Antonio Contestabile

Roles of NMDA receptor activity and nitric oxide production in brain development

The concept that neural activity is important for brain maturation has focused much research interest on the developmental role of the NMDA receptor, a key mediator of experience-dependent synaptic plasticity. However, a mechanism able to link spatial and temporal parameters of synaptic activity during development emerged as a necessary condition to explain how axons segregate into a common brain region and make specific synapses on neuronal sub-populations. To comply with this developmental constraint, it was proposed that nitric oxide (NO), or other substances having similar chemical and biological characteristics, could act as short-lived, activity-dependent spatial signals, able to stabilize active synapses by diffusing through a local volume of tissue. The present article addresses this issue, by reviewing the experimental evidence for a correlated role of the activity of the NMDA receptor and the production of NO in key steps of neural development. Evidence for such a functional coupling emerges not only concerning synaptogenesis and formation of neural maps, for which it was originally proposed, but also for some earlier phases of neurogenesis, such as neural cell proliferation and migration. Regarding synaptogenesis and neural map formation in some cases, there is so far no conclusive experimental evidence for a coupled functional role of NMDA receptor activation and NO production. Some technical problems related to the use of inhibitors of NO formation and of gene knockout animals are discussed. It is also suggested that other substances, known to act as spatial signals in adult synaptic plasticity, could have a role in developmental plasticity. Concerning the crucial developmental phase of neuronal survival or elimination through programmed cell death, the well-documented survival role related to NMDA receptor activation also starts to find evidence for a concomitant requirement of downstream NO production. On the basis of the reviewed literature, some of the major controversial issues are addressed and, in some cases, suggestions for possible future experiments are proposed.

Partial neuroprotection of in vivo excitotoxic brain damage by chronic administration of the red wine antioxidant agent, trans-resveratrol in rats

The antioxidant compound trans-resveratrol, is found in substantial amount in several types of red wine and is considered one of the substances responsible for the lower incidence of coronary heart diseases among regular consumers of such wines, an effect also known as the French paradox. It has also been proposed that resveratrol may have beneficial effects against neurodegenerative diseases. We report here that chronic administration of resveratrol to young-adult rats, significantly protects from the damage caused by systemic injection of the excitotoxin kainic acid, in the olfactory cortex and the hippocampus. The same treatment, however, is not able to give any significant protection in an ex vivo model of simulated ischemia on hippocampal slices in vitro. This first evidence of a partial neuroprotective action of chronic administration of resveratrol in vivo, suggests that other models of neurodegenerative injury, and in particular of excitotoxic brain damage, should be investigated in order to assess the potentiality for resveratrol to be used as a pharmacological tool for neuroprotection.

Cerebellar granule cells as a model to study mechanisms of neuronal apoptosis or survivalin vivo andin 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 primaryin 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. Thein 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 primaryin 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 developmentin vivo andin 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.

Biochemical, Molecular and Epigenetic Mechanisms of Valproic Acid Neuroprotection

Valproic acid (VPA, 2-propylpentanoic acid) has been widely used as an antiepileptic drug and for the therapy of bipolar disorders for several years. Its mechanism of action was initially found to be primarily related to neurotransmission and modulation of intracellular pathways. More recently, it emerged as an anti-neoplastic agent as well, by acting on cell growth, differentiation and apoptosis. Here, it mainly exerts its effect by regulating gene expression at the molecular level, through epigenetic mechanisms. In particular, it has been demonstrated the effect of VPA in chromatin remodeling, as VPA directly inhibits histone deacetylases (HDACs) activity. Interestingly, it has been observed that these biochemical and molecular pathways are involved not only in beneficial effect of VPA against epilepsy and malignancies, but they are also responsible for more general neuroprotective mechanisms. In particular, it has been demonstrated that VPA is neuroprotective in several models of neurodegenerative diseases. Moreover, due to the involvement of the VPA-affected mechanisms in complex behaviors, VPA is increasingly used as a psychotherapeutic agent. This review summarizes the more recent data on VPA neuroprotective mechanisms at the biochemical, molecular and epigenetic levels, focusing on both in vitro and in vivo models of neurodegenerative diseases. In particular, attention is paid to mechanisms by which VPA affects neuronal survival/apoptosis and proliferation/differentiation balance, as well as synaptic plasticity, by acting both directly on neurons and indirectly through glial cells. Perspective applications of the VPA neuroprotective potential in human neurodegenerative diseases are discussed, when relevant.

Role of nitric oxide in the regulation of neuronal proliferation, survival and differentiation.

Nitric oxide (NO), an important cellular messenger, has been linked to both neurodegenerative and neuroprotective actions. In the present review, we focus on recent data establishing a survival and differentiation role for NO in several neural in vitro and in vivo models. Nitric oxide has been found to be essential for survival of neuronal cell lines and primary neurons in culture under various death challenges. Furthermore, its lack may aggravate some neuropathological conditions in experimental animals. Several cellular pathways and signaling systems subserving this neuroprotective role of NO are considered in the review. Survey of recent data related to the developmental role of NO mainly focus on its action as a negative regulator of neuronal precursor cells proliferation and on its role of promotion of neuronal differentiation. Discussion on discrepancies arising from the literature is focused on the Janus-faced properties of the molecule and it is proposed that most controversial results are related to the intrinsic property of NO to compensate among functionally opposed effects. As an example, the increased proliferation of neural cell precursors under conditions of NO shortage may be, later on in the development, compensated by increased elimination through programmed cell death as a consequence of the lack of the survival-promoting action of the molecule. To elucidate these complex, and possibly contrasting, effects of NO is indicated as an important task for future researches.

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.

Inhibition of free radical production or free radical scavenging protects from the excitotoxic cell death mediated by glutamate in cultures of cerebellar granule neurons

Glutamate kills sensitive neurons through several steps downstream to receptor activation: increased free Ca2+ levels, activation of various enzymes and accumulation of reactive oxygen species (ROS). We have evaluated in a well established model of neuronal cultures the neuroprotective effects of blocking these mechanisms, either singularly or by combining multiple enzyme inhibition and/or ROS scavenging. In vitro cultures of cerebellar granule cells exposed to a toxic concentration of glutamate (100 microM for 15 min in the absence of Mg2+) combined with several pharmacological treatments. Inhibition of nitric oxide synthase (NOS) and phospholipase A2 (PLA2) were effective in decreasing cell death and the combined treatments showed some degree of additivity. By contrast, inhibition of xanthine oxidase (XO) with allopurinol was uneffective. Antioxidants (in particular vitamin e or vitamin E analogs). protected neurons up to more than 50%. A synergistic effect was demonstrated by the combination of vitamin E and C. On the other hand, antioxidants did not increase the protection granted by enzyme inhibitors, suggesting that they act downstream to NOS and PLA2. In conclusion, NOS and PLA2 activated by Ca2+ influx give rise to reactive oxygen species whose deleterious action can be counteracted either by inhibiting these enzymes or by scavenging the excess of free radicals produced by them. Finally, a moderate protection was obtained by blocking protein synthesis with cycloheximide, suggesting a partial contribution of apoptotic mechanisms to the excitotoxic cell death.

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.

Subchronic Rolipram Delivery Activates Hippocampal CREB and Arc, Enhances Retention and Slows Down Extinction of Conditioned Fear

Rolipram, a type IV-specific phosphodiesterase inhibitor, is known to improve memory under various learning tasks. Moreover, Rolipram treatments have been shown to increase expression and phosphorylation of a key factor for hippocampal memory consolidation, the cAMP-dependent response element-binding protein, CREB. However, the exact correlation between hippocampal CREB phosphorylation and memory improvement induced by Rolipram has not yet been determined in a CREB-dependent type of hippocampal-related learning in normogenic, intact rodents. Here, we report that subchronic Rolipram delivery by using osmotic minipumps increased the basal rat hippocampal expression and phosphorylation of CREB, as well as the expression of the cAMP-dependent, memory-related protein, Arc. In parallel, the same treatment improved memory consolidation of conditioned fear. Furthermore, the increase of CREB phosphorylation and Arc expression consequent to the learning experience was enhanced in Rolipram-treated rats, compared to controls. By evaluating the time course of memory extinction over 10 days after the initial learning test, we also observed significant slowing down of the memory extinction rate in Rolipram-treated rats. This effect could be attributed to CREB phosphorylation and memory having been initially higher, as osmotic minipumps stopped to release Rolipram the first day after the initial learning test. Our data define the conditions through which the pharmacological manipulation of hippocampal CREB expression and activation result in memory amelioration in normogenic, intact animals. These results are relevant for the study of molecular correlates of memory, and may also be important in view of the efforts to design new pharmacological treatments, targeting the CREB pathway and leading to enhancement of learning and memory, even in the absence of patent neuropathology.

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 (1). 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.