Localisation of Formyl-Peptide Receptor 2 in the Rat Central Nervous System and Its Role in Axonal and Dendritic Outgrowth

Arachidonic acid and docosahexaenoic acid (DHA) released by the action of phospholipases A₂ (PLA₂) on membrane phospholipids may be metabolized by lipoxygenases to the anti-inflammatory mediators lipoxin A4 (LXA4) and resolvin D1 (RvD1), and these can bind to a common receptor, formyl-peptide receptor 2 (FPR2). The contribution of this receptor to axonal or dendritic outgrowth is unknown. The present study was carried out to elucidate the distribution of FPR2 in the rat CNS and its role in outgrowth of neuronal processes. FPR2 mRNA expression was greatest in the brainstem, followed by the spinal cord, thalamus/hypothalamus, cerebral neocortex, hippocampus, cerebellum and striatum. The brainstem and spinal cord also contained high levels of FPR2 protein. The cerebral neocortex was moderately immunolabelled for FPR2, with staining mostly present as puncta in the neuropil. Dentate granule neurons and their axons (mossy fibres) in the hippocampus were very densely labelled. The cerebellar cortex was lightly stained, but the deep cerebellar nuclei, inferior olivary nucleus, vestibular nuclei, spinal trigeminal nucleus and dorsal horn of the spinal cord were densely labelled. Electron microscopy of the prefrontal cortex showed FPR2 immunolabel mostly in immature axon terminals or ‘pre-terminals’, that did not form synapses with dendrites. Treatment of primary hippocampal neurons with the FPR2 inhibitors, PBP10 or WRW4, resulted in reduced lengths of axons and dendrites. The CNS distribution of FPR2 suggests important functions in learning and memory, balance and nociception. This might be due to an effect of FPR2 in mediating arachidonic acid/LXA4 or DHA/RvD1-induced axonal or dendritic outgrowth.     

Specific differential expression of phospholipase A2 subtypes in rat cerebellum

Phospholipase A₂ (PLA₂) is a family of enzymes playing diverse roles in lipid signaling in neurons and glia cells. In this study, we examined the expression of subtypes of PLA₂ in the cerebellum using immunolabeling and in situ hybridization methods. Two Ca²⁺-dependent cytosolic subtypes (cPLA₂α and cPLA₂β), one Ca²⁺-independent cytosolic subtype (iPLA₂), and two secretory subtypes (sPLA₂IIA and sPLA₂V) were detected in the cerebellum. cPLA₂α is present in somata and dendrites of Purkinje cells, while sPLA₂IIA is associated with the endoplasmic reticulum in perinuclear regions of Purkinje cell somata. iPLA₂ is present in granule cells, stellate cells and also in the nucleus of Purkinje cells. In addition, cPLA₂β is localized in granule cells, and sPLA₂V in Bergmann glia cells. These results provide an important basis for identifying functional roles of PLA₂s in the cerebellum.     

Phospholipase A2 and its Molecular Mechanism after Spinal Cord Injury

Phospholipases A₂ (PLA₂s) are a diverse family of lipolytic enzymes which hydrolyze the acyl bond at the sn-2 position of glycerophospholipids to produce free fatty acids and lysophospholipids. These products are precursors of bioactive eicosanoids and platelet-activating factor which have been implicated in pathological states of numerous acute and chronic neurological disorders. To date, more than 27 isoforms of PLA₂ have been found in the mammalian system which can be classified into four major categories: secretory PLA₂, cytosolic PLA₂, Ca²⁺-independent PLA₂, and platelet-activating factor acetylhydrolases. Multiple isoforms of PLA₂ are found in the mammalian spinal cord. Under physiological conditions, PLA₂s are involved in diverse cellular responses, including phospholipid digestion and metabolism, host defense, and signal transduction. However, under pathological situations, increased PLA₂ activity, excessive production of free fatty acids and their metabolites may lead to the loss of membrane integrity, inflammation, oxidative stress, and subsequent neuronal injury. There is emerging evidence that PLA₂ plays a key role in the secondary injury process after traumatic spinal cord injury. This review outlines the current knowledge of the PLA₂ in the spinal cord with an emphasis being placed on the possible roles of PLA₂ in mediating the secondary SCI.     

Inhibition of sPLA2-IIA Prevents LPS-Induced Neuroinflammation by Suppressing ERK1/2-cPLA2α Pathway in Mice Cerebral Cortex

Neuroinflammation is involved in various central nervous system (CNS) disorders, including brain infections, ischemia, trauma, stroke, and degenerative CNS diseases. In the CNS inflammation, secretory phospholipase A₂-IIA (sPLA₂-IIA) acts as a mediator, resulting in the generation of the precursors of pro-inflammatory lipid mediators, such as prostaglandins (PGs) and leukotrienes (LTs). However, the role of sPLA₂-IIA in neuroinflammation is more complicated and remains unclear yet. In the present study, we investigated the effect of sPLA₂-IIA inhibition by specific inhibitor SC-215 on the inflammation in LPS-induced mice cerebral cortex and primary astrocytes. Our results showed that the inhibition of sPLA₂-IIA alleviated the release of PGE₂ by suppressing the activation of ERK1/2, cPLA₂α, COX-2 and mPGES-1. These findings demonstrated that sPLA₂-IIA showed the potential to regulate the neuroinflammation in vivo and in vitro, indicating that sPLA₂-IIA might be a novel target for the treatment of acute neuroinflammation.     

Phospholipase A2-like activity of human bocavirus VP1 unique region

Human bocavirus (HBoV) is a new parvovirus first discovered in 2005, which is associated with acute respiratory infection. Analysis of sequence homology has revealed that a putative phospholipase A₂ (PLA₂) motif exists in the VP1 unique region of HBoV. However, little is known about whether the VP1 unique region of HBoV has PLA₂ enzymatic activity and how these critical residues contribute to its PLA₂ activity. To address these issues, the VP1 unique region protein and four of its mutants, were expressed in Eschericha coli. The purified VP1 unique protein (VP1U) showed a typical Ca²⁺-dependent secreted PLA₂-like (sPLA₂) activity, which was inhibited by sPLA₂-specific inhibitors in a time-dependent manner. Mutation of one of the amino acids (21Pro, 41His, 42Asp or 63Asp) in VP1U almost eliminated the sPLA₂ activity of HBoV VP1U. These data indicate that VP1U of HBoV has sPLA₂-like enzymatic activity, and these residues are crucial for its sPLA₂-like activity. Potentially, VP1U may be a target for the development of anti-viral drugs for HBoV.     

Impaired phospholipases A2 production by stimulated macrophages from patients with acute respiratory distress syndrome

The aim of this study was to investigate whether early phase of acute respiratory distress syndrome (ARDS) is associated with changes in immune response, either systemic or localized to the lung. ARDS and control mechanically ventilated patients, as well as healthy volunteers were studied. Alveolar macrophages (AMΦ) and blood monocytes (BM) were treated ex vivo with lipopolysaccharide (LPS), interferon-γ (IFNγ), and surfactant. Phospholipase A₂ (PLA₂) activity and TLR4 expression were evaluated as markers of cell response. AMΦ from ARDS patients did not respond upon treatment with either LPS or IFN-γ by inducing PLA₂ production. On the contrary, upon stimulation, in control patients the intracellular PLA₂, (mainly cPLA₂) levels were increased, but secretion of PLA₂ (mainly sPLA₂-IIA) was observed only after treatment with LPS. Surfactant suppressed PLA₂ production in cells from both groups of patients. Increased relative changes of total PLA₂ activity and an upregulation of TLR4 expression upon stimulation was observed in BM from primary ARDS, control patients and healthy volunteers. In BM from secondary ARDS patients, however, no PLA₂ induction was observed, with a concomitant down-regulation of TLR4 expression. Cytosolic PLA₂, its activated form, p-cPLA_2, and sPLA₂-IIA were the predominant PLA₂ types within the cells, while extracellularly only sPLA₂-IIA was identified. These results support the concept of down-regulated innate immunity in early ARDS that is compartmentalized in primary and systemic in secondary ARDS. PLA₂ isoforms could serve as markers of the immunity status in ARDS. Finally, our data highlight the role of surfactant in controlling inflammation.     

Phospholipase A2 in astrocytes

Astrocytes comprise the major cell type in the central nervous system (CNS) and they are essential for support of neuronal functions by providing nutrients and regulating cell-to-cell communication. Astrocytes also are immune-like cells that become reactive in response to neuronal injury. Phospholipases A₂ (PLA ₂) are a family of ubiquitous enzymes that degrade membrane phospholipids and produce lipid mediators for regulating cellular functions. Three major classes of PLA ₂ are expressed in astrocytes: group IV calcium-dependent cytosolic PLA ₂ (cPLA₂), group VI calcium-independent PLA ₂ (iPLA₂), and group II secretory PLA ₂ (sPLA₂). Upregulation of PLA ₂ in reactive astrocytes has been shown to occur in a number of neurodegenerative diseases, including stroke and Alzheimer’s disease. This review focuses on describing the effects of oxidative stress, inflammation, and activation of G protein-coupled receptors on PLA ₂ activation, arachidonic acid (AA) release, and production of prostanoids in astrocytes.     

Thematic Review Series: Phospholipases: Central Role in Lipid Signaling and Disease: A new era of secreted phospholipase A2

Among more than 30 members of the phospholipase A₂ (PLA₂) superfamily, secreted PLA₂ (sPLA₂) enzymes represent the largest family, being Ca²⁺-dependent low-molecular-weight enzymes with a His-Asp catalytic dyad. Individual sPLA₂s exhibit unique tissue and cellular distributions and enzymatic properties, suggesting their distinct biological roles. Recent studies using transgenic and knockout mice for nearly a full set of sPLA₂ subtypes, in combination with sophisticated lipidomics as well as biochemical and cell biological studies, have revealed distinct contributions of individual sPLA₂s to various pathophysiological events, including production of pro- and anti-inflammatory lipid mediators, regulation of membrane remodeling, degradation of foreign phospholipids in microbes or food, or modification of extracellular noncellular lipid components. In this review, we highlight the current understanding of the in vivo functions of sPLA₂s and the underlying lipid pathways as revealed by a series of studies over the last decade.     

Suppression of murine endotoxic shock by sPLA2 inhibitor,indoxam, through group IIA sPLA2-independent mechanisms

Endotoxic shock is a systemic inflammatory process, involving a variety of proinflammatory mediators. Two types of secretory phospholipase A₂ (sPLA₂) have been implicated in this process. Group IB sPLA₂ (PLA₂-IB) binds to the PLA₂ receptor (PLA₂R), and PLA₂R-deficient mice exhibit resistance to endotoxin-induced lethality with reduced plasma levels of proinflammatory cytokines, such as TNF-α. Group IIA sPLA₂ (PLA₂-IIA) is found in many tissues and cell types, and local and systemic levels are elevated under numerous inflammatory conditions including sepsis. In this study, we investigated the effect of a specific sPLA₂ inhibitor, indoxam, on murine endotoxic shock. Indoxam suppressed the elevation of plasma TNF-α with a similar potency in PLA₂-IIA-expressing and PLA₂-IIA-deficient mice after LPS challenge. In PLA₂-IIA-deficient mice, indoxam also suppressed the elevation of plasma IL-1β, IL-6 and NO, and prolonged survival after LPS challenge. Indoxam was found to block the PLA₂-IB binding to murine PLA₂R with a high potency (K_i=30 nM). The inhibitory effects of indoxam on the LPS-induced elevation of plasma TNF-α levels could not be observed in mice deficient in PLA₂R. These findings suggest that indoxam blocks the production of proinflammatory cytokines during endotoxemia through PLA₂-IIA-independent mechanisms, possibly via blockade of the PLA₂R function.     

Proteolysis of Apolipoprotein A-I by Secretory Phospholipase A2: A NEW LINK BETWEEN INFLAMMATION AND ATHEROSCLEROSIS*

In the acute phase of the inflammatory response, secretory phospholipase A₂ (sPLA₂) reaches its maximum levels in plasma, where it is mostly associated with high density lipoproteins (HDL). Overexpression of human sPLA₂ in transgenic mice reduces both HDL cholesterol and apolipoprotein A-I (apoA-I) plasma levels through increased HDL catabolism by an unknown mechanism. To identify unknown PLA₂-mediated activities on the molecular components of HDL, we characterized the protein and lipid products of the PLA₂ reaction with HDL. Consistent with previous studies, hydrolysis of HDL phospholipids by PLA₂ reduced the particle size without changing its protein composition. However, when HDL was destabilized in the presence of PLA₂ by the action of cholesteryl ester transfer protein or by guanidine hydrochloride treatment, a fraction of apoA-I, but no other proteins, dissociated from the particle and was rapidly cleaved. Incubation of PLA₂ with lipid-free apoA-I produced similar protein fragments in the range of 6–15 kDa, suggesting specific and direct reaction of PLA₂ with apoA-I. Mass spectrometry analysis of isolated proteolytic fragments indicated at least two major cleavage sites at the C-terminal and the central domain of apoA-I. ApoA-I proteolysis by PLA₂ was Ca²⁺-independent, implicating a different mechanism from the Ca²⁺-dependent PLA₂-mediated phospholipid hydrolysis. Inhibition of proteolysis by benzamidine suggests that the proteolytic and lipolytic activities of PLA₂ proceed through different mechanisms. Our study identifies a previously unknown proteolytic activity of PLA₂ that is specific to apoA-I and may contribute to the enhanced catabolism of apoA-I in inflammation and atherosclerosis.     

Recent progress in phospholipase A₂ research: from cells to animals to humans

Mammalian genomes encode genes for more than 30 phospholipase A₂s (PLA₂s) or related enzymes, which are subdivided into several classes including low-molecular-weight secreted PLA₂s (sPLA₂s), Ca²⁺-dependent cytosolic PLA₂s (cPLA₂s), Ca²⁺-independent PLA₂s (iPLA₂s), platelet-activating factor acetylhydrolases (PAF-AHs), lysosomal PLA₂s, and a recently identified adipose-specific PLA. Of these, the intracellular cPLA₂ and iPLA₂ families and the extracellular sPLA₂ family are recognized as the “big three”. From a general viewpoint, cPLA₂α (the prototypic cPLA₂) plays a major role in the initiation of arachidonic acid metabolism, the iPLA₂ family contributes to membrane homeostasis and energy metabolism, and the sPLA₂ family affects various biological events by modulating the extracellular phospholipid milieus. The cPLA₂ family evolved along with eicosanoid receptors when vertebrates first appeared, whereas the diverse branching of the iPLA₂ and sPLA₂ families during earlier eukaryote development suggests that they play fundamental roles in life-related processes. During the past decade, data concerning the unexplored roles of various PLA₂ enzymes in pathophysiology have emerged on the basis of studies using knockout and transgenic mice, the use of specific inhibitors, and information obtained from analysis of human diseases caused by mutations in PLA₂ genes. This review focuses on current understanding of the emerging biological functions of PLA₂s and related enzymes.     

LPA receptor expression in the central nervous system in health and following injury

Lysophosphatidic acid (LPA) is released from platelets following injury and also plays a role in neural development but little is known about its effects in the adult central nervous system (CNS). We have examined the expression of LPA receptors 1-3 (LPA_1–3) in intact mouse spinal cord and cortical tissues and following injury. In intact and injured tissues, LPA₁ was expressed by ependymal cells in the central canal of the spinal cord and was upregulated in reactive astrocytes following spinal cord injury. LPA₂ showed low expression in intact CNS tissue, on grey matter astrocytes in spinal cord and in ependymal cells lining the lateral ventricle. Following injury, its expression was upregulated on astrocytes in both cortex and spinal cord. LPA₃ showed low expression in intact CNS tissue, viz. on cortical neurons and motor neurons in the spinal cord, and was upregulated on neurons in both regions after injury. Therefore, LPA_1–3 are differentially expressed in the CNS and their expression is upregulated in response to injury. LPA release following CNS injury may have different consequences for each cell type because of this differential expression in the adult nervous system.     

The expression pattern of Follistatin-like 1 in mouse central nervous system development

Follistatin-like 1 (Fstl1), also named TSC-36 (TGF-β-stimulated clone 36), was first cloned from the mouse osteoblastic MC3T3-E1 cell line and can be up-regulated by TGF-β. To better study the function of Fstl1 during the development of the mouse central nervous system (CNS), we examined Fstl1 expression in the developing mouse CNS, in detail, by in situ hybridization. Our results show that Fstl1 is strongly expressed in the telencephalon, diencephalon, brainstem, limbic system and spinal cord. In the telencephalon, Fstl1 positive cells are mainly located in the ventricular zone (VZ) and the subventricular zone (SVZ); a relatively weak signal was observed in layers II and III of the neocortex at postnatal stages. Fstl1 expression is robust in the developing hippocampus and persists to P20. In the developing diencephalon and hindbrain, abundant Fstl1 signals were also detected in nuclei including the medial habenular nucleus, the medial dorsal nucleus, the cochlear nuclei and so on. In addition, a strong expression of Fstl1 was detected in the thalamencephalic signal center, as well as in the olfactory cortex from E14.5 to P0. Meanwhile, Fstl1 was expressed in the septal area and the cingulate gyrus of the limbic system after birth. A high level of expression was also observed in the ventral horn of the spinal cord. These results indicate that Fstl1 may play an important role during CNS development in the mouse.     

Biochemical characteristics of immune-associated phospholipase A<Subscript>2</Subscript> and its inhibition by an entomopathogenic bacterium, <Emphasis Type="Italic">Xenorhabdus nematophila</Emphasis>

An entomopathogenic bacterium, Xenorhabdus nematophila, induces an immunosuppression of target insects by inhibiting phospholipase A₂ (PLA₂) activity. Recently, an immune-associated PLA₂ gene was identified from the red flour beetle, Tribolium castaneum. This study cloned this PLA₂ gene in a bacterial expression vector to produce a recombinant enzyme. The recombinant T. castaneum PLA₂ (TcPLA₂) exhibited its characteristic enzyme activity with substrate concentration, pH, and ambient temperature. Its biochemical characteristics matched to a secretory type of PLA₂ (sPLA₂) because its activity was inhibited by dithiothreitol (a reducing agent of disulfide bond) and bromophenacyl bromide (a specific sPLA₂ inhibitor) but not by methylarachidonyl fluorophosphonate (a specific cytosolic type of PLA₂). The X. nematophila culture broth contained PLA₂ inhibitory factor(s), which was most abundant in the media obtained at a stationary bacterial growth phase. The PLA₂ inhibitory factor(s) was heat-resistant and extracted in both aqueous and organic fractions. Effect of a PLA₂-inhibitory fraction on the immunosuppression of T. castaneum was equally comparable with that resulted from inhibition of the TcPLA₂ gene expression by RNA interference.     

Platelet‐activating factor: role in long‐term potentiation and in brain damage

Brain platelet‐activating factor (PAF) is a lipid mediator involved in neurotransmission and in LTP. It has been reported that the induction of LTP by high frequency stimulation increases the activity of the enzymes responsible for its synthesis by a still unknown mechanism ( 1 ). One of the two biosynthetic pathways is Ca²⁺‐dependent and transforms a membrane ether phospholipid into PAF by a sequence of two reactions being the first one, catalyzed by a phospholipase A₂ (PLA₂), rate limiting. Overproduction of PAF, taking place in pathological conditions, contributes to brain damage. Various PLA₂s are present in brain tissue and, particularly, sPLA₂‐IIA is very likely involved in the production of PAF as its expression increases in pathological conditions. Recently, we have found the release of sPLA₂‐IIA from rat brain cortex mitochondria and its association with nuclear membranes, which might be an intracellular target for the enzyme.     

Group IID,IIE, IIF and III secreted phospholipase A2s

Among the 11 members of the secreted phospholipase A₂ (sPLA₂) family, group IID, IIE, IIF and III sPLA₂s (sPLA₂-IID, -IIE, -IIF and -III, respectively) are “new” isoforms in the history of sPLA₂ research. Relative to the better characterized sPLA₂s (sPLA₂-IB, -IIA, -V and -X), the enzymatic properties, distributions, and functions of these “new” sPLA₂s have remained obscure until recently. Our current studies using knockout and transgenic mice for a nearly full set of sPLA₂s, in combination with comprehensive lipidomics, have revealed unique and distinct roles of these “new” sPLA₂s in specific biological events. Thus, sPLA₂-IID is involved in immune suppression, sPLA₂-IIE in metabolic regulation and hair follicle homeostasis, sPLA₂-IIF in epidermal hyperplasia, and sPLA₂-III in male reproduction, anaphylaxis, colonic diseases, and possibly atherosclerosis. In this article, we overview current understanding of the properties and functions of these sPLA₂s and their underlying lipid pathways in vivo.     

Assessing Phospholipase A2 Activity toward Cardiolipin by Mass Spectrometry

Cardiolipin, a major component of mitochondria, is critical for mitochondrial functioning including the regulation of cytochrome c release during apoptosis and proper electron transport. Mitochondrial cardiolipin with its unique bulky amphipathic structure is a potential substrate for phospholipase A₂ (PLA₂) in vivo. We have developed mass spectrometric methodology for analyzing PLA₂ activity toward various cardiolipin forms and demonstrate that cardiolipin is a substrate for sPLA₂, cPLA₂ and iPLA₂, but not for Lp-PLA₂. Our results also show that none of these PLA₂s have significant PLA₁ activities toward dilyso-cardiolipin. To understand the mechanism of cardiolipin hydrolysis by PLA₂, we also quantified the release of monolyso-cardiolipin and dilyso-cardiolipin in the PLA₂ assays. The sPLA₂s caused an accumulation of dilyso-cardiolipin, in contrast to iPLA₂ which caused an accumulation of monolyso-cardiolipin. Moreover, cardiolipin inhibits iPLA₂ and cPLA₂, and activates sPLA₂ at low mol fractions in mixed micelles of Triton X-100 with the substrate 1-palmitoyl-2-arachidonyl-sn-phosphtidylcholine. Thus, cardiolipin functions as both a substrate and a regulator of PLA₂ activity and the ability to assay the various forms of PLA₂ is important in understanding its function.     

Genes encoding phospholipases A2 mediate insect nodulation reactions to bacterial challenge

We propose that expression of four genes encoding secretory phospholipases A₂ (sPLA₂) mediates insect nodulation responses to bacterial infection. Nodulation is the quantitatively predominant cellular defense reaction to bacterial infection. This reaction is mediated by eicosanoids, the biosynthesis of which depends on PLA₂-catalyzed hydrolysis of arachidonic acid (AA) from cellular phospholipids. Injecting late instar larvae of the red flour beetle, Tribolium castaneum, with the bacterium, Escherichia coli, stimulated nodulation reactions and sPLA₂ activity in time- and dose-related manners. Nodulation was inhibited by pharmaceutical inhibitors of enzymes involved in eicosanoid biosynthesis, and the inhibition was rescued by AA. We cloned five genes encoding sPLA₂ and expressed them in E. coli cells to demonstrate these genes encode catalytically active sPLA₂s. The recombinant sPLA₂s were inhibited by sPLA₂ inhibitors. Injecting larvae with double-stranded RNAs specific to each of the five genes led to reduced expression of the corresponding sPLA₂ genes and to reduced nodulation reactions to bacterial infections for four of the five genes. The reduced nodulation was rescued by AA, indicating that expression of four genes encoding sPLA₂s mediates nodulation reactions. A polyclonal antibody that reacted with all five sPLA₂s showed the presence of the sPLA₂ enzymes in hemocytes and revealed that the enzymes were more closely associated with hemocyte plasma membranes following infection. Identifying specific sPLA₂ genes that mediate nodulation reactions strongly supports our hypothesis that sPLA₂s are central enzymes in insect cellular immune reactions.