Independent Phospholipase A2 Research Articles

The Effect of Resveratrol on the Composition and State of Lipids and the Activity of Phospholipase A2 During the Excitation and Regeneration of Somatic Nerves.

It has been shown that in the somatic nerve’s lipids, both during excitation and transection, changes occur with the composition of individual phospholipids and in phospholipids fatty acids, which changes the phase state of the myelin and nerve fiber axolemma lipid bilayer. A main contribution in the nerve degenerative processes is dependent on the composition phospholipid’s fatty acid changes during the activation of both Ca2+-dependent and Ca2+-independent phospholipase A2 forms. At the same time, we studded changes in phosphoinisitol (PI) and diacylglycerol (DAG), which depend on the phosphoinositide cycle function during nerve excitation and degeneration processes. It was found that myelin lipids and nerve fiber axolemmas are involved not only in the functioning of the peripheral nerves, but also the pathological processes underlying deep functional and structural disorders. The effect of resveratrol on regeneration processes in the damaged rat sciatic nerve has also been investigated.

Combined targeted Omic and Functional Assays Identify Phospholipases A₂ that Regulate Docking/Priming in Calcium-Triggered Exocytosis.

The fundamental molecular mechanism underlying the membrane merger steps of regulated exocytosis is highly conserved across cell types. Although involvement of Phospholipase A2 (PLA2) in regulated exocytosis has long been suggested, its function or that of its metabolites—a lyso-phospholipid and a free fatty acid—remain somewhat speculative. Here, using a combined bioinformatics and top-down discovery proteomics approach, coupled with lipidomic analyses, PLA2 were found to be associated with release-ready cortical secretory vesicles (CV) that possess the minimal molecular machinery for docking, Ca2+ sensing and membrane fusion. Tightly coupling the molecular analyses with well-established quantitative fusion assays, we show for the first time that inhibition of a CV surface calcium independent intracellular PLA2 and a luminal secretory PLA2 significantly reduce docking/priming in the late steps of regulated exocytosis, indicating key regulatory roles in the critical step(s) preceding membrane merger.

β-Lactones: A Novel Class of Ca2+-Independent Phospholipase A2 (Group VIA iPLA2) Inhibitors with the Ability To Inhibit β-Cell Apoptosis.

Ca2+-independent phospholipase A2 (GVIA iPLA2) has gained increasing interest recently as it has been recognized as a participant in biological processes underlying diabetes development and autoimmune-based neurological disorders. The development of potent GVIA iPLA2 inhibitors is of great importance because only a few have been reported so far. We present a novel class of GVIA iPLA2 inhibitors based on the β-lactone ring. This functionality in combination with a four-carbon chain carrying a phenyl group at position-3 and a linear propyl group at position-4 of the lactone ring confers excellent potency. trans-3-(4-Phenylbutyl)-4-propyloxetan-2-one (GK563) was identified as being the most potent GVIA iPLA2 inhibitor ever reported ( XI(50) 0.0000021, IC50 1 nM) and also one that is 22 000 times more active against GVIA iPLA2 than GIVA cPLA2. It was found to reduce β-cell apoptosis induced by proinflammatory cytokines, raising the possibility that it can be beneficial in countering autoimmune diseases, such as type 1 diabetes.

Hyperoxidation of Peroxiredoxin 6 Induces Alteration from Dimeric to Oligomeric State.

Peroxiredoxins(Prdx), the family of non-selenium glutathione peroxidases, are important antioxidant enzymes that defend our system from the toxic reactive oxygen species (ROS). They are thiol-based peroxidases that utilize self-oxidation of their peroxidatic cysteine (Cp) group to reduce peroxides and peroxidized biomolecules. However, because of its high affinity for hydrogen peroxide this peroxidatic cysteine moiety is extremely susceptible to hyperoxidation, forming peroxidase inactive sulfinic acid (Cys-SO2H) and sulfonic acid (Cys-SO3H) derivatives. With the exception of peroxiredoxin 6 (Prdx6), hyperoxidized sulfinic forms of Prdx can be reversed to restore peroxidase activity by the ATP-dependent enzyme sulfiredoxin. Interestingly, hyperoxidized Prdx6 protein seems to have physiological significance as hyperoxidation has been reported to dramatically upregulate its calcium independent phospholipase A2 activity. Using biochemical studies and molecular dynamic (MD) simulation, we investigated the roles of thermodynamic, structural and internal flexibility of Prdx6 to comprehend the structural alteration of the protein in the oxidized state. We observed the loosening of the hydrophobic core of the enzyme in its secondary and tertiary structures. These changes do not affect the internal dynamics of the protein (as indicated by root-mean-square deviation, RMSD and root mean square fluctuation, RMSF plots). Native-PAGE and dynamic light scattering experiments revealed the formation of higher oligomers of Prdx6 under hyperoxidation. Our study demonstrates that post translational modification (like hyperoxidation) in Prdx6 can result in major alterations of its multimeric status.

Amorphous solid dispersion of Berberine mitigates apoptosis via iPLA2β/Cardiolipin/Opa1 pathway in db/db mice and in Palmitate-treated MIN6 β-cells.

Aims: Berberine (BBR) improves beta-cell function in Type 2 diabetes (T2D) because of its anti-apoptotic activity, and our laboratory developed a new preparation named Huang-Gui Solid Dispersion (HGSD) to improve the oral bioavailability of BBR. However, the mechanism by which BBR inhibits beta-cell apoptosis is unclear. We hypothesized that the Group VIA Ca2+-Independent Phospholipase A2 (iPLA2β)/Cardiolipin(CL)/Opa1 signaling pathway could exert a protective role in T2D by regulating beta-cell apoptosis and that HGSD could inhibit β-cell apoptosis through iPLA2β/CL/Opa1 upregulation.Methods: We examined how iPLA2β and BBR regulated apoptosis and insulin secretion through CL/Opa1 in vivo and in vitro. In in vitro studies, we developed Palmitate(PA)-induced apoptotic cell death model in mouse insulinoma cells (MIN6). iPLA2β overexpression and silencing technology were used to examine how the iPLA2β/CL/Opa1 interaction may play an important role in BBR treatment. In in vivo studies, db/db mice were used as a diabetic animal model. The pancreatic islet function and morphology, beta-cell apoptosis and mitochondrial injury were examined to explore the effects of HGSD. The expression of iPLA2β/CL/Opa1 was measured to explore whether the signaling pathway was damaged in T2D and was involved in HGSD treatment.Results: The overexpression of iPLA2β and BBR treatment significantly attenuated Palmitate- induced mitochondrial injury and apoptotic death compared with Palmitate-treated MIN6 cell. In addition, iPLA2β silencing could simultaneously partly abolish the anti-apoptotic effect of BBR and decrease CL/Opa1 signaling in MIN6 cells. Moreover, HGSD treatment significantly decreased beta-cell apoptosis and resulted in the upregulation of iPLA2β/CL/Opa1 compared to those of the db/db mice.Conclusion: The results indicated that the regulation of iPLA2β/CL/Opa1 by HGSD may prevent beta-cell apoptosis and may improve islet beta-cell function in Type 2 diabetic mice and in palmitate-treated MIN6 cells.

The role of prostaglandins and polymorphisms of A2 phospholipase genes in the occurrence of early uterine contractions during pregnancy

Abstract This study focused on the role of polymorphisms in prostaglandin expression regulating genes in the occurrence of early uterine contractions during pregnancy. The analyzed genes were: PLA2G4C, encoding calcium independent phospholipase A2, and PLA2G4C, encoding IVD phospholipase A2. It was examined if known reference polymorphisms in these genes (rs1366442, [A/C/T] for PLA2G4C; and rs4924618, [A/T] for PLA2G4D), have any influence on preterm birth. Additionally, other biological, genetic and socio-economic factors were taken into account and analyzed, based on their role in induction of early resolve of pregnancy in the study group. Blood samples were taken from 20 patients. 15 of them gave birth preterm, 5 gave birth at the predicted date of pregnancy resolve. DNA was isolated from the samples, and subjected to PCR, with obtained amplified samples separated using electrophoresis on 1,5% agarose gel. Resulting material was subjected to high-throughput sequencing. Statistical analysis was performed using Statistica 13 software. Analyses have shown that the discussed biological, genetic and societal-economic factors have statistically significant influence on preterm birth. Sequencing results presented the suspected presence of the analyzed SNPs in most women from the studied groups, while not showing their presence in any of the controls. The biological, genetic, and socio-economic factors analyzed have a significant influence on pre-term birth. Presence of SNPs in PLA2G4C and PLA2G4D genes may increase the risk of early resolve of pregnancy. However, as the control and study groups were relatively small, it is suggested to repeat the studies on bigger samples to validate the results.

A non-venomous sPLA2 of a lepidopteran insect: Its physiological functions in development and immunity

Eicosanoids are oxygenated C20 polyunsaturated fatty acids that mediate various physiological processes in insects. Eicosanoid biosynthesis begins with a C20 precursor, arachidonic acid (5,8,11,14-eicosatetraenoic acid: AA). AA is usually released from phospholipids at sn-2 position by catalytic activity of phospholipase A2 (PLA2). Although various PLA2s classified into 16 gene families (= Groups) are known in various biological systems, few PLA2s are known in insects. Only two PLA2s involved in intracellular calcium independent PLA2 (iPLA2) group have been identified in lepidopteran insects with well known eicosanoid physiology. This study reports the first secretory PLA2 (sPLA2) in lepidopteran insects. A partial open reading frame (ORF) of PLA2 was obtained by interrogating Spodoptera exigua transcriptome. Subsequent 3′-RACE resulted in a full ORF (Se-sPLA2A) encoding 194 amino acid sequence containing signal peptide, calcium-binding domain, and catalytic site. Phylogenetic analysis indicated that Se-sPLA2A was clustered with other Group III sPLA2s. Se-sPLA2A was expressed in most larval instars except late last instar. Its expression was inducible by immune challenge and juvenile hormone analog injection. RNA interference of Se-sPLA2A significantly suppressed cellular immunity and impaired larval development. These results suggest that non-venomous sPLA2 plays a crucial role in immune and developmental processes in S. exigua, a lepidopteran insect.

Unveiling anti-oxidative and anti-inflammatory effects of docosahexaenoic acid and its lipid peroxidation product on lipopolysaccharide-stimulated BV-2 microglial cells

BackgroundPhospholipids in the central nervous system are enriched in n-3 and n-6 polyunsaturated fatty acids (PUFA), especially docosahexaenoic acid (DHA) and arachidonic acid (ARA). These PUFA can undergo enzymatic reactions to produce lipid mediators, as well as reaction with oxygen free radicals to produce 4-hydroxyhexenal (4-HHE) from DHA and 4-hydroxynonenal (4-HNE) from ARA. Recent studies demonstrated pleiotropic properties of these peroxidation products through interaction with oxidative and anti-oxidant response pathways. In this study, BV-2 microglial cells were used to investigate ability for DHA, 4-HHE, and 4-HNE to stimulate the anti-oxidant stress responses involving the nuclear factor erythroid-2-related factor 2 (Nrf2) pathway and synthesis of heme oxygenase (HO-1), as well as to mitigate lipopolysaccharide (LPS)-induced nitric oxide (NO), reactive oxygen species (ROS), and cytosolic phospholipase A2 (cPLA2). In addition, LC-MS/MS analysis was carried out to examine effects of exogenous DHA and LPS stimulation on endogenous 4-HHE and 4-HNE levels in BV-2 microglial cells.MethodsEffects of DHA, 4-HHE, and 4-HNE on LPS-induced NO production was determined using the Griess reagent. LPS-induced ROS production was measured using CM-H2DCFDA. Western blots were used to analyze expression of p-cPLA2, Nrf2, and HO-1. Cell viability and cytotoxicity were measured using the WST-1 assay, and cell protein concentrations were measured using the BCA protein assay kit. An ultra-high-performance liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis was used to determine levels of free 4-HHE and 4-HNE in cells.ResultsDHA (12.5–100 μM), 4-HHE (1.25–10 μM), and 4-HNE (1.25–10 μM) dose dependently suppressed LPS-induced production of NO, ROS, and as p-cPLA2 in BV-2 microglial cells. With the same concentrations, these compounds could enhance Nrf2 and HO-1 expression in these cells. Based on the estimated IC50 values, 4-HHE and 4-HNE were five- to tenfold more potent than DHA in inhibiting LPS-induced NO, ROS, and p-cPLA2. LC-MS/MS analysis indicated ability for DHA (10–50 μM) to increase levels of 4-HHE and attenuate levels of 4-HNE in BV-2 microglial cells. Stimulation of cells with LPS caused an increase in 4-HNE which could be abrogated by cPLA2 inhibitor. In contrast, bromoenol lactone (BEL), a specific inhibitor for the Ca2+-independent phospholipase A2 (iPLA2), could only partially suppress levels of 4-HHE induced by DHA or DHA + LPS.ConclusionsThis study demonstrated the ability of DHA and its lipid peroxidation products, namely, 4-HHE and 4-HNE at 1.25–10 μM, to enhance Nrf2/HO-1 and mitigate LPS-induced NO, ROS, and p-cPLA2 in BV-2 microglial cells. In addition, LC-MS/MS analysis of the levels of 4-HHE and 4-HNE in microglial cells demonstrates that increases in production of 4-HHE from DHA and 4-HNE from LPS are mediated by different mechanisms.

Peroxiredoxin 6 Inhibits Osteogenic Differentiation and Bone Formation Through Human Dental Pulp Stem Cells and Induces Delayed Bone Development.

Aims: Peroxiredoxins (PRDXs) are thiol-specific antioxidant enzymes that regulate redox balance that are critical for maintaining the cellular potential for self-renewal and stemness. Stem cell-based regenerative medicine is a promising approach in tissue reconstruction. However, to obtain functional cells for use in clinical applications, stem cell technology still requires improvements. Results: In this study, we found that PRDX6 levels were decreased during osteogenic differentiation in human dental pulp stem cells (hDPSCs). hDPSCs stably expressing Myc-PRDX6 (hDPSC/myc-PRDX6) inhibited cell growth in hDPSCs during osteogenic differentiation and impaired osteogenic phenotypes such as alkaline phosphatase (ALP) activity, mineralized nodule formation, and osteogenic marker genes [ALP and osteocalcin (OCN)]. hDPSC cell lines stably expressing mutant glutathione peroxidase (PRDX6(C47S)) and independent phospholipase A2 (PRDX6(S32A)) were also generated. Each mutant form of PRDX6 abolished the impaired osteogenic phenotypes, the transforming growth factor-β-mediated Smad2 and p38 pathways, and RUNX2 expression. Furthermore, in vivo experiments revealed that hDPSC/myc-PRDX6 suppressed hDPSC-based bone regeneration in calvarial defect mice, and newborn PRDX6 transgenic mice exhibited delayed bone development and reduced RUNX2 expression. Innovation and Conclusion: These findings illuminate the effects of PRDX6 during osteogenic differentiation of hDPSCs, and also suggest that regulating PRDX6 may improve the clinical utility of stem cell-based regenerative medicine for the treatment of bone diseases. Antioxid. Redox Signal. 30, 1969-1982.

β-Nicotinamide Adenine Dinucleotide (β-NAD) Inhibits ATP-Dependent IL-1β Release from Human Monocytic Cells.

While interleukin-1β (IL-1β) is a potent pro-inflammatory cytokine essential for host defense, high systemic levels cause life-threatening inflammatory syndromes. ATP, a stimulus of IL-1β maturation, is released from damaged cells along with β-nicotinamide adenine dinucleotide (β-NAD). Here, we tested the hypothesis that β-NAD controls ATP-signaling and, hence, IL-1β release. Lipopolysaccharide-primed monocytic U937 cells and primary human mononuclear leukocytes were stimulated with 2′(3′)-O-(4-benzoyl-benzoyl)ATP trieethylammonium salt (BzATP), a P2X7 receptor agonist, in the presence or absence of β-NAD. IL-1β was measured in cell culture supernatants. The roles of P2Y receptors, nicotinic acetylcholine receptors (nAChRs), and Ca2+-independent phospholipase A2 (iPLA2β, PLA2G6) were investigated using specific inhibitors and gene-silencing. Exogenous β-NAD signaled via P2Y receptors and dose-dependently (IC50 = 15 µM) suppressed the BzATP-induced IL-1β release. Signaling involved iPLA2β, release of a soluble mediator, and nAChR subunit α9. Patch-clamp experiments revealed that β-NAD inhibited BzATP-induced ion currents. In conclusion, we describe a novel triple membrane-passing signaling cascade triggered by extracellular β-NAD that suppresses ATP-induced release of IL-1β by monocytic cells. This cascade links activation of P2Y receptors to non-canonical metabotropic functions of nAChRs that inhibit P2X7 receptor function. The biomedical relevance of this mechanism might be the control of trauma-associated systemic inflammation.

PPAR-α agonists acutely inhibit Ca2+-independent PLA2 to reduce H2O2-induced contractions in aortae of spontaneously hypertensive rats.

Hypertension is associated with endothelial dysfunction, which favors the release of endothelium-derived contracting factors, including vasoconstrictor prostanoids and reactive oxygen species. Peroxisome proliferator-activated receptor-α (PPAR-α) agonists, clinically used as lipid-lowering drugs, possess antioxidant properties and exert beneficial effects in the vascular system. The present study aimed to identify the mechanism(s) underlying the acute effects of the PPAR-α agonists Wy14643 and fenofibate on endothelium-dependent contractions, in particular those related to oxidative stress, in the aorta of the spontaneously hypertensive rat (SHR). Aortic rings with and without endothelium of male SHRs and normotensive Wistar-Kyoto rats were suspended in organ chambers for isometric tension measurements and homogenized for enzyme activity assays. Contractions to acetylcholine in quiescent SHR aortae with endothelium were reduced by tiron (superoxide anion scavenger), diethyldithiocarbamic acid (superoxide dismutase inhibitor), and acute treatment with either Wy14643 or fenofibrate. Similarly to contractions evoked by acetylcholine, H2O2-induced increases in tension in SHR aortae involved, in succession, phospholipase A2 (PLA2), cyclooxygenase, and thromboxane-prostanoid receptors. Wy14643 or fenofibrate, by decreasing the activity of endothelial Ca2+-independent PLA2, attenuated the contractions to H2O2. In conclusion, the increased oxidative stress in the SHR aorta (mainly increased production of H2O2 and its partially reduced product, hydroxyl radical) contributed to acetylcholine-induced, endothelium-dependent contractions; PPAR-α agonists likely inhibit the H2O2-mediated contractions by inhibiting endothelial Ca2+-independent PLA2. The present study highlights the prospective therapeutic effects of PPAR-α agonists in improving endothelial function in hypertension and other vascular implications due to oxidative stress. NEW & NOTEWORTHY Peroxisome proliferator-activated receptor-α agonists, which are used clinically as lipid-lowering drugs, acutely reduce H2O2-induced contractions in aortae of hypertensive rats by inhibiting the activity of endothelial Ca2+-independent phospholipase A2. These vascular effects of peroxisome proliferator-activated receptor-α agonists suggest that they may help to prevent vascular complications under pathological conditions associated with oxidative stress.

PARK14 PLA2G6 mutants are defective in preventing rotenone-induced mitochondrial dysfunction, ROS generation and activation of mitochondrial apoptotic pathway.

Mutations in the gene encoding Ca2+-independent phospholipase A2 group 6 (PLA2G6) cause the recessive familial type 14 of Parkinson’s disease (PARK14). Mitochondrial dysfunction is involved in the pathogenesis of Parkinson’s disease (PD). PLA2G6 is believed to be required for maintaining mitochondrial function. In the present study, rotenone-induced cellular model of PD was used to investigate possible molecular pathogenic mechanism of PARK14 mutant PLA2G6-induced PD. Overexpression of wild-type (WT) PLA2G6 ameliorated rotenone-induced apoptotic death of SH-SY5Y dopaminergic cells. PARK14 mutant (D331Y), (G517C), (T572I), (R632W), (N659S) or (R741Q) PLA2G6 failed to prevent rotenone-induced activation of mitochondrial apoptotic pathway and exert a neuroprotective effect. WT PLA2G6, but not PARK14 mutant PLA2G6, prevented rotenone-induced mitophagy impairment. In contrast to WT PLA2G6, PARK14 mutant PLA2G6 was ineffective in attenuating rotenone-induced decrease in mitochondrial membrane potential and increase in the level of mitochondrial superoxide. WT PLA2G6, but not PARK14 PLA2G6 mutants, restored enzyme activity of mitochondrial complex I and cellular ATP content in rotenone-treated SH-SY5Y dopaminergic cells. In contrast to WT PLA2G6, PARK14 mutant PLA2G6 failed to prevent rotenone-induced mitochondrial lipid peroxidation and cytochrome c release. These results suggest that PARK14 PLA2G6 mutants lose their ability to maintain mitochondrial function and are defective inpreventing mitochondrial dysfunction, ROS production and activation of mitochondrial apoptotic pathway in rotenone-induced cellular model of PD.

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.

Phospholipase A2 of Peroxiredoxin 6 Plays a Critical Role in Cerebral Ischemia/Reperfusion Inflammatory Injury.

Microglia-mediated inflammation is an important step in the progression of cerebral ischemia/reperfusion injury and the associated production of receptors of immunomoudulation, including Toll-like receptors (TLRs). Peroxiredoxin 6 (Prdx6) has been demonstrated as the endogenous antioxidant protein for its peroxidase properties. However, the role of the independent phospholipase A2 (iPLA2) activity of Prdx6 in stroke has not been well studied. In this study, we evaluated whether blocking the calcium-iPLA2 activity of Prdx6 using siRNA and inhibitors (1-hexadecyl-3-(trifluoroethgl)-sn-glycerol-2 phosphomethanol, MJ33) would have a critical effect on inflammatory brain damage. We conducted oxygen-glucose deprivation (OGD)/recovery (R) in vitro and middle cerebral artery occlusion (MCAO) in vivo in a microglia/neuron co-culture system and in rats. In vitro, we found that Prdx6-iPLA2 activity was associated with the secretion of neurotoxic inflammatory mediators interleukin1β (IL-1β), interleukin-17 (IL-17) and interleukin-23 (IL-23) and elevated expression of Toll-like receptor 2/4 (TLR2/4), leading to the formation of nuclear factor-kappa B (NF-κB), inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) in microglial cells. In vivo, combined treatment with Prdx6-iPLA2 activity inhibitor MJ33 showed a greater diminution in neurologic deficits, cerebral infarction, brain water content and inflammatory molecules than Prdx6-siRNA treatment alone. Our findings provide new insight into Prdx6-iPLA2 function in the brain. Inhibition of Prdx6-iPLA2 activity by gene therapy and/or pharmacology may constitute a promising new therapeutic approach to the treatment of stroke.

Lysophosphatidic acid-induced itch is mediated by signalling of LPA5 receptor, phospholipase D and TRPA1/TRPV1.

Lysophosphatidic acid (LPA) is an itch mediator, but not a pain mediator by a cheek injection model. Dorsal root ganglion neurons directly respond to LPA depending on transient receptor potential ankyrin 1 (TRPA1) and vanilloid 1 (TRPV1). LPA-induced itch-related behaviours are decreased in TRPA1-knockout (KO), TRPV1KO or TRPA1TRPV1 double KO mice. TRPA1 and TRPV1 channels are activated by intracellular LPA, but not by extracellular LPA following LPA5 receptor activation with an activity of Ca2+ -independent phospholipase A2 and phospholipase D. Intracellular LPA interaction sites of TRPA1 are KK672-673 and KR977-978 (K: lysine, R: arginine). Intractable and continuous itch sensations often accompany diseases such as atopic dermatitis, neurogenic lesions, uremia and cholestasis. Lysophosphatidic acid (LPA) is an itch mediator found in cholestatic itch patients and it induces acute itch and pain in experimental rodent models. However, the molecular mechanism by which LPA activates peripheral sensory neurons remains unknown. In this study, we used a cheek injection method in mice to reveal that LPA induced itch-related behaviours but not pain-related behaviours. The LPA-induced itch behaviour and cellular effects were dependent on transient receptor potential ankyrin 1 (TRPA1) and vanilloid 1 (TRPV1), which are important for itch signal transduction. We also found that, among the six LPA receptors, the LPA5 receptor had the greatest involvement in itching. Furthermore, we demonstrated that phospholipase D (PLD) plays a critical role downstream of LPA5 and that LPA directly and intracellularly activates TRPA1 and TRPV1. These results suggest a unique mechanism by which cytoplasmic LPA produced de novo could activate TRPA1 and TRPV1. We conclude that LPA-induced itch is mediated by LPA5 , PLD, TRPA1 and TRPV1 signalling, and thus targeting TRPA1, TRPV1 or PLD could be effective for cholestatic itch interventions.

Neurons and astrocytes in an infantile neuroaxonal dystrophy (INAD) mouse model show characteristic alterations in glutamate-induced Ca2+ signaling

INAD (infantile neuroaxonal dystrophy, OMIM#256600), an autosomal recessive inherited degenerative disease, is associated with PLA2G6 mutations. PLA2G6 encodes Ca2+-independent phospholipase A2 (VIA iPLA2). However, it is unclear how the PLA2G6-mutations lead to disease. Non-canonical functions, which were suggested for VIA iPLA2, such as regulation of cellular and mitochondrial Ca2+ are promising candidates. Therefore, we investigate glutamate (Glu)-evoked Ca2+ signals in neurons and astrocytes in co-culture obtained from three INAD mouse model strains with Pla2g6 mutations, (i) hypomorphic Pla2g6 allele with reduced transcript levels, (ii) knocked-out Pla2g6, and (iii) (G373R)-point mutation with inactive VIA iPLA2 enzyme. Homozygous offspring from these strains develop pathology similar to that observed in INAD patients. We found that in mouse neurons the Pla2g6 mutation disrupted the dependency of Glu-induced extracellular Ca2+ influx on mitochondrial Ca2+ uptake. Thus, in neurons with Pla2g6 mutation we did not detect the characteristic reduction in Glu-induced Ca2+ influx upon treatment with Ru360, a blocker of mitochondrial Ca2+ uniporter, or with rotenone. In contrast to neurons, in astrocytes, both with Pla2g6 mutation or wild-type cells, the treatment with Ru360 or rotenone reduced the rate of Glu-induced Ca2+ influx ∼2-fold. This Ca2+ influx in astrocytes represents capacitative Ca2+ entry. In astrocytes with Pla2g6 mutation, the Glu-induced Ca2+ influx was ∼2-fold lower than in wild-type controls. We suggest that this is the mechanism for strongly decreased durations of Glu-induced Ca2+ responses in astrocytes with Pla2g6 mutation. We could mimic the mutation by pharmacological inhibition of iPLA2 using S-BEL. Thus, lack of VIA iPLA2 activity caused effects in astrocytes. In summary, three INAD mouse models show comparable changes in Glu-induced Ca2+ signaling, but specific for neurons or astrocytes. This finding helps to identify pathways altered during INAD and highlights non-canonical VIA iPLA2 functions, like regulation of cellular Ca2+ fluxes by mitochondria or capacitative Ca2+-entry.

Regulatory Functions of Phospholipase A2.

Phospholipase A2 (PLA2) plays crucial roles in diverse cellular responses, including phospholipid digestion and metabolism, host defense and signal transduction. PLA2 provides precursors for generation of eicosanoids, such as prostaglandins (PGs) and leukotrienes (LTs), when the cleaved fatty acid is arachidonic acid, platelet-activating factor (PAF) when the sn-1 position of the phosphatidylcholine contains an alkyl ether linkage and some bioactive lysophospholipids, such as lysophosphatidic acid (lysoPA). As overproduction of these lipid mediators causes inflammation and tissue disorders, it is extremely important to understand the mechanisms regulating the expression and functions of PLA2. Recent advances in molecular and cellular biology have enabled us to understand the molecular nature, possible function, and regulation of a variety of PLA2 isozymes. Mammalian tissues and cells generally contain more than one enzyme, each of which is regulated independently and exerts distinct functions. Here we classify mammalian PLA2s into three large groups, namely, secretory (sPLA2), cytosolic (cPLA2), and Ca2+-independent PLA2s, on the basis of their enzymatic properties and structures and focus on the general undestanding of the possible regulatory functions of each PLA2 isozyme. In particular, the roles of type II sPLA2 and cPLA2 in lipid mediator generation are discussed.

Pharmacological Characterization of the Native Store-Operated Calcium Channels of Cortical Neurons from Embryonic Mouse Brain.

In the murine brain, the first post-mitotic cortical neurons formed during embryogenesis express store-operated channels (SOCs) sensitive to Pyr3, initially proposed as a blocker of the transient receptor potential channel of C type 3 (TRPC3 channel). However, Pyr3 does not discriminate between Orai and TRPC3 channels, questioning the contribution of TRPC3 in SOCs. This study was undertaken to clarify the molecular identity and the pharmacological profile of native SOCs from E13 cortical neurons. The mRNA expression of STIM1-2 and Orai1-3 was assessed by quantitative reverse transcription polymerase chain reaction. E13 cortical neurons expressed STIM1-2 mRNAs, with STIM2 being the predominant isoform. Only transcripts of Orai2 were found but no Orai1 and Orai3 mRNAs. Blockers of Orai and TRPC channels (Pyr6, Pyr10, EVP4593, SAR7334, and GSK-7975A) were used to further characterize the endogenous SOCs. Their activity was recorded using the fluorescent Ca2+ probe Fluo-4. Cortical SOCs were sensitive to the Orai blockers Pyr6 and GSK-7975A, as well as to EVP4593, zinc, copper, and gadolinium ions, the latter one being the most potent SOCs blocker tested (IC50 ∼10 nM). SOCs were insensitive to the TRPC channel blockers Pyr10 and SAR7334. In addition, preventing mitochondrial Ca2+ uptake inhibited SOCs which were unaffected by inhibitors of the Ca2+-independent phospholipase A2. Altogether, Orai2 channels are present at the beginning of the embryonic murine cortico-genesis and form the core component of native SOCs in the immature cortex. This Ca2+ route is likely to play a role in the formation of the brain cortex.

Phospholipase A2 Isoforms as Novel Targets for Prevention and Treatment of Inflammatory and Oncologic Diseases.

Phospholipase A2s (PLA2s) are group of enzymes, which cleave phospholipids specifically at sn-2 position to liberate free fatty acid, mostly arachidonic acid (AA) and lysophospholipids (LPLs). Inhibition of PLA2 prevents the liberation of AA and LPLs. Hence, researchers have been considering PLA2s could be a better therapeutic target than the downstream enzymes cyclooxygense and lipoxygenase. Several isoforms of PLA2s exist; they are mainly divided into secretory PLA2s (sPLA2), cytosolic PLA2s (cPLA2), and calcium independent PLA2s (iPLA2), platelet activating factor- acyl hydrolase (PAF-AH), lysosomal PLA2 (LPLA2), adipose-specific PLA2 (Ad- PLA). Each isoform of PLA2s is different in its chemical structure and physiological functions. sPLA2s (Groups IIA, V and X) are well characterized as proinflammatory mediating enzymes but their role in cancer is controversial. Groups IVA, IVB and IVC cPLA2s are present in humans but only Group IVA cPLA2 plays key role in pathophysiology of various cancers and inflammation. The role of iPLA2 in inflammation and cancer is limited. Lipoprotein associated PLA2 (Group VIIA PLA2), a PAF-AH isoform, has key role in atherosclerosis. Several isoform specific PLA2 inhibitors have been developed and some of the PLA2s inhibitors are currently under clinical trials for various inflammatory and oncologic diseases. This review focuses on the recent experimental evidences to support the notion that PLA2s are causally implicated in the pathobiology of cancer and inflammatory related disorders and discuss the potential utility of isoform specific PLA2 inhibitors as preventive and/or therapeutic agents.

Translationally Controlled Tumor Protein Stimulates Dopamine Release from PC12 Cells via Ca2+-Independent Phospholipase A₂ Pathways.

The translationally controlled tumor protein (TCTP), initially identified as a tumor- and growth-related protein, is also known as a histamine-releasing factor (HRF). TCTP is widely distributed in the neuronal systems, but its function is largely uncharacterized. Here, we report a novel function of TCTP in the neurotransmitter release from a neurosecretory, pheochromocytoma (PC12) cells. Treatment with recombinant TCTP (rTCTP) enhanced both basal and depolarization (50 mM KCl)-evoked [3H]dopamine release in concentration- and time-dependent manners. Interestingly, even though rTCTP induced the increase in intracellular calcium levels ([Ca2+]i), the rTCTP-driven effect on dopamine release was mediated by a Ca2+-independent pathway, as evidenced by the fact that Ca2+-modulating agents such as Ca2+ chelators and a voltage-gated L-type Ca2+-channel blocker did not produce any changes in rTCTP-evoked dopamine release. In a study to investigate the involvement of phospholipase A2 (PLA2) in rTCTP-induced dopamine release, the inhibitor for Ca2+-independent PLA2 (iPLA2) produced a significant inhibitory effect on rTCTP-induced dopamine release, whereas this release was not significantly inhibited by Ca2+-dependent cytosolic PLA2 (cPLA2) and secretory PLA2 (sPLA2) inhibitors. We found that rTCTP-induced dopamine release from neuronal PC12 cells was modulated by a Ca2+-independent mechanism that involved PLA2 in the process, suggesting the regulatory role of TCTP in the neuronal functions.