The Onset of Brain Injury and Neurodegeneration Triggers the Synthesis of Docosanoid Neuroprotective Signaling

Bioactive lipid messengers are formed through phospholipase-mediated cleavage of specific phospholipids from membrane reservoirs. Effectors that activate the synthesis of lipid messengers, include ion channels, neurotransmitters, membrane depolarization, cytokines, and neurotrophic factors. In turn, lipid messengers regulate and interact with multiple pathways, participating in the development, differentiation, function (e.g., long-term potentiation and memory), protection, and repair of cells of the nervous system. Overall, bioactive lipids participate in the regulation of synaptic function and dysfunction. Platelet-activating factor (PAF) and COX-2-synthesized PGE₂ modulate synaptic plasticity and memory. Oxidative stress disrupts lipid signaling, fosters lipid peroxidation, and initiates and propagates neurodegeneration. Lipid messengers participate in the interactions among neurons, astrocytes, oligodendrocytes, microglia, cells of the microvasculature, and other cells. A conglomerate of interrelated cells comprises the neurovascular unit. Signaling at the neurovascular unit is clearly altered in the early stages of cerebrovascular disease as well as in neurodegenerations. Here we will provide examples of how signaling by lipids regulates critical events essential for neuronal survival. We will highlight a newly identified, DHA-derived messenger, neuroprotectin D1, which attenuates oxidative stress-induced apoptosis. The specificity and potency of this novel docosanoid (neuroprotectin D1) indicate a potentially important target for therapeutic intervention.     

Synaptic lipid signaling: significance of polyunsaturated fatty acids and platelet-activating factor

Neuronal cellular and intracellular membranes are rich in specialized phospholipids that are reservoirs of lipid messengers released by specific phospholipases and stimulated by neurotransmitters, neurotrophic factors, cytokines, membrane depolarization, ion channel activation, etc. Secretory phospholipases A2 may be both intercellular messengers and generators of lipid messengers. The highly networked nervous system includes cells (e.g., astrocytes, oligodendrocytes, microglial cells, endothelial microvascular cells) that extensively interact with neurons; several lipid messengers participate in these interactions. This review highlights modulation of postsynaptic membrane excitability and long-term synaptic plasticity by cyclooxygenase-2-generated prostaglandin E2, arachidonoyldiacylcylglycerol, and arachidonic acid-containing endocannabinoids. The peroxidation of docosahexaenoic acid (DHA), a critical component of excitable membranes in brain and retina, is promoted by oxidative stress. DHA is also the precursor of enzyme-derived, neuroprotective docosanoids. The phospholipid platelet-activating factor is a retrograde messenger of long-term potentiation, a modulator of glutamate release, and an upregulator of memory formation. Lipid messengers modulate signaling cascades and contribute to cellular differentiation, function, protection, and repair in the nervous system. Lipidomic neurobiology will advance our knowledge of the brain, spinal cord, retina, and peripheral nerve function and diseases that affect them, and new discoveries on networks of signaling in health and disease will likely lead to novel therapeutic interventions.     

Lipid signaling: sleep, synaptic plasticity, and neuroprotection

Increasing evidence indicates that bioactive lipids participate in the regulation of synaptic function and dysfunction. We have demonstrated that signaling mediated by platelet-activating factor (PAF) and cyclooxygenase (COX)-2-synthesized PGE2 is involved in synaptic plasticity, memory, and neuronal protection [Clark GD, Happel LT, Zorumski CF, Bazan NG. Enhancement of hippocampal excitatory synaptic transmission by platelet-activating factor. Neuron 1992; 9:1211; Kato K, Clark GD, Bazan NG, Zorumski CF. Platelet-activating factor as a potential retrograde messenger in CA1 hippocampal long-term potentiation. Nature 1994; 367:175; Izquierdo I, Fin C, Schmitz PK, et al. Memory enhancement by intrahippocampal, intraamygdala or intraentorhinal infusion of platelet-activating factor measured in an inhibitory avoidance. Proc Natl Acad Sci USA 1995; 92:5047; Chen C, Magee CJ, Bazan NG. Cyclooxygenase-2 regulates prostaglandin E2 signaling in hippocampal long-term synaptic plasticity. J Neurophysiol 2002; 87:2851]. Recently, we found that prolonged continuous wakefulness (primarily rapid eye movement (REM)-sleep deprivation, SD) causes impairments in hippocampal long-term synaptic plasticity and hippocampus-dependent memory formation [McDermott CM, LaHoste GJ, Chen C, Musto A, Bazan NG, Magee JC. Sleep deprivation causes behavioral, synaptic, and membrane excitability alterations in hippocampal neurons. J Neurosci 2003; 23:9687]. To explore the mechanisms underlying SD-induced impairments, we have studied several bioactive lipids in the hippocampus following SD. It appears that SD causes increases in prostaglandin D2 (PGD2) and 2-arachidonylglycerol (2-AG), and a decrease in PGE2, suggesting that these lipid messengers participate in memory consolidation during REM sleep. We have also explored the formation of endogenous neuroprotective lipids. Toward this aim, we have used ischemia-reperfusion damage and LC-PDA-ESI-MS-MS-based lipidomic analysis and identified docosanoids derived from synaptic phospholipid-enriched docosahexaenoic acid. Some of the docosanoids exert potent neuroprotective bioactivity [Marcheselli VL, Hong S, Lukiw WJ, et al. Novel docosanoids inhibit brain ischemia-reperfusion-mediated leukocyte infiltration and pro-inflammatory gene expression. J Biol Chem 2003; 278:43807; Mukherjee PK, Marcheselli VL, Serhan CN, Bazan, NG. Neuroprotectin D1: A docosahexaenoic acid-derived docosatriene protects human retinal pigment epithelial cells from oxidative stress. Proc Nat Acad Sci USA 2004; 101:8491). Taken together, these observations that signaling lipids participate in synaptic plasticity, cognition, and survival indicate that lipid signaling is closely associated with several functions (e.g; learning and memory, sleep, and experimental stroke) and pathologic events. Alterations in endogenous signaling lipids or their receptors resulting from drug abuse lead to changes in synaptic circuitry and induce profound effects on these important functions. In the present article, we will briefly review bioactive lipids involved in sleep, synaptic transmission and plasticity, and neuroprotection, focusing mainly on our experimental studies and how these signaling molecules are related to functions and implicated in some neurologic disorders.     

Novel docosanoids inhibit brain ischemia-reperfusion-mediated leukocyte infiltration and pro-inflammatory gene expression

Ischemic stroke triggers lipid peroxidation and neuronal injury. Docosahexaenoic acid released from membrane phospholipids during brain ischemia is a major source of lipid peroxides. Leukocyte infiltration and pro-inflammatory gene expression also contribute to stroke damage. In this study using lipidomic analysis, we have identified stereospecific messengers from docosahexaenoate-oxygenation pathways in a mouse stroke model. Aspirin, widely used to prevent cerebrovascular disease, activates an additional pathway, which includes the 17R-resolvins. The newly discovered brain messenger 10,17S-docosatriene potently inhibited leukocyte infiltration, NFkappaB, and cyclooxygenase-2 induction in experimental stroke and elicited neuroprotection. In addition, in neural cells in culture, this lipid messenger also inhibited both interleukin 1-beta-induced NFkappaB activation and cyclooxygenase-2 expression. Thus, the specific novel bioactive docosanoids generated in vivo counteract leukocyte-mediated injury as well as pro-inflammatory gene induction. These results challenge the view that docosahexaenoate only participates in brain damage and demonstrate that this fatty acid is also the endogenous precursor to a neuroprotective signaling response to ischemia-reperfusion.     

Docosanoids are multifunctional regulators of neural cell integrity and fate: significance in aging and disease

The identification of neuroprotectin D1 (NPD1), a biosynthetic product of docosahexaenoic acid (DHA), in brain and retina as well as the characterization of its bioactivity, is generating a renewed interest in the functional role and pathophysiological significance of omega-3 fatty acids in the central nervous system. Neurotrophins, particularly pigment epithelium-derived factor (PEDF), induce NPD1 synthesis and its polarized apical secretion, implying paracrine and autocrine bioactivity of this lipid mediator. Also, DHA and PEDF synergistically activate NPD1 synthesis and antiapoptotic protein expression and decreased proapoptotic Bcl-2 protein expression and caspase 3 activation during oxidative stress. In experimental stroke, endogenous NPD1 synthesis was found to be upregulated, and the infusion of the lipid mediator into the brain under these conditions revealed neuroprotective bioactivity of NPD1. The hippocampal CA1 region from Alzheimer's disease (AD) patients (rapidly sampled) shows a major reduction in NPD1. The interplay of DHA-derived neuroprotective signaling aims to counteract proinflammatory, cell-damaging events triggered by multiple, converging cytokine and amyloid peptide factors, as in the case of AD. Generation of NPD1 from DHA thereby appears to redirect cellular fate toward successful preservation of retinal pigment epithelial (RPE)-photoreceptor cell integrity and brain cell aging. The Bcl-2 pro- and antiapoptotic proteins, neurotrophins, and NPD1, lie along a cell fate-regulatory pathway whose component members are highly interactive, and have potential to function cooperatively in cell survival. Agents that stimulate NPD1 biosynthesis, NPD1 analogs, or dietary regimens may be useful as new preventive/therapeutic strategies for neurodegenerative diseases.     

Endogenous Signaling by Omega-3 Docosahexaenoic Acid-derived Mediators Sustains Homeostatic Synaptic and Circuitry Integrity

The harmony and function of the complex brain circuits and synapses are sustained mainly by excitatory and inhibitory neurotransmission, neurotrophins, gene regulation, and factors, many of which are incompletely understood. A common feature of brain circuit components, such as dendrites, synaptic membranes, and other membranes of the nervous system, is that they are richly endowed in docosahexaenoic acid (DHA), the main member of the omega-3 essential fatty acid family. DHA is avidly retained and concentrated in the nervous system and known to play a role in neuroprotection, memory, and vision. Only recently has it become apparent why the surprisingly rapid increases in free (unesterified) DHA pool size take place at the onset of seizures or brain injury. This phenomenon began to be clarified by the discovery of neuroprotectin D1 (NPD1), the first-uncovered bioactive docosanoid formed from free DHA through 15-lipoxygenase-1 (15-LOX-1). NPD1 synthesis includes, as agonists, oxidative stress and neurotrophins. The evolving concept is that DHA-derived docosanoids set in motion endogenous signaling to sustain homeostatic synaptic and circuit integrity. NPD1 is anti-inflammatory, displays inflammatory resolving activities, and induces cell survival, which is in contrast to the pro-inflammatory actions of the many of omega-6 fatty acid family members. We highlight here studies relevant to the ability of DHA to sustain neuronal function and protect synapses and circuits in the context of DHA signalolipidomics. DHA signalolipidomics comprises the integration of the cellular/tissue mechanism of DHA uptake, its distribution among cellular compartments, the organization and function of membrane domains containing DHA phospholipids, and the precise cellular and molecular events revealed by the uncovering of signaling pathways regulated by docosanoids endowed with prohomeostatic and cell survival bioactivity. Therefore, this approach offers emerging targets for prevention, pharmaceutical intervention, and clinical translation involving DHA-mediated signaling.     

Neuroprotectin D1 Induces Neuronal Survival and Downregulation of Amyloidogenic Processing in Alzheimer’s Disease Cellular Models

The mediator neuroprotectin D1 (NPD1) is an enzymatic derivative of the omega-3 essential fatty acid docosahexaenoic acid. NPD1 stereoselectively and specifically binds to human retinal pigment epithelium (RPE) cells and neutrophils. In turn, this lipid mediator induces dephosphorylation of Bcl-x_L in a PP2A-dependent manner and induces PI3K/Akt and mTOR/p70S6K pathways leading to RPE cell survival during oxidative stress-induced apoptosis. As a proof of principle of its systemic in vivo bioactivity, NPD1 attenuates laser-induced choroidal neovascularization in mice. Using human neural cells transfected with amyloid precursor protein (APP)sw (Swedish double mutation APP695sw, K595N, M596L), NPD1 was shown to regulate secretase-mediated production of Aβ peptide, downregulates pro-inflammatory gene expression, and promotes cell survival. In human neural cells overexpressing beta-amyloid precursor protein (βAPP), the lipid mediator suppressed Aβ42 shedding by downregulating β-secretase (BACE1) while activating the α-secretase (ADAM10), thus shifting the βAPP cleavage from the noxious amyloidogenic pathway into a non-amyloidogenic, neurotrophic pathway. Furthermore, downregulation of Aβ42 peptide release by NPD1 may be dependent upon PPARγ activation. In conclusion, NPD1 exhibits anti-inflammatory, anti-amyloidogenic, and anti-apoptotic bioactivities in human neural cells in part via PPARγ signaling and through the targeting of α- and β-secretase systems.     

Hypoxia signaling to genes: significance in Alzheimer's disease

Aberrations in neural signaling, converging to and diverging from oxidative metabolism and blood supply, contribute to the initiation and maintenance of inflammatory responses, neuronal degeneration, and age-related cognitive decline in Alzheimer's disease (AD). Hypoxia/ischemia triggers phospholipase A2, leading to the accumulation of free arachidonic and docosahexaenoic acids (AA, DHA), as well as that of lysophospholipids. Some of these bioactive lipid messengers in turn give rise to several downstream lipid messengers, such as platelet-activating factor (PAF) and ecosanoids (prostaglandins and leukotrienes). Eicosanoid synthesis is highly regulated in hypoxia and in reperfusion subsequent to ischemia. As one of the consequences, mitochondrial function is disrupted and reactive oxygen species (ROS) both contribute to the expansion of cellular inflammatory responses and reduce the expression of genes required to maintain synaptic structure and function. On the other hand, pro-inflammatory genes are up-regulated. One of these, the inducible cyclooxygenase-2 (COX-2), along with oxygen-starved mitochondria, comprise the major sources of ROS in the brain during hypoxia, ischemia, and reperfusion. One outcome is a sustained metabolic stress that drives progressive dysfunction, apoptosis and/or necrosis, and brain cell death. How hypoxia modulates oxygen-sensitive gene expression is not well understood. Pro-inflammatory gene families that contribute to neurodegeneration are transiently activated in part by the heterodimeric oxygen-sensitive DNA-binding proteins nuclear factor for kappa B (NF-kappaB) and hypoxia-inducible factor-alpha (HIF-1alpha). Here the authors summarize current studies supporting the hypothesis that synaptically-derived lipid messengers play significant roles in ischemic stroke and that hypoxia is an important contributor to the onset and progression of AD neurodegeneration.     

A single enzyme catalyzes both platelet-activating factor production and membrane biogenesis of inflammatory cells. Cloning and characterization of acetyl-CoA:LYSO-PAF acetyltransferase

Platelet-activating factor (PAF) is a potent proinflammatory lipid mediator eliciting a variety of cellular functions. Lipid mediators, including PAF are produced from membrane phospholipids by enzymatic cascades. Although a G protein-coupled PAF receptor and degradation enzymes have been cloned and characterized, the PAF biosynthetic enzyme, aceyl-CoA:lyso-PAF acetyltransferase, has not been identified. Here, we cloned lyso-PAF acetyltransferase, which is critical in stimulus-dependent formation of PAF. The enzyme is a 60-kDa microsomal protein with three putative membrane-spanning domains. The enzyme was induced by bacterial endotoxin (lipopolysaccharide), which was suppressed by dexamethasone treatment. Surprisingly, the enzyme catalyzed not only biosynthesis of PAF from lyso-PAF but also incorporation of arachidonoyl-CoA to produce PAF precursor membrane glycerophospholipids (lysophosphatidylcholine acyltransferase activity). Under resting conditions, the enzyme prefers arachidonoyl-CoA and contributes to membrane biogenesis. Upon acute inflammatory stimulation with lipopolysaccharide, the activated enzyme utilizes acetyl-CoA more efficiently and produces PAF. Thus, our findings provide a novel concept that a single enzyme catalyzes membrane biogenesis of inflammatory cells while producing a prophlogistic mediator in response to external stimuli.     

The docosanoid neuroprotectin D1 induces homeostatic regulation of neuroinflammation and cell survival

The onset of neurodegenerations and nervous system injury both trigger cell signaling perturbations that lead to damage of neuronal circuits and synapic connections, as well as protective signaling that aims to halt disease onset. Here we review recent findings that support the role of the docosanoid mediator neuroprotectin D1 (NPD1) as an early response or sentinel during the initial phase of nervous system damage. NPD1 is derived from docosahexaenoic acid that is selectively concentrated and retained in the nervous system. The protein misfolding triggers the biosynthesis of NPD1 which in turn downregulates pathways that lead to cell death and changes the outcome to cell survival. Proteotoxic stress as a result of protein misfolding is a widespread event in many neurodegenerative diseases. Therefore, mechanisms and mediators such as NPD1 that curtail consequences of these events are of interest as leads in the search for novel preventive and or therapeutic approaches.     

The platelet activating factor (PAF) signaling cascade in systemic inflammatory responses

The platelet-activating factor (PAF) signaling cascade evolved as a component of the repertoire of innate host defenses, but is also an effector pathway in inflammatory and thrombotic diseases. This review focuses on the PAF signaling cascade in systemic inflammatory responses and, specifically, explores its activities in experimental and clinical sepsis and anaphylaxis in the context of the basic biochemistry and biology of signaling via this lipid mediator system.     

Synaptic signaling by lipids in the life and death of neurons

Synaptic activity promotes the regulated formation of lipid messengers through phospholipase-mediated cleavage of specific phospholipid reservoirs from membranes. Multiple effectors trigger the formation of lipid messengers, including neurotransmitters, membrane depolarization, ion channels, cytokines, and neurotrophic factors. Lipid messengers in turn modulate and interact with other signaling cascades, contributing to the development, differentiation, function (e.g., long-term potentiation [LTP] and memory), protection, and repair of cells in the nervous system. These relationships with other signaling cascades remain largely to be investigated. Oxidative stress disrupts lipid signaling, enhances lipid peroxidation, and initiates and propagates neurodegeneration. There is growing evidence that lipid messengers participate in the extensive interactions among neurons, astrocytes, oligodendrocytes, microglia, cells of the microvasculature, and other cells. This article provides an example of how signaling by lipids regulates critical events essential for neuronal survival and reviews the recent identification of a novel endogenous neuroprotective signaling pathway involving a docosahexaneoic acid-derived mediator.     

Rescue and repair during photoreceptor cell renewal mediated by docosahexaenoic acid-derived neuroprotectin D1

Retinal degenerative diseases result in retinal pigment epithelial (RPE) and photoreceptor cell loss. These cells are continuously exposed to the environment (light) and to potentially pro-oxidative conditions, as the retina''s oxygen consumption is very high. There is also a high flux of docosahexaenoic acid (DHA), a PUFA that moves through the blood stream toward photoreceptors and between them and RPE cells. Photoreceptor outer segment shedding and phagocytosis intermittently renews photoreceptor membranes. DHA is converted through 15-lipoxygenase-1 into neuroprotectin D1 (NPD1), a potent mediator that evokes counteracting cell-protective, anti-inflammatory, pro-survival repair signaling, including the induction of anti-apoptotic proteins and inhibition of pro-apoptotic proteins. Thus, NPD1 triggers activation of signaling pathway/s that modulate/s pro-apoptotic signals, promoting cell survival. This review provides an overview of DHA in photoreceptors and describes the ability of RPE cells to synthesize NPD1 from DHA. It also describes the role of neurotrophins as agonists of NPD1 synthesis and how photoreceptor phagocytosis induces refractoriness to oxidative stress in RPE cells, with concomitant NPD1 synthesis.     

New insights into the retinal circulation: inflammatory lipid mediators in ischemic retinopathy

Ischemic proliferative retinopathy develops in various retinal disorders, including retinal vein occlusion, diabetic retinopathy and retinopathy of prematurity. Ischemic retinopathy remains a common cause of visual impairment and blindness in the industrialized world due to relatively ineffective treatment. Oxygen-induced retinopathy (OIR) is an established model of retinopathy of prematurity associated with vascular cell injury culminating in microvascular degeneration, which precedes an abnormal neovascularization. The retina is a tissue particularly rich in polyunsaturated fatty acids and the ischemic retina becomes highly sensitive to lipid peroxidation initiated by oxygenated free radicals. Consequently, the retina constitutes an excellent model for testing the functional consequences of membrane lipid peroxidation. Retinal tissue responds to physiological and pathophysiological stimuli by the activation of phospholipases and the consequent release from membrane phospholipids of biologically active metabolites. Activation of phospholipase A(2) is the first step in the synthesis of two important classes of lipid second messengers, the eicosanoids and a membrane-derived phospholipid mediator platelet-activating factor (PAF). These lipid mediators accumulate in the retina in response to injury and a physiologic role of these metabolites in retinal vasculature remains for the most part to be determined; albeit proposed roles have been suggested for some. The eicosanoids, in particular the prostanoids, thromboxane (TXA2) and PAF are abundantly generated following an oxidant stress and contribute to neurovascular injury. TXA2 and PAF play an important role in the retinal microvacular degeneration of OIR by directly inducing endothelial cell death and potentially could contribute to the pathogenesis of ischemic retinopathies. Despite these advances there are still a number of important questions that remain to be answered before we can confidently target pathological signals. This review focuses on mechanisms that precede the development of neovascularization, most notably regarding the role of lipid mediators that partake in microvascular degeneration.     

Omega-3 Essential Fatty Acids Modulate Initiation and Progression of Neurodegenerative Disease

The significance of the selective enrichment in omega-3 essential fatty acids in photoreceptors and synaptic membranes of the nervous system has remained, until recently, incompletely understood. While studying mechanisms of cell survival in neural degeneration, we discovered a docosanoid synthesized from unesterified docosahexaenoic acid (DHA) by a 15-lipoxygenase (15-LOX), which we called neuroprotectin D1 (NPD1; 10R,17S-dihydroxy-docosa-4Z,7Z,11E,13E,15E,19Z hexaenoic acid). This lipid mediator is a docosanoid because it is derived from the 22 carbon (22C) precursor DHA, unlike eicosanoids, which are derived from the 20 carbon (20C) arachidonic acid (AA) family member of essential fatty acids. We discovered that NPD1 is promptly made in response to oxidative stress, as a response to brain ischemia–reperfusion, and in the presence of neurotrophins. NPD1 is neuroprotective in experimental brain damage, in oxidative-stressed retinal pigment epithelial (RPE) cells, and in human brain cells exposed to amyloid-β (Aβ) peptides. We thus envision NPD1 as a protective sentinel, one of the very first defenses activated when cell homeostasis is threatened by imbalances in normal neural function. We provide here, in three sections, recent experimental examples that highlight the specificity and potency of NPD1 spanning beneficial bioactivity during initiation and early progression of neurodegeneration: (1) during retinal signal phototransduction, (2) during brain ischemia–reperfusion, and (3) in Alzheimer's disease (AD) and stressed human brain cell models of AD. From this experimental evidence, we conclude that DHA-derived NPD1 regulation targets upstream events of brain cell apoptosis, as well as neuro-inflammatory signaling, promoting and maintaining cellular homeostasis, and restoring neural and retinal cell integrity.     

Enhancement of hippocampal excitatory synaptic transmission by platelet-activating factor.

The biologically active lipid platelet-activating factor (1-O-alkyl-2-acetyl-sn-glycero-3-phosphorylcholine; PAF) is a mediator of inflammatory and immune responses, and it accumulates in the brain during convulsions or ischemia. We have examined whether PAF may play a second messenger role in the central nervous system by studying effects on synaptic transmission in cultured hippocampal neurons. Carbamyl-PAF, a nonhydrolyzable PAF analog with a similar pharmacologic profile, augmented glutamate-mediated, evoked excitatory synaptic transmission and increased the frequency of spontaneous miniature excitatory synaptic events without increasing their amplitude or altering their time course. This compound had no significant effect on gamma-aminobutyric acid-mediated inhibitory synaptic responses. Lyso-PAF, the biologically inactive metabolic intermediate, had no effect on synaptic transmission. Moreover, the enhancement of excitatory synaptic transmission by carbamyl-PAF was blocked by a PAF receptor antagonist. These results indicate a specific presynaptic effect of PAF in enhancing excitatory synaptic transmission in cultured rat hippocampal neurons.     

Platelet activating factor-induced neuronal apoptosis is initiated independently of its G-protein coupled PAF receptor and is inhibited by the benzoate orsellinic acid

The bioactive lipid mediator platelet activating factor (PAF) is recognized as a key effecter of neuronal apoptosis, yet it is not clear whether its G-protein coupled receptor (PAFR) initiates or prevents PAF neurotoxicity. Using PAFR-/- and congenic wild-type mice, we show that PAF triggers caspase-3/7 activity and neuronal death in PAFR-/- but not PAFR+/+ cerebellar granule neurons. Restoring receptor expression by recombinant adenoviral infection protected cells from PAF challenge. Neuronal death was not mediated by nitric oxide or N-methyl-d-aspartate receptor signaling given that N-nitro-l-arginine methyl ester and MK-801 did not inhibit PAF-induced neuronal loss in PAFR-/- neurons. To intervene in PAFR-independent neurotoxicity, the anti-apoptotic actions of three structurally distinct PAF antagonists were compared to a panel of plant and fungal benzoic acid derivatives. We found that the PAF antagonist BN 52021 but not FR 49175 or CV 3988 inhibited PAFR-independent neurotoxicity. Orsellinic acid, a fungal-derived benzoic acid, blocked PAF-mediated neuronal apoptosis without affecting PAFR-mediated neuroprotection. These findings demonstrate that PAF can transduce apoptotic death in primary neurons independently of its G-protein coupled receptor, that PAFR activation is neuroprotective, and that orsellinic acid effectively attenuates PAFR-independent neuronal apoptosis.     

Docosahexaenoic acid-derived neuroprotectin D1 induces neuronal survival via secretase- and PPARγ-mediated mechanisms in Alzheimer's disease models

Neuroprotectin D1 (NPD1) is a stereoselective mediator derived from the omega-3 essential fatty acid docosahexaenoic acid (DHA) with potent inflammatory resolving and neuroprotective bioactivity. NPD1 reduces Aβ42 peptide release from aging human brain cells and is severely depleted in Alzheimer's disease (AD) brain. Here we further characterize the mechanism of NPD1's neurogenic actions using 3xTg-AD mouse models and human neuronal-glial (HNG) cells in primary culture, either challenged with Aβ42 oligomeric peptide, or transfected with beta amyloid precursor protein (βAPP)(sw) (Swedish double mutation APP695(sw), K595N-M596L). We also show that NPD1 downregulates Aβ42-triggered expression of the pro-inflammatory enzyme cyclooxygenase-2 (COX-2) and of B-94 (a TNF-α-inducible pro-inflammatory element) and apoptosis in HNG cells. Moreover, NPD1 suppresses Aβ42 peptide shedding by down-regulating β-secretase-1 (BACE1) while activating the α-secretase ADAM10 and up-regulating sAPPα, thus shifting the cleavage of βAPP holoenzyme from an amyloidogenic into the non-amyloidogenic pathway. Use of the thiazolidinedione peroxisome proliferator-activated receptor gamma (PPARγ) agonist rosiglitazone, the irreversible PPARγ antagonist GW9662, and overexpressing PPARγ suggests that the NPD1-mediated down-regulation of BACE1 and Aβ42 peptide release is PPARγ-dependent. In conclusion, NPD1 bioactivity potently down regulates inflammatory signaling, amyloidogenic APP cleavage and apoptosis, underscoring the potential of this lipid mediator to rescue human brain cells in early stages of neurodegenerations.     

NPD1 Induction of Retinal Pigment Epithelial Cell Survival Involves PI3K/Akt Phosphorylation Signaling

Neuroprotectin D1 (NPD1), a docosahexaenoic acid (DHA)-derived lipid mediator, promotes survival in cells exposed to oxidative stress by inducing the activity of anti-inflammatory mediators and suppressing the expression of pro-inflammatory genes. Though retinal pigment epithelial (RPE) cells naturally produce NPD1 from DHA, investigating the mechanisms through which exogenous NPD1 induces cell survival is essential to assess mechanisms of actions and the potential of this lipid mediator for treatment of retinal degenerative diseases. The PI3K/Akt and mTOR/p70S6K pathways are responsible for supporting cell survival upon exposure to oxidative stress. In human ARPE-19 cells pretreated with NPD1 then exposed to varying concentrations of oxidative stress or repeated exposures to oxidative stress, Akt, mTOR, and p70S6K were phosphorylated to a greater extent and for a greater duration than cells not pretreated with NPD1. In addition to increased phosphorylation, a subsequent decreased rate of apoptosis was observed upon NPD1 treatment. Thus NPD1 bioactivity in RPE cells enhances activation of these pathways and promotes cell integrity and survival.     

A role for the mouse 12/15-lipoxygenase pathway in promoting epithelial wound healing and host defense

The surface of the eye actively suppresses inflammation while maintaining a remarkable capacity for epithelial wound repair. Our understanding of mechanisms that balance inflammatory/reparative responses to provide effective host defense while preserving tissue function is limited, in particular, in the cornea. Lipoxin A(4) (LXA(4)) and docosahexaenoic acid-derived neuroprotectin D1 (NPD1) are lipid autacoids formed by 12/15-lipoxygenase (LOX) pathways that exhibit anti-inflammatory and neuroprotective properties. Here, we demonstrate that mouse corneas generate endogenous LXA(4) and NPD1. 12/15-LOX (Alox15) and LXA(4) receptor mRNA expression as well as LXA(4) formation were abrogated by epithelial removal and restored during wound healing. Amplification of these pathways by topical treatment with LXA(4) or NPD1 (1 microg) increased the rate of re-epithelialization (65-90%, n = 6-10, p < 0.03) and attenuated the sequelae of thermal injury. In contrast, the proinflammatory eicosanoids, LTB(4) and 12R-hydroxyeicosatrienoic acid, had no impact on corneal re-epithelialization. Epithelial removal induced a temporally defined influx of neutrophils into the stroma as well as formation of the proinflammatory chemokine KC. Topical treatment with LXA(4) and NPD1 significantly increased PMNs in the cornea while abrogating KC formation by 60%. More importantly, Alox15-deficient mice exhibited a defect in both corneal re-epithelialization and neutrophil recruitment that correlated with a 43% reduction in endogenous LXA(4) formation. Collectively, these results identify a novel action for the mouse 12/15-LOX (Alox15) and its products, LXA(4) and NPD1, in wound healing that is distinct from their well established anti-inflammatory properties.