Bokkyoo Jun

Novel aspirin-triggered neuroprotectin D1 attenuates cerebral ischemic injury after experimental stroke

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, 48 h, 72 h, and 7 days. At 3h post-stroke onset, an intravenous administration of 333 μg/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 11.7 T 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, 48 h, 72 h 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.

NPD1-mediated stereoselective regulation of BIRC3 expression through cREL is decisive for neural cell survival.

Neuroprotectin D1 (NPD1), a docosahexaenoic acid (DHA)-derived mediator, induces cell survival in uncompensated oxidative stress (OS), neurodegenerations or ischemic stroke. The molecular principles underlying this protection remain unresolved. We report here that, in retinal pigment epithelial cells, NPD1 induces nuclear translocation and cREL synthesis that, in turn, mediates BIRC3 transcription. NPD1 activates NF-κB by an alternate route to canonical signaling, so the opposing effects of TNFR1 and NPD1 on BIRC3 expression are not due to interaction/s between NF-κB pathways. RelB expression follows a similar pattern as BIRC3, indicating that NPD1 also is required to activate cREL-mediated RelB expression. These results suggest that cREL, which follows a periodic pattern augmented by the lipid mediator, regulates a cluster of NPD1-dependent genes after cREL nuclear translocation. BIRC3 silencing prevents NPD1 induction of survival against OS. Moreover, brain NPD1 biosynthesis and selective neuronal BIRC3 abundance are increased by DHA after experimental ischemic stroke followed by remarkable neurological recovery. Thus, NPD1 bioactivity governs key counter-regulatory gene transcription decisive for retinal and brain neural cell integrity when confronted with potential disruptions of homeostasis.

Real-time structural transitions are coupled to chemical steps in ATP hydrolysis by Eg5 kinesin

At the biochemical level, motor proteins are enzymatic molecules that function by converting chemical energy into mechanical motion. The key element for energy transduction and a major unresolved question common for all motor proteins is the coordination between the chemical and conformational steps in ATP hydrolysis. Here we show time-lapse monitoring of an in vitro ATP hydrolysis reaction by the motor domain of a human Kinesin-5 protein (Eg5) using difference Fourier transform infrared spectroscopy and UV photolysis of caged ATP. In this first continuous observation of a biological reaction coordinate from substrate to product, direct spectral markers for two catalytic events are measured: proton abstraction from nucleophilic water by the catalytic base and formation of the inorganic phosphate leaving group. Simultaneous examination of conformational switching in Eg5, in parallel with catalytic steps, shows structural transitions in solution consistent with published crystal structures of the prehydrolytic and ADP-bound states. In addition, we detect structural modifications in the Eg5 motor domain during bond cleavage between the β- and γ-phosphates of ATP. This conclusion challenges mechanochemical models for motor proteins that utilize only two stages of the catalytic cycle to drive force and motion.

Elovanoids are novel cell-specific lipid mediators necessary for neuroprotective signaling for photoreceptor cell integrity

Docosahexaenoic acid (DHA, 22:6 n-3) is abundant in the retina and is enzymatically converted into pro-homeostatic docosanoids. The DHA- or eicosapentaenoic acid (EPA)-derived 26 carbon fatty acid is a substrate of elongase ELOVL4, which is expressed in photoreceptor cells and generates very long chain (≥C28) polyunsaturated fatty acids including n-3 (VLC-PUFAs,n-3). While ELOVL4 mutations are linked to vision loss and neuronal dysfunctions, the roles of VLC-PUFAs remain unknown. Here we report a novel class of lipid mediators biosynthesized in human retinal pigment epithelial (RPE) cells that are oxygenated derivatives of VLC-PUFAs,n-3; we termed these mediators elovanoids (ELV). ELVs have structures reminiscent of docosanoids but with different physicochemical properties and alternatively-regulated biosynthetic pathways. The structures, stereochemistry, and bioactivity of ELVs were determined using synthetic materials produced by stereo-controlled chemical synthesis. ELVs enhance expression of pro-survival proteins in cells undergoing uncompensated oxidative stress. Our findings unveil a novel autocrine/paracrine pro-homeostatic RPE cell signaling that aims to sustain photoreceptor cell integrity and reveal potential therapeutic targets for retinal degenerations.

Defining a mechanistic link between pigment epithelium-derived factor, docosahexaenoic acid and corneal nerve regeneration

The cornea is densely innervated to sustain the integrity of the ocular surface. Corneal nerve damage produced by aging, diabetes, refractive surgeries, and viral or bacterial infections impairs tear production, the blinking reflex, and epithelial wound healing, resulting in loss of transparency and vision. A combination of the known neuroprotective molecule, pigment epithelium–derived factor (PEDF) plus docosahexaenoic acid (DHA), has been shown to stimulate corneal nerve regeneration, but the mechanisms involved are unclear. Here, we sought to define the molecular events of this effect in an in vivo mouse injury model. We first confirmed that PEDF + DHA increased nerve regeneration in the mouse cornea. Treatment with PEDF activates the phospholipase A2 activity of the PEDF-receptor (PEDF-R) leading to the release of DHA; this free DHA led to enhanced docosanoid synthesis and induction of bdnf, ngf, and the axon growth promoter semaphorin 7a (sema7a), and as a consequence, their products appeared in the mouse tears. Surprisingly, corneal injury and treatment with PEDF + DHA induced transcription of neuropeptide y (npy), small proline-rich protein 1a (sprr1a), and vasoactive intestinal peptide (vip) in the trigeminal ganglia (TG). The PEDF-R inhibitor, atglistatin, blocked all of these changes in the cornea and TG. In conclusion, we uncovered here an active cornea–TG axis, driven by PEDF-R activation, that fosters axon outgrowth in the cornea.

Loss of diacylglycerol kinase epsilon in mice causes endothelial distress and impairs glomerular Cox-2 and PGE2 production

Thrombotic microangiopathy (TMA) is a disorder characterized by microvascular occlusion that can lead to thrombocytopenia, hemolytic anemia, and glomerular damage. Complement activation is the central event in most cases of TMA. Primary forms of TMA are caused by mutations in genes encoding components of the complement or regulators of the complement cascade. Recently, we and others have described a genetic form of TMA caused by mutations in the gene diacylglycerol kinase-ε (DGKE) that encodes the lipid kinase DGKε (Lemaire M, Fremeaux-Bacchi V, Schaefer F, Choi MR, Tang WH, Le Quintrec M, Fakhouri F, Taque S, Nobili F, Martinez F, Ji WZ, Overton JD, Mane SM, Nurnberg G, Altmuller J, Thiele H, Morin D, Deschenes G, Baudouin V, Llanas B, Collard L, Majid MA, Simkova E, Nurnberg P, Rioux-Leclerc N, Moeckel GW, Gubler MC, Hwa J, Loirat C, Lifton RP. Nat Genet 45: 531-536, 2013; Ozaltin F, Li BH, Rauhauser A, An SW, Soylemezoglu O, Gonul II, Taskiran EZ, Ibsirlioglu T, Korkmaz E, Bilginer Y, Duzova A, Ozen S, Topaloglu R, Besbas N, Ashraf S, Du Y, Liang CY, Chen P, Lu DM, Vadnagara K, Arbuckle S, Lewis D, Wakeland B, Quigg RJ, Ransom RF, Wakeland EK, Topham MK, Bazan NG, Mohan C, Hildebrandt F, Bakkaloglu A, Huang CL, Attanasio M. J Am Soc Nephrol 24: 377-384, 2013). DGKε is unrelated to the complement pathway, which suggests that unidentified pathogenic mechanisms independent of complement dysregulation may result in TMA. Studying Dgke knockout mice may help to understand the pathogenesis of this disease, but no glomerular phenotype has been described in these animals so far. Here we report that Dgke null mice present subclinical microscopic anomalies of the glomerular endothelium and basal membrane that worsen with age and develop glomerular capillary occlusion when exposed to nephrotoxic serum. We found that induction of cyclooxygenase-2 and of the proangiogenic prostaglandin E2 are impaired in Dgke null kidneys and are associated with reduced expression of the antithrombotic cell adhesion molecule platelet endothelial cell adhesion molecule-1/CD31 in the glomerular endothelium. Notably, prostaglandin E2 supplementation was able to rescue motility defects of Dgke knockdown cells in vitro and to restore angiogenesis in a test in vivo. Our results unveil an unexpected role of Dgke in the induction of cyclooxygenase-2 and in the regulation of glomerular prostanoids synthesis under stress.

Retinal Pigment Epithelium and Photoreceptor Preconditioning Protection Requires Docosanoid Signaling

Omega-3 and omega-6 polyunsaturated fatty acids (PUFAs) are necessary for functional cell integrity. Preconditioning (PC), as we define it, is an acquired protection or resilience by a cell, tissue, or organ to a lethal stimulus enabled by a previous sublethal stressor or stimulus. In this study, we provide evidence that the omega-3 fatty acid docosahexaenoic acid (DHA) and its derivatives, the docosanoids 17-hydroxy docosahexaenoic acid (17-HDHA) and neuroprotectin D1 (NPD1), facilitate cell survival in both in vitro and in vivo models of retinal PC. We also demonstrate that PC requires the enzyme 15-lipoxygenase-1 (15-LOX-1), which synthesizes 17-HDHA and NPD1, and that this is specific to docosanoid signaling despite the concomitant release of the omega-6 arachidonic acid and eicosanoid synthesis. These findings advocate that DHA and docosanoids are protective enablers of PC in photoreceptor and retinal pigment epithelial cells.

Circulating inflammation-resolving lipid mediators RvD1 and DHA are decreased in patients with acutely symptomatic carotid disease

BACKGROUND Efficient biomarkers for early prediction and diagnosis of an acutely symptomatic carotid plaque rupture event are currently lacking, impairing the ability to diagnose and treat patients with an acute plaque rupture events in a timely fashion. Resolvins are endogenous inflammation-resolving lipid mediators that are induced by inflammatory insults. We hypothesized that resolvin and other lipid profiles in sera likely mark the process towards plaque rupture. METHODS Circulating lipids associated with plaque rupture events were quantitatively profiled via targeted mediator-lipidomics using ultraperformance liquid chromatography tandem mass spectrometry in patients with acutely symptomatic and asymptomatic carotid disease. RESULTS Resolvin D1 (RvD1, 82 ± 11pM vs. 152 ± 17pM, p = 0.001) and docosahexaenoic acid (DHA) (0.052 ± 0.007µM versus 0.076 ± 0.008µM, p = 0.025) levels are decreased in the sera of patients presenting with an acutely symptomatic carotid plaque rupture event (n = 21) compared to patients with asymptomatic (n = 24) high-grade carotid stenosis. Circulating arachidonic acid (AA) levels, however, were higher (0.429 ± 0.046µM versus 0.257 ± 0.035µM, p < 0.01) in acutely symptomatic compared to asymptomatic carotid patients. ROC curve analysis demonstrates that the serum ratio AA:RvD1 (AUC 0.84, sensitivity 0.71, specificity 0.92) and AA:DHA (AUC 0.86, sensitivity 0.90, specificity 0.71) are biomarkers for the risk of atherosclerotic plaque rupture. CONCLUSIONS A circulating pro-inflammatory lipid profile, characterized by high AA:RvD1 and AA:DHA, is associated with acutely symptomatic carotid disease and stroke.

Stereoselective Total Synthesis of Macrophage-Produced Prohealing 14,21-Dihydroxy Docosahexaenoic Acids

Synthesis of 14S,21R- and 14S,21S-dihydroxy-DHA (diHDHA) among the four possible stereoisomers of 14,21-diHDHA was studied. Methyl (R)-lactate (>97% ee), selected as a C20-C22 fragment (DHA numbering), was converted to the C17-C22 phosphonium salt, which was subjected to a Wittig reaction with racemic C16-aldehyde of the C12-C16 part with the TMS and TBS-oxy groups at C12 and C14, yielding the C12-C22 derivative with 14R/S and 21R chirality. Kinetic resolution using Sharpless asymmetric epoxidation of the TBS-deprotected allylic alcohol with l-(+)-DIPT/Ti(O-i-Pr)4 afforded 14S-epoxy alcohol and 14R-allylic alcohol with >99% diastereomeric excess (de) for both. The CN group was introduced to the epoxy alcohol by reaction with Et2AlCN. The 14R-allylic alcohol was also converted to the nitrile via Mitsunobu inversion. Reduction of the nitrile with DIBAL afforded the key aldehyde corresponding to the C11-C22 moiety. The Wittig reaction of this aldehyde with a phosphonium salt of the remaining C1-C10 part followed by functional group manipulation gave 14S,21R-diHDHA. Similarly, ethyl (S)-lactate (>99% ee) was converted to 14S,21S-diHDHA. The chiral LC-UV-MS/MS analysis demonstrated that each of these two 14,21-diHDHAs synthesized using the presented total organic synthesis was highly stereoselective and identical to the macrophage-produced counterpart.

Enteral Arg-Gln Dipeptide Administration Increases Retinal Docosahexaenoic Acid and Neuroprotectin D1 in a Murine Model of Retinopathy of Prematurity

Purpose Low levels of the long chain polyunsaturated fatty acid (LCPUFA) docosahexaenoic acid (DHA) have been implicated in retinopathy of prematurity (ROP). However, oral DHA suffers from poor palatability and is associated with increased bleeding in premature infants. We asked whether oral administration of the neutraceutical arginine-glutamine (Arg-Glu) could increase retinal DHA and improve outcomes in a mouse model of oxygen-induced retinopathy (OIR). Methods Postnatal day 7 (P7) pups were maintained at 75% oxygen for 5 days and then returned to room air on P12. Pups were gavaged twice daily with Arg-Gln or vehicle from P12 to P17 and eyes were harvested for analysis on P17. Vaso-obliteration and vascular density were assessed on retinal flat mounts and preretinal neovascularization was assessed on retinal cross sections. Retinas were used for measurement of DHA and 10,17S-docosatriene (neuroprotectin D1, NPD1), a key DHA-derived lipid, and for analysis by reverse-phase protein array (RPPA). Results With Arg-Gln treatment, retinal DHA and NPD1 levels were increased in OIR pups. Arg-Gln reduced preretinal neovascularization by 39 ± 6% (P < 0.05) relative to vehicle control. This was accompanied by a restoration of vascular density of the retina in the pups treated with Arg-Gln (73.0 ± 3.0%) compared to vehicle (53.1 ± 3.4%; P < 0.05). Arg-Gln dipeptide restored OIR-induced signaling changes toward normoxia and was associated with normalization of insulin-like growth factor receptor 1 signaling and reduction of apoptosis and an increase in anti-apoptosis proteins. Conclusions Arg-Gln may serve as a safer and easily tolerated nutraceutical agent for prevention or treatment of ROP.