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The Interplay Between Retinal Pathways of Cholesterol Output and Its Effects on Mouse Retina

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The Interplay Between Retinal Pathways of Cholesterol Output and Its Effects on Mouse Retina biomolecules Article The Interplay between Retinal Pathways of Cholesterol Output and Its Effects on Mouse Retina Alexey M. Petrov , Artem A. Astafev, Natalia Mast, Aicha Saadane, Nicole El-Darzi and Irina A. Pikuleva * Department of Ophthalmology and Visual Sciences, Case Western Reserve University, Cleveland, OH 44106, USA; [email protected] (A.M.P.); [email protected] (A.A.A.); [email protected] (N.M.); [email protected] (A.S.); [email protected] (N.E.-D.) * Correspondence: [email protected]; Tel.: +1-216-368-3823 Received: 13 November 2019; Accepted: 10 December 2019; Published: 12 December 2019 Abstract: In mammalian retina, cholesterol excess is mainly metabolized to oxysterols by cytochromes P450 27A1 (CYP27A1) and 46A1 (CYP46A1) or removed on lipoprotein particles containing apolipoprotein E (APOE). In contrast, esterification by sterol-O-acyltransferase 1 (SOAT) plays only a minor role in this process. Accordingly, retinal cholesterol levels are unchanged / / / / in Soat1− − mice but are increased in Cyp27a1− −Cyp46a1− − and Apoe− − mice. Herein, we / / / / / / characterized Cyp27a1− −Cyp46a1− −Soat1− − and Cyp27a1− −Cyp46a1− −Apoe− − mice. In the former, retinal cholesterol levels, anatomical gross structure, and vasculature were normal, yet the / / / electroretinographic responses were impaired. Conversely, in Cyp27a1− −Cyp46a1− −Apoe− − mice, retinal cholesterol levels were increased while anatomical structure and vasculature were unaffected with only male mice showing a decrease in electroretinographic responses. Sterol profiling, qRT-PCR, proteomics, and transmission electron microscopy mapped potential compensatory mechanisms / / / / / / in the Cyp27a1− −Cyp46a1− −Soat1− − and Cyp27a1− −Cyp46a1− −Apoe− − retina. These included decreased cholesterol biosynthesis along with enhanced formation of intra- and extracellular vesicles, possibly a reserve mechanism for lowering retinal cholesterol. In addition, there was altered / / / abundance of proteins in Cyp27a1− −Cyp46a1− −Soat1− − mice that can affect photoreceptor function, survival, and retinal energy homeostasis (glucose and fatty acid metabolism). Therefore, the levels of retinal cholesterol do not seem to predict retinal abnormalities, and it is rather the network of compensatory mechanisms that appears to determine retinal phenotype. Keywords: CYP27A1; CYP46A1; SOAT1; APOE; cholesterol; oxysterols; vesicular traffic; glucose metabolism; fatty acid oxidation; synaptic function; inflammation; photoreceptors; cilia; age-related macular degeneration 1. Introduction Cholesterol is abundant in mammalian retina, a multilayered light-sensitive tissue lining the back of the eye. The neurosensory retina is underlined by the retinal pigment epithelium (RPE) and then choroid, which nourish the retina. Evidence suggests that retinal cholesterol dyshomeostasis is linked to age-related macular degeneration (AMD), a cause of vision loss in ~200 million older adults worldwide [1]. First, in aging humans, cholesterol accumulates on either side of the RPE and represents a significant component of drusen and subretinal drusenoid deposits [2–6], the AMD hallmarks. Second, genetic studies identified several cholesterol-related genes (CETP, ABCA1, LIPC, LPL, VLDLR, LRP6, and APOE, which encodes apolipoprotein E) as risk factors for AMD and suggested that these gene effects are independent of plasma lipid profiles [7–11]. Lastly, the two prospective clinical trials of statins, drugs prescribed for reduction of plasma cholesterol, showed a decreased risk Biomolecules 2019, 9, 867; doi:10.3390/biom9120867 www.mdpi.com/journal/biomolecules Biomolecules 2019, 9, 867 2 of 22 of progressionBiomolecules 2019 of, non-advanced 9, x AMD in specific patient populations [12,13]. Moreover, evidence2 of 22 was obtained that intensive statin therapy may even remove drusen in early stages of AMD [13]. These resultsdecreased underscore risk theof progression importance of of non-advanced understanding AM theD in mechanisms specific patient by which populations retinal [12,13]. cholesterol is managed.Moreover, evidence was obtained that intensive statin therapy may even remove drusen in early Thestages major of AMD pathways [13]. These of results cholesterol underscore input the andimportance output ofin understanding the retina seemthe mechanisms to be identified by (Figurewhich1)[14 retinal–20]. cholesterol Input is dependent is managed. on cholesterol biosynthesis in di fferent retinal cells and uptake The major pathways of cholesterol input and output in the retina seem to be identified (Figure from the systemic circulation; the latter is likely from the choriocapillaris, as cholesterol delivery 1) [14−20]. Input is dependent on cholesterol biosynthesis in different retinal cells and uptake from fromthe the systemic inner retinal circulation; blood the vessels latter is islikely currently from the speculative choriocapillaris, [20]. as Output cholesterol includes delivery metabolism from the to oxysterolsinner retinal by the blood cytochrome vessels is P450 currently enzymes speculative CYP27A1 [20]. and Output CYP46A1, includes the metabolism photoreceptor to oxysterols phagocytosis, by and etheffl uxcytochrome to lipoprotein P450 enzymes particles CYP27A1 (LP), which and CYP46A1 circulate, the in photoreceptor the intraretinal phag space.ocytosis, LP and then efflux deliver cholesterolto lipoprotein to different particles retinal (LP), cells which as well circulate as the in systemic the intraretinal circulation, space. probably LP then deliver through cholesterol the RPE [to14 –20]. Intracellulardifferent esterificationretinal cells as by well sterol-O-acyltransferase as the systemic circulation, 1 (SOAT1, probably named through previously the RPE as[14 acyl-CoA−20]. cholesterolIntracellular acyltransferase esterification 1 orby ACAT1,sterol-O-acyltransferase Text S1) represents 1 (SOAT1, themeans namedof previously cholesterol as storageacyl-CoA and a formcholesterol of cholesterol acyltransferase output. Cholesterol 1 or ACAT1, esters Text form S1) represents lipid droplets, the means cellular of cholesterol organelles, storage which and provide a a uniqueform separation of cholesterol of the output. aqueous Cholesterol and organic esters form phases lipid of droplets, the cell [cellular21]. Cholesterol organelles, esterswhich areprovide found in a unique separation of the aqueous and organic phases of the cell [21]. Cholesterol esters are found the photoreceptors outer segments, drusen, and Bruch’s membrane [2,3,22]. in the photoreceptors outer segments, drusen, and Bruch’s membrane [2,3,22]. FigureFigure 1. Schematic1. Schematic representation representation ofof thethe known chol cholesterol-relatedesterol-related pathways pathways contributing contributing to to cholesterolcholesterol homeostasis homeostasis in in the the retina. retina. Local Local biosynthesisbiosynthesis accounts accounts for for the the majority majority (~72%) (~72%) of retinal of retinal cholesterolcholesterol input, input, at least at least in in mice, mice, with with the the remaining remaining cholesterol cholesterol (~28%) (~28%) being being taken taken up by up the by retina the retina fromfrom the systemicthe systemic circulation circulation [ 23[23].]. A small small fraction fraction of ofretinal retinal cholesterol cholesterol (up to (up 15%) to is 15%) esterified is esterified by by SOAT1SOAT1 [24[24,25],25] andand isis storedstored in the form of of lipid lipid droplets. droplets. Retinal Retinal choles cholesterolterol output output includes includes metabolismmetabolism by CYP27A1by CYP27A1 and and CYP46A1, CYP46A1, the the photoreceptorphotoreceptor phagocytosis, phagocytosis, and and removal removal on lipoprotein on lipoprotein particlesparticles containing containing APOE. APOE. The The relative relative quantitative quantitative contribution of of these these three three pathways pathways to the to total the total cholesterol output in the retina is currently unknown. 27HC and 24HC are 27-hydroxycholesterol and cholesterol output in the retina is currently unknown. 27HC and 24HC are 27-hydroxycholesterol and 24-hydroxycholesterol, respectively, the products of CYP27A1 and CYP46A1; EC, esterified 24-hydroxycholesterol, respectively, the products of CYP27A1 and CYP46A1; EC, esterified cholesterol, cholesterol, the product of SOAT1; LP, lipoprotein particles. the product of SOAT1; LP, lipoprotein particles. To ascertain retinal significance of cholesterol removal by metabolism and LP, we previously Tocomprehensively ascertain retinal characterized significance the retina of cholesterol of Cyp27a1 removal−/−Cyp46a1 by−/− metabolism mice and mice and lacking LP, we APOE, previously a / / comprehensivelymajor apolipoprotein characterized in the retina the retina[22,26,27]. of Cyp27a1In the former,− −Cyp46a1 total cholesterol− − mice was and increased mice lacking up to 2- APOE, a majorfold, apolipoprotein and all of the cholesterol in the retina surplus [22,26 was,27]. converted In the former, to cholesterol total cholesterol esters, which was were increased mainly up to 2-fold,accumulated and all of in the the cholesterolphotoreceptor surplus outer segments was converted [22]. In addition, to cholesterol there we esters,re focal which accumulations were mainly accumulatedof free (unesterified) in the photoreceptor cholesterol outer in the segments Cyp27a1 [−/22−Cyp46a1]. In addition,−/− retina, there impairments were focal in accumulationsthe retinal function (as assessed by electroretinographic recordings,/ ERG),/ and pathologic
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