Rod-specific downregulation of omega-3 very-long-chain polyunsaturated fatty acid pathway in age-related macular degeneration.

Docosahexaenoic acid (DHA; 22:6) plays a key role in vision and is the precursor for very-long-chain polyunsaturated fatty acids (VLC-PUFAs). The release of 32- and 34-carbon VLC-PUFAs and DHA from sn-1 and sn-2 of phosphatidylcholine (PC) leads to the synthesis of cell-survival mediators, the elovanoids (ELVs) and neuroprotectin D1 (NPD1), respectively. Macula and periphery from age-related macular degeneration (AMD) donor retinas were assessed for the availability of DHA-related lipids by LC-MS/MS-based lipidomic analysis and MALDI-molecular imaging. We found reduced retina DHA and VLC-PUFA pathways to synthesize omega-3 ELVs from precursors that likely resulted in altered disks and photoreceptor loss. Additionally, we compared omega-3 (n-3) fatty acid with DHA (22:6) and omega-6 (n-6) fatty acid with arachidonic acid (AA; 20:4) pathways. n-3 PC(22:6/22:6, 44:12) and n-6 PC(20:4/20:4, 40:8) showed differences among male/female, macula/periphery, and normal/AMD retinas. Periphery of AMD retina males increased 44:12 abundance, while normal females increased 40:8 (all macula had an upward 40:8 tendency). We also showed that female AMD switched from n-3 to n-6 fatty acids; most changes in AMD occurred in the periphery of female AMD retinas. DHA and VLC-PUFA release from PCs leads to conversion in pro-survival NPD1 and ELVs. The loss of the neuroprotective precursors of ELVs in the retina periphery from AMD facilitates uncompensated stress and cell loss. In AMD, the female retina loses peripheral rods VLC-PUFAs to about 33% less than in males limiting ELV formation and its protective bioactivity.

Inverse correlation between fatty acid transport protein 4 and vision in Leber congenital amaurosis associated with RPE65 mutation.

Fatty acid transport protein 4 (FATP4), a transmembrane protein in the endoplasmic reticulum (ER), is a recently identified negative regulator of the ER-associated retinal pigment epithelium (RPE)65 isomerase necessary for recycling 11-cis-retinal, the light-sensitive chromophore of both rod and cone opsin visual pigments. The role of FATP4 in the disease progression of retinal dystrophies associated with RPE65 mutations is completely unknown. Here we show that FATP4-deficiency in the RPE results in 2.8-fold and 1.7-fold increase of 11-cis- and 9-cis-retinals, respectively, improving dark-adaptation rates as well as survival and function of rods in the Rpe65 R91W knockin (KI) mouse model of Leber congenital amaurosis (LCA). Degradation of S-opsin in the proteasomes, but not in the lysosomes, was remarkably reduced in the KI mouse retinas lacking FATP4. FATP4-deficiency also significantly rescued S-opsin trafficking and M-opsin solubility in the KI retinas. The number of S-cones in the inferior retinas of 4- or 6-mo-old KI;Fatp4-/- mice was 7.6- or 13.5-fold greater than those in age-matched KI mice. Degeneration rates of S- and M-cones are negatively correlated with expression levels of FATP4 in the RPE of the KI, KI;Fatp4+/- , and KI;Fatp4-/- mice. Moreover, the visual function of S- and M-cones is markedly preserved in the KI;Fatp4-/- mice, displaying an inverse correlation with the FATP4 expression levels in the RPE of the three mutant lines. These findings establish FATP4 as a promising therapeutic target to improve the visual cycle, as well as survival and function of cones and rods in patients with RPE65 mutations.

Elovanoids counteract oligomeric ß-amyloid-induced gene expression and protect photoreceptors.

The onset of neurodegenerative diseases activates inflammation that leads to progressive neuronal cell death and impairments in cognition (Alzheimer's disease) and sight (age-related macular degeneration [AMD]). How neuroinflammation can be counteracted is not known. In AMD, amyloid ß-peptide (Aß) accumulates in subretinal drusen. In the 5xFAD retina, we found early functional deficiencies (ERG) without photoreceptor cell (PRC) death and identified early insufficiency in biosynthetic pathways of prohomeostatic/neuroprotective mediators neuroprotectin D1 (NPD1) and elovanoids (ELVs). To mimic an inflammatory milieu in wild-type mouse, we triggered retinal pigment epithelium (RPE) damage/PRC death by subretinally injected oligomeric ß-amyloid (OAß) and observed that ELVs administration counteracted their effects, protecting these cells. In addition, ELVs prevented OAß-induced changes in gene expression engaged in senescence, inflammation, autophagy, extracellular matrix remodeling, and AMD. Moreover, as OAß targets the RPE, we used primary human RPE cell cultures and demonstrated that OAß caused cell damage, while ELVs protected and restored gene expression as in mouse. Our data show OAß activates senescence as reflected by enhanced expression of p16INK4a, MMP1, p53, p21, p27, and Il-6, and of senescence-associated phenotype secretome, followed by RPE and PRC demise, and that ELVs 32 and 34 blunt these events and elicit protection. In addition, ELVs counteracted OAß-induced expression of genes engaged in AMD, autophagy, and extracellular matrix remodeling. Overall, our data uncovered that ELVs downplay OAß-senescence program induction and inflammatory transcriptional events and protect RPE cells and PRC, and therefore have potential as a possible therapeutic avenue for AMD.

Membrane-type frizzled-related protein regulates lipidome and transcription for photoreceptor function.

Molecular decision-makers of photoreceptor (PRC) membrane organization and gene regulation are critical to understanding sight and retinal degenerations that lead to blindness. Using Mfrprd6 mice, which develop PRC degeneration, we uncovered that membrane-type frizzled-related protein (MFRP) participates in docosahexaenoic acid (DHA, 22:6) enrichment in a manner similar to adiponectin receptor 1 (AdipoR1). Untargeted imaging mass spectrometry demonstrates cell-specific reduction of phospholipids containing 22:6 and very long-chain polyunsaturated fatty acids (VLC-PUFAs) in Adipor1-/- and Mfrprd6 retinas. Gene expression of pro-inflammatory signaling pathways is increased and gene-encoding proteins for PRC function decrease in both mutants. Thus, we propose that both proteins are necessary for retinal lipidome membrane organization, visual function, and to the understanding of the early pathology of retinal degenerative diseases.

Bioavailability and spatial distribution of fatty acids in the rat retina after dietary omega-3 supplementation.

Spatial changes of FAs in the retina in response to different dietary n-3 formulations have never been explored, although a diet rich in EPA and DHA is recommended to protect the retina against the effects of aging. In this study, Wistar rats were fed for 8 weeks with balanced diet including either EPA-containing phospholipids (PLs), EPA-containing TGs, DHA-containing PLs, or DHA-containing TGs. Qualitative changes in FA composition of plasma, erythrocytes, and retina were evaluated by gas chromatography-flame ionization detector. Following the different dietary intakes, changes to the quantity and spatial organization of PC and PE species in retina were determined by LC coupled to MS/MS and MALDI coupled to MS imaging. The omega-3 content in the lipids of plasma and erythrocytes suggests that PLs as well as TGs are good omega-3 carriers for retina. However, a significant increase in DHA content in retina was observed, especially molecular species as di-DHA-containing PC and PE, as well as an increase in very long chain PUFAs (more than 28 carbons) following PL-EPA and TG-DHA diets only. All supplemented diets triggered spatial organization changes of DHA in the photoreceptor layer around the optic nerve. Taken together, these findings suggest that dietary omega-3 supplementation can modify the content of FAs in the rat retina.

Ciliary neurotrophic factor (CNTF) protects retinal cone and rod photoreceptors by suppressing excessive formation of the visual pigments.

The retinal pigment epithelium (RPE)-dependent visual cycle provides 11-cis-retinal to opsins in the photoreceptor outer segments to generate functional visual pigments that initiate phototransduction in response to light stimuli. Both RPE65 isomerase of the visual cycle and the rhodopsin visual pigment have recently been identified as critical players in mediating light-induced retinal degeneration. These findings suggest that the expression and function of RPE65 and rhodopsin need to be coordinately controlled to sustain normal vision and to protect the retina from photodamage. However, the mechanism controlling the development of the retinal visual system remains poorly understood. Here, we show that deficiency in ciliary neurotrophic factor (CNTF) up-regulates the levels of rod and cone opsins accompanied by an increase in the thickness of the outer nuclear layers and the lengths of cone and rod outer segments in the mouse retina. Moreover, retinoid isomerase activity, expression levels of RPE65 and lecithin:retinol acyltransferase (LRAT), which synthesizes the RPE65 substrate, were also significantly increased in the Cntf-/- RPE. Rod a-wave and cone b-wave amplitudes of electroretinograms were increased in Cntf-/- mice, but rod b-wave amplitudes were unchanged compared with those in WT mice. Up-regulated RPE65 and LRAT levels accelerated both the visual cycle rate and recovery rate of rod light sensitivity in Cntf-/- mice. Of note, rods and cones in Cntf-/- mice exhibited hypersusceptibility to light-induced degeneration. These results indicate that CNTF is a common extracellular factor that prevents excessive production of opsins, the photoreceptor outer segments, and 11-cis-retinal to protect rods and cones from photodamage.

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.

Fatty acid transport protein 4 (FATP4) prevents light-induced degeneration of cone and rod photoreceptors by inhibiting RPE65 isomerase.

Although rhodopsin is essential for sensing light for vision, it also mediates light-induced apoptosis of photoreceptors in mouse. RPE65, which catalyzes isomerization of all-trans retinyl fatty acid esters to 11-cis-retinol (11cROL) in the visual cycle, controls the rhodopsin regeneration rate and photoreceptor susceptibility to light-induced degeneration. Mutations in RPE65 have been linked to blindness in affected children. Despite such importance, the mechanism that regulates RPE65 function remains unclear. Through unbiased expression screening of a bovine retinal pigment epithelium (RPE) cDNA library, we have identified elongation of very long-chain fatty acids-like 1 (ELOVL1) and fatty acid transport protein 4 (FATP4), which each have very long-chain fatty acid acyl-CoA synthetase (VLCFA-ACS) activity, as negative regulators of RPE65. We found that the VLCFA derivative lignoceroyl (C24:0)-CoA inhibited synthesis of 11cROL, whereas palmitoyl (C16:0)-CoA promoted synthesis of 11cROL. We further found that competition of FATP4 with RPE65 for the substrate of RPE65 was also involved in the mechanisms by which FATP4 inhibits synthesis of 11cROL. FATP4 was predominantly expressed in RPE, and the FATP4-deficient RPE showed significantly higher isomerase activity. Consistent with these results, the regeneration rate of 11-cis-retinaldehyde and the recovery rate for rod light sensitivity were faster in FATP4-deficient mice than wild-type mice. Moreover, FATP4-deficient mice displayed increased accumulation of the cytotoxic all-trans retinaldehyde and hypersusceptibility to light-induced photoreceptor degeneration. Our findings demonstrate that ELOVL1, FATP4, and their products comprise the regulatory elements of RPE65 and play important roles in protecting photoreceptors from degeneration induced by light damage.

Receptor interacting protein kinase-mediated necrosis contributes to cone and rod photoreceptor degeneration in the retina lacking interphotoreceptor retinoid-binding protein.

Interphotoreceptor retinoid-binding protein (IRBP) secreted by photoreceptors plays a pivotal role in photoreceptor survival with an unknown mechanism. A mutation in the human IRBP has been linked to retinitis pigmentosa, a progressive retinal degenerative disease. Mice lacking IRBP display severe early and progressive photoreceptor degeneration. However, the signaling pathway(s) leading to photoreceptor death in IRBP-deficient mice remains poorly understood. Here, we show that amounts of tumor necrosis factor-α (TNF-α) in the interphotoreceptor matrix and retinas of Irbp(-/-) mice were increased more than 10-fold and fivefold, respectively, compared with those in wild-type mice. Moreover, TNF-α receptor 1, an important membrane death receptor that mediates both programmed apoptosis and necrosis, was also significantly increased in Irbp(-/-) retina, and was colocalized with peanut agglutinin to the Irbp(-/-) cone outer segments. Although these death signaling proteins were increased, the caspase-dependent and independent apoptotic pathways were mildly activated in the Irbp(-/-) retinas, suggesting that other cell death mechanism(s) also contributes to the extensive photoreceptor degeneration in Irbp(-/-) retina. We found that receptor interacting protein 1 and 3 (RIP1 and RIP3) kinases, the intracellular key mediators of TNF-induced cellular necrosis, were elevated at least threefold in the Irbp(-/-) retinas. Moreover, pharmacological inhibition of RIP1 kinase significantly prevented cone and rod photoreceptor degeneration in Irbp(-/-) mice. These results reveal that RIP kinase-mediated necrosis strongly contributes to cone and rod degeneration in Irbp(-/-) mice, implicating the TNF-RIP pathway as a potential therapeutic target to prevent or delay photoreceptor degeneration in patients with retinitis pigmentosa caused by IRBP mutation.

Spatial organization of lipids in the human retina and optic nerve by MALDI imaging mass spectrometry.

MALDI imaging mass spectrometry (IMS) was used to characterize lipid species within sections of human eyes. Common phospholipids that are abundant in most tissues were not highly localized and observed throughout the accessory tissue, optic nerve, and retina. Triacylglycerols were highly localized in accessory tissue, whereas sulfatide and plasmalogen glycerophosphoethanolamine (PE) lipids with a monounsaturated fatty acid were found enriched in the optic nerve. Additionally, several lipids were associated solely with the inner retina, photoreceptors, or retinal pigment epithelium (RPE); a plasmalogen PE lipid containing DHA (22:6), PE(P-18:0/22:6), was present exclusively in the inner retina, and DHA-containing glycerophosphatidylcholine (PC) and PE lipids were found solely in photoreceptors. PC lipids containing very long chain (VLC)-PUFAs were detected in photoreceptors despite their low abundance in the retina. Ceramide lipids and the bis-retinoid, N-retinylidene-N-retinylethanolamine, was tentatively identified and found only in the RPE. This MALDI IMS study readily revealed the location of many lipids that have been associated with degenerative retinal diseases. Complex lipid localization within retinal tissue provides a global view of lipid organization and initial evidence for specific functions in localized regions, offering opportunities to assess their significance in retinal diseases, such as macular degeneration, where lipids have been implicated in the disease process.

Secretory defect and cytotoxicity: the potential disease mechanisms for the retinitis pigmentosa (RP)-associated interphotoreceptor retinoid-binding protein (IRBP).

Interphotoreceptor retinoid-binding protein (IRBP) secreted by photoreceptors plays a pivotal role in photoreceptor survival and function. Recently, a D1080N mutation in IRBP was found in patients with retinitis pigmentosa, a frequent cause of retinal degeneration. The molecular and cellular bases for pathogenicity of the mutation are unknown. Here, we show that the mutation abolishes secretion of IRBP and results in formation of insoluble high molecular weight complexes via disulfide bonds. Co-expression of protein disulfide isomerase A2 that regulates disulfide bond formation or introduction of double Cys-to-Ala substitutions at positions 304 and 1175 in D1080N IRBP promoted secretion of the mutated IRBP. D1080N IRBP was not transported to the Golgi apparatus, but accumulated in the endoplasmic reticulum (ER), bound with the ER-resident chaperone proteins such as BiP, protein disulfide isomerase, and heat shock proteins. Splicing of X-box-binding protein-1 mRNA, expression of activating transcription factor 4 (ATF4), and cleavage of ATF6 were significantly increased in cells expressing D1080N IRBP. Moreover, D1080N IRBP induced up-regulation and nuclear translocation of the C/EBP homologous protein, a proapoptotic transcription factor associated with the unfolded protein response. These results indicate that loss of normal function (nonsecretion) and gain of cytotoxic function (ER stress) are involved in the disease mechanisms of D1080N IRBP. Chemical chaperones and low temperature, which help proper folding of many mutated proteins, significantly rescued secretion of D1080N IRBP, suggesting that misfolding is the molecular basis for pathogenicity of D1080N substitution and that chemical chaperones are therapeutic candidates for the mutation-caused blinding disease.

Microglial ramification and redistribution concomitant with the attenuation of choroidal neovascularization by neuroprotectin D1.

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.

Docosahexaenoic acid signalolipidomics in nutrition: significance in aging, neuroinflammation, macular degeneration, Alzheimer's, and other neurodegenerative diseases.

Essential polyunsaturated fatty acids (PUFAs) are critical nutritional lipids that must be obtained from the diet to sustain homeostasis. Omega-3 and -6 PUFAs are key components of biomembranes and play important roles in cell integrity, development, maintenance, and function. The essential omega-3 fatty acid family member docosahexaenoic acid (DHA) is avidly retained and uniquely concentrated in the nervous system, particularly in photoreceptors and synaptic membranes. DHA plays a key role in vision, neuroprotection, successful aging, memory, and other functions. In addition, DHA displays anti-inflammatory and inflammatory resolving properties in contrast to the proinflammatory actions of several members of the omega-6 PUFAs family. This review discusses DHA signalolipidomics, comprising the cellular/tissue organization of DHA uptake, its distribution among cellular compartments, the organization and function of membrane domains rich in DHA-containing phospholipids, and the cellular and molecular events revealed by the uncovering of signaling pathways regulated by DHA and docosanoids, the DHA-derived bioactive lipids, which include neuroprotectin D1 (NPD1), a novel DHA-derived stereoselective mediator. NPD1 synthesis agonists include neurotrophins and oxidative stress; NPD1 elicits potent anti-inflammatory actions and prohomeostatic bioactivity, is anti-angiogenic, promotes corneal nerve regeneration, and induces cell survival. In the context of DHA signalolipidomics, this review highlights aging and the evolving studies on the significance of DHA in Alzheimer's disease, macular degeneration, Parkinson's disease, and other brain disorders. DHA signalolipidomics in the nervous system offers emerging targets for pharmaceutical intervention and clinical translation.

New Retinal Pigment Epithelial Cell Model to Unravel Neuroprotection Sensors of Neurodegeneration in Retinal Disease.

Retinal pigment epithelial (RPE) cells sustain photoreceptor integrity, and when this function is disrupted, retinal degenerations ensue. Herein, we characterize a new cell line from human RPE that we termed ABC. These cells remarkably recapitulate human eye native cells. Distinctive from other epithelia, RPE cells originate from the neural crest and follow a neural development but are terminally differentiated into "epithelial" type, thus sharing characteristics with their neuronal lineages counterparts. Additionally, they form microvilli, tight junctions, and honeycomb packing and express distinctive markers. In these cells, outer segment phagocytosis, phagolysosome fate, phospholipid metabolism, and lipid mediator release can be studied. ABC cells display higher resistance to oxidative stress and are protected from senescence through mTOR inhibition, making them more stable in culture. The cells are responsive to Neuroprotectin D1 (NPD1), which downregulates inflammasomes and upregulates antioxidant and anti-inflammatory genes. ABC gene expression profile displays close proximity to native RPE lineage, making them a reliable cell system to unravel signaling in uncompensated oxidative stress (UOS) and retinal degenerative disease to define neuroprotection sites.

Spatial correlation of mouse photoreceptor-RPE thickness between SD-OCT and histology.

Spectral Domain Optical Coherence Tomography (SD-OCT) applied to the mouse retina has been limited due to inherent movement artifacts and lack of resolution. Recently, SD-OCT scans from a commercially available imaging system have yielded retinal thickness values comparable to histology. However, these measurements are based on single point analysis of images. Here we report that using the Spectralis HRA + OCT Spectral Domain OCT and Fluorescein Angiography system (Heidelberg Engineering, Heidelberg, Germany), retinal thickness of linear expanses from SD-OCT data can be accurately assessed. This is possible by the development of a Spectralis-compatible ImageJ plug-in that imports 8-bit SLO and 32-bit OCT B-scan images, retaining scale and segmentation data and enabling analysis and 3D reconstruction. Moreover, mouse retinal layer thickness values obtained with this plug-in exhibit a high correlation to thickness measurements from histology of the same retinas. Thus, use of this ImageJ plug-in results in reliable quantification of long retinal expanses from in vivo SD-OCT images.

Cellular and 3D optical coherence tomography assessment during the initiation and progression of retinal degeneration in the Ccl2/Cx3cr1-deficient mouse.

Retinal pathologies common to human eye diseases, including abnormal retinal pigment epithelial (RPE) cells, drusen-like accumulation, photoreceptor atrophy, and choroidal neovascularization, have been reported in the Ccl2/Cx3cr1-deficient mouse. The Ccl2 gene encodes the pro-inflammatory chemokine CCL2 (MCP-1), which is responsible for chemotactic recruitment of monocyte-derived macrophages to sites of inflammation. The Cx3cr1 gene encodes the fractalkine receptor, CX3CR1, and is required for accumulation of monocytes and microglia recruited via CCL2. Chemokine-mediated inflammation is implicated in retinal degenerative diseases such as diabetic retinopathy, age-related macular degeneration, retinitis pigmentosa, and uveoretinitis, and proper chemokine signaling from the RPE, Müller glia, and astrocytes is necessary to regulate leukocyte trafficking. Therefore, this mouse, possessing aberrant chemokine signaling coupled with retinal degenerative pathologies, presents an ideal opportunity to investigate the effect of altered signaling on retinal homeostasis and photoreceptor degeneration. Since this mouse is a recent development, more data covering the onset, location, and progression rate of pathologies is needed. In the present study we establish these parameters and show two photoreceptor cell death processes. Our observations of decreased glutamine synthetase and increased glial fibrillary acidic protein suggest that Müller cells respond very early within regions where lesions are forming. Finally, we suggest that retinal angiomatous proliferation contributes to pathological angiogenesis in this Ccl2/Cx3cr1-deficient mouse.

Identification of histones as endogenous antibiotics in fish and quantification in rainbow trout (Oncorhynchus mykiss) skin and gill.

Antimicrobial polypeptides (AMPPs) are increasingly recognized as a critical component of innate host defense. Among the AMPPs, polypeptides related to histones have been identified from many animals. Using peptide mapping, we further confirm the identity of two histone-like proteins from fish as members of the H2B (sunshine bass) and H1 (rainbow trout) histone groups. We optimized the conditions for measuring rainbow trout HLP-1/H2B via sandwich ELISA. We used two antibodies, one to the amino terminus and one to the carboxyl terminus, of trout histone H2B, as the capture antibodies, and we used peroxidase-labeled antibody raised to calf histone H2B as the secondary antibody. Specificity of the detecting antibody was confirmed by specific reactivity with histone H2B in tissue extracts via western blotting. The test was reproducible and capable of detecting as little as 5 ng of histone H2B (0.05 µg/ml). Histone H2B levels expressed in gill tissue of juvenile, healthy rainbow trout were well within concentrations that are lethal to important fish pathogens. However, there was a significant, age (size)-dependent decline in histone H2B concentrations as fish matured, until levels became virtually undetectable in market-size fish. In contrast, levels in skin appeared to remain high and unchanged in small versus large fish. Antibacterial activity in skin and gill tissues was closely correlated with histone H2B concentration measured via ELISA, which supports our previous finding that histones are the major AMPPs in rainbow trout skin and gill.

Estrogenic Modulation of Retinal Sensitivity in Reproductive Female Túngara Frogs.

Although mate searching behavior in female túngara frogs (Physalaemus pustulosus) is nocturnal and largely mediated by acoustic cues, male signaling includes visual cues produced by the vocal sac. To compensate for these low light conditions, visual sensitivity in females is modulated when they are in a reproductive state, as retinal thresholds are decreased. This study tested whether estradiol (E2) plays a role in this modulation. Female túngara frogs were injected with either human chorionic gonadotropin (hCG) or a combination of hCG and fadrozole. hCG induces a reproductive state and increases retinal sensitivity, while fadrozole is an aromatase inhibitor that blocks hCG-induced E2 synthesis. In an analysis of scotopic electroretinograms (ERGs), hCG treatment lowered the threshold for eliciting a b-wave response, whereas the addition of fadrozole abolished this effect, matching thresholds in non-reproductive saline-injected controls. This suggests that blocking E2 synthesis blocked the hCG-mediated reproductive modulation of retinal sensitivity. By implicating E2 in control of retinal sensitivity, our data add to growing evidence that the targets of gonadal steroid feedback loops include sensory receptor organs, where stimulus sensitivity may be modulated, rather than more central brain nuclei, where modulation may affect mechanisms involved in motivation.

Elovanoids downregulate SARS-CoV-2 cell-entry, canonical mediators and enhance protective signaling in human alveolar cells.

The pro-homeostatic lipid mediators elovanoids (ELVs) attenuate cell binding and entrance of the SARS-CoV-2 receptor-binding domain (RBD) as well as of the SARS-CoV-2 virus in human primary alveoli cells in culture. We uncovered that very-long-chain polyunsaturated fatty acid precursors (VLC-PUFA, n-3) activate ELV biosynthesis in lung cells. Both ELVs and their precursors reduce the binding to RBD. ELVs downregulate angiotensin-converting enzyme 2 (ACE2) and enhance the expression of a set of protective proteins hindering cell surface virus binding and upregulating defensive proteins against lung damage. In addition, ELVs and their precursors decreased the signal of spike (S) protein found in SARS-CoV-2 infected cells, suggesting that the lipids curb viral infection. These findings open avenues for potential preventive and disease-modifiable therapeutic approaches for COVID-19.

Age-related changes in brain phospholipids and bioactive lipids in the APP knock-in mouse model of Alzheimer's disease.

Sustained brain chronic inflammation in Alzheimer's disease (AD) includes glial cell activation, an increase in cytokines and chemokines, and lipid mediators (LMs), concomitant with decreased pro-homeostatic mediators. The inflammatory response at the onset of pathology engages activation of pro-resolving, pro-homeostatic LMs followed by a gradual decrease. We used an APP knock-in (App KI) AD mouse that accumulates ß-amyloid (Aß) and presents cognitive deficits (at 2 and 6 months of age, respectively) to investigate LMs, their precursors, biosynthetic enzymes and receptors, glial activation, and inflammatory proteins in the cerebral cortex and hippocampus at 2-, 4-, 8- and 18-month-old in comparison with wild-type (WT) mice. We used LC-mass-spectrometry and MALDI molecular imaging to analyze LMs and phospholipids, and immunochemistry for proteins. Our results revealed an age-specific lipid and cytokine profile, and glial activation in the App KI mice. Despite an early onset of Aß pathology, pro-inflammatory and pro-resolving LMs were prominently increased only in the oldest age group. Furthermore, the LM biosynthetic enzymes increased, and their receptor expression decreased in the aged App KI mice. Arachidonic acid (AA)-containing phospholipid molecular species were elevated, correlating with decreased cPLA2 activity. MALDI molecular imaging depicted differential distribution of phospholipids according to genotype in hippocampal layers. Brain histology disclosed increased microglia proliferation starting from young age in the App KI mice, while astrocyte numbers were enhanced in older ages. Our results demonstrate that the brain lipidome is modified preferentially during aging as compared to amyloid pathology in the model studied here. However, alterations in phospholipids signal early pathological changes in membrane composition.