Olivier Dauteuil
email: olivier.dauteuil@univ-rennes1.fr

C Homberg

M Rocher

M Amarouche
email: amarouche_mohamed@hotmail.fr

Jean-Jacques J Kermarrec
email: jean-jacques.kermarrec@univ-rennes1.fr

Y Jegat

Rheological-layered basin under strike-slip deformation: analogue approach at crustal scale

Keywords: Rheological layering, Strike-slip, Thinning, Thickening, Boudinage, Faulting pattern

de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

Introduction

Sedimentary basins are subsiding domains filled with various deposits, where the dynamics are driven by material supply, deformation, climate and base level variations. Although the regional boundary conditions control the types of basins (e.g., strike-slip, flexural, rift, marginal and foreland basins- [START_REF] Allen | Basin Analysis: Principles and Applications[END_REF], the sediment supply controls the nature of the infilling. Low inputs allow the precipitation of autogenic carbonates, whereas larger terrigenous fluxes result in the accumulation of clays, sands or conglomerates. During diagenesis and burial, the deposits acquire various mechanical behaviours. For instance, sandstones and carbonates tend to have generally brittle behaviour, whereas clays and evaporites are more viscous depending on the stress conditions (strain rate and deviatoric stresses). Furthermore, differences in compaction may generate some layers with high fluid pressure, which favours viscous behaviour. This may increase the contrast with upper and lower brittle layers. The horizontal layering resulting from such variations in lithology constitutes a major mechanical heterogeneity, which has an impact on the propagation and shape of faults and thus the overall structure of the basin [START_REF] Mcclay | Experimental models of hangingwall deformation in ramp-flat listric extensional fault systems[END_REF][START_REF] Nalpas | Influence de la charge sédimentaire sur le développement d'anticlinaux synsédimentaires. Modélisation analogique et exemple de terrain (bordure sud du bassin de Jaca)[END_REF][START_REF] Withjack | Active normal faulting beneath a salt layer: an experimental study of deformation patterns in the cover sequence[END_REF]. Improving the mechanical understanding of such a rheological-layered system is of major interest for the management of natural resources trapped or stored in reservoirs and sealed by clayey layers and for the intermediate-depth or deep geological disposal of nuclear waste in clayey rocks.

Previous works on extensional systems [START_REF] Mcclay | Experimental models of hangingwall deformation in ramp-flat listric extensional fault systems[END_REF][START_REF] Withjack | Active normal faulting beneath a salt layer: an experimental study of deformation patterns in the cover sequence[END_REF][START_REF] Roche | Fault displacement profiles in multilayer systems: from fault restriction to fault propagation[END_REF], 2013] and compressive systems [START_REF] Nalpas | Influence de la charge sédimentaire sur le développement d'anticlinaux synsédimentaires. Modélisation analogique et exemple de terrain (bordure sud du bassin de Jaca)[END_REF][START_REF] Barrier | Influence of syntectonic sedimentation on thust geometry. Field examples from the Iberian Chain (Spain) and analogue modelling[END_REF] have shown the impact of rheological layering on the deformation of sedimentary basins. The fault dip angle (higher or lower compared to a typical dip), the fault geometry (planar, flat and ramp, curve, three-dimensional shape), the number of faults and their connection are impacted by mechanical layering. Moreover, this deformation of multilayered systems can also generate associated structures such as extensional folds [START_REF] Withjack | Active normal faulting beneath a salt layer: an experimental study of deformation patterns in the cover sequence[END_REF]Callaway, 2000, Homberg et al., 2017] or long wavelength flexures.

Many modelling studies (see the review of Dooley and Schreurs, 2012) were performed within a purely strike-slip context using different kinds of materials: clay [Riedel, 1929, Cloos, 1928, 1955, Tchalenko, 1970, Wilcox et al., 1973, Soula, 1984, Lazarte and Bray, 1996, Atmaoui et al., 2006], dry sand [Emmons, 1969, Naylor et al., 1986, Richard et al., 1989, Richard, 1990, 1995, Burbidge and Braun 1998, Schöpfer and Steyrer, 2001[START_REF] Viola | Analogue modelling of reverse fault reactivation in strike-slip and transpressive regimes: application to the Giudicarie fault system, Italian Eastern Alps[END_REF][START_REF] Le Guerroué | Influence of erosion and sedimentation on strike-slip fault systems: insights from analogue models[END_REF], and rheologically stratified materials [Wilcox et al., 1973, Odonne and Vialon, 1983, Richard et al., 1989[START_REF] Dauteuil | Faulting pattern, ductile deformation, and vertical motion in strikeslip provinces: analog modeling[END_REF][START_REF] Casas | Analogue models of transpressive systems[END_REF]. These works reveal a complex faulting pattern varying along the strike-slip boundary even in experiments with uniform mechanical layers. Several works investigated the parameters driving the fault patterns in pull-apart basins [START_REF] Mcclay | Analogue models of pull-apart basins[END_REF][START_REF] Dooley | Analog modeling of pull-apart basins[END_REF][START_REF] Schreurs | Analogue modelling of faulting in zones of continental transpression and transtension[END_REF][START_REF] Sims | Role of a ductile décollement in the development of pull-apart basins: Experimental results and natural examples[END_REF][START_REF] Wu | 4D analogue modelling of transtensional pull-apart basins[END_REF][START_REF] Corti | Modelling the influence of pre-existing brittle fabrics on the development and architecture pull-apart basins[END_REF]. The addition of a viscous layer allows producing en échelon folds [Odonne and Vialon, 1983] or along-strike bulging [START_REF] Dauteuil | Faulting pattern, ductile deformation, and vertical motion in strikeslip provinces: analog modeling[END_REF], that is, the genesis of associated features that In this study, we investigated the structures generated by strike-slip boundary conditions to provide a 3D view of the deformation pattern that could be present in a nonuniform mechanical system. We used analogue modelling because of the scale investigated (basin in the upper crust), the nonlinear mechanical properties of the rocks and the 3D nature of the structures associated with this context [Richard et al., 1991, Dauteuil and[START_REF] Dauteuil | Faulting pattern, ductile deformation, and vertical motion in strikeslip provinces: analog modeling[END_REF]]. The results provide some guidelines to interpret features generated in a strike-slip context.

Experimental set-up

Earth materials exhibit two main mechanical behaviours (brittle and viscous) depending on the lithology, temperature, pressure, deformation rate and fluid pressure [START_REF] Kirby | Rheology of the lithosphere[END_REF][START_REF] Kohlstedt | Strength of the lithosphere: constraints imposed by laboratory experiments[END_REF]. The variation in these parameters with depth generates a rheological layering that can be seen as the succession of brittle and ductile layers approximated in analogue modelling using adapted materials [START_REF] Davy | Experiments on shortening of a 4-layer model of the continental lithosphere[END_REF][START_REF] Graveleau | Experimental modelling of orogenic wedges: a review[END_REF][START_REF] Schellart | A review of analogue modelling of geodynamic processes: approaches, scaling, materials and quantification, with an application to subduction experiments[END_REF]. The brittle behaviour was simulated with Fontainebleau sand and manufactured glass microbeads and the ductile behaviour with two types of silicone putty (pink and transparent). Table 1 summarizes the different mechanical properties. The rheological dimensioning of these materials was described in previous works [START_REF] Davy | Indentation tectonics in nature and experiment. 1 Experiments scaled for gravity[END_REF][START_REF] Le Guerroué | Influence of erosion and sedimentation on strike-slip fault systems: insights from analogue models[END_REF][START_REF] Panien | Mechanical behaviour of granular materials used in analogue modelling: insights from grain characterisation, ring-shear tests and analogue experiments[END_REF][START_REF] Vasquez | 3D geometries of normal faults in a brittle-ductile sedimentary cover: Analogue modelling[END_REF]. For the scaling of physical parameters, we followed the methodology described by [START_REF] Davy | Experiments on shortening of a 4-layer model of the continental lithosphere[END_REF] and [START_REF] Vasquez | 3D geometries of normal faults in a brittle-ductile sedimentary cover: Analogue modelling[END_REF]. The physical scaling used is as follows: 1 km in nature corresponds to 1 cm in the model and 1 cm/yr in nature to 1 cm/h in the model.

The models are constructed in a 1 m long and 40 cm wide box designed with two drawers: one fixed and one mobile pushed by a step-by-step motor (Figure 1). The sliding border of the two drawers defines a strike-slip velocity discontinuity above which deformation occurs in the model. Five experiments are conducted. All of them comprise five layers. Models Decro-1, Decro-2, Decro-4, and Decro-5 have three layers of 1 cm thick Fontainebleau sand and two layers of either 1 cm thick or 0.5 cm thick silicone putty. Model Decro-3 consists of alternating silica powder and sand layers. All models are 5 cm in height except Decro-4, which is 4 cm in height. Two different pulling rates are tested: 1 cm/h and 3 cm/h. The change in rate modifies the strength of the silicone putty: the higher the rate, the stronger the silicone. The rate does not modify the mechanical properties of the sand [START_REF] Kirby | Rheology of the lithosphere[END_REF][START_REF] Ranalli | Rheological stratification of the lithosphere[END_REF][START_REF] Kohlstedt | Strength of the lithosphere: constraints imposed by laboratory experiments[END_REF]. Surface and oblique pictures are taken during the experiment. Finally, the models are cut into several vertical cross-sections every 4 cm to provide 3D data sets. The properties of the five experiments are Table 1. Physical properties and scaling are described in Tables 1 and2. Mean deviatoric stresses 4.8 3.15 2.2 2.3 0.9

Table 2. Experimental conditions

Note: The thickness row displays the thickness of each layer from top to bottom; the number in bold corresponds to intermediate layers. In the rheological layering row, the section in dark grey is silicone 1, medium grey is silicone 2 and light grey is silica powder. The whole given strength is relative (without units) and corresponds to the area of the rheological profile from the weakest (1) to the strongest (5). The mean of deviatoric stresses is the average of the differences in deviatoric stresses from one layer to another. Thus, high values indicate high rheological contrasts.

summarized in Table 2, snapshots of surface views and selected cross-sections are displayed in Figure 2 and larger views are presented in the Supplementary material.

Faults in brittle layers

Regardless of rheological layering, the analogue experiments display deformations with some common 2 for the experimental setup). For each experiment, the surface view (right column) and two or three line drawings of the sections are given (left column). The dotted zones inside the silicone layer trace the path of viscous deformation. The label of the section includes the distance from the border without the motor.

characteristics, which we describe first. Surface views and cross-sections show that the deformation is focused into a narrow shear band affected by numerous faults (Figure 2). In horizontal view, the faults systematically display an en échelon pattern with a mean 9 • -14 • obliquity relative to the basal strike-slip discontinuity (Figure 3) corresponding to R-faults in the Riedel shear system. The highest angles are close to the experiment border, resulting from edge effects.

The fault azimuths roughly decrease as their lengths increase (Figure 3). This means that the faults tend to become parallel to the velocity discontinuity as they grow. Likewise, a positive correlation between the width of the deformed band and the fault azimuth indicates that the fault orientation drives the width of the deformed band. In the vertical plane, the faults are difficult or impossible to detect because they laterally offset the layers and have very small or no vertical throw. However, they are often associated with a change in layer thickness or in layer dip (flexure and fold). We did not systematically observe the genesis of one type of associated feature depending on rheological layering. Folds and flexures change along the strike of the deformed zone and exist at different depths. In all of the experiments, the faults grow upward from the basal strike-slip discontinuity. The number of faults decreases with depth as previously described in Le [START_REF] Le Guerroué | Influence of erosion and sedimentation on strike-slip fault systems: insights from analogue models[END_REF]. In the experiments with silicone layers (Decro-1, Decro-2, Decro-3, and Decro-5), the lowest sand layer is only affected by one fault, whereas the uppermost layer is affected by up to five faults. New faults appear at the top of each silicone layer, which react as a local velocity discontinuity. The fault dip does not change significantly at each layer interface. Secondary faults appear in the surface layer. In the experiment with silica powder (Decro-3; Figure 2), two main faults with opposite dip are nucleated at the strike-slip discontinuity located at the base of the experiment, and they propagate upward. The fault dip slightly changes at each interface separating the sand layers from the layers of glass microbeads. Therefore, a change in the friction angle of cohesive layers modifies the fault dip, which does not act as a velocity discontinuity. These results show that rheological layering controls the geometry and upward propagation of the faults: (i) the fault dip when the faults cut through layers with different friction angles (a decrease in the friction angle, flattening the fault dip) and (ii) the nucleation of new faults at each interface between brittle and viscous layers (the fault number increases at each interface).

The faults mainly accommodate the strike-slip motion as imposed by the boundary conditions. However, some of them show a small vertical throw generated with either normal or reverse throw. As expected in such a strike-slip context, the vertical displacements (up to 0.6 mm) along the faults are lower than the horizontal displacements (7 cm), that is, less than two orders of magnitude. This vertical component is worth mentioning because even if it is small, it is observed at the basin scale, on seismic profiles or in the field. In the same experiment, some sections display faults with reverse components and other sections show faults with normal components. Furthermore, some faults with reverse and normal components may coexist within the same section. Therefore, the type of fault (strike slip with a normal or a reverse component) varies along the strike of the deformed band and through the depth despite the boundary and kinetic conditions remaining similar during the experiment. The multilayered medium deformed by horizontal shearing applied at the boundaries generates intrinsic conditions, allowing upward and downward displacements.

Changes in layer shape

In addition to the brittle deformation described above, the five layers display other types of deformation within the shear band: thinning, thickening and folding. Thinning is accommodated by normal oblique-slip faults in the sand and silica layers and by boudins in the silicone and cohesive layers. Reverse oblique-slip faults in the sand layers as well as thick bulges in both types of layers accommodate thickening. No folding occurs when there are only cohesive layers such as those in the Decro-3 experiment.

Surprisingly, different deformation styles coexist within the same section except in the experiment without a viscous layer (Decro-3). One layer may be thinned, while another is thickened or folded. This means that there is strong decoupling of deformation between layers having different rheological behaviours. Therefore, strike-slip boundary conditions can simultaneously induce thinning and thickening, indicating that such a deformation pattern is not symptomatic of extensive and compressive contexts, respectively. This observation has important implications for the interpretation of geological crosssections or seismic lines in geophysical surveys. Although opposite deformation styles are frequently interpreted as belonging to different tectonic conditions in a polyphase history, the present work reveals that they may have occurred during a single deformation stage. The experiment comprising only cohesive layers is deformed by positive or negative flower structures as previously described by Le Guerroué and Cobbold [2006].

In addition to the changes in deformation pattern with depth, the deformation also evolves along strike. The different sections of the same experiment display changes in deformation style. Thinned, stretched or folded domains succeed each other within the same layer (Figure 4). The variations in layer thickness display a roughly periodic pattern. The wavelength is approximately 16 cm, that is, ±3.2 times the whole thickness of the experimental structure. This ratio is consistent with that from the literature [START_REF] Martinod | Periodic instabilities during compression of the lithosphere: 2. Analogue experiments[END_REF][START_REF] Martinod | Periodic instabilities during compression of the lithosphere: 2. Analogue experiments[END_REF]Cobbold, 1988, Dauteuil [START_REF] Dauteuil | Faulting pattern, ductile deformation, and vertical motion in strikeslip provinces: analog modeling[END_REF]], which predicts that the deformation wavelength ranges from 3 to 5 times the thickness of the layer involved in the deformation. In the experiments with viscous layers (Figure 4), the thickness for the basal silicone layer displays longer wavelengths than the thicknesses for the middle layers, and the thickness for the upper sand layer shows shorter wavelengths than the thicknesses for the middle layers. The deformation wavelength roughly displays an opposite phase shift between the viscous and cohesive layers. A thinning of the viscous layer fits with a thickening of the cohesive layer. The amplitudes of deformation wavelength in the sand are often higher than those in the silicone. The viscous layers are more often thinned than thickened, while the cohesive layers thicken more easily than the viscous layers.

In the experiment with five cohesive layers (Decro-3), the layer thickness also displays variations with wavelengths that have an opposite shift (Figure 4). The wavelength amplitude is smaller for the more cohesive layer (silica layer). The wavelength corresponds to a succession of positive and negative flower structures in which the vertical throw decreases downward to become null at the base of the system. Previous experiments with one mechanical layer only display positive flower structures [START_REF] Richard | Experiments on simultaneous faulting and folding above a basement wrench fault[END_REF][START_REF] Mcclay | Analog models of restraining stepovers in strike-slip fault systems[END_REF][START_REF] Le Guerroué | Influence of erosion and sedimentation on strike-slip fault systems: insights from analogue models[END_REF]. In our experiment, negative flower structures are associated with the thinning of the basal layer. [START_REF] Al-Zoubi | Geological structure of the eastern side of the lower Jordan valley/Dead Sea rift: reflection seismic evidence[END_REF], [START_REF] Hang | Geometry, kinematics and displacement characteristics of strike-slip faults in the northern slope of Tazhong uplift in Tarim Basin: A study based on 3D seismic data[END_REF] and [START_REF] Carlini | The role of mechanical stratigraphy on the refraction of strike-slip fault[END_REF] described natural cases with alternation positive and negative structures strike on deformed zones.

Physical parameters driving the deformation

This experimental modelling reveals a more complex deformation pattern in a multilayer system affected by simple strike-slip boundary conditions than in a single-layer system (e.g., [START_REF] Richard | Experiments on simultaneous faulting and folding above a basement wrench fault[END_REF][START_REF] Mcclay | Analog models of restraining stepovers in strike-slip fault systems[END_REF][START_REF] Le Guerroué | Influence of erosion and sedimentation on strike-slip fault systems: insights from analogue models[END_REF]. These previous analogue modelling experiments explored the structural pattern in simplelayer experiments: only positive flower structures were generated. In our experimental study using a small-scale model with different cohesive layers, we generated positive and negative flower structures associated with different deformation modes in intermediate layers. The thickness variations in the most cohesive layers (glass microbeads) display smaller amplitudes. In other words, the less cohesive layers (Fontainebleau sand) are the most deformed layers. Thus, we advance the hypothesis that the difference in cohesion strength between two layers controls the changes in layer thickness, whereas the friction angle does not largely influence the fault geometry as indicated in the experiment with the sand and silica powder. In consequence, a deformation pattern characterized in a vertical section is not symptomatic of the boundary conditions. Folding or bulging does not necessarily result from regional compression, for instance, or a transpressive component imposed on the fault zone. However, the association of different deformation patterns both through depth (observed in a section) and along strike can be attributed to strike-slip boundary conditions in a multilayer setup. [START_REF] Nemser | Downdip segmentation of strike-slip fault zones in the brittle crust[END_REF] described a vertical change in fault pattern, which occurs where the lithology changes abruptly. We suggest that the rheological contrast between cohesive layers may drive the capacity to generate one type or two types of flower structure (positive or negative). It is possible that the local heterogeneities induce stress changes, which promote local shortening or thinning in a global context of horizontal shearing.

All the structures that affect the different layers are summarized in the 3D block diagram of Figure 5: strike-slip faults, normal oblique-slip faults and reverse oblique-slip faults for the cohesive layers and boudins, bulges and folds for the viscous layers. The spatial distribution of these features evolve along both strike and depth. The organization of the structures generates variations in layer thickness (thinning and thickening) out of phase between cohesive and viscous layers. The amplitude of the wavelength is different in each experiment and is directly linked to the strength ratio between cohesive and viscous layers (Figure 6). The higher the amplitude, the greater the strength ratio. The variations in the wavelength for the layer thickness clearly illustrate the importance of mechanical layering and of the coupling between different layers in the deformation distribution and pattern (Figure 6). The Decro-1 experiment, which has the highest mean strength ratio between the viscous and cohesive layers, displays the highest wavelength range, whereas the Decro-3 experiment, with the lowest mean strength ratio, displays a very small range of amplitudes for the wavelength deformation. Therefore, the main process that drives the deformation is the strength contrast between the different mechanical layers. The wavelength amplitude increases as the strength coupling decreases.

Conclusions

This experimental work highlights the fundamental role of rheological layering of a system in a strike-slip setting. The layering drives several characteristics of the deformed zone: the width of the shear zone, the fault pattern, the vertical displacements and the variations in thickness layers deformed in the shear zone. In the same experiment, thinning, thickening and folding domains alternate with a wavelength that is proportional to the whole thickness of the model. Rheological layering increases the number of faults generated relative to the experiment with only one layer. The amplitude of layer thinning/thickening is driven by the rheological coupling between different layers. Therefore, the interpretation of geological sections and seismic lines in a strike-slip setting is questioned. The proposed multiphase history must be reconsidered with caution if no 3D data set is available to constrain the 3D deformation pattern. A guideline for detecting unexpected strike-slip boundary conditions is the coexistence of thickening and thinning features in the same vertical section.

Figure 1 .

 1 Figure 1. Oblique view and schematic block diagram of the experimental apparatus. The apparatus placed on a rigid board is composed of two grey plexiglass drawers freely sliding on the board. The right drawer is pulled by a step-by-step motor. The model comprises five layers, the description of which is given in Table1. Physical properties and scaling are described in Tables1 and 2.

Figure 2 .

 2 Figure 2. Five analogue experiments at the end of the deformation (see Table2for the experimental setup). For each experiment, the surface view (right column) and two or three line drawings of the sections are given (left column). The dotted zones inside the silicone layer trace the path of viscous deformation. The label of the section includes the distance from the border without the motor.

Figure 3 .

 3 Figure 3. Relations among the fault length, the fault azimuth and the width of the deformed band. The upper plot displays the negative relationships between the length and the azimuth and the lower plot shows the positive relation between the width of the deformed band and the fault azimuth.

Figure 4 .

 4 Figure 4. Variations in thicknesses of the five layers along the velocity discontinuity in each experiment. Note the wavelength for the thickness that is in opposite shift.

Figure 5 .

 5 Figure 5. Schematic 3D block diagram showing the various structures affecting the layers in a strike-slip context. This work reveals that all these features coexist in the same experiment. The association of extensive and compressive features is symptomatic of strike-slip boundary conditions.

Figure 6 .

 6 Figure 6. Strength ratio between layers (colour symbols) compared to the wavelength amplitude (grey vertical bars at bottom) for the five experiments. The strength ratio is estimated at each interface between two layers by dividing the deviatoric stresses of the sand layer by the deviatoric stresses of the silicone/microbead layer. The inset at top right shows the location where the strength ratios are estimated. Decro-1 displays the lowest ratio because of the low viscosity of the viscous layers and Decro-3 has the highest ratio because of the absence of viscous layers.

Table 1 .

 1 Rheological properties of the material

	Brittle layers	Cohesion (Pa) Friction angle ( • ) Density Grain size (µm)
	Sand	≈10	30-35	1.4	250-300
	(Fontainebleau quartz-Sibelco, France)
	Glass microbeads	≈300	20-22	1.5	180-200
	(CVP, France)
	Viscous layers	Viscosity (Pa/s) @ 20 • C	Density
	Transparent silicone putty	3 × 10 4		1
	(GS1 RG 70 009-Rhône-Poulenc)
	Pink silicone putty	6 × 10 4		1.3
	(SGM 36-Rhône-Poulenc)
	can be symptomatic of strike-slip context. Although
	these studies focused on the specific areas of strike-
	slip domains such as pull-apart basins or on oblique
	(transpressive or transtensive) boundaries, the role of
	rheological layering in the strike-slip setting remains
	largely unexplored.

C. R. Géoscience, 0000, 1, n 0, 000-000

Acknowledgements

This work was cofunded by IRSN/CNRS/UPMC in a collaboration programme. The experimental modelling was conducted in the Experimental Laboratory of Géosciences Rennes at the University of Rennes.

Supplementary data

Supporting information for this article is available on the journal's website under https://doi.org/10.5802/ crgeos.38 or from the author.


Alejandra Daruich

Quentin Le Rouzic

Laurent Jonet

Marie-Christine Naud

Laura Kowalczuk

Jean-Antoine Pournaras

Jeffrey H Boatright

Aurélien Thomas

Natacha Turck

Alexandre Moulin

Francine Behar-Cohen
email: francine.behar@gmail.com

Emilie Picard

Iron is neurotoxic in retinal

MAIN TEXT Introduction

Retinal detachment is an ocular emergency, as separation of the neuroretina from the underlying retinal pigment epithelium (RPE) (Fig. 1A) disturbs photoreceptor (PR) metabolism causing PR death and subsequent vision loss [START_REF] Murakami | Photoreceptor cell death and rescue in retinal detachment and degenerations[END_REF]. In rhegmatogenous retinal detachment (RRD), a retinal tear (arrowhead, Fig. 1A) induces subretinal fluid (SRF) accumulation in a space formed between the neuroretina and the RPE. The incidence of adult RRD varies between 10 to 55 per 100,000 individuals/year, with higher occurrence in patients who also have myopia or pseudophakia [START_REF] Van De Put | Dutch Rhegmatogenous Retinal Detachment Study Group, The incidence of rhegmatogenous retinal detachment in The Netherlands[END_REF]. This incidence will likely rise with myopia increasing worldwide [START_REF] Holden | Global Prevalence of Myopia and High Myopia and Temporal Trends from 2000 through 2050[END_REF]. Intraocular surgery is commonly used to repair RRD but must be realized rapidly after detachment, as the duration of macular detachment is one of the major visual prognosis factors [START_REF] Mitry | Long-term visual acuity and the duration of macular detachment: findings from a prospective population-based study[END_REF]. The principles of surgery are based on retinal breaks closure (i.e., by laser therapy or cryotherapy) and internal or external temporary tamponade to maintain the retina attached during wound healing. When the procedure is performed internally, vitreous is removed and SRF is eventually extracted. Despite tremendous progress made in surgical techniques and the high repair rate, 60% of patients with RRD involving the macula (macula-OFF RD, 50% of the cases) recover less than 20/40 vision (4), and have poor vision-related quality of life [START_REF] Van De Put | Postoperative vision-related quality of life in macula-off rhegmatogenous retinal detachment patients and its relation to visual function[END_REF]. Improving vision after retinal detachment surgery is thus a critical therapeutic challenge.

Experimental models of retinal detachment (RD) correlate well with human pathology, making them appropriate to study mechanisms and pharmacotherapeutic strategies. First changes are observed at the RPE/PR interface [START_REF] Anderson | Retinal detachment in the cat: the pigment epithelial-photoreceptor interface[END_REF], followed by PR cell death that peaks at 2-3 days through apoptosis, necrosis and autophagic cell death mechanisms [START_REF] Murakami | Photoreceptor cell death and rescue in retinal detachment and degenerations[END_REF]. Interestingly, caspase inhibitors do not prevent cell death in RRD models , but rather promote receptor interacting protein kinase (RIP)-kinase dependent necrosis, referred to as necroptosis [START_REF] Trichonas | Receptor interacting protein kinases mediate retinal detachment-induced photoreceptor necrosis and compensate for inhibition of apoptosis[END_REF]. Subsequent inflammatory reactions, including macrophage/microglial activation contribute to a neurotoxic environment and to subsequent gliosis [START_REF] Lewis | Rapid changes in the expression of glial cell proteins caused by experimental retinal detachment[END_REF]. Factors initiating cell death during RD remain undetermined, and the identification of a common target of the different death mechanisms is mandatory for developing an effective neuroprotective strategy for RD.

Iron is an important biological component for catalyzing enzymatic reactions. But when it is poorly liganded, iron generates reactive oxygen species and damaging cellular components [START_REF] Eid | Iron mediated toxicity and programmed cell death: A review and a re-examination of existing paradigms[END_REF]. Iron-mediated retinal cell death has been suspected to occur in various forms of retinal degeneration [START_REF] He | Iron homeostasis and toxicity in retinal degeneration[END_REF], including age-related macular degeneration [START_REF] He | Iron homeostasis and toxicity in retinal degeneration[END_REF], the leading cause of blindness in developed countries, but no correlation between iron and visual function was possible and the iron sequestration capacity in human ocular fluids has never been evaluated in relation to retinal diseases. The control of ocular iron homeostasis is tightly controlled by retinal cell proteins, and the iron transporter, transferrin (TF), is one major protein found in the vitreous. We previously showed that the sequestration of two ferric iron ions by TF [START_REF] Picard | The protective role of transferrin in Muller glial cells after iron-induced toxicity[END_REF] prevents the formation of free radical-mediated stress and glial retinal cell death in culture.

Our previous studies also showed, in various animal models, that treatment with TF exerted retinal neuroprotective effects through the reduction of apoptosis, inflammation and oxidative stress [START_REF] Picard | Targeting iron-mediated retinal degeneration by local delivery of transferrin[END_REF][START_REF] Picard | Overexpressed or intraperitoneally injected human transferrin prevents photoreceptor degeneration in rd10 mice[END_REF].

However, the molecular mediators and pathways involved in the protective effects of TF have never been investigated in depth.

In our study, we evaluated for the first time the iron and transferrin status in ocular fluids from RRD patients and correlated those parameters to visual acuity recovery and duration of RRD. Retinal explants are a common tool used for RD studies, recapitulating pathological processes found in the detached retina [START_REF] Winkler | The retinal organ culture-a model system for the examination of the early cytoskeletal reaction pattern after retinal detachment[END_REF]. Thus, we established an ex vivo model of RRD by placing rodent retinas in iron-overload culture conditions and investigated whether TF protects the retina by evaluating apoptosis, necroptosis, and oxidative stress cell markers. Transgenic mice expressing human TF in the retina were used to observe TF neuroprotective efficiency in an experimentally induced RD model [START_REF] Matsumoto | Retinal detachment model in rodents by subretinal injection of sodium hyaluronate[END_REF], and using an RNA sequencing approach to explored the pathways implied in these effects. Intravitreal injection of TF in rats following experimental RD was used to determine the potential of TF as an adjuvant for RD surgery.

Results

In ocular fluids from patients with RRD, iron increases with detachment duration and correlates with poor visual recovery

Total iron, TF, TF iron-binding capacity (TIBC), and percentage of iron saturation (TSAT) were evaluated in undiluted vitreous and SRF collected from patients with RRD (age: 64.3 years; SD: 9.6; 11 females, 24 males, n=35), and from control patients (age: 70.2 years; SD: 9.5, 5 females, 4 males, n=9).

In the vitreous, the level of iron was significantly higher in patients with RRD as compared to control subjects without retinal detachment (143.1 ng/ml ±14.4 vs 81.7 ng/ml ±16.8, p=0.046) (Fig. 1B). The TIBC values did not differ in eyes with or without RRD (4.5 ±0.7 and 4.3 ±0.6 µmol/L of TF, p>0.5) but TSAT increased from 22.8% ±3.8 in control eyes to 89.91% ±11.4 in eyes with RRD, reaching almost complete saturation (p=0.006) (Fig. 1C andD). In the SRF, the high rate of TF saturation (53.5% ±12.6) (Fig. 1E) also reflected iron overload. Higher levels of iron in the SRF were associated with longer duration of detachment (Fig. 1E, left graph, p=0.038) and correlated with lower visual recovery one month after surgery (Fig. 1E, right graph, p=0.047). Though the data are correlative, these results suggest that iron toxicity could contribute to the functional outcome in RRD.

Iron deposits, evaluated in post-mortem ocular tissues sections of RD, were found in the remaining PR layer, inside cells in the subretinal space very likely to be inflammatory cells (arrows) and in RPE (Fig. 1F) using Perl's staining. Inductively coupled plasma mass spectrometer (ICP-MS) analysis confirmed elevated iron content throughout the retina sections (Fig. 1G, green), with higher concentrations in focal areas (red, arrowheads). These observations suggest that iron could mediate, at least partially, PR damage during RRD.

Iron induces photoreceptors death in rat retinal explants ex vivo

To evaluate iron neurotoxicity, rat neural retina explants were cultured in low-serum medium and PR cells were immediately exposed to iron at a range of concentrations similar to those measured in SRF from RRD patients. After 2 days of exposure to 1 mM FeSO4 (fig. S1A), no increase in caspasedependent apoptosis was observed (caspase 3 activation and TUNEL positive cells were similar in control and iron conditions) (fig. S1B andC). Caspase-independent apoptosis and autophagy evaluated by the expression of AIF, LEI/L-DNase II, and LC3B I/II proteins were also not activated by iron exposure (data not shown). However, lactate dehydrogenase (LDH) release, an early marker of necrosis, was concentration-dependently increased as early as one-day post-iron overload exposure (fig. S1D).

After 2 days of iron overload, necroptosis was confirmed with an increase of RIP kinase protein expression and staining of PR nuclei with propidium iodide (fig. S1E andF). The two days of iron exposure leads to the loss of both rods and cones PR, as shown by the decreased rhodopsin protein level with increasing concentration of FeSO4, (Fig. 2B) and nearly no arrestin-positive cones remaining after 1 mM FeSO4 exposure (Fig. 2C, arrows). After iron overload, progressive histologic damage appeared over time, leading to complete retinal layer disorganization and shortening of the residual PR segments (Fig. 2D). While iron staining was not observed in the retina during iron exposure (data not shown), 7 days after iron removal, iron diffusely stained the retinal explants, particularly in the remaining PR segments, and was concentrated in inflammatory cells type (brown, arrows) as observed in human retinal detachment sections (Fig. 2E). The direct contact of iron with PR led to immediate necroptosis that increased with higher iron concentrations and with longer iron exposure times.

Transferrin protects against iron-induced toxicity in ex vivo mouse and rat retinal explants

In transgenic (TG) mice expressing human TF (hTF) in retinal cells [START_REF] Picard | The protective role of transferrin in Muller glial cells after iron-induced toxicity[END_REF], hTF levels increased in the medium with the duration of retinal explants culture (fig. S2 Subsequently, we investigated whether TF was protective against iron overload combined with RD. Iron overload conditions were applied to TG and WT retinal explants (Fig. 3A). LDH release, evaluated after one day, was reduced by 24.4% in retinal explants from TG mice as compared with explants from WT mice (p=0.03) (Fig. 3B). After 6 days of iron exposure, the number of cones was 55.5% higher and the outer segment lengths were 58.2% longer in retinas from TG mice as compared with WT (p<0.0001 for both) (Fig. 3C). In addition, reduced Müller glial cell activation and oxidative stress markers (fig. S3 A to C) were observed in retinal explants from TG mice. The expression of hTF in TG mice retinas reduced iron accumulation as indicated by a reduction in ferritin light-chain (LFt) staining (Fig. 3D) [START_REF] Picard | Targeting iron-mediated retinal degeneration by local delivery of transferrin[END_REF]. The TG mouse retinas were thus protected from iron-induced oxidative stress and necroptosis.

We then evaluated the effect of TF added to rat retinal explants after 2 days of iron exposure to more closely mimic the therapeutic time-course common in human RD treatment. After 2 days, iron was removed and exogenous hTF (50 mg/ml) was added in the medium (Fig. 3E). TF treatment rescued not only rod-specific protein (61.7% greater rhodopsin level (p=0.021) (Fig. 3F) and the lengths of rod outer segments (Fig. 3G, asterisk), but also cones (83.8% greater number of cones versus untreated (p=0.04)) (Fig. 3G, arrows). Two days after iron exposure, the RIP kinase protein level remained elevated, but the presence of the cleaved form of RIP kinase proteins (Fig. 3H) suggested an activation of caspase 8, leading to a delayed activation of apoptosis [START_REF] Trichonas | Receptor interacting protein kinases mediate retinal detachment-induced photoreceptor necrosis and compensate for inhibition of apoptosis[END_REF]. Treatment with hTF significantly reduced the level of cleaved RIP kinase by 76.1% (Fig. 3H

Transferrin rescues the detached mouse retina in vivo

We induced partial retinal detachment in mice and rats (around 50-70% and 30-50% of the retinal surface, respectively). After 7 days, the detached retina was thickened, the outer nuclear layer (ONL) was disorganized, PR nuclei were pyknotic and outer segments were absent, and RPE cells were enlarged in the detached area (fig. S5). Similar to human RD, iron accumulated in the detached rodent outer retinas (fig. S6 A andB) and in rat ocular fluids (52.54% increase in total iron levels after 14 days of detachment, p=0.042) (fig. S6C). In TG mice (Fig. 4), hTF protected the detached retina from edematous thickening (21.7% decrease in retinal thickness of the detached retina/non-detached retina; p=0.045, designated by brackets in Fig. 4A, quantified in the left graph of Fig. 4B), from ONL thinning (20.3% decrease; p=0.033, Fig. 4B, right graph) and prevented PR outer segment shortening (50% decrease; p=0.047, graph in Fig. 4C and arrowheads in Fig. 4A and4C). TG detached retinas had 311.3% more cones than WT (p=0.025; Fig. 4D). Activation of Müller glial cells, observed not only in the areas of detached retinas but also in attached regions of WT mice (data not shown), was significantly reduced in TG mouse retinas (Fig. 4E, arrows). Compared with WT retinas, hTF expressed in TG retinas decreased caspase 8 mRNA expression by 48.9% (p=0.008), the level of the cleaved form of caspase 3 protein by 57.1% (p=0.03), and the number of TUNEL positive cells in the ONL of detached retinas by 56.3% (p=0.03), demonstrating the anti-apoptotic effect of TF in the mouse RD model (Fig. 4F). RIP kinase protein levels were reduced by 35.4% in the TG detached retina compared with the WT (p=0.03) (Fig. 4G) suggesting that hTF reduced both apoptotic and necrotic cell death in the mouse RD model.

Molecular signature of hTF overexpression in the mouse retinal detachment model

We performed a genome-wide analysis (RNA-Seq) of neural retinas from TG and WT mice 4 days after induction of retinal detachment. From a total of 412 genes differentially regulated between TG and WT mice (1.5 fold-change, p≤0.05), 149 were up-regulated and 263 were down-regulated. An extensive list is provided in data File S1. The 20 most up-and down-regulated genes are listed in table S1. Gene ontology (GO) (biological process, molecular function, cellular compartments categories) and reactome pathways enrichment analysis demonstrated significantly affected categories in genes down-regulated or up-regulated in TG mice as compared with WT (Fig. 5; see data File S1 for details). We regrouped the significantly enriched pathways into 10 groups according to their biological functions: Retina homeostasis, Neurological process, Phagocytosis, Immune system, Remodeling of extracellular matrix (ECM), Mechanisms of cell death, Metabolism, DNA/RNA regulation, Intracellular signaling, and others, which comprise other biological functions such as cellular proliferation and differentiation (Fig. 5). The Retina homeostasis group was solely composed with the most significantly enriched GO term: "Photoreceptor cell maintenance" which included genes related to the connector cilium linking the inner segment to the outer segment and playing a major role in rhodopsin transport toward outer segments disks. Pathways relative to the Neurological process group were especially involved in the regulation of neuron development and cognition. The Phagocytosis group was composed of the most significant biological process according to the reactome analysis ("ER-phagosome pathway") that was also represented in GO analysis associated with the "Receptor-mediated endocytosis" and the "Regulation of vesicle fusion" pathways. Interestingly, the GO molecular function analysis revealed that "binding" was the most significantly enriched term (16 of a total of 29 terms) (data File S1). Furthermore, of the top 20 of genes up-and down-regulated, half of these were associated with "protein binding" Molecular Function (GO term classification) (table S1). Pathways regrouped in the Immune system group were also highly represented in the two enrichment analyses, and genes included in those were mostly upregulated. Pathways related to the Mechanism of cell death group were represented by two GO biological process terms involved in the regulation of apoptotic processes. In the 40 most significantly regulated genes, 12 were implied in cell death processes (table S1). The GO analysis of the cellular components showed 19 significant enrichments which can be regrouped in 3 equal cellular compartments: the nucleus, the cytoplasm, and membranes (data File S1), reflecting cross talk between the cell membrane and the nucleus.

Among the 412 genes differentially regulated between TG and WT mice, 2 genes have direct interactions with TF, Tfrc and Igfbp3 [START_REF] Miljuš | IGFBP-3/transferrin/transferrin receptor 1 complexes as principal mediators of IGFBP-3 delivery to colon cells in non-cancer and cancer tissues[END_REF]. The insulin-like growth factor binding protein 3 (Igfbp3) transcript was significantly increased in TG mouse retina compared with WT 4 days after RD induction (fold-change: 3.27, p=0.086, table S1). In the normal retina, the IGFBP3 protein was localized in PR segments (arrowhead), in retinal capillaries and in the outer plexiform layer (arrow) (fig. S7A). Western blots analysis confirmed an increase of 46.7% of IGFBP3 protein level in the retinas of TG mice compared with WT mice 7 days after RD (p=0.028) (fig. S7B).

Local ocular injection of transferrin preserves detached rat retinas in vivo

In a therapeutic perspective trial, intra-vitreous injection of hTF (50 mg/ml) showed protective effects in a rat RD model (Fig. 6A). The retinal thickness of the detached retina/non-detached retina (Fig. 6A, brackets and Fig 6B, upper graph) was 14.2% lower in TF-treated rats (p=0.003), whereas the ONL thickness was preserved (30.6% reduction, p=0.013, Fig. 6B, middle graph), and immunostaining of rods and cones PRs (Fig. 6C) revealed preserved morphology. There was a 37.6% reduction of outer segment length (arrowhead, Fig. 6B andC) in BSS RD rats compared to TF-treated rats (p=0.004, Fig.

6B, lower graph)

. Therapeutic TF thus reduced edema of the detached retina and preserved PR morphology. Taken together, these results indicate that TF, even if administrated at the time of RD, can prevent further retinal alterations and cell death.

Discussion

Iron accumulation-induced oxidative stress has been observed in neurodegenerative and retinal diseases [START_REF] Eid | Iron mediated toxicity and programmed cell death: A review and a re-examination of existing paradigms[END_REF] and causes rapid PR death [START_REF] He | Iron homeostasis and toxicity in retinal degeneration[END_REF], but no correlation between intraocular iron and visual function has been demonstrated in a human disease. In our study, we show for the first time in human eyes increased iron levels and TF saturation in the vitreous and SRF fluid, reflecting the accumulation of free iron in ocular tissues (RPE, PRs and inflammatory cells like) upon retinal detachment. Our results confirmed previous analyses which demonstrate increased oxidative stress in vitreous from RRD patients [START_REF] Cederlund | Vitreous levels of oxidative stress biomarkers and the radical-scavenger α1microglobulin/A1M in human rhegmatogenous retinal detachment[END_REF].

Increased iron following retinal detachment could result from a combination of factors. In physiologic conditions, retinal iron entry is actively controlled through TF uptake by its receptor at the RPE basal membrane that forms the outer retinal barrier. After detachment, acute RPE alteration (6) may favor free iron entry. Similarly, a breakdown of the inner blood-retinal barrier in an animal model of type 2 macular telangiectasia, a rare human disease, led to iron accumulation in the retina and PR degeneration [START_REF] Baumann | Conditional Müller Cell Ablation Leads to Retinal Iron Accumulation[END_REF].

However, iron accumulation was also shown in retinal dystrophies, especially in PRs, without obvious barrier breakdowns [START_REF] Jeanny | Iron and regulatory proteins in the normal and pathological retina[END_REF] suggesting that other mechanisms contribute to iron increases. PRs and RPE have high iron content because it is a cofactor in phototransduction enzymes and have a major role in iron retinal homeostasis [START_REF] Jeanny | Iron and regulatory proteins in the normal and pathological retina[END_REF]. RPE cells, through the phagocytosis of shed outer segments, recycles iron and exports it from the retina [START_REF] Jeanny | Iron and regulatory proteins in the normal and pathological retina[END_REF]. During RD, the loss of contact between PR and RPE leads to intracellular iron accumulation, a mechanism described in the RCS dystrophic rat, a genetic model of defective outer segment phagocytosis [START_REF] Jeanny | Iron and regulatory proteins in the normal and pathological retina[END_REF]. PRs are extremely sensitive to iron excess, but the exact mechanisms of this toxicity have not been fully determined. We used organotypic retinal cultures to show that direct contact with iron causes rapid PR necrosis. Peroxidation by iron of polyunsaturated fatty acids (PUFAs) in the outer segments could be one necrotic mechanism [START_REF] Guajardo | Lipid-protein modifications during ascorbate-Fe2+ peroxidation of photoreceptor membranes: protective effect of melatonin[END_REF]. Furthermore, when iron has accumulated in the retina explant and after its removal from medium, apoptotic cell death observed could result from intracellular molecular iron-induced oxidation through the Fenton/Haber-Weiss reaction cycle (DNA, RNA, lipids, proteins) (9), as suggested by DNA breaks (8-HGN) and lipid peroxidation (4-HNE) (Fig. S4). Thus, labile iron exerts deleterious extracellular and delayed intracellular effects. The fact that vision recovery in patients with RRD was inversely correlated with ocular iron levels suggests that iron toxicity could be one of the mechanisms of PR loss over the time course of RRD.

The observation of fully saturated TF in ocular fluids and of intracellular iron accumulation in the retina both suggest that iron loading capacities are saturated. Therefore, the use of iron appears as a relevant therapeutic therapy. Chemical chelators (deferiprone, deferoxamine, or deferasirox) have already been evaluated in neurogenerative diseases after systemic administration [START_REF] Devos | Targeting chelatable iron as a therapeutic modality in Parkinson's disease[END_REF]. However, long-term iron chelation therapy, routinely used for instance in beta-thalassemia and chronic iron overload diseases impacts negatively patient's quality of life, and induced ocular toxicity, including cataracts, maculopathy, and optic neuritis [START_REF] Parakh | Neurological Complications and Cataract in a Child With Thalassemia Major Treated With Deferiprone[END_REF][START_REF] Di Nicola | Functional and Structural Abnormalities in Deferoxamine Retinopathy: A Review of the Literature[END_REF]. Thus, the use of such chemical chelators in RD patients without systemic iron overload could be deleterious for the eye. Moreover, intraocular local administration of chemical iron chelators would require repeated intravitreal injections due to their short half-life [START_REF] Mobarra | A Review on Iron Chelators in Treatment of Iron Overload Syndromes[END_REF]. Such local administration might cause excessive intracellular iron depletion due to high chelator to iron ratio.

Based on these finding, we proposed to use local ocular TF to neutralize iron toxicity. We previously showed that TF is safe for ocular use even at high doses, prevents iron accumulation in RPE and PRs and that iron-loaded (holoTF) as well as iron-free TF egresses the retina through the TF receptor at RPE apical side [START_REF] Picard | Targeting iron-mediated retinal degeneration by local delivery of transferrin[END_REF]. Using various models of retinal degeneration, we have previously showed that apoTF has neuroprotective effect [START_REF] Picard | Targeting iron-mediated retinal degeneration by local delivery of transferrin[END_REF][START_REF] Picard | Overexpressed or intraperitoneally injected human transferrin prevents photoreceptor degeneration in rd10 mice[END_REF], but involved mechanisms have not been yet fully elucidated. In this paper, using two RD models, we confirmed that apoTF efficiently removes iron, and exerts anti-oxidant and neuroprotective effect. However, recent studies have demonstrated that the blockade of apoptosis or necroptosis was required to protect the detached retina [START_REF] Trichonas | Receptor interacting protein kinases mediate retinal detachment-induced photoreceptor necrosis and compensate for inhibition of apoptosis[END_REF]. Using transgenic mice that express human TF in the retina, or using therapeutic regimens with exogenous TF, we demonstrated that TF reduced both the direct iron-mediated necroptosis through RIP kinase down-regulation, as well as the delayed iron-induced apoptosis.

The biologic effects of TF, present at high concentration in the normal vitreous, is not limited to iron metabolism and its neuroprotective properties result from numerous targeted pathways. Lesnikov 27) and vesicle trafficking [START_REF] Longatti | TBC1D14 regulates autophagosome formation via Rab11-and ULK1-positive recycling endosomes[END_REF]. Amongst the numerous pathways involved in neuroprotection regulated by TF (table S2), the IGFBP3 pathway is of particular interest. The upregulation of IGFBP3 by TF was confirmed at the protein level, and Figure S8 illustrates IGFBP3 pathways and the genes in this pathway that were up-(red underlined) or down-(green underlined) regulated by TF. The main role of IGFBP3 is to regulate the various functions of IGFs [START_REF] Johnson | IGFBP-3: A cell fate pivot in cancer and disease[END_REF]. Interestingly, when iron levels are high, TF forms complexes with IGFBP3 [START_REF] Miljuš | IGFBP-3/transferrin/transferrin receptor 1 complexes as principal mediators of IGFBP-3 delivery to colon cells in non-cancer and cancer tissues[END_REF], preventing binding to its receptors and subsequent apoptosis and growth inhibition [START_REF] Johnson | IGFBP-3: A cell fate pivot in cancer and disease[END_REF]. IGFBP3-TF complex internalization allows IGFBP3 to be released to cellular organelles [START_REF] Storch | Transferrin binds insulin-like growth factors and affects binding properties of insulin-like growth factor binding protein-3[END_REF] and mediates neuroprotective effects [START_REF] Johnson | IGFBP-3: A cell fate pivot in cancer and disease[END_REF][START_REF] Baxter | Nuclear Actions of Insulin-like Growth Factor Binding Protein-3[END_REF][START_REF] Lee | Cellular internalization of insulin-like growth factor binding protein-3: distinct endocytic pathways facilitate re-uptake and nuclear localization[END_REF] (see Fig. S8 for more details).

From the analysis of ocular fluids of RRD patients, we have demonstrated for the first time the oversaturation of TF and iron overload in a human ocular disease. Despite a high TF concentration in the vitreous, iron accumulates in the retina in other human diseases as observed in Age-related macular degeneration (AMD) [START_REF] He | Iron homeostasis and toxicity in retinal degeneration[END_REF]; thus, TF over-saturation could be a general mechanism of PR cell death.

Therefore, TF supplementation is a credible strategy for further translational clinical trials in eligible patients. As compared with other iron chelators, TF treatment not only restores iron homeostasis but also activates major neuroprotective pathways such as the IGFBP3 pathway. Future work elucidating the iron-unrelated pathways of TF will be required. We have used retinal detachment as a "model disease" in which neuroprotective treatments could show functional endpoints in a reasonable time frame. On the basis of the results obtained from experimental models and from human pathology, and because TF is an endogenous molecule, clinical studies should be performed to evaluate TF in conjunction with retinal detachment surgery. Once validated, TF could be proposed as a treatment for other diseases associated with retinal detachment, such as in myopia, or those associated with iron overload such as AMD [START_REF] He | Iron homeostasis and toxicity in retinal degeneration[END_REF].

Materials and Methods

Human samples

Consecutive patients presenting with RRD at Jules-Gonin Eye Hospital (Lausanne, Switzerland) from December 1, 2014, to July 31, 2015, were included according to the protocol approved by the ethics committee and diagnosed through normal standard clinical care at first examination. Upon presentation with RRD, the main inclusion criterion was the necessity of performing intraocular surgery to limit visual impairment, in the absence of other ocular conditions. Among 44 vitreous samples, 35 samples were obtained from patients with macula OFF RD. Nine sex-matched and age-matched patients presented with macular holes (n=4) or with epiretinal membranes (n=5), and no other ocular disease were included during the same period as control subjects for vitreous humor sampling.

Undiluted vitreous samples were collected by manual aspiration through a syringe connected to a 23G vitrectomy cutter (Alcon, Rotkreuz ZG, Switzerland before infusion opening). SRF was collected by active aspiration through a retinal tear (Advanced Backflush DSP cannula, Alcon, Rotkreuz ZG, Switzerland). Samples were coded and stored in a biobank at -80°C until biochemical analysis.

Duration of RRD (mean: 6.7 ± 5.9 days) was defined as the time from the onset of symptoms to surgery.

Visual acuities were recorded before and one month after surgery and converted to the logarithm of the minimum angle of resolution (LogMAR). Visual recovery was defined as the difference between postoperative and pre-operative LogMAR visual acuity.

Frozen human post-mortem eye tissues (10 donors) with non-hemorrhagic retinal detachment retrieved from the pathological files of Jules-Gonin Eye Pathology Laboratory were used to stained iron by Perl's method. A block of tissue ranging from 1 to 1.5 cm long and 0.5 cm wide containing the RD was processed as serial 5 µm-thick paraffin sections.

Animals

Adult male Long-Evans rats (Janvier laboratory, Le Genest St Isle, France) (n=32), adult male Wistar rats (Janvier laboratory) (n=21), adult male C57BL6/J mice (Wild-type (WT)) (Janvier laboratory) (n=40), and adult male transgenic mice expressing human transferrin (TG) (Centre d'exploration fonctionnelle du Centre de Recherche des Cordeliers, UMRS1138, Paris France) (n=40) were fed with a standard laboratory diet and ad libitum tap water in a room maintained at 21-23°C with a 12 h light/dark cycle (6 am-6 pm). Mice were anesthetized with an intraperitoneal injection of ketamine (80 mg/kg; Virbac, Carros, France) and xylazine (12 mg/kg; Bayer, Lyon, France); rats were anesthetized with an intramuscular injection of ketamine (35 mg/kg) and xylazine (5 mg/kg). All animals were sacrificed by carbon dioxide inhalation.

Surgical Induction of RD in rodents and study design

Features of retinal degeneration secondary to detachment were observed in animal models in which the retina is mechanically detached by the subretinal injection of sodium hyaluronate. Once anesthetized Long-Evans rats or WT and TG mice received topical anesthesia with a drop of 1% tetracaïne (Faure, Europhta laboratories, Monaco). Pupil dilatation was obtained by dropwise instillation of 2 mg/0.4 ml mydriaticum (Théa, Clermont-Ferrand, France) and 10% phenylephrine hydrochloride (Faure, Europhta laboratories, Monaco). Retinal detachment was induced by a subretinal injection of 10 µl (mice) or 30 µl (rats) of sodium hyaluronate (5 mg/ml, Healon, Uppsala, Sweden) under an operating microscope using a 34G needle through the sclera for a total of 50 animals. The induced retinal detachment occupied approximately 30-70% of the fundus. Eyes with hemorrhagic complications were excluded (nearly 15%). Eyes receiving no injection were used as controls.

Intravitreal injections in rats was performed immediately after RD induction with 5 µl of human apo transferrin solution (50 mg/ml in Balanced Salt Solution (BSS), Sigma Aldrich Chemical Co, Saint-Quentin en Yvelines, France) or control solution (BSS) as previously described [START_REF] Picard | Targeting iron-mediated retinal degeneration by local delivery of transferrin[END_REF].

Fundus and Spectral-Domain Optical coherence tomography (SD-OCT) were simultaneously realized with a Micron III retinal imaging microscope (Phoenix Research Labs, Pleasanton, CA, USA) in anesthetized animals as described, (12) before and after RD induction and 4 or 7 days later. At 4 days, some mouse eyes were collected for RNA sequencing (n=4 eyes per group). At 7 days, eyes from rats and mice were collected for histology and immunostaining (n=5 and 7 per group and per analysis for rats and mice, respectively). At 14 days, (5 per group) rat eyes were collected for assay of iron in ocular fluids.

Histology and thickness layers measurement

Fixed eyes were embedded in historesin (Leica, Nanterre, France) to facilitate the cutting of 5 µm-thick sections through the detached area. Sections were stained with 1% Toluidine Blue solution. Sections were observed on a Leitz microscope. Sections with the most detached retinal area were photographed (20X) with a Leica camera for measurements (n=5 WT mice; n=5 TG mice; n=5 BSS-treated rats; n=5 TF-treated rats). Retinal thicknesses (from the outer segments to the inner part of the ganglion cell layer nuclei), the outer nuclear layer (ONL), and the outer and inner segments were measured every 100 µm (mice) and 300 µm (rats) using Image J software by two different blind experimenters. Retinas were considered detached when an empty space was observed between the RPE and the outer segments and attached when any signs of histological disturbance was observed. The retinal thickness ratio was calculated as the thickness in the detached retina/ the thickness in the attached retina. For the ONL and segment thickness ratio, layer measurements were reported as the total retinal thickness of the corresponding retinal area using the formula "(ONL detached/total retina detached)/(ONL attached/total retina attached)".

Immunohistochemistry and fluorescence intensity evaluation

Fixed eyes were mounted in Tissue Tek® O.C.T. (Siemens Medical, Puteaux, France) in order to pass through the detached area. Explants were rinsed in 1X phosphate-buffered saline (PBS, Thermo scientific, Cergy Pontoise, France), fixed for 20 min with 4% paraformaldehyde (PAF, Inland Europe, Conflans sur Lanterne, France), infiltrated in a sucrose gradient series and then, mounted in Tissue Tek O.C.T. Immunohistochemistry was performed on 10 µm-thick sections as previously described [START_REF] Picard | Targeting iron-mediated retinal degeneration by local delivery of transferrin[END_REF]. Primary antibodies were: rabbit polyclonal anti-TF receptor 1 (Serotec, Oxford, UK), rabbit polyclonal specific for the Light subunit of ferritin (P. Santambrogio); rabbit anti-glial fibrillary acid protein (Dako, Thermo Scientific); rabbit anti-ionized calcium-binding adapter molecule 1 (Iba1, Wako Pure Chemical Industries, Neuss, Germany); mouse anti-CD68 (Bio-Rad AbD Serotec GmbH, Colmar, Fr); anti-8 Hydroxyguanosine (8-HGN, Abcam, Cambridge, UK); anti-4 Hydroxynonenal (4-HNE, Abcam); antirhodopsin (Rho4D2, Abcam), anti-cone arrestin (Millipore, Saint Quentin En Yvelines, Fr), and anti-IGFBP3 (Santa Cruz). Control sections were incubated with rabbit non-immune serum (Thermo scientific) or without primary antibodies. The corresponding Alexa-conjugated secondary antibodies (Thermo scientific) were used to reveal the primary antibodies, and sections were counterstained with 4.6-diamidino-2-phenylindole DAPI; Sigma). The terminal deoxynucleotidyl transferase-mediated biotinylated UTP nick end labeling (TUNEL) reaction was performed, along with DAPI staining using the manufacturer's protocol (Roche Diagnostics, Meylan, France) as described [START_REF] Picard | Targeting iron-mediated retinal degeneration by local delivery of transferrin[END_REF]. Propidium iodine at 0.5 µg/ml was incubated directly with sections for one hour and the nuclei were counterstained with DAPI.

The sections were photographed with a fluorescence microscope (BX51, Olympus, Rungis, France) using identical exposure parameters for all compared samples. Blind quantifications were realized on photographs acquired at 40x magnification with Image J software as follows: Quantification of cone nuclei (arrestin staining) or TUNEL positives cells (20x magnifications) in mouse (n=5 per group) or rat (n=5 per group) models of RD was performed using 3 photographs of the attached retina and 3 photographs of the detached retina. Measurement of the outer segment length in rat retina (n=5 per group) was realized in 6 photographs by delimiting Rho4D2 staining and calculating the mean OS length in each retina area. The OS length ratio was then calculated as described for histological measurement.

For the explants model, all quantifications were performed on 4 photographs (n=3 explants per group).

The OS length measurement was realized by measuring at 3 equidistant points on the photographs.

Fluorescence intensity staining was evaluated on photographs and reported as the mean intensity under control conditions. Each sample measurement was averaged and differences between the groups were analyzed.

Retinal explant cultures

Adult Wistar rat retinas were isolated from fresh enucleated eyes, divided into 4 parts, and transferred to 0.2 mm polycarbonate membranes (Millipore) with the ganglion cell layer facing the membrane (Fig. 2). Mouse retinal explants were isolated as indicated above, with one explant corresponding to one eye.

The insert was placed into a six-well culture plate containing Dulbecco's modified Eagles medium with L-glutamine and 5% serum (Thermo Scientific) corresponding to day 0. To analyze the effects of iron exposure on retinal explants, 30 µl of medium containing FeSO4 (7H2O, Sigma) was added to the photoreceptor side of the explants (upper chamber). To explore transferrin's therapeutic effects, medium in the upper chamber was removed after 2 days; PBS was added for washing, and 30 µl of medium containing 0.5, 5 or 50 mg/ml of human apoTF (Sigma) was placed on the retinas. LDH release and ELISA directed against TF were measured in medium collected from the lower chamber as described previously [START_REF] Picard | Targeting iron-mediated retinal degeneration by local delivery of transferrin[END_REF][START_REF] Picard | Overexpressed or intraperitoneally injected human transferrin prevents photoreceptor degeneration in rd10 mice[END_REF]. Histology, iron staining, immunohistochemistry, and western blotting were performed on explants as described below and in the time workflow legends.

Iron status

Vitreous and SRF samples from humans, ocular fluids from RD rats, and medium from rat explants (n=6 per group) were diluted with 1% HNO3 solution containing 10 ng/mL Rh and 10 ng/mL indium as internal standards. In addition, each analytical batch of study samples was processed with laboratory controls, including method blanks and standard reference materials to continuously monitor method performance. Samples were analyzed using an inductively coupled plasma mass spectrometer (ICP-MS, 7700 Series, Agilent, Santa Clara, CA, USA). Quantitative analysis of iron (Fe) was carried out by external calibration using 7 standards, with concentrations ranging from 10 to 1.000 ng/ml. The method was assessed using internal and external quality controls, analysis of initial calibration, a verification standard, procedural blanks, and duplicate samples.

Transferrin and proteins were quantified using an ELISA kit (AssayPro, St-Charles, USA) and a Micro BCA assay kit (Thermo Scientific), respectively. For human samples, heme iron contamination was verified with a hemoglobin colorimetric assay (AssayPro). Saturation of transferrin was calculated as the total iron content (µmol/l)/ TIBC (µmol/l). TIBC measures the amount of iron that can potentially be stored in TF and corresponds to the capacity of 1 mol of TF (80,000 Da) to bind 2 mol of ferric iron and was calculated (µmol/l) as: TF concentration (g/l) X 10^6 X (2/80,000).

Iron detection-staining

Perl's reaction staining was performed by incubating in a solution containing 4% potassium ferrocyanide (Sigma) in 4% aqueous hydrochloric acid (Sigma) for 30 min at room temperature to yield a Prussian blue reaction product. Sensitivity for iron detection was enhanced by incubation with a solution of 5 mg/ml 3,3'-Diaminobenzidine Tetrahydrochloride (DAB, Sigma) containing 1% H202 for 30 min at room temperature. For nucleus counterstaining, incubation with a solution of 1% nuclear red or toluidine blue (Sigma) was performed for 30 s. Stained sections were mounted in aqueous medium. Control of amplification was realized by incubation of sections with DAB without the preceding Perl's reaction step.

Imaging LA-ICP-MS measurements were performed on a quadrupole instrument (Agilent 7700 ICP series, Darmstadt, Germany) hyphenated to a laser ablation system (NWR-213, New Wave, Fremont, USA). Material on tissue sections was ablated by a Nd:YAG/213 nm laser focused at 18 µm diameter prior to transport towards the ICP ion source by a helium carrier gas with a flow-rate of 800 mL/min. The LA-ICP MS parameters were optimized to provide the best signal-to-noise ratio while keeping the maximum reproducibility between analyses. In this way, the energy output was set at 23%, corresponding to a laser fluency of 0.35 J/cm 2 . The lateral resolution was fixed at 20 µm in the raster mode with a scan speed and a repetition frequency of 20 Hz and 20 µm/sec, respectively. 56 Fe + was monitored for a duty cycle of 1 s. Raw data were acquired using MassHunter software (Agilent, Darmstadt, Germany). Data files (.d) of the IMS experiments were successively converted in mzML and imzML formats using msconvert and imzMLConverter tools, respectively. Ion images were reconstructed and visualized using the MSIReader v0.06 software.

Western blot analysis

Explants (n=4 per group) were suspended in lysis MPER buffer (Thermo Scientific) and then centrifuged for 10 min at 4°C. Protein concentrations were calculated using the Micro BCA protein assay (Thermo Scientific). Five to ten µg of total extract was mixed with protein loading buffer (Thermo Scientific) per manufacturer's instructions. Samples were loaded on 4-12% Bis-Tris gel (Thermo Scientific) and proteins were transferred onto nitrocellulose membranes. Non-specific binding was blocked with 5% non-fat dry milk in Tween/Tris-buffered saline, and then membranes were incubated overnight at 4 °C with the primary antibody against Rhodopsin (1:5,000; Abcam (ab98887)), human TF (1:1,000; Abcam (ab88165), IGFBP3 (clone E9, 1:1,000, Santa Cruz), caspase 3 (clone L-18, 1:500, Santa Cruz), Bcl2 (clone N-19, 1:500, Santa Cruz), RIP (clone 38, 1:500, BD transduction Laboratories), RIP3 (1:1,000, Sigma (PRS2283)), LC3B (1:1,000, Cell Signaling (2775)), or actin (1:4,000, Sigma), followed by incubation with the supplier-recommended dilution of HRP-conjugated secondary antibody for one hour (VectorLabs, Eurobio, Les Ulis, Fr). Protein bands were visualized by an enhanced chemiluminescence reaction (Thermo Scientific) using a BioImaging system (MicroChemi 4.2, Berthold, Fr). The gray values of specific bands were analyzed using Image J and the protein signals of interest were reported relative to the actin signal or to their respective cleaved forms (for caspase 3 and RIP) for each sample.

Real-time RT-PCR

Neural retinas were isolated on ice and directly frozen until RNA isolation. Tissues were homogenized in tubes prefilled with beads (Bertin Technologies, Ozyme, Saint Quentin en Yvelines, Fr) containing RLT lysis buffer (RNeasy mini kit, Qiagen, Courtaboeuf, Fr) for 3 cycles of 30 s at 5,500 rpm in a Precellys Evolution homogenizer (Bertin Technologies). Total RNA was isolated with a RNeasy mini kit (Qiagen, Courtaboeuf, France) according to the manufacturer's protocols. RNA concentration, purity, and integrity were determined with an Agilent Bio-analyzer. All RNA used had an RNA integrity number superior to 8. First-strand cDNA was generated by reverse transcription using 88 ng total RNA and a QuantiTect Reverse Transcription Kit (Qiagen). Quantitative PCR using duplicate technical replicates was performed on a Quant Studio 5 PCR System (Thermo Scientific) using SYBR Green gene expression assay probes (QuantiTech Primer Assay, Qiagen) of the mouse target genes Caspase 8 (Casp8, NM_009812; QT00171437), Insulin-like growth factor binding protein 3 (Igfbp3, NM_0008343; QT00493332), an endogenous control Actin b (Actb, NM_007393; QT01136772), and QuantiFast SYBR Green PCR Master Mix (Thermo Scientific). The expression levels of individual genes were normalized with Actb in the same sample by calculation of the ΔCt value, and relative quantification was performed using the ΔΔCt method with NLE retina serving as a control.

RNA sequencing

Retinas (n=4 per group) were frozen immediately after isolation. Total RNA was extracted using a Precellys Homogenizer (Ozyme, Bertin) and an RNeasy Mini Kit (Qiagen). Samples were sent for sequencing to the iGenSeq transcriptomic platform of the Brain and Spine Institute (Paris, France). The RNA quality was evaluated by capillary electrophoresis (Agilent 2100 Bioanalyzer system) and RNA with integrity numbers (RIN) ranging from 7.8 to 8.2. was accepted for library generation. The cDNA library of each sample was prepared with a KAPA mRNA Hyper Prep (Roche) for 75 bp paired-end reads, according to the manufacturer's instructions. Each of the cDNA libraries was indexed for multiplexing (2x60 million reads/sample), and indexed libraries were sequenced on one lane of the Illumina Nextseq 500 device. Data were recorded in the FASTQ format.

RNA-seq data analysis RNA-Seq data analysis was performed by GenoSplice technology (www.genosplice.com). Sequencing, data quality, reads repartition (e.g., for potential ribosomal contamination), and insert size estimation were performed using FastQC, Picard-Tools, Samtools and rseqc. Reads were mapped using STARv2.4.0 on the mm10 Mouse genome assembly. A gene expression regulation study was performed as previously described [START_REF] Noli | Discordant Growth of Monozygotic Twins Starts at the Blastocyst Stage: A Case Study[END_REF]. Briefly, for each gene present in the Mouse FAST DB v2016_1 annotations, reads aligning on constitutive regions (that are not prone to alternative splicing) were counted. Based on these read counts, normalization and differential gene expression were performed using DESeq2 on R (v.3.2.5). Only genes expressed in at least one of the two compared experimental conditions were further analyzed. Genes were considered as expressed if their rpkm value was greater than 93% of the background rpkm value, based on intergenic regions. Results were considered statistically significant for uncorrected p-values ≤ 0.05 and fold-changes ≥ 1.5. GenBank accession number: GSE102824. Pathway/Gene Ontology (GO) analysis and Transcription Factor analysis Clusterings and heatmaps have been performed on unlabeled data using "dist" and "hclust" functions in R, using Euclidean distance and the Ward agglomeration method. Analysis for enriched GO terms, KEGG pathways and REACTOME pathways were performed using the DAVID Functional Annotation Tool (v6.8). GO terms and pathways were considered enriched if the fold enrichment ≥ 2.0, the uncorrected p-value ≤ 0.05, and the minimum number of regulated genes in the pathway/term ≥ 2.0.

Analysis was performed three times: using all regulated genes, using up-regulated genes, and using down-regulated genes only. The union of these three analyses was performed to provide a single list of results. Analysis for transcription factors was performed using orthologous genes between the mouse and the human with the DAVID Functional Annotation Tool (v6.8). Orthologous genes are aggregated in FAST DB from Ensembl compara. The same thresholds and methods were used as GO terms and Pathway analysis but with human data.

Statistical analysis

Results are presented as the mean ± standard error of the mean (SEM). Analyses were performed using GraphPad Prism 5 software. Normal distribution of data was confirmed using the Shapiro-Wilk test.

Comparisons between 2 groups of human ocular fluid samples were analyzed by an unpaired two-tailed Student's t-test. Multiples comparisons were performed using one-way analysis of variance (ANOVA) followed by the Bonferroni post-test as appropriate. Non-normally distributed data were analyzed using a nonparametric Mann Whitney test to compare the two groups. Unadjusted linear regression and twotailed Pearson correlation analysis were performed to determine whether a relationship existed between iron concentration and delta logMAR visual acuity or RD duration. p<0.05 was considered statistically significant. Over-representation pathway analysis using gene ontology (GO) terms enrichment (biological process clustering) (A) and reactome biosource (B). Color codes were used to associate pathways with common biological effects. Comparatives analysis were done in TG versus WT retinas 4 days after retinal detachment (n=4). GO terms and pathways were considered as enriched if fold enrichment ≥ 2.0, uncorrected p-value ≤ 0.05, and the minimum number of regulated genes in pathway/term ≥ 2.0.

Figures and Tables

  A to C). TG-derived explants exhibited fewer signs of degeneration and oxidative stress in low-serum medium compared with WT explants. After 6 days of culture, retinal glial Müller cells activation, identified by glial fibrillary acidic protein (GFAP) distribution (16) (fig. S2D, arrow), and markers of oxidative stress (8-hydroxyguanosine [8-HGN] and 4-hydroxynonenal [4-HNE]) (fig. S2 E and F) were reduced in retinal explants from TG mice, compared with those from WT mice.

  , p=0.028), increased the anti-apoptotic protein Bcl2 by 83.3% (Fig. 3I, p=0.028), and reduced the number of TUNEL positive cells (Fig 3J). As expected, LFt staining intensity was reduced (Fig. 3K), in parallel to the reduction of iron deposits in the retina. Exogenous hTF prevented the activation of resident macrophages/microglial cells (fig. S4A, CD68, arrowheads), and decreased Müller glial cell activation (fig. S4B, GFAP, arrows). The iron-induced oxidative stress markers 4-HNE and 8-HGN were also decreased with hTF treatment (fig. S4 C and D). Taken together, these results demonstrate that treatment with exogenous TF efficiently protects the retina from two types of iron-induced toxicity: direct and early necroptosis and delayed apoptosis that could result from secondary iron-related effects.

  et al. previously demonstrated that upon binding to the Transferrin receptor 2 (TFR2), TF protected against Fas-induced cell death via the mitochondrial pathway by reducing BID cleavage, inhibiting caspase-3 and -9 activations, and up-regulating survival signals such as Bcl-xL (26). The retinal transcriptome of TG RD mice compared with WT RD mice highlighted 7 major groups of pathways regulated by TF: Phagocytosis, Immune systems, Retina homeostasis, Neurological process, Remodeling of the extracellular matrix, Mechanisms of cell death, and Metabolism. TF up-regulated the expression of receptors involved in endocytosis (Cd36, Cd14, Siglech), and down-regulated the expression of vesicle trafficking inhibitors (Tbc1d14, Evi5, Tbc1d30, Usp6nl). As a cargo protein, TF facilitates endocytosis/phagocytosis (

Figure 1 :

 1 Figure 1: Iron accumulates during retinal detachment in humans

Figure 2 :

 2 Figure 2: Iron toxicity for photoreceptors in rat retinal explants

Figure 4 :

 4 Figure 4: Transferrin expression preserves the detached retina in mice

Figure 5 :

 5 Figure 5: High-throughput analyses of the transcriptome of TG mouse retinas following retinal

Table 1 : Top 20 up-and down-regulated genes in TG compared with WT mice following retinal detachment

 1 were reported relative to WT explants. Mann Whitney test (n=3), *p<0.05; **p<0.01; **p<0.001. GCL: Ganglion cell layer; ONL: Outer nuclear layer; INL: Inner nuclear layer; S: Segments. Scale bar: 100 µm. All values are represented as the mean ± SEM. R= Retina.

	Supplementary Gene Symbol	Gene Name	Fold-	P-Value	Entrez
				Change		Gene ID
			Genes up-regulated
	Gm19554		predicted gene, 19554	88,67	1,04E-19 100503099
	Serpina3h	serine (or cysteine) peptidase inhibitor, clade A, member 3H	22,11	6,88E-05	546546
	Snord15a		small nucleolar RNA, C/D box 15A	14,88	3,51E-02	449630
	Gab3	growth factor receptor bound protein 2-associated protein 3	4,83	1,14E-02 100503280
	Lilrb4a	leukocyte immunoglobulin-like receptor, subfamily B, member 4A	4,48	1,38E-03	14728
	Hmox1		heme oxygenase 1	4,28	1,84E-03	15368
	Mmp12		matrix metallopeptidase 12	4,27	1,45E-02	17381
	Glycam1		glycosylation dependent cell adhesion molecule 1	4,19	3,36E-04	14663
	Cym		chymosin	3,87	8,42E-03	229697
	Ms4a6d	membrane-spanning 4-domains, subfamily A, member 6D	3,60	1,49E-02	68774
	Igfbp3		insulin-like growth factor binding protein 3	3,27	5,28E-05	16009
	Fndc1		fibronectin type III domain containing 1	3,20	4,84E-11	68655
	Msr1		macrophage scavenger receptor 1	3,00	1,30E-02	20288
	Cd36		CD36 antigen	2,85	6,88E-07	12491
	Gal		galanin	2,84	1,75E-02	14419
	Tmem221		transmembrane protein 221	2,78	1,74E-03	434325
	Ccl17		chemokine (C-C motif) ligand 17	2,78	1,06E-02	20295
	Sphk1		sphingosine kinase 1	2,75	4,88E-03	20698
	Il2rg		interleukin 2 receptor, gamma chain	2,74	4,94E-03	16186
	H2-Q7		histocompatibility 2, Q region locus 7	2,74	6,11E-06	15018
			Genes down-regulated
	S100a9		S100 calcium binding protein A9 (calgranulin B)	8,34	3,94E-03	20202
	G530011O06Rik		RIKEN cDNA G530011O06 gene	6,38	1,03E-02	--
	Sik1		salt inducible kinase 1	3,73	9,62E-03	17691

Acknowledgments

General:

We thank Drs. Yves Courtois, Alicia Torriglia, Imène Jaadane and Marc Abitbol (UMRS1138, Centre de Recherche des Cordeliers, INSERM) for their guidance, stimulating discussion and manuscript reviews. We thank the Institut du cerveau et de la Moelle épinière -ICM (Plateforme de Séquençage -iGenSeq; Hôpital de la Salpetrière) for RNA sequencing, and Genosplice for data analysis. The authors wish to thank Paolo Santambrogio (Department of Biological and Technological Research, Instituto de Ricovero e Cure a Carattere Scientifico, San Rafaelle, Milan, Italy) for the rabbit polyclonal antibody specific for the High and Light subunits of Ferritin. And we also wish to thank MSGraphique and Sébastien Maroillez) for illustrations.

Funding:

This study was supported by INSERM, ANR JCJC 2015 Transfiron, and NIH P30EY006360, VA RR&D I21RX001924 and C9246C, Research to Prevent Blindness, Inc., and the Abraham J. & Phyllis Katz Foundation. We would like to thank Editage (www.editage.com) for English language editing.

AM: conducting human iron staining experiments.

FBC: designing research studies, conducting experiments, analyzing data, and writing the manuscript.

EP: designing research studies, conducting experiments, acquiring data, analyzing data, and writing the manuscript.

Study approval

The study involving human subjects adhered to the tenets of the Declaration of Helsinki and was approved by the local Ethics Committee of the Swiss Department of Health on research involving human subjects (CER-VD N°340/15). For vitreous and sub-retinal fluids, written informed consent was obtained from all patients after detailed explanation of the purpose and methods of the study. All experimental procedures performed using rats and mice were approved by the local ethics committee of the Charles Darwin European Council, University of Paris-Descartes. Experiments were performed in accordance with the Association for Research in Vision and Ophthalmology (ARVO) statement for the use of animals in Ophthalmic and Vision Research.

H2: Supplementary Materials

References and Notes

Author contributions: AD: conducting experiments and surgeries, acquiring data, analyzing data, and writing the manuscript.

Competing interests:

The authors declare no competing interests.

Data and materials availability:

GenBank accession number: GSE102824.

Supplementary Materials

Supplementary Figure 1

Supplementary Figure 8: Proposed role of IGFBP3 in the protective effect of transferrin

Based on genes up-and down-regulated in transcriptomic analysis, this illustration highlights the cellular protective mechanism involving the IGFBP3-transferrin (TF) interaction. The formation of the IGFBP3-TF complexes may be expected: i) to reduce IGFBP3 binding on its cell-surface specific receptors, IGFBP-3R and low-density lipoprotein receptor-related protein-1 (LRP1), and inhibits caspase-dependent apoptosis and growth inhibition through the dephosphorization of the insulin receptor substrate (IRS) by a Ser/Thr-specific protein phosphatase. ii) to enhance the interaction of IGFBP3 with multiple extracellular components including glycosaminoglycan (GAG) or receptors for its cellular uptake. IGFBP3 binding to the extracellular matrix promotes sphingosine-1-phosphate (S1P) production by inhibiting sphingomyelinase (SMase) and increasing sphingosine-1-phosphatase (SphK1). S1P interaction with S1PR receptors leads to multi pathways like preservation of blood barrier integrity, and also transactivation of EGFR leading to a caveolin-mediated endocytosis of the complex EGFR-IGFBP3. The IGFBP3-TF complex is taken up by the TF 1 receptor and internalized by a clathrin endocytosis vesicle. IGFBP3 and its binding partners (TF or EGFR) may be then found in the late endosome/multivesicular body for redistribution of molecules in the cell. iii) Nuclear import of IGFBP3 is mediated by importin beta, where it can form nuclear complexes. Phosphorylation of nuclear IGFBP3 by DNA-PKcs, resulting in activation of IGBP3-EGFR-DNA-PK complexes and favoring double-strand DNA break repair by non-homologous end-joining (NHEJ). The IGFBP3-RXR alpha interaction increased RXRalpha homodimer transcriptional activity whereas it prevented formation of the RXRalpha-RARalpha, and RXRalpha-PPRAgamma-RXRalpha-Nur77 heterodimers. The endoplasmic reticulum (ER) is one of the probable sources of the autophagosomal double membrane in mammals, and the ER-resident chaperone GPR78 is required for stress-induced autophagy. The interaction IGFBP3 with GPR78 (iv) stimulates the autophagic survival response in case of nutrient deprivation or hypoxia.

(for literature review [START_REF] Johnson | IGFBP-3: A cell fate pivot in cancer and disease[END_REF][START_REF] Baxter | Nuclear Actions of Insulin-like Growth Factor Binding Protein-3[END_REF][START_REF] Lee | Cellular internalization of insulin-like growth factor binding protein-3: distinct endocytic pathways facilitate re-uptake and nuclear localization[END_REF])

Supplementary Table 2: Pathways and genes regulated by Transferrin and implicated in photoreceptors rescues

Data files S1: Complete transcriptomic analysis

The file is composed with a list of the regulated genes, the GO analysis and the analysis of splicing factors and others genes encoding for RNA-binding proteins (transcription Factors)

Pathways

Genes regulated by TF Functions References

Retina homeostasis

Cdhr1, Pcdh15, Mak, Rp1, Ush1g

found in PR cilium for protein trafficking through the connecting cilium [START_REF] Cosgrove | Usher protein functions in hair cells and photoreceptors[END_REF] Tbc1d14, Evi5, Tbc1d30, Usp6nl

negative regulator of Rab proteins family, implicated in rhodopsin transport in cilium photoreceptors [START_REF] Longatti | TBC1D14 regulates autophagosome formation via Rab11-and ULK1-positive recycling endosomes[END_REF] Immune systems Serpina3h, Glycam1 [START_REF] Trichonas | Receptor interacting protein kinases mediate retinal detachment-induced photoreceptor necrosis and compensate for inhibition of apoptosis[END_REF]