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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.

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.

Prostaglandins and nitric oxide (NO) are among the numerous substances released by activated microglial cells, the brain resident macrophages, and they mediate several important microglial functions. We have previously shown that cyclooxygenase-2 (COX-2) and inducible NO synthase (iNOS), the two key enzymes in prostaglandin and NO synthesis, respectively, are rapidly co-induced in rat neonatal microglial cultures activated by bacterial endotoxin (lipopolysaccharide [LPS]) and that COX-2 expression appears to be under the negative control of endogenous as well as exogenous NO. In this study we show that exogenous prostaglandin E2 (PGE2), which is known to increase cyclic adenosine monophosphate (cAMP) levels in microglial cells, downregulates LPS-induced iNOS expression in a dose-dependent manner. The involvement of cAMP in the PGE2-dependent inhibition of iNOS is supported by several pieces of evidence. First, iNOS expression was also inhibited by agents such as isoproterenol and forskolin, which cause an elevation of cAMP levels, and by dibutyryl cAMP (dbcAMP), a cAMP stable analogue. Second, the inhibitory effect of PGE2 was mimicked by 11-deoxy-16,16-dm PGE2, a selective agonist at the PGE2 receptor subtype EP2, coupled to the activation of adenylyl cyclase, but not by sulprostone, a potent agonist at receptor subtypes EP3 and EP1, associated with an inhibition of adenylyl cyclase activity and intracellular Ca2+ elevation, respectively. Third, the inhibitory effect of PGE2 on NO synthesis was blocked by SQ 22,536, a specific inhibitor of adenylyl cyclase. Interestingly, the abrogation of endogenous prostanoid production by several COX inhibitors caused a reduction of iNOS expression, suggesting a positive modulatory effect of endogenous prostanoids of iNOS expression, as opposed to the inhibitory effect of exogenous PGE2.

1 Activated microglial cells are believed to play an active role in most brain pathologies, during which they can contribute to host defence and repair but also to the establishment of tissue damage. These actions are largely mediated by microglial secretory products, among which are prostaglandins (PGs) and nitric oxide (NO). 2 The anti-in¯ammatory protein, lipocortin 1 (LC1) was reported to have neuroprotective action and to be induced by glucocorticoids in several brain structures, with a preferential expression in microglia. In this paper we tested whether the neuroprotective eect of LC1 could be explained by an inhibitory eect on microglial activation. 3 We have previously shown that bacterial endotoxin (LPS) strongly stimulates PGE 2 and NO production in rat primary microglial cultures, by inducing the expression of the key enzymes cyclooxygenase-2 (COX-2) and inducible nitric oxide synthase (iNOS), respectively. 4 Dexamethasone (DEX, 1 ± 100 nM) and LC1-derived N-terminus peptide (peptide Ac2-26, 1 ± 100 mg ml 71) dose-dependently inhibited the production of both PGE 2 and NO from LPS-stimulated microglia. The inhibitory eects of DEX on NO and of the peptide on NO and PGE 2 synthesis were partially abrogated by a speci®c antiserum, raised against the N-terminus of human LC1. The peptide Ac2-26 did not aect arachidonic acid release from control and LPS-stimulated microglial cultures. 5 Western blot experiments showed that the LPS-induced expression of COX-2 and iNOS was eectively down-regulated by DEX (100 nM) and peptide Ac2-26 (100 mg ml 71 ). 6 In conclusion, our ®ndings support the hypothesis that LC1 may foster neuroprotection by limiting microglial activation, through autocrine and paracrine mechanisms.

Cyclooxygenase-2, the inducible isoform of cyclooxygenase, is highly expressed in microglial cells activated by bacterial lipopolysaccharide and is a major regulatory factor in the synthesis of prostanoids, such as prostaglandins, prostacyclin and thromboxanes. Since prostanoids are potent modulators of inflammation, immune responses and neurotoxicity, the regulation of their synthesis may be crucial for balancing microglial neuroprotective and neurotoxic activities. The present study shows that expression of cyclooxygenase-2 and prostanoid production in cultured rat microglia activated by lipopolysaccharide is up-regulated by cyclic AMP (cAMP), as indicated by experiments performed in the presence of adenylyl cyclase activators, cAMP analogues and protein kinase A-specific inhibitors. Exogenous prostaglandin E2 (PGE2), which elevates the cAMP level in microglial cells, also increased the lipopolysaccharide-induced expression of cyclooxygenase-2 and production of thromboxane in a dose- and time-dependent manner. The observations that the lipopolysaccharide-induced prostanoid production was specifically increased by 11-deoxy-16,16-dm PGE2, a selective agonist at the PGE2 receptor EP2 coupled to the activation of adenylyl cyclase, and that the enhancing effect of PGE2 was partially prevented by specific inhibitors of adenylyl cyclase and protein kinase A, suggest that the up-regulation of cyclooxygenase-2 expression by PGE2 is mediated by cAMP, through a putative microglial EP2 receptor. Unexpectedly, non-steroidal anti-inflammatory drugs such as indomethacin and 6-methoxy naphthalene acetic acidic, which inhibit cyclooxygenase enzymatic activity and abrogate prostanoid synthesis, caused a moderate but consistent up-regulation of cyclooxygenase-2 expression. In conclusion, while the strong up-regulation of cyclooxygenase-2 expression by exogenous PGE2 appears to be mediated by EP2 receptors and cAMP, the limited down-regulation caused by anti-inflammatory drug treatments may be either due to arachidonic acid metabolites other than PGE2, or to PGE2 itself, acting through a distinct cAMP-independent signalling pathway.

The microglia are the immune cell population of the nervous system and play important roles both in normal function and in disease. Reciprocal neuron-microglia interactions are not well understood, in particular those concerning the crosstalk between the two cell populations when neuronal damage does occur. We have used a well-established model of apoptosis in cerebellar granule neurons to test the effect of co-culturing microglial cells with them or of exposing them to microglia-conditioned medium. Microglial cells, derived from cortical or cerebellar mixed glial cultures and plated over cerebellar granule neurons, protected these neurons from apoptosis induced by shifting them, at 7 days in vitro, for 24 h from a depolarizing (high-potassium) to a nondepolarizing (low-potassium) medium. The same result was achieved when microglial cells obtained from mixed glial cortical cultures were plated over a membrane well insert in the culture chamber, permitting medium exchange without physical contact with granule neurons. A similar result was obtained when the low-potassium, apoptosis-inducing medium was conditioned by 48-h exposure to microglial cells; 24-h exposure to microglial cells was not enough to confer neuroprotective capability to the conditioned medium. However in double-conditioned medium experiments, in which the medium was first exposed to apoptotic neurons and then to microglial cells, unknown signal(s) released by apoptotic neurons, conferred to the 24-h conditioned medium a strong neuroprotective action, similar to that observed in the co-cultures experiments. This finding, together with the results from co-culture experiments, is explained by admitting that molecules released in the medium by apoptotic neurons potentiate the anti-apoptotic activity of microglia. Our results, therefore, demonstrate not only that normally microglial cells release in the medium molecule(s) able to rescue neurons from apoptotic death, but that unknown diffusible signal(s) from apoptotic neurons enhance(s) microglial neuroprotective properties as well.

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