3.2.1. General Iron Homeostasis

Under physiological conditions, almost all non-heme iron (Fe<sup>3</sup>+) in the circulation is transported bound to TF (transferrin-bound iron: TBI) with high a ffinity (for review on cellular iron metabolism [16]). At cellular level, the TF with two Fe3<sup>+</sup> (holo-TF) is bound by its receptor (TFR1), and the complex is internalized. The Fe3<sup>+</sup> is released from TF in the endosome under the e ffect of acidification through the action of an ATP-dependent proton pump. Iron is then reduced into the ferrous form, Fe2+, by endosomal ferrireductase six transmembrane epithelial antigen of the prostate 3 (STEAP3) and exported by divalent metal transporter 1 (DMT1) in the cytosol where it contributes to the labile iron pool (LIP). In case of iron overload, TF is saturated with iron, and the non-transferrin-bound ferrous iron (NTBI) could be up-taken by iron importers such as the ZRT/IRT-like proteins (ZIPs) or DMT1 and joined intracellular LIP. The LIP consists of a transitory pool of iron species associated with a variety of ligands with low a ffinity (citrate, phosphate, inorganics irons) and easily oxidized, in transit to be distributed to the organelles (in particularly in mitochondria or nucleus) for cell metabolism in iron requiring proteins, stored or released.

In non-erythroid cells, the majority of iron is stored in ferritin (FT). Composed of 24 subunits of both heavy (HFT) and light (LFT) chains, FT forms a tissue-specific heterocomplex which can store up to 4500 Fe3+. Fe2<sup>+</sup> from LIP is transported to FT by Poly(rC)-binding proteins (PCBP1 and PCBP2) and is oxidized by ferroxidase activity of HFT in an oxygen-dependent manner. Then Fe3<sup>+</sup> is stored in the cavity formed by LFT. Iron is released from FT in a controlled manner by autophagy involving a cargo receptor, the nuclear receptor coactivator 4 (NCOA4).

The only known mammalian iron exporter is ferroportin (FPN), a transmembrane protein which requires a multicopper ferroxidase to convert Fe2<sup>+</sup> to Fe3+, allowing binding to TF. Hephaestin (HEPC), ceruloplasmin (CP), amyloid-beta precursor protein (APP), and the newly identified zyloklopen (ZP) are co-localized with FPN at the surface membrane or secreted.

### 3.2.2. Iron Flux in Retina

A local retinal homeostasis of iron, independent from the systemic regulation, is suspected by the fact that main proteins involved in iron homeostasis, previously confined to systemic expression, are locally synthesized in the retina (Table 1). In addition, the outer and inner BRB prevents the entry of large quantities of iron into the eye in case of systemic iron overload. The study of mouse models invalidated for iron homeostasis proteins (Table 1) led to a hypothetical model in which iron entry and homeostasis in the retina is divided into two compartments, carried by the RPE and MGC, delimited by the external limiting membrane, and having limited exchanges in physiological condition.


**Table 1.** Proteins involved in iron homeostasis of the retina.



**Legend Table 1**: Proteins localization were obtained from immunostaining on sections of mouse/rat retinas. The mouse models presented are limited to those with retinal changes in iron homeostasis and retinal abnormalities, if any. The corresponding human diseases were obtained by searching the UniProt site. ND: Not determined. Legends: β2M: β-2-Microglobulin; CP: ceruloplasmin; CEC: choroidal endothelial cell; FT: ferritin; GCL: ganglion cells layer; HFE: hereditary hemochromatosis protein, HFT: ferritin heavy chain; IPL: inner plexiform layer; IRE: iron responsive element; IS: inner segments; LFT: ferritin light chain; MGC: Müller's glial cell; OPL: outer plexiform layer; OS: outer segments; PR: photoreceptor; REC: retinal endothelial cell; RPE: retinal pigment epithelium; TFR1: transferrin receptor 1. Transferrin-Bound Iron Transport in the Retina

The RPE imports iron bound to TF from the choriocapillaries through the transcytosis of TFR1 present at the basal membrane of the RPE (Figure 2). The transcytosis of the TF/TFR1 complex along microtubules via galectin 4 and Rab11a [42] has been described in vitro. The presence of TFR1 on the apical side of RPE is ambiguous and suggests that TF/TFR1 transcytosis or a potential iron-TF uptake by RPE could egress iron from the outer retina to choriocapillaries. Six hours after an intravitreal injection of holoTF tagged with a fluorochrome, TF is localized in RPE and choroid, which favors the later hypothesis [43]. Another iron entry in RPE is the phagocytosis of PR outer segments which contains high quantities of iron [8]. Once in RPE cytosol, iron is stored in FT and in melanosomes [44]. The release of iron from cell is possible through FPN present at the basal membrane of RPE and a multicopper ferroxidase. HEPH, CP, and APP but not ZP are expressed in RPE [26].

**Figure 2.** Iron uptake from capillaries and transport in the retina. **Legend Figure 2:** Under physiological condition, non-heme iron (Fe<sup>3</sup>+) in the circulation is transported bound to transferrin (TF). A. At the

choroidal side, Fe3<sup>+</sup> linked to TF is captured by its receptor 1 (TFR1) (1) at the basolateral level of the retinal pigment epithelium (RPE) (blue arrows). The internalized TF/TFR complex is transported to the apical pole by transcytosis (T) (2) or to the endosome (E) (3). In this case, Fe3<sup>+</sup> is released from TF and reduced by the metaloreductase six transmembrane epithelial antigen of the prostate 3 (STEAP3) to ferrous iron (Fe<sup>2</sup>+) and then exported to the cytosol by the transporter divalent metal transporter 1 (DMT1) where it constitutes the free iron pool (LIP) (4). TF and TRF1 are recycled to membrane (5). Iron is then transported from LIP to the organelles as needed, either stored in ferritin (FT) and melanosomes (Me), or exported by ferroportin (FPN) coupled to ferroxidases such as ceruloplasmin (CP) or hephaestin (HEPH), amyloid-beta precursor protein (APP) or zyloklopen (ZP) (black arrows). Hemochromatosis protein (HFE) and beta-2 microglobulin (B2M) associated to TFR1, shift to TFR2 in case of iron overload (saturation of TF) and activate hepcidin (HEPC) transcription (6). B. At retinal capillaries side, Fe3<sup>+</sup> bound to TF is up-taken by TFR1 at the luminal side of retinal endothelial cells (REC) (a), and TF/TFR1 pass directly through transcytosis into the retina (b) or endocytosed then exported by FPN (c). TF synthetized by RPE, Müller glial cells (MGC) or photoreceptors (PR) up-taken retinal iron (d) and distributed it throughout the retina, especially to PR. Phototransduction performed on the outer segments of PR is a highly iron-dependent process. PR uptake Fe3<sup>+</sup> bound to TF by TFR1 presents in inner segments and export it by FPN or by phagocytosis (P) of the outer segments of PR by RPE. TF-independent iron delivery to the retina can occur, especially in case of systemic iron dysregulation (black dotted lines). Serum FT has a specific receptor, the scavenger receptor class A, member 5 (SCARA5) localizes at the basal membrane of RPE, luminal side of REC, PR and MGC. Lactoferrin (LF), a member of TF superfamily and its receptors (LFR) are present in RPE. Fe3<sup>+</sup> captured by a siderophore (2,5-dihydroxybenzoic acid (2,5-DHBA)) is bound by lipocalin 2 (LCN2) and its receptors (24p3R) in RPE. The non-TF-bound iron (NTBI) is up-taken by MGC, REC, PR and RPE by DMT1 or ZRT/IRT-like proteins (ZIP) importers. BM: Bruch's membrane; CEC: choroidal endothelial cell; E: endosome; Me: melanosome; MGC: Müller's glial cell; MI: mitochondria; P: phagosome; PR: photoreceptor; REC: retinal endothelial cell; RPE: retinal pigment epithelium; T: transcytosis.

In the inner retinal layers, iron is imported through retinal endothelial cells (REC) which express TFR1 at their luminal side [45]. Two mechanisms of iron transfer across the abluminal membrane of REC into the retina are evoked: TF/TFR1 transcytosis or/and TF/TFR1 endocytosis following by iron released from endosome and iron export by FPN. The abluminal membrane of REC expresses FPN colocalized with HEPC, CP, and APP [24,31]. Iron is bound by TF and distributed to the retina or the vitreous. With its unique position extending from the vitreous to PRs and its capacity to synthetize TF [46] and to express FPN [24], MGC plays a crucial role in the distribution of iron from the inner retina to the inner segmen<sup>t</sup> of the PR.

Iron presents in the non-vascularized subretinal space, between the apical side of RPE and the PR, is mainly bound to TF, secreted by PRs and RPE, and up-taken by PRs for their highly metabolism activities, by TFR1 express at inner segments. The PR inner segmen<sup>t</sup> is the iron storage compartment for PR segments, where both FT chains and mitochondrial FT are highly concentrated [30]. Iron export from the PR is ensured mostly by FPN also present in inner segments, CP and HEPH being poorly involved in favor of APP [26].

### Non-Transferrin-Bound Iron Transport in the Retina

Whereas iron-bound TF is the main transport system to cross the BRB, transferrin-independent iron delivery to the retina can occur (Figure 1). Serum FT, exclusively composed of LFT has specific receptor, the scavenger receptor class A, member 5 (SCARA5) expressed in cytoplasm and nucleus in retinal endothelial cells, ganglion cells, astrocytes, the inner nuclear layer, MGC, microglia, outer nuclear layer, cones segments and RPE. After intravenous injection, serum FT remained confined in retinal endothelial cells in the inner retina [47]. Another protein possibly involved in iron transport is lactoferrin (LF), a multifunctional protein which shares 65% of homology with TF. It is synthesized in various human ocular tissues mainly in the RPE but not in the neural retina [18]. LF receptors have

not been studied in the eye but are present in the brain [48]. Lipocalin 2 (LCN2) does not bind to iron directly, but through interaction with siderophores (catecholate and carboxylate) as cofactors [49] could also be implicated in iron transport in the retina [20]. β-hydroxybutyrate dehydrogenase-2 (Bdh2), an enzyme that is critical for the synthesis of 2,5-dihydroxybenzoic acid (2,5-DHBA), the mammalian siderophore, is found throughout the retina in all cell layers, including ganglion cells, MGC, and RPE cells [50]. Two major membrane-bound receptors for LCN2, megalin and 24p3R, have been identified in RPE [21,22]. Although LCN2 is being recognized as an important factor in retinal diseases [21], its exact contribution in iron retinal transport in health and diseases remain to be determined.

In case of iron overload, the NTBI could be up taken by iron importer ZIP or DMT1. This could explain why in retinal iron overload models, iron continues to accumulate despite the reduced expression of TFR1 in retina and RPE [31]. The specific localization of DMT1 in PRs and bipolar and horizontal cells suggests that it could be involved in providing iron to these cells, for phototransduction or neurotransmitter synthesis [30] ZIP8 and ZIP14 are expressed in RPE, choroid, REC, choroidal endothelial cells (CEC), ganglion cells, PRs, and MGC. At a high degree of TF saturation in the retina, there is a decreased ZIP14 expression whereas ZIP8 expression remains stable [31].
