Rescue of the Stargardt Disease phenotype in Abca4 knockout mice through dietary modulation of the vitamin A receptor RBPR2

Rescue of the Stargardt Disease phenotype in Abca4 knockout mice through dietary modulation of the vitamin A receptor RBPR2 | bioRxiv /* */ /* */ <!-- <!-- /*! * yepnope1.5.4 * (c) WTFPL, GPLv2 */ (function(a,b,c){function d(a){return"[object Function]"==o.call(a)}function e(a){return"string"==typeof a}function f(){}function g(a){return!a||"loaded"==a||"complete"==a||"uninitialized"==a}function h(){var a=p.shift();q=1,a?a.t?m(function(){("c"==a.t?B.injectCss:B.injectJs)(a.s,0,a.a,a.x,a.e,1)},0):(a(),h()):q=0}function i(a,c,d,e,f,i,j){function k(b){if(!o&&g(l.readyState)&&(u.r=o=1,!q&&h(),l.onload=l.onreadystatechange=null,b)){"img"!=a&&m(function(){t.removeChild(l)},50);for(var d in y[c])y[c].hasOwnProperty(d)&&y[c][d].onload()}}var j=j||B.errorTimeout,l=b.createElement(a),o=0,r=0,u={t:d,s:c,e:f,a:i,x:j};1===y[c]&&(r=1,y[c]=[]),"object"==a?l.data=c:(l.src=c,l.type=a),l.width=l.height="0",l.onerror=l.onload=l.onreadystatechange=function(){k.call(this,r)},p.splice(e,0,u),"img"!=a&&(r||2===y[c]?(t.insertBefore(l,s?null:n),m(k,j)):y[c].push(l))}function j(a,b,c,d,f){return q=0,b=b||"j",e(a)?i("c"==b?v:u,a,b,this.i++,c,d,f):(p.splice(this.i++,0,a),1==p.length&&h()),this}function k(){var a=B;return a.loader={load:j,i:0},a}var l=b.documentElement,m=a.setTimeout,n=b.getElementsByTagName("script")[0],o={}.toString,p=[],q=0,r="MozAppearance"in l.style,s=r&&!!b.createRange().compareNode,t=s?l:n.parentNode,l=a.opera&&"[object Opera]"==o.call(a.opera),l=!!b.attachEvent&&!l,u=r?"object":l?"script":"img",v=l?"script":u,w=Array.isArray||function(a){return"[object Array]"==o.call(a)},x=[],y={},z={timeout:function(a,b){return b.length&&(a.timeout=b[0]),a}},A,B;B=function(a){function b(a){var a=a.split("!"),b=x.length,c=a.pop(),d=a.length,c={url:c,origUrl:c,prefixes:a},e,f,g;for(f=0;f<d;f++)g=a[f].split("="),(e=z[g.shift()])&&(c=e(c,g));for(f=0;f<b;f++)c=x[f](c);return c}function g(a,e,f,g,h){var i=b(a),j=i.autoCallback;i.url.split(".").pop().split("?").shift(),i.bypass||(e&&(e=d(e)?e:e[a]||e[g]||e[a.split("/").pop().split("?")[0]]),i.instead?i.instead(a,e,f,g,h):(y[i.url]?i.noexec=!0:y[i.url]=1,f.load(i.url,i.forceCSS||!i.forceJS&&"css"==i.url.split(".").pop().split("?").shift()?"c":c,i.noexec,i.attrs,i.timeout),(d(e)||d(j))&&f.load(function(){k(),e&&e(i.origUrl,h,g),j&&j(i.origUrl,h,g),y[i.url]=2})))}function h(a,b){function c(a,c){if(a){if(e(a))c||(j=function(){var a=[].slice.call(arguments);k.apply(this,a),l()}),g(a,j,b,0,h);else if(Object(a)===a)for(n in m=function(){var b=0,c;for(c in a)a.hasOwnProperty(c)&&b++;return b}(),a)a.hasOwnProperty(n)&&(!c&&!--m&&(d(j)?j=function(){var a=[].slice.call(arguments);k.apply(this,a),l()}:j[n]=function(a){return function(){var b=[].slice.call(arguments);a&&a.apply(this,b),l()}}(k[n])),g(a[n],j,b,n,h))}else!c&&l()}var h=!!a.test,i=a.load||a.both,j=a.callback||f,k=j,l=a.complete||f,m,n;c(h?a.yep:a.nope,!!i),i&&c(i)}var i,j,l=this.yepnope.loader;if(e(a))g(a,0,l,0);else if(w(a))for(i=0;i (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0];var j=d.createElement(s);var dl=l!='dataLayer'?'&l='+l:'';j.src='//www.googletagmanager.com/gtm.js?id='+i+dl;j.type='text/javascript';j.async=true;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-M677548'); Skip to main content Home About Submit ALERTS / RSS Search for this keyword Advanced Search New Results Rescue of the Stargardt Disease phenotype in Abca4 knockout mice through dietary modulation of the vitamin A receptor RBPR2 Rakesh Radhakrishnan , Matthias Leung , Drew Yochim , Heidi Roehrich , Scott W. McPherson , Glenn P. Lobo doi: https://doi.org/10.1101/2025.06.27.662034 Rakesh Radhakrishnan 1 Department of Ophthalmology and Visual Neurosciences, University of Minnesota, Lions Research Building , 2001 6th Street SE, Minneapolis, MN 55455, USA Find this author on Google Scholar Find this author on PubMed Search for this author on this site Matthias Leung 1 Department of Ophthalmology and Visual Neurosciences, University of Minnesota, Lions Research Building , 2001 6th Street SE, Minneapolis, MN 55455, USA Find this author on Google Scholar Find this author on PubMed Search for this author on this site Drew Yochim 1 Department of Ophthalmology and Visual Neurosciences, University of Minnesota, Lions Research Building , 2001 6th Street SE, Minneapolis, MN 55455, USA Find this author on Google Scholar Find this author on PubMed Search for this author on this site Heidi Roehrich 1 Department of Ophthalmology and Visual Neurosciences, University of Minnesota, Lions Research Building , 2001 6th Street SE, Minneapolis, MN 55455, USA Find this author on Google Scholar Find this author on PubMed Search for this author on this site Scott W. McPherson 1 Department of Ophthalmology and Visual Neurosciences, University of Minnesota, Lions Research Building , 2001 6th Street SE, Minneapolis, MN 55455, USA Find this author on Google Scholar Find this author on PubMed Search for this author on this site Glenn P. Lobo 1 Department of Ophthalmology and Visual Neurosciences, University of Minnesota, Lions Research Building , 2001 6th Street SE, Minneapolis, MN 55455, USA Find this author on Google Scholar Find this author on PubMed Search for this author on this site For correspondence: lobo0023{at}umn.edu Abstract Full Text Info/History Metrics Supplementary material Preview PDF ABSTRACT Mutations in the ABCA4 gene in Stargardt disease (STGD1) causes accumulation of cytotoxic lipofuscin, resulting in RPE atrophy and photoreceptor dysfunction. One component of lipofuscin is the all- trans -retinal derivative, bisretinoid N- retinylidene- N- retinylethanolamine (A2E). Since ocular A2E biosynthesis relies on circulating all- trans -retinol bound to retinol binding protein 4 (RBP4-ROL), we hypothesized that modulating vitamin A receptors, such as the retinol binding protein receptor 2, RBPR2, which regulate serum RBP4-ROL concentration, should attenuate A2E production. In-silico analysis revealed multiple retinoic acid response element (RARE) binding sites on the murine Rbpr2 gene promotor, which was confirmed in vitro by EMSA and ChIP assays. In vitro luciferase assays showed that Rbpr2 promotor activity was induced by exogenous β-carotene (BC) metabolites. Dietary BC supplementation of Abca4 −/− mice, a mouse model for STGD1, increased hepatic all- trans -retinoic acid and 9- cis -retinoic acid production, which induced Rbpr2 mRNA expression. This mechanism decreased serum RBP4 protein levels, fundus autofluorescence (AF) and ocular A2E accumulation, altogether improving photoreceptor and RPE function. Conversely, such a rescue was not observed in either Abca4 −/− mice fed a diet devoid of BC or in double knockout Rbpr2 −/− ; Abca4 −/− mice. Thus, there was a significant inverse correlation between dietary BC supplementation and Rbpr2 gene presence in Abca4 −/− mice, to that of lipofuscin accumulation in Abca4 −/− mice on diets devoid of BC or in Rbpr2 −/− ; Abca4 −/− mice. Our results provide impetus to pursue BC supplemented diets as therapeutic interventions for STGD1 patients with ABCA4 gene mutations and identifies a novel role for the vitamin A receptor RBPR2 in this process. 1. INTRODUCTION Stargardt disease (STGD1) is one of the most common forms of inherited retinal dystrophies, or inherited retinitis pigmentosa (RP) known, affecting approximately 1 in 10,000 people 1 . STGD1 is characterized as a cone-rod photoreceptor dystrophy, and differs from the typical presentation of RP in that the cones degrade prior to rod degeneration 2 . STGD1 is a childhood-onset disease, with a median age of onset at 8.5 years 3 and typically presents initially as loss of bilateral visual acuity as a result of macular atrophy, a “darkened” choroid as a result of blockage of choroid fluorescence by accumulated lipofuscin, the presence of yellowish-white flecks of lipofuscin at the posterior pole of the retinal pigment epithelium (RPE) as seen through ophthalmoscopy or fundus autofluorescence (AF), disrupted visual function as measured through electroretinography, and RPE atrophy enlargement 3 – 8 . The causative gene of STGD1 is the ABCA4 gene, which encodes the A TP- b inding c assette subfamily A member 4 transporter (ABCA4) 7 , 8 . ABCA4 protein is expressed in the rim of the disc segments within photoreceptors, and functions as a flippase to transport accumulated N -retinylidene-phosphatidylethanolamine ( N -ret-PE) outside the photoreceptor discs. N -ret-PE is metabolite resulting from the Schiff base formation between phosphatidylethanolamine (PE), a common phospholipid found within mammalian cell membranes, and the retinoid all- trans -retinal (atRAL), the consequential product after photoisomerization of the critical chromophore 11- cis -retinal in the visual cycle 9 – 11 . While most atRAL continues along the visual cycle through reduction to all- trans -retinol (ROL) through the action of retinol dehydrogenase 8 (RDH8), eventually leading to the regeneration of 11- cis -retinal chromophore, a large quantity of this atRAL reacts with PE to form the N -ret-PE metabolite 12 – 14 . Following the formation of N -ret-PE, another molecule of atRAL may react with N -ret-PE to form dihydropyridinium-A2PE, which subsequently undergoes a proton shift and proton elimination reaction to form A2PE. This can undergo a phosphate hydrolysis reaction catalyzed by phospholipase D to form N -retinyl- N -retinylidene ethanolamine (pyridinium bisretinoid-A2E). A2E is one of the main components of lipofuscin, a primary component of the previously mentioned yellowish-white flecks and contributes to the “darkened” choroid in STGD1. A2E is highly cytotoxic, and its accumulation in STGD1 has been directly attributed to its macular atrophic effects. There are several hypotheses as to how A2E exert its cytotoxic effects. First, bisretinoids such as A2E exhibit mild detergent like characteristics, which may disrupt membrane bilayers, including lysosomal membranes, potentially leading to disrupted proteolytic capabilities 15 , 16 . Second, the photodegradation products of A2E have been implicated in the degenerative effects associated with bisretinoid accumulation, including singlet oxygen species, epoxides, as well as oxo-aldehydes 14 , 15 , 17 . A2E, being derived from atRAL originating from the visual cycle and its vitamin A/all- trans -retinol/ROL based intermediates, is therefore intrinsically linked to the broader system of dietary vitamin A homeostasis present in mammalian systems. Dietary obtained vitamin A, much like many other nutrients, must be maintained at homeostatic levels in an organism 10 . This is especially true for vitamin A/ROL since ROL is directly used to generate the critical light sensitive chromophore, 11- cis -retinal, that stimulates the photoreceptor to generate an electrical impulse. Consequently, modulating the biochemical components within serum ROL homeostasis could potentially influence the generation and accumulation of A2E containing lipofuscin within STGD1. Maintenance of serum ROL homeostasis requires an extensive system of receptors, binding proteins, specialized cells, and other biochemical mechanisms 10 , 11 . In the past, our laboratory has elucidated the role of the non-ocular vitamin A receptor, Retinol Binding Protein 4 Receptor 2 (RBPR2), in the maintenance of serum RBP4-ROL and whole-body ROL homeostasis within the murine system. RBPR2 is a relatively novel vitamin A receptor, and was identified in 2013 by the Graham laboratory 18 . RBPR2 is one of two known vitamin A receptors and functions to transport circulatory ROL into the cell when bound to its transporter Retinol Binding Protein 4 (RBP4-ROL) 18 , 19 . Through its expression within the liver, the main storage organ for retinoids, and its specific interactions with serum RBP4-ROL, RBPR2 likely serves as a key regulator for whole-body vitamin A homeostasis by modulating serum RBP4-ROL homeostasis 20 . Since lipofuscin/A2E generation is dependent upon the supply of ROL to ocular tissue via circulatory ROL bound RBP4 complex, which in turn is regulated in the liver by RBPR2, we have sought evidence that by modulating Rbpr2 gene expression within the liver will affect the availability of RBP4-ROL precursors for ocular A2E production and accumulation in Abca4 −/− mice, a mouse model of STGD1. 2. MATERIALS AND METHODS 2.1 Materials All chemicals unless stated otherwise were purchased from Sigma-Aldrich (St. Louis, MO, USA) and were of molecular or cell culture grade quality. Cell culture reagents including Fetal Bovine Serum (FBS) were purchased from Gibco/ThermoFisher (Waltham, MA, USA). High Performance Liquid Chromatography (HPLC) standards were purchased from Toronto Research Chemicals (ON, Canada) or Sigma-Aldrich (St. Louis, MO, USA). 2.2 In-silico Rbpr2 gene Promoter Analysis We used NUBIScan version 2.0 software ( http://www.nubiscan.unibas.ch/software ), AliBaba 2.1 ( http://www.gene-regulation.com ), and EPD (Eukaryotic Promoter Database; https://epd.expasy.org/epd/ ) to identify putative nuclear receptor Retinoic Acid Receptor (RAR) and Retinoid X Receptor (RXR) binding sites on the mouse Rbpr2 gene promoter region, as previously described by us 21 . 2.3 Dual-Luciferase Reporter Gene Assay The murine Rbpr2 promoter region (∼2.2 kb) was cloned using oligonucleotides listed in Table 1 and inserted into the pGL3 luciferase reporter vector (Promega). 2 µg of purified plasmid DNA ( Rbpr2 promoter reporter vector or the empty pGL3 vector) was incubated with 4 µL of FuGENE HD Transfection reagent (Cat. No. E2311 Promega) for 15 min at RT and transfected individually in to COS1 cells (at ∼50% confluence) as per manufacturer’s protocol. At ∼12 h post-transfection, individually transfected COS1 cells were treated with either 2 µM all- trans -retinoic acid (RA), 2 µM 9- cis -retinoic acid, 2 µM β-carotene (BC), 2 µM 9- cis -retinol, 2 µM all- trans -retinol (ROL), or with vehicle control (DMSO). Post 24 h treatment the cells were harvested and the Rbpr2 promoter activity in response to the individual retinoids was determined by Luciferase assay using the Pierce™ Renilla-Firefly Luciferase Dual Assay Kit (ThermoFisher). The cells were washed and harvested with 1X Lysis buffer and 20 mL loaded on a black opaque 96-well plate with 50 μL working solution; after incubating the mix at room temperature for 10-15 min, the luminescence was measured on a SpectraMax Gemini EM microplate spectrofluorometer (Molecular Devices). The data were normalized with blank and pGL3 basic vector samples and plotted in GraphPad Prism version 10.1. View this table: View inline View popup Download powerpoint TABLE 1 Oligonucleotide Sequences: Murine Rbpr2 promoter cloning in the pGL3 plasmid 2.4 Chromatin Immunoprecipitation Experiment COS1 cells were cultured in DMEM medium with 10% FBS for five days. At ∼50% confluence, COS1 cells were transfected with 3 µg of pGL3-promoter plasmid containing the 2.2 kB Rbpr2 promoter. Approximately 40 h post-transfection, cells were treated with either 2.5 µm ROL or 2.5 µm BC, for 24 h. Cells were then treated with 1% formaldehyde at 37 0 C with gentle swirling for 10 min to enable crosslinking of nuclear proteins with genomic DNA, followed by sonication to shear and obtain DNA fragment sizes between 150 and 250 bp in length, as described 22 . The Chromatin Immunoprecipitation (ChIP) assay was then performed as described by the manufacturer (Millipore), to evaluate binding of RARs and RXRs to the murine Rbpr2 gene promotor. Approximately 4 µg of RARγ and RXRα antibody (Santa Cruz Biotechnologies or ThermoFisher) was used for immunoprecipitation separately, while 4 µg of IgG antibody was used as the negative control. Immunoprecipitated DNA was PCR amplified with the Rbpr2 gene promoter oligonucleotides (ChIP-8 and ChIP-9) listed in Table 2 . PCR products were electrophoresed on 2% agarose gels for visualization of amplified products. View this table: View inline View popup Download powerpoint TABLE 2 Oligonucleotide Sequences: Chromatin Immunoprecipitation (ChIP) Assay Only ChIP-8 and ChIP-9 oligonucleotide pairs were used for this assay. 2.5 Electrophoresis Mobility Shift Assay The mouse Rbpr2 promoter (∼2.2 kb) was subdivided into eight overlapping DNA fragments. Each fragment was PCR amplified from wild-type (WT) mouse genomic DNA prepared from liver with the oligonucleotides listed in Table 3 . An aliquot of each PCR product was evaluated on 2% agarose gels to determine its size and purity. Electrophoresis Mobility Shift Assay (EMSA) was performed with SYBR™ Green & SYPRO™ Ruby EMSA stains (ThermoFisher). Liver extracts (∼5 µg) from C57BL/6 mice were immunoprecipitated with RXRα and RXRγ monoclonal antibodies and then incubated with individual Rbpr2 promoter fragments (∼300 bp; 20 ng) for 1 h at 4°C. The protein-DNA complex was mixed with loading buffer and electrophoresed on 6% Tris-Borate-EDTA buffer PAGE gels. The DNA staining with SYBR Green and protein binding SYPRO Ruby staining signal images were captured sequentially as per manufacturer’s protocol using a ChemiDoc Imaging System (BioRad). The Rbpr2 gene promoter fragments with various molecular weight complexes in the shifted bands were quantified (densitometry) using Image J software version 1.54. and normalized with only promoter DNA control band. All EMSA images in relevant figures are representative of three independent experiments. View this table: View inline View popup Download powerpoint TABLE 3 Electrophoresis Mobility Shift Assay (EMSA). Oligonucleotide sequences for generation of individual Rbpr2 gene promotor fragments. 2.6 Animal handling and Carotenoid Feeding Experiment Four-week-old WT mice (C57BL/6J) were purchased from Jackson (JAX) laboratories (RRID:IMSR_JAX:000664). Rbpr2 -knockout ( Rbpr2 −/− ) mice used in the study have been previously described 20 . Abca4 −/− founders on a C57BL/6J background were purchased from Jackson laboratories (RRID:IMSR_JAX:026800). Breeding pairs and litters of Rbpr2 −/− , Abca4 −/− , double knockout Abca4 −/− ; Rbpr2 −/− , and WT mice were genotyped and found to be negative for the known Rd8 and Rd1 gene mutations, as previously described by us 20 . Breeding pairs were fed purified chow diets containing 8 IU of vitamin A/g (Research Diets, New Brunswick, NJ) and provided water ad libitum and maintained at 24°C in a 12:12 hour light-dark cycle. All animal experiments were approved by the Institutional Animal Care and Use Committee (IACUC) of the University of Minnesota (protocol #2312-41637A) and performed in compliance with the ARVO Statement for the use of Animals in Ophthalmic and Vision Research. Post weaning (P21), equal numbers of male and female mice were randomly distributed to chow diets containing 4 IU vitamin A/g (VAS diet) or β-carotene (BC supplemented diet) feeding groups. For experiments, WT, Abca4 −/− , or double knockout Abca4 −/− ; Rbpr2 −/− mice (both males and females) were fed purified rodent diets (AIN-93G; Research Diets, New Brunswick, NJ) containing the recommended 4 IU of vitamin A/g (VAS) or β-carotene (BC) supplemented diets containing 0.150 mg/g based on the AIN-93G diet (Research Diets, New Brunswick, NJ) for up to 6-months. 2.7 Optical Coherence Tomography and Fundus Imaging Mice from various cohorts were sedated with 5% isoflurane with adjustable room airflow from Kent Scientific’s mouse anesthesia machine. After sedation, the isoflurane was reduced to 2%, and the mouse eyes were dilated with a 1:1 mix of Tropicamide 1.0% Ophthalmic Solution (Cat. No. NDC 61314-355-02 Sandoz) and Phenylephrine HCl 2.5% Solution (Cat. No. NDC 17478-201-15 Akorn). Alcon Systane Lubricant Eye Gel was applied to moisturize the eye, and the Phoenix Micron IV image guided OCT2 instrument, equipped with a specialized objective lens, was used to capture OCT images. After capturing the OCT images, the lens of the instrument was replaced with a fundus-contact lens, and fundus images and autofluorescence of the fundus (AF) was captured. The A2E lipofuscin in-vivo excitation spectra is in range of 400-590nm 23 , the instrument excitator filter was opened for the excitation wavelength range on the fundus and barrier filter on the instrument for fluorescence was used to capture emission wavelength. 2.8 RNA Isolation and Quantitative Real-Time Reverse Transcription PCR After euthanizing mice, total RNA from ∼200 mg liver samples were extracted using a total RNA isolation kit (Qiagen). Four µg of isolated RNA with purity 260/280 nm ratio of 2.0 was reverse transcribed with an RNA to cDNA system (Applied Biosystems). An aliquot of cDNA (∼1 µL from a 20 µL reaction mix) was then subjected to qRT-PCR using TaqMan™ Fast Advanced Master Mix and ThermoTaq probes for Rbpr2 (GenBank: AK004855.1 ) and Gapdh genes (GenBank: AK002273.1 ). The reaction was performed in a BioRad qPCR instrument and quantified using the ΔΔCt method. The ΔCt value was calculated by (Ct value (gene of interest) – Ct value (housekeeping gene)), then ΔΔCt was calculated by (ΔCt value (BC diet)-ΔCt value (VAS diet)). Then the fold change was calculated by 2-ΔΔCt. All qRT-PCR experiments were performed in triplicate and repeated thrice, using freshly synthesized cDNA. Rbpr2 qRT-PCR values were normalized to 18S RNA, and the ΔΔCt method was employed to calculate fold changes. Data of qRT-PCR were expressed as mean ± standard error of mean (SEM). 2.9 High-Performance Liquid Chromatography analyses of retinoids Retinoid isolation procedures were performed under a dim red safety light (600 nm) in a dark room, as previously established by us 20 . Animals were first euthanized with CO 2 asphyxiation, and pertinent tissues were removed from the carcass. Various murine tissues (liver, serum, kidney) were homogenized separately in 0.9% saline with a handheld tissue grinder, consisting of a glass tube and glass pestle. Methanol (2 mL) was added to the homogenate to precipitate the proteins. The retinoid content from the homogenate was then extracted with 10 mL of hexane (twice), with the aqueous layer subsequently removed. The combined hexane extracts were then evaporated with a vacuum evaporator, re-suspended in 100 μL of hexane, then injected into an HPLC for analysis. HPLC analysis was performed on an Agilent 1260 Infinity HPLC with a UV detector. The HPLC conditions employed two normal-phase Zorbax Sil (5 μm, 4.6 × 150 mm) columns (Agilent, Santa Clara, CA, USA), connected in series within the Multicolumn Thermostat compartment. Chromatographic separation was achieved by isocratic flow of mobile phase containing 1.4% 1-Octanol/2% 1,4-Dioxane/11.2% Ethyl Acetate/85.4% Hexane, at a flow rate of 1 ml/min for 40 min. Retinaldehydes, retinol, and retinyl esters were detected at 325 nm using a UV-Vis DAD detector, while the UV absorbance spectra was collected from 200 nm – 700 nm. For quantifying molar amounts of retinoids, the HPLC was previously calibrated with synthesized standards and as previously described by us 20 , 23 . Calculation of concentration (µM): Standards were injected in concentrations ranging from 0-3.5 µM prepared solutions in the mobile phase. The plotted concentrations were fit through linear regression to obtain R-equation (y=mx+c) where y is the peak area (mAU*sec), m is the slope of the calibration curve, and c is the y-intercept. The area from the HPLC peaks of the samples were interpolated into concentration and expressed as picomoles. For eyes the values are expressed as picomoles/eye; for liver and kidney the values are expressed as picomoles/mg of tissue; for serum the ROL values are expressed as picomoles/ microliter. 2.10 Biomimetic Synthesis of Bisretinoid Standards Bisretinoids were synthesized by incubating 32 µg ethanolamine and approximately higher equivalent of ∼0.3 mg or more quantity of all- trans -retinal in 3 mL of ethanol stirred and incubated at room temperature for 3-7 days in the dark 24 . 2.11 Spectrophotometric quantification of synthesized bisretinoid standards and Standard Curve Generation To quantify the concentration of synthesized bisretinoid standard, we first separated and resolved the bisretinoids utilizing the HPLC method described below and collected the bisretinoid fractions using an HPLC fraction collector. Utilizing the Beer-Lambert Law and the molar absorptivity of the pertinent bisretinoid, we quantified the concentration of the collected fractions. The molar absorptivity coefficient for A2E in methanol at 439 nm is 36,900, while the molar absorptivity coefficient for isoA2E in methanol at 426 nm is 31,000 24 . Standards were then injected in amounts ranging from 250-2500 picograms prepared solutions in the mobile phase. The plotted concentrations were fit through linear regression to obtain R-equation (y=mx+c) where y is the peak area (mAU*sec), m is the slope of the calibration curve, and c is the y-intercept. The area from the HPLC peaks of the samples (mAU*sec) are interpolated into concentration and expressed as picomoles. For eyes, the values are expressed as picomoles/ eye. 2.12 Bisretinoid extraction from Murine Eyes All bisretinoid extractions were performed in a dark room illuminated by dim red light (600 nm) to minimize potential photoisomerization. Mice were euthanized through CO 2 asphyxiation, and whole eye globes were removed and homogenized in 0.5 mL Chloroform / 0.5 mL Methanol / 0.25 mL 1X PBS with a manual tissue grinder. The homogenate was then centrifuged to separate out the supernatant. The supernatant was removed and vacuum evaporated and resuspended in 0.1% TFA in methanol. This mixture was centrifuged again, and the final supernatant injected in to the HPLC for analysis. 2.13 High Performance Liquid Chromatography analysis and Quantification of Bisretinoids in Murine Eyes High Performance Liquid Chromatography (HPLC) analysis was performed on an Agilent 1260 Infinity HPLC with a UV/Vis detector. The ZORBAX Eclipse Plus C18 (4.6 x 250 mm, 5 µm) column (Agilent, Santa Clara, CA, USA) was used to resolve bisretinoids, with the column compartment set to 20°C. Chromatographic separation was achieved using a gradient flow facilitated by a binary pump system, with acetonitrile in one pump, and 0.1% TFA in water in the other. The gradient flow was as follows: 50% A and 50% B (0-4 min), 20% A and 80% B (4-6 min), 10% A and 90% B (6-10min), 100% B (10-20 min). The flow rate was set to 0.5 mL/min for the duration of the run. Bisretinoids were best detected at 440 nm using a UV-Vis DAD detector, while the UV absorbance spectra was collected from 200 nm – 700nm. 2.14 Confocal Microscopy for Lipofuscin/ A2E granule visualization in murine RPE Eye globes from mice in the respective cohorts were harvested and fixed in Davidson’s fixative overnight. The globes were then embedded in OCT compound (Tissue-Tek) and flash frozen. 10 microns thick tissue slices were sectioned with a microtome and layered on to a microscope glass slide. The slides were washed with 1X PBS then mounted with VectaShield Antifade Mounting Medium with DAPI for nuclear staining. Images were captured using 60X water immersion objectives. The A2E autofluorescence emission spectrum, spanning a continuous wavelength range from 520 to 800 nm, was detected using Laser settings of 445 nm and 405 nm for A2E excitation, resulting in emission filter signals at 570-616 nm and 607-850 nm. Additionally, 405 nm was used for the DAPI nuclear staining emission signal, with a wavelength of ∼460 nm 23 , 26 . The images were processed and calibrated with the ZEISS ZEN 3.4 software package. The autofluorescence signal in RPE layer from all the groups were quantified using Image J or Fiji (NIH) version 1.54, and the data-plotted in GraphPad Prism version 10.3.1. 2.15 Western Blot Analysis and Densitometry for Protein Quantification Total protein from mouse serum was solubilized using RIPA lysis buffer containing protease inhibitors (Roche, Indianapolis, IN). Approximately 25 µg of total protein was electrophoresed on 15% SDS-PAGE gels and transferred to PVDF membranes. Membranes were stained with Ponceau S and images captured. The blots were then washed, destained, and probed with primary antibodies against RBP4 (1:250, Polyclonal Rabbit anti-human RBP4) in buffer (0.2% Triton X-100, 2% BSA, 1X PBS). HRP conjugated secondary antibodies (BioRad) were used at 1:5,000 dilution. Protein expression was detected, and images captured using a ChemiDoc Imaging System (BioRad). The relative intensities of each protein band were quantified (densitometry) using Image J software version 1.54 and normalized to loading control (Ponceau S staining) images. Each western blot analysis was repeated thrice. 2.16 Immunohistochemistry and Fluorescence Imaging Mice, at 7-months of age in the different cohorts, were euthanized by CO 2 asphyxiation and cervical dislocation. Eyes were enucleated and fixed in Davidson’s fixative at RT for 24 h. Paraffin-embedded retinal sections (∼10 microns) were processed for antigen retrieval and immunofluorescence. Primary antibodies: anti-rhodopsin 1D4 for mouse rod opsin (1:200; ab5417 Abcam), anti-Red/Green Opsin Polyclonal Antibody for mouse cone opsin (1:200; OSR00222W ThermoFisher Scientific) were diluted in blocking solution for overnight incubation with the samples. All secondary antibodies (Alexa Fluor 488 or Alexa Fluor 594) were used at 1:5000 concentration (Molecular Probes). 4′,6-diamidino-2-phenylendole DAPI; VECTASHIELD® Mounting Medium (H-1200 Vector Laboratories) was used to label nuclei. The retinal section Z-stack images were obtained using a 60X objective and sectioning on the Keyence BZ-X800 microscope. The images were processed using the Z-projection Max Intensity method. The fluorescence intensities and various length measurements of retinal sections were quantified using ImageJ or Fiji (NIH), and the data were plotted in GraphPad Prism. 2.16 Statistical Analysis Data were expressed as means ± standard error mean, statistical analysis by ANOVA and Student t-test. Differences between means were assessed by Tukey’s honestly significant difference (HSD) test. P-values below 0.05 (P<0.05) were considered statistically significant. Statistical analysis was carried out using GraphPad Prism v 10.1. 3. RESULTS 3.1 In-silico analysis of murine Rbpr2 promotor region reveals putative RARE binding sites RARs and RXRs function by binding to specific retinoic acid response elements (RAREs) within regulatory/ promoter regions of target genes. We used NubiScan Version 2.0 and AliBaba 2.1 software to identify putative RAREs in the murine Rbpr2 gene promoter, as achieved previously by us for other gene promoters 21 , 22 . These programs predicted multiple direct repeat (DR-2) RAREs sites located upstream of the ATG start codon of the Rbpr2 gene promoter region ( Supplementary Figure S1A ). Additionally, promoter analysis from the eukaryotic promoter database UCSC genome browser for the presence of RAR and RXR binding elements further confirmed presence of RARE regulatory elements on the murine Rbpr2 promoter ( Supplementary Figures S1B and S1C ). 3.2 RAR and RXR interacts with the murine Rbpr2 promoter region in Chromatin Immunoprecipitation Assays To investigate whether RARs and RXRs associate with the DR-2 RARE sites on the murine Rbpr2 gene promoter region, we performed chromatin immunoprecipitation (ChIP) assays in COS1 cells using antibodies specific for RARγ and RXRα and Rbpr2 gene oligonucleotide pairs designed for detection of the putative RARE sites in immune-precipitated DNA fractions ( Figure 1A ). Here we observed that PCR products were clearly detectable in chromatin fractions immunoprecipitated with individual RARγ and RXRα antibodies ( Figure 1B ). In contrast, no PCR products were detected when IgG antibody was used as a control in this assay ( Figure 1B ). Thus, our analysis provides evidence that RARs and RXRs bind to the computer predicted RARE elements within the murine Rbpr2 gene promotor. Download figure Open in new tab Figure 1: ChIP and EMSA assays identify multiple RAR and RXR binding sites on the murine Rbpr2 gene promoter. ( A ) Schematic representation of ∼2.2 kb of the mouse Rbpr2 promoter. ( A ) For electromobility shift assays (EMSA), genomic DNA was isolated from a wild-type/WT mouse liver and using oligonucleotide pairs listed in Table 3 , the full-length Rbpr2 gene promoter was PCR amplified into eight overlapping fragments (F1-F8) as indicated. Individual fragment sizes and positions are relevant to the Rbpr2 gene ATG start site. Position of the two separate oligonucleotide pairs used in the chromatin immunoprecipitation (ChIP) assay are indicated (ChIP-8 and ChIP-9). ( B ) To show binding of RAR and RXR to the RARE sites on the mouse Rbpr2 promoter, COS1 cells were transfected with pGL3 vector containing the full-length Rbpr2 -promoter fragment (∼2.2 kb). After 40 h transfection, COS1 cells were incubated with either 2 µM all- trans -retinoic acid (atRA), β-carotene (BC), or 9- cis -retinoic acid (9cRA) for 24 h. ChIP assays in COS1 cells were performed with commercial anti-RARγ and anti-RXRα antibodies. The 10% of initial cell extract was used as input and IgG antibody was used as a control in the pull-down. Two oligonucleotide pairs flanking the F6 and F7 Rbpr2 gene promoter region was designed for its detection in precipitated DNA fractions, namely ChIP-8, oligonucleotide pair 1, and ChIP-9, oligonucleotide pair 2, as shown in panel A. In EMSAs, individual Rbpr2 gene promoter fragments were incubated with either RXRα ( C ) or RARγ ( D ) protein. Rbpr2 promoter fragments F3-F8, each capable of interacting with RXRα or RARγ protein is indicated by the complex/shift (white arrows) and faster migrating unbound DNA probes are shown as free probes by the black arrow. Protein-DNA complexes were resolved on 6% PAGE gels, and results were obtained by Chemiluminescence. Shifts of the RAR and RXR protein bound Rbpr2 -probe complexes are indicated by arrows. Although the free unbound probe migrated rapidly upon electrophoreses, the shifted RAR- Rbpr2 and RXR- Rbpr2 bound probe migrated slower and above the labeled free DNA probe. The figures show representative images from at least three independent experiments. 3.3 Electrophoresis Mobility Shift Assays of the Rbpr2 promoter reveals multiple RARE binding sites To confirm the observations obtained from the In-silico computer analysis and ChIP assay above, we used Electrophoresis Mobility Shift Assays (EMSA) to determine if a physical interaction exists between RAR and RXR and the RARE sites on the murine Rbpr2 promotor. EMSA assays with individual Rbpr2 promotor DNA fragments ( Figure 1A ) and RARγ and RXRα protein revealed that both RAR and RXR bound six of the eight Rbpr2 gene promotor fragments in COS1 cells, wherein slower DNA-protein migration patterns were observed (arrows in Figures 1C and 1D ). Thus, our EMSA analysis provides further evidence that RAR and RXR can physically bind to the distal RARE sites on the promoter region of murine Rbpr2 gene. 3.4 Luciferase assays of mouse Rbpr2 promoter reveals induction by exogenous carotenoids We next tested the in vitro effects of various retinoids and carotenoids on murine Rbpr2 gene promoter activity using luciferase assays. To achieve this, a full-length ∼2.2 kb region of the murine Rbpr2 promoter region was PCR amplified using gene specific oligonucleotides and cloned into the pGL3 luciferase reporter plasmid, as outlined in the methods section. The pGL3- Rbpr2 plasmid or empty pGL3 plasmid was transfected into COS1 cells and then treated individually with retinoids, all- trans -retinoic acid (atRA), 9- cis -retinoic acid (9cRA), 9- cis -retinol (9cROL), all- trans -retinol (atROL), and carotenoids (β-carotene). The influence of individual retinoids and carotenoids on Rbpr2 gene promoter activity was determined by Luciferase assay. As expected, the empty pGL3 plasmid transfected COS1 cells, empty pGL3 transfected COS1 cells with atRA, and empty pGL3 transfected COS cells with vehicle (DMSO), showed no luciferase activity ( Supplementary Figures S2A and S2B ). Interestingly, we observed that pGL3- Rbpr2 expressing cells treated with exogenous BC showed a significant induction of Rbpr2 mRNA activity, while the individual retinoids (atRA, 9cRA, atROL, and 9cROL) treated cells caused a suppression of Rbpr2 promoter activity ( Supplementary Figures S2A and S2B ). To determine the mechanism for these observations we isolated total retinoids from individual COS1 treated cells and subjected them to HPLC analysis to determine the retinoid profiles. As expected, no retinoids were detectable in control COS1 cells (empty pGL3 plasmid and treated with DMSO) ( Supplementary Figure S2C ). Conversely, cells expressing pGL3- Rbpr2 gene plasmid and treated with atROL or BC showed a retinoid profile on HPLC analysis. Herein, we observed that exogenous BC treated cells showed significantly higher production and concentration of retinoic acid isomers (atRA, 9cRA, and 13cRA), compared to the all- trans -ROL treated cells ( Supplementary Figure S2D vs. S2E; individual retinoic acid isomers quantified and shown in Supplementary Figures S2F-S2H ). 3.5 Dietary β-carotene induces hepatic Rbpr2 expression and modulates serum RBP4-ROL levels in wild-type mice Based on our in vitro observations above, we tested whether a similar mechanism could exist in vivo. For this, cohorts of 4-week-old wild-type (WT) mice were fed a diet supplemented with BC or a diet with vitamin A/ retinyl palmitate (VAS), the latter a source for all- trans -retinol. After 3-months of feeding, mice were euthanized, and their liver and serum were collected. Quantitative PCR (qRT-PCR) analysis showed that hepatic Rbpr2 mRNA levels were significantly induced (∼5.4 fold) in WT mice fed a BC supplemented diet, compared to those fed a VAS diet ( Supplementary Figure S3A ). Western blot analysis showed that serum RBP4 protein levels were significantly lower in WT mice fed a BC diet, compared to those fed a VAS or chow diet ( Supplementary Figure S3B and quantified in S3C ). HPLC analysis for hepatic retinoids showed a similar trend to those observed above in COS1 cells, wherein, we observed an increased production and higher concentration of retinoic acid isomers (atRA, 9cRA, and 13cRA) in the liver of BC supplemented WT mice, compared to those fed a VAS diet ( Supplementary Figure S3D vs. S3E, and quantified in Supplementary Figures S3F-S3H ). Taken together, our in vitro and in vivo results support the observation that exogenous BC in COS1 cells and dietary BC supplementation in WT mice can induce Rbpr2 mRNA expression and activity via increased RA synthesis. 3.6 β-carotene supplementation of Abca4 −/− mice increases hepatic retinoic acid production and induces Rbpr2 mRNA expression Since all- trans -RAL is a primary reactant in the biogenesis of ocular lipofuscin/A2E, we tested the strategy identified above of reducing serum RBP4 availability in Abca4 −/− mice 25 – 28 . This we hypothesized would limit serum ROL precursors for ocular all- trans -RAL formation in mouse models that can accumulate cytotoxic lipofuscin/A2E. To achieve this, we obtained Abca4 −/− mice and crossed them with Rbpr2 −/− mice. Then, individual cohorts of age-matched WT, Rbpr2 −/− , Abca4 −/− , and the double knockout Rbpr2 −/− ; Abca4 −/− mice were fed a vitamin A sufficient/VAS diet (without BC) or a BC supplemented diet, starting at 1-month to 7-months of age. At 1-month of age, we collected blood to quantify serum RBP4 protein. After 3-months (4-month-old mice) and at 6-month (7-month-old mice) of feeding, mice in the individual cohorts were subjected to non-invasive tests for visual function and then euthanized, and their tissues and serum were collected. As we observed above in WT animals, qRT-PCR analysis showed that hepatic Rbpr2 mRNA levels were significantly induced and upregulated (∼6.2 fold) in BC supplemented Abca4 −/− mice, compared to VAS Abca4 −/− mice ( Figure 2A ). Western blot analysis showed that serum RBP4 protein levels were significantly lower in BC supplemented Abca4 −/− mice, compared to VAS Abca4 −/− mice, at the 7-month of age ( Figures 2B and 2C ). Interestingly, as these mice age, serum RBP4 protein levels continued to rise in VAS Abca4 −/− mice, conversely, it was observed to be lower in BC supplemented Abca4 −/− mice ( Figures 2B and 2C ; 1-month-old vs. 7-month-old mice). HPLC analysis for hepatic retinoids in BC supplemented Abca4 −/− mice showed a similar trend to those observed in WT mice. Together this data showed increased production and higher concentration of BC and retinoic acid isomers (atRA, 9- cis -RA, and 13- cis -RA) in the livers of BC supplemented Abca4 −/− mice, compared to VAS Abca4 −/− , Rbpr2 −/− , or double knockout Rbpr2 −/− ; Abca4 −/− mice ( Figure 2D vs. 2E, individual RA isomers quantified in Figures 2F - 2K , and BC quantified in Supplementary Figure S4 ). Download figure Open in new tab Figure 2: Long-term dietary β-carotene supplementation of Abca4 −/− mice induces hepatic Rbpr2 mRNA expression and increases hepatic retinoid production. ( A ) Quantitative RT-PCR analysis of Rbpr2 mRNA expression in liver of Abca4 −/− mice at 7-months of age, fed either a VAS or BC supplemented diet. ( B ) RBP4 protein expression in serum of Abca4 −/− mice fed a VAS diet or BC supplemented diet, determined by western blot analysis and ( C ) protein quantified by densitometry. Ponceau S was used to confirm equal protein loading among samples. Approximately 50 µg of protein was loaded. The 1-month timepoint reflects start of the dietary intervention (either VAS or BC). ( D , E ) HPLC analysis of hepatic retinoic acid isomers extracted from Abca4 −/− mice fed either a BC supplemented diet or a diet devoid of BC (VAS diet), respectively. ( F-K ) Quantification of various retinoic acid isomers in liver of mice from different cohorts fed with dietary BC or a diet devoid of BC (VAS diet). VAS, vitamin A sufficient diet; BC, β-carotene supplemented diet. n=4-6 mice per cohort and dietary condition. atRA, all- trans -retinoic acid; 9cisRA, 9- cis -retinoic acid; 13cRA, 13- cis -retinoic acid. Student t-test; *P<0.05; **P<0.01. n=4-6 mice per cohort. 3.7 Longitudinal recordings of 488 nm Fundus Autofluorescence intensity in Abca4 −/− mice A feature of ABCA4 -mediated retinal and macular dystrophies is the accumulation of auto fluorescence (AF) lipofuscin/A2E pigments in the RPE 29 . We therefore investigated the changes in fundus AF levels over time in WT, Abca4 −/− , Rbpr2 −/− , and Rbpr2 −/− ;Abca4 −/− mice, previously fed a BC supplemented or a diet devoid of BC (VAS diet). Repeated fundus recordings in the same animal cohorts were performed at 4-months of age (3-months post dietary BC supplementation or VAS diet) and then at 7-months of age (6-months post dietary BC supplementation or VAS diet). In control VAS WT and BC supplemented WT mice, 488 nm fundus AF was not detected at either 4– or 7-months of age ( Figures 3A and 3B ), likewise no AF was detected in adult VAS Rbpr2 −/− mice at 7-months of age, indicating that loss of the vitamin A receptor Rbpr2 −/− does not result in fundus AF lipofuscin accumulation ( Figure 3B ). At 4-months of age, fundus AF in double knockout Rbpr2 −/− ;Abca4 −/− mice was significantly higher to that in VAS Abca4 −/− mice, indicating that genetic loss of Rbpr2 can accelerate AF accumulation in the fundus of these mice ( Figures 3A and 3A ’ ). Thereafter, 488 nm fundus AF rose significantly in both Abca4 −/− and double knockout Rbpr2 −/− ;Abca4 −/− mice fed a VAS diet, compared to either WT or BC supplemented Abca4 −/− mice ( Figure 3A vs 3B ). Download figure Open in new tab Figure 3: Long-term dietary β-carotene supplementation of Abca4 −/− mice reduces fundus autofluorescence (AF) intensity. Repeated fundus recordings in the same animal cohorts were performed at ( A ) 4-months of age (3-months post dietary supplementation) and then at ( B ) 7-months of age (6-months post dietary supplementation). Shown are the changes in fundus autofluorescence (AF) levels over time in WT, Abca4 −/− , Rbpr2 −/− , and double knockout Rbpr2 −/− ;Abca4 −/− mice fed either a β-carotene supplemented diet or a diet devoid of any β-carotene (VAS diet), which were evaluated at 488 nm. The relative intensity comparison was performed via pseudo-color Look Up Table on 8-bit images in Image J, showing the distribution of autofluorescence intensities in Fundus images. Red represents high intensity and Blue represents low intensity AF, in the respective 4– and 7-month cohorts. ( A’ ) Fold change mean intensity of AF levels among cohorts at 4-months of age, and ( B’ ) fold change mean intensity of AF levels among cohorts at 7-months of age. n=4-6 mice per cohort. VAS, vitamin A sufficient diet; BC, β-carotene supplemented diet. ANOVA, **P<0.01; ***P<0.005; ****P<0.001. 3.8 Comparison of Fundus Autofluorescence recordings in Abca4 −/− mice A2E is regarded as a major fluorophore of lipofuscin in the ocular fundus 25 – 28 . We therefore assessed if A2E levels in Abca4 −/− and Rbpr2 −/− ;Abca4 −/− mice fed a VAS diet increased in parallel with 488 nm autofluorescence (AF) levels, in comparison to BC supplemented Abca4 −/− mice. AF ratios between Abca4 −/− and Rbpr2 −/− ;Abca4 −/− fed a VAS diet and BC supplemented Abca4 −/− mice confirmed results from the longitudinal data set. At the 4-months of age timepoint, 488 nm AF levels in the double knockout Rbpr2 −/− ;Abca4 −/− mice were significantly higher to those in VAS Abca4 −/− mice ( Figure 3A , quantified in Figure 3A ’ ). At the 7-months of age, 488 nm AF continued to rise in Rbpr2 −/− ;Abca4 −/− mice and were higher than those observed and quantified in VAS Abca4 −/− mice ( Figure 3B , quantified in Figure 3B ’ ). BC supplemented Abca4 −/− mice showed significantly lower fundus AF, compared to both Rbpr2 −/− ;Abca4 −/− and Abca4 −/− mice fed a VAS diet at the 4-month time point ( Figures 3A and 3A ’ ), and this observation remained consistent at the 7-month-of age timepoint ( Figures 3B and 3B ’ ). Thus, BC supplementation and the presence of the Rbpr2 gene attenuated AF lipofuscin accumulation in eyes of Abca4 −/− mice. 3.9 Qualitative Optical Coherence Tomography findings in RPE of Abca4 −/− mice We next conducted a qualitative analysis of retinal pigmented epithelium (RPE) structure in various mice cohorts from above using Optical Coherence Tomography (OCT). In all mice groups a reliable evaluation of RPE skip lesions, thickening, and atrophy was possible. At the 7-month of age, BC supplemented WT and Abca4 −/− mice showed normal/ unremarkable RPE structure ( Figure 4 ). Conversely, and at the 7-month of age time-point, Abca4 −/− and Rbpr2 −/− ;Abca4 −/− mice that were fed VAS diets, showed RPE skip lesions (indicated by * in Figure 4 ) and RPE aggregates and thickening (indicated by arrows in Figure 4 ), compared to BC supplemented Abca4 −/− mice. Thus, long-term dietary BC supplementation of Abca4 −/− mice not only attenuated ocular AF lipofuscin accumulation, but also subsequently improved RPE structure, which was dependent on the presence of the Rbpr2 gene. Download figure Open in new tab Figure 4: Long-term dietary β-carotene supplementation of Abca4 −/− mice rescues RPE phenotypes. Optical coherence tomography (OCT) was used to evaluate RPE skip lesions, thickening, and atrophy in various cohorts of mice fed either a β-carotene (BC) supplemented or a diet devoid of any β-carotene (VAS diet), at 7-months of age. n=4-6 mice per cohort. VAS, vitamin A sufficient diet; BC, β-carotene supplemented diet. *, RPE skip lesions; arrows, RPE thickening and RPE aggregates. 3.10 β-carotene supplemented Abca4 −/− mice show a significant improvement of rod and cone photoreceptor cell function We then measured rod and cone photoreceptor cell responses by scotopic and photopic flash ERG full-field potentials in the various mice cohorts. After 3– and 6-months of BC supplementation in Abca4 −/− mice, scotopic and photopic ERG responses were measured relative to WT, Abca4 −/− , and Rbpr2 −/− ;Abca4 −/− mice fed a VAS diet of the same age (4– and 7-months-old). We found no significant a -wave or b -wave differences relative to age-matched WT and Abca4 −/− mice under any feeding conditions in either mouse cohort at 4-months of age ( Supplementary Figures S5A and S5B ). However, at 7-months of age, BC supplemented Abca4 −/− mice showed a significant improvement of rod photoreceptor cell function, compared to either Abca4 −/− or double knockout Rbpr2 −/− ;Abca4 −/− mice fed a VAS diet ( Figures 5A - 5H , Supplementary Figure S5C ). Comparison of b -wave amplitudes among mice cohorts at the 7-month of age, showed no significant differences between BC supplemented and VAS fed Abca4 −/− mice ( Supplementary Figures S5D, S6A, and S6B ). However, double knockout Rbpr2 −/− ;Abca4 −/− mice fed a VAS diet showed lower b -wave amplitudes compared to Abca4 −/− mice fed a VAS or BC supplemented diet ( Supplementary Figures S6C-S6F ). Download figure Open in new tab Figure 5: Long-term dietary β-carotene supplementation of Abca4 −/− mice improves rod photoreceptor cell function. Dark-adapted scotopic electroretinographic (ERG) a -wave analysis in mice at 7-months of age. ( A-D ) shows scotopic ERG recordings at 1 cd.s/m 2 and ( E-H ) at 10 cd.s/m 2 . n=4-6 mice per cohort. VAS, vitamin A sufficient diet; BC, β-carotene supplemented diet. Student t-test; *P<0.05; **P<0.01; ***P<0.005. We next determined ERG responses in light-adapted mice fed either the BC supplemented or VAS diet under different light source color (green, red, white, and UV/blue). Under photopic light conditions, ERG responses of Abca4 −/− mice under green, white, and UV color sources were significantly diminished at the 7-month timepoint, while red light source ERG responses were not changed, when compared with BC supplemented Abca4 −/− mice ( Supplementary Figures S7A-S7D ). Light-adapted ERG responses for blue ( Supplementary Figures S7A, S7A’, and S7A’’ ), green ( Supplementary Figures S7B, S7B’, and S7B’’ ), and white ( Supplementary Figures S7C, S7C’, and S7C’’ ) light intensity improved in BC supplemented Abca4 −/− mice at the 7-month timepoint, but remained significantly diminished under red-light exposure ( Supplementary Figure S7D ). Thus, BC supplemented Abca4 −/− mice showed an overall improvement of rod and cone photoreceptor cell function, compared to Abca4 −/− mice fed a VAS diet or double knockout Rbpr2 −/− ;Abca4 −/− mice. 3.11 β-carotene supplementation of Abca4 −/− mice improves RPE function A key pathological feature of STGD1 is the progressive accumulation of fluorescent lipofuscin/A2E pigments in the retinal pigmented epithelium (RPE), which could potentially affect RPE function 30 and be causative of photoreceptor dysfunction observed above. We therefore used c -wave ERGs to test for RPE function in the various mice cohorts. At 7-months of age BC supplemented Abca4 −/− mice showed an improvement of RPE function, compared to either Abca4 −/− or double knockout Rbpr2 −/− ;Abca4 −/− mice fed a VAS diet ( Figures 6A - 6C ). Additionally, double knockout Rbpr2 −/− ;Abca4 −/− mice showed an even more severe decrease in RPE function, compared to VAS Abca4 −/− mice ( Figure 6D ). Download figure Open in new tab Figure 6: Long-term dietary β-carotene supplementation of Abca4 −/− mice improves RPE cell function. ( A-D ) Dark adapted c -wave analysis for RPE cell function in 7-month-old mice among different genotypes and dietary conditions. n=4-6 mice per cohort. VAS, vitamin A sufficient diet; BC, β-carotene supplemented diet. Student t-test; *P<0.05; **P<0.01. 3.12 Quantification of A2E isomers in mice eyes by High Performance Liquid Chromatography Since loss of ABCA4 has been associated with accumulation of lipofuscin pigments in the eye, we quantified A2E, a bisretinoid component of lipofuscin, by integrating HPLC peaks and normalizing to a calibration curve obtained from an A2E and iso-A2E standard (Supplementary Figure S8) . HPLC analysis revealed that A2E and iso-A2E isomer concentrations in 7-month-old BC supplemented Abca4 −/− mice were significantly lower, compared to those fed a VAS diet ( Figures 7A - 7E ), which also collated with the fundus AF images observed in these mice cohorts ( Figure 3B ). Comparison of A2E and iso-A2E isomer concentrations in 7-month-old Abca4 −/− and double knockout Rbpr2 −/− ;Abca4 −/− mice fed a VAS diet, showed no significant differences (P<0.0601; Figure 7B ). Download figure Open in new tab Figure 7: HPLC quantification of A2E and iso-A2E isomers in mice eyes. ( A ) HPLC traces of A2E and iso-A2E isomers in Abca4 −/− and Rbpr2 −/− ;Abca4 −/− mice eyes, fed a VAS or BC supplement diet, at 7-months of age. ( B ) Total ocular A2E concentrations shown in pmole/eye among various mice cohorts. ( C ) Total A2E, ( D ) iso-A2E, and ( E ) A2E, in eyes of Abca4 −/− mice fed either a VAS or BC supplemented diet. n=4-6 mice per cohort. VAS, vitamin A sufficient diet; BC, β-carotene supplemented diet. One way ANOVA or Student t-test; *P<0.05; **P<0.01; ***P<0.005; ****P<0.001. 3.13 Fluorescence Microscopy of Mice Retinal Sections for Lipofuscin Accumulation We next used retinal sections and fluorescence microscopy to visualize A2E/lipofuscin accumulation in the RPE of various mice cohorts, fed either a VAS or BC supplemented diet. This analysis revealed that A2E/ lipofuscin fluorescence in 7-month-old Abca4 −/− mice supplemented with dietary BC were significantly lower to those on a VAS diet ( Figure 8A ). This observation also collated with the fundus AF images observed in these mice cohorts ( Figure 3B ) and with the HPLC quantification of A2E isomers ( Figure 7 ). Comparison of A2E florescence among mice cohorts, showed that double knockout Rbpr2 −/− ;Abca4 −/− mice displayed higher A2E/lipofuscin fluorescence intensity, compared to VAS Abca4 −/− mice ( Figure 8B ). Thus, this data supports the hypothesis that the systemic vitamin A receptor ( Rbpr2 ) in synergy with a carotenoid (β-carotene) supplemented diet, plays a beneficial role in attenuating lipofuscin/A2E accumulation, which leads to an improvement of photoreceptor and RPE function in Abca4 −/− mice. Download figure Open in new tab Figure 8: Fluorescent microscopy detects autofluorescent lipofuscin/ A2E granules in RPE of mice eyes. Autofluorescent lipofuscin/ A2E granules in RPE of mice fed either a VAS or BC supplemented diet, at 7-months of age. ( A ) Two channels were used with 405 nm excitation and emission of 570-616 nm and 607-850 nm to detect A2E fluorescence in RPE/retinas from various mice cohorts. ( B ) Quantification of autofluorescent lipofuscin/ A2E granules in RPE. n=4-6 mice per cohort. VAS, vitamin A sufficient diet; BC, β-carotene supplemented diet. One way ANOVA, *P<0.05; ***P<0.005; *****P<0.001. 3.14 β-carotene supplemented Abca4 −/− mice show rescue of cone photoreceptor phenotypes Since lipofuscin/A2E accumulation in the absence of ABCA4 has been linked to macular degeneration, which can specifically affect integrity of cone photoreceptors, we next performed immunohistology analysis of retinas in the different mice cohorts for cone photoreceptor morphology using R/G cone opsin staining. This analysis showed that VAS Abca4 −/− mice and double knockout Rbpr2 −/− ;Abca4 −/− mice at 7-months of age had shorter and misshaped cone outer segments (OS), compared to WT mice at the same age ( Figure 9A ). Interestingly, double knockout Rbpr2 −/− ;Abca4 −/− mice showed an even shorter cone photoreceptor OS phenotype, compared to Abca4 −/− mice ( Figure 9 , cone length quantified in Figure 9B ). Conversely, BC supplemented Abca4 −/− mice showed a rescue of cone OS phenotypes, indicating that long-term BC supplemented diet and presence of the vitamin A receptor, Rbpr2 , can protect against cone photoreceptor OS phenotypes in Abca4 −/− mice ( Figures 9A and 9B ). Download figure Open in new tab Figure 9: Long-term dietary β-carotene supplementation of Abca4 −/− mice rescues cone photoreceptor phenotypes. ( A ) Retinal sections from various mouse cohorts at 7-months of age were subjected to immunohistochemistry using 1D4 (rhodopsin; green) and R/G cone opsin (red) antibodies. DAPI (blue) in the mounting solution was used to stain nuclei. ( B ) Quantification of cone photoreceptor length from various mouse cohorts. n=3-5 retinal sections from n=3-6 mice eyes per cohort. VAS, vitamin A sufficient diet; BC, β-carotene supplemented diet. One way ANOVA, ****P<0.001. 4. DISCUSSION Here we identify a synergistic role between long-term dietary β-carotene (BC) supplementation and presence of the non-ocular vitamin A receptor Rbpr2 gene as a therapeutic modality in attenuating cytotoxic lipofuscin/A2E accumulation in Abca4 −/− mice, a well-established mouse model of Stargardt disease/STGD1 ( Figure 10 ). Since serum all- trans -retinol (ROL) serves as the first step precursor for ocular all- trans -retinal synthesis, a component of cytotoxic A2E, we explored in vivo mechanisms that could regulate serum ROL bound RBP4 (RBP4-ROL) concentration in Abca4 −/− mice. We identify multiple RAR and RXR sites on the vitamin A receptor Rbpr2 gene promotor, which can be induced in vitro and in vivo by BC metabolites, all- trans -retinoic acid (RA), 9- cis -retinoic acid (9cRA), and 13-cis-retinoic acid (13cRA). In vivo, this mechanism enhanced hepatic retinoic acid production and significantly induced Rbpr2 mRNA expression, reducing serum RBP4 levels in WT and Abca4 −/− mice. Subsequently, long-term dietary BC supplementation of Abca4 −/− mice, reduced fundus auto fluorescence (AF) levels, decreased ocular A2E and iso-A2E isomer accumulation in the RPE, which led to an improvement of rod and cone photoreceptor and RPE cell function. Conversely, neither Abca4 −/− or double knockout Rbpr2 −/− ;Abca4 −/− mice fed a diet devoid of BC, showed such a rescue of ocular phenotypes. Thus, we identified a novel mechanism, where long-term dietary BC supplementation and subsequent metabolism to RA isomers influences activity of the systemic vitamin A receptor Rbpr2, which in turn regulates serum RBP4-ROL precursors required for cytotoxic A2E production in Abca4 −/− mice. This mechanism we hypothesize could benefit STGD1 patients with ABCA4 gene mutations. Download figure Open in new tab Figure 10: Genetics and the Diet attenuate ocular Lipofuscin/A2E production and accumulation and improves visual and RPE function in Abca4 −/− mice. Abca4 −/− mice were fed a β-carotene (BC) supplemented or vitamin A sufficient (VAS) diet for up to 7-months of age. BC supplemented Abca4 −/− mice showed a higher production of retinoic acid isomers (atRA, 9 cis RA, and 13 cis RA) and BC concentrations in the liver, which led to an induction of hepatic Rbpr2 gene activity via RAR and RXR regulatory elements on its promoter. This mechanism increased reuptake of serum ROL bound RBP4 by Rbpr2 , in turn reducing serum RBP4 concentrations. In the long-term, this mechanism reduced ocular A2E and iso-A2E production and accumulation, overall improving rod and cone photoreceptor, and RPE function in Abca4 −/− mice fed BC supplemented diets. BC, β-carotene; VAS, vitamin A sufficient; atRA, all- trans -retinoic acid; 9 cis RA, 9- cis -retinoic acid; 13 cis RA, 13- cis -retinoic acid; BCMO1, β-carotene monooxygenase; LRAT, Lecithin Retinol Acyltransferase; atROL, all- trans -retinol; RDH, retinol dehydrogenase; RALDH, retinal dehydrogenase; RAR, retinoic acid receptor; RXR, retinoid X receptor; 4Oxo-RA, 4-oxo-retinoic acid; RBP4, retinol binding protein 4; RBPR2, retinol binding protein 4 receptor 2; SR-B1, Scavenger receptor class B receptor 1; STRA6, stimulated by retinoic acid 6; STGD1, Stargar d t D is ease; A bca4 , ATP-binding cassette subfamily A member 4 transporter. Schematic figure created in BioRender (BioRender.com/a90mdtq). First, we confirmed and expanded on the previously proposed transcriptional regulation of the systemic vitamin A receptor Rbpr2 18 . We and others have shown that Rbpr2 is highly expressed in the murine liver, intestine, and adipose tissue, followed by moderate to lower expression in other non-ocular tissues 10 , 18 , 20 , 31 . Because these tissues mediate uptake, storage, and distribution of all- trans -retinol (ROL) to peripheral tissues including the eye, these observations suggest that RBPR2 could play a physiological role in whole-body ROL/retinoid homeostasis 10 , 18 , 20 . Additionally, the Graham laboratory had previously suggested negative regulation of the murine Rbpr2 gene promotor activity via retinoic acid response elements (RARE) on its promotor, which could be relevant in regulating liver ROL stores 18 . Our detailed analysis of the Rbpr2 gene promotor region revealed multiple RAR and RXR sites, which were transcriptionally regulated by retinoids. We also confirmed previous observations that exogenous all- trans -ROL can downregulate Rbpr2 mRNA expression and further discovered that exogenous BC metabolites induce Rbpr2 mRNA expression in cultured cells. The latter observation was then confirmed in vivo in BC fed WT mice. The mechanism for induction of hepatic Rbpr2 gene activity in BC supplemented WT and Abca4 -/ - mice we attributed to significantly higher production of hepatic RA metabolites (atRA, 9cRA, and 13cRA) derived from BC metabolism, compared to VAS fed animals. Interestingly and more recently, hepatic Rbpr2 mRNA expression was also found to be significantly induced in BC fed animals of a different genetic background, which led to increased accumulation of hepatic retinoids, further indicating that induction of hepatic Rbpr2 gene expression can lead to increased re-absorption of circulatory ROL from RBP4 32 . Thus, based on our findings here and those previously reported by other investigators, exogenous and dietary BC metabolites induces Rbpr2 gene activity via RARE elements on its promoter region, which plays a physiological role in modulating circulatory ROL bound RBP4 levels ( Figure 10 ). Stargardt disease (STGD1) is the phenotype most caused by ABCA4 gene mutations. A key pathological feature of STGD1 is the buildup of fundus autofluorescence (AF) lipofuscin/bisretinoid A2E pigments in the retinal pigmented epithelium (RPE), a feature previously reported in Abca4 −/− mice. We note that the effects of genetic deletion of the vitamin A receptor, Rbpr2 , on the Abca4 −/− background (double knockout Rbpr2 −/− ;Abca4 −/− mice) was not only additive to the Abca4 −/− mice ocular phenotypes, but was also causative of earlier appearance and detection of fundus AF lipofuscin. This observation was made by comparing the fundus AF images from Abca4 −/− and Rbpr2 −/− ;Abca4 −/− mice fed a VAS diet, at the earlier 4-month of age and then at 7-month of age time-point. Specifically at the 4-month of age, fundus AF levels in Rbpr2 −/− ;Abca4 −/− mice fed a diet devoid of BC (VAS diet) were observed to be significantly higher, to those noted in Abca4 −/− mice also fed the same VAS diet, indicating that loss of the Rbpr2 receptor in these mice was additive to the retinal phenotypes. Among the explanations for this observation could be the unregulated and higher levels of serum RBP4-ROL in VAS Rbpr2 −/− ;Abca4 −/− mice, which could have provided additional precursors for accelerating ocular A2E accumulation. Conversely, Abca4 −/− mice fed a BC diet showed increased hepatic Rbpr2 gene expression and decreased serum RBP4 levels, which was reflected in reduced fundus AF levels, indicating that both genetics ( Rbpr2 gene presence) and the diet (BC) acts as a therapeutic modality, in modulating serum RBP4-ROL levels in these mice. In fact, long-term BC supplementation, but not VAS diets, in Abca4 −/− mice, showed a significant reduction in A2E/lipofuscin accumulation in the RPE of these animals, which improved photoreceptor and RPE cell function. Our study has certain limitations, which can be addressed by generating novel mouse models in the future. The reductions in serum RBP4-ROL in BC supplemented WT and Abca4 −/− mice, might be a due to a combination of different mechanisms, either due to increased hepatic re-uptake of circulatory ROL from RBP4 via BC metabolite production and induction of Rbpr2 gene expression (observed herein), or due to increased storage of vitamin A (as retinyl esters) in the intestine, or due to increased efflux of ROL from the intestine by RBPR2 ( Figure 10 ). Since, RBPR2 has been shown by us and others to be highly expressed in the murine intestine (presumably basolateral) and in the liver, we are currently establishing tissue-specific liver ( Rbpr2 −/− ;Alb-Cre+ ) and intestine ( Rbpr2 −/− ;Villin-Cre+ ) mice to answer this question. Importantly, since vitamin A content in western diets predominantly consists of high-levels of preformed retinoids (>75%), it can be hypothesized that the un-regulated and excess ocular uptake of circulatory preformed retinoids, could contribute to accelerated A2E/lipofuscin biogenesis in humans 33 , 34 . Based on these observations, we propose that dietary β -carotene derived RA regulation of systemic Rbpr2 receptor activity might serve to limit excess circulatory and ocular ROL precursor availability. This regulatory mechanism via vitamin A transporters could attenuate accelerated toxic ocular A2E biosynthesis in patients with ABCA4 mutations, thus preserving visual function in STGD1 patients on long-term carotenoid sufficient diets 35 . In summary, we have shown that long-term dietary BC supplementation in Abca4 −/− mice, reduced serum RBP4 levels via Rbpr2 gene activity, which translated to robust reduction of cytotoxic A2E and iso-A2E accumulation in the RPE, which in the long-term improved photoreceptor and RPE function ( Figure 10 ). Importantly, these positive effects of BC diet in Abca4 −/− mice, were dependent on the presence of the non-ocular vitamin A receptor Rbpr2 . Thus, our approach could not only benefit STGD1 patients with ABCA4 gene mutations, but in the future could also be explored as a therapeutic modality in other mouse models of macular degeneration, such as MnSod −/− , Abcr −/− , and Elvol4 −/− mice 36 – 41 , where A2E/lipofuscin pigmented granule accumulation is the major cause of RPE and photoreceptor cell degeneration in these mutant mouse models of STGD1. AUTHOR CONTRIBUTIONS Conceptualization, G.P.L.; methodology, G.P.L., R.R., M.L., H.R.; software, R.R.; validation, R.R., G.P.L., M.L.; reagents, G.P.L., S.M.; formal analysis, R.R., G.P.L., D.Y., H.R., M.L.; investigation, R.R., G.P.L., M.L.; resources, G.P.L., S.M., H.R.; data curation, R.R., G.P.L., M.L.; writing-original draft preparation, G.P.L.; manuscript writing, review, and editing, R.R., G.P.L., M.L., D.Y., S.M.; supervision, G.P.L.; project administration, G.P.L.; funding acquisition, G.P.L. All authors have read and agreed to the published version of the manuscript. CONFLICTS OF INTEREST The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results. DATA AVAILABILITY STATEMENT Data sharing is not applicable to this article, as no datasets were generated or analyzed in this study. Reagents are available from the corresponding author upon reasonable request. ACKNOWLEDGMENTS This work was supported by NIH-NEI grants (EY030889 and 3R01EY030889-03S1 G.P.L. and EY033328 to S.W.M.) and in part by the University of Minnesota start-up funds to G.P.L. Funder Information Declared NIH-NEI , EY030889 , 3R01EY030889-03S1 , EY033328 University of Minnesota start-up funds Footnotes ↵ # joint first authors Abbreviations ABCA4 ATP-binding cassette transporter subfamily A4 A2E N -retinyl- N -retinylidene ethanolamine (pyridinium bisretinoid) BC β-carotene ERG Electroretinogram RAR retinoic acid receptor RARE retinoic acid response element RBPR2 retinol binding protein 4 receptor 2 RBP4 retinol binding protein 4 ROL all- trans -retinol RA all- trans -retinoic acid RPE retinal pigmented epithelium STGD1 Stargardt Disease Type 1 HPLC High Performance Liquid Chromatography REFERENCES (1). ↵ Sahel , J.-A. ; Marazova , K. ; Audo , I . 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