Ординатура / Офтальмология / Английские материалы / Eye, Retina, and Visual System of the Mouse_Chalupa, Williams_2008

Within the visual cycle, 11-cis-retinal, the chromophore of visual pigment in photoreceptor cells, absorbs a photon of light, is isomerized to all-trans-retinal, and is then regenerated through a series of biochemical reactions within the retinal pigment epithelium (RPE). Not all of the highly reactive all-trans-retinal generated by photoisomerization of 11-cis-retinal within photoreceptor cells remains within the visual cycle, however; a minor portion inadvertently reacts to form retinoid-derived fluorophores that reside in photoreceptor outer segments until the latter are phagocytosed by RPE cells, whereupon the compounds accumulate as the lipofuscin of RPE (Sparrow and Boulton, 2005). Accordingly, all of the RPE lipofuscin fluorophores that have been isolated to date, including A2E, the A2E photoisomer isoA2E, minor cis-isomers of A2E, and the all-trans-retinal dimerconjugates, all-trans-retinal dimer-phosphatidylethanolamine (atRAL-dimer-PE), and all-trans-retinal dimer-ethanolamine (atRAL-dimer-E) (figure 44.1), are generated by reactions of all-trans-retinal. Because of their unusual structures, including that of a pyridinium bisretinoid in the case of A2E, these pigments cannot be enzymatically degraded, and thus they accumulate. The excessive accumulation of the retinoid-derived nondegradable lipofuscin in RPE cells is considered the primary cause of RPE atrophy in autosomal recessive Stargardt disease (arSTGD), a form of macular degeneration having onset in the early decades of life; RPE lipofuscin may also be a factor contributing to age-related macular degeneration (Sparrow and Boulton, 2005).

Rpe65 −/− mouse: Absence of all-trans-retinal-derived fluorophores

Important insight into the origin of RPE lipofuscin as a by-product of the visual cycle was provided by studies of mice carrying a null mutation in Rpe65 (Rpe65−/−) (Redmond et al., 1998), an essential protein of the visual

cycle that is reported to be the isomerohydrolase responsible for generating 11-cis-retinol from all-trans-retinyl esters (Jin et al., 2005; Moiseyev et al., 2006). Without Rpe65, the 11-cis-retinal chromophore of rhodopsin is not generated, photoisomerization does not occur, and all-trans-retinal is not produced. We compared the constituents present in chloroform-methanol extracts of eyecups obtained from 3- month-old wild-type C57BL/6J mice to that from agematched Rpe65 −/− mice (figure 44.2), the latter lacking the 11-cisRAL and atRAL chromophores (Redmond et al., 1998). When eluent was monitored for absorbance at 440 nm using a C4 column (figure 44.2A and B), Rpe65 −/− samples generated a chromatographic profile of reduced complexity as compared to C57BL/6J mice samples. Representative chromatograms are presented in figure 44.2. By simple visual subtraction analysis it was apparent that there were several peaks in the C57BL/6J profile for which there was no match in chromatograms generated from Rpe65 −/− mice samples. In particular, peaks in the C57BL/6J profile were attributed to the bisretinoid lipofuscin fluorophores A2E (λmax 335, 437 nm), atRAL dimer-PE (λmax 271, 518 nm), and the A2E precursors, NRPE (λmax 440 nm) and A2PE (λmax 332, 450 nm; 333, 448 nm), all of which were identified by coelution with synthetic standard and all of which we have previously identified. None of these pigments was detectable in Rpe65 −/− eyes. Further analysis of the eyecup extracts with a reverse phase C18 column (figure 44.2C and D) revealed peaks corresponding to all-trans- retinal (λmax 378 nm), A2E (λmax 335, 439 nm), iso-A2E (λmax 330, 445 nm), and a conjugate of all-trans-retinal with ethanolamine (NRE; λmax 440 nm), all of which were absent in Rpe65 −/− mice. These results are consistent with the observation that lipofuscin-specific autofluorescence in the RPE is reduced by more than 90% in Rpe65 −/− mice (Katz and Redmond, 2001), and they indicate that chloroform/meth- anol-soluble RPE lipofuscin fluorophores are derived primarily from retinoid precursors (Sparrow, Fishkin, et al.,

Figure 44.1 RPE lipofuscin pigments, precursors, and photooxidation products. Shown are the structures of A2E and its photoisomer, iso-A2E, and the precursor of the latter pigments, A2PE, all-trans-retinal dimer-phosphatidylethanolamine (atRAL dimer-

Figure 44.2 HPLC detection of RPE lipofuscin pigments in C57BL/6J mouse eyecups and the absence of these compounds in Rpe65 null mutant mice. Shown are chromatograms generated from hydrophobic extracts of eyecups (6 eyecups per sample) from 3-month-old C57BL/6J (A and C ) and Rpe65 −/− (B and D) mice using reverse phase C4 (A and B) and C18 (C and D) columns with monitoring at 440 nm. Compounds were identified by UV/visible

2003). Indeed, a number of observations over the years have also shown that the deposition of lipofuscin fluorophores in RPE is dependent on vitamin A availability (Katz et al., 1986, 1987). Significantly, in patients with early-onset retinal dystrophy associated with mutations in RPE65, RPE lipofuscin is also lacking (Lorenz et al., 2004).

PE), all-trans-retinal dimer-ethanolamine (atRAL dimer-E), and the precursor all-trans-retinal dimer (atRAL dimer). Monofurano-A2E and peroxy-A2E are photo-oxidation products of A2E.

absorbance and by coinjection of authentic standards. The two peaks attributable to A2PE (A) have retention times and UV/visible absorbance that correspond to the synthesized standard dipalmi- toyl-A2PE and likely reflect A2PE of varying fatty acid composition. (Rpe65−/− mice were a gift from Dr. Michael Redmond, National Eye Institute, Bethesda, MD.)

Stargardt macular degeneration and the Abca4/Abcr null mutant mice

The relationship between Stargardt macular degeneration and RPE lipofuscin was elucidated by studies in Abca4/Abcr null mutant mice (Weng et al., 1999; Mata et al., 2000, 2001, 2002; Kim et al., 2004) (figure 44.3). ABCA4/ABCR (rim

Figure 44.3 Autofluorescence of RPE lipofusin in Abca4/Abcr−/− mouse retina. Cross section of mouse retina viewed by epifluorescence microscopy (excitation 425 ± 45 nm, emission 510 nm long pass). Nuclei are stained with DAPI. The yellow-gold autofluorescence in RPE cells is associated with lipofuscin in RPE cells. The less pronounced autofluorescence in photoreceptor outer segments (POS) is attributable to lipofuscin precursors that form in this location. The albino mouse was 8 months old and homozygous for leucine at amino acid 450 of Rpe65. GC, ganglion cell layer; INL, inner nuclear layer; IPL, inner plexiform layer; ONL, outer nuclear layer; OPL, outer plexiform layer; RPE, retinal pigment epithelium. See color plate 44.

Figure 44.4 RPE lipofuscin pigments in a mouse model of Stargardt disease. A, A2E/iso-A2E (left) increases with age in Abca4/ Abcr−/− mice; A2PE, the precursor of A2E in photoreceptor outer segments, does not accrue with age. Compared with A2E/iso-A2E, atRAL dimer-PE and atRAL dimer-E (right) exhibit increase with

protein) is an ATP-binding-cassette transporter and the protein product of the Stargardt disease gene, ABCA4/ABCR (Allikmets et al., 1997). Studies of purified and reconstituted ABCA4/ABCR protein (Sun and Nathans, 1997, 2001a, 2001b; Sun et al., 1999; Ahn et al., 2000; Beharry et al., 2004), together with work in Abca4/Abcr−/− mice (Weng et al., 1999), have shown that this photoreceptor-specific protein aids in the movement of all-trans-retinal to the cytosolic side of the disc membrane so that it can be reduced to all-trans-retinol by retinol dehydrogenase. The ligand recognized by ABCA4/ABCR is probably N-retinylidene- phosphatidylethanolamine (NRPE), the conjugate formed by Schiff base reaction between a single all-trans-retinal molecule and phosphatidylethanolamine. The reaction of NRPE with a second molecule of all-trans-retinal leads to a series of random/nonenzymatic reactions and the formation of an unstable dihydropyridinium (dihydro-A2PE) (Kim et al., 2007a) (see figure 44.1) compound. Simulation by timedependent density functional theory reveals that dihydroA2PE exhibits a UV-visible spectrum with two absorbance maxima at approximately 494 and 344 nm. Dihydro-A2PE is an unstable intermediate that automatically undergoes oxidative aromatization to yield A2PE (see figure 44.1), the precursor of A2E detectable in photoreceptor outer segments (Parish et al., 1998; Liu et al., 2000). A2PE is internalized by RPE cells concomitant with outer segment phagocytosis; within RPE cell lysosomes, A2PE subsequently undergoes phosphate cleavage to generate A2E. Due to its unusual pyridinium bisretinoid structure (Sakai et al., 1996), A2E cannot be enzymatically degraded and thus is amassed with age (figure 44.4A). Conversely, A2PE does not accumulate with age; this would be expected of a precursor that is continually being cleaved to generate A2E (see figure 44.4A).

age. B, A2E increases with age and is more abundant in Abca4/ Abcr−/− mice than in wild-type mice (Abca4/Abcr+/+). Compounds were quantified from HPLC chromatograms and external standards to calibrate analyte peak areas.

Chromatographic studies of extracts of eyecups harvested from Abca4/Abcr null mutant mice have consistently demonstrated that the availability of all-trans-retinal resulting from a loss of Abca4/Abcr activity leads to levels of A2E that are increased manyfold relative to age-matched wild-type eyes (Parish et al., 1998; Mata et al., 2000; Kim et al., 2004, 2006; Fishkin et al., 2005) (figure 44.4B). As a model of Stargardt disease, these increases in A2E in Abca4/Abcr−/− mice parallel similar increases in individuals diagnosed with Stargardt disease (Delori et al., 1995).

A2E has an amphiphilic structure that is conferred by hydrophobic side-arms extending from a positively charged aromatic head group. A number of different approaches have shown that A2E can perturb cell membrane integrity when present in sufficient concentration (Sparrow et al., 1999, 2006; De and Sakmar, 2002; Sparrow, Fishkin, et al., 2003). This detergent-like behavior may be one means by which lipofuscin constitutents exert adverse effects on RPE cells.

A2E also has an absorbance maximum in the blue range of the visible spectrum (ca. 440 nm). Photosensitization of A2E leads to the generation of singlet oxygen and perhaps other reactive forms of oxygen. In the process, A2E is photo-oxidized along the retinoid-derived side-arms of the molecule, with the result that reactive photolytic products are generated (Sparrow et al., 2000, 2002; Schutt et al., 2000; Ben-Shabat et al., 2002; Sparrow, Vollmer-Snarr, et al., 2003; Dillon et al., 2004; Jang et al., 2006). Studies in Abca4/Abcr−/− mice have enabled detection of photooxidized forms of A2E. Specifically, analysis of eyecups of Abca4/Abcr−/− mice ( Jang et al., 2006) by liquid chromatog- raphy-mass spectrometry permitted the detection of both monofuran-A2E (Dillon et al., 2004), a product generated by the addition of one atom of oxygen, and monoperoxyA2E, resulting from the addition of molecular oxygen (O2) at two carbon-carbon double bonds (see figure 44.1). In vitro studies indicate that the photo-oxidation of A2E can involve the incorporation of as many as nine oxygens into the retinoid-derived side-arms of A2E; thus we anticipate that a complex mixture of oxidized products may form in vivo, with multiple oxygen-containing moieties situated within an A2E molecule. Moreover, mass spectral studies also demonstrate that irradiation-induced oxidation can lead to A2E fragmentation. Evidence indicates that the production of reactive oxidized forms of A2E may account at least in part for the cellular damage ensuing from photochemical mechanisms initiated by blue light excitation of A2E (Sparrow, Vollmer-Snarr, et al., 2003). Indeed, the amount of A2E that undergoes photo-oxidation and cleavage in a lifetime may be significant: high performance liquid chromatography (HPLC) quantitation of A2E and its precursor, A2PE, in posterior eyecups of young mice (9 weeks) revealed that the amounts of A2PE that form are

greater than is reflected in the levels of A2E that accumulate, an observation that indicates that some portion of A2E may undergo degradation (Kim et al., 2006), perhaps subsequent to oxidation.

Work in the Abca4/Abcr−/− mouse has further served in the detection and characterization of diretinal RPE lipofuscin constituents other than A2E. In particular, we found that condensation reactions between two all-trans-retinal leads to the generation of an aldehyde-bearing dimer (all-trans-retinal dimer, atRAL dimer), which then forms conjugates with primary amines such as phosphatidylethanolamine via Schiff base linkages (Fishkin et al., 2004, 2005; Kim et al., 2007b) (see figure 44.1). The pigment all-trans-retinal dimer-PE that forms through this unique biosynthetic pathway has a structure that is distinct from A2E/isoA2E and an absorbance maximum in the visible spectrum that is red-shifted relative to A2E (A2E/iso-A2E, λmax ca. 435 nm; atRAL dimer-PE, λmax ca. 510 nm). Another ca. 510 nm absorbing dimerconjugate, atRAL dimer-E, forms by phosphate cleavage of atRAL dimer-PE. Both atRAL dimer-PE and atRAL dimer-E have been identified in the lipofuscin-filled RPE of Abca4/Abcr−/− mice and are present at higher levels in the mutant mice than in wild-type mice (see figure 44.4A).

The Leu450Met variant in murine Rpe65 is associated with reduced levels of retinal pigment epithelial lipofuscin

As part of our effort to understand factors that modulate RPE lipofuscin formation, we have studied the relationship between the kinetics of the visual cycle and A2E formation. The Rpe65 polymorphism present in inbred strains of mice is such that the amino acid variant at residue 450 is either leucine or methionine (Danciger et al., 2000). From biochemical and electroretinographic studies it was apparent that in C57BL/6J mice that express methionine at codon 450, regeneration of rhodopsin is slowed and photon catch is reduced as compared to BALB/cByJ mice, which have leucine at that position (Wenzel et al., 2001, 2003; Nusinowitz et al., 2003). To determine whether the reduced availability of all-trans-retinal is accompanied by a decrease in RPE lipofuscin formation, we relied on quantitative HPLC to measure A2E and iso-A2E levels in mice homozygous for either the Leu-450 or Met-450 allele (Kim et al., 2004) (figure 44.5). Accordingly, whether the comparison was made between C57BL/6J-c2J (Met-450) and BALB/cByJ (Leu-450) mice or between Leu-450 and Met-450 in Abca4/ Abcr−/− mice, the levels of A2E in the presence of the methionine variant were consistently 25%–30% of that occurring with the leucine variant. The difference in the quantity of A2E associated with the Leu-450 variant versus the Met-450 variant occurred in pigmented and albino mice and in mice of varying ages. It is likely that the reduced flux of all-

trans-retinal that accompanies slowing of the visual cycle is responsible for the decreased formation of A2E in the presence of the methionine variant in C57BL/6J-c2J mice. The slowing of the visual cycle at the stage involving RPE65, a protein that has a rate-determining role in the visual cycle, can clearly provide protection against RPE lipofuscin formation.

Figure 44.5 The levels of A2E and iso-A2E in mouse eyecups varies in relation to the Rpe65 Leu450Met variant. A2E and isoA2E were measured in wild-type (BALB/cByJ and C57BL/6J-c2J) mice (A) and in Abca4/Abcr−/− mice expressing either the Leu-450 or Met-450 variant of Rpe65 (B). Levels (mean ± SEM) were determined from HPLC chromatograms as integrated peak areas normalized to an external standard of A2E.

Figure 44.6 Nonretinoid RPE65 antagonists suppress A2E/isoA2E formation in a mouse model of recessive Stargardt disease. Shown is the quantitation of A2E and iso-A2E in eyecups of Abca4/ Abcr−/− mice. Abca4/Abcr−/− mice were treated with TDT (50 mg/kg twice weekly, intraperitoneally) for 1 or 2 months beginning at age 10 weeks, and A2E and iso-A2E levels at the end of treatment (age

Targeting the visual cycle to inhibit the formation of retinal pigment epithelial lipofuscin fluorophores

Given the adverse behavior of the diretinoid pigments that form RPE lipofuscin (Sparrow et al., 1999, 2000; Schutt et al., 2000; De and Sakmar, 2002; Sparrow et al., 2002; Sparrow, Fishkin, et al., 2003; Sparrow, Vollmer-Snarr, et al., 2003; Sparrow et al., 2006), there is considerable interest in retarding A2E formation as a means to prevent vision loss in Stargardt disease and perhaps in age-related macular degeneration. Accordingly, isotretinoin (13-cis-retinoic acid), the acne medication that induces night blindness and protects against light damage by retarding 11-cis-retinal regeneration (Sieving et al., 2001), can reduce A2E deposition in RPE of Abca4/ Abcr−/− mice (Radu et al., 2003). Isotretinoin is suggested to act, at least partially, by inhibiting 11-cis-retinol dehydrogenase (Sieving et al., 2001). Nonetheless, 13-cis-retinoic acid has severe side effects, including teratogenicity (Sparrow, 2003), and thus is not appropriate for long-term therapy.

The evidence garnered from studies of the Rpe65 Leu450Met variant suggested that RPE65 would be an excellent target for therapeutic interventions aimed at reducing A2E formation in arSTGD and other retinal disorders characterized by aberrant RPE lipofuscin accumulation. Thus, in a collaborative effort, we tested nonretinoid isoprenoid compounds for their ability to compete with retinyl esters for binding to RPE65, thereby interfering with visual cycle kinetics (Maiti et al., 2006). Following a single intraperitoneal injection (50 mg/kg) of the compounds TDT (figure 44.6) and TDH (not shown) in mice, recovery of darkadapted ERG b-wave amplitudes to prebleach levels was delayed and 11-cis-retinal regeneration was slowed. Moreover, twice-weekly treatment of Abca4/Abcr−/− mice for 1 or 2 months resulted in substantial decreases in A2E accumulation (see figure 44.6). Thus, these nontoxic isoprenoid RPE65

14 weeks or 18 weeks) were compared to levels in 10-week-old vehicle-treated (control) mice and 14or 18-week-old vehicle-treated (control) mice. A2E/iso-A2E levels were determined from HPLC chromatograms by integrating peak areas and normalizing to external standards. Values are expressed as picomoles per eye and are based on single samples obtained by pooling data from four eyes.

antagonists are candidates for the treatment of forms of macular degeneration in which lipofuscin accumulation is an important risk factor.

In an alternative approach, the excessive accumulation of A2E in Abca4/Abcr−/− mice has been reduced by daily administration of the retinoic acid analogue N-(4-hydroxy- phenyl)retinamide (HPR) for 1 month (Radu et al., 2005). As shown by the investigators, HPR acts by competing for binding sites on retinol-binding protein, thus reducing serum retinol levels. As a result, retinol uptake by the eye is reduced and visual cycle retinoids are decreased. The accompanying decrease in atRAL leads to retarded A2E formation.

Future directions

Mouse models of retinal disease have elucidated the relationship between the visual cycle and RPE lipofuscin formation and will continue to be central to the development of geneand drug-based therapies that can combat vision loss in disorders in which aberrant lipofuscin accumulation threatens RPE cell function and survival. Studies have shown that RPE lipofuscin in ABCR-associated disorders is similar in composition to age-related lipofuscin, although in ABCR-associated disorders the formation and accumulation occur at a faster rate. Work in mice should also clarify mechanisms involved in the higher levels of RPE lipofuscin reported in autosomal dominant Stargardt-like (STGD3) macular degeneration (Karan et al., 2005), an early-onset disorder due to mutations in ELOVL4 (Zhang et al., 2001; Bernstein et al., 2001; Maugeri et al., 2004), and in Best vitelliform macular dystrophy (BMD), an autosomal dominant form of macular degeneration caused by mutations in VMD2 (Petrukhin et al., 1998), the gene encoding bes- trophin-1 (hBest1), a protein in RPE (Marmorstein et al., 2000).

acknowledgments Work was supported by National Institutes of Health grant no. EY 12951, Foundation Fighting Blindness, the American Health Assistance Foundation, the Steinbach Fund, and Research to Prevent Blindness. JRS is a recipient of an Alcon Research Institute Award.

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Diabetic retinopathy is a progressive retinal disease that threatens patients with blindness. The retinopathy develops slowly, over decades in humans, making investigation of its pathogenesis challenging. Many animal models have been developed to study the evolution of the retinopathy (Kern and Mohr, 2008). The rate of development of the early stages of the retinopathy seems to vary among species, with at least the early stages of the retinal disease apparently progressing faster in smaller species such as the mouse. The relatively short interval until pathology presents, coupled with the ability to genetically engineer the mouse, has prompted an increased interest in mouse models. This chapter reviews mouse models of diabetic retinopathy, compares pathological changes in the retina of the diabetic mouse with those in humans, and highlights advances made using mice to understand the pathogenesis of the retinopathy.

Diabetic retinopathy in humans

Diabetic retinopathy has classically referred to alterations in the retinal microvasculature visualized on ophthalmoscopic examination (Davis et al., 1997). Over the course of disease progression, two stages of retinal pathology have been defined:

1. Retinal lesions observed clinically in early retinopathy, or the nonproliferative stage of retinopathy (previously referred to as background retinopathy), consist of nonperfused capillaries, microaneurysms, retinal hemorrhages, cotton wool spots, exudates, and edema. Each of these lesions is believed to represent local alteration in the microvascular circulation of the retina, which in some cases results in damage to the surrounding retinal cells.

Histological studies using techniques to isolate the retinal vasculature have demonstrated that the vascular histopathology includes capillary microaneurysms, pericytedeficient capillaries, basement membrane thickening, and degenerate (acellular) capillaries (Aguilar et al., 2003; Cogan and Kuwabara, 1967; Engerman, 1989; Kohner and Henkind, 1970). In addition, histological analysis of the human retina has demonstrated changes in the nonvascular retina as well, including ganglion cell loss (Barber et al., 1998; Bloodworth and Molitor, 1965; Bresnick and Palta, 1987a,

1987b; Lieth et al., 2000). Currently, several different therapies, including intensive insulin therapy and blood pressure medications, are available to inhibit the development of the nonproliferative stage of diabetic retinopathy (The Diabetes Control and Complications Trial Research Group, 1993; Jandeleit-Dahm and Cooper, 2006; Zhang et al., 2001).

2. Subsequently, a proliferative retinopathy develops in about 10%–15% of diabetic patients, with marked growth of new blood vessels growing out of the existing retinal vasculature onto the surface of the retina and into the vitreous. These newly formed blood vessels (as well as other blood vessels in the retina) leak, occasionally resulting in vitreous hemorrhages, which can obstruct vision (Cunha-Vaz et al., 1975). In addition, with the development of these new vessels a preretinal fibrovascular membrane can form that also can obstruct vision. The neovascularization, preretinal hemorrhage, and membranes currently can be corrected using photocoagulation or vitrectomy, respectively.

In addition to vitreous hemorrhages, other late retinal changes that lead to visual impairment in diabetic retinopathy include retinal detachments (caused by traction of the fibrovascular membrane associated with new vessel formation) and retinal edema secondary to breakdown of the blood-retinal barrier and leakage of plasma from the microvasculature. The evolution of these stages of diabetic retinopathy in humans occurs over years, with nonproliferative changes first detected at 5–10 years of diabetes and proliferative changes developing later.

Diabetic retinopathy in mice

The desire to investigate the pathogenesis of diabetic retinopathy has driven the development and evaluation of many animal models of diabetic retinopathy. Of course, the better the human disease is replicated, the better the animal model. To date, the diabetic dog is the animal model that best reproduces the nonproliferative stages of diabetic retinopathy, reproducibly developing lesions seen rarely or not at all in rodent models (Engerman, 1989; Engerman and Bloodworth, 1965; Kern and Mohr, 2007). Because retinopathy in the dog requires 3–5 years to develop, however, the associated time and cost make using this model cumbersome.