Transgenic mouse in vivo library of human Down syndrome critical region 1: association between DYRK1A overexpression, brain development abnormalities, and cell cycle protein alteration.

Down syndrome is the most frequent genetic cause of mental retardation, having an incidence of 1 in 700 live births. In the present study we used a transgenic mouse in vivo library consisting of 4 yeast artificial chromosome (YAC) transgenic mouse lines, each bearing a different fragment of the Down syndrome critical region 1 (DCR-1), implicated in brain abnormalities characterizing this pathology. The 152F7 fragment, in addition to genes also located on the other DCR-1 fragments, bears the DYRK1A gene, encoding for a serine-threonine kinase. The neurobehavioral analysis of these mouse lines showed that DYRK1A overexpressing 152F7 mice but not the other lines display learning impairment and hyperactivity during development. Additionally, 152F7 mice display increased brain weight and neuronal size. At a biochemical level we found DYRK1A overexpression associated with a development-dependent increase in phosphorylation of the transcription factor FKHR and with high levels of cyclin B1, suggesting for the first time in vivo a correlation between DYRK1A overexpression and cell cycle protein alteration. In addition, we found an altered phosphorylation of transcription factors of CREB family. Our findings support a role of DYRK1A overexpression in the neuronal abnormalities seen in Down syndrome and suggest that this pathology is linked to altered levels of proteins involved in the regulation of cell cycle.

Paracetamol effectively reduces prostaglandin E2 synthesis in brain macrophages by inhibiting enzymatic activity of cyclooxygenase but not phospholipase and prostaglandin E synthase.

Epidemiological studies indicate that nonsteroidal anti-inflammatory drugs (NSAIDs) are neuroprotective, although the mechanisms underlying their beneficial effect remain largely unknown. Given their well-known adverse effects, which of the NSAIDs is the best for neurodegenerative disease management remains a matter of debate. Paracetamol is a widely used analgesic/antipyretic drug with low peripheral adverse effects, possibly related to its weak activity as inhibitor of peripheral cyclooxygenase (COX), the main target of NSAIDs. As microglia play an important role in CNS inflammation and pathogenesis of neurodegenerative diseases, we investigate the effect of paracetamol on rat microglial cultures. Although less potent than other NSAIDs, (indomethacin approximately NS-398 > flurbiprofen approximately piroxicam > paracetamol approximately acetylsalicylic acid), paracetamol completely inhibited the synthesis of prostaglandin E(2) (PGE(2)) in lipopolysaccharide-stimulated microglia, when used at concentrations comparable to therapeutic doses. The drug did not affect the expression of the enzymes involved in PGE(2) synthesis, i.e., COX-1, COX-2, and microsomal PGE synthase, or the release of the precursor arachidonic acid (AA). Paracetamol inhibited the conversion of exogenous AA, but not PGH(2), into PGE(2) indicating that the target of the drug is COX activity. Consistently, paracetamol inhibited with similar IC(50) the synthesis of PGF(2alpha) and thromboxane B(2), two other COX metabolites. Finally, none of the NSAIDs affected the productions of nitric oxide and tumor necrosis factor(alpha), two inflammatory mediators released by activated microglia. As paracetamol was reported to inhibit PG synthesis in peripheral macrophages with an IC(50) at least three orders of magnitude higher than in microglia, we suggest that this drug represents a good tool for treating brain inflammation without compromising peripheral PG synthesis.

Modulation of PGE(2) and TNFalpha by nitric oxide and LPS-activated RAW 264.7 cells.

Prostaglandins (PGs), the arachidonic acid (AA) metabolites of the cyclooxygenase (COX) pathway, and the cytokine TNFalpha 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 TNFalpha 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 TNFalpha 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.

Expression of phosphatidylserine receptor and down-regulation of pro-inflammatory molecule production by its natural ligand in rat microglial cultures.

Exposure of phosphatidylserine (PS), an aminophospholipid normally sequestered in the inner leaflet of plasma membrane, is one of the crucial steps in the recognition and ingestion of apoptotic cells by macrophages. The recognition of PS on apoptotic cells by peripheral macrophages is mediated by a phosphatidylserine-specific receptor (PtdSerR), which has recently been cloned. In spite of the important role of apoptosis in the CNS, the process of apoptotic neuron recognition by microglia is poorly understood. Because recent studies suggest that engagement of PS with a not yet characterized microglial receptor is necessary for apoptotic neuron uptake, we investigated the expression of PtdSer-R and its functional role in neonatal rat brain microglial cultures. Semi-quantitative RT-PCR analysis revealed that PtdSerR mRNA was detectable in unstimulated cultures and enhanced in LPS activated microglia. The presence of PS-liposomes strongly reduced the release of pro-inflammatory molecules such as nitric oxide, interleukin-1beta, and tumor necrosis factor-alpha by LPS-activated microglia. At variance, the immunoregulatory cytokines interleukin-10 and transforming growth factor-beta1 were moderately decreased or unaffected. The activity of PS-liposomes was mimicked by the PS head group phospho-L-serine, but not by phosphatidylcholine-containing liposomes. Our data suggest that, as for peripheral macrophages, PS through its receptor can modulate microglial activation toward an anti-inflammatory phenotype.

Opposite regulation of prostaglandin E2 synthesis by transforming growth factor-beta1 and interleukin 10 in activated microglial cultures.

We have recently shown that prostaglandin E2 (PGE2) synthesis in activated microglia is tightly regulated by several substances (NO, neurotransmitters, pro-inflammatory cytokines), that might originate from intrinsic brain cells or from hematogenous cells infiltrating the brain in the course of inflammatory diseases. In view of the important immunoregulatory and neuroprotective functions recently attributed to PGE2, in the present study we extended our analysis of factors regulating PGE2 synthesis in rat microglial cultures to two anti-inflammatory and immunosuppressive cytokines, transforming growth factor beta1 (TGF-beta1) and interleukin 10 (IL-10), which share with PGE2 the ability to strongly deactivate peripheral macrophages and microglial cells. Moreover, we looked at the effect of the two cytokines on nitric oxide (NO) synthesis, another important microglial effector, whose synthesis is linked to that of PGE2 by complex feed-back mechanisms. We found that while both cytokines inhibited LPS-induced NO release, they had distinct and opposite regulatory activities on PGE2 production. In fact, while TGF-beta1 enhanced LPS-induced PGE2 synthesis, IL-10 showed an inhibitory effect. The two cytokines acted mainly by regulating the LPS-induced expression of the rate limiting enzymes of the two metabolic pathways, cyclooxygenase-2 (COX-2) and inducible NO synthase (iNOS). Moreover, TGF-beta1 counteracted the effect of the pro-inflammatory cytokine interferon-gamma, which in the same cultures has been shown to downregulate PGE2 and to upregulate NO synthesis. Although the present in vitro observations cannot be directly extrapolated to the in vivo situation, they may provide a novel clue for understanding the specific role of TGF-beta1 and IL-10 in several neurological diseases such as multiple sclerosis, in which their cerebral level was found to be elevated.