Calcium-independent Phospholipase Research Articles

Deficiency of peroxiredoxin 6 or inhibition of its phospholipase A2 activity impair the in vitro sperm fertilizing competence in mice

Prdx6−/− male mice are subfertile, and the deficiency or inactivation of Peroxiredoxins (PRDXs) is associated with human male infertility. We elucidate the impact of the lack of PRDX6 or inhibition of its calcium-independent phospholipase A2 (Ca2+-iPLA2) activity by MJ33 on fertilization competence of mouse spermatozoa. Sperm motility, viability, fertilization and blastocyst rates were lower in Prdx6−/− spermatozoa than in C57BL/6J wild-type (WT) controls (p ≤ 0.05). MJ33 inhibited the PRDX6 Ca2+-iPLA2 activity and reduced these parameters in WT spermatozoa compared with controls (p ≤ 0.05). Levels of lipid peroxidation and of superoxide anion (O2•─) were higher in Prdx6−/− than in WT spermatozoa (p ≤ 0.05). MJ33 increased the levels of lipid peroxidation and mitochondrial O2•─ production in treated versus non-treated WT spermatozoa. Acrosome reaction, binding to zona pellucida and fusion with the oolemma were lower in Prdx6−/− capacitated spermatozoa than WT capacitated controls and lower in WT spermatozoa treated with the PRDX6 inhibitor. In conclusion, the inhibition of the PRDX6 Ca2+-iPLA2 activity promotes an oxidative stress affecting viability, motility, and the ability of mouse spermatozoa to fertilize oocytes. Thus, PRDX6 has a critical role in the protection of the mouse spermatozoon against oxidative stress to assure fertilizing competence.

A new PLA2G6 mutation in a family with infantile neuroaxonal dystrophy

Phospholipase A2-associated neurodegeneration (PLAN), a syndrome of Neurodegeneration with Brain Iron Accumulation (NBIA), is an autosomal recessive disorder caused by mutations in PLA2G6 gene. This gene encodes a calcium-independent group VI phospholipase A2 (iPLA-VI) critical in cell membrane homeostasis.PLAN syndrome encompasses a group of phenotypes with a different age of onset: classic infantile neuroaxonal dystrophy (INAD), atypical neuroaxonal dystrophy of childhood-onset (atypical NAD) and adult-onset PLA2G6-related dystonia-parkinsonism (PARK14).INAD is a severe progressive psychomotor disorder characterized by the presence of axonal spheroids throughout the central and peripheral nervous system. Here we report clinical, genetic and histopathological findings of an INAD consanguineous-family from Senegal. Sanger sequencing analysis revealed a new homozygous PLA2G6-mutation in the proband (c.1483C>T) and the co-segregation of the mutation in this family.Electron microscopy on skin biopsy showed degenerated axons confirming the phenotype.This study contributes to enrich the landscape of PLA2G6-associated INAD mutations and enforce the genotype-phenotype correlation.

A novel calcium-independent phospholipase A2 and its physiological roles in development and immunity of a lepidopteran insect, Spodoptera exigua

Phospholipase A2 (PLA2) catalyzes hydrolysis of ester linkage at sn-2 position of phospholipids. At least 15 groups (I-XV) of PLA2 gene superfamily are associated with various physiological processes such as digestion, secretion, immunity, and maintenance of membrane integrity. This study suggests that various insects encode putative Group VI PLA2s representing intracellular and calcium-independent PLA2s (iPLA2). These insect iPLA2s are separated into at least two subgroups: iPLA2A (Group VIA-like) and iPLA2B (non-Group VIA). Most insects encode genes of iPLA2B type, although their biological functions are currently unknown. This study predicted a novel iPLA2 from Spodoptera exigua (a lepidopteran insect) (SeiPLA2B) and analyzed its physiological functions by RNA interference (RNAi). SeiPLA2B encodes 336 amino acid sequence with a predicted size of about 36.6 kDa and an isoelectric point at pH 8.61. It possesses a lipase catalytic site, but does not have ankyrin repeats in the amino terminal region. Phylogenetic analysis indicated that SeiPLA2B was clustered with other Group VI iPLA2s, in which SeiPLA2B was closely associated with Group VIF gene while SeiPLA2A was closely related to Group VIA gene. SeiPLA2B was expressed in all developmental stages of S. exigua. In larval stage, SeiPLA2B was expressed in fat body, hemocyte, and epidermis, but not in digestive tract. SeiPLA2B RNAi significantly reduced PLA2 enzyme activities and resulted in developmental retardation and immunosuppression. Though RNAi treatment did not significantly change fatty acid composition in fat body lipids, it significantly increased lipid peroxidation. Taken together, our results suggest that SeiPLA2B plays important roles in the development and immunity of S. exigua.

The clinical characteristics and molecular genetics of infantile neuroaxonal dystrophy: The current research progress

Infantile neuroaxonal dystrophy (INAD) is a rare autosome-recessive disease characterized by progressive motor and cognitive regression. The PLA2G6 gene is its causative gene, which encodes calcium-independent phospholipase A2 enzyme (iPLA2-VIA). The diagnosis of INAD is difficult because of its clinical heterogeneity, and the rate of misdiagnosis is high. The purpose of this study is to describe the clinical characteristics, molecular genetics, treatment and prognosis of INAD to improve the acknowledgement of INAD in medical workers and to help make an early diagnosis of INAD. Key words: Neuroaxonal dystrophies/DI/TH/GE; Infant; Editorial

Change of plasmalogen content of red blood cells in myocardial hypoxia and acidosis

Background: The contribution of hypoxic conditions to the chemical composition of membranes is not completely established. Plasmalogens, containing an alkenyl group with aldehydogenic ether linkage, are significant components of membrane lipids and their level can change in oxygen deficiency.Methods: Analysis of plasmalogens in red blood cells was performed in patients (n = 17) with coronary heart disease, stable angina (functional class II–III) and coronary atherosclerosis. The control group consisted of 17 healthy volunteers. In addition, isolated blood samples of seven healthy volunteers were analysed before and after 180 min incubation at 37 °C.Fatty acid ethyl esters and diethyl acetals of fatty aldehydes, obtained during sample preparation from red blood cells, were analysed by capillary gas–liquid chromatography. Quantitative assessment of the change of the plasmalogen levels was evaluated as change of the share of fatty aldehyde diethyl acetals in the total sum of fatty aldehyde diethyl acetals and fatty acid ethyl esters.Results: In comparison with the healthy volunteers, an increase in plasmalogen content of red blood cells and a reduction of the pH of the blood plasma in the group of patients with coronary heart disease were detected. In experimental hypoxia, there was an increase in the plasmalogen content of the red blood cells and a plasma pH decrease in all samples subjected to the incubation.Conclusions: The results indicate changes in the physicochemical properties of the cell membrane in hypoxia. One of the most likely reasons of the increase of plasmalogen content in the membranes may be a more significant increase in activity of calcium-dependent phospholipases in comparison with the activity of calcium-independent plasmalogen phospholipases.

Oligomeric amyloid-beta induces MAPK-mediated activation of brain cytosolic and calcium-independent phospholipase A2 in a spatial-specific manner

Alzheimer’s disease (AD) is histopathologically characterized by the build-up of fibrillar amyloid beta (Aβ) in the form of amyloid plaques and the development of intraneuronal neurofibrillary tangles consisting of aggregated hyperphosphorylated Tau. Although amyloid fibrils were originally considered responsible for AD pathogenesis, recent convincing evidence strongly implicates soluble oligomeric Aβ as the primary neurotoxic species driving disease progression. A third largely ignored pathological hallmark, originally described by Alois Alzheimer, is the presence of “adipose inclusions”, suggestive of aberrant lipid metabolism. The molecular mechanisms underlying these “lipoid granules”, as well as their potential link to soluble and/or fibrillar Aβ remain largely unknown. Seeking to better-understand these conundrums, we took advantage of the powerful technology of multidimensional mass spectrometry-based shotgun lipidomics and an AD transgenic mouse model overexpressing mutant amyloid precursor protein (APP E693Δ-Osaka-), where AD-like pathology and neurodegeneration occur as a consequence of oligomeric Aβ accumulation in the absence of amyloid plaques. Our results revealed for the first time that APP overexpression and oligomeric Aβ accumulation lead to an additive global accumulation of nonesterified polyunsaturated fatty acids (PUFAs) independently of amyloid plaques. Furthermore, we revealed that this accumulation is mediated by an increase in phospholipase A2 (PLA2) activity, evidenced by an accumulation of sn-1 lysophosphatidylcholine and by MAPK-mediated phosphorylation/activation of group IV Ca2+-dependent cytosolic (cPLA2) and the group VI Ca2+-independent PLA2 (iPLA2) independently of PKC. We further revealed that Aβ-induced oxidative stress also disrupts lipid metabolism via reactive oxygen species-mediated phospholipid cleavage leading to increased sn-2 lysophosphatidylcholine as well as lipid peroxidation and the subsequent accumulation of 4-hydroxynonenal. Brain histological studies implicated cPLA2 activity with arachidonic acid accumulation within myelin-rich regions, and iPLA2 activity with docosahexaenoic acid accumulation within pyramidal neuron-rich regions. Taken together, our results suggest that PLA2-mediated accumulation of free PUFAs drives AD-related disruption of brain lipid metabolism.

The overall fatty acid absorption controlled by basolateral chylomicron excretion under regulation of p-JNK1

Suppression of fatty acid absorption is one goal to fight obesity. However, the responsible molecular mechanism is poorly understood. Aim of the present study was the search for the key regulator of the overall fatty acid absorption mechanism and its pharmaceutical modulation. As experimental tool we employed the polarized human intestinal tumor derived cell line CaCo2. Here we showed that influx of fatty acids is mediated by an apical heterotetrameric plasma membrane protein complex of which the calcium-independent membrane phospholipase A2 (iPLA2ß) is one constituent. The newly synthesized bile acid-phospholipid conjugate ursodeoxycholate-lysophosphatidylethanolamide (UDCA-LPE) blocked iPLA2ß, which structurally disrupted the fatty acid-uptake complex. Furthermore, the inhibition of iPLA2ß lead to reduction of cytosolic lysophosphatidylcholine (LPC) production which suppressed p-JNK1, as a central regulator of metabolism. In a concerted action low p-JNK1 levels prohibited synthesis of the members of the fatty acid uptake complex as well as of apolipoprotein B and the connected members of the basolateral vesicular chylomicron excretion machinery, thereby inhibiting cellular lipid excretion. The basolateral chylomicron release was shown to determine the overall fatty acid-absorption capacity as rate limiting step, whereas apical uptake replenishes the cellular stores, enabling continuous transcellular movement of fatty acids. In conclusion, the UDCA-LPE mediated inhibition of p-JNK1 represents a powerful tool to control intestinal absorption of fatty acids and, thus may be employed as a drug to treat obesity.

The phospholipase iPLA2γ is a major mediator releasing oxidized aliphatic chains from cardiolipin, integrating mitochondrial bioenergetics and signaling

Cardiolipin (CL) is a dimeric phospholipid with critical roles in mitochondrial bioenergetics and signaling. Recently, inhibition of the release of oxidized fatty acyl chains from CL by the calcium-independent phospholipase A2γ (iPLA2γ)-selective inhibitor (R)-BEL suggested that iPLA2γ is responsible for the hydrolysis of oxidized CL and subsequent signaling mediated by the released oxidized fatty acids. However, chemical inhibition by BEL is subject to off-target pharmacologic effects. Accordingly, to unambiguously determine the role of iPLA2γ in the hydrolysis of oxidized CL, we compared alterations in oxidized CLs and the release of oxidized aliphatic chains from CL in experiments with purified recombinant iPLA2γ, germ-line iPLA2γ-/- mice, cardiac myocyte-specific iPLA2γ transgenic mice, and wild-type mice. Using charge-switch high mass accuracy LC-MS/MS with selected reaction monitoring and product ion accurate masses, we demonstrated that iPLA2γ is the major enzyme responsible for the release of oxidized aliphatic chains from CL. Our results also indicated that iPLA2γ selectively hydrolyzes 9-hydroxy-octadecenoic acid in comparison to 13-hydroxy-octadecenoic acid from oxidized CLs. Moreover, oxidative stress (ADP, NADPH, and Fe3+) resulted in the robust production of oxidized CLs in intact mitochondria from iPLA2γ-/- mice. In sharp contrast, oxidized CLs were readily hydrolyzed in mitochondria from wild-type mice during oxidative stress. Finally, we demonstrated that CL activates the iPLA2γ-mediated hydrolysis of arachidonic acid from phosphatidylcholine, thereby integrating the production of lipid messengers from different lipid classes in mitochondria. Collectively, these results demonstrate the integrated roles of CL and iPLA2γ in lipid second-messenger production and mitochondrial bioenergetics during oxidative stress.

Effect of calcium-independent phospholipase A(2) inhibitor in reducing hepatocyte lipoapoptosis and improving insulin resistance

Objective: To investigate the effect of calcium-independent phospholipase A(2) (iPLA(2)) inhibitor in reducing hepatocyte lipoapoptosis and improving insulin resistance. Methods: A total of 28 male Sprague-Dawley rats were randomly divided into the following three groups: 12 rats in group I (normal control group) were given normal diet for 18 weeks; 8 rats in group II (non-alcoholic fatty liver disease model group) were given high-fat diet for 18 weeks; 8 rats in group III (iPLA(2) inhibitor group) were given high-fat diet for 18 weeks and intraperitoneal injection of the iPLA(2) inhibitor bromoenol lactone 150 μg/kg once every other day since week 15 (14 times of injection in total). All the rats were sacrificed at the same time, and body weight and liver weight were measured. Blood lipids, serum enzymes, fasting blood glucose, fasting insulin, free fatty acid, and serum iPLA(2) concentration were measured in each group, and liver pathological changes were evaluated. The terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling was used to measure the level of hepatocyte apoptosis and the apoptotic index was calculated. Quantitative PCR was used to measure the mRNA expression of iPLA(2). The Student-Newman-Keuls test and the chi-square test were used for comparison of parameters between groups I, II, and III. P < 0.05 was considered statistically significant. Results: Compared with group I, group II had significant increases in triglyceride (0.75±0.05 mmol/L vs 1.20±0.13 mmol/L, P < 0.05), cholesterol (1.50±0.12 mmol/L vs 2.94±0.34 mmol/L, P < 0.05), low-density lipoprotein (0.65±0.06 mmol/L vs 1.30±0.16 mmol/L, P < 0.05), free fatty acid (0.58±0.09 mEq/L vs 0.80±0.20 mEq/L, P < 0.05), fasting blood glucose (4.85±0.22 mmol/L vs 6.94±0.65 mmol/L, P < 0.05), and fasting insulin (0.89±0.52 mmol/L vs 1.29±0.52 mmol/L, P < 0.05), and a significant reduction in the insulin sensitivity index (0.52±0.21 vs 0.27±0.11, P < 0.05); group II also had significant inflammation and fatty degeneration shown by liver pathology, and compared with group I, group II had significant increases in apoptotic cells and apoptotic index (0.58%±0.17% vs 39.69%±4.96%, P < 0.05). Compared with group I, group II had significant increases in serum iPLA(2) concentration (2.92±0.08 ng/ml vs 3.28±0.14 ng/ml, P < 0.05) and the mRNA expression of iPLA(2) in the liver (1.07±0.18 vs 7.68±0.49, P < 0.05). Compared with group II, group III had a lower level of hepatocyte apoptosis, a significant reduction in apoptotic index (39.69%±4.96% vs 24.80%±2.53%, P < 0.05), significant reductions in serum iPLA(2) concentration (3.28±0.14 ng/ml vs 2.64±0.24 ng/ml, P < 0.05) and the mRNA expression of iPLA(2) in the liver (7.68±0.49 vs 2.60±0.36, P < 0.05), significant reductions in fasting insulin (1.29±0.52 mmol/L vs 0.80±0.09 mmol/L, P < 0.05) and fasting blood glucose (6.94±0.65 mmol/L vs 5.18±0.35 mmol/L, P < 0.05), and a significant increase in insulin sensitivity index (0.27±0.11 vs 0.45±0.09, P < 0.05). Conclusion: There is a significant increase in the expression of iPLA(2) in rats with non-alcoholic fatty liver disease, and iPLA2 inhibitor can reduce hepatocyte lipoapoptosis and improve insulin resistance.

Impaired embryonic development in glucose-6-phosphate dehydrogenase-deficient Caenorhabditis elegans due to abnormal redox homeostasis induced activation of calcium-independent phospholipase and alteration of glycerophospholipid metabolism

Glucose-6-phosphate dehydrogenase (G6PD) deficiency is a commonly pervasive inherited disease in many parts of the world. The complete lack of G6PD activity in a mouse model causes embryonic lethality. The G6PD-deficient Caenorhabditis elegans model also shows embryonic death as indicated by a severe hatching defect. Although increased oxidative stress has been implicated in both cases as the underlying cause, the exact mechanism has not been clearly delineated. In this study with C. elegans, membrane-associated defects, including enhanced permeability, defective polarity and cytokinesis, were found in G6PD-deficient embryos. The membrane-associated abnormalities were accompanied by impaired eggshell structure as evidenced by a transmission electron microscopic study. Such loss of membrane structural integrity was associated with abnormal lipid composition as lipidomic analysis revealed that lysoglycerophospholipids were significantly increased in G6PD-deficient embryos. Abnormal glycerophospholipid metabolism leading to defective embryonic development could be attributed to the increased activity of calcium-independent phospholipase A2 (iPLA) in G6PD-deficient embryos. This notion is further supported by the fact that the suppression of multiple iPLAs by genetic manipulation partially rescued the embryonic defects in G6PD-deficient embryos. In addition, G6PD deficiency induced disruption of redox balance as manifested by diminished NADPH and elevated lipid peroxidation in embryos. Taken together, disrupted lipid metabolism due to abnormal redox homeostasis is a major factor contributing to abnormal embryonic development in G6PD-deficient C. elegans.

Sumoylation-deficient Prdx6 gains protective function by amplifying enzymatic activity and stability and escapes oxidative stress-induced aberrant Sumoylation

Aberrant Sumoylation of protein(s) in response to oxidative stress or during aging is known to be involved in etiopathogenesis of many diseases. Upon oxidative stress, Peroxiredoxin (Prdx) 6 is aberrantly Sumoylated by Sumo1, resulting in loss of functions and cell death. We identified lysines (K) 122 and 142 as the major Sumo1 conjugation sites in Prdx6. Intriguingly, the mutant Prdx6 K122/142 R (arginine) gained protective efficacy, increasing in abundance and promoting glutathione (GSH) peroxidase and acidic calcium-independent phospholipase A2 (aiPLA2) activities. Using lens epithelial cells derived from targeted inactivation of Prdx6−/− gene and relative enzymatic and stability assays, we discovered dramatic increases in GSH-peroxidase (30%) and aiPLA2 (37%) activities and stability in the K122/142 R mutant, suggesting Sumo1 destabilized Prdx6 integrity. Prdx6−/−LECs with EGFP-Sumo1 transduced or co-expressed with mutant TAT-HA-Prdx6K122/142 R or pGFP-Prdx6K122/142 R were highly resistant to oxidative stress, demonstrating mutant protein escaped and interrupted the Prdx6 aberrant Sumoylation-mediated cell death pathway. Mutational analysis of functional sites showed that both peroxidase and PLA2 active sites were necessary for mutant Prdx6 function, and that Prdx6 phosphorylation (at T177 residue) was essential for optimum PLA2 activity. Our work reveals the involvement of oxidative stress-induced aberrant Sumoylation in dysregulation of Prdx6 function. Mutant Prdx6 at its Sumo1 sites escapes and abates this adverse process by maintaining its integrity and gaining function. We propose that the K122/142R mutant of Prdx6 in the form of a TAT-fusion protein may be an easily applicable intervention for pathobiology of cells related to aberrant Sumoylation signaling in aging or oxidative stress.

Chemokines (CCL3, CCL4, and CCL5) Inhibit ATP-Induced Release of IL-1β by Monocytic Cells.

Chemokines and ATP are among the mediators of inflammatory sites that can enter the circulation via damaged blood vessels. The main function of chemokines is leukocyte mobilization, and ATP typically triggers inflammasome assembly. IL-1β, a potent inflammasome-dependent cytokine of innate immunity, is essential for pathogen defense. However, excessive IL-1β may cause life-threatening systemic inflammation. Here, we hypothesize that chemokines control ATP-dependent secretion of monocytic IL-1β. Lipopolysaccharide-primed human monocytic U937 cells were stimulated with the P2X7 agonist BzATP for 30 min to induce IL-1β release. CCL3, CCL4, and CCL5 dose dependently inhibited BzATP-stimulated release of IL-1β, whereas CXCL16 was ineffective. The effect of CCL3 was confirmed for primary mononuclear leukocytes. It was blunted after silencing CCR1 or calcium-independent phospholipase A2 (iPLA2) by siRNA and was sensitive to antagonists of nicotinic acetylcholine receptors containing subunits α7 and α9. U937 cells secreted small factors in response to CCL3 that mediated the inhibition of IL-1β release. We suggest that CCL chemokines inhibit ATP-induced release of IL-1β from U937 cells by a triple-membrane-passing mechanism involving CCR, iPLA2, release of small mediators, and nicotinic acetylcholine receptor subunits α7 and α9. We speculate that whenever chemokines and ATP enter the circulation concomitantly, systemic release of IL-1β is minimized.

Role of Calcium-Independent Phospholipase A2 VIA in Mediating Neurological Disorder and Cancer

Calcium-independent phospholipase A2 (iPLA2) belongs to the group VI family of phospholipase superfamily (PLA2) that catalyses the hydrolysis of glycerophospholipids at the sn-2 ester bond, producing unesterified fatty acids and 2-lysophospholipids. Research interests on iPLA2 have not been as significant as those on secretary PLA2 and cytosolic PLA2. However, more efforts have been made recently on understanding the expression, regulation and biological function of iPLA2. iPLA2 plays important roles in several biological processes, including signal transduction, phospholipid remodelling, eicosanoid formation, cell proliferation, cell differentiation and apoptosis. Modulation of iPLA2 activity can have prominent effects on cellular metabolism, central nervous system and cardiovascular functions. Thus, dysregulation iPLA2 can play a vital role in the pathogenesis of several diseases. The aim of this review is to provide the current understanding of the structure, function and regulation of group VI iPLA2 and highlight its potential mechanisms of action in mediating several neurological disorders and cancer.

2-Oxoamides based on dipeptides as selective calcium-independent phospholipase A2 inhibitors

Calcium-independent phospholipase A2 (GVIA iPLA2) has recently attracted interest as a medicinal target. The number of known GVIA iPLA2 inhibitors is limited to a handful of synthetic compounds (bromoenol lactone and polyfluoroketones). To expand the chemical diversity, a variety of 2-oxoamides based on dipeptides and ether dipeptides were synthesized and studied for their in vitro inhibitory activity on human GVIA iPLA2 and their selectivity over the other major intracellular GIVA cPLA2 and the secreted GV sPLA2. Structure-activity relationship studies revealed the first 2-oxoamide derivative (GK317), which presents potent inhibition of GVIA iPLA2 (XI(50) value of 0.007) and at the same time significant selectivity over GIVA cPLA2 and GV sPLA2.

Novel Mutation in PLA2G6 Gene in a Patient with Infantile Neuroaxonal Dystrophy

AbstractInfantile neuroaxonal dystrophy (INAD) is an autosomal recessive disorder that causes psychomotor regression. Clinicopathological features include truncal hypotonia followed by spastic quadriparesis, strabismus, nystagmus, cerebellar ataxia, bulbar dysfunction, and cerebellar atrophy. INAD is associated with a variety of mutations in the PLA2G6 gene that encodes the group VI calcium-independent phospholipase A2 protein which is important in cell membrane homeostasis. Defects in this protein cause axonal dystrophy. We report a 2-year-old boy with INAD who was found to have a novel deletion c.1019_1025del7 leading to a frame shift in the PLA2G6 gene. We suggest that this change may be an addition to the array of mutations causing INAD.

127 - Abnormal Lipid Metabolism and Lipid Peroxidation in G6PD Deficiency-Induced Embryonic Lethality in Caenorhabditis Elegans embryo

Complete Glucose 6-phosphate dehydrogenase (G6PD) deficiency in mouse and C. elegans causes embryonic lethality, yet the mechanism is unclear. In this study, membrane-associated abnormalities, including defective permeability, loss of polarity and cytokinesis, were found in G6PD-deficient embryos, which were accompanied by impaired eggshell structure. Such loss of membrane-structural integrity was associated with significantly increased lysoglycerophospholipids as revealed by lipidomic analysis in G6PD-deficient embryos. Abnormal glycerophospholipid metabolism could be, in part, attributed to the increased activity of calcium-independent phospholipase A (iPLA) in G6PD-deficient embryos. Suppression of multiple iPLAs partially rescued the embryonic defects in G6PD-deficient C. elegans embryos provides further support to this notion. In addition, elevated lipid peroxidation had been found in G6PD-deficient embryos. Taken together, disrupted lipid metabolism due to abnormal redox homeostasis is a major factor contributing to defective embryonic development in G6PD-deficient C. elegans.

Regulation of Integrin α6 Recycling by Calcium-independent Phospholipase A2 (iPLA2) to Promote Microglia Chemotaxis on Laminin

Microglia are the immune effector cells that are activated in response to pathological changes in the central nervous system. Microglial activation is accompanied by the alteration of integrin expression on the microglia surface. However, changes of integrin expression upon chemoattractant (ADP) stimulation still remain unknown. In this study, we investigated whether ADP induces the alteration of integrin species on the cell surface, leading to changes in chemotactic ability on different extracellular matrix proteins. Flow cytometry scans and on-cell Western assays showed that ADP stimulation induced a significant increase of α6 integrin-GFP, but not α5, on the surface of microglia cells. Microglia also showed a greater motility increase on laminin than fibronectin after ADP stimulation. Time lapse microscopy and integrin endocytosis assay revealed the essential role of calcium-independent phospholipase A2 activity for the recycling of α6 integrin-GFP from the endosomal recycling complex to the plasma membrane. Lack of calcium-independent phospholipase A2 activity caused a reduced rate of focal adhesion formation on laminin at the leading edge. Our results suggest that the alteration of integrin-mediated adhesion may regulate the extent of microglial infiltration into the site of damage by controlling their chemotactic ability.

Role of prefrontal cortical calcium-independent phospholipase A2 in antinociceptive effect of the norepinephrine reuptake inhibitor antidepresssant maprotiline

The prefrontal cortex is essential for executive functions such as decision-making and planning. There is also accumulating evidence that it is important for the modulation of pain. In this study, we investigated a possible role of prefrontal cortical calcium-independent phospholipase A2 (iPLA2) in antinociception induced by the norepinephrine reuptake inhibitor (NRI) and tetracyclic (tricyclic) antidepressant, maprotiline. Intraperitoneal injections of maprotiline increased iPLA2 mRNA and protein expression in the prefrontal cortex. This treatment also reduced grooming responses to von-Frey hair stimulation of the face after facial carrageenan injection, indicating decreased sensitivity to pain. The antinociceptive effect of maprotiline was abrogated by iPLA2 antisense oligonucleotide injection to the prefrontal cortex, indicating a role of this enzyme in antinociception. In contrast, injection of iPLA2 antisense oligonucleotide to the somatosensory cortex did not reduce the antinociceptive effect of maprotiline. Lipidomic analysis of the prefrontal cortex showed decrease in phosphatidylcholine species, but increase in lysophosphatidylcholine species, indicating increased PLA2 activity, and release of docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA) after maprotiline treatment. Differences in sphingomyelin/ceramide were also detected. These changes were not observed in maprotiline-treated mice that received iPLA2 antisense oligonucleotide to the prefrontal cortex. Metabolites of DHA and EPA may help to strengthen a known supraspinal antinociceptive pathway from the prefrontal cortex to the periaqueductal gray. Together, results indicate a role of prefrontal cortical iPLA2 and its enzymatic products in the antinociceptive effect of maprotiline.

P.1.b.006 - Investigation of long-term potentiation in young and old hyperthyroid rats

Docosahexaenoic acid (DHA) is a 22 carbon, six cis-double bonded (22:6, w3) omega-3 polyunsaturated acid (PUFA), found highly enriched with neuronal membranes, and believed to play a critical role in synaptic plasticity and cognitive correlates of learning and memory. DHA is released from the neuronal membrane via the action of the cytostolic calcium-independent phospholipase A2 (iPLA2) enzyme. Previous studies have demonstrated that inhibition of iPLA2 by bromoenol lactone (BEL), results in inhibition of CA1 long-term potentiation (LTP), restored following acute application of DHA. In the present study, we investigated the effect of selective iPLA2 inhibition and acute application of DHA on corticostriatal synaptic plasticity. We demonstrate that acute application of 30 μM DHA facilitates cotricostriatal LTP, whilst long-term depression (LTD), basal transmission, and paired-pulse facilitation (PPF) are unaffected. Conversely, selective inhibition of iPLA2, via acute application of 10 μM BEL, inhibits the expression of corticostriatal LTP, with no effect on LTD. Furthermore, we show that 10 μM BEL inhibition of LTP is reversed following acute application of 30 μM DHA. Finally, we demonstrate that 10 μM BEL inhibits depotentiation of corticostriatal LTP, which is restored following acute application of 30 μM DHA. Our findings indicate that appropriate release of DHA is a critical facet of corticostriatal LTP and depotentiation, and thus provides an exciting cellular target for the positive facilitation of cognitive function observed following DHA dietary supplementation.

Cytosolic and Calcium-Independent Phospholipases A2 Activation and Prostaglandins E2 Are Associated with Escherichia coli-Induced Reduction of Insulin Secretion in INS-1E Cells.

It is suspected that microbial infections take part in the pathogenesis of diabetes mellitus type 1 (T1DM). Glucose-induced insulin secretion is accompanied by the release of free arachidonic acid (AA) mainly by cytosolic- and calcium independent phospholipases A2 (cPLA2 and iPLA2). Insulinoma cell line (INS-1E) was infected with E. coli isolated from the blood culture of a patient with sepsis. Invasion assay, Scanning Electron Microscopy and Transmission Electron Microscopy demonstrated the capacity of E. coli to enter cells, which was reduced by PLA2 inhibitors. Glucose-induced insulin secretion was significantly increased after acute infection (8h) but significantly decreased after chronic infection (72h). PLA2 activities, cPLA2, iPLA2, phospho-cPLA2, and COX-2 expressions were increased after acute and, even more, after chronic E. coli infection. The silencing of the two isoforms of PLA2s, with specific cPLA2- or iPLA2-siRNAs, reduced insulin secretion after acute infection and determined a rise in insulin release after chronic infection. Prostaglandins E2 (PGE2) production was significantly elevated in INS-1E after long-term E. coli infection and the restored insulin secretion in presence of L798106, a specific EP3 antagonist, and NS-398, a COX-2 inhibitor, and the reduction of insulin secretion in presence of sulprostone, a specific EP3 agonist, revealed their involvement in the effects triggered by bacterial infection. The results obtained demonstrated that cPLA2 and iPLA2 play a key role in insulin secretion process after E. coli infection. The high concentration of AA released is transformed into PGE2, which could be responsible for the reduced insulin secretion.