Triglyceride-derived fatty acids reduce autophagy in a model of retinal angiomatous proliferation

Dyslipidemia and autophagy have been implicated in the pathogenesis of blinding neovascular age-related macular degeneration (NV-AMD). VLDL receptor (VLDLR), expressed in photoreceptors with a high metabolic rate, facilitates the uptake of triglyceride-derived fatty acids. Since fatty acid uptake is reduced in Vldlr–/– tissues, more remain in circulation, and the retina is fuel deficient, driving the formation in mice of neovascular lesions reminiscent of retinal angiomatous proliferation (RAP), a subtype of NV-AMD. Nutrient scarcity and energy failure are classically mitigated by increasing autophagy. We found that excess circulating lipids restrained retinal autophagy, which contributed to pathological angiogenesis in the Vldlr–/– RAP model. Triglyceride-derived fatty acid sensed by free fatty acid receptor 1 (FFAR1) restricted autophagy and oxidative metabolism in photoreceptors. FFAR1 suppressed transcription factor EB (TFEB), a master regulator of autophagy and lipid metabolism. Reduced TFEB, in turn, decreased sirtuin-3 expression and mitochondrial respiration. Metabolomic signatures of mouse RAP-like retinas were consistent with a role in promoting angiogenesis. This signature was also found in human NV-AMD vitreous. Restoring photoreceptor autophagy in Vldlr–/– retinas, either pharmacologically or by deleting Ffar1, enhanced metabolic efficiency and suppressed pathological angiogenesis. Dysregulated autophagy by circulating lipids might therefore contribute to the energy failure of photoreceptors driving neovascular eye diseases, and FFAR1 may be a target for intervention.

One-way ANOVA with Tukey's and Dunn's multiple comparisons test.  (F) Western blots of TFEB protein expression in nuclear and cytoplasmic 661W cell fractions, treated or not with GW9508 (30µM, 17h). n = 5-6 experiments per group.

Human retina collection
All patients previously diagnosed with type 3 neovascular membrane (or retinal angiomatous proliferation -RAP) were treated with Bevacizumab 1.25 mm/0.05ml and followed by a single

Mice retina collection
All retinas and eyes were collected at the same time of day (10 am) in order to limit variation in autophagy caused by circadian rhythm, with the exception of experiments that required starvation (8 hours) and their corresponding controls that were performed during the day.
Littermate controls were used for each experiment.

Cell drug treatments
Cells were equally distributed into 6-, 12-or 24-well plates and cultured to 80% confluence. Cells

Dual luciferase Assay
Cells with stable expression of Tfeb-Luciferase reporter were, if necessary, transfected with siRNA against Luciferase activity was measured using the Dual-Luciferase® Reporter Assay System (Promega) according to the manufacturer's protocol. Each independent experiment was repeated at least three times and normalized using the Renilla signal.

Immunoprecipitation
Cells were cultured to 80% confluence and treated 17h with MCT (0.4%, Nestlé), washed with PBS and their protein were collected in RIPA buffer containing protease inhibitors (ratio 1:100, Primer sequences are reported in supplemental Table S3.

Immunoblotting
Retinal lysates (P16, 25-50 μg) were obtained from mice that had a confirmed neovascular phenotype in the contralateral eye; they were loaded on SDS-PAGE gels and electro-blotted onto a actin or Tubulin (Cell Signaling) was used as a loading control. All antibodies were used according to the manufacturer's instructions. Revelation was done using ECL reagent from Thermofisher.
Refer to Table S4.

Single-cell drop-seq
Single-cell suspensions were prepared from C57Bl/6 control retinas at P14, as reported

Single cell RNA sequencing analysis
Unique molecular identifier (UMI) counts for WT and/or Vldlr -/-scRNAseq replicates were merged into one single Digital Gene Expression (DGE) matrix and processed using the "Seurat" package (2). Cells expressing less than 500 genes and more than 10% of mitochondrial genes were filtered out. Single cell transcriptomes were normalized by dividing by the total number of nm laser. Slides were imaged at room temperature (~22˚C). Post-acquisition image analysis was performed with LAS-X and ImageJ software.

Autophagic flux quantification
Eyes from CAG-RFP-EGFP-LC3 and CAG-RFP-EGFP-LC3/Vldlr -/mice P16 were fixed in 4% paraformaldehyde at room temperature 1h, cryoprotected with a sucrose gradient, and embedded using Surgipath FSC22 Clear (Leica). Cryosections (10µm) were prepared from a minimum of 6 mice per group and 1 section with corresponding volume of 0.226 µm 3 for each eye were analyzed. Autophagic flux was quantified using Imaris software and the 3D object colocalization tool.
Images were then loaded on ImageJ, color images were separated and analyzed using the region of interest (ROI) manager. ROI were selected based on Dapi signal (corresponding to nucleus) and were superposed onto eGFP signal image, defining nuclear eGFP intensity.
Samples were centrifuged (9,000 x g, 10 min, 4°C) and supernatants were collected for direct analysis using negative ion mode profiling and were 5-fold diluted using acetonitrile/methanol/formic acid (74.9:24.9:0. Additional MS settings were: ion spray voltage, -3.0 kV; capillary temperature, 350°C; probe heater temperature, 325 °C; sheath gas, 55; auxiliary gas, 10; and S-lens RF level 50. Postiive ion mode data were acquired by injecting extracts (10 µl) onto a 150 x 2 mm, 3 µm Atlantis HILIC column (Waters; Milford, MA). The column was eluted isocratically at a flow rate of 250 µl/min with 5% mobile phase A (10 mM ammonium formate and 0.1% formic acid in water) for 0.5 minute followed by a linear gradient to 40% mobile phase B (acetonitrile with 0.1% formic acid) over 10 minutes. MS analyses were carried out using electrospray ionization in the positive ion mode using full scan analysis over 70-800 m/z at 70,000 resolution and 3 Hz data acquisition rate.
Other MS settings were: sheath gas 40, sweep gas 2, spray voltage 3.5 kV, capillary temperature 350°C, S-lens RF 40, heater temperature 300°C, microscans 1, automatic gain control target 1e6, and maximum ion time 250 ms. Raw data were processed using TraceFinder 3.3 and 4.1 software (Thermo Scientific; Waltham, MA) and Progenesis QI (Nonlinear Dynamics; Newcastle upon Tyne, UK). For each method, metabolite identities were confirmed using authentic reference standards.
Human and mouse metabolomics profile were analyzed using MetaboAnalyst (11). Data was filtered based on interquartile range, then normalized using quantile normalization and autoscaling. Outliers, detected by Random Forest algorithm, were removed and Principal component analysis (PLSDA) was performed on normalized data. Metabolite Set Enrichment Analysis was also performed using MetaboAnalyst. Correlated metabolites between mouse and human samples were identified and visualized using R/3.4.0 and the ShinyHeatmap package (12).