Phospholipase A2 in the central nervous system: implications for neurodegenerative diseases

Phospholipase A2 (PLA2) belongs to a family of enzymes that catalyze the cleavage of fatty acids from the sn-2 position of phospholipids. There are more than 19 different isoforms of PLA2 in the mammalian system, but recent studies have focused on three major groups, namely, the group IV cytosolic PLA2, the group II secretory PLA2 (sPLA2), and the group VI Ca(2+)-independent PLA2. These PLA2s are involved in a complex network of signaling pathways that link receptor agonists, oxidative agents, and proinflammatory cytokines to the release of arachidonic acid (AA) and the synthesis of eicosanoids. PLA2s acting on membrane phospholipids have been implicated in intracellular membrane trafficking, differentiation, proliferation, and apoptotic processes. All major groups of PLA2 are present in the central nervous system (CNS). Therefore, this review is focused on PLA2 and AA release in neural cells, especially in astrocytes and neurons. In addition, because many neurodegenerative diseases are associated with increased oxidative and inflammatory responses, an attempt was made to include studies on PLA2 in cerebral ischemia, Alzheimer's disease, and neuronal injury due to excitotoxic agents. Information from these studies has provided clear evidence for the important role of PLA2 in regulating physiological and pathological functions in the CNS.     

Role of Cytosolic Calcium-Dependent Phospholipase A2 in Alzheimer's Disease Pathogenesis

Phospholipases (PLA2s) are a superfamily of enzymes characterized by the ability to specifically hydrolyze the sn-2 ester bond of phospholipids generating arachidonic acid, utilized in inflammatory responses, and lysophospholipids involved in the control of cell membrane remodeling and fluidity. PLA2s have been so far considered a crucial element in the etiopathogenesis of several neurological diseases such as cerebral ischemia, multiple sclerosis, Parkinson's disease, and Alzheimer's disease (AD). In AD, the role of beta-amyloid (Aβ) fragments is well established although still more elusive are the molecular events of the cascade that from the Aβ accumulation leads to neurodegeneration with its clinical manifestations. However, it is well known that inflammation and alteration of lipid metabolism are common features of AD brains. Findings obtained from in vitro studies, animal models, and human brain imaging analysis point towards cPLA2 as a key molecule in the onset and maintenance of the neurodegenerative mechanism(s) of AD. In this review, we have focused on the molecular and biological evidence of the involvement of cPLA2s in the pathogenesis of AD. An insight into the molecular mechanism(s) underlying the action and the regulation of cPLA2 is of tremendous interest in the pharmaceutical and biotechnology industry in developing selective and potent inhibitors able to modulate the onset and/or the outcome of AD.     

Development of a second generation of inhibitors of microsomal prostaglandin E synthase 1 expression bearing the gamma-hydroxybutenolide scaffold

Petrosaspongiolide M (PM), a marine sesterterpene metabolite bearing the gamma-hydroxybutenolide scaffold and displaying a potent inhibitory activity toward PLA(2) enzyme, was selected by us as an attractive target in order to explore its mechanism of action at molecular level. In the course of our investigations we decided to synthetically modify the parent compound to clarify the structural determinants responsible for the activity; in fact, very recently, our research group reported the synthesis and the pharmacological properties of a first collection of PM analogues generated by Ludi approach. The synthesized compounds showed a poor or moderate activity toward PLA(2) enzymes, nevertheless we discovered a potent and selective modulator of the expression of microsomal prostaglandin E synthase 1 (mPGES-1), an enzyme highly involved in the inflammatory response, which represents an interesting target for the development of a new class of anti-inflammatory agents. In this paper we report the synthesis of a further collection of nine analogues, having the same scaffold of PM, the gamma-hydroxybutenolide, and bearing, as side chain, more complex aromatic portions, in substitution of the sesterterpene moiety. Their pharmacological behavior against PLA(2) enzymes as well as to modulate the expression of inducible cyclooxygenase 2 (COX-2) and mPGES-1 enzymes is also described.     

Phospholipases A2 and neural membrane dynamics: implications for Alzheimer's disease

Phospholipases A(2) (PLA(2)s) are essential enzymes in cells. They are not only responsible for maintaining the structural organization of cell membranes, but also play a pivotal role in the regulation of cell functions. Activation of PLA(2) s results in the release of fatty acids and lysophospholipids, products that are lipid mediators and compounds capable of altering membrane microdomains and physical properties. Although not fully understood, recent studies have linked aberrant PLA(2) activity to oxidative signaling pathways involving NADPH oxidase that underlie the pathophysiology of a number of neurodegenerative diseases. In this paper, we review studies describing the involvement of cytosolic PLA(2) in oxidative signaling pathways leading to neuronal impairment and activation of glial cell inflammatory responses. In addition, this review also includes information on the role of cytosolic PLA(2) and exogenous secretory PLA(2) on membrane physical properties, dynamics, and membrane proteins. Unraveling the mechanisms that regulate specific types of PLA(2)s and their effects on membrane dynamics are important prerequisites towards understanding their roles in the pathophysiology of Alzheimer's disease, and in the development of novel therapeutics to retard progression of the disease.     

Asp49 phospholipase A(2)-elaidoylamide complex: a new mode of inhibition

The inhibition of phospholipase A(2)s (PLA(2)s) is of pharmacological and therapeutic interest because these enzymes are involved in several inflammatory diseases. Elaidoylamide is a powerful inhibitor of a neurotoxic PLA(2) from the Vipera ammodytes meridionalis venom. The X-ray structure of the enzyme-inhibitor complex reveals a new mode of Asp49 PLA(2) inhibition by a fatty acid hydrocarbon chain. The structure contains two identical homodimers in the asymmetric unit. In each dimer one subunit is rotated by 180 degrees with respect to the other and the two molecules are oriented head-to-tail. One molecule of elaidoylamide is bound simultaneously to the substrate binding sites of two associated neurotoxic phospholipase A(2) molecules. The inhibitor binds symmetrically to the hydrophobic channels of the two monomers. The structure can be used to design anti-inflammatory drugs.     

Secretory phospholipases A2 induce beta-glucuronidase release and IL-6 production from human lung macrophages

Secretory phospholipases A2 (sPLA2s) are a group of extracellular enzymes that release fatty acids at the sn-2 position of phospholipids. Group IIA sPLA2 has been detected in inflammatory fluids, and its plasma level is increased in inflammatory diseases. To investigate a potential mechanism of sPLA2-induced inflammation we studied the effect of group IA (from cobra venom) and group IIA (human synovial) sPLA2s on human macrophages. Both sPLA2s induced a concentration- and Ca2+-dependent, noncytotoxic release of beta-glucuronidase (16.2 +/- 2.4% and 13.1 +/- 1.5% of the total content with groups IA and IIA, respectively). Both sPLA2s also increased the rate of secretion of IL-6 and enhanced the expression of IL-6 mRNA. Preincubation of macrophages with inhibitors of the hydrolytic activity of sPLA2 or cytosolic PLA2 did not influence the release of beta-glucuronidase. Incubation of macrophages with p-aminophenyl-mannopyranoside-BSA (mp-BSA), a ligand of the mannose receptor, also resulted in beta-glucuronidase release. However, while preincubation of macrophages with mp-BSA had no effect on beta-glucuronidase release induced by group IIA sPLA2, it enhanced that induced by group IA sPLA2. A blocking Ab anti-mannose receptor inhibited both mp-BSA- and group IIA-induced beta-glucuronidase release. Taken together, these data indicate that group IA and IIA sPLA2s activate macrophages with a mechanism independent from their enzymatic activities and probably related to the activation of the mannose receptor or sPLA2-specific receptors. The secretion of enzymes and cytokines induced by sPLA2s from human macrophages may play an important role in inflammation and tissue damage associated with the release of sPLA2s.     

Human retinal pigment epithelium secretes a phospholipase A2 and contains two novel intracellular phospholipases A2.

The sensitivity of different phospholipase A2 (PLA2)-active fractions eluted from cation-exchange chromatography to para-bromophenacylbromide (pBPB), Ca2+-EGTA, DTT, heat, and H2SO4 indicates that human cultured retinal pigment epithelial (hRPE) cells probably contain two different intracellular PLA2 enzymes. Control experiments using "back-and-forth" thin-layer chromatography confirmed that, in our assay conditions, the generation of free fatty acids originated solely from PLA2 activity. Together with immunoblot experiments where no cross-reactivity was observed between the hRPE cytosolic PLA2 enzymes and several antisera directed against secretory PLA2s (sPLA2s) and cytosolic PLA2 (cPLA2), these findings suggest that intracellular hRPE PLA2s are different from well-known sPLA2s, cPLA2, and Ca2+-independent PLA2s. We also report an additional hRPE-PLA2 enzyme that is secreted and that exhibits sensitivity to pBPB, Ca2+-EGTA, DTT, heat, and H2SO4, which is characteristic of sPLA2 enzymes. This approximately 22-kDa PLA2 cross-reacted weakly with an antiserum directed against porcine pancreatic group I sPLA2 but strongly with an antiserum directed against N-terminal residues 1-14 of human synovial group II sPLA2, suggesting that this extracellular enzyme is a member of the sPLA2 class of enzymes. We thus conclude that there are three distinct PLA2 enzymes in cultured hRPE cells, including two novel intracellular PLA2s and a 22-kDa secreted sPLA2 enzyme.     

Control of phospholipase A2 activities for the treatment of inflammatory conditions

Phospholipase-A2 (PLA2) enzymes hydrolyze cell membrane phospholipids to produce arachidonic acid (AA) and lyso-phospholipids (LysoPL), playing a key role in the production of inflammatory lipid mediators, mainly eicosanoids. They are therefore considered pro-inflammatory enzymes and their inhibition has long been recognized as a desirable therapeutic target. However, attempts to develop suitable PLA2 inhibitors for the treatment of inflammatory diseases have yet to succeed. This is due to their functional and structural diversity, and their homeostatic and even anti-inflammatory roles in certain circumstances. In the present review we outline the diversity and functions of PLA2 isoforms, and their interplay in the induction and inhibition of inflammatory processes, with emphasis on discussing approaches for therapeutic manipulation of PLA2 activities.     

The mechanisms of action of interleukin-1 on rabbit intestinal epithelial cells

Interleukin-1 (IL-1) is an inflammatory mediator that increases Cl- secretion in intestinal epithelial cells. To identify the signal transduction pathway(s) involved in IL-1's action, cells were treated with IL-1 and the levels of cyclooxygenase (COX) enzymes, prostaglandin E2 (PGE2) and phospholipase A2-activating protein (PLAP), and the activity of phospholipase A2 (PLA2) were measured. IL-1 caused concentration- and time-dependent increases in the levels of PLA2 activity, and/or in the levels of PLAP, COX-2 and PGE2. The IL-induced increase in PGE2 levels was biphasic, with the first peak due to the increase in PLAP levels, and the second peak due to the increase in COX-2 levels. This increase in PGE2 levels may provide a mechanism for acute and chronic inflammation in the intestine.     

Effects of betamethasone,sulindac and quinacrine drugs on the inflammatory neoangiogenesis response induced by polyurethane sponge implanted in mouse

In this study, we showed the effect of the betamethasone, sulindac and quinacrine alone or combined, on the inflammatory angiogenesis promoted by polyurethane sponge on mice. The main finding reported here is that the formation of new blood vessels was strongly inhibited by low concentration of betamethasone, sulindac or quinacrine, whether alone or in combination. It is known that steroidal anti-inflammatory drugs inhibit the enzymes required for the production of prostaglandins through a nuclear glucocorticoid receptor (GR) mediated mechanism. This mechanism may occur in endothelial cells as well. Considering that activity of cyclo-oxigenases 1 and 2 is inhibited by sulindac, and that these enzymes are located in the stromal tissue, we propose that the anti-angiogenic effect of these agents may occur via inhibition of both COX isoforms. On the other hand, quinacrine inhibited PLA2 activity, and we propose here that the anti-angiogenic effect occurs via inhibition of the enzyme PLA2. The potentiated effect of the association of betamethasone, sulindac and quinacrine may have some therapeutic benefit in the control of pathological angiogenesis. Further studies are required to validate these propositions.     

Phospholipase A(2) isoforms: a perspective

Several new PLA(2)s have been identified based on their nucleotide gene sequences. They were classified mainly into three groups: cytosolic PLA(2) (cPLA(2)), secretary PLA(2) (sPLA(2)), and intracellular PLA(2) (iPLA(2)). They differ from each other in terms of substrate specificity, Ca(2+) requirement and lipid modification. The questions that still remain to be addressed are the subcellular localization and differential regulation of the isoforms in various cell types and under different physiological conditions. It is required to identify the downstream events that occur upon PLA(2) activation, particularly target protein or metabolic pathway for liberated arachidonic acid or other fatty acids. Understanding the same will greatly help in the development of potent and specific pharmacological modulators that can be used for basic research and clinical applications.The information of the human and other genomes of PLA(2)s, combined with the use of proteomics and genetically manipulated mouse models of different diseases, will illuminate us about the specific and potentially overlapping roles of individual phospholipases as mediators of physiological and pathological processes. Hopefully, such understanding will enable the development of specific agents aimed at decreasing the potential contribution of individual secretary phospholipases to vascular diseases.The signaling cascades involved in the activation of cPLA(2) by mitogen activated protein kinases (MAPKs) is now evident. It has been demonstrated that p44 MAPK phosphorylates cPLA(2) and increases its activity in cells and tissues. The phosphorylation of cPLA(2) at ser505 occurs before the increase in intracellular Ca(2+) that facilitate the binding of the lipid binding domain of cPLA(2) to phospholipids, promoting its translocation to cellular membranes and AA release. Recently, a negative feed back loop for cPLA(2) activation by MAPK has been proposed. If PLA(2) activation in a given model depends on PKC, PKA, cAMP, or MAPK then inhibition of these phosphorylating enzymes may alter activities of PLA(2) isoforms during cellular injury. Understanding the signaling pathways involved in the activation/deactivation of PLA(2) during cellular injury will point to key events that can be used to prevent the cellular injury. Furthermore, to date, there is limited information available regarding the regulation of iPLA(2) or sPLA(2) by these pathways.     

Emerging role and therapeutic implication of mTOR signalling in intervertebral disc degeneration

Intervertebral disc degeneration (IDD), an important cause of chronic low back pain (LBP), is considered the pathological basis for various spinal degenerative diseases. A series of factors, including inflammatory response, oxidative stress, autophagy, abnormal mechanical stress, nutritional deficiency, and genetics, lead to reduced extracellular matrix (ECM) synthesis by intervertebral disc (IVD) cells and accelerate IDD progression. Mammalian target of rapamycin (mTOR) is an evolutionarily conserved serine/threonine kinase that plays a vital role in diverse degenerative diseases. Recent studies have shown that mTOR signalling is involved in the regulation of autophagy, oxidative stress, inflammatory responses, ECM homeostasis, cellular senescence, and apoptosis in IVD cells. Accordingly, we reviewed the mechanism of mTOR signalling in the pathogenesis of IDD to provide innovative ideas for future research and IDD treatment.     

Bactericidal properties of group IIA and group V phospholipases A2

Group V phospholipase A(2) (PLA(2)) is a recently characterized 14-kDa secretory PLA(2) of mammalian heart and macrophage-derived cells. Group IIA PLA(2), which is structurally close to group V PLA(2), has been shown to kill Gram-positive bacteria in vitro and to prevent symptoms of Gram-positive infection in vivo. We studied the antibacterial properties of fully active recombinant rat group IIA and V PLA(2)s. Both group IIA and V PLA(2)s were highly bactericidal against Gram-positive bacteria, including methicillin-resistant staphylococci and vancomycin-resistant enterococci. Only high concentrations of group IIA PLA(2) showed some bactericidal effect against the Gram-negative bacterium Escherichia coli. Our results confirm that group IIA PLA(2) is a potent antibacterial enzyme against Gram-positive bacteria. Moreover, we show here that group V PLA(2) is a novel antibacterial mammalian protein, but is less potent than group IIA PLA(2). Both enzymes may be considered as future therapeutic agents against bacterial infections.     

Assay of phospholipases A(2) and their inhibitors by kinetic analysis in the scooting mode

Several cellular processes are regulated by interfacial catalysis on biomembrane surfaces. Phospholipases A(2) (PLA(2)) are interesting not only as prototypes for interfacial catalysis, but also because they mobilize precursors for the biosynthesis of eicosanoids and platelet activating factor, and these agents ultimately control a wide range of secretory and inflammatory processes. Since PLA(2) carry out their catalytic function at membrane surfaces, the kinetics of these enzymes depends on what the enzyme 'sees' at the interface, and thus the observed rate is profoundly influenced by the organization and dynamics of the lipidwater interface ('quality of the interface'). In this review we elaborate the advantages of monitoring interfacial catalysis in the scooting mode, that is, under the conditions where the enzyme remains bound to vesicles for several thousand catalytic turnover cycles. Such a highly processive catalytic turnover in the scooting mode is useful for a rigorous and quantitative characterization of the kinetics of interfacial catalysis. This analysis is now extended to provide insights into designing strategy for PLA(2) assays and screens for their inhibitors.     

Cholera toxin B accelerates disease progression in lupus-prone mice by promoting lipid raft aggregation

Infectious agents, including bacteria and viruses, are thought to provide triggers for the development or exacerbation of autoimmune diseases such as systemic lupus erythematosus in the genetically predisposed individual. Molecular mimicry and engagement of TLRs have been assigned limited roles that link infection to autoimmunity, but additional mechanisms are suspected to be involved. In this study we show that T cells from lupus-prone mice display aggregated lipid rafts that harbor signaling, costimulatory, inflammatory, adhesion, and TLR molecules. The percentage of T cells with clustered lipid rafts increases with age and peaks before the development of lupus pathology. We show that cholera toxin B, a component of Vibrio cholerae, promotes autoantibody production and glomerulonephritis in lupus-prone mice by enhancing lipid raft aggregation in T cells. In contrast, disruption of lipid raft aggregation results in delay of disease pathology. Our results demonstrate that lipid rafts contribute significantly to the pathogenesis of lupus and provide a novel mechanism whereby aggregated lipid rafts represent a potential link between infection and autoimmunity.     

Role of cytochrome P450 in phospholipase A2- and arachidonic acid-mediated cytotoxicity

Phospholipases A2 (PLA2) comprise a set of extracellular and intracellular enzymes that catalyze the hydrolysis of the sn-2 fatty acyl bond of phospholipids to yield fatty acids and lysophospholipids. The PLA2 reaction is the primary pathway through which arachidonic acid (AA) is released from phospholipids. PLA2s have an important role in cellular death that occurs via necrosis or apoptosis. Several reports support the hypothesis that unesterified arachidonic acid in cells is a signal for the induction of apoptosis. However, most of the biological effects of arachidonic acid are attributable to its metabolism by mainly three different groups of enzymes: cytochromes P450, cyclooxygenases, and lipoxygenases. In this review we will focus on the role of cytochrome P450 in AA metabolism and toxicity. The major pathways of arachidonic acid metabolism catalyzed by cytochrome P450 generate metabolites that are subdivided into two groups: the epoxyeicosatrienoic acids, formed by CYP epoxygenases, and the arachidonic acid derivatives that are hydroxylated at or near the omega-terminus by CYP omega-oxidases. In addition, autoxidation of AA by cytochrome P450-derived reactive oxygen species produces lipid hydroperoxides as primary oxidation products. In some cellular models of toxicity, cytochrome P450 activity exacerbates PLA2- and AA-dependent injury, mainly through the production of oxygen radicals that promote lipid peroxidation or production of metabolites that alter Ca2+ homeostasis. In contrast, in other situations, cytochrome P450 metabolism of AA is protective, mainly by lowering levels of unesterified AA and by production of metabolites that activate antiapoptotic pathways. Several lines of evidence point to the combined action of phospholipase A2 and cytochrome P450 as central in the mechanism of cellular injury in several human diseases, such as alcoholic liver disease and myocardial reperfusion injury. Inhibition of specific PLA2 and cytochrome P450 isoforms may represent novel therapeutic strategies against these diseases.     

Singlet oxygen is essential for neutrophil extracellular trap formation

Neutrophil extracellular traps (NETs) that bind invading microbes are pivotal for innate host defense. There is a growing body of evidence for the significance of NETs in the pathogenesis of infectious and inflammatory diseases, but the mechanism of NET formation remains unclear. Previous observation in neutrophils of chronic granulomatous disease (CGD) patients, which defect NADPH oxidase (Nox) and fail to produce reactive oxygen species (ROS), revealed that ROS contributed to the formation of NETs. However, the active species were not identified. In this study, we discovered that singlet oxygen, one of the ROS, mediated Nox-dependent NET formation upon stimulation with phorbol myristate acetate. We also revealed that singlet oxygen itself could induce NET formation by a distinct system generating singlet oxygen with porfimer sodium (Photofrin) in CGD neutrophils, as well as healthy neutrophils. This was independent of Nox activation. These results show that singlet oxygen is essential for NET formation, and provide novel insights into the pathogenesis of infectious and inflammatory diseases.     

Endoplasmic reticulum- and Golgi-localized phospholipase A2 plays critical roles in Arabidopsis pollen development and germination

The phospholipase A(2) (PLA(2)) superfamily of lipolytic enzymes is involved in a number of essential biological processes, such as inflammation, development, host defense, and signal transduction. Despite the proven involvement of plant PLA(2)s in many biological functions, including senescence, wounding, elicitor and stress responses, and pathogen defense, relatively little is known about plant PLA(2)s, and their genes essentially remain uncharacterized. We characterized three of four Arabidopsis thaliana PLA(2) paralogs (PLA(2)-β, -γ, and -δ) and found that they (1) are expressed during pollen development, (2) localize to the endoplasmic reticulum and/or Golgi, and (3) play critical roles in pollen development and germination and tube growth. The suppression of PLA(2) using the RNA interference approach resulted in pollen lethality. The inhibition of pollen germination by pharmacological PLA(2) inhibitors was rescued by a lipid signal molecule, lysophosphatidyl ethanolamine. Based on these results, we propose that plant reproduction, in particular, male gametophyte development, requires the activities of the lipid-modifying PLA(2)s that are conserved in other organisms.     

Negative feedback between secretory and cytosolic phospholipase A2 and their opposing roles in ovalbumin-induced bronchoconstriction in rats

Phospholipase A2 (PLA2) hydrolyzes cell membrane phospholipids (PL) to produce arachidonic acid and lyso-PL. The PLA2 enzymes include the secretory (sPLA2) and cytosolic (cPLA2) isoforms, which are assumed to act synergistically in production of eicosanoids that are involved in inflammatory processes. However, growing evidence raises the possibility that in airways and asthma-related inflammatory cells (eosinophils, basophils), the production of the bronchoconstrictor cysteinyl leukotrienes (CysLT) is linked exclusively to sPLA2, whereas the bronchodilator prostaglandin PGE2 is produced by cPLA2. It has been further reported that the capacity of airway epithelial cells to produce CysLT is inversely proportional to PGE2 production. This seems to suggest that sPLA2 and cPLA2 play opposing roles in asthma pathophysiology and the possibility of a negative feedback between the two isoenzymes. To test this hypothesis, we examined the effect of a cell-impermeable extracellular sPLA2 inhibitor on bronchoconstriction and PLA2 expression in rats with ovalbumin (OVA)-induced asthma. It was found that OVA-induced bronchoconstriction was associated with elevation of lung sPLA2 expression and CysLT production, concomitantly with suppression of cPLA2 expression and PGE2 production. These were reversed by treatment with the sPLA2 inhibitor, resulting in amelioration of bronchoconstriction and reduced CysLT production and sPLA2 expression, concomitantly with enhanced PGE2 production and cPLA2 expression. This study demonstrates, for the first time in vivo, a negative feedback between sPLA2 and cPLA2 and assigns opposing roles for these enzymes in asthma pathophysiology: sPLA2 activation induces production of the bronchoconstrictor CysLT and suppresses cPLA2 expression and the subsequent production of the bronchodilator PGE2.