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.

Hydroxytyrosol (2-(3,4dihydroxyphenyl)ethanol, (DPE), a phenolic compound present in oliveoil, is known to have antioxidant properties. The aim of this study was to investigate the effectof 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 lipidperoxidation 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, weobserved 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 restoresthe values to the control levels. Noteworthy, mitochondrial activities of SOD and CAT werestrongly reduced by cadmium treatment both in spleen and testes, and DPE was not be able torestore their activity. Overall, the results from this study indicated that the DPE has differentantioxidant efficiency in spleen and testis of cadmium intoxicated rats.

To find out whether and how the adenine nucleotide translocator-1 (ANT-1) inhibition due to NH2htau and A?1-42 is due to an interplay between these two Alzheimer's peptides, ROS and ANT-1 thiols, use was made of mersalyl, a reversible alkylating agent of thiol groups that are oriented toward the external hydrophilic phase, to selectively block and protect, in a reversible manner, the -SH groups of ANT-1. The rate of ATP appearance outside mitochondria was measured as the increase in NADPH absorbance which occurs, following external addition of ADP, when ATP is produced by oxidative phosphorylation and exported from mitochondria in the presence of glucose, hexokinase and glucose-6-phosphate dehydrogenase. We found that the mitochondrial superoxide anions, whose production is induced at the level of Complex I by externally added A?1-42 and whose release from mitochondria is significantly reduced by the addition of the VDAC inhibitor DIDS, modify the thiol group/s present at the active site of mitochondrial ANT-1, impair ANT-1 in a mersalyl-prevented manner and abrogate the toxic effect of NH2htau on ANT-1 when A?1-42 is already present. A molecular mechanism is proposed in which the pathological A?-NH2htau interplay on ANT-1 in Alzheimer's neurons involves the thiol redox state of ANT-1 and the A?1-42-induced ROS increase. This result represents an important innovation because it suggests the possibility of using various strategies to protect cells at the mitochondrial level, by stabilizing or restoring mitochondrial function or by interfering with the energy metabolism providing a promising tool for treating or preventing AD.

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.

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.

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

Here we investigate the effect of ?-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 ?-amyloid, and Alzheimer synaptic-enriched brain samples. We found that ?-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 ?-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 ?-amyloid with the identified targets, by stabilizing or by restoring mitochondrial function or by interfering with the energy metabolism.

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 aminoacids 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.

The involvement of thioredoxin/thioredoxinreductase system has been investigated in cerebellar granulecells (CGCs), a cellular system in which neurons areinduced in apoptosis by the physiological stimulus oflowering extracellular potassium. Clarifying the sequenceof events that occur during apoptosis is a critical issue as itcan lead to the identification of those key events that, ifblocked, can slow down or reverse the death process. Theresults reported in this work show that TrxR is involved inthe early phase of CGC apoptosis with an increase inactivity that coincides with the increased expression of theTrxR1 isoform and guarantees the maintenance of adequatelevel of Trx in its reduced, active form. However, in lateapoptosis, when about 50 % of cells are dead, partialproteolysis of TrxR1 by calpain occurs and the reduction ofTrxR1 mRNA, together with the overall decrease in TrxRactivity, contribute to increase the levels of the oxidizedform of Trx. When the reduced form of Trx is externallyadded to apoptotic cultures, a significant reduction in celldeath is achieved confirming that a well-functioning thioredoxin/thioredoxin reductase system is required for survivalof CGCs.

To investigate the role of cytochrome c (cyt c) release in yeast acetic acid-induced programmed celldeath (AA-PCD), wild type (wt) and cells lacking metacaspase (Dyca1), cytochrome c (Dcyc1,7) andboth (Dcyc1,7Dyca1) were compared for AA-PCD occurrence, hydrogen peroxide (H2O2) productionand caspase activity. AA-PCD occurs in Dcyc1,7 and Dcyc1,7Dyca1 cells slower than in wt, but similarto that in Dyca1 cells, in which no cytochrome c release occurs. Both H2O2 production and caspaseactivation occur in these cells with early and extra-activation in Dcyc1,7 cells. We conclude thatalternative death pathways can be activated in yeast AA-PCD, one dependent on cyt c release, whichrequires YCA1, and the other(s) independent on it.