Search

Supplementary Data from DIPG Harbors Alterations Targetable by MEK Inhibitors, with Acquired Resistance Mechanisms Overcome by Combinatorial Inhibition

The survival of children with diffuse intrinsic pontine glioma (DIPG) remains dismal, with new treatments desperately needed. In a prospective biopsy-stratified clinical trial, we combined detailed molecular profiling and drug screening in newly established patient-derived models in vitro and in vivo. We identified in vitro sensitivity to MEK inhibitors in DIPGs harboring MAPK pathway alterations, but treatment of patient-derived xenograft models and a patient at relapse failed to elicit a significant response. We generated trametinib-resistant clones in a BRAFG469V model through continuous drug exposure and identified acquired mutations in MEK1/2 with sustained pathway upregulation. These cells showed hallmarks of mesenchymal transition and expression signatures overlapping with inherently trametinib-insensitive patient-derived cells, predicting sensitivity to dasatinib. Combined trametinib and dasatinib showed highly synergistic effects in vitro and on ex vivo brain slices. We highlight the MAPK pathway as a therapeutic target in DIPG and show the importance of parallel resistance modeling and combinatorial treatments for meaningful clinical translation.Significance:We report alterations in the MAPK pathway in DIPGs to confer initial sensitivity to targeted MEK inhibition. We further identify for the first time the mechanism of resistance to single-agent targeted therapy in these tumors and suggest a novel combinatorial treatment strategy to overcome it in the clinic.This article is highlighted in the In This Issue feature, p. 587

Maternal obesity affects 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 prenatal adverse 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 and gene expression analysis of selected neuroinflammatory and plasticity-related markers in the hippocampus as 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 comparable effects, reducing brain anti-oxidant defenses (Nrf-2), 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.

BackgroundDiffuse midline gliomas (DMG) H3K27M-mutant, including diffuse intrinsic pontine glioma (DIPG), are pediatric brain tumors associated with grim prognosis. Although GD2-CAR T-cells demonstrated significant anti-tumor activity against DMG H3K27M-mutant in vivo, a multimodal approach may be needed to more effectively treat patients. We investigated GD2 expression in DMG/DIPG and other pediatric high-grade gliomas (pHGG) and sought to identify chemical compounds that would enhance GD2-CAR T-cell anti-tumor efficacy.MethodsImmunohistochemistry in tumor tissue samples and immunofluorescence in primary patient-derived cell lines were performed to study GD2 expression. We developed a high-throughput cell-based assay to screen 42 kinase inhibitors in combination with GD2-CAR T-cells. Cell viability, western blots, flow-cytometry, real time PCR experiments, DIPG 3D culture models, and orthotopic xenograft model were applied to investigate the effect of selected compounds on DIPG cell death and CAR T-cell function.ResultsGD2 was heterogeneously, but widely, expressed in the tissue tested, while its expression was homogeneous and restricted to DMG/DIPG H3K27M-mutant cell lines. We identified dual IGF1R/IR antagonists, BMS-754807 and linsitinib, able to inhibit tumor cell viability at concentrations that do not affect CAR T-cells. Linsitinib, but not BMS-754807, decreases activation/exhaustion of GD2-CAR T-cells and increases their central memory profile. The enhanced anti-tumor activity of linsitinib/GD2-CAR T-cell combination was confirmed in DIPG models in vitro, ex vivo, and in vivo.ConclusionOur study supports the development of IGF1R/IR inhibitors to be used in combination with GD2-CAR T-cells for treating patients affected by DMG/DIPG and, potentially, by pHGG.

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.

The survival of children with diffuse intrinsic pontine glioma (DIPG) remains dismal, with new treatments desperately needed. In a prospective biopsy-stratified clinical trial, we combined detailed molecular profiling and drug screening in newly established patient-derived models in vitro and in vivo. We identified in vitro sensitivity to MEK inhibitors in DIPGs harboring MAPK pathway alterations, but treatment of patient-derived xenograft models and a patient at relapse failed to elicit a significant response. We generated trametinib-resistant clones in a BRAFG469V model through continuous drug exposure and identified acquired mutations in MEK1/2 with sustained pathway upregulation. These cells showed hallmarks of mesenchymal transition and expression signatures overlapping with inherently trametinib-insensitive patient-derived cells, predicting sensitivity to dasatinib. Combined trametinib and dasatinib showed highly synergistic effects in vitro and on ex vivo brain slices. We highlight the MAPK pathway as a therapeutic target in DIPG and show the importance of parallel resistance modeling and combinatorial treatments for meaningful clinical translation. Significance: We report alterations in the MAPK pathway in DIPGs to confer initial sensitivity to targeted MEK inhibition. We further identify for the first time the mechanism of resistance to single-agent targeted therapy in these tumors and suggest a novel combinatorial treatment strategy to overcome it in the clinic. This article is highlighted in the In This Issue feature, p. 587

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.

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.

Docosahexaenoic acid (DHA) is an essential omega-3 fatty acid known to be neuroprotective in several models of human diseases, including multiple sclerosis. The protective effects of DHA are largely attributed to its ability to interfere with the activity of transcription factors controlling immune and inflammatory responses, including the agonist-dependent transcription factor peroxisome proliferator-activated receptor-γ (PPAR-γ). In this study, we used primary oligodendrocyte progenitor (OP) cultures from neonatal rat brain to investigate whether DHA could influence OP maturation and directly promote myelination, as previously reported for selective PPAR-γ agonists. We show that, similarly to the selective PPAR-γ agonist pioglitazone (PGZ), DHA promotes OP maturation and counteracts the maturational arrest induced by TNF-α, used to mimic inflammatory conditions. The PPAR-γ antagonist GW9662 prevented both DHA-induced OP maturation and PPAR-γ nuclear translocation, supporting the hypothesis that DHA acts through the activation of PPAR-γ. In addition, both PGZ and DHA induced the phosphorylation of extracellular signal-regulated-kinase 1-2 (ERK1/2), in a PPAR-γ-dependent manner. ERK1/2 activity is known to regulate the transition from OPs to immature oligodendrocytes and the presence of specific inhibitors of ERK1/2 phosphorylation (U0126 or PD98059) prevented the differentiating effects of both DHA and PGZ. These results indicate that DHA might influence the process of OP maturation through its PPAR-γ agonistic activity and provide novel molecular mechanisms for the action of this dietary fatty acid, further supporting the nutritional intervention in demyelinating diseases such as multiple sclerosis.

Adverse psychosocial experiences have been shown to modulate individual responses to immune challenges and affect mitochondrial functions. The aim of this study was to investigate inflammation and immune responses as well as mitochondrial bioenergetics in an experimental model of Paediatric Autoimmune Neuropsychiatric Disorders Associated with Streptococcus (PANDAS). Starting in adolescence (postnatal day 28), male SJL/J mice were exposed to five injections (interspaced by two weeks) with Group-A beta-haemolytic streptococcus (GAS) homogenate. Mice were exposed to chronic psychosocial stress, in the form of protracted visual exposure to an aggressive conspecific, for four weeks. Our results indicate that psychosocial stress exacerbated individual response to GAS administrations whereby mice exposed to both treatments exhibited altered cytokine and immune-related enzyme expression in the hippocampus and hypothalamus. Additionally, they showed impaired mitochondrial respiratory chain complexes IV and V, and reduced adenosine triphosphate (ATP) production by mitochondria and ATP content. These brain abnormalities, observed in GAS-Stress mice, were associated with blunted titers of plasma corticosterone. Present data support the hypothesis that challenging environmental conditions, in terms of chronic psychosocial stress, may exacerbate the long-term consequences of exposure to GAS processes through the promotion of central immunomodulatory and oxidative stress.

Mutations of Fused in sarcoma (FUS), a ribonucleoprotein involved in RNA metabolism, have been found associated with both familial and sporadic cases of amyotrophic lateral sclerosis (ALS). Notably, besides mutations in the coding sequence, also mutations into the 3′ untranslated region, leading to increased levels of the wild-type protein, have been associated with neuronal death and ALS pathology, in ALS models and patients. The mechanistic link between altered FUS levels and ALS-related neurodegeneration is far to be elucidated, as well as the consequences of elevated FUS levels in the modulation of the inflammatory response sustained by glial cells, a well-recognized player in ALS progression. Here, we studied the effect of wild-type FUS overexpression on the responsiveness of mouse and human neural progenitor-derived astrocytes to a pro-inflammatory stimulus (IL1β) used to mimic an inflammatory environment. We found that astrocytes with increased FUS levels were more sensitive to IL1β, as shown by their enhanced expression of inflammatory genes, compared with control astrocytes. Moreover, astrocytes overexpressing FUS promoted neuronal cell death and pro-inflammatory microglia activation. We conclude that overexpression of wild-type FUS intrinsically affects astrocyte reactivity and drives their properties toward pro-inflammatory and neurotoxic functions, suggesting that a non-cell autonomous mechanism can support neurodegeneration in FUS-mutated animals and patients.

An increasing number of studies show that both inflammation and neural plasticity act as key players in the vulnerability and recovery from psychiatric disorders and neurodegenerative diseases. However, the interplay between these two players has been limitedly explored. In fact, while a few studies reported an immune activation, others conveyed an immune suppression, associated with an impairment in neural plasticity. Therefore, we hypothesized that deviations in inflammatory levels in both directions may impair neural plasticity. We tested this hypothesis experimentally, by acute treatment of C57BL/6 adult male mice with different doses of two inflammatory modulators: lipopolysaccharide (LPS), an endotoxin, and ibuprofen (IBU), a nonselective cyclooxygenase inhibitor, which are respectively a pro- and an anti-inflammatory agent. The results showed that LPS and IBU have different effects on behavior and inflammatory response. LPS treatment induced a reduction of body temperature, a decrease of body weight and a reduced food and liquid intake. In addition, it led to increased levels of inflammatory markers expression, both in the total hippocampus and in isolated microglia cells, including Interleukin (IL)-1β, and enhanced the concentration of prostaglandin E2 (PGE2). On the other hand, IBU increased the level of anti-inflammatory markers, decreased tryptophan 2,3-dioxygenase (TDO2), the first step in the kynurenine pathway known to be activated during inflammatory conditions, and PGE2 levels. Though LPS and IBU administration differently affected mediators related with pro- or anti-inflammatory responses, they produced overlapping effects on neural plasticity. Indeed, higher doses of both LPS and IBU induced a statistically significant decrease in the amplitude of long-term potentiation (LTP), in Brain-Derived Neurotrophic Factor (BDNF) expression levels and in the phosphorylation of the AMPA (α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid) receptor subunit GluR1, compared to the control group. Such effect appears to be dose-dependent since only the higher, but not the lower, dose of both compounds led to a plasticity impairment. Overall, the present findings indicate that acute treatment with pro- and anti-inflammatory agents impair neural plasticity in a dose dependent manner.

Neuroinflammation, mainly sustained by microglial activation, is one of the hallmarks of many neurodegenerative diseases, including Parkinson’s disease, Huntington’s disease, Alzheimer’s disease, and amyotrophic lateral sclerosis. A broad spectrum of functionally distinct microglial phenotypes has been described, differently affecting the central nervous system (CNS) homeostasis. Manipulating the activation state of microglia toward neuroprotective functions can thus be of therapeutic benefit in a number of CNS diseases.

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.

Background The CXCL12/CXCR4 pathway regulates tumor cell proliferation, metastasis, angiogenesis and the tumor-microenvironment cross-talk in several solid tumors, including glioblastoma (GBM), the most common and fatal brain cancer. In the present study, we evaluated the effects of peptide R, a new specific CXCR4 antagonist that we recently developed by a ligand-based approach, in an in vitro and in vivo model of GBM. The well-characterized CXCR4 antagonist Plerixafor was also included in the study. Methods The effects of peptide R on CXCR4 expression, cell survival and migration were assessed on the human glioblastoma cell line U87MG exposed to CXCL12, by immunofluorescence and western blotting, MTT assay, flow cytometry and transwell chamber migration assay. Peptide R was then tested in vivo, by using U87MG intracranial xenografts in CD1 nude mice. Peptide R was administered for 23 days since cell implantation and tumor volume was assessed by magnetic resonance imaging (MRI) at 4.7 T. Glioma associated microglia/macrophage (GAMs) polarization (anti-tumor M1 versus pro-tumor M2 phenotypes) and expressions of vascular endothelial growth factor (VEGF) and CD31 were assessed by immunohistochemistry and immunofluorescence. Results We found that peptide R impairs the metabolic activity and cell proliferation of human U87MG cells and stably reduces CXCR4 expression and cell migration in response to CXCL12 in vitro. In the orthotopic U87MG model, peptide R reduced tumor cellularity, promoted M1 features of GAMs and astrogliosis, and hindered intra-tumor vasculature. Conclusions Our findings suggest that targeting CXCR4 by peptide R might represent a novel therapeutic approach against GBM, and contribute to the rationale to further explore in more complex pre-clinical settings the therapeutic potential of peptide R, alone or in combination with standard therapies of GBM. Electronic supplementary material The online version of this article (doi:10.1186/s13046-016-0326-y) contains supplementary material, which is available to authorized users.

J. Neurochem. (2010) 115, 450–459. Abstract 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-β (TGF-β) 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 Ca2+ 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-β 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-β treatment of mixed glial cultures were clearly distinct. LPS induced a set of classical pro-inflammatory genes, whereas TGF-β increased the expression of genes associated with atypical microglial phenotype, namely arginase-1 and TGF-β 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.

We have extended our previous findings and shown that human immunodeficiency virus Tat protein, in addition to nitric oxide (NO), stimulated rat microglial cultures to release pro-inflammatory cytokine interleukin-1β and tumour necrosis factor-α in a nuclear factor (NF)-κB-dependent manner. At the same time, Tat stimulated the accumulation of free radicals, as indicated by the increased levels of isoprostane 8-epi-prostaglandin F2α (8-epi-PGF2α), a reliable marker of lipid peroxidation and oxidative stress, by a mechanism unrelated to NF-κB activation. The presence of free radical scavengers abrogated Tat-induced 8-epi-PGF2α accumulation without affecting NO and cytokine production. Consistently, Tat-induced IκBα degradation – an index of NF-κB activation – was not affected by free radical scavengers, but was prevented by an NF-κB-specific inhibitor. Our observations indicate that NF-κB plays a key role in Tat-dependent microglial activation, and that oxidative stress and NF-κB activation induced by Tat occur by independent mechanisms.

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.

Using intracerebral microdialysis, we reported previously that acute in vivo activation of NMDA glutamate receptors triggers rapid and transient releases of prostaglandin E2 (PGE2) and F2-isoprostane 15-F2t-IsoP in the hippocampus of freely moving rats. The formation of the two metabolites – produced through cyclo-oxygenase (COX) enzymatic activity and free radical-mediated peroxidation of arachidonic acid (AA), respectively, – was prevented by the specific NMDA antagonist MK-801, and was largely dependent on COX-2 activity. Here, we demonstrate that besides COX-2, which is the prominent COX isoform in the brain and particularly in the hippocampus, the constitutive isoform, COX-1 also contributes to prostaglandin (PG) synthesis and oxidative damage following in vivo acute activation of hippocampal NMDA glutamate receptors. The relative contribution of the two isoforms is dynamically regulated, as the COX-2 selective inhibitor NS398 immediately prevented PGE2 and 15-F2t-IsoP formation during the application of NMDA, whereas the COX-1 selective inhibitor SC560 was effective only 1 h after agonist infusion. Our data suggest that, although COX-2 is the prominent isoform, COX-1 activity may significantly contribute to excitotoxicity, particularly when considering the amount of lipid peroxidation associated with its catalytic cycle. We suggest that both isoforms should be considered as possible therapeutic targets to prevent brain damage caused by excitotoxicity.

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-β, and nerve growth factor), whereas the synthesis of pro-inflammatory molecules (tumor necrosis factor-α 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.

The human immunodeficiency virus type-1 (HIV-1) regulatory protein Tat is produced in the early phase of infection and is essential for virus replication. Together with other viral products, Tat has been implicated in the pathogenesis of HIV-1-associated dementia (HAD). As HIV-1 infection in the brain is very limited and macrophage/microglial cells are the only cellular type productively infected by the virus, it has been proposed that many of the viral neurotoxic effects are mediated by microglial products. We and others have shown that Tat affects the functional state of microglial cells, supporting the hypothesis that activated microglia play a role in the neuropathology associated with HIV-1 infection. This review describes the experimental evidence indicating that Tat stimulates microglia to synthesize potentially neurotoxic molecules, including proinflammatory cytokines and free radicals, and interferes with molecular mechanisms controlling cAMP levels, intracellular [Ca2+], and ion channel expression.

In the central nervous system (CNS), apoptosis plays an important role during development and is a primary pathogenic mechanism in several adult neurodegenerative diseases. A main feature of apoptotic cell death is the efficient and fast removal of dying cells by macrophages and nonprofessional phagocytes, without eliciting inflammation in the surrounding tissue. Apoptotic cells undergo several membrane changes, including the externalization of so-called "eat me" signals whose cognate receptors are present on professional phagocytes. Among these signals, the aminophospholipid phosphatidylserine (PS) appears to have a crucial and unique role in preventing the classical pro-inflammatory activation of macrophages, thus ensuring the silent and safe removal of apoptotic cells. Although extensively studied in the peripheral organs, the process of recognition and removal of apoptotic cells in the brain has only recently begun to be unraveled. Here, we summarize the evidence suggesting that upon interaction with PS-expressing apoptotic neurons, microglia may no longer promote the inflammatory cascade, but rather facilitate the elimination of damaged neurons through antiinflammatory and neuroprotective functions. We propose that the anti-inflammatory microglial phenotype induced through the activation of the specific PS receptor (PtdSerR), expressed by resting and activated microglial cells, could be relevant to the final outcome of neurodegenerative diseases, in which apoptosis seems to play a crucial role.

During inflammatory or degenerative processes microglial cells are likely to be exposed to activating agents that persist in brain parenchyma for prolonged periods. As our knowledge on microglial activation is largely based on in vitro studies in which microglial cultures are activated by a single administration of pro-inflammatory stimuli, we investigated the effects of repeated endotoxin (LPS) challenges on microglial functional state. Primary rat microglial cultures were subjected to one, two or three consecutive LPS-stimulation and the production of tumor necrosis factor-alpha (TNF-alpha), nitric oxide (NO), prostaglandin E2 (PGE2) and 15-deoxy-Delta12,14-PGJ2 (15d-PGJ2) measured. The ability of microglial cells to produce NO, TNF-alpha and 15d-PGJ2 upon the first LPS challenge rapidly declined after the second and the third stimulations, whereas PGE2 synthesis remained constantly elevated. Accordingly, the expression of inducible NO synthase decreased whereas cyclooxygenase-2 and microsomal PGE synthase remained up-regulated. The signaling pathways evoked by single or multiple LPS-stimulation were also profoundly different, when considering the activation of the transcription factors nuclear factor-kappa B and CREB, and of the p38 MAPK. Our observations suggest that prolonged exposure to LPS, and likely other activating agents, induces in microglia a functional state clearly distinct from that triggered by acute stimulation. The progressive down-regulation of pro-inflammatory molecules and the sustained release of PGE2 could have important implications for the resolution of brain inflammation.

We have previously reported that rat primary microglial cultures express the nuclear receptor peroxisome proliferator-activated receptor-gamma (PPAR-gamma) and that several functions associated with the activation of these cells, including nitric oxide (NO) and tumor necrosis factor-alpha synthesis, are down-regulated by 15-deoxy-delta12,14-prostaglandin J2 (15d-PGJ2) and ciglitazone, two specific PPAR-gamma agonists. Here we demonstrate that microglial cells not only express a functionally active PPAR-gamma, but also synthesize large amounts of 15d-PGJ2 upon stimulation with lipopolysaccharide (LPS). In addition, we show that, although 15d-PGJ2 and ciglitazone were equally effective in reducing microglial activation when used at 1-5 microm concentrations, 15d-PGJ2, but not of ciglitazone, reduced PGE2 production at low concentration (0.1 microm) and induced a time-dependent microglial impairment and apoptosis at high concentration (10 microm). Interestingly, the inhibition of PGE2 production was achieved mainly through the inhibition of cycloxygenase-2 enzymatic activity, as the expression of this enzyme and that of the microsomal isoform of PGE synthase remained unaltered. These findings suggest that 15d-PGJ2 affects the functional state and the survival of activated microglia through mechanisms only in part dependent on PPAR-gamma and that the concentration of 15d-PGJ2 is crucial in determining the particular microglial function affected.

In the last few years, the interaction between phosphatidylserine (PS), a phospholipid that becomes permanently exposed on the external cell surface in the early phases of apoptosis, and its specific receptor (PtdSerR) has emerged as a crucial event for the engulfing of apoptotic cells and for preventing the acquisition of pro-inflammatory functions by peripheral macrophages. Recently, we demonstrated that PtdSerR is expressed in microglial cultures purified from neonatal rat brain, and that PS-liposomes, used to mimic apoptotic cells, strongly reduce the lipopolysaccharide (LPS)-induced release of inflammatory mediators. Here, we show that in resting microglia, PS-liposomes induce cyclic AMP responding element binding protein (CREB) phosphorylation but do not activate nuclear factor-κB (NF-κB) and p38 mitogen-activated protein kinase (p38), in line with the non-inflammatory consequences of the recognition and removal of apoptotic cells by macrophages. In LPS-activated microglia, PS-liposomes did not affect NF-κB activation but inhibited the phosphorylation of p38 and delayed that of CREB. To our knowledge, this is the first biochemical evidence of the molecular signaling evoked by PS/PtdSerR interaction possibly related to repression of pro-inflammatory activities in microglial cells.

Prostaglandins (PGs), the arachidonic acid (AA) metabolites of the cyclooxygenase (COX) pathway, and the cytokine TNFα play major roles in inflammation and they are synthesised mainly by macrophages. Their syntheses have been shown to be regulated by several factors, including nitric oxide, a further important macrophage product. Since both positive and negative regulations of PGs and TNFα synthesis by NO have been reported, we sought to understand the mechanisms underlying these opposite NO effects by using a recent class of NO releasing compounds, the NONOates, which have been shown to release NO in a controlled fashion. To this aim, we analysed the effect of NO released from PAPA/NO (t1/2 15 min) and DETA/NO (t1/2 20 h) in RAW 264.7 cells. Both NONOates were used at the same concentrations allowing the cell cultures to be exposed either at high levels of NO for brief time (PAPA/NO) or at low levels of NO for long time (DETA/NO). We found that the two NONOates had opposite effect on basal TNFα release, being increased by PAPA/NO and decreased by DETA/NO, while they did not affect the release stimulated by LPS. At variance, both NONOates increased the basal PGE 2 production, while the LPS-stimulated production was slightly increased only by PAPA/NO. The modulation of PGE 2 synthesis was the result of the distinct effects of the two NO-donors on either arachidonic acid (AA) release or cyclooxygense-2 (COX-2) expression, the precursor and synthetic enzyme of PGs, respectively. Indeed, in resting cultures AA release was enhanced only by PAPA/NO whereas COX-2 expression was moderately upregulated by both donors. In LPS activated cells, both NONOates induced AA release, although with different kinetics and potencies, but only DETA/NO significantly increased COX-2 expression. In conclusion, by comparing the activities of these two NONOates, our observations indicate that level and time of exposure to NO are both crucial in determining the molecular target and the final result of the interactions between NO and inflammatory molecules.

Cyclooxygenases (COX) are a family of enzymes involved in the biosynthesis of prostaglandin (PG) and thromboxanes. The inducible enzyme cyclooxygenase-2 (COX-2) is the major isoform found in normal brain, where it is constitutively expressed in neurons and is further up-regulated during several pathological events, including seizures and ischaemia. Emerging evidence suggests that COX-2 is implicated in excitotoxic neurodegenerative phenomena. It remains unclear whether PGs or other products associated to COX activity take part in these processes. Indeed, it has been suggested that reactive oxygen species, produced by COX, could mediate neuronal damage. In order to obtain direct evidence of free radical production during COX activity, we undertook an in vivo microdialysis study to monitor the levels of PGE2 and 8-epi-PGF2α following infusion of N-methyl-d-aspartate (NMDA). A 20-min application of 1 mm NMDA caused an immediate, MK-801-sensitive increase of both PGE2 and 8-epi-PGF2α basal levels. These effects were largely prevented by the specific cytosolic phospholipase A2 (cPLA2) inhibitor arachidonyl trifluoromethyl ketone (ATK), by non- selective COX inhibitors indomethacin and flurbiprofen or by the COX-2 selective inhibitor NS-398, suggesting that the NMDA-evoked prostaglandin synthesis and free radical-mediated lipid peroxidation are largely dependent on COX-2 activity. As several lines of evidence suggest that prostaglandins may be potentially neuroprotective, our findings support the hypothesis that free radicals, rather than prostaglandins, mediate the toxicity associated to COX-2 activity.

International audience; Phytoprostanes (PhytoP's) are formed in higher plants from α-linolenic acid via a nonenzymatic free radical-catalyzed pathway and act as endogenous mediators capable of protecting cells from damage under various conditions related to oxidative stress. Humans are exposed to PhytoP's, as they are present in relevant quantities in vegetable food and pollen. The uptake of PhytoP's through the olfactory epithelium of the nasal mucosa, upon pollen grain inhalation, is of interest as the intranasal pathway is regarded as a direct route of communication between the environment and the brain. On this basis, we sought to investigate the potential activities of PhytoP's on immature cells of the central nervous system, which are particularly susceptible to oxidative stress. In neuroblastoma SH-SY5Y cells, used as a model for undifferentiated neurons, B1-PhytoP's, but not F1-PhytoP's, increased cell metabolic activity and protected them from oxidant damage caused by H2O2. Moreover, B1-PhytoP's induced a moderate depolarization of the mitochondrial inner membrane potential. These effects were prevented by the PPAR-γ antagonist GW9662. When SH-SY5Y cells were induced to differentiate toward a more mature phenotype, they became resistant to B1-PhytoP activities. B1-PhytoP's also influenced immature cells of an oligodendroglial line, as they increased the metabolic activity of oligodendrocyte progenitors and strongly accelerated their differentiation to immature oligodendrocytes, through mechanisms at least partially dependent on PPAR-γ activity. However, B1-PhytoP's did not protect oligodendrocyte progenitors against oxidant injury. Taken together, these data suggest that B1-PhytoP's, through novel mechanisms involving PPAR-γ, can specifically affect immature brain cells, such as neuroblasts and oligodendrocyte progenitors, thereby conferring neuroprotection against oxidant injury and promoting myelination.

The serotonin receptor 7 (5-HT7-R), is very abundant in the thalamus, hypothalamus, dorsal raphe nucleus and hippocampus; at lower levels it is found also in the cortex, striatum and amygdala. This neurochemical receptor system plays important functional roles in learning and memory, in regulation of mood and circadian rhythmicity. Recently, many agonist drugs selective for this receptor have been developed and their effects reported. Presently, we review some recent studies we have conducted aimed to evaluate the modulatory effect of a subchronic regimen with LP-211, a new selective agonist compound belonging to 1-arylpiperazine category, on behavioural and molecular parameters. At low dose (0.25 mg/kg, i.p.), LP-211 induced in adult mice a six-hour anticipated wake up with no temporal landmark (constant light) acting as non-photic stimulus for “internal clock” resetting. In standard 12:12h L/D cycle, we observed a subchronic effect (5-6 days at 0.25 mg/kg, once/day), on delay of wake up associated with a reduction of peak locomotor activity and any evidence for a real cellular proliferation after ex vivo analysis. The other studies, conduct on rats, were aimed to investigate long-term effects of subchronic LP-211 administration into adulthood development. Subchronic LP-211 (0.125 mg/kg, i.p. once/day) during the prepuberal phase reduced L-Glutamate, NMDAR1 and DAT, within the ventral striatum. Moreover, we observed a clear reduction of the immunoreactivity of serotonin transporter (SERT) and dopaminergic D2 receptors at dorsal striatum. These results represent a starting point to explore the neurophysiology of 5-HT7-R: subchronic LP-211 in rats and mice appears a suitable tool for studying the role of 5-HT7-R in sleep disorders, emotional and motivational regulations, attentive processes and executive functions.

Astrocytosis is a common feature of amyloid plaques, the hallmark of Alzheimer's disease (AD), along with activated microglia, neurofibrillary tangles, and beta-amyloid (beta A) deposition. However, the relationship between astrocytosis and neurodegeneration remains unclear. To assess whether beta A-stimulated astrocytes can damage neurons and contribute to beta A neurotoxicity, we studied the effects of beta A treatment in astrocytic/neuronal co-cultures, obtained from rat embryonic brain tissue. We found that in neuronal cultures conditioned by beta A-treated astrocytes, but not directly in contact with beta A, the number of apoptotic cells increased, doubling the values of controls. In astrocytes, beta A did not cause astrocytic cell death, nor did produce changes in nitric oxide or prostaglandin E(2) levels. In contrast, S-100 beta expression was remarkably increased. Our data show for the first time that beta A--astrocytic interaction produces a detrimental effect on neurons, which may contribute to neurodegeneration in AD.

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.

Summary The contribution that oxidative damage to DNA and/or RNA makes to the aging process remains undefined. In this study, we used the hMTH1-Tg mouse model to investigate how oxidative damage to nucleic acids affects aging. hMTH1-Tg mice express high levels of the hMTH1 hydrolase that degrades 8-oxodGTP and 8-oxoGTP and excludes 8-oxoguanine from both DNA and RNA. Compared to wild-type animals, hMTH1-overexpressing mice have significantly lower steady-state levels of 8-oxoguanine in both nuclear and mitochondrial DNA of several organs, including the brain. hMTH1 overexpression prevents the age-dependent accumulation of DNA 8-oxoguanine that occurs in wild-type mice. These lower levels of oxidized guanines are associated with increased longevity and hMTH1-Tg animals live significantly longer than their wild-type littermates. Neither lipid oxidation nor overall antioxidant status is significantly affected by hMTH1 overexpression. At the cellular level, neurospheres derived from adult hMTH1-Tg neural progenitor cells display increased proliferative capacity and primary fibroblasts from hMTH1-Tg embryos do not undergo overt senescence in vitro. The significantly lower levels of oxidized DNA/RNA in transgenic animals are associated with behavioral changes. These mice show reduced anxiety and enhanced investigation of environmental and social cues. Longevity conferred by overexpression of a single nucleotide hydrolase in hMTH1-Tg animals is an example of lifespan extension associated with healthy aging. It provides a link between aging and oxidative damage to nucleic acids.

International audience; Metalloproteinases (MMPs) and their endogenous inhibitors (TIMPs), produced in the brain by cells of non-neural and neural origin, including neural progenitors (NPs), are emerging as regulators of nervous system development and adult brain functions. In the present study, we explored whether MMP-2, MMP-9, and TIMP-2, abundantly produced in the brain, modulate NP developmental properties. We found that treatment of NPs, isolated from the murine fetal cerebral cortex or adult subventricular zone, with the clinically tested broad-spectrum MMP inhibitor Marimastat profoundly affected the NP differentiation fate. Marimastat treatment allowed for an enrichment of our cultures in neuronal cells, inducing NPs to generate higher percentage of neurons and a lower percentage of astrocytes, possibly affecting NP commitment. Consistently with its proneurogenic effect, Marimastat early downregulated the expression of Notch target genes, such as Hes1 and Hes5. MMP-2 and MMP-9 profiling on proliferating and differentiating NPs revealed that MMP-9 was not expressed under these conditions, whereas MMP-2 increased in the medium as pro-MMP-2 (72 kDa) during differentiation; its active form (62 kDa) was not detectable by gel zymography. MMP-2 silencing or administration of recombinant active MMP-2 demonstrated that MMP-2 does not affect NP neuronal differentiation, nor it is involved in the Marimastat proneurogenic effect. We also found that TIMP-2 is expressed in NPs and increases during late differentiation, mainly as a consequence of astrocyte generation. Endogenous TIMP-2 did not modulate NP neurogenic potential; however, the proneurogenic action of Marimastat was mediated by TIMP-2, as demonstrated by silencing experiments. In conclusion, our data exclude a major involvement of MMP-2 and MMP-9 in the regulation of basal NP differentiation, but highlight the ability of TIMP-2 to act as key effector of the proneurogenic response to an inducing stimulus such as Marimastat.

Maternal-fetal HIV-1 transmission can be prevented by administration of AZT, alone or in combination with other antiretroviral drugs to pregnant HIV-1-infected women and their newborns. In spite of the benefits deriving from this life-saving prophylactic therapy, there is still considerable uncertainty on the potential long-term adverse effects of antiretroviral drugs on exposed children. Clinical and experimental studies have consistently shown the occurrence of mitochondrial dysfunction and increased oxidative stress following prenatal treatment with antiretroviral drugs, and clinical evidence suggests that the developing brain is one of the targets of the toxic action of these compounds possibly resulting in behavioral problems. We intended to verify the effects on brain and behavior of mice exposed during gestation to AZT, the backbone of antiretroviral therapy during human pregnancy. We hypothesized that glutamate, a neurotransmitter involved in excitotoxicity and behavioral plasticity, could be one of the major actors in AZT-induced neurochemical and behavioral alterations. We also assessed the antioxidant and neuroprotective effect of L-acetylcarnitine, a compound that improves mitochondrial function and is successfully used to treat antiretroviral-induced polyneuropathy in HIV-1 patients. We found that transplacental exposure to AZT given per os to pregnant mice from day 10 of pregnancy to delivery impaired in the adult offspring spatial learning and memory, enhanced corticosterone release in response to acute stress, increased brain oxidative stress also at birth and markedly reduced expression of mGluR1 and mGluR5 subtypes and GluR1 subunit of AMPA receptors in the hippocampus. Notably, administration during the entire pregnancy of L-acetylcarnitine was effective in preventing/ameliorating the neurochemical, neuroendocrine and behavioral adverse effects induced by AZT in the offspring. The present preclinical findings provide a mechanistic hypothesis for the neurobehavioral effects of AZT and strongly suggest that preventive administration of L-acetylcarnitine might be effective in reducing the neurological side-effects of antiretroviral therapy in fetus/newborn.

International audience; 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.

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-β (TGF-β) 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-β 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-β treatment of mixed glial cultures were clearly distinct. LPS induced a set of classical pro-inflammatory genes, whereas TGF-β increased the expression of genes associated with atypical microglial phenotype, namely arginase-1 and TGF-β 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.

J. Neurochem. (2010) 113, 1060–1072. Abstract Inflammation is regarded as a main obstacle to brain regeneration. Major detrimental effects are attributed to microglial/macrophagic products, such as TNF-α and interleukin (IL)-6. The role of cytokines of the IL-1 family, particularly of IL-1α, in the modulation of neural precursor cell (NPC) properties is less characterized. IL-1α is one of the most abundant cytokines released upon acute stimulation of microglia with lipopolysaccharide and is down-regulated upon chronic stimulation. As we recently demonstrated, acutely activated microglia reduces NPC survival, prevent neuronal differentiation and promote glial differentiation. Chronically activated microglia are instead permissive to NPC survival and neuronal differentiation, and less effective in promoting astrocytic differentiation. We thus investigated whether IL-1α could contribute to the effects of acutely activated microglia on NPC. We found that NPC express functional IL-1 receptors and that exposure to recombinant IL-1α strongly enhances NPC differentiation into astrocytes, without affecting cell viability and neuronal differentiation. In the same conditions, recombinant IL-1β has pro-gliogenic effects at concentrations 10-fold higher than those found in activated microglial conditioned media. Interestingly, immunodepletion of IL-1α in activated microglial conditioned media fails to revert microglial pro-gliogenic action and slightly enhances neuronal differentiation, revealing that other microglial-derived factors contribute to the modulation of NPC properties.

Inflammation is a self-defensive reaction that may develop into a chronic state and become a causative factor in the pathogenesis of a broad range of disabling diseases. Similar to peripheral inflammation, brain inflammation is increasingly being viewed as a target for treating neurological diseases, not only infectious and immune-mediated disorders such as meningitis or multiple sclerosis but also stroke, trauma, and neurodegenerative diseases that were originally not considered to be inflammatory. Microglial cells, the resident macrophages of brain parenchyma, are generally viewed as major sources of pro-inflammatory and potentially neurotoxic molecules in the damaged brain, However, a direct link between activated microglia and tissue damage has not been univocally demonstrated in vivo, and recent studies have rather documented exacerbation of injury following selective microglial ablation or anti-inflammatory treatments. Recent studies have implicated inflammation in the regulation of adult neurogenesis, thus broadening the therapeutic potential of strategies aimed at controlling neuroinflammation. This chapter summarizes the main evidence supporting both detrimental and protective roles of inflammation in acute and chronic brain diseases.

Early phases of Parkinson's disease (PD) are characterized by a mild reduction of dopamine (DA) in striatum and by emergence of psychiatric disturbances that precede overt motor symptoms. In order to characterize the neurochemical re-arrangements induced by such striatal impairment, we used a mouse model in which a low dose of 6-hydroxydopamine (6-OHDA) was bilaterally injected into the dorsal striatum. These mice showed a DA reduction of about 40% that remained stable up to 12 weeks after injection. This reduction was accompanied by changes in DA metabolite levels, such as HVA, transiently reduced at 4 weeks, and DOPAC, decreased at 12 weeks. No change in the 5-hydroxytryptamine (5-HT) levels was found but the 5-hydroxyindoleacetic acid (5-HIAA)/5-HT ratio was increased at 4 weeks. In addition, at the same time-point, the levels of 15-F(2t)-IsoP, an index of oxidative stress, and of PGE(2), a major product of cyclooxygenase-2, were decreased in different brain areas while BDNF levels were increased. These neurochemical changes were accompanied by altered behavioral responses concerning the emotional reactivity. Overall, the present findings suggest that a change of 5-HT metabolism and a modification of oxidative stress levels may play a role in the early PD degeneration phases.

The contribution of microglia to the modulation of neurogenesis under pathological conditions is unclear. Both pro- and anti-neurogenic effects have been reported, likely reflecting the complexity of microglial activation process. We previously demonstrated that prolonged (72 hr) in vitro exposure to lipopolysaccharide (LPS) endows microglia with a potentially neuroprotective phenotype, here referred as to "chronic". In the present study we further characterized the chronic phenotype and investigated whether it might differently regulate the properties of embryonic and adult neural precursor cells (NPC) with respect to the "acute" phenotype acquired following a single (24 hr) LPS stimulation. We show that the LPS-dependent induction of the proinflammatory cytokines interleukin (IL)-1 alpha, IL-1 beta, IL-6, and tumor necrosis factor (TNF)-alpha was strongly reduced after chronic stimulation of microglia, as compared with acute stimulation. Conversely, the synthesis of the anti-inflammatory cytokine IL-10 and the immunomodulatory prostaglandin E2 (PGE2) was still elevated or further increased, after chronic LPS exposure, as revealed by real time PCR and ELISA techniques. Acutely activated microglia, or their conditioned medium, reduced NPC survival, prevented neuronal differentiation and strongly increased glial differentiation, likely through the release of proinflammatory cytokines, whereas chronically activated microglia were permissive to neuronal differentiation and cell survival, and still supported glial differentiation. Our data suggest that, in a chronically altered environment, persistently activated microglia can display protective functions that favor rather than hinder brain repair processes.

Non steroidal anti-inflammatory drugs (NSAIDs) are therapeutic agents of first choice for the treatment of inflammation, pain, and fever. Neuroscience research of the last decades has pointed out the important role of inflammation in the pathogenesis of several brain disorders, and epidemiological and experimental evidence has suggested a beneficial role of NSAIDs in both chronic and acute neuropathologies. More recently NSAIDs have gained further attention as potential tools to enhance neuroregenerative processes in the adult mammalian brain. The rational behind their use arises from the notion that inflammatory processes that accompany brain damage would exert a major detrimental effect on endogenous neurogenesis. However, inflammation and glial responses to acute or chronic injuries constitute a complex and multifaceted process by which, besides potentially harmful and cytotoxic activities, beneficial responses can be initiated in the attempt to re-establish the lost tissue integrity. The individuation of optimal timing and type of pharmacological intervention able to potentiate the beneficial aspects of inflammation rather than to suppress it as a whole, would allow the achievement of enhanced and successful regenerative responses. In the present article, we will review the current literature on the effects of NSAIDs on neurogenesis and briefly discuss the cellular or molecular mechanisms by which these drugs can modulate brain restorative processes.

Activation and mobilization of microglia are early events in the majority of brain pathologies. Among the signalling molecules that can affect microglial behaviour, we investigated whether nerve growth factor (NGF) was able to influence microglial motility. We found that NGF induced chemotaxis of microglial cells through the activation of TrkA receptor. In addition, NGF chemotactic activity was increased in the presence of low concentrations (or =0.2 ng/ml) of transforming growth factor-beta (TGF-beta), which at this concentration showed chemotactic activity per se. On the contrary, NGF-induced microglial migration was reduced in the presence of chemokinetic concentration of TGF-beta (or =2 ng/ml). Finally, both basal and NGF-induced migratory activity of microglial cells was increased after a long-term exposure of primary mixed glial cultures to 2 ng/ml of TGF-beta. Our observations suggest that both NGF and TGF-beta contribute to microglial recruitment. The chemotactic activities of these two pleiotropic factors could be particularly relevant during chronic diseases in which recruited microglia remove apoptotic neurons in the absence of a typical inflammatory reaction.

Partial and generalized status epilepticus (pSE and gSE) trigger the same level of progenitor cell proliferation in adult dentate gyrus, but survival of new neurons is poor after gSE. Here, we show markedly elevated levels of prostaglandin E2 (PGE2) and brain-derived neurotrophic factor (BDNF) in rat hippocampal formation at 7 days following pSE but not gSE. Administration of the cyclooxygenase (COX) inhibitor flurbiprofen for 1 week, starting at day 8 post-SE, abated PGE2 and decreased BDNF levels, but did not affect survival of new neurons 4 weeks later. Thus, high PGE2 and BDNF levels induced by pSE are probably not of major importance for survival of new neurons during the first days after formation. We propose that they modulate other aspects of synaptic and cellular plasticity, and thereby may influence epileptogenesis.