Microglia and the Blood-Brain Barrier: An External Player in Acute and Chronic Neuroinflammatory Conditions.

Microglia are the resident immune cells of the central nervous system that guarantee immune surveillance and exert also a modulating role on neuronal synaptic development and function. Upon injury, microglia get activated and modify their morphology acquiring an ameboid phenotype and pro- or anti-inflammatory features. The active role of microglia in blood-brain barrier (BBB) function and their interaction with different cellular components of the BBB-endothelial cells, astrocytes and pericytes-are described. Here, we report the specific crosstalk of microglia with all the BBB cell types focusing in particular on the involvement of microglia in the modulation of BBB function in neuroinflammatory conditions that occur in conjunction with an acute event, such as a stroke, or in a slow neurodegenerative disease, such as Alzheimer's disease. The potential of microglia to exert a dual role, either protective or detrimental, depending on disease stages and environmental conditioning factors is also discussed.

Neurobiological links between depression and AD: The role of TGF-ß1 signaling as a new pharmacological target.

In the last several years a large number of studies have demonstrated the neurobiological and clinical continuum between depression and Alzheimer's disease (AD). Depression is a risk factor for the development of AD, and the presence of depressive symptoms significantly increases the conversion of Mild Cognitive Impairment (MCI) into AD. Common pathophysiological events have been identified in depression and AD, including neuroinflammation with an aberrant Tumor Necrosis Factor-α (TNF-α) signaling, and an impairment of Brain-Derived Neurotrophic Factor (BDNF) and Transforming-Growth-Factor-ß1 (TGF-ß1) signaling. TGF-ß1 is an anti-inflammatory cytokine that exerts neuroprotective effects against amyloid-ß (Aß)-induced neurodegeneration, and it has a key role in memory formation and synaptic plasticity. TGF-ß1 plasma levels are reduced in major depressed patients (MDD), correlate with depression severity, and significantly contribute to treatment resistance in MDD. The deficit of Smad-dependent TGF-ß1 signaling is also an early event in AD pathogenesis, which contributes to inflammaging and cognitive decline in AD. A long-term treatment with antidepressants such as selective-serotonin-reuptake inhibitors (SSRIs) is known to reduce the risk of AD in patients with depression and, SSRIs, such as fluoxetine, increase the release of TGF-ß1 from astrocytes and exert relevant neuroprotective effects in experimental models of AD. We propose the TGF-ß1 signaling pathway as a common pharmacological target in depression and AD, and discuss the potential rescue of TGF-ß1 signaling by antidepressants as a way to prevent the transition from depression to AD.

Astrocytes contribute to Aß-induced blood-brain barrier damage through activation of endothelial MMP9.

The blood-brain barrier (BBB) plays an important role in the maintenance of the brain homeostasis, and its proper functions are warranted by the interplay between different cellular components (endothelial cells, astrocytes and pericytes). BBB dysfunctions in pathological conditions, and particularly in Alzheimer's disease, have been documented. Here, using an in vitroBBB model, the interaction between endothelial cells and astrocytes exposed to Aß1-42 was investigated. Human endothelial cells, cultured in monolayer or co-cultured with astrocytes, were exposed to Aß1-42 (2 µM for 18 h). Aß induced dysfunction of endothelial barrier, as assessed by enhanced permeability to FITC-conjugated dextran and reduced expression of claudin-5; these modifications were observed in the co-culture model, but not in endothelial cells cultured in monolayer. Similarly, Aß-induced damage at the barrier was observed when endothelial cells were challenged in the presence of conditioned medium generated by astrocytes previously exposed to Aß (ACM Aß). Endothelial barrier damages were associated with enhanced matrix metalloprotease 9 (MMP9) activity, known to mediate claudin-5 disruption. These events were not related to the direct effects played by Aß on endothelial cells, but they were rather the consequence of Aß-induced vascular endothelial growth factor (VEGF) expression in astrocytes. Indeed, when vascular endothelial growth factor expression was down-regulated in astrocytes, neither barrier properties or MMP9 expression in endothelial cells were affected after Aß exposure both in the co-culture model or in the presence of ACM Aß. These data point out the importance of astrocytes' mediation in inducing endothelial sensitivity to Aß1-42.

Identification of 5-Methoxyflavone as a Novel DNA Polymerase-Beta Inhibitor and Neuroprotective Agent against Beta-Amyloid Toxicity.

Cell-cycle reactivation is a core feature of degenerating neurons in Alzheimer's disease (AD) and Parkinson's disease (PD). A variety of stressors, including ß-amyloid (Aß) in the case of AD, can force neurons to leave quiescence and to initiate an ectopic DNA replication process, leading to neuronal death rather than division. As the primary polymerase (pol) involved in neuronal DNA replication, DNA pol-ß contributes to neuronal death, and DNA pol-ß inhibitors may prove to be effective neuroprotective agents. Currently, specific and highly active DNA pol-ß inhibitors are lacking. Nine putative DNA pol-ß inhibitors were identified in silico by querying the ZINC database, containing more than 35 million purchasable compounds. Following pharmacological evaluation, only 5-methoxyflavone (1) was validated as an inhibitor of DNA pol-ß activity. Cultured primary neurons are a useful model to investigate the neuroprotective effects of potential DNA pol-ß inhibitors, since these neurons undergo DNA replication and death when treated with Aß. Consistent with the inhibition of DNA pol-ß, 5-methoxyflavone (1) reduced the number of S-phase neurons and the ensuing apoptotic death triggered by Aß. 5-Methoxyflavone (1) is the first flavonoid compound able to halt neurodegeneration via a definite molecular mechanism rather than through general antioxidant and anti-inflammatory properties.

HDAC and HAT inhibitors differently affect analgesia mediated by group II metabotropic glutamate receptors.

BACKGROUND: Histone deacetylases (HDACs) and histone acetyltransferases (HATs) are key players in epigenetic regulation of gene expression. Analgesic activity by HDAC inhibitors has been reported in different pain models including inflammatory and neuropathic pain. These drugs interfere with gene expression through different mechanisms including chromatin remodeling and/or activation of transcription factors. Among other targets, HDAC inhibitors regulate metabotropic glutamate receptors type 2 (mGlu2) expression in central and peripheral central nervous system. However whether inhibition of HAT activity also regulates mGlu2 expression has not been reported. FINDINGS: Here we report that curcumin (CUR), a naturally occurring compound endowed with p300/CREB-binding protein HAT inhibitory activity, is able to induce a drastic down-regulation of the mGlu2 receptor in the mouse spinal cord after systemic administration together with a marked hypoacetylation of histones H3 and H4 in dorsal root ganglia (DRG). Furthermore, the analgesic activity of the mGlu2/3 agonist, LY379268 is lost after a 3-day treatment with CUR. Conversely the analgesic activity of LY379268 is potentiated in mice pretreated for 5 consecutive days with the HDAC inhibitor, Suberoylanilide Hydroxamic Acid (SAHA), known to induce mGlu2-upregulation. CONCLUSIONS: Our results demonstrate that systemically injected CUR is able to inhibit H3 and H4 acetylation in the DRG and to down-regulate mGlu2 receptors in the spinal cord. We also demonstrate that long term modification of the mGlu2 expression affects the analgesic properties of the orthosteric mGlu2/3 agonist, LY379268. These data open up the possibility that epigenetic modulators might be given in combination with "traditional" drugs in a context of a multi target approach for a better analgesic efficacy.

Estrogen activates matrix metalloproteinases-2 and -9 to increase beta amyloid degradation.

Estrogen is known to affect different aspects of ß-amyloid (Aß) synthesis and degradation. The present work was undertaken to evaluate specifically whether matrix metalloproteinases (MMP) -2 and -9 are involved in Aß degradation induced by estrogen and whether this is relevant to estrogen-induced neuroprotection. In SH-SY5Y human neuroblastoma cells, 10 nM 17ß-estradiol (17ß-E2) increases mRNA and intracellular protein expression of MMP-2 and -9, as well as the levels of the active forms of both enzymes released in the medium. Specificity of the effect is proved by prevention with the estrogen receptor (ER) antagonist ICI 182,780 (1 µM) and involvement of the ERα subtype is confirmed by the use of selective ERα or ERß agonists (PPT, DPN) and antagonists (MPP, PHTPP). 17ß-E2 significantly increases the degradation of Aß, either transferred with the conditioned medium of H4-APPSw human neuroglioma cells, engineered to overproduce Aß(1-40) and Aß(1-42), or exogenously added as 2 µM Aß(1-42). Both these effects are completely prevented by preexposure to the broad spectrum MMP inhibitor GM6001 (5 µM). Importantly, the 17ß-E2-induced rescue of neuroblastoma cells challenged with 2 µM Aß(1-42), an effect prevented by ICI 182,780 (1 µM), is mediated by MMPs, as it appears significantly reduced by GM6001 (5 µM) as well as by both MMP-2 (200 nM) and MMP-9 (200nM) selective inhibitors. In conclusion, the present study shows for the first time that MMP-2 and -9 give a main contribution to estrogen's neuroprotective effect.

Purinergic ionotropic P2X7 and metabotropic glutamate mGlu5 receptors crosstalk influences pro-inflammatory conditions in microglia.

Microglia represent the resident immune system in the brain. They mediate neuroinflammatory processes and have been described as important regulators of homeostasis in the central nervous system (CNS). Among several players and mechanisms contributing to microglial function in inflammation, ATP and glutamate have been shown to be involved in microgliosis. In this study, we focused on receptor subtypes that respond to these neurotransmitters, purinergic ionotropic P2X7 receptor and metabotropic glutamate mGlu5 receptor. We found that both receptors are functionally expressed in a murine microglia cell line, BV2 cells, and we performed patch-clamp experiments to measure purinergic ionotropic P2X7 receptor ion flux in control condition and after metabotropic glutamate mGlu5 receptor activation. The selective purinergic ionotropic P2X7 receptor agonist, 2'(3')-O-(4-benzoylbenzoyl)adenosine-5'-triphosphate (BzATP, 100 µM), elicited a robust current that was prevented by the selective purinergic ionotropic P2X7 receptor antagonist A438079 (10 µM). When BV2 cells were acutely stimulated with the selective metabotropic glutamate mGlu5 agonist, (RS)-2-chloro-5-hydroxyphenylglycine (CHPG, 200 µM), purinergic ionotropic P2X7 receptor current was increased. This positive modulation was prevented by the selective metabotropic glutamate mGlu5 receptor antagonist 3-((2-Methyl-4-thiazolyl)ethynyl)pyridine (MTEP, 1 µM). Moreover, nitric oxide synthesis elicited by purinergic ionotropic P2X7 receptor activation was enhanced by metabotropic glutamate mGlu5 receptor co-stimulation. Taken together, our results suggest an important crosstalk between ATP and glutamate in inflammation. Pro-inflammatory effects mediated by purinergic ionotropic P2X7 receptor might be exacerbated by simultaneous exposure of microglia to ATP and glutamate, suggesting new pharmacological targets to modulate neuroinflammation.

Melatonin Activates Anti-Inflammatory Features in Microglia in a Multicellular Context: Evidence from Organotypic Brain Slices and HMC3 Cells.

Melatonin (MEL) is a neurohormone endowed with neuroprotective activity, exerted both directly on neuronal cells and indirectly through modulation of responsive glial cells. In particular, MEL's effects on microglia are receptor-mediated and in part dependent on SIRT1 activation. In the present study, we exploited the highly preserved cytoarchitecture of organotypic brain cultures (OC) to explore the effects of MEL on hippocampal microglia in a 3D context as compared to a single cell type context represented by the human HMC3 cell line. We first evaluated the expression of MEL receptor MT1 and SIRT1 and then investigated MEL action against an inflammatory stimulation with LPS: OCs were cultured for a total of 2 weeks and during this time exposed to 0.1 µg/mL of LPS for 24 h either on day 1 (LPS 1°) or on day 11 (LPS 11°). MEL was added immediately after plating and kept for the entire experiment. Under these conditions, both MEL and LPS induced amoeboid microglia. However, the same round phenotype matched different polarization features. LPS increased the number of nuclear-NF-kB+ round cells and MEL alone or in combination with LPS increased BDNF+ round microglia. In addition, MEL contrasted LPS effects on NF-kB expression. Data from HMC3 microglia confirmed MEL's anti-inflammatory effects against LPS in terms of CASP1 induction and BDNF release, identifying SIRT1 as a mediator. However, no effects were evident for MEL alone on HMC3 microglia. Overall, our results point to the importance of the multicellular context for full MEL activity, especially in a preventive view, and support the use of OCs as a favorable model to explore inflammatory responses.

Increased Heat Pain Tolerance but Hyperalgesia to Tonic Inflammatory Pain in the CRND8 Mouse Model of Alzheimer's Disease.

BACKGROUND: The effects of Alzheimer's disease (AD) pathology on the experience of pain are poorly understood. OBJECTIVE: To understand the pathophysiological mechanisms underlying pain sensory transmission in the transgenic mouse model of AD, CRND8. METHODS: We explored AD-related pathology in the spinal cord and dorsal root ganglia of 18-week-old female CRND8 mice. We assessed nociceptive responses to both acute heat stimuli and persistent inflammatory pain in CRND8 mice and non-transgenic (non-Tg) littermates. In addition, we searched for differences in biochemical correlates of inflammatory pain between CRND8 and non-Tg mice. Finally, we investigated the excitability of dorsal horn noc iceptive neurons in spinal cord slices from CRND8 and non-Tg mice. RESULTS: We demonstrated the presence of intracellular AD-like pathology in the spinal cord and in the dorsal root ganglia nociceptive sensory neurons of CRND8 mice. We found that CRND8 mice had a reduced susceptibility to acute noxious heat stimuli and an increased sensitivity to tonic inflammatory pain. Tonic inflammatory pain correlated with a lack of induction of pro-opiomelanocortin in the spinal cord of CRND8 mice as compared to non-Tg mice. Electrophysiological recording in acute spinal cord slice preparations indicated an increased probability of glutamate release at the membrane of dorsal horn nociceptive neurons in CRND8 mice. CONCLUSION: This study suggests that an increased thermal tolerance and a facilitation of nociception by peripheral inflammation can coexist in AD.

Estrogen receptors and type 1 metabotropic glutamate receptors are interdependent in protecting cortical neurons against ß-amyloid toxicity.

We examined the interaction between estrogen receptors (ERs) and type 1 metabotropic glutamate receptors (mGlu1 receptors) in mechanisms of neurodegeneration/neuroprotection using mixed cultures of cortical cells challenged with ß-amyloid peptide. Both receptors were present in neurons, whereas only ERα but not mGlu1 receptors were found in astrocytes. Addition of 17ß-estradiol (17ßE2) protected cultured neurons against amyloid toxicity, and its action was mimicked by the selective ERα agonist, 1,3,5-tris(4-hydroxyphenyl)-4-propyl-1H-pyrazole (PPT) as well as by a cell-impermeable bovine serum albumin conjugate of 17ßE2. The selective ERß agonist, diarylpropionitrile (DPN), was only slightly neuroprotective. The mGlu1/5 receptor agonist, 3,5-dihydroxyphenylglycine (DHPG), was also neuroprotective against amyloid toxicity, and its action was abolished by the mGlu1 receptor antagonist, (3,4-dihydro-2H-pyrano[2,3-b]quinolin-7-yl)-(cis-4-methoxycyclohexyl)-methanone (JNJ 16259685). Neuroprotection by 17ßΕ2 or PPT (but not DPN) and DHPG was less than additive, suggesting that ERα and mGlu1 receptors activate the same pathway of cell survival. More important, neuroprotection by 17ßΕ2 was abolished not only by the ER antagonist fulvestrant (ICI 182,780) but also by JNJ 16259685, and neuroprotection by DHPG was abolished by ICI 182,780. ERα and mGlu1 receptors were also interdependent in activating the phosphatidylinositol-3-kinase pathway, and pharmacological blockade of this pathway abolished neuroprotection by 17ßE2, DHPG, or their combination. These data provide the first evidence that ERα and mGlu1 receptors critically interact in promoting neuroprotection, information that should be taken into account when the impact of estrogen on neurodegeneration associated with central nervous system disorders is examined.

Dysfunction of TGF-ß1 signaling in Alzheimer's disease: perspectives for neuroprotection.

Alzheimer's disease (AD) is a neurodegenerative disorder that affects about 35 million people worldwide. Current drugs for AD only treat the symptoms and do not interfere with the underlying pathogenic mechanisms of the disease. AD is characterized by the presence of ß-amyloid (Aß) plaques, neurofibrillary tangles, and neuronal loss. Identification of the molecular determinants underlying Aß-induced neurodegeneration is an essential step for the development of disease-modifying drugs. Recently, an impairment of the transforming growth factor-ß1 (TGF-ß1) signaling pathway has been demonstrated to be specific to the AD brain and, particularly, to the early phase of the disease. TGF-ß1 is a neurotrophic factor responsible for the initiation and maintenance of neuronal differentiation and synaptic plasticity. The deficiency of TGF-ß1 signaling is associated with Aß pathology and neurofibrillary tangle formation in AD animal models. Reduced TGF-ß1 signaling seems to contribute both to microglial activation and to ectopic cell-cycle re-activation in neurons, two events that contribute to neurodegeneration in the AD brain. The neuroprotective features of TGF-ß1 indicate the advantage of rescuing TGF-ß1 signaling as a means to slow down the neurodegenerative process in AD.

Sphingosine-1-phosphate and Sphingosine-1-phosphate receptors in the cardiovascular system: pharmacology and clinical implications.

Sphingosine-1-phosphate (S1P) is a lipid that binds and activates five distinct receptor subtypes, S1P1, S1P2, S1P3, S1P4, S1P5, widely expressed in different cells, tissues and organs. In the cardiovascular system these receptors have been extensively studied, but no drug acting on them has been approved so far for treating cardiovascular diseases. In contrast, a number of S1P receptor agonists are approved as immunomodulators, mainly for multiple sclerosis, because of their action on lymphocyte trafficking. This chapter summarizes the available information on S1P receptors in the cardiovascular system and discusses their potential for treating cardiovascular conditions and/or their role on the clinical pharmacology of drugs so far approved for non-cardiovascular conditions. Basic research has recently produced data useful to understand the molecular pharmacology of S1P and S1P receptors, regarding biased agonism, S1P storage, release and vehiculation and chaperoning by lipoproteins, paracrine actions, intracellular non-receptorial S1P actions. On the other hand, the approval of fingolimod and newer generation S1P receptor ligands as immunomodulators, provides information on a number of clinical observations on the impact of these drugs on cardiovascular system which need to be integrated with preclinical data. S1P receptors are potential targets for prevention and treatment of major cardiovascular conditions, including hypertension, myocardial infarction, heart failure and stroke.

Decreased Astrocytic CCL2 Accounts for BAF-312 Effect on PBMCs Transendothelial Migration Through a Blood Brain Barrier in Vitro Model.

Disruption of the blood brain barrier (BBB) is a common event in several neurological diseases and in particular, in multiple sclerosis (MS), it contributes to the infiltration of the central nervous system by peripheral inflammatory cells. Sphingosine-1-phosphate (S1P) is a bioactive molecule with pleiotropic effects. Agonists of S1P receptors such as fingolimod and siponimod (BAF-312) are in clinical practice for MS and have been shown to preserve BBB function in inflammatory conditions. Using an in vitro BBB model of endothelial-astrocytes co-culture exposed to an inflammatory insult (tumor necrosis factor-α and interferon-γ; T&I), we show that BAF-312 reduced the migration of peripheral blood mononuclear cells (PBMCs) through the endothelial layer, only in the presence of astrocytes. This effect was accompanied by decreased expression of the adhesion molecule ICAM-1. BAF-312 also reduced the activation of astrocytes, by controlling NF-kB and NLRP3 induction and preventing the increase of proinflammatory cytokine and chemokines. Reduction of CCL2 by BAF-312 may be responsible for the observed effects and, accordingly, addition of exogenous CCL2 was able to counteract BAF-312 effects and rescued T&I responses on PBMC migration, ICAM-1 expression and astrocyte activation. The present results further point out BAF-312 effects on BBB properties, suggesting also the key role of astrocytes in mediating drug effects on endothelial function.

Microglia Contributes to BAF-312 Effects on Blood-Brain Barrier Stability.

Microglia, together with astrocytes and pericytes, cooperate to ensure blood-brain barrier (BBB) stability, modulating endothelial responses to inflammatory insults. Agonists of the sphingosine 1 phosphate (S1P) receptors, such as siponimod (BAF-312), are important pharmacological tools in multiple sclerosis and other inflammatory diseases. Modulation of S1P receptors may result in a reduced inflammatory response and increased BBB stability. An in vitro BBB model was reproduced using human-derived endothelial cells, astrocytes and microglia. Co-cultures were exposed to inflammatory cytokines (TNFα, 10 UI and IFNγ, 5 UI) in the presence of BAF-312 (100 nM), and the BBB properties and microglia role were evaluated. The drug facilitated microglial migration towards endothelial/astrocyte co-cultures, involving the activity of the metalloprotease 2 (MMP2). Microglia actively cooperated with astrocytes in the maintenance of endothelial barrier stability: in the triple co-culture, selective treatment of microglial cells with BAF-312 significantly prevented cytokines' effects on the endothelial barrier. In conclusion, BAF-312, modulating S1P receptors in microglia, may contribute to the reinforcement of the endothelial barrier at the BBB, suggesting an additional effect of the drug in the treatment of multiple sclerosis.

Microglial polarization differentially affects neuronal vulnerability to the ß-amyloid protein: Modulation by melatonin.

Microglial cells play a central but yet debated role in neuroinflammatory events occurring in Alzheimer's disease (AD). We here explored how microglial features are modulated by melatonin following ß-amyloid (Aß42)-induced activation and examined the cross-talk with Aß-challenged neuronal cells. Human microglial HMC3 cells were exposed to Aß42 (200 nM) in the presence of melatonin (MEL; 1 µM) added since the beginning (MELco) or after a 72 h-exposure to Aß42 (MELpost). In both conditions, MEL favored an anti-inflammatory activation and rescued SIRT1 and BDNF expression/release. Caspase-1 up-regulation and phospho-ERK induction following a prolonged exposure to Aß42 were prevented by MEL. In addition, MEL partially restored proteasome functionality that was altered by long-term Aß42 treatment, re-establishing both 20S and 26S chymotrypsin-like activity. Differentiated neuronal-like SH-SY5Y cells were exposed to Aß42 (200 nM for 24 h) in basal medium or in the presence of conditioned medium (CM) collected from microglia exposed for different times to Aß42 alone or in combination with MELco or MELpost. Aß42 significantly reduced pre-synaptic proteins synaptophysin and VAMP2 and mean neuritic length. These effects were prevented by CM from anti-inflammatory microglia (Aß42 for 6 h), or from MELco and MELpost microglia, but the reduction of neuritic length was not rescued when the SIRT1 inhibitor EX527 was added. In conclusion, our data add to the concept that melatonin shows a promising anti-inflammatory action on microglia that is retained even after pro-inflammatory activation, involving modulation of proteasome function and translating into neuroprotective microglial effects.

Inverse correlation between the expression of AMPK/SIRT1 and NAMPT in psoriatic skin: A pilot study.

PURPOSE: Epidermal hyperplasia and the involvement of immune cells characterize the clinical picture of psoriasis. Among the several factors involved, attention has been focused on sirtuin 1 (SIRT1) - a deacetylase endowed with a variety of functions including the control of metabolic and inflammatory processes-, and on nicotinamide phosphoribosyltransferase (NAMPT), important for SIRT1 activation and involved in inflammatory events. The aim of the study was to analyze changes of SIRT1 and NAMPT expression in psoriatic skin. PATIENTS AND METHODS: Samples from healthy controls and psoriatic patients were subjected to immunohistochemical analysis. RESULTS: A strong downregulation of SIRT1 expression was observed in skin samples from psoriatic patients compared to healthy controls. This was accompanied by a parallel reduction of adenosine monophosphate-activated kinase (AMPK) expression and, more strikingly, by the disappearance of cells immunolabeled for its active, phosphorylated form (pAMPK). In both cases, analysis of the distribution of immunopositive cells revealed a shift towards reduced intensity of staining. In contrast, NAMPT expression was upregulated in psoriatic samples in line with its pro-inflammatory role. This was again more visible with an intensity-based distribution analysis that evidenced a shift towards more intensely immunostained cell populations. CONCLUSIONS: The present data correlate in the same samples the expression of SIRT1, pAMPK/AMPK and NAMPT in psoriasis and open the way for novel pharmacological targets in the treatment of the disease.

Targeting group II metabotropic glutamate (mGlu) receptors for the treatment of psychosis associated with Alzheimer's disease: selective activation of mGlu2 receptors amplifies beta-amyloid toxicity in cultured neurons, whereas dual activation of mGlu2 and mGlu3 receptors is neuroprotective.

Dual orthosteric agonists of metabotropic glutamate 2 (mGlu2) and mGlu3 receptors are being developed as novel antipsychotic agents devoid of the adverse effects of conventional antipsychotics. Therefore, these drugs could be helpful for the treatment of psychotic symptoms associated with Alzheimer's disease (AD). In experimental animals, the antipsychotic activity of mGlu2/3 receptor agonists is largely mediated by the activation of mGlu2 receptors and is mimicked by selective positive allosteric modulators (PAMs) of mGlu2 receptors. We investigated the distinct influence of mGlu2 and mGlu3 receptors in mixed and pure neuronal cultures exposed to synthetic ß-amyloid protein (Aß) to model neurodegeneration occurring in AD. The mGlu2 receptor PAM, N-4'-cyano-biphenyl-3-yl)-N-(3-pyridinylmethyl)-ethanesulfonamide hydrochloride (LY566332), devoid of toxicity per se, amplified Aß-induced neurodegeneration, and this effect was prevented by the mGlu2/3 receptor antagonist (2S,1'S,2'S)-2-(9-xanthylmethyl)-2-(2'-carboxycyclopropyl)glycine (LY341495). LY566332 potentiated Aß toxicity regardless of the presence of glial mGlu3 receptors, but it was inactive when neurons lacked mGlu2 receptors. The dual mGlu2/3 receptor agonist, (-)-2-oxa-4-aminobicyclo[3.1.0]exhane-4,6-dicarboxylic acid (LY379268), was neuroprotective in mixed cultures via a paracrine mechanism mediated by transforming growth factor-ß1. LY379268 lost its protective activity in neurons grown with astrocytes lacking mGlu3 receptors, indicating that protection against Aß neurotoxicity was mediated entirely by glial mGlu3 receptors. The selective noncompetitive mGlu3 receptor antagonist, (3S)-1-(5-bromopyrimidin-2-yl)-N-(2,4-dichlorobenzyl)pyrrolidin-3-amine methanesulfonate hydrate (LY2389575), amplified Aß toxicity on its own, and, interestingly, unmasked a neurotoxic activity of LY379268, which probably was mediated by the activation of mGlu2 receptors. These data indicate that selective potentiation of mGlu2 receptors enhances neuronal vulnerability to Aß, whereas dual activation of mGlu2 and mGlu3 receptors is protective against Aß-induced toxicity.

Hyperalgesic activity of kisspeptin in mice.

BACKGROUND: Kisspeptin is a neuropeptide known for its role in the hypothalamic regulation of the reproductive axis. Following the recent description of kisspeptin and its 7-TM receptor, GPR54, in the dorsal root ganglia and dorsal horns of the spinal cord, we examined the role of kisspeptin in the regulation of pain sensitivity in mice. RESULTS: Immunofluorescent staining in the mouse skin showed the presence of GPR54 receptors in PGP9.5-positive sensory fibers. Intraplantar injection of kisspeptin (1 or 3 nmol/5 µl) induced a small nocifensive response in naive mice, and lowered thermal pain threshold in the hot plate test. Both intraplantar and intrathecal (0.5 or 1 nmol/3 µl) injection of kisspeptin caused hyperalgesia in the first and second phases of the formalin test, whereas the GPR54 antagonist, p234 (0.1 or 1 nmol), caused a robust analgesia. Intraplantar injection of kisspeptin combined with formalin enhanced TRPV1 phosphorylation at Ser800 at the injection site, and increased ERK1/2 phosphorylation in the ipsilateral dorsal horn as compared to naive mice and mice treated with formalin alone. CONCLUSION: These data demonstrate for the first time that kisspeptin regulates pain sensitivity in rodents and suggest that peripheral GPR54 receptors could be targeted by novel drugs in the treatment of inflammatory pain.

Neurotoxic properties of the anabolic androgenic steroids nandrolone and methandrostenolone in primary neuronal cultures.

Anabolic-androgenic steroid (AAS) abuse is associated with multiple neurobehavioral disturbances. The sites of action and the neurobiological sequels of AAS abuse are unclear at present. We investigated whether two different AASs, nandrolone and methandrostenolone, could affect neuronal survival in culture. The endogenous androgenic steroid testosterone was used for comparison. Both testosterone and nandrolone were neurotoxic at micromolar concentrations, and their effects were prevented by blockade of androgen receptors (ARs) with flutamide. Neuronal toxicity developed only over a 48-hr exposure to the steroids. The cell-impermeable analogues testosterone-BSA and nandrolone-BSA, which preferentially target membrane-associated ARs, were also neurotoxic in a time-dependent and flutamide-sensitive manner. Testosterone-BSA and nandrolone-BSA were more potent than their parent compounds, suggesting that membrane-associated ARs were the relevant sites for the neurotoxic actions of the steroids. Unlike testosterone and nandrolone, toxicity by methandrostenolone and methandrostenolone-BSA was insensitive to flutamide, but it was prevented by the glucocorticoid receptor (GR) antagonist RU-486. Methandrostenolone-BSA was more potent than the parent compound, suggesting that its toxicity relied on the preferential activation of putative membrane-associated GRs. Consistently with the evidence that membrane-associated GRs can mediate rapid effects, a brief challenge with methandrostenolone-BSA was able to promote neuronal toxicity. Activation of putative membrane steroid receptors by nontoxic (nanomolar) concentrations of either nandrolone-BSA or methandrostenolone-BSA became sufficient to increase neuronal susceptibility to the apoptotic stimulus provided by ß-amyloid (the main culprit of AD). We speculate that AAS abuse might facilitate the onset or progression of neurodegenerative diseases not usually linked to drug abuse.

Protective effect of the sphingosine-1 phosphate receptor agonist siponimod on disrupted blood brain barrier function.

Sphingosine 1 phosphate (S1P) is a bioactive sphingolipid that exerts several functions in physiological and pathological conditions. The modulation of one of its receptors, S1P1, plays an important role in the egress of lymphocytes from lymph nodes and is a useful target in multiple sclerosis (MS) treatment. A new drug, siponimod (BAF-312) has been recently approved for the treatment of secondary progressive MS and has affinity for two S1P receptors, S1P1 and S1P5. The two receptors are expressed by endothelial cells that, as components of the blood-brain barrier (BBB), prevent the access of solutes and lymphocytes into the central nervous system, function often compromised in MS. Using an in vitro BBB model exposed to inflammatory cytokines (TNFα and IFNγ, 5 UI and 10 UI respectively), we evaluated the effects of BAF-312 (100 nM) on expression and function of endothelial tight junctional proteins (Zo-1 and claudin-5), regulation of transendothelial electrical resistance (TEER) and permeability to FITC-conjugated dextran. Zo-1 expression, as well as TEER values, were promptly recovered (24 h) when both S1P1 and S1P5 were activated by BAF-312. In contrast, at this time point, activation of S1P5 with the selective agonist UC-42-WP04 (300 nM) or with BAF-312, under blockade of S1P1 with the selective antagonist NIBR-0213 (1 µM), resulted in recovery of expression and localization of claudin-5 and reduction of TNFα/INFγ-induced expression of metalloproteinase 9. Only after a prolonged BAF-312 exposure (48 h), S1P1 was involved through activation of the PI3K/Akt pathway. The PI3K inhibitor LY294002 (10 µM) prevented in fact the effects of BAF-312 on all the parameters examined. In conclusion, BAF-312, by modulating both S1P1 and S1P5, may strengthen BBB properties, thus providing additional effects in the treatment of MS.