Iron is neurotoxic in retinal detachment and transferrin confers neuroprotection

Abstract

In retinal detachment (RD), photoreceptor death and permanent vision loss are caused by neurosensory retina separating from the retinal pigment epithelium because of subretinal fluid (SRF), and successful surgical reattachment is not predictive of total visual recovery. As retinal iron overload exacerbates cell death in retinal diseases, we assessed iron as a predictive marker and therapeutic target for RD. In the vitreous and SRF from patients with RD, we measured increased iron and transferrin (TF) saturation that is correlated with poor visual recovery. In ex vivo and in vivo RD models, iron induces immediate necrosis and delayed apoptosis. We demonstrate that TF decreases both apoptosis and necroptosis induced by RD, and using RNA sequencing, pathways mediating the neuroprotective effects of TF are identified. Since toxic iron accumulates in RD, we propose TF supplementation as an adjunctive therapy to surgery for improving the visual outcomes of patients with RD.

Fig. 1. Iron accumulates during RD in humans.

(A) Schematic representation of RRD. RPE strictly interacted with PRs (rods and cones), supporting their function and maintaining retinal physiology. During RRD, separation of the neural retina from the RPE disrupts the metabolism of PRs and induces permanent cellular damage, SRF accumulation through the retinal tear (arrowhead), and inflammatory cells in subretinal space. (B to D) Iron level (B), total iron binding capacity (TIBC) (C), and transferrin saturation (TSAT) (D) were quantified in vitreous from control patients and in vitreous and SRF from patients with RRD. Unpaired t test (n = 9 to 35 for vitreous and n = 30 for SRF), *P = 0.046 and **P = 0.006. ns, not significant. (E) Iron level in SRF was correlated to duration of the RRD (n = 30) and to the visual recovery 1 month following surgical treatment (n = 10). Pearson correlation test, *P < 0.05. (F) Perl’s staining (blue) on retinal sections from patients with nonhemorrhagic RD (asterisk shows space between retina and underlying RPE) revealed iron deposits in the retina and the RPE (arrows). Scale bars, 500 μm. (G) Iron distribution map realized by inductively coupled plasma mass spectrometry (ICP-MS) on the retina from a patient with nonhemorrhagic RD revealed iron deposits (arrowheads). An optical image of the analyzed retina section (left), the corresponding ICP-MS image of Fe distribution (medium), and the superposition of both (right). The color spectrum represents an ion intensity map of Fe. GCL, ganglion cell layer; INL, inner nuclear layer; ONL, outer nuclear layer; m/z, mass/charge ratio; ppm, parts per million. All values are represented as the mean ± SEM.

Fig. 2. Iron toxicity for PRs in rat retinal explants.

(A) Illustration of the retinal explant model and the timed workflow for iron exposure protocol: Adult rat retinas were dissected, placed on membranes with PRs facing up, and immediately exposed for 2 days to iron (FeSO₄). Control explants were cultured with medium alone. Some explants were harvested, and Western blotting and immunostaining were performed. Afterward, the medium was completely replaced, and explants were maintained in culture for 4 or 7 days for histological analysis and iron staining, respectively. (B) Western blotting and quantitative analysis of a rod protein showed a decreased rhodopsin level in explants exposed to increasing concentrations of FeSO₄. The molecular masses of the immunolabeled fragments were indicated in the right margin. Mann-Whitney test (n = 3, *P = 0.028). (C) Immunostaining of arrestin (red, arrows) and subsequent quantification showed very few cones present in explants exposed to 1 mM FeSO₄. Mann-Whitney test (n = 3, ***P < 0.0001). (D) Semithin sections of explants showed disorganization of the ONL and segments after 1 mM FeSO₄ exposure. (E) 3,3′-Diaminobenzidine (DAB) amplified Perl’s reaction revealed iron deposits (arrows) in explants exposed to 1 mM FeSO₄. GCL, ganglion cell layer; ONL, outer nuclear layer; INL, inner nuclear layer; S, segments. Scale bars, 100 μm. All values are represented as the mean ± SEM.

Fig. 3. Expression of TF protects retina explants exposed to iron.

(A) Illustration of timed workflow for retinal explants from WT and TG mice expressing hTF continuously exposed to 1 mM FeSO₄. LDH release was measured after 1 day and immunostaining after 6 days. (B) LDH release was lower in culture medium from TG explanted mice compared to WT explants. Mann-Whitney test (n = 6), *P = 0.03. (C) Number of cones stained by arrestin (arrow) and length of rod segments stained by rhodopsin (Rho4D2) were quantified and were higher in TG explants. Mann-Whitney test (n = 3), ***P < 0.001. (D) Quantification of immunostaining intensity for markers of iron storage LFt was decreased in TG explants compared with WT explants. Mann-Whitney test (n = 3), **P < 0.01. (E) Illustration of timed workflow for TF treatment on iron-exposed explants: Adult rat retinas were exposed for 2 days with 1 mM FeSO₄. Control explants were cultured with medium alone. Afterward, the medium was completely replaced and explants were treated with hTF (50 mg/ml) for 2 or 4 days. (F) Quantitative analysis of rhodopsin protein by Western blotting show increased rhodopsin protein expression in TF-treated iron-exposed explants (Fe + TF). Mann-Whitney test (n = 3), *P = 0.021. (G) Rhodopsin and arrestin immunostaining revealed protection of rod segments (Rho4D2, asterisk) and cones (arrow) by TF treatment (Fe + TF). Quantification of cone nuclei showed more cones in explants treated with TF than those exposed to FeSO₄. Mann-Whitney test (n = 3), *P = 0.036. (H) Western blotting and quantitative analysis of RIP kinase demonstrated higher full form and cleaved form of the proteins in iron-exposed retinal explants. The cleaved form of RIP reported on RIP full form was reduced when TF was used to treat iron-exposed explants. Mann-Whitney test (n = 3), *P = 0.028. (I) Antiapoptotic Bcl2 protein, detected by Western blotting, was increased in TF-treated iron-exposed explants. Mann-Whitney test (n = 3), *P = 0.028. (J) TUNEL-positive cells in the ONL were reduced by TF treatment. (K) Immunostaining of iron storage marker ferritin light chain was significantly lower in explants treated with TF (Fe + TF) than without treatment (Fe). Fluorescence intensity was reported relative to control conditions. One-way analysis of variance (ANOVA), Bonferroni post test (n = 3), *P < 0.05. Scale bars, 100 μm. All values are represented as mean ± SEM.

Fig. 4. TF expression preserves the detached retina in mice.

(A) Semithin retinal sections from control WT mice without RD and WT and TG mice expressing hTF 7 days after RD (red asterisks). In TG mice, the histology of the detached retina (brackets) was less disrupted than in WT mice, with remaining OS (arrowheads). Nuclei were stained with toluidine blue. Measurements of total retinal thickness in the detached area were reported to undetached retina thickness. (B) Expression of hTF in mice (TG) reduced thickening of the total retina and ONL. Mann-Whitney test (n = 5), *P < 0.05. (C) Rhodopsin staining in OS was conserved in TG compared with WT mice (arrowheads) 7 days after RD. The length of OS measured on semithin sections was higher in TG mice compared with WT mice. Mann-Whitney test (n = 6), *P = 0.047 (D) Arrestin staining revealed cones in retinal sections of TG mice (arrows) 7 days after RD. Cone number was higher in TG compared with WT mice. Mann-Whitney test (n = 6), *P = 0.025. (E) Müller glial cell activation revealed by GFAP expression was lower in TG mice compared with WT mice (arrows). Mann-Whitney test (n = 5), **P = 0.0056. (F) Cellular markers of apoptosis were lower in TG compared to WT mouse retinas. Caspase 8 mRNA level by reverse transcription quantitative polymerase chain reaction (RT-qPCR), and the ratio of cleaved/pro–caspase 3 protein levels was determined by Western blotting performed 4 days after RD. TUNEL staining was performed in eyes collected 7 days after RD. Mann-Whitney test (n = 5), **P = 0.008 and *P = 0.028. (G) Necrotic RIP kinase protein level, detected by Western blotting, was reduced in TG mice compared with WT mice. Mann-Whitney test (n = 3), *P = 0.028. IS, inner segment. Scale bars, 100 μm (A, E, and F) and 50 μm (C and D). All values are represented as the mean ± SEM.

Fig. 5. High-throughput analyses of the transcriptome of TG mouse retinas following RD.

Overrepresentation pathway analysis using GO terms enrichment (biological process clustering) (A) and reactome biosource (B). Color codes were used to associate pathways with common biological effects. Comparative analyses were carried out in TG versus WT retinas 4 days after RD (n = 4). GO terms and pathways were considered as enriched if fold enrichment is ≥2.0, uncorrected P value is ≤0.05, and the minimum number of regulated genes in pathway/term is ≥2.0. MHC, major histocompatibility complex; ER, endoplasmic reticulum.

Fig. 6. Local administration of TF preserves neural retina in the rat model of RD.

(A) Rat semithin retinal sections without RD (control) and with RD, treated by either intravitreal injection of a control solution [balanced salt solution (BSS)] or TF (50 mg/ml). Retinal histology in the detached area (asterisks) was more preserved in animals treated with TF compared with those receiving BSS. Higher magnifications showed less ONL disorganization (arrow) and longer OSs (arrowheads) in TF-treated RD rats. Nuclei were stained with toluidine blue. Measurements of thickness in detached retinas were reported relative to undetached retina thickness. (B) Treatment with TF reduced thickening of the total retina and the ONL. Mann-Whitney test (n = 5), *P = 0.013 and **P ≤ 0.01. The length of OSs was higher in TF-injected RD rats. Mann-Whitney test (n = 5), **P = 0.007. (C) Rhodopsin (green) and arrestin (red) staining in rats 7 days after RD. OSs were preserved in TF-treated animals (arrowhead). Scale bars, 20 μm (A and C) and 50 μm [(A) high magnification]. All values are represented as the mean ± SEM.