Estudi dels efectes del liti sobre el metabolisme dels fosfolípids i l'alliberació de senyals químics en cultius d'astròcits

Descripció del recurs: el 26 de març de 2010El desenvolupament de noves teràpies per als trastorns bipolars, caracteritzats per l'alternança d'episodis de depressió i d'eufòria exacerbada, es veu dificultat pel desconeixement de l'etiologia de la malaltia i del mecanisme d'acció dels fàrmacs que estabilitzen l'estat d'ànim del pacient, com el valproat, la carbamazepina, la lamotrigina, i el liti. En aquest sentit, una possibilitat que ha estat poc explorada és que les cèl·lules diana d'aquests fàrmacs siguin els astròcits, un tipus de glia que darrerament està emergent com a element actiu en els processos de transmissió sinàptica, fet que ens ha portat a estudiar els efectes del liti en cultius d'astròcits. Hem caracteritzat l'increment que produeix el liti en la velocitat de síntesi de fosfatidilcolina (PC) en astròcits, que ja apareix amb el tractament agut amb liti, però és especialment significatiu a partir de les 24 hores de tractament. El liti potencia la ruta de Kennedy per a la síntesi de PC, un efecte que es veu parcialment revertit per l'addició d'inositol, indicant que la inhibició de la IMPasa per part del liti hi estaria implicada. Els efectes del liti no es limiten a la PC sinó que es produeix una alteració general del metabolisme lipídic dels astròcits. El liti inhibeix la síntesi de fosfatidilinositol i de fosfatidiletanolamina, un efecte que es dóna de manera similar amb valproat o carbamazepina que, de manera oposada al liti, també provoquen una forta inhibició en la síntesi de PC. Paral·lelament, el tractament crònic amb liti sembla reduïr la síntesi de novo d'àcids grassos i de colesterol. Globalment, els efectes observats ens fan pensar que en incrementar la síntesi de PC el liti podria produir una reducció en els nivells de diacilglicerol cel·lulars, fet que podria estar implicat amb el mecanisme terapèutic d'aquest ió. Si la diana terapèutica del liti es troba en els astròcits, és probable que actuï a nivell de les molècules que aquests alliberen per tal de modular la transmissió sinàptica. En aquest sentit hem comprovat que el tractament amb liti, a dosis lleugerament superiors a les terapèutiques, incrementa l'alliberació de TNF-, òxid nítric, i prostaglandina E2 (PGE2) en cultius d'astròcits estimulats amb lipopolisacàrid bacterià (LPS). Sorprenentment en emprar una dosi de liti inferior, dins del rang terapèutic del fàrmac, s'observa una reducció en l'alliberació de PGE2, un efecte reproduït pel valproat, la carbamazepina i la lamotrigina. L'efecte dual del liti en funció de la concentració aplicada, es deu a una reducció en la producció d'àcid araquidònic en resposta a senyals de Ca2+ citosòlic, paral·lela a un increment en la inducció de la ciclooxigenasa-2 (COX-2) en astròcits estimulats amb LPS. Aquest fet podria estar relacionat amb l'estreta finestra terapèutica d'aquest fàmac, i contribueix a relacionar els astròcits amb el mecanisme d'acció dels estabilitzadors de l'estat d'ànim.Bipolar disorder is a common disease characterized by an alternating pattern of depression and mania episodes. Bipolar patients are treated with the mood stabilizing drugs, like lithium, valproate and carbamazepine, however the action mechanism of these drugs remains unknown. As most of the studies on this issue have been focused in neurons, we decided to analyze the effects of lithium in cultured astrocytes, a type of glial cells that are emerging as active elements in the regulation of synaptic transmission. We have characterized the increase on phosphatidylcholine (PC) synthesis induced by lithium in cultured astrocytes, an effect that appears with the acute treatment and is enhanced after 24 hours of treatment with lithium. Lithium potentiates the Kennedy pathway for the synthesis of PC, in an IMPase (inositol monophosphatase) inhibition dependent manner. Lithium effects are not limited to PC but it produces a broad alteration on astrocytic lipid metabolism. Lithium inhibits phosphatidylinositol and phosfatidylethanolamine synthesis, an effect also induced by the treatment with valproate or carbamazepine, which don't stimulate PC synthesis as lithium but inhibit it. On the other hand, chronic lithium treatment reduces fatty acids and cholesterol "de novo" synthesis. Overall, we hypothesized that lithium, increasing PC synthesis, induces a reduction on cellular diacylglycerol levels, which in turn could be related with the therapeutic mechanism of this ion

Orientación agrícola en la escuela primaria

La agricultura nos rodea, la industria agrícola es la principal industria nacional; sus productos forman la base de nuestra alimentación; el bienestar y la riqueza del país dependen casi exclusivamente de la explotación del suelo, y sin embargo, se observa con caracteres alarmantes el fenómeno de ausencia de tradición agrícola. La falta de tradición se explicaría por una falla de la psicología social y hasta, si se quiere dentro de la población nativa, como consecuencia de prejuicios heredados.Departamento de Ciencias de la Educació

Lipid droplet growth: regulation of a dynamic organelle

Intracellular lipid droplets (LDs) are remarkably dynamic and complex organelles that enact regulated storage and release of lipids to fulfil their fundamental roles in energy metabolism, membrane synthesis and provision of lipid-derived signaling molecules. Although small LDs are observed in all types of eukaryotic cells, it is adipocytes that present the widest range of sizes up to the massive unilocular droplet of a white adipocyte. Our knowledge of the proteins and associated processes that control LD dynamics is improving. The dynamic expression of LD-associated proteins is vital for controlling LD biology and is most apparent during adipocyte differentiation. Recent findings on the molecular mechanisms of lipid droplet enlargement reveal the importance of distinct functional groups of proteins and phospholipids

Amyloid-β reduces the expression of neuronal FAIM-L, thereby shifting the inflammatory response mediated by TNFα from neuronal protection to death

The brains of patients with Alzheimer's disease (AD) present elevated levels of tumor necrosis factor-α (TNFα), a cytokine that has a dual function in neuronal cells. On one hand, TNFα can activate neuronal apoptosis, and on the other hand, it can protect these cells against amyloid-β (Aβ) toxicity. Given the dual behavior of this molecule, there is some controversy regarding its contribution to the pathogenesis of AD. Here we examined the relevance of the long form of Fas apoptotic inhibitory molecule (FAIM) protein, FAIM-L, in regulating the dual function of TNFα. We detected that FAIM-L was reduced in the hippocampi of patients with AD. We also observed that the entorhinal and hippocampal cortex of a mouse model of AD (PS1M146LxAPP751sl) showed a reduction in this protein before the onset of neurodegeneration. Notably, cultured neurons treated with the cortical soluble fractions of these animals showed a decrease in endogenous FAIM-L, an effect that is mimicked by the treatment with Aβ-derived diffusible ligands (ADDLs). The reduction in the expression of FAIM-L is associated with the progression of the neurodegeneration by changing the inflammatory response mediated by TNFα in neurons. In this sense, we also demonstrate that the protection afforded by TNFα against Aβ toxicity ceases when endogenous FAIM-L is reduced by short hairpin RNA (shRNA) or by treatment with ADDLs. All together, these results support the notion that levels of FAIM-L contribute to determine the protective or deleterious effect of TNFα in neuronal cell

Epigenetic Regulation of B Lymphocyte Differentiation, Transdifferentiation, and Reprogramming

B cell development is a multistep process that is tightly regulated at the transcriptional level. In recent years, investigators have shed light on the transcription factor networks involved in all the differentiation steps comprising B lymphopoiesis. The interplay between transcription factors and the epigenetic machinery involved in establishing the correct genomic landscape characteristic of each cellular state is beginning to be dissected. The participation of "epigenetic regulator-transcription factor" complexes is also crucial for directing cells during reprogramming into pluripotency or lineage conversion. In this context, greater knowledge of epigenetic regulation during B cell development, transdifferentiation, and reprogramming will enable us to understand better how epigenetics can control cell lineage commitment and identity. Herein, we review the current knowledge about the epigenetic events that contribute to B cell development and reprogramming

Lipid droplet biogenesis induced by stress involves triacylglycerol synthesis that depends on group VIA phospholipase A2

This work investigates the metabolic origin of triacylglycerol (TAG) formed during lipid droplet (LD) biogenesis induced by stress. Cytotoxic inhibitors of fatty acid synthase induced TAG synthesis and LD biogenesis in CHO-K1 cells, in the absence of external sources of fatty acids. TAG synthesis was required for LD biogenesis and was sensitive to inhibition and down-regulation of the expression of group VIA phospholipase A2 (iPLA2-VIA). Induction of stress with acidic pH, C2-ceramide, tunicamycin, or deprivation of glucose also stimulated TAG synthesis and LD formation in a manner dependent on iPLA2-VIA. Overexpression of the enzyme enhanced TAG synthesis from endogenous fatty acids and LD occurrence. During stress, LD biogenesis but not TAG synthesis required phosphorylation and activation of group IVA PLA2 (cPLA2α). The results demonstrate that iPLA2-VIA provides fatty acids for TAG synthesis while cPLA2α allows LD biogenesis. LD biogenesis during stress may be a survival strategy, recycling structural phospholipids into energy-generating substrates

Nurr1 protein is required for N-Methyl-d-aspartic Acid (NMDA) receptor-mediated neuronal survival

NMDA receptor (NMDAR) stimulation promotes neuronal survival during brain development. Cerebellar granule cells (CGCs) need NMDAR stimulation to survive and develop. These neurons differentiate and mature during its migration from the external granular layer to the internal granular layer, and lack of excitatory inputs triggers their apoptotic death. It is possible to mimic this process in vitro by culturing CGCs in low KCl concentrations (5 mm) in the presence or absence of NMDA. Using this experimental approach, we have obtained whole genome expression profiles after 3 and 8 h of NMDA addition to identify genes involved in NMDA-mediated survival of CGCs. One of the identified genes was Nurr1, a member of the orphan nuclear receptor subfamily Nr4a. Our results report a direct regulation of Nurr1 by CREB after NMDAR stimulation. ChIP assay confirmed CREB binding to Nurr1 promoter, whereas CREB shRNA blocked NMDA-mediated increase in Nurr1 expression. Moreover, we show that Nurr1 is important for NMDAR survival effect. We show that Nurr1 binds to Bdnf promoter IV and that silencing Nurr1 by shRNA leads to a decrease in brain-derived neurotrophic factor (BDNF) protein levels and a reduction of NMDA neuroprotective effect. Also, we report that Nurr1 and BDNF show a similar expression pattern during postnatal cerebellar development. Thus, we conclude that Nurr1 is a downstream target of CREB and that it is responsible for the NMDA-mediated increase in BDNF, which is necessary for the NMDA-mediated prosurvival effect on neurons

Group IVA phospholipase A2 is necessary for the biogenesis of lipid droplets

Lipid droplets (LD) are organelles present in all cell types, consisting of a hydrophobic core of triacylglycerols and cholesteryl esters, surrounded by a monolayer of phospholipids and cholesterol. This work shows that LD biogenesis induced by serum, by long-chain fatty acids, or the combination of both in CHO-K1 cells was prevented by phospholipase A2 inhibitors with a pharmacological profile consistent with the implication of group IVA cytosolic phospholipase A2 (cPLA2α). Knocking down cPLA2α expression with short interfering RNA was similar to pharmacological inhibition in terms of enzyme activity and LD biogenesis. A Chinese hamster ovary cell clone stably expressing an enhanced green fluorescent protein-cPLA2α fusion protein (EGFP-cPLA2) displayed higher LD occurrence under basal conditions and upon LD induction. Induction of LD took place with concurrent phosphorylation of cPLA2α at Ser505. Transfection of a S505A mutant cPLA2α showed that phosphorylation at Ser505 is key for enzyme activity and LD formation. cPLA2α contribution to LD biogenesis was not because of the generation of arachidonic acid, nor was it related to neutral lipid synthesis. cPLA2α inhibition in cells induced to form LD resulted in the appearance of tubulo-vesicular profiles of the smooth endoplasmic reticulum, compatible with a role of cPLA2α in the formation of nascent LD from the endoplasmic reticulum

TNFα sensitizes neuroblastoma cells to FasL-, cisplatin- and etoposide-induced cell death by NF-κB-mediated expression of Fas

Background Patients with high-risk neuroblastoma (NBL) tumors have a high mortality rate. Consequently, there is an urgent need for the development of new treatments for this condition. Targeting death receptor signaling has been proposed as an alternative to standard chemo- and radio-therapies in various tumors. In NBL, this therapeutic strategy has been largely disregarded, possibly because ~50-70% of all human NBLs are characterized by caspase-8 silencing. However, the expression of caspase-8 is detected in a significant group of NBL patients, and they could therefore benefit from treatments that induce cell death through death receptor activation. Given that cytokines, such as TNFα, are able to upregulate Fas expression, we sought to address the therapeutic relevance of co-treatment with TNFα and FasL in NBL. Methods For the purpose of the study we used a set of eight NBL cell lines. Here we explore the cell death induced by TNFα, FasL, cisplatin, and etoposide, or a combination thereof by Hoechst staining and calcein viability assay. Further assessment of the signaling pathways involved was performed by caspase activity assays and Western blot experiments. Characterization of Fas expression levels was achieved by qRT-PCR, cell surface biotinylation assays, and cytometry. Results We have found that TNFα is able to increase FasL-induced cell death by a mechanism that involves the NF-κB-mediated induction of the Fas receptor. Moreover, TNFα sensitized NBL cells to DNA-damaging agents (i.e. cisplatin and etoposide) that induce the expression of FasL. Priming to FasL-, cisplatin-, and etoposide-induced cell death could only be achieved in NBLs that display TNFα-induced upregulation of Fas. Further analysis denotes that the high degree of heterogeneity between NBLs is also manifested in Fas expression and modulation thereof by TNFα. Conclusions In summary, our findings reveal that TNFα sensitizes NBL cells to FasL-induced cell death by NF-κB-mediated upregulation of Fas and unveil a new mechanism through which TNFα enhances the efficacy of currently used NBL treatments, cisplatin and etoposide

X-linked Inhibitor of Apoptosis Protein negatively regulates neuronal differentiation through interaction with cRAF and Trk

Altres ajuts: CIBERNED CB06/05/0042 i CB06/05/1104, RENEVAS RD06/0026/1009 i Juan de la CiervaX-linked Inhibitor of apoptosis protein (XIAP) has been classically identified as a cell death regulator. Here, we demonstrate a novel function of XIAP as a regulator of neurite outgrowth in neuronal cells. In PC12 cells, XIAP overexpression prevents NGF-induced neuronal differentiation, whereas NGF treatment induces a reduction of endogenous XIAP levels concomitant with the induction of neuronal differentiation. Accordingly, downregulation of endogenous XIAP protein levels strongly increases neurite outgrowth in PC12 cells as well as axonal and dendritic length in primary cortical neurons. The effects of XIAP are mediated by the mitogen-activated protein kinase (MEK)/extracellular signal-regulated kinases (ERKs) pathway since blocking this pathway completely prevents the neuritogenesis mediated by XIAP downregulation. In addition, we found that XIAP binds to cRaf and Trk receptors. Our results demonstrate that XIAP plays a new role as a negative regulator of neurotrophin-induced neurite outgrowth and neuronal differentiation in developing neurons