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.

Chronic valproic acid administration impairs contextual memory and dysregulates hippocampal GSK-3ß in rats.

Valproic acid (VPA), a long-standing anti-epileptic and anti-manic drug, exerts multiple actions in the nervous system through various molecular mechanisms. Neuroprotective properties have been attributed to VPA in different models of neurodegeneration, but contrasting results on its improvement of learning and memory have been reported in non-pathologic conditions. In the present study, we have tested on a hippocampal-dependent learning test, the contextual fear conditioning, the effect of chronic VPA administration through alimentary supplementation that allows relatively steady concentrations to be reached by a drug otherwise very rapidly eliminated in rodents. Contextual fear memory was significantly impaired in rats chronically treated with VPA for 4 weeks. To understand the cellular and molecular correlates of this amnesic effect with particular regard to hippocampus, we addressed three putatively memory-related targets of VPA action in this brain area, obtaining the following main results: i) chronic VPA promoted an increase of post-translational modifications of histone H3 (acetylation and phosphorylation) known to favor gene transcription; ii) adult neurogenesis in the dentate gyrus, which has been controversially reported to be affected by VPA, was unchanged; and iii) GSK-3ß, a kinase playing a key role in hippocampal plasticity, as well as in learning and memory, was dysregulated by VPA treatment. These results point at GSK-3ß dysregulation in the hippocampus as an important parameter in the amnesic effect of VPA. The VPA amnesic effect in the animal model here reported is also supported by some observations in patients and, therefore, it should be taken into account and monitored in VPA-based therapies.

Histone acetylation in neurodevelopment.

Post-translational modification of histones is a primary mechanism through which epigenetic regulation of DNA transcription does occur. Among these modifications, regulation of histone acetylation state is an important tool to influence gene expression. Epigenetic regulation of neurodevelopment contributes to the structural and functional shaping of the brain during neurogenesis and continues to impact on neural plasticity lifelong. Alterations of these mechanisms during neurodevelopment may result in later occurrence of neuropsychatric disorders. The present paper reviews and discusses available data on histone modifications, in particular histone acetylation, in neurogenesis considering results obtained in culture systems of neural progenitors as well as in in vivo studies. Possible teratogenic effects of altered histone acetylation state during development are also considered. The use during pregnancy of drugs such as valproic acid, which acts as a histone deacetylase inhibitor, may result during postnatal development in autistic-like symptoms. The effect of gestational administration of the drug has been, therefore, tested on adult hippocampal neurogenesis in animals showing behavioral impairment as a consequence of the drug administration at a specific stage of pregnancy. These experimental results show that adult neurogenesis in the hippocampal dentate gyrus is not quantitatively altered by gestational valproic acid administration. Future steps and goals of research on the role and mechanisms of histone acetylation in neurodevelopment are briefly discussed.

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.

The history of the cholinergic hypothesis.

The cholinergic hypothesis of cognitive impairment and Alzheimer's disease has been for decades a "polar star" for studies on dementia and neurodegenerative diseases. Aim of the present article is to briefly summarize its birth and its evolution throughout years and discoveries. Putting the cholinergic hypothesis in an historical perspective, allows to appreciate the enormous amount of experimental and clinical research that it has stimulated over years and the impressive extent of knowledge generated by this research. While some of the assumptions at the basis of its original formulation are disputable in the light of recent developments, the cholinergic hypothesis has, however, constituted an invaluable stimulus to better understand not only the anatomy and the biochemistry of the cholinergic systems of brain connections but also its developmental biology, its complex relationships with trophic factors, its role in cognitive functions. Thus, rather than being consigned to history, the cholinergic hypothesis will likely contribute to further understanding dementia and neurodegenerative diseases and will hopefully be integrated in novel therapies and treatments.

Nitric oxide control of MYCN expression and multi drug resistance genes in tumours of neural origin.

Nitric oxide (NO) exerts its function in several cell and organ compartments. Recently, several lines of evidence have been accrued showing that NO can play a critical role in oncogenesis. Here we summarize some of these findings and highlight the role of NO as a possible target for antineoplastic drugs. Specifically, NO appears to affect some aspects of neuronal tumour progression, particularly the chemoresistance phenotype, through inhibition of MYC activity and expression of a large set of ATP binding cassette transporters. Here we provide lines of evidence supporting the view that MYCN can alter expression of several members of the ABC transporter family thus influencing the chemoresistance phenotype of neuroblastoma cells. Furthermore, we show that increased intracellular NO concentration either through addition of NO donors to culture medium or through forced expression of nNOS in neuroblastoma cells leads to decreased expression of MYCN and ABC drug transporter genes. Overall, data reviewed here and novel results presented, unveil a NO-MYCN-ABC transporters axis with important implication on development and control of the chemoresistance phenotype in neuronal tumours.

Nitric oxide control of proliferation in nerve cells and in tumor cells of nervous origin.

Recent evidence suggests that nitric oxide (NO) has a remarkable anti-proliferative action towards dividing neural precursor cells as well as towards cells giving rise to neural-derived tumors. The present paper summarizes essential literature-derived information on this issue and provides novel experimental evidence for these NO-mediated actions regarding a well characterized population of neuronal precursors, the cerebellar granule cell precursors and a cell line of medulloblastoma, a pediatric tumor originating from these same precursor cells undergoing deregulated proliferation. Evidence is presented regarding the NO-mediated regulation of proliferation of neuronal precursor cells both during developmental and adult neurogenesis. Then, the role of NO in the control of proliferation of neural-derived tumor cells, such as PC12 and neuroblastoma cells, is discussed. Novel experimental data are provided documenting the anti-proliferative action of NO towards basal and mitogen-stimulated division of rat cerebellar granule cell precursors, as well as towards medulloblastoma DAOY cells. Finally, some molecular correlates of NO action on cell cycle regulation are discussed. Overall, the data presented and discussed here highlight similarities at the molecular level between physiologic processes regulating normal proliferation of neural precursors and pathologic deregulation of these processes leading to tumor formation.

Transcriptional profiling in the lumbar spinal cord of a mouse model of amyotrophic lateral sclerosis: a role for wild-type superoxide dismutase 1 in sporadic disease?

Mice bearing mutations of copper-zinc-superoxide dismutase recapitulate spinal cord motor neuron degeneration and disease progression occurring in human amyotrophic lateral sclerosis. We have investigated the relationship between disease progression and altered gene expression by comparing the transcriptional profiles in lumbar spinal cord, fronto-parietal cortex and hippocampus of mutant G93A-SOD1, wild-type SOD1 transgenic and non-transgenic mice. Gene expression was evaluated at 55 and 110 days of age, representing pre-symptomatic and advanced disease stages of G93A mice, respectively. Whereas no significant variations were detectable in cortical and hippocampal areas, several mutation-related changes were detected in the lumbar spinal cord at the symptomatic stage, consistent with a condition of neuronal distress. Also, at both ages, we found a number of transgene-related changes, i.e. variations occurring in both transgenic groups independently of the G93A mutation, with wild-type SOD1- and G93A-SOD1-overexpressing mice displaying global transcriptional similarity at 110 days of age. Some of the changes in common between the two transgenic groups involve genes implicated in oxidative stress, inflammation, spinocerebellar degeneration and other neurodegenerative disorders. The finding that gene expressional alterations potentially associated to cellular distress are shared by wild-type and mutant human SOD1-overexpressing mice raises the possibility that mutated (in familial ALS) or otherwise dysregulated (in sporadic ALS) SOD1 expression is a common pathogenetic substrate of the disease.

Valproic acid is neuroprotective in the rotenone rat model of Parkinson's disease: involvement of alpha-synuclein.

Valproic acid (VPA), an established antiepileptic and antimanic drug, has recently emerged as a promising neuroprotective agent. Among its many cellular targets, VPA has been recently demonstrated to be an effective inhibitor of histone deacetylases. Accordingly, we have adopted a schedule of dietary administration (2% VPA added to the chow) that results in a significant inhibition of histone deacetylase activity and in an increase of histone H3 acetylation in brain tissues of 4 weeks-treated rats. We have tested this schedule of VPA treatment in an animal model of Parkinson's disease (PD), in which degeneration of nigro-striatal dopaminergic neurons is obtained through sub-chronic administration of the mitochondrial toxin, rotenone, via osmotic mini pumps implanted to rats. The decrease of the dopaminergic marker tyrosine hydroxylase in substantia nigra and striatum caused by 7 days toxin administration was prevented in VPA-fed rats. VPA treatment also significantly counteracted the death of nigral neurons and the 50% drop of striatal dopamine levels caused by rotenone administration. The PD-marker protein alpha-synuclein decreased, in its native form, in substantia nigra and striatum of rotenone-treated rats, while monoubiquitinated alpha-synuclein increased in the same regions. VPA treatment counteracted both these alpha-synuclein alterations. Furthermore, monoubiquitinated alpha-synuclein increased its localization in nuclei isolated from substantia nigra of rotenone-treated rats, an effect also prevented by VPA treatment. Nuclear localization of alpha-synuclein has been recently described in some models of PD and its neurodegenerative effect has been ascribed to histone acetylation inhibition. Thus, the ability of VPA to increase histone acetylation is a novel candidate mechanism for its neuroprotective action.

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.

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 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.

Chronic dietary administration of valproic acid protects neurons of the rat nucleus basalis magnocellularis from ibotenic acid neurotoxicity.

Valproic acid (VPA) has been used for many years as a drug of choice for epilepsy and mood disorders. Recently, evidence has been proposed for a wide spectrum of actions of this drug, including antitumoral and neuroprotective properties. Valproic acid-mediated neuroprotection in vivo has been so far demonstrated in a limited number of experimental models. In this study, we have tested the neuroprotective potential of chronic (4 + 1 weeks) dietary administration of VPA on degeneration of cholinergic and GABAergic neurons of the rat nucleus basalis magnocellularis (NBM), injected with the excitotoxin, ibotenic acid (IBO), an animal models that is relevant for Alzheimer's disease-like neurodegeneration. We show that VPA treatment significantly protects both cholinergic and GABAergic neurons present in the injected area from the excitotoxic insult. A significant level of neuroprotection, in particular, is exerted towards the cholinergic neurons of the NBM projecting to the cortex, as demonstrated by the substantially higher levels of cholinergic markers maintained in the target cortical area of VPA-treated rats after IBO injection in the NBM. We further show that chronic VPA administration results in increased acetylation of histone H3 in brain, consistent with the histone deacetylase inhibitory action of VPA and putatively linked to a neuroprotective action of the drug mediated at the epigenetic level.

Long-term dietary administration of valproic acid does not affect, while retinoic acid decreases, the lifespan of G93A mice, a model for amyotrophic lateral sclerosis.

Mice bearing the mutated gene for Cu/Zn superoxide dismutase (G93A) are a good model for human amyotrophic lateral sclerosis (ALS). They develop progressive limb paralysis paralleled by loss of motor neurons of the cervical and lumbar spinal cord, which starts at 3-3.5 months of age and ends with death at 4-5 months. Several treatments have been attempted to delay clinical symptoms and to extend lifespan, and some have had modest beneficial effects. One such treatment, based on long-term administration of valproic acid (VPA), resulted in controversial results. We report here that, while dietary supplementation with high VPA dosage slows down motor neuron death, as assessed by measurement of a specific marker for cholinergic neurons in the spinal cord, it has no significant effect on lifespan. Recently, the hypothesis has been put forward that a deficiency of retinoic acid (RA) and its signaling may have a role in ALS. We report that long-term dietary supplementation with RA has no effect on the decrease of the cholinergic marker in the spinal cord, but it significantly shortens lifespan of G93A mice.

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.