Maternal suboptimal selenium intake and low-level lead exposure affect offspring's microglial immune profile and its reactivity to a subsequent inflammatory hit.

Micronutrients such as selenium (Se) are essentials since prenatal life to support brain and cognitive development. Se deficiency, which affects up to 1 billion people worldwide, can interact with common adverse environmental challenges including (Pb), exacerbating their toxic effects. Exploiting our recently validated rat model of maternal Se restriction and developmental low Pb exposure, our aims were to investigate: (i) the early consequences of suboptimal Se intake and low-Pb exposure on neuroinflammation in neonates' whole brains; (ii) the potential priming effect of suboptimal Se and low-Pb exposure on offspring's glial reactivity to a further inflammatory hit. To these aims female rats were fed with suboptimal (0.04 mg/kg; Subopt) and optimal (0.15 mg/kg; Opt) Se dietary levels throughout pregnancy and lactation and exposed or not to environmentally relevant Pb dose in drinking water (12.5 µg/mL) since 4 weeks pre-mating. We found an overall higher basal expression of inflammatory markers in neonatal brains, as well as in purified microglia and organotypic hippocampal slice cultures, from the Subopt Se offspring. Subopt/Pb cultures were highly activated than Subopt cultures and showed a higher susceptibility to the inflammatory challenge lipopolysaccharide than cultures from the Opt groups. We demonstrate that even a mild Se deficiency and low-Pb exposure during brain development can influence the neuroinflammatory tone of microglia, exacerbate the toxic effects of Pb and prime microglial reactivity to subsequent inflammatory stimuli. These neuroinflammatory changes may be responsible, at least in part, for adverse neurodevelopmental outcomes.

Docosahexaenoic acid promotes oligodendrocyte differentiation via PPAR-γ signalling and prevents tumor necrosis factor-α-dependent maturational arrest.

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.

NGF promotes microglial migration through the activation of its high affinity receptor: modulation by TGF-beta.

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.

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-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.

Effects of phosphatidylserine on p38 mitogen activated protein kinase, cyclic AMP responding element binding protein and nuclear factor-kappaB activation in resting and activated microglial cells.

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-kappaB (NF-kappaB) 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-kappaB 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.

The presence of astrocytes enhances beta amyloid-induced neurotoxicity in hippocampal cell cultures.

A characteristic feature of neuritic plaques in Alzheimer's disease is represented by the presence of activated astrocytes, surrounding dystrophic neurons and beta-amyloid deposition. To explore the role of astrocytes in in vitro beta-amyloid neurotoxicity, we studied the effect of beta-amyloid treatment in hippocampal neurons in two different cell models: pure cultures, where neurons were grown in absence of astrocytes and mixed cultures, where neurons were seeded on a confluent layer of astrocytes. We evaluated two characteristic aspects of in vitro beta-amyloid neurotoxicity: reduction of cell viability and degeneration of the neuritic tree. We demonstrated that neurons growing on astrocytes were more prone to the detrimental effect of the amyloid peptide, with respect to neurons grown in absence of the glial component. Our results support the hypothesis that beta-amyloid-astrocyte interaction can adversely condition neurons and contribute to neuronal damage in Alzheimer's disease.

In vivo activation of N-methyl-D-aspartate receptors in the rat hippocampus increases prostaglandin E(2) 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 PGE(2) and 8-epi-PGF(2alpha) 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 PGE(2) and 8-epi-PGF(2alpha) basal levels. These effects were largely prevented by the specific cytosolic phospholipase A(2) (cPLA(2) ) 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.

Differential effects of the nonsteroidal antiinflammatory drug flurbiprofen and its nitric oxide-releasing derivative, nitroflurbiprofen, on prostaglandin E(2), interleukin-1beta, and nitric oxide synthesis by activated microglia.

Increasing experimental, clinical, and epidemiological studies point to the pivotal role of inflammation in the pathogenesis of acute and chronic neurodegenerative diseases and to the protective effects of nonsteroidal antiinflammatory drug (NSAID) therapies. Nonetheless, NSAID long-term therapies are limited by their significant adverse effects on gastrointestinal tract and kidneys. Nitroflurbiprofen (NO-flurbiprofen) belongs to a novel class of antiinflammatory agents obtained by derivatization of conventional NSAIDs with a nitric oxide (NO)-releasing moiety, which strongly reduces their untoward side effects without altering the antiinflammatory effectiveness. The recent evidence of neuroprotective effects of NO-NSAIDs in animal models of chronic brain inflammation prompted us to investigate the activities of NO-flurbiprofen and its parent molecule flurbiprofen on activated rat microglia, the brain resident macrophages. We found that NO-flurbiprofen was as potent as flurbiprofen in preventing prostaglandin E(2) synthesis in lipopolysaccharide-activated microglial cultures. At variance with previous observations on peripheral macrophages is that NO-flurbiprofen did not show any additional capacity to inhibit interleukin-1beta synthesis compared with flurbiprofen. Moreover, NO enhanced the expression of the inducible NO synthase; this effect was most likely attributable to the NO released from the drug, as suggested by experiments performed in the presence of the NO donor Deta-NONOate, which similarly to NO-flurbiprofen is characterised by a slow and long-lasting release. Our findings indicate that NO-NSAIDs may differently affect peripheral and brain macrophages. Given their potential therapeutic role in brain inflammation, further in vivo and in vitro studies are required to understand fully their mechanism of action in the CNS.

Human immunodeficiency virus type-1 Tat protein induces nuclear factor (NF)-kappaB activation and oxidative stress in microglial cultures by independent mechanisms.

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-1beta and tumour necrosis factor-alpha in a nuclear factor (NF)-kappaB-dependent manner. At the same time, Tat stimulated the accumulation of free radicals, as indicated by the increased levels of isoprostane 8-epi-prostaglandin F(2alpha) (8-epi-PGF(2alpha)), a reliable marker of lipid peroxidation and oxidative stress, by a mechanism unrelated to NF-kappaB activation. The presence of free radical scavengers abrogated Tat-induced 8-epi-PGF(2alpha) accumulation without affecting NO and cytokine production. Consistently, Tat-induced IkappaBalpha degradation - an index of NF-kappaB activation - was not affected by free radical scavengers, but was prevented by an NF-kappaB-specific inhibitor. Our observations indicate that NF-kappaB plays a key role in Tat-dependent microglial activation, and that oxidative stress and NF-kappaB activation induced by Tat occur by independent mechanisms.

Astrocytes contribute to neuronal impairment in beta A toxicity increasing apoptosis in rat hippocampal neurons.

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.

Modulation of acetylcholinesterase and voltage-gated Na(+) channels in choline acetyltransferase- transfected neuroblastoma clones.

Neurotransmitters appear early in the developing embryo and may play a role in the regulation of neuronal differentiation. To study potential effects of acetylcholine production in neuronal differentiation, we used the FB5 subclone of N18TG2 murine neuroblastoma cells stably transfected with cDNA for choline acetyltransferase. We tested whether the forced acetylcholine production can modify the expression or the cellular localization of different neuronal markers. We studied the activity, localization, and secretion of acetylcholinesterase in view of its possible role in the modulation of the morphogenetic action of acetylcholine and of its proposed role of a regulator of neurite outgrowth. FB5 cells are characterized by a high level of acetylcholinesterase, predominantly released into the culture medium. Acetylcholinesterase secretion into the medium was lower in choline acetyltransferase-transfected clones than in nontransfected and antisense-transfected controls. Moreover, sequential extraction of acetylcholinesterase revealed that detergent-extracted, i.e., membrane-associated, activity was higher in the transfected clones expressing choline acetyltransferase activity than in both control groups. These observations suggest that a shift occurs in the utilization of acetylcholinesterase in choline acetyltransferase-transfected clones from a secretion pathway to a pathway leading to membrane localization. In addition, the choline acetyltransferase-positive clones showed higher densities of voltage-gated Na(+) channels and enhanced high-affinity choline uptake, suggesting the accomplishment of a more advanced differentiated neuronal phenotype. Finally, binding experiments demonstrated the presence of muscarinic acetylcholine receptors in all examined clones. This observation is consistent with the proposed existence of an autocrine loop, which may be important for the enhancement in the expression of neurospecific traits.