Elisabetta Ciani

Early Pharmacotherapy Restores Neurogenesis and Cognitive Performance in the Ts65Dn Mouse Model for Down Syndrome

Down syndrome (DS) is a genetic pathology characterized by intellectual disability and brain hypotrophy. Widespread neurogenesis impairment characterizes the fetal and neonatal DS brain, strongly suggesting that this defect may be a major determinant of mental retardation. Our goal was to establish, in a mouse model for DS, whether early pharmacotherapy improves neurogenesis and cognitive behavior. Neonate Ts65Dn mice were treated from postnatal day (P) 3 to P15 with fluoxetine, an antidepressant that inhibits serotonin (5-HT) reuptake and increases proliferation in the adult Ts65Dn mouse (Clark et al., 2006). On P15, they received a BrdU injection and were killed after either 2 h or 1 month. Results showed that P15 Ts65Dn mice had notably defective proliferation in the hippocampal dentate gyrus, subventricular zone, striatum, and neocortex and that proliferation was completely rescued by fluoxetine. In the hippocampus of untreated P15 Ts65Dn mice, we found normal 5-HT levels but a lower expression of 5-HT1A receptors and brain-derived neurotrophic factor (BDNF). In Ts65Dn mice, fluoxetine treatment restored the expression of 5-HT1A receptors and BDNF. One month after cessation of treatment, there were more surviving cells in the dentate gyrus of Ts65Dn mice, more cells with a neuronal phenotype, more proliferating precursors, and more granule cells. These animals were tested for contextual fear conditioning, a hippocampus-dependent memory task, and exhibited a complete recovery of memory performance. Results show that early pharmacotherapy with a drug usable by humans can correct neurogenesis and behavioral impairment in a model for DS.

Neurogenesis Impairment and Increased Cell Death Reduce Total Neuron Number in the Hippocampal Region of Fetuses with Down Syndrome

We previously obtained evidence for reduced cell proliferation in the dentate gyrus (DG) of fetuses with Down syndrome (DS), suggesting that the hippocampal hypoplasia seen in adulthood may be caused by defective early neuron production. The goal of this study was to establish whether DS fetuses (17–21 weeks of gestation) exhibit reduction in total cell number in the DG, hippocampus and parahippocampal gyrus (PHG). Volumes of the cellular layers and cell number were estimated with Cavalieris principle and the optical fractionator method, respectively. We found that in DS fetuses all investigated structures had a reduced volume and cell number. Analysis of cell phenotype showed that DS fetuses had a higher percentage of cells with astrocytic phenotype but a smaller percentage of cells with neuronal phenotype. Immunohistochemistry for Ki‐67, a marker of cycling cells, showed that DS fetuses had less proliferating cells in the germinal zones of the hippocampus and PHG. We additionally found that in the hippocampal region of DS fetuses there was a higher incidence of apoptotic cell death. Results show reduced neuron number in the DS hippocampal region and suggest that this defect is caused by disruption of neurogenesis and apoptosis, two fundamental processes underlying brain building.

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.

RESEARCH ARTICLE: Neurogenesis Impairment and Increased Cell Death Reduce Total Neuron Number in the Hippocampal Region of Fetuses with Down Syndrome

We previously obtained evidence for reduced cell proliferation in the dentate gyrus (DG) of fetuses with Down syndrome (DS), suggesting that the hippocampal hypoplasia seen in adulthood may be caused by defective early neuron production. The goal of this study was to establish whether DS fetuses (17–21 weeks of gestation) exhibit reduction in total cell number in the DG, hippocampus and parahippocampal gyrus (PHG). Volumes of the cellular layers and cell number were estimated with Cavalieris principle and the optical fractionator method, respectively. We found that in DS fetuses all investigated structures had a reduced volume and cell number. Analysis of cell phenotype showed that DS fetuses had a higher percentage of cells with astrocytic phenotype but a smaller percentage of cells with neuronal phenotype. Immunohistochemistry for Ki‐67, a marker of cycling cells, showed that DS fetuses had less proliferating cells in the germinal zones of the hippocampus and PHG. We additionally found that in the hippocampal region of DS fetuses there was a higher incidence of apoptotic cell death. Results show reduced neuron number in the DS hippocampal region and suggest that this defect is caused by disruption of neurogenesis and apoptosis, two fundamental processes underlying brain building.

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.

APP-dependent up-regulation of Ptch1 underlies proliferation impairment of neural precursors in Down syndrome

Mental retardation in Down syndrome (DS) appears to be related to severe neurogenesis impairment during critical phases of brain development. Recent lines of evidence in the cerebellum of a mouse model for DS (the Ts65Dn mouse) have shown a defective responsiveness to Sonic Hedgehog (Shh), a potent mitogen that controls cell division during brain development, suggesting involvement of the Shh pathway in the neurogenesis defects of DS. Based on these premises, we sought to identify the molecular mechanisms underlying derangement of the Shh pathway in neural precursor cells (NPCs) from Ts65Dn mice. By using an in vitro model of NPCs obtained from the subventricular zone and hippocampus, we found that trisomic NPCs had an increased expression of the Shh receptor Patched1 (Ptch1), a membrane protein that suppresses the action of a second receptor, Smoothened (Smo), thereby maintaining the pathway in a repressed state. Partial silencing of Ptch1 expression in trisomic NPCs restored cell proliferation, indicating that proliferation impairment was due to Ptch1 overexpression. The overexpression of Ptch1 in trisomic NPCs resulted from increased levels of AICD [a transcription-promoting fragment of amyloid precursor protein (APP)] and increased AICD binding to the Ptch1 promoter. Our data provide novel evidence that Ptch1 overexpression underlies derangement of the Shh pathway in trisomic NPCs with consequent proliferation impairment. The demonstration that Ptch1 overexpression in trisomic NPCs is due to an APP fragment provides a link between this trisomic gene and the defective neuronal production that characterizes the DS brain.

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.

Structural, neurochemical and behavioural consequences of neonatal blockade of NMDA receptor through chronic treatment with CGP 39551 or MK-801

Recent evidence suggests that NMDA receptors may be involved in survival of neurons and establishment of correct connectivity during development. We have treated rat pups from postnatal day 1 to 22 with daily s.c. injections of a competitive (CGP 39551) and a non-competitive (MK-801) antagonist of the NMDA receptor. Body weight of treated rats was decreased by 50-65% at postnatal day 24 and by 25-32% at 70 days of age. Brain weight was decreased by 16-24% at both ages. Among the different brain regions, the cerebellum and striatum appeared more decreased in size than the cortex and hippocampus. Only few minor, and in some cases transient, differences were measured in the cerebellum, the hippocampus and the cortex for a battery of neurochemical markers related to cholinergic, GABAergic and glutamatergic transmission as well as to astrocyte and oligodendrocyte activity. When tested in actometric cages from postnatal days 28 to 60, treated rats exhibited a dramatic increase of spontaneous locomotor activity which was maximal in 28-day-old animals (380% and 250% of control values in CGP 39551 and MK-801 groups, respectively) and was still significant at 60 days of age. Therefore, long-lasting alteration of motor behaviour is obtained by the schedule of chronic treatment adopted for the present experiments. Our results suggest that blockade of NMDA receptors during the critical period of brain maturation may result in permanent alteration of neural circuits.