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Mitochondrial RTG-dependent retrograde signaling, whose regulators have been characterized in Saccharomyces cerevisiae, plays a recognized role under various environmental stresses. Of special significance, the activity of the transcriptional complex Rtg1/3 has been shown to be modulated by Hog1, the master regulator of the high osmolarity glycerol pathway, in response to osmotic stress. The present work focuses on the role of RTG signaling in salt-induced osmotic stress and its interaction with HOG1. Wild-type and mutant cells, lacking HOG1 and/or RTG genes, are compared with respect to cell growth features, retrograde signaling activation and mitochondrial function in the presence and in the absence of high osmostress. We show that RTG2, the main upstream regulator of the RTG pathway, contributes to osmoadaptation in an HOG1-dependent manner and that, with RTG3, it is notably involved in a late phase of growth. Our data demonstrate that impairment of RTG signaling causes a decrease in mitochondrial respiratory capacity exclusively under osmostress. Overall, these results suggest that HOG1 and the RTG pathway may interact sequentially in the stress signaling cascade and that the RTG pathway may play a role in inter-organellar metabolic communication for osmoadaptation.

The neurodegeneration of cerebellar granule cells, after low potassium induced apoptosis, is known to be temporally divided into an early and a late phase. Voltage-dependent anion channel-1 (VDAC1) protein, changing from the closed inactive state to the active open state, is central to the switch between the early and late phase. It is also known that: (i) VDAC1 can undergo phosphorylation events and (ii) AMP-activated protein kinase (AMPK), the sensor of cellular stress, may have a role in neuronal homeostasis. In the view of this, the involvement of AMPK activation and its correlation with VDAC1 status and activity has been investigated in the course of cerebellar granule cells apoptosis. The results reported in this study show that an increased level of the phosphorylated, active, isoform of AMPK occurs in the early phase, peaks at 3 h and guarantees an increase in the phosphorylation status of VDCA1, resulting in a reduced activity of this latter. However this situation is transient in nature, since, in the late phase, AMPK activation decreases as well as the level of phosphorylated VDAC1. In a less phosphorylated status, VDAC1 fully recovers its gating activity and drives cells along the death route.

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.

The metabolism of benthic aquatic invertebrates, populating transitional water ecosystems, is influenced by both physiological and environmental factors, thus involving an adjustment of physiological processes which has a metabolic cost. In order to discover changes in metabolic pathways in response to specific factors, it's firstly necessary characterizing the principal cellular metabolic activities of the small benthic aquatic organisms. We approach here the bioenergetic state issue of two benthic organisms, i.e. Lekanesphaera monodi and Gammarus insensibilis, evidencing that no apparent and statistically significative differences between them in aerobic as well in glycolytic capacities are detected, except for COX activity.

Cystic fibrosis (CF) is associated to impaired Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) channel also causing decreased glutathione (GSH) secretion, defective airway bacterial clearance and inflammation. Here we checked the main ROS-producing and ROS-scavenging enzymes as potential additional factors involved in CF pathogenesis. We found that CFBE41o-cells, expressing F508del CFTR, have increased NADPH oxidase (NOX) activity and expression level, mainly responsible of the increased ROS production, and decreased glutathione reductase (GR) activity, not dependent on GR protein level decrease. Furthermore, defective CFTR proved to cause both extracellular and intracellular GSH level decrease, probably by reducing the amount of extracellular GSH-derived cysteine required for cytosolic GSH synthesis. Importantly, we provide evidence that defective CFTR and NOX/GR activity imbalance both contribute to NADPH and GSH level decrease and ROS overproduction in CF cells.

// Fulvio Florenzano 1,* , Corsetti Veronica 2,* , Gabriele Ciasca 3,* , Maria Teresa Ciotti 4 , Anna Pittaluga 5 , Gunedalina Olivero 5 , Marco Feligioni 1,6 , Filomena Iannuzzi 1 , Valentina Latina 2 , Michele Francesco Maria Sciacca 7 , Alessandro Sinopoli 7 , Danilo Milardi 7 , Giuseppe Pappalardo 7 , De Spirito Marco 3 , Massimiliano Papi 3 , Anna Atlante 8,9 , Antonella Bobba 8,9 , Antonella Borreca 4 , Pietro Calissano 1 and Giuseppina Amadoro 1,2 1 European Brain Research Institute, Rome, Italy 2 Institute of Translational Pharmacology, CNR, Rome, Italy 3 Institute of Physics, Catholic University of the Sacred Heart, Largo F Vito 1, Rome, Italy 4 Institute of Cellular Biology and Neuroscience, CNR, IRCSS Santa Lucia Foundation, Rome, Italy 5 Department of Pharmacy, Pharmacology and Toxicology Section, University of Genoa, Genoa, Viale Cembrano, Italy 6 Department of Neurorehabilitation Sciences, Casa Cura Policlinico, Milan, Italy 7 Institute of Biostructures and Bioimaging, CNR, Catania, Italy 8 Institute of Biomembranes and Bioenergetics, CNR, Bari, Italy 9 Center of Excellence for Biomedical Research, University of Genoa, Genoa, Viale Benedetto XV, Italy * These authors have equally contributed to this work Correspondence to: Giuseppina Amadoro, email: // Keywords : extracellular tau, tau cleavage, Alzheimer’s disease, neurodegeneration, synapse(s), Gerotarget Received : March 07, 2017 Accepted : April 11, 2017 Published : April 22, 2017 Abstract 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 2 -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 2 tau 26-44 (aka NH 2 htau) -which is the minimal active moiety of neurotoxic 20-22kDa peptide accumulating in vivo 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 2+ 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 2 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.

After more than 80years 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 beta-amyloid (Abeta)-dependent apoptosis. However, in the late phase, while most AD brain cells die in response to Abeta toxicity, only small populations of neurons, exhibiting increased glucose uptake and glycolytic flux, are able to survive as they are resistant to Abeta. 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 Abeta toxicity is an AD hallmark-are sent into quiescence, this likely contributing to activate mechanisms of resistance to Abeta-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 Abeta-induced neurotoxicity and leading to cell death and dementia. The understanding of how certain nerve cells become resistant to Abeta toxicity, while the majority dies, is an attractive challenge toward the identification of novel possible targets for AD therapy.

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 adenine nucleotide-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's 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.

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 24 h 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.

In this paper, we discuss the interplay between beta-amyloid (A) 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.

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.

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.

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.

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.

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.

A metabolic shift from oxidative phosphorylation to glycolysis (i.e. the Warburg effect) occurs in Alzheimer’s disease accompanied by an increase of both activity and level of HK-I. The findings reported here demonstrate that in the early phase of apoptosis VDAC1 activity, but not its protein level, progressively decreases, in concomitance with the physical interaction of HK-I with VDAC1. In the late phase of apoptosis, glucose-6-phosphate accumulation in the cell causes the dissociation of the two proteins, the re-opening of the channel and the recovery of VDAC1 function, resulting in a reawakening of the mitochondrial function, thus inevitably leading to cell death.

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.

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.

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.

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. In addition, a 130% increase of the GSSG/GSH ratio was measured in Alzheimer brains with respect to age-matched controls. Knowing 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.

Summary We have applied a double tagging system in order to study whether purified myelin basic protein is able to adhere to normal human peripheral T lymphocytes without the need to purify cells. Evaluation of myelin basic protein adherence to peripheral blood mononuclear cells was determined with biotinylated myelin basic protein and fluoresceinated avidin, and lymphocyte population was identified by the corresponding phycoerythrinated monoclonal antibody. The observed adherence of myelin basic protein to T lymphocytes was found to depend on protein conformation.

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.

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.

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.

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.

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.

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.

The analysis of short tandem repeat (STR) systems usually relies on polyacrylamide gel electrophoresis analysis followed by visualization with silver staining or autoradiography. Both these techniques may not be suitable for clinical laboratories. We developed a simple procedure based on the visualization of STR alleles by ethidium bromide staining. The 4-bp STR system analysed is located in the human phenylalanine hydroxylase gene. Alleles differing by 4 bp are clearly separated independently of the size of the amplified fragments and homozygous samples are easily identified by comparison of the relative intensity of the electrophoretic bands. This method could be applied to the analysis of other STR systems located in different genetic loci by carefully changing the electrophoretic conditions.

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.

Haplotype analysis using microsatellite markers is a useful indicator of specific mutations and is often exploited as the first large-scale screening technique to carry out the molecular characterization of the disease gene in probands from a specific population. However, the methodologies available are still cumbersome and require the use of either radioactive compounds or specialized equipment suitable to follow fluorescent dyes. Both these techniques may not be available for newly-developing clinical laboratories. We have set up a sensitive and easy-to-use protocol to characterize five closely spaced, highly polymorphic microsatellite polymorphisms (CA repeats) that span the Wilson Disease (WD) region i.e., D13S316, D13S133, D13S301, D13S314, D13S315. The technique described here for the analysis of the WD gene microsatellite system relies on the quick detection method of silver staining, avoiding the use of toxic or sophisticated equipment. This approach could be the method of choice to implement molecular genetic testing in clinical laboratories, even those not especially equipped for DNA analysis and in particular in newly-developed molecular genetics centers in countries whose population has not yet been characterized for WD-causing ATP7B gene mutations.

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.

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.

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.

The excitatory neurotransmitter glutamate plays a major role in determining certain neurological disorders. This situation, referred to as ‘glutamate neurotoxicity’ (GNT), is characterized by an increasing damage of cell components, including mitochondria, leading to cell death. In the death process, reactive oxygen species (ROS) are generated. The present study describes the state of art in the field of GNT with a special emphasis on the oxidative stress and mitochondria. In particular, we report how ROS are generated and how they affect mitochondrial function in GNT. The relationship between ROS generation and cytochrome c release is described in detail, with the released cytochrome c playing a role in the cell defense mechanism against neurotoxicity.

Cytochrome c (cyt c) release was investigated in cerebellar granule cells used as an in vitro neuronal model of apoptosis. We have found that cyt c is released into the cytoplasm as an intact, functionally active protein, that this event occurs early, in the commitment phase of the apoptotic process, and that after accumulation, this protein is progressively degraded. Degradation, but not release, is fully blocked by benzyloxycarbonyl-Val-Ala-Asp-fluoromethylchetone (z-VAD-fmk). On the basis of previous findings obtained in the same neuronal population undergoing excitotoxic death, it is hypothesized that release of cyt c may be part of a cellular attempt to maintain production of ATP via cytochrome oxidase, which is reduced by cytosolic NADH in a cytochrome b5-soluble cyt c-mediated fashion.

The major cause of congenital adrenal hyperplasia (CAH), a common recessive genetic disease, is the deficiency of steroid 21-hydroxylase (21OH), a microsomal enzyme encoded by the CYP21 gene. Although several CAH causing mutations have been identified in the CYP21 gene of patients with 21OH deficiency, genotyping of the 21OH locus is quite complex because of the high frequency of gene conversion and the presence of multiple mutations on single CAH alleles. In order to perform the complete characterisation of the CYP21 gene coding region more simply, we developed a highly sensitive, non-radioactive method allowing DNA single strand conformation polymorphism (DNA-SSCP) analysis. This method was applied to the characterisation of all the exons and intron-exon junctions of the CYP21 gene in five patients affected by the simple virilising form and one affected by the salt wasting form. In all samples showing SSCP signals, direct sequence analysis showed the presence of more than one single sequence variant. In particular, four mutations which are already known to cause the disease, 16 polymorphisms, and one newly identified C to T transition at position 849 were detected. A random sequence analysis, performed on 31 out of 81 exons showing a normal SSCP pattern, shows the method to be highly sensitive: no sequence variant was detected, thus confirming the validity of this non-radioactive DNA-SSCP analysis in characterising the CYP21 gene in patients with steroid 21OH deficiency. Notwithstanding the complete characterisation of all exons and exon/intron junctions of the CYP21 gene, no complete genotype/phenotype correlation was found in the panel of patients analysed, thus suggesting that characterisation of CAH alleles must be extended to outside the coding region of the CYP21 gene, most probably into the promoter region.

With the aim of studying the involvement of the mitochondrial genome in the impairment of heart function, mitochondrial DNA was analyzed by modified primer shift-polymerase chain reaction in a panel of young patients affected by primary cardiomyopathies. Mitochondrial DNA molecules harboring the 7436 bp deletion were specifically found in cardiomyopathic patients as compared with a panel of control subjects. The 4977 bp deletion was commonly detected among the subjects analyzed whereas none of the specific tRNA gene point mutations generally associated with the cardiomyopathic trait were detected. The presence of the 7436 bp deletion as a consequence of a premature aging of the heart muscle, secondary to heart dysfunction, is discussed.

Zn2+ appears to stabilize the myelin sheath but the mechanism of this effect is unknown. In a previous report we have shown that zinc binds to CNS myelin basic protein (MBP) in the presence of phosphate and this results in MBP aggregation. For this paper we used a solid phase zinc blotting assay to identify which myelin proteins bind zinc. MBP and a 58 kDa band were found to be the major targets of 65Zn binding. Moreover, using fluorescence, light scattering and electron microscopy we investigated the binding of zinc and other cations to purified MBP in solution. Among the cations tested for their ability to interfere with the binding of zinc, the most effective were cadmium, mercury and copper, but only cadmium and mercury increased the scattering intensity, whereas MBP aggregation was not inhibited by copper ions. Thus, the effect of zinc on the formation of MBP clusters seems to be specific.

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.

Publisher Summary Myelin is a membrane surrounding the nerve axon, which is very important because implicated in some neurological diseases. This chapter discusses the selective extraction of myelin proteins from bovine brain myelin by detergents. The most effective detergents used were the cationic sarcosine and cetyltrimethylammoniurn bromide (CTAB), the nonionic Triton X-100 (TX-100) in the presence of 0.25-0.50 M NaCl, and the bile salt sodium deoxycholate (Na-DOC). The chapter focuses on the selective extraction of the three major proteins of myelin: the basic protein (MBP, 19KD), the proteolipid (MPL, 25 KD), and the acidic Wolfgram protein (MWP, 52-54 KD). Selective extraction of the Wolfgram protein was only possible at the lower concentration of the chaotropic agent urea. On the other hand, a fraction of the proteolipid could be selectively solubilized only by Triton. The basic protein was extracted more easily and near to purity by a number of detergents: Triton X-100 at pH 3 in the absence of salts, CTAB at low concentrations, and sodium cholate. The basic protein was further purified in all these detergents, the proteolipid in Triton X-100 only. The localization of both proteolipid and basic protein in the myelin membrane can be described by studying the type of interaction of these proteins with the Triton X-100 micelles. A particular feature of both solubilized proteins is their nonadsorption to hydroxyapatite.

Myelin basic protein, only in association with certain detergents, is able to bind irreversibly to the usual gel filtration media. While this binding is greatly advantageous in purification of proteolipid (the other major myelin protein), the question arises of the uncommon, nonphysiological behaviour of the basic protein. A relationship between binding property and basic protein structure is suggested.

An unknown protein has been isolated from bovine brain myelin. This protein, purified in the nonionic detergent n-octylpolydisperse oligooxyethylene, reveals on SDS gel electrophoresis a large number of bands in the higher MW region. However, chemical analysis and gel chromatography indicate the presence of a single, small protein containing large amounts of bound phosphatidylserine. N-terminal and C-terminal sequences, aminoacid composition, and the anomalous electrophoretic behaviour led us to exclude the protein as a fragment of other already known myelin proteins.

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.

Disarrangement in functions and quality control of mitochondria at synapses are early events in Alzheimer's disease (AD) pathobiology. We reported that a 20-22 kDa NH2-tau fragment mapping between 26 and 230 amino acids of the longest human tau isoform (aka NH2htau): (i) is detectable in cellular and animal AD models, as well in synaptic mitochondria and cerebrospinal fluids (CSF) from human AD subjects; (ii) is neurotoxic in primary hippocampal neurons; (iii) compromises the mitochondrial biology both directly, by inhibiting the ANT-1-dependent ADP/ATP exchange, and indirectly, by impairing their selective autophagic clearance (mitophagy). Here, we show that the extensive Parkin-dependent turnover of mitochondria occurring in NH2htau-expressing post-mitotic neurons plays a pro-death role and that UCHL-1, the cytosolic Ubiquitin-C-terminal hydrolase L1 which directs the physiological remodeling of synapses by controlling ubiquitin homeostasis, critically contributes to mitochondrial and synaptic failure in this in vitro AD model. Pharmacological or genetic suppression of improper mitophagy, either by inhibition of mitochondrial targeting to autophagosomes or by shRNA-mediated silencing of Parkin or UCHL-1 gene expression, restores synaptic and mitochondrial content providing partial but significant protection against the NH2htau-induced neuronal death. Moreover, in mitochondria from human AD synapses, the endogenous NH2htau is stably associated with Parkin and with UCHL-1. Taken together, our studies show a causative link between the excessive mitochondrial turnover and the NH2htau-induced in vitro neuronal death, suggesting that pathogenetic tau truncation may contribute to synaptic deterioration in AD by aberrant recruitment of Parkin and UCHL-1 to mitochondria making them more prone to detrimental autophagic clearance.