[Show abstract][Hide abstract] ABSTRACT: Alzheimer’s disease (AD) and cancer proceed via one or more common molecular mechanisms: a metabolic shift from oxidative phosphorylation to glycolysis—corresponding to the activation of the Warburg effect—occurs in both diseases. The findings reported in this paper demonstrate that, in the early phase of apoptosis, glucose metabolism is enhanced, i.e. key proteins which internalize and metabolize glucose—glucose transporter, hexokinase and phosphofructokinase—are up-regulated, in concomitance with a parallel decrease in oxygen consumption by mitochondria and increase of l-lactate accumulation. Reversal of the glycolytic phenotype occurs in the presence of dichloroacetate, inhibitor of the pyruvate dehydrogenase kinase enzyme, which speeds up apoptosis of cerebellar granule cells, reawakening mitochondria and then modulating glycolytic enzymes. Loss of the adaptive advantage afforded by aerobic glycolysis, which occurs in the late phase of apoptosis, exacerbates the pathological processes underlying neurodegeneration, leading inevitably the cell to death. In conclusion, the data propose that both aerobic, i.e.
Warburg effect, essentially due to the protective numbness of mitochondria, and anaerobic glycolysis, rather due to the mitochondrial impairment, characterize the entire time frame of apoptosis, from the early to the late phase, which mimics the development of AD.
[Show abstract][Hide abstract] ABSTRACT: The involvement of thioredoxin/thioredoxin reductase system has been investigated in cerebellar granule cells (CGCs), a cellular system in which neurons are induced in apoptosis by the physiological stimulus of lowering extracellular potassium. Clarifying the sequence of events that occur during apoptosis is a critical issue as it can lead to the identification of those key events that, if blocked, can slow down or reverse the death process. The results reported in this work show that TrxR is involved in the early phase of CGC apoptosis with an increase in activity that coincides with the increased expression of the TrxR1 isoform and guarantees the maintenance of adequate level of Trx in its reduced, active form. However, in late apoptosis, when about 50 % of cells are dead, partial proteolysis of TrxR1 by calpain occurs and the reduction of TrxR1 mRNA, together with the overall decrease in TrxR activity, contribute to increase the levels of the oxidized form of Trx. When the reduced form of Trx is externally added to apoptotic cultures, a significant reduction in cell death is achieved confirming that a well-functioning thioredoxin/thioredoxin reductase system is required for survival of CGCs.
[Show abstract][Hide abstract] ABSTRACT: Apoptosis in neuronal tissue is an efficient mechanism which contributes to both normal cell development and pathological cell death. The present study explores the effects of extracellular ADP on low [K(+)]-induced apoptosis in rat cerebellar granule cells. ADP, released into the extracellular space in brain by multiple mechanisms, can interact with its receptor or be converted, through the actions of ectoenzymes, to adenosine. The findings reported in this paper demonstrate that ADP inhibits the proapoptotic stimulus supposedly via: i) inhibition of ROS production during early stages of apoptosis, an effect mediated by its interaction with cell receptor/s. This conclusion is validated by the increase in SOD and Catalase activities as well as by the GSSG/GSH ratio value decrease, in conjunction with the drop of ROS level and the prevention of the ADP protective effect by pyridoxalphosphate-6-azophenyl-2',4'-disulfonic acid (PPADS), a novel functionally selective antagonist of purine receptor; ii) safeguard of the functionality of the mitochondrial adeninnucleotide-1 translocator (ANT-1), which is early impaired during apoptosis. This effect is mediated by its plausible internalization into cell occurring as such or after its hydrolysis, by means of plasma membrane nucleotide metabolizing enzymes, and resynthesis into the cell. Moreover, the findings that ADP also protects ANT-1 from the toxic action of the two Alzheimer disease peptides, i.e. Aβ1-42 and NH2htau, which are known to be produced in apoptotic cerebellar neurons, further corroborate the molecular mechanism of neuroprotection by ADP, herein proposed.
[Show abstract][Hide abstract] ABSTRACT: Evidence suggests a striking causal relationship between changes in quality control of neuronal mitochondria and numerous devastating human neurodegenerative diseases, including Parkinson's disease, Alzheimer's disease, Huntington's disease, and amyotrophic lateral sclerosis. Contrary to replicating mammalian cells with a metabolism essentially glycolytic, post-mitotic neurons are distinctive owing to (i) their exclusive energetic dependence from mitochondrial metabolism and (ii) their polarized shape, which entails compartmentalized and distinct energetic needs. Here, we review the recent findings on mitochondrial dynamics and mitophagy in differentiated neurons focusing on how the exceptional characteristics of neuronal populations in their morphology and bioenergetics needs make them quite different to other cells in controlling the intracellular turnover of these organelles.
[Show abstract][Hide abstract] ABSTRACT: Abstract Hydroxytyrosol (2-(3,4dihydroxyphenyl)ethanol, (DPE), a phenolic compound present in olive oil, is known to have antioxidant properties. The aim of this study was to investigate the effect of DPE on oxidative stress induced by cadmium injections (CdCl2 2.5 mg/kg body weight) in spleen and testes of adult male rats. Oxidative stress was evaluated by measuring lipid peroxidation by thiobarbituric acid reactive substances (TBARS) as well as superoxide dismutase (SOD) and catalase (CAT) activities in cytosol and mitochondria. We found that in spleen no TBARS formation was detected following CdCl2 injections; however, DPE induces decrease in TBARS level in treated and untreated rats. On the contrary, we observed that DPE showed no effect on cadmium-induced lipid peroxidation in testes. Cytosolic activities of SOD and CAT decreased significantly only in spleen, where DPE restores the values to the control levels. Noteworthy, mitochondrial activities of SOD and CAT were strongly reduced by cadmium treatment both in spleen and testes, and DPE was not be able to restore their activity. Overall, the results from this study indicated that the DPE has different antioxidant efficiency in spleen and testis of cadmium intoxicated rats.
Drug and Chemical Toxicology 01/2014; 37(4). DOI:10.3109/01480545.2013.878950 · 1.23 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: To find out whether and how the adenine nucleotide translocator-1 (ANT-1) inhibition due to NH2htau and Aβ1-42 is due to an interplay between these two Alzheimer's peptides, ROS and ANT-1 thiols, use was made of mersalyl, a reversible alkylating agent of thiol groups that are oriented toward the external hydrophilic phase, to selectively block and protect, in a reversible manner, the -SH groups of ANT-1. The rate of ATP appearance outside mitochondria was measured as the increase in NADPH absorbance which occurs, following external addition of ADP, when ATP is produced by oxidative phosphorylation and exported from mitochondria in the presence of glucose, hexokinase and glucose-6-phosphate dehydrogenase. We found that the mitochondrial superoxide anions, whose production is induced at the level of Complex I by externally added Aβ1-42 and whose release from mitochondria is significantly reduced by the addition of the VDAC inhibitor DIDS, modify the thiol group/s present at the active site of mitochondrial ANT-1, impair ANT-1 in a mersalyl-prevented manner and abrogate the toxic effect of NH2htau on ANT-1 when Aβ1-42 is already present. A molecular mechanism is proposed in which the pathological Aβ-NH2htau interplay on ANT-1 in Alzheimer's neurons involves the thiol redox state of ANT-1 and the Aβ1-42-induced ROS increase. This result represents an important innovation because it suggests the possibility of using various strategies to protect cells at the mitochondrial level, by stabilizing or restoring mitochondrial function or by interfering with the energy metabolism providing a promising tool for treating or preventing AD.
[Show abstract][Hide abstract] ABSTRACT: Here we investigate the effect of β-amyloid on mitochondrial respiratory function, i.e. mitochondrial oxygen consumption and membrane potential generation as well as the individual activities of both the mitochondrial complexes I-IV, that compose mitochondrial electron transport chain, and the ATP synthase, by using homogenate from cerebellar granule cells, treated with low concentrations of β-amyloid, and Alzheimer synaptic-enriched brain samples. We found that β-amyloid caused both a selective defect in Complex I activity associated with an increase (5 fold) of intracellular reactive oxygen species and an impairment of Complex IV likely due to membrane lipid peroxidation. Add to that, a 130 % increase of the GSSG/GSH ratio measured in Alzheimer brains with respect to age-matched controls. Know the mechanisms of action of β-amyloid could allow to mitigate or even to interrupt the toxic cascade that leads a cell to death. The results of this study represent an important innovation because they offer the possibility to act at mitochondrial level and on specific sites to protect cells, for example by preventing the interaction of β-amyloid with the identified targets, by stabilizing or by restoring mitochondrial function or by interfering with the energy metabolism.
[Show abstract][Hide abstract] ABSTRACT: In this paper, we discuss the interplay between beta-amyloid (Aβ) peptide, Tau fragments, oxidative stress, and mitochondria in the neuronal model of cerebellar granule neurons (CGNs) in which the molecular events reminiscent of AD are activated. The identification of the death route and the cause/effect relationships between the events leading to death could be helpful to manage the progression of apoptosis in neurodegeneration and to define antiapoptotic treatments acting on precocious steps of the death process. Mitochondrial dysfunction is among the earliest events linked to AD and might play a causative role in disease onset and progression. Recent studies on CGNs have shown that adenine nucleotide translocator (ANT) impairment, due to interaction with toxic N-ter Tau fragment, contributes in a significant manner to bioenergetic failure and mitochondrial dysfunction. These findings open a window for new therapeutic strategies aimed at preserving and/or improving mitochondrial function.
International Journal of Alzheimer's Disease 09/2010; 2010(11-12). DOI:10.4061/2010/621870
[Show abstract][Hide abstract] ABSTRACT: To investigate the role of cytochrome c (cyt c) release in yeast acetic acid-induced programmed cell death (AA-PCD), wild type (wt) and cells lacking metacaspase (Deltayca1), cytochrome c (Deltacyc1,7) and both (Deltacyc1,7Deltayca1) were compared for AA-PCD occurrence, hydrogen peroxide (H(2)O(2)) production and caspase activity. AA-PCD occurs in Deltacyc1,7 and Deltacyc1,7Deltayca1 cells slower than in wt, but similar to that in Deltayca1 cells, in which no cytochrome c release occurs. Both H(2)O(2) production and caspase activation occur in these cells with early and extra-activation in Deltacyc1,7 cells. We conclude that alternative death pathways can be activated in yeast AA-PCD, one dependent on cyt c release, which requires YCA1, and the other(s) independent on it.
[Show abstract][Hide abstract] ABSTRACT: We have investigated the ability of certain dietary flavonoids, known to exert beneficial effects on the central nervous system, to affect neuronal apoptosis. We used cerebellar granule cells undergoing apoptosis due to potassium deprivation in a serum-free medium in either the absence or presence of the flavonoids genistein and daidzein, which are present in soy, and of catechin and epicatechin, which are present in cocoa. These compounds were used in a blood dietary concentration range. We found that genistein and daidzein, but not catechin and epicatechin, prevented apoptosis, with cell survival measured 24h after the induction of apoptosis being higher than that of the same cells incubated in flavonoid free medium (80% and 40%, respectively); there was no effect in control cells. A detailed investigation of the effect of these compounds on certain mitochondrial events that occur in cells en route to apoptosis showed that genistein and daidzein prevented the impairment of glucose oxidation and mitochondrial coupling, reduced cytochrome c release, and prevented both impairment of the adenine nucleotide translocator and opening of the mitochondrial permeability transition pore. Interestingly, genistein and daidzein were found to reduce the levels of reactive oxygen species, which are elevated in cerebellar granule cell apoptosis. These findings strongly suggest that the prevention of apoptosis depends mainly on the antioxidant properties of genistein and daidzein. This could lead to the development of a flavonoid-based therapy in neuropathies.
[Show abstract][Hide abstract] ABSTRACT: Having confirmed that adenovirus-mediated overexpression of NH(2)-tau fragment lacking the first 25 aminoacids evokes a potent neurotoxic effect, sustained by protracted stimulation of NMDA receptors, in primary neuronal cultures we investigated whether and how chemically synthesized NH(2)-derived tau peptides, i.e. NH(2)-26-44 and NH(2)-1-25 fragments, affect mitochondrial function. We tested both fragments on each step of the processes leading to ATP synthesis via oxidative phosphorylation: i) electron flow via the respiratory chain from physiological substrates to oxygen with the activity of each individual complex of the respiratory chain investigated in some detail, ii) membrane potential generation arising from externally added succinate and iii) the activity of both the adenine nucleotide translocator and iv) ATP synthase. Oxidative phosphorylation is not affected by NH(2)-1-25 tau fragment, but dramatically impaired by NH(2)-26-44 tau fragment. Both cytochrome c oxidase and the adenine nucleotide translocator are targets of NH(2)-26-44 tau fragment, but adenine nucleotide translocator is the unique mitochondrial target responsible for impairment of oxidative phosphorylation by the NH(2)-26-44 tau fragment, which then exerts deleterious effects on cellular availability of ATP synthesized into mitochondria.
[Show abstract][Hide abstract] ABSTRACT: An early increase in ROS production is characteristic of cerebellar granule cells undergoing apoptosis in the presence of 5 mM KCl. However, the sources of this increase have not been investigated in detail. In particular whether there is a single enzymatic source or the increase in ROS production is the consequence of the involvement of different enzymes has not been studied in depth. Different enzymatic pathways may indeed contribute to the up-regulation of intracellular ROS production either directly or via side-chain reactions and a number of candidate enzymes are known to be involved in the apoptotic process in various cell types. The aim of this study was to identify the cellular sources of the ROS generated by CGCs undergoing apoptosis by low K+. A panel of specific inhibitors against phospholipase, cytochromes P450, cyclooxygenase, lipoxygenase, xanthine oxidase, ribonucleotide reductase and NADPH oxidase were used. We provide evidence that no single source of ROS can be identified in apoptotic CGCs, but the ROS generated through the arachidonic acid (AA) pathways, mainly via lipoxygenase activities, seems to be the most prominent.
International Journal of Molecular Medicine 07/2008; 21(6):737-45. DOI:10.3892/ijmm.21.6.737 · 2.09 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Having confirmed that externally added L-lactate can enter cerebellar granule cells, we investigated whether and how L-lactate is metabolized by mitochondria from these cells under normal or apoptotic conditions. (1) L-lactate enters mitochondria, perhaps via an L-lactate/H+ symporter, and is oxidized in a manner stimulated by ADP. The existence of an L-lactate dehydrogenase, located in the inner mitochondrial compartment, was shown by immunological analysis. Neither the protein level nor the Km and Vmax values changed en route to apoptosis. (2) In both normal and apoptotic cell homogenates, externally added L-lactate caused reduction of the intramitochondrial pyridine cofactors, inhibited by phenylsuccinate. This process mirrored L-lactate uptake by mitochondria and occurred with a hyperbolic dependence on L-lactate concentrations. Pyruvate appeared outside mitochondria as a result of external addition of L-lactate. The rate of the process depended on L-lactate concentration and showed saturation characteristics. This shows the occurrence of an intracellular L-lactate/pyruvate shuttle, whose activity was limited by the putative L-lactate/pyruvate antiporter. Both the carriers were different from the monocarboxylate carrier. (3) L-lactate transport changed en route to apoptosis. Uptake increased in the early phase of apoptosis, but decreased in the late phase with characteristics of a non-competitive like inhibition. In contrast, the putative L-lactate/pyruvate antiport decreased en route to apoptosis with characteristics of a competitive like inhibition in early apoptosis, and a mixed non-competitive like inhibition in late apoptosis.
[Show abstract][Hide abstract] ABSTRACT: The involvement and the role of nitric oxide (NO) as a signaling molecule in the course of neuronal apoptosis, whether unique or modulated during the progression of the apoptotic program, has been investigated in a cellular system consisting of cerebellar granule cells (CGCs) where apoptosis can be induced by lowering extracellular potassium. Several parameters involved in NO signaling pathway, such as NO production, neuronal nitric oxide synthase (nNOS) expression, and cyclic GMP (cGMP) production were examined in the presence or absence of different inhibitors. We provide evidence that nitric oxide has dual and opposite effects depending on time after induction of apoptosis. In an early phase, up to 3 h of apoptosis, nitric oxide supports survival of CGCs through a cGMP-dependent mechanism. After 3 h, nNOS expression and activity decreased resulting in shut down of NO and cGMP production. Residual NO then contributes to the apoptotic process by reacting with rising superoxide anions leading to peroxynitrite production and protein inactivation. We conclude that whilst NO over-production protects neurons from death in the early phase of neuronal damage, its subsequent reduction may contribute to neuronal degeneration and ultimate cell death.
[Show abstract][Hide abstract] ABSTRACT: To find out whether and how proteasome is involved in plant programmed cell death (PCD) we measured proteasome function in tobacco cells undergoing PCD as a result of heat shock (HS-PCD). Reactive oxygen species (ROS) production, cytochrome c levels and caspase-3-like protease activation were also measured in the absence or presence of MG132, a proteasome inhibitor. We show that proteasome activation occurs in early phase of HS-PCD upstream of the caspase-like proteases activation; moreover inhibition of proteasome function by MG132 results in prevention of PCD perhaps due to the prevention of ROS production, cytochrome c release and caspase-3-like protease activation.
[Show abstract][Hide abstract] ABSTRACT: To gain some insight into the mechanism of plant programmed cell death, certain features of cytochrome c (cyt c) release were investigated in heat-shocked tobacco (Nicotiana tabacum) Bright-Yellow 2 cells in the 2- to 6-h time range. We found that 2 h after heat shock, cyt c is released from intact mitochondria into the cytoplasm as a functionally active protein. Such a release did not occur in the presence of superoxide anion dismutase and catalase, thus showing that it depends on reactive oxygen species (ROS). Interestingly, ROS production due to xanthine plus xanthine oxidase results in cyt c release in sister control cultures. Maximal cyt c release was found 2 h after heat shock; later, activation of caspase-3-like protease was found to increase with time. Activation of this protease did not occur in the presence of ROS scavenger enzymes. The released cyt c was found to be progressively degraded in a manner prevented by either the broad-range caspase inhibitor (zVAD-fmk) or the specific inhibitor of caspase-3 (AC-DEVD-CHO), which have no effect on cyt c release. In the presence of these inhibitors, a significant increase in survival of the cells undergoing programmed cell death was found. We conclude that ROS can trigger release of cyt c, but do not cause cell death, which requires caspase-like activation.
[Show abstract][Hide abstract] ABSTRACT: We investigated ADP/ATP exchange mediated by the adenine nucleotide translocator and opening of the mitochondrial permeability transition pore in homogenates from cerebellar granule cells en route to apoptosis induced by low potassium. We showed that, in the first 3 h of apoptosis, when maximum cytochrome c release had already occurred, adenine nucleotide translocator function was impaired owing to the action of reactive oxygen species, but no permeability transition pore opening occurred. Over 3-8 h of apoptosis, the permeability transition pore progressively opened, owing to caspase action, and further ADP/ATP translocator impairment occurred. The kinetics of transport and permeability transition pore opening were inversely correlated, both in the absence and presence of inhibitors of antioxidant and proteolytic systems. We conclude that, en route to apoptosis, alteration of the adenine nucleotide translocator occurs, resulting in permeability transition pore opening. This process depends on the action of caspase on pore component(s) other than the ADP/ATP translocator, because no change in either amount or molecular weight of the latter protein was noted during apoptosis, as measured by western blotting. Cell death occurs via apoptosis in the presence of cyclosporin A, the permeability transition pore inhibitor, thus showing that permeability transition pore opening, not needed for cytochrome c release, is also unnecessary for apoptosis to occur.
Journal of Neurochemistry 06/2006; 97(4):1166-81. DOI:10.1111/j.1471-4159.2006.03820.x · 4.28 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: We investigated the occurrence of the plant Uncoupling Protein (UCP) in mitochondria isolated from both fresh (f-JAM) and aged-dehydrated (a-d-JAM) slices of Jerusalem artichoke tubers (Helianthus tuberosus L.). The presence of UCP was shown by immunological analysis and its function was investigated by measuring the decrease of the mitochondrial membrane potential due to linoleic acid (LA) and its inhibition by purine nucleotides under conditions in which the adenine nucleotide translocator (ANT) was inhibited by atractyloside (Atr). f-JAM and a-d-JAM had the same protein content, but differed from one another with respect to purine nucleotide inhibition, substrate specificity, and sensitivity to ROS. Hydrogen peroxide and superoxide anion, generated in situ by xanthine plus xanthine oxidase, caused a significant increase in the UCP function in a-d-JAM, but not in f-JAM. This occurred in a manner sensitive to ATP, but not to Atr, thus showing that ANT has no role in the process. The dependence of the rate of membrane potential decrease on increasing LA concentrations, either in the absence or the presence of ROS, showed a sigmoidal saturation both in f-JAM and a-d-JAM. However, addition of ROS in a-d-JAM resulted in about 40% increase of the Vmax value, with no change in the K0.5 (about 20 microM), whereas in f-JAM no effect on either the Vmax or K0.5 (about 28 microM) was found. Furthermore, a decreased ROS production as a result of LA addition was found in both f-JAM and a-d-JAM, the effect being more marked in a-d-JAM.
[Show abstract][Hide abstract] ABSTRACT: Mutational analysis of the cystic fibrosis transmembrane regulator (CFTR) gene was performed in 98 unrelated CF chromosomes from 49 Lithuanian CF patients through a combined approach in which the p.F508del mutation was first screened by allele-specific PCR while CFTR mutations in nonp.F508del chromosomes have been screened for by denaturing gradient gel electrophoresis analysis. A CFTR mutation was characterized in 62.2% of CF chromosomes, two of which (2.0%) have been previously shown to carry a large gene deletion CFTRdele2,3(21 kb). The most frequent Lithuanian CF mutation is p.F508del (52.0%). Seven CFTR mutations, p.N1303K (2.0%), p.R75Q (1.0%), p.G314R (1.0%), p.R553X (4.2%), p.W1282X (1.0%), and g.3944delGT (1.0%), accounted for 10.1% of Lithuanian CF chromosomes. It was not possible to characterize 35.8% of the CF Lithuanian chromosomes. Analysis of intron 8 (TG)mTn and M470V polymorphic loci did not permit the characterization of the CFTR dysfunction underlying the CF phenotype in the patients for which no CFTR mutation was identified. Thus, screening of the eight CFTR mutations identified in this study and of the large deletion CFTRdele2,3(21 kb) allows the implementation of an early molecular or confirmatory CF diagnosis for 65% of Lithuanian CF chromosomes.