the bisretinoids of retinal pigment epithelium

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Progress in Retinal and Eye Research 31 (2012) 121e135

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Progress in Retinal and Eye Research

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The bisretinoids of retinal pigment epithelium

Janet R. Sparrowa,b,*, Emily Gregory-Roberts a, Kazunori Yamamoto a, Anna Blonska a,Shanti Kaligotla Ghosh a, Keiko Ueda a, Jilin Zhou a

aDepartment of Ophthalmology, Columbia University, 630 W. 168th Street, New York, NY 10032, United StatesbDepartment of Pathology and Cell Biology, Columbia University, 630 W. 168th Street, New York, NY 10032, United States

a r t i c l e i n f o

Article history:Available online 22 December 2011

Keywords:A2EAll-trans-retinalBisretinoidRetinal pigment epitheliumMacular degenerationRetina

Abbreviations: ABCA4, ATP-binding cassette, sub-fam3-phosphate dehydrogenase;GSH, glutathione;mSOD2RDH, retinol dehydrogenase; RPE, retinal pigment epith* Corresponding author. Columbia University, Depa

305 9638.E-mail address: [email protected] (J.R. Sparrow

1350-9462/$ e see front matter � 2012 Elsevier Ltd.doi:10.1016/j.preteyeres.2011.12.001

a b s t r a c t

The retina exhibits an inherent autofluorescence that is imaged ophthalmoscopically as fundus auto-fluorescence. In clinical settings, fundus autofluorescence examination aids in the diagnosis and follow-upof many retinal disorders. Fundus autofluorescence originates from the complex mixture of bisretinoidfluorophores that are amassed by retinal pigment epithelial (RPE) cells as lipofuscin. Unlike the lipofuscinfound in other cell-types, this material does not form as a result of oxidative stress. Rather, the formation isattributable to non-enzymatic reactions of vitamin A aldehyde in photoreceptor cells; transfer to RPE occursupon phagocytosis of photoreceptor outer segments. These fluorescent pigments accumulate even inhealthy photoreceptor cells and are generated as a consequence of the light capturing function of the cells.Nevertheless, the formation of this material is accelerated in some retinal disorders including recessiveStargardt disease and ELOVL4-related retinal degeneration. As such, these bisretinoid side-products areimplicated in the disease processes that threaten vision. In this article, we reviewour current understandingof the composition of RPE lipofuscin, the structural characteristics of the various bisretinoids, their relatedspectroscopic features and the biosynthetic pathways by which they form. We will revisit factors knownto influence the extent of the accumulation and therapeutic strategies being used to limit bisretinoidformation. Given their origin from vitamin A aldehyde, an isomer of the visual pigment chromophore, it isnot surprising that the bisretinoids of retina are light sensitive molecules. Accordingly, we will discussrecent findings that implicate the photodegradation of bisretinoid in the etiology of age-related maculardegeneration.

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Contents

1. The origin of RPE lipofuscin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .1222. The composition of RPE lipofuscin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .122

2.1. Known bisretinoids of retina . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1222.2. Topographic distribution . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1232.3. Structural features of bisretinoids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 123

3. Reactions in photoreceptor outer segments leading to bisretinoid formation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .1233.1. N-retinylidene-PE . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1233.2. Formation of A2PE and A2E . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1243.3. Formation of A2-DHP-PE . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1253.4. Formation of all-trans-retinal dimer and conjugates . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1263.5. Formation of A2-GPE . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 126

4. Absorbance and emission spectra . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .1264.1. Structural features underlying bisretinoid absorbance and fluorescence emission . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 126

ily A,member 4; DHA, docosahexaenoic acid; ESI-MS, electrospray ionizationmass spectrometry; GAPDH, glyceraldehyde,mitochondrial superoxidedismutase-2;NRPE,N-retinylidene-phosphatidylethanolamine; PE, phosphatidylethanolamine;elium.rtment of Ophthalmology, 630 W. 168th Street, New York, NY 10032, United States. Tel.: þ1 212 305 9944; fax: þ1 212

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J.R. Sparrow et al. / Progress in Retinal and Eye Research 31 (2012) 121e135122

4.2. Comparing the fluorescence emission spectra of human lipofuscin and A2E . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1274.3. Fluorescence emission spectra of lipofuscin in RPE of Abca4 null mutant mice . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1274.4. Changes in fluorescence emission with irradiation of bisretinoid . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 127

5. Photodegradation of RPE bisretinoids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .1286. Retinal bisretinoids and fundus autofluorescence . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .1297. Learning from human disease and animal models . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .130

7.1. ABCA4-deficiency . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1307.2. Light exposure and vitamin A supplementation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1307.3. Modulation of retinal bisretinoid formation by RPE65 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1317.4. Mutations in retinol dehydrogenase (RDH) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1317.5. ELOVL4-related macular dystrophy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1317.6. Senescence-accelerated mice . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1317.7. Age-related macular degeneration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1317.8. Conditions of oxidative stress . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 132

8. Conclusions and future directions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .132Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 132References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 132

1. The origin of RPE lipofuscin

The lipofuscin of retinal pigment epithelial (RPE) cells is amassedwith age in organelles of the lysosomal compartment of the cells,in both healthy and diseased retina. It is generally agreed thatthis material originates, for the most part, from ingestion of shedphotoreceptor outer segment membrane. Historically, however,opinions as to the molecular composition of RPE lipofuscin, havediffered (Chio et al., 1969; Chio and Tappel, 1969; Chowdhury et al.,2004; Eldred and Katz, 1991, 1989), with one notion being that itconsists of oxidatively modified lipid. Carboxyethyl pyrrole protein(CEP)-adducts, modifications that are generated from the oxidationof docosahexaenoate-containing lipids in photoreceptor cells havebeen detected in RPE lipofuscin (Ng et al., 2008). Presumably, thephotooxidative process responsible for generating these carbox-yethyl pyrrole-protein adducts could occur in photoreceptor cellsbefore RPE phagocytosis of outer segment membrane or could occurwithin the lysosomal bodies in which RPE lipofuscin is stored, orboth. The notion that oxidation processes are otherwise responsiblefor the formation of RPE lipofuscin, is not consistent with what isknown regarding the composition of this material. For instance, thespectral properties of the blueegreen emitting fluorescent productsof lipid oxidation are substantially different (excitation maximaw350, emission maxima w435 nm) (Rein and Tappel, 1998) thanspectra generated fromRPE lipofuscin (Eldred and Katz,1991; Eldredet al., 1982). Specifically, spectra obtained with intact RPE cells,RPE extracts (Eldred and Katz, 1991; Eldred et al., 1982), explants ofadult human eyes (Delori et al., 1995) and suspensions of lipofuscinstorage bodies (Boulton et al., 1990; Feeney-Burns and Eldred,1983),have all demonstrated that RPE lipofuscin emits with an emissionmaximum of approximately 590e600 nm (yelloweorange) whenexcited by light in the ‘blue’ region of the spectrum. It has also beenassumed that RPE lipofuscin consists of cross-linked oxidativelymodified proteins (Brunk and Terman, 2002) derived from phago-cytosed photoreceptor outer segments. However, Feeney-Burns andcolleagues (Eldred and Katz, 1991; Eldred et al., 1982) and Boultonand colleagues (Boulton, 2009) have pointed out that the presence ofphotoreceptor proteins in preparations enriched in lipofuscin gran-ules (lysosomal organelles) (Schutt et al., 2002; Warburton et al.,2005), is attributable to contamination with phagosomes. More-over, a proteomic study of purified lipofuscin granules revealed thatthe amino acid content was only 2% (w/w) (Ng et al., 2008). It is alsoworth noting that the view that RPE lipofuscin accumulates becauseof inhibition of lysosomal enzymes, cannot be reconciled with theaccumulation of this material in all healthy eyes even at young ages.

Instead, it is likely that RPE lysosomal enzymes thatwould otherwisedegrade the bisretinoid, do not recognize the unprecedented struc-tures that constitute this material.

2. The composition of RPE lipofuscin

2.1. Known bisretinoids of retina

Considerable evidence has accumulated that, unlike the lipofuscinthat accumulates in other non-dividing cells (Brunk and Terman,2002), the lipofuscin pigments in RPE originate in photoreceptorcell outer segments (Katz et al., 1986) from random non-enzymaticreactions of retinaldehyde (Katz et al., 1987) (Fig. 1). Indeed, all ofthe constituents of RPE lipofuscin that have been isolated and char-acterized have been shown to form in this manner (Sparrow, 2007b;Sparrow et al., 2010a). The transfer from photoreceptor cell to RPEoccurswith phagocytosis of shed outer segmentmembrane. Currentlyat least 25 bisretinoid pigments can be identified chromatographicallyand by mass spectrometry; these compounds can be grouped within4 families: 1) A2E, the first RPE lipofuscin constituent to be described;2) A2-glycerophosphoethanolamine (A2-GPE) a recently character-ized pigment (Yamamoto et al., 2011); 3) A2-dihydropyridine-phosphatidylethanolamine (A2-DHP-PE); 4) all-trans-retinal dimer,all-trans-retinal dimer-phosphatidylethanolamine and all-trans-retinal dimer-ethanolamine ADD SAKAI (Ben-Shabat et al., 2002b;Fishkin et al., 2005; Kim et al., 2007a, 2007b; Liu et al., 2000; Parishet al., 1998; Wu et al., 2009; Yamamoto et al., 2011) (Fig. 1).Other chromatographic peaks having fluorescence and absorbanceproperties suggestive of bisretinoid, can be observed but the struc-tures of the corresponding compounds are not yet known. The 25peaks mentioned above include known isomers (e.g. cis isomersof A2E), some of the photooxidized forms of these bisretinoidsand biosynthetic intermediates such as A2PE (Fig.1, compound 7) anddihydropyridinium-A2PE (Fig. 1, compound 5) (Section 3.2) (Fig. 1).Not included in this number, however, are bisretinoids differing interms of the variety of fatty acid moieties that constitute thephospholipid-derived tails of someof thesefluorophores (Section 3.2).The bisretinoids in RPE that retain the phospholipid moiety includeall-trans-retinal dimer-PE and A2-DHP-PE (Fig. 1, compounds 6and 10). A2E (Fig. 1, compound 8) on the other hand, does not carryfatty acids, since it is generated when its immediate precursor, thephosphatidylethanolamine-bisretinoid A2PE, undergoes phospholi-pase D-mediated enzymatic hydrolysis (Ben-Shabat et al., 2002b; Liuet al., 2000; Sparrow et al., 2008).

Fig. 1. Proposed biosynthetic schemes for bisretinoid formation. All-trans-retinal that is released from opsin after photoisomerization of ground state 11-cis-retinal reacts withphosphatidylethanolamine (PE) in the disk membrane to produce the N-retinylidene-phosphatidylethanolamine Schiff base (NRPE). NRPE undergoes a [1,6] H-shift producingtautomer x. Reaction with a second molecule of all-trans-retinal leads to path a and the formation of a bisretinoid phosphatidyl-dihydropyridinium molecule (dihydropyr-idinium-A2PE). Dihydropyridinium-A2PE automatically loses one hydrogen to produce A2-dihydropyridine-phosphatidylethanolamine (A2-DHP-PE) or it can eliminate 2hydrogens to form A2PE, a phosphatidyl-pyridinium bisretinoid. Hydrolysis of the phosphate ester of A2PE, probably by the lysosomal enzyme phospholipase D, yieldsA2E. Alternatively, all-trans-retinal can add to tautomer x and after ring closure all-trans-retinal dimer will form (path b). Subsequent Schiff base reaction with PE yieldsall-trans-retinal dimer-PE. Dashed arrows indicate multiple steps in the pathway. Note that OP indicates retention of phosphatidic acid (glycerol, phosphate and fatty acids)originating from PE.

J.R. Sparrow et al. / Progress in Retinal and Eye Research 31 (2012) 121e135 123

2.2. Topographic distribution

RPE lipofuscin, measured by recording autofluorescence, can bedetected throughout the human retina, with the content increasingfrom the equator to the posterior pole except for a dip at the fovea(Weiter et al., 1986; Wing et al., 1978). Similarly, when humanRPE/choroid is sampledbymeans of 4-mmtrephine punches followedby chromatographic (430 nm detection) and mass spectrometric(m/z 592) analysis, A2E is found to accumulate centrally and in each ofthe 4 quadrants (Fig. 2). We have analyzed bisretinoid composition inhuman,mouse, rat andbovine eyes. In all cases, the samepigments areobserved, although the relative levels of one bisretinoid to another canvary amongst these species.

2.3. Structural features of bisretinoids

Common to all of the known bisretinoids, is a ring structure inthe core of themolecule. For instance, A2E and its isomers (Liu et al.,2000; Parish et al., 1998), A2-GPE (Yamamoto et al., 2011) and A2PE(Liu et al., 2000) are characterized by a central pyridinium ring thathouses a quaternary amine nitrogen (Fig. 1, compounds 7,8,9). Thenitrogen does not undergo deprotonation (Parish et al., 1998;Sparrow et al., 1999) and the positive charge on the pyridiniumnitrogen is neutralized by a counter ion (probably chloride) makingA2E a salt. While the double bonds along the side-arms of A2E areall in the trans (E) position, Z-isomers of A2E have double bonds atthe C13/14 (isoA2E), C9/90-10/100 and C11/110-12/120 positions.A2-DHP-PE has a non-charged dihydropyridine ring at its core (Fig.1,compound 10); this structure was confirmed by high performance

liquid chromatography-electrospray ionization-tandem mass spec-trometry with corroboration by Fourier transform infrared spec-troscopy and modeling using density functional theory (Wu et al.,2009). On the other hand, the compound all-trans-retinal dimercontains a cyclohexadiene ring from which extend two polyenearms e 7 double-bond conjugations on the long arm and 4 on theshort arm. All-trans-retinal dimer-E and all-trans-retinal dimer-PEare dimers of all-trans-retinal attached to PE via an imine functiongroup (eC]Ne) with a protonation state that is pH-dependent(Fishkin et al., 2005) (Fig. 1, compound 6).

3. Reactions in photoreceptor outer segments leading tobisretinoid formation

3.1. N-retinylidene-PE

All-trans-retinal that participates in bisretinoid biosynthesis isgenerated by photoisomerization of 11-cis-retinal, following captureof a photon of light. In what is probably a mechanism for chaper-oning all-trans-retinal, at least some of this aldehyde when releasedfrom photoactivated rhodopsin, reacts with phosphatidylethanol-amine (PE) (1:1 ratio) in the disc membrane to form the adductN-retinylidene-phosphatidylethanolamine (NRPE) (Fig.1, compound3) via an imine (Schiff base) linkage (Anderson andMaude,1970; Liuet al., 2000; Poincelot et al., 1969; Sun et al., 1999;Weng et al., 1999).The sequestration of all-trans-retinal as NRPE may be an importantmechanism limiting the toxicity of this reactive aldehyde.

NRPE is well known to be the ligand recognized by ABCA4,a member of the ABC (ATP-binding cassette) family of lipid

Fig. 2. A2E accumulates in central and peripheral retina of adult human eye. RPE/choroid samples obtained fromhuman eye using 4mm trephine. A. RPE/choroid from central (centeredon fovea) retina. Tissue was analyzed by ultra performance liquid chromatography/mass spectrometry (MS) analysis (electrospray ion multi-mode ionization, ESI). Upper trace,chromatogramwith absorbancemonitoring at 430nm. Inset, absorbance spectra of A2E and isoA2E. Lower trace, ESI-MSatm/z 592, themolecularweight of A2E. B. RPE/choroid fromeachof four retinal quadrants. Shown are absorbance spectra and molecular weights (m/z) for A2E and isoA2E obtained by analysis of samples from each quadrant. C. Chloroform/methanolextract of human RPE/choroid showing water soluble and chloroform/methanol soluble phases.


transporters, that is thought to serve in the delivery of all-trans-retinal to cytosolic retinol dehydrogenases, a group of enzymesthat reduce vitamin A aldehyde to the non-reactive alcohol form(Beharry et al., 2004; Molday et al., 2000; Molday andMolday, 1979;Papermaster et al., 1978; Sun et al., 1999; Sun and Nathans, 1997,2001a, 2001b; Weng et al., 1999). ABCA4 is the gene responsible forrecessive Stargardt disease (Allikmets et al., 1997). The Schiff base ofNRPE can exist in either a protonated or unprotonated state but it isconsidered to be protonated when present in the acidic milieu ofthe disk lumen (Molday et al., 2009). The unprotonated formofNRPEbinds to ABCA4 (Molday et al., 2009) and it is reported that in wild-type mice the unprotonated form predominates while Abca4�/�

mice are associated with the protonated form (Weng et al., 1999).NRPE can readily undergo hydrolysis to release all-trans-retinal;however, when NRPE reacts non-enzymatically with a secondmolecule of all-trans-retinal, the irreversible formationof bisretinoidoccurs (Fig. 1).

As shown in Fig. 3, NRPE is afluorescentmolecule; at an excitationof 430 nm, the fluorescence efficiency of NRPE is 30% of A2E (A2Efluorescence/absorbance, 0.18; NRPE fluorescence/absorbance, 0.05).Analysis of eye extracts obtained from Abca4�/� mice reveals thatNRPE can form by reactionwith diacyl-PE comprised of various fattyacids and degrees of saturation, for instance 22:6 (docosahexaenoicacid, DHA) and 18:0 (stearic acid) (Fig. 3). Not surprisingly, DHA isa predominant constituent of these adducts due to its abundance inouter segment membrane (Fliesler and Anderson, 1983). In addition,however, we found that NRPE can form as a Schiff base conjugatebetween all-trans-retinal and both ether-PE (e.g. C22:5 and 18:0,Fig. 3) and PE-based plasmalogens (e.g. C22:5 and 18:1, Fig. 2); ether-PE and ethanolamine plasmalogens are both lipid constituentsof outer segments (Anderson and Maude, 1970). PE may not be theonly amine-bearing molecule that interacts with all-trans-retinalhowever, since we have also observed that pairs of all-trans-retinal(A2) can covalently attach to the lysine residues of rhodopsin (Fishkinet al., 2003). As yet, however, we have not detected A2-peptides inRPE lipofuscin.

3.2. Formation of A2PE and A2E

The formation of NRPE from all-trans-retinal and PE is the firststep in the multi-step process by which at least some retinal bis-retinoids are generated (Fig. 1). For instance, A2E forms when NRPEreacts non-enzymatically with a secondmolecule of all-trans-retinaland the biosynthetic pathway continues with the generation ofa dihydropyridinium molecule (dihydropyridinium-A2PE) (Fig. 1,compound 5). Dihydropyridinium-A2PE subsequently undergoesautooxidation to eliminate two hydrogens (Ben-Shabat et al., 2002b;Kim et al., 2007a; Liu et al., 2000; Parish et al., 1998) and yield A2PE(Fig. 1, compound 7), a positively charged phosphatidyl-pyridiniumbisretinoid. Phospholipase D-mediated cleavage of A2PE generatesA2E and establishes A2PE as the immediate precursor of A2E(Ben-Shabat et al., 2002b; Liu et al., 2000). This hydrolytic activity ispresent in lysosomal fractions of RPE (Sparrow et al., 2008).

The formation of bisretinoid in photoreceptor outer segments(POS) can be observed by analyzing POS isolated from bovine eyes.As presented in Fig. 4, one of these, the pigment A2PE, forms inouter segments as the immediate precursor of A2E. A2PE is nota single molecule but a complex mixture consisting of fatty acids ofvarying lengths and degrees of unsaturation, for instance 22:6(docosahexaenoic, DHA) and 18:0 (stearic acid) (Fig. 4). Incubationof bovine outer segments with all-trans-retinal mimics bisretinoidformation in vivo and by intensifying the detection of theseA2PE species (Fig. 4C), confirms their formation by all-trans-retinalreactivity.

In a series of experiments, we varied the ratio of all-trans-retinalto PE in synthetic mixtures to determine how these two precursorsmight affect A2PE formation (Fig. 5). We found that the yield ofA2PE increased when the ratio of equivalents of all-trans-retinal toPE (egg-PE as starting material) was increased from 1:2 to 2:2 andthen 4:2 equivalents (Fig. 5A). No further increase was observedat 8 equivalents of all-trans-retinal (8:2 ratio) indicating thatA2-adduct formation saturated at an equivalent ratio of 4:2 (all-trans-retinal to PE). On the other hand, increasing the concentration

Fig. 3. Ultra performance liquid chromatography/mass spectrometry analysis (electrospray ionmulti-mode ionization) of eyes obtained from Abca4�/�mice (2e3months of age, 8 eyes)with detection ofN-retinylidene-PE (NRPE), the Schiff base adduct of all-trans-retinal and phosphatidylethanolamine. UPLC operatedwith BEH phenyl� C18 reversed phase columnwitha mobile phase of acetonitrile/methanol (1:1) in water with 0.1% formic acid. Monitoring of 430 nm absorbance, fluorescence and molecular weight (m/z). NRPE is detected as multiplepeaks reflecting differences in length of fatty acid chains and in unsaturation. The derivedm/z values correspond to the lipid moieties indicated in parentheses. Insets above, UVevisibleabsorbance spectra of indicated eluting compounds. Structures, NRPE and plasmalogen-NRPE. NRPE has the usual ester bonds at the carbon 1 (sn-1) and carbon 2 (sn-2) positionsof glycerol. In plasmalogen-NRPE the first position of the glycerol backbone is bonded to a vinyl-ethermoiety (i.e. a carbonecarbon double bond on one side of a ether (CeOeC) linkage).


of PE from an equivalents ratio of 4:1 through 4:8 (all-trans-retinalto PE) resulted in a steady increase in A2PE synthesis product yield(Fig. 5B). These findings indicate that the concentration of PE hasa pronounced effect on the rate of A2PE synthesis. In particular,the first step in the synthetic pathway, the formation of NRPE, isexpected to be the most facile reaction in the pathway. Interest-ingly, increasing PE in the reaction mixture also promoted all-trans-retinal dimer formation (Fig. 5C), an observation supporting ourprevious suggestion that PE is an early participant in reactionsleading to all-trans-retinal dimer synthesis (Fishkin et al., 2005).These findings are significant since in the Abca4�/� mouse, a modelinwhich bisretinoid forms in abundance, the level of PE in the outersegment membrane is reported to be increased (Weng et al., 1999).Pepperberg and colleagues have suggested that the higher level ofPE in Abca4�/� mice may facilitate sequestering of all-trans-retinalas NRPE, thereby accelerating recovery of the rod photoresponseafter a 30e40% bleach (Pawar et al., 2008).

Since 22:6n-3 (DHA) is an abundant unsaturated fatty acid inphotoreceptor cell outer segments and is acquired through dietaryintake, we also compared the yield of A2PE when 22:6n-3-containing PE was precursor versus egg-PE (a mixture of fatty acidmoieties) (Fig. 5D). A2PE production was greater with egg-PE. On

the other hand, if free DHAwas added to the egg-PE/all-trans-retinalreaction mixture, the yield of A2PE was increased (Fig. 5E). DHAcontent has been shown to have several effects on retina (Tanitoet al., 2009) including susceptibility to light damage, but whethermodulation of bisretinoid formation is involved, it is not known.We also found that under deprotonating conditions associated withthe addition of base (triethylamine) to the reactants, the generationof A2PE was increased (Fig. 5F).

3.3. Formation of A2-DHP-PE

While elimination of two hydrogens from dihydropyridinium-A2PE leads to the formation of A2PE in the A2E syntheticpath, hydrogen transfer and one hydrogen elimination yieldsthe uncharged and stable dihydropyridine compound, A2-DHP-PE(A2-dihydropyridine-phosphatidylethanolamine) (Wu et al., 2009)(Fig. 1, compound 10). Conditions that favor A2-DHP-PE formationrather than A2PE, are not yet known. A2-DHP-PE is amongst thebisretinoids detected in human,mouse and bovine RPE and as is thecase for other bisretinoids of RPE lipofuscin, it accumulates withage. In the human eye, the ratio of A2E to A2-DHP-PE is approxi-mately 1:1 (Wu et al., 2009).

Fig. 4. Detection of the bisretinoid A2PE in isolated bovine photoreceptor outer segments (POS). Ultra performance liquid chromatography/mass spectrometry analysis (electro-spray ion multi-mode ionization) as in Fig. 2. A and B. In POS extract, a series of chromatographic peaks (A) exhibitm/z 1322.9 (B). UVevisible absorbances (Insets in A) are consistentwith A2PE; the multiple peaks with same m/z are indicative of A2PE isomers. C. Incubation of POS with all-trans-retinal accentuates peak height (peaks 1e3 in A are increased inC), indicating that peaks 1e3 reflect compounds forming by reaction of all-trans-retinal. D and E. The fatty acid species in the A2PE detected in POS (A) are identified as stearic acid(18:0) and docosahexaenoic acid (DHA, 22:6) since A2PE synthesized from 1-stearoyl-2-docosahexaenoyl-sn-glycero-3-phosphoethanolamine has the same retention time (D) andm/z (E) as the compound in A and B.


3.4. Formation of all-trans-retinal dimer and conjugates

A dimer of all-trans-retinal (all-trans-retinal dimer) is producedwhen two molecules of all-trans-retinal condense; this aldehyde-bearing dimer can then proceed to react with the amine of PE toforma conjugatewith PE (all-trans-retinal dimer-PE) via a Schiff baselinkage that is protonated (Fishkin et al., 2005; Kim et al., 2007b)(Fig. 1, compound 4 and 6). We have proposed a mechanism bywhich all-trans-retinal dimer may form after reaction of NRPE witha secondmolecule of all-trans-retinal (Fishkin et al., 2005). All-trans-retinal dimer-PE is particularly abundant in eyes harvested fromAbca4 null mutant (Abca4�/�) mice (Kim et al., 2007b) (Section 7.1).

3.5. Formation of A2-GPE

The most recent RPE bisretinoid to be identified, exists asa conjugate of two all-trans-retinal and glycerophosphoethanol-amine (GPE) (Fig. 1, compound 9) (Yamamoto et al., 2011). A2-GPE

was found to accumulate with age in human and mouse eyes andwas more abundant in Abca4�/� mice, a model of recessive Star-gardt disease (Section 7.1). We have suggested that A2-GPE mayform by direct bisretinoid adduction on GPE or it may be generatedsecondary to A2PE catabolism. Direct bisretinoid adduct formationon GPE would indicate that in addition to A2-adducts on PE,glycerophosphoethanolamine is available for reaction.

4. Absorbance and emission spectra

4.1. Structural features underlying bisretinoid absorbance andfluorescence emission

All of the isolated bisretinoids of RPE lipofuscin exhibit a central6-membered ring from which extends dual polyene arms termi-nating in b-ionine rings (Fig. 1). Each of the arms constitutesa separate system of double bond conjugations, and as such, eachserves as a chromophore, one arm absorbing in the ultraviolet and

Fig. 5. A2PE and all-trans-retinal dimer synthesis: modulating factors. Reaction mixtures of all-trans-retinal and phosphatidylethanolamine (PE). Presented as equivalent ratios.A. A2PE formation when concentration of all-trans-retinal is varied. B. A2PE formation when concentration of PE is varied. C. All-trans-retinal dimer synthesis with varying PEconcentration. D. A2PE yield using egg yolk-PE versus docosahexaenoic-PE (DHA-PE) as precursor. E. A2PE yield with varying levels of egg-PE and in the presence/absence of freeDHA fatty acid (8 equivalents). F. A2PE yield is facilitated in presence of triethylamine; all-trans-retinal:PE ratio was 4:2. Synthetic reaction mixtures were prepared in chloroform/methanol (3:1) at 37 �C. G. Detection of NRPE, all-trans-retinal dimer (atRAL-dimer) and A2PE by HPLC analysis (C4 column). Quantitation by integrating chromatographic peakareas. DHA-PE, 1,2-didocosahexaenoyl-sn-glycero-3-phosphoethanolamine. Major fatty acids in egg yolk-PE are 16:0, 18:0, 18:1, 18:2, 20:4 and 22:6.


the other in the visible region of the spectrum. Absorbances in thevisible spectrum are significant since these wavelengths reach theretina.

The wavelengths at which bisretinoids absorb are determined bythe length of the systems of alternating double and single bonds,including the double bonds in the b-ionone and central rings, ifcontinuous with the chain conjugations. For instance, A2E has anabsorbance maximum in the visible spectrum at w440 nm that canbe assigned to the long arm and a shorter wavelength absorbanceat w340 nm that is generated within the short arm. The additionalred-shift to 510 nm absorbance exhibited by all-trans-retinal dimer-PE and all-trans-retinal dimer-E, occurs due to protonation ofthe Schiff base nitrogen (Fishkin et al., 2005; Kim et al., 2007b).Accordingly, the bisretinoid pigments exhibit differences in theirabsorbancemaxima: A2E-A2E/isoA2E, lmaxw340, 440 nm; all-trans-retinal dimer, lmaxw290, 432 nm; all-trans-retinal dimer-E/all-trans-retinal dimer-PE, lmaxw290, 510 nm; and A2-DHP-PE, lmaxw333,490 nm. As noted above, some of the bisretinoids retain a phospho-lipid moiety, but this portion of the molecule does not makea contribution to absorbance at wavelengths greater than 250 nm.

4.2. Comparing the fluorescence emission spectra of humanlipofuscin and A2E

Given the varying absorbances presented above, it is notsurprising that fluorescence emission spectra can be recorded fromRPE lipofuscin over a range of excitation wavelengths. As shownin Fig. 6A, if the fluorescence emission spectrum obtained fromthe lipofuscin in the human RPE monolayer is compared with thespectrum obtained from cells containing A2E only, the spectra arerevealed to be similar in shapewith 488 nmexcitation. The emissionmaxima for both are approximately 590 nm, the same maximum

originally reported by Eldred for whole RPE lipofuscin (Eldredand Katz, 1991; Eldred et al., 1982). The spectral width (at half-maximal intensity) for A2E and whole lipofuscin are also similar(133 nm and 136 nm for A2E and human lipofuscin, respectively)in this recording. A difference is evident however with 561 nmexcitation in that a fluorescence spectrum can be generated fromwhole human lipofuscin but A2E exhibited little fluorescence at thelonger wavelength excitation (561 nm). It is worth noting that at561 nm excitation the peak emission recorded for human lipofuscinis red-shifted to 620 nm and the spectral width is narrower (96 nm).These features are indicative of a lipofuscin composed of amixture ofpigments with different, but overlapping, subsets of fluorophoresbeing excited as the excitation wavelength is changed.

4.3. Fluorescence emission spectra of lipofuscin in RPE ofAbca4 null mutant mice

In Abca4�/� mice, the accumulation of bisretinoid lipofuscin isaccentuated (Kim et al., 2004; Weng et al., 1999). The emissionspectrum of RPE lipofuscin measured using histological sectionsof Abca4�/� mice can also be obtained using a range of excitationwavelengths (Fig. 6B). The fluorescence emission intensity increaseswith age from 2 to 8 months in the mutant mice.

4.4. Changes in fluorescence emission with irradiation of bisretinoid

When excited at 430 nm, A2E and isoA2E have relatively broademission spectra with maxima at w600 nm (Sparrow et al., 1999)(Fig. 6C), an orange fluorescence. After irradiation (430 nm) for2 min however, A2E fluorescence intensity (with 440 nm excita-tion) decreases and exhibits a blue-shifted emission maximum(Fig. 6C). The spectral width, measured at half-maximal intensity,

Fig. 6. Fluorescence emission spectra of RPE lipofuscin and cell-based A2E. Data recorded using confocal laser scanning fluorescence microscope (Nikon A1R MP) with a 60� objectivein 6-nm increments while exciting with lasers at 488 and 561 nm. A. Emission spectra obtained from RPE monolayer (posterior) in cryostat sections of human eye (age 45 years)(field size 512� 128 pixels, 0.21 um per pixel) and from A2E that had accumulated in ARPE-19 cells (field size 512 � 512 pixels, 0.42 microns per pixel). Emission data adjusted for laserpower and pixel size. B. Emission spectra obtained from cryostat sections of eyes obtained from Abca4 null mutant mice at ages indicated in months (m). Field size was 512 � 64 pixels,0.09 microns per pixel. Spectra were adjusted for pixel size and laser power. C. Fluorescence emission of A2E (in DMSO/buffered saline) when not irradiated (red trace) and whenirradiated for the times indicated (blue traces). Emission peak wavelengths are indicated adjacent to each trace. Note the decrease in peak height and the peak shift to shorterwavelengths with increasing duration of irradiation. D. Emission spectra of RPE bisretinoid A2-GPE (diretinal glycerophosphoethanolamine). Note red-shift with increasing excitationwavelength. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)


also increases, the extension of the spectral profile occurringtoward shorter wavelengths. The latter change reflects an increas-ingly complex mixture due to photooxidation of A2E (Section 5).

5. Photodegradation of RPE bisretinoids

Efforts to understand the damaging effects of RPE lipofuscinhave lead to the study of lipofuscin photoreactivity. Early on, photo-dependent uptake of oxygenwas detected in both suspensions of RPEcells and in lipofuscin granules (Pawlak et al., 2002; Rozanowskaet al., 1995, 2002, 2004) and the rate of oxygen uptake was shownto increase with age (Rozanowska et al., 1995, 2002, 2004). Produc-tion of singlet oxygen and superoxide anion was also demonstrated(Gaillard et al., 1995; Reszka et al., 1995; Rozanowska et al., 1995,1998). Luminescence studies based on the detection of a character-istic phosphorescence at 1270 nm, and bymeasuring singlet-oxygen-mediated production of cholesterol hydroperoxides, demonstratedthat photoexcitation of A2E leads to singlet oxygen production(Ben-Shabat et al., 2002a; Pawlak et al., 2003). Measurements of thequantum yield of singlet oxygen production by A2E have providedwidely varying results (Kanofsky et al., 2003; Lamb et al., 2001), inlarge part because the conjugated double bond structure serves toquench the singlet oxygen as generated (Ben-Shabat et al., 2002a;Roberts et al., 2002). Singlet oxygen and superoxide anionproductionand singlet oxygen quenching are even more pronounced forunconjugated all-trans-retinal dimer than for A2E (Kim et al., 2007b)and the photooxidation of these bisretinoids (Fig. 7) leads to theformation of epoxides, furanoid moieties and endoperoxides (Janget al., 2005; Kim et al., 2007b).

Photo-exposure of A2E-containing RPE can lead to cell damageand death (Schutt et al., 2000; Sparrow et al., 2000). Given the shortlife-time of singlet oxygen and the realization that singlet oxygenis quenched by the polyene chains of the bisretinoids (Ben-Shabatet al., 2002a; Roberts et al., 2002), it is unlikely that cellulardamage occurs as a direct consequence of singlet oxygen generation.Instead, the deleterious effects are mediated by the photooxidation(Ben-Shabat et al., 2002a; Sparrow et al., 2002), and photo-degradation products of bisretinoid that result from singlet oxygenaddition at the carbonecarbon double bonds (Fig. 7). We demon-strated the reactivity of these photoproducts by assaying the activityof cellular GAPDH (glyceraldehyde 3-phosphate dehydrogenase),awell known redox sensitive thiol-containing enzyme that is subjectto oxidation related inhibition (Ferri et al., 1978; Lee et al., 2005).Wemeasured the cellular activity of GAPDH using an assay wherebyNADH generated from GAPDH activity converts the chromogenicsubstrate 2-(4-iodophenyl)-3-(4-nitrophenyl)-5-phenyl tetrazoliumto formazan. In ARPE-19 cells that had accumulated A2E and wereirradiated at 430 nm to induce photooxidation/photofragmentationof A2E, GADPH activity was decreased by approximately 50% (Fig. 8).

Utilizing liquid chromatography (LC) coupled to electrosprayionization mass spectrometry (ESI-MS) together with tandem massspectrometry (MS/MS), we have shown that A2E (Wu et al., 2010b)and all-trans-retinal dimer (Fig. 7) undergo photooxidation-induceddegradation at sites of singlet molecular oxygen addition therebyreleasing aldehyde-bearing cleavage products (Wu et al., 2010b)(Fig. 7). Importantly, the photodegradation products of A2E andall-trans-retinal dimer include methylglyoxal and glyoxal, small,reactive oxo-aldehydes with the capacity to form advanced glyca-tion end (AGE) products (Fig. 9). In chronic diseases such as diabetes

Fig. 7. Photooxidation and photodegradation of all-trans-retinal dimer. Analysis by electrospray ion multi-mode ionization (ESI). Samples of all-trans-retinal were unirradiated(A) or irradiated (B) at 430 nm (hv). The series of peaks from m/z 551 to 679 reflect photooxidation at carbonecarbon double bonds in all-trans-retinal dimer. Lower mass peaks(<m/z 592) correspond to all-trans-retinal dimer photodegradation products. C. Proposed structures of the largest of the photodegradation products. Upper left, all-trans-retinaldimer exhibits absorbance peaks at 432 nm and 292 nm that can be assigned to long and short arms of the molecule, respectively.

Fig. 8. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) activity is decreased incells that have accumulated A2E and are irradiated at 430 nm (1.38 mW/cm2). ARPE-19cells accumulate A2E into the lysosomal compartment. With this experimental model,the incidence of cell death is not elevated in A2E-containing cells versus untreatedcells.


and atherosclerosis, methylglyoxal and glyoxal form as intermedi-ates in the non-enzymatic glycation and oxidation reactions thatmodify proteins by AGEs. AGE-modified proteins are also detectedin deposits (drusen) that accumulate below RPE cells in vivo; drusenare a major risk factor for AMD progression (Farboud et al., 1999;Glenn et al., 2007; Handa et al., 1999; Ishibashi et al., 1998). Bisre-tinoid photocleavage upon exposure to wavelengths of light thatreach the retina, is a previously unknown source of methylglyoxaland glyoxal and likely serves as the origin of the dicarbonylsthat play a role in drusen formation. These findings also indicatea link between RPE lipofuscin photooxidation and drusen formation.The photolysis of bisretinoid molecules following photooxidationexplains the observation that photooxidized forms of A2E do notaccumulate with age (Grey et al., 2011).

It is important to note that the photooxidative processesdescribed above occur in vivo. Specifically, mono- and bis-peroxy-A2E, mono- and bis-furano-A2E, mono- and bis-peroxy-all-trans-retinal dimer and mono- and bis-furano-all-trans-retinal dimer aredetected in extracts from human and mouse eyes (Jang et al., 2005;Kim et al., 2007b).

6. Retinal bisretinoids and fundus autofluorescence

The retina, even under healthy conditions, exhibits a naturalautofluorescence (fundus autofluorescence) that can be imagednon-invasively for clinical examination and study. The spectralcharacteristics and age-dependent intensification of this auto-fluorescence are consistent with the source being the bisretinoidsof retina (Delori et al., 2007, 2011; Sparrow, 2007a; Sparrow et al.,2010c). Studies of the tissue distribution have localizedthe autofluorescence primarily to the RPE monolayer in thehealthy eye.

Although RPE bisretinoids exhibit a variety of excitationmaxima(Section 4.1), they all emit a fluorescence that is centered approx-imately around 600 nm. This emission is similar to the maximumemission of fundus autofluorescence (Delori et al., 2007). As is alsothe case for fundus autofluorescence (Delori et al., 2007) and RPElipofuscin (Sparrow et al., 2010b), individual retinal bisretinoids

exhibit a small but characteristic red-shift with increasing excita-tion wavelength (Fig. 5).

Abnormal patterns of autofluorescence are a feature of someretinal disorders. In terms of actual changes in bisretinoid levels,regions of reduced or absent fundus autofluorescence can reflectRPE cell atrophy or death (Holz et al., 2001; Robson et al., 2008b).Parafoveal rings of intense autofluorescence are often observedin patients with retinitis pigmentosa (Robson et al., 2008b)while outward-expanding autofluorescent rings can be visualized inABCA4-related retinal disease (Michaelides, 2009; Robson et al.,2008a, 2008b). Under these conditions, aberrant autofluorescencefrom excessive production of bisretinoid by impaired photoreceptorcells may serve as an additional source of fundus autofluorescence(Sparrow et al., 2010c). Alterations in fundus autofluorescence thatdo not reflect changes in bisretinoid level could be accounted for byRPE cell migration and clumping.

Efforts are currently ongoing to extend the resolution of fundus AFimaging through the incorporation of adaptive optics to scanninglaser ophthalmoscopy. Surprisingly, preclinical studies innon-human

Fig. 9. Photodegradation of the RPE bisretinoids A2E and all-trans-retinal dimer (atRAL-dimer) releases the dicarbonyls methylglyoxal and glyoxal. Potential cleavage sites atcarbonecarbon double bonds are presented (dotted lines) Depending on the photodegradation patterns, each molecule of A2E and all-trans-retinal dimer could release 2e3molecules of methylglyoxal or glyoxal.


primates have revealed that the use of light levels (�247 J/cm2)below light safety standards results in photobleaching of RPE lip-ofuscin along with notable RPE cell damage (Hunter et al., 2012;Morgan et al., 2009). Since the photobleaching observed reflectsbisretinoid photooxidation and degradation (Hunter et al., 2012),these observations call attention to the role of these photoreactivepigments in light damage and indicate that the RPE may be moresusceptible to light damage than previously recognized. Also unan-swered are questions concerning the involvement of RPE versusphotoreceptor cells in photochemical damage. Furthermore, since atconsiderably lower light levels (�5 J/cm2) photobleaching can alsooccur in the absence of RPE cell damage, it is clear that the processesof photooxidation and photodegradation can be ongoing withoutacute cell death.

Compounds other than those present in RPE lipofuscin havebeen suggested as the source of fundus autofluorescence. Forinstance, cellular flavoproteins (Field et al., 2008) in the oxidizedstate absorb at 460 nm and emit at 530 nm (Kindzewlskii and Petty,2004). Flavoproteins participate in mitochondrial electron trans-port and their fluorescence has been used to monitor the metabolicstatus of cells, the amount of flavoprotein autofluorescence beinginversely related to mitochondrial activity. However, it should bekept in mind that flavoproteins are not specific to RPE and thuswould emit from any or all cells of retina, a pattern that is nottypical of fundus autofluorescence.

There has also been considerable interest in whether drusen arethe source of fundus autofluorescence. Although autofluorescencelevels at sites of drusen, are not appreciably different from back-ground (Holz et al., 2001; Lois et al., 2002; vonRuckmann et al.,1997),in histological sections of human macula viewed under a fluores-cence microscope, some sub-RPE deposits have a blue-green fluo-rescence; the latter reflects a shift toward shorter wavelengthsrelative to RPE lipofuscin (Marmorstein et al., 2002). Detailed analysisof the topography of autofluorescence over drusen has shown thatfor both hard and soft drusen (60e175mm in size), there is a distinctannulus of hyperfluorescence over the outer edge of the druse andlower autofluorescence over the center. This pattern is thoughtto reflect drusen-associated displacement and thinning of RPE cells(Delori et al., 2000).

7. Learning from human disease and animal models

7.1. ABCA4-deficiency

>As discussed in Section 3.2, excessive bisretinoid lipofuscinaccumulation is a feature of diseases caused by ABCA4 genemutations in humans. Bisretinoid pigments likely also account for

the lipofuscin-like autofluorescence that can be visualized in thephotoreceptor cell membrane in some forms of ABCA4-linkeddisease (Birnbach et al., 1994; Bunt-Milam et al., 1983; Szamierand Berson, 1977). Mutations in the ABCA4 gene are responsiblefor recessive Stargardt macular degeneration, recessive cone-roddystrophy and recessive retinitis pigmentosa (Cremers et al.,1998). While the severity of the disease phenotype is suggestedto be inversely related to the level of residual protein activity(Shroyer et al., 1999), it is also noted that some mutations, partic-ularly those in the C-terminus, are associated with misfoldedprotein that is retained in the endoplasmic reticulum, so there isthe possibility that simple loss of function does not always accountfor disease severity (Cideciyan et al., 2009; Zhong et al., 2009). Theabundant RPE lipofuscin observed in ABCA4-related disease inhumans, is recapitulated in the Abca4 null mutant mouse (Kimet al., 2004; Weng et al., 1999). Studies in the Abca4�/� mice havealso revealed an association between excessive RPE lipofuscinaccumulation and photoreceptor cell death. In particular, analysisof outer nuclear layer thickness, as an indicator of photoreceptorcell survival has revealed that albino Abca4�/� mice displayprogressive photoreceptor cell loss that is clearly detectable at 8months of age and that has worsened by 12 and 13 months of age(Wu et al., 2010a). Photoreceptor cell degeneration was indepen-dently observed in 11-month-old albino Abca4�/� mice (Radu et al.,2008). Delivery of the human ABCA4 gene via lentiviral (Kong et al.,2008), adeno-associated virus (Allocca et al., 2008) vector to thesubretinal space of Abca4�/� mice, reduces disease-associated A2Eaccumulation.

7.2. Light exposure and vitamin A supplementation

Since the bisretinoid lipofuscin fluorophores form fromrandom inadvertent reactions between all-trans-retinal and amine-containing compounds, and since the production of all-trans-retinal is light-dependent, it is not surprising to find that exposureto light influences the formation of these vitamin A-relatedpigments. Specifically, it has been shown that bright light augmentsthe formation of A2PE in photoreceptor outer segments (Ben-Shabat et al., 2002b) while dark rearing of mice depresses thedeposition of A2E in the RPE (Mata et al., 2000). Mice, bothwild-type and Abca4 null mutant, that are given a vitamin A sup-plemented diet also exhibit increased levels of A2E as comparedto mice fed a control diet (Radu et al., 2008). The investigatorssuggested that this increasewas likely attributable to a faster rate ofvisual chromophore regeneration and a concomitant increase inthe rate of 11-cis-retinal photoisomerization to all-trans-retinal,yielding heightened bisretinoid formation.


7.3. Modulation of retinal bisretinoid formation by RPE65

In contrast to the abundant RPE lipofuscin formation associatedwith ABCA4 deficiency, knock-out of the RPE65 gene in mice leadsto an absence or pronounced decrease in RPE lipofuscin (Katz andRedmond, 2001). Similarly, in Lebers congenital amaurosis due toRPE65 mutations, there is an absence or pronounced decrease inRPE lipofuscin as detected by fundus autofluorescence imaging(Lorenz et al., 2004). Lebers congenital amaurosis is a severe form ofretinal degeneration with onset in children. The function of RPE65In the visual cycle, is to serve in the conversion of all-trans-retinylesters to 11-cis-retinol (Jin et al., 2005; Moiseyev et al., 2005;Redmond et al., 2005). The absence of RPE65 results in a failure toproduce the 11-cis-retinal chromophore, so all-trans-retinal that isthe essential precursor for bisretinoid formation, is not generatedand bisretinoid lipofuscin does not form. An amino acid change(Leu450Met) that reduces levels of Rpe65 in mouse RPE (Lyubarskyet al., 2005; Nusinowitz et al., 2003), also lowers A2E formation(Kim et al., 2004), as can small molecule inhibitors that targetRPE65 function (Golczak et al., 2005; Maiti et al., 2006).

7.4. Mutations in retinol dehydrogenase (RDH)

Other visual cycle genes also impact RPE lipofuscin formation. Forinstance, mutant mice deficient in enzymes that reduce all-trans-retinal to the alcohol form (e.g. retinol dehydrogenase-8, Rdh8;Rdh12) exhibit pronounced increases in A2E as compared to wild-type mice (Chrispell et al., 2009; Maeda et al., 2008). This abnor-mality occurs because the photoreceptor cell is deprived of avenuesfor inactivating all-trans-retinal. Mutations in human RDH12 geneare responsible for a subset of cases of Leber Congenital Amaurosis(Janecke et al., 2004).

7.5. ELOVL4-related macular dystrophy

Mutations in ELOVL4 (elongation of very long chain fatty acidsprotein 4), the gene responsible for early-onset dominant Stargardt-like macular degeneration, are also reported to result in increasedlevels of RPE lipofuscin. Frame-shift mutations in ELOVL4 resultin premature termination of the protein, aggregate-formation withthe wild-type protein and mis-localization of the protein (Graysonand Molday, 2005; Karan et al., 2005b; Vasireddy et al., 2005;Zhang et al., 2001). Transgenic mice expressing the mutant proteinexhibit modest elevations in RPE lipofuscin, measured as A2E(Karan et al., 2005a; Vasireddy et al., 2009, 2005). ELOVL4 isrequired for the synthesis of C28 and C30 saturated fatty acids, andthe synthesis of C28eC38 very long chain polyunsaturated fatty

Fig. 10. Senescence-accelerated mice. These mice carry the Leu450 variant in Rpe65. A. Qu(SAMR) and senescence-accelerated prone (SAMP8) mice at age 6 month. Measurements arsuperior hemispheres. Values are mean � SEM of 4 eyes. B,C. Light micrographs of SAMRD. HPLC measurements of A2E and isoA2E in eyecups of SAMR and SAMP8 mice. Values ar

acids, the latter being abundant in retina (Agbaga et al., 2008).Nevertheless, the link to enhanced bisretinoid formation is notunderstood.

7.6. Senescence-accelerated mice

Bisretinoids accumulate in RPE cells with age because the cell isnot able to metabolize this material. However, cellular aging, perse, is not the cause of RPE bisretinoid formation. For instance,lipofuscin can be detected in human RPE during the first decade oflife (Wing et al., 1978) and can also bemeasured in youngmice (Kimet al., 2007b; Yamamoto et al., 2011). Nevertheless, to evaluatewhether aging itself might aggravate bisretinoid formation weexamined mice that exhibit accelerated senescence (senescence-accelerated mouse-prone, SAMP) (Fig. 10). These mice exhibitearly retina changes such as Bruch’s membrane thickening and lossof photoreceptor cells (Majji et al., 2000; Nomura et al., 2004). InHPLC analysis of A2E/isoA2E levels in the mouse eyes, we observedan early increase in the bisretinoid that was demonstrable by 4months of age and then declined. By measuring outer nuclear layer(ONL) thickness in retinal sections, we observed an appreciabledecrease in the ONL at 6 months of age relative to control mice(senescence-accelerated mouse-resistant; SAMR).

7.7. Age-related macular degeneration

Dysregulation of complement activation is considered tounderlie the association between susceptibility to age-relatedmacular degeneration and certain genetic variants in complementfactors (Edwards et al., 2005; Hageman et al., 2005; Haines et al.,2005; Klein et al., 2005). In terms of initiators of complementactivation, we have shown using differentiated cultures of humanfetal RPE (Zhou et al., 2009) and ARPE-19 cells (Zhou et al., 2006)that photooxidation products of A2E and all-trans-retinal dimer canactivate complement while depletion of factor B reduces comple-ment activation as does an inhibitor of complement 3 (C3) (Zhouet al., 2006, 2009). The addition of C-reactive protein in theseassays also suppresses complement activation. In support of thesefindings, vigorous complement activation has been observed in theAbca4�/� mouse, a model defined by pronounced RPE bisretinoid(Radu et al., 2011). While AMD has onset in the elder years it likelydevelops for several years before diagnosis. Complement activationtriggered by photooxidation products of RPE bisretinoid lipofuscincould begin early in life and generate low-grade inflammatoryprocesses that gradually, along with other factors, predispose themacula to disease.

antification of outer nuclear layer (ONL) thickness in senescence-accelerated resistante plotted as a function of distance from the optic nerve head (ONH) in the inferior andand SAMP8 central retinas, 6 months of age. RPE is not included in the micrographs.e mean � SEM of 2e7 samples, 6 eyes per sample.


7.8. Conditions of oxidative stress

Ribozyme-mediated knock-down of MnSOD2 in the mouseis associated with an increase in A2E when measured by highperformance liquid chromatography (Justilien et al., 2007). Theantioxidant enzymeMnSOD2 is encoded by the nuclear gene SOD2,the protein is localized to mitochondria and the enzyme catalyzesthe conversion of superoxide anion (generated by aerobic respira-tion) to hydrogen peroxide. The mechanism by which A2E forma-tion is increased in the presence of a deficiency in SOD2 is notclear. The formation of A2E involves an automatic oxidation stepwhereby two hydrogens are eliminated from dihydropyrdinium-A2PE (Parish et al., 1998). We have shown that deoxygenationunder argon allows dihydropyridinium-A2PE to collect and A2Eformation is reduced (Kim et al., 2007a). However, diatomic groundstate oxygen would readily accept these hydrogens and one wouldnot expect the A2E synthetic pathway to fluctuate with changinglevels of reactive forms of oxygen. Thus one has also to considerother mechanisms. It is conceivable that in the setting of SOD2deficiency, compensatorymechanisms come into play. For instance,the reduced form of glutathione (GSH) can detoxify superoxideanion radical by reacting with it (Dickinson and Forman, 2002).Oxidized glutathione (GSSG) is subsequently converted back to GSHin a redox cycle involving glutathione reductase and the electronacceptor NADPH (nicotinamide adenine dinucleotide). Perhapsdepletion of NADPH in this way, could interfere with NADPH-dependent reduction of all-trans-retinal to retinol, the availabilityof the former allowing for increased A2E formation.

8. Conclusions and future directions

It is generally considered that in juvenile onset recessiveStargardt disease, the overproduction of bisretinoid and its accu-mulation as lipofuscin in RPE cells, are the causes of RPE atrophy andphotoreceptor cell degeneration. Corroborating evidence of thiscausality could come from the analysis of clinical outcomes achievedwith therapeutic strategies aimed at reducing bisretinoid formationby limiting the visual cycle. RPE cell density in the human eye is alsoreduced with age (Del Priore et al., 2002), although a relationshipbetween this loss and the amassing of bisretinoid has not beenshown. Healthy retinas of similar age vary substantially in terms oflevels of bisretinoid levels (Delori et al., 2001, 2011), but the factorsresponsible for these differences have not been elucidated. Alsounknown is the pathway from a mutation in ELOVL4 to elevatedbisretinoid accumulation (Section 7.5).

RPE lipofuscin consists of a mixture of bisretinoids, only some ofwhich have been characterized. Missing from our understanding areconditions that favor the formation of one bisretinoid over another.Efforts to clarify the composition of RPE lipofuscin are importantsince these compounds are targets of gene-based and drug therapiesthat aim to alleviate ABCA4-related retinal disease. Importantly,the various bisretinoid pigments have different properties (Sparrowet al., 2010a). For instance, while whole RPE lipofuscin exhibitsfluorescencewhen excitedwith 561 nm light (Fig. 6A), A2E does not.Consequently, at the wavelength (568 nm) used to record fundusautofluorescence by adaptive optics/scanning laser ophthalmoscopy(Section 6), there is unlikely to be appreciable A2E excitation. Thusthe RPE damaged elicited by 568 nm exposure must involvedphotoreactive processes involving bisretinoids other than A2E.

We have provided evidence that bisretinoid photodegradationcan serve as a source of the dicarbonyl molecules responsiblefor AGE-modification of protein, such as is observed in drusen(Wu et al., 2010b). It is clear that bisretinoid photooxidation isongoing in in vivo retina since the photooxidized forms of A2E andall-trans-retinal dimer are detected in human and mouse RPE

(Jang et al., 2005; Kim et al., 2007b). As might be expected,however, these photooxidized species do not accumulate withage (Grey et al., 2011), since the oxidation lead to bisretinoid frag-mentation. The processes of photooxidation and photodegradationmechanisms such as these may point to links amongst factorsposited as being associated with AMD including oxidativeprocesses, complement dysregulation, light exposure and drusenformation.

Given thewide-spreaduse of fundus autofluorescence imaging inthe diagnosis of retinal disease, continued efforts to better under-stand RPE lipofuscin will also inform interpretations of theseimages (Sparrow et al., 2010b, 2010c). Progress has beenmade in theimplementation of methods to quantify fundus autofluorescence soas to correlate levels of fundus autofluorescence with diseaseseverity (Delori et al., 2011). The assumption is that AF intensity isameasure of the amount of lipofuscin in the RPEmonolayer and thuscan be used to gauge the health of the cells. Theremay be exceptionsto this interpretation however, since in the case of some patterns ofAF, high intensity fluorescence may be attributable to anomalousbut greatly elevated lipofuscin formation in impaired photoreceptorcells (Sparrow et al., 2010c). Moreover, given the evidence for pho-todegradation of bisretinoid, measurements of AF are unlikely to beindicative of amounts of bisretinoid accumulated over a life-time ofaccumulation and therefore are not indicative of the total lipofuscinburden. Indeed the amount lost and therefore not measurable maybe more informative of RPE cell status.

Acknowledgments

This work was supported by National Institutes of Healthgrants EY12951 (JRS) and P30EY019007 and a grant from Researchto Prevent Blindness to the Department of Ophthalmology. The CarlMarshall Reeves and Mildred Almen Reeves Foundation providedfunds for equipment. EGRwas supported by an Endeavour AustraliaResearch Fellowship and Lucy Falkiner Fellowship. Drs. Yalin Wu,Emiko Yanase, Chul Young Kim and Kee Dong Yoon are acknowl-edged for contributions.

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the bisretinoids of retinal pigment epithelium

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