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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.

The S-100: A protein family in search of a function

The S-100 is a group of low molecular weight (10-12 kD) calcium-binding proteins highly conserved among vertebrates. It is present in different tissues as dimers of homologous or different subunits (alpha, beta). In the nervous system, the S-100 exists as a mixture composed of beta beta and alpha beta dimers with the monomer beta represented more often. Its intracellular localisation is mainly restricted to the glial cytoplasmic compartment with a small fraction bound to membranes. In this compartment the S-100 acts as a potent inhibitor of phosphorylation on several substrates including the synaptosomal C-Kinase and Tau, a microtubule-associated protein. The S-100 in particular conditions, after binding with specific membrane sites (Kd = 0.2 microM; Bmax = 4.5 nM), is able to modify the activity of adenylate cyclase, probably via G-proteins. In addition, the Ca2+ homeostasis is also modulated by S-100 via an increase of specific membrane conductance and/or Ca2+ release from intracellular stores. In vitro and in vivo experiments showed that lower (nM) concentrations of extracellular S-100 beta act on glial and neuronal cells as a growth-differentiating factor. On the other hand, higher concentrations of the protein induce apoptosis of some cells such as the sympathetic-like PC12 line. Finally, data obtained from physiological (development, ageing) or pathological (dementia associated with Downs syndrome, Alzheimers disease) conditions showed that a relationship could be established between the S-100 levels and some aspects of the statii.

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.

Glutamate Neurotoxicity in Rat Cerebellar Granule Cells: A Major Role for Xanthine Oxidase in Oxygen Radical Formation

Abstract: To gain insight into the mechanism through which the neurotransmitter glutamate causally participates in several neurological diseases, in vitro cultured cerebellar granule cells were exposed to glutamate and oxygen radical production was investigated. To this aim, a novel procedure was developed to detect oxygen radicals; the fluorescent dye 2′,7′‐dichlorofluorescein was used to detect production of peroxides, and a specific search for the possible conversion of the enzyme xanthine dehydrogenase into xanthine oxidase after the excitotoxic glutamate pulse was undertaken. A 100 µM glutamate pulse administered to 7‐day‐old cerebellar granule cells is accompanied by the onset of neuronal death, the appearance of xanthine oxidase, and production of oxygen radicals. Xanthine oxidase activation and superoxide (O2•−) production are completely inhibited by concomitant incubation of glutamate with MK‐801, a specific NMDA receptor antagonist, or by chelation of external calcium with EGTA. Partial inhibition of both cell death and parallel production of reactive oxygen species is achieved with allopurinol, a xanthine oxidase inhibitor, leupeptin, a protease inhibitor, reducing agents such as glutathione or dithiothreitol, antioxidants such as vitamin E and vitamin C, and externally added superoxide dismutase. It is concluded that glutamate‐triggered, NMDA‐mediated, massive Ca2+ influx induces rapid conversion of xanthine dehydrogenase into xanthine oxidase with subsequent production of reactive oxygen species that most probably have a causal involvement in the initial steps of the series of intracellular events leading to neuronal degeneration and death.

The Mode of Action of Nerve Growth Factor in PC12 Cells

This review deals with the mechanism of nerve growth factor action. In view of the many and diversified effects of this growth factor, and since it could utilize different mechanism(s) in distinct types of cells, we have confined our analysis to the best characterized and more extensively studied target, the clonal cell line PC12.When exposed to NGF in vitro, these neoplastic cells recapitulate the last major steps of neuronal differentiation, i.e., the commitment to become a neuron and the acquisition of the neuronal phenotype. This is characterized by electrically excitable neurites, a display of a highly organized cytoskeleton, and the specific chemical and molecular neuronal properties. These effects are elicited upon the interaction of NGF with a receptor whose gene has been cloned and whose kinetic properties are now relatively well characterized. It is not yet clear, on the contrary, if and which of the several potential second messengers (cAMP, Ca, or phosphoinositides) that undergo marked fluctuations following NGF binding, transduce and amplify the NGF message. Among both the early and late effects of NGF is the modulation of expression of several genes. Some of the products of these genes are mainly restricted to nerve cells and others appear to play a crucial role in regulating the proper assembly of cytoskeletal elements.It is hypothesized that this complex array of chemical, molecular, and ultrastructural changes is triggered by NGF, not through activation of a single pathway, but more likely via combinatorial processes whereby several intracellular signals interplay before the irreversible commitment of becoming a neuron is undertaken.

Nerve growth factor as a paradigm for other polypeptide growth factors

The last decade has witnessed a veritable explosion in the number of studies on polypeptide growth factors (PGFs) because of their ability to promote the proliferation and/or differentiation of different cell types 1. A PGFs typical effect is to cause growth in its target cells, where growth may be defined as an increase in the number (hyperplasia), in the size (hypertrophy) of cells, or, in the case of neurons in the extension of axons and dendrites. By the end of this growth process, cells have usually undergone terminal differentiation. The object of this article is to review recent developments in the study of one of the PGFs nerve growth factor (NGF). This factor was initially considered a specific effector molecule for two neural crest derivatives, sensory and sympathetic nerve cells. Work during the last decade has shown that NGF activity also extends to CNS neurons and to non-neuronal cells such as chromaffin and mast cells.

The brain protein S-100ab induces apoptosis in PC12 cells

Incubation of PC12 cells with S-100 protein induces a rapid (0.5-1.0 min) rise of intracellular Ca2+ which lasts for the whole period of incubation. This effect is abolished in a Ca(2+)-free medium or in the presence of 1.0 microM Ni2+, an inhibitor of calcium channels. The rise in intracellular Ca2+ is followed by a progressive increase of cells undergoing degeneration and death. This event is accompanied by the appearance of apoptotic bodies and DNA fragmentation typical of the process known as apoptosis. S-100-induced cell death is prevented by 1 microM Ni2+ or by 0.1 nM cycloheximide, suggesting the involvement of new protein synthesis. It is postulated that the binding of S-100ab to specific sites present in PC12 cells is followed by the formation of Ca2+ channels and/or the stimulation of pre-existing ones with consequent increase of Ca2+ influx and activation of a process of cell death.

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.

Testosterone induction of estero-proteolytic activity in the mouse submaxillary gland.

Abstract Testosterone injections sharply increase the estero-proteolytic activity in submaxillary gland extracts of female and of adolescent mice. A major form of hormone-dependent enzyme has been isolated and purified from male gland extracts. The enzyme has a molecular weight of about 32000 and appears to be a peptide hydrolase with strong esterolytic activity. A specific antiserum to the purified enzyme was prepared and used for immunochemical titrations. The results indicate that the increased enzyme activity in the gland following testosterone treatment is due to an increased concentration of enzyme molecules. Incorporation studies gave additional evidence that the hormone stimulates the rate of the enzyme synthesis in the gland.

The S-100 protein causes an increase of intracellular calcium and death of PC12 cells

The S-100 protein-PC12 cell interaction has been studied as a model system of the possible physiological role played by S-100 protein in the nervous system. The data reported demonstrate that S-100 exerts a cytotoxic action which eventually leads to PC12 cell death, regardless of the cell cycle phase. The effect is specific for the S-100 isoforms, which are made up of two identical subunits and is abolished by a monoclonal antibody directed against the same isoforms. Other isoforms and/or calcium-binding proteins, such as troponin or calmodulin, do not induce the same effects. The action of S-100 on cell viability is not detectable in other cell lines of different embryological origin, such as 3T3, L1210, GH3. S-100 causes a rapid and considerable increase (two- to three-fold) of intracellular Ca2+ concentration in PC12 cells accompanied by cytostatic and cytotoxic action. It is postulated that this action also occurs in vivo, as part of the physiological action of this protein.