n-3 fatty acid-derived lipid mediators in the brain: new weapons against oxidative stress and inflammation.

Neuroprotectins, resolvins, and maresins are subfamilies of endogenous oxygenated metabolites derived from n-3 or ω-3 fatty acids (eicosapentaenoic and docosahexaenoic acids). These metabolites are associated with signal transduction processes involved in downregulation of oxidative stress, neuroinflammation and apoptosis. Eicosapentaenoic acid-derived E-series resolvins (RvE1 and RvE2) and docosahexaenoic acid-derived D-series resolvins (RvD1 and RvD2) and neuroprotectins have potent anti-inflammatory and proresolution, and antioxidant properties. They not only retard excessive inflammatory process, but also promote resolution by enhancing clearance of apoptotic cells and debris from inflamed brain tissue and vasculature leading to tissue homeostasis. These actions may underlie the beneficial effects of eicosapentaenoic acid and docosahexaenoic acid in normal human health, neurotraumatic and neurodegenerative diseases. Aspirin initiates resolution not only by exerting antithrombotic actions, but also triggering biosynthesis of specific and stereoselective epimers of resolvins, protectins, and maresins. In addition during the onset of resolution, these lipid mediators also display potent protective roles in neural systems, liver, lungs, and eyes. Potent anti-inflammatory actions of resolvins, and protectins in models of chronic human diseases indicate that down-regulation in resolution pathways may contribute to the decrease in the intensity of many chronic neurodegenerative and visceral diseases.

Increased expression of acyl-coenzyme A: cholesterol acyltransferase-1 and elevated cholesteryl esters in the hippocampus after excitotoxic injury.

Significant increases in levels of cholesterol and cholesterol oxidation products are detected in the hippocampus undergoing degeneration after excitotoxicity induced by the potent glutamate analog, kainate (KA), but until now, it is unclear whether the cholesterol is in the free or esterified form. The present study was carried out to examine the expression of the enzyme involved in cholesteryl ester biosynthesis, acyl-coenzyme A: cholesterol acyltransferase (ACAT) and cholesteryl esters after KA excitotoxicity. A 1000-fold greater basal mRNA level of ACAT1 than ACAT2 was detected in the normal brain. ACAT1 mRNA and protein were upregulated in the hippocampus at 1 and 2 weeks after KA injections, at a time of glial reaction. Immunohistochemistry showed ACAT1 labeling of oligodendrocytes in the white matter and axon terminals in hippocampal CA fields of normal rats, and loss of staining in neurons but increased immunoreactivity of oligodendrocytes, in areas affected by KA. Gas chromatography-mass spectrometry analyses confirmed previous observations of a marked increase in level of total cholesterol and cholesterol oxidation products, whilst nuclear magnetic resonance spectroscopy showed significant increases in cholesteryl ester species in the degenerating hippocampus. Upregulation of ACAT1 expression was detected in OLN93 oligodendrocytes after KA treatment, and increased expression was prevented by an antioxidant or free radical scavenger in vitro. This suggests that ACAT1 expression may be induced by oxidative stress. Together, our results show elevated ACAT1 expression and increased cholesteryl esters after KA excitotoxicity. Further studies are necessary to determine a possible role of ACAT1 in acute and chronic neurodegenerative diseases.

Involvement of cytosolic phospholipase A(2), calcium independent phospholipase A(2) and plasmalogen selective phospholipase A(2) in neurodegenerative and neuropsychiatric conditions.

Enzymes belonging to the PLA(2) superfamily catalyze the hydrolysis of unsaturated fatty acids from the sn-2 position of glycerol moiety of neural membrane phospholipids. The PLA(2) superfamily is classified into cytosolic PLA(2) (cPLA(2)), calcium-independent PLA(2) (iPLA(2)), plasmalogen-selective PLA(2) (PlsEtn-PLA(2)) and secretory PLA(2) (sPLA(2)). PLA(2) paralogs/splice variants/isozymes are part of a complex signal transduction network that maintains cross-talk among excitatory amino acid and dopamine receptors through the generation of second messengers. Individual paralogs, splice variants and multiple forms of PLA(2) may have unique enzymatic properties, tissue and subcellular localizations and role in various physiological and pathological situations, hence tight regulation of all PLA(2) isoforms is essential for normal brain function. Quantitative RT-PCR analyses show significantly higher relative level of expression of iPLA(2) than cPLA(2) in all regions of the rat brain. Upregulation of the cPLA(2) family is involved in degradation of neural membrane phospholipids and generation of arachidonic acid-derived lipid metabolites that have been implicated in nociception, neuroinflammation, oxidative stress and neurodegeneration. In contrast, studies using a selective iPLA(2) inhibitor, bromoenol lactone, or antisense oligonucleotide indicate that iPLA(2) is an important "housekeeping" enzyme under basal conditions, whose activity is required for the prevention of vacuous chewing movements, a rodent model for tardive dyskinesia, and deficits in the prepulse inhibition of the auditory startle reflex, a common finding in schizophrenia. These studies support the view that PLA(2) activity may not only play a crucial role in neurodegeneration but depending on the isoform, could also be essential in prevention of neuropsychiatric diseases. The findings could open new doors for understanding and treatment of neurodegenerative and neuropsychiatric diseases.

Induction of astrocytic cytoplasmic phospholipase A2 and neuronal death after intracerebroventricular carrageenan injection, and neuroprotective effects of quinacrine.

Glial reaction is often associated with nervous tissue injury, but thus far, few studies have examined whether it can be a cause of neuronal injury. We now study the effect of intracerebroventricular injection of a carrageenan on cytoplasmic phospholipase A(2) (cPLA(2)) expression and neuronal injury in the hippocampus. The enzyme cPLA(2) hydrolyzes neural membrane glycerophospholipids and generates precursors for proinflammatory mediators. An induction of cPLA(2) in astrocytes and death of neurons in the hippocampus were observed following glial reaction induced by intracerebroventricular injections of carrageenan. cPLA(2) levels and neuronal death were modulated by daily intraperitoneal injections of quinacrine, an inhibitor of phospholipase A(2) that can cross the blood brain barrier. These observations support a role for astrocytic cPLA(2) in mediating neuronal death.

Group IIA secretory phospholipase A2 stimulates exocytosis and neurotransmitter release in pheochromocytoma-12 cells and cultured rat hippocampal neurons.

Recent evidence shows that secretory phospholipase A2 (sPLA2) may play a role in membrane fusion and fission, and may thus affect neurotransmission. The present study therefore aimed to elucidate the effects of sPLA2 on vesicle exocytosis. External application of group IIA sPLA2 (purified crotoxin subunit B or purified human synovial sPLA2) caused an immediate increase in exocytosis and neurotransmitter release in pheochromocytoma-12 (PC12) cells, detected by carbon fiber electrodes placed near the cells, or by changes in membrane capacitance of the cells. EGTA and a specific inhibitor of sPLA2 activity, 12-epi-scalaradial, abolished the increase in neurotransmitter release, indicating that the effect of sPLA2 was dependent on calcium and sPLA2 enzymatic activity. A similar increase in neurotransmitter release was also observed in hippocampal neurons after external application of sPLA2, as detected by changes in membrane capacitance of the neurons. In contrast to external application, internal application of sPLA2 to PC12 cells and neurons produced blockade of neurotransmitter release. Our recent studies showed high levels of sPLA2 activity in the normal rat hippocampus, medulla oblongata and cerebral neocortex. The sPLA2 activity in the hippocampus was significantly increased, after kainate-induced neuronal injury. The observed effects of sPLA2 on neurotransmitter release in this study may therefore have a physiological, as well as a pathological role.

Secretory phospholipase A2 activity in the normal and kainate injected rat brain, and inhibition by a peptide derived from python serum.

The present study aimed to elucidate sPLA(2) activity in the normal and kainate-lesioned hippocampus using selective inhibitors of sPLA(2). In normal rats the highest levels of sPLA(2) were observed in the hippocampus, pons, and medulla, followed by the cerebral neocortex and caudate nucleus. After intracerebroventricular kainate injections an increase in total PLA(2) activity was observed in the rat hippocampus. Using a selective sPLA(2) inhibitor 12-epi-scalaradial, sPLA(2) activity was found to be significantly increased by 2.5-fold on the side of the intracerebroventricular injection compared to the contralateral side. A peptide P-NT.II, derived from the amino acid sequence of "PLA(2)-inhibitory protein," discovered in the serum of the reticulated python, also showed potent sPLA(2) inhibitory activity in homogenates from the kainate-injected hippocampus. These results show that there is a high level of sPLA(2) activity in the normal hippocampus, pons, and medulla oblongata, and that the level increases further in the hippocampus after kainate-induced excitotoxic injury. The increased PLA(2) activity was inhibited by P-NT.II, indicating a potential use of this peptide as a PLA(2) inhibitory agent in the brain.

Effect of D609 on phosphatidylcholine metabolism in the nuclei of LA-N-1 neuroblastoma cells: a key role for diacylglycerol.

In our previous studies, TPA treatment of LA-N-1 cells stimulated the production of diacylglycerol in nuclei, probably through the activation of a phospholipase C. Stimulation of the synthesis of nuclear phosphatidylcholine by the activation of CTP:phosphocholine cytidylyltransferase was also observed. The present data show that both effects were inhibited by the pretreatment of the cells with D609, a selective phosphatidylcholine-phospholipase C inhibitor, indicating that the diacylglycerol produced through the hydrolysis of phosphatidylcholine in the nuclei is reutilized for the synthesis of nuclear phosphatidylcholine and is required for the activation of CTP:phosphocholine cytidylyltransferase.

Neurochemical consequences of kainate-induced toxicity in brain: involvement of arachidonic acid release and prevention of toxicity by phospholipase A(2) inhibitors.

In kainate-induced neurotoxicity, the stimulation of kainate receptors results in the activation of phospholipase A(2) and a rapid release of arachidonic acid from neural membrane glycerophospholipids. This process raises arachidonic acid levels and produces alterations in membrane fluidity and permeability. These result in calcium influx and stimulation of lipolysis and proteolysis, production of lipid peroxides, depletion of ATP, and loss of reduced glutathione. As well as the above neurochemical changes, stimulation of ornithine decarboxylase, altered activities of protein kinase C isozymes, and expression of immediate early genes, cytokines, growth factors, and heat shock proteins have also been reported. Kainate-induced stimulation of arachidonic acid release, calcium influx, accumulation of lipid peroxides and products of their decomposition, especially 4-hydroxynonenal (4-HNE), along with alterations in cellular redox state and ATP depletion may play important roles in kainate-induced cell death. Thus the consequences of altered glycerophospholipid metabolism in kainate-induced neurotoxicity can lead to cell death. Kainate-induced neurotoxicity initiates apoptotic as well as necrotic cell death depending upon the intensity of oxidative stress and abnormality in mitochondrial function. Other neurochemical changes may be related to synaptic reorganization following kainate-induced seizures and may be involved in recapitulation of hippocampal development and synaptogenesis.

Plasmalogens: workhorse lipids of membranes in normal and injured neurons and glia.

Plasmalogens are unique glycerophospholipids because they have an enol ether double bond at the sn-1 position of the glycerol backbone. They are found in all mammalian tissues, with ethanolamine plasmalogens 10-fold higher than choline plasmalogens except in muscles. The enol ether double bond at the sn-1 position makes plasmalogens more susceptible to oxidative stress than the corresponding ester-bonded glycerophospholipids. Plasmalogens are not only structural membrane components and a reservoir for second messengers but may also be involved in membrane fusion, ion transport, and cholesterol efflux. Plasmalogens may also act as antioxidants, thus protecting cells from oxidative stress. Receptor-mediated degradation of plasmalogens by plasmalogen-selective phospholipase A2 results in the generation of arachidonic acid, eicosanoids, and platelet activating factor. Low levels of these metabolites have trophic effects, but at high concentration they are cytotoxic and may be involved in allergic response, inflammation, and trauma. Levels of plasmalogens are decreased in several neurological disorders including Alzheimer's disease, ischemia, and spinal cord trauma. This may be due to the stimulation of plasmalogen-selective phospholipase A2. A deficiency of plasmalogens in peroxisomal disorders and Niemann-Pick type C disease indicates that this deficiency may be due to the decreased activity of plasmalogen synthesizing enzymes that occur in peroxisomes.

Plasmalogens, phospholipase A2, and docosahexaenoic acid turnover in brain tissue.

Plasmalogens are glycerophospholipids of neural membranes containing vinyl ether bonds. Their synthetic pathway is located in peroxisomes and endoplasmic reticulum. The rate-limiting enzymes are in the peroxisomes and are induced by docosahexaenoic acid (DHA). Plasmalogens often contain arachidonic acid (AA) or DHA at the sn-2 position of the glycerol moiety. The receptor-mediated hydrolysis of plasmalogens by cytosolic plasmalogen-selective phospholipase A2 generates AA or DHA and lysoplasmalogens. AA is metabolized to eicosanoids. The mechanism of signaling with DHA is not known. The plasmalogen-selective phospholipase A2 differs from other intracellular phospholipases A2 in molecular mass, kinetic properties, substrate specificity, and response to glycosaminoglycans, gangliosides, and sialoglycoproteins. A major portion of [3H]DHA incorporated into neural membranes is found at the sn-2 position of ethanolamine glycerophospholipids. Studies with a mutant cell line defective in plasmalogen biosynthesis indicate that the incorporation of DHA is reduced in this RAW 264.7 cell line by 50%. In contrast, the incorporation of AA remains unaffected. This is reversed completely when the growth medium is supplemented with sn-1-hexadecylglycerol, suggesting that DHA can be selectively targeted for incorporation into plasmalogens. We suggest that deficiencies of DHA and plasmalogens in peroxisomal disorders, Alzheimer's disease (AD), depression, and attention deficit hyperactivity disorders (ADHD) may be responsible for abnormal signal transduction associated with learning disability, cognitive deficit, and visual dysfunction. These abnormalities in the signal-transduction process can be partially corrected by supplementation with a diet enriched with DHA.

Effect of retinoic acid on the Ca2+-independent phospholipase A2 in nuclei of LA-N-1 neuroblastoma cells.

LA-N-1 neuroblastoma cell cultures contain Ca2+-independent phospholipases A2 hydrolyzing phosphatidylethanolamine and ethanolamine plasmalogens. These enzymes differ from each other in their molecular mass, substrate specificity, and kinetic properties. Subcellular distribution studies have indicated that the activity of these phospholipases is not only localized in the cytosol but also in non-nuclear membranes and in nuclei. The treatment of LA-N-1 neuroblastoma cell cultures with retinoic acid results in a marked stimulation of Ca2+-independent phospholipases A2 hydrolyzing phosphatidylethanolamine and plasmenylethanolamine. The increase of the activities of both enzymes was first observed in nuclei followed by those present in the cytosol. No effect of retinoic acid on either phospholipase activity could be observed in non-nuclear membranes. The stimulation of these enzymes may be involved in the generation and regulation of arachidonic acid and its metabolites during differentiation.

Differential effects of calcium-dependent and calcium-independent phospholipase A(2) inhibitors on kainate-induced neuronal injury in rat hippocampal slices.

Brain tissue contains multiple forms of intracellular phospholipase A(2) (PLA(2)) activity that differ from each other in many ways including their response to specific inhibitors. The systemic administration of kainic acid to rats produces a marked increase in cPLA(2) activity in neurons and astrocytes. This is associated with increased lipid peroxidation as evidenced by accumulation of 4-hydroxynonenal (4-HNE) modified proteins. The present study describes the effect of specific inhibitors of Ca(2+)-dependent or Ca(2+)-independent PLA(2) on kainite-induced excitotoxic injury in rat hippocampal slices. Specific inhibitors of Ca(2+)-dependent PLA(2) prevented the decrease of a neuronal marker, GluR1, and increase in cPLA(2) and 4-HNE immunoreactivities in slices treated with kainate. This shows that cPLA(2) plays an important role in kainite-induced neurotoxicity and that cPLA(2) inhibitors can be used to protect hippocampal slices from damage induced by kainate.

Review of current evidence for apoptosis after spinal cord injury.

The initial mechanical tissue disruption of spinal cord injury (SCI) is followed by a period of secondary injury that increases the size of the lesion. The secondary injury has long been thought to be due to the continuation of cellular destruction through necrotic (or passive) cell death. Recent evidence from brain injury and ischemia suggested that cellular apoptosis, an active form of programmed cell death seen during development, could play a role in CNS injury in adulthood. Here, we review the evidence that apoptosis may be important in the pathophysiology of SCI. There is now strong morphological and biochemical evidence from a number of laboratories demonstrating the presence of apoptosis after SCI. Apoptosis occurs in populations of neurons, oligodendrocytes, microglia, and, perhaps, astrocytes. The death of oligodendrocytes in white matter tracts continues for many weeks after injury and may contribute to post-injury demyelination. The mediators of apoptosis after SCI are not well understood, but there is a close relationship between microglia and dying oligodendrocytes, suggesting that microglial activation may be involved. There is also evidence for the activation of important intracellular pathways known to be involved in apoptosis in other cells and systems. For example, some members of the caspase family of cysteine proteases are activated after SCI. It appears that the evolution of the lesion after SCI involves both necrosis and apoptosis. It is likely that better understanding of apoptosis after SCI will lead to novel strategies for therapeutic interventions that can diminish secondary injury.

Deacylation and reacylation of neural membrane glycerophospholipids.

The deacylation-reacylation cycle is an important mechanism responsible for the introduction of polyunsaturated fatty acids into neural membrane glycerophospholipids. It involves four enzymes, namely acyl-CoA synthetase, acyl-CoA hydrolase, acyl-CoA: lysophospholipid acyltransferase, and phospholipase A2. All of these enzymes have been purified and characterized from brain tissue. Under normal conditions, the stimulation of neural membrane receptors by neurotransmitters and growth factors results in the release of arachidonic acid from neural membrane glycerophospholipids. The released arachidonic acid acts as a second messenger itself. It can be further metabolized to eicosanoids, a group of second messengers involved in a variety of neurochemical functions. A lysophospholipid, the second product of reactions catalyzed by phospholipase A2, is rapidly acylated with acyl-CoA, resulting in the maintenance of the normal and essential neural membrane glycerophospholipid composition. However, under pathological situations (ischemia), the overstimulation of phospholipase A2 results in a rapid generation and accumulation of free fatty acids including arachidonic acid, eicosanoids, and lipid peroxides. This results in neural inflammation, oxidative stress, and neurodegeneration. In neural membranes, the deacylation-reacylation cycle maintains a balance between free and esterified fatty acids, resulting in low levels of arachidonic acid and lysophospholipids. This is necessary for not only normal membrane integrity and function, but also for the optimal activity of the membrane-bound enzymes, receptors, and ion channels involved in normal signal-transduction processes.

Glycerophospholipids in brain: their metabolism, incorporation into membranes, functions, and involvement in neurological disorders.

Neural membranes contain several classes of glycerophospholipids which turnover at different rates with respect to their structure and localization in different cells and membranes. The glycerophospholipid composition of neural membranes greatly alters their functional efficacy. The length of glycerophospholipid acyl chain and the degree of saturation are important determinants of many membrane characteristics including the formation of lateral domains that are rich in polyunsaturated fatty acids. Receptor-mediated degradation of glycerophospholipids by phospholipases A(l), A(2), C, and D results in generation of second messengers such as arachidonic acid, eicosanoids, platelet activating factor and diacylglycerol. Thus, neural membrane phospholipids are a reservoir for second messengers. They are also involved in apoptosis, modulation of activities of transporters, and membrane-bound enzymes. Marked alterations in neural membrane glycerophospholipid composition have been reported to occur in neurological disorders. These alterations result in changes in membrane fluidity and permeability. These processes along with the accumulation of lipid peroxides and compromised energy metabolism may be responsible for the neurodegeneration observed in neurological disorders.

Immunocytochemical localization of cPLA2 in rat and monkey spinal cord.

Rat spinal cord contains a high level of calcium-dependent cytosolic phospholipase A2 (PLA2) activity. A dense immunoreactivity is present in motor neurons from cervical, thoracic, lumbar, and sacral regions of rat spinal cord. Under normal conditions, this enzyme liberates arachidonic acid, a polyunsaturated fatty acid that is a second messenger itself, and a precursor for eicosanoids. However, under pathological conditions during spinal cord injury, intracellular calcium increases so the cytosolic PLA2 may also be involved in the release and accumulation of arachidonic acid, eicosanoids, and lipid peroxides.

Distribution of cytoplasmic phospholipase A2 in the normal rat brain.

The calcium-dependent cytoplasmic PLA2 (cPLA2) is an 85-kDa cytosolic enzyme that has been detected in cytosolic fractions from rat brain. With immunocytochemical methods, this cPLA2 is distributed throughout rat brain. Very dense immunostaining is observed in the superior olivary nucleus, periolivary nucleus, facial motor nucleus and dorsal cochlear nucleus in hindbrain whereas light immunostaining is seen in forebrain and midbrain areas. Assays of cPLA2 activity in forebrain, midbrain and hindbrain show the highest specific activity in the hindbrain. The distribution of cPLA2 coincides with that of protein kinase C activity in rat brain. The presence of cPLA2 and PKC in hindbrain suggests that these enzymes play a central role in neurotransmitter release, long-term potentiation and neuritogenesis in this area under normal conditions.

Inhibitors of intracellular phospholipase A2 activity: their neurochemical effects and therapeutical importance for neurological disorders.

Intracellular phospholipases A2 (PLA2) are a diverse group of enzymes with a growing number of members. These enzymes hydrolyze membrane phospholipids into fatty acid and lysophospholipids. These lipid products may serve as intracellular second messengers or can be further metabolized to potent inflammatory mediators, such as eicosanoids and platelet-activating factors. Several inhibitors of nonneural intracellular PLA2 have been recently discovered. However, nothing is known about their neurochemical effects, mechanism of action or toxicity in human or animal models of neurological disorders. Elevated intracellular PLA2 activities, found in neurological disorders strongly associated with inflammation and oxidative stress (ischemia, spinal cord injury, and Alzheimer's disease), can be treated with specific, potent and nontoxic inhibitors of PLA2 that can cross blood-brain barrier without harm. Currently, potent intracellular PLA2 inhibitors are not available for clinical use in human or animal models of neurological disorders, but studies on this interesting topic are beginning to emerge. The use of nonspecific intracellular PLA2 inhibitors (quinacrine, heparin, gangliosides, vitamin E) in animal model studies of neurological disorders in vivo has provided some useful information on tolerance, toxicity, and effectiveness of these compounds.