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1.  NEUROTROPHINS INDUCE NEUROPROTECTIVE SIGNALING IN THE RETINAL PIGMENT EPITHELIAL CELL BY ACTIVATING THE SYNTHESIS OF THE ANTI-INFLAMMATORY AND ANTI-APOPTOTIC NEUROPROTECTIN D1 
1. SUMMARY
The integrity of retinal pigment epithelial cells is critical for photoreceptor cell survival and vision. The essential omega-3 fatty acid, docosahexaenoic acid, attains its highest concentration in the human body in photoreceptors. Docosahexaenoic acid is the essential precursor of neuroprotectin D1 (NPD1). NPD1 acts against apoptosis mediated by A2E, a byproduct of phototransduction that becomes toxic when it accumulates in aging retinal pigment epithelial (RPE) cells and in some inherited retinal degenerations. Here we also describe that neurotrophins, mainly pigment epithelium-derived factor, induce NPD1 synthesis and its polarized apical secretion, suggesting paracrine and autocrine bioactivity of this lipid mediator. In addition, DHA elicits a concentration-dependent and selective potentiation of pigment epithelial-derived factor-stimulated NPD1 synthesis and release through the apical RPE cell surface. The bioactivity of signaling activated by PEDF and DHA demonstrates synergistic cytoprotection when cells were challenged with oxidative stress, resulting in concomitant NPD1 synthesis. Also, DHA and PEDF synergistically activate anti-apoptotic protein expression and decreased pro-apoptotic Bcl-2 protein expression and caspase 3 activation during oxidative stress. Thus, DHA-derived NPD1 protects against RPE cell damage mediated by aging/disease-induced A2E accumulation. Also, neurotrophins are regulators of NPD1 synthesis and of its polarized apical efflux from RPE cells. Therefore, NPD1 may elicit autocrine and paracrine bioactivity in cells located in the proximity of the interphotoreceptor matrix.
PMCID: PMC2645073  PMID: 18188926
2.  Cellular and molecular events mediated by docosahexaenoic acid-derived neuroprotectin D1 signaling in photoreceptor cell survival and brain protection 
Deficiency in docosahexaenoic acid (DHA) is associated with impaired visual and neurological postnatal development, cognitive decline, macular degeneration, and other neurodegenerative diseases. DHA is an omega-3 polyunsaturated fatty acyl chain concentrated in phospholipids of brain and retina, with photoreceptor cells displaying the highest content of DHA of all cell membranes. The identification and characterization of neuroprotectin D1 (NPD1, 10R, 17S-dihydroxy-docosa-4Z, 7Z, 11E, 13E, 15Z, 19Z-hexaenoic acid) contributes to understanding the biological significance of DHA. In oxidative stress-challenged human retinal pigment epithelial (RPE) cells, human brain cells, or rat brains undergoing ischemia-reperfusion, NPD1 synthesis is enhanced as a response for sustaining homeostasis. Thus, neurotrophins, Aβ peptide 42 (Aβ42), calcium ionophore A23187, interleukin (IL)-1 β, or DHA supply enhances NPD1 synthesis. NPD1, in turn, up-regulates the anti-apoptotic proteins of the Bcl-2 family and decreases the expression of pro-apoptotic Bcl-2 family members. Moreover, NPD1 inhibits IL-1 β-stimulated expression of cyclooxygenase-2 (COX-2). Because both RPE and photoreceptors are damaged and then die in retinal degenerations, elucidating how NPD1 signaling contributes to retinal cell survival may lead to a new understanding of disease mechanisms. In human neural cells, DHA attenuates amyloid-β (Aβ) secretion, resulting in concomitant formation of NPD1. NPD1 was found to be reduced in the Alzheimer’s disease (AD) CA1 hippocampal region, but not in other areas of the brain. The expression of key enzymes for NPD1 biosynthesis, cytosolic phospholipase A2 (cPLA2), and 15-lipoxygenase (15-LOX) was found altered in the AD hippocampal CA1 region. NPD1 repressed Aβ42-triggered activation of pro-inflammatory genes and upregulated the anti-apoptotic genes encoding Bcl-2, Bcl-xl, and Bfl-1(A1) in human brain cells in culture. Overall, these results support the concept that NPD1 promotes brain and retina cell survival via the induction of anti-apoptotic and neuroprotective gene-expression programs that suppress Aβ42-induced neurotoxicity and other forms of cell injury, which in turn fosters homeostasis during development in aging, as well as during the initiation and progression of neurodegenerative diseases.
doi:10.1016/j.plefa.2009.05.024
PMCID: PMC2756692  PMID: 19520558
n-3 (omega-3) fatty acid; n-6 (omega-6) fatty acid; retinal pigment epithelial cell; Aβ42; Bcl-2 proteins; eicosanoids; docosanoids; inflammation; photoreceptor renewal; liver; neurotrophins; aging; Alzheimer’s disease; macular degeneration
3.  Ataxin-1 Poly(Q)-induced Proteotoxic Stress and Apoptosis Are Attenuated in Neural Cells by Docosahexaenoic Acid-derived Neuroprotectin D1* 
The Journal of Biological Chemistry  2012;287(28):23726-23739.
Background: Neurodegenerative diseases involve proteotoxic stress and apoptosis.
Results: NPD1 inhibits proteotoxic stress-induced apoptosis.
Conclusion: NPD1 synthesis is an early response to proteotoxic stress.
Significance: This might be one of the first survival defenses activated in neurodegenerations.
Neurodegenerative diseases share two common features: enhanced oxidative stress and cellular inability to scavenge structurally damaged abnormal proteins. Pathogenesis of polyglutamine (poly(Q)) diseases involves increased protein misfolding, along with ubiquitin and chaperon protein-containing nuclear aggregates. In spinocerebellar ataxia, the brain and retina undergo degeneration. Neuroprotectin D1 (NPD1) is made on-demand in the nervous system and retinal pigment epithelial (RPE) cells in response to oxidative stress, which activates prosurvival signaling via regulation of gene expression and other processes. We hypothesized that protein misfolding-induced proteotoxic stress triggers NPD1 synthesis. We used ARPE-19 cells as a cellular model to assess stress due to ataxin-1 82Q protein expression and determine whether NPD1 prevents apoptosis. Ectopic ataxin-1 expression induced RPE cell apoptosis, which was abrogated by 100 nm docosahexaenoic acid, 10 ng/ml pigment epithelium-derived factor, or NPD1. Similarly, NPD1 was protective in neurons and primary human RPE cells. Furthermore, when ataxin-1 82Q was expressed in 15-lipoxygenase-1-deficient cells, apoptosis was greatly enhanced, and only NPD1 (50 nm) rescued cells from death. NPD1 reduced misfolded ataxin-1-induced accumulation of proapoptotic Bax in the cytoplasm, suggesting that NPD1 acts by preventing proapoptotic signaling pathways from occurring. Finally, NPD1 signaling interfered with ataxin-1/capicua repression of gene expression and decreased phosphorylated ataxin-1 in an Akt-independent manner, suggesting that NPD1 signaling modulates formation or stabilization of ataxin-1 complexes. These data suggest that 1) NPD1 synthesis is an early response induced by proteotoxic stress due to abnormally folded ataxin-1, and 2) NPD1 promotes cell survival through modulating stabilization of ataxin-1 functional complexes and pro-/antiapoptotic and inflammatory pathways.
doi:10.1074/jbc.M111.287078
PMCID: PMC3390647  PMID: 22511762
Apoptosis; Lipids; Neurodegenerative Diseases; Oxidative Stress; Retinal Degeneration
4.  Docosahexaenoic Acid-Derived Neuroprotectin D1 Induces Neuronal Survival via Secretase- and PPARγ-Mediated Mechanisms in Alzheimer's Disease Models 
PLoS ONE  2011;6(1):e15816.
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 APP695sw, 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.
doi:10.1371/journal.pone.0015816
PMCID: PMC3016440  PMID: 21246057
5.  Endogenous Signaling by Omega-3 Docosahexaenoic Acid-derived Mediators Sustains Homeostatic Synaptic and Circuitry Integrity 
Molecular Neurobiology  2011;44(2):216-222.
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.
doi:10.1007/s12035-011-8200-6
PMCID: PMC3180614  PMID: 21918832
Epilepsy; Neuroprotectin D1; Photoreceptors; Retinal pigment epithelial cells; Liver
6.  Novel aspirin-triggered Neuroprotectin D1 attenuates cerebral ischemic injury after experimental stroke 
Experimental neurology  2012;236(1):122-130.
Acute ischemic stroke triggers complex neurovascular, neuroinflammatory and synaptic alterations. Aspirin and docosahexaenoic acid (DHA), an omega-3 essential fatty acid family member, have beneficial effects on cerebrovascular diseases. DHA is the precursor of neuroprotectin D1 (NPD1), which downregulates apoptosis and, in turn, promotes cell survival. Here we have tested the effect of aspirin plus DHA administration and discovered the synthesis of aspirin-triggered NPD1 (AT-NPD1) in the brain. Then we performed the total chemical synthesis of this molecule and tested in the setting of 2h middle cerebral artery occlusion (MCAo) in Sprague-Dawley rats. Neurological status was evaluated at 24h, 48h, 72h, and 7 days. At 3h post-stroke onset, an intravenous administration of 333μg/kg of AT-NPD1 sodium salt (AT-NPD1-SS) or methyl-ester (AT-NPD1-ME) or vehicle (saline) as treatment was given. On day 7, ex vivo magnetic resonance imaging (MRI) of the brains was conducted on 11.7T MRI. T2WI, 3D volumes, and apparent diffusion coefficient (ADC) maps were generated. In addition, infarct volumes and number of GFAP (reactive astrocytes), ED-1 (activated microglia/macrophages) and SMI-71-positive vessels were counted in the cortex and striatum at the level of the central lesion. All animals showed similar values for rectal and cranial temperatures, arterial blood gases, and plasma glucose during and after MCAo. Treatment with both AT-NPD1-SS and AT-NPD1-ME significantly improved neurological scores compared to saline treatment at 24h, 48h, 72h and 7 days. Total lesion volumes computed from T2WI images were significantly reduced by both AT-NPD1-SS and AT-NPD1-ME treatment in the cortex (by 44% and 81%), striatum (by 61% and 77%) and total infarct (by 48% and 78%, respectively). Brain edema, computed from T2WI in the cortex (penumbra) and striatum (core), was elevated in the saline group. In contrast, both AT-NPD1 decreased water content in the striatum on day 7. 3D volumes, computed from T2WI, were dramatically reduced with both AT-NPD1 and the lesion was mostly localized in the subcortical areas. Treatment with both AT-NPD1-SS and AT-NPD1-ME significantly reduced cortical (by 76% and 96%), subcortical (by 61% and 70%) and total (69% and 84%, respectively) infarct volumes as defined by histopathology. In conclusion, a novel biosynthetic pathway that leads to the formation of AT-NPD1 mediator in the brain was discovered. In addition, administration of synthetic AT-NPD1, in either its sodium salt or as the methyl ester, was able to attenuate cerebral ischemic injury which leads to a novel approach for pharmaceutical intervention and clinical translation.
doi:10.1016/j.expneurol.2012.04.007
PMCID: PMC3409566  PMID: 22542947
Neuroprotectin D1; Docosahexaenoic acid; Aspirin; Stroke; Middle cerebral artery occlusion; Cerebral ischemia; Behavior; Histopathology; Rat
7.  Neuroprotectin D1 Synthesis and Corneal Nerve Regeneration after Experimental Surgery and Treatment with PEDF plus DHA 
This study revealed that pigment epithelial–derived growth factor in combination with docosahexaenoic acid (DHA) enhances the regeneration of corneal nerves damaged after surgery.
Purpose.
This study was conducted to define whether pigment epithelial–derived growth factor (PEDF), together with docosahexaenoic acid (DHA), enhances the synthesis of neuroprotectin D1 (NPD1) and the regeneration of corneal nerves damaged after surgery.
Methods.
Corneal stromal dissection was performed in the left eyes of adult New Zealand rabbits treated with DHA+PEDF, PEDF, or DHA for 6 weeks. In vivo confocal images of the corneas were obtained at 2, 4, and 8 weeks, and nerve areas were quantified. At 8 weeks after treatment, corneas were stained with tubulin βIII antibody, and the epithelial nerve area and the sub-basal and stromal nerve plexus were quantified. At 1 week and 2 weeks after treatment, lipids were extracted from corneas, and the synthesis of NPD1 was analyzed by mass spectrometry. Epithelial cell density was quantified by confocal microscopy 8 weeks after surgery.
Results.
In vivo confocal images at 2 and 4 weeks after surgery showed a 2.5-fold increase in corneal nerve area in PEDF+DHA–treated animals compared with control animals. Increased nerve surface areas in epithelia, subepithelia, and stroma were observed in rabbits treated for 8 weeks with PEDF+DHA. PEDF or DHA alone did not produce a significant increase. NPD1 synthesis peaked at 1 week and was four times higher in the PEDF+DHA–treated group than in the controls.
Conclusions.
PEDF+DHA promotes the regeneration of corneal nerves. Neurotrophin-mediated NPD1 synthesis is suggested to precede nerve regeneration by demonstration of its accumulation upon addition of DHA and PEDF at earlier time points. Therefore, this signaling mechanism upregulates corneal nerve regeneration and may be targeted in neurotrophic keratitis, dry eye after refractive surgery, and other corneal diseases.
doi:10.1167/iovs.09-3641
PMCID: PMC2868452  PMID: 19797230
8.  A role for docosahexaenoic acid–derived neuroprotectin D1 in neural cell survival and Alzheimer disease 
Journal of Clinical Investigation  2005;115(10):2774-2783.
Deficiency in docosahexaenoic acid (DHA), a brain-essential omega-3 fatty acid, is associated with cognitive decline. Here we report that, in cytokine-stressed human neural cells, DHA attenuates amyloid-β (Aβ) secretion, an effect accompanied by the formation of NPD1, a novel, DHA-derived 10,17S-docosatriene. DHA and NPD1 were reduced in Alzheimer disease (AD) hippocampal cornu ammonis region 1, but not in the thalamus or occipital lobes from the same brains. The expression of key enzymes in NPD1 biosynthesis, cytosolic phospholipase A2 and 15-lipoxygenase, was altered in AD hippocampus. NPD1 repressed Aβ42-triggered activation of proinflammatory genes while upregulating the antiapoptotic genes encoding Bcl-2, Bcl-xl, and Bfl-1(A1). Soluble amyloid precursor protein-α stimulated NPD1 biosynthesis from DHA. These results indicate that NPD1 promotes brain cell survival via the induction of antiapoptotic and neuroprotective gene-expression programs that suppress Aβ42-induced neurotoxicity.
doi:10.1172/JCI25420
PMCID: PMC1199531  PMID: 16151530
9.  Neuroprotectin D1/Protectin D1 Stereoselective and Specific Binding With Human Retinal Pigment Epithelial Cells and Neutrophils 
Retinal pigment epithelial (RPE) cells, derived from the neuroectoderm, biosynthesize the novel lipid mediator neuroprotectin D1 (NPD1) from docosahexaenoic acid (DHA) in response to oxidative stress or to neurotrophins, and in turn, elicits cytoprotection. Here, we report the identification of a 16,17-epoxide-containing intermediate in the biosynthesis of NPD1 in ARPE-19 cells from 17S-hydro-(peroxy)-docosahexaenoic acid. We prepared and isolated tritium-labeled NPD1 ([3H]-NPD1) and demonstrate specific and high-affinity stereoselective binding to ARPE-19 cells (Kd = 31.3 ± 13.1 pmol/mg of cell protein). The stereospecific NPD1 interactions with these cells in turn gave potent protection against oxidative stress-induced apoptosis, and other structurally related compounds were weak competitors of NPD1 specific binding. This [3H]-NPD1/PD1 also displayed specific and selective high affinity binding with isolated human neutrophils (Kd = ~25 nM). Neither resolvin E1 nor lipoxin A4 competed for [3H]-NPD1/PD1 specific binding with human neutrophils. Together, these results provide evidence for stereoselective specific binding of NPD1/PD1 with retinal pigment epithelial cells as well as human neutrophils. Moreover, they suggest specific receptors for this novel mediator in both the immune and visual systems.
doi:10.1016/j.plefa.2009.10.010
PMCID: PMC2822836  PMID: 19931440
Mediators; Neuroprotectin; Inflammation; Resolution; Photoreceptors; Binding
10.  Docosahexaenoic Acid and the Aging Brain123 
The Journal of nutrition  2008;138(12):2510-2514.
The dietary essential PUFA docosahexaenoic acid [DHA; 22:6(n-3)] is a critical contributor to cell structure and function in the nervous system, and deficits in DHA abundance are associated with cognitive decline during aging and in neurodegenerative disease. Recent studies underscore the importance of DHA-derived neuroprotectin D1 (NPD1) in the homeostatic regulation of brain cell survival and repair involving neurotrophic, antiapoptotic and antiinflammatory signaling. Emerging evidence suggests that NPD1 synthesis is activated by growth factors and neurotrophins. Evolving research indicates that NPD1 has important determinant and regulatory interactions with the molecular-genetic mechanisms affecting b-amyloid precursor protein (bAPP) and amyloid beta (Ab) peptide neurobiology. Deficits in DHA or its peroxidation appear to contribute to inflammatory signaling, apoptosis, and neuronal dysfunction in Alzheimer disease (AD), a common and progressive age-related neurological disorder unique to structures and processes of the human brain. This article briefly reviews our current understanding of the interactions of DHA and NPD1 on bAPP processing and Ab peptide signaling and how this contributes to oxidative and pathogenic processes characteristic of aging and AD pathology.
doi:10.3945/jn.108.096016
PMCID: PMC2666388  PMID: 19022980
11.  Neuroprotectin D1 Restores Corneal Nerve Integrity and Function After Damage From Experimental Surgery 
Purpose.
To investigate if topical treatment of neuroprotectin D1 (NPD1) increases regeneration of functional nerves after lamellar keratectomy.
Methods.
An 8-mm stromal dissection was performed in the left eye of each rabbit. The rabbits were treated with NPD1, pigment epithelial-derived factor (PEDF) in combination with docosahexaenoic acid (DHA) or vehicle for 6 weeks, and corneas were obtained at 8 weeks. After fixation, corneal wholemounts were stained with mouse monoclonal anti-βIII-tubulin antibody and double stained with chicken anti-calcitonin gene-related peptide (CGRP) antibody. Corneal sensitivity and tear secretion were measured using the Cochet-Bonnet esthesiometer and the Schirmer's test, respectively. Additional rabbits were treated with NPD1, PEDF+DHA, or vehicle, and corneal sections were stained with a rat monoclonal anti-neutrophil antibody. Cultures of trigeminal ganglia from 5-day-old mice were treated with NPD1, PEDF+DHA, lipoxin A4 (LXA4), 12- or 15-hydroxyeicosatetraenoic acid (12[S] or 15[S]-HETE), and nerve growth factor (NGF) as positive control.
Results.
NPD1 increased subepithelial corneal nerve area three times compared with vehicle-treated rabbits. The effect was similar to PEDF+DHA–treated animals. There was recovery of CGRP-positive neurons and an increase in corneal sensitivity and tear secretion in NPD1-treated animals. NPD1 decreased neutrophil infiltration after 2 and 4 days of treatment. In the in vitro cultures, NPD1 and PEDF+DHA induced a 3-fold increase in neurite outgrowth compared with cultures without supplementation. Treatments with LXA4, 12(S)-, and 15(S)- HETE did not stimulate neurite outgrowth.
Conclusions.
NPD1 has anti-inflammatory and nerve regenerative properties. This study demonstrates that NPD1 may offer an effective treatment for neurotrophic corneas.
We show, for the first time, that the docosanoid neuroprotectin D1 (NPD1) is able to regenerate corneal nerves after damage from lamellar keratectomy in an animal model and reduce polymorphonuclear neutrophil infiltration induced by injury.
doi:10.1167/iovs.13-12075
PMCID: PMC3681478  PMID: 23702780
neuroregeneration; corneal sensitivity; pigment epithelial-derived factor; neuroprotectin D1; dry eye
12.  Neuroprotectin D1 Reduces the Severity of Herpes Simplex Virus–Induced Corneal Immunopathology 
Purpose.
Neuroprotectin D1 (NPD1) is an anti-inflammatory and proresolving lipid mediator biosynthesized from the omega-3-polyunsaturated fatty acid docosahexaenoic acid (DHA). The purpose of this study is to test the therapeutic potential of NPD1 for the treatment of herpes simplex virus (HSV)–induced stromal keratitis (SK) using a mouse model.
Methods.
C57BL/6 mice were infected ocularly with HSV-1 strain RE. Infected animals were treated topically with methyl ester prodrug NPD1 (300 ng/eye, 5-μL drop). Development of SK lesions, infiltration of inflammatory cells into the cornea, and production of proinflammatory cytokines, chemokines, and angiogenic factors were compared to untreated animals using slit-lamp biomicroscopy, flow cytometry, ELISA, and quantitative PCR (qPCR).
Results.
Topical administration of NPD1 resulted in a significant reduction in the severity and incidence of SK, as well as the extent of corneal neovascularization in the NPD1-treated animals compared to their untreated counterparts. Infiltration of fewer neutrophils and pathogenic CD4+ T cells into the cornea, along with a lower number of cells that could be induced ex vivo to produce IFN-γ and IL-17, occurred with NPD1 treatment. Additionally, treatment with NPD1 diminished the production of proinflammatory cytokines, chemokines, and angiogenic factors, such as IL-6, CXCL1, CXCL-10, CCL-20, VEGF-A, MMP-2, and MMP-9 in the corneas of infected animals. Importantly, treatment with NPD1 increased the production of the anti-inflammatory cytokine, IL-10.
Conclusions.
Our novel findings demonstrate that NPD1 treatment could represent a valuable therapeutic approach to control SK lesions.
In this report, we demonstrate the efficacy of topical therapy with neuroprotectin D1 against ocular disease caused by HSV.
doi:10.1167/iovs.13-12152
PMCID: PMC3776714  PMID: 23942967
NPD1; immunopathology; HSV-1; herpes stromal keratitis
13.  Docosahexaenoic Acid Neurolipidomics 
Mediator lipidomics is a field of study concerned with the characterization, structural elucidation and bioactivity of lipid derivatives generated by enzymatic activity. Omega-3 fatty acids have beneficial effects for vision, brain function, cardiovascular function, and immune-inflammatory responses. Docosahexaenoic acid [DHA; 22:6(n-3)], the most abundant essential omega-3 fatty acid in the human body, is selectively enriched and avidly retained in the central nervous system as an acyl chain of phospholipids. Brain-ischemia reperfusion and seizures trigger rapid release of DHA and of arachidonic acid (AA) as free, unesterified fatty acids. AA in turn generates eicosanoids, and DHA forms docosanoids. The stereoselective docosanoid neuroprotectin D1 (NPD1; 10R,17S-dihydroxy-docosa-4Z,7Z,11E,15E,19Z hexaenoic acid) is formed early in brain-ischemia reperfusion. Supplementation of NPD1 (intracerebroventricularly; i.c.v.) or of DHA (i.c.v. or systemically) results in decreased infarct size, polymorphonuclear neutrophil infiltration, ischemia-induced nuclear factor kappa B (NFκB) activation, and cyclooxygenase-2 (COX-2) induction. DHA involvement in cell function includes enhancing Akt translocation and activation, and binding to a peroxisome proliferator-activated receptor-gamma (PPAR-γ) family of ligand-activated nuclear receptors. Here we present an overview of recent DHA-mediator lipidomic studies in experimental brain ischemia-reperfusion and other conditions.
doi:10.1016/j.prostaglandins.2009.09.005
PMCID: PMC2905848  PMID: 19804838
14.  Docosahexaenoic Acid Signaling Modulates Cell Survival in Experimental Ischemic Stroke Penumbra and Initiates Long-Term Repair in Young and Aged Rats 
PLoS ONE  2012;7(10):e46151.
Background
Docosahexaenoic acid, a major omega-3 essential fatty acid family member, improves behavioral deficit and reduces infarct volume and edema after experimental focal cerebral ischemia. We hypothesize that DHA elicits neuroprotection by inducing AKT/p70S6K phosphorylation, which in turn leads to cell survival and protects against ischemic stroke in young and aged rats.
Methods and Results
Rats underwent 2 h of middle cerebral artery occlusion (MCAo). DHA, neuroprotectin D1 (NPD1) or vehicle (saline) was administered 3 h after onset of stroke. Neurological function was evaluated on days 1, 2, 3, and 7. DHA treatment improved functional recovery and reduced cortical, subcortical and total infarct volumes 7 days after stroke. DHA also reduced microglia infiltration and increased the number of astrocytes and neurons when compared to vehicle on days 1 and 7. Increases in p473 AKT and p308 AKT phosphorylation/activation were observed in animals treated with DHA 4 h after MCAo. Activation of other members of the AKT signaling pathway were also observed in DHA treated animals including increases in pS6 at 4 h and pGSK at 24 h. DHA or NPD1 remarkably reduced total and cortical infarct in aged rats. Moreover, we show that in young and aged rats DHA treatment after MCAo potentiates NPD1 biosynthesis. The phosphorylation of p308 AKT or pGSK was not different between groups in aged rats. However, pS6 expression was increased with DHA or NPD1 treatment when compared to vehicle.
Conclusions
We suggest that DHA induces cell survival, modulates the neuroinflammatory response and triggers long term restoration of synaptic circuits. Both DHA and NPD1 elicited remarkable protection in aged animals. Accordingly, activation of DHA signaling might provide benefits in the management of ischemic stroke both acutely as well as long term to limit ensuing disabilities.
doi:10.1371/journal.pone.0046151
PMCID: PMC3484151  PMID: 23118851
15.  Hippocampal Neuro-Networks and Dendritic Spine Perturbations in Epileptogenesis Are Attenuated by Neuroprotectin D1 
PLoS ONE  2015;10(1):e0116543.
Purpose
Limbic epileptogenesis triggers molecular and cellular events that foster the establishment of aberrant neuronal networks that, in turn, contribute to temporal lobe epilepsy (TLE). Here we have examined hippocampal neuronal network activities in the pilocarpine post-status epilepticus model of limbic epileptogenesis and asked whether or not the docosahexaenoic acid (DHA)-derived lipid mediator, neuroprotectin D1 (NPD1), modulates epileptogenesis.
Methods
Status epilepticus (SE) was induced by intraperitoneal administration of pilocarpine in adult male C57BL/6 mice. To evaluate simultaneous hippocampal neuronal networks, local field potentials were recorded from multi-microelectrode arrays (silicon probe) chronically implanted in the dorsal hippocampus. NPD1 (570 μg/kg) or vehicle was administered intraperitoneally daily for five consecutive days 24 hours after termination of SE. Seizures and epileptiform activity were analyzed in freely-moving control and treated mice during epileptogenesis and epileptic periods. Then hippocampal dendritic spines were evaluated using Golgi-staining.
Results
We found brief spontaneous microepileptiform activity with high amplitudes in the CA1 pyramidal and stratum radiatum in epileptogenesis. These aberrant activities were attenuated following systemic NPD1 administration, with concomitant hippocampal dendritic spine protection. Moreover, NPD1 treatment led to a reduction in spontaneous recurrent seizures.
Conclusions
Our results indicate that NPD1 displays neuroprotective bioactivity on the hippocampal neuronal network ensemble that mediates aberrant circuit activity during epileptogenesis. Insight into the molecular signaling mediated by neuroprotective bioactivity of NPD1 on neuronal network dysfunction may contribute to the development of anti-epileptogenic therapeutic strategies.
doi:10.1371/journal.pone.0116543
PMCID: PMC4305283  PMID: 25617763
16.  Neuroprotectin D1 Attenuates Laser-induced Choroidal Neovascularization in Mouse 
Molecular Vision  2010;16:320-329.
Purpose
To examine the effects of neuroprotectin D1 (NPD1), a stereospecific derivative of docosahexaenoic acid, on choroidal neovascularization (CNV) in a laser-induced mouse model. Specifically, this was assessed by clinically grading laser-induced lesions, measuring leakage area, and volumetrically quantifying vascular endothelial cell proliferation.
Methods
C57Bl/6 mice were treated with vehicle control or NPD1, and choroidal neovascularization was induced by laser rupture of Bruch's membrane; treatment was administered throughout the first week of recovery. One and two weeks after CNV induction, fundus fluorescein angiography was performed. Angiograms were clinically graded to assess leakage severity, while leakage area was measured by image analysis of angiograms. Proliferation of vascular endothelial cells was evaluated volumetrically by three-dimensional laser confocal immunofluorescent microscopy of cytoskeletal, nuclear, and endothelial cell markers.
Results
At seven days after CNV induction, NPD1-treated mice had 60% fewer clinically relevant lesions than controls, dropping to 80% fewer by 14 days. NPD1 mice exhibited 25% smaller leakage area than controls at 7 days and 44% smaller area at 14 days. Volumetric immunofluorescence revealed 46% less vascular endothelial cell volume in 7-day NPD1-treated mice than in 7-day controls, and by 14 days NPD1 treatment was 68% lower than controls. Furthermore, comparison of 7- and 14-day volumes of NPD1-treated mice revealed a 50% reduction at 14 days.
Conclusions
NPD1 significantly inhibits choroidal neovascularization. There are at least two possible mechanisms that could explain the neuroprotective action of NPD1. Ultimately, nuclear factor-κB could be inhibited with a reduction in cyclooxygenase-2 (COX-2) to reduce vascular endothelial growth factor (VEGF) expression, and/or activation of the resolution phase of the inflammatory response/survival pathways could be upregulated. Moreover, NPD1 continues to be effective after treatment is concluded, suggesting sustained protection and highlighting the potential applicability of this lipid mediator in preventing or ameliorating endothelial cell growth in pathoangiogenesis.
PMCID: PMC2834569  PMID: 20216940
17.  Omega-3 Fatty acids in Dry Eye and Corneal Nerve Regeneration after Refractive Surgery 
Dry eye (DE) is a multifactorial condition that affects the surface of the eye and induces an inflammatory response. Corneal nerves play an important role in the maintenance of a healthy ocular surface. Here we review corneal structure, nerve architecture, DE conditions, and nerve regeneration following corneal surgery and discuss how n-3 fatty acids affect the health of the cornea. Animal studies show that resolvins, compounds derived from eicosapentaenoic acid (EPA), increase tear volume and decrease inflammation induced by DE. After corneal surgery in rabbits, treatment with nerve growth factor (NGF) or pigment epithelial derived factor (PEDF) in conjunction with docosahexaenoic acid (DHA) increase nerve density and corneal epithelial cell proliferation. Increased synthesis of the novel docosanoid, neuroprotectin D1 (NPD1), was found in corneas after the animals were treated with PEDF and DHA. Topical application of these lipids derived from n-3 fatty acids could be useful in treating DE and prevent clinical complications such as cornea erosion and ulcerations.
doi:10.1016/j.plefa.2010.02.004
PMCID: PMC2856794  PMID: 20202804
18.  Docosahexaenoic Acid Therapy of Experimental Ischemic Stroke 
Translational Stroke Research  2010;2(1):33-41.
We examined the neuroprotective efficacy of docosahexaenoic acid (DHA), an omega-3 essential fatty acid family member, in acute ischemic stroke; studied the therapeutic window; and investigated whether DHA administration after an ischemic stroke is able to salvage the penumbra. In each series described below, SD rats underwent 2 h of middle cerebral artery occlusion (MCAo). In series 1, DHA or saline was administered i.v. at 3, 4, 5, or 6 h after stroke. In series 2, MRI was conducted on days 1, 3 and 7. In series 3, DHA or saline was administered at 3 h, and lipidomic analysis was conducted on day 3. Treatment with DHA significantly improved behavior and reduced total infarct volume by a mean of 40% when administered at 3 h, by 66% at 4 h, and by 59% at 5 h. Total lesion volumes computed from T2-weighted images were reduced in the DHA group at all time points. Lipidomic analysis showed that DHA treatment potentiates neuroprotectin D1 (NPD1) synthesis in the penumbra 3 days after MCAo. DHA administration provides neurobehavioral recovery, reduces brain infarction and edema, and activates NPD1 synthesis in the penumbra when administered up to 5 h after focal cerebral ischemia in rats.
doi:10.1007/s12975-010-0046-0
PMCID: PMC3037476  PMID: 21423332
Focal ischemia; Magnetic resonance imaging; Neuroprotection; Animal models
19.  Resolving TRPV1 and TNF-α Mediated Spinal Cord Synaptic Plasticity and Inflammatory Pain with Neuroprotectin D1 
Mechanisms of inflammatory pain are not fully understood. We investigated the role of TRPV1 and TNF-α, two critical mediators for inflammatory pain, in regulating spinal cord synaptic transmission. We found in mice lacking Trpv1 the frequency but not the amplitude of spontaneous EPSCs (sEPSCs) in lamina II neurons of spinal cord slices is reduced. Further, C-fiber-induced spinal long-term potentiation (LTP) in vivo is abolished in Trpv1 knockout mice. TNF-α also increases sEPSC frequency but not amplitude in spinal lamina IIo neurons, and this increase is abolished in Trpv1 knockout mice. Single-cell PCR analysis revealed that TNF-α-responding neurons in lamina IIo are exclusively excitatory (vGluT2+) neurons. Notably, neuroprotectin-1 (NPD1), an anti-inflammatory lipid mediator derived from omega-3 polyunsaturated fatty acid (docosahexaenoic acid) blocks TNF-α- and capsaicin-evoked sEPSC frequency increases but has no effect on basal synaptic transmission. Strikingly, NPD1 potently inhibits capsaicin-induced TRPV1 current (IC50=0.4 nM) in dissociated dorsal root ganglion neurons, and this IC50 is ≈ 500 times lower than that of AMG9810, a commonly used TRPV1 antagonist. NPD1 inhibition of TRPV1 is mediated by GPCRs, since the effects were blocked by pertussis toxin. In contrast, NPD1 had not effect on mustard oil-induced TRPA1 currents. Spinal injection of NPD1, at very low doses (0.1–10 ng), blocks spinal LTP and reduces TRPV1-dependent inflammatory pain, without affecting baseline pain. NPD1 also reduces TRPV1-independent but TNF-α-dependent pain hypersensitivity. Our findings demonstrate a novel role of NPD1 in regulating TRPV1/TNF-α-mediated spinal synaptic plasticity and identify NPD1 as a novel analgesic for treating inflammatory pain.
doi:10.1523/JNEUROSCI.2443-11.2011
PMCID: PMC3202339  PMID: 22016541
central sensitization; docosahexaenoic acid; long-term potentiation; omega-3 polyunsaturated fatty acid; primary sensory neurons; single-cell PCR; spontaneous EPSCs; TRPA1
20.  Neuroinflammation and proteostasis are modulated by endogenously biosynthetized Neuroprotectin D1 
Molecular neurobiology  2012;46(1):221-226.
Neurodegenerative diseases encompass complex cell signaling disturbances that initially damage neuronal circuits and synapses. Due to multiple protective mechanisms that are enacted to counteract the onset of neurodegenerative diseases, there is often a prolonged period without noticeable impairments during their initiation. Since severe cognitive deficit or vision loss takes place after that period there is an opportunity to harness endogenous protective mechanisms as potential therapeutic approaches. The activation of the biosynthesis of the docosanoid mediator neuroprotectin D1 (NPD1) is an early response to the upsurge of protein misfolding and other neuroinflammatory events. This overview discusses the potent neuroprotective and inflammation-modulating bioactivity of NPD1. This lipid mediator represents an early response to neurodegenerations, aiming to restore homeostasis.
doi:10.1007/s12035-012-8322-5
PMCID: PMC3477672  PMID: 22956271
Misfolding; retinal degenerations; Alzheimer’s disease; Huntington’s disease; epilepsy; docosahexaenoic acid; ataxin1; huntingtin; CAG repeats; APP; Bcl-2 proteins
21.  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.
doi:10.1016/j.plefa.2012.08.008
PMCID: PMC3538114  PMID: 23022417
Misfolding; Alzheimer’s disease; docosahexaenoic acid; ataxin-1; huntingtin; CAG repeats; APP; Retinal pigment epithelial cell
22.  Novel Pro-resolving Aspirin-Triggered DHA Pathway 
Chemistry & biology  2011;18(8):976-987.
Summary
Endogenous mechanisms in the resolution of acute inflammation are of interest since excessive inflammation underlies many pathologies. We report a new aspirin-triggered DHA metabolome that biosynthesizes a potent product in inflammatory exudates and human leukocytes, namely aspirin-triggered Neuroprotectin D1/Protectin D1 [AT-(NPD1/PD1)]. The complete stereochemistry of AT-(NPD1/PD1) proved to be 10R,17R-dihydroxydocosa- 4Z,7Z,11E,13E,15Z,19Z-hexaenoic acid. The chirality of hydroxyl groups and geometry of the conjugated triene system essential for bioactivity were established by matching biological materials with stereochemically pure isomers prepared by organic synthesis. AT-(NPD1/PD1) reduced neutrophil (PMN) recruitment in murine peritonitis in a dose-dependent fashion where neither a Δ15-trans-isomer nor DHA was effective. With human cells, AT-(NPD1/PD1) decreased transendothelial PMN migration as well as enhanced efferocytosis of apoptotic human PMN by macrophages. These results indicate that AT-(NPD1/PD1) is a potent antiinflammatory-pro-resolving molecule.
doi:10.1016/j.chembiol.2011.06.008
PMCID: PMC3164791  PMID: 21867913
23.  Docosahexaenoic Acid, Protectins and Dry Eye 
Purpose of review
To report recent data on the potential role of omega-3 fatty acids, in particular docosahexaenoic acid (DHA) and its derivatives, in the treatment of dry eye syndrome.
Recent findings
Dietary supplementation with polyunsaturated fatty acids (PUFAs) yields positive results in the improvement of dry eye signs and symptoms. Although several studies have shown this, evidence is still lacking as to which fatty acid or what combination constitutes the most effective treatment. Studies show that treatment with alpha-linoleic acid reduces dry eyeinduced inflammation. Eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) derivatives, particularly resolvin E1 (RvE1) and neuroprotectin D1 (NPD1), appear to be responsible for DHA’s anti-inflammatory effect. This is supported in a study where topical RvE1 resulted in decreased inflammation in a mouse dry eye model. Topical administration of pigment epithelium derived factor (PEDF) in combination with DHA accelerates the regeneration of corneal nerves after their damage during corneal surgery, promoting the return of sensitivity and reducing the signs of dry eye. This combined treatment also reduces objective signs of dry eye, such as rose bengal staining.
Summary
No firm recommendations can be made regarding optimal dietary supplementation of essential fatty acids that benefit dry eye patients. Based on animal data and preliminary human studies, DHA and its derivatives appear to be a safe, effective topical treatment for dry eye patients. This may result from their role in the resolution of inflammation and the regeneration of damaged corneal nerves.
doi:10.1097/MCO.0b013e328342bb1a
PMCID: PMC3971926  PMID: 21157308
Dry eye; docosahexaenoic acid; omega-3 fatty acids; omega-6 fatty acids; corneal nerves; neuroprotectin D1
24.  PI3K/Akt and mTOR/p70S6K Pathways Mediate Neuroprotectin D1-Induced Retinal Pigment Epithelial Cell Survival during Oxidative Stress-Induced Apoptosis 
Experimental eye research  2010;90(6):718-725.
The initiation and progression of several forms of retinal degenerations involve excessive, repetitive, and/or sustained oxidative stress that, in turn, mediate photoreceptor cell damage and death. Since phosphatidylinositol 3-kinase (PI3K)/Akt and mTOR/p70S6-kinase pathways are part of survival signaling in cells confronted with oxidative stress, we asked whether or not docosahexaenoic acid-derived neuroprotectin D1 (NPD1) mediates survival upon single-dose and/or repetitive oxidative stress through this pathway. For this purpose, we used human retinal pigment epithelial (ARPE-19) cells challenged by exposure to hydrogen peroxide (H2O2) plus tumor necrosis factor alpha (TNF-α). We found that in single-dose oxidative stress-induced apoptosis, phosphorylation of Akt, mTOR, and p70S6K was both time- and dose-dependent. Inhibition of PI3K or mTOR/p70S6K by wortmannin and rapamycin, respectively, increased apoptosis and inhibited phosphorylation of Akt and p70S6K induced by single-dose oxidative stress. While two exposures of a low-dose, non-damaging oxidation induced apoptosis and upregulation of Akt, mTOR, and p70S6K, longer treatment of the cells with three exposures of low dose to low-dose stress showed no changes in the levels of Akt, mTOR, or p70S6K, and resulted in enhanced apoptosis compared to higher doses. Removing the oxidative stress-inducing agents following the single-dose or short term repetitive oxidative stress at the peak of Akt, mTOR, and p70S6K phosphorylation (i.e, 30 minutes after induction) led to recovery, with no apoptosis after 16 hours of incubation. Cells that were induced with three low doses of stress did not show recovery when oxidative stress was removed 30 minutes after the last exposure. NPD1 protected the RPE cells against both single-dose and repetitive oxidative stress-induced apoptosis and promoted higher levels of phosphorylated Akt, mTOR, and p70S6K. Together, our results show that a) repetitive oxidative stress is dose dependent and may not be recovered by removing the oxidative stress-inducing agents, b) PI3K/Akt and mTOR/p70S6K pathways play a major role in the protection against oxidative stress-induced apoptosis in ARPE-19 cells, and c) NPD1 exerts protection under these conditions by inducing PI3K/Akt and mTOR/p70S6K pathways.
doi:10.1016/j.exer.2010.03.002
PMCID: PMC2873108  PMID: 20230819
phosphorylation; neurodegenerative diseases; photoreceptor cells; docosahexaenoic acid; retinal degenerations
25.  Microglial ramification and redistribution concomitant with the attenuation of choroidal neovascularization by neuroprotectin D1 
Molecular Vision  2013;19:1747-1759.
Purpose
Neuroprotectin D1 (NPD1) attenuates laser-induced choroidal neovascularization (CNV) when administered intraperitoneally. Due to its lipophilicity and low molecular weight, NPD1 is well suited for topical delivery; thus, we investigated the efficacy of topically applied NPD1 in attenuating CNV. We also examined the effect of NPD1 on the recruitment and activation of microglia surrounding CNV lesions.
Methods
Mice were given laser-induced CNV and treated with NPD1 eye drops. CNV was evaluated by fluorescein leakage using a novel image analysis method and by isolectin B4 immunofluorescence of neovasculature. Microglia; recruitment was assessed by quantification. Using form factor, solidity, convexity, and fractal dimension, microglial activation was quantitatively assessed by two-dimensional, and for the first time, three-dimensional morphology. An ImageJ plugin, 3D Shape, was developed to enable this analysis.
Results
NPD1 attenuated leakage and neovascularization. The proximity of microglia to CNV lesions was significantly closer with NPD1. Consistent with the cellular ramification, microglia in NPD1-treated eyes were larger and exhibited a lower form factor and higher fractal dimension.
Conclusions
Our data show that NPD1 signaling induces a ramified, non-injury-inducing microglial phenotype coincident with attenuation of CNV. Since microglia are crucial participants in neurodegenerative diseases, the discovery that microglia are potential targets of NPD1 signaling warrants further investigation.
PMCID: PMC3733904  PMID: 23922492

Results 1-25 (1008250)