Antonella Bobba

Glutamate neurotoxicity, oxidative stress and mitochondria

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 Is Released from Mitochondria in a Reactive Oxygen Species (ROS)-dependent Fashion and Can Operate as a ROS Scavenger and as a Respiratory Substrate in Cerebellar Neurons Undergoing Excitotoxic Death

In rat cerebellar granule cells both reactive oxygen species production and release of cytochrome c take place during glutamate toxicity. This investigation was aimed (i) to ascertain whether and how these two processes are related and (ii) to gain insight into the role played by the released cytochrome c in the onset of neurotoxicity. Cytochrome c release takes place owing to the generation of reactive oxygen species both in glutamate-treated cerebellar granule cells and in sister control cultures incubated in the presence of the reactive oxygen species-generating system consisting of xanthine plus xanthine oxidase. In the early phase of neurotoxicity (30-min glutamate exposure) about 40% of the maximum (as measured at 3 h of glutamate exposure) cytochrome c release was found to occur in cerebellar granule cells from mitochondria that were essentially coupled and intact and that had a negligible production of oxygen free radicals. Contrarily, mitochondria from cells treated with glutamate for 3 h were mostly uncoupled and produced reactive oxygen species at a high rate. The cytosolic fraction containing the released cytochrome c was able to transfer electrons from superoxide anion to molecular oxygen via the respiratory chain and was found to partially prevent glutamate toxicity when added externally to cerebellar neurons undergoing necrosis. In the light of these findings, we propose that in the early phase of neurotoxicity, cytochromec release can be part of a cellular and mitochondrial defense mechanism against oxidative stress.

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

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.

Early release and subsequent caspase‐mediated degradation of cytochrome c in apoptotic cerebellar granule cells

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 b 5‐soluble cyt c‐mediated fashion.

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

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.

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

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.

A peptide containing residues 26-44 of tau protein impairs mitochondrial oxidative phosphorylation acting at the level of the adenine nucleotide translocator.

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.

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

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 (Δyca1), cytochrome c (Δcyc1,7) and both (Δcyc1,7Δyca1) were compared for AA‐PCD occurrence, hydrogen peroxide (H2O2) production and caspase activity. AA‐PCD occurs in Δcyc1,7 and Δcyc1,7Δyca1 cells slower than in wt, but similar to that in Δyca1 cells, in which no cytochrome c release occurs. Both H2O2 production and caspase activation occur in these cells with early and extra‐activation in Δcyc1,7 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.

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

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.

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

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.