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

Edge-related degeneration is evident in all species in which it has been studied (rats, mice, and humans; reviewed in Stone et al., 2005). In humans, the edge of the retina is eroded throughout life by a process called cystoid degeneration (Bell and Stenstrom, 1983; Vrabec, 1967). The degeneration is most severe at the edge of the retina, where the layered structure of the retina breaks down; cysts form within the retina, and eventually, pigmented cells invade from the RPE. This invasion mimics the pigmentation of the prematurely degenerating retina, which led to the term retinitis pigmentosa.

In the area of degeneration, vessels are absent, a symptom of raised oxygen levels. Central to the cystoid degeneration, extending some millimeters toward the center of the retina, the layered structure of the retina is intact, but vessels are absent or reduced, and the morphology of photoreceptors is degraded (Stone et al., 2005). More centrally, the morphology of photoreceptors and their content, the metabolic enzyme cytochrome oxidase, and the retinal circulation are normal. This grading of the degeneration from the edge of the retina suggests that the source of stress is at the edge. Further, the extent of the degeneration increases with age (Stone et al., 2005), indicating, as we originally suggested, that the normal retina is unstable under hyperoxic stress and the degeneration is progressive.

Oxygen stress during development: The concept of a critical period in photoreceptor development

The relationship between photoreceptor stability and retinal oxygen levels described in the previous section—that photoreceptors are maximally stable in normoxia and are destabilized by both hypoxia and hyperoxia—is true for the adult. Different relationships hold during development, however. In early postnatal life in mouse (Mervin and Stone, 2002a) and rat (Maslim et al., 1997), a period of accelerated photoreceptor death has been described. This “critical period” of development (P15–P20 in the mouse, P15–P25 in the rat) coincides with a period of physiological hypoxia caused by a rapid increase in photoreceptor metabolism (Graymore, 1960). This period of physiological hypoxia (Chan-Ling et al., 1995a) is critical in inducing angiogenesis of the retinal circulation.

Testing whether the death of photoreceptors in the developing retina is driven by hypoxia, Maslim and colleagues (1997) demonstrated that the rate is sharply increased by hypoxia and decreased by hyperoxia. Mervin and Stone (2002) extended these observations to the C57BL/6 mouse. After about P30 in both species, the effects of oxygen levels on photoreceptor death shift subtly but significantly, in two ways. First, photoreceptors become relatively resistant to variations in oxygen levels; some still unidentified stabilizing factor ends the developmental period of high naturally

occurring death rates. Second, the sign of the effect of hyperoxia changes. During the critical period, hyperoxia is protective; in the adult, hyperoxia is toxic.

Finally, it has become clear that many forms of mutationinduced retinal degeneration accelerate during the critical period, suggesting an interaction between environmental or cell biological factors (the critical period) and the gene mutation (RCS rat: Valter et al., 1998; P23H-3 rat: Walsh et al., 2004b; the rd mouse: Hafezi et al., 1998).

In our original (1999) review, we speculated on the teleological significance of a period of development during which photoreceptors are hypoxia sensitive. We suggested that, like most classes of retinal cells, photoreceptors are overproduced during development and then culled to adult populations by a process of cell death. In photoreceptors but not other retinal cells, the culling is driven by the supply of oxygen to the retina, with the effect (we suggested) of matching the photoreceptor population to the supply of oxygen available.

Implications for the retinal degenerations

The analysis just developed draws attention to environmental factors, such as tissue oxygen levels, that influence the course of retinal degenerations. How powerful is this influence? Can purely environmental factors precipitate degenerations? Can they provide effective therapy?

Evidence that environmental factors can precipitate degenerations has come from two sources. Earlier we referred to evidence that age-related macular degeneration may be preceded by significant loss of rods. Curcio and colleagues (2000) suggest that the rod loss follows age-related changes in the recycling of retinoids, that rod loss contributes somehow to the degeneration of the cone-rich macula, and that improving rod survival could be the key to the stability of the macula. Considering the possibility of significant environmentally induced photoreceptor degeneration early in life, we (Stone et al., 2001) examined a cohort of cases of retinal degeneration, separating them into groups in which a familial pattern could and could not be detected. Our working hypothesis was that perinatal stress, which is associated with a wide range of adult-onset diseases, might also contribute to retinal degenerations, perhaps by precipitating degeneration in the developing photoreceptor population. A history of perinatal stress was significantly more common in the nonfamilial group, suggesting that in a proportion of nonfamilial cases, perinatal stress (which includes hypoxic stress) contributes to the cause of photoreceptor degenerations.

We have mentioned the ability of light restriction not only to slow photoreceptor loss in a degenerative model, the P23H-3 rat, but also to restore photoreceptor structure and function. This recovery of function stems from a capacity of

individual cells to repair their outer segments and restore their functional organization. Obviously, this self-repair capacity has therapeutic potential; but is it special to this model? In normal (wild-type) retina, photoreceptors also respond to sustained (over weeks) increases in ambient light by decreasing the length of their outer segments. The result is a drop in rhodopsin levels in the retina and a reduction in the dark current and a-wave. The effects are reversible, and the effect and its reversal have been summarized in the principle of photostasis (reviewed in Williams, 1998), which states that the retina regulates the amount of active rhodopsin deployed to keep the daily quantum catch constant.

The effect of light on wild-type outer segments goes beyond shortening and lengthening, however. Reports of the effect of ambient light on normal photoreceptors (Kuwabara, 1970; Penn and Anderson, 1991) describe significant disorganization of the outer segment membrane, very like that seen in the P23H-3 model. This damage is also reversed, as the cell replaces its outer segment membrane. It thus seems likely that the ability of photoreceptors to self-repair is general, and likely to be available in any retina.

These examples of environmentally induced damage, degeneration, and recovery of photoreceptors are unlikely to be all oxygen mediated. The level of oxygen in the outer retina is only one measure of the impact of light, photoreceptor depletion, and age-related factors such as the recycling of retinoids on the stability of photoreceptors. Nevertheless, oxidative stress is prominent in many recent ideas of the mechanisms that precipitate photoreceptor death. Photoreceptors produce reactive oxygen species when exposed to light (especially blue light; Yang et al., 2003), and their production follows the activation of rhodopsin, before the downstream events of visual transduction (Demontis et al., 2002). Thus, oxidative stress may be intrinsically linked to the absorption of visible light by rhodopsin, and photoreceptors are rich in antioxidant molecules, such as docosahexaenoic acid (Rotstein et al., 2003). As a consequence, measures such as light restriction, filtering out just blue light (Margrain et al., 2004), dietary supplementation with antioxidants (Militante and Lombardini, 2004), and infrared radiation to repair oxidatively damaged mitochondria (Eells et al., 2003) are being debated and studied. As emphasized in this chapter, environmental factors can add significantly to the stresses that cause photoreceptor degeneration, and consequently, management of these factors can be valuable therapeutically.

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MICHAEL DANCIGER

The understanding of music is in the understanding of a single note.

—Unknown

Retinal degeneration (RD) can be caused by a number of different factors: gene mutations, such as those responsible for retinitis pigmentosa (RP) or other forms of RD; environmental exposures, such as toxic levels of light; and a combination of environmental factors and genetic tendencies, such as those producing age-related macular degeneration (AMD). Regardless of cause, the end stage is loss of photoreceptors due to apoptosis. Therefore, RD in experimental animals (in this case, mice) caused by toxic levels of light is a good model in which to study photoreceptor death because it is easy to manage in the laboratory. In addition, patients with some retinal diseases who are exposed to high levels of light for extended periods, such as fishermen or skiers, may have their disease course worsened (Taylor et al., 1990; Cruickshanks et al., 1993; Simons, 1993; Cideciyan et al., 1998; Mata et al., 2000). Supporting this is the fact that in some animal models of inherited RD, the disease process is accelerated by light exposures (Sanyal and Hawkins, 1986; Wang et al., 1997; LaVail et al., 1999; Organisciak et al., 1999). Therefore, it is not surprising that there has been a tremendous amount of work on light damage to the retina in many different fields. In this chapter, we cover work on the complex genetics of light-induced RD in the mouse model.

In the late 1980s, LaVail and colleagues published two studies on photoreceptor (PR) light damage of albino mice (LaVail et al., 1987a, 1987c). Several albino strains were exposed to constant light at about 115–130 foot-candles (ft-c) (1,265–1,400 lux) for 2 weeks. After exposure, the PR outer nuclear layer (ONL) was measured and used as an indicator of PR loss. Compared to unexposed control mouse retinal ONLs, the B6(Cg)-Tyrc-2J/J albino (B6alb) still had 80% of the PR ONL, while the other strains (NZWLacJ, A/J, BALB/c, AKR, Ma/My, RF/J, RIIIs/J) had only 40% or less (LaVail et al., 1987a, 1987c). The B6alb strain is coisogenic with the standard pigmented C57BL/6J strain; the only genetic difference is the homozygous mutation in the tyrosinase gene carried by B6alb, causing it to be albino. (Co-isogenic or congenic refers to nearly identical strains of an organism that vary at only a single locus.) Why were B6alb

PRs so much more resistant to light insult than those of the other strains? Insofar as the mice were all raised and maintained under the same circumstances, the answer had to be genetics. Of course, this is what the LaVail group thought and demonstrated in a second study in which they performed the same type of light damage protocol on B6alb, BALB/c (C), and F1 mice (in this case, F1 = progeny of a B6alb × C cross). The results confirmed the substantial and significant difference in loss of PRs to light insult between the B6alb and C strains, with an intermediate response from the F1s (skewed a bit toward the C phenotype), demonstrating the genetic influence on the trait (LaVail et al., 1987b). We became interested in this work and set up a collaboration with Dr. LaVail (Beckman Research Center, University of California–San Francisco) to try to identify the gene (or genes) and alleles responsible for this remarkable difference in light-induced PR loss between the C and B6alb strains.

Identification and verification of a gene modifier that has a strong influence on photoreceptor light damage

We started with the simplest explanation: a single gene is responsible for the difference in phenotype between the strains. The B6alb allele protects the PRs from light insult, and the C allele makes them more sensitive. We performed a backcross with the B6alb and C strains and attempted to score the ONL of the progeny after the same constant light exposure as before. The term backcross refers to a cross of the F1 of two strains (for example, strains A and B) with one of the original strains. Thus, F1(A × B) × B or F1(A × B) × A, or, in our case, F1(B6alb × C) × B6alb. If one gene was responsible for the difference, there should have been only two phenotypes, resistant (B6alb/B6alb) and sensitive (B6alb/C). Linkage analysis with genetic markers evenly spanning the genome at regular intervals of ≤30 centimorgans (cM) within chromosomes and ≤15 cM from the telomere or centromere (mouse chromosomes are acro- centric—they have the centromere at one end) should have revealed the chromosomal location of the gene. It did not work. First, it was difficult in many individual cases

to score the light damage phenotype as resistant or sensitive, and second, no marker cosegregated with the phenotype; that is, no marker was B6alb/B6alb for all mice with the resistant phenotype and B6alb/C for all mice with the sensitive phenotype (data not published). Therefore, we hypothesized that PR damage in response to constant light exposure is a complex trait.

The definition of a complex trait in the circumstance in which the environment is controlled—that is, it is not a differentiating factor—is a trait or phenotype that is governed by two or more genes. Since the number of genes that govern the trait is unknown at the outset, the number of phenotypes is unknown as well. For example, one gene governing a trait with alleles that are additive would produce three phenotypes. Additive means that the alleles are not dominant or recessive but instead, when both are present, the phenotype is intermediate. Two additive genes would produce nine phenotypes, three genes 27 phenotypes, and so on. Therefore, the phenotype is measured or quantified. In this case, the phenotype was the average of 54 measurements of the ONL thickness along a central retinal section running far superior through the optic nerve head to far inferior. To do the experiment, we used the same backcross protocol and light exposure as before, but instead of linkage analysis, quantitative genetics analysis was performed, using the Map Manager QTX program (Manly et al., 2001). This software identifies quantitative trait loci (QTL) by regression analysis. Thus, it computes the degree of significance for the association of a marker with the phenotype by evaluating the genotype of all markers comprising a genome-wide scan in all progeny animals and comparing that with the phenotypes of all progeny animals. Significance is identified when a high proportion of progeny with a particular genotype for a marker (for example, B6alb/B6alb) tend to have a particular phenotype, and at the same time when the genotype is different for that marker (B6alb/C, in this case) the phenotype tends to be opposite. In this way, utilizing the Map Manager program, we identified several QTL that influenced PR light damage in this cross. The most significant of these was on distal chromosome 3, which accounted for nearly 50% of the genetic effect and had a probability of 10−19 of being a random, chance phenomenon (LOD score was ca. 19) (Danciger et al., 2000).

When we examined that locus on the gene map for candidates, we found the Rpe65 gene, a gene present in the retinal pigmented epithelium (RPE) but known to be involved in the response of the PR cell to light. When we sequenced the Rpe65 cDNA, we found an A for the first nucleotide of codon 450 in B6alb and a C in the BALB/c strain, predicting a methionine for B6alb and a leucine for the BALB/c. Since both methionine and leucine are nonpolar amino acids, the change from one to the other is conservative and would not predict the normal function of RPE65 to be

affected. This was supported by studies showing normal responses of B6alb mice in water mazes and by studies showing normal electrophysiological responses of B6alb retinas (Balkema and Dräger, 1991; Hayes and Balkema, 1993; Nusisnovitz et al., 2003). Our hypothesis was that under the oxidative conditions of constant light, however, the methionine would be susceptible to oxidation to methionine sulfoxide, rendering the RPE65 protein more susceptible to degradation, while the leucine 450 would not be. Oxidation of key methionines in other proteins has been shown to disrupt function (Vogt, 1995). Lower levels of RPE65 would decrease the cycles of rhodopsin regeneration and thereby protect the PRs by slowing down the vision transduction process and its concomitant hyperpolarization and depolarization cycling. The PR response to light involves the stimulation of rhodopsin involving the conversion of 11-cis-retinal to all-trans-retinal and separation from the opsin protein. The activated protein interacts with the G protein transducin as the first step in a biochemical pathway resulting in the hyperpolarization of the PR cell with a concomitant alteration in neurotransmission to the next level of neurons in the retina. To stop the process, both rhodopsin kinase and arrestin interact with rhodopsin, stopping its interaction with transducin and preparing it for ligation with 11-cis-retinal. 11-cis-retinal is recycled by a pathway that involves RPE65 in the RPE cells and appropriate transport from and to the PRs (for reviews, see Chabre and Deterre, 1989; Chen, 2005).

Support for our hypothesis came from two publications demonstrating the same PR sensitivity to perturbations in the visual transduction process under different circumstances. In one case, PRs were protected from intense light exposures by the absence of RPE65 protein in the corresponding knockout mice (Grimm et al., 2000), and in the other, PRs degenerated even in the presence of dim light, owing to the absence of rhodopsin kinase (Grk1) in the corresponding knockout mice (Chen et al., 1999a). In the first case, the cycling of rhodopsin and the hyperpolarization and depolarization cycles of PRs were stopped, and in the second case the PRs were overstimulated because activated rhodopsin could not be shut off in a timely way. Verification of the hypothesis and a more precise explanation of the protective effect of the RPE65 met450 variant were demonstrated in studies by Wenzel et al. (2001b, 2003). The sum of the two studies (done on wild-type and c-FOS −/− backgrounds) was severalfold. Mice homozygous for RPE65 leu450, for met450, or heterozygous were exposed to intense light after dilation of their pupils producing a bleach of a high number of PRs (a high percentage of the retinal rhodopsin) and then held in the dark. At various time points the amount of rhodopsin regenerated was measured spectrophotometrically and compared to the baseline before the bleach application. The presence of the met450 variant significantly slowed

the regeneration of rhodopsin. The speed of regeneration was directly correlated to the amount of RPE65 protein present in the RPE measured by Western blot and to the PR damage to the retina measured in stained histological sections. Thus, mice homozygous for RPE65 met450 had lower levels of RPE65 protein in their RPE, slower regeneration of rhodopsin, and less PR loss after intense light exposure (Wenzel et al., 2001b, 2003). The amount of Rpe65 mRNA, however, was the same in eyes with RPE65 met450 and RPE65 leu450 (Danciger et al., 2000; Wenzel et al., 2001b, 2003).

An additional piece of information was the fact that the protective effect of the met/met genotype was not great enough to account for the resistance of B6 mice to damage following toxic light exposure (Wenzel et al., 2001b). This substantiated the original quantitative genetics study demonstrating the presence of several other B6alb gene alleles that also protect against light-induced damage (Danciger et al., 2000). In addition, several other QTL were identified in a light damage study of an F1 intercross of the BALB/c and 129S1/SvImJ strains (Danciger et al., 2004). These QTL were on chromosomes 1, 4, 6, and 2, representing a completely different set of QTL than in the original light damage study of BALB/c and B6alb. However, in the earlier study the mice were exposed to 2 weeks of constant light at an intensity of approximately 115–130 ft-c (1,265–1,400 lux) and sacrificed immediately after exposure. In the 129 × BALB/c study the mice were exposed (with dilated pupils) to 1 hour of 15,000 lux, allowed to stay in the dark for 16 hours, and then kept in dim cyclic light for about 12 days before evaluation of retinal damage by measurement of the amount of rhodopsin remaining (Danciger et al., 2004).

To evaluate the argument that the visual transduction response was slowed in the PRs of mice carrying the RPE65 met450 variant at the physiological level, an electroretinogram study was carried out as follows: BALB/c and B6alb mice were exposed to a bleaching light and then placed in the dark. At various time points, the mice were tested for the rod response to light and compared to normal. For both the a-wave and b-wave, the BALB/c retinas recovered significantly sooner than those of B6alb (Nusinowitz et al., 2003). The slower physiological recovery of the response to light in B6alb mice compared to BALB/c mice corresponded perfectly to results of the previous biochemical studies showing a slower regeneration of rhodopsin (Wenzel et al., 2001b). A further substantiation of the RPE65 met/leu450 variant as a modifier of PR light damage was demonstrated when RPE65 protein activity and concentration were measured in the eyes of various breeding combinations of BALB/c, B6 and RPE65−/− (knockout) mice (the knockout mice were on a mixed background of 129S1/SvImJ and B6). The order of concentration of RPE65 in the eye from highest to lowest was BALB/c (leu/leu) > (BALB/c × B6)F1 (leu/met) > BALB ×

KO (leu/−) > B6alb (met/met) > B6alb × KO (met/−). The rate of rhodopsin regeneration matched that order, but the rate of rhodopsin regeneration per unit RPE65 protein was not influenced by the leu/met variant; it was the different amount of RPE65 protein present (Lyubarsky et al., 2005). This same conclusion can be extracted from two very recent cell expression studies. In the first case, it took 5–10 × met450 cDNA to express the same amount of RPE65 protein as leu450 cDNA in 293T-LC cells, but both RPE65 proteins had the same activity (Jin et al., 2006). In the second case, amounts of RPE65 protein expressed by cell transfection of cDNA were directly related to the presence of the met450 or leu450 variant, whether it was in cDNA from mouse or from dog. Based on predicted protein structure, the authors of this work hypothesized that the RPE65 met450 variant made the protein much less stable than the leu450 variant (Redmond et al., 2007).

The evidence cited so far demonstrated that the met450 variant of RPE65 reduced the amount of RPE65 protein present in the eye relative to the leu450 variant. This reduction protected the PRs from light-induced damage by slowing the recycling of rhodopsin, and therefore slowing the recovery of the capability to respond to light physiologically. There was, however, a fly in the ointment (something inconsistent). The met450 variant of RPE65 is rare (figure 47.1). However, the albino NZW/LacJ mouse (NZW) carries the met variant yet is almost as sensitive to light-induced retinal damage as BALB/c mice, and far more sensitive than B6alb mice (LaVail et al., 1987a, 1987c; Danciger et al., 2005). We could think of three explanations for this:

(1) The met450 variant of RPE65 is not responsible for the retinal light damage resistance found in B6alb. It is instead a nonfunctional sequence variant that segregates with the actual nearby gene responsible for the effect. The protective allele of this gene is present in B6alb and the sensitive allele in BALB/c and NZW. (2) There is a second change in the NZW Rpe65 gene or a nearby sequence that suppresses the protective effect of RPE65 met450. (3) There are other NZW susceptibility gene alleles that overcome the protective effect of the met450 variant.

To evaluate these possibilities, we repeated the constant light–induced retinal damage protocol on a small test cross between B6alb and NZW. If the first or second explanation was true, we would expect a strong and highly significant QTL at distal chromosome 3 with a B6alb protective allele because the NZW allele of the gene in this QTL would not be present either because met450 was suppressed or because the allele of the true gene responsible for the QTL was not present. We found no such distal chromosome 3 QTL and did find other QTL supporting the third explanation (Danciger et al., 2005). In addition, we bred the met450 variant from NZW to BALB/c to the N8 generation and tested it under the same conditions of light exposure and by

the same ONL thickness measurements described earlier in the text. Figure 47.2 shows that the RPE65 met450 coming from NZW and operating in the BALB/c background now protected the PRs from light-induced damage. To make the N8 generation, BALB/c mice were crossed with NZW. The offspring (F1) were crossed into BALB/c mice to produce N2 mice. Tail DNAs from these mice were tested for the presence of the met450 variant by PCR, and those that were positive (RPE65 heterozygous met/leu450) were bred to BALB/c again to produce N3 offspring. The same process was repeated until N8. N refers to the number of generations of one strain (in this case BALB/c) bred into the F1 such that the higher the N, the greater the percentage of the mouse that is BALB/c while still carrying the selected allele—in this case, around 99.6% BALB/c background and heterozygous for met/leu450. The N8, BALB/c, RPE65 met/leu450 mice were intercrossed to produce mice homozygous for leu450, homozygous for met450, and heterozygous.

Glazier et al. (2002) recently published a set of criteria that must be demonstrated to establish that a gene is responsible for a QTL (quantitative trait gene, or QTG). These include:

(1) the demonstration of a linkage or association between the locus of the gene and the trait; (2) a significant sequence variant in the QTG between two strains with different phenotypes; (3) functional tests of the candidate QTG variants; and (4) an explanation of any complicating or confounding factors. A QTL on distal mouse chromosome 3, the locus of the Rpe65 gene, was demonstrated to be associated with differences in a retinal light damage phenotype between the BALB/c and B6alb albino mouse strains (point 1). The BALB/c sequence of the Rpe65 gene has the CTG code for leucine at residue 450, while the B6alb sequence has ATG for methionine (point 2). There is less RPE65 protein when met450 is present (demonstrated by Western blot), resulting

in a slower recovery of the visual transduction response of the PR demonstrated physiologically, a slower regeneration of rhodopsin after bleach demonstrated biochemically, and a decreased level of PR damage after light insult demonstrated histologically (point 3). The presence of the “protective” met450 variant of RPE65 in the NZW strain that is relatively sensitive to light insult was explained by the presence of other QTL genes that negate the protective effect (point 4). The types and quality of data cited in this review match or exceed the criteria for establishing a variant gene to be one QTG or one modifier of several comprising the genetic portion of the complex trait of PR light damage.

Some genes identified in microarray expression studies and studies of single genes are candidates for light damage quantitative trait loci

Differential or comparative studies of mRNA expression and studies of individual genes bring much information about the light damage process in their own right, and also provide candidates for QTL based on chromosomal map locations. In the first approach, the upregulation (or downregulation) of retinal mRNAs in response to damaging levels of light is determined by comparing expression before and after light exposure, generally using microarray chips. The second approach involves measuring the effect on light damage of an increase or decrease or elimination of the expression of a gene or genes. The point of these studies is to identify the genes that govern the response to light insult, the protective defensive response, and the pathophysiological process of PR death. Three quantitative genetics studies designed to identify the chromosomal loci of genes that are involved in the retinal light damage process have been carried out, and, along with the chromosome 3 QTL (shown to be the Rpe65

Figure 47.2 The number of mice measured for ONL was 15 for leu/leu, 18 for met/leu, and 5 for met/met. Probabilities derived from the student’s unpaired t-test were 0.03 between leu/leu and

met/met, 0.31 between leu/leu and met/leu, and 0.01 between met/leu and met/met.