Effects of a gut-selective integrin-targeted therapy in male mice exposed to early immune activation, a model for the study of autism spectrum disorder.

To clarify the role of gut mucosal immunity in ASD, we evaluated, in the early-life immune activation (EIA) mouse model, the effects of administration of a monoclonal antibody directed against the integrin alpha4 beta7 (α4ß7 mAb), blocking the leukocyte homing into the gut mucosa. EIA is a double-hit variant of the maternal immune-activation (MIA) model, including both prenatal (Poly I:C) and postnatal (LPS) immune challenges. In C57BL6/J EIA male adult offspring mice, IL-1ß and IL-17A mRNA colonic tissue content increased when compared with controls. Cytofluorimetric analyses of lymphocytes isolated from mesenteric lymph-nodes (MLN) and spleens of EIA mice show increased percentage of total and CD4+α4ß7+, unstimulated and stimulated IL-17A+ and stimulated IFN-γ+ lymphocytes in MLN and CD4+α4ß7+ unstimulated and stimulated IL-17A+ and stimulated IFN-γ+ lymphocytes in the spleen. Treatment with anti-α4ß7 mAb in EIA male mice was associated with colonic tissue IL-1ß, and IL-17A mRNA content and percentage of CD4+ IL-17A+ and IFN-γ+ lymphocytes in MLN and spleens comparable to control mice. The anti-α4ß7 mAb treatment rescue social novelty deficit showed in the three-chamber test by EIA male mice. Increased levels of IL-6 and IL-1ß and decreased CD68 and TGF-ß mRNAs were also observed in hippocampus and prefrontal cortex of EIA male mice together with a reduction of BDNF mRNA levels in all brain regions examined. Anti-α4ß7 mAb treatment restored the expression of BDNF, TGF-ß and CD68 in hippocampus and prefrontal cortex. Improvement of the gut inflammatory status, obtained by a pharmacological agent acting exclusively at gut level, ameliorates some ASD behavioral features and the neuroinflammatory status. Data provide the first preclinical indication for a therapeutic strategy against gut-immune activation in ASD subjects with peripheral increase of gut-derived (α4ß7+) lymphocytes expressing IL-17A.

Bdnf-Nrf-2 crosstalk and emotional behavior are disrupted in a sex-dependent fashion in adolescent mice exposed to maternal stress or maternal obesity.

Maternal obesity has been recognized as a stressor affecting the developing fetal brain, leading to long-term negative outcomes comparable to those resulting from maternal psychological stress, although the mechanisms have not been completely elucidated. In this study, we tested the hypothesis that adverse prenatal conditions as diverse as maternal stress and maternal obesity might affect emotional regulation and stress response in the offspring through common pathways, with a main focus on oxidative stress and neuroplasticity. We contrasted and compared adolescent male and female offspring in two mouse models of maternal psychophysical stress (restraint during pregnancy - PNS) and maternal obesity (high-fat diet before and during gestation - mHFD) by combining behavioral assays, evaluation of the hypothalamic-pituitary-adrenal (HPA) axis reactivity, immunohistochemistry and gene expression analysis of selected markers of neuronal function and neuroinflammation in the hippocampus, a key region involved in stress appraisal. Prenatal administration of the antioxidant N-acetyl-cysteine (NAC) was used as a strategy to protect fetal neurodevelopment from the negative effects of PNS and mHFD. Our findings show that these two stressors produce overlapping effects, reducing brain anti-oxidant defenses (Nrf-2) and leading to sex-dependent impairments of hippocampal Bdnf expression and alterations of the emotional behavior and HPA axis functionality. Prenatal NAC administration, by restoring the redox balance, was able to exert long-term protective effects on brain development, suggesting that the modulation of redox pathways might be an effective strategy to target common shared mechanisms between different adverse prenatal conditions.

Maternal immune activation induces autism-like changes in behavior, neuroinflammatory profile and gut microbiota in mouse offspring of both sexes.

Autism Spectrum Disorder (ASD) is a sex-biased neurodevelopmental disorder with a male to female prevalence of 4:1, characterized by persistent deficits in social communication and interaction and restricted-repetitive patterns of behavior, interests or activities. Microbiota alterations as well as signs of neuroinflammation have been also reported in ASD. The involvement of immune dysregulation in ASD is further supported by evidence suggesting that maternal immune activation (MIA), especially during early pregnancy, may be a risk factor for ASD. The present study was aimed at characterizing the effects of MIA on behavior, gut microbiota and neuroinflammation in the mouse offspring also considering the impact of MIA in the two sexes. MIA offspring exhibited significant ASD-like behavioral alterations (i.e., deficits in sociability and sensorimotor gating, perseverative behaviors). The analysis of microbiota revealed changes in specific microbial taxa that recapitulated those seen in ASD children. In addition, molecular analyses indicated sex-related differences in the neuroinflammatory responses triggered by MIA, with a more prominent effect in the cerebellum. Our data suggest that both sexes should be included in the experimental designs of preclinical studies in order to identify those mechanisms that confer different vulnerability to ASD to males and females.

Inflammatory Signatures of Maternal Obesity as Risk Factors for Neurodevelopmental Disorders: Role of Maternal Microbiota and Nutritional Intervention Strategies.

Obesity is a main risk factor for the onset and the precipitation of many non-communicable diseases. This condition, which is associated with low-grade chronic systemic inflammation, is of main concern during pregnancy leading to very serious consequences for the new generations. In addition to the prominent role played by the adipose tissue, dysbiosis of the maternal gut may also sustain the obesity-related inflammatory milieu contributing to create an overall suboptimal intrauterine environment. Such a condition here generically defined as "inflamed womb" may hold long-term detrimental effects on fetal brain development, increasing the vulnerability to mental disorders. In this review, we will examine the hypothesis that maternal obesity-related gut dysbiosis and the associated inflammation might specifically target fetal brain microglia, the resident brain immune macrophages, altering neurodevelopmental trajectories in a sex-dependent fashion. We will also review some of the most promising nutritional strategies capable to prevent or counteract the effects of maternal obesity through the modulation of inflammation and oxidative stress or by targeting the maternal microbiota.

Critical Role of Maternal Selenium Nutrition in Neurodevelopment: Effects on Offspring Behavior and Neuroinflammatory Profile.

Research in both animals and humans shows that some nutrients are important in pregnancy and during the first years of life to support brain and cognitive development. Our aim was to evaluate the role of selenium (Se) in supporting brain and behavioral plasticity and maturation. Pregnant and lactating female rats and their offspring up to postnatal day 40 were fed isocaloric diets differing in Se content-i.e., optimal, sub-optimal, and deficient-and neurodevelopmental, neuroinflammatory, and anti-oxidant markers were analyzed. We observed early adverse behavioral changes in juvenile rats only in sub-optimal offspring. In addition, sub-optimal, more than deficient supply, reduced basal glial reactivity in sex dimorphic and brain-area specific fashion. In female offspring, deficient and sub-optimal diets reduced the antioxidant Glutathione peroxidase (GPx) activity in the cortex and in the liver, the latter being the key organ regulating Se metabolism and homeostasis. The finding that the Se sub-optimal was more detrimental than Se deficient diet may suggest that maternal Se deficient diet, leading to a lower Se supply at earlier stages of fetal development, stimulated homeostatic mechanisms in the offspring that were not initiated by sub-optimal Se. Our observations demonstrate that even moderate Se deficiency during early life negatively may affect, in a sex-specific manner, optimal brain development.

Repurposing Dipyridamole in Niemann Pick Type C Disease: A Proof of Concept Study.

Niemann Pick type C disease (NPC) is a rare disorder characterized by lysosomal lipid accumulation that damages peripheral organs and the central nervous system. Currently, only miglustat is authorized for NPC treatment in Europe, and thus the identification of new therapies is necessary. The hypothesis addressed in this study is that increasing adenosine levels may represent a new therapeutic approach for NPC. In fact, a reduced level of adenosine has been shown in the brain of animal models of NPC; moreover, the compound T1-11, which is able to weakly stimulate A2A receptor and to increase adenosine levels by blocking the equilibrative nucleoside transporter ENT1, significantly ameliorated the pathological phenotype and extended the survival in a mouse model of the disease. To test our hypothesis, fibroblasts from NPC1 patients were treated with dipyridamole, a clinically-approved drug with inhibitory activity towards ENT1. Dipyridamole significantly reduced cholesterol accumulation in fibroblasts and rescued mitochondrial deficits; the mechanism elicited by dipyridamole relies on activation of the adenosine A2AR subtype subsequent to the increased levels of extracellular adenosine due to the inhibition of ENT1. In conclusion, our results provide the proof of concept that targeting adenosine tone could be beneficial in NPC.

Systemic depletion of histone macroH2A1.1 boosts hippocampal synaptic plasticity and social behavior in mice.

Gene expression and epigenetic processes in several brain regions regulate physiological processes such as cognitive functions and social behavior. MacroH2A1.1 is a ubiquitous variant of histone H2A that regulates cell stemness and differentiation in various organs. Whether macroH2A1.1 has a modulatory role in emotional behavior is unknown. Here, we employed macroH2A1.1 knock-out (-/- ) mice to perform a comprehensive battery of behavioral tests, and an assessment of hippocampal synaptic plasticity (long-term potentiation) accompanied by whole hippocampus RNA sequencing. MacroH2A1.1-/- mice exhibit a stunningly enhancement both of sociability and of active stress-coping behavior, reflected by the increased social behavior in social activity tests and higher mobility time in the forced swim test, respectively. They also display an increased hippocampal synaptic plasticity, accompanied by significant neurotransmission transcriptional networks changes. These results suggest that systemic depletion of histone macroH2A1.1 supports an epigenetic control necessary for hippocampal function and social behavior.

Integration of Multiple Platforms for the Analysis of Multifluorescent Marking Technology Applied to Pediatric GBM and DIPG.

The intratumor heterogeneity represents one of the most difficult challenges for the development of effective therapies to treat pediatric glioblastoma (pGBM) and diffuse intrinsic pontine glioma (DIPG). These brain tumors are composed of heterogeneous cell subpopulations that coexist and cooperate to build a functional network responsible for their aggressive phenotype. Understanding the cellular and molecular mechanisms sustaining such network will be crucial for the identification of new therapeutic strategies. To study more in-depth these mechanisms, we sought to apply the Multifluorescent Marking Technology. We generated multifluorescent pGBM and DIPG bulk cell lines randomly expressing six different fluorescent proteins and from which we derived stable optical barcoded single cell-derived clones. In this study, we focused on the application of the Multifluorescent Marking Technology in 2D and 3D in vitro/ex vivo culture systems. We discuss how we integrated different multimodal fluorescence analysis platforms, identifying their strengths and limitations, to establish the tools that will enable further studies on the intratumor heterogeneity and interclonal interactions in pGBM and DIPG.

Beneficial Effects of Fingolimod on Social Interaction, CNS and Peripheral Immune Response in the BTBR Mouse Model of Autism.

Autism Spectrum Disorders (ASD) are neurodevelopmental disorders characterized by social communication deficits and repetitive/stereotyped behaviours. We evaluated the effects of a chronic treatment with the immunomodulator drug Fingolimod (FTY720 - a non-selective Sphingosine 1-Phosphate Receptor ligand) in an ASD model, the BTBR T+tf/J (BTBR) mouse strain. In adult BTBR males, chronic FTY720 treatment (4 weeks) increased social and vocal response during a male-female interaction and hippocampal expression of BDNF and Neuregulin 1, two trophic factors reduced in BTBR when compared to control C57 mice. FTY720 also re-established the expression of IL-1ß and MnSOD in the hippocampus, whereas it did not modify IL-6 mRNA content. In addition to its central effect, FTY720 modulated the activation state of peripheral macrophages in the BTBR model, both in basal conditions and after stimulation with an immune challenge. Furthermore, IL-6 mRNA colonic content of BTBR mice, reduced when compared with C57 mice, was normalized by chronic treatment with FTY720. Our study, while indicating FTY720 as a tool to attenuate relevant alterations of the BTBR neurobehavioural phenotype, emphasizes the importance of gut mucosal immune evaluation as an additional target that deserve to be investigated in preclinical studies of anti-inflammatory therapeutic approaches in ASD.

Fingolimod: A Disease-Modifier Drug in a Mouse Model of Amyotrophic Lateral Sclerosis.

Fingolimod phosphate (FTY720), the first approved oral therapy for multiple sclerosis, primarily acts as an immunomodulator. Its concomitant effects in the central nervous system, however, indicate a potentially broader spectrum of activity in neurodegenerative diseases. In the present study, we investigated the possible effects of fingolimod in a mouse model of amyotrophic lateral sclerosis (ALS), a neurodegenerative disease characterized by a strong neuroinflammatory component. Fingolimod (0.1 and 1 mg/kg i.p.) was administered to mSOD1G93A mice, a well-characterized mouse model of ALS, starting from the onset of motor symptoms to the end stage of the disease. The drug was able to improve the neurological phenotype (p < 0.05) and to extend the survival (p < 0.01) of ALS mice. The beneficial effect of fingolimod administration was associated with a significant modulation of neuroinflammatory and protective genes (CD11b, Foxp3, iNOS, Il1ß, Il10, Arg1, and Bdnf) in motor cortex and spinal cord of animals. Our data show, for the first time, that fingolimod is protective in ALS mice and that its beneficial effects are accompanied by a modulation of microglial activation and innate immunity. Considering that the treatment was started in already symptomatic mice, our data strongly support fingolimod as a potential new therapeutic approach to ALS.

Glycogen synthase kinase 3 is part of the molecular machinery regulating the adaptive response to LPS stimulation in microglial cells.

Repeated stimulation of TLR4 signaling by lipopolysaccharide (LPS) in microglia induces a state of tolerance/sensitization consisting in the reprogramming of the expression of pro-inflammatory genes in favor of anti-inflammatory ones. The molecular mechanisms underlying this adaptive response are far to be elucidated. Glycogen synthase kinase 3 (GSK3) has emerged as crucial regulator of TLR signaling, mediating the balance between pro- and anti-inflammatory functions in both periphery and central nervous system. The present study extends this notion identifying GSK3 as part of the molecular machinery regulating the LPS-adaptive response in microglial cells, by using primary microglial cultures and organotypic hippocampal slices (OHSCs). We found that lithium chloride (LiCl), a widely used GSK3 inhibitor and the mainstay treatment for bipolar disorder, reinforced the LPS adaptive response by enhancing both downregulation of pro-inflammatory genes (inducible nitric oxide synthase, interleukin 1ß, interleukin 6, tumor necrosis factor α), and upregulation of genes typically associated to anti-inflammatory functions (interleukin 10 and MRC1). The effects of GSK3 inhibition were mimicked by Wnt3a, added exogenously, and reversed by Inhibitor of Wnt-Response-1-endo, a pharmacological disruptor of the canonical Wnt/ß-catenin pathway, and GW9662, a selective peroxisome proliferator activated receptor γ antagonist, suggesting that these two pathways are involved in the regulation of LPS-tolerance/sensitization by GSK. Finally, LiCl treatment of OHSCs enhanced the protective functional consequences of the microglial adaptive response to LPS on oligodendrocyte maturation, as indicated by MBP mRNA upregulation. These results further indicate GSK3 as key component in the orchestration of neuroinflammation and target for neuroprotective strategies.

The mitochondrial uncoupling protein-2 is a master regulator of both M1 and M2 microglial responses.

Microglial activation is a dynamic process, central to neuroinflammation, which can have beneficial or pathogenic effects to human health. Mitochondria are key players in neuroinflammatory and neurodegenerative processes, common to most brain diseases. To the best of our knowledge on the role of mitochondria in the modulation of neuroinflammation, we focused on the mitochondrial uncoupling protein-2 (UCP2), known to control mitochondrial functions and to be implicated in a variety of physiological and pathological processes. In primary microglial cultures, the M1 stimulus lipopolysaccharide induced an early and transitory decrease in UCP2 levels. The initial UCP2 down-regulation was paralleled by mitochondrial inner membrane potential (mMP) depolarization and increased mitochondrial reactive oxygen species production. The key role of UCP2 in controlling mMP and reactive oxygen species production was confirmed by both pharmacological inhibition and down-regulation by RNA interference. Additionally, UCP2-silenced microglia stimulated with lipopolysaccharide showed an enhanced inflammatory response, characterized by a greater production of nitric oxide and interleukin-6. UCP2 was differently regulated by M2 stimuli, as indicated by its persistent up-regulation by interleukin-4. In UCP2-silenced microglia, interleukin-4 failed to induce M2 genes (mannose receptor 1 and interleukin-10) and to reduce M1 genes (inducible nitric oxide synthase and tumour necrosis factor-α). Our findings indicate that UCP2 is central to the process of microglial activation, with opposite regulation of M1 and M2 responses, and point to UCP2 manipulation as a potential strategy for redirecting microglial response towards protective phenotypes in several brain diseases where neuroinflammation is recognized to contribute to neurodegeneration. We show that the mitochondrial uncoupling protein-2 (UCP2) is central to the process of microglial activation, with opposite regulation of M1 and M2 responses. In UCP2-silenced microglia, lipopolysaccharide (LPS) triggers an enhanced inflammatory response characterized by a greater expression of M1 genes, whereas interleukin-4 (IL-4) fails in inducing M2 genes and reducing M1 genes. We propose UCP2 manipulation as a potential strategy for redirecting microglial response towards protective phenotypes.

Microglial polarization and plasticity: evidence from organotypic hippocampal slice cultures.

Increasing evidence indicates that "functional plasticity" is not solely a neuronal attribute but a hallmark of microglial cells, the main brain resident macrophage population. Far from being a univocal phenomenon, microglial activation can originate a plethora of functional phenotypes, encompassing the classic M1 proinflammatory and the alternative M2 anti-inflammatory phenotypes. This concept overturns the popular view of microglial activation as a synonym of neurotoxicity and neurogenesis failure in brain disorders. The characterization of the alternative programs is a matter of intense investigation, but still scarce information is available on the course of microglial activation, on the reversibility of the different commitments and on the capability of preserving molecular memory of previous priming stimuli. By using organotypic hippocampal slice cultures as a model, we developed paradigms of stimulation aimed at shedding light on some of these aspects. We show that persistent stimulation of TLR4 signaling promotes an anti-inflammatory response and microglial polarization toward M2-like phenotype. Moreover, acute and chronic preconditioning regimens permanently affect the capability to respond to a later challenge, suggesting the onset of mechanisms of molecular memory. Similar phenomena could occur in the intact brain and differently affect the vulnerability of mature and newborn neurons to noxious signals.

The nuclear receptor peroxisome proliferator-activated receptor-γ promotes oligodendrocyte differentiation through mechanisms involving mitochondria and oscillatory Ca2+ waves.

Peroxisome proliferator-activated receptor-γ (PPAR-γ) is one of the most studied nuclear receptor since its identification as a target to treat metabolic and neurological diseases. In addition to exerting anti-inflammatory and neuroprotective effects, PPAR-γ agonists, such as the insulin-sensitizing drug pioglitazone, promote the differentiation of oligodendrocytes (OLs), the myelin-forming cells of the central nervous system (CNS). In addition, PPAR-γ agonists increase OL mitochondrial respiratory chain activity and OL's ability to respond to environmental signals with oscillatory Ca2+ waves. Both OL maturation and oscillatory Ca2+ waves are prevented by the mitochondrial inhibitor rotenone and restored by PPAR-γ agonists, suggesting that PPAR-γ promotes myelination through mechanisms involving mitochondria.

Branched-chain amino acids influence the immune properties of microglial cells and their responsiveness to pro-inflammatory signals.

The branched-chain amino acids (BCAAs) valine, leucine and isoleucine are essential amino acids involved in several important brain functions. Although commonly used as nutritional supplements, excessive intake of BCAAs might favour the establishment of neurotoxic conditions as indicated by the severe neurological symptoms characterising inherited disorders of BCAA catabolism such as maple syrup urine disease (MSUD). Recent evidence indicates that BCAAs induce excitotoxicity through mechanisms that require the presence of astrocytes. In the present study, we evaluated the effects of BCAAs on microglia, the main immune cells of the brain. As an experimental model we used primary microglial cells harvested from mixed glial cultures that had been kept in normal or high BCAA medium (H-BCAA). We show that H-BCAA microglial cells exhibit a peculiar phenotype characterized by a partial skewing toward the M2 state, with enhanced IL-10 expression and phagocytic activity but also increased free radical generation and decreased neuroprotective functions. We suggest that such an intermediate M1/M2 phenotype might result in a less efficient microglial response, which would promote the establishment of a low grade chronic inflammation and increase the likelihood of neurodegeneration. Although based on in vitro evidence, our study adds on to an increasing literature indicating that the increasing use of dietary integrators might deserve consideration for the possible drawbacks. In addition to excitotoxicity, the altered immune profile of microglia might represent a further mechanism by which BCAAs might turn into toxicants and facilitate neurodegeneration.

Docosahexaenoic acid modulates inflammatory and antineurogenic functions of activated microglial cells.

The complex process of microglial activation encompasses several functional activation states associated either with neurotoxic/antineurogenic or with neurotrophic/proneurogenic properties, depending mainly on the extent of activation and the nature of the activating stimuli. Several studies have demonstrated that acute exposure to the prototypical activating agent lipopolysaccharide (LPS) confers antineurogenic properties upon microglial cells. Acutely activated microglia ortheir conditioned media (CM) reduce neural stem progenitor cell (NPC) survival and prevent NPC differentiation into neurons. The present study tested the hypothesis that docosahexaenoic acid (DHA), a long-chain polyunsatured fatty acid (L-PUFA) with potent immunomodulatory properties, could dampen microglial proinflammatory functions and modulate their antineurogenic effect. We demonstrate that DHA dose dependently inhibits the synthesis of inflammatory products in activated microglia without inducing an alternative antiinflammatory phenotype. Among the possible DHA mechanisms of action, we propose the inhibition of p38 MAPK phosphorylation and the activation of the nuclear receptor peroxisome proliferator activated receptor (PPAR)-γ. The attenuation of M1 proinflammatory phenotype has relevant consequences for the survival and differentiation of NPC, because DHA reverses the antineurogenic activities of conditioned media from LPS-activated microglia. Our study identifies new relevant potentially protective and proneurogenic functions of DHA, exerted through the modulation of microglial functions, that could be exploited to sustain or promote neuroregenerative processes in damaged/aged brain.

Peroxisome proliferator-activated receptor γ agonists accelerate oligodendrocyte maturation and influence mitochondrial functions and oscillatory Ca(2+) waves.

We have previously shown that natural (15-deoxy-Δ-prostaglandin J2) and synthetic (pioglitazone) agonists of peroxisome proliferator-activated receptor γ (PPAR-γ) strengthen the intrinsic cellular mechanisms protecting oligodendrocyte (OL) progenitors (OPs) from oxidative insults and promote their differentiation. Here, we demonstrate that repeated administrations of PPAR-γ agonists to OP cultures accelerate their differentiation to OLs, as indicated by increased numbers of O4- and O1-positive cells that show increased myelin basic protein expression, elaborated cholesterol-enrichedmembranes and have increased peroxisomes. Moreover, PPAR-γ agonist-treated OLs show increased activity of the mitochondrial respiratory chain Complex IV and an increased ability to respond to environmental signals, such as adenosine diphosphate (ADP), with oscillatory Ca waves; the latter closely correlated with the presence of mitochondria and were inhibited by the mitochondrial respiratory chain Complex I inhibitor rotenone. Because Ca oscillations and mitochondrial respiratory chain activity play crucial roles in OL differentiation, these findings suggest that PPAR-γ agonists could protect OLs and promote myelination through several mechanisms, including those involving mitochondrial functions. Our studies support the therapeutic potential of PPAR-γ agonists in brain diseases in which mitochondrial alteration, oxidative stress, and demyelination occur and point to the need for a better understanding of the role of PPAR-γ and its agonists in OL biology.

TGF-ß and LPS modulate ADP-induced migration of microglial cells through P2Y1 and P2Y12 receptor expression.

Nucleotides act as early signals for microglial recruitment to sites of CNS injury. As microglial motility and activation can be influenced by several local factors at the site of the lesion, we investigated the effects of interferon-gamma, lipopolysaccharide (LPS) or transforming growth factor-ß (TGF-ß) addition to mixed glial cell cultures, on microglial migration in response to ADP, P2Y12 and P2Y1 mRNA expression as well as on the expression of an array of genes associated with the process of microglial activation. First, we demonstrated, by pharmacological inhibition and by using small interfering RNAs, that in addition to P2Y12, P2Y1 is involved in ADP-stimulated microglial migration. The ability of specific agonists to induce Ca(2+) mobilization further confirmed the expression of functional P2Y receptors in microglia. Then, we found that migratory capability and expression of both P2Y receptors were abrogated in microglial cells from LPS-stimulated mixed glial cultures, while TGF-ß increased ADP-induced migration and the expression of P2Y12 and P2Y1 receptors. Interferon-gamma did not influence receptor expression or microglial migration. Finally, the patterns of gene expression induced in microglia by LPS or TGF-ß treatment of mixed glial cultures were clearly distinct. LPS induced a set of classical pro-inflammatory genes, whereas TGF-ß increased the expression of genes associated with atypical microglial phenotype, namely arginase-1 and TGF-ß genes. These results imply that both P2Y1 and P2Y12 may guide microglia toward the lesion. They also suggest that the modulation of microglial purinergic receptors expression by local factors, through direct and/or astrocyte-mediated actions, may represent a novel mechanism affecting neuroinflammatory response.

Microglial activation in chronic neurodegenerative diseases: roles of apoptotic neurons and chronic stimulation.

In chronic neurodegenerative diseases, microglial activation is an early sign that often precedes neuronal death. Increasing evidence indicates that in these chronic pathologies activated microglia sustain a local inflammatory response. Nonetheless, the potential detrimental or protective roles of such reaction remain to date not fully understood, mainly because of the lack of direct evidence of the functional properties acquired by microglia in the course of chronic diseases. Purified microglial cultures have been extensively used to investigate microglial functions associated with activation, but they are often criticized for some experimental constrains, including the abrupt addition of activators, the limited time of stimulation, and the absence of interactions with neurons or other elements of brain parenchyma. To limit these confounding factors, we developed in vitro models in which microglial cells were repeatedly challenged with lipopolysaccharide or co-cultured with healthy, apoptotic, or necrotic neuronal cells. We found that chronic stimulation and interaction with phosphatidylserine-expressing apoptotic cells induced microglial cells to release immunoregulatory and neuroprotective agents (prostaglandin E(2), transforming growth factor-beta, and nerve growth factor), whereas the synthesis of pro-inflammatory molecules (tumor necrosis factor-alpha and nitric oxide) was inhibited. These findings suggest that signals that are relevant to chronic diseases lead to a progressive down-regulation of pro-inflammatory microglial functions and may help in understanding the atypical microglial activation that begins to be recognized in some chronic neuropathologies.

Activation of alpha7 nicotinic acetylcholine receptor by nicotine selectively up-regulates cyclooxygenase-2 and prostaglandin E2 in rat microglial cultures.

BACKGROUND: Nicotinic acetylcholine (Ach) receptors are ligand-gated pentameric ion channels whose main function is to transmit signals for the neurotransmitter Ach in peripheral and central nervous system. However, the alpha7 nicotinic receptor has been recently found in several non-neuronal cells and described as an important regulator of cellular function. Nicotine and ACh have been recently reported to inhibit tumor necrosis factor-alpha (TNF-alpha) production in human macrophages as well as in mouse microglial cultures. In the present study, we investigated whether the stimulation of alpha7 nicotinic receptor by the specific agonist nicotine could affect the functional state of activated microglia by promoting and/or inhibiting the release of other important pro-inflammatory and lipid mediator such as prostaglandin E2. METHODS: Expression of alpha7 nicotinic receptor in rat microglial cell was examined by RT-PCR, immunofluorescence staining and Western blot. The functional effects of alpha7 receptor activation were analyzed in resting or lipopolysaccharide (LPS) stimulated microglial cells pre-treated with nicotine. Culture media were assayed for the levels of tumor necrosis factor, interleukin-1beta, nitric oxide, interleukin-10 and prostaglandin E2. Total RNA was assayed by RT-PCR for the expression of COX-2 mRNA. RESULTS: Rat microglial cells express alpha7 nicotinic receptor, and its activation by nicotine dose-dependently reduces the LPS-induced release of TNF-alpha, but has little or no effect on nitric oxide, interleukin-10 and interleukin-1beta. By contrast, nicotine enhances the expression of cyclooxygenase-2 and the synthesis of one of its major products, prostaglandin E2. CONCLUSIONS: Since prostaglandin E2 modulates several macrophage and lymphocyte functions, which are instrumental for inflammatory resolution, our study further supports the existence of a brain cholinergic anti-inflammatory pathway mediated by alpha7 nicotinic receptor that could be potentially exploited for novel treatments of several neuropathologies in which local inflammation, sustained by activated microglia, plays a crucial role.