Oxidation of methionine 35 reduces toxicity of the amyloid beta-peptide(1-42) in neuroblastoma cells (IMR-32) via enzyme methionine sulfoxide reductase A expression and function.

The beta amyloid peptide (Abeta), the major protein component of brain senile plaques in Alzheimer's disease, is known to be directly responsible for the production of free radicals that may lead to neurodegeneration. Our recent evidence suggest that the redox state of methionine residue in position 35 (Met-35) of Abeta has the ability to deeply modify peptide's neurotoxic actions. Reversible oxidation of methionine in proteins involving the enzyme methionine sulfoxide reductase type A (MsrA) is postulated to serve a general antioxidant role and a decrease in MsrA has been implicated in Alzheimer's disease. In rat neuroblastoma cells (IMR-32), we used Abeta(1-42), in which the Met-35 is present in the reduced state, with a modified peptide with oxidized Met-35 (Abeta(1-42)Met35(OX)), as well as an Abeta-derivative in which Met-35 is substituted with norleucine (Abeta(1-42)Nle35) to investigate the relationship between Met-35 redox state, expression and function of MsrA and reactive oxygen species (ROS) generation. The obtained results shown that MsrA activity, as well as mRNA levels, increase in IMR-32 cells treated with Abeta(1-42)Met35(OX), differently to that shown by the reduced derivative. The increase in MsrA function and expression was associated with a decline of ROS levels. None of these effects were observed when cells were exposed to Abeta containing oxidized Met35 (Abeta1-42)Met35(OX). Taken together, the results of the present study indicate that the differential toxicity of Abeta peptides containing reduced or oxidised Met-35 depends on the ability of the latter form to reduce ROS generation by enhancing MsrA gene expression and function and suggests the therapeutic potential of MsrA in Alzheimer's disease.

Derangement of erythrocytic AE1 in beta-thalassemia by caspase 3: pathogenic mechanisms and implications in red blood cell senescence.

Considering its complex molecular pathophysiology, beta-thalassemia could be a good in vivo model to study some aspects related to erythrocyte functions with potential therapeutic implications not only within the frame of this particular hemoglobinopathy but also with respect to conditions in which the cellular milieu, altered by a deranged anion exchanger, could display a significant pathogenetic role (i.e., erythrocyte senescence, complications of red cell storage, renal tubular acidosis and some abnormal protein thesaurismosis). This work evaluates the anionic influx across band 3 protein in normal and beta-thalassemic red blood cells (RBCs) and ghosts. Since redox-mediated injury is an important pathway in the destruction of beta-thalassemic RBCs, we studied the anion transport and the activity of caspase 3 in the absence and presence of t-butylhydroperoxide in order to evaluate the effect of an increase of cellular oxidative stress. Interestingly, beta-thalassemic erythrocytes show a faster rate of anion exchange than normal RBCs and absence of any modulation mechanism of anion influx. These findings led us to formulate a hypothesis about the metabolic characteristics of beta-thalassemic erythrocytes, outlining that one of the main targets of caspase 3 in RBCs is the cytoplasmic domain of band 3 protein.

Amyloid peptide inhibits ATP release from human erythrocytes.

The oxygen required to meet metabolic needs of all tissues is delivered by the erythrocyte, a small, flexible cell, which, in mammals, is devoid of a nucleus and mitochondria. Despite its simple appearance, this cell has an important role in its own distribution, enabling the delivery of oxygen to precisely meet localized metabolic need. When an erythrocyte enters in a hypoxic area, a signalling pathway is activated within the cell resulting in the release of ATP in amounts adequate to activate purinergic receptors on vascular endothelium, which trigger secretion of nitric oxide and other factors resulting in vasodilatation. Among other mechanisms, binding of deoxyhemoglobin to the cytoplasmic domain of the anion-exchange protein band 3 is probably involved in this pathway. The present study investigates the effect of amyloid beta peptide exposure on this molecular mechanism. We report that deoxygenated human erythrocytes fail to release ATP following 24 h exposure to amyloid beta peptide. Concurrently, amyloid beta peptide induces caspase 3 activation. Preincubation of amyloid beta peptide treated erythrocytes with a specific inhibitor of caspase 3 prevents amyloid-induced caspase 3 activation and restores the erythrocyte's ability to release ATP under deoxygenated conditions. Since the activity of red cell phosphofructokinase, a key step in glycolytic flux, is not modified within the red cell following amyloid peptide exposure, it is likely that ATP release reduction is not dependent on glycolytic flux alterations. It has also been suggested that the heterotrimeric G protein, Gi, and adenylyl cyclase are downstream critical components of the pathway responsible for ATP release. We show that cAMP synthesis and ATP release are not failed in amyloid-peptide-treated erythrocytes in response to incubation with mastoparan 7 or forskolin plus 3-isobutyl-1-methyl xanthine, agents that stimulate cAMP synthesis. In conclusion, these results indicate that amyloid beta peptide inhibits ATP release from deoxygenated erythrocytes by activating red cell caspase 3, suggesting a pathophysiologic role for vascular amyloid peptide in Alzheimer's disease.

Oxidative effects of gemfibrozil on anion influx and metabolism in normal and Beta-thalassemic erythrocytes: physiological implications.

To further clarify some peculiar molecular mechanisms related to the physiology and pathophysiology of erythrocytes with respect to oxygen binding and release, metabolism and senescence, we investigated the oxidative effects of gemfibrozil in normal and beta-thalassemic red blood cells. Our results showed that the oxidative stress promoted by the drug, through a direct interaction with hemoglobin, may lead to activation of caspase 3, which in turn influences the band 3 anion flux and glucose metabolism. In a comparative context, we also evaluated the effect on band 3 and caspase 3 activation of orthovanadate (a phosphatase inhibitor) and t-butylhydroperoxide (a known oxidant). The results support the hypothesis that gemfibrozil influences band 3 function through several mechanisms of action, centered on oxidative stress, which induces significant alterations of glucose metabolism.

Mitochondrial oxygen consumption inhibition importance for TMT-dependent cell death in undifferentiated PC12 cells.

The evolving role of mitochondria as a target for different death-inducing noxae prompted us to investigate trimethyltin (TMT)-dependent effects on mitochondrial functionality. For this purpose, we used a homogeneous cell culture model represented by undifferentiated PC12 cells. Mitochondria isolated from PC12 cells treated with TMT for 6, 12 and 24h, showed a time-dependent inhibition of ADP-stimulated oxygen consumption using succinate or glutamate/malate as substrate. Using a fluorescent assay, the effect of TMT on mitochondrial membrane potential (delta Psi) in PC12 cells was also determined. After 24h in culture, a strong loss of mitochondrial membrane potential (delta Psi) was observed in TMT-treated cells. Collapse of mitochondrial membrane potential correlated with an increased expression of bax/bcl-2 ratio, as evaluated by polymerase chain reaction. Western blotting and spectrophotometric analysis showed that cytochrome c release and activation of caspase 3 were concurrently induced. Our findings suggest that inhibition of mitochondrial respiration represents the early toxic event for cell death in PC12 due to trimethyltin.

Amyloid-beta peptide affects the oxygen dependence of erythrocyte metabolism: a role for caspase 3.

Human erythrocyte metabolism is modulated by the cell oxygenation state. Among other mechanisms, competition of deoxyhemoglobin and some glycolytic enzymes for the cytoplasmic domain of band 3 is probably involved in modulation. This metabolic modulation is connected to variations in intracellular NADPH and ATP levels as a function of the oxygenation state of the cell, and, consequently, it should have physiologic relevance. The present study investigates the effect of amyloid-beta peptide exposure on this metabolic modulation and its relationship with the activity of erythrocyte caspase 3. Metabolic differences between erythrocytes incubated at high and low oxygen saturation disappear following to 24 h exposure to amyloid-beta peptide. Western blotting analysis shows that caspase 3 is concurrently activated. Pre-incubation of amyloid-beta peptide-treated erythrocytes with a specific inhibitor of caspase 3, partially restores the oxygen-dependent modulation. This finding suggests that human erythrocytes following to exposure to amyloid-beta peptide show a complete loss of the oxygen-dependent metabolic modulation, which is partially restored by caspase 3 inhibitor-treatment.

Fragment 31-35 of beta-amyloid peptide induces neurodegeneration in rat cerebellar granule cells via bax gene expression and caspase-3 activation. A crucial role for the redox state of methionine-35 residue.

The amyloid beta-peptide (AbetaP) is the major protein component of brain senile plaques in Alzheimer's disease. The redox state of methionine-35 residue plays a critical role in peptide neurotoxic actions. We used the fragment 31-35 of AbetaP [AbetaP(31-35)], containing a single methionine-35 residue (Met-35), to investigate the relationship between the oxidative state of Met-35 and neurotoxic and pro-apoptotic actions induced by the peptide; in rat cerebellar granule cells (CGC), we compared the effects of AbetaP(31-35), in which the Met-35 is present in the reduced state, with those of a modified peptide with oxidized Met-35 [AbetaP(31-35)Met-35(OX)](,) as well as an AbetaP-derivative with Met-35 substituted by norleucine [AbetaP(31-35)Nle-35]. AbetaP(31-35) induced a time-dependent decrease in cell viability. AbetaP(31-35)Met-35(OX) was significantly less potent, but still induced a significant decrease in cell viability compared to control. No toxic effects were observed after treatment with AbetaP(31-35)Nle-35. AbetaP(31-35) induced a 2-fold increase in bax mRNA levels after 4h, whereas AbetaP(31-35)Met-35(OX) raised bax mRNA levels by 41% and AbetaP(31-35)Nle-35 had no effect. Finally, AbetaP(31-35) caused a 43% increase in caspase-3 activity after 24h; AbetaP(31-35)Met-35(OX) caused only a 18% increase, and AbetaP(31-35)Nle-35 had no effect. These findings suggest that AbetaP(31-35)-induced neurodegeneration in CGC is mediated by a selective early increase in bax mRNA levels followed by delayed caspase-3 activation; the redox state of the single Met-35 residue is crucial in the occurrence and extent of the above phenomena.

Abeta(31-35) and Abeta(25-35) fragments of amyloid beta-protein induce cellular death through apoptotic signals: Role of the redox state of methionine-35.

In order to clarify the basis of neuronal toxicity exerted by the shortest active peptides of amyloid beta-protein (Abeta), the toxic effects of Abeta(31-35) and Abeta(25-35) peptides on isolated rat brain mitochondria were investigated. The results show that exposure of isolated rat brain mitochondria to Abeta(31-35) and Abeta(25-35) peptides determines: (i) release of cytochrome c; (ii) mitochondrial swelling and (iii) a significant reduction in mitochondrial oxygen consumption. In contrast, the amplitude of these events resulted attenuated in isolated brain mitochondria exposed to the Abeta(31-35)Met35(OX) in which methionine-35 was oxidized to methionine sulfoxide. The Abeta peptide derivative with norleucine substituting Met-35, i.e., Abeta(31-35)Nle-35, had not effect on any of the biochemical parameters tested. We have further characterized the action of Abeta(31-35) and Abeta(25-35) peptides on neuronal cells. Taken together our result indicate that Abeta(31-35) and Abeta(25-35) peptides in non-aggregated form, i.e., predominantly monomeric, are strongly neurotoxic, having the ability to enter within the cells, determining mitochondrial damage with an evident trigger of apoptotic signals. Such a mechanism of toxicity seems to be dependent by the redox state of methionine-35.