Fernando Picatoste

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.

Opposed effects of lithium on the MEK-ERK pathway in neural cells: inhibition in astrocytes and stimulation in neurons by GSK3 independent mechanisms.

Lithium is widely used in the treatment of bipolar disorder, but despite its proven therapeutic efficacy, the molecular mechanisms of action are not fully understood. The present study was undertaken to explore lithium effects of the MEK/ERK cascade of protein kinases in astrocytes and neurons. In asynchronously proliferating rat cortical astrocytes, lithium decreased time‐ and dose‐dependently the phosphorylation of MEK and ERK, with 1 mm concentrations achieving 60 and 50% inhibition of ERK and MEK, respectively, after a 7‐day exposure. Lithium also inhibited [3H]thymidine incorporation into DNA and induced a G2/M cell cycle arrest. In serum‐deprived, quiescent astrocytes, pre‐exposure to lithium resulted in the inhibition of cell cycle re‐entry as stimulated by the mitogen endothelin‐1: under this experimental setting, lithium did not affect the rapid, peak phosphorylation of MEK taking place after 3–5 min, but was effective in inhibiting the long‐term, sustained phosphorylation of MEK. Lithium inhibition of the astrocyte MEK/ERK pathway was independent of inositol depletion. Further, compound SB216763 inhibited Tau phosphorylation at Ser396 and stabilized cytosolic β‐catenin, consistent with the inhibition of glycogen synthase kinase‐3β (GSK‐3β), but failed to reproduce lithium effects on MEK and ERK phosphorylation and cell cycle arrest. In cerebellar granule neurons, millimolar concentrations of lithium enhanced MEK and ERK phosphorylation in a concentration‐dependent manner, again through an inositol and GSK‐3β independent mechanism. These opposing effects in astrocytes and neurons make lithium treatment a promising strategy to favour neural repair and reduce reactive gliosis after traumatic injury.

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.

Effects of Oxidative Stress on Phospholipid Signaling in Rat Cultured Astrocytes and Brain Slices

Although reactive oxygen species (ROS) are conventionally viewed as toxic by‐products of cellular metabolism, a growing body of evidence suggests that they may act as signaling molecules. We have studied the effects of hydrogen peroxide (H2O2)‐induced oxidative stress on phospholipid signaling in cultured rat cortical astrocytes. H2O2 stimulated the formation of phosphatidic acid and the accumulation of phosphatidylbutanol, a product of the phospholipase D (PLD)‐catalyzed transphosphatidylation reaction. The effect of exogenous H2O2 on the PLD response was mimicked by menadione‐induced production of endogenous H2O2. Oxidative stress also elicited inositol phosphate accumulation resulting from phosphoinositide phospholipase C (PLC) activation. The PLD response to H2O2 was totally suppressed by chelation of both extracellular and cytosolic Ca2+ with EGTA and BAPTA/AM, respectively. Furthermore, H2O2‐induced PLD stimulation was completely abolished by the protein kinase C (PKC) inhibitors bisindolylmaleimide and chelerythrine and by PKC down‐regulation. Activation of PLD by H2O2 was also inhibited by the protein‐tyrosine kinase inhibitor genistein. Finally, H2O2 also stimulated both PLC and PLD in rat brain cortical slices. These results show for the first time that oxidative stress elicits phospholipid breakdown by both PLC and PLD in rat cultured astrocytes and brain slices.

JNK and Ceramide Kinase Govern the Biogenesis of Lipid Droplets through Activation of Group IVA Phospholipase A2

The biogenesis of lipid droplets (LD) induced by serum depends on group IVA phospholipase A2 (cPLA2α). This work dissects the pathway leading to cPLA2α activation and LD biogenesis. Both processes were Ca2+-independent, as they took place after pharmacological blockade of Ca2+ transients elicited by serum or chelation with 1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid tetrakis(acetoxymethyl ester). The single mutation D43N in cPLA2α, which abrogates its Ca2+ binding capacity and translocation to membranes, did not affect enzyme activation and formation of LD. In contrast, the mutation S505A did not affect membrane relocation of the enzyme in response to Ca2+ but prevented its phosphorylation, activation, and the appearance of LD. Expression of specific activators of different mitogen-activated protein kinases showed that phosphorylation of cPLA2α at Ser-505 is due to JNK. This was confirmed by pharmacological inhibition and expression of a dominant-negative form of the upstream activator MEKK1. LD biogenesis was accompanied by increased synthesis of ceramide 1-phosphate. Overexpression of its synthesizing enzyme ceramide kinase increased phosphorylation of cPLA2α at Ser-505 and formation of LD, and its down-regulation blocked the phosphorylation of cPLA2α and LD biogenesis. These results demonstrate that LD biogenesis induced by serum is regulated by JNK and ceramide kinase.

Cell Survival during Complete Nutrient Deprivation Depends on Lipid Droplet-fueled β-Oxidation of Fatty Acids

Background: Cellular stress leading to cell death induces the formation of lipid droplets. Results: Nutrient deprivation induces LD biogenesis and mobilization, fueling fatty acid oxidation to sustain cell viability. Conclusion: β-Oxidation requires biogenesis and mobilization of LD. Significance: The role of LD in cell survival and β-oxidation might provide new potential targets for antitumor therapy. Cells exposed to stress of different origins synthesize triacylglycerols and generate lipid droplets (LD), but the physiological relevance of this response is uncertain. Using complete nutrient deprivation of cells in culture as a simple model of stress, we have addressed whether LD biogenesis has a protective role in cells committed to die. Complete nutrient deprivation induced the biogenesis of LD in human LN18 glioblastoma and HeLa cells and also in CHO and rat primary astrocytes. In all cell types, death was associated with LD depletion and was accelerated by blocking LD biogenesis after pharmacological inhibition of Group IVA phospholipase A2 (cPLA2α) or down-regulation of ceramide kinase. Nutrient deprivation also induced β-oxidation of fatty acids that was sensitive to cPLA2α inhibition, and cell survival in these conditions became strictly dependent on fatty acid catabolism. These results show that, during nutrient deprivation, cell viability is sustained by β-oxidation of fatty acids that requires biogenesis and mobilization of LD.

Group I metabotropic glutamate receptors mediate phospholipase D stimulation in rat cultured astrocytes.

Abstract: We have studied the activation of phospholipase D (PLD) by glutamate in rat cultured astrocytes by measuring the PLD‐catalyzed formation of [32P]phosphatidylbutanol in [32P]Pi‐prelabeled cells, stimulated in the presence of butanol. Glutamate elicited the activation of PLD in cortical astrocytes but not in cortical neurons, whereas similar glutamate activation of phosphoinositide phospholipase C was found in both astrocytes and neurons. The extent of PLD stimulation by glutamate was similar in astrocytes from brain cortex and hippocampus, but no effect was found in cerebellar astrocytes. In cortical astrocytes, the glutamate response was insensitive to antagonists of ionotropic glutamate receptors and was reproduced by agonists of metabotropic glutamate receptors (mGluRs) with a rank order of agonist potency similar to that reported for group I mGluR‐mediated phosphoinositide phospholipase activation [quisqualate > (S)‐3,5‐dihydroxyphenylglycine > (1S,3R)‐1‐aminocyclopentane‐1,3‐dicarboxylic acid]. The response to (1S,3R)‐1‐aminocyclopentane‐1,3‐dicarboxylic acid was inhibited by the mGluR antagonist (S‐α‐methyl‐4‐carboxyphenylglycine and, less potently, by 1‐aminoindan‐1,5‐dicarboxylic acid and 4‐carboxyphenylglycine, two antagonists of group I mGluRs that display higher potency on mGluR1 than on mGluR5. The mGluR5‐selective agonist (RS)‐2‐chloro‐5‐hydroxyphenylglycine also activated PLD in astrocytes. These findings indicate the involvement of group I mGluRs, most likely mGluR5, in the glutamate activation of PLD in cultured rat cortical astrocytes.

Histamine Stimulation of Cyclic AMP Accumulation in Astrocyte-Enriched and Neuronal Primary Cultures from Rat Brain

Abstract: Histamine stimulates cyclic AMP accumulation in astrocyte‐enriched and neuronal primary cultures from rat brain in the presence of the phosphodiesterase inhibitor isobutylmethylxanthine. The response in the astrocyte cultures (Emax= 304 ± 44% over basal, EC50= 43 ± 5 μM) was much higher than in neuronal cultures (Emax= 24 ±2%, EC50= 14 ± 7 μM). The histamine effect in astrocytes was competitively inhibited by the H2 antagonists cimetidine (Ki=1.1 ± 0.2 μM) and ranitidine (Ki‐ 46 ± 10 nM) but was insensitive to the H1 antagonist mepyramine (1 μM). The two selective H2 agonists impromidine and dimaprit behaved as partial agonists and showed relative potencies (139 and 0.5, respectively) consistent with an interaction with H2 receptors. The more selective H1 agonist 2‐thiazolylethylamine (0.01–1 mM) did not potentiate the response to impromidine (10 μM). Thus, in contrast to what is generally observed in intact cell preparations from brain, the histamine‐induced cyclic AMP accumulation in astroglial cells is mediated solely by H2 receptors. The small effect shown in neuronal cultures also appears to be mediated by H2 receptors.

The presence of two cellular pools of rat brain histamine

HISTAMINE (HA) is now believed to be a neurotransmitter in the central nervous system of mammals (for a review see SNYDER & TAYLOR, 1972; SCHWARTZ, 1975). In adult rat brain, although HA has been found to be concentrated in fractions enriched in synaptosomes (CARLINI & GREEN, 1963; MICHAELSON & COFFMAN, 1967; KATAOKA & DE ROBERTIS, 1967; KUHAR et al., 1971b; SNYDER et al., 1974), around 30% of the amine has been reported to be present in the crude nuclear fraction (SCHWARTZ, 1975) in contrast to the low amounts of other neurotransmitters found in that fraction (DE ROBERTIS et al., 1962; KUHAR et al., 1971a; COYLE & KUHAR, 1974). If brain HA is only considered a neurotransmitter, its ontogenic development in rat brain would seem anomalous since HA levels are found to be 5-6 times higher in the 5-day-old rat brain than in the adult brain (PEARCE & SCHANBERG, 1969; RONNBERG & SCHWARTZ, ~ ~ ~ ~ ; T I L L E M E N T et al., 1971). This pattern differs markedly from that described for other neurotransmitters (ABDEL-LATIF et al., 1970; COYLE & AXELROD, 1971; TISARI & RAUNU, 1975) whose development with age parallels synaptogenesis (AGHAJANIAN & BLOOM, 1967). In contrast, the development of L-histidine decarboxylase activity (HD), the HA synthesizing enzyme, follows a pattern (SCHWARTZ et al., 1971) similar to that described for other neurotransmitter synthesizing enzymes (SCHMIDT & SANDERS-BUSH, 1971 ; COYLE & AXELROD, 1972). During the first week of postnatal life, rat brain H D activity is very low increasing later to reach adult values during the fourth week. The discrepancies between the developmental patterns of HA and HD activity suggested the presence of a pool of brain HA other than the synaptosomal one. In the neonatal rat brain, HA has been found to be concentrated in the crude nuclear fraction (YOUNG et al., 1971). These findings could be related to the recently reported presence of mast cells, known to be rich in HA and poor in H D activity (SCHAYER, 1966), in rat brain (IBRAHIM, 1974; KRUGER, 1974; DROPP, 1976). In the present work we compare the subcellular distribution of HA in neonatal and adult rat brain with that of peritoneal mast cells homogenized together with brain tissue.

Histamine H1-receptors mediate phosphoinositide hydrolysis in astrocyte-enriched primary cultures

Astrocyte-enriched primary cultures of newborn rat brain hemispheres, prelabeled with [3H]inositol, accumulated [3H]inositol phosphate but not [3H]inositol bis- and tris-phosphate, after exposure to histamine for 60 min in the presence of 10 mM LiCl. The response to histamine was not a function of contaminating meningeal fibroblasts since no accumulation of [3H]inositol phosphate was elicited by histamine in meningeal cultures. The stimulation of phosphoinositide hydrolysis by histamine in astrocytes was dose-dependent (EC50 = 1.7 microM, maximal effect = 345% over basal levels) and was mimicked by several H1-receptor agonists. The use of selective receptor antagonists confirmed that the histamine response was the result of activation of H1-receptors. The histamine-induced [3H]inositol phosphate accumulation was completely abolished by omission of Ca2+ from the incubation medium. Astrocyte membranes specifically bound the radiolabeled H1-antagonist, [3H]mepyramine with an affinity (Kd = 5.9 nM) and a density of binding sites (Bmax = 113 fmol/mg protein) similar to rat brain. These results demonstrate the presence of functional histamine H1-receptors in rat brain astrocytes and suggest a role for histamine as a neuromodulator of astrocyte function.