i.p., administered four times) partially prevented all decreases in GSH of KA‐treated rats. These neuroprotective effects of melatonin may result from a sparing of glutathione reductase, which decreased in KA‐treated but not in KA/melatonin‐treated animals. Moreover, KA caused a rapid decrease in the GSH content of cultured cerebellar granule neurons but not astrocytes. These cell types both express functional KA receptors, but only the former are sensitive to reactive oxygen species‐dependent KA injury. Melatonin counteracted the changes in GSH induced by KA in cultured cerebellar granule neurons. Our results suggest that melatonin prevents the neurotoxic effects of reactive oxygen species linked to KA receptor activation by maintaining cellular GSH homeostasis.—Floreani, M., Skaper, S. D., Facci, L., Lipartiti, M., Giusti, P. Melatonin maintains glutathione homeostasis in kainic acid‐exposed rat brain tissues. FASEB J. 11, 1309–1315 (1997)

A Comparison Between Different Methods for the Determination of Reduced and Oxidized Glutathione in Mammalian Tissues

In this study, three rapid assay techniques for the determination of glutathione, one enzymatic, one fluorometric and one newly patented colorimetric method, were compared by measuring reduced (GSH) and oxidized (GSSG) glutathione in guinea-pig heart and liver. The HPLC technique was used as a standard, since it is considered the most reliable assay method. In heart, all methods measured the same levels of GSH (about 1 mumole/g wet tissue), whereas in liver the fluorometric assay gave GSH levels about half as high as those measured by the other methods (about 3 vs. 7 mumoles/g wet tissue). Conversely, the fluorometric assay grossly overestimated GSSG concentration (by 5 to 8 times) in both heart and liver. These results confirm previous doubts about the use of the fluorometric technique for GSSG determination in mammalian tissues and also raise some questions about its use for the measurement of GSH in liver. In this tissue, the GSH concentration determined by the fluorometric method was shown to be inversely correlated with the size of the sample, suggesting the presence of some quenching material.

Antioxidant defences in cybrids harboring mtDNA mutations associated with Leber's hereditary optic neuropathy

Oxidative stress and imbalance between free radical generation and detoxification may play a pivotal role in the pathogenesis of Lebers hereditary optic neuropathy (LHON). Mitochondria, carrying the homoplasmic 11778/ND4, 3460/ND1 and 14484/ND6 mtDNA point mutations associated with LHON, were used to generate osteosarcoma‐derived cybrids. Enhanced mitochondrial production of reactive oxygen species has recently been demonstrated in these cybrids [Beretta S, Mattavelli L, Sala G, Tremolizzo L, Schapira AHV, Martinuzzi A, Carelli V & Ferrarese C (2004) Brain127, 2183–2192]. The aim of this study was to characterize the antioxidant defences of these LHON‐affected cells. The activities of glutathione peroxidase (GPx), glutathione reductase (GR), superoxide dismutases (SOD) and catalase, and the amounts of glutathione (GSH) and oxidized glutathione (GSSG) were measured in cybrids cultured both in glucose‐rich medium and galactose‐rich medium. The latter is known to cause oxidative stress and to trigger apoptotic death in these cells. In spite of reduced SOD activities in all LHON cybrids, and of low GPx and GR activities in cells with the most severe 3460/ND1 and 11778/ND4 mutations, GSH and GSSG content were not significantly modified in LHON cybrids cultured in glucose medium. In contrast, in galactose, GSSG concentrations increased significantly in all cells, indicating severe oxidative stress, whereas GR and MnSOD activities further decreased in all LHON cybrids. These data suggest that, in cells carrying LHON mutations, there is a decrease in antioxidant defences, which is especially evident in cells with mutations associated with the most severe clinical phenotype. This is magnified by stressful conditions such as exposure to galactose.

Cytochrome P450 1A2 is a major determinant of lidocaine metabolism in vivo: Effects of liver function

This study was designed (1) to evaluate the effect of a cytochrome P450 (CYP) 1A2 inhibitor, fluvoxamine, on the pharmacokinetics of intravenous lidocaine and its 2 pharmacologically active metabolites, monoethylglycinexylidide (MEGX) and glycinexylidide (GX), to confirm recent in vitro results indicating that CYP1A2 is the main isoform responsible for lidocaine biotransformation and (2) to assess whether liver function has any influence on the fluvoxamine‐lidocaine interaction.

Amiloride: Relationship between cardiac effects and inhibition of Na+/Ca2+ exchange

The potassium sparing diuretic amiloride at concentrations ranging between 0.1-0.8 mM inhibited the Na+/Ca2+ exchange in sarcolemmal vesicles isolated from beef heart. The rate of exchange activity was 50% reduced by 0.35 mM amiloride. In spontaneously beating atria isolated from normal and reserpinized guinea-pigs, amiloride produced a concentration-dependent positive inotropic effect and negative chronotropic effect (EC50 = 0.7 mM). Amiloride protected spontaneously beating atria and left atria driven at 1 Hz from digitalis cardiotoxicity assessed in terms of a raised end-diastolic tension. It is suggested that the positive inotropic effect, negative chronotropic effect of amiloride and heart protection against digitalis toxicity are related to the observed inhibition of sarcolemmal Na+/Ca2+ exchange activity.

In vitro and in vivo protection against kainate-induced excitotoxicity by melatonin.

Abstract: In this study, the protective effect of melatonin against kainate (KA)‐induced neurotoxicity was evaluated in vitro and in vivo. In rat brain synaptosomes, KA‐induced oxidative stress was measured as shown by significant increases in both the basal generation of reactive oxygen species (ROS), assessed by a fluorescent method, and lipid peroxidation, evaluated as malondialdehyde (MDA) levels. Melatonin decreased, in a concentration‐dependent manner, KA‐induced lipid peroxidation. The intrinsic fluorescence of melatonin molecule hindered the evaluation of its protective effect against KA‐induced ROS generation. However, melatonin was able to reduce FeSO4/ascorbate‐induced ROS generation. The melatonin protective effect was confirmed by in vivo experiments: 73% of rats injected with KA (10 mg/kg i.p.) died within 5 days; melatonin administration i.p. significantly reduced mortality of the animals. The present results suggest that melatonin might be considered a pharmacological agent for the treatment of neurodegenerative pathologies.

Glutathione Transferases as Targets for Cancer Therapy

Besides catalyzing the inactivation of various electrophile-producing anticancer agents via conjugation to the tripeptide glutathione, some cytosolic proteins belonging to the glutathione transferase (formerly glutatione-S-transferase; GST) superfamily are emerging as negative modulators of stress/drug-induced cell apoptosis through the interaction with specific signaling kinases. In addition, several data link the overexpression of some GSTs, in particular GSTP1-1, to both natural and acquired resistance to various structurally unrelated anticancer drugs. Tumor overexpression of these proteins has provided a rationale for the search of GST inhibitors and GST-activated cytotoxic prodrugs. In the present review we discuss the current structural and pharmacological knowledge of both types of GST-targeting compounds.