Francesco Misiti

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

Taken together our result indicate that Aβ(31–35) and Aβ(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.

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.

Role of methionine 35 in the intracellular Ca2+ homeostasis dysregulation and Ca2+-dependent apoptosis induced by amyloid β-peptide in human neuroblastoma IMR32 cells

Amyloid β‐peptide (Aβ) plays a fundamental role in the pathogenesis of Alzheimer’s disease. We recently reported that the redox state of the methionine residue in position 35 of amyloid β‐peptide (Aβ) 1–42 (Met35) strongly affects the peptide’s ability to trigger apoptosis and is thus a major determinant of its neurotoxicity. Dysregulation of intracellular Ca2+ homeostasis resulting in the activation of pro‐apoptotic pathways has been proposed as a mechanism underlying Aβ toxicity. Therefore, we investigated correlations between the Met35 redox state, Aβ toxicity, and altered intracellular Ca2+ signaling in human neuroblastoma IMR32 cells. Cells incubated for 6–24u2003h with 10u2003μM Aβ1–42 exhibited significantly increased KCl‐induced Ca2+ transient amplitudes and resting free Ca2+ concentrations. Nifedipine‐sensitive Ca2+ current densities and Cav1 channel expression were markedly enhanced by Aβ1–42. None of these effects were observed when cells were exposed to Aβ containing oxidized Met35 (Aβ1–42Met35‐Ox). Cell pre‐treatment with the intracellular Ca2+ chelator 1,2‐bis(2‐aminophenoxy)ethane‐N,N,N’,N’‐tetraacetic acid acetoxymethyl ester (1u2003μM) or the Cav1 channel blocker nifedipine (5u2003μM) significantly attenuated Aβ1–42‐induced apoptosis but had no effect on Aβ1–42Met35‐Ox toxicity. Collectively, these data suggest that reduced Met35 plays a critical role in Aβ1–42 toxicity by rendering the peptide capable of disrupting intracellular Ca2+ homeostasis and thereby provoking apoptotic cell death.

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 Alzheimers 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 peptides 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 Alzheimers 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 Alzheimers disease.

Protective effect of rhubarb derivatives on amyloid beta (1-42) peptide-induced apoptosis in IMR-32 cells: a case of nutrigenomic.

Amyloid beta (1-42) peptide is considered responsible for the formation of senile plaques that accumulate in the brains of patients with Alzheimers disease (AD). In the last years considerable attention has been focused on identifying natural food products, such as phytochemicals that prevent or almost retard the appearance of amyloid beta (1-42)-related neurotoxic effects. In this study, human neuroblastoma cells (IMR-32) was used as system model to evaluate the protective role of rhaponticin (3,3,5-trihydroxy-4-methoxystilbene 3-O-d-glucoside) a stilbene glucoside extracted from rhubarb roots (Rhei rhizoma) and rhapontigenin, its aglycone metabolite, against amyloid beta (1-42)-dependent toxicity. The obtained results show that rhapontigenin maintains significant cell viability in a dose-dependent manner and it exerts a protective effect on mitochondrial functionality, as evidenced by mitochondrial oxygen consumption experiments. A similar behaviour, but to a lesser extent, has been shown by rhaponticin. The protective mechanism mediated by the two stilbenes could be related to their effect on bcl-2 gene family expression. Bax, a pro-apoptotic gene, resulted down-regulated by the treatment with rhaponticin and rhapontigenin compared with the results obtained in the presence of amyloid beta (1-42) peptide. Conversely, bcl-2, an anti-apoptotic gene, highly down-regulated by amyloid beta (1-42) treatment, resulted expressed in the presence of stilbenes similarly to that shown by control cells. The obtained results support the hypothesis that amyloid beta (1-42)-induced neurotoxicity occurs via bax over-expression, bcl-2 down-regulation, firstly indicating that rhaponticin and its aglycone moiety may alter this cell death pathway. Based on these studies, we suggest that rhaponticin and its main metabolite could be developed as agents for the management of AD.

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.

Antiepileptic carbamazepine drug treatment induces alteration of membrane in red blood cells: possible positive effects on metabolism and oxidative stress.

Carbamazepine (CBZ) is an iminostilbene derivative commonly used for treatment of neuralgic pain and bipolar affective disorders. CBZ blood levels of treated patients are within the range of micromolar concentrations and therefore, significant interactions of this drug with erythrocytes are very likely. Moreover, the lipid domains of the cell membrane are believed to be one of the sites where iminostilbene derivatives exert their effects. The present study aimed to deeply characterize CBZ effects on erythrocytes, in order to identify extra and/or cytosolic cell targets. Our results indicate that erythrocyte morphological changes promoted by the drug, may be triggered by an alteration in band 3 functionality i.e. at the level of anionic flux. In addition, from a metabolic point of view this perturbation could be considered, at least in part, as a beneficial event because it could favour the CO2 elimination. Since lipid peroxidation, superoxide and free radical scavenging activities, caspase 3 activity and hemoglobin (Hb) functionality were not modified within the CBZ treated red blood cell (RBC), band 3 protein (B3) may well be a specific membrane target for CBZ and responsible for CBZ-induced toxic effects in erythrocytes. However some beneficial effects of this drug have been evidenced; among them an increased release of ATP and nitric oxide (NO) derived metabolites from erythrocytes to lumen, leading to an increased NO pool in the vasculature. In conclusion, these results indicate that CBZ, though considered responsible for toxic effects on erythrocytes, can also exhibit effects that at least in some conditions may be seen as beneficial.

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.

β-amyloid decreases detectable endothelial nitric oxide synthase in human erythrocytes: a role for membrane acetylcholinesterase

Until few years ago, many studies of Alzheimers disease investigated the effects of this syndrome in the central nervous system. Only recently, the detection of amyloid beta peptide (Aβ) in the blood has evidenced the necessity to extend studies on extraneuronal cells, particularly on erythrocytes. Aβ is also present in brain capillaries, where it interacts with the erythrocytes, inducing several metabolic and functional alterations. Recently, functionally active endothelial type nitric oxide synthase (eNOS) was discovered in human erythrocytes. The goal of the present study was to evidence the effect of Aβ on erythrocyte eNOS. We found that Aβ following to 24‐h exposure causes a decrease in the immune staining of erythrocyte eNOS. Concurrently, Aβ alters erythrocyte cell morphology, decreases nitrites and nitrates levels, and affects membrane acetylcholinesterase activity. Propidium, an acetylcholinesterase inhibitor, was able to reverse the effects elicited by Aβ. These events could contribute to the vascular alterations associated with Alzheimers disease disease. Copyright

Involvement of acetylcholinesterase and protein kinase C in the protective effect of caffeine against β-amyloid-induced alterations in red blood cells.

It is well known the role of oxidative stress in the pathophysiology of Alzheimers disease (AD) and of other neurodegenerative pathologies. We have previously documented that Amyloid beta peptide (1-42) (Abeta) dependent-oxidative modifications affect red blood cell (RBC) morphology and function. Experimental studies show that caffeine (CF) consumption is inversely correlated with AD. In this study, we investigated the role played by RBC in the protective mechanism elicited by CF against Abeta mediated toxicity. PS exposure levels by FACS analysis, as well as protein band 3 functionality analysis, indicated that CF at 100xa0μM protected against Abeta-mediated membrane alterations, which are known to occur in AD. Moreover, CF counteracts inhibition of ATP release from RBC by Abeta, restoring its ability to modulate vasodilation. Concurrently, analysis of protein kinase C (PKC) and caspase 3 activities, responsible for cytoskeleton alterations, revealed that unlike to caspase 3, PKCα activation induced by Abeta was fully abolished by CF through a mechanism involving Acetylcholinesterase (AChE), located on external face of RBC plasma membrane. These results provide support for the hypothesis concerning the protective role of CF in AD patients could include also a peripheral mechanism involving RBC.