Maria Antonietta Ajmone-Cat

Activation of α7 nicotinic acetylcholine receptor by nicotine selectively up-regulates cyclooxygenase-2 and prostaglandin E2 in rat microglial cultures

BackgroundNicotinic 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 α7 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-α (TNF-α) production in human macrophages as well as in mouse microglial cultures. In the present study, we investigated whether the stimulation of α7 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.MethodsExpression of α7 nicotinic receptor in rat microglial cell was examined by RT-PCR, immunofluorescence staining and Western blot. The functional effects of α7 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-1β, nitric oxide, interleukin-10 and prostaglandin E2. Total RNA was assayed by RT-PCR for the expression of COX-2 mRNA.ResultsRat microglial cells express α7 nicotinic receptor, and its activation by nicotine dose-dependently reduces the LPS-induced release of TNF-α, but has little or no effect on nitric oxide, interleukin-10 and interleukin-1β. By contrast, nicotine enhances the expression of cyclooxygenase-2 and the synthesis of one of its major products, prostaglandin E2.ConclusionsSince 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 α7 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.

In vitro neuronal and glial differentiation from embryonic or adult neural precursor cells are differently affected by chronic or acute activation of microglia.

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)‐1α, IL‐1β, IL‐6, and tumor necrosis factor (TNF)‐α 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.

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.

Cyclo-oxygenase-1 and -2 differently contribute to prostaglandin E2 synthesis and lipid peroxidation after in vivo activation of N-methyl-D-aspartate receptors in rat hippocampus

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 vivo activation of N-methyl-d-aspartate receptors in the rat hippocampus increases prostaglandin E2 extracellular levels and triggers lipid peroxidation through cyclooxygenase-mediated mechanisms

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.

Atypical antiinflammatory activation of microglia induced by apoptotic neurons: possible role of phosphatidylserine-phosphatidylserine receptor interaction.

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.

Prolonged exposure of microglia to lipopolysaccharide modifies the intracellular signaling pathways and selectively promotes prostaglandin E2 synthesis

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‐α (TNF‐α), nitric oxide (NO), prostaglandin E2 (PGE2) and 15‐deoxy‐Δ12,14‐PGJ2 (15d‐PGJ2) measured. The ability of microglial cells to produce NO, TNF‐α 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.

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. GLIA 2013;61:1698–1711

Astrocytes contribute to neuronal impairment in βA toxicity increasing apoptosis in rat hippocampal neurons

Astrocytosis is a common feature of amyloid plaques, the hallmark of Alzheimers disease (AD), along with activated microglia, neurofibrillary tangles, and β‐amyloid (βA) deposition. However, the relationship between astrocytosis and neurodegeneration remains unclear. To assess whether βA‐stimulated astrocytes can damage neurons and contribute to βA neurotoxicity, we studied the effects of βA treatment in astrocytic/neuronal co‐cultures, obtained from rat embryonic brain tissue. We found that in neuronal cultures conditioned by βA‐treated astrocytes, but not directly in contact with βA, the number of apoptotic cells increased, doubling the values of controls. In astrocytes, βA did not cause astrocytic cell death, nor did produce changes in nitric oxide or prostaglandin E2 levels. In contrast, S‐100β expression was remarkably increased. Our data show for the first time that βA–astrocytic interaction produces a detrimental effect on neurons, which may contribute to neurodegeneration in AD. GLIA 34:68–72, 2001.

Nuclear receptor peroxisome proliferator-activated receptor-gamma is activated in rat microglial cells by the anti-inflammatory drug HCT1026, a derivative of flurbiprofen.

The peroxisome proliferator‐activated receptor‐γ (PPAR‐γ) is constitutively expressed in primary cultures of rat microglia, the main population of brain resident macrophages, and its ligand‐dependent activation leads to the repression of several microglial functions. A few non‐steroidal anti‐inflammatory drugs (NSAIDs), e.g. indomethacin and ibuprofen, show PPAR‐γ agonistic properties. It has been proposed that PPAR‐γ activation contributes to the potential benefits of the long‐term use of certain NSAIDs in delaying the progression of Alzheimers disease (AD). Previous data have shown that the NSAID HCT1026 [2‐fluoro‐α‐methyl(1,1′‐biphenyl)4‐acetic acid‐4‐(nitrooxy)butyl ester], a derivative of flurbiprofen which releases nitric oxide (NO), reduces the number of reactive microglial cells in a variety of models. This evidence together with the chemical analogy with ibuprofen led us to investigate whether flurbiprofen and HCT1026 interact with PPAR‐γ and interfere with microglial activation. We found that a low concentration (1 µm) of HCT1026, but not flurbiprofen, activated PPAR‐γ in primary cultures of rat microglia, with kinetics similar to those of the synthetic agonist ciglitazone. The PPAR‐γ antagonist GW9662 (2‐chloro‐5‐nitrobenzanilide) prevented the activation of PPAR‐γ by HCT1026. Interestingly, unlike other NSAIDs that activate PPAR‐γ at concentrations higher than those required for cyclooxygenase inhibition, HCT1026 activated PPAR‐γ and inhibited prostaglandin E2 synthesis at the same low concentration (1 µm). The results suggest that HCT1026 may exert additional anti‐inflammatory actions through PPAR‐γ activation, allowing a more effective control of microglial activation and brain inflammation.