Isopropyl‐phloroglucinol‐DHA protects outer retinal cells against lethal dose of all‐trans‐retinal

Abstract All‐trans‐retinal (atRAL) is a highly reactive carbonyl specie, known for its reactivity on cellular phosphatidylethanolamine in photoreceptor. It is generated by photoisomerization of 11‐cis‐retinal chromophore linked to opsin by the Schiff's base reaction. In ABCA4‐associated autosomal recessive Stargardt macular dystrophy, atRAL results in carbonyl and oxidative stress, which leads to bisretinoid A2E, accumulation in the retinal pigment epithelium (RPE). This A2E‐accumulation presents as lipofuscin fluorescent pigment, and its photooxidation causes subsequent damage. Here we describe protection against a lethal dose of atRAL in both photoreceptors and RPE in primary cultures by a lipidic polyphenol derivative, an isopropyl‐phloroglucinol linked to DHA, referred to as IP‐DHA. Next, we addressed the cellular and molecular defence mechanisms in commonly used human ARPE‐19 cells. We determined that both polyunsaturated fatty acid and isopropyl substituents bond to phloroglucinol are essential to confer the highest protection. IP‐DHA responds rapidly against the toxicity of atRAL and its protective effect persists. This healthy effect of IP‐DHA applies to the mitochondrial respiration. IP‐DHA also rescues RPE cells subjected to the toxic effects of A2E after blue light exposure. Together, our findings suggest that the beneficial role of IP‐DHA in retinal cells involves both anti‐carbonyl and anti‐oxidative capacities.

11cRAL to atRAL in POS, the atRAL Schiff base is hydrolysed, yielding the photochemically inactive opsin protein and the freed atRAL. 2 Removal of the latter is required to avoid acute toxicity, and delayed clearance of atRAL after light exposure contributes to light-induced retinal degeneration. 3 The atRAL is cleared from disc membranes of POS by retinal ATP-binding cassette (ABCA4) transporter proteins to the cytoplasm where atRAL-dehydrogenase (RDH) catalyses its reduction to the much less reactive all-trans-retinol (atROL).
Mutations in the ABCA4 gene are found in patients with Stargardt macular dystrophy (STGD1), cone-rod dystrophy and recessive retinitis pigmentosa, and variants in ABCA4 increased susceptibility to age-related macular degeneration (AMD). 4 Mechanisms of acute toxicity of atRAL were previously studied by Palczewski and coworkers. 3,5 They first reported that NADPH oxidase at the plasma membrane can be activated by an increase in atRAL levels via the phospholipase C/ inositol 1,4,5-triphosphate pathway, resulting in overproduction of reactive oxygen species (ROS). The respiratory chain in mitochondria also participates in ROS production (the more reactive being HO · ) within the cell in reply to atRAL accumulation. 3 More recently, atRAL has been shown to induce mitochondrial transmembrane potential loss and endoplasmic reticulum (ER) stress that ultimately trigger programmed cell death by activating apoptotic Bax-and caspase-dependent cascades. 6,7 Free atRAL is itself a reactive carbonyl compound through its all trans-polyene conjugated aldehyde that is toxic to cells. 8,9 In ABCA4-associated pathologies, atRAL accumulates due to delayed clearance by the defective ABCR transporter. Nickell et al 10 reported that rhodopsin is present at a concentration of 4.62 mmol/L in disc membranes of rod outer segments. Therefore, the level of atRAL released after photoactivation of rhodopsin can range from 25 to 100 μmol/L in the disc membranes following photobleaching of only 0.5%-2%. This level of atRAL is toxic in cultured retinal cells. 7,9 Excess atRAL is a potent photosensitizer which can mediate light-induced oxidation. 11 However, atRAL condenses on the PE by a double mechanism of carbonyl and oxidative stress. 12,13 This leads to decrease atRAL levels and to the formation of bisretinoid adducts such as A2E and RAL dimer, which are pigments of retinal pigment epithelium (RPE) autofluorescent lipofuscin. 14 These pigments are sensitive to visible blue light and are photo-oxidized and fragmented accordingly. 15 The oxidized metabolites are reactive carbonyl and oxidative species that would have toxic effects in the RPE. 16 Based on epidemiology studies, natural antioxidants such as polyphenols appear as efficient protectors against oxidative stress.
This activity may be related to their capacity to block the formation and accumulation of ROS or to stimulate the enzymatic antioxidant defences of the organism. 17 Literature also addressed the efficiency of polyphenols to act as anti-carbonyl stressor agents by trapping reactive toxic carbonyl entities. 18 We previously reported in vitro cytoprotective effects of the polyphenol phloroglucinol, a natural monomer of phlorotannins abundantly present in Ecklonia cava (edible brown algae), in outer retinal cells by scavenging ROS and trapping atRAL. 9 Because of its low bioavailability, phloroglucinol was then structurally modified by the addition of polyunsaturated fatty acid (PUFA) and isopropyl substituents. 13 In the present study, we investigated the protective effect of the medicinal chemical compound, isopropyl-phloroglucinol-DHA (IP-DHA), also called lipophenol, against atRAL-related carbonyl and oxidative stresses (COS). We first analysed primary cultures of outer retina to demonstrate the dose-dependent protective effect against lethal dose of atRAL. We then used ARPE-19 cells as a standard cellular model to study the mechanisms of cell death and protection.
We demonstrate that each of the structural part, that is isopropyl and PUFA, is essential for the full action of the lipophenol with selectivity for LA and DHA. We compared the capacities of phloroglucinol and IP-DHA to reverse the effects of atRAL and finally discuss how to understand the enhanced protective effects of IP-DHA compared to phloroglucinol.

| Synthesis of phloroglucinol lipophenols
To evaluate the influence of different lipid chains and of the isopropyl substituent, several lipophenols were synthesized: five lipophenols with an isopropyl-phloroglucinol (IP) core linked to various fatty acid, IP-DHA, IP-EPA, IP-ALA, IP-LA and IP-C22, and three lipophenols using only the phloroglucinol without alkyl substituent, P-DHA, P-EPA and P-LA. All the lipophenols were synthesized according to the chemical strategy developed by Crauste et al. 13 Briefly, one hydroxyl group of the phloroglucinol or IP is protected by triisopropylsilyl (TIPS) groups using triflate reagent (TIPS-OTf) and diisopropylethylamine (DIPEA) as a base to obtain the protected derivative. The coupling reactions between the protected polyphenol and the different fatty acids, docosahexaenoic acid (DHA), eicosapentaenoic acid (EPA), α-linolenic acid (ALA), linoleic acid (LA) and behenic acid (C22), were initiated using dicyclohexylcarbodiimide and dimethylaminopyridine (DCC/DMAP) as coupling reagents to access the protected lipophenols. Deprotection of the TIPS groups by Et 3 N-3HF in dry tetrahydrofuran (THF) yielded final lipophenols compounds, IP-DHA, IP-EPA, IP-ALA, IP-LA, IP-C22, P-DHA, P-EPA and P-LA. A quality control assessment was established by a complete 1 H and 13 C NMR spectral analysis for each synthesized compound (chemical structure, general procedure, yield and NMR analysis are reported in Supporting Information).

| Cell cultures and atRAL treatment
Primary rat RPE and mouse neural retina (NR) cultures were obtained as previously described. 9 RPE cells were cultured for 3 days until they reached 80%-85% confluency, and NR was cultured for 10 days until glial cells were confluent in the bottom layer and neural cells with a neurite outgrowth in the upper layer. Human RPE-like cells, ARPE-19, were seeded at 100 000 cells/cm 2 and grown to confluency before being assayed as instruct by ATCC.
Pre-and co-treatment procedures with lipophenol were carried out. During pre-treatments, rat RPE primary cultures received a medium containing lipophenol at different concentrations (40-320 μmol/L) for 24 hours. The medium was then removed and replaced by a serum-free culture medium containing 25 μmol/L atRAL for 4 hours. During co-treatments, RPE and NR cells received a serum-free medium containing 25 μmol/L atRAL and/or IP-DHA (10-320 μmol/L) for 4 hours. NR cells were refreshed with serum-free medium the next day.
The cell cultures were treated with serum-free DMEM/F12 medium without phenol red containing IP-DHA at different concentrations (0-80 μmol/L) for 1 hour. A2E was then added to a final concentration of 20 μmol/L for 6 hours before rinsing with medium. Control cells were incubated with 0.2% DMSO with or without A2E. The cells were exposed to intense blue light (4600 lux) for 30 minutes to induce phototoxicity of A2E and incubated at 37°C. Irradiation was achieved using a LED device with blue emission wavelengths from 430 to 470 nm and a dimmable luminance (Roleadro lighting).
Control ambient white light was less than 300 lux. Without blue light exposure, A2E-loaded cell survival was not affected. The cell viability was determined 16-20 hours later using a MTT colorimetric assay. Results are expressed in percentage of viable cells normalized to control conditions in the absence of lipophenol and stressor.

| Cell viability
Cell viability in primary RPE and ARPE-19 was determined by MTT assay in 96-well plates as described. 9 To distinguish viable cells from

| Mitochondrial respirometry
Mitochondrial respiration was measured in ARPE-19 cultured in six-well plates after 4-hour exposure to 25 μmol/L atRAL and/or 40 μmol/L lipophenol. Respiration was measured on 10 6 cells permeabilized by incubation for 2 minutes with 15 µg digitonin and resuspended in a respiratory buffer (pH 7.4, 10 mmol/L KH 2 PO 4 , 300 mmol/L mannitol, 10 mmol/L KCl and 5 mmol/L MgCl 2 ). The respiratory rates were recorded at 37°C in 2-mL glass chambers using a high-resolution Oxygraph respirometer (Oroboros) as recently described. 20 Assays were initiated in the presence of 5 mmol/L malate/pyruvate to measure basal respiration. (state 2), Complex I-coupled state 3 respiration was measured by adding 0.5 mmol/L NAD + /1.5 mmol/L ADP. Then, 10 mmol/L succinate was added to reach maximal coupled respiration, and 10 μmol/L rotenone was injected to obtain the CII-coupled state 3 respiration. Oligomycin (8 µg/mL) was added to determine the uncoupled state 4 respiration rate. Finally, carbonyl cyanide-4-(trifluoromethoxy) phenylhydrazone (1 μmol/L) was added to control the permeabilization of the tissues.
The respiration rate driven by complex IV was measured starting from CII-coupled state 3 and the addition of antinomycin A (1 μmol/L), which inhibited complex III, and of ascorbate/TMPD redox couple to reduce cytochrome c.

| Mitochondrial enzymatic activities
The enzymatic activity of the mitochondrial respiratory chain complexes (RCC) was measured on cell homogenates as described previously. 21 Briefly, ARPE-19 was grown in six-well plates and treated for 4 hours with 25 μmol/L atRAL and/or 40 μmol/L lipophenol. Cells were scraped, rinsed with DPBS and resuspended on ice with cell buffer (250 mmol/L saccharose, 20 mmol/L tris[hydroxymethyl]aminomethane, 2 mmol/L EGTA, and 1 mg/mL bovine serum albumin (BSA), pH 7.2). Cell was disrupted by two freezing-thawing cycles, centrifuged at 16 000 g for one minute and suspended in the cell buffer (50 µL/10 6 cells). The cellular protein content was determined with the Bicinchoninic assay kit (Pierce) using BSA as standard. The initial kinetics of enzymatic activities were monitored by spectrophotometry (UV-SAFAS spectrophotometer, SAFAS monaco). Complex I (NADH ubiquinone reductase) activity was measured as described elsewhere 22 and adapted using 2, 6 dichloroindophenol (DCPIP) to avoid inhibition of complex I activity by decylubiquinol. 23 Complex II (succinate ubiquinone reductase) activity was measured according to James et al. 24 Specific enzymatic activities of complexes I and II were expressed in mIU (ie nanomoles of DCPIP/min/mg protein).
Complex IV (cytochrome c oxidase) activity was recorded according to a method by Rustin et al, 25 adapted in a 50 mmol/L KH 2 PO 4 buffer, using 15 μmol/L reduced cytochrome c. Specific enzymatic activity was expressed in mIU (ie nanomoles of cyt c/min/mg protein).

| ROS production
ROS were measured in ARPE-19 using the H 2 DCFDA probe as described 9 with minor modifications. Radicals such as peroxyl, alkoxyl, NO 2 · , carbonate or HO · are able to oxidize H 2 DCFDA and thus to be quantified by this assay. 14

| Catalase activity
ARPE-19 cells were seeded in six-well plates and treated as aforementioned. Immediately after treatment, cells were scrapped on ice,

| Western blot analysis
ARPE-19 cells were lysed in RIPA buffer containing protease inhibitors, homogenized and then centrifuged at 9600 g for 3 minutes.
Twenty-five micrograms of the protein lysates in Laemmli buffer were separated on 10% SDS-PAGE (Mini-PROTEAN ® TGX™ gels, Bio-Rad) and electrotransferred to PVDF membranes (Trans-Blot ® Turbo™ Transfer System, Bio-Rad). After blocking, membranes were blotted overnight at 4°C with primary antibodies. After incubation with the corresponding HRP-conjugated secondary antibodies, detection was performed using an enhanced chemiluminescence kit (Pierce ECL, Thermo Scientific) and recorded by the V3 Western Worflow™ system (Bio-Rad). The bands were semi-quantified using densitometry by ImageJ software. Commercial antibodies were used to assess protein expression as follows: monoclonal mouse anti-GAPDH (Sigma-Aldrich ® , G8795 diluted to 1:5000, 37 kD); mouse anti-α-tubulin (Sigma-Aldrich ® T5168, diluted to 1:4000,

| XCELLigence assay
The xCELLigence system (Roche and ACEA Biosciences) was used to monitor cell adhesion, proliferation and cytotoxicity. The xCELLigence system was connected and tested by Resistor Plate before the RTCA Single Plate station was placed inside the incubator at 37°C and 5% CO 2 . First, the optimal seeding concentration for the prolif-

| Statistics
Statistical analyses were performed using GraphPad Prism 5.0.
Software. Data were first analysed with Shapiro-Wilk normality test, and then, two-tailed P-values were determined using either the unpaired Student's t test or the non-parametric Mann-Whitney test. A P-value < .05 was considered significant. The linear correlation was measured by Pearson's r correlation coefficient.

| IP-DHA protects retinal primary cultures against atRAL toxicity
ABCA4-associated retinopathies often affect both photoreceptor and RPE in a manner that is not fully elucidated, 5,14 but which originally involves a defective retinal clearance from the photoreceptor. We tested the protection with IP-DHA of atRAL-challenged primary cultures of rat RPE ( Figure 1A,B) and mouse NR enriched in photoreceptor cells ( Figure 1C). These analyses confirmed significant protective effects of IP-DHA against atRAL, regardless of the mode of treatment (pre-vs co-treatment Figure 1A,B), the density ( Figure 1BB1,BB2), or the cell type (RPE and photoreceptor, Figure 1A,B,C). Furthermore, IP-DHA did not show cytotoxicity on the primary RPE at a concentration of 320 μmol/L up to eight times higher than the one we previously reported using the ARPE-19 cell line. 13 Thus, IP-DHA is able to protect both RPE and photoreceptor from the toxic effects of atRAL overload without adverse effect.

| Structure-function relationship of IP-DHA
A selection of fatty acids (omega-6, omega-3 and saturated fatty acids) that were conjugated to IP showed a structural selectivity for protection efficacy of ARPE-19 cells challenged with a toxic dose of atRAL ( Figure 2A). The rank of efficacy was LA ≥ DHA > EPA = ALA > C22.
This order was not correlated with the level of fatty acid unsaturation (DHA > EPA > ALA > LA > C22), nor with the rank of the toxicity of free fatty acid (EPA > DHA > ALA > LA > C22, Figure 2B). As oxidation levels are correlated with cell toxicity, PUFA toxicity may come from lipid peroxidation. 28 However, coupling polyunsaturated FA (PUFA) to IP or phloroglucinol (P) significantly reduced PUFA toxicity ( Figure 2C,D, respectively). These data highlight that isopropyl does not alter the low toxicity of lipophenols. Regardless of the fatty acid, isopropyl function was necessary for effective protection, the protective effect was lost upon use of non-alkylated lipophenols (P-fatty acid, Figure 2E). These results demonstrated that both PUFA and isopropyl are essential for lipophenol activity.
The choice to use IP-DHA throughout this study rather than IP-LA, despite the latter's showing better protection against atRAL is justified in view of planned in vivo evaluations. DHA has general and specific transporters to concentrate DHA in the photoreceptors. Therefore, the use of DHA seems more appropriate to improve the uptake of IP-DHA by the retina. In addition, an omega-3 such as DHA, which can be released by the enzyme esterase, can have beneficial effects on human retinal diseases that omega-6, known for their superior pro-inflammatory properties, cannot reproduce.

| Cell-based assays of IP-DHA protection
Dynamic cellular biology was first monitored using the xCELLigence System in ARPE-19 before and after treatment with atRAL and/or IP-DHA. The system measures electrical impedance which provides quantitative information about the biological status of the cells, including cell number, viability and morphology. The xCELLigence read-out is a dimensionless parameter called Cell Index (CI) that was normalized with the time-point before the treatment ~16.5 hours after plating ( Figure S1). The addition of 25 μmol/L atRAL significantly decreased the CI, which then stabilized 4 hours later at 11 ± 2% ( Figure S1; Table S1). Co-incubation with 40 μmol/L IP-DHA limited this decrease to 56 ± 11% two hours after the beginning of treatment, and this effect lasted throughout the analysis (37 hours).
We conclude that IP-DHA responds rapidly against the toxicity of atRAL with persisting protective effect.
Consequently, we applied a 4-hour co-treatment with atRAL and IP-DHA to assess the protection against detrimental effects induced by atRAL overload in ARPE-19 ( Figure 3). As shown in Figure 3A Morphologic changes in ARPE-19 cells were observed following atRAL exposure ( Figure 3C) and an apoptotic caspase 3-cleavage signal was detected by Western blot (Figure 3D,E). Long (24 hour's pre-) and short (4-hour co-) IP-DHA treatment restored healthy morphology and abolished the caspase-dependent apoptosis. Previous reports revealed that atRAL could directly act on and elicit a poisonous effect in mitochondria. 3,7 We therefore performed respirometry to assess the functionality of mitochondrial RCC ( Figure 4A showed that all RCC were impaired by atRAL and partially rescued by IP-DHA treatment ( Figure 4C). Thus, the protective effect of IP-DHA seems to apply directly to the mitochondrial respiration essential to the cell viability.

| Molecular and cellular mechanisms of IP-DHA protection
Natural polyphenols were reported as potent against COS involved in age-related diseases, 29 either as sequestrating agents of F I G U R E 1 Protection of retinal primary cultures by IP-DHA against atRAL. A, pre-treatment of RPE cells with IP-DHA inhibits atRALinduced cell death. A1, rat primary RPE cells were cultured in 96-well plates and pre-treated with increasing concentrations of IP-DHA for 24 h, washed and exposed to 25 μmol/L atRAL for 4 h. A2, RPE cultures were incubated for 24 h with increasing concentrations of IP-DHA. Cell viability was determined by MTT assay. The data are represented as mean ± SD (n = 7). B, co-treatment with IP-DHA and atRAL protects RPE cells. Sub-confluent (B1) and low-density (twofold less) (B2) cultures of rat primary RPE cells were cultured in 96-well plates and co-incubated with increasing concentrations of IP-DHA and 25 μmol/L atRAL for 4 h. Cell viability was determined by MTT assay. The data are represented as mean ± SD (n = 3-6). C, long-lasting effect of atRAL and IP-DHA on NR and photoreceptors. NR primary cultures were incubated with increasing concentrations of IP-DHA for 1 h, and 50 μmol/L atRAL was added for an additional 4 h. The medium was refreshed for the next 20 h. MTT assay measured cell survival (C1) and Rhodopsin-IR (rhodopsin-immunoreactivity positive cells) revealed the number of photoreceptor-derived primary cells (C2). The data are presented as mean ± SEM (n = 3-4). All data are expressed as a percentage of untreated cells (CTL) *P < .05, **P < .01, ***P < .001 vs atRAL-treated cells reactive aldehydes and scavengers of reactive species, or by the activation of Nrf2 transcription factor that promotes expression of many phase II detoxifying enzymes. 30 We recently showed that phloroglucinol acts as an anti-COS agent trapping atRAL and scavenging ROS produced by H 2 O 2 treatment (identified by DCFDA probes. 9,27 Here, we consider the anti-COS capacity of IP-DHA compared to phloroglucinol in RPE cells ( Figure 5). IP-DHA reduced both free atRAL and ROS produced by atRAL treatment ( Figure 5A and 5B, respectively). AtRAL treatment is able to alter the respiratory chain in mitochondria and potentially disrupts homeostasis in the ER, thereby initiating ER stress, which in turn induces ROS generation such as O 2 −· and then HO · . 31 Figure 6).
A 4-hour treatment with IP-DHA increased the catalase activity and prevented its decrease by atRAL ( Figure 6A), whereas it did not regulate its expression ( Figure 6B). The 24 hours pre-treatment with IP-DHA increased in a dose-dependent manner the expression of Nrf2 and its nuclear translocation in ARPE-19 cells ( Figure 6C). The same treatment increased the GSH/GSSG ratio ( Figure 6D) and the expression of NQO-1 ( Figure 6E), suggesting an up-regulation of redox regulating and detoxifying enzymes.
These results support the notion that the protective role of IP-DHA in retinal cells involves both molecular (atRAL reduction) and cellular (enzymatic) mechanisms.

| IP-DHA rescues cells under toxic effect of blue light-exposed A2E
The daily shedding of the distal tips of the outer segment followed by their phagocytosis in RPE cells leads to accumulation of bisretinoids in lysosomes and formation of A2E. 33

| D ISCUSS I ON
The pathophysiological mechanisms of STGD1, first involve alterations in the photoreceptors due to mutations in the ATP-binding cassette transporter ABCA4 gene, delay in atRAL reduction, and accumulation of autofluorescent bisretinoids in photoreceptors by condensation of atRAL and phosphatidylethanolamine. 34 At this stage, atRAL reactivity is responsible for COS. 9,13 Later, phagocytosis transfers bisretinoid-burdened POS to the RPE where bisretinoids can account for autofluorescence of lipofuscin, lightdependent COS and consequently death of RPE. 33 Therefore, COS play a crucial role throughout the disease from its onset in the photoreceptors to its progression in the RPE. Thus, it is highly relevant to develop new therapeutic compounds capable of limiting COS in the outer retina.
Polyphenols have long been recognized as antioxidant and more recently as anti-carbonyl stress derivatives, and their application in the treatment of neurodegenerative diseases has been widely acknowledged in the past few years. 35,36 Among them, phloroglucinol is a monomer of phlorotannins, which also displays therapeutic potential for neurodegenerative diseases. 37,38 Neurodegeneration is a multifactorial process and polyphenols present pleiotropic effects (antioxidant, anti-inflammatory, immunomodulatory properties) due to their ability to modulate the activity of multiple targets involved in pathogenesis, thereby halting the progression of these diseases.
We previously reported cytoprotective effects of phloroglucinol in  In the prospective treatment of patients with IP-DHA, we assume that the release of free DHA and IP may be part of the mechanism of action of the compound, as the ester bond could be cleaved by a plasma and/or cellular esterase. This is not a drawback as many studies show the beneficial effects of DHA supplementation in AMD and STGD1 45,46 and dietary polyphenols in AMD against oxidative stress and beyond. 47 In addition, the oxidation of DHA not only causes deleterious effects (lipid peroxidation), but should also contribute to the release of cellular mediators (neuroprostane, neuroprotectin D1) helping the cell to fight oxidation. In addition, the Catalase expression was quantified by Western blot analysis with a monoclonal rabbit anti-catalase antibody and enhanced chemiluminescence (ECL) detection using densitometry and ImageJ software. GAPDH expression was used as a loading control. Results are expressed as mean ± SEM (n ≥ 3). C, Nrf2 expression and nuclear translocation were explored by immunofluorescence with a rabbit monoclonal anti-Nrf2 antibody and Alexa488-conjugated anti-rabbit. Nuclei were stained with the blue fluorescent Hoechst dye. Confocal imaging revealed increased green spots in the nuclei after 24-h treatment with IP-DHA. Similar ARPE-19 treatments were performed, and GSH/GGSSG ratio (D) and NQO-1 expression (E) were quantified. Results are expressed as mean ± SEM (n = 4). # P < .05, ### P < .001 vs untreated CTL and *P < .05, **P < .01, ***P < .001 vs atRAL-treated cells antioxidant activity of phloroglucinol would enhance the beneficial effect on vision.
In conclusion, our data show that IP-DHA is effective to protect outer retinal cells against lethal dose of atRAL. The beneficial role of IP-DHA in retinal cells involves both anti-carbonyl and anti-oxidative capacities. This suggests potential effects of lipophenols in the prevention of macular degeneration associated with COS, such as STGD1 and AMD. Additional studies will be necessary to examine the effect of IP-DHA in animal models of macular degeneration.

ACK N OWLED G EM ENTS
We would like to thank the ARPEGE Pharmacology Screening-

CO N FLI C T O F I NTE R E S T
The authors have declared that no conflict of interest exists.

DATA AVA I L A B I L I T Y S TAT E M E N T
The data that support the findings of this study are available from the corresponding author upon reasonable request.