Biomedical subjects

Antonio Contestabile

Publications and source records attributed to Antonio Contestabile.

25 records · Page 2Linked to original sources

PubMed2002

Nitric oxide protects neuroblastoma cells from apoptosis induced by serum deprivation through cAMP-response element-binding protein (CREB) activation.

The transcription factor cAMP-response element-binding protein (CREB) mediates survival in many cells, including neurons. Recently, death of cerebellar granule neurons due to nitric oxide (NO) deprivation was shown to be accompanied by down-regulation of CREB activity (). We now provide evidence that overproduction of endogenous NO or supplementation with exogenous NO renders SK-N-BE human neuroblastoma cells more resistant to apoptosis induced by serum deprivation. Parental cells underwent apoptosis after 24 h of serum deprivation, an outcome largely absent in clones overexpressing human neuronal nitric oxide synthase (nNOS). This protective effect was reversed by the inhibition of NOS itself or soluble guanylyl cyclase, pointing at cGMP as an intermediate effector of NO-mediated rescue. A slow-releasing NO donor protected parental cells to a significant extent, thus confirming the survival effect of NO. The impaired viability of serum-deprived parental cells was accompanied by a strong decrease of CREB phosphorylation and transcriptional activity, effects significantly attenuated in nNOS-overexpressing clones. To confirm the role of CREB in survival, the ectopic expression of CREB and/or protein kinase A largely counteracted serum deprivation-induced cell death of SK-N-BE cells, whereas transfection with a CREB negative mutant was ineffective. These experiments indicate that CREB activity is an important step for NO-mediated survival in neuronal cells.

Apoptosis↗

PubMed2002

Regional alterations of the NO/NOS system in the aging brain: a biochemical, histochemical and immunochemical study in the rat.

We have used several approaches (immunohistochemistry and enzyme histochemistry, Western blotting, biochemical assay of Ca(2+)-dependent catalytic activity) in order to detect differences in neuronal nitric oxide synthase (nNOS) expression and activity in various brain regions of young-adult (4-month-old) and aged (28-month-old) rats. In most of the brain regions examined (striatum, neocortex, olfactory cortex and hippocampus) some significant decrease in the density per unit area of nNOS neurons, detected either through immunohistochemistry or enzyme histochemistry, was observed in aged rats. However, only in the striatum and olfactory cortex this was accompanied by a significant decrease of the catalytic activity of the constitutive, Ca(2+)-dependent NOS form. In these two regions, the relative level of expression of nNOS protein was also significantly decreased, as assessed by Western blotting of proteic extracts from young-adult and aged rats. Other observed differences were a paler stain of neurons in some brain areas of the aged rats and differences of cellular compartmentalization of the protein in the same rats, as assessed through confocal microscopy. The present observations demonstrate that the expression and activity of nNOS show regionally-specific alterations in the brain of aged healthy rats, with a trend towards decrease, rather than toward increase as suggested by some previous reports. Therefore, hypotheses implicating nitric oxide increase in brain aging should be reconsidered on the basis of a clear-cut distinction between the physiological and the pathological aspects of the aging process.

Aging↗

PubMed2002

NMDA receptor-dependent CREB activation in survival of cerebellar granule cells during in vivo and in vitro development.

During both in vivo and in vitro development, cerebellar granule cells depend on the activity of the NMDA glutamate receptor subtype for survival and full differentiation. With the present results, we demonstrate that CREB activation, downstream of the NMDA receptor, is a necessary step to ensure survival of these neurons. The levels of CREB expression and activity increase progressively during the second week of postnatal cerebellar development and the phosphorylated form of CREB is localized selectively to cerebellar granule cells during the critical developmental stages examined. Chronically blocking the NMDA receptor through systemic administration of the competitive antagonist, CGP 39551, during the in vivo critical developmental period, between 7-11 postnatal days, results in increased apoptotic elimination of differentiating granule neurons in the cerebellum [Monti & Contestabile, Eur. J. Neurosci., 12, 3117-3123 (2000)]. We report here that this event is accompanied by a significant decrease of CREB phosphorylation in the cerebellum of treated rat pups. When cerebellar granule neurons are explanted and maintained in dissociated cultures, the levels of CREB phosphorylation increase with differentiation, similar to that which happens during in vivo development. When granule cells are kept in non-trophic conditions, their viability is affected and both CREB phosphorylation and transcriptional activity are decreased significantly. The neuronal viability and the deficiency of CREB activity, are both rescued by the pharmacological activation of the NMDA receptor. These results provide good circumstantial evidence for a functional link between the NMDA receptor and CREB activity in promoting neuronal survival during development.

2-Amino-5-phosphonovalerate↗

PubMed2002

Akt pathway mediates a cGMP-dependent survival role of nitric oxide in cerebellar granule neurones.

Apoptotic death results from disrupting the balance between anti-apoptotic and pro-apoptotic cellular signals. The inter- and intracellular messenger nitric oxide is known to mediate either death or survival of neurones. In the present work, cerebellar granule cells were used as a model to assess the survival role of nitric oxide and to find novel signal transduction pathways related to this role. It is reported that sustained inhibition of nitric oxide production induces apoptosis in differentiated cerebellar granule neurones and that compounds that slowly release nitric oxide significantly revert this effect. Neuronal death was also reverted by a caspase-3-like inhibitor and by a cyclic GMP analogue, thus suggesting that nitric oxide-induced activation of guanylate cyclase is essential for the survival of these neurones. We also report that the Akt/GSK-3 kinase system is a transduction pathway related to the survival action of nitric oxide, as apoptosis caused by nitric oxide deprivation is accompanied by down-regulation of this, but not of other, kinase systems. Conversely, treatments able to rescue neurones from apoptosis also counteracted this down-regulation. Furthermore, in transfection experiments, overexpression of the Akt gene significantly decreased nitric oxide deprivation-related apoptosis. These results are the first evidence for a mechanism where endogenous nitric oxide promotes neuronal survival via Akt/GSK-3 pathway.

Animals↗

PubMed2002

Nitric oxide regulates cGMP-dependent cAMP-responsive element binding protein phosphorylation and Bcl-2 expression in cerebellar neurons: implication for a survival role of nitric oxide.

Nitric oxide (NO) is a small, diffusible, highly reactive molecule with a dichotomous regulatory role in the brain: an intra- and intercellular messenger under physiological conditions and a neurodegenerative agent under pathological conditions. We have recently demonstrated that long-lasting exposure to an neuronal nitric oxide synthase (nNOS) inhibitor down-regulated serine/threonine kinase (Akt) survival pathway and caused apoptosis in cerebellar granule cell cultures. The present study further substantiates the role of NO in neuronal survival by demonstrating that blocking its production down-regulates the activity of cAMP-responsive element binding protein (CREB), a transcription factor involved in cell survival and synaptic plasticity. Pharmacological dissection of the pathway linking NO to CREB shows that cGMP and its kinase are intermediate effectors. We also identify Bcl-2 as one of the anti-apoptotic genes down-regulated by NO shortage and decreased CREB phosphorylation. These results not only confirm the role of CREB in neuronal survival but also provide circumstantial evidence for a novel link among NO, CREB activation and survival.

Animals↗

PubMed2002

Reciprocal interactions between microglia and neurons: from survival to neuropathology.

Microglia represent a major cellular component of the brain, where they constitute a widely distributed network of immunoprotective cells. During the last decades, it has become clear that the functions traditionally ascribed to microglia, i.e. to dispose of dead cells and debris and to mediate brain inflammatory states, are only a fraction of a much wider repertoire of functions spanning from brain development to aging and neuropathology. The aim of the present survey is to critically discuss some of these functions, focusing in particular on the reciprocal microglia-neuron interactions and on the complex signaling systems subserving them. We consider first some of the functional interactions dealing with invasion, proliferation and migration of microglia as well as with the establishment of the initial blueprint of neural circuits in the developing brain. The signals related to the suppression of immunological properties of microglia by neurons in the healthy brain, and the derangement from this physiological equilibrium in aging and diseases, are then examined. Finally, we make a closer examination of the reciprocal signaling between damaged neurons and microglia and, on these bases, we propose that microglial activation, consequent to neuronal injury, is primarily aimed at neuroprotection. The loss of specific communication between damaged neurons and microglia is viewed as responsible for the turning of microglia to a hyperactivated state, which allows them to escape neuronal control and to give rise to persistent inflammation, resulting in exacerbation of neuropathology. The data surveyed here point at microglial-neuron interactions as the basis of a complex network of signals conveying messages with high information content and regulating the most important aspects of brain function. This network shares similar features with some fundamental principles governing the activity of brain circuits: it is provided with memory and it continuously evolves in relation to the flow of time and information.

Animals↗

PubMedDate not supplied

Cerebellar granule cells as a model to study mechanisms of neuronal apoptosis or survival in vivo and in vitro.

Granule cells of the cerebellum constitute the largest homogeneous neuronal population of mammalian brain. Due to their postnatal generation and the feasibility of well characterized primary in vitro cultures, cerebellar granule cells are a model of election for the study of cellular and molecular correlates of mechanisms of survival/apoptosis and neurodegeneration/neuroprotection. The present review mainly deals with recent data on mechanisms and factors promoting survival or apoptotic elimination of cerebellar granule neurons, with a particular focus on the molecular correlates at the level of gene expression and induction of cellular signal pathways. The in vivo development is first analysed with particular reference to the role played by several neurotrophic factors and by the NMDA subtype of glutamate receptor. Then, mechanisms of survival/apoptosis are examined in the model of primary in vitro cultures, where the role of neurotrophins acting on cerebellar granule cells is followed by the large deal of data coming from the paradigm of potassium/serum withdrawal. The role of some key genes of the Bcl family, of some kinase systems and of transcriptional factors is primarily highlighted. Furthermore, the involvement of mitochondria, free radicals and proteases of the caspase family is considered. Finally, the use of cerebellar granule neurons in primary culture to experimentally address the issue of neurodegeneration and pharmacological neuroprotection is considered, with some comments on models at the borderline between necrosis and apoptosis, such as the excitotoxic neuronal damage. The overlapping of cellular signal pathways activated in granule neurons by apparently unrelated stimuli, such as neurotrophins and neurotransmitters/neuromodulators is stressed to put into light the special 'trophic' role played by activity in neurons. Finally, the advantage of designing and performing conceptually equivalent experiments on cerebellar granule neurons during development in vivo and in vitro, is stressed. On the basis of the reviewed material, it is concluded that cerebellar granule neurons have acquired a special position in modern neuroscience as one of the most reliable models for the study of neural development, function and pathology.

Animals↗