Role of nitric oxide in cerebellar development and function: focus on granule neurons.

More than 20 years of research have firmly established important roles of the diffusible messenger molecule, nitric oxide (NO), in cerebellar development and function. Granule neurons are main players in every NO-related mechanism involving cerebellar function and dysfunction. Granule neurons are endowed with remarkable amounts of the Ca(2+)-dependent neuronal isoform of nitric oxide synthase and can directly respond to endogenously produced NO or induce responses in neighboring cells taking advantage of the high diffusibility of the molecule. Nitric oxide acts as a negative regulator of granule cell precursor proliferation and promotes survival and differentiation of these neurons. Nitric oxide is neuroprotective towards granule neurons challenged with toxic insults. Nitric oxide is a main regulator of bidirectional plasticity at parallel fiber-Purkinje neuron synapses, inducing long-term depression (LTD) or long-term potentiation (LTP) depending on postsynaptic Ca(2+) levels, thus playing a central role in cerebellar learning related to motor control. Granule neurons cooperate with glial cells, in particular with microglia, in the regulation of NO production through the respective forms of NOS present in the two cellular types. Aim of the present paper is to review the state of the art and the improvement of our understanding of NO functions in cerebellar granule neurons obtained during the last two decades and to outline possible future development of the research.

Neuronal-glial Interactions Define the Role of Nitric Oxide in Neural Functional Processes.

Nitric oxide (NO) is a versatile cellular messenger performing a variety of physiologic and pathologic actions in most tissues. It is particularly important in the nervous system, where it is involved in multiple functions, as well as in neuropathology, when produced in excess. Several of these functions are based on interactions between NO produced by neurons and NO produced by glial cells, mainly astrocytes and microglia. The present paper briefly reviews some of these interactions, in particular those involved in metabolic regulation, control of cerebral blood flow, axonogenesis, synaptic function and neurogenesis. Aim of the paper is mainly to underline the physiologic aspects of these interactions rather than the pathologic ones.

Regulation of transcription factors by nitric oxide in neurons and in neural-derived tumor cells.

Nitric oxide (NO), a diffusible molecule acting as an intercellular and intracellular messenger in many tissues, plays multiple roles in the nervous system. In addition to regulating proliferation, survival and differentiation of neurons, NO is also involved in synaptic activity, neural plasticity and memory formation. Long-lasting effects of NO, a simple and unstable molecule, occur through regulation of transcription factors and modulation of gene expression. cAMP-response-element-binding (CREB) protein is an important transcription factor that regulates the expression of several genes involved in survival and neuroprotection as well as in synaptic plasticity and memory formation. Nitric oxide promotes survival and differentiation of neural cells, both activating through cGMP signaling CREB phosphorylation-dependent transcriptional activity and promoting S-nitrosylation of nuclear proteins that favor CREB binding to its promoters on target genes. Among oncogenic transcription factors, N-Myc is important in neurogenesis and in regulating proliferation of neural-derived tumor cells, such as neuroblastomas and medulloblastomas. Nitric oxide negatively regulates the proliferation of neuronal precursors, as well as the proliferation of neuroblastoma cells, by downregulating N-Myc expression through cGMP signaling. Other oncogenic transcription factors, such as c-fos and c-jun, zinc-finger transcription factors, such as egr-1, and NF-kappaB are regulated by NO signaling in cGMP-dependent way or through nitrosative conformational changes. The present survey of how NO signaling influences neural cells through regulation of transcription factors allows us to predict that better knowledge of these interactions will provide a better understanding of the physiological role of NO in the nervous system in order to conceive novel therapies for neural-derived tumors.

Neuroprotection of microglial conditioned medium on 6-hydroxydopamine-induced neuronal death: role of transforming growth factor beta-2.

Microglia, the immune cells of the CNS, play essential roles in both physiological and pathological brain states. Here we have used an in vitro model to demonstrate neuroprotection of a 48 h-microglial conditioned medium (MCM) towards cerebellar granule neurons (CGNs) challenged with the neurotoxin 6-hydroxydopamine, which induces a Parkinson-like neurodegeneration, and to identify the protective factor(s). MCM nearly completely protects CGNs from 6-hydroxydopamine neurotoxicity and at least some of the protective factor(s) are peptidic in nature. While the fraction of the medium containing molecules < 30 kDa completely protects CGNs, fractions containing molecules < 10 kDa or > 10 kDa are not neuroprotective. We further demonstrate that microglia release high amounts of transforming growth factor-beta2 (TGF-beta2) and that its exogenous addition to the fraction of the medium not containing it (< 10 kDa) fully restores the neuroprotective action. Moreover, MCM neuroprotection is significantly counteracted by an inhibitor of TGF-beta2 transduction pathway. Our results identify TGF-beta2 as an essential neuroprotective factor released by microglia in its culture medium that requires to be fully effective the concomitant presence of other factor(s) of low molecular weight.

Benefits of caloric restriction on brain aging and related pathological States: understanding mechanisms to devise novel therapies.

Long term caloric restriction is known to counteract aging and extend lifespan in several organisms from yeasts to mammals. Recent research has provided solid ground to the concept that limiting calorie intake slows down brain aging and protects from age-related neurodegenerative diseases. The present review summarizes the most relevant among these data and highlights some genetic and molecular mechanisms responsible for caloric restriction-related neuroprotection. To understand these mechanisms is important because this information makes them potential targets for therapeutic intervention aimed at reproducing the metabolic, genetic and molecular features responsible for the beneficial effect of caloric restriction. Most promising among these targets are neurotrophins, such as BDNF, transcription factors, such as FoxO and PPAR, anti-aging proteins, such as sirtuins, and caloric restriction mimetics acting on oxidative stress and energy metabolism. Notwithstanding the complexity of any therapeutic strategy aimed at reproducing the beneficial effects of caloric restriction, due to multiplicity of the cellular pathways involved in the responses, a great expansion of medicinal chemistry research in this field is expected in the next future.

Memory-enhancing drugs: a molecular perspective.

Neurodegenerative diseases associated with dementia are characterized by cognitive deficits and memory impairment, thus stimulating research for memory enhancing drugs. We survey here the state of the art of research and clinical trials on these drugs from cholinesterase inhibitors and drugs acting on neurotransmitter receptors to drugs acting on gene expression.

Neuroprotection of microglia conditioned media from apoptotic death induced by staurosporine and glutamate in cultures of rat cerebellar granule cells.

Microglia, the immune cells of the mammalian CNS, have often been indicated as dangerous effector cells for their activation in response to traumatic CNS injuries or immunological stimuli and for their involvement in many chronic neurodegenerative diseases. Recently, several in vitro and in vivo studies have emphasized that microglial activity is essential in promoting neuronal survival. We have tested the efficacy of media directly conditioned by microglia or conditioned by microglia after having been exposed to apoptotic neurons, towards neuroprotection of rat cerebellar granule cells (CGCs) challenged with staurosporine or glutamate. Apoptotic death of CGC caused by staurosporine, as well as by a mild excitotoxic stimulus delivered through sub-chronic glutamate treatment, was significantly counteracted by microglia conditioned media. On the other hand, an acute excitotoxic insult delivered through a short pulse of glutamate exposure in the absence of magnesium and resulting in a mix of apoptotic and necrotic death was only marginally counteracted by microglia conditioned media. The present results extend the available information regarding the neuroprotective role of microglia and support the usefulness of employing the culture approach for perspective identification of neuroprotective factors released by these cells. Furthermore, the use of media previously exposed to apoptotic neurons to elicit the neuroprotective response of microglia, indicate the feasibility to re-create also in the isolated culture conditions, at least some of the elements at the basis of neuron/microglia cross-talk.

In vitro and in vivo toxicity of type 2 ribosome-inactivating proteins lanceolin and stenodactylin on glial and neuronal cells.

Lanceolin and stenodactylin, new type 2 ribosome-inactivating proteins (RIPs) from Adenia plants were recently isolated and their high cytotoxicity was described. Present experiments were performed to investigate the effect of these toxins on neural cells in culture and their in vivo retrograde transport and neurotoxicity in the central nervous system. The concentrations of lanceolin and stenodactylin inhibiting by 50% protein synthesis were in the 10(-11) and 10(-12) (cerebellar granule neurons), 10(-12) and 10(-13) (astrocytes), and 10(-13) (microglia) molar range, respectively. Both RIPs resulted toxic for glial cells in culture by MTT test, killing 50% of microglia, the most sensitive cell type, at concentrations around 10(-14)M. Stenodactylin was highly neurotoxic in vivo, when injected intracerebrally, and was retrogradely transported through axons projecting to the injected region. Stereotaxic injection of 1.3 ng toxin into the left dorsal hippocampus resulted in loss of cholinergic neurons in the ipsilateral medial septal nucleus, where cell bodies of neurons providing cholinergic input to the hippocampus are located. The retrograde transport of RIPs along neurons allows to perform experiments of target-selective lesioning, and can be exploited also to perform specific experiments of immunolesioning of selected neuronal populations.

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.

Overactivation of LPS-stimulated microglial cells by co-cultured neurons or neuron-conditioned medium.

Microglial activation represents a well known aspect of several neuropathological diseases. However, little is known concerning the role of neurons in starting and modulating this process. In the present report, we demonstrate that differentiated, healthy neurons constitutively release in the culture medium substance(s) that are able to induce a state of overactivation in LPS-stimulated microglial cells. The neuronal factors synergize with LPS in stimulating synthesis and release of interleukin-1beta (IL-1beta) and nitric oxide by microglial cells. Prolonged exposure (72 h) to neuron-conditioned media in the presence of LPS induced microglial apoptosis, thus suggesting that neuronal overactivation of stimulated microglia favors their subsequent apoptotic elimination as part of a safety mechanism.

Regional and temporal alterations of ODC/polyamine system during ALS-like neurodegenerative motor syndrome in G93A transgenic mice.

Natural polyamines (putrescine, spermidine and spermine) are ubiquitous molecules known to regulate a number of physiological processes and suspected to play a role also in various pathological conditions. Changes in polyamine levels and in their biosynthetic enzymes have been described for some neurodegenerative diseases but the available data are incomplete and somewhat contradictory. We report here alterations of the key enzyme of the polyamine pathway, ornithine decarboxylase (ODC) catalytic activity and polyamine levels in different CNS areas from SOD1 G39A transgenic mice, an animal model for amyotrophic lateral sclerosis (ALS). ODC catalytic activity, was found significantly increased both in the cervical and lumbar spinal cord and, to a lesser extent in the brain stem of transgenic mice at a symptomatic stage of the disease (125-day-old mice), while no differences were present at a pre-symptomatic stage (55-day-old mice). In parallel with the increase of ODC activity putrescine levels were several times increased in both cervical and lumbar spinal cord and in the brain stem of 125-day-old SOD1 G39A mice. Higher order polyamines were not increased except for a significant increase of spermidine in the cervical spinal cord. The present data demonstrate considerable alterations of the ODC/polyamine system in a reliable animal model of ASL, consistent with their role in neurodegeneration and in particular in motor neuron diseases.

Zinc supplementation in rats impairs hippocampal-dependent memory consolidation and dampens post-traumatic recollection of stressful event.

Zinc is a trace element important for synaptic plasticity, learning and memory. Zinc deficiency, both during pregnancy and after birth, impairs cognitive performance and, in addition to memory deficits, also results in alterations of attention, activity, neuropsychological behavior and motor development. The effects of zinc supplementation on cognition, particularly in the adult, are less clear. We demonstrate here in adult rats, that 4 week-long zinc supplementation given by drinking water, and approximately doubling normal daily intake, strongly impairs consolidation of hippocampal-dependent memory, tested through contextual fear conditioning and inhibitory avoidance. Furthermore, the same treatment started after memory consolidation of training for the same behavioral tests, substantially dampens the recall of the stressful event occurred 4 weeks before. A molecular correlate of the amnesic effect of zinc supplementation is represented by a dysregulated function of GSK-3ß in the hippocampus, a kinase that participates in memory processes. The possible relevance of these data for humans, in particular regarding post-traumatic stress disorders, is discussed in view of future investigation.

Changing paradigm to target microglia in neurodegenerative diseases: from anti-inflammatory strategy to active immunomodulation.

INTRODUCTION: The importance of microglia in most neurodegenerative pathologies, from Parkinson's disease to amyotrophic lateral sclerosis and Alzheimer's disease, is increasingly recognized. Until few years ago, microglial activation in pathological conditions was considered dangerous to neurons due to its causing inflammation. Today we know that these glial cells also play a crucial physiological and neuroprotective role, which is altered in neurodegenerative conditions. AREAS COVERED: The neuroinflammatory hypothesis for neurodegenerative diseases has led to the trial of anti-inflammatory agents as therapeutics with largely disappointing results. New information about the physiopathological role of microglia has highlighted the importance of immunomodulation as a potential new therapeutic approach. This review summarizes knowledge on microglia as a potential therapeutic target in the most common neurodegenerative diseases, with focus on compounds directed toward the modulation of microglial immune response through specific molecular pathways. EXPERT OPINION: Here we support the innovative concept of targeting microglial cells by modulating their activity, rather than simply trying to counteract their inflammatory neurotoxicity, as a potential therapeutic approach for neurodegenerative diseases. The advantage of this therapeutic approach could be to reduce neuroinflammation and toxicity, while at the same time strengthening intrinsic neuroprotective properties of microglia and promoting neuroregeneration.

Disease-related regressive alterations of forebrain cholinergic system in SOD1 mutant transgenic mice.

Transgenic mice carrying the human mutated SOD1 gene with a glycine/alanine substitution at codon 93 (G93A) are a widely used model for the fatal human disease amyotrophic lateral sclerosis (ALS). In these transgenic mice, we carried out a neurochemical study not only restricted to the primarily affected regions, the cervical and lumbar segments of the spinal cord, but also to several other brain regions. At symptomatic (110 and 125 days of age), but not at pre-symptomatic (55 days of age) stages, we found significant decreases in catalytic activity of the cholinergic enzyme, choline acetyltransferase (ChAT) in the hippocampus, olfactory cortex and fronto-parietal cortex. In parallel, we observed a decreased number of basal forebrain cholinergic neurons projecting to these areas. No alterations of the cholinergic markers were noticed in the striatum and the cerebellum. A widespread marker for GABAergic neurons, glutamate decarboxylase (GAD), was unaffected in all the areas examined. Alteration of cholinergic markers in forebrain areas was paralleled by concomitant alterations in the spinal cord and brainstem, as a consequence of progressive apoptotic elimination of cholinergic motor neuron. Gestational supplementation of choline, while able to result in long-term enhancement of cholinergic activity, did not improve transgenic mice lifespan nor counteracted cholinergic impairment in brain regions and spinal cord.

Neurochemical correlates of nicotine neurotoxicity on rat habenulo-interpeduncular cholinergic neurons.

Chronic administration of high doses of nicotine results in axonal degeneration in the central core of the fasciculus retroflexus, a fiber tract connecting the habenulae (Hb) to the interpeduncular nucleus (IPN). An important part of this connection is cholinergic and neurons of origin are located in the medial Hb. We have undertaken the present investigation in order to ascertain whether the cholinergic Hb-IPN neurons are the actual target of nicotine toxicity and to begin studying molecular correlates of this action. In the present report, we demonstrate that 7-day-long continuous administration of nicotine through osmotic minipumps, results in a significant (-13%) decrease in the volume of the medial Hb, where cholinergic neurons projecting to the IPN are located, and in a drop of a specific marker for cholinergic neurons, choline acetyltransferase (ChAT), in Hb (-36%) and IPN (-28%). At various intervals (2-6 days) during continuous nicotine administration, some apoptotic neurons were visualized in the medial Hb by the TUNEL technique. The chronic nicotine treatment also resulted, after 2 days of continuous administration in significant activation of the transcription factor CREB and the ERK/MAPK survival kinase in the Hb, suggesting that these alterations in expression are in some way related to the neurodegenerative/neuroreparative process. The present observations demonstrate that the cholinergic Hb-IPN neurons are a target for nicotine neurotoxicity and confirm the usefulness of the experimental model used here not only to study the consequences of chronic stimulant abuse, but also to study the neurochemistry of the affected neural systems and the role of signaling factors in neurodegenerative and repair mechanisms. Medical relevance of the data on unique vulnerability of the Hb-IPN connection to nicotine in relation to heavy smoking habits, is briefly discussed.

Neuron-conditioned media differentially affect the survival of activated or unstimulated microglia: evidence for neuronal control on apoptotic elimination of activated microglia.

It is presently unknown what types of neuronal signals maintain microglial cells resting in the normal brain or control their activation in neuropathology. Recent data suggest that microglia activation induces apoptosis and that healthy neurons are controllers of the activation state and immune functions of microglia. In the present study we have evaluated, on microglial cells in cultures, whether neurons are able to affect their survival in resting conditions or upon activation with the bacterial endotoxin, lipopolysaccharide (LPS). We report that neuron-conditioned culture media induced apoptosis of LPS-stimulated, but not of unstimulated, microglia. This effect was, however, only present when conditioned media had been exposed to differentiated neurons and not to immature ones, and was absent when glutamate receptors had been pharmacologically blocked in neuronal cultures. The effect was also blocked by heat-inactivation of the conditioned media. Media conditioned with either differentiated or undifferentiated cerebellar granule neurons positively affected the survival of unstimulated microglial cells when the standard concentration of fetal bovine serum (10%) was included in the culture media. Our results highlight the ability of differentiated neurons to maintain a controlled inflammatory state through production of factor(s) favoring the apoptotic elimination of activated microglia. They also suggest that immature neurons may, on the contrary, favor the survival of microglia during development.

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.

Brain nitric oxide and its dual role in neurodegeneration/neuroprotection: understanding molecular mechanisms to devise drug approaches.

Nitric oxide (NO) has been established as an important messenger molecule in various steps of brain physiology, from development to synaptic plasticity, learning and memory. However, NO has also been viewed as a major agent of neuropathology when, escaping controlled production it may directly or indirectly promote oxidative and nitrosative stress. The exact borderline between physiological, and therefore neuroprotective, and pathological, and therefore neurodegenerative, actions of NO is a matter of controversy among researchers in the field. This is reflected in the present status of drug research, that is focused on finding ways to block NO production, and therefore limit neuropathology, as well as on finding ways to increase NO availability and therefore elicit neuroprotection. As an unavoidable consequence, both classes of drugs are reported to have neurodegenerative or neuroprotective effects, depending on the models in which they are tested. Aim of the present paper is to provide the reader with a survey, as much complete as possible, on the main aspects of NO biology, from biochemistry and chemical reactivity to the molecular signals elicited in neural cells target of its neurodegenerative or neuroprotective action. In doing that, many controversial aspects related to basic biology and to neuropathology of NO are taken into account. In the final sections, main classes of drugs able to interfere with NO physiopathology are examined, in order to try to devise possible directions for future NO-based therapeutical strategies.

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.