Studies of ApoD-/- and ApoD-/-ApoE-/- mice uncover the APOD significance for retinal metabolism, function, and status of chorioretinal blood vessels

Abstract

Apolipoprotein D (APOD) is an atypical apolipoprotein with unknown significance for retinal structure and function. Conversely, apolipoprotein E (APOE) is a typical apolipoprotein with established roles in retinal cholesterol transport. Herein, we immunolocalized APOD to the photoreceptor inner segments and conducted ophthalmic characterizations of ApoD^-/- and ApoD^-/-ApoE^-/- mice. ApoD^-/- mice had normal levels of retinal sterols but changes in the chorioretinal blood vessels and impaired retinal function. The whole-body glucose disposal was impaired in this genotype but the retinal glucose metabolism was unchanged. ApoD^-/-ApoE^-/- mice had altered sterol profile in the retina but apparently normal chorioretinal vasculature and function. The whole-body glucose disposal and retinal glucose utilization were enhanced in this genotype. OB-Rb, both leptin and APOD receptor, was found to be expressed in the photoreceptor inner segments and was at increased abundance in the ApoD^-/- and ApoD^-/-ApoE^-/- retinas. Retinal levels of Glut4 and Cd36, the glucose transporter and scavenger receptor, respectively, were increased as well, thus linking APOD to retinal glucose and fatty acid metabolism and suggesting the APOD-OB-Rb-GLUT4/CD36 axis. In vivo isotopic labeling, transmission electron microscopy, and retinal proteomics provided additional insights into the mechanism underlying the retinal phenotypes of ApoD^-/- and ApoD^-/-ApoE^-/- mice. Collectively, our data suggest that the APOD roles in the retina are context specific and could determine retinal glucose fluxes into different pathways. APOD and APOE do not play redundant, complementary or opposing roles in the retina, rather their interplay is more complex and reflects retinal responses elicited by lack of these apolipoproteins.

Keywords: Apolipoprotein; Cholesterol; Fatty acids; Glucose; Isotopic labeling; Leptin receptor.

Fig. 1

Immunolocalization of APOD in mouse retina. Nuclei are in blue (stained with DAPI), and immunoreactivity for APOD is in green. PIS preimmune serum, used as control stains. GCL ganglion cell layer, INL inner nuclear layer, ONL outer nuclear layer, IS photoreceptor inner segments, OS photoreceptor outer segments, RPE retinal pigment epithelium, WT wild type (C57BL/6J). All images are representative: n = 3 mice (6–8 months old) per genotype. Scale bars 50 μm

Fig. 2

Serum and retinal sterols. a–d, Sterol quantifications. Results are mean ± SD of the measurements in individual mice (3–6 animals/genotype/sex; number of mice equals the number of retinas); animals were 6-months old. Statistical significance was assessed by two-way ANOVA with Bonferroni correction. Black line and asterisk indicate statistically significant differences between sexes of the same genotype; the results of all other comparisons are summarized in Supplemental Tables S2–S5. ***P ≤ 0.001. e–g Retinal distribution of unesterified cholesterol as assessed by filipin (cyan) staining. All images are representative: n = 3 mice per genotype. Scale bars 50 μm. GCL ganglion cell layer, IPL inner plexiform layer, INL inner nuclear layer, OPL outer plexiform layer, ONL outer nuclear layer, IS photoreceptor inner segments, OS photoreceptor outer segments, RPE retinal pigment epithelium, WT wild type

Fig. 3

Retinal in vivo imaging of mice on regular diet. Representative (n = 5/genotype) assessments by spectral domain-optical coherence tomography (SD-OCT), fundus color imaging (Fundus), fundus autofluorescence (Autofluorescence), and fundus fluorescein angiography (FA) after an injection with sodium fluorescein. The SD-OCT panels show a fundus image and two retinal cross-sections (from left to right), the latter is a Doppler flow, which reflects the direction of the blood flow. The FA panels show an early, intermediate and late stage fundus fluorescence (from left to right) with the laser beam being focused either on the outer (Outer) or inner (Inner) retina. Yellow arrowheads point to retinal abnormalities. No sex-based differences were detected, hence only images of male mice are shown

Fig. 4

Electroretinography responses in 6-month-old male mice. Results are mean ± SEM of the measurements in 7–15 animals; the number of animals (n) is indicated in each upper panel. Statistical significance was assessed at various light intensities for two mouse genotypes by repeated measures two-way ANOVA with Bonferroni’s correction for multiple comparisons. *P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.001; ****P ≤ 0.0001

Fig. 5

Retinal ultrastructure as assessed by transmission electron microscopy. Longitudinal cross-sections through the photoreceptor inner segments are shown. a–c Black rectangles indicate regions that are shown at a higher magnification in the upper left corner. Black and magenta arrowheads point to individual intracellular vesicles or multivesicular bodies, respectively. Orange arrowheads point to autophagosomes. d–f, Yellow circles indicate mitochondrial pinching, yellow rectangle indicates mitochondrial rupture, yellow arrowheads indicate wavy plasma membranes. Images are representative of two male mice per genotype; all mice were 7-months old. Scale bars 1 μm

Fig. 6

Retinal glucose homeostasis. a Retinal gene expression. Results are mean ± SD of the triplicate measurements in pooled samples of the retinas from 5–7 wild type (WT, C57BL/6J), ApoD^−/−, and ApoD^−/−ApoE^−/− mice; all mice were 6–8-month-old males. b Representative images (n = 3–4 mice/genotype) of OB-Rb immunolocalization in mouse retina. Nuclei are in blue (stained with DAPI), and immunoreactivity for OB-Rb is in red. PIS, preimmune serum, used as control stains. 2^o, another control stain with secondary antibody. GCL ganglion cell layer, INL inner nuclear layer, OPL outer plexiform layer, ONL outer nuclear layer, IS photoreceptor inner segments, OS photoreceptor outer segments, RPE retinal pigment epithelium. Scale bars 50 μm. c and e, retinal and serum [U-¹³C]glucose molar percent enrichment (MPE) and glucose concentrations, respectively. d Retinal glucose metabolism (n = 7–9/genotype). Blue circles represent ¹³C-labeling of glucose (6 carbons) and subsequent ¹³C incorporation into the intermediates of various metabolic pathways. Numbers next to each metabolite represent its absolute flux (× 10⁻³, nmol/mg of protein). Ala alanine, Asp aspartate, Gln glutamine, Glu glutamate, αKG α-ketoglutarate, OAA oxaloacetate, TCA the tricarboxylic acid cycle. c–e All results are means ± SD of the measurements in pooled samples of two retinas from individual mice. a, c–e The statistical significance was assessed by a two-tailed, unpaired Student’s t test vs the WT retina; *P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.001

Fig. 7

Glucose tolerance test. Results are means ± SD of the measurements in blood samples from individual animals (n = 5–8/genotype/age group). The statistical significance was assessed by a two-way ANOVA followed by the Bonferroni correction; *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001

Fig. 8

Retinal in vivo imaging and immunohistochemistry of mice on high- fat high-sugar (HFHS) diet. a, b Representative (n = 5/genotype) assessments by spectral domain-optical coherence tomography (SD-OCT), fundus color imaging (Fundus), fundus autofluorescence (Autofluorescence), and fundus fluorescein angiography (FA) after an injection with sodium fluorescein. The SD-OCT panels show a fundus image and two retinal cross-sections (from left to right), the latter is a Doppler flow, which reflects the direction of the blood flow. The FA panels show an early, intermediate and late stage fundus fluorescence (from left to right) with the laser beam being focused either on the outer (Outer) or inner (Inner) retina. Yellow ovals outline some of the areas in the retina with increased permeability of the blood vessels. Yellow and magenta arrowheads point to the blood vessel leakage in different locations. No sex-based differences were detected, hence only images of male mice are shown. c Representative (n = 3-5/genotype) immunohistochemistry stains for albumin to assess vascular permeability. PIS preimmune serum, used as control stains. Nuclei are in blue (stained with DAPI), and immunoreactivity for albumin is in red. Green arrowheads point towards some of the choroidal neovessels growing toward the outer retina. Green rectangle in the upper left panel denotes the region shown at a higher magnification on the right. Scale bars 50 μm