Nausicaa Poullet

Ivan Golushko

Vladimir Lorman

Jana Travnickova

Dmitryi Chalin

Sergei Rochal

Andrea Parmeggiani

Karima Kissa
email: karima.kissa-marin@umontpellier.fr

Mechanical instabilities of aorta drive blood stem cell production: a live study

Introduction

Transplantation of human blood cells is essential to regularly save lives on a large-scale.

However, this method requires an exogeneous allogenic source to avoid histoincompatibility as well as graft versus host disease associated problems. Currently, haematopoietic stem/progenitor cells (HSPCs) can only be produced in vitro by methods involving genetic cellular reprogramming. Different groups succeeded in it, however these approaches remain scientifically and technically challenging [START_REF] Lis | Conversion of adult endothelium to immunocompetent haematopoietic stem cells[END_REF][START_REF] Batta | Direct Reprogramming of Murine Fibroblasts to Hematopoietic Progenitor Cells[END_REF][START_REF] Riddell | Reprogramming Committed Murine Blood Cells to Induced Hematopoietic Stem Cells with Defined Factors[END_REF][START_REF] Lancrin | The haemangioblast generates haematopoietic cells through a haemogenic endothelium stage[END_REF] . Moreover, the presence of transgenes in the genome of reprogrammed human HSPCs represents an important clinical risk [START_REF] Ivanovs | Human haematopoietic stem cell development: from the embryo to the dish[END_REF] .

In order to develop new methods to generate HSPCs in vitro and control their fate after transplantation, we need to further deepen our knowledge on blood cells ontogenesis at tissue and organism levels, considering also novel features like biomechanical forces experienced by HSPCs in physiological conditions. Indeed, the 3D in vivo structure of the tissue from which HSPCs are generated is subjected to mechanical forces that influence cellular properties and processes like cell migration, adhesion and polarity [START_REF] Hamill | Molecular Basis of Mechanotransduction in Living Cells[END_REF][START_REF] Eyckmans | A Hitchhiker's Guide to Mechanobiology[END_REF] . Moreover, mechanical stress is known to have a major impact on gene expression modulation and consequently on developmental processes [START_REF] Modesto | Myocardial mechanics in cardiomyopathies[END_REF] , inflammation and cancer [START_REF] Desprat | Tissue Deformation Modulates Twist Expression to Determine Anterior Midgut Differentiation in Drosophila Embryos[END_REF] .

In this article, we address for the first time the question of HSPCs production from the aorta in relationship with the growth of the zebrafish embryo, the most widely used animal model to study developmental processes in real time. We discuss and underline that, together with haemodynamic forces, the growth of the whole embryo generate mechanical stresses on the aorta and play a crucial role in blood production.

Previously, we and colleagues have demonstrated that HSPCs emerge in the Aorta Gonad Mesonophros (AGM) region [START_REF] Gering | Hedgehog Signaling Is Required for Adult Blood Stem Cell Formation in Zebrafish Embryos[END_REF][START_REF] Tavian | Embryonic development of the human hematopoietic system[END_REF][START_REF] Murayama | Tracing hematopoietic precursor migration to successive hematopoietic organs during zebrafish development[END_REF][START_REF] Kissa | Live imaging of emerging hematopoietic stem cells and early thymus colonization[END_REF] , from the ventral wall of the dorsal aorta (DA) [START_REF] Kissa | Blood stem cells emerge from aortic endothelium by a novel type of cell transition[END_REF][START_REF] Bertrand | Haematopoietic stem cells derive directly from aortic endothelium during development[END_REF][START_REF] Robin | Human Placenta Is a Potent Hematopoietic Niche Containing Hematopoietic Stem and Progenitor Cells throughout Development[END_REF] . We named this process Endothelial-to-Haematopoietic Transition or EHT [START_REF] Kissa | Blood stem cells emerge from aortic endothelium by a novel type of cell transition[END_REF] . In zebrafish, EHT takes place during a specific time window between 30 and 65 hours post fertilization (h.p.f.) [START_REF] Murayama | Tracing hematopoietic precursor migration to successive hematopoietic organs during zebrafish development[END_REF][START_REF] Kissa | Blood stem cells emerge from aortic endothelium by a novel type of cell transition[END_REF][START_REF] Kimmel | Stages of embryonic development of the zebrafish[END_REF] Systematic tracking of aortic endothelial cells (EC) in live embryos showed that HSPCs emerge from the aortic ventral floor through a process that involves a strong shape change followed by the egress of single cells from the aortic ventral wall into the sub-aortic space [START_REF] Kissa | Blood stem cells emerge from aortic endothelium by a novel type of cell transition[END_REF] . Moreover, we and colleagues observed that the extrusion of HSPCs was aborted when the runx1 transcription factor essential for HPSCs emergence was inhibited [START_REF] Kissa | Blood stem cells emerge from aortic endothelium by a novel type of cell transition[END_REF][START_REF] Chen | Runx1 is required for the endothelial to haematopoietic cell transition but not thereafter[END_REF] . Surprisingly, this inhibition affected neither aorta formation nor any events preceding HSPCs extrusion, such as aorta radius dilation and contraction. A more recent study has then put in evidence with wealth of details the cytoskeletal processes occurring in the single cell dynamics during the HSPC egress from the aorta [START_REF] Lancino | Anisotropic organization of circumferential actomyosin characterizes hematopoietic stem cells emergence in the zebrafish[END_REF] .

In the present study, with the help of 4D confocal microscopy, we follow and quantify at tissue level, the whole aorta behaviour as well as the one of its cells throughout EHT between 24 and 72 h.p.f. We put in evidence important structural changes of the aorta and the collective migration of its lateral cells down to the aorta floor prior to HSPC egress. These phenomena result in a global aorta remodelling in terms of number and localisation of endothelial cells, thus showing an ongoing global cellular reorganisation of the tissue that assures aorta integrity during EHT. We then analyse the role of the actin cytoskeleton both in emerging and neighbouring cells during the HSPCs extrusion process.

Based on these observations and applying general principles of mechanics to the novel context of EHT, we relate the overall aorta remodelling and EHT egress to mechanical instabilities. This cross-disciplinary analysis indeed reveals that mechanical instabilities, induced by different stresses arising from the inhomogeneous growth of the aorta and its interaction with surrounding growing tissues, play a key non-specific role in HSPCs extrusion. Thus, not only haemodynamic forces, but also stresses induced by the global zebrafish embryonic growth [START_REF] Kimmel | Stages of embryonic development of the zebrafish[END_REF] are essential for haematopoiesis.

Results

EHT is associated with important aorta remodelling. To investigate the mechanisms underlying EHT, we first carried out a 4D confocal microscopy imaging of this process in a physiological context. For that, we image the trunk of zebrafish embryos (Fig. 1a) including diverse structures such as the DA, the cardinal vein (CV), the notochord and the muscles surrounding the DA (Fig. 1a-c) between 30 and 65 h.p.f. The imaging revealed strong changes of the morphology of the whole DA with time (Fig. 1bf, Supplementary Movie S1). From 24 to 42.5 h.p.f., we observed a drastic increase of the aorta diameter from 24±0.8 µm to 32±0.9 µm (Fig. 1c,1g) followed by the emergence of a pattern with alternating thinner and thicker diameter regions (Fig. 1c-d, 1g-h) with a relative amplitude variation ranging from 17% to 33% the average aorta diameter. At 42.5 h.p.f., the average diameter of the DA starts to decrease (Fig. 1f,1g) and at 65 h.p.f., the aorta original cylindrical shape and diameter are restored which corresponds to the end of EHT (Fig. 1d-f, 1g). To determine if there is a causal link between the change in aorta diameter and cell extrusion, we followed the behaviour of individual cells leaving the DA (Fig. 1e-f, arrowheads correspond to regions where HSPCs emerged). Our observations showed that interestingly, HSPC extrusion rate peaks between 42.5 and 52 h.p.f. precisely when the aorta diameter starts to decrease.

Aorta dilation is not associated with cell mitosis.

To further assess EC behaviour during aorta dilation and HSPC extrusion, we looked at EC division and whether an increase of cell number can explain vessel expansion or compensate for HSPC emerging from the aorta floor.

We used a zebrafish transgenic line that expresses a marker of the S/G2/M phase of the cell cycle, mVenus-zGeminin [START_REF] Fukuhara | Visualizing the cell-cycle progression of endothelial cells in zebrafish[END_REF] . This marker was specifically expressed under the kdrl promoter, allowing visualization of ECs during mitosis [START_REF] Fukuhara | Visualizing the cell-cycle progression of endothelial cells in zebrafish[END_REF] . We quantified the number of EC divisions in the trunk and in the tail between 27 and 72 h.p.f. We found that many division events occur in the tail region (33.0±2.8 EC divisions at 48 h.p.f), allowing development of the Caudal Haematopoietic Tissue (CHT) (Fig. 1i). In contrast, at the peak of EHT (at around 48 h.p.f.), we quantified a mean of 2.2±0.6 ECs dividing in the whole AGM area (Fig. 1i), far under what would have been needed to compensate for the estimated 25 HSPCs emerging from the aorta during the whole AGM [START_REF] Kissa | Blood stem cells emerge from aortic endothelium by a novel type of cell transition[END_REF] . Overall, between 27 and 72 h.p.f., we observed only a few EC divisions in the trunk region: the total number of cells in this area essentially decreases throughout the EHT process. Thus, additional surface area, for the aorta dilation and later for integrity maintenance during HSPC extrusion, should be provided exclusively by cell deformation.

HSPCs migrate collectively from the sides towards the ventral part of the aorta. To quantify the precise contribution of each EC to the EHT process, we followed the fate and behaviour of individual cells between 30 and 60 h.p.f. in the whole trunk region of the DA. To do so, we used the double transgenic line kdrl:nls-GFP/kdrl:caax-mCherry, allowing respective visualization of nucleus and membrane of ECs through time. We discovered that ECs undergo substantial rearrangements in the trunk region between 30 and 45 h.p.f. (Fig. 2ab). Interestingly, during this time window ECs migrate massively from the lateral side of the DA to the floor (Fig. 2a-h with arrows). The number of cells in the DA floor peaks at about 40 h.p.f., when cell migration from the sides fully compensate for cell egress, and then steadily declines afterwards (Fig. 2i) as lateral cells stop migrating towards the DA floor. The decline is due to the extrusion of cells from the aorta floor into the sub-aortic space. The number of ECs forming the dorsal aorta in the trunk region therefore strongly decreases from the start to the end of the process (from 12.1±0.9 cells/somite at 30 h.p.f. to 7.9±0.3 cells/somite at 60 h.p.f., p-value=0.009) (Fig. 2a-c, 2i-j). Remarkably, the number of ECs localized in the DA roof (facing the notochord) is essentially constant during the whole process (Fig. 2i).

HSPCs extrusion requires collective EC morphology changes.

In order to determine the contribution of neighbouring ECs to the extrusion of HSPCs, we compared the morphology of the emerging cells versus the morphology of their neighbours during EHT. Imaging of the double transgenic line kdrl:utrophin-CH-GFP/kdr:nls-GFP allowed to visualize the boundaries of each individual EC together with its nucleus. Quantification of the cell area between 40 and 60 h.p.f. showed that both emerging cells and their neighbours undergo drastic and rapid morphology changes (Fig. 3). The area of cells prior to their extrusion significantly reduces and shape changes from flat endothelial to a round plate-like cell (-69%±5% decrease in cell area) (Fig. 3a-b, 3e-g). In contrast, neighbouring lateral cells increase their area comparably (+60%±9% increase in cell area) (Fig. 3c-d, 3e-f and3h).

These data confirm that in the absence of divisions in the trunk region, the loss of cells through EHT is mainly compensated by the deformation of the lateral ECs thus assuring global aorta integrity.

Extrusion is finalized by actin ring closure around the emerging HSPC. We then focused on the cytoskeletal activity of ECs to understand the mechanism of this dramatic cell-to-tissue reorganization process. First, we used the kdrl:utrophin-CH-GFP line to mark stable F-actin in ECs [START_REF] Burkel | Versatile fluorescent probes for actin filaments based on the actin-binding domain of utrophin[END_REF] . 4D confocal imaging during the period of rearrangement (42-60 h.p.f.) showed that EC junctions are highly dynamic and have tight cell-to-cell membrane boundaries (Supplementary Movie S2). Because of the significant vascular cell deformation during EHT, we then analysed the interplay between cytoskeleton activity and HSPCs shape during the extrusion process. We imaged a double transgenic line kdrl:utrophin-CH-GFP/kdrl:caax-mCherry showing respectively stable actin filaments and EC membranes. Live imaging revealed that the aorta diameter decreases while HSPC extrusion in the sub-aortic space are driven by the formation and closure of an actin ring surrounding the emerging cells (Fig. 4, Supplementary Movie S1, Supplementary Movie S3) as also recently observed by Lancino et al [START_REF] Lancino | Anisotropic organization of circumferential actomyosin characterizes hematopoietic stem cells emergence in the zebrafish[END_REF] . Transverse view of the aorta shows that the emerging cell membrane and the actin cytoskeleton co-localize to form a perfect circle that closes as the cell exits the DA floor (Fig.

4a-c Supplementary Movie S3). As the kdrl:utrophin-CH-GFP line displays a mosaic

expression, not all ECs express Utrophin-CH-GFP and we were therefore able to follow EHT in cases where only the neighbouring cells expressed the GFP probe. In this scenario, we still clearly observed the formation (Fig. 4a,4d) and closure of the actin ring (Fig. 4b-c, e-f), suggesting that the neighbouring cells also actively participate in the ring formation and actin contractile dynamics.

In order to visualize all F-actin in the system, we also used a transgenic line expressing Lifeact (a 17-amino-acid peptide that binds to filamentous actin) fused to green fluorescent protein (GFP) [START_REF] Helker | The zebrafish common cardinal veins develop by a novel mechanism: lumen ensheathment[END_REF] . Vascular expression of Lifeact-GFP was driven by the VE-cadherin promoter and this transgenic line was used together with kdrl:caax-mCherry to visualize EC membrane as well. Imaging of VE-cad: Lifeact-GFP was consistent with the results obtained with kdrl:utrophin-CH-GFP and we could follow the formation and closure of the actin ring during EHT (data not shown).

To confirm the role of actin polymerization in HSPC extrusion, we treated embryos with drugs blocking actin assembly, Latrunculin B or Rac inhibitor NSC23766. In treated embryos, despite the presence of the aorta diameter modulation, the actin cytoskeleton was highly disrupted and the formation of the actin ring surrounding the emerging cells was not observed (Supplementary Fig. 2). Interestingly, the formation of typical plate-like cells in the aorta floor preceding HSPCs extrusion was also not observed (data not shown). We also found that in treated embryos some cells undergoing EHT bent toward the DA lumen: 33%±9% of cells emerged toward DA lumen in Latrunculin B-treated embryos (Supplementary Fig. 2). These results show the importance of actin polymerization for the acquisition of cell rigidity and the direction of HSPCs extrusion from the aorta in the sub-aortic space.

We further looked at the role of the acto-myosin contraction in HSPC extrusion by subjecting embryos to drugs blocking myosin contraction, Blebbistatin and ROCK inhibitor Y-27632.

The action of these drugs was very similar to that of the actin-blocking ones. The actin ring surrounding emerging cells was not observed and HSPC exit took longer with some cells undergoing fragmentation during the process (Supplementary Fig. 2).

To quantify the effect of actin polymerization and contraction blocking on haematopoietic organs colonization such as the CHT by HSPCs, we treated cd41:GFP embryos, which express GFP in HSPCs and thrombocytes [START_REF] Lin | Analysis of thrombocyte development in CD41-GFP transgenic zebrafish[END_REF] , with Latrunculin B, Rho-kinase inhibitor Y-27632, Blebbistatin and Rac inhibitor NSC23766. For all drugs, except with Rho-kinase inhibitor Y-27632, we found that the number of HSPCs colonizing the CHT (cd41+ cells) between 52 and 72 h.p.f. was significantly lower in treated embryos compared to control (Supplementary Table 1). Live imaging of EHT reveals that HSPCs bend without achieving complete extrusion. Part of them bents in the wrong direction, i.e. in the aorta lumen, while others cells burst as previously observed after runx1 gene inactivation [START_REF] Kissa | Blood stem cells emerge from aortic endothelium by a novel type of cell transition[END_REF] . Interestingly, none of the drugs blocking actin polymerisation or contraction was found to affect EC rearrangement occurring during the earlier stages of EHT (data not shown).

All together these results show that the acto-myosin cytoskeleton plays an essential role mainly in the final step of EHT, precisely to complete the extrusion of HSPCs from the aorta floor to the sub-aortic space.

Cellular and tissular levels are dynamically coordinated during EHT. Structuring up our observations, the EHT transition is a collective phenomenon organized on both tissular and cellular spatial scales (Fig. 5).

At tissue level, EHT is organized in two phases. In the first phase, from 28 to 42.5 h.p.f. the DA diameter increases (Fig. 1g and5a). At 40 h.p.f. aorta's shape of straight cylinder is distorted by an average diameter modulation along the aorta longitudinal direction (Fig. 1h and5a).

During this first tissular phase, between 30 and 40 h.p.f., and concurrent with the diameter modulation, at cellular level ECs reorganize themselves spatially and cells, localized on the side of the aorta, migrate towards its floor. This cell migration is a quick process, which takes on average 5.9h±0.7h (counted for 18 cells in 15 individuals).

The second tissular phase corresponds to the diameter reduction and the disappearing of the diameter modulation up to the end of EHT (from about 42.5 to 65 h.p.f.). Here, global morphological reorganization of DA is coupled with the dynamics of cells that, once localized at the aorta floor, undergo a drastic morphological change, shaping from a flat EC to a round plate-like cell. As cells round up, they undergo a strong antero-posterior contraction. In parallel, neighbouring ECs increase their area and compensate for the surface reduction of emerging HPSC cells and eventually for their extrusion. The whole cellular dynamics assures aorta integrity while DA diameter decreases. This important morphology and identity tissular and cellular remodelling take on average 9.2h±1.5h (counted for 6 cells in 6 individuals) and occur between 37 and 55 h.p.f. The closure of the actin rings around the emerging cells allows for their individualization from the aorta. The cellular extrusion and release from the aorta take on average 7.5h±1.3h (counted for 10 cells in 10 individuals). Remnant ECs finally complete the remodelling of the aorta by fully recovering its initial diameter and cylindrical geometry (Fig. 1e-f and5).

Discussion

In this article, we study and characterise the EHT process at the cellular and tissular level. We quantify the temporal sequences of cells rearrangements, shape modifications, and their coordination with cellular migration events, whilst the DA tissue takes a peculiar tridimensional shape in the trunk region. Thanks to 4D imaging and drug treatment, we have organized the EHT process in two different phases of DA dilation and contraction, and analysed the cellular events occurring.

To decipher the mechanisms that control the DA shape at various stages of EHT, we discuss here why the mechanical properties of the DA endothelium play an important role in the global control of the spatiotemporal organisation of the DA.

In the last century, theoretical methods of classical elasticity have been successful to describe instabilities in metallic pipes and similar mechanical systems that, once subjected to a critical load, change their shape and buckle. More recently, the generalization and adaptation of linear elasticity theory for the needs of biophysical systems has allowed the mechanical description of living matter like lipid membranes, cells or tissues [START_REF] Golushko | Complex instability of axially compressed tubular lipid membrane with controlled spontaneous curvature[END_REF][START_REF] Alstrøm | Instability and "Sausage-String" Appearance in Blood Vessels during High Blood Pressure[END_REF][START_REF] Li | Surface wrinkling of mucosa induced by volumetric growth: Theory, simulation and experiment[END_REF] . Moreover, high susceptibility of biological systems, residing near the critical point to the variation of external parameters, is supposed to be often used in nature to control and regulate various processes [START_REF] Muñoz | Colloquium : Criticality and dynamical scaling in living systems[END_REF] .

We argue here that mechanical instabilities are involved in the DA shape transition during EHT. More precisely, global variations of aorta shape in the trunk region assisting EHT are the result of instabilities driven by mechanical stresses of various natures acting on the DA. These stresses emerge spontaneously from the DA inhomogeneous growth and its interaction with surrounding tissues rather than from genetically preprogrammed features of embryogenesis. As a matter of fact, the specific diameter modulation in the lower part of the DA appears even when the morphogenetic program of EHT is strongly altered under inhibition of the runx1 transcription factor [START_REF] Kissa | Blood stem cells emerge from aortic endothelium by a novel type of cell transition[END_REF] . Moreover, cytoskeleton-blocking drugs only affect the final step of extrusion of HPSC, but not at all the previous phases involving the cellular tissue remodeling and the global shape change of the DA.

The DA is formed by a single monolayer of cells contrary to an adult aorta multilayer complex organization [START_REF] Kissa | Blood stem cells emerge from aortic endothelium by a novel type of cell transition[END_REF][START_REF] Santoro | Characterization of vascular mural cells during zebrafish development[END_REF] . Therefore, in first approximation, DA can be considered as a thin cylindrical elastic shell. DA is much softer than metallic pipes, and its shape is finally defined by the opposition of different active factors. These factors include: the inner hemodynamic forces, the outer compressions exerted by the surrounding tissues due to embryonic growth and development [START_REF] Kimmel | Stages of embryonic development of the zebrafish[END_REF] and the in-plane stresses generated in the aorta tissue by ECs shaping and migration as observed in our experiments. The typical timescales for such developmental events are of several hours, thus much longer than the typical time of aortic elastic response to the heart-beat (fractions of seconds), for instance [START_REF] Campàs | A toolbox to explore the mechanics of living embryonic tissues[END_REF][START_REF] Wyatt | A question of time: tissue adaptation to mechanical forces[END_REF] .

Classical mechanics predicts that destabilization of a tubular membrane can be associated with three main modes corresponding to various membrane deformations. These modes are:

Euler buckling (a tube's longitudinal axis bends while cross-section remains circular, Fig. 5b), transverse buckling (axis of a tube remains straight, whereas its cross-section takes an oval shape Fig. 5c) and "corrugation instability" (a tube preserves its rotational symmetry along the main axis, but its radius is modulated along the main axis, Fig. 5d).

Euler buckling of the cylindrical tube can be invoked by the compressive stress along the tube main axis. If the surrounding tissues were absent, the DA stability with respect to this mode would have been determined almost exclusively by the bending rigidity of the tube wall.

However, as it is proven by the experiment, long-wave buckling instability is irrelevant for the aorta, because notochord and other tissues surrounding the DA prevent its longitudinal axis from bending (Fig. 1 and5).

Transverse buckling of the cylindrical shell occurs when transverse isotropic (i.e. possessing the rotational symmetry of the tube) stress in its walls reaches a negative critical value.

Stability with respect to transverse buckling is independent on the tension applied along the tube axis and is determined by material constants characterizing the system. This type of mechanical instability also occurs in ordinary rings [START_REF] Landau | Statistical physics[END_REF] . Slightly elongated shape of aorta in the dorsal-ventral direction is preserved throughout the whole process and in our opinion this shape is due to an anisotropic compression applied by the muscles located on the sides of the aorta rather than to the transverse buckling instability with spontaneous symmetry breaking.

On the contrary, deformation appearing in the DA prior to HSPCs extrusion has a wellpronounced space periodicity and amplitude (Fig. 1c-e,1h, and Fig. 5) which makes it similar to the corrugation deformation of an axially compressed thin-walled rigid pipe [START_REF] Timoshenko | Theory of Elasticity[END_REF] . Importantly, space period and amplitude of the DA diameter modulation lay beyond the cell diameter and thickness. As it was mentioned before, the blood pressure creates positive stress in DA walls.

Its longitudinal component can be calculated as σ=R ΔP/2h, where ΔP is a pressure difference between the interior and the exterior of DA, R and h are DA's radius and thickness respectively. This positive stress cannot induce corrugation since it makes DA even more stable. However, negative compressive stress (inducing the corrugation in the system) can originate from the difference between the growth rates of the DA and the tissues around it. As it is shown by our data at about 30 h.p.f., this negative stress eventually overpowers the positive one associated with blood pressure and leads to the corrugation of the aorta. This deformation occurs mainly in the ventral part of the DA where the migrating cells converge, and the compressive stress is maximal. Later on, as the zebrafish embryo develops, exit of the cells undergoing EHT decreases the effective stress due to the decrease of the equilibrium surface area of the DA. Consequently, at about 65 h.p.f. the stress drops below the critical value and the tube regains its initial not-deformed shape (Fig. 1f, Fig. 5).

As we show experimentally, chemical perturbation or inhibition of acto-myosin contractility machinery seriously affects only the final event of EHT, i.e. the extrusion of the EHT cell from the aorta endothelium. Very interestingly, we stress that the initial phases of EHT resulting in DA shape distortion still occurs in presence of chemical perturbations. Moreover, the deformation of HSPCs that are preparing to leave the aorta also occurs. What is different is that, in the drug-treated specimens where the actin rings surrounding the cells are absent, many cells do not bend outside (as usual) but do bend inside of the aorta. Based on this observation, we hypothesize that the EHT process is also associated with additional shape instability of individual cells forming the aorta endothelium. We believe that this second instability is also provoked by the stress exerted on the DA tissue, whereas the polymerization of the actin ring and the actin cytoskeleton activity insures the right direction of future HSPC bending and further facilitates its extrusion in the sub-aortic space toward the cardinal vein.

In conclusion, by using 4D fluorescence microscopy, we have characterized qualitatively and quantitatively different dynamical phases of EHT leading to the generation of circulating hematopoietic stem cells from the DA. From the analysis of our observations on wild-type, genetically-modified and chemically-treated zebrafish, we confirmed the important role of the acto-myosin system in EHT single cell final extrusion [START_REF] Lancino | Anisotropic organization of circumferential actomyosin characterizes hematopoietic stem cells emergence in the zebrafish[END_REF][START_REF] Guillot | Mechanics of Epithelial Tissue Homeostasis and Morphogenesis[END_REF] . Other processes involving the externalisation of a single cell from a cell layer, such as apoptotic extrusion from the epithelial layer of the zebrafish embryo epidermis, confirm that actin/myosin contraction is essential in this process [START_REF] Rosenblatt | An epithelial cell destined for apoptosis signals its neighbors to extrude it by an actin-and myosin-dependent mechanism[END_REF] .

In particular, we profiled a general mechanism based on mechanical instabilities that prepare and support the whole EHT prior to a specific genetic control of the process. Importantly, our interpretation suggests a generic and self-organized mechanism that drives unique collective events of tissue reorganization such as EHT in the development and growth of complex organisms.

Further on, it will be interesting to develop a more precise mathematical model for the description of aorta dynamics and study EHT transition in other model systems. Since the aorta of the zebrafish embryo consists in a limited number of cells, which decreases even more during the EHT process, it will be important to combine an analytical continuum model with a coarse-grained approach allowing for the description of individual cells, similarly to ones used in [START_REF] Bi | Motility-Driven Glass and Jamming Transitions in Biological Tissues[END_REF][START_REF] Farhadifar | The influence of cell mechanics, cell-cell interactions, and proliferation on epithelial packing[END_REF][START_REF] Merkel | Triangles bridge the scales: Quantifying cellular contributions to tissue deformation[END_REF][START_REF] Krajnc | Fluidization of epithelial sheets by active cell rearrangements[END_REF] , but generalized for a curved 2-dimensional surface in 3-dimensional space.

We believe that further studies of EHT will shed light on complex HSPC genesis, a fundamental example of developmental process with important applications in tissue engineering and regenerative therapies, but also on mechanical processes resulting in the development of pathologies. Finally, does mechanics prepare the tissue before genetic reprogramming? It is a debate that is developed in this study in an illustrated way with the example of HSPCs ontogenesis.

Methods.

Zebrafish husbandry

Tg(kdrl:Has.HRAS-mCherry) (here cited as kdrl:caax-mCherry) [START_REF] Chi | Foxn4 directly regulates tbx2b expression and atrioventricular canal formation[END_REF] , Tg(kdrl:utrophin-CH-GFP), Tg(Cdh5:Gal4//UAS:lifeact:GFP) [START_REF] Helker | The zebrafish common cardinal veins develop by a novel mechanism: lumen ensheathment[END_REF] , Tg(kdrl:nls-GFP) [START_REF] Blum | Complex cell rearrangements during intersegmental vessel sprouting and vessel fusion in the zebrafish embryo[END_REF] , Tg(flk-1:mV-zGem) [START_REF] Fukuhara | Visualizing the cell-cycle progression of endothelial cells in zebrafish[END_REF] and Tg(cd41:eGFP) [START_REF] Lin | Analysis of thrombocyte development in CD41-GFP transgenic zebrafish[END_REF] were maintained, crossed, raised and staged as described previously [START_REF] Kimmel | Stages of embryonic development of the zebrafish[END_REF][START_REF] Westerfield | The Zebrafish Book. A Guide for the Laboratory Use of Zebrafish (Danio rerio)[END_REF] .

All animal experiments described in the present study were conducted at the University of Montpellier according to European Union guidelines for handling of laboratory animals (http://ec.europa.eu/environment/chemicals/lab_animals/home_en.htm) and were approved by the Direction Sanitaire et Vétérinaire de l'Hérault and Comité d'Ethique pour l'Expérimentation Animale under reference CEEA-LR-13007.

Drug treatments

From 24 h.p.f. onwards, embryos were grown in PTU-containing medium to block pigmentation. Embryos were dechorionated and treated with different drugs after the start of circulation at 26 h.p.f. Drugs were dissolved in DMSO as stock solution and diluted in E3 medium up to 1% DMSO final concentration. Control embryos were subjected to the same concentration of DMSO as treated embryos. Embryos were treated with 2µM Latrunculin B, 0.8µM Blebbistatin, 50µM Rac inhibitor NSC23766 for or 50µM Rho-kinase (ROCK) inhibitor Y-27632. Embryos were kept in drug solution until image acquisition and appropriate concentrations of drugs were added to the mounting medium for time-lapse acquisitions.

Quantification of HSPC colonization using Tg(cd41:eGFP)

To quantify cd41+ cells in the CHT, embryos were imaged under a fluorescent binocular scope Zeiss V12 at 100X at 52 h.p.f. and 72 h.p.f. Cd41+ cells were then counted using ImageJ.

Microscopy

Fluorescence images of transgenic embryos were acquired using Zeiss LSM510 at 20X. Time-lapse imaging was performed using Zeiss LSM510 at 20X or 40X magnification essentially as described [START_REF] Renaud | Studying cell behavior in whole zebrafish embryos by confocal live imaging: application to hematopoietic stem cells[END_REF] . Embryos were anesthetised with tricaine (0.016%) and mounted on a glass petri dish with 0.7 % low melting agarose and covered with standard E3 medium supplemented with tricaine and 1-phenyl-2-thiourea (PTU) (0.003%) to prevent pigment formation. Temperature was maintained at 28°C by placing the dish in a temperature-control chamber during time-lapse acquisitions. Images were analysed using ImageJ and Imaris (Bitplane). The EHT transition starts first by a global aorta dilation (as soon as the heart starts to beat), taking place from 24 to about 42,5 h.p.f., followed by the aorta contraction, occurring from about 42,5 to 65 h.p.f. While the embryo is growing and developing, the first phase of aorta dilation is essentially characterized by cells that start to increase their area, while others, in particular those located on the sides of the aorta, migrate towards the ventral part (aorta floor). During this phase the characteristic modulation of the aorta diameter, the corrugation instability (see main text and below), appears.

Figure 2 .

 2 Figure 2. Cells undergoing EHT are recruited from the side of the aorta and migrate to its floor prior to extrusion. a-c. From 30 to 60 h.p.f., still frames of time-lapse imaging of Tg(kdrl:ns-GFP)/Tg(kdrl:caax-mCherry) embryo. Maximum projection from 40 z-stack spaced by 1µm. Stars indicate the nucleus of the ECs in the dorsal aorta in the 2 central somites of the image. d-h. Still frames of time-lapse imaging of Tg(kdrl:nls-GFP)/Tg(kdrl:caax-mCherry) embryo. Maximum projection from 40 z-stack spaced by 0.6µm. Cells migrating from the side of the aorta toward the floor are numbered 1 and 2 in white. i. Graph showing the number of nuclei counted in 3 different zones of the dorsal aorta: roof, side and floor, n=5 cells. j. Schematic representation of cell rearrangement occurring from 30 to 60 h.p.f. Colour coding of the cells corresponds to the graph in i. DA: dorsal aorta; CV: cardinal vein. Scale bar: 25 µm (a-c), 10µm (d-h).

Figure 3 .

 3 Figure 3. Endothelial cells adopt collaborative behaviour during EHT: cellular contractions and extensions to maintain aorta integrity. a-d. Still frames of time-lapse imaging of Tg(kdrl:nls-GFP)/Tg(kdrl:utrophin-CH-GFP) embryo between 40 and 60 h.p.f. Maximum projection from 40 z-stack spaced by 0.6µm. Cell A (highlighted in orange) is an endothelial cell located in the floor of the aorta and starting to undergo EHT. Cell A surface reduces importantly between 42 h.p.f. (a) and 60 h.p.f. (b). Cell B (highlighted in blue) is located in the side of the aorta neighbouring a cell undergoing EHT (star). Cell B surface increases between 42 h.p.f. (c) and 54 h.p.f. (d). e-f Schematic representation of EC surfaces and positions at t1 (e) and t2 > t1 (f). Colour-code and letters correspond to the panel a-d. g. Graph showing the temporal evolution of EC starting to emerge from the aorta floor in percentage of difference compared to area at 40 or 42 h.p.f. Each line represents the measurements for one cell. 6 ECs starting to emerge from the aorta floor were analysed in 5 different embryos. For details on cell area (µm²) calculation, see Material and Methods section. h. Graph showing temporal evolution of cell area of lateral EC in percentage of difference compared to area at 40 or 42 h.p.f. Each line represents the measurements for one cell. 5 ECs on the side of the aorta were analysed in 5 different embryos. Scale bar: 10µm.

Figure 4 .

 4 Figure 4. Final contraction in EHT is actin-dependent and coordinated with surrounding cells. a-c. Horizontal reconstructed view from a maximum projection of a series of z-stack of a Tg(kdrl:utr-CH-GFP)/Tg(kdrl:caax-mCherry) embryo during EHT at 42, 44 and 46 h.p.f. Upper panels are kdrl:utrophin-CH-GFP alone and lower panels are merged images with kdrl:caax-mCherry. Closure of the actin ring by the neighbouring cells is clearly observable in the upper panels. See also Supplementary Movie S3. d-f. Single z-stacks of time-lapse imaging of the same embryo as in a-c. Upper panels are kdrl:utrophin-CH-GFP alone and middle panels are merged images with kdrl:caax-mCherry. Actin ring (arrows) closes around emerging cell (arrowhead). Note that in this case the emerging cell does not express Utrophin-CH-GFP due to mosaic labelling, allowing us to confirm the role of the neighbouring cells in actin ring closure.

Figure 5 .

 5 Figure 5. Overall schematic representation of Endothelial-to-Haematopoietic Transition with mechanical instabilities. a. The EHT transition starts first by a global aorta dilation (as

  In the contraction phase, cells localized at the aorta floor undergo a drastic morphological change, shaping from flat ECs to round plate-like cells. As cells round up, they undergo a strong antero-posterior contraction. In parallel, neighbouring ECs compensate for the surface reduction of emerging cells, and eventually for the extrusion of the latter cells from the aorta, by increasing importantly their surface. Finally emerging round cells extrude and individualize from the aorta. This phase coincides with the decrease of the diameter modulation, recovering the initial aorta diameter and its cylindrical geometry. b-c-d. The three main mechanical instability modes of a thin cylindrical soft pipe (membrane) under mechanical stresses: b. Longitudinal buckling; c. Transverse buckling; d. Corrugation.

Figure 1 .

 1 Figure 1. Aorta remodelling during EHT: embryo anatomy and spatiotemporal organisation of aorta diameter a b

Figure 2 .Figure 3 .Figure 4 .

 234 Figure 2. Cells undergoing EHT are recruited from the middle of the aorta and migrate to its floor prior to extrusion.

Figure 5 .

 5 Figure 5. Overall schematic representation of Endothelial-to-Haematopoietic Transition with mechanical instabilities

Acknowledgements

We thank Etienne Lelièvre for his critical reading of the manuscript, A. Sahuquet, C. Chevalier, V. Diakou for their assistance and the MRI facility, N. Abdellaoui for management of zebrafish facility. D. Stainier lab for Tg(Cdh5:Gal4//UAS:lifeact:GFP), S. Shulte-Merker lab for Tg(kdrl:utrophin-CH-GFP) and Tg(kdrl:nls-GFP) and National Bioresource Project Zebrafish for Tg(flk-1:mV-zGem). This work was supported by the ARC, FRM, ATIP-Avenir fellowships and a fellowship from the Région Languedoc-Roussillon, Chercheur d'Avenir. NP was supported by a fellowship from the ATIP-Avenir, SR and IG are grateful to the RSF grant N 19-12-00032, AP, IG and SR acknowledge NUMEV (AAP-2016-2-025) for financial support. I.G.'s thesis was funded by Campus France (Vernadsky Fellowship) and the France-Russia Cooperation Program, and JT by a fellowship from the MESR and the FRM.

Aorta diameter measurement

To measure aorta diameter Tg(kdrl:caax-mCherry) embryos were imaged using Zeiss LSM510 as described above. Aorta diameter was measured manually using ImageJ at 10 different points along the trunk which were then averaged. To show aorta diameter evolution through time, the aorta was measured every 2.5 hours in 5 embryos.

Cell surface area measurements

Cell surface area was measured using the Surface Contour tool of the Imaris software (Bitplane). Briefly, the contour of a given cell was outlined on the different stacks where the cell was visible using the Click drawing mode. The software then calculated automatically the total cell surface area (µm²). A total of 6 ECs starting to emerge from the aorta floor and 5

ECs on the side of the aorta were analysed in 5 different embryos.

Statistical analysis

Normal distributions were analysed using Shapiro-Wilk test. Non-Gaussian data were analysed using Wilcoxon or Kruskal-Wallis test, Gaussian with Student's t-test or analysis of variance followed by Holm's multiple comparison. P<0.05 was considered as statistically significant. Statistical analyses were performed using R software.

Bibliography.

Disclosure of Conflicts of Interest

The authors declare no competing financial interests.

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(which was not peer-reviewed) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint . http://dx.doi.org/10.1101/509190 doi: bioRxiv preprint first posted online Jan. 1, 2019;


Virginie Naël

Karine Pérès

Jean-François Dartigues

Luc Letenneur

Hélène Amieva

Angelo Arleo

Anne-Catherine Scherlen

Christophe Tzourio

Claudine Berr

Isabelle Carrière

Cécile Delcourt

Catherine Helmer

Geir Bertelsen

Suzanne Cosh

Audrey Cougnard-Grégoire

Piers Dawes

Fofi Constantinidou

M Arfan Ikram

Caroline Cw Klaver

Iracema Leroi

Asri Maharani

Magda Meester- Smor

Unal Mutlu

Virginie Nael
email: virginie.nael@gmail.com

Neil Pendleton

Henrik Schirmer

Gindo Tampubolon

Henning Tiemeier

Therese Von Hanno

Vision loss and 12-year risk of dementia in older adults: the 3C cohort study

Keywords: Vision loss, Dementia, Cohort study, Epidemiology

To analyze the longitudinal relationships between vision loss and the risk of dementia in the first 2 years, from 2 to 4 years and beyond 4 years) after inclusion and to determine the roles of depressive symptomatology and engagement in cognitively stimulating activities in these associations. This study is based on the Three-City (3C) study, a populationbased cohort of 7736 initially dementia-free participants aged 65 years and over with 12 years of follow-up. Near visual impairment (VI) was measured and distance visual function (VF) loss was self-reported. Dementia was diagnosed and screened over the 12-year period. At baseline, 8.7% had mild near VI, 4.2% had moderate to severe near VI, and 5.3% had distance VF loss. Among the 882 dementia cases diagnosed over the 12-year follow-up period, 140 cases occurred in the first 2 years, 149 from 2 to 4 years and 593 beyond 4 years after inclusion. In Cox multivariate analysis, moderate to severe near VI was associated with an increased risk of dementia in the first 2 years (HR= 2.0, 95%CI: 1.2 to 3.3) and from 2 to 4 years (HR=1.8, 95%CI: 1.1 to 3.1) but the association was not significant beyond 4 years after inclusion even if pointing in similar direction (HR=1.3, 95%CI: 0.95 to 1.9). Mild near VI was associated with an increased risk of dementia only in the first 2 years (HR=1.6, 95%CI: 1.1 to 2.5). Moreover, self-reported distance VF loss was associated with an increased risk beyond 4 years after inclusion (HR= 1.5, 95%CI: 1.1 to 2.0) but the association was no longer significant after taking into account baseline cognitive performances. Further adjustment for engagement in cognitively stimulating activities only slightly decreased these associations. However, there was an interaction between vision loss and depressive symptomatology, with vision loss associated with dementia only among participants with depressive symptomatology. These results suggest that poor vision, in particular near vision loss, may represent an indicator of dementia risk at short and middle-term, mostly in depressed elderly people.

INTRODUCTION

Vision loss (VL) is common in older adults and increases with age. The leading cause of VL is uncorrected refractive error [START_REF] Bourne | Causes of vision loss worldwide, 1990-2010: a systematic analysis[END_REF], easily avoidable with corrective eyeglasses, lenses or refractive surgery. VL has been associated with cognitive impairment, but mainly in cross-sectional studies [START_REF] Chen | Association of Vision Loss With Cognition in Older Adults[END_REF][START_REF] Spierer | Correlation Between Vision and Cognitive Function in the Elderly: A Cross-Sectional Study[END_REF][START_REF] Mine | Association of Visual Acuity and Cognitive Impairment in Older Individuals: Fujiwara-kyo Eye Study[END_REF][START_REF] Garin | Visual impairment and multimorbidity in a representative sample of the Spanish population[END_REF][START_REF] Ong | Visual impairment, age-related eye diseases, and cognitive function: the Singapore Malay Eye study[END_REF][START_REF] Tay | Sensory and cognitive association in older persons: findings from an older Australian population[END_REF][START_REF] Clemons | Age-Related Eye Disease Study Research Group. Cognitive impairment in the Age-Related Eye Disease Study: AREDS report no. 16[END_REF][START_REF] De Kok | What is the relationship between visual impairment and cognitive function in octogenarians?[END_REF][START_REF] Diaz | Cognitive profile of elders in an ophthalmic ambulatory setting[END_REF], whereas longitudinal studies have shown conflicting results [START_REF] Hong | Visual Impairment, Hearing Loss and Cognitive Function in an Older Population: Longitudinal Findings from the Blue Mountains Eye Study[END_REF][START_REF] Fischer | Age-Related Sensory Impairments and Risk of Cognitive Impairment[END_REF][START_REF] Valentijn | Change in sensory functioning predicts change in cognitive functioning: results from a 6-year follow-up in the maastricht aging study[END_REF][START_REF] Reyes-Ortiz | Near vision impairment predicts cognitive decline: data from the Hispanic Established Populations for Epidemiologic Studies of the Elderly[END_REF][START_REF] Sloan | Effects of changes in self-reported vision on cognitive, affective, and functional status and living arrangements among the elderly[END_REF][START_REF] Lin | Vision impairment and combined vision and hearing impairment predict cognitive and functional decline in older women[END_REF]. Furthermore, little is known about the longitudinal association between VL and dementia, with only one previous study among participants aged 71 and older, showing that those who perceived that their vision was good or excellent had a lower risk of developing dementia [START_REF] Rogers | Untreated poor vision: a contributing factor to late-life dementia[END_REF].

Several hypotheses have been proposed to explain the association between VL and cognitive impairment: 1) VL may act through factors known to be associated with cognitive decline and dementia, in particular engagement in activities and depression [START_REF] Katon | Effect of depression and diabetes mellitus on the risk for dementia: a national population-based cohort study[END_REF][START_REF] Köhler | Depression, vascular factors, and risk of dementia in primary care: a retrospective cohort study[END_REF][START_REF] Dartigues | Playing board games, cognitive decline and dementia: a French population-based cohort study[END_REF]. Thus, VL may decrease the engagement in cognitively stimulating activities or participation in social life or increase the risk of depression, which may subsequently increase the risk of dementia [START_REF] Ivers | Sensory impairment and driving: the Blue Mountains Eye Study[END_REF][START_REF] Laitinen | A Nationwide Population-Based Survey on Visual Acuity, Near Vision, and Self-Reported Visual Function in the Adult Population in Finland[END_REF][START_REF] Carrière | A prospective study of the bi-directional association between vision loss and depression in the elderly[END_REF][START_REF] Hong | Visual impairment and depressive symptoms in an older Australian cohort: longitudinal findings from the Blue Mountains Eye Study[END_REF][START_REF] Kiely | Dual sensory loss and depressive symptoms: the importance of hearing, daily functioning, and activity engagement[END_REF][START_REF] Tsai | Association between visual impairment and depression in the elderly[END_REF]. 2) A lack of adequate sensory input could lead to neuronal atrophy and thus cognitive impairment [START_REF] Lindenberger | Sensory functioning and intelligence in old age: a strong connection[END_REF]. 3) Visually impaired people may need to allocate more resources to perceive and interpret sensory information and thus have fewer resources for other cognitive tasks [START_REF] Baltes | Emergence of a powerful connection between sensory and cognitive functions across the adult life span: a new window to the study of cognitive aging?[END_REF]. 4) Alternatively, VL and dementia may share common risk factors as aging [START_REF] Lindenberger | Sensory functioning and intelligence in old age: a strong connection[END_REF]. 5) Finally, vision loss may be one of the early symptoms of dementia as, Alzheimer's disease (AD), the major cause of dementia, can affect the visual pathway and result in visual deficits [START_REF] Armstrong | Alzheimer's Disease and the Eye[END_REF].

In the present study, we aimed to investigate the longitudinal association between vision loss and the incidence of dementia. In particular, thanks to a 12-year follow-up period within a large population-based cohort we aimed to study the associations for several periods of time after inclusion. Moreover, we aimed to explore the roles of depressive symptomatology and engagement in cognitively stimulating activities in these associations.

METHODS

This study forms part of the SENSE-Cog multi-phase research program, funded by the European Union Horizon 2020 program. SENSE-Cog aims to promote mental well-being in older adults with sensory and cognitive impairments (http://www.sense-cog.eu/). The first part of this project aims to better understand the links between sensory, cognitive and mental health in older Europeans.

Study population

This study was based on the Three-City study (3C), a French population-based cohort of 9294 community-dwelling older adults aged 65 years and over who enrolled between 1999 and 2001. The aim of 3C is to assess the risk of dementia and cognitive decline due to vascular risk factors. The methodology of the 3C study has been described elsewhere [START_REF] Study | Vascular factors and risk of dementia: design of the Three-City Study and baseline characteristics of the study population[END_REF]. Briefly, participants were recruited from the electoral rolls of three French cities: Bordeaux (n=2104), Dijon (n=4931) and Montpellier (n=2259). Data were collected during face-to-face interviews; trained neuropsychologists administered standardized questionnaires and performed clinical examinations at baseline and 2, 4, 7, 10 and 12 years later. Sociodemographic characteristics, lifestyle, cardiovascular risk factors, vision, and depressive symptomatology were assessed at each interview. A complete functional and cognitive evaluation with systematic screening for dementia was also conducted. Moreover, blood samples and participation in leisure activities were collected at baseline. Among the 8250 participants who were without prevalent dementia at baseline and were followed up at least once during the 12-year follow-up period, 7736 had baseline data for both distance and near vision loss and were thus, included in this study (Figure 1).

Diagnosis of dementia

Dementia was actively diagnosed at baseline and at each follow-up visit using a 3-step procedure. The first step consisted of a cognitive evaluation made by the neuropsychologist through a series of psychometric tests including at a minimum the MMSE [START_REF] Folstein | Mini-mental state". A practical method for grading the cognitive state of patients for the clinician[END_REF], the Isaacs set test and the Benton Visual Retention Test [START_REF] Isaacs | The Set test as an aid to the detection of dementia in old people[END_REF][START_REF] Benton | Manuel pour l'application du test de retention visuelle[END_REF]. Participants suspected of having dementia, based on either their neuropsychological performance or decline relative to a previous examination, were then examined by a senior neurologist to establish a clinical diagnosis. Finally, an independent committee of neurologists and geriatricians reviewed all potential cases of dementia with all available information in order to obtain a consensus on the diagnosis and etiology, according to the DSM-IV and the NINCDS-ADRDA criteria [START_REF]Diagnostic and Statistical Manual of Mental Disorders: DSM-IV[END_REF][START_REF] Mckhann | Clinical diagnosis of Alzheimer's disease: report of the NINCDS-ADRDA Work Group under the auspices of Department of Health and Human Services Task Force on Alzheimer's Disease[END_REF].

Vision loss

Binocular near visual acuity was assessed using the Parinaud scale (a Jaeger-like reading test commonly used in France). Assessments were carried out using presenting vision with usual optical correction (i.e., their personal spectacles) where applicable, with a standardized reading distance of 33 cm. Mild near visual impairment (VI) was classified by Parinaud 3 or 4 (Snellen equivalent 20/30-20/60) and moderate to severe near VI by Parinaud > 4 (Snellen equivalent < 20/60). Distance visual function (VF) loss was self-reported, defined as an inability or difficulty in recognizing a familiar face at 4 meters, using presenting optical correction if any.

Leisure activities

Leisure activities were assessed at baseline using two different self-administered questionnaires. In Bordeaux, 28 activities were assessed, 12 of which were considered cognitively stimulating activities: going to the cinema, painting, sculpting, going to the theater, reading literature, reading newspapers, acting as a director of an association (sporting, cultural or political), playing board games, doing crossword puzzles, and travelling. One point was awarded for each activity performed [START_REF] Foubert-Samier | Education, occupation, leisure activities, and brain reserve: a population-based study[END_REF]. In Dijon and Montpellier, 6 cognitively stimulating activities were considered among 19 activities assessed: reading, doing crossword puzzles, playing cards, going to the cinema/theater, practicing an artistic activity and managing an association (sporting, cultural or political). Participants were asked about the monthly frequency (0= never or rarely; 1= 1-3 per month; 2= 1 per week; 3= ≥2 per week) that they engaged in each activity, except for reading, for which participants were asked about the daily frequency (0= <1 hour per day; 1= 1-2 hours per day; 2= >2 hours per day). Cognitively stimulating activities scores were calculated by summing the item scores [START_REF] Akbaraly | Leisure activities and the risk of dementia in the elderly: results from the Three-City Study[END_REF].

Due to the difference in assessments between the centers, cognitively stimulating activities scores were standardized (Zscore) for the analyses.

Depressive symptomatology

Depressive symptomatology at baseline was evaluated using the Center for Epidemiologic Studies Depression Scale (CESD) questionnaire, a 20-item self-report rating scale designed to evaluate the frequency of depressive symptoms experienced over the past week. Each item is scored from 0 (rarely) to 3 (most of the time). Thus, the total CESD score ranges from 0 to 60, increasing with the level of severity of depressive symptomatology. As previously validated, scored of ≥17 for men and ≥23 for women were used to define depressive symptomatology [START_REF] Radloff | The CES-D Scale A Self-Report Depression Scale for Research in the General Population[END_REF][START_REF] Fuhrer | La version française de l'échelle CES-D (Center for epidemiologic studies-depression scale). Description et traduction de l'échelle d'autoévaluation[END_REF].

Potential confounders

The following sociodemographic factors were considered: age, gender, educational level (elementary school without diploma, short secondary school and higher levels), living alone and monthly income (<1500€, 1500-2300€, >€2300, refusal to answer). As other sensory impairments may also have an impact on dementia risk [START_REF] Lin | Hearing Loss and Incident Dementia[END_REF], self-reported hearing loss, classified in three categories (no, mild and moderate to severe) was also considered. Other potential confounders included cardiovascular risk factors: hypertension (systolic blood pressure ≥140 mmHg and/or diastolic blood pressure ≥90 mmHg using the mean of two measures and/or antihypertensive medication), history of cardiovascular ischemic disease, history of stroke, hypercholesterolemia (cholesterol ≥6.20 mmol/L and/or hypolipemiant treatment), hypertriglyceridemia (triglycerides ≥1.7 mmol/L), diabetes (normal, hyperglycemia (fasting glycemia [6.1-7.0[ mmol/L, diabetes (fasting glycemia ≥7.0 mmol/L and/or antidiabetic treatment)), smoking habits (never, former, current), and body mass index (BMI) (weight/height 2 ) in four categories (<21, 27-29.9, ≥30 vs 21-26.9). Additionally, APOE genotype (at least one ɛ4 allele vs no ɛ4) was also taken into account.

Statistical analysis

Cox proportional hazards models with delayed entry (using age as time-scale) were used to compare the baseline characteristics of participants according to incident dementia and to estimate the risk of dementia associated with vision loss, providing hazard ratios (HR) and 95% confidence intervals (CI). For participants who developed dementia, time of event was determined at the middle of the interval between the visit when dementia was diagnosed and the last visit prior to dementia diagnosis. Participants who did not develop dementia were censored at the last follow-up visit. Near and distance vision loss were analyzed in separate models. In order to assess whether vision loss had a constant effect over time, vision loss was modelled by a time-dependent variable represented by a step function. The effects of vision loss on the risk of dementia were estimated in the first 2 years, from 2 to 4 years and beyond 4 years after inclusion. Supplementary analysis using other periods of time (the first 4 years, from 4 to 7 years and beyond 7 years after inclusion) was also performed. The multivariate model included the following covariates: center, gender, educational level, self-reported hearing loss, living alone, income, cardiovascular risk factors and ApoE4 genotype. A total of 276 participants had missing data for at least one covariate. To avoid excluding these participants and limit thus selection bias we performed multiple imputation for missing values [START_REF] Schafer | Multiple imputation: a primer: Stat Methods Med Res[END_REF].

To further assess the robustness of our analyses to potential reverse causation, we carried out a sensitivity analysis additionally adjusted for baseline cognitive status. A z-score of global cognition at baseline was computed, including performances on the MMSE, Isaacs Set Test and Benton Visual Retention Test.

We further studied the effects of engagement in cognitively stimulating activities and depressive symptomatology on the relationships between vision and dementia, these two factors being potentially associated with both vision and dementia. We first searched for a potential interaction between these two factors and vision loss on the risk of dementia and then presented accordingly either adjusted or stratified analyses. The analyses were performed using SAS software (version 9.3; SAS Institute Inc., Cary, NC, USA).

RESULTS

Study sample

At baseline, the mean age of the 7736 participants was 73.9 years; 61.3% were women, 39.5% had a high educational level, and 12.6% had depressive symptomatology (Table 1). The mean MMSE score was 27.4 (SD 1.9). A total of 882 participants developed dementia over the 12-year follow-up period (median= 9.1 years, range= 0.6 -13.5) with an incidence of 14.5 cases per 1000 person-years. Participants who ultimately developed dementia were older, less educated, had lower income, more self-reported hearing loss, more vascular risk factors, more depressive symptomatology, reported less engagement in cognitively stimulating activities, were more often APOE4 carriers and had lower MMSE scores at baseline. They had also more often near VI and distance VF loss.

Vision loss and risk of incident dementia

 Main analysis

Among the 7736 participants, 671 (8.7%) had mild near VI, 325 (4.2%) moderate to severe near VI and 413 (5.3%) distance VF loss. Among the 882 cases of dementia occurred over the 12-year follow-up, 140 cases occurred before the first follow-up time ( in the first 2 years), 149 between the first and the second follow-up time ( from 2 to 4 years) and 593 after the second follow-up time (beyond 4 years). In the analyses adjusted for all the potential confounders (Table 2), mild near VI was associated with an increased risk of dementia only in the first 2 years (HR=1.6, 95%CI: 1.1 to 2.5).

Moderate to severe near VI was associated with an increased risk of dementia in the first 2 years (HR= 2.0, 95%CI: 1.2 to 3.3) and from 2 to 4 years (HR=1.8, 95%CI: 1.1 to 3.1), but the risk was not significantly increased beyond 4 years although there was a trend (HR= 1.3, 95%CI: 0.95 to 1.9). Distance VF loss was associated with an increased risk of dementia only beyond 4 years (HR= 1.5, 95%CI: 1.1 to 2.0). In sensitivity analysis additionally adjusted for baseline global cognition, results were almost unchanged for near vision although the association between mild near VI and dementia in the first 2 years was no longer significant (Supplementary table 1, HR=1.5, 95%CI: 0.98 to 2.4, p=0.06).

However, distance VF loss was no longer significantly associated with dementia beyond 4 years (HR= 1.3, 95%CI: 0.94 to 1.8). In the supplementary analysis using other cut-offs (<4 years, 4-7 years and ≥7 years) we found a significant effect of vision loss on dementia for moderate to severe near VI only in the first 4 years after inclusion (HR=1.88, 95%CI 1.29;2.73, p=0.001); after 7 years the increased risk associated with distance VF loss was only borderline significant (HR=1.55, 95%CI 0.99;2.44, p=0.055) (Supplementary table 2).

 Engagement in cognitively stimulating activities

There was no interaction between vision loss and cognitively stimulating activities, thus to analyze the effects of engagement in cognitively stimulating activities on the associations between vision loss and dementia we additionally adjusted for these activities. Engagement in cognitively stimulating activities was evaluated for 7089 participants, this relatively large number of missing data (8.4%) being due to the evaluation by self-questionnaire. At baseline, participants with near VI had lower mean scores of engagement in cognitively stimulating activities, with scores decreasing as the severity of VI increased (0.08 (95% CI: 0.06 to 0.11) for no near VI, -0.14 (95% CI: -0.22 to -0.06) for mild VI, -0.38 (95% CI:-0.49 to -0.26)for moderate to severe VI, p<0.0001 adjusted for age). After additional adjustment for these activities in the multivariate model (Table 3, model B), mild near VI was still associated with an increased risk of dementia in the first 2 years (HR=1.7, 95%CI: 1.0 to 2.8). Moderate to severe near VI was also associated with an increased risk of dementia from 2 to 4 years (HR=1.8, 95%CI: 1.1 to 3.3) but the risk was no longer significant beyond 4 years (HR=1.7, 95%CI: 0.9 to 3.2). As in the previous model distance VF loss was associated with an increased risk only beyond 4 years (HR=1.4, 95%CI 1.0 to 2.0). Compared to results observed in the same sample (Table 3, Model A), HRs were only slightly decreased after this adjustment for cognitively stimulating activities.

 Depressive symptomatology

Analyses stratified on depressive symptomatology showed an interaction between depressive symptomatology and vision loss. At baseline, distribution of depressive symptomatology differed according to near VI (p=0.0040): participants with moderate to severe near VI had more often depressive symptomatology (15.6% (95%CI; 11.7 to 20.1) vs 12.4% (95%CI: 11.6 to 13.2) for those without near VI) and had a higher proportion of missing evaluations of their depressive symptomatology (5.2% (95% CI: 3.1 to 8.2) vs 2.1% (95%CI: 1.8 to 2.5)). Among the 6610 participants without depressive symptoms at baseline, 700 developed a dementia over the follow-up compared to 150 of the 951 participants with depressive symptoms. The risks of dementia associated with visual loss were significantly increased only among participants with depressive symptomatology and not in those without. Indeed, moderate to severe near VI was associated with an increased risk in the first 2 years (HR=2.9. 95%CI 1.0 to 7.9) and beyond 4 years (HR=3.1, 95%CI 1.5 to 6.5); the risk from 2 to 4 years tended to be increased but not significantly (HR=2.6, 95%CI 0.9 to 7.9).

Distance VF loss was associated with an increased risk of dementia only beyond 4 years (HR=2.8, 95%CI 1.5 to 5.3).

DISCUSSION

Within a large population-based cohort followed up over 12 years, we found that moderate to severe near VI was associated with an increased risk of dementia in the first 2 years and from 2 to 4 years after adjusting for multiple potential confounders, whereas mild near VI was associated with dementia only in the first 2 years. Less engagement in cognitively stimulating activities only slightly decreased these associations. However, stratified analyses on depressive symptomatology showed an interaction between vision loss and depressive symptomatology, such that moderate to severe near visually impaired participants had an increased risk of dementia in the first 2 years, from 2 to 4 years (at the limits of the significance) and beyond 4 years only when depressive symptomatology was present, with a nearly threefold increased risk. Participants self-reporting distance VF loss had an increased risk of dementia only beyond 4 years, but that association was no longer significant after taking into account baseline cognitive performances.

Most of the previous research in this area has focused on cognition rather than dementia [START_REF] Chen | Association of Vision Loss With Cognition in Older Adults[END_REF][START_REF] Spierer | Correlation Between Vision and Cognitive Function in the Elderly: A Cross-Sectional Study[END_REF][START_REF] Mine | Association of Visual Acuity and Cognitive Impairment in Older Individuals: Fujiwara-kyo Eye Study[END_REF][START_REF] Garin | Visual impairment and multimorbidity in a representative sample of the Spanish population[END_REF][START_REF] Ong | Visual impairment, age-related eye diseases, and cognitive function: the Singapore Malay Eye study[END_REF][START_REF] Tay | Sensory and cognitive association in older persons: findings from an older Australian population[END_REF][START_REF] Clemons | Age-Related Eye Disease Study Research Group. Cognitive impairment in the Age-Related Eye Disease Study: AREDS report no. 16[END_REF][START_REF] De Kok | What is the relationship between visual impairment and cognitive function in octogenarians?[END_REF][START_REF] Diaz | Cognitive profile of elders in an ophthalmic ambulatory setting[END_REF][START_REF] Hong | Visual Impairment, Hearing Loss and Cognitive Function in an Older Population: Longitudinal Findings from the Blue Mountains Eye Study[END_REF][START_REF] Fischer | Age-Related Sensory Impairments and Risk of Cognitive Impairment[END_REF][START_REF] Valentijn | Change in sensory functioning predicts change in cognitive functioning: results from a 6-year follow-up in the maastricht aging study[END_REF][START_REF] Reyes-Ortiz | Near vision impairment predicts cognitive decline: data from the Hispanic Established Populations for Epidemiologic Studies of the Elderly[END_REF][START_REF] Sloan | Effects of changes in self-reported vision on cognitive, affective, and functional status and living arrangements among the elderly[END_REF][START_REF] Lin | Vision impairment and combined vision and hearing impairment predict cognitive and functional decline in older women[END_REF]. Within cross-sectional studies, the results show either a significant association between VI and cognition [START_REF] Chen | Association of Vision Loss With Cognition in Older Adults[END_REF][START_REF] Spierer | Correlation Between Vision and Cognitive Function in the Elderly: A Cross-Sectional Study[END_REF][START_REF] Mine | Association of Visual Acuity and Cognitive Impairment in Older Individuals: Fujiwara-kyo Eye Study[END_REF][START_REF] Garin | Visual impairment and multimorbidity in a representative sample of the Spanish population[END_REF][START_REF] Ong | Visual impairment, age-related eye diseases, and cognitive function: the Singapore Malay Eye study[END_REF][START_REF] Tay | Sensory and cognitive association in older persons: findings from an older Australian population[END_REF][START_REF] Clemons | Age-Related Eye Disease Study Research Group. Cognitive impairment in the Age-Related Eye Disease Study: AREDS report no. 16[END_REF] or no association [START_REF] De Kok | What is the relationship between visual impairment and cognitive function in octogenarians?[END_REF][START_REF] Diaz | Cognitive profile of elders in an ophthalmic ambulatory setting[END_REF]. In longitudinal studies, significant associations between VI and cognitive decline have been found [START_REF] Fischer | Age-Related Sensory Impairments and Risk of Cognitive Impairment[END_REF][START_REF] Reyes-Ortiz | Near vision impairment predicts cognitive decline: data from the Hispanic Established Populations for Epidemiologic Studies of the Elderly[END_REF][START_REF] Sloan | Effects of changes in self-reported vision on cognitive, affective, and functional status and living arrangements among the elderly[END_REF][START_REF] Lin | Vision impairment and combined vision and hearing impairment predict cognitive and functional decline in older women[END_REF], using different assessments of VI, either a measure of contrast sensitivity [START_REF] Fischer | Age-Related Sensory Impairments and Risk of Cognitive Impairment[END_REF], a measure of presenting near VA [START_REF] Reyes-Ortiz | Near vision impairment predicts cognitive decline: data from the Hispanic Established Populations for Epidemiologic Studies of the Elderly[END_REF] or presenting distance VA [START_REF] Lin | Vision impairment and combined vision and hearing impairment predict cognitive and functional decline in older women[END_REF] or self-reported decline in near or distance vision [START_REF] Sloan | Effects of changes in self-reported vision on cognitive, affective, and functional status and living arrangements among the elderly[END_REF]. Regarding distance vision, however, other studies using measures of presenting distance VA [START_REF] Reyes-Ortiz | Near vision impairment predicts cognitive decline: data from the Hispanic Established Populations for Epidemiologic Studies of the Elderly[END_REF] or best-corrected distance VA [START_REF] Hong | Visual Impairment, Hearing Loss and Cognitive Function in an Older Population: Longitudinal Findings from the Blue Mountains Eye Study[END_REF][START_REF] Valentijn | Change in sensory functioning predicts change in cognitive functioning: results from a 6-year follow-up in the maastricht aging study[END_REF] have not found any association, or they found an association only when decline in distance vision was considered [START_REF] Valentijn | Change in sensory functioning predicts change in cognitive functioning: results from a 6-year follow-up in the maastricht aging study[END_REF]. However, the cognitive tests have differed between studies, with some including items requiring vision [START_REF] Fischer | Age-Related Sensory Impairments and Risk of Cognitive Impairment[END_REF][START_REF] Sloan | Effects of changes in self-reported vision on cognitive, affective, and functional status and living arrangements among the elderly[END_REF][START_REF] Lin | Vision impairment and combined vision and hearing impairment predict cognitive and functional decline in older women[END_REF] whereas others have used blind versions of cognitive tests [START_REF] Hong | Visual Impairment, Hearing Loss and Cognitive Function in an Older Population: Longitudinal Findings from the Blue Mountains Eye Study[END_REF][START_REF] Reyes-Ortiz | Near vision impairment predicts cognitive decline: data from the Hispanic Established Populations for Epidemiologic Studies of the Elderly[END_REF]. Only one study presented results according to several cognitive tests. The authors found significant associations between measured decline in distance vision and several cognitive tests exploring speed, executive functions and memory, but all of them required visual capacities. In contrast, there was no association with the only test not requiring vision, i.e., the verbal fluency test [START_REF] Valentijn | Change in sensory functioning predicts change in cognitive functioning: results from a 6-year follow-up in the maastricht aging study[END_REF].

In addition, to our knowledge, only one study has explored the longitudinal association between VL and risk of dementia [START_REF] Rogers | Untreated poor vision: a contributing factor to late-life dementia[END_REF], focusing only on self-reported VL without exploring the potential factors involved in that association. In this American retrospective study on 625 older adults aged 71 years and older, who were followed up over 8.5 years, the authors found that participants who reported their corrected (if applicable) vision as good or excellent at baseline had a reduced 63% risk of dementia after adjusting for potential confounders. However, there was no indication about the kind of vision (near or distance) studied and the authors did not evaluate the effect of vision loss on dementia risk over time.

Several hypotheses have been proposed to explain the association between vision and dementia, either in favor of a direct role of VL or via confounding factors, measurement bias and common processes [START_REF] Lindenberger | Sensory functioning and intelligence in old age: a strong connection[END_REF][START_REF] Baltes | Emergence of a powerful connection between sensory and cognitive functions across the adult life span: a new window to the study of cognitive aging?[END_REF]. To limit the impact of potential confounders, we adjusted for numerous factors, including age, socio-economic factors, hearing impairment and vascular factors. Although residual confounds cannot be totally excluded, none of these adjustments explained the association. Moreover, as previous authors suggested that VL could be one of the first symptoms of dementia [START_REF] Bowen | The Prevalence of Visual Impairment in People with Dementia (the PrOVIDe study)[END_REF], we adjusted for baseline cognitive performance, and we assessed whether vision loss had a constant effect over time, by modeling the effect of vision loss on the risk of dementia in the first 2 years, from 2 to 4 years and beyond 4 years after inclusion. The risk of dementia associated with near vision loss was indeed higher in the two first periods than beyond 4 years. Thus, reverse causation cannot be excluded and some participants were probably in a pre-dementia phase with their visual impairment being one of the symptoms of this pre-dementia phase. However, although not significant, there was a trend to an association between near vision loss and dementia beyond 4 years in the main analysis. Moreover, near vision loss was associated with a significantly increased risk beyond 4 years among participants with depressive symptoms, with a three-fold increased risk. In addition, distance VF loss tended also to be associated with an increased risk beyond 4 years, suggesting that vision loss could be a risk factor of dementia.

Previous studies have also suggested that the effect of VL on dementia may be achieved through intermediate factors, in particular decreased engagement in cognitively stimulating activities [START_REF] Laitinen | A Nationwide Population-Based Survey on Visual Acuity, Near Vision, and Self-Reported Visual Function in the Adult Population in Finland[END_REF][START_REF] Kiely | Dual sensory loss and depressive symptoms: the importance of hearing, daily functioning, and activity engagement[END_REF] or an increased level of depressive symptomatology [START_REF] Carrière | A prospective study of the bi-directional association between vision loss and depression in the elderly[END_REF][START_REF] Hong | Visual impairment and depressive symptoms in an older Australian cohort: longitudinal findings from the Blue Mountains Eye Study[END_REF]. In our population, in spite of decreased engagement in cognitively stimulating activities by participants with near VI, taking into account these activities only slightly explained the association between near VI and dementia. We cannot exclude that the measurement of activities was not accurate enough or occurred not timely in the relationship between VI and dementia, but it seems not to be the main factor involved. On the contrary, depressive symptomatology seems to be particularly involved in the relationship between vision loss and dementia. Indeed, an interaction between vision loss and depressive symptomatology was found with a risk of dementia for visually impaired participants significantly increased only for those with depressive symptomatology. As such, VL could worsen the risk of dementia in participants with pre-existing depressive symptoms. The mechanisms of this interaction need to be further explored to understand if there are potential underlying pathophysiological mechanisms, or if it acts through environmental or social factors, for example, social isolation and its consequences on recourse to care. Moreover, whether depression is a risk factor or a prodromal symptom of dementia is still unknown [START_REF] Li | Hearing Impairment Associated With Depression in US Adults, National Health and Nutrition Examination Survey 2005-2010[END_REF]. Thus, this increased risk of dementia in participants with vision loss and depressive symptoms could also reflect at least partly the onset of dementia more so than a real increased risk. Furthermore, the direction of the relation between visual impairment and depression is still uncertain, but previous papers from the 3C-Study showed an increased risk of depressive symptomatology or suicidal ideation in participants with visual loss ( [START_REF] Carrière | A prospective study of the bi-directional association between vision loss and depression in the elderly[END_REF][START_REF] Cosh | Sensory loss and suicide ideation in older adults: findings from the Three-City cohort study[END_REF]).

Our results have some limitations. Even after attempting to explore possible factors involved and in spite of a longitudinal design with 12 years of follow-up, the temporal sequence between visual loss, engagement in cognitively stimulating activities and depression is difficult to evaluate. Indeed, all of these factors can change over time. Moreover, distance vision was self-reported using a single question; thus, it is less accurate than other standard measures. Indeed, the meaning of 4-meters distance is really difficult to estimate and may vary from one participant to another. Moreover self-reported ability of vision loss probably varies according to cognitive capacities. However, surprisingly the association between distance VF loss and dementia was observed only beyond 4 years, even if it was no longer significant after adjustment for global cognition. For near vision, the test used (the Parinaud chart) required the ability to read, which can potentially be impaired in cognitive impairment. However, the association between near VI and dementia remained after additionally adjusting for baseline cognition. In addition, other parameters of vision, such as contrast sensitivity or visual field, are probably important to study but were not available in our study. Moreover, we did not have information about best-corrected visual acuity neither about the cause of the decreased near vision loss, which could be due to under corrected refractive error or eye-diseases such as glaucoma or age-related macular degeneration. Indeed, some studies have suggested that dementia and some eye diseases could share common agerelated pathogenesis [START_REF] Baker | Early age-related macular degeneration, cognitive function, and dementia: the Cardiovascular Health Study[END_REF]. However, our objective was to study the association between vision loss (evaluated with visual acuity used in daily life) and dementia, whatever the cause of vision loss. Beyond clinical diagnosis of dementia, it would be of great interest to document whether vision loss is associated with imaging markers of Alzheimer's disease and dementia. Within a subsample of the 3C-Bordeaux participants we failed to find a cross-sectional association between vision loss and hippocampus volume. However, these analyses deserve to be further explored and replicated on a larger sample. Finally, engagement in cognitively stimulating activities was assessed using self-administered questionnaires, with some missing data. As expected, the participants answering the questionnaire were younger, more educated, had less cardiovascular risk factors and better cognition at baseline. However, the associations were almost unchanged in this subsample.

The strengths of our results are attributable to a large population-based cohort with a long period of follow-up, a baseline measure of presenting binocular near visual acuity using a standardized scale and an adjustment for numerous major potential confounding factors. Moreover, we actively screened for dementia using validation by an independent committee. We explored factorsnamely, depressive symptomatology and engagement in cognitively stimulating activitiesthat may be involved in the association between VL and risk of dementia. Finally, we assessed the effect of vision loss on dementia over time.

Conclusions

This longitudinal population-based study suggests that moderate to severe near VI could represent an indicator of dementia risk in the subsequent years, particularly in people suffering from depression. These results need to be replicated in other large longitudinal studies with both measure of near and distance visual acuity, and potential mediators need to be further explored. A large part of VL is correctable or preventable [START_REF] Naël | Prevalence and Associated Factors of Uncorrected Refractive Error in Older Adults in a Population-Based Study in France[END_REF] . However, future researches and interventional studies are needed to further evaluate whether VL is only an indicator of future dementia or whether the improvement of VL, may represent a promising opportunity for dementia prevention.

COMPLIANCE WITH ETHICAL STANDARDS

Conflict of interest

Research involving Human Participants

All procedures performed in studies involving human participants were in accordance with the ethical standards of the Kremlin-Bicêtre University Hospital and Sud-Mediterranée III committee and with the 1964 Helsinki declaration and its later amendments or comparable ethical standards.

Informed consent

Informed consent was obtained from all individual participants included in the study.

Incident

  Authors V. Naël and AC Scherlen are Essilor International employees. Author C. Delcourt is a consultant for Bausch & Lomb, Novartis, and Laboratoires Théa and has received research grants from Laboratoires Théa. The authors K. Pérès, JF. Dartigues, L. Letenneur, H. Amieva, A. Arleo, I. Carrière, C. Tzourio, C. Berr and C. Helmer declare that they have no conflict of interest.

Table 2 . Risk of dementia by vision loss in the first 2 years, from 2 to 4 years and beyond 4 years after inclusion: The Three-City (3C) cohort study (n=7736)

 2 Cox models for the risk of dementia with age used as the time-scale except for age, which was tested using a 2-tailed T

		No incident	P a	Total
	dementia	demented
	(n=882)	(n=6854)

a a Adjusted for center, age, gender, educational level, for hearing loss, living alone, income, depressive symptomatology at baseline, smoking habits, body mass index, hypertension, history of stroke, history of cardiovascular disease, hypercholesterolemia, hypertriglyceridemia, diabetes and APOE4 genotype b Separate models

Table 3 . Risk of dementia by vision loss, with supplementary adjustment for engagement in cognitively stimulating activities: The Three-City (3C) cohort study

 3 Model re-run among participants without missing data on cognitively stimulating activities b Model adjusted for center, age, gender, educational level, hearing loss, living alone, income, smoking habits, body mass index, hypertension, history of stroke, history of cardiovascular disease, hypercholesterolemia, hypertriglyceridemia, diabetes and APOE4 genotype c Separate models

	(n=7089)
		<2 years (n=611)			2-4 years (n=1190)			≥4 years (n=5288)
	No of	HR	95%CI	P a	No of	HR	95%CI	P a	No of	HR	95%CI	P a
	cases				cases				cases

a

Table 4 . Risk of dementia stratified by depressive symptomatology: The Three-City (3C) cohort study (n=7561) No depressive symptoms (n=6610)

 4

			<2 years (n=552)			2-4 years (n=1080)			≥4 years (n=4978)
		No of	HR	95%CI	P a	No of	HR	95%CI	P a	No of	HR	95%CI	P a
		cases				cases				cases
	Near visual impairment	100				113				487
	No	71	Ref			86	Ref			406	Ref
	Mild	20	1.62	0.98 -2.68	0.06	16	1.18 0.69 -2.03	0.54	58	1.02	0.76 -1.36	0.90
	Moderate to severe	9	1.48	0.73 -2.97	0.28	11	1.70 0.90 -3.20	0.10	23	1.07	0.70 -1.63	0.77
	Distance visual function	6	0.80	0.35 -1.83	0.60	10	1.26 0.66 -2.42	0.49	35	1.25	0.88 -1.77	0.22
	loss b
					Depressive symptoms (n=951)

<2 years (n=115) 2-4 years (n=193) ≥4 years (n=643)

	Supplementary
		No of	HR	95%CI	P a	No of	HR	95%CI	P a	No of	HR	95%CI	P a
		cases				cases				cases
	Near visual impairment	30				28				92
	No	19	Ref			21	Ref			73	Ref
	Mild	6	2.11	0.83 -5.39	0.12	3	1.07 0.31 -3.63	0.92	10	1.18	0.59 -2.37	0.65
	Moderate to severe	5	2.87	1.04 -7.94	0.04	4	2.62 0.87 -7.89 0.087	9	3.07	1.46 -6.47	0.003

Table 2 . Risk of dementia by vision loss in the first 4 years, from 4 to 7 years and beyond 7 years after inclusion: The Three-City (3C) cohort study

 2 Adjusted for center, age, gender, educational level, for hearing loss, living alone, income, depressive symptomatology at baseline, smoking habits, body mass index, hypertension, history of stroke, history of cardiovascular disease, hypercholesterolemia, hypertriglyceridemia, diabetes and APOE4 genotype

	(n=7736)
			<4 years (n=2009)			4-7 years (n=902)			≥7 years (n=4825)
		No of	HR	95%CI	P a	No of	HR	95%CI	P a No of	HR	95%CI	P a
		cases				cases				cases
	Near visual impairment	289				331				262
	No	209	Ref			276	Ref			213	Ref
	Mild	47	1.38	1.00 -1.91	0.0504	35	0.88 0.62 -1.26	0.50	33	1.17 0.81 -1.70	0.41
	Moderate to severe	33	1.88	1.29 -2.73	0.001	20	1.22 0.77 -1.93	0.39	16	1.52 0.91 -2.53	0.11
	Distance visual function	27	1.17	0.79 -1.75	0.44	29	1.45 0.99 -2.12	0.0597	21	1.55 0.99 -2.44	0.0547
	loss b
	Abbreviations: CI= Confidence Interval; HR= Hazard Ratio

a b Separate models

Table 1. Baseline characteristics according to incident dementia over the 12-year follow-up: The Three-City (3C)

cohort study (n=7736)

ACKNOWLEDGMENTS

Study funding: The Three-City study is conducted under a partnership agreement between the Institut National de la Santé et de la Recherche Médicale (INSERM), the University Bordeaux 2 Victor Segalen and Sanofi-Aventis. The Fondation pour la Recherche Médicale funded the preparation and initiation of the study. The Three-City study is also supported by the Caisse Nationale Maladie des Travailleurs Salariés, Direction Générale de la Santé, MGEN, Institut de la Longévité, Conseils Régionaux d'Aquitaine et Bourgogne, Fondation de France, Ministry of Research-INSERM Programme "Cohortes et collections de données biologiques", Agence Nationale de la Recherche ANR PNRA 2006 and LongVie 2007, the "Fondation Plan Alzheimer" (FCS 2009-2012) and the Caisse Nationale de Solidarité pour l'Autonomie (CNSA). None of the sponsors participated in the collection, management, statistical analysis and interpretation of the data, nor in the preparation, review or approval of the present manuscript. SENSE-Cog has received funding from the European Union's Horizon 2020 research and innovation program under grant agreement No 668648.