Table 1.
Primer pairs sequences for quantitative q-RT- PCR.1–3
Figure 1.
Scara5 was expressed in mouse retinal cells.
A: The expression of SCARA5 mRNA in the retina was evaluated by q-RT-PCR. Agarose gel electrophoresis of q-RT-PCR products confirmed that SCARA5 single amplicon with 108 bp was generated. ACTB and GAPDH were used as housekeeping genes. B: Western blotting analysis revealed a specific band with a molecular weight of 48 KDa, confirming the presence of Scara5 receptors in the retina. α-tubulin was used as a loading control. C: Retinal immunolabeling with a specific antibody in a histological section, along the eye axis through the optic disc and cornea, showed that Scara5 was expressed throughout the retina, mainly at the level of ganglion cell layer, inner nuclear layer, outer nuclear layer and RPE. D: Immunohistochemical negative control, where the primary antibody was omitted. Ret, retina; Liv, liver; -, no-template control; GL, ganglion cell layer; INL, inner nuclear layer; ONL, outer nuclear layer; RPE, retinal pigment epithelium. Scale bars: 28 µm (A); 28 µm (B).
Figure 2.
Scara5 was found both in the nucleus and cytoplasm of retinal cells.
A: Scara5 expression was observed at nuclear (arrow) and cytoplasmic (arrowhead) compartments in mouse retinal histological sections immunolabeled with a specific anti-Scara5 antibody. Nuclei were marked with Hoechst Stain Solution. B: Western blotting analysis of nuclear and cytoplasmic protein fractions samples confirmed the nuclear and cytoplasmic Scara5 content in the retinal cells. α-tubulin was used as a loading control. Topoisomerase-I (Topo I) was used to assure that nuclear protein was not present in the cytoplasmic fraction. GL, ganglion cell layer; INL, inner nuclear layer; ONL, outer nuclear layer. Scale bar: 28 µm.
Figure 3.
Scara5 was expressed in retinal neurons.
A: Labeled retinal ganglion cells with Brn3a showed a nuclear intense Scara5 signal. B: PKC positive bipolar cells expressed Scara5. C: Photoreceptors presented a high expression of Scara5. The labeling with PNA lectin, which is specific for cone inner segments, showed a more intense signal in cones than in rods. Nuclei were counterstained with ToPro3. ga, retinal ganglion cells; bi, bipolar cells; co, cone inner segments. Scale bars: 3,8 µm (A); 5,4 µm (B); 12,5 µm (C).
Figure 4.
Scara5 was expressed in retinal glia.
A: Labeled astrocytes with GFAP showed Scara5 expression. B: GS positive Müller cells presented intense Scara5 signal both at the nuclear and cytoplasmic compartments. C: Microglial cells revealed a more intense Scara5 signal in nucleus than in cytoplasm. Nuclei were counterstained with ToPro3. as, astrocyte; mü, Müller cell; mi, microglial cell. GL, ganglion cell layer; INL, inner nuclear layer; ONL, outer nuclear layer. Scale bars: 33 µm (A); 33 µm (B); 14,7 µm (C).
Figure 5.
L-ferritin expression in mouse retina.
A: The expression of FTL1 mRNA in the retina was evaluated by q-RT-PCR. Agarose gel electrophoresis of q-RT-PCR products confirmed that FTL1 single amplicon with 144 bp was generated. ACTB and GAPDH were used as housekeeping genes. B: Western blotting analysis showed a specific band with a molecular weight of 19 KDa, confirming the presence of L-ferritin in the retina. α-tubulin was used as a loading control. C: Analysis of nuclear and cytoplasmic protein fractions samples showed that L-ferritin was present in both cellular compartments. α-tubulin was used as a loading control. Topo I was used to assure that nuclear protein was not present in the cytoplasmic fraction sample. D: As expected, L-ferritin immunolabeling distribution pattern (arrows) was in accordance with the Scara5 signal (arrows) in paraffin-embedded retinal sections. Ret, retina; Liv, liver; -, no-template control; GL, ganglion cell layer; INL, inner nuclear layer; ONL, outer nuclear layer. Scale bar: 29 µm.
Figure 6.
Retinal vasculature expressed Scara5 receptors.
A: Cells surrounded by the blood basement membrane marked with collagen IV showed intense Scara5 signal. B and C: Dual immunolabeling with Scara5 and with α-SMA and CD34, respectively, confirmed that vascular smooth muscle cells and endothelial cells expressed Scara5 receptors. D: Whole mount retinas immunohistochemically marked with collagen IV and Scara5 showed that perivascular astrocyte-like cells intensively expressed Scara5 in their vascular end-feet. Nuclei were counterstained with ToPro3. en, endothelial cell; sm, smooth muscle cell; v, blood vessel; as: astrocyte-like cell. Scale bars: 10 µm (A); 9 µm (B); 9 µm (C); 12 µm (D).
Figure 7.
Intravenously injected HSF crossed the BRB.
A: Six hours after the intravenous injection of HSF, western blotting analysis revealed that HSF was present in the retina. As expected, a marked increase of L-ferritin content was also confirmed. B: The immunolabeling with a specific anti-HSF antibody showed that HSF crossed the inner BRB and accumulated in mouse retina. HSF was internally lining the retinal blood vessels. C and D: The double staining with anti-HSF and with anti-Scara5 antibodies showed that L-ferritin co-localized with endothelial cytoplasmic Scara5 (arrowhead), but no content of HSF was observed in RPE cells, suggesting a differential function of the inner and outer component of BRB. Nuclei were counterstained with ToPro3. Con, non-injected control; Inj, injected; V, blood vessel; GL, ganglion cell layer; INL, inner nuclear layer; ONL, outer nuclear layer; RPE, retinal pigment epithelium; CH, choroid. Scale bars: 24 µm (B); 10 µm (C); 8 µm (D).
Figure 8.
HSF influx into the retina changed Scara5, TfR1, and transferrin gene and protein expression.
A: Relative expression of SCARA5, TFRC, and TRF obtained from q-RT-PCR analysis. Y-axis indicates the relative expression distribution of a ratio of injected mice retinas versus non-injected control mice retinas with ACTB and GAPDH as housekeeping genes. Boxes represent interquartile range, the median value is indicated by horizontal dotted line, and whiskers represent the minimum and maximum observation. Statistical significance calculated by Rest 2009 (http://www.REST.de.com) is indicated by *p<0,05, and ***p<0,001 (n = 12). Ratios over one indicate genes (SCARA5 and TFRC) with higher expression in injected mice retinas relative to non-injected control mice retinas, and ratio less than one indicates gene (TRF) with lower expression in injected mice retinas opposed to non-injected control mice retinas. Western blotting and immunohistochemistry analyses confirmed that HSF accumulation in the retina was accompanied with an increased expression of Scara5 and TfR1 (B,C,D), and a slightly decrease of transferrin (B,E). Nuclei were counterstained with ToPro3. Con, non-injected control; Inj, injected; GL, ganglion cell layer; INL, inner nuclear layer; ONL, outer nuclear layer. Scale bars: 25 µm (C); 28 µm (D); 30 µm (E).
Figure 9.
Murine model of retinopathy with photoreceptor degeneration.
A: Forty-eight hours after sodium iodate injection, retinas analyzed by western blotting showed an increased expression of GFAP. α-tubulin was used as a loading control. B: Paraffin-embedded retinal sections stained with hematoxylin-eosin or immunolabeled with a specific anti-GFAP antibody revealed photoreceptor alterations and gliosis, indicating that retinopathy was well established. Nuclei were counterstained with ToPro3. GL, ganglion cell layer; INL, inner nuclear layer; ONL, outer nuclear layer. Scale bar: 35 µm.
Figure 10.
Scara5 expression decreased during retinopathy.
A: Retinas of a sodium iodate murine model were analyzed by western blotting. Scara5 expression reduced to about half during the treatment. B: Graph representing the optical density quantification of the Western blotting analysis for Scara5, after normalization with respect to α-tubulin. C: The immunolabeling of paraffin-embedded retinal sections with anti-Scara5 antibody confirmed that Scara5 expression decreased throughout the parenchyma. However, several positive Scara5 cells were found in the outer nuclear layer (arrowhead). D: During retinopathy, 2F8 positive cells were also observed in the outer nuclear layer (arrowhead), with a disposition compatible with that of Scara5 positive cells. 2F8 was revealed by DAB reaction and histological sections were counterstained with hematoxylin. GL, ganglion cell layer; INL, inner nuclear layer; ONL, outer nuclear layer. Scale bars: 45 µm (C); 20 µm (D).
Figure 11.
Scara5 was expressed in human retinal cells.
Laser confocal analysis of double-stained paraffin-embedded human retinal sections with anti-Scara5 and anti-collagen IV, anti-GFAP, anti-GS and anti-Iba1 antibodies revealed that Scara5 was present throughout the retina, including endothelial cells, astroctyes, Müller cells and microglial cells, following the distribution pattern observed in mouse retinas. D1, D2 and D3, 42, 78 and 86-years-old healthy human donors, respectively; GL, ganglion cell layer; INL, inner nuclear layer; ONL, outer nuclear layer.: 22 µm (A); 21 µm (B); 24 µm (C); 28 µm (D).