DOI: 10.5061/dryad.gxd25480h
title: Retinal polyunsaturated fatty acid supplementation reverses aging-related vision decline in mice

description: The retina is uniquely enriched in polyunsaturated fatty acids (PUFAs),
 primarily localized in cell membranes, where they govern membrane
 biophysical properties. During aging, alterations in lipid metabolism lead
 to reduced content of very long-chain PUFAs (VLC-PUFAs) in the retina,
 which is associated with normal age-related reductions in contrast
 sensitivity, diminished photoreceptor function and delayed rod-mediated
 dark adaptation recovery, and pathological age-related macular
 degeneration (AMD). ELOVL2 (Elongation of very-long-chain fatty acids-like
 2) encodes a transmembrane protein that produces precursors to
 docosahexaenoic acid (DHA) and VLC-PUFAs. The methylation status of the
 ELOVL2  promoter is currently one of the best predictors of
 chronological age. Here, we show that lower VLC-PUFA abundance in the aged
 mouse retina is accompanied by a reduction in visual function. Similarly,
 mice lacking ELOVL2-specific enzymatic activity (Elovl2C234W) demonstrate
 reduced contrast sensitivity and slower rod-mediated dark adaptation.
 Intravitreal supplementation with the direct product of ELOVL2, 24:5n-3,
 in aged animals improved visual function for up to 4 weeks and reduced
 accumulation of ApoE- and C3d-positive sub-RPE deposits. The gene
 expression pattern observed in supplemented retinas exhibited a partial
 rejuvenation profile, including decreased expression of aging-related
 genes and a transcriptomic signature resembling younger retinas. Finally,
 we present human genetic data from the IAMGDC and UK Biobank showing an
 association of two variants in the ELOVL2 locus with the onset of
 intermediate AMD, underlining the translational importance of our
 findings. Our work highlights VLC-PUFA supplementation as potential
 therapeutic opportunity and defines ELOVL2 as a promising target for
 interventions to prevent age-related vision loss.
description: # Retinal polyunsaturated fatty acid supplementation reverses
 aging-related vision decline in mice Dataset DOI:
 [10.5061/dryad.gxd25480h](10.5061/dryad.gxd25480h) ## Description of the
 data and file structure To investigate molecular changes retinas in aging
 and after lipid supplementation, total fatty acid, global lipidomic,
 transcriptomic, and targeted proteomic analyses were performed on mouse
 retinas and photoreceptor outer segments. The data in this file was used
 to produce panels in Figure 1, 3, 5 and 6 and Supplementary Figure 1, 3, 5
 and 6 of the associated publication. In the associated publication, we
 utilized p-values from the multiple t test or 2-way ANOVA. This file can
 be opened with any text editing software. In the present study, this file
 was included in analyses that utilized the Prism software. ### Files and
 variables #### File: retina_tFA-different_age_stages.csv
 **Description:** Total fatty acid analysis was performed on mouse retinas
 collected from different age stages. The data in this file was used to
 produce panels in Figure 1 of the associated publication. The statistical
 analysis was performed by utilizing 2-way ANOVA. This file can be opened
 with any text editing software. In the present study, this file was
 included in analyses that utilized the Prism software. ##### Variables *
 FAs: Names of the fatty acids detected in the analysis. * Chemical
 formula: Molecular formula of each fatty acid. * m/z: Mass-to-charge ratio
 of the detected fatty acid ion. tR/min: Retention time in minutes during
 chromatographic separation. Sample Columns * Columns labeled by age group
 and replicate (e.g., 3mo-1, 3mo-2, …, 23mo-5) represent fatty acid
 abundances measured from mouse retinas at specific ages: - 3mo: 3 months
 old - 6mo: 6 months old - 12mo: 12 months old - 18mo: 18 months old -
 23mo: 23 months old The suffix (e.g., -1, -2, -3, etc.) indicates
 biological replicates within each age group Missing values "0"
 indicate not detected species #### File: retina_tFA_Elovl2.csv
 **Description:** To investigate molecular changes in Elovl2 mouse, total
 fatty acid analysis was performed on mouse retinas collected from age
 matched wild type and Elovl2 mutant mice. The data in this file was used
 to produce panels in Figure 3 of the associated publication. In the
 associated publication, we utilized p-values from the multiple t test .
 This file can be opened with any text editing software. In the present
 study, this file was included in analyses that utilized the Prism
 software. ##### Variables * FAs: Names of the fatty acids detected in the
 analysis. * Chemical formula: Molecular formula of each fatty acid. * m/z:
 Mass-to-charge ratio of the detected fatty acid ion. * tR/min: Retention
 time in minutes during chromatographic separation. Sample Columns *
 WT1–WT4: Biological replicates from wild-type mouse retinas. *
 Elovl2_1–Elovl2_4: Biological replicates from Elovl2 knockout mouse
 retinas. #### File: retina_lipidomics_3mo_vs_18mo.csv **Description:** To
 investigate molecular changes retinas in aging, global lipidomic analysis
 was performed on mouse retinas collected from 3-month-old and 18-month-old
 mice. The data in this file was used to produce panels in Figure 1 and
 Supplementary Figure 1 of the associated publication. In the associated
 publication, we utilized p-values from the multiple t test. This file can
 be opened with any text editing software. In the present study, this file
 was included in analyses that utilized the Prism software. ##### Variables
 * LipidMolec: Names of the lipid molecules detected in the analysis. *
 Class: Lipid class each molecule belongs to. * Formula: Molecular formula
 of each lipid. * BaseRt: Retention time at which the lipid elutes during
 chromatographic separation. Sample Columns * 3mo-1 to 3mo-5: Biological
 replicates from mouse retinas collected at 3 months of age. * 18mo-1 to
 18mo-5: Biological replicates from mouse retinas collected at 18 months of
 age. #### File: retina_lipidomics_Elovl2.csv **Description:**  To
 investigate molecular changes in Elovl2 mouse, global lipidomic analysis
 was performed on mouse retinas collected from age matched wild type and
 Elovl2 mutant mice. The data in this file was used to produce panels in
 Figure 3 and  Supplementary Figure 3 of the associated publication. In the
 associated publication, we utilized p-values from the multiple t test.
 This file can be opened with any text editing software. In the present
 study, this file was included in analyses that utilized the Prism
 software. ##### Variables * LipidMolec: Names of the lipid molecules
 detected in the analysis. * Class: Lipid class each molecule belongs to
 (e.g., phospholipids, sphingolipids). * Formula: Molecular formula of each
 lipid. * BaseRt: Retention time at which the lipid elutes during
 chromatographic separation. Sample Columns * WT1–WT3: Biological
 replicates from wild-type mouse retinas. * Elovl2_1–Elovl2_2: Biological
 replicates from Elovl2 knockout mouse retinas. #### File:
 retina_lipidomics_Elovl2-2-M.csv **Description:** To investigate molecular
 changes in Elovl2 mouse, global lipidomic analysis was performed on mouse
 retinas collected from age matched wild type and Elovl2 mutant male mice.
 The data in this file was used to produce panels in Figure 3 and
 Supplementary Figure 3 of the associated publication. In the associated
 publication, we utilized p-values from the multiple t test. This file can
 be opened with any text editing software. In the present study, this file
 was included in analyses that utilized the Prism software. ##### Variables
 * LipidMolec: Names of the lipid molecules detected in the analysis. *
 Class: Lipid class each molecule belongs to (e.g., phospholipids,
 sphingolipids). * Formula: Molecular formula of each lipid. * BaseRt:
 Retention time at which the lipid elutes during chromatographic
 separation. Sample Columns * WT1–WT4: Biological replicates from wild-type
 mouse retinas. * Elovl2_1–Elovl2_2: Biological replicates from Elovl2
 knockout mouse retinas. #### File: retina_lipidomics_Elovl2-3-F.csv
 **Description:** To investigate molecular changes in Elovl2 mouse, global
 lipidomic analysis was performed on mouse retinas collected from age
 matched wild type and Elovl2 mutant female mice. The data in this file was
 used to produce panels in Figure 3 and Supplementary Figure 3 of the
 associated publication. In the associated publication, we utilized
 p-values from the multiple t test. This file can be opened with any text
 editing software. In the present study, this file was included in analyses
 that utilized the Prism software. ##### Variables * LipidMolec: Names of
 the lipid molecules detected in the analysis. * Class: Lipid class each
 molecule belongs to (e.g., phospholipids, sphingolipids). * Formula:
 Molecular formula of each lipid. * BaseRt: Retention time at which the
 lipid elutes during chromatographic separation. Sample Columns * WT1–WT2:
 Biological replicates from wild-type mouse retinas. * Elovl2_1–Elovl2_3:
 Biological replicates from Elovl2 knockout mouse retinas. #### File:
 POS_lipidomics_245_supp-5d.csv **Description:** To investigate molecular
 changes retinas after lipid supplementation, global lipidomics was
 performed on mouse photoreceptor outer segments isolated from retina
 receiving vehicle and 24:5 supplementation 5 days post injection. The data
 in this file was used to produce panels in Figure 5 and Supplementary
 Figure 5 of the associated publication. In the associated publication, we
 utilized p-values from the multiple t test . This file can be opened with
 any text editing software. In the present study, this file was included in
 analyses that utilized the Prism software. ##### Variables * LipidMolec:
 Names of the lipid molecules detected in the analysis. * Class: Lipid
 class each molecule belongs to (e.g., phospholipids, sphingolipids). *
 Formula: Molecular formula of each lipid. * BaseRt: Retention time at
 which the lipid elutes during chromatographic separation. * MainIon: The
 primary ion (mass-to-charge signal) detected for the lipid species. Sample
 Columns * veh_G45, veh_G47, veh_105M, veh_105F: Vehicle-treated samples
 from different groups/conditions, with replicate numbers indicated by the
 suffix (e.g., veh_G45_1, veh_G45_2). * 24:5_G47, 24:5_105M, 24:5_105F,
 24:5_G45: Samples treated with fatty acid 24:5 in the respective
 groups/conditions, with replicate numbers indicated by the suffix. ####
 File: POS_lipidomics_245_supp-4w.csv **Description:** To investigate
 molecular changes retinas after lipid supplementation, global lipidomics
 was performed on mouse photoreceptor outer segments isolated from retina
 receiving vehicle and 24:5 supplementation 4 weeks post injection. The
 data in this file was used to produce panels in Figure 6 of the associated
 publication. In the associated publication, we utilized p-values from the
 multiple t test. This file can be opened with any text editing software.
 In the present study, this file was included in analyses that utilized the
 Prism software. ##### Variables * LipidMolec: Names of the lipid molecules
 detected in the analysis (prefix “VEH” indicates vehicle dataset). *
 Class: Lipid class each molecule belongs to (e.g., phospholipids,
 sphingolipids). * Formula: Molecular formula of each lipid. * Base245_t:
 Retention time at which the lipid elutes during chromatographic
 separation. Sample Columns * VEH_2, VEH_4, VEH_5, VEH_6, VEH_7, VEH_8:
 Vehicle-treated samples, with replicate numbers indicated by the suffix. *
 245_2, 245_4, 245_5, 245_6, 245_7, 245_8: Samples treated with compound
 245, with replicate numbers indicated by the suffix. #### File:
 POS_lipidomics_245_supp-9w.csv **Description:** To investigate molecular
 changes retinas after lipid supplementation, global lipidomics was
 performed on mouse photoreceptor outer segments isolated from retina
 receiving vehicle and 24:5 supplementation 9 weeks post injection. The
 data in this file was used to produce panels in Figure 6 of the associated
 publication. In the associated publication, we utilized p-values from the
 multiple t test. This file can be opened with any text editing software.
 In the present study, this file was included in analyses that utilized the
 Prism software. ##### Variables * LipidMolec: Names of the lipid molecules
 detected in the analysis. - Class: Lipid class each molecule belongs to
 (e.g., phospholipids, sphingolipids). - Calc Mass: Calculated monoisotopic
 mass of the lipid molecule. - Formula: Molecular formula of each lipid. -
 BaseRt: Retention time at which the lipid elutes during chromatographic
 separation. - MainIon: Primary ion (mass-to-charge signal) detected for
 the lipid species. Sample Columns * VEH2, VEH3, VEH4, VEH5, VEH6, VEH7,
 VEH10: Vehicle-treated samples, with replicate numbers indicated by the
 suffix. * 245_2, 245_3, 245_4, 245_5, 245_6, 245_7, 245_10: Samples
 treated with compound 245, with replicate numbers indicated by the suffix.
 #### File: ROS_lipidomics_3mo_vs_18mo.csv **Description:** To investigate
 molecular changes retinas in aging, global lipidomic analysis was
 performed on photoreceptor outer segments isolated from retinas collected
 from 3-month-old and 18-month-old mice. The data in this file was used to
 produce panels in Figure 1 and Supplementary Figure 1 of the associated
 publication. In the associated publication, we utilized p-values from the
 multiple t test. This file can be opened with any text editing software.
 In the present study, this file was included in analyses that utilized the
 Prism software. ##### Variables * LipidMolec: Names of the lipid molecules
 detected in the analysis. * Class: Lipid class each molecule belongs to
 (e.g., phospholipids, sphingolipids). * Formula: Molecular formula of each
 lipid. * BaseRt: Retention time at which the lipid elutes during
 chromatographic separation. Sample Columns * 3mo-1 to 3mo-10: Biological
 replicates from mouse retinas collected at 3 months of age (10 replicates
 total). * 18mo-1 to 18mo-10: Biological replicates from mouse retinas
 collected at 18 months of age (10 replicates total). #### File:
 retina_proteins_quantification_3mo_vs_18mo.csv **Description:** To address
 whether age-related visual decline involves changes in the
 phototransduction cascade, we quantified proteins involved in signal
 transduction in POS, (Rho, Pde6a, Pde6b, Gnat1 and Gbb1) in retinas
 collected from 3-month-old and 18-month-old mouse using a stable
 isotope-labeled (SIL) peptides-based absolute quantification method. The
 data in this file was used to produce panels in Figure 1  and
 Supplementary Figure 1 of the associated publication. The statistical
 analysis was performed by utilizing multiple t test. This file can be
 opened with any text editing software. In the present study, this file was
 included in analyses that utilized the Prism software. ##### Variables *
 IS: Stable isotope–labeled (SIL) peptides used as internal standards for
 quantification of PDE6A, PDE6B, GNAT1, GBB and Rho. * sample: Biological
 sample from 3-month-old and 18-month-old mice which protein-derived
 peptides were quantified. * Area-peptide: Peak area corresponding to the
 endogenous (unlabeled) peptide. * Area-IS: Peak area corresponding to the
 SIL internal standard peptide. * IS conc (mg/mL): Concentration of the
 internal standard stock solution in mg/mL. * MW: Molecular weight of the
 peptide. * purity: Chemical purity of the SIL peptide. * dilution:
 Dilution factor applied to the internal standard solution. * IS volume/µL:
 Volume of internal standard solution added to the sample (in µL). * IS
 conc (mol): Final molar concentration of the internal standard in the
 assay. * peptide (mol): Calculated molar amount of the endogenous peptide
 in the sample. #### File: FPKM_12movs18mo_Elovl2.txt **Description:** Bulk
 RNAsequencing analysis was performed on mouse retinas collected from
 different age stages and genotypes. The data in this file was used to
 produce panels in Figure 3 of the associated publication. Differential
 expression analysis was performed using DESeq2. This file can be opened
 with any text editing software. In the present study, this file was
 included in analyses that utilized the Prism software. ##### Variables *
 gene_id: Unique identifier for each gene. * WT1_12mo, WT2_12mo, WT3_12mo:
 Biological replicates from wild-type mouse retinas at 12 months of age. *
 Mut1_12mo, Mut2_12mo, Mut3_12mo: Biological replicates from mutant mouse
 retinas at 12 months of age. * WT1_18mo, WT2_18mo, WT3_18mo, WT4_18mo:
 Biological replicates from wild-type mouse retinas at 18 months of age. *
 gene_name: Standard gene symbol. * gene_chr: Chromosome location of the
 gene. * gene_start: Start coordinate of the gene on the chromosome. *
 gene_end: End coordinate of the gene on the chromosome. #### File:
 RAWCOUNTS_18mo_VEHvsSUPPL.txt **Description:** Bulk RNAsequencing analysis
 was performed on vehicle- and 24:5-supplemented mouse retinas. The data in
 this file was used to produce panels in Figure 5 of the associated
 publication. Differential expression analysis was performed using DESeq2.
 This file can be opened with any text editing software. In the present
 study, this file was included in analyses that utilized the Prism
 software. ##### Variables * neid: Unique identifier for each gene or
 feature measured (depending on the dataset context). - 18mo_supp_1,
 18mo_supp_2, 18mo_supp_3: Biological replicates from 18-month-old mice in
 the supplemented group. - 18mo_veh_1, 18mo_veh_2, 18mo_veh_3: Biological
 replicates from 18-month-old mice in the vehicle-treated group. ##
 Code/software The software was used for statistical analysis and
 generating figures is Prism 10, Version 10.1.2 (324) ## Access information
 Other publicly accessible locations of the data: * Bulk RNA-sequencing
 data generated in this study have been deposited in the NCBI Gene
 Expression Omnibus (GEO) under accession number GSE303302. *
 snRNA-sequencing data generated in this study have been deposited under
 accession number GSE303793. * The mass spectrometry proteomics data have
 been deposited to the ProteomeXchange Consortium via the PRIDE partner
 repository with the dataset identifier PXD066430 Data was derived from the
 following sources: * International AMD Genomics Consortium (IAMGDC,
 n=2,407, mean age at diagnosis: 74.1 years) * Incident AMD cases from the
 UK Biobank (n=1,309, mean age of onset: 62.8 years)