Endoplasmic Reticulum Stress Delays Choroid Development in the HCAR1 Knockout Mouse

Abstract

The subretina, composed of the choroid and the retinal pigment epithelium (RPE), plays a critical role in proper vision. In addition to phagocytosis of photoreceptor debris, the RPE shuttles oxygen and nutrients to the neuroretina. For their own energy production, RPE cells mainly rely on lactate, a major by-product of glycolysis. Lactate, in turn, conveys most of its biological effects via the hydroxycarboxylic acid receptor 1 (HCAR1). Herein, the lactate-specific receptor, HCAR1, was found to be exclusively expressed in the RPE cells within the subretina, and Hcar1^-/- mice exhibited a substantially thinner choroidal vasculature during development. Notably, the angiogenic properties of lactate on the choroid were impacted by the absence of Hcar1. HCAR1-deficient mice exhibited elevated endoplasmic reticulum stress along with eukaryotic translation initiation factor 2α phosphorylation, a significant decrease in the global protein translation rate, and a lower proliferation rate of choroidal vasculature. Strikingly, inhibition of the integrated stress response using an inhibitor that reverses the effect of eukaryotic translation initiation factor 2α phosphorylation restored protein translation and rescued choroidal thinning. These results provide evidence that lactate signalling via HCAR1 is important for choroidal development/angiogenesis and highlight the importance of this receptor in establishing mature vision.

Graphical abstract

Figure 1

HCAR1 expression in retinal pigment epithelium (RPE) cells promotes choroidal angiogenesis. A: Representative confocal images of subretinas from PT12 pups, stained with anti-HCAR1 (red) and anti-RPE65 (green); nuclei were counterstained with DAPI (blue). HCAR1 and RPE signals exhibit strong colocalization. The RPE cell layer is delineated with a dashed line. B: mRNA levels of Hcar1 in the RPE/choroid layers were measured at PT7, PT9, PT12, and PT15 by quantitative RT-PCR. C: Left panels: Representative confocal microscopy images of choroidal cross-sections from PT12 pups, stained with rhodamine-conjugated Griffonia simplicifolia lectin I (red). Choroidal thickness is delineated with a dashed line; examples of measured thicknesses are shown with plain white brackets. Right panels: Spider graphs reveal quantification of the choroid thickness at indicated distance (μm) from the optic nerve (position 0) in PT12 pups. The values of choroid thickness are provided in μm. D: Quantifications of central choroidal thickness at PT7, PT9, PT12, PT15, and PT30 in wild-type (WT) and Hcar1-knockout (KO) mice. E: Left panels: Representative images of subretinas stained with rhodamine-conjugated Griffonia simplicifolia lectin I (red) from PT12 mice injected intravitreally with 10 mmol/L lactate at PT9. Right panel: Quantification of the choroidal vasculature thickness on lactate injection. Choroidal thickness is delineated with a dashed line; examples of measured thicknesses are shown with plain white brackets. F: Top panels: Representative images of ex vivo choroid sprout assays using isolated choroid or RPE/choroid punches from WT mice, and RPE/choroid from KO mice treated with vehicle or 10 mmol/L lactate for 48 hours. Bottom panels: Quantification of the choroid sprout assays treated with vehicle, 10 mmol/L lactate, or 80 μmol/L 3, 5-dihydroxybenzoic acid (DHBA) for 48 hours. The explant sprouting area at 48 hours was normalized to the choroidal explant area at 0 hours. Data are presented as means ± SEM (B–F). n = 4 to 6 per group. ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001, and ∗∗∗∗P < 0.0001. Scale bars: 50 μm (A, C, and E); 1 mm (F). BF, bright field; NS, not significant.

Figure 2

HCAR1 deficiency impairs cell proliferation and protein synthesis. A: Quantification of number of Ki-67–positive cells per 0.5 mm² in central choroid at PT7, PT9, PT12, and PT15 pups; representative images are shown in Supplemental Figure 4A. B: Heat map of expression levels of several growth factors involved in cellular proliferation in wild-type (WT) and Hcar1-knockout (KO) mice at PT9 and PT12. Protein levels were analyzed by protein microarray, and the signal intensity of each growth factor was normalized to the internal positive control; data were not subsequently transformed. C: mRNA levels of 10 angiogenic factors were measured by quantitative RT-PCR; data presented as fold changes of the KO relative to the WT pups, at PT9 (left panel) and PT12 (right panel). (The dotted line indicates the average gene expression in WT that was considered as 1.) D: O-propargyl-puromycin (OPP) incorporation, as a proxy of protein translation rate, was revealed by the AZDye 488 Azide. The AZDye 488 signal was quantified on confocal microscopy images and adjusted relatively to the intensity of the Hoechst DNA counterstain; representative images are shown in Supplemental Figure 4C. Data are presented as means ± SEM (A, C, and D). n = 3 to 6 per group. ∗P < 0.05, ∗∗P < 0.01, and ∗∗∗P < 0.001. ANGPT2, angiopoietin 2; APOE, apolipoprotein E; AREG, amphiregulin; CSF, colony-stimulating factor; EGF, epidermal growth factor; EGFR, epidermal growth factor receptor; FGF, fibroblast growth factor; HGF, hepatocyte growth factor; HGFR, hepatocyte growth factor receptor; IGF, insulin-like growth factor; IGF1R, insulin-like growth factor 1 receptor; IGFBP, insulin-like growth factor binding protein; KITL, KIT ligand; NGF, nerve growth factor; PDGF, platelet-derived growth factor; PLGF, placental growth factor; SEMA3F, semaphorin 3F; SERPINF1, serpin family F member 1; TGFB1, transforming growth factor beta 1; TSP1, thrombospondin 1; VEGF, vascular endothelial growth factor; VEGFR, vascular endothelial growth factor receptor.

Figure 3

HCAR1 deficiency leads to a higher level of oxidative stress in the outer retina. A: Reactive oxygen species (ROS) and reactive nitrogen species (RNS) were measured in freshly isolated retinal pigment epithelium (RPE)/choroid from wild-type (WT) and Hcar1-knockout (KO) pups at PT7, PT9, PT12, and PT15. ROS and RNS levels were adjusted to the amount of tissue collected. B: Superoxide dismutase (SOD) activity in freshly isolated RPE/choroid from WT and KO pups at PT7, PT9, PT12, and PT15. C: Left panel: Quantification. Right panel: Representative Western blot analysis images of the stress sensor nuclear factor erythroid 2-related factor 2 (NRF2) in the RPE/choroid from WT and KO pups at PT7, PT9, PT12, and PT15, relative to heat shock protein 90 (Hsp90) expression. Data are presented as means ± SEM (A–C). n = 4 to 6 per group (A–C). ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001, and ∗∗∗∗P < 0.0001.

Figure 4

HCAR1 maintains endoplasmic reticulum (ER) homeostasis in the retinal pigment epithelium (RPE)/choroid complex. A: Representative Western blot analysis images of the ER stress markers binding immunoglobulin protein (BiP) and protein disulfide isomerase (PDI), from the subretina of wild-type (WT) and Hcar1-knockout (KO) mice at PT7, PT9, PT12, and PT15. Immediately to the right is the Western blot analysis quantification for BiP and PDI. B: Representative confocal images of subretinas from PT12 pups, stained with anti-PDI (red). C: Quantification of the phosphorylated IRE-1α (P-IRE-1α) relative to the total IRE-1α signal, in the subretina of WT and Hcar1^−/− mice at PT7, PT9, PT12, and PT15. Representative images of IRE-1α are in Supplemental Figure 5B. D: Quantification of the level of spliced XBP1 (XBP-1s) relative to the level of β-tubulin, in the subretina of WT and Hcar1^−/− mice at PT7, PT9, PT12, and PT15; for representative image of XBP-1s, see Supplemental Figure 5C. E: Representative Western blot analyses of phosphorylated ER stress signal protein kinase RNA-like ER kinase (PERK), from the subretina of WT and Hcar1^−/− mice at PT7, PT9, PT12, and PT15. Immediately adjacent (right) is the quantification of phosphorylated PERK (P-PERK) relative to total PERK. F: Representative Western blot analysis of phosphorylated eukaryotic translation initiation factor 2α (P-eIF2α) relative to total eIF2α in subretina of WT and Hcar1^−/− mice at PT7, PT9, PT12, and PT15. Histogram (right) is the quantification of eIF2α phosphorylation Western blot analysis, presented as the ratio of the phosphorylated eIF2α signal/the total eIF2α signal. G: Representative Western blot analysis and quantifications (histogram) of activating transcription factor 4 (ATF4), from the subretina of WT and Hcar1-KO (KO) mice at PT7, PT9, PT12, and PT15. H: Quantitative RT-PCR quantifications of ATF4 downstream target gene expression in the subretina of Hcar1^−/− and WT mice at PT12. Data are presented as means ± SEM (A and C–H). n = 4 (A, B, F, G, and H) to 6 (C–E) per group. ∗P < 0.05, ∗∗P < 0.01, and ∗∗∗P < 0.001. Scale bars = 50 μm (B). ASNS, asparagine synthetase; CHOP, C/EBP homologous protein; Hsp90, heat shock protein 90; TRIB3, tribbles homolog 3.

Figure 5

Integrated stress response inhibitor (ISRIB) rescues the choroidal involution in Hcar1-knockout (KO) mice. A: Representative confocal images of a rhodamine-conjugated Griffonia simplicifolia lectin I staining of choroidal sections from PT9, PT12, or PT15 mice intravitreally injected with ISRIB (200 nmol/L) or vehicle at PT7, PT9, and PT12. Choroidal region is delineated with a dashed line; examples of measured thicknesses are shown with plain white brackets. To the immediate right are spider graph quantifications of the choroid thickness at indicated distance (μm) from the optic nerve (position 0) in the four groups [wild type (WT)-ISRIB, WT-vehicle, KO-ISRIB, and KO-vehicle] at ages indicated (PT9, PT12, and PT15). B: Growth factor expression at PT12 on injection of ISRIB at PT9 in KO mice, relative to similarly treated WT animals. (The dotted line indicates the average growth factor expression in KO vehicle that was considered as 1.) Data are presented as means ± SEM (B). n = 3 to 6 per group ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001, and ∗∗∗∗P < 0.0001; ^††P < 0.01, ^†††P < 0.001, and ^††††P < 0.0001 compared with KO-ISRIB and KO-vehicle groups. Scale bars = 50 μm (A). AREG, amphiregulin; CSF, colony-stimulating factor; EGF, epidermal growth factor; EGFR, epidermal growth factor receptor; FGF, fibroblast growth factor; HGF, hepatocyte growth factor; HGFR, hepatocyte growth factor receptor; IGF, insulin-like growth factor; IGF1R, insulin-like growth factor 1 receptor; IGFBP, insulin-like growth factor binding protein; KITL, KIT ligand; NGF, nerve growth factor; PDGF, platelet-derived growth factor; PLGF, placental growth factor; TGFB1, transforming growth factor beta 1; VEGF, vascular endothelial growth factor; VEGFR, vascular endothelial growth factor receptor.