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.     

Integrating Cytosolic Phospholipase A2 with Oxidative/Nitrosative Signaling Pathways in Neurons: A Novel Therapeutic Strategy for AD

The pathophysiology of Alzheimer's disease (AD) is comprised of complex metabolic abnormalities in different cell types in the brain. To date, there are not yet effective drugs that can completely inhibit the pathophysiological event, and efforts have been devoted to prevent or minimize the progression of this disease. Much attention has focused on studies to understand aberrant functions of the ionotropic glutamate receptors, perturbation of calcium homeostasis, and toxic effects of oligomeric amyloid beta peptides (Aβ) which results in production of reactive oxygen and nitrogen species and signaling pathways, leading to mitochondrial dysfunction and synaptic impairments. Aberrant phospholipase A(2) (PLA(2)) activity has been implicated to play a role in the pathogenesis of many neurodegenerative diseases, including AD. However, mechanisms for their modes of action and their roles in the oxidative and nitrosative signaling pathways have not been firmly established. In this article, we review recent studies providing a metabolic link between cytosolic PLA(2) (cPLA(2)) and neuronal excitation due to stimulation of ionotropic glutamate receptors and toxic Aβ peptides. The requirements for Ca(2+) binding together with its posttranslational modifications by protein kinases and possible by the redox-based S-nitrosylation, provide strong support for a dynamic role of cPLA(2) in serving multiple functions to neurons and glial cells under abnormal physiological and pathological conditions. Therefore, understanding mechanisms for cPLA(2) in the oxidative and nitrosative pathways in neurons will allow the development of novel therapeutic targets to mitigate the detrimental effects of AD.     

The roles of NADPH oxidase and phospholipases A2 in oxidative and inflammatory responses in neurodegenerative diseases

Reactive oxygen species (ROS) are produced in mammalian cells through enzymic and non-enzymic mechanisms. Although some ROS production pathways are needed for specific physiological functions, excessive production is detrimental and is regarded as the basis of numerous neurodegenerative diseases. Among enzymes producing superoxide anions, NADPH oxidase is widespread in mammalian cells and is an important source of ROS in mediating physiological and pathological processes in the cardiovascular and the CNS. ROS production is linked to the alteration of intracellular calcium homeostasis, activation of Ca(2+)-dependent enzymes, alteration of cytoskeletal proteins, and degradation of membrane glycerophospholipids. There is evolving evidence that ROS produced by NADPH oxidase regulate neuronal functions and degrade membrane phospholipids through activation of phospholipases A(2) (PLA(2)). This review is intended to cover recent studies describing ROS generation from NADPH oxidase in the CNS and its downstream activation of PLA(2), namely, the group IV cytosolic cPLA(2) and the group II secretory sPLA(2). A major focus is to elaborate the dual role of NADPH oxidase and PLA(2) in mediating the oxidative and inflammatory responses in neurodegenerative diseases, including cerebral ischemia and Alzheimer's disease. Elucidation of the signaling pathways linking NADPH oxidase with the multiple forms of PLA(2) will be important in understanding the oxidative and degradative mechanisms that underline neuronal damage and glial activation and will facilitate development of therapeutic intervention for prevention and treatment of these and other neurodegenerative diseases.     

Targeting NADPH Oxidase and Phospholipases A2 in Alzheimer’s Disease

Alzheimer’s disease (AD) is marked by an increase in the production of extracellular beta amyloid plaques and intracellular neurofibrillary tangles associated with a decline in brain function. Increases in oxidative stress are regarded as an early sign of AD pathophysiology, although the source of reactive oxygen species (ROS) and the mechanism(s) whereby beta amyloid peptides (Aβ) impact oxidative stress have not been adequately investigated. Recent studies provide strong evidence for the involvement of NADPH oxidase and its downstream oxidative signaling pathways in the toxic effects elicited by Aβ. ROS produced by NADPH oxidase activate multiple signaling pathways leading to neuronal excitotoxicity and glial cell-mediated inflammation. This review describes recent studies demonstrating the neurotoxic effects of Aβ in conjunction with ROS produced by NADPH oxidase and the downstream pathways leading to activation of cytosolic phospholipase A₂ (PLA₂) and secretory PLA₂. In addition, this review also describes recent studies using botanical antioxidants to protect against oxidative damage associated with AD. Investigating the metabolic and signaling pathways involving Aβ NADPH oxidase and PLA₂ can help understand the mechanisms underlying the neurodegenerative effects of oxidative stress in AD. This information should provide new therapeutic approaches for prevention of this debilitating disease.     

Deletion of cytosolic phospholipase A2 promotes striated muscle growth

Generation of arachidonic acid by the ubiquitously expressed cytosolic phospholipase A2 (PLA2) has a fundamental role in the regulation of cellular homeostasis, inflammation and tumorigenesis. Here we report that cytosolic PLA2 is a negative regulator of growth, specifically of striated muscle. We find that normal growth of skeletal muscle, as well as normal and pathologic stress-induced hypertrophic growth of the heart, are exaggerated in Pla2g4a-/- mice, which lack the gene encoding cytosolic PLA2. The mechanism underlying this phenotype is that cytosolic PLA2 negatively regulates insulin-like growth factor (IGF)-1 signaling. Absence of cytosolic PLA2 leads to sustained activation of the IGF-1 pathway, which results from the failure of 3-phosphoinositide-dependent protein kinase (PDK)-1 to recruit and phosphorylate protein kinase C (PKC)-zeta, a negative regulator of IGF-1 signaling. Arachidonic acid restores activation of PKC-zeta, correcting the exaggerated IGF-1 signaling. These results indicate that cytosolic PLA2 and arachidonic acid regulate striated muscle growth by modulating multiple growth-regulatory pathways.     

Calcium-independent phospholipase A2 through arachidonic acid mobilization is involved in Caco-2 cell growth

Several studies indicate that phospholipase A(2) (PLA(2)) expression and/or activation account for the high levels of arachidonic acid (AA) detected in cancer and, together with the elevated expression of cyclooxygenase-2, lead to cell proliferation and tumor formation. Using Caco-2 cells, a human colorectal carcinoma cell, we studied the role of high-molecular-weight PLA(2)s, cytosolic PLA(2) (cPLA(2)), and calcium-independent PLA(2) (iPLA(2)) in the AA cascade and in cell growth. Treatment with an antisense oligonucleotide against cPLA(2)alpha decreased [(3)H]AA release induced by ionophore A23187 or by a phorbol ester but did not affect the release of [(3)H]AA, [(3)H]thymidine incorporation, or Caco-2 growth induced by fetal calf serum (FCS). However, these parameters were significantly modified by iPLA(2) inhibitors and by an antisense oligonucleotide against iPLA(2)beta. Our results show that iPLA(2) was involved in AA release and the subsequent prostaglandin production induced by serum. Moreover, these data indicate that iPLA(2) may be involved in the signaling pathways involved in the control of Caco-2 proliferation.     

Structure, function, and regulation of group V phospholipase A(2)

The hydrolysis of membrane phospholipid by phospholipase A(2) (PLA(2)) is a key step in the production of inflammatory eicosanoids. Recent cell studies have shown that secretory group V PLA(2) (gVPLA(2)) is involved in agonist-induced eicosanoid biosynthesis in mouse P388D1 cell line, mast cells, and transfected HEK 293 cells. gVPLA(2) is homologous to other group II PLA(2) family members but has distinctive enzymatic properties, including its activity to effectively hydrolyze phosphatidylcholine (PC) vesicles and the outer plasma membrane of mammalian cells. Mutational studies showed that gVPLA(2) has a unique structure that allows effective binding to PC membranes and efficient catalysis of an active-site-bound PC substrate. Thanks to this unique structure and activity, exogenously added gVPLA(2) can induce the eicosanoid biosynthesis in unstimulated inflammatory cells, including human neutrophils and eosinophils, suggesting that it might be able to trigger inflammatory responses under certain physiological conditions. Extensive structure-function and cell studies showed that gVPLA(2) could act directly on the outer plasma membranes of neutrophils and eosinophils. The release of fatty acids and lysophospholipids from the cell surfaces induces the translocation and activation of cytosolic PLA(2) and 5-lipoxygenase, resulting in the leukotriene synthesis. In case of neutrophils, induction of leukotriene B(4) synthesis by gVPLA(2) leads to the phosphorylation of cytosolic PLA(2) by a leukotriene B(4) receptor and MAP kinase-mediated mechanism. Finally, heparan sulfate proteoglycans in neutrophils appear to play a role of internalizing and degrading the cell surface-bound gVPLA(2) to protect the cells from extensive lipolytic damage.     

Van Gogh-like2 (Strabismus) and its role in planar cell polarity and convergent extension in vertebrates

In the past two years, studies of Stbm genes (also known as Vangl2) and the proteins that they encode in mice, flies, frogs and fish have shown that they have a crucial role in regulating planar cell polarity and convergent extension movements. Combined genetic and biochemical analyses have pointed to signaling pathways where Stbm (Vangl2) proteins might act, and have identified several interacting proteins that form a crucial multi-protein signaling complex at the membrane. These studies show that these proteins have a pivotal role in a signaling cascade(s) that has been highly conserved in evolution. This review will summarize recent findings documenting the involvement of Stbm (Vangl2) and associated proteins in planar cell polarity, non-canonical Wnt signaling and convergent extension movements.     

Studies on a mechanism by which cytosolic phospholipase A2 regulates the expression and function of type IIA secretory phospholipase A2

Although it has been proposed that arachidonate release by several secretory phospholipase A2 (sPLA2) isozymes is modulated by cytosolic PLA2 (cPLA2), the cellular component(s) that intermediates between these two signaling PLA2s remains unknown. Here we provide evidence that 12- or 15-lipoxygenase (12/15-LOX), which lies downstream of cPLA2, plays a pivotal role in cytokine-induced gene expression and function of sPLA2-IIA. The sPLA2-IIA expression and associated PGE2 generation induced by cytokines in rat fibroblastic 3Y1 cells were markedly attenuated by antioxidants that possess 12/15-LOX inhibitory activity. 3Y1 cells expressed 12/15-LOX endogenously, and forcible overexpression of 12/15-LOX in these cells greatly enhanced cytokine-induced expression of sPLA2-IIA, with a concomitant increase in delayed PG generation. Moreover, studies using 293 cells stably transfected with sPLA2-IIA revealed that stimulus-dependent hydrolysis of membrane phospholipids by sPLA2-IIA was enhanced by overexpression of 12/15-LOX. These results indicate that the product(s) generated by the cPLA2-12/15-LOX pathway following cell activation may play two roles: enhancement of sPLA2-IIA gene expression and membrane sensitization that leads to accelerated sPLA2-IIA-mediated hydrolysis.     

Cellular components that functionally interact with signaling phospholipase A(2)s

Accumulating evidence has suggested that cytosolic phospholipase A(2) (cPLA(2)) and several secretory PLA(2) (sPLA(2)) isozymes are signaling PLA(2)s that are functionally coupled with downstream cyclooxygenase (COX) isozymes for prostaglandin (PG) biosynthesis. Arachidonic acid (AA) released by cPLA(2) and sPLA(2)s is supplied to both COX-1 and COX-2 in the immediate, and predominantly to COX-2 in the delayed, PG-biosynthetic responses. Vimentin, an intermediate filament component, acts as a functional perinuclear adapter for cPLA(2), in which the C2 domain of cPLA(2) associates with the head domain of vimentin in a Ca(2+)-sensitive manner. The heparin-binding signaling sPLA(2)-IIA, IID and V bind the glycosylphosphatidylinositol-anchored heparan sulfate proteoglycan glypican, which plays a role in sorting of these isozymes into caveolae and perinuclear compartments. Phospholipid scramblase, which facilitates transbilayer movement of anionic phospholipids, renders the cellular membranes more susceptible to signaling sPLA(2)s. There is functional cooperation between cPLA(2) and signaling sPLA(2)s in that prior activation of cPLA(2) is required for the signaling sPLA(2)s to act properly. cPLA(2)-derived AA is oxidized by 12/15-lipoxygenase, the products of which not only augment the induction of sPLA(2) expression, but also cause membrane perturbation, leading to increased cellular susceptibility to the signaling sPLA(2)s. sPLA(2)-X, a heparin-non-binding sPLA(2) isozyme, is capable of releasing AA from intact cells in the absence of cofactors. This property is attributed to its ability to avidly hydrolyze zwitterionic phosphatidylcholine, a major phospholipid in the outer plasma membrane. sPLA(2)-V can also utilize this route in several cell types. Taken together, the AA-releasing function of sPLA(2)s depends on the presence of regulatory cofactors and interfacial binding to membrane phospholipids, which differ according to cell type, stimuli, secretory processes, and subcellular distributions.     

Secreted phospholipase A2 revisited

Phospholipase A(2) (PLA(2)) catalyses the hydrolysis of the sn-2 position of glycerophospholipids to yield fatty acids and lysophospholipids. So far, more than 30 enzymes that possess PLA(2) or related activity have been identified in mammals. About one third of these enzymes belong to the secreted PLA(2) (sPLA(2)) family, which comprises low molecular weight, Ca(2+) requiring, secreted enzymes with a His/Asp catalytic dyad. Individual sPLA(2)s display distinct localizations and enzymatic properties, suggesting their specialized biological roles. However, in contrast to intracellular PLA(2)s, whose roles in signal transduction and membrane homoeostasis have been well documented, the biological roles of sPLA(2)s in vivo have remained obscure until recently. Over the past decade, information fuelled by studies employing knockout and transgenic mice as well as specific inhibitors, in combination with lipidomics, has clarified when and where the different sPLA(2) isoforms are expressed, which isoforms are involved in what types of pathophysiology, and how they exhibit their specific functions. In this review, we highlight recent advances in PLA(2) research, focusing mainly on the physiological functions of sPLA(2)s and their modes of action on 'extracellular' phospholipid targets versus lipid mediator production.     

Biophysical mechanisms of phospholipase A2 activation and their use in liposome-based drug delivery

Secretory phospholipase A2 (PLA2) is a ubiquitous water-soluble enzyme found in venom, pancreatic, and cancerous fluid. It is also known to play a role in membrane remodeling processes as well as in cellular signaling cascades. PLA2 is interfacially active and functions mainly on organized types of substrate, e.g. micelles and lipid bilayers. Hence the activity of the enzyme is modulated by the lateral organization and the physical properties of the substrate, in particular the structure in the nanometer range. The evidence for nano-scale structure and lipid domains in bilayers is briefly reviewed. Results obtained from a variety of experimental and theoretical studies of PLA2 activity on lipid-bilayer substrates are then presented which provide insight into the biophysical mechanisms of PLA2 activation on lipid bilayers and liposomes of different composition. The insight into these mechanisms has been used to propose a novel principle for liposomal drug targeting, release, and absorption triggered by secretory PLA2.     

Stimulation of Golgi membrane tubulation and retrograde trafficking to the ER by phospholipase A(2) activating protein (PLAP) peptide.

Recent pharmacological studies using specific antagonists of phospholipase A(2) (PLA(2)) activity have suggested that the formation of Golgi membrane tubules, 60-80 nm in diameter and up to several microns long, both in vivo and in a cell-free cytosol-dependent reconstitution system, requires the activity of a cytoplasmic Ca(2+)-independent PLA(2). We confirm and extend these studies by demonstrating that the stimulators of PLA(2), melittin and PLA(2) activating protein peptide (PLAPp), enhance cytosol-dependent Golgi membrane tubulation. Starting with preparations of bovine brain cytosol (BBC), or a fraction of BBC that is highly enriched in tubulation activity, called the gel filtration (GF) fraction, that are at subsaturating concentrations for inducing tubulation in vitro, we found that increasing concentrations of melittin or PLAPp produced a linear and saturable stimulation of Golgi membrane tubulation. This stimulation was inhibited by cytosolic PLA(2) antagonists, including the Ca(2+)-independent PLA(2)-specific antagonist, bromoenol lactone. The stimulatory effect of PLAPp, and its inhibition by PLA(2) antagonists, was reproduced using a permeabilized cell system, which reconstitutes both cytosol-dependent Golgi membrane tubulation and retrograde trafficking to the endoplasmic reticulum (ER). Taken together, these results are consistent with the idea that cytosolic PLA(2) activity is involved in the formation of Golgi membrane tubules, which can serve as trafficking intermediates in Golgi-to-ER retrograde movement.     

The biology of a prostate cancer metastasis suppressor protein: Raf kinase inhibitor protein

Raf kinase inhibitor protein (RKIP) was originally identified as a protein that bound membrane phospholipids and was named phosphatidylethanolamine binding protein-2 (PEBP-2). RKIP was than identified as a protein that bound Raf and blocked its ability to phosphorylate MEK, thus earning its new name of RKIP. Subsequent to identification of its role in the Raf:MEK pathway, RKIP has been demonstrated to regulate several other signaling pathways including G-protein signaling and NF-kappaB signaling. Its involvement in several signaling pathways has engendered RKIP to contribute to several physiological processes including membrane biosynthesis, spermatogenesis, neural development, and apoptosis. RKIP is expressed in many tissues including brain, lung, and liver and thus, dysregulation of RKIP expression or function has potential to contribute to pathophysiology in these tissues. Loss of RKIP expression in prostate cancer cells confers a metastatic phenotype on them. Additionally, restoration of RKIP expression in a metastatic prostate cancer cell line does not effect primary tumor growth, but it does inhibit prostate cancer metastasis. These parameters identify RKIP as a metastasis suppressor gene. In this review, the biology and pathophysiology of RKIP is described.     

PLA2R1 kills cancer cells by inducing mitochondrial stress

Little is known about the biological functions of the phospholipase A2 receptor (PLA2R1) except that it has the ability to bind a few secreted phospholipases A2 (sPLA2′s). We have previously shown that PLA2R1 regulates senescence in normal human cells. In this study, we investigated the ability of PLA2R1 to control cancer cell growth. Analysis of expression in cancer cells indicates a marked PLA2R1 decrease in breast cancer cell lines compared to normal or nontransformed human mammary epithelial cells. Accordingly, PLA2R1 ectopic expression in PLA2R1-negative breast cancer cell lines led to apoptosis, whereas a prosenescence response was predominantly triggered in normal cells. PLA2R1 structure–function studies and the use of chemical inhibitors of sPLA2-related signaling pathways suggest that the effect of PLA2R1 is sPLA2-independent. Functional experiments demonstrate that PLA2R1 regulation of cell death is driven by a reactive oxygen species (ROS)-dependent mechanism. While screening for ROS-producing complexes involved in PLA2R1 biological responses, we identified a critical role for the mitochondrial electron transport chain in PLA2R1-induced ROS production and cell death. Taken together, this set of data provides evidence for an important role of PLA2R1 in controlling cancer cell death by influencing mitochondrial biology.     

Intraluminal calcium as a primary regulator of endoplasmic reticulum function

The concentration of Ca2+ inside the lumen of endoplasmic reticulum (ER) regulates a vast array of spatiotemporally distinct cellular processes, from intracellular Ca2+ signals to intra-ER protein processing and cell death. This review summarises recent data on the mechanisms of luminal Ca2+-dependent regulation of Ca2+ release and uptake as well as ER regulation of cellular adaptive processes. In addition we discuss general biophysical properties of the ER membrane, as trans-endomembrane Ca2+ fluxes are subject to basic electrical forces, determined by factors such as the membrane potential of the ER and the ease with which Ca2+ fluxes are able to change this potential (i.e. the resistance of the ER membrane). Although these electrical forces undoubtedly play a fundamental role in shaping [Ca2+](ER) dynamics, at present there is very little direct experimental information about the biophysical properties of the ER membrane. Further studies of how intraluminal [Ca2+] is regulated, best carried out with direct measurements, are vital for understanding how Ca2+ orchestrates cell function. Direct monitoring of [Ca2+](ER) under conditions where the cytosolic [Ca2+] is known may also help to capture elusive biophysical information about the ER, such as the potential difference across the ER membrane.     

Inhibition of phospholipase A(2) as a therapeutic target

The hydrolysis of cell membrane phospholipids by phospholipase A(2) (PLA(2)) leads to the production of numerous lipid mediators of diverse pathological conditions, mainly inflammatory diseases. These include lysophospholipids and their derivatives, and arachidonic acid and its derivatives (the eicosanoids). Both these groups of mediators are produced predominantly by the secretory PLA(2)s (sPLA(2)s) which hydrolyze the phospholipids of the cell surface membrane. Protection of cell membrane from these 'inflammatory enzymes' can therefore be used for the treatment of inflammatory processes. A prototype of cell-impermeable PLA(2) inhibitors, which protect the cell membrane from different sPLA(2)s without affecting vital phospholipid metabolism, is presented and discussed in the present review.