DOI: 10.5061/dryad.59zw3r233
title: Data from: Elovanoids counteract oligomeric β-Amyloid-induced gene expression and protect photoreceptors

description: The onset of neurodegenerative diseases activates inflammation that leads
 to progressive neuronal cell death and impairments in cognition
 (Alzheimer’s disease, AD) and sight (age-related macular degeneration,
 AMD). How neuroinflammation can be counteracted is not known. In AMD,
 amyloid β-peptide (Aβ) 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 pro-homeostatic/neuroprotective mediators, neuroprotectin D1
 (NPD1) and elovanoids (ELVs). To mimic an inflammatory milieu in wild-type
 (WT) mouse, we triggered retinal pigment epithelium (RPE) damage/PRC death
 by subretinally injected oligomeric β-Amyloid (OAβ) and observed that ELVs
 administration counteracted their effects, protecting these cells. In
 addition, ELVs prevented OAβ-induced changes in gene expression engaged in
 senescence, inflammation, autophagy, extracellular matrix remodeling and
 AMD. Moreover, since OAβ target the RPE, we used primary human RPE cell
 cultures and demonstrated that OAβ caused cell damage, while ELVs
 protected and restored gene expression as in mouse. Our data show OAβ
 activates senescence as reflected by enhanced expression of p16INK4a,
 MMP1, p53, p21, p27 and Il-6 and of senescence-associated secretory
 phenotype (SASP) secretome, followed by RPE and PRC demise and that
 elovanoids 32 and 34 blunt these events and elicits protection. In
 addition, ELVs counteracted OAβ-induced expression of genes engaged in
 AMD, autophagy and extracellular matrix (ECM) remodeling. Overall, our
 data uncovered that ELVs downplay OAβ-senescence program induction and
 inflammatory transcriptional events and protect RPE cells and PRC, and
 therefore have potential as a possible therapeutic avenue for AMD.
description: <b>Animals.
 </b>All animal experiments
 were performed according to ARVO Statement for the Use of Animals in
 Ophthalmic and Vision Research, and the protocol was approved by LSU
 Health New Orleans’ Institutional Animal Care and Use Committee
 (IACUC).  6-month-old 5xFAD mice (stock number: 34848-JAX, The Jackson Laboratory, Bar Harbor, ME, USA), co-overexpress FAD mutant forms of human amyloid precursor protein (the Swedish mutation: K670N, M671L; the Florida mutation: I716V; and the London mutation: V717I) and presenilin 1 (PS1, encoded by Psen1: M146L, L286V) transgenes under transcriptional control of the neuron-specific mouse Thy1 promoter. 5xFAD mice were hemizygotes with respect to the transgenes and non-transgenic WT littermates. Genotyping was performed by PCR of tail DNA. All analyses were carried out blind with respect to the mice genotype. For the subretinal injection, 6-month-old C57BL/6J mice were anesthetized by an intraperitoneal injection of ketamine/xylazine, pupils dilated with 1.0% tropicamide (Akorn, IL, USA); and 0.5% Proparacaine Hydrochloride (Akorn) was applied for topical anesthesia. Eyes were punctured with a 30-gauge needle between the corneoscleral junction and the <i>ora serrata</i> into the vitreous cavity without disturbing the lens. Compounds were delivered to the subretinal region using a 33-gauge blunt needle attached to a 5μl Hamilton syringe (Hamilton Company, Reno, NV, USA) under a dissecting microscope. Non-injected mice were used as negative control while the PBS-injected mice were used for sham. The injection volume was 2µl containing PBS, 10μM of OAβ, 10μM of OAβ + 200ng ELV-N32, 200ng ELV-N32 alone, 10μM of OAβ + 200ng ELV-N34 or 200ng ELV-N34 alone (<i>n</i>=12/group). All groups received topical drops: PBS only or ELV-N32 (200nM) or ELV-N34 (200nM), twice a day for 3 or 7 days. <b>Lipid extraction and LC-MS/MS-based lipidomic analysis. </b>Retina or RPE/choroid were homogenized in 3ml of MeOH followed by adding 6ml of CHCl<sub>3</sub> and 5μl of an internal standard mixture of deuterium-labeled lipids (AA-d8 (5ng/μl), PGD2-d4 (1ng/μl), EPA-d5 (1ng/μl), 15-HETE-d8 (1ng/μl), and LTB4-d4 (1ng/μl)). Samples were sonicated for 30 min and stored at −80°C overnight. Then supernatant collected, pellet washed with 1ml of CHCl<sub>3</sub>/MeOH (2:1) and centrifuged, and supernatants combined. Two ml of distilled water, pH 3.5, was added to the supernatant, vortexed, and centrifuged, and then the pH of the upper phase adjusted to 3.5–4.0 with 0.1 N HCl. The lower phase was dried down under N<sub>2</sub> and then resuspended in 1ml of MeOH. LC-MS/MS analysis was performed in a Xevo TQ equipped with Acquity I class UPLC with a flow-through needle (Waters, Milford, MA, USA). For PC and PE molecular species analysis, samples were dried under N<sub>2</sub> and then resuspended in 20μl of the sample solvent (acetonitrile/chloroform/methanol, 90:5:5 by volume). The Acquity UPLC BEH HILIC 1.7-μm, 2.1×100-mm column was used with a mixture of solvent A (acetonitrile/water, 1:1; 10mM ammonium acetate, pH 8.3) and solvent B (acetonitrile/water, 95:5; 10mM ammonium acetate, pH 8.3) as the mobile phase (0.5ml/min). Solvent B (100%) was isocratically run for the first 5 min and then run in a gradient to 20% of solvent A for 8 min, increased to 65% of solvent A for 0.5 min, run isocratically at 65% of solvent A for 3 min, and then returned to 100% of solvent B for 3.5 min for equilibration. The column temperature was set to 30°C. The amount for each PC and PE species was calculated as % of the total PCs and PEs/sample. For analysis of fatty acids and their derivatives, six retinas or six RPE/Choroid were pooled and homogenized as above. Samples (in 1ml of MeOH) were mixed with 9ml of H<sub>2</sub>O at pH 3.5, loaded onto C18 columns (Agilent, Santa Clara, CA, USA), and then eluted with 10ml of methyl formate, dried under N2, resuspended in 50μl of MeOH/H<sub>2</sub>O (1:1), and injected into an Acquity UPLC HSS T3 1.8-μm 2.1×50-mm column. Mobile phase 45% solvent A -H<sub>2</sub>O + 0.01% acetic acid- and 55% solvent B -MeOH + 0.01% acetic acid-, 0.4ml/min flow initially, and then a gradient to 15% solvent A for the first 10 min, a gradient to 2% solvent A for 18 min, 2% solvent A run isocratically until 25 min, and then a gradient back to 45% solvent A for re-equilibration until 30 min. Lipid standards (Cayman, Ann Arbor, MI, USA) were used for tuning and optimization, as well as to create calibration curves for each compound. <b>Primary human RPE culture.</b> All experiments with primary human RPE cells were approved by the Institutional Review Board of LSUHNO and conducted in accordance with NIH guidelines. Cells were collected from anonymous donors provided by eye banks. Briefly, globes of a 19-year-old Caucasian male, without eye pathology were obtained from NDRI within 24 hours after death from head trauma. Globes were opened, and RPE cells harvested and cultured (59, 60) and grown in MEM medium supplemented with 10% FBS, 5% NCS, non-essential amino acids, Penicillin-Streptomycin (100U/mL), human fibroblast growth factor 10ng/ml and incubated at 37°C with a constant supply of 5% CO<sub>2</sub>. Cells integrity was validated as in previous study (9). For oligomeric Aβ treatment, cells were seeded in the 6-well plates, 30.000 cells/cm<sup>2</sup>. After 2 days, sub-confluent cells were treated with 10μM OAβ or with PBS (vehicle control). <b>Aβ (1-42) oligomerization. </b>Aβ (1-42) (HFIP-treated, ANASPEC Company, Fremont, CA, USA, Cat AS-64129) was resuspended by adding 1%NH<sub>4</sub>OH/Water and DMSO to obtain a concentration 500μM and sonicated for 10 min. Then oligomerization was performed by diluting t Aβ (1-42) with sterile phosphate buffer in low-binding polypropylene micro-centrifuge tube for 24 h at 4<sup>o</sup>C. Oligomerization was verified by Western blot using mouse monoclonal 6E10 antibody (<i>SI Appendix, </i>Fig. S6). <b>Senescence-Associated β-Galactosidase (SA-β-Gal)</b> <b>staining. </b>Cells were visualized using SA-β-Gal staining kit (Cat 9860, Cell Signaling Technology, MA, USA). Briefly, RPE cells were washed with PBS, fixed with 4% paraformaldehyde (PFA) for 15 min, then washed again with PBS and incubated in staining solution mix overnight at 37<sup>o</sup>C (no CO<sub>2</sub>), the presence of CO<sub>2</sub> can cause changes to the pH which may affect staining results. Pictures were taken under brightfield microscope (Nikon Eclipse TS100) 200X magnification after the development of blue color, and cells counted in 10 different random fields per well. <b>Protein extraction and Western blot analysis. </b>Samples were lysed by RIPA buffer and protein determined by Bradford assay (Bio-Rad, Hercules, CA, USA). After denaturation, 20μl of each medium sample or 30μg of total protein for cell/tissue sample was separated by SDS-PAGE (4-12% gradient) gel (Thermo Fisher Scientific, Waltham, MA, USA) and transferred to nitrocellulose membranes (Bio-Rad). The membranes were blocked by 5% non-fat dry milk in PBST, probed with primary antibodies (<i>SI Appendix, </i>Table S1) for 1 h, washed 3x by PBST, probed with secondary antibodies (GE Healthcare, Chicago, IL, USA) for 1 h, and washed 3x by PBST. Proteins bands were visualized using a LAS 4000 imaging system (GE Healthcare). Densitometry data were statistically analyzed at 95% confidence level. <b>RNA isolation and qPCR analysis. </b>Cell culture media was removed, cells were wash with PBS 1X and samples were collected using cell scraper. Total RNA was isolated using RNeasy Plus Mini Kit (Qiagen, Hilden, Germany). For the <i>in vivo </i>experiments, eyeballs were enucleated and anterior segment containing the cornea, lens and iris removed and the retina separated from the rest of the eyecup (RPE/choroid). Total retinal and eyecup (RPE/choroid) RNA were isolated using RNeasy Plus Mini Kit (Qiagen). One µg of total RNA was reverse transcribed using an iScript cDNA Synthesis Kit (Bio-Rad) and the reaction carried out with BrightGreen 2X qPCR MasterMix (Applied Biological Materials Inc., Richmond, BC, Canada) and validated primers (<i>SI Appendix, </i>Table S2). Quantitative PCR was performed in a CFX-384 Real-Time PCR system (Bio-Rad). The expression of target genes was normalized to the geometric mean of housekeeping genes and relative expression was calculated by the comparative threshold cycle method (ΔΔCT). <b>Immunofluorescence and confocal microscopy.</b> For the whole mount RPE staining, eyeballs were enucleated and pre-fixed in 4% PFA for 15 min. Then the eye cup containing RPE sheet were fixed in 4% PFA for 30 min, washed in PBS 3x following the blocking step for 1 h at room temperature. The immunostaining was performed by incubating primary antibody (ZO-1) for 48 h at 4°C. Then the eye cups were washed 3x with PBS and incubated with the secondary antibody for 12 h at 4°C. The primary human RPE cells as well as mouse eye cups were embedded in ProLong™ Gold Antifade Mounting medium (Thermo Fisher Scientific) between two glass coverslips. Pictures were taken with Olympus FV1200 microscope (Olympus, Japan). Images were analyzed by software ImageJ (rsb.info.nih.gov/ij/). <b>Spectral Domain-Optical Coherence Tomography imaging and analysis. </b>7 days post-injection, mice were anesthetized with ip ketamine/xylazine, pupil dilated by topical 1.0% tropicamide and placed in a custom-built holder for OCT imaging (body temperature maintained at 38°C with a heat pad). Retinas were imaged along the horizontal meridian through the optic nerve head using a Heidelberg Spectralis HRA OCT system (Heidelberg Engineering, Heidelberg, Germany). Axial resolution is 7mm optical and 3.5mm digital. The raw OCT B-scans cross-sectional images were exported with the scale in µm and opened in ImageJ (http//imagej.nih.gov/ij). The PRC layer thickness was defined as the width from the tip of the outer nuclear layer, right after the outer plexiform layer, to the outer segments of PRC. Three measurements were made on the same scan and averaged. Mean and standard error of the mean (SEM) were calculated (<i>n</i>=4/group). Students’ T-test was used to calculate statistical significance and a <i>P</i>-value less than 0.05 was considered significant. <b>Statistics. </b>Data are expressed as mean ± SEM of three or more independent experiments. The data were analyzed by one-way ANOVA followed by Tukey HSD post-hoc test at 95% confidence level to compare the different groups and considered significant with a <i>P</i> ˂ 0.05. The Pearson relation analysis was used to analyze the relationship between factors. Statistical analysis was performed by using BioVinci software (Bioturing INC., San Diego, CA, USA).