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Thematic Review Series Thematic Review Series: Seeing 2020: Lipids and Lipid-Soluble Molecules in the Eye| Volume 62, 100057, January 01, 2021

Cholesterol homeostasis in the vertebrate retina: biology and pathobiology

  • Sriganesh Ramachandra Rao
    Affiliations
    Departments of Ophthalmology and Biochemistry and Neuroscience Graduate Program, Jacobs School of Medicine and Biomedical Sciences, State University of New York- University at Buffalo, Buffalo, NY, USA

    Research Service, VA Western NY Healthcare System, Buffalo, NY, USA
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  • Steven J. Fliesler
    Correspondence
    For correspondence: Steven J. Fliesler.
    Affiliations
    Departments of Ophthalmology and Biochemistry and Neuroscience Graduate Program, Jacobs School of Medicine and Biomedical Sciences, State University of New York- University at Buffalo, Buffalo, NY, USA

    Research Service, VA Western NY Healthcare System, Buffalo, NY, USA
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Open AccessPublished:March 01, 2021DOI:https://doi.org/10.1194/jlr.TR120000979

      Abstract

      Cholesterol is a quantitatively and biologically significant constituent of all mammalian cell membrane, including those that comprise the retina. Retinal cholesterol homeostasis entails the interplay between de novo synthesis, uptake, intraretinal sterol transport, metabolism, and efflux. Defects in these complex processes are associated with several congenital and age-related disorders of the visual system. Herein, we provide an overview of the following topics: (a) cholesterol synthesis in the neural retina; (b) lipoprotein uptake and intraretinal sterol transport in the neural retina and the retinal pigment epithelium (RPE); (c) cholesterol efflux from the neural retina and the RPE; and (d) biology and pathobiology of defects in sterol synthesis and sterol oxidation in the neural retina and the RPE. We focus, in particular, on studies involving animal models of monogenic disorders pertinent to the above topics, as well as in vitro models using biochemical, metabolic, and omic approaches. We also identify current knowledge gaps and opportunities in the field that beg further research in this topic area.

      Supplementary key words

      Abbreviations:

      7DHC (7-dehydrocholesterol), A2E (N-Retinylidene-N-retinylethanolamine (2-[2,6-dimethyl-8-(2,6,6-trimethyl-1-cyclohexen-1- yl)-1E,3E, 5E,7E-octatetraenyl]-1-(2-hydroxyethyl)-4-[4-methyl-6-(2,6,6-trimethyl-1-cyclohexen-1-yl)-1E, 3E, 5E-hexatrienyl]-pyridinium)), AMD (age-related macular degeneration), CD36 (cluster of differentiation 36), CNV (choroidal neovascularization), DHCR7 (7-dehydrocholesterol reductase), DHCR24 (24-dehydrocholesterol reductase), ERG (electroretinogram), GCL (Ganglion cell layer), HMGCR (HMG-CoA reductase), iPSC (induced pluripotent stem cell), IS (inner segment), LAL (lysosomal acid lipase), LOX-1 (OxLDL receptor), LRP1 (LDL-related particle 1), MVK (mevalonate kinase), NPC-I/II (Niemann-Pick type C I and II), OS (outer segment(s)), PFO (perfringolysin O), PN (postnatal), ROS (reactive oxgen species), RPE (retinal pigment epithelium), SQLE (squalene monooxygenase), SRB-I/II (class B scavenger receptor (I and II)), TSPO (translocator protein 18 kDa), VEGF (vascular endothelial growth factor)
      Sterols represent a diverse class of biologically significant lipids that are found ubiquitously in all eukaryotic cells, primarily in the plasma membrane (
      • Bloch K.
      Sterol structure and function.
      ). Cholesterol is, by far, the dominant sterol normally found in mammalian cells and tissues. Maintaining optimal levels of cholesterol is a requisite for normal cellular function and viability, and represents a delicate balance between endogenous de novo synthesis, exogenous uptake, and efflux of sterols. Mechanisms governing the process of cholesterol homeostasis have been investigated extensively, given the role they play both in normal biology and in several significant human clinical disorders, such as Alzheimer's disease, cardiovascular disease, and age-related macular degeneration (AMD) (
      • Luo J.
      • Yang H.
      • Song B.L.
      Mechanisms and regulation of cholesterol homeostasis.
      ). Herein, we review the mechanisms governing cholesterol homeostasis in the neural retina, and the pathological mechanisms that underlie certain ocular diseases where this homeostasis is disturbed.
      Cholesterol in the central nervous system (of which the retina is a part) exists mostly in the unesterified form, in nerve myelin and in the plasma membranes of both neuronal and non-neuronal (e.g., glial) cells. The brain relies exclusively on its own de novo synthesis of sterols because the blood-brain barrier excludes circulating lipoproteins (
      • Bjorkhem I.
      • Meaney S.
      Brain cholesterol: long secret life behind a barrier.
      ). By comparison, sterol homeostasis in the retina is somewhat more complex because its sterol pool is derived from both local de novo synthesis and extraretinal uptake. The retina has many advantages for experimental studies, such as its layered organization and ease of accessibility, and represents one of the best-studied parts of the central nervous system. Cholesterol and its biogenic sterol precursors can undergo both enzymatic and nonenzymatic oxidation, which is particularly relevant, given the pro-oxidative environment of the retina, yielding a variety of oxysterol products, some of which are highly toxic to cells (
      • Olkkonen V.M.
      • Beaslas O.
      • Nissila E.
      Oxysterols and their cellular effectors.
      ,
      • Griffiths W.J.
      • Wang Y.
      Oxysterol research: a brief review.
      ). Sterols and sterol metabolites may play causative roles in several neurodegenerative conditions, including certain retinopathies (
      • Griffiths W.J.
      • Wang Y.
      Oxysterol research: a brief review.
      ,
      • Bjorkhem I.
      • Cedazo-Minguez A.
      • Leoni V.
      • Meaney S.
      Oxysterols and neurodegenerative diseases.
      ,
      • Massey J.B.
      • Pownall H.J.
      Structures of biologically active oxysterols determine their differential effects on phospholipid membranes.
      ).
      In part, this review is an extension of a prior review in a similar Thematic Issue series in this journal published a decade ago (
      • Fliesler S.J.
      • Bretillon L.
      The ins and outs of cholesterol in the vertebrate retina.
      ). A subsequent review by other authors highlighted the role of sterol homeostatic processes in AMD (
      • Pikuleva I.A.
      • Curcio C.A.
      Cholesterol in the retina: the best is yet to come.
      ). The scope of this review encompasses the following topics: (a) cholesterol biosynthesis in the neural retina (in vitro and in vivo isotopomer techniques, pharmacological inhibition of sterol biosynthesis, and pharmacological/genetic modeling of monogenic diseases affecting cholesterol biosynthesis); (b) cholesterol up-take by the neural retina and the retinal pigment epithelium (RPE), and intraretinal sterol transport (in vitro and in vivo modeling of lipoprotein uptake and monogenic disease affecting lipoprotein synthesis/uptake, LDL-tagging methods); (c) cholesterol efflux in the neural retina (role of LXRα/β, ATP binding cassette (ABC) transporters, cytochrome P450 (CYP) enzymes, related monogenic diseases, and mechanisms of drusen formation in AMD, isotopic and pharmacologic approaches to measure the retinal sterol turnover rate); and (d) biology of lipid peroxidation in the neural retina and the RPE, and the effects of oxysterols and oxidized LDL (OxLDL) in vitro and in vivo (uptake and metabolism of OxLDL and oxysterols and its relevance in retinopathies).
      We will focus on studies that, for the most part, have been conducted using either laboratory animals (e.g., mice, rats) or animal-derived cells in vitro, augmented by the use of selective inhibitors of enzymes in the cholesterol synthesis pathway or by genetic manipulation of the genes encoding those enzymes. Although these experimental systems are admittedly highly simplified compared with the complexity of human ocular anatomy and physiology and cannot fully model multifactorial disorders such as AMD, they nonetheless have provided fundamental insights into cholesterol homeostasis in the vertebrate retina. In addition, the knowledge derived from such models has provided potentially useful tools for developing effective therapeutic interventions for human diseases that are caused by defects in the cholesterol pathway that impact the structure and function of the retina.

      Overview of retinal architecture

      The neural retina is the photon-sensory tissue lining the inner posterior segment of the eye (see Fig. 1, and http://webvision.med.utah.edu/). There are about 150 million cells in a human retina, about 85% of which are neurons, including, c.a., six to seven million cone photoreceptors and, c.a., 110–125 million rod photoreceptors. Photoreceptor cells are anatomically segregated into “inner segment” (IS) and “outer segment” (OS) compartments; the IS consists of mitochondria and houses the biosynthetic machinery of the cell, whereas the OS serves as the membrane residence for the phototransduction cascade (
      • Arshavsky V.Y.
      • Wensel T.G.
      Timing is everything: GTPase regulation in phototransduction.
      ). Other neuronal cell types in the retina include the bipolar, horizontal, amacrine, and ganglion cells, forming the neuronal circuitry involved in ultimately relaying visual information originating in the photoreceptor cells to the brain. The nonneuronal cell types include Müller glia, microglia, astrocytes, and the RPE, involved in meeting tissue homeostatic requirements. This review discusses the roles played by these cell types in retinal sterol homeostasis.
      Figure thumbnail gr1
      Fig. 1Histological organization of a model vertebrate retina. Schematic representation of individual retinal cell types is superimposed on a light microscopy image of a normal C57Bl/6J mouse retina (Toluidine blue-stained). The retinal pigment epithelium (RPE) forms a cellular monolayer interface between the neural retina and the choriocapillaris (the elements of the choroidal blood supply most proximal to the RPE). The RPE junctional complex network comprises the outer blood-retinal barrier, restricting the flow of blood-borne substances from the choroid to the outer retina. The photoreceptor layer (containing rods and cones) spans nearly half the total neural retina thickness and is comprised of the photoreceptor outer segment (OS) and inner segment (IS) layers, the outer nuclear layer (ONL, containing the rod and cone nuclei), and the outer plexiform layer (OPL), the latter containing the axonal processes and presynaptic endings of the photoreceptor cells, along with the postsynaptic processes of the bipolar cells and dendritic extensions of the horizontal cells. The inner nuclear layer (INL) consists of the nuclei and cell bodies of bipolar cells, amacrine cells, and horizontal cells, as well the Müller glia. The inner plexiform layer (IPL) consists of the axonal processes and synaptic termini of bipolar and amacrine cells, along with the dendritic arbors of the ganglion cells; the latter form the ganglion cell layer (GCL) of the neural retina. The collective axons of the ganglion cells form the nerve fiber layer (NFL) and exit the eye as the optic nerve en route to the visual cortex of the brain. The inner retinal cells are nourished by the retinal vasculature; the tight junctions of its constituent endothelial cells comprise the inner blood-retinal barrier. Microglia normally reside in the IPL and GCL, but migrate into the INL and outer retinal layers when activated. Note: The schematic does not depict some features specific to human or primate retinas, such as the macula or cone-rich fovea. ELM, external limiting membrane; ILM, internal limiting membrane. (Modified and adapted, with permission, from (
      • Wolkow N.
      • Song D.
      • Song Y.
      • Chu S.
      • Hadziahmetovic M.
      • Lee J.C.
      • et al.
      Ferroxidase hephaestin's cell-autonomous role in the retinal pigment epithelium.
      )).
      The OS membrane is comprised of approximately equal amounts of lipids and proteins, by weight (
      • Fliesler S.J.
      • Anderson R.E.
      Chemistry and metabolism of lipids in the vertebrate retina.
      ): the dominant lipids are glycerophospholipids (80%–85%), whereas cholesterol represents only about 8–10 mol % of the total lipid (which is only about a third of the level of cholesterol found in the plasma membrane of most cells); the overwhelming majority of the protein content of OS membranes (>90%) is accounted for by the visual pigment apoprotein, opsin. Diurnally shed photoreceptor outer segment disk membranes are then phagocytized and degraded by the adjacent underlying RPE (
      • Molday R.S.
      • Moritz O.L.
      Photoreceptors at a glance.
      ,
      • Young R.W.
      Visual cells and the concept of renewal.
      ). The compensatory synthesis and incorporation of OS membranes at its base contributes to the large demand for membrane constituents (including sterols) in photoreceptors (
      • Young R.W.
      Visual cells and the concept of renewal.
      ). Such demands of the retina may be met by a combination of de novo synthesis and receptor-mediated uptake from two separate blood supplies—the choroidal vasculature (supplying the outer retina, notably the photoreceptor cells) and the inner retinal vasculature—in conjunction with an internal auxiliary source represented by Müller glia. In addition, synaptic connections in the outer and inner plexiform layers, respectively, also necessitate a high rate of turnover of cholesterol pools in retinal neurons because of assembly and recycling of the synaptic vesicles that contain neurotransmitters. Below, we will consider the various potential contributors to overall cholesterol homeostasis in the retina.

      General considerations: cholesterol synthesis, uptake, and efflux

      A general schematic of the cholesterol biosynthesis pathway is provided in Fig. 2. The rate-limiting step of the cholesterol synthesis pathway (a.k.a. the mevalonate pathway) is catalyzed by HMG-CoA reductase (HMGCR; OMIM# 142910, EC 1.1.1.88). A secondary regulatory locus in this pathway is at the level of squalene-2,3-epoxidase [a.k.a. squalene monooxygenase (SQLE) OMIM# 602019, E.C. 1.14.99.7] (
      • Chua N.K.
      • Coates H.W.
      • Brown A.J.
      Squalene monooxygenase: a journey to the heart of cholesterol synthesis.
      ).
      Figure thumbnail gr2
      Fig. 2Schematic representation of the mevalonate pathway. Acetyl-CoA is converted in two steps, sequentially catalyzed by ACAT1 and ACAT2 and HMGCS1 (HMG-CoA synthase 1), to mevalonate, whose formation is the main rate-limiting step in the pathway, catalyzed by HMGCR (HMG-CoA reductase; inhibited by statins). The presqualene portion of the pathway generates a series of acyclic isoprenoid compounds, with a critical branch point at the level of farnesyl diphosphate (FPP) generation. The committed step toward sterol synthesis involves epoxidation of squalene to squalene-2,3-epoxide, catalyzed squalene epoxidase (SQLE; inhibited by NB-598), which then undergoes cyclization to form the first sterol intermediate (lanosterol; 4α,4β,14α-trimethyl-cholesta-8(9),24-dien-3β-ol) in the postsqualene portion of the pathway. This is followed by a series of demethylation and double-bond isomerization and reduction reactions, with ultimate engagement of either the Bloch Pathway or the Kandutsch-Russell Pathway to form cholesterol. Reduction of the side-chain double bonds in desmosterol (cholesta-5,24-dien-3β-ol) and 7-dehydrodesmosterol (cholesta-5,7,24-trien-3β-ol) is catalyzed by DHCR24 (inhibited by U18666A), whereas reduction of the ring B nuclear double bond in 7-dehydrocholesterol (7DHC; cholesta-5,7-dien-3β-ol) and 7-dehydrodesmosterol are catalyzed by DHCR7 (inhibited by AY9944). The mevalonate pathway is involved in the synthesis of several other important isoprenoid metabolites, including ubiquinone (coenzyme Q), dolichols, vitamin-D, and steroid hormones. Mutations in the DHCR24 gene lead to desmosterolosis, whereas such defects in the DHCR7 gene cause Smith-Lemli-Opitz syndrome (SLOS). Inset: Chemical structure of cholesterol (cholest-5-en-3β–ol). DHCR24, 24-dehydrocholesterol reductase; DHCR7, 7DHC reductase.
      The mevalonate pathway generates linear isoprenoid products such as farnesyl diphosphate and geranylgeranyl diphosphate (used for prenylation of proteins), dolichol and its phosphorylated derivatives (required for protein N-glycosylation), and squalene (the committed acyclic intermediate required for sterol synthesis), to name a few (
      • Miziorko H.M.
      Enzymes of the mevalonate pathway of isoprenoid biosynthesis.
      ,
      • Schroepfer Jr., G.J.
      Sterol biosynthesis.
      ,
      • Schroepfer Jr., G.J.
      Sterol biosynthesis.
      ). Sterol intermediates are then generated by cyclization of squalene through the Kandutsch-Russell pathway [lathosterol, 7-dehydrocholesterol (7DHC)] and the Bloch pathway (lanosterol, 7-dehydrodesmosterol, desmosterol). Desmosterol and 7DHC undergo reduction of double bonds at the C24 (Δ24) and C7 (Δ7) positions by 7DHC reductase (DHCR7; OMIM# 602858, EC 1.3.1.21) and 24-dehydrocholesterol reductase (DHCR24; OMIM# 606418, EC 1.3.1.72), respectively, to generate cholesterol. Genetic mutations in any step of this pathway can result in pathologies, typically recessive and often lethal, because of the buildup of intermediate sterols and their metabolites (
      • Porter F.D.
      • Herman G.E.
      Malformation syndromes caused by disorders of cholesterol synthesis.
      ,
      • Platt F.M.
      • Wassif C.
      • Colaco A.
      • Dardis A.
      • Lloyd-Evans E.
      • Bembi B.
      • et al.
      Disorders of cholesterol metabolism and their unanticipated convergent mechanisms of disease.
      ). Examples of such congenital disorders and the affected enzymes include Smith-Lemli-Opitz syndrome (SLOS; DHCR7, OMIM# 270400), desmosterolosis (DHCR24, OMIM# 602398), lathosterolosis (sterol-C5-desaturase, OMIM# 607330), and mevalonate kinase (MVK, OMIM# 251170) deficiency (see Fig. 2) (
      • Porter F.D.
      • Herman G.E.
      Malformation syndromes caused by disorders of cholesterol synthesis.
      ). The impact of such defects on the structure and function of the retina will be discussed.
      As an alternative to de novo synthesis of cholesterol, cells may import cholesteryl esters by membrane receptor–mediated endocytosis of blood-borne, liver-derived lipoproteins (VLDL, LDL, IDL) (
      • Siri-Tarino P.W.
      • Krauss R.M.
      The early years of lipoprotein research: from discovery to clinical application.
      ,
      • Herz J.
      • Bock H.H.
      Lipoprotein receptors in the nervous system.
      ). Lysosomal acid lipase (LAL) generates free cholesterol in the lysosomal lumen, which is then trafficked to the ER by Niemann-Pick type C I and II (NPC-I/NPC-II) protein complex. Mutations in NPC-I/NPC-II, causing Niemann-Pick disease, alter cholesterol trafficking and subsequent accumulation of free cholesterol in lysosomes (
      • Platt F.M.
      • Wassif C.
      • Colaco A.
      • Dardis A.
      • Lloyd-Evans E.
      • Bembi B.
      • et al.
      Disorders of cholesterol metabolism and their unanticipated convergent mechanisms of disease.
      ,
      • Subramanian K.
      • Balch W.E.
      NPC1/NPC2 function as a tag team duo to mobilize cholesterol.
      ). Alternatively, cholesterol uptake can involve class B scavenger receptor I (SRB-I)–mediated selective uptake of cholesteryl esters from HDL particles. OxLDL is endocytosed by the cell via cluster of differentiation 36 (CD36) and OxLDL receptor (LOX-1) receptors. The role of these sterol uptake machineries in the neural retina and the RPE is discussed.
      Cellular cholesterol is effluxed to naïve apolipoprotein (APO)A1-containing HDL by ABC transporters, which represent a large class of biologically important molecules involved in efflux of diverse substrates, such as ions, peptides and proteins, membrane lipids, and lipid-soluble molecules (
      • Linton K.J.
      • Higgins C.F.
      Structure and function of ABC transporters: the ATP switch provides flexible control.
      ,
      • Oram J.F.
      • Vaughan A.M.
      ATP-Binding cassette cholesterol transporters and cardiovascular disease.
      ). Several of these, including ABCA1, ABCG1, and ABCG4, play important roles in cholesterol efflux from peripheral tissues to naïve APO-A1–containing HDL particles and, to a lesser extent, to APO-E–containing LDL particles (
      • Oram J.F.
      • Vaughan A.M.
      ATP-Binding cassette cholesterol transporters and cardiovascular disease.
      ,
      • Phillips M.C.
      Is ABCA1 a lipid transfer protein?.
      ,
      • Phillips M.C.
      Molecular mechanisms of cellular cholesterol efflux.
      ). Defects in ABC transporters can result in severe, often chronic, pathologies [including those that impact the structure and function of the retina (see below)]; for example, defects in ABCA1-mediated efflux have been implicated in atherogenesis and neurological disorders, such as Alzheimer's disease (
      • Oram J.F.
      • Vaughan A.M.
      ATP-Binding cassette cholesterol transporters and cardiovascular disease.
      • Tang C.
      • Oram J.F.
      The cell cholesterol exporter ABCA1 as a protector from cardiovascular disease and diabetes.
      ,
      • Rebeck G.W.
      Cholesterol efflux as a critical component of Alzheimer's disease pathogenesis.
      ). Tangier disease (OMIM# 205400) is caused by recessive mutations in ABCA1 and is characterized by hypoalphalipoproteinemia (low serum APO-A1-HDL levels), mild neuropathy, atherosclerosis, retinopathy, corneal infiltrates and scarring, lipid deposits in the conjunctiva, and cataract formation (
      • Fredrickson D.S.
      The inheritance of high density lipoprotein deficiency (Tangier disease).
      ). (The reader is referred to a recent review that provides detailed analysis of the specific role of HDL in age-related retinopathies (
      • Betzler B.K.
      • Rim T.H.
      • Sabanayagam C.
      • Cheung C.M.G.
      • Cheng C.Y.
      High-density lipoprotein cholesterol in age-related ocular diseases.
      ).) This review discusses the role of sterol efflux in retinal physiology and pathophysiology.
      The transcriptional regulation of sterol efflux-related ABC transporters occurs through the LXR-α response element (
      • Nakamura K.
      • Kennedy M.A.
      • Baldan A.
      • Bojanic D.D.
      • Lyons K.
      • Edwards P.A.
      Expression and regulation of multiple murine ATP-binding cassette transporter G1 mRNAs/isoforms that stimulate cellular cholesterol efflux to high density lipoprotein.
      ). Endogenous LXR-α agonists and antagonists play an important modulatory role in cellular sterol efflux (
      • Lehmann J.M.
      • Kliewer S.A.
      • Moore L.B.
      • Smith-Oliver T.A.
      • Oliver B.B.
      • Su J.L.
      • et al.
      Activation of the nuclear receptor LXR by oxysterols defines a new hormone response pathway.
      ,
      • Escher G.
      • Krozowski Z.
      • Croft K.D.
      • Sviridov D.
      Expression of sterol 27-hydroxylase (CYP27A1) enhances cholesterol efflux.
      ). Sterols undergo CYP enzyme-mediated (CYP27A1, CYP46A1, CYP11A1, CYP39A1, CYP7A1) oxidation or hydroxylation, generating sterol metabolites, which function as potent LXR-α agonists (
      • Theofilopoulos S.
      • Griffiths W.J.
      • Crick P.J.
      • Yang S.
      • Meljon A.
      • Ogundare M.
      • et al.
      Cholestenoic acids regulate motor neuron survival via liver X receptors.
      ,
      • Endo-Umeda K.
      • Yasuda K.
      • Sugita K.
      • Honda A.
      • Ohta M.
      • Ishikawa M.
      • et al.
      7-Dehydrocholesterol metabolites produced by sterol 27-hydroxylase (CYP27A1) modulate liver X receptor activity.
      ). LXR activation stimulates sterol efflux by upregulating the expression of ABC transporters (
      • Javitt N.B.
      25R,26-Hydroxycholesterol revisited: synthesis, metabolism, and biologic roles.
      ,
      • Griffiths W.J.
      • Crick P.J.
      • Meljon A.
      • Theofilopoulos S.
      • Abdel-Khalik J.
      • Yutuc E.
      • et al.
      Additional pathways of sterol metabolism: evidence from analysis of Cyp27a1-/- mouse brain and plasma.
      ). Mutations in a critical mitochondrial CYP enzyme, CYP27A1, causes cerebrotendinous xanthomatosis (OMIM# 213700), an autosomal recessive disorder clinically characterized by chronic diarrhea, neuronal dysfunction, early-onset of atherosclerosis, and visual system deficits such as cataract formation (
      • Salen G.
      • Steiner R.D.
      Epidemiology, diagnosis, and treatment of cerebrotendinous xanthomatosis (CTX).
      ,
      • Nie S.
      • Chen G.
      • Cao X.
      • Zhang Y.
      Cerebrotendinous xanthomatosis: a comprehensive review of pathogenesis, clinical manifestations, diagnosis, and management.
      ). The CYP enzymes act on a broad spectrum of substrates including sterols and oxysterols, generating biologically important metabolites. CYP7A1 is known to metabolize 7DHC to 7-oxocholesterol and 7-ketocholesterol (7KChol), contributing to SLOS and cerebrotendinous xanthomatosis pathology (
      • Bjorkhem I.
      • Diczfalusy U.
      • Lovgren-Sandblom A.
      • Starck L.
      • Jonsson M.
      • Tallman K.
      • et al.
      On the formation of 7-ketocholesterol from 7-dehydrocholesterol in patients with CTX and SLO.
      ,
      • Shinkyo R.
      • Xu L.
      • Tallman K.A.
      • Cheng Q.
      • Porter N.A.
      • Guengerich F.P.
      Conversion of 7-dehydrocholesterol to 7-ketocholesterol is catalyzed by human cytochrome P450 7A1 and occurs by direct oxidation without an epoxide intermediate.
      ). Mitochondrial CYP27A1 metabolizes cholesterol to 27-COOH-Chol and 27-OH-Chol, and 7KChol to 27-COOH-7KChol and 27-OH-7KChol, via acid hydroxylation (
      • Griffiths W.J.
      • Crick P.J.
      • Meljon A.
      • Theofilopoulos S.
      • Abdel-Khalik J.
      • Yutuc E.
      • et al.
      Additional pathways of sterol metabolism: evidence from analysis of Cyp27a1-/- mouse brain and plasma.
      ,
      • Pikuleva I.A.
      • Babiker A.
      • Waterman M.R.
      • Bjorkhem I.
      Activities of recombinant human cytochrome P450c27 (CYP27) which produce intermediates of alternative bile acid biosynthetic pathways.
      ), which stimulate ABCA1/G1-mediated cellular cholesterol efflux as discussed below (
      • Marengo B.
      • Bellora F.
      • Ricciarelli R.
      • De Ciucis C.
      • Furfaro A.
      • Leardi R.
      • et al.
      Oxysterol mixture and, in particular, 27-hydroxycholesterol drive M2 polarization of human macrophages.
      ,
      • Anderson A.
      • Campo A.
      • Fulton E.
      • Corwin A.
      • Jerome 3rd, W.G.
      • O'Connor M.S.
      7-Ketocholesterol in disease and aging.
      ). Hereby, we discuss the dynamic interplay of de novo synthesis, uptake, efflux, and metabolism of cholesterol in the retina to maintain steady-state content of cholesterol (i.e., cholesterol homeostasis), and retinopathies that arise upon perturbing these important homeostatic processes.

      Retinal de novo cholesterol synthesis and uptake

      Insights into cholesterol synthesis and uptake in the neural retina

      Early in vitro experiments by Fliesler and Schroepfer, using either bovine retina cell-free homogenates (10,000g supernatant fraction that contains microsome and cytosol) (
      • Fliesler S.J.
      • Schroepfer Jr., G.J.
      Metabolism of mevalonic acid in cell-free homogenates of bovine retinas. Formation of novel isoprenoid acids.
      ) or intact whole bovine retinas in organ culture (
      • Fliesler S.J.
      • Schroepfer Jr., G.J.
      In vitro metabolism of mevalonic acid in the bovine retina.
      ) incubated with [3H]mevalonate, demonstrated sterol synthesis in the vertebrate neural retina. However, the radiolabel was primarily incorporated in mevalonate pathway intermediates such as 15- and 20-carbon isoprenoid acids, while conversion to cholesterol was rather limited. The first in vivo investigations of cholesterol synthesis in the vertebrate retina utilized intravitreal injection of [3H]acetate in rats followed by monitoring its incorporation into cholesterol, in the presence and absence of lovastatin, an inhibitor of HMGCR (
      • Fliesler S.J.
      • Florman R.
      • Rapp L.M.
      • Pittler S.J.
      • Keller R.K.
      In vivo biosynthesis of cholesterol in the rat retina.
      ,
      • Pittler S.J.
      • Fliesler S.J.
      • Rapp L.M.
      Novel morphological changes in rat retina induced by intravitreal injection of lovastatin.
      ). The neural retina showed [3H]cholesterol formation within 6 h, with little accumulation in intermediates, and its formation was fully inhibited upon coinjection with lovastatin (
      • Fliesler S.J.
      • Florman R.
      • Rapp L.M.
      • Pittler S.J.
      • Keller R.K.
      In vivo biosynthesis of cholesterol in the rat retina.
      ,
      • Pittler S.J.
      • Fliesler S.J.
      • Rapp L.M.
      Novel morphological changes in rat retina induced by intravitreal injection of lovastatin.
      ,
      • Pittler S.J.
      • Fliesler S.J.
      • Fisher P.L.
      • Keller P.K.
      • Rapp L.M.
      In vivo requirement of protein prenylation for maintenance of retinal cytoarchitecture and photoreceptor structure.
      ). In the same study, inhibiting the postsqualene phase of the pathway using NB-598 (an inhibitor of squalene 2–3 epoxidase) (SQLE; Fig. 3) caused the accumulation of radiolabeled squalene, as predicted. Similarly, [3H]farnesol injected intravitreally in rats resulted in formation of [3H]cholesterol, in an NB-598–sensitive manner (
      • Fliesler S.J.
      • Keller R.K.
      Metabolism of [3H]farnesol to cholesterol and cholesterogenic intermediates in the living rat eye.
      ). These results first qualitatively demonstrated the presence of a functional de novo sterol synthesis pathway in the whole retina. However, calculation of absolute rates of cholesterol synthesis using this metabolic approach is not possible because of nonuniform cellular uptake and incorporation of radiolabeled de novo precursors into cholesterol, acetyl-CoA hydrolysis, and the pleiotropic effects of statins (
      • Laufs U.
      • Liao J.K.
      Isoprenoid metabolism and the pleiotropic effects of statins.
      ,
      • Dietschy J.M.
      • McGarry J.D.
      Limitations of acetate as a substrate for measuring cholesterol synthesis in liver.
      ,
      • Previs S.F.
      • Herath K.
      • Nawrocki A.R.
      • Rodriguez C.G.
      • Slipetz D.
      • Singh S.B.
      • et al.
      Using [(2)H]water to quantify the contribution of de novo palmitate synthesis in plasma: enabling back-to-back studies.
      ). Intravitreal injection of lovastatin led to early changes in the structural organization of the neural retina characterized by formation of rosette-like arrangements of photoreceptors and eventually necrosis of the retina by 4 days (
      • Pittler S.J.
      • Fliesler S.J.
      • Rapp L.M.
      Novel morphological changes in rat retina induced by intravitreal injection of lovastatin.
      ,
      • Pittler S.J.
      • Fliesler S.J.
      • Fisher P.L.
      • Keller P.K.
      • Rapp L.M.
      In vivo requirement of protein prenylation for maintenance of retinal cytoarchitecture and photoreceptor structure.
      ). However, contrary to initial expectations, such effects of lovastatin, were found to be due to defective protein prenylation in the retina, rather than to disruption of cholesterol synthesis (
      • Roosing S.
      • Collin R.W.
      • den Hollander A.I.
      • Cremers F.P.
      • Siemiatkowska A.M.
      Prenylation defects in inherited retinal diseases.
      ). Such pharmacological targeting of HMGCR and SQLE also provided critical evidence for a functional presqualene and postsqualene pathways in the rodent retina. To date, there have been no published studies regarding the operation of the mevalonate shunt pathway in the retina.
      In vivo measurements of absolute rates of tissue cholesterol synthesis are achieved by chronic administration of deuterated water ([2H]water) and subsequent MS analysis of the cholesterol isotopomer distribution (
      • Dietschy J.M.
      • Spady D.K.
      Measurement of rates of cholesterol synthesis using tritiated water.
      ,
      • Jones P.J.
      • Leitch C.A.
      • Li Z.C.
      • Connor W.E.
      Human cholesterol synthesis measurement using deuterated water. Theoretical and procedural considerations.
      ,
      • Lee W.N.
      • Bassilian S.
      • Ajie H.O.
      • Schoeller D.A.
      • Edmond J.
      • Bergner E.A.
      • et al.
      In vivo measurement of fatty acids and cholesterol synthesis using D2O and mass isotopomer analysis.
      ,
      • Castro-Perez J.
      • Previs S.F.
      • McLaren D.G.
      • Shah V.
      • Herath K.
      • Bhat G.
      • et al.
      In vivo D2O labeling to quantify static and dynamic changes in cholesterol and cholesterol esters by high resolution LC/MS.
      ). A recent investigation adapting the isotopomer approach suggested that majority (>70%) of retinal sterol arises from de novo synthesis (
      • Lin J.B.
      • Mast N.
      • Bederman I.R.
      • Li Y.
      • Brunengraber H.
      • Bjorkhem I.
      • et al.
      Cholesterol in mouse retina originates primarily from in situ de novo biosynthesis.
      ). However, proper estimation of tissue sterol synthesis rates using this technique requires detailed assessment of several critical factors, such as molar fraction distribution in the tissue, molar enrichment in deuterated sterol (average number of [2H] atoms per newly synthesized sterol molecule), experimental verification of steady-state synthesis of [2H]cholesterol, and experimental determination of the correction factor to account for any newly synthesized cholesterol without [2H] incorporation (
      • Dietschy J.M.
      • Spady D.K.
      Measurement of rates of cholesterol synthesis using tritiated water.
      ,
      • Jones P.J.
      • Leitch C.A.
      • Li Z.C.
      • Connor W.E.
      Human cholesterol synthesis measurement using deuterated water. Theoretical and procedural considerations.
      ,
      • Lee W.N.
      • Bassilian S.
      • Ajie H.O.
      • Schoeller D.A.
      • Edmond J.
      • Bergner E.A.
      • et al.
      In vivo measurement of fatty acids and cholesterol synthesis using D2O and mass isotopomer analysis.
      ,
      • Castro-Perez J.
      • Previs S.F.
      • McLaren D.G.
      • Shah V.
      • Herath K.
      • Bhat G.
      • et al.
      In vivo D2O labeling to quantify static and dynamic changes in cholesterol and cholesterol esters by high resolution LC/MS.
      ). Parallel quantification of retinal cholesterol uptake was measured in mice maintained on chow supplemented with 0.3% w/w [2H]cholesterol for 2 weeks. Sterol uptake in the retina (after 1 week) was estimated to be about 3.6% of the total cholesterol content (
      • Lin J.B.
      • Mast N.
      • Bederman I.R.
      • Li Y.
      • Brunengraber H.
      • Bjorkhem I.
      • et al.
      Cholesterol in mouse retina originates primarily from in situ de novo biosynthesis.
      ). This experimental approach would be significantly strengthened by inclusion of a weaning experiment (i.e., weaning from [2H]water after 2 weeks back to normal water [t = 0]) to experimentally determine the true half-life (and hence, the absolute turnover rate) of labeled cholesterol in the retina.
      Systemically administered simvastatin was shown to exhibit the highest bioavailability compared with other statins (after 6 weeks) in the neural retina of mice (
      • Mast N.
      • Bederman I.R.
      • Pikuleva I.A.
      Retinal cholesterol content is reduced in Simvastatin-treated mice due to inhibited local biosynthesis albeit increased uptake of serum cholesterol.
      ) and also was significantly higher than that of the brain tissue, suggesting that simvastatin is permeable to the blood-retinal barrier. Such treatment of adult mice led to a significant decrease (by about 20%, after 6 weeks) in retinal cholesterol content, as well as a reduction in sterol intermediates, but did not alter total retinal cholesterol uptake. Given the estimated cholesterol turnover rate (c.a., 54 days) in the retina, and the estimated contribution of endogenous (retina-derived) biosynthesis to the total retinal cholesterol pool (c.a., 72%) (
      • Lin J.B.
      • Mast N.
      • Bederman I.R.
      • Li Y.
      • Brunengraber H.
      • Bjorkhem I.
      • et al.
      Cholesterol in mouse retina originates primarily from in situ de novo biosynthesis.
      ), it was concluded that systemic simvastatin treatment led to partial inhibition of retinal HMGCR activity (
      • Mast N.
      • Bederman I.R.
      • Pikuleva I.A.
      Retinal cholesterol content is reduced in Simvastatin-treated mice due to inhibited local biosynthesis albeit increased uptake of serum cholesterol.
      ,
      • Zheng W.
      • Mast N.
      • Saadane A.
      • Pikuleva I.A.
      Pathways of cholesterol homeostasis in mouse retina responsive to dietary and pharmacologic treatments.
      ). This further verifies the local activity of the mevalonate pathway in the retina.
      In another study, the de novo synthesis of both cholesterol and dolichol in frog retina was assessed using the same fundamental principles, but with two essential differences: the study was performed in vitro, rather than in vivo; and [3H]water (rather than [2H]water) was used, with separate, parallel incubations using [3H]acetate as the radiolabeled de novo precursor (
      • Keller R.K.
      • Fliesler S.J.
      • Nellis S.W.
      Isoprenoid biosynthesis in the retina. Quantitation of the sterol and dolichol biosynthetic pathways.
      ). The specific activity of radiolabeled products was determined by radio-HPLC. The majority of the [3H]acetate was incorporated into squalene, rather than into sterols; in addition, the frog retina was found to contain significant steady-state levels of squalene mass (unlike other vertebrate retinas). Hence, the flux of acetate into new cholesterol molecules was trapped in the squalene pool. The absolute rate of cholesterol synthesis was found to be only 3.4 pmol/h. This suggests that the de novo biosynthetic of sterol products in the retinas of amphibians (poikilotherms) are lower than those of warm-blooded species (homeotherms); hence, they cannot be compared directly with results obtained with rodent retinas. This was subsequently verified in vivo by intravitreal injection of [3H]acetate in frogs. Although acetyl CoA incorporation to mevalonate pathway is limited because of its hydrolysis (
      • Crabtree B.
      • Gordon M.J.
      • Christie S.L.
      Measurement of the rates of acetyl-CoA hydrolysis and synthesis from acetate in rat hepatocytes and the role of these fluxes in substrate cycling.
      ), [3H]acetate was mostly incorporated into [3H]squalene, as well as [3H]cholesterol, detected both in whole retinas and in isolated rod OS membranes derived therefrom (
      • Fliesler S.J.
      • Florman R.
      • Keller R.K.
      Isoprenoid lipid metabolism in the retina: dynamics of squalene and cholesterol incorporation and turnover in frog rod outer segment membranes.
      ). Taken together, the in vitro and in vivo systems described above reflect squalene and sterol biosynthetic capacity in the neural retina of rodents and amphibians. It should be appreciated that these results apply to total retinal sterol synthesis but do not address or exclude the possibility that some portion of the sterols utilized by retinal neurons, including photoreceptor cells, may be derived from glia (i.e., Müller cells), as is the case in the CNS (
      • Mauch D.H.
      • Nagler K.
      • Schumacher S.
      • Goritz C.
      • Muller E.C.
      • Otto A.
      • et al.
      CNS synaptogenesis promoted by glia-derived cholesterol.
      ,
      • Barres B.A.
      • Smith S.J.
      Neurobiology. Cholesterol--making or breaking the synapse.
      ). Future investigation of the mevalonate pathway in a retinal cell type–specific manner might provide additional critical insights into this aspect of retinal sterol homeostasis.
      We now turn our discussion to the activity of postsqualene branch of the mevalonate pathway in the neural retina. Such investigations have involved the pharmacological targeting of the Kandutsch-Russell pathway or the Bloch pathway (see Fig. 2), followed by an assessment of the impact of such treatments on retinal structure and function. This mimics what occurs in some relatively rare hereditary disorders where the synthesis of cholesterol is decreased and its immediate precursor accumulates (
      • Porter F.D.
      • Herman G.E.
      Malformation syndromes caused by disorders of cholesterol synthesis.
      ). A prime example of this is SLOS, the most common recessive disorder affecting the mevalonate pathway. The key biochemical signature of this disease is the accumulation of 7DHC in bodily tissues and fluids and clinically characterized by dysmorphologies, such as 2–3 toe syndactyly, craniofacial malformations, cognitive defects (autism spectrum), as well as rod and cone function deficits (
      • Porter F.D.
      Smith-Lemli-Opitz syndrome: pathogenesis, diagnosis and management.
      ,
      • Yu H.
      • Patel S.B.
      Recent insights into the Smith-Lemli-Opitz syndrome.
      ,
      • Garry D.
      • Hansen R.M.
      • Moskowitz A.
      • Elias E.R.
      • Irons M.
      • Fulton A.B.
      Cone ERG responses in patients with Smith-Lemli-Opitz Syndrome (SLOS).
      ,
      • Elias E.R.
      • Hansen R.M.
      • Irons M.
      • Quinn N.B.
      • Fulton A.B.
      Rod photoreceptor responses in children with Smith-Lemli-Opitz syndrome.
      ). The latter point serves as prima facie evidence indicating a requirement for cholesterol to support normal retinal function (
      • Bloch K.
      Sterol structure and function.
      ). The mevalonate pathway intermediate 7DHC is highly prone to oxidation (
      • Xu L.
      • Korade Z.
      • Porter N.A.
      Oxysterols from free radical chain oxidation of 7-dehydrocholesterol: product and mechanistic studies.
      ) and generates a spectrum of cytotoxic oxysterol metabolites, implicating them in the observed pathology (
      • Yin H.
      • Xu L.
      • Porter N.A.
      Free radical lipid peroxidation: mechanisms and analysis.
      ,
      • Korade Z.
      • Xu L.
      • Shelton R.
      • Porter N.A.
      Biological activities of 7-dehydrocholesterol-derived oxysterols: implications for Smith-Lemli-Opitz syndrome.
      ,
      • Xu L.
      • Davis T.A.
      • Porter N.A.
      Rate constants for peroxidation of polyunsaturated fatty acids and sterols in solution and in liposomes.
      ). A pharmacological model of SLOS has been generated by systemic treatment of rats with AY9944 [(trans-1,4-bis(2-dichlorobenzylamino-ethyl)) cyclohexane dihydrochloride], a DHCR7 inhibitor (
      • Fliesler S.J.
      • Peachey N.S.
      • Richards M.J.
      • Nagel B.A.
      • Vaughan D.K.
      Retinal degeneration in a rodent model of Smith-Lemli-Opitz syndrome: electrophysiologic, biochemical, and morphologic features.
      ). AY9944-treated rat retinas exhibit significant accumulation of 7DHC (7DHC/Chol mole ratio > 5) compared with control rat retinas (7DHC/Chol mole ratio < 0.1). Increased steady-state levels of 7DHC in the neural retina were associated with a progressive, caspase 3–independent, photoreceptor-specific retinal degeneration characterized by pyknotic (and TUNEL positive) photoreceptor nuclei, progressive shortening of rod OS, and thinning of the photoreceptor layer of the retina, defective clearance of shed rod OS tips by the RPE, and markedly decreased and delayed responses to light stimulation, indicating both rod and cone visual transduction defects (
      • Fliesler S.J.
      • Peachey N.S.
      • Richards M.J.
      • Nagel B.A.
      • Vaughan D.K.
      Retinal degeneration in a rodent model of Smith-Lemli-Opitz syndrome: electrophysiologic, biochemical, and morphologic features.
      ,
      • Ramachandra Rao S.
      • Pfeffer B.A.
      • Mas Gomez N.
      • Skelton L.A.
      • Keiko U.
      • Sparrow J.R.
      • et al.
      Compromised phagosome maturation underlies RPE pathology in cell culture and whole animal models of Smith-Lemli-Opitz syndrome.
      ,
      • Tu C.
      • Li J.
      • Jiang X.
      • Sheflin L.G.
      • Pfeffer B.A.
      • Behringer M.
      • et al.
      Ion-current-based proteomic profiling of the retina in a rat model of Smith-Lemli-Opitz syndrome.
      ).
      Pharmacological targeting of DHCR7 also has been used to assess retinal cholesterol uptake. AY9944-treated Sprague-Dawley rats were maintained on a cholesterol-free diet until postnatal (PN) day 28 and then randomized into two dietary groups: one continued to receive a cholesterol-free diet, whereas the other was fed the same chow base, but supplemented with 2% (w/w) cholesterol, and then both groups were continued on this treatment until PN day 74 (
      • Fliesler S.J.
      • Vaughan D.K.
      • Jenewein E.C.
      • Richards M.J.
      • Nagel B.A.
      • Peachey N.S.
      Partial rescue of retinal function and sterol steady-state in a rat model of Smith-Lemli-Opitz syndrome.
      ). 7DHC was the predominant sterol species in the retinas of AY9944-treated rats fed the cholesterol-free diet (7DHC/Chol mole ratio, 5.65), whereas cholesterol was the dominant sterol in retinas of those maintained on a cholesterol-enriched diet (7DHC/Chol mole ratio, 1.40). However, the total sterol content of the retina did not change appreciably under the conditions used, indicating that there was a 1:1 molar replacement of 7DHC by diet-derived, blood-borne cholesterol in the retina. (At the time, this was a striking finding because it had been assumed that the blood-retina barrier was similar to the blood-brain barrier in that it would exclude the uptake of blood-borne cholesterol.) The serum 7DHC/Chol mole ratio was 7.30 in the cholesterol-free dietary group and 0.10 in the cholesterol-enriched dietary group (
      • Fliesler S.J.
      • Vaughan D.K.
      • Jenewein E.C.
      • Richards M.J.
      • Nagel B.A.
      • Peachey N.S.
      Partial rescue of retinal function and sterol steady-state in a rat model of Smith-Lemli-Opitz syndrome.
      ). These findings are in fair agreement with the above discussed metabolic approach using dietary [2H]cholesterol supplementation; both clearly demonstrate that blood-borne cholesterol is able to cross the blood-retina barrier and be taken up by the neural retina (
      • Lin J.B.
      • Mast N.
      • Bederman I.R.
      • Li Y.
      • Brunengraber H.
      • Bjorkhem I.
      • et al.
      Cholesterol in mouse retina originates primarily from in situ de novo biosynthesis.
      ,
      • Fliesler S.J.
      • Vaughan D.K.
      • Jenewein E.C.
      • Richards M.J.
      • Nagel B.A.
      • Peachey N.S.
      Partial rescue of retinal function and sterol steady-state in a rat model of Smith-Lemli-Opitz syndrome.
      ). This has been verified through another independent approach, using intravenous injection of human LDL particles “doped” with cholestatrienol (cholesta-5,7,9(11)-trien-3β-ol), a naturally fluorescent derivative of cholesterol (
      • Tserentsoodol N.
      • Sztein J.
      • Campos M.
      • Gordiyenko N.V.
      • Fariss R.N.
      • Lee J.W.
      • et al.
      Uptake of cholesterol by the retina occurs primarily via a low density lipoprotein receptor-mediated process.
      ). The incorporation of cholestatrienol into the retina was followed as a function of the postinjection time, using confocal fluorescence microscopy; in parallel, rats were injected intravenously with human LDL particles containing [2H]cholesterol, and its uptake was followed by LC-MS analysis of retinal lipid extracts. Cholestatrienol fluorescence was observed in the choroid, RPE, and the distal outer neural retina within 2 h after injection of derivatized LDL; within 4 h, the entire outer retina fluoresced (including the photoreceptor cells), and by 6 h, the entire neural retina exhibited the brilliant blue fluorescence characteristic of UV-excited cholestatrienol. LC-MS confirmed the presence of [2H]cholesterol, in parallel, in the neural retina, increasing as a function of the post-injection time (
      • Tserentsoodol N.
      • Sztein J.
      • Campos M.
      • Gordiyenko N.V.
      • Fariss R.N.
      • Lee J.W.
      • et al.
      Uptake of cholesterol by the retina occurs primarily via a low density lipoprotein receptor-mediated process.
      ). Taken together, these independent lines of evidence demonstrate conclusively that cholesterol carried by LDL particles in the blood can be taken up by the retina and be broadly distributed throughout the neural retina (see also discussion below).
      The previously discussed in vivo pharmacological and metabolic approaches have provided insights into cholesterol synthesis and uptake at the steady state in the mature neural retina, notably in rodents. However, the retina consists of both neuronal and glial cell types, arranged in discrete histological layers (see Fig. 1). An important question that remains to be investigated is the relative contributions of each of those cell types to overall retinal sterol homeostasis. The neural retina expresses the signature proteins involved in sterol synthesis and the lipoprotein uptake pathway. Quantitative PCR analysis has demonstrated the expression of presqualene and postsqualene enzymes, including MVK, HMGCR, DHCR24, and DHCR7 in the neural retina (
      • Zheng W.
      • Mast N.
      • Saadane A.
      • Pikuleva I.A.
      Pathways of cholesterol homeostasis in mouse retina responsive to dietary and pharmacologic treatments.
      ,
      • Zheng W.
      • Reem R.E.
      • Omarova S.
      • Huang S.
      • DiPatre P.L.
      • Charvet C.D.
      • et al.
      Spatial distribution of the pathways of cholesterol homeostasis in human retina.
      ). However, immunohistochemical analysis of murine retina revealed strongest expression of HMGCR in the inner retinal layers, rather than in the outer retina (where the photoreceptor cells reside) (
      • Zheng W.
      • Mast N.
      • Saadane A.
      • Pikuleva I.A.
      Pathways of cholesterol homeostasis in mouse retina responsive to dietary and pharmacologic treatments.
      ,
      • Zheng W.
      • Reem R.E.
      • Omarova S.
      • Huang S.
      • DiPatre P.L.
      • Charvet C.D.
      • et al.
      Spatial distribution of the pathways of cholesterol homeostasis in human retina.
      ). Correlative in situ hybridization analysis of key enzymes of the presqualene and postsqualene steps of the mevalonate pathway in other species may provide a morphological context to retinal cell type–specific differences in mevalonate pathway regulation. In this regard, a recent study performed single-cell RNA-Seq experiments on 90-day-old human retinal organoids derived from human embryonic stem cells (
      • Collin J.
      • Zerti D.
      • Queen R.
      • Santos-Ferreira T.
      • Bauer R.
      • Coxhead J.
      • et al.
      CRX Expression in pluripotent stem cell-derived photoreceptors marks a transplantable subpopulation of early cones.
      ). The human embryonic stem cells were genetically engineered to drive GFP expression under the control of the CRX promoter (cone-rod homeobox gene), which is expressed in developing precursor and mature photoreceptor cells, as well as inner retinal neurons such as CHX10-positive bipolar cells (
      • Glubrecht D.D.
      • Kim J.H.
      • Russell L.
      • Bamforth J.S.
      • Godbout R.
      Differential CRX and OTX2 expression in human retina and retinoblastoma.
      ,
      • Prasov L.
      • Glaser T.
      Pushing the envelope of retinal ganglion cell genesis: context dependent function of Math5 (Atoh7).
      ,
      • Furukawa A.
      • Koike C.
      • Lippincott P.
      • Cepko C.L.
      • Furukawa T.
      The mouse Crx 5'-upstream transgene sequence directs cell-specific and developmentally regulated expression in retinal photoreceptor cells.
      ), to be able to selectively sort out expression of mevalonate pathway enzymes in CRX-GFP–positive retinal neurons cells versus other (GFP-negative) retinal cell types. HMGCR, SQLE, MVK, and other players of the mevalonate pathway were significantly enriched in the CRX-negative cluster, while GFP-positive cells (photoreceptors) only expressed basal levels of mevalonate pathway genes (
      • Collin J.
      • Zerti D.
      • Queen R.
      • Santos-Ferreira T.
      • Bauer R.
      • Coxhead J.
      • et al.
      CRX Expression in pluripotent stem cell-derived photoreceptors marks a transplantable subpopulation of early cones.
      ), possibly because of the requirement for de novo nonsterol isoprenoid synthesis, for example, dolichols (
      • Pittler S.J.
      • Fliesler S.J.
      • Fisher P.L.
      • Keller P.K.
      • Rapp L.M.
      In vivo requirement of protein prenylation for maintenance of retinal cytoarchitecture and photoreceptor structure.
      ). Furthermore, a recent in silico modeling of retinal sterol homeostasis suggests that photoreceptors acquire sterol from exogenous sources, rather than mevalonate pathway (
      • Zekavat S.M.
      • Lu J.
      • Maugeais C.
      • Mazer N.A.
      An in silico model of retinal cholesterol dynamics (RCD model): insights into the pathophysiology of dry AMD.
      ).
      Hence, taken together, these results suggest that de novo synthesis of cholesterol in photoreceptors, per se, is likely minimal. This is curious and unexpected, given the prodigious daily rate of membrane assembly and turnover of photoreceptor OS membranes, which requires a continuous supply of lipids (including cholesterol) and proteins (
      • Young R.W.
      Visual cells and the concept of renewal.
      ,
      • Young R.W.
      • Bok D.
      Participation of the retinal pigment epithelium in the rod outer segment renewal process.
      ). Also, it should be appreciated that a significant level of lipid (including cholesterol) synthesis is required during retinal development and maturation. Future investigations into retinal sterol homeostasis during retinal development may provide important insights into congenital disorders involving defective sterol homeostasis. Such investigations are now possible because of the recent development of a transgenic mouse line (mRX-Cre) that exhibits retina-specific Cre-recombinase expression (starting at day E8.5), driven by the Rx promoter (retina and anterior neural fold homeobox (RAX)), the earliest retinal determinant factor (
      • Klimova L.
      • Lachova J.
      • Machon O.
      • Sedlacek R.
      • Kozmik Z.
      Generation of mRx-Cre transgenic mouse line for efficient conditional gene deletion in early retinal progenitors.
      ). Investigations into retinal neuronal- and glial-specific inhibition of the mevalonate pathway, using targeted gene-deletion methods, also should shed light on neuron-glial interactions in the retina that contribute to maintenance of retinal cholesterol homeostasis.

      Intraretinal cholesterol exchange: role of Müller glial cells

      Intravitreal injection of radiolabeled amino acids, followed by tracking the fate of newly synthesized retinal proteins by light and electron microscopy-level autoradiography, first demonstrated the diurnal process of photoreceptor OS renewal and the intimate involvement of RPE cells in this process (
      • Young R.W.
      Visual cells and the concept of renewal.
      ,
      • Young R.W.
      • Bok D.
      Participation of the retinal pigment epithelium in the rod outer segment renewal process.
      ). A similar approach was adapted to demonstrate the continual synthesis and distribution of APO-E, a signature protein associated with VLDL and IDL particles, in the rabbit neural retina, and in a primary Müller glial cell culture model (
      • Amaratunga A.
      • Abraham C.R.
      • Edwards R.B.
      • Sandell J.H.
      • Schreiber B.M.
      • Fine R.E.
      Apolipoprotein E is synthesized in the retina by Muller glial cells, secreted into the vitreous, and rapidly transported into the optic nerve by retinal ganglion cells.
      ). Radiolabeled amino acids were faithfully incorporated into APO-E within 3–6 h after intravitreal injection. SDS-PAGE autoradiography of immunoprecipitated APO-E showed its presence mostly in the vitreous and in the neural retina, with little incorporation in the optic nerve (
      • Amaratunga A.
      • Abraham C.R.
      • Edwards R.B.
      • Sandell J.H.
      • Schreiber B.M.
      • Fine R.E.
      Apolipoprotein E is synthesized in the retina by Muller glial cells, secreted into the vitreous, and rapidly transported into the optic nerve by retinal ganglion cells.
      ). This agrees with the results of neuron-glia coculture studies, which showed that cholesterol, but not APO-E, is required for retinal ganglion cell synaptogenesis (
      • Mauch D.H.
      • Nagler K.
      • Schumacher S.
      • Goritz C.
      • Muller E.C.
      • Otto A.
      • et al.
      CNS synaptogenesis promoted by glia-derived cholesterol.
      ,
      • Barres B.A.
      • Smith S.J.
      Neurobiology. Cholesterol--making or breaking the synapse.
      ). Transcriptomic analysis of purified mouse Müller glial cells and in situ hybridization of mouse retinal tissue sections also demonstrated APO-E expression in Müller glia, along with other classic markers, such as aquaporin-4, RLBP1, and GLUL (
      • Roesch K.
      • Jadhav A.P.
      • Trimarchi J.M.
      • Stadler M.B.
      • Roska B.
      • Sun B.B.
      • et al.
      The transcriptome of retinal Muller glial cells.
      ). Additional evidence shows synthesis and export of APO-E– and APO-J–containing lipoproteins, varying in density from 1.006 to 1.180 g/cc and diameters of 14–45 nm, in primary rabbit Müller glia (
      • Shanmugaratnam J.
      • Berg E.
      • Kimerer L.
      • Johnson R.J.
      • Amaratunga A.
      • Schreiber B.M.
      • et al.
      Retinal Muller glia secrete apolipoproteins E and J which are efficiently assembled into lipoprotein particles.
      ). This is consistent with the results of studies using CNS-derived astrocytes, which also secrete APO-E and APO-J and are thought to play a role in retinal sterol homeostasis (
      • Amaratunga A.
      • Abraham C.R.
      • Edwards R.B.
      • Sandell J.H.
      • Schreiber B.M.
      • Fine R.E.
      Apolipoprotein E is synthesized in the retina by Muller glial cells, secreted into the vitreous, and rapidly transported into the optic nerve by retinal ganglion cells.
      ,
      • Shanmugaratnam J.
      • Berg E.
      • Kimerer L.
      • Johnson R.J.
      • Amaratunga A.
      • Schreiber B.M.
      • et al.
      Retinal Muller glia secrete apolipoproteins E and J which are efficiently assembled into lipoprotein particles.
      ,
      • Fagan A.M.
      • Holtzman D.M.
      • Munson G.
      • Mathur T.
      • Schneider D.
      • Chang L.K.
      • et al.
      Unique lipoproteins secreted by primary astrocytes from wild type, apoE (-/-), and human apoE transgenic mice.
      ). Thus, Müller glia have the capacity to assemble and secrete lipoproteins or lipoprotein-like particles, which then can be utilized by adjacent cells (e.g., photoreceptors or inner retinal neurons), and may serve as a local, intraretinal source of cholesterol. Retinas from APO-E–null mice have been reported to have a significant increase (2.8-fold) in their unesterified cholesterol content, and this increase was observed despite a compensatory increase in retinal APO-B levels. This suggests an important role for retinal APO-E synthesis for tissue sterol redistribution (
      • Saadane A.
      • Petrov A.
      • Mast N.
      • El-Darzi N.
      • Dao T.
      • Alnemri A.
      • et al.
      Mechanisms that minimize retinal impact of apolipoprotein E absence.
      ). The requirement of Müller cells in retinal cholesterol homeostasis could be validated by cell type–specific ablation of genes coding for postsqualene pathway enzymes; for example, breeding mice harboring floxed mevalonate pathway genes with mice expressing Cre recombinase under the control of a Müller cell–specific promoter (
      • Ueki Y.
      • Ash J.D.
      • Zhu M.
      • Zheng L.
      • Le Y.Z.
      Expression of Cre recombinase in retinal Muller cells.
      ,
      • Shen W.
      • Fruttiger M.
      • Zhu L.
      • Chung S.H.
      • Barnett N.L.
      • Kirk J.K.
      • et al.
      Conditional Mullercell ablation causes independent neuronal and vascular pathologies in a novel transgenic model.
      ).
      A study using APO-E knockout mice reported electrophysiological deficits, accompanied by possible dropout of Müller glial cells, by PN 25 weeks of age (
      • Ong J.M.
      • Zorapapel N.C.
      • Rich K.A.
      • Wagstaff R.E.
      • Lambert R.W.
      • Rosenberg S.E.
      • et al.
      Effects of cholesterol and apolipoprotein E on retinal abnormalities in ApoE-deficient mice.
      ). In another study, the lack of APO-E also reportedly lead to an appreciable (3-fold) increase in retinal cholesterol content compared with age-matched controls, with the majority of the total retinal cholesterol being unesterified (
      • Saadane A.
      • Petrov A.
      • Mast N.
      • El-Darzi N.
      • Dao T.
      • Alnemri A.
      • et al.
      Mechanisms that minimize retinal impact of apolipoprotein E absence.
      ). (We have not been able to reproduce those findings independently; c.f., Fliesler, S.J., M.J. Richards, N.S. Peachey, K. Kauser. Hypercholesterolemia does not alter retinal sterol composition or compromise retinal structure or function in APO-E–knockout mice. Invest. Ophthalmol. Vis. Sci. (ARVO Abstr.) 2000; 41:S199.) Also, a compensatory increase in the retinal expression of other lipoproteins, such as APO-A4 and APO-B, was observed upon APO-E gene ablation (
      • Saadane A.
      • Petrov A.
      • Mast N.
      • El-Darzi N.
      • Dao T.
      • Alnemri A.
      • et al.
      Mechanisms that minimize retinal impact of apolipoprotein E absence.
      ).
      APO receptors also play an important role in retinal development. Undifferentiated neuronal precursors express APO-A1 and the SRB-I (
      • Kurumada S.
      • Onishi A.
      • Imai H.
      • Ishii K.
      • Kobayashi T.
      • Sato S.B.
      Stage-specific association of apolipoprotein A-I and E in developing mouse retina.
      ). Rod photoreceptor differentiation, maturation, and synaptogenesis temporally coincide with Müller glial development and their synthesis of APO-E (
      • Kurumada S.
      • Onishi A.
      • Imai H.
      • Ishii K.
      • Kobayashi T.
      • Sato S.B.
      Stage-specific association of apolipoprotein A-I and E in developing mouse retina.
      ). In the CNS, a major receptor for APO-E is LDL-related particle 1 (LRP1), which is required for synaptogenesis, oligodendrocyte progenitor cell differentiation, and myelination (
      • Lin J.P.
      • Mironova Y.A.
      • Shrager P.
      • Giger R.J.
      LRP1 regulates peroxisome biogenesis and cholesterol homeostasis in oligodendrocytes and is required for proper CNS myelin development and repair.
      ,
      • Landowski L.M.
      • Pavez M.
      • Brown L.S.
      • Gasperini R.
      • Taylor B.V.
      • West A.K.
      • et al.
      Low-density lipoprotein receptor-related proteins in a novel mechanism of axon guidance and peripheral nerve regeneration.
      ). LRP1 is expressed in primary RPE cells (
      • Hollborn M.
      • Birkenmeier G.
      • Saalbach A.
      • Iandiev I.
      • Reichenbach A.
      • Wiedemann P.
      • et al.
      Expression of LRP1 in retinal pigment epithelial cells and its regulation by growth factors.
      ), as well as in retinal endothelial and Müller glial cells (
      • Mao H.
      • Lockyer P.
      • Townley-Tilson W.H.
      • Xie L.
      • Pi X.
      LRP1 regulates retinal angiogenesis by inhibiting PARP-1 activity and endothelial cell proliferation.
      ,
      • Barcelona P.F.
      • Ortiz S.G.
      • Chiabrando G.A.
      • Sanchez M.C.
      alpha2-Macroglobulin induces glial fibrillary acidic protein expression mediated by low-density lipoprotein receptor-related protein 1 in Muller cells.
      ). However, the specific requirement of LRP1 for sterol homeostasis in retinal neuronal cell types remains to be assessed. Taken together, the evidence extant suggests a role for APO secretion by Müller glia in maintaining cholesterol homeostasis in the neural retina. In vivo evaluation of neuronal uptake of Müller glia–derived sterols by surrounding neurons is not possible using conventional metabolic approaches. This is due to the inability to “tag” the de novo–synthesized sterol with a fluor and follow its trafficking, secretion and uptake because sterols (unlike proteins) are not coded by genes. However, an alternative approach might be targeted deletion of enzymes of the postsqualene pathway in Müller glia, such as sterol-C5-desaturase, DHCR24, or DHCR7, and follow-up assessment of uptake and incorporation of the biogenic cholesterol precursor into neighboring retinal neurons (e.g., photoreceptor cells) (see Fig. 3).

      Mevalonate pathway activity in the RPE

      The above results pertain only to de novo cholesterol synthesis and uptake in the neural retina. We will now specifically consider the mevalonate pathway in the RPE. Although immunohistochemical analysis has shown the presence of HMGCR in human and murine RPE cells (
      • Zheng W.
      • Mast N.
      • Saadane A.
      • Pikuleva I.A.
      Pathways of cholesterol homeostasis in mouse retina responsive to dietary and pharmacologic treatments.
      ,
      • Zheng W.
      • Reem R.E.
      • Omarova S.
      • Huang S.
      • DiPatre P.L.
      • Charvet C.D.
      • et al.
      Spatial distribution of the pathways of cholesterol homeostasis in human retina.
      ), investigating RPE cholesterol synthesis rates in vivo is extremely challenging because of the technical difficulties involved in the metabolic approaches and the need for targeted cell type–specific inhibition of the mevalonate pathway. As a potentially more tractable and fruitful alternative, RPE in vitro models of genetic diseases pertaining to the mevalonate pathway and related pharmacological models as well as the conventional metabolic approach may be of utility to investigate RPE de novo sterol synthesis. Recently, we generated a human induced pluripotent stem cell (iPSC)–derived RPE in vitro model of SLOS (point mutations in DHCR7, leading to hampered reduction of 7DHC to cholesterol), comparing SLOS RPE cells (generated from iPSCs from fibroblasts isolated from patients with well-characterized SLOS) with iPSC-derived RPE cells from normal human controls (
      • Ramachandra Rao S.
      • Pfeffer B.A.
      • Mas Gomez N.
      • Skelton L.A.
      • Keiko U.
      • Sparrow J.R.
      • et al.
      Compromised phagosome maturation underlies RPE pathology in cell culture and whole animal models of Smith-Lemli-Opitz syndrome.
      ). SLOS-RPE cells cultured in delipidated serum showed elevated steady-state levels of 7DHC (∼40% of total sterol content), unlike the control RPE cells (which had minimal 7DHC content), indicating an active cholesterol synthesis pathway (
      • Ramachandra Rao S.
      • Pfeffer B.A.
      • Mas Gomez N.
      • Skelton L.A.
      • Keiko U.
      • Sparrow J.R.
      • et al.
      Compromised phagosome maturation underlies RPE pathology in cell culture and whole animal models of Smith-Lemli-Opitz syndrome.
      ). Other studies have demonstrated in vitro incorporation of radiolabeled acetate into cholesterol in ARPE-19 cells, an immortalized human RPE–derived cell line (
      • Biswas L.
      • Zhou X.
      • Dhillon B.
      • Graham A.
      • Shu X.
      Retinal pigment epithelium cholesterol efflux mediated by the 18 kDa translocator protein, TSPO, a potential target for treating age-related macular degeneration.
      ). In vivo study utilizing RNASeq analysis suggest that diurnal changes occur in the expression of mevalonate pathway genes, such as Hmgcr, Dhcr24, and Sqle, in the RPE of 10- to 13-week-old mice (
      • Louer E.M.M.
      • Yi G.
      • Carmone C.
      • Robben J.
      • Stunnenberg H.G.
      • den Hollander A.I.
      • et al.
      Genes involved in energy metabolism are differentially expressed during the day-night cycle in murine retinal pigment epithelium.
      ). These results qualitatively demonstrate the ability of RPE cells to synthesize cholesterol autonomously but do not permit the calculation of absolute sterol synthetic rates.

      Role for RPE in retinal cholesterol uptake

      Uptake of blood-borne LDL particles by the RPE was first demonstrated utilizing tail vein injection of Rhodamine-labeled LDL particles and subsequent monitoring of their uptake by the RPE using fluorescence microscopy (
      • Gordiyenko N.
      • Campos M.
      • Lee J.W.
      • Fariss R.N.
      • Sztein J.
      • Rodriguez I.R.
      RPE cells internalize low-density lipoprotein (LDL) and oxidized LDL (oxLDL) in large quantities in vitro and in vivo.
      ). However, fluorescent tags are not ideal tracers because their conjugation to LDL may be unstable in vivo. In vitro experiments utilizing immortalized ARPE-19 cells demonstrated LDL receptor (LDLR)-mediated uptake of LDL (
      • Gordiyenko N.
      • Campos M.
      • Lee J.W.
      • Fariss R.N.
      • Sztein J.
      • Rodriguez I.R.
      RPE cells internalize low-density lipoprotein (LDL) and oxidized LDL (oxLDL) in large quantities in vitro and in vivo.
      ). As described above, RPE-mediated uptake of circulating, LDL-bound cholesterol was demonstrated in rats using intravenous injection of human LDL equilibrated with unesterified cholestatrienol, monitoring the incorporation of the fluorescent sterol by confocal fluorescence microscopy (
      • Tserentsoodol N.
      • Sztein J.
      • Campos M.
      • Gordiyenko N.V.
      • Fariss R.N.
      • Lee J.W.
      • et al.
      Uptake of cholesterol by the retina occurs primarily via a low density lipoprotein receptor-mediated process.
      ). In that study, LDL uptake also was monitored by immunohistochemistry and correlative Western blot analysis of the neural retina with a monospecific antibody against human APO-B (importantly, with no cross-reactivity against rat APO-B). Immunohistochemistry confirmed the presence of human APO-B immunoreactivity in the RPE and the neural retina, whereas Western blot analysis showed an immunoreactive 80-kDa APO-B peptide (consistent with partially degraded human APO-B), indicating endocytic uptake and degradation of LDL particles by the RPE and the neural retina (
      • Tserentsoodol N.
      • Sztein J.
      • Campos M.
      • Gordiyenko N.V.
      • Fariss R.N.
      • Lee J.W.
      • et al.
      Uptake of cholesterol by the retina occurs primarily via a low density lipoprotein receptor-mediated process.
      ,
      • Yin L.
      • Shi Y.
      • Liu X.
      • Zhang H.
      • Gong Y.
      • Gu Q.
      • et al.
      A rat model for studying the biological effects of circulating LDL in the choriocapillaris-BrM-RPE complex.
      ). On the other hand, serum deprivation of ARPE-19 cells has been shown to result in transcriptional upregulation of mevalonate pathway genes, as well as accumulation of cellular free cholesterol (
      • Mishra S.
      • Peterson K.
      • Yin L.
      • Berger A.
      • Fan J.
      • Wistow G.
      Accumulation of cholesterol and increased demand for zinc in serum-deprived RPE cells.
      ). LDLR expression has been observed in the RPE, as well as in the ganglion cell layer (GCL) and in the endothelial cells of the retinal vasculature in human and monkey retinas (
      • Zheng W.
      • Mast N.
      • Saadane A.
      • Pikuleva I.A.
      Pathways of cholesterol homeostasis in mouse retina responsive to dietary and pharmacologic treatments.
      ,
      • Tserentsoodol N.
      • Sztein J.
      • Campos M.
      • Gordiyenko N.V.
      • Fariss R.N.
      • Lee J.W.
      • et al.
      Uptake of cholesterol by the retina occurs primarily via a low density lipoprotein receptor-mediated process.
      ,
      • Zheng W.
      • Reem R.E.
      • Omarova S.
      • Huang S.
      • DiPatre P.L.
      • Charvet C.D.
      • et al.
      Spatial distribution of the pathways of cholesterol homeostasis in human retina.
      ). In another study, LDLR knockout mice were generated on an ApoB100 background (ApoB100 LDLR−/−), and their retinas were probed with filipin, a naturally fluorescent macrolide antibiotic molecule that binds to the 3β-hydroxyl group of sterols, to localize cholesterol in various cell types (
      • Bretillon L.
      • Acar N.
      • Seeliger M.W.
      • Santos M.
      • Maire M.A.
      • Juaneda P.
      • et al.
      ApoB100,LDLR-/- mice exhibit reduced electroretinographic response and cholesteryl esters deposits in the retina.
      ). ApoB100 LDLR−/− mice exhibited deficits in scotopic (rod-driven) electroretinogram (ERG) responses, as well as accumulation of esterified cholesterol on the basolateral surface of the RPE, consistent with inefficient uptake of LDL particles (
      • Bretillon L.
      • Acar N.
      • Seeliger M.W.
      • Santos M.
      • Maire M.A.
      • Juaneda P.
      • et al.
      ApoB100,LDLR-/- mice exhibit reduced electroretinographic response and cholesteryl esters deposits in the retina.
      ). The acid lipase-dependent processing of endocytosed lipoproteins in RPE cells is sensitive to buildup of bisretinoid adducts such as A2E, which displaces cholesterol from lipid rafts (
      • Lakkaraju A.
      • Finnemann S.C.
      • Rodriguez-Boulan E.
      The lipofuscin fluorophore A2E perturbs cholesterol metabolism in retinal pigment epithelial cells.
      ). Taken together, the results suggest the involvement of LDLR-mediated uptake of LDL by the RPE and the neural retina, the subsequent endolysosomal processing of LDL, and ultimate incorporation of LDL-derived free cholesterol into the neural retina.
      Mutations in the NPC-I gene lead to accumulation of free cholesterol in lysosomes because of deficient NPC-I–mediated transfer of free cholesterol from lysosomes to the ER (
      • Ory D.S.
      The niemann-pick disease genes; regulators of cellular cholesterol homeostasis.
      ). In a genetic mouse model of NPC-I disease, significant accumulation of free cholesterol (determined by filipin binding) was observed in the RPE and in the outer plexiform layer of the retina (
      • Claudepierre T.
      • Paques M.
      • Simonutti M.
      • Buard I.
      • Sahel J.
      • Maue R.A.
      • et al.
      Lack of Niemann-Pick type C1 induces age-related degeneration in the mouse retina.
      ). Also, retinal degeneration is observed in NPC-I–modified genetic mouse models, characterized by an age-dependent decrease in dark-adapted (rod-driven) a-wave and b-wave ERG responses, as well as by progressive, irreversible photoreceptor-specific cell death (observed as exclusive TUNEL -positive labeling in the outer nuclear layer) (
      • Claudepierre T.
      • Paques M.
      • Simonutti M.
      • Buard I.
      • Sahel J.
      • Maue R.A.
      • et al.
      Lack of Niemann-Pick type C1 induces age-related degeneration in the mouse retina.
      ,
      • Yan X.
      • Ma L.
      • Hovakimyan M.
      • Lukas J.
      • Wree A.
      • Frank M.
      • et al.
      Defects in the retina of Niemann-pick type C 1 mutant mice.
      ). This animal model further exhibited defective autophagy, accumulation of membranous and lipid inclusion bodies, aberrant dendritic arborization, and neurite stratification defects in retinal neurons, but without increased filipin staining in retinal neurons (
      • Yan X.
      • Ma L.
      • Hovakimyan M.
      • Lukas J.
      • Wree A.
      • Frank M.
      • et al.
      Defects in the retina of Niemann-pick type C 1 mutant mice.
      ). Spectral domain-optical coherence tomography imaging of the neural retina of NPC-I patients has revealed significant thinning of the nerve fiber layer and axonal degeneration (
      • Havla J.
      • Moser M.
      • Sztatecsny C.
      • Lotz-Havla A.S.
      • Maier E.M.
      • Hizli B.
      • et al.
      Retinal axonal degeneration in Niemann-Pick type C disease.
      ). Surprisingly, however, patients with LAL deficiency, which results in accumulation of lysosomal cholesteryl esters, do not exhibit retinopathies (
      • Cohen J.L.
      • Burfield J.
      • Valdez-Gonzalez K.
      • Samuels A.
      • Stefanatos A.K.
      • Yudkoff M.
      • et al.
      Early diagnosis of infantile-onset lysosomal acid lipase deficiency in the advent of available enzyme replacement therapy.
      ). Although LAL is required for RPE cholesterol homoeostasis (
      • Elner V.M.
      Retinal pigment epithelial acid lipase activity and lipoprotein receptors: effects of dietary omega-3 fatty acids.
      ), the effect of conditional deletion of LAL in retinal neurons remains to be directly investigated. Overall, these defects reflect a neuronal requirement for glia-derived cholesterol in the retina, as discussed earlier.
      Cholesterol uptake also occurs through SRB-I and SRB-II, which are involved in the uptake of HDL and OxLDL, respectively. Binding studies using [125I]-labeled LDL and acetylated-LDL particles and bovine RPE cells have demonstrated that RPE cells possess both LDLR and scavenger receptor activity (
      • Hayes K.C.
      • Lindsey S.
      • Stephan Z.F.
      • Brecker D.
      Retinal pigment epithelium possesses both LDL and scavenger receptor activity.
      ). When challenged with excess unlabeled LDL or acetylated LDL in vitro, cultured bovine RPE cells responded by downregulating the LDLR, but not scavenger receptors, which is typical of macrophages and arterial endothelial cells. Primary human RPE cells also have been shown to express SRBs (
      • Duncan K.G.
      • Bailey K.R.
      • Kane J.P.
      • Schwartz D.M.
      Human retinal pigment epithelial cells express scavenger receptors BI and BII.
      ). The transcript and protein level expression of SRB-I/SRB-II receptors in primary RPE cells was examined using RT-PCR, and by analyzing incorporation of radiolabeled amino acids into newly synthesized SRB-I (
      • Duncan K.G.
      • Bailey K.R.
      • Kane J.P.
      • Schwartz D.M.
      Human retinal pigment epithelial cells express scavenger receptors BI and BII.
      ). A comprehensive study showed the differential expression of APOs and SRB reporters in the monkey retina. Although both the RPE and the GCL showed APO-A1, SRB-I, and SRB-II immunoreactivity, photoreceptors expressed only class B scavenger reporters (
      • Tserentsoodol N.
      • Sztein J.
      • Campos M.
      • Gordiyenko N.V.
      • Fariss R.N.
      • Lee J.W.
      • et al.
      Uptake of cholesterol by the retina occurs primarily via a low density lipoprotein receptor-mediated process.
      ). These findings suggest a role for complex intraretinal lipoprotein transport mechanisms in maintaining sterol homeostasis in the neural retina and in the RPE (
      • Tserentsoodol N.
      • Sztein J.
      • Campos M.
      • Gordiyenko N.V.
      • Fariss R.N.
      • Lee J.W.
      • et al.
      Uptake of cholesterol by the retina occurs primarily via a low density lipoprotein receptor-mediated process.
      ,
      • Duncan K.G.
      • Bailey K.R.
      • Kane J.P.
      • Schwartz D.M.
      Human retinal pigment epithelial cells express scavenger receptors BI and BII.
      ).
      SRB-I is involved in cellular uptake of cholesteryl esters from HDL particles, as well as lutein uptake by the RPE (
      • Sato Y.
      • Kondo Y.
      • Sumi M.
      • Takekuma Y.
      • Sugawara M.
      Intracellular uptake mechanism of lutein in retinal pigment epithelial cells.
      ). SRB-I also participates in cholesterol efflux from extrahepatic tissues, thereby performing both uptake and efflux roles (
      • Shen W.J.
      • Azhar S.
      • Kraemer F.B.
      SR-B1: a unique multifunctional receptor for cholesterol influx and efflux.
      ). Further understanding of SRB-I requirement for proper retinal functioning has been achieved using a global SRB-I knockout model (
      • Provost A.C.
      • Vede L.
      • Bigot K.
      • Keller N.
      • Tailleux A.
      • Jais J.P.
      • et al.
      Morphologic and electroretinographic phenotype of SR-BI knockout mice after a long-term atherogenic diet.
      ). This model exhibits hypolipoproteinemia and concurrent increase in serum cholesterol levels, which is further exacerbated by feeding a high-fat diet. SRB-I knockout mice fed a normal chow diet exhibited mild decreases in dark-adapted a-wave and b-wave ERG responses, as compared with controls, but without any observable retinal structure abnormalities (
      • Provost A.C.
      • Vede L.
      • Bigot K.
      • Keller N.
      • Tailleux A.
      • Jais J.P.
      • et al.
      Morphologic and electroretinographic phenotype of SR-BI knockout mice after a long-term atherogenic diet.
      ). However, those mice fed a high-fat diet exhibited photoreceptor layer disorganization, sparse sub-RPE lipid deposits, ERG abnormalities, and significant thickening of Bruch's membrane (the extracellular matrix interface between the choriocapillaris and the RPE (
      • Provost A.C.
      • Vede L.
      • Bigot K.
      • Keller N.
      • Tailleux A.
      • Jais J.P.
      • et al.
      Morphologic and electroretinographic phenotype of SR-BI knockout mice after a long-term atherogenic diet.
      )).

      Sequestration of cholesterol in storage depots

      A key mechanism in cellular cholesterol homeostasis is the esterification of excess free cholesterol and its storage in lipid droplets (
      • Martin S.
      • Parton R.G.
      Caveolin, cholesterol, and lipid bodies.
      ,
      • Olofsson S.O.
      • Bostrom P.
      • Andersson L.
      • Rutberg M.
      • Perman J.
      • Boren J.
      Lipid droplets as dynamic organelles connecting storage and efflux of lipids.
      ). Three enzymes catalyze the esterification of ACAT1 and ACAT2 and LCAT (
      • Rogers M.A.
      • Liu J.
      • Song B.L.
      • Li B.L.
      • Chang C.C.
      • Chang T.Y.
      Acyl-CoA:cholesterol acyltransferases (ACATs/SOATs): enzymes with multiple sterols as substrates and as activators.
      ). Both human and macaque retinas express ACAT1 and LCAT (
      • Tserentsoodol N.
      • Sztein J.
      • Campos M.
      • Gordiyenko N.V.
      • Fariss R.N.
      • Lee J.W.
      • et al.
      Uptake of cholesterol by the retina occurs primarily via a low density lipoprotein receptor-mediated process.
      ,
      • Li C.M.
      • Presley J.B.
      • Zhang X.
      • Dashti N.
      • Chung B.H.
      • Medeiros N.E.
      • et al.
      Retina expresses microsomal triglyceride transfer protein: implications for age-related maculopathy.
      ). ACAT1 immunolocalization also has been reported in the murine retina in the photoreceptor OS layer, outer plexiform layer, GCL, and in the RPE (
      • Zheng W.
      • Reem R.E.
      • Omarova S.
      • Huang S.
      • DiPatre P.L.
      • Charvet C.D.
      • et al.
      Spatial distribution of the pathways of cholesterol homeostasis in human retina.
      ). However, LCAT appears to localize to Müller glial cells, rather than to retinal neurons (
      • Saadane A.
      • Mast N.
      • Dao T.
      • Ahmad B.
      • Pikuleva I.A.
      Retinal hypercholesterolemia triggers cholesterol accumulation and esterification in photoreceptor cells.
      ). Also, LCAT ablation does not lead to retinal degeneration or dysfunction (
      • Sakai N.
      • Vaisman B.L.
      • Koch C.A.
      • Hoyt Jr., R.F.
      • Meyn S.M.
      • Talley G.D.
      • et al.
      Targeted disruption of the mouse lecithin:cholesterol acyltransferase (LCAT) gene. Generation of a new animal model for human LCAT deficiency.
      ). Inhibition of cholesterol efflux, such as that observed in CYP27A1/46A1 double-knockout mice, with resultant increase in the photoreceptor cholesterol content, exhibits ACAT1-dependent esterification of sterols in photoreceptors, notably in their OSs (
      • Saadane A.
      • Mast N.
      • Dao T.
      • Ahmad B.
      • Pikuleva I.A.
      Retinal hypercholesterolemia triggers cholesterol accumulation and esterification in photoreceptor cells.
      ). The latter finding is curious, considering the fact that, historically, extensive lipid composition analyses across multiple vertebrate species have failed to detect such molecules in purified photoreceptor OS membrane preparations (
      • Fliesler S.J.
      • Anderson R.E.
      Chemistry and metabolism of lipids in the vertebrate retina.
      ).

      Cholesterol efflux in the neural retina and RPE

      Role of ABC transporters in retinal cholesterol efflux

      ABC transporters ABCA1 and ABCG1 generally account for the majority of cellular sterol efflux, depending on tissue expression levels (
      • Oram J.F.
      • Vaughan A.M.
      ATP-Binding cassette cholesterol transporters and cardiovascular disease.
      ). ABCA1 and ABCG1 are expressed widely in most tissues, including brain, retina, and macrophages (
      • Nakamura K.
      • Kennedy M.A.
      • Baldan A.
      • Bojanic D.D.
      • Lyons K.
      • Edwards P.A.
      Expression and regulation of multiple murine ATP-binding cassette transporter G1 mRNAs/isoforms that stimulate cellular cholesterol efflux to high density lipoprotein.
      ), whereas ABCG4 is predominantly expressed in the brain (
      • Bojanic D.D.
      • Tarr P.T.
      • Gale G.D.
      • Smith D.J.
      • Bok D.
      • Chen B.
      • et al.
      Differential expression and function of ABCG1 and ABCG4 during development and aging.
      ). ABCG1 additionally caters to efflux of sterols derived from OxLDL to HDL particles in macrophages, and plays a protective role in atherosclerosis (
      • Xu M.
      • Zhou H.
      • Tan K.C.
      • Guo R.
      • Shiu S.W.
      • Wong Y.
      ABCG1 mediated oxidized LDL-derived oxysterol efflux from macrophages.
      ). ABCG1 is expressed in the developing and the mature retina (
      • Nakamura K.
      • Kennedy M.A.
      • Baldan A.
      • Bojanic D.D.
      • Lyons K.
      • Edwards P.A.
      Expression and regulation of multiple murine ATP-binding cassette transporter G1 mRNAs/isoforms that stimulate cellular cholesterol efflux to high density lipoprotein.
      ,
      • Bojanic D.D.
      • Tarr P.T.
      • Gale G.D.
      • Smith D.J.
      • Bok D.
      • Chen B.
      • et al.
      Differential expression and function of ABCG1 and ABCG4 during development and aging.
      ). Brain tissue from Abcg1-Abcg4 double-knockout mice show significant accumulation cholesterol and lathosterol (cholest-7-en-3β-ol), as well as 24-OH-Chol, 25-OH-Chol, and 27-OH-Chol by 8 months of age, compared with age-matched controls. Therefore, ABCG1 and ABCG4 play a key role in brain sterol efflux (
      • Bojanic D.D.
      • Tarr P.T.
      • Gale G.D.
      • Smith D.J.
      • Bok D.
      • Chen B.
      • et al.
      Differential expression and function of ABCG1 and ABCG4 during development and aging.
      ). ABCG1 and ABCG4 are expressed in all the layers of the neural retina and in the RPE, as well as in primary cultures of Müller glial cells and ganglion cells (
      • Nakamura K.
      • Kennedy M.A.
      • Baldan A.
      • Bojanic D.D.
      • Lyons K.
      • Edwards P.A.
      Expression and regulation of multiple murine ATP-binding cassette transporter G1 mRNAs/isoforms that stimulate cellular cholesterol efflux to high density lipoprotein.
      ,
      • Bojanic D.D.
      • Tarr P.T.
      • Gale G.D.
      • Smith D.J.
      • Bok D.
      • Chen B.
      • et al.
      Differential expression and function of ABCG1 and ABCG4 during development and aging.
      ,
      • Ananth S.
      • Gnana-Prakasam J.P.
      • Bhutia Y.D.
      • Veeranan-Karmegam R.
      • Martin P.M.
      • Smith S.B.
      • et al.
      Regulation of the cholesterol efflux transporters ABCA1 and ABCG1 in retina in hemochromatosis and by the endogenous siderophore 2,5-dihydroxybenzoic acid.
      ). Retinal histological maturation appears normal in Abcg1-Abcg4 double-knockout mice, which exhibit mild retinal dysfunction accompanied by a relatively small increase in lathosterol content (unlike the accumulation of oxysterols and cholesterol in the brain) (
      • Bojanic D.D.
      • Tarr P.T.
      • Gale G.D.
      • Smith D.J.
      • Bok D.
      • Chen B.
      • et al.
      Differential expression and function of ABCG1 and ABCG4 during development and aging.
      ).
      ABCA1 and ABCG1 expression in the neural retina increased upon treatment with the LXR-α agonist T0901317, suggesting a role for both LXR-α regulation and ABC transporters in retinal sterol efflux (
      • Zheng W.
      • Mast N.
      • Saadane A.
      • Pikuleva I.A.
      Pathways of cholesterol homeostasis in mouse retina responsive to dietary and pharmacologic treatments.
      ). Rod photoreceptor–specific knockout of ABCA1, using Rho-iCre mice (
      • Li S.
      • Chen D.
      • Sauve Y.
      • McCandless J.
      • Chen Y.J.
      • Chen C.K.
      Rhodopsin-iCre transgenic mouse line for Cre-mediated rod-specific gene targeting.
      ), leads to appreciable lipid droplet accumulation and age-related retinal dysfunction at around PN 12 months (
      • Ban N.
      • Lee T.J.
      • Sene A.
      • Dong Z.
      • Santeford A.
      • Lin J.B.
      • et al.
      Disrupted cholesterol metabolism promotes age-related photoreceptor neurodegeneration.
      ). Rod photoreceptor–specific ABCA1/G1 knockout (unlike ABCG1/G4 knockout) leads to increased levels of retinal cholesterol, 7KChol, and 24-, 25-, and 27-OH-Chol by PN 12 months (
      • Ban N.
      • Lee T.J.
      • Sene A.
      • Dong Z.
      • Santeford A.
      • Lin J.B.
      • et al.
      Disrupted cholesterol metabolism promotes age-related photoreceptor neurodegeneration.
      ). Maintaining rod-specific ABCA1/G1 knockouts on a high-fat diet accelerates lipid accumulation and retinal degeneration (
      • Ban N.
      • Lee T.J.
      • Sene A.
      • Dong Z.
      • Santeford A.
      • Lin J.B.
      • et al.
      Disrupted cholesterol metabolism promotes age-related photoreceptor neurodegeneration.
      ). Similar age-related retinal dysfunction and cholesterol accumulation (around PN 10–14 months) occur upon LXR-α deletion, with subsequent reduction in ABC transporter levels (
      • Choudhary M.
      • Ismail E.N.
      • Yao P.L.
      • Tayyari F.
      • Radu R.A.
      • Nusinowitz S.
      • et al.
      LXRs regulate features of age-related macular degeneration and may be a potential therapeutic target.
      ). By contrast, LXR-β knockout did not lead to photoreceptor dysfunction (even up to PN 10 months). However, a lack of LXR-α and/or LXR-β leads to lipid accumulation in the RPE (
      • Choudhary M.
      • Ismail E.N.
      • Yao P.L.
      • Tayyari F.
      • Radu R.A.
      • Nusinowitz S.
      • et al.
      LXRs regulate features of age-related macular degeneration and may be a potential therapeutic target.
      ). Also, LXR-β knockout causes slow, progressive loss of ganglion cells over the course of PN 18 months, accompanied by decreased retinal aquaporin-4 expression as well as microglial activation (indicative of neuroinflammation) (
      • Song X.Y.
      • Wu W.F.
      • Gabbi C.
      • Dai Y.B.
      • So M.
      • Chaurasiya S.P.
      • et al.
      Retinal and optic nerve degeneration in liver X receptor beta knockout mice.
      ). Curiously, the onset of retinal dysfunction upon inhibition of cholesterol efflux at the level of LXR-α/β, or ABC transporters (also CYP hydroxylase knockouts, discussed later) leads to a slow retinal degeneration phenotype that manifests by about 1 year. This is unlike inhibition of retinal de novo cholesterol biosynthesis either through statin treatment (which most likely is due to protein prenylation compromise, rather than blockade of sterol synthesis) or as observed in the AY9944-induced model of SLOS (see below), which leads to retinal degeneration on a timescale of weeks (
      • Fliesler S.J.
      • Peachey N.S.
      • Richards M.J.
      • Nagel B.A.
      • Vaughan D.K.
      Retinal degeneration in a rodent model of Smith-Lemli-Opitz syndrome: electrophysiologic, biochemical, and morphologic features.
      ,
      • Ban N.
      • Lee T.J.
      • Sene A.
      • Dong Z.
      • Santeford A.
      • Lin J.B.
      • et al.
      Disrupted cholesterol metabolism promotes age-related photoreceptor neurodegeneration.
      ,
      • Choudhary M.
      • Ismail E.N.
      • Yao P.L.
      • Tayyari F.
      • Radu R.A.
      • Nusinowitz S.
      • et al.
      LXRs regulate features of age-related macular degeneration and may be a potential therapeutic target.
      ,
      • Song X.Y.
      • Wu W.F.
      • Gabbi C.
      • Dai Y.B.
      • So M.
      • Chaurasiya S.P.
      • et al.
      Retinal and optic nerve degeneration in liver X receptor beta knockout mice.
      ). In sum, these observations independently indicate significant differences in the rates of retinal sterol synthesis and turnover, in agreement with the results of previous studies (
      • Lin J.B.
      • Mast N.
      • Bederman I.R.
      • Li Y.
      • Brunengraber H.
      • Bjorkhem I.
      • et al.
      Cholesterol in mouse retina originates primarily from in situ de novo biosynthesis.
      ,
      • Fliesler S.J.
      • Vaughan D.K.
      • Jenewein E.C.
      • Richards M.J.
      • Nagel B.A.
      • Peachey N.S.
      Partial rescue of retinal function and sterol steady-state in a rat model of Smith-Lemli-Opitz syndrome.
      ).

      Cholesterol efflux in the RPE

      ABCA1 and ABCG1 expression has been reported for the RPE, in addition to the neural retina (
      • Zheng W.
      • Reem R.E.
      • Omarova S.
      • Huang S.
      • DiPatre P.L.
      • Charvet C.D.
      • et al.
      Spatial distribution of the pathways of cholesterol homeostasis in human retina.
      ,
      • Ananth S.
      • Gnana-Prakasam J.P.
      • Bhutia Y.D.
      • Veeranan-Karmegam R.
      • Martin P.M.
      • Smith S.B.
      • et al.
      Regulation of the cholesterol efflux transporters ABCA1 and ABCG1 in retina in hemochromatosis and by the endogenous siderophore 2,5-dihydroxybenzoic acid.
      ,
      • Storti F.
      • Klee K.
      • Todorova V.
      • Steiner R.
      • Othman A.
      • van der Velde-Visser S.
      • et al.
      Impaired ABCA1/ABCG1-mediated lipid efflux in the mouse retinal pigment epithelium (RPE) leads to retinal degeneration.
      ). Human and murine RPE/choroid express all of the known components of the cholesterol efflux mechanism (LXR-α/β, ABC transporters, APO-A1, APO-E, APO-B) as well as players in intracellular sterol transport (NPC-I, translocator protein 18 kDa [TSPO]) (
      • Storti F.
      • Raphael G.
      • Griesser V.
      • Klee K.
      • Drawnel F.
      • Willburger C.
      • et al.
      Regulated efflux of photoreceptor outer segment-derived cholesterol by human RPE cells.
      ). Native RPE cells and ARPE-19 cells express microsomal triglyceride transfer protein and APO-B, suggesting their capability to assemble their own lipoprotein-like particles (presumably for export) (
      • Li C.M.
      • Presley J.B.
      • Zhang X.
      • Dashti N.
      • Chung B.H.
      • Medeiros N.E.
      • et al.
      Retina expresses microsomal triglyceride transfer protein: implications for age-related maculopathy.
      ). ARPE-19 cells cultured with [3H]oleate have been show to secrete [3H]-labeled cholesteryl esters and triglycerides into the culture medium, and the lipoprotein-like particles isolated from the culture medium had physical characteristics (e.g., d << 1.21 g/ml) comparable with plasma lipoproteins (
      • Li C.M.
      • Presley J.B.
      • Zhang X.
      • Dashti N.
      • Chung B.H.
      • Medeiros N.E.
      • et al.
      Retina expresses microsomal triglyceride transfer protein: implications for age-related maculopathy.
      ). APO-A1 expression has been documented in the human and monkey RPE, as well as in the neural retina (
      • Tserentsoodol N.
      • Sztein J.
      • Campos M.
      • Gordiyenko N.V.
      • Fariss R.N.
      • Lee J.W.
      • et al.
      Uptake of cholesterol by the retina occurs primarily via a low density lipoprotein receptor-mediated process.
      ,
      • Kurumada S.
      • Onishi A.
      • Imai H.
      • Ishii K.
      • Kobayashi T.
      • Sato S.B.
      Stage-specific association of apolipoprotein A-I and E in developing mouse retina.
      ,
      • Simo R.
      • Garcia-Ramirez M.
      • Higuera M.
      • Hernandez C.
      Apolipoprotein A1 is overexpressed in the retina of diabetic patients.
      ,
      • Li C.M.
      • Chung B.H.
      • Presley J.B.
      • Malek G.
      • Zhang X.
      • Dashti N.
      • et al.
      Lipoprotein-like particles and cholesteryl esters in human Bruch's membrane: initial characterization.
      ). RPE-specific double knockout of ABCA1 and ABCG1 leads to accumulation of cholesteryl ester–rich lipid droplets in the RPE, accompanied by frank degeneration of the neural retina by PN 6 months (
      • Storti F.
      • Klee K.
      • Todorova V.
      • Steiner R.
      • Othman A.
      • van der Velde-Visser S.
      • et al.
      Impaired ABCA1/ABCG1-mediated lipid efflux in the mouse retinal pigment epithelium (RPE) leads to retinal degeneration.
      ). Furthermore, RPE-specific ABCA1 knockout was sufficient to cause lipid droplet accumulation, suggesting an important role for ABCA1 in RPE cholesterol efflux (
      • Storti F.
      • Klee K.
      • Todorova V.
      • Steiner R.
      • Othman A.
      • van der Velde-Visser S.
      • et al.
      Impaired ABCA1/ABCG1-mediated lipid efflux in the mouse retinal pigment epithelium (RPE) leads to retinal degeneration.
      ). ABCA1-dependent cholesterol efflux in the RPE is sensitive to treatment with probucol (a potent bis-phenol antioxidant that also inhibits ABCA1) and ABCA1 antibodies (
      • Storti F.
      • Raphael G.
      • Griesser V.
      • Klee K.
      • Drawnel F.
      • Willburger C.
      • et al.
      Regulated efflux of photoreceptor outer segment-derived cholesterol by human RPE cells.
      ,
      • Lyssenko N.N.
      • Haider N.
      • Picataggi A.
      • Cipollari E.
      • Jiao W.
      • Phillips M.C.
      • et al.
      Directional ABCA1-mediated cholesterol efflux and apoB-lipoprotein secretion in the retinal pigment epithelium.
      ). Poorly polarized ARPE-19 cells fail to stimulate basolateral cholesterol efflux to APO-A1, unlike well-differentiated and polarized human primary RPE cells (with healthy transepithelial electrical resistance). Polarized RPE can engage in ABCA1-mediated sterol transfer to APO-A1 on both the apical and basolateral sides of the cell (
      • Storti F.
      • Raphael G.
      • Griesser V.
      • Klee K.
      • Drawnel F.
      • Willburger C.
      • et al.
      Regulated efflux of photoreceptor outer segment-derived cholesterol by human RPE cells.
      ). Furthermore, RPE cells can successfully efflux cholesterol derived from photoreceptor OS membranes on both the apical and basolateral side of polarized RPE in an APO-A1–dependent manner (
      • Storti F.
      • Raphael G.
      • Griesser V.
      • Klee K.
      • Drawnel F.
      • Willburger C.
      • et al.
      Regulated efflux of photoreceptor outer segment-derived cholesterol by human RPE cells.
      ). It is important to meet the following criteria when using primary, iPSC-derived, or transformed RPE cells for these kinds of studies: (1) the cells should be well polarized (i.e., have defined apical and basolateral compartments); (2) the culture medium should have a defined lipid content (as well as the lactate content); (3) cells should have proper trans-epithelial electrical resistance; (4) the cytoskeleton and microtubule alignments should be comparable with those observed in normal RPE cells in vivo; (5) the genotype should be confirmed when using human donor primary or iPSC-derived cells; and (6) differences in lipid-related genes should be documented between the various models used (
      • Pilgrim M.G.
      • Lengyel I.
      • Lanzirotti A.
      • Newville M.
      • Fearn S.
      • Emri E.
      • et al.
      Subretinal pigment epithelial deposition of drusen components including hydroxyapatite in a primary cell culture model.
      ,
      • Pfeffer B.A.
      • Philp N.J.
      Cell culture of retinal pigment epithelium: special issue.
      ).
      Conditional ablation of Abca1 and Abcg1 in macrophages leads to thickening of Bruch's membrane and lipid droplet accumulation in the RPE as well as in the subretinal space (
      • Ban N.
      • Lee T.J.
      • Sene A.
      • Choudhary M.
      • Lekwuwa M.
      • Dong Z.
      • et al.
      Impaired monocyte cholesterol clearance initiates age-related retinal degeneration and vision loss.
      ). Under these conditions, both esterified and unesterified cholesterol content increase in the retina and RPE/choroid, as compared with age-matched controls (
      • Ban N.
      • Lee T.J.
      • Sene A.
      • Choudhary M.
      • Lekwuwa M.
      • Dong Z.
      • et al.
      Impaired monocyte cholesterol clearance initiates age-related retinal degeneration and vision loss.
      ). Using this type of model, concomitant age-related retinal degeneration was observed, as characterized by decreased ERG responses and the appearance of macrophages in the subretinal space and choroid by PN 12 months. These are common features observed in several retinal degeneration animal (primarily mouse) models, as well as in human AMD (
      • Ban N.
      • Lee T.J.
      • Sene A.
      • Choudhary M.
      • Lekwuwa M.
      • Dong Z.
      • et al.
      Impaired monocyte cholesterol clearance initiates age-related retinal degeneration and vision loss.
      ,
      • Zhao L.
      • Zabel M.K.
      • Wang X.
      • Ma W.
      • Shah P.
      • Fariss R.N.
      • et al.
      Microglial phagocytosis of living photoreceptors contributes to inherited retinal degeneration.
      ,
      • Levy O.
      • Calippe B.
      • Lavalette S.
      • Hu S.J.
      • Raoul W.
      • Dominguez E.
      • et al.
      Apolipoprotein E promotes subretinal mononuclear phagocyte survival and chronic inflammation in age-related macular degeneration.
      ,
      • Ramachandra Rao S.
      • Skelton L.A.
      • Wu F.
      • Onysk A.
      • Spolnik G.
      • Danikiewicz W.
      • et al.
      Retinal degeneration caused by rod-specific Dhdds ablation occurs without concomitant inhibition of protein N-glycosylation.
      ). Collectively, these findings suggest a role for macrophage interactions with the RPE in efficient cholesterol efflux across the outer blood-retinal barrier.

      CYP enzyme-catalyzed sterol hydroxylation and oxidation in the neural retina

      Two CYP genes are expressed in the neural retina: CYP27A1 and CYP46A1. The oxidized cholesterol derivatives 27-COOH-Chol and 27-OH-Chol (metabolites of CYP27A1) are the predominant oxysterol species found in human and bovine retinas, which stimulate LXRα-dependent cholesterol efflux (
      • Mast N.
      • Reem R.
      • Bederman I.
      • Huang S.
      • DiPatre P.L.
      • Bjorkhem I.
      • et al.
      Cholestenoic Acid is an important elimination product of cholesterol in the retina: comparison of retinal cholesterol metabolism with that in the brain.
      ). CYP27A1 expression was observed in ARPE-19 cells, as well as photoreceptor ISs, ganglion cells, and RPE of the monkey retina (
      • Lee J.W.
      • Fuda H.
      • Javitt N.B.
      • Strott C.A.
      • Rodriguez I.R.
      Expression and localization of sterol 27-hydroxylase (CYP27A1) in monkey retina.
      ). 27OH-7KCh, a product of CYP27A1-mediated metabolism of 7KChol, was found to be significantly less cytotoxic to ARPE-19 cells than 7KChol (
      • Lee J.W.
      • Fuda H.
      • Javitt N.B.
      • Strott C.A.
      • Rodriguez I.R.
      Expression and localization of sterol 27-hydroxylase (CYP27A1) in monkey retina.
      ). Under conditions of elevated oxidative stress and lipid peroxidation, CYP27A1 undergoes modification by lipid peroxide products, such as isolevuglandins, leading to reduced enzymatic activity, in turn contributing to altered cholesterol homeostasis (
      • Charvet C.
      • Liao W.L.
      • Heo G.Y.
      • Laird J.
      • Salomon R.G.
      • Turko I.V.
      • et al.
      Isolevuglandins and mitochondrial enzymes in the retina: mass spectrometry detection of post-translational modification of sterol-metabolizing CYP27A1.
      ).
      TSPO is a transmembrane protein involved in translocation of cholesterol from the outer to the inner mitochondrial membrane (
      • Rupprecht R.
      • Papadopoulos V.
      • Rammes G.
      • Baghai T.C.
      • Fan J.
      • Akula N.
      • et al.
      Translocator protein (18 kDa) (TSPO) as a therapeutic target for neurological and psychiatric disorders.
      ,
      • Costa B.
      • Da Pozzo E.
      • Martini C.
      18-kDa translocator protein association complexes in the brain: from structure to function.
      ). Thereby, TSPO regulates sterol substrate availability to inner mitochondrial membrane resident CYP27A1 and plays a regulatory role in sterol efflux (
      • Graham A.
      Mitochondrial regulation of macrophage cholesterol homeostasis.
      ). Activating ligands of TSPO, such as FGIN-1-27, increase RPE cholesterol efflux and decrease cellular cholesterol and phospholipid levels (
      • Biswas L.
      • Zhou X.
      • Dhillon B.
      • Graham A.
      • Shu X.
      Retinal pigment epithelium cholesterol efflux mediated by the 18 kDa translocator protein, TSPO, a potential target for treating age-related macular degeneration.
      ). TSPO knockdown sensitizes ARPE-19 cells to OxLDL challenge, leading to increased reactive oxgen species (ROS) generation and expression of inflammatory cytokines, such as interleukin-1β and TNF-α (
      • Biswas L.
      • Zhou X.
      • Dhillon B.
      • Graham A.
      • Shu X.
      Retinal pigment epithelium cholesterol efflux mediated by the 18 kDa translocator protein, TSPO, a potential target for treating age-related macular degeneration.
      ). Immunohistochemical analysis suggests expression of TSPO in the RPE and GCLs of the mouse retina. RPE TSPO expression levels decline with age and correlate with accumulation of cholesterol in the cell (
      • Biswas L.
      • Zhou X.
      • Dhillon B.
      • Graham A.
      • Shu X.
      Retinal pigment epithelium cholesterol efflux mediated by the 18 kDa translocator protein, TSPO, a potential target for treating age-related macular degeneration.
      ). The increase in RPE ROS levels also is accompanied by increase in the GSSG:GSH ratio (an indicator of oxidative stress), accumulation of free fatty acids, and decreased cellular ATP and NADH content (
      • Alamri A.
      • Biswas L.
      • Watson D.G.
      • Shu X.
      Deletion of TSPO resulted in change of metabolomic profile in retinal pigment epithelial cells.
      ).
      Other CYP enzymes involved in lipid efflux also affect cellular cholesterol homeostasis. For example, cholesteryl ester-laden lipid droplet accumulation and autophagic defects also have been observed in an iPSC-derived RPE model of Bietti's crystalline dystrophy, which is caused by mutations in the gene encoding CYP4V2 (
      • Nakano M.
      • Kelly E.J.
      • Wiek C.
      • Hanenberg H.
      • Rettie A.E.
      CYP4V2 in Bietti's crystalline dystrophy: ocular localization, metabolism of omega-3-polyunsaturated fatty acids, and functional deficit of the p.H331P variant.
      ,
      • Hata M.
      • Ikeda H.O.
      • Iwai S.
      • Iida Y.
      • Gotoh N.
      • Asaka I.
      • et al.
      Reduction of lipid accumulation rescues Bietti's crystalline dystrophy phenotypes.
      ). CYP4V2 is required for ω-oxidation of fatty acids, and the RPE lipid accumulation observed in the Bietti's crystalline dystrophy in vitro model was partially relieved by cyclodextrin treatment (
      • Hata M.
      • Ikeda H.O.
      • Iwai S.
      • Iida Y.
      • Gotoh N.
      • Asaka I.
      • et al.
      Reduction of lipid accumulation rescues Bietti's crystalline dystrophy phenotypes.
      ). This finding suggests a role for CYP hydroxylase–mediated fatty acid oxidation in RPE lipid efflux.
      ER-resident cholesterol-24S-hydroxylase (CYP46A1), which catalyzes the rate-limiting step in brain cholesterol efflux, metabolizes cholesterol to 24S-hydroxycholesterol (24S-OH-Chol) (
      • Lutjohann D.
      • Breuer O.
      • Ahlborg G.
      • Nennesmo I.
      • Siden A.
      • Diczfalusy U.
      • et al.
      Cholesterol homeostasis in human brain: evidence for an age-dependent flux of 24S-hydroxycholesterol from the brain into the circulation.
      ,
      • Bjorkhem I.
      • Lutjohann D.
      • Breuer O.
      • Sakinis A.
      • Wennmalm A.
      Importance of a novel oxidative mechanism for elimination of brain cholesterol. Turnover of cholesterol and 24(S)-hydroxycholesterol in rat brain as measured with 18O2 techniques in vivo and in vitro.
      ,
      • Russell D.W.
      • Halford R.W.
      • Ramirez D.M.
      • Shah R.
      • Kotti T.
      Cholesterol 24-hydroxylase: an enzyme of cholesterol turnover in the brain.
      ). In the retina, CYP46A1 is expressed predominantly in the inner retinal layers and in the RPE but is comparatively low in the photoreceptor layer (
      • Bretillon L.
      • Diczfalusy U.
      • Bjorkhem I.
      • Maire M.A.
      • Martine L.
      • Joffre C.
      • et al.
      Cholesterol-24S-hydroxylase (CYP46A1) is specifically expressed in neurons of the neural retina.
      ). Intravitreal injection of albino rats with voriconazole, a CYP46A1 inhibitor, did not lead to retinal degeneration or altered dark-adapted ERG responses (
      • Gao H.
      • Pennesi M.E.
      • Shah K.
      • Qiao X.
      • Hariprasad S.M.
      • Mieler W.F.
      • et al.
      Intravitreal voriconazole: an electroretinographic and histopathologic study.
      ). However, intraperitoneal injection of voriconazole led to a significant decrease in retinal 24S-OH-Chol levels without concomitant changes in brain or serum levels within 5 days (
      • Fourgeux C.
      • Martine L.
      • Acar N.
      • Bron A.M.
      • Creuzot-Garcher C.P.
      • Bretillon L.
      In vivo consequences of cholesterol-24S-hydroxylase (CYP46A1) inhibition by voriconazole on cholesterol homeostasis and function in the rat retina.
      ). This is very surprising, given that CYP46A1-synthesized 24S-OH-Chol is the predominant cholesterol elimination product in the brain, unlike the retina, which strongly depends on CYP27A1-dependent metabolism for cholesterol efflux (
      • Lutjohann D.
      • Breuer O.
      • Ahlborg G.
      • Nennesmo I.
      • Siden A.
      • Diczfalusy U.
      • et al.
      Cholesterol homeostasis in human brain: evidence for an age-dependent flux of 24S-hydroxycholesterol from the brain into the circulation.
      ,
      • Bjorkhem I.
      • Lutjohann D.
      • Breuer O.
      • Sakinis A.
      • Wennmalm A.
      Importance of a novel oxidative mechanism for elimination of brain cholesterol. Turnover of cholesterol and 24(S)-hydroxycholesterol in rat brain as measured with 18O2 techniques in vivo and in vitro.
      ,
      • Liao W.L.
      • Heo G.Y.
      • Dodder N.G.
      • Reem R.E.
      • Mast N.
      • Huang S.
      • et al.
      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.
      ,
      • Zhang J.
      • Akwa Y.
      • el-Etr M.
      • Baulieu E.E.
      • Sjovall J.
      Metabolism of 27-, 25- and 24-hydroxycholesterol in rat glial cells and neurons.
      ). A global knockout mouse model of CYP46A1 exhibited a significant, compensatory increase in retinal cholestenoic acid (a by-product of 27-OH-Chol oxidation by CYP27A1), and subsequent activation of LXRα/β and their gene targets (
      • Saadane A.
      • Mast N.
      • Trichonas G.
      • Chakraborty D.
      • Hammer S.
      • Busik J.V.
      • et al.
      Retinal Vascular abnormalities and microglia activation in mice with deficiency in cytochrome P450 46A1-mediated cholesterol removal.
      ). However, no significant changes in retina cholesterol content or ERG rod- or cone-driven responses were observed in 6-month-old CYP46A1 knockout mice compared with age-matched controls at PN 6 months (
      • Saadane A.
      • Mast N.
      • Trichonas G.
      • Chakraborty D.
      • Hammer S.
      • Busik J.V.
      • et al.
      Retinal Vascular abnormalities and microglia activation in mice with deficiency in cytochrome P450 46A1-mediated cholesterol removal.
      ). The retinas of CYP46A1 knockout mice exhibited leaky vasculature and microglial activation (
      • Saadane A.
      • Mast N.
      • Trichonas G.
      • Chakraborty D.
      • Hammer S.
      • Busik J.V.
      • et al.
      Retinal Vascular abnormalities and microglia activation in mice with deficiency in cytochrome P450 46A1-mediated cholesterol removal.
      ).
      CYP27A1/CYP46A1 global double-knockout mice (CYP27A1−/−-CYP46A1−/−) exhibit elevation in retinal cholesteryl ester content in lipid droplets. As discussed above, remarkably, such lipid droplet accumulation was observed in the OS layer (
      • Saadane A.
      • Mast N.
      • Dao T.
      • Ahmad B.
      • Pikuleva I.A.
      Retinal hypercholesterolemia triggers cholesterol accumulation and esterification in photoreceptor cells.
      ). The total cholesteryl ester and 7KChol content was significantly elevated in the retina, liver, brain, and lungs of the double-knockout mice (
      • Saadane A.
      • Mast N.
      • Dao T.
      • Ahmad B.
      • Pikuleva I.A.
      Retinal hypercholesterolemia triggers cholesterol accumulation and esterification in photoreceptor cells.
      ,
      • Saadane A.
      • Mast N.
      • Charvet C.D.
      • Omarova S.
      • Zheng W.
      • Huang S.S.
      • et al.
      Retinal and nonocular abnormalities in Cyp27a1(-/-)Cyp46a1(-/-) mice with dysfunctional metabolism of cholesterol.
      ), which also exhibited aberrant angiogenesis and retinal vasculature defects (
      • Saadane A.
      • Mast N.
      • Charvet C.D.
      • Omarova S.
      • Zheng W.
      • Huang S.S.
      • et al.
      Retinal and nonocular abnormalities in Cyp27a1(-/-)Cyp46a1(-/-) mice with dysfunctional metabolism of cholesterol.
      ). The accumulation of retinal cholesteryl esters was fully inhibited by deletion of Acat1 (required for sterol esterification) on the double-knockout background (
      • Saadane A.
      • Mast N.
      • Dao T.
      • Ahmad B.
      • Pikuleva I.A.
      Retinal hypercholesterolemia triggers cholesterol accumulation and esterification in photoreceptor cells.
      ). The sterol profile in triple-knockout (CYP27A1−/−-CYP46A1−/−-ACAT1−/−) mice was comparable with that of controls. (
      • Saadane A.
      • Mast N.
      • Dao T.
      • Ahmad B.
      • Pikuleva I.A.
      Retinal hypercholesterolemia triggers cholesterol accumulation and esterification in photoreceptor cells.
      ,
      • Saadane A.
      • Mast N.
      • Trichonas G.
      • Chakraborty D.
      • Hammer S.
      • Busik J.V.
      • et al.
      Retinal Vascular abnormalities and microglia activation in mice with deficiency in cytochrome P450 46A1-mediated cholesterol removal.
      ). Despite normalization of the sterol profile, photoreceptor cell death was observed (using TUNEL labeling) (
      • Saadane A.
      • Mast N.
      • Dao T.
      • Ahmad B.
      • Pikuleva I.A.
      Retinal hypercholesterolemia triggers cholesterol accumulation and esterification in photoreceptor cells.
      ,
      • Saadane A.
      • Mast N.
      • Trichonas G.
      • Chakraborty D.
      • Hammer S.
      • Busik J.V.
      • et al.
      Retinal Vascular abnormalities and microglia activation in mice with deficiency in cytochrome P450 46A1-mediated cholesterol removal.
      ). Future investigations into ABC transporter activity in CYP27A1−/−-CYP46A1−/−-ACAT1−/− and CYP27A1−/−-CYP46A1−/− models may provide additional new insights into the retinal cholesterol efflux mechanism. The accumulation of cholesteryl esters in the retina and RPE in the above discussed animal models has been observed using Oil Red O staining, filipin labeling, and fluorescence imaging of retinal tissue sections (plus and minus cholesteryl esterase treatment), as well as transmission electron microscopy (
      • Saadane A.
      • Mast N.
      • Dao T.
      • Ahmad B.
      • Pikuleva I.A.
      Retinal hypercholesterolemia triggers cholesterol accumulation and esterification in photoreceptor cells.
      ,
      • Storti F.
      • Klee K.
      • Todorova V.
      • Steiner R.
      • Othman A.
      • van der Velde-Visser S.
      • et al.
      Impaired ABCA1/ABCG1-mediated lipid efflux in the mouse retinal pigment epithelium (RPE) leads to retinal degeneration.
      ,
      • Saadane A.
      • Mast N.
      • Charvet C.D.
      • Omarova S.
      • Zheng W.
      • Huang S.S.
      • et al.
      Retinal and nonocular abnormalities in Cyp27a1(-/-)Cyp46a1(-/-) mice with dysfunctional metabolism of cholesterol.
      ). These methods along with lipidomic analysis of retinal tissue to quantify total and free sterol (and, by difference, esterified cholesterol) have provided compelling validation of retinal sterol esterification (
      • Saadane A.
      • Mast N.
      • Charvet C.D.
      • Omarova S.
      • Zheng W.
      • Huang S.S.
      • et al.
      Retinal and nonocular abnormalities in Cyp27a1(-/-)Cyp46a1(-/-) mice with dysfunctional metabolism of cholesterol.
      ).

      Ox-Ldl and oxysterols on the neural retina and the RPE

      We have previously discussed the critical role oxysterols play as LXR agonists, thus facilitating ABC transporter–mediated cellular cholesterol efflux. Therefore, oxysterols act as regulators of cholesterol biosynthesis and metabolism (
      • Schroepfer Jr., G.J.
      Oxysterols: modulators of cholesterol metabolism and other processes.
      ,
      • Brown A.J.
      • Jessup W.
      Oxysterols: sources, cellular storage and metabolism, and new insights into their roles in cholesterol homeostasis.
      ). Elevated oxysterol levels in membranes lead to organelle dysfunctions such as mitochondrial dysfunction, ER stress, and lysosomal membrane permeabilization (
      • Massey J.B.
      Membrane and protein interactions of oxysterols.
      ). High-intensity light exposure leads to nonenzymatic lipid peroxidation and generation of 4-hydroxynonenal (4-HNE, a by-product of oxidation of omega-6 PUFAs) and, subsequently, oxidatively modified retinal proteins (
      • Tanito M.
      • Haniu H.
      • Elliott M.H.
      • Singh A.K.
      • Matsumoto H.
      • Anderson R.E.
      Identification of 4-hydroxynonenal-modified retinal proteins induced by photooxidative stress prior to retinal degeneration.
      ). Similar conditions can promote the nonenzymatic oxidation of cholesterol and other sterols, generating cytotoxic oxysterols (
      • Barnaba C.
      • Rodriguez-Estrada M.T.
      • Lercker G.
      • Garcia H.S.
      • Medina-Meza I.G.
      Cholesterol photo-oxidation: a chemical reaction network for kinetic modeling.
      ,
      • Iuliano L.
      Pathways of cholesterol oxidation via non-enzymatic mechanisms.
      ,
      • Murphy R.C.
      • Johnson K.M.
      Cholesterol, reactive oxygen species, and the formation of biologically active mediators.
      ,
      • Carvalho J.F.
      • Silva M.M.
      • Moreira J.N.
      • Simoes S.
      • Sa E.M.M.L.
      Selective cytotoxicity of oxysterols through structural modulation on rings A and B. Synthesis, in vitro evaluation, and SAR.
      ,
      • O'Callaghan Y.C.
      • Woods J.A.
      • O'Brien N.M.
      Comparative study of the cytotoxicity and apoptosis-inducing potential of commonly occurring oxysterols.
      ). In addition to conditions that cause photo-oxidative stress, alterations in iron homeostasis also have been shown to promote lipid peroxidation (
      • Braughler J.M.
      • Duncan L.A.
      • Chase R.L.
      The involvement of iron in lipid peroxidation. Importance of ferric to ferrous ratios in initiation.
      ,
      • Latunde-Dada G.O.
      Ferroptosis: role of lipid peroxidation, iron and ferritinophagy.
      ), including in the retina (
      • He X.
      • Hahn P.
      • Iacovelli J.
      • Wong R.
      • King C.
      • Bhisitkul R.
      • et al.
      Iron homeostasis and toxicity in retinal degeneration.
      ,
      • Loh A.
      • Hadziahmetovic M.
      • Dunaief J.L.
      Iron homeostasis and eye disease.
      ). The chemistry of oxysterols and their biophysical effects on the plasma membrane have been discussed above, as well as in other reviews (
      • Filomenko R.
      • Fourgeux C.
      • Bretillon L.
      • Gambert-Nicot S.
      Oxysterols: influence on plasma membrane rafts microdomains and development of ocular diseases.
      ,
      • Fliesler S.J.
      • Xu L.
      Oxysterols and retinal degeneration in a rat model of Smith-Lemli-Opitz syndrome: implications for an improved therapeutic intervention.
      ). We will now discuss the uptake mechanisms of OxLDL and the biological effects of oxysterols and OxLDL in the RPE and the retina.

      OxLDL and oxysterols in the RPE

      The RPE cell is a unique epithelial cell type because it is a postmitotic, long-lived professional, stationary phagocyte, while also possessing the classic characteristics of a polarized epithelial cell and participating in barrier functions like other epithelial cells. Key insights into RPE cholesterol homeostasis have arisen from studies performed using in vitro models, given the ease of establishment and long-term maintenance of primary RPE cultures, availability of transformed RPE-derived cell lines (RPE-J, ARPE-19, etc.), and recent advancements in iPSC-derived RPE in vitro cell models (
      • Pfeffer B.A.
      • Philp N.J.
      Cell culture of retinal pigment epithelium: special issue.
      ,
      • Kuznetsova A.V.
      • Kurinov A.M.
      • Aleksandrova M.A.
      Cell models to study regulation of cell transformation in pathologies of retinal pigment epithelium.
      ,
      • Fronk A.H.
      • Vargis E.
      Methods for culturing retinal pigment epithelial cells: a review of current protocols and future recommendations.
      ). However, it should be kept in mind that ARPE-19 cells, in addition to be immortalized, are not fully differentiated and have some distinct differences from primary RPE cells that may limit the direct applicability of results obtained with their use to the normal biology of the RPE (
      • Pfeffer B.A.
      • Philp N.J.
      Cell culture of retinal pigment epithelium: special issue.
      ,
      • Fronk A.H.
      • Vargis E.
      Methods for culturing retinal pigment epithelial cells: a review of current protocols and future recommendations.
      ). However, in vitro oxysterol treatment leads to direct free oxysterol incorporation into the plasma membrane or entry into the cell, by-passing the canonical endocytic uptake pathway.
      Uptake of OxLDL occurs primarily via a receptor-mediated endocytic uptake pathway dependent on either SRB CD36 or the lectin-like LOX-1 (
      • Gordiyenko N.
      • Campos M.
      • Lee J.W.
      • Fariss R.N.
      • Sztein J.
      • Rodriguez I.R.
      RPE cells internalize low-density lipoprotein (LDL) and oxidized LDL (oxLDL) in large quantities in vitro and in vivo.
      ). In the retina, a primary function of SRB-II is its role in the uptake of shed photoreceptor OS tips by the RPE during the daily process of photoreceptor membrane turnover (
      • Ryeom S.W.
      • Sparrow J.R.
      • Silverstein R.L.
      CD36 participates in the phagocytosis of rod outer segments by retinal pigment epithelium.
      ). Expression of SRB-II has been observed in primary RPE cells, ARPE-19 cells, and in animal models (
      • Tserentsoodol N.
      • Sztein J.
      • Campos M.
      • Gordiyenko N.V.
      • Fariss R.N.
      • Lee J.W.
      • et al.
      Uptake of cholesterol by the retina occurs primarily via a low density lipoprotein receptor-mediated process.
      ,
      • Gordiyenko N.
      • Campos M.
      • Lee J.W.
      • Fariss R.N.
      • Sztein J.
      • Rodriguez I.R.
      RPE cells internalize low-density lipoprotein (LDL) and oxidized LDL (oxLDL) in large quantities in vitro and in vivo.
      ,
      • Duncan K.G.
      • Bailey K.R.
      • Kane J.P.
      • Schwartz D.M.
      Human retinal pigment epithelial cells express scavenger receptors BI and BII.
      ). Both CD36 and internalize various cargoes, such as cholesteryl esters and phosphatidylserine-rich membranes. RPE cells exhibit CD36-dependent phagocytic uptake of OS phospholipids (
      • Ryeom S.W.
      • Sparrow J.R.
      • Silverstein R.L.
      CD36 participates in the phagocytosis of rod outer segments by retinal pigment epithelium.
      ,
      • Finnemann S.C.
      • Silverstein R.L.
      Differential roles of CD36 and alphavbeta5 integrin in photoreceptor phagocytosis by the retinal pigment epithelium.
      ,
      • Ryeom S.W.
      • Silverstein R.L.
      • Scotto A.
      • Sparrow J.R.
      Binding of anionic phospholipids to retinal pigment epithelium may be mediated by the scavenger receptor CD36.
      ). On the other hand, the lipid peroxide species generated by photo-oxidation of OS membranes (e.g., of their constituent PUFA-containing phospholipids, as well as sterols) serve as potent ligands for the CD36-mediated diurnal uptake of OS by the RPE (
      • Sun M.
      • Finnemann S.C.
      • Febbraio M.
      • Shan L.
      • Annangudi S.P.
      • Podrez E.A.
      • et al.
      Light-induced oxidation of photoreceptor outer segment phospholipids generates ligands for CD36-mediated phagocytosis by retinal pigment epithelium: a potential mechanism for modulating outer segment phagocytosis under oxidant stress conditions.
      ). Furthermore, OxLDL and lipid peroxide products competitively inhibit CD36-mediated uptake of OS by RPE cells (
      • Sun M.
      • Finnemann S.C.
      • Febbraio M.
      • Shan L.
      • Annangudi S.P.
      • Podrez E.A.
      • et al.
      Light-induced oxidation of photoreceptor outer segment phospholipids generates ligands for CD36-mediated phagocytosis by retinal pigment epithelium: a potential mechanism for modulating outer segment phagocytosis under oxidant stress conditions.
      ), and uptake of OxLDL mediated by CD36 is also blocked by antibodies against CD36 (
      • Gnanaguru G.
      • Choi A.R.
      • Amarnani D.
      • D'Amore P.A.
      Oxidized lipoprotein uptake through the CD36 receptor activates the NLRP3 inflammasome in human retinal pigment epithelial cells.
      ).
      Treatment of RPE-J cells with OxLDL or oxidized OS membranes significantly decreased the degradation of phagocytosed OS by RPE cells (
      • Hoppe G.
      • Marmorstein A.D.
      • Pennock E.A.
      • Hoff H.F.
      Oxidized low density lipoprotein-induced inhibition of processing of photoreceptor outer segments by RPE.
      ). This was due to inefficient phagosome maturation observed as a lack of co-compartmentalization of opsin (the visual pigment apoprotein) with markers of the endolysosomal system, such as Cathepsin-D or Rab5 (
      • Hoppe G.
      • Marmorstein A.D.
      • Pennock E.A.
      • Hoff H.F.
      Oxidized low density lipoprotein-induced inhibition of processing of photoreceptor outer segments by RPE.
      ,
      • Hoppe G.
      • O'Neil J.
      • Hoff H.F.
      • Sears J.
      Accumulation of oxidized lipid-protein complexes alters phagosome maturation in retinal pigment epithelium.
      ,
      • Hoppe G.
      • O'Neil J.
      • Hoff H.F.
      • Sears J.
      Products of lipid peroxidation induce missorting of the principal lysosomal protease in retinal pigment epithelium.
      ).
      Primary RPE and transformed ARPE-19 cells challenged with OxLDL (100 μg/ml) undergo cell death, with transformed RPE cells exhibiting sensitivity at lower concentrations (
      • Yu A.L.
      • Lorenz R.L.
      • Haritoglou C.
      • Kampik A.
      • Welge-Lussen U.
      Biological effects of native and oxidized low-density lipoproteins in cultured human retinal pigment epithelial cells.
      ). RPE cell death was accompanied by elevated expression of vascular endothelial growth factor (VEGF), proapoptotic mediators like Bax (causes cell death upon mitochondrial membrane permeabilization), and generation of ROS (
      • Yin L.
      • Wu X.
      • Gong Y.
      • Shi Y.
      • Qiu Y.
      • Zhang H.
      • et al.
      OX-LDL up-regulates the vascular endothelial growth factor-to-pigment epithelium-derived factor ratio in human retinal pigment epithelial cells.
      ,
      • Kim J.H.
      • Lee S.J.
      • Kim K.W.
      • Yu Y.S.
      • Kim J.H.
      Oxidized low density lipoprotein-induced senescence of retinal pigment epithelial cells is followed by outer blood-retinal barrier dysfunction.
      ,