3.2.3. Iron Regulation in Retina

Cellular iron uptake and release and the intracellular LIP size are tightly controlled. Transcriptional, post-transcriptional, and post-translational processes regulate iron homeostatic proteins (for a review, see [51]). The main mechanisms of intra- and extra-cellular regulation of iron levels are limited to two extremely controlled systems.

The first system includes iron regulatory proteins (IRP) 1 and 2—intracellular iron regulatory proteins which, depending on the amount of iron, bind iron responsive element (IRE) sequences present on the mRNAs of iron homeostasis proteins such as FPN, TFR1, FT, and DMT1. Depending on the position of the IRE site, IRP controls their translation or degradation. Under conditions of increased cellular iron, IRP1 loses its IRE-binding activity by acquiring an iron in the 4Fe–4S cluster, whereas IRP2, is degraded by proteasome. In this condition, *tfr1* and *dmt1* mRNA are degraded, whereas *ft*, *fpn*, and *hif-2*α mRNA are translated. The localization of IRP1 and IRP2 has not ye<sup>t</sup> been identified in the retina but their expressions are ubiquitous in mammalian cells. Mice with *Irp1*+/− *Irp2*−/− genotype show more severe neurodegenerative disease than *Irp2*−/− animals [30]. These IRP deficient retinas have increased FPN and FT in the inner segments, MGC endfeet, and inner retina compared to age and strain matched wild type retinas, suggesting that FPN and FT levels are regulated by IRPs in the retina [23]. In a model of light induced retinal degeneration, 2 h after light exposure, *Irp2* but not *Irp1* mRNA increased in the retina [32].

The second system focuses on hepcidin (HEPC), a peptide hormone principally synthetized by the liver. However, HEPC is also synthesized by PR, RPE, and MGC [39]. It is activated by two cellular signaling pathways induced by excess of iron, the transferrin receptor 2 (TFR2)/Human homeostatic iron regulator protein (HFE) pathway and the Bone Morphogenetic protein (BMP6)/Mothers against decapentaplegic homolog 1 (SMAD) pathway. When the TF saturation is high at the basolateral level of the RPE, the HFE is released from TFR1 and binds to TFR2, which activates the transcription of HEPC. BMP6 secreted by the retina and the RPE, binds to its receptors coupled to hemojuvelin (HJV) protein at the apical level of RPE in order to activate the synthesis of HEPC [35]. HEPC binds to the extracellular domain of FPN on the cell surface, leading to its internalization and degradation, effectively preventing cellular iron export and limiting the amount of iron that gets into the extracellular fluid. The specific deletion of HEPC in the retina does not lead to age-associated retinal iron accumulation, whereas liver-specific HEPC silencing leads to early serum, RPE, and retina iron accumulation followed by retinal degeneration [52].

Finally, the hypoxia inducible factor (HIF) acts as a transcription factor for certain iron homeostasis genes such as the *Tf*, *tfr1*, *Dmt1*, *Fpn*, and *Cp* genes by binding to a specific hypoxia-responsive element (HRE) site present on their mRNAs. Expression and degradation of HIF are also dependent on iron. In fact, Fe2<sup>+</sup> is the cofactor of prolyl hydroxylase involved in the degradation of HIF-1 α, and at the same time HIF-2 α has an IRE sequence in the 5UTR of its mRNA, which in the condition of iron deficiency, inhibits its translation. Nuclear staining of HIF-1 α was observed in the GCL, the inner nuclear layer and the outer nuclear layer in human and rat [41]. Under retinal hypoxia, both HIF-1 α and HIF-2 α are activated but have cell specific expression within the inner retina. Specifically HIF-2 α activation seems to play a key role in regulating the response of MGC to hypoxia [53].

### **4. Physiopathological Role of Iron in the Retina**

### *4.1. Iron in Cellular Metabolism*/*Functions*

### 4.1.1. Iron as a Fe-S Structural Motif Involved in Various Cellular Machinery Proteins

Iron sulfur (Fe-S) proteins are characterized by the presence of Fe-S clusters localized in di fferent cell compartments (for review [54]). IRP1 is a Fe-S cluster that participates in sensing and regulating iron homeostasis in the retina. Frataxin is a nuclear-encoded mitochondrial protein involved in Fe-S cluster assembly, heme synthesis, and intracellular iron homeostasis. Frataxin is an allosteric activator which binds to this assembly complex [55]. It is present in the retina [56] and in the RPE [57] and could be responsible for retinal neurodegeneration induced by defective mitochondrial function [58]. In addition, Fe-S clusters may act as biological sensors by their binding properties to molecular oxygen and nitric oxide [59] both critical for the retinal physiology and pathology.

### 4.1.2. Iron in Nucleic Acids Machinery, Cell Proliferation, and DNA Repair

A recent review has reported the multiple implications of iron in DNA synthesis and repair, as well as in RNA metabolism [60]. Cytosolic and nuclear Fe-S proteins intervene in the genome stability [61]. Iron has been implicated in DNA synthesis and repair as a cofactor of sirtuin 2, an histone deacetylase, involved in iron homeostasis [62]. Sirtuin 2 maintains cellular iron levels by binding the nuclear factor erythroid-2-related factor 2 (NRF2) leading to a reduction in total and nuclear NRF2 levels. NRF2 is a transcription factor that plays key roles in retinal antioxidant and detoxification responses and has been linked with the development of age-related macular degeneration (AMD) [63].

Mitochondria are a major source of ROS and mitochondrial DNA is very susceptible to oxidative damage [64]. In RPE cells, mitochondrial DNA is damaged by hydrogen peroxide [65]. Deletions in mitochondrial DNA occurred in function of age in human neural retina [66], and the accumulation of age-related mitochondrial mutations in the eye has been correlated with a decrease in ATP production and increase ROS output, leading to oxidative stress, inflammation, and degradation [67].

### 4.1.3. Iron in Oxygen Transport and Regulation

Hemoglobin is synthetized in the retina [68]. It is one of the main protein synthesized in primary cultures of human RPE and secreted in vivo through the basolateral membrane [69].

Under physiological condition, free hemoglobin is bound by haptoglobin, but in case of massive hemolysis, hemoglobin releases free heme which binds hemopexin. Both hemopexin and haptoglobin have been described in the human retina [70,71]. The mRNAs for both haptoglobin and hemopexin were detected in the neural retina and PR as well as ganglion cells but not in RPE cells.

Neuroglobin is a highly conserved oxygen-binding protein reviewed in [72] and highly expressed in the retina. Its role is to facilitate oxygen metabolism, being localized in mitochondria. Hemin, the ferric chloride salt of heme enhances neuroglobin expression and protects animal model of N-methyl-N-nitrosourea-induced retinal degeneration [73]. In this model, hemin protects also cones from apoptosis. Neuroglobin has also been associated with retinal damage induced by light [74] which may reflect the changes in iron metabolism first described with light on retina [32]. It has also been associated with VEGF expression and thus could participate in retinal angiogenesis [75].

Heme, Fe2<sup>+</sup> protoporphyrin IX, the prosthetic group of hemoproteins including hemoglobin, neuroglobin, oxidases/peroxidases, or cytochromes can be released after auto-oxidation. Heme transporter proteins also intervene in iron metabolism in the retina, and their dysregulation could potentially cause oxidative cell damage. All three heme transporters feline leukemia virus subgroup C receptor (FLVCR), breast cancer resistance protein (BCRP), and proton-coupled folate transporter (PCFT/HCP-1) are expressed in the retina and RPE. In the RPE, the expression of FLVCR is restricted to the apical membrane and the expression of BCRP and PCFT to the basolateral membrane. In cases of iron overload, the expression of FLVCR and PCFT is upregulated and BCRP is downregulated, suggesting an important role of heme transporter proteins in retinal iron regulation [76].

### 4.1.4. Iron and Visual Function

The involvement of iron in the vision cycle was discovered with the characterization of the enzyme RPE65, as an iron-dependent isomerohydrolase [77]. RPE65, abundant in the RPE [78], ensures the isomerization and hydrolysis of all-*trans* retinyl ester to 11-*cis* retinol. RPE65 is essential for vision, and mutations in *rpe65* genes induce Leber congenital amaurosis, a form of retinitis pigmentosa that leads to blindness [79]. Recently, RPE65 was also shown to intervene in the production of meso-zeaxantin, an ocular specific carotenoid which protects the fovea from oxidative stress [80].

An alternate pathway for 11-*cis* retinol recycling has been described in MGC by isomerases 1 or 2 that also appear to be iron dependent [81]. Few studies have analyzed the iron flux in the retina with the diurnal cycle conversely to what has been performed in brain in mice [82,83]. Among the sensory guanylate cyclase proteins and signaling network, guanylyl cyclase activating protein 5 is the only protein that binds strongly Fe2<sup>+</sup> in zebrafish [84]. It is proposed as redox sensor in visual transduction.

Phototransduction depends on the phagocytosis of outer segments from PR by the RPE. The constant release of the outer segments from PR and their digestion during phagocytosis by RPE implies membrane biogenesis, a process which needs iron as a cofactor of fatty acid desaturase [85]. Royal College Surgeon (RCS) rats invalided for the phagocytosis protein Myeloid-epithelial-reproductive tyrosine kinase (MERTK) have increased iron in retina and particularly in RPE phagosomes and also increased retinal FT and TF expression [86].

Iron is also involved in neurotransmitters secretion as it regulates glutamate secretion by RPE cells via the cytosolic aconitase pathway [87]. Dopamine biosynthesis in specialized amacrine cells results from the conversion of the amino acid L-tyrosine in L-3,4-dihydroxyphenylalanine (L-DOPA) using oxygen and Fe2<sup>+</sup> [88]. Synaptosomal nerve-associated protein 25 (SNAP-25) is a Fe-S protein involved in synapse vesicle fusion with plasma membranes highly present in retina [89].

A significant number of ATP binding cassette (ABC) transporters, involved in lipid trafficking in retinal cells, have been linked to severe genetic ocular diseases [90]. ABCA4 is present in the PR and transports 11-*cis* and all-*trans* isomers of *N*-retinylidene-phosphatidylethanolamine across disc membranes, preventing the accumulation of toxic bisretinoid lipofuscin compounds in PR and RPE cells. In *Abca4* null mutant mouse which presents accumulation of N-retinylidineN-ethanolamine (A2E) bisretinoids and lipofuscin in the RPE, intracellular iron accumulation is also observed which contributes to enhancing oxidative cell death [91]. The intracellular accumulation of iron in cells of the RPE in culture decreases the expression of the transporters of cholesterol ABCA1/ABCG1, increasing the level of pro-inflammatory cholesterol in retina [50].

### *4.2. The Dark Side of Iron*

### 4.2.1. The Crucial Role of Iron in Oxidative Stress-Mediated Damages in the Retina

The ability of iron to change easily its valence and switch between the Fe2<sup>+</sup> and Fe3<sup>+</sup> forms, providing or accepting electrons, respectively, ensures a privileged position in living matter as mediator of key biochemical reactions. However, the presence of free labile iron in cell or NTBI in circulation is prone to generate highly ROS in the Fenton/Haber–Weiss reaction.

$$\begin{array}{rcl}Fe^{2+} + H\_2O\_2 & \rightarrow Fe^{3+} + OH^- + HO^\cdot \text{ : Factor reaction} \\ O\_2{}^\cdot + Fe^{3+} & \rightarrow O\_2 + Fe^{2+} \text{ : Haber-Weiss reaction} \\ O\_2{}^\cdot + H\_2O\_2 & \stackrel{Fe^{2+} \text{ : Fe^{3+}}}{\rightarrow} O\_2 + OH^- + HO^\cdot \text{ : Factor/Haber-Weiss reaction} \end{array}$$

The toxicity of free iron has been extensively studied on neuronal and retinal cells, and they are not sensitive to the same doses of iron [46,92,93], the cones being the most sensitive to iron [94]. In RPE cells, the interaction of iron with bisretinoids and lipofuscin induces cell damage and retinal degeneration [91]. Conversely, melanin can bind large amounts of iron to preserve the RPE and the choroid from a pro-oxidant environment, intensified by light exposure. However, with age, the accumulation of iron in melanosomes associated with a reduction in the amount of melanin in RPE promotes the formation of free radicals [95]. Exposure of RPE cells to high non-lethal doses of iron leads to a decrease in phagocytic and lysosomal activity [15], favoring the accumulation of breakdown products of Vitamin A (lipofuscin) leading to the formation of glycation end products (AGE) present in drusen, RPE, and Bruch's membrane of AMD patients [91]. In addition, phagocytosis of PR discs, peroxidized by ferrous ions, damage the membranes of phagosomes and lysosomes in RPE cells in culture [15,96].

In hypoxic conditions, an e fflux of iron from RPE to the basolateral direction [97] could explain, at least in part, that PRs tolerate better hypoxia than hyperoxia [98]. Fe2<sup>+</sup> contributes also to light-induced PR cell death through the production of hydroxyl radicals [99]. The ascorbate-Fe2+ complex induce lipid peroxidation in rod outer segmen<sup>t</sup> membranes and subsequently damage proteins such as rhodopsin by carbonylation or loss of thiol groups [100]. Finally, free heme can be also a source of redox-active iron and therefore highly toxic for the retina and for RPE cells [101].

In optic neuropathy, such as glaucoma, several mechanisms involved in ganglion cell death seem to be enhanced by iron-dependent oxidative stress [102,103].

Iron is thus a key component of oxidative-induced damages in the retina and in the RPE and involved in major cell death mechanisms.

### 4.2.2. Retinal Cells Death Mechanisms in Iron Overload

Iron overload, induced experimentally by the implantation of iron particles in rat vitreous cavity caused apoptosis (TUNEL-positive nuclei) in the outer nuclear layer after only 2 days [104]. Rat retinal explant exposed to iron showed an early increase of necrotic markers, such as lactate deshydrogenase, receptor-interacting serine/threonine-protein (RIP) kinase, and incorporation of propidium iodide, even before intraretinal iron accumulation was detected. Using retinal organo-culture, it was observed that iron deposits in retinal explants induced a shift from necrosis to apoptosis with activation of caspase 3 and TUNEL-positive nuclei [105]. Increased intraocular iron levels following intravitreal FeSO4 injection caused oxidative damage of PR, as shown by the increase of superoxide radicals; hydroxynonenal, a marker of lipid peroxidation; and increased expression of heme oxygenase 1 [94]. Retinal iron overload also activates the NOD-like receptor family, pyrin domain containing 3 (NLRP3) inflammasome signaling pathway. In fact, the expression levels of NLRP3, activated caspase-1, a downstream target of NLRP3, and interleukin (IL) 1ß were higher in the retinas of HFE KO mice, a model of genetic iron overload [106]. Ferroptosis, a newly characterized form of necrosis, is induced by the accumulation of iron in degenerative diseases and has been described in RPE cells in culture subjected to oxidative stress [107]. Glutathione depletion also induced ferroptosis, autophagy, and premature senescence in RPE cells [108].
