Publications by authors named "Antonella Bobba"

23 Publications

  • Page 1 of 1

Functional Foods: An Approach to Modulate Molecular Mechanisms of Alzheimer's Disease.

Cells 2020 10 23;9(11). Epub 2020 Oct 23.

European Brain Research Institute (EBRI), Viale Regina Elena 295, 00161 Rome, Italy.

A new epoch is emerging with intense research on nutraceuticals, i.e., "food or food product that provides medical or health benefits including the prevention and treatment of diseases", such as Alzheimer's disease. Nutraceuticals act at different biochemical and metabolic levels and much evidence shows their neuroprotective effects; in particular, they are able to provide protection against mitochondrial damage, oxidative stress, toxicity of β-amyloid and Tau and cell death. They have been shown to influence the composition of the intestinal microbiota significantly contributing to the discovery that differential microorganisms composition is associated with the formation and aggregation of cerebral toxic proteins. Further, the routes of interaction between epigenetic mechanisms and the microbiota-gut-brain axis have been elucidated, thus establishing a modulatory role of diet-induced epigenetic changes of gut microbiota in shaping the brain. This review examines recent scientific literature addressing the beneficial effects of some natural products for which mechanistic evidence to prevent or slowdown AD are available. Even if the road is still long, the results are already exceptional.
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http://dx.doi.org/10.3390/cells9112347DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7690784PMC
October 2020

An Intriguing Involvement of Mitochondria in Cystic Fibrosis.

J Clin Med 2019 Nov 6;8(11). Epub 2019 Nov 6.

Istituto di Biomembrane, Bioenergetica e Biotecnologie Molecolari -CNR, Via G. Amendola 122/O, 70126 Bari, Italy.

Cystic fibrosis (CF) occurs when the cystic fibrosis transmembrane conductance regulator (CFTR) protein is not synthetized and folded correctly. The CFTR protein helps to maintain the balance of salt and water on many body surfaces, such as the lung surface. When the protein is not working correctly, chloride becomes trapped in cells, then water cannot hydrate the cellular surface and the mucus covering the cells becomes thick and sticky. Furthermore, a defective CFTR appears to produce a redox imbalance in epithelial cells and extracellular fluids and to cause an abnormal generation of reactive oxygen species: as a consequence, oxidative stress has been implicated as a causative factor in the aetiology of the process. Moreover, massive evidences show that defective CFTR gives rise to extracellular GSH level decrease and elevated glucose concentrations in airway surface liquid (ASL), thus encouraging lung infection by pathogens in the CF advancement. Recent research in progress aims to rediscover a possible role of mitochondria in CF. Here the latest new and recent studies on mitochondrial bioenergetics are collected. Surprisingly, they have enabled us to ascertain that mitochondria have a leading role in opposing the high ASL glucose level as well as oxidative stress in CF.
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http://dx.doi.org/10.3390/jcm8111890DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6912654PMC
November 2019

Extracellular truncated tau causes early presynaptic dysfunction associated with Alzheimer's disease and other tauopathies.

Oncotarget 2017 Sep 22;8(39):64745-64778. Epub 2017 Apr 22.

European Brain Research Institute, Rome, Italy.

The largest part of tau secreted from AD nerve terminals and released in cerebral spinal fluid (CSF) is C-terminally truncated, soluble and unaggregated supporting potential extracellular role(s) of NH -derived fragments of protein on synaptic dysfunction underlying neurodegenerative tauopathies, including Alzheimer's disease (AD). Here we show that sub-toxic doses of extracellular-applied human NH tau 26-44 (aka NH htau) -which is the minimal active moiety of neurotoxic 20-22kDa peptide accumulating at AD synapses and secreted into parenchyma- acutely provokes presynaptic deficit in K -evoked glutamate release on hippocampal synaptosomes along with alteration in local Ca dynamics. Neuritic dystrophy, microtubules breakdown, deregulation in presynaptic proteins and loss of mitochondria located at nerve endings are detected in hippocampal cultures only after prolonged exposure to NH htau. The specificity of these biological effects is supported by the lack of any significant change, either on neuronal activity or on cellular integrity, shown by administration of its reverse sequence counterpart which behaves as an inactive control, likely due to a poor conformational flexibility which makes it unable to dynamically perturb biomembrane-like environments. Our results demonstrate that one of the AD-relevant, soluble and secreted N-terminally truncated tau forms can early contribute to pathology outside of neurons causing alterations in synaptic activity at presynaptic level, independently of overt neurodegeneration.
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http://dx.doi.org/10.18632/oncotarget.17371DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5630290PMC
September 2017

A disease with a sweet tooth: exploring the Warburg effect in Alzheimer's disease.

Biogerontology 2017 06 17;18(3):301-319. Epub 2017 Mar 17.

Institute of Translational Pharmacology (IFT), National Research Council (CNR), Via Fosso del Cavaliere 100, 00133, Rome, Italy.

After more than 80 years from the revolutionary discoveries of Otto Warburg, who observed high glucose dependency, with increased glycolysis and lactate production regardless of oxygen availability in most cancer cells, the 'Warburg effect' returns to the fore in neuronal cells affected by Alzheimer's disease (AD). Indeed, it seems that, in the mild phase of AD, neuronal cells "prefer" to use the energetically inefficient method of burning glucose by glycolysis, as in cancer, proving to become resistant to β-amyloid (Aβ)-dependent apoptosis. However, in the late phase, while most AD brain cells die in response to Aβ toxicity, only small populations of neurons, exhibiting increased glucose uptake and glycolytic flux, are able to survive as they are resistant to Aβ. Here we draw an overview on the metabolic shift for glucose utilization from oxidative phosphorylation to glycolysis, focusing on the hypothesis that, as extreme attempt to oppose the impending death, mitochondria-whose dysfunction and central role in Aβ toxicity is an AD hallmark-are sent into quiescence, this likely contributing to activate mechanisms of resistance to Aβ-dependent apoptosis. Finally, the attempt turns out fruitless since the loss of the adaptive advantage afforded by elevated aerobic glycolysis exacerbates the pathophysiological processes associated with AD, making the brain susceptible to Aβ-induced neurotoxicity and leading to cell death and dementia. The understanding of how certain nerve cells become resistant to Aβ toxicity, while the majority dies, is an attractive challenge toward the identification of novel possible targets for AD therapy.
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http://dx.doi.org/10.1007/s10522-017-9692-xDOI Listing
June 2017

Characterization of mitochondrial function in cells with impaired cystic fibrosis transmembrane conductance regulator (CFTR) function.

J Bioenerg Biomembr 2016 06 5;48(3):197-210. Epub 2016 May 5.

Department of Biosciences, Biotechnology and Biopharmaceutics, University of Bari, Via E. Orabona 4, 70126, Bari, Italy.

Evidence supporting the occurrence of oxidative stress in Cystic Fibrosis (CF) is well established and the literature suggests that oxidative stress is inseparably linked to mitochondrial dysfunction. Here, we have characterized mitochondrial function, in particular as it regards the steps of oxidative phosphorylation and ROS production, in airway cells either homozygous for the F508del-CFTR allele or stably expressing wt-CFTR. We find that oxygen consumption, ΔΨ generation, adenine nucleotide translocator-dependent ADP/ATP exchange and both mitochondrial Complex I and IV activities are impaired in CF cells, while both mitochondrial ROS production and membrane lipid peroxidation increase. Importantly, treatment of CF cells with the small molecules VX-809 and 4,6,4'-trimethylangelicin, which act as "correctors" for F508del CFTR by rescuing the F508del CFTR-dependent chloride secretion, while having no effect per sè on mitochondrial function in wt-CFTR cells, significantly improved all the above mitochondrial parameters towards values found in the airway cells expressing wt-CFTR. This novel study on mitochondrial bioenergetics provides a springboard for future research to further understand the molecular mechanisms responsible for the involvement of mitochondria in CF and identify the proteins primarily responsible for the F508del-CFTR-dependent mitochondrial impairment and thus reveal potential novel targets for CF therapy.
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http://dx.doi.org/10.1007/s10863-016-9663-yDOI Listing
June 2016

Morphological and bioenergetic demands underlying the mitophagy in post-mitotic neurons: the pink-parkin pathway.

Front Aging Neurosci 2014 18;6:18. Epub 2014 Feb 18.

European Brain Research Institute Rome, Italy.

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.
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http://dx.doi.org/10.3389/fnagi.2014.00018DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3927396PMC
March 2014

Antioxidant role of hydroxytyrosol on oxidative stress in cadmium-intoxicated rats: different effect in spleen and testes.

Drug Chem Toxicol 2014 Oct 20;37(4):420-6. Epub 2014 Jan 20.

Department of Veterinary Medicine, University of Bari "Aldo Moro" , Str. Prov. per Casamassima km 3 , Valenzano, Italy .

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.
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http://dx.doi.org/10.3109/01480545.2013.878950DOI Listing
October 2014

Alzheimer's proteins, oxidative stress, and mitochondrial dysfunction interplay in a neuronal model of Alzheimer's disease.

Int J Alzheimers Dis 2010 Sep 2;2010. Epub 2010 Sep 2.

Istituto di Biomembrane e Bioenergetica, CNR, Via Amendola 165/A, 70126 Bari, Italy.

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.
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http://dx.doi.org/10.4061/2010/621870DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2939402PMC
September 2010

Yeast acetic acid-induced programmed cell death can occur without cytochrome c release which requires metacaspase YCA1.

FEBS Lett 2010 Jan;584(1):224-8

CNR, Istituto di Biomembrane e Bioenergetica, Bari, Italy.

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.
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http://dx.doi.org/10.1016/j.febslet.2009.11.072DOI Listing
January 2010

Genistein and daidzein prevent low potassium-dependent apoptosis of cerebellar granule cells.

Biochem Pharmacol 2010 Mar 12;79(5):758-67. Epub 2009 Oct 12.

Istituto di Biomembrane e Bioenergetica, CNR, Via G. Amendola 165/A, 70126 Bari, Italy.

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.
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http://dx.doi.org/10.1016/j.bcp.2009.10.005DOI Listing
March 2010

Different sources of reactive oxygen species contribute to low potassium-induced apoptosis in cerebellar granule cells.

Int J Mol Med 2008 Jun;21(6):737-45

Istituto di Biomembrane e Bioenergetica, Consiglio Nazionale delle Ricerche, I-70126 Bari, Italy.

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.
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June 2008

Transport and metabolism of L-lactate occur in mitochondria from cerebellar granule cells and are modified in cells undergoing low potassium dependent apoptosis.

Biochim Biophys Acta 2007 Nov 15;1767(11):1285-99. Epub 2007 Sep 15.

Istituto di Biomembrane e Bioenergetica, Consiglio Nazionale delle Ricerche, Via G Amendola, 165/A-70126, Bari, Italy.

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.
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http://dx.doi.org/10.1016/j.bbabio.2007.08.003DOI Listing
November 2007

Nitric oxide has dual opposite roles during early and late phases of apoptosis in cerebellar granule neurons.

Apoptosis 2007 Sep;12(9):1597-610

Istituto di Biomembrane e Bioenergetica, Consiglio Nazionale delle Ricerche, Via Amendola 165/A, Bari 70126, Italy.

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.
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http://dx.doi.org/10.1007/s10495-007-0086-4DOI Listing
September 2007

Proteasome function is required for activation of programmed cell death in heat shocked tobacco Bright-Yellow 2 cells.

FEBS Lett 2007 Mar 6;581(5):917-22. Epub 2007 Feb 6.

Istituto di Biomembrane e Bioenergetica, Consiglio Nazionale delle Ricerche, Via Amendola 165/A, I-70126 Bari, Italy.

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.
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http://dx.doi.org/10.1016/j.febslet.2007.01.071DOI Listing
March 2007

Caspase-dependent alteration of the ADP/ATP translocator triggers the mitochondrial permeability transition which is not required for the low-potassium-dependent apoptosis of cerebellar granule cells.

J Neurochem 2006 May 5;97(4):1166-81. Epub 2006 Apr 5.

Istituto di Biomembrane e Bioenergetica, Consiglio Nazionale delle Ricerche, Bari, Italy.

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.
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http://dx.doi.org/10.1111/j.1471-4159.2006.03820.xDOI Listing
May 2006

Cytochrome c is released in a reactive oxygen species-dependent manner and is degraded via caspase-like proteases in tobacco Bright-Yellow 2 cells en route to heat shock-induced cell death.

Plant Physiol 2006 May 10;141(1):208-19. Epub 2006 Mar 10.

Istituto di Biomembrane e Bioenergetica, Consiglio Nazionale delle Ricerche, I-70126 Bari, Italy.

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.
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http://dx.doi.org/10.1104/pp.106.078683DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1459318PMC
May 2006

Plant uncoupling protein in mitochondria from aged-dehydrated slices of Jerusalem artichoke tubers becomes sensitive to superoxide and to hydrogen peroxide without increase in protein level.

Biochimie 2006 Feb 19;88(2):179-88. Epub 2005 Aug 19.

Dipartimento di Scienze Animali, Vegetali e dell'Ambiente, Facoltà di Agraria, Università del Molise, Via De Sanctis, 86100 Campobasso, Italy.

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.
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http://dx.doi.org/10.1016/j.biochi.2005.07.009DOI Listing
February 2006

An increase in the ATP levels occurs in cerebellar granule cells en route to apoptosis in which ATP derives from both oxidative phosphorylation and anaerobic glycolysis.

Biochim Biophys Acta 2005 Jun 7;1708(1):50-62. Epub 2005 Feb 7.

Istituto di Biomembrane e Bioenergetica, CNR, Via G. Amendola, 165/A-70126 Bari, Italy.

Although it is recognized that ATP plays a part in apoptosis, whether and how its level changes en route to apoptosis as well as how ATP is synthesized has not been fully investigated. We have addressed these questions using cultured cerebellar granule cells. In particular, we measured the content of ATP, ADP, AMP, IMP, inosine, adenosine and L-lactate in cells undergoing apoptosis during the commitment phase (0-8 h) in the absence or presence of oligomycin or/and of citrate, which can inhibit totally the mitochondrial oxidative phosphorylation and largely the substrate-level phosphorylation in glycolysis, respectively. In the absence of inhibitors, apoptosis was accompanied by an increase in ATP and a decrease in ADP with 1:1 stoichiometry, with maximum ATP level found at 3 h apoptosis, but with no change in levels of AMP and its breakdown products and with a relatively low level of L-lactate production. Consistently, there was an increase in the cell energy charge and in the ratio ([ATP][AMP])/[ADP](2). When the oxidative phosphorylation was completely blocked by oligomycin, a decrease of the ATP content was found both in control cells and in cells undergoing apoptosis, but nonetheless cells still died by apoptosis, as shown by checking DNA laddering and by death prevention due to actinomycin D. In this case, ATP was provided by anaerobic glycolysis, as suggested by the large increase of L-lactate production. On the other hand, citrate itself caused a small decrease in ATP level together with a huge decrease in L-lactate production, but it had no effect on cell survival. When ATP level was further decreased due to the presence of both oligomycin and citrate, death occurred via necrosis at 8 h, as shown by the lack of DNA laddering and by death prevention found due to the NMDA receptor antagonist MK801. However, at a longer time, when ATP level was further decreased, cells died neither via apoptosis nor via glutamate-dependent necrosis, in a manner similar to something like to energy catastrophe. Our results shows that cellular ATP content increases in cerebellar granule cell apoptosis, that the role of oxidative phosphorylation is facultative, i.e. ATP can also derive from anaerobic glycolysis, and that the type of cell death depends on the ATP availability.
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http://dx.doi.org/10.1016/j.bbabio.2005.01.009DOI Listing
June 2005

Apoptosis and cytochrome c release in cerebellar granule cells.

In Vivo 2004 May-Jun;18(3):335-44

Istituto di Biomembrane e Bioenergetica, CNR, Via G Amendola, 165/A - 70126 Bari.

In the light of both the major role played by released cytochrome c in apoptosis of a variety of cells and the availability of cerebellar granule cells as a model system to investigate apoptosis as a function of time from induction to cell death, we review data aimed at elucidating the events dealing with cytochrome c release from mitochondria as well as its role outside mitochondria. We report cytochrome c release in the apoptosis time course as dependent on the function of both the antioxidant and proteolytic systems. We show that, beside the role played by cytochrome c in participating in apoptosome formation and in triggering the caspase cascade, at least in cerebellar granule cells, released cytochrome c can maintain its ability to work as an electron carrier, being a scavenger of reactive oxygen species and an electron donor to cytochrome oxidase, thus driving the ATP synthesis.
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March 2005

Mitochondrial impairment induces excitotoxic death in cerebellar granule cells.

Int J Mol Med 2004 Jun;13(6):873-6

Istituto di Biomembrane e Bioenergetica, CNR, I-70126 Bari, Italy.

A close relationship links mitochondria to cell death with mitochondrial function-impairment considered a major biochemical event in the process of both apoptosis and necrosis. We have used different inhibitors of oxidative phosphorylation, i.e. mitochondrial respiratory chain and ATP synthesis inhibitors, and an uncoupler to investigate the mode of cell death caused by these compounds in cerebellar granule cells. This study shows that in cultured cerebellar granule cells either oxidative phosphorylation inhibitors or uncoupler induce an excitotoxic-like reaction which is mediated by activation of NMDA receptors and is likely due to the release of glutamate. Consistently, survival may occur if the toxic action of glutamate is prevented.
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June 2004

Cytochrome c, released from cerebellar granule cells undergoing apoptosis or excytotoxic death, can generate protonmotive force and drive ATP synthesis in isolated mitochondria.

J Neurochem 2003 Aug;86(3):591-604

Istituto di Biomembrane e Bioenergetica, CNR, Via G. Amendola, Bari, Italy.

In rat cerebellar granule cells, cytochrome c release takes place during glutamate toxicity and apoptosis due to deprivation of depolarising levels of potassium. We show that, as in necrosis, the released cytochrome c present in the cytosolic fraction obtained from cerebellar granule cells undergoing apoptosis can operate as a reactive oxygen species (ROS) scavenger and as a respiratory substrate. The capability of the cytosolic fraction containing cytochrome c, obtained from cerebellar granule cells undergoing either necrosis or apoptosis, to energise coupled mitochondria isolated by the same cells is also investigated. We show that, in both cases, the cytosolic fraction containing cytochrome c, added to mitochondria, can cause proton ejection, and membrane potential generation and can drive ATP synthesis and export in the extramitochondrial phase, as photometrically measured via the ATP detecting system. Cytochrome c, separated immunologically from the cytosolic fraction of apoptotic cells when added to mitochondria, is found to cause proton ejection to generate membrane potential and to drive ATP synthesis and export in a manner not sensitive to the further addition of the cytosolic fraction depleted of cytochrome c, which failed to do this. In the light of these findings we propose that in apoptosis the released cytochrome c can contribute to provide ATP required for the cell programmed death to occur.
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http://dx.doi.org/10.1046/j.1471-4159.2003.01863.xDOI Listing
August 2003

The apoptosis/necrosis transition in cerebellar granule cells depends on the mutual relationship of the antioxidant and the proteolytic systems which regulate ROS production and cytochrome c release en route to death.

J Neurochem 2003 Mar;84(5):960-71

Istituto di Biomembrane e Bioenergetica CNR, Via Amendola 165/A, 70126 Bari, Italy.

We investigate the death route induced by potassium depletion in cerebellar granule cells in 0-15 h time range and study whether and how mutual relationship occurs between the cell antioxidant and proteolytic system. To achieve this, we incubated cells in the absence or presence of inhibitors of the antioxidant system, including superoxide dismutase and catalase, and of the proteolytic system, consisting of proteasomes and caspases, and investigated whether and how (i) cell survival, (ii) reactive oxygen species (ROS) production and (iii) antioxidant enzyme and caspase-3 activity change as a function of time after the apoptotic stimulus. The involvement of both antioxidant and proteolytic system on cytochrome c release was also investigated. Cell survival was found to increase in the presence of either proteasome or caspase inhibitors. On the contrary, as a result of the antioxidant system impairment, shift from apoptosis to necrosis occurs. We show that the antioxidant system, which exhibits a huge activity increase up to 3 h after apoptosis induction, is subjected to the proteasome-dependent proteolysis and that the increase in the antioxidant system found in the absence of proteasome activity is accompanied by ROS production decrease. Consistently, the early ROS-dependent release of cytochrome c was found to be prevented when the activity of the antioxidant system increased. Finally, caspase-3 activation was prevented by the inhibitors of both antioxidant system and proteasome.
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http://dx.doi.org/10.1046/j.1471-4159.2003.01613.xDOI Listing
March 2003

Proteasome inhibitors prevent cytochrome c release during apoptosis but not in excitotoxic death of cerebellar granule neurons.

FEBS Lett 2002 Mar;515(1-3):8-12

Centro di Studio sui Mitocondri e Metabolismo Energetico, CNR, Via Amendola 165/A, 70126, Bari, Italy.

In order to find out whether and how proteasomes participate in the processes leading cerebellar granule cells to death either in necrosis, due to glutamate neurotoxicity, or in apoptosis, due to K(+) shift, we measured the three proteasome activities by using specific fluorescent probes and investigated the effect of several proteasome inhibitors, including MG132, on the cytochrome c release taking place in the early phase of both apoptosis and necrosis. We show that differently from apoptosis, the early phase of necrosis does not require proteasome activation. Inhibition of proteasome activity can prevent cytochrome c release in cerebellar granule cells undergoing apoptosis, thus improving cell survival, but not necrosis. These findings show that proteasomes play an important role in the early phase of apoptosis but not that of necrosis, and that these two types of cell death differ from each other in their mechanism of cytochrome c release.
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http://dx.doi.org/10.1016/s0014-5793(02)02231-7DOI Listing
March 2002
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