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• W2518362517 abstract "The process of vision is impossible without the photoreceptor cells, which have a unique structure and specific maintenance of cholesterol. Herein we report on the previously unrecognized cholesterol-related pathway in the retina discovered during follow-up characterizations of Cyp27a1−/−Cyp46a1−/− mice. These animals have retinal hypercholesterolemia and convert excess retinal cholesterol into cholesterol esters, normally present in the retina in very small amounts. We established that in the Cyp27a1−/−Cyp46a1−/− retina, cholesterol esters are generated by and accumulate in the photoreceptor outer segments (OS), which is the retinal layer with the lowest cholesterol content. Mouse OS were also found to express the cholesterol-esterifying enzyme acyl-coenzyme A:cholesterol acyltransferase (ACAT1), but not lecithin-cholesterol acyltransferase (LCAT), and to differ from humans in retinal expression of ACAT1. Nevertheless, cholesterol esters were discovered to be abundant in human OS. We suggest a mechanism for cholesterol ester accumulation in the OS and that activity impairment of ACAT1 in humans may underlie the development of subretinal drusenoid deposits, a hallmark of age-related macular degeneration, which is a common blinding disease. We generated Cyp27a1−/−Cyp46a1−/−Acat1−/− mice, characterized their retina by different imaging modalities, and confirmed that unesterified cholesterol does accumulate in their OS and that there is photoreceptor apoptosis and OS degeneration in this line. Our results provide insights into the retinal response to local hypercholesterolemia and the retinal significance of cholesterol esterification, which could be cell-specific and both beneficial and detrimental for retinal structure and function. The process of vision is impossible without the photoreceptor cells, which have a unique structure and specific maintenance of cholesterol. Herein we report on the previously unrecognized cholesterol-related pathway in the retina discovered during follow-up characterizations of Cyp27a1−/−Cyp46a1−/− mice. These animals have retinal hypercholesterolemia and convert excess retinal cholesterol into cholesterol esters, normally present in the retina in very small amounts. We established that in the Cyp27a1−/−Cyp46a1−/− retina, cholesterol esters are generated by and accumulate in the photoreceptor outer segments (OS), which is the retinal layer with the lowest cholesterol content. Mouse OS were also found to express the cholesterol-esterifying enzyme acyl-coenzyme A:cholesterol acyltransferase (ACAT1), but not lecithin-cholesterol acyltransferase (LCAT), and to differ from humans in retinal expression of ACAT1. Nevertheless, cholesterol esters were discovered to be abundant in human OS. We suggest a mechanism for cholesterol ester accumulation in the OS and that activity impairment of ACAT1 in humans may underlie the development of subretinal drusenoid deposits, a hallmark of age-related macular degeneration, which is a common blinding disease. We generated Cyp27a1−/−Cyp46a1−/−Acat1−/− mice, characterized their retina by different imaging modalities, and confirmed that unesterified cholesterol does accumulate in their OS and that there is photoreceptor apoptosis and OS degeneration in this line. Our results provide insights into the retinal response to local hypercholesterolemia and the retinal significance of cholesterol esterification, which could be cell-specific and both beneficial and detrimental for retinal structure and function. Photoreceptor (PR) 2The abbreviations used are: PR, photoreceptor; ACAT, acyl-coenzyme A:cholesterol acyltransferase; AMD, age-related macular degeneration; EC, esterified cholesterol; GCL, ganglion cell layer; GC-MS, gas chromatography-mass spectrometry; GS, glutamine synthase; IS, inner segments; LCAT, lecithin-cholesterol acyltransferase; LCM, laser capture microdissection; OPL, outer plexiform layer; OS, outer segment; RPE, retinal pigment epithelium; SDD, subretinal drusenoid deposits; SD-OCT, spectral domain optical coherence tomography; TC, total cholesterol; TEM, transmission electron microscopy; UC, unesterified cholesterol; qRT-PCR, quantitative RT-PCR. cells, rods and cones, are highly specialized neurons that initiate the transmission of the visual signal to the brain. These cells are localized exclusively to the retina, a multilayered structure lining the inner surface of the eye. Vertebrate PR cells have a unique polarized morphology that includes the outer segment (OS), formed by stacks of discs responsible for light capture and the transmission of visual signal; the inner segment (IS), rich in mitochondria to provide the cell with energy; the nucleus-containing soma; the axon; and the synaptic terminal (1Swaroop A. Kim D. Forrest D. Transcriptional regulation of photoreceptor development and homeostasis in the mammalian retina.Nat. Rev. Neurosci. 2010; 11: 563-576Crossref PubMed Scopus (376) Google Scholar). Because PR cells are essential for light perception, their dysfunction affects vision, and their loss due to degeneration causes irreversible blindness (2Jayakody S.A. Gonzalez-Cordero A. Ali R.R. Pearson R.A. Cellular strategies for retinal repair by photoreceptor replacement.Prog. Retin. Eye Res. 2015; 46: 31-66Crossref PubMed Scopus (93) Google Scholar). Not only do the PR cells have a unique structure, but they also have very specific maintenance of cholesterol as indicated by the lack of the key proteins of cholesterol biosynthesis, uptake, metabolism, efflux, and regulation (3Zheng W. Reem R.E. Omarova S. Huang S. DiPatre P.L. Charvet C.D. Curcio C.A. Pikuleva I.A. Spatial distribution of the pathways of cholesterol homeostasis in human retina.PloS One. 2012; 7: e37926Crossref PubMed Scopus (84) Google Scholar, 4Zheng W. Mast N. Saadane A. Pikuleva I.A. Pathways of cholesterol homeostasis in mouse retina responsive to dietary and pharmacologic treatments.J. Lipid Res. 2015; 56: 81-97Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar). Cholesterol distribution within PR cells is also unique; it is uneven (3Zheng W. Reem R.E. Omarova S. Huang S. DiPatre P.L. Charvet C.D. Curcio C.A. Pikuleva I.A. Spatial distribution of the pathways of cholesterol homeostasis in human retina.PloS One. 2012; 7: e37926Crossref PubMed Scopus (84) Google Scholar, 5Pikuleva I.A. Curcio C.A. Cholesterol in the retina: the best is yet to come.Prog. Retin. Eye Res. 2014; 41: 64-89Crossref PubMed Scopus (176) Google Scholar) and forms a gradient with a higher sterol concentration at the IS/OS border and a lower cholesterol concentration at the tip of the OS embraced by apical processes of the retinal pigment epithelium (RPE) (6Boesze-Battaglia K. Hennessey T. Albert A.D. Cholesterol heterogeneity in bovine rod outer segment disk membranes.J. Biol. Chem. 1989; 264: 8151-8155Abstract Full Text PDF PubMed Google Scholar). The reason for such unique maintenance and distribution of cholesterol in the PR cells is currently unknown but could be due to the inhibitory effect of cholesterol on the efficiency of the phototransduction cascade initiated in the OS (7Boesze-Battaglia K. Albert A.D. Cholesterol modulation of photoreceptor function in bovine retinal rod outer segments.J. Biol. Chem. 1990; 265: 20727-20730Abstract Full Text PDF PubMed Google Scholar, 8Albert A.D. Boesze-Battaglia K. The role of cholesterol in rod outer segment membranes.Prog. Lipid Res. 2005; 44: 99-124Crossref PubMed Scopus (90) Google Scholar). OS phagocytosis by the RPE could be a factor as well, because it accounts for ∼10% of daily OS renewal (9Bok D. Young R.W. Phagocytic Properties of the Retinal Pigment Epithelium. Harvard University Press, Cambridge, MA1979Google Scholar). Accordingly, the tip of the OS has adapted to contain low cholesterol content to minimize daily retinal cholesterol loss from phagocytosis and the amount of cholesterol that has to be replenished (3Zheng W. Reem R.E. Omarova S. Huang S. DiPatre P.L. Charvet C.D. Curcio C.A. Pikuleva I.A. Spatial distribution of the pathways of cholesterol homeostasis in human retina.PloS One. 2012; 7: e37926Crossref PubMed Scopus (84) Google Scholar). Cholesterol maintenance in the retina has been linked to age-related macular degeneration (AMD) (5Pikuleva I.A. Curcio C.A. Cholesterol in the retina: the best is yet to come.Prog. Retin. Eye Res. 2014; 41: 64-89Crossref PubMed Scopus (176) Google Scholar), a devastating blinding disease in the elderly of the industrialized world (10Pascolini D. Mariotti S.P. Pokharel G.P. Pararajasegaram R. Etya'ale D. Négrel A.D. Resnikoff S. 2002 global update of available data on visual impairment: a compilation of population-based prevalence studies.Ophthalmic Epidemiol. 2004; 11: 67-115Crossref PubMed Scopus (350) Google Scholar). Drusen and subretinal drusenoid deposits (SDD), the two major hallmarks of AMD, contain large amounts of cholesterol (11Curcio C.A. Millican C.L. Bailey T. Kruth H.S. Accumulation of cholesterol with age in human Bruch's membrane.Invest. Ophthalmol. Vis. Sci. 2001; 42: 265-274PubMed Google Scholar, 12Curcio C.A. Presley J.B. Malek G. Medeiros N.E. Avery D.V. Kruth H.S. Esterified and unesterified cholesterol in drusen and basal deposits of eyes with age-related maculopathy.Exp. Eye Res. 2005; 81: 731-741Crossref PubMed Scopus (192) Google Scholar13Oak A.S. Messinger J.D. Curcio C.A. Subretinal drusenoid deposits: further characterization by lipid histochemistry.Retina. 2014; 34: 825-826Crossref PubMed Scopus (54) Google Scholar) and develop below the RPE (14Wolter J.R. Falls H.F. Bilateral confluent drusen.Arch. Ophthalmol. 1962; 68: 219-226Crossref PubMed Scopus (36) Google Scholar) and at the OS/RPE interface, respectively (15Zweifel S.A. Spaide R.F. Curcio C.A. Malek G. Imamura Y. Reticular pseudodrusen are subretinal drusenoid deposits.Ophthalmology. 2010; 117: 303-312Abstract Full Text Full Text PDF PubMed Scopus (334) Google Scholar). Accordingly, the OS may be involved in the biogenesis of SDD (15Zweifel S.A. Spaide R.F. Curcio C.A. Malek G. Imamura Y. Reticular pseudodrusen are subretinal drusenoid deposits.Ophthalmology. 2010; 117: 303-312Abstract Full Text Full Text PDF PubMed Scopus (334) Google Scholar, 16Zweifel S.A. Imamura Y. Spaide T.C. Fujiwara T. Spaide R.F. Prevalence and significance of subretinal drusenoid deposits (reticular pseudodrusen) in age-related macular degeneration.Ophthalmology. 2010; 117: 1775-1781Abstract Full Text Full Text PDF PubMed Scopus (247) Google Scholar17Curcio C.A. Messinger J.D. Sloan K.R. McGwin G. Medeiros N.E. Spaide R.F. Subretinal drusenoid deposits in non-neovascular age-related macular degeneration: morphology, prevalence, topography, and biogenesis model.Retina. 2013; 33: 265-276Crossref PubMed Scopus (271) Google Scholar). In SDD, cholesterol is mostly unesterified (13Oak A.S. Messinger J.D. Curcio C.A. Subretinal drusenoid deposits: further characterization by lipid histochemistry.Retina. 2014; 34: 825-826Crossref PubMed Scopus (54) Google Scholar), whereas drusen contain both unesterified cholesterol (UC) and esterified cholesterol (EC) (11Curcio C.A. Millican C.L. Bailey T. Kruth H.S. Accumulation of cholesterol with age in human Bruch's membrane.Invest. Ophthalmol. Vis. Sci. 2001; 42: 265-274PubMed Google Scholar, 12Curcio C.A. Presley J.B. Malek G. Medeiros N.E. Avery D.V. Kruth H.S. Esterified and unesterified cholesterol in drusen and basal deposits of eyes with age-related maculopathy.Exp. Eye Res. 2005; 81: 731-741Crossref PubMed Scopus (192) Google Scholar). Retinal sensitivity in eyes with SDD is reduced much more than in eyes with typical drusen (18Mrejen S. Sato T. Curcio C.A. Spaide R.F. Assessing the cone photoreceptor mosaic in eyes with pseudodrusen and soft drusen in vivo using adaptive optics imaging.Ophthalmology. 2014; 121: 545-551Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar, 19Querques G. Massamba N. Srour M. Boulanger E. Georges A. Souied E.H. Impact of reticular pseudodrusen on macular function.Retina. 2014; 34: 321-329Crossref PubMed Scopus (67) Google Scholar), and SDD accumulation has been found to be a risk factor for AMD advancement and disease severity (20Huisingh C. McGwin Jr, G. Neely D. Zarubina A. Clark M. Zhang Y. Curcio C.A. Owsley C. The association between subretinal drusenoid deposits in older adults in normal macular health and incident age-related macular degeneration.Invest. Ophthalmol. Vis. Sci. 2016; 57: 739-745Crossref PubMed Scopus (17) Google Scholar21Zarubina A.V. Neely D.C. Clark M.E. Huisingh C.E. Samuels B.C. Zhang Y. McGwin Jr., G. Owsley C. Curcio C.A. Prevalence of subretinal drusenoid deposits in older persons with and without age-related macular degeneration, by multimodal imaging.Ophthalmology. 2016; 123: 1090-1100Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar, 22Marsiglia M. Boddu S. Bearelly S. Xu L. Breaux Jr, B.E. Freund K.B. Yannuzzi L.A. Smith R.T. Association between geographic atrophy progression and reticular pseudodrusen in eyes with dry age-related macular degeneration.Invest. Ophthalmol. Vis. Sci. 2013; 54: 7362-7369Crossref PubMed Scopus (133) Google Scholar, 23Xu L. Blonska A.M. Pumariega N.M. Bearelly S. Sohrab M.A. Hageman G.S. Smith R.T. Reticular macular disease is associated with multilobular geographic atrophy in age-related macular degeneration.Retina. 2013; 33: 1850-1862Crossref PubMed Scopus (78) Google Scholar24Steinberg J.S. Auge J. Jaffe G.J. Fleckenstein M. Holz F.G. Schmitz-Valckenberg S. GAP Study Group Longitudinal analysis of reticular drusen associated with geographic atrophy in age-related macular degeneration.Invest. Ophthalmol. Vis. Sci. 2013; 54: 4054-4060Crossref PubMed Scopus (34) Google Scholar). SDD are also associated with a higher mortality rate (25Klein R. Meuer S.M. Knudtson M.D. Iyengar S.K. Klein B.E. The epidemiology of retinal reticular drusen.Am. J. Ophthalmol. 2008; 145: 317-326Abstract Full Text Full Text PDF PubMed Scopus (179) Google Scholar), which might be a manifestation of some systemic, perhaps inflammatory or vascular, diseases (26Pumariega N.M. Smith R.T. Sohrab M.A. Letien V. Souied E.H. A prospective study of reticular macular disease.Ophthalmology. 2011; 118: 1619-1625Abstract Full Text Full Text PDF PubMed Scopus (128) Google Scholar). As part of our investigation of retinal cholesterol homeostasis, we generated Cyp27a1−/−Cyp46a1−/− mice (27Saadane A. Mast N. Charvet C.D. Omarova S. Zheng W. Huang S.S. Kern T.S. Peachey N.S. Pikuleva I.A. Retinal and non-ocular abnormalities in Cyp27a1−/− Cyp64a1−/− mice with dysfunctional metabolism of cholesterol.Am. J. Pathol. 2014; 184: 2403-2419Abstract Full Text Full Text PDF PubMed Scopus (33) Google Scholar) lacking cytochromes P450 CYP27A1 and CYP46A1, responsible for the majority of cholesterol metabolism in the retina (28Liao W.L. Heo G.Y. Dodder N.G. Pikuleva I.A. Turko I.V. Optimizing the conditions of a multiple reaction monitoring assay for membrane proteins: quantification of cytochrome P450 11A1 and adrenodoxin reductase in bovine adrenal cortex and retina.Anal. Chem. 2010; 82: 5760-5767Crossref PubMed Scopus (27) Google Scholar29Mast N. Reem R. Bederman I. Huang S. DiPatre P.L. Bjorkhem I. Pikuleva I.A. Cholestenoic acid is an important elimination product of cholesterol in the retina: comparison of retinal cholesterol metabolism with that in the brain.Invest. Ophthalmol. Vis. Sci. 2011; 52: 594-603Crossref PubMed Scopus (71) Google Scholar, 30Wang M. Heo G.Y. Omarova S. Pikuleva I.A. Turko I.V. Sample prefractionation for mass spectrometry quantification of low-abundance membrane proteins.Anal. Chem. 2012; 84: 5186-5191Crossref PubMed Scopus (20) Google Scholar31Liao W.L. Heo G.Y. Dodder N.G. Reem R.E. Mast N. Huang S. Dipatre P.L. Turko I.V. Pikuleva I.A. Quantification of cholesterol-metabolizing P450s CYP27A1 and CYP46A1 in neural tissues reveals a lack of enzyme-product correlations in human retina but not human brain.J. Proteome Res. 2011; 10: 241-248Crossref PubMed Scopus (39) Google Scholar). These animals have a 1.8- and 2-fold increase in total retinal cholesterol in females and males, respectively, with more than half of this total cholesterol (TC) being esterified. This is very unusual for the neural retina, in which cholesterol is mostly (∼85%) unesterified under normal conditions (29Mast N. Reem R. Bederman I. Huang S. DiPatre P.L. Bjorkhem I. Pikuleva I.A. Cholestenoic acid is an important elimination product of cholesterol in the retina: comparison of retinal cholesterol metabolism with that in the brain.Invest. Ophthalmol. Vis. Sci. 2011; 52: 594-603Crossref PubMed Scopus (71) Google Scholar, 32Bretillon L. Thuret G. Grégoire S. Acar N. Joffre C. Bron A.M. Gain P. Creuzot-Garcher C.P. Lipid and fatty acid profile of the retina, retinal pigment epithelium/choroid, and the lacrimal gland, and associations with adipose tissue fatty acids in human subjects.Exp. Eye Res. 2008; 87: 521-528Crossref PubMed Scopus (78) Google Scholar). Significant cholesterol esterification in the Cyp27a1−/−Cyp46a1−/− retina provided the impetus for the present work, in which we investigated retinal localization and the origin of EC in Cyp27a1−/−Cyp46a1−/− mice. We found that EC is localized mainly in the Cyp27a1−/−Cyp46a1−/− OS, thereby revealing a previously unrecognized, photoreceptor-specific mechanism for handling retinal cholesterol excess. Subsequent investigation of human retinas suggested that the data obtained may be of clinical relevance because they provide novel insight into the biogenesis of SDD, which is only now beginning to be investigated (13Oak A.S. Messinger J.D. Curcio C.A. Subretinal drusenoid deposits: further characterization by lipid histochemistry.Retina. 2014; 34: 825-826Crossref PubMed Scopus (54) Google Scholar, 33Rudolf M. Malek G. Messinger J.D. Clark M.E. Wang L. Curcio C.A. Sub-retinal drusenoid deposits in human retina: organization and composition.Exp. Eye Res. 2008; 87: 402-408Crossref PubMed Scopus (151) Google Scholar). Normally, EC accounts for only 14% of TC in the retina of female and male mice (Fig. 1A). Yet, in the retina of Cyp27a1−/−Cyp46a1−/− mice, in which TC is increased from 1.8- to 2-fold depending on the gender (Fig. 1A), EC represents from 55% (females) to 76% (males) of TC as established by our previous measurements by gas chromatography-mass spectrometry (GC-MS) (27Saadane A. Mast N. Charvet C.D. Omarova S. Zheng W. Huang S.S. Kern T.S. Peachey N.S. Pikuleva I.A. Retinal and non-ocular abnormalities in Cyp27a1−/− Cyp64a1−/− mice with dysfunctional metabolism of cholesterol.Am. J. Pathol. 2014; 184: 2403-2419Abstract Full Text Full Text PDF PubMed Scopus (33) Google Scholar). To localize EC in the Cyp27a1−/−Cyp46a1−/− retina, we used the fluorescent antibiotic filipin, which interacts with the 3β-hydroxyl in EC released by tissue pretreatment with cholesterol esterase (34Castanho M.A. Coutinho A. Prieto M.J. Absorption and fluorescence spectra of polyene antibiotics in the presence of cholesterol.J. Biol. Chem. 1992; 267: 204-209Abstract Full Text PDF PubMed Google Scholar, 35Rudolf M. Curcio C.A. Esterified cholesterol is highly localized to Bruch's membrane, as revealed by lipid histochemistry in wholemounts of human choroid.J. Histochem. Cytochem. 2009; 57: 731-739Crossref PubMed Scopus (62) Google Scholar). As compared with the Cyp27a1+/+Cyp46a1+/+ retina, which showed no fluorescent signal for EC (Fig. 2, A–C), the Cyp27a1−/−Cyp46a1−/− retina had a strong fluorescent signal for EC in the OS and much weaker fluorescent signals in the ganglion cell layer (GCL) and outer plexiform layer (OPL) (Fig. 2, D–F).FIGURE 2.Histochemical detection of retinal EC with filipin. Filipin is a fluorescent antibiotic that interacts with the 3β-hydroxyl group of UC and other sterols. This allows for the visualization of EC after tissue pretreatment with 70% aqueous ethanol to extract UC followed by cholesterol esterase to release the 3β-hydroxyl group in remaining EC. The left section in each panel is a phase contrast image, and the right section is a histochemistry image. A and D, control stains for completeness of UC removal. These sections were extracted with ethanol and treated with filipin. B and E, control stains for background fluorescence. These sections were extracted with ethanol but not treated with filipin or cholesterol esterase. C and F, stains for EC. These sections were extracted with ethanol and then sequentially treated with cholesterol esterase and filipin. All images are representative: n = 3 mice (3–7 months old)/genotype, with one retina from each mouse. Scale bars: 100 μm.View Large Image Figure ViewerDownload Hi-res image Download (PPT) We next isolated the PR layer, containing both OS and IS, from retinal cross-sections of Cyp27a1+/+Cyp46a1+/+ and Cyp27a1−/−Cyp46a1−/− mice by laser capture microdissection (LCM) and analyzed this layer for cholesterol content by GC-MS. TC was increased ∼3-fold in the Cyp27a1−/−Cyp46a1−/− PR cells relative to the Cyp27a1+/+Cyp46a1+/+ PR cells (Fig. 3A), a higher TC increase than in the whole retina (1.8–2-fold, Fig. 1A) suggesting that PR cells are a site for cholesterol accumulation in the Cyp27a1−/−Cyp46a1−/− retina. Furthermore, an increase in TC in the Cyp27a1−/−Cyp46a1−/− PR cells was because of an increase in EC, which accounted for 79% of TC in this genotype. For comparison, EC represented only 22% of TC in the Cyp27a1+/+Cyp46a1+/+ PR cells. Thus, sterol quantifications in mouse PR cells confirmed EC detection by histochemistry (Fig. 2F). We then used transmission electron microscopy (TEM) following tannic acid and para-phenylenediamine treatment of the osmicated retina (11Curcio C.A. Millican C.L. Bailey T. Kruth H.S. Accumulation of cholesterol with age in human Bruch's membrane.Invest. Ophthalmol. Vis. Sci. 2001; 42: 265-274PubMed Google Scholar, 36Guyton J.R. Klemp K.F. Ultrastructural discrimination of lipid droplets and vesicles in atherosclerosis: value of osmium-thiocarbohydrazide-osmium and tannic acid-paraphenylenediamine techniques.J. Histochem. Cytochem. 1988; 36: 1319-1328Crossref PubMed Scopus (71) Google Scholar). Tannic acid preserves the lipids (phospholipids and UC) of vesicular membranes, whereas para-phenylenediamine preserves the lipids (neutral lipids such as EC) in droplets (36Guyton J.R. Klemp K.F. Ultrastructural discrimination of lipid droplets and vesicles in atherosclerosis: value of osmium-thiocarbohydrazide-osmium and tannic acid-paraphenylenediamine techniques.J. Histochem. Cytochem. 1988; 36: 1319-1328Crossref PubMed Scopus (71) Google Scholar). Lipid accumulations were found in the Cyp27a1−/−Cyp46a1−/− OS and RPE but not in the Cyp27a1+/+Cyp46a1+/+ retina (Fig. 4). These accumulations were in the form of droplets of varied size, from 50 to 300 nm in diameter, present inside and outside the PR cells as singlets or in clusters (Fig. 4, B and C). The linear track of droplets in some of the Cyp27a1−/−Cyp46a1−/− OS (Fig. 4D) could indicate that droplets are formed inside the PR cell and are then expelled into the interphotoreceptor space following membrane disruption. Lipid droplets were also found in the Cyp27a1−/−Cyp46a1−/− RPE, cytosol, and phagosomes (Fig. 4, F, H, and I) but in apparently smaller amounts than in the Cyp27a1−/−Cyp46a1−/− OS. Unlike the OS, the Cyp27a1−/−Cyp46a1−/− RPE contained large semi-electron-lucent areas with content, which included phagosomes, vesicular membranes, and membranous debris (Fig. 4, F, G, and I). Because EC was not detectable in the Cyp27a1−/−Cyp46a1−/− RPE by filipin (Fig. 2F), and we had found previously that the Cyp27a1−/−Cyp46a1−/− RPE may contain focal deposits of UC (27Saadane A. Mast N. Charvet C.D. Omarova S. Zheng W. Huang S.S. Kern T.S. Peachey N.S. Pikuleva I.A. Retinal and non-ocular abnormalities in Cyp27a1−/− Cyp64a1−/− mice with dysfunctional metabolism of cholesterol.Am. J. Pathol. 2014; 184: 2403-2419Abstract Full Text Full Text PDF PubMed Scopus (33) Google Scholar), it is possible that these large semi-electron-lucent areas represent focal deposits of UC. Studies by TEM are supported by our previous light microscopy stains with oil red O, a dye that interacts mainly with neutral lipids and fatty acids. As found from analysis by TEM, the oil red O stains also revealed lipid accumulation in the Cyp27a1−/−Cyp46a1−/− photoreceptors (27Saadane A. Mast N. Charvet C.D. Omarova S. Zheng W. Huang S.S. Kern T.S. Peachey N.S. Pikuleva I.A. Retinal and non-ocular abnormalities in Cyp27a1−/− Cyp64a1−/− mice with dysfunctional metabolism of cholesterol.Am. J. Pathol. 2014; 184: 2403-2419Abstract Full Text Full Text PDF PubMed Scopus (33) Google Scholar). In mammals, cholesterol esterification is carried out by only three enzymes: two isoforms of acyl-coenzyme A:cholesterol acyltransferase (ACAT1 and ACAT2) and lecithin-cholesterol acyltransferase (LCAT) (37Chang C.C. Huh H.Y. Cadigan K.M. Chang T.Y. Molecular cloning and functional expression of human acyl-coenzyme A:cholesterol acyltransferase cDNA in mutant Chinese hamster ovary cells.J. Biol. Chem. 1993; 268: 20747-20755Abstract Full Text PDF PubMed Google Scholar38Cases S. Novak S. Zheng Y.W. Myers H.M. Lear S.R. Sande E. Welch C.B. Lusis A.J. Spencer T.A. Krause B.R. Erickson S.K. Farese Jr., R.V. ACAT-2, a second mammalian acyl-CoA:cholesterol acyltransferase: its cloning, expression, and characterization.J. Biol. Chem. 1998; 273: 26755-26764Abstract Full Text Full Text PDF PubMed Scopus (332) Google Scholar, 39Anderson R.A. Joyce C. Davis M. Reagan J.W. Clark M. Shelness G.S. Rudel L.L. 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Biol. 2006; 26: 1684-1686Crossref PubMed Scopus (31) Google Scholar), whereas LCAT is a secreted protein, which acts on cholesterol in high-density lipoproteins (43Glomset J.A. The mechanism of the plasma cholesterol esterification reaction: plasma fatty acid transferase.Biochim. Biophys. Acta. 1962; 65: 128-135Crossref PubMed Scopus (211) Google Scholar). ACAT1 is ubiquitous, whereas the expression of ACAT2 and LCAT is limited to specific tissues (40Oelkers P. Behari A. Cromley D. Billheimer J.T. Sturley S.L. Characterization of two human genes encoding acyl coenzyme A:cholesterol acyltransferase-related enzymes.J. Biol. Chem. 1998; 273: 26765-26771Abstract Full Text Full Text PDF PubMed Scopus (334) Google Scholar, 41Buhman K.K. Chen H.C. Farese Jr., R.V. The enzymes of neutral lipid synthesis.J. Biol. Chem. 2001; 276: 40369-40372Abstract Full Text Full Text PDF PubMed Scopus (134) Google Scholar, 44Uelmen P.J. Oka K. Sullivan M. Chang C.C. Chang T.Y. Chan L. Tissue-specific expression and cholesterol regulation of acyl-coenzyme A:cholesterol acyltransferase (ACAT) in mice: molecular cloning of mouse ACAT cDNA, chromosomal localization, and regulation of ACAT in vivo and in vitro.J. Biol. Chem. 1995; 270: 26192-26201Abstract Full Text Full Text PDF PubMed Scopus (117) Google Scholar). Previously, human and monkey retina was found to contain transcripts for ACAT1 and LCAT but not ACAT2 (45Li C.M. Presley J.B. Zhang X. Dashti N. Chung B.H. Medeiros N.E. Guidry C. Curcio C.A. Retina expresses microsomal triglyceride transfer protein: implications for age-related maculopathy.J. Lipid Res. 2005; 46: 628-640Abstract Full Text Full Text PDF PubMed Scopus (83) Google Scholar, 46Li C.M. Chung B.H. Presley J.B. Malek G. Zhang X. Dashti N. Li L. Chen J. Bradley K. Kruth H.S. Curcio C.A. Lipoprotein-like particles and cholesteryl esters in human Bruch's membrane: initial characterization.Invest. Ophthalmol. Vis. Sci. 2005; 46: 2576-2586Crossref PubMed Scopus (114) Google Scholar) and also to express LCAT as a protein (47Tserentsoodol N. Gordiyenko N.V. Pascual I. Lee J.W. Fliesler S.J. Rodriguez I.R. Intraretinal lipid transport is dependent on high density lipoprotein-like particles and class B scavenger receptors.Mol. Vis. 2006; 12: 1319-1333PubMed Google Scholar). We investigated whether Acat2 was also at low abundance in mouse retina. We used qRT-PCR and quantified the expression of Acat1, Acat2, and β-actin in wild type and Cyp27a1−/−Cyp46a1−/− retinas. In both genotypes, the mean Ct numbers (wild type/Cyp27a1−/−Cyp46a1−/−) for each of these proteins were very similar and equal to: Acat1, 23.9/23.0; Acat2, 35.5/34.9, and β-actin, 18.5/18.7. Very high Ct numbers for Acat2 suggest that in mouse retina, the ACAT2 protein levels are quite small if present at all. Therefore, we investigated the retinal expression of ACAT1 and LCAT only following the assessment of the quality of different anti-ACAT1 antibodies (Fig. 5); the anti-LCAT Ab has been characterized previously (47Tserentsoodol N. Gordiyenko N.V. Pascual I. Lee J.W. Fliesler S.J. Rodriguez I.R. Intraretinal lipid transport is dependent on high density lipoprotein-like particles and class B scavenger receptors.Mol. Vis. 2006; 12: 1319-1333P" @default.
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• W2518362517 title "Retinal Hypercholesterolemia Triggers Cholesterol Accumulation and Esterification in Photoreceptor Cells" @default.
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