Structures and biogenetic analysis of lipofuscin bis-retinoids

Structures and biogenetic analysis of lipofuscin bis-retinoids 1 2 first-author †E-mail:yalinw@zju.edu.cn †E-mail:yalinw@zju.edu.cn yalinw@zju.edu.cn 1 2 College of Pharmaceutical Sciences, Zhejiang University, Hangzhou 310058, China College of Pharmaceutical Sciences, Zhejiang University, Hangzhou 310058, China Eye Center, the Second Affiliated Hospital, School of Medicine, Zhejiang University, Hangzhou 310009, China Eye Center, the Second Affiliated Hospital, School of Medicine, Zhejiang University, Hangzhou 310009, China Age-related macular degeneration (AMD) is still an incurable blinding eye disease because of complex pathogenic mechanisms and unusual diseased regions. With the use of chemical biology tools, great progress has been achieved in improving the understanding of AMD pathogenesis. The severity of AMD is, at least in part, linked to the non-degradable lipofuscin bis-retinoids in retinal pigment epithelial (RPE). This material is thought to result from the lifelong accumulation of lysosomal residual bodies containing the end products derived from the daily phagocytosis of rod outer segments by RPE cells. Here, we present previously recognized bis-retinoids with focus on structures and biosynthetic pathways. In addition to a brief discussion on the mutual conversion relationships of bis-retinoids, future perspectives and the medical relevance of such studies on these lipofuscin constituents are also highlighted. Age-related macular degeneration (AMD) Lipofuscin Retinal pigment epithelial (RPE) Bis-retinoids Structures Biogenesis Conversion relationships Introduction 1 Time-dependent lipofuscin accumulation in retinal pigment epithelial (RPE) cells has been notoriously deemed as a major risk factor in association with age-related macular degeneration (AMD) (Bressler et al., 1988 1988 ; Eldred and Katz, 1988 1988 ; Eldred and Lasky, 1993 1993 ; Kliffen et al., 1997 1997 ; Sarks et al., 1999 1999 ; Delori et al., 2000 2000 ). AMD, the leading cause of blindness in elderly people, is a degenerative disease of the eye (Bressler et al., 1988 1988 ; Mata et al., 2000 2000 ; Telander, 2011 2011 ), for which there is thus far no remedy. Increasing accumulation of lipofuscin granules in RPE is not a feature of aging RPE alone given that excessive amounts of these fluorophores are also remarkably detected in the RPE of juvenile patients with Stargardt’s disease (Eagle et al., 1980 1980 ; Feeney-Burns et al., 1984 1984 ; Rabb et al., 1986 1986 ; Birnbach et al., 1994 1994 ; Kennedy et al., 1995 1995 ) caused by mutations in Abca4 gene (Delori et al., 1995 1995 ; Shroyer et al., 1999 1999 ). RPE lipofuscin is an enzymatically non-degradable heterogeneous mixture of numerous biomolecules (Ng et al., 2008 2008 ), in which extensive attention has been drawn to the indigestible bis-retinoid constituents that contribute to the etiology of inherited and AMD (Broniec et al., 2005 2005 ; Sparrow et al., 2010a 2010a ). Vitamin A cycling leading to vision occurs in the retina, and is also regarded as the origin of adverse lipofuscin bis-retinoids (Fig. 1 1 ) (Borhan et al., 2000 2000 ; Sparrow et al., 2010d 2010d ). After capturing a photon, the inherent visual chromophore 11- cis -retinal bound to lysine 296 of opsin, with a protonated covalent Schiff base bond, isomerizes to all- trans -retinal (Rando, 1996 1996 ; Borhan et al., 2000 2000 ). A sequence of conformational rearrangements causes the dark-adapted rhodopsin to undergo metarhodopsin II (Meta II) transition (Sakmar, 1997 1997 ; Gelasco et al., 2000 2000 ; Burns and Baylor, 2001 2001 ; Okada et al., 2001 2001 ; Isin et al., 2006 2006 ). Given that Meta II comprises all- trans -retinal and opsin that bind together by a deprotonated Schiff base linkage, the absorbance of this light-activated intermediate is at 380 nm, reflecting around 120-nm blue shift relative to the maximal absorbance ( λ max ) value of native rhodopsin (500 nm) (Borhan et al., 2000 2000 ; Liu et al., 2011 2011 ). A hydrolysis of Meta II results in the release of a free all- trans -retinal, followed by a reduction in the latter to all- trans -retinol under the action of all- trans -retinol dehydrogenases (atRDHs). As an alternative, some all- trans -retinals react with either phosphatidylethanolamine (PE) or glycerophosphoethanolamine (GPE) distributed in the lipid bilayer to generate two covalent adducts, N -retinylidene-phosphatidylethanolamine (NR-PE) and N -retinylidene-glycerophosphoethanolamine (NR-GPE). After that, ABCA4 (ATP-binding cassette, sub-family A (ABC1), member 4) facilitates transfer of NR-PE/NR-GPE trapped inside the disk as a charged species out to the cytoplasmic surface (Fig. 2 2 ), where they hydrolyze to leave PE/GPE and all- trans- retinal (Molday et al., 2006 2006 ). Subsequent reduction of the latter by atRDHs generates all- trans -retinol. Within the RPE cells, following the esterification of all- trans -retinol by the enzyme lecithin retinol acyl transferase (LRAT), RPE65, the visual cycle retinol isomerase, isomerizes the resulting ester from the all- trans configuration to the 11- cis -retinol. By 11- cis- retinol dehydrogenase (11cRDH), the gained alcohol is finally oxidized to 11- cis -retinal; this chromophore will bind opsin to regenerate the fresh rhodopsin (Jäger et al., 1996 1996 ; Kim et al., 2004 2004 ; Sparrow et al., 2010d 2010d ). On the other hand, since some NR-PEs/NR-GPEs evade the degradation to create all- trans -retinal and PE, the pathway for formation of bis-retinoids starts with the condensation reaction of NR-PE/NR-GPE and a second molecule of all- trans -retinal. As depicted in Fig. 1 1 , a multi-step pathway leads to the formation of dihydropyridinium N -retinylidene- N -retinyl-phosphatidylethanolamine (A2PE) and dihydropyridinium N -retinylidene- N -retinyl-glycerophosphoethanolamine (A2-GPE). Automatic oxidation of these two intermediates generates A2PE and A2-GPE, the immediate precursors of N -retinylidene- N -retinyl-ethanolamine (A2E). Loss of one hydrogen (–H) from dihydropyridinium A2PE generates N -retinylidene- N -retinyl-dihydropyridine-phosphatidylethanolamine (A2-DHP-PE); phosphate hydrolysis of the latter produces N -retinylidene- N -retinyl-dihydropyridine-ethanolamine (A2-DHP-E). Via an alternative path, all- trans -retinal dimer (ATR dimer) forms from the condensation of two molecules of all- trans -retinal. Reaction of ATR dimer with PE by formation of a protonated Schiff base linkage generates ATR-dimer-PE; phosphate hydrolysis of the latter yields ATR dimer-E. The toxic bis-retinoids form in rod outer segments that consist of nearly 1 500 thin disks (Fig. 2 2 ). Daily phagocytosis of spent photoreceptor outer segment debris by RPE cells is extremely critical for vision and, as a consequence, leads to the transfer of bis-retinoid pigments to the lysosomal bodies of RPE cells (Feeney-Burns, 1980 1980 ; Feeney-Burns et al., 1984 1984 ; Clancy et al., 2000 2000 ; Sparrow and Boulton, 2005 2005 ; Wu et al., 2011 2011 ). According to the proposed scheme that was published previously (Sparrow et al., 2010d 2010d ), we here expand the schematic diagram regarding the formation of lipofuscin bis-retinoids; the leading feature is the formation of a novel bis-retinoid compound N -retinylidene- N -retinyl-glycerophosphoethanolamine (A2-GPE) (Yamamoto et al., 2011 2011 ). Biogenesis of the latter starts with one molecule of all- trans -retinal incubated with GPE, followed by a [1,6]H-shift to give rise to N -retinylidene-glycerophosphoethanolamine (NR-GPE). Via a multi-step cascade, a proposed intermediate, dihydropyridinium A2-GPE is generated, and undergoes auto-oxidation to yield A2-GPE. Phosphate hydrolysis of the latter by phospholipase D (PLD) produces N -retinylidene- N -retinyl-ethanolamine (A2E), indicating that A2-GPE, like N -retinylidene- N -retinyl-phosphatidylethanolamine (A2PE), may also serve as the precursor of A2E All- trans -retinal (ATR) comes from the isomerization of 11- cis -retinal that is released from light-activated rhodopsin in the retina. Incubation of ATR with phosphatidylethanolamine (PE)/glycerophosphoethanolamine (GPE) would give rise to N -retinylidene-phosphatidylethanolamine (NR-PE)/ N -retinylidene-glycerophosphoethanolamine (NR-GPE) that can be delivered to the cytoplasmic side of disk membranes by ABCA4. When the activity of this transporter attenuates or becomes extinct, NR-PE and NR-GPE have no choice but to reside in the rod outer segments, and react with a second molecule of ATR to form various bis-retinoids including N -retinylidene- N -retinyl-phosphatidylethanolamine (A2PE), N -retinylidene- N -retinyl-dihydropyridine-phosphatidylethanolamine (A2-DHP-PE), N -retinylidene- N -retinyl-glycerophosphoethanolamine (A2-GPE), ATR dimer, and ATR dimer-PE via a multi-step cascade. Rod outer segments are routinely discarded on a daily basis, and subsequently are phagocytosed by RPE cells, resulting in the transfer of these di-retinal pigments to RPE where PLD, a lysosomal acid enzyme, can cleave A2PE, A2-GPE, A2-DHP-PE, and ATR dimer-PE to produce N -retinylidene- N -retinyl-ethanolamine (A2E), N -retinylidene- N -retinyl-dihydropyridine-ethanolamine (A2-DHP-E), and ATR dimer-E, respectively. However, ATR dimer remains intact due to the inability to be further digested by the lysosomal enzymes Up to the present time, at least eleven bis-retinoid pigments (Fig. 3 3 ) present in human RPE and mouse eyecups have been characterized structurally and chromatographically. Given significant implications of lipofuscin bis-retinoids in AMD, this review will mainly focus on the advances in both structure and biogenetic analysis of these inadvertent fluorophores. Structures, UV-visible absorbance (nm), and electronic transition assignments (↔) are shown. All of these fluorescent di-retinal pigments have dual conjugation systems located in two side-arms, conferring light-induced absorbance in both the ultraviolet and visible regions Bis-retinoids with a pyridinium ring 1 To date, the identified pyridinium bis-retinoids with a positive charge on the nitrogen atom residing in the six-membered heterocyclic ring include A2E, isoA2E (13′- cis ), A2PE, oxidized derivatives of A2E and A2-GPE. A2E, an unprecedented pyridinium bis-retinoid was the most studied ocular age pigment, and was structurally characterized by Sakai et al. ( 1996 1996 ). Ren et al. ( 1997 1997 ) completed its first total synthesis. One year later, Parish et al. ( 1998 1998 ) optimized in vitro A2E synthesis, which led to the one-step biomimetic preparation of this pigment in 49% yield. Given that A2E has two hydrophobic side-arms derived from all- trans -retinal, together with a hydrophilic pyridinium head group, this molecule is conferred with an unusual amphiphilic property (Sparrow et al., 2003a 2003a ). Evidence from several fronts (Poincelot et al., 1969 1969 ; Anderson and Maude, 1970 1970 ; Mata et al., 2000 2000 ; Sparrow et al., 2010a 2010a ) denotes that the biosynthetic route of A2E (Fig. 4 4 ) begins in the photoreceptor outer segment membrane as a condensation reaction between PE and a single molecule of all- trans -retinal that generates a Schiff base conjugate NR-PE. NR-PE goes through a [1,6]-proton tautomerization to yield an N -retinylidene-phosphatidylenamine (Fig. 4 4 ). It is postulated that subsequent condensation of the latter with a second molecule of all- trans -retinal and 6π- aza -electrocyclization will produce an imperative phosphatidyl-dihydropyridinium bis-retinoid intermediate (dihydropyridinium A2PE) (Fig. 4 4 ) (Kim et al., 2007 2007 ; Sparrow et al., 2012 2012 ). Automatic oxidation of dihydropyridinium A2PE with loss of two hydrogens (−2H) yields A2PE, the immediate precursor of A2E and isoA2E (13′- cis ). It is interesting to observe that A2E and isoA2E (13′- cis ) would reach photo-equilibrium at a ratio of 4:1 when exposed to room light (Parish et al., 1998 1998 ). The (430±20) nm blue light irradiation of A2E-laden ARPE-19 cells in a dark room generated two detectable types of oxidized A2E: monofuran-A2E and monoperoxy-A2E, and they also were found in aged human RPE and in eyecups of Abca4 −/− mice, an animal model of Stargardt’s disease (Jang et al., 2005 2005 ). These two bis-retinoids form from a proposed uniform intermediate, dihydropyridinium N -retinylidene- N -retinyl-phosphatidylethanolamine (A2PE). The latter is enclosed by a rectangle and is not detectable in the eye and biomimetic chemical reactions, probably resulting from the facile auto-oxidation of a unique dihydropyridinium ring, from which one or double particular hydrogen is eliminated with the electronic rearrangements. Hydrogens in blue and green represent the active atoms involved in the shifting and eliminating processes in the biosynthesis of these two heterocyclic fluorophores (Note: for interpretation of the references to color in this figure legend, the reader is referred to the web version of this article) Most recently, Yamamoto et al. ( 2011 2011 ) mentioned that Sparrow and his colleagues illustrated a fresh bis-retinoid of retina, A2-GPE, to be more abundant than A2E in the eyecups of Abca4 −/− mice and to form from incubation of two molecules of all- trans -retinal and GPE. Due to the similarity in structure between PE and GPE, the biosynthesis of A2-GPE (Fig. 5 5 ), slightly different from that of A2E, starts with a condensation reaction between GPE and all- trans -retinal, which creates a Schiff base conjugate NR-GPE. Subsequently, a sequence of chemical processes including [1,6]-proton tautomerization, condensation reaction with a second molecule of all- trans -retinal and 6π- aza -electrocyclization, yields dihydropyridinium A2-GPE, the proposed intermediate in the biosynthetic pathway. With loss of two hydrogens (−2H) arising from the automatic oxidation, dihydropyridinium A2-GPE is converted into A2-GPE containing a pyridinium ring. An in vitro enzyme assay manifested that phospholipase D (PLD) activity can cleave A2-GPE to release A2E, thereby reflecting that A2-GPE, like A2PE, may also serve as a precursor of A2E (Sparrow et al., 2008 2008 ). Like NR-PE acting as a key intermediate to form A2PE, NR-GPE also plays an important role in the biosynthetic pathway of A2-GPE. To corroborate A2-GPE biogenesis, it will be highly urgent to detect NR-GPE in mouse eyecups and human RPE. It is also worth noting that dihydropyridinium A2PE enclosed with a rounded rectangle (Fig. 4 4 ) and dihydropyridinium A2-GPE (Fig. 5 5 ) were not detectable in eyes and biomimetic chemical reactions on account of the transient auto-oxidation behavior. N -retinylidene-glycerophosphoethanolamine (NR-GPE) and dihydropyridinium A2-GPE, like N -retinylidene-phosphatidylethanolamine (NR-PE) and dihydropyridinium N -retinylidene- N -retinyl-phosphatidylethanolamine (A2PE) in the N -retinylidene- N -retinyl-ethanolamine (A2E) biosynthetic route, ought to play important roles in the biogenesis of A2-GPE. Due to the susceptibility of the dihydropyridinium ring to auto-oxidation, it is concluded that dihydropyridinium A2-GPE, like dihydropyridinium A2PE, is not found in the eye and biomimetic chemical reactions, from which NR-GPE is, however, possible to be trapped by high-performance liquid chromatography (HPLC) Bis-retinoids with a dihydropyridine ring 1 In addition to pyridinium bis-retinoids, Wu et al. ( 2009 2009 ) identified two novel but correlated di-retinal pigments (A2-DHP-PE and A2-DHP-E) that were distinguished by containing a dihydropyridine ring rather than a pyridinium ring. As shown in Fig. 4 4 , A2-DHP-PE biogenesis is closely associated with that of A2E. Following the generation of an estimated intermediate (dihydropyridinium A2PE), there is a bifurcation in the pathway. As mentioned above, one way gives rise to A2E and its isomers (Liu et al., 2000 2000 ; Mata et al., 2000 2000 ; Sparrow et al., 2010a 2010a ). Conversely, another path to form A2-DHP-PE is involved with [1,3]-proton tautomerization and elimination of a hydrogen (Wu et al., 2009 2009 ; Sparrow et al., 2010a 2010a ). PLD-mediated hydrolysis of A2-DHP-PE yields A2-DHP-E, revealing that A2-DHP-PE may act as the precursor of A2-DHP-E (Wu et al., 2009 2009 ). Bis-retinoids with a cyclohexadiene ring 1 In comparison with the nitrogen-containing heterocyclic bis-retinoids, all- trans -retinal dimer series, including ATR dimer, ATR dimer-PE, and ATR dimer-E (Fishkin et al., 2005 2005 ), are absolutely distinctive, given that their chemical structures contain a simpler cyclohexadiene ring in which no nitrogen atom resides. Cyclohexa-1,3-diene-carbaldehydes, incredibly similar to ATR dimer, could be released from a base-catalyzed condensation reaction between α,β-unsaturated aldehydes and a β-methyl substituent. Thus, the proposed pathway for ATR dimer biogenesis (Fig. 6 6 ) is rooted in the reported mechanism relevant to this type of transformation (Thomas and Guntzdubini, 1976 1976 ; Duhamel et al., 1991 1991 ). Like A2PE and A2-DHP-PE, ATR dimer is supposed to form in the photoreceptor cell outer segments (Fig. 2 2 ). All bis-retinoids appear to form from the same tautomer ( N -retinylidene-phosphatidylenamine), probably arising from a [1,6] H-shift of NR-PE/NR-GPE (Liu et al., 2000 2000 ; Mata et al., 2000 2000 ; Wu et al., 2009 2009 ; Sparrow et al., 2010a 2010a ). Via a Michael-type addition reaction (Michael, 1887 1887 ; Mather et al., 2006 2006 ), the C 20 of NR-PE/NR-GPE would tie with C 13 of a second molecule of all- trans -retinal. After a Mannich reaction to close the ring (Mannich and Krösche, 1912 1912 ), the elimination of amine group in PE/GPE yields ATR dimer, along with an electronic rearrangement in the six-membered ring (Fishkin et al., 2005 2005 ). Apart from the nucleophilicity of C 20 residing in N -retinylidene-phosphatidylenamine (Fig. 6 6 ), this carbon also possesses the ability to participate in the [1,4] conjugate addition. These analyses connote that ATR dimer ought to form in the rod outer segments rather than in RPE. Incubation of ATR dimer with PE produces ATR dimer-PE with creation of a protonated Schiff base linkage (Fishkin et al., 2005 2005 ). ATR dimer-E is released from the phosphate hydrolysis of ATR dimer-PE by PLD (Fishkin et al., 2005 2005 ). In addition to A2-DHP-PE, examination of the 7-d reaction mixture between all- trans -retinal and 1,2-diacyl- sn -glycero-3-phosphatidylethanolamine from egg yolk (egg-PE) by high-performance liquid chromatography (HPLC) also exhibited the formations of ATR dimer and ATR dimer-PE (Wu et al., 2009 2009 ). Evidence gleaned from HPLC chromatograms of mouse eyecup extracts showed that ATR dimer-PE is more readily detectable than ATR dimer in the Abca4 −/− mice. These findings, together with evidence that an incubation of ATR dimer with PE produces ATR dimer-PE, corroborate that ATR dimer is obliged to form firstly and functions as a precursor of ATR dimer-PE (Figs. 1 1 and 6 6 ). It is thought that ATR dimer series, N -retinylidene- N -retinyl-ethanolamine (A2E) and N -retinylidene- N -retinyl-dihydropyridine-phosphatidylethanolamine (A2-DHP-PE), form from the same intermediate N -retinylidene-phosphatidylenamine (see structure in red). Also, it is interesting to know that ATR dimer-E ( λ max 290, 510 nm) and saturated ATR dimer-PE ( λ max 290, 510 nm) exhibited a visible absorption maximum of about 80 nm red shifted from that of ATR dimer ( λ max 290, 430 nm) because of a protonated Schiff base linkage formed in the position of aldehyde moiety (Note: for interpretation of the references to color in this figure legend, the reader is referred to the web version of this article) Structural features and properties 1 A common feature in structures of these bis-retinoids is two side-arms consisting of alternating single and double bonds that begin from an aromatic head group and extend into terminal β-ionone rings. The systems of conjugated double bonds in these bischromophores confer their own absorbance maxima in the visible region. It is noted that all of the bis-retinoids listed in Fig. 3 3 possess fluorescent properties and are the source of fundus autofluorescence (Sparrow et al., 2010b 2010b ; 2010c 2010c ). Moreover, it seems that these bis-retinoids form by the random condensation reactions of two molecules of all- trans- retinal, and are highly susceptible to photooxidation (Ben-Shabat et al., 2002 2002 ; Sparrow et al., 2002 2002 ; Dillon et al., 2004 2004 ; Fishkin et al., 2005 2005 ; Jang et al., 2005 2005 ; Wang et al., 2006 2006 ; Kim et al., 2008 2008 ; Wu et al., 2010 2010 ). RPE cells, different from other types of cells, are constantly exposed to sunlight strongest at 400‒700 nm. Given that the human eye is taking full advantage of the solar energy distribution, we envision that light-mediated actions on the malignant bis-retinoids accumulated in RPE with age, will of necessity influence RPE cells to some extent. Indeed, events initiated by the blue-light illumination of some bis-retinoids allow for the release of epoxides and cleavage products that have been verified to damage RPE cells (Sparrow et al., 2000 2000 ; 2002 2002 ; 2003b 2003b ; Sparrow and Cai, 2001 2001 ). Relations between ABCA4 transporter and bis-retinoid formation 1 ABCA4 (formerly known as ATP-binding cassette transporter, retina-specific (ABCR)) is a member of the ABCA subfamily of adenosine triphosphate (ATP) binding cassette (ABC) transporters expressed in vertebrate rod and cone cells, and is localized to the outer segment disk edges. Allikmets et al. ( 1997 1997 ) first cloned and characterized Abca4 gene. Mutations in this gene have been verified to account for all cases of early- and late-onset autosomal recessive Stargardt macular degeneration and the major part of autosomal recessive cone-rod dystrophies (Gerber et al., 1998 1998 ; Maugeri et al., 2000 2000 ; Briggs et al., 2001 2001 ; Ducroq et al., 2002 2002 ) as well as some exceptional cases of retinitis pigmentosa (Martinez-Mir et al., 1998 1998 ; Rozet et al., 1999 1999 ; Fukui et al., 2002 2002 ). These hereditarily degenerative diseases would cause severe vision loss in affected individuals with age and reflect a continuum of disease phenotype associated with severity of gene mutations. As a critical intermediate to form bis-retinoids, NR-PE is the putative ligand translocated from the lumen to the cytoplasmic side of the disk membrane by the ABCA4 acting as a flippase (Fig. 2 2 ) (Sun et al., 1999 1999 ; Weng et al., 1999 1999 ). According to a recent finding of A2-GPE by the Sparrow group (Yamamoto et al., 2011 2011 ), we here propose its biosynthetic pathway (Fig. 5 5 ), in which a newly estimated intermediate named NR-GPE is considered to form. Due to the abundance of A2-GPE in the eyecups of ABCA4 null mutant mice, ABCA4 is also considered to be the carrier of NR-GPE. Following the hydrolysis of NR-PE and NR-GPE, all- trans -retinal is released and reduced to all- trans -retinol. The retinol binding proteins carry vitamin A to RPE where it is isomerized and re-oxidized to 11- cis -retinal. This chromophore is subsequently reincorporated into opsin, yielding ground state rhodopsin (Fig. 2 2 ). When ABCA4 activity is reduced or absent, NR-PE and NR-GPE abundantly accumulate in the disk membrane, and trigger further reaction with an additional molecule of all- trans -retinal to generate A2PE, A2-DHP-PE, A2-GPE, ATR dimer, and ATR dimer-PE (Fig. 2 2 ). Daily phagocytosis of the shed outer segment membrane by RPE cells would take these bis-retinoid pigments to RPE lysosomal bodies where the PLD, an acidic hydrolase in the lysosomes of RPE cells, serves as a blade to cleave to A2PE, A2-GPE, A2-DHP-PE and ATR dimer-PE to give A2E, A2-DHP-E, and ATR dimer-E, respectively (Fig. 2 2 ). Medical relevance and future perspective 1 With regard to treatment for AMD, some attempts at mediating the retinoid cycling rate have been made by utilizing the exogenous small-molecule inhibitors of 11cRDH (Radu et al., 2003 2003 ), RPE65 (Maiti et al., 2006 2006 ; Golczak et al., 2008 2008 ), retinol binding protein and transthyretin (Radu et al., 2005 2005 ). Indeed, these compounds slow lipofuscin accumulation in RPE cells of the eye. Thus, it will be a promising strategy to screen a large body of low-molecular-weight antagonists limiting the visual cycle with an eventual aim of developing clinical medicines in favor of alleviating AMD. In contrast, no remedies can reverse malicious accumulation of lipofuscin bis-retinoids refractory to lysosomal enzyme degradation, once it has already occurred. In view of this fact, it would be beneficial if there are some exogenous enzymes that could be delivered to RPE cells and directly degrade these aging pigments. A promising finding by Wu et al. ( 2011 2011 ) attested to the ability of horseradish peroxidase (HRP) to degrade A2E loaded in RPE cells. To contribute towards a better understanding of biosynthetic pathways of toxic bis-retinoids, a mutual conversion relationship among the bis-retinoids is expected to be clarified. As illustrated in Fig. 7 7 , a future objective will be to establish whether either A2-DHP-PE or A2-DHP-E can be converted into A2E and to investigate whether A2-DHP-PE can undergo further transformation to form A2PE under certain conditions. On the other hand, continuing research and advances in this field should be directed towards characterizing those unrecognized compositions of RPE lipofuscin, understanding the properties of each lipofuscin constituent, and clarifying in vivo mechanisms by which they form and the adverse effects of these pigments on retina. 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This article does not contain any studies with human or animal subjects performed by any of the authors. 10.1631/jzus.B1300051 Project supported by the National Natural Science Foundation of China (Nos. 21202146 and 81271018), the Fundamental Research Funds for the Central Universities, China, and the Zhejiang Key Laboratory Fund of China (No. 2011E10006) 2013-02-19 2013-08-13 2013-07-01 2022-01-05 Copyright © Zhejiang University and Springer-Verlag Berlin Heidelberg 2013 2013 Journal of Zhejiang University-SCIENCE B (Biomedicine and Biotechnology) 09 14