Lipopolysaccharide-induced murine embryonic resorption involves nitric oxide-mediated inhibition of the NAD+-dependent 15-hydroxyprostaglandin dehydrogenase.

The initial inactivation of prostaglandins (PGs) is mediated by 15-hydroxyprostaglandin dehydrogenase (15-PGDH). PGs are potent mediators of several biological processes, including inflammation and reproduction. In uterus, PGs play a key role in infection-induced pregnancy loss, in which concentration of this mediator increased. This process is accompanied with the induction of nitric oxide synthase expression and a marked increase in uterine levels of nitric oxide. There is no information concerning nitric oxide contribution to potential changes in PG catabolism, but experimental evidence suggests that nitric oxide modulates PG pathways. The specific objectives of the study were to evaluate the protein expression of HPGD (15-PGDH) and to characterize the nitric oxide-dependent regulation of this enzyme in a model of lipopolysaccharide (LPS)-induced embryonic resorption. Results show that LPS decreased HPGD protein expression and augmented PGE synthase activity; therefore, PGE2 levels increased in uterus in this inflammatory condition. Just as LPS, the treatment with a nitric oxide donor diminished HPGD protein expression in uterine tissue. In contrast, the inhibition of nitric oxide synthesis both in control and in LPS-treated mice increased 15-PGDH levels. Also, we have found that this enzyme and PGE2 levels are not modulated by peroxynitrite, an oxidant agent derived from nitric oxide. This study suggests that LPS and nitric oxide promote a decrease in the ability of the uterus for PG catabolism during bacterially triggered pregnancy loss in mice.

17beta-oestradiol and progesterone regulate anandamide synthesis in the rat uterus.

Anandamide is an endocannabinoid known to participate in reproductive processes. This study observed that 17beta-oestradiol and progesterone modulated the production of anandamide and its metabolizing enzymes in the rat uterus. Anandamide production was highest at the oestrous stage and 17beta-oestradiol and progesterone stimulated its synthesis in ovariectomized rats. During early pregnancy, anandamide production remained constant on days 1-5 of gestation and diminished towards day 6. On day 6, implantation sites showed lower synthesis compared with interimplantation sites. In the delayed implantation model, 17beta-oestradiol inhibited anandamide synthesis compared with progesterone. During pseudopregnancy, anandamide production did not decrease towards day 6 as occurred during normal gestation. The administration of 17beta-oestradiol augmented anandamide production in rats on day 5 of pseudopregnancy; the treatment with mifepristone did not produce any change in anandamide synthesis. Anandamide-metabolizing enzymes were regulated by progesterone and 17beta-oestradiol. The effect of ovarian hormones on the synthesis of anandamide depends on different physiological conditions, oestrous cycle and early pregnancy, and on the presence of the activated blastocyst. Thus, ovarian hormones, as signals that emanate from the mother, operate in conjunction with the blastocyst intrinsic programme, regulating the synthesis of anandamide in a specific manner during crucial reproductive events that may compromise pregnancy outcome.

Neddylation inhibition impairs spine development, destabilizes synapses and deteriorates cognition.

Neddylation is a ubiquitylation-like pathway that controls cell cycle and proliferation by covalently conjugating Nedd8 to specific targets. However, its role in neurons, nonreplicating postmitotic cells, remains unexplored. Here we report that Nedd8 conjugation increased during postnatal brain development and is active in mature synapses, where many proteins are neddylated. We show that neddylation controls spine development during neuronal maturation and spine stability in mature neurons. We found that neddylated PSD-95 was present in spines and that neddylation on Lys202 of PSD-95 is required for the proactive role of the scaffolding protein in spine maturation and synaptic transmission. Finally, we developed Nae1(CamKIIα-CreERT2) mice, in which neddylation is conditionally ablated in adult excitatory forebrain neurons. These mice showed synaptic loss, impaired neurotransmission and severe cognitive deficits. In summary, our results establish neddylation as an active post-translational modification in the synapse regulating the maturation, stability and function of dendritic spines.

Behavioral phenotyping of Nestin-Cre mice: implications for genetic mouse models of psychiatric disorders.

Genetic mouse models based on the Cre-loxP system have been extensively used to explore the influence of specific gene deletions on different aspects of behavioral neurobiology. However, the interpretation of the effects attributed to the gene deletion might be obscured by potential side effects secondary to the Cre recombinase transgene insertion or Cre activity, usually neither controlled nor reported. Here, we performed a comprehensive behavioral analysis of endophenotypes of neuropsychiatric disorders in the extensively used Nestin(Cre) mouse line, commonly employed to restrict genetic modifications to the CNS. We observed no alterations in locomotion, general exploratory activity, learning and memory, sociability, startle response and sensorimotor gating. Although the overall response to stimuli triggering anxiety-like behaviors remained unaltered in Nestin(Cre) mice, a strong impairment in the acquisition of both contextual- and cued-conditioned fear was observed. These results underline the importance of adequately controlling the behavioral performance of the employed Cre-lines per-se in pre-clinical neurobehavioral research.

MicroRNA-9 controls dendritic development by targeting REST.

MicroRNAs (miRNAs) are conserved noncoding RNAs that function as posttranscriptional regulators of gene expression. miR-9 is one of the most abundant miRNAs in the brain. Although the function of miR-9 has been well characterized in neural progenitors, its role in dendritic and synaptic development remains largely unknown. In order to target miR-9 in vivo, we developed a transgenic miRNA sponge mouse line allowing conditional inactivation of the miR-9 family in a spatio-temporal-controlled manner. Using this novel approach, we found that miR-9 controls dendritic growth and synaptic transmission in vivo. Furthermore, we demonstrate that miR-9-mediated downregulation of the transcriptional repressor REST is essential for proper dendritic growth.

Progesterone is essential for protecting against LPS-induced pregnancy loss. LIF as a potential mediator of the anti-inflammatory effect of progesterone.

Lipopolysaccharide (LPS) administration to mice on day 7 of gestation led to 100% embryonic resorption after 24 h. In this model, nitric oxide is fundamental for the resorption process. Progesterone may be responsible, at least in part, for a Th2 switch in the feto-maternal interface, inducing active immune tolerance against fetal antigens. Th2 cells promote the development of T cells, producing leukemia inhibitory factor (LIF), which seems to be important due to its immunomodulatory action during early pregnancy. Our aim was to evaluate the involvement of progesterone in the mechanism of LPS-induced embryonic resorption, and whether LIF can mediate hormonal action. Using in vivo and in vitro models, we provide evidence that circulating progesterone is an important component of the process by which infection causes embryonic resorption in mice. Also, LIF seems to be a mediator of the progesterone effect under inflammatory conditions. We found that serum progesterone fell to very low levels after 24 h of LPS exposure. Moreover, progesterone supplementation prevented embryonic resorption and LPS-induced increase of uterine nitric oxide levels in vivo. Results show that LPS diminished the expression of the nuclear progesterone receptor in the uterus after 6 and 12 h of treatment. We investigated the expression of LIF in uterine tissue from pregnant mice and found that progesterone up-regulates LIF mRNA expression in vitro. We observed that LIF was able to modulate the levels of nitric oxide induced by LPS in vitro, suggesting that it could be a potential mediator of the inflammatory action of progesterone. Our observations support the view that progesterone plays a critical role in a successful pregnancy as an anti-inflammatory agent, and that it could have possible therapeutic applications in the prevention of early reproductive failure associated with inflammatory disorders.

Progesterone reverts LPS-reduced FAAH activity in murine peripheral blood mononuclear cells by a receptor-mediated fashion.

Increased anandamide concentrations are associated with pregnancy failure. Anandamide levels are regulated by the fatty acid amide hydrolase (FAAH). The aim of the study was to investigate the role of progesterone (P) on FAAH modulation in murine peripheral blood mononuclear cells (PBMC) under septic conditions. We observed that in vivo administration of LPS to non-pregnant (NP) mice decreased FAAH activity of PBMC while in pregnant mice no changes in FAAH activity were observed. NP animals administered with P had a similar response to LPS as the pregnant animals. Also, NP mice injected with P antagonist and P showed that the effect of P on LPS-reduced FAAH activity was impaired. Furthermore, LPS produced a decrease in the ratio of PR-B/PR-A in NP animals. Our results showed that, in our model the endotoxin decreased PBMC's FAAH activity and this condition was reverted by P in a receptor-mediated fashion.

Opposite effects of methanandamide on lipopolysaccharide-induced prostaglandin E2 and F2α synthesis in uterine explants from pregnant mice.

Prostaglandins (PG) are effective abortifacients and are important mediators of lipopolisaccharide (LPS)-induced embryonic resorption (ER). Besides, anandamide (AEA) has been described as one of the major endocannabinoids present in the uterus suggesting that it might play a role in reproduction. It has been reported that high levels of AEA are associated with pregnancy failure and that LPS increases AEA production. Also, it has been observed that AEA modulates PG production in different tissues. In this sense, we studied whether LPS-induced PG production is modulated by AEA and we also assessed the effect of this endocannabinoid on PG metabolism in an in vitro model. Uterine explants from BALB/c implantation sites were cultured in the presence of LPS plus cannabinoid receptor (CB) specific antagonists and PG production was assessed. Then, we studied the effect of exogenous AEA on different steps of PG metabolic pathway. We showed that AEA is involved in LPS-induced PG biosynthesis. Also, we observed that AEA exerts opposite effects on PGE(2) and PGF(2α) biosynthesis, by inhibiting PGE(2) production and increasing PGF(2α) levels. We suggest that AEA could be involved in the mechanisms implicated in LPS-induced ER. A better understanding of how AEA could be affecting ER could help developing specific interventions to prevent this pathology.