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

gene), 11 other QTL have been described (Danciger et al., 2000, 2004, 2005). Are any of the genes implicated in light damage and discovered by microarray or single-gene studies located in the loci of these 11 QTL?

Three parallel studies asked what changes occur in levels of mRNA after a toxic light exposure (Chen et al., 2004; Roca et al., 2004; Rattner and Nathans, 2005). The BALB/c albino was the mouse strain, the retina was the source of the mRNA, and a comprehensive Affymetrix (Santa Clara, Calif.) genome chip was used for mRNA probe hybridization in all three studies. The conditions before and after light exposure and the intensity and duration of light differed. Dark adaptation ranged from 24 hours (Chen et al., 2004) to 1–2 weeks (Rattner and Nathans, 2005) to rearing in the dark (Roca et al., 2004). Light exposures ranged from 1,500– 2,000 lux for 3 hours to 6,000 lux for 6 hours to 10,000 lux for 7 hours. Mice were sacrificed immediately (Chen et al., 2004; Roca et al., 2004) or kept in the dark for 24 hours first (Rattner and Nathans, 2005). Despite differences in the light exposure protocol, many genes were upregulated in common, particularly in the category of transcription factors. Many of the genes most highly upregulated appeared to be induced in Müller cells, even though light damage occurs primarily in PRs (Rattner and Nathans, 2005). At least part of the function of retinal Müller cells is thought to be to help maintain PRs and protect them from stress. The total number of upregulated genes in the three studies was greater than 300. Searching among 300 genes for those that are located in any of 11 chromosomal QTL loci would certainly provide candidate genes just by chance. In one of the studies, only 15 genes were upregulated (Roca et al., 2004); in another study, well over 200 were (Rattner and Nathans, 2005), and in the third study, about 70 genes were upregulated (Chen et al.,

2004). The first study had more stringent criteria for considering a gene upregulated. Therefore, we considered the 15 genes most highly upregulated (the only 15 genes, in one case) from each study. Of the 45 genes, 6 were upregulated in more than one study and 12 were located within the loci of retinal light damage QTL. One gene (Cebpd) was shown to be upregulated in all three microarray reports. This gene was located in a QTL from one retinal light damage study. Two more upregulated genes were each located in QTL present in two light damage studies (table 47.1).

Examples of single-gene studies show that many genes influence retinal light damage. Bax and Bak1 double knockout mice were protected from intense light exposure (Hahn et al., 2004). Bax and Bak1 are pro-apoptotic genes of the bcl-2 family. Inhibition of the apoptotic factor AP-1 also protected PR cells from light-induced damage (Wenzel et al., 2001a). AP-1 is either a heterodimer of any of several Fos and Jun proteins or a homodimer of any of several Jun proteins (Curran and Franza, 1988). The absence of the antioxidant glutathione peroxidase-1 (Gpx1) in knockout mice left the retinas more sensitive to light-induced damage (Gosbell et al., 2006). The absence of the apoptotic regulator p53 (Trp53) or the absence of the Müller cell neurotrophin receptor p75 (Ngfr) in knockout mice had no effect on retinal light damage (Marti et al., 1998; Rohrer et al., 2003). Overexpression of erythropoietin (Epo) by hypoxic preconditioning protected the retina (Grimm et al., 2004), while overexpression of the EAT/mcl-1 gene (Mcl1), another member of the bcl-2 family, worsened light damage to the retina (Shinoda et al., 2001). These examples are apoptotic or other genes that are widely expressed and have a function that is not specific to the retina. On the other hand, the mouse orthologue of human RDH12 (Rdh12), a gene involved

in the specific retinal function of visual transduction, particularly in the vitamin A recycling system (Thomson et al., 2005), protected the mouse retina from light-induced damage (Maeda et al., 2006). Specifically, the absence of mouse Rdh12 did not cause RD in the mouse, but it did make the mouse retina more sensitive to intense light. The absence of Rdh12 caused 11-cis-retinal to be regenerated more rapidly under excessive light (Maeda et al., 2006), increasing the cycles of rhodopsin regeneration and increasing the levels of all-trans-retinaldehyde in the inner segments. The increased levels of all-trans-retinal made PRs more sensitive to light. On the other hand, an opposite mechanism was involved when RPE65 protein was removed (Grimm et al., 2000) or decreased due to the met450 variant (Danciger et al., 2000; Wenzel et al., 2001b, 2003) with opposite effect. As mentioned earlier, mice homozygous for RPE65 met450 or with no RPE65 had lower or no levels of RPE65 protein in their RPE, slower regeneration of rhodopsin, and less PR loss after intense light exposure (Danciger et al., 2000; Grimm et al., 2000; Wenzel et al., 2001b, 2003).

In summary, quantitative genetics studies have demonstrated the presence of many genes that affect light damage by the discovery of QTL. Microarray studies show the upregulation of many genes after damaging light exposure. Some of these genes are located in QTL and may be considered light damage effector gene candidates. Not surprisingly, single-gene knockout or enhanced gene expression studies have implicated apoptotic genes in PR light damage. Such genes are involved in cell death initiated in many other tissues as well. So far, only one specific eye gene expressed in the RPE, Rpe65, has been shown to be a naturally occurring complex trait gene or gene modifier. The influence of the met450 variant of RPE65 is to protect PRs from light damage by slowing down the visual cycle (Wenzel et al., 2001b, 2003). However, this same mechanism is in play in several genetically modified mice. The absence of Rdh12 speeds up regeneration of rhodopsin during intense light exposure, exacerbating PR damage (Thompson et al., 2005). Absence of the α -subunit of transducin (Gnat1) protects PRs from light damage under some circumstances (Hao et al., 2002). α-Transducin is an integral part of the visual cycle, and without it there is no hyperpolarization-depolarization cycle. The absence of either rhodopsin kinase (Grk1) (Chen et al., 1999a) or arrestin (Sag) (Chen et al., 1999b), both needed to shut off rhodopsin, brings about RD even in dim cyclic light because the visual cycle is kept on. Therefore, one component of the complex trait of light damage to PRs is perturbation of the very biochemical-physiological cycle that mediates vision under normal circumstances. Thus, in the retina of a mouse expressing the naturally occurring but rare RPE65 met450 modifier, damage is modulated under the stressed conditions of toxic light, while under nonstressed conditions the retina is normal. This is a very nice example

of an allele or gene variant that perturbs a system only under abnormal conditions and thereby modifies the effect of the abnormal conditions.

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Age-related eye diseases are the leading causes of vision impairment and blindness throughout the world. With the world’s population of senior citizens growing rapidly, the issue of vision loss in the older population is of paramount importance. Age-related eye diseases are costly to treat, threaten the ability of older adults to live independently, and increase the risk for accidents and falls. Of the age-related eye diseases, age-related macular degeneration (AMD) increases dramatically with age in men and women and is the most important cause of irreversible visual loss in the elderly. Cataracts are the leading cause of blindness in the world, affecting nearly 20.5 million Americans age 40 and older (Congdon et al., 2004; Blindness and visual impairment [editorial], 2004).

From the available data, it appears that age-related eye diseases are caused by environmental factors triggering disease in genetically susceptible subjects. Identifying the genetic factors would contribute to understanding the pathogenesis. If those at risk could be identified, it might be possible to modify lifestyle or develop novel therapies in the presymptomatic stage to prevent disease or decrease severity. However, direct research on human ocular conditions is impeded by the poor availability of tissues and the impossibility of performing genetic manipulation in humans. Human eye tissue (including biopsy material) in most ocular diseases is seldom available, because it is difficult to obtain eye tissue samples without the risk of damaging the patient’s vision. Since genetic and biochemical experiments in human patients are not possible, animal models serve an important and unique role. Historically, mouse models have been especially useful in determining biochemical mechanisms in human ocular diseases. In this chapter, I focus on mouse age-related retinal degeneration disorders as possible models for human AMD and mouse age-related cataract mutants as models for human cataracts.

Mice as models for human age-related eye diseases

The mouse lens and retina are remarkably similar in structure to the human lens and retina, and both species experience similar ocular disorders (Chang et al., 2005). Not only are developmental and invasive studies possible in mice, but the mouse’s accelerated life span and generation time (one mouse year equals about 30 human years, based on a ratio of average life span for each species) make it possible to

follow the natural progression of eye diseases in a relatively brief period. Studying aging diseases in mice has many advantages: typically mice live 2 years and thus age quickly, and they are relatively inexpensive and easy to maintain. The availability of inbred strains provides a population that is genetically the same from one mouse to the rest; each inbred strain is like an infinite set of monozygotic or identical twins. All mice of the same inbred strain generally can be expected to have the same phenotype. By contrast, differing environmental factors combined with many genetic variations between affected and unaffected individuals make it difficult to identify specific genes responsible for age-related eye diseases in humans. Environmental factors can play an especially strong confounding role in the etiology of lateonset eye diseases in humans because of the long period of life with exposure to different variables before any agerelated eye diseases are detected. In the mouse, environmental factors can be controlled to a high degree by raising mice in standard conditions of diet, light-dark cycle, cagechanging schedules, experienced animal handlers, and constant and clean air exchange in the mouse rooms, and with a high standard of disease prevention and animal health care. With environmental variance controlled, researchers can focus on the underlying genetic causes. Genetically homogeneous inbred mice, accessible at any age, offer an opportunity to study the histopathological and biochemical changes that occur during age-related eye disease formation and permit controlled study of single gene variations in a constant genetic background. The high level of homology between mouse and human genomes (>95% of genes are conserved) means that similar disease manifestations are often identified in mice and humans. Preservation of gene function is the reason that mice are often useful in studies of disease mechanisms in humans (Davisson et al., 1991; Nadeau et al., 1992; Quiring et al., 1994). Thus, the high degree of conservation between human and mouse chromosomes makes the mouse a powerful research tool with which to identify human genes, which can then be used in presymptomatic testing and for developing preventive treatment.

The search for age-related eye diseases in mice

The Jackson Laboratory (TJL), having the world’s largest collection of mouse mutant stocks and genetically diverse inbred strains—more than 3,000 strains in 2006—is an ideal

place to discover genetically determined eye variations and disorders. While screening mouse strains and stocks at TJL for genetic mouse models of human ocular disorders, we have identified numerous spontaneous or naturally occurring mutations (table 48.1). However, the search for lateonset eye disease has never been done, and the best place to do this is at TJL. TJL has the best collection of genetically unrelated inbred strains available anywhere. For clinical characterization, a primary search for gross eye abnormalities has been done by examining the eyelids, globe, cornea, and iris, first with visual inspection and then using a Nikon biomicroscope (slit lamp). The cornea is checked for clarity, size (bupthalmos vs. microcornea), surface texture, and vascularization. The iris is examined for pupil size, constriction, reflected luminescence, and synechiae. The eye is then dilated with 1% atropine and the lens is examined for cataracts. Finally, an indirect ophthalmoscope is used to examine the fundus for signs of retinal degeneration. In mice, the typical changes are retinal vessel constriction or retinal pigment epithelial disturbance, drusen, or other retinal deposits. Mice with a suspected abnormality are followed up with a secondary examination that includes electroretinography (ERG) and histological investigation. For genetic characterization, an initial genetic analysis to determine the mode of inheritance is carried out by making outcrosses and backcrosses or intercrosses. Once an eye disorder has been shown to be due to a new mutation, a linkage cross is set up to determine the mutant gene’s chromosomal location by doing a genome scan of DNA markers using PCR analysis. Mice with age-related eye diseases often are past the breeding age. Because all mice in an inbred strain are genetically identical, this can be overcome by doing the genetic crosses with young mice from the same strain, because they will acquire the same eye disease. Dominant and semidominant mutations are mapped by backcrosses or outcrosses to wildtype (+/+) mice. Recessive mutations are mapped using intercrosses, which are more efficient, because each F2 mouse obtains two potentially recombinant chromosomes, one from each parent. Modifying genes often can be detected in the initial cross by finding more than one chromosomal region associated with the disease phenotype. Polygenic traits are recognized by phenotype loss in outcrosses. If a disease turns out to be polygenic—that is, influenced by more genes than can be identified in standard genetic crosses—mice of the strain are still histologically and clinically characterized to provide a model for clinically similar human ocular disorders.

Age-related retinal degenerations in human and mouse eyes

For many people, retirement means more time to read, watch television, sew, play cards, or drive to places they have

always longed to visit. Yet by the time they reach age 65, many retirees find they no longer have those options because they have lost much of their vision to age-related eye disorders, such as age-related macular degeneration (AMD) and many forms of late-onset retinal degeneration, the most common uncorrectable causes of vision loss in the elderly. Although mice do not have a macula, mice do have homologues of genes that are associated with human AMD, and mutations in such genes in mice likely cause age-related retinal degeneration (ARRD). For example, partial loss of the ABCA4 or rim protein is sufficient to cause a phenotype in mice similar to recessive Stargardt disease and AMD, a common cause of visual loss in the human elderly (Mata et al., 2001). In the Abca4tm1Ght homozygous mouse model, the presence of A2E and lipofuscin granules is seen, along with shortening of the photoreceptor outer segments (Weng et al., 1999; Mata et al., 2000; Radu et al., 2004). Another example is that the ELOVL4 gene is associated with two related forms of human autosomal dominant macular dystrophy, and in situ hybridization on mouse retinal sections shows a strong uniform signal in the photoreceptor layer, particularly in the region corresponding to the photoreceptor inner segments (Zhang et al., 2001). Mutant mice carrying an Elovl4 transgene demonstrate an accumulation of A2E and lipofuscinlike material, but they also show photoreceptor outer segment disc disorganization and geographic atrophy (Karan et al., 2005). Mice with a 5 bp deletion knock-in of Elovl4 develop progressive photoreceptor degeneration (Vasireddy et al., 2006). Recently, a known polymorphism in human complement factor H (CFH), T → C substitution in exon 9, which resulted in the substitution of an uncharged tyrosine with a positively charged histidine (Y402H), was shown to be associated with increased risk for AMD (Edwards et al., 2005; Hageman et al., 2005; Haines et al., 2005; Klein et al., 2005; Zareparsi et al., 2005). The human and mouse CFH proteins contain 20 repetitive units of 60 amino acids, referred to as the short consensus repeat or the complement control protein module, arranged in a continuous fashion like a string of 20 beads (Rodriguez de Cordoba et al., 2004). In human and mouse, the CFH expression pattern was found to be similar, with the highest level of expression in the liver. In ocular tissue, CFH was detected in the distalmost optic nerve (3 mm) cut from the scleral surface of the eyeball, sclera, RPE-choroid, retina, lens, and ciliary body. In mouse, Cfh expression was observed from early embryonic stages, and in the eye, its expression increased with age (Mandal and Ayyagari, 2006).

Historically, mouse models have been especially useful in determining biochemical mechanisms in retinal diseases. For example, invaluable information about the molecular and pathological basis of some of these diseases has been provided by the discoveries of gene mutations in several mouse retinal degeneration models. Discovery of the mouse

retinal degeneration 1 (rd1) mutation in the gene for the β-subunit of cGMP-phosphodiesterase (Pde6b) (Bowes et al., 1990) led to the identification of mutations in the human homologue (PDE6B) in similar human disorders (McLaughlin et al., 1993), and identification of the tubby (tub) mouse gene family (Noben-Trauth et al., 1996) led to the finding of tubby-like protein 1 (TULP1) alterations in individuals affected with autosomal recessive RP (Hagstrom et al., 1998; Banerjee et al., 1998). Similarly, discovery of the mutations Rds (retinal degeneration slow) in the peripherin 2 (Prph2) (Travis et al., 1989) and shaker 1 (sh1) in the myosin 7A (Myo7a) (Gibson et al., 1995) mouse genes paved the way for the identification of defects in the RDS-periph- erin gene in patients with autosomal dominant RP, and various cone, cone-rod, and macular dystrophies (Keen and

Inglehearn, 1996) and in MYO7A in patients with Usher’s syndrome type 1b (Weil et al., 1995).

Discovery of 15 Strains of Mice with Age-Related

Retinal Degeneration At TJL we have a productive screening and evaluation program for identifying new mouse models of ocular diseases. A component of this program, the study of age-related genetic eye diseases in mice, was funded by the Foundation Fighting Blindness. We aged 10 mice from each of 35 different inbred strains and screened them from 6 months to 2 years of age and up for late-onset retinal disorders. The 35 strains listed in table 48.2 have known abnormalities (low ERG, iris atrophy, cataracts) as strain characteristcs or are wild-derived strains that have not been characterized for age-related eye diseases.

the 15 strains in which ARRD was found were blind at 2 years of age as confirmed by ERG (no response) and histology (no outer nuclear layer, ONL), and mice of the other 12 strains experienced slow retinal degeneration with very poor vision, as confirmed by low ERG response and reduced ONL (2–6 layers compared to the normal 10 layers of ONL) at 2 years of age. The data presented here document the

disease phenotype in seven wild-derived ARRD strains with retinal degeneration (figure 48.1) and eight laboratory ARRD strains of mice with ONL cell loss (figure 48.2). In six of these laboratory strains the retinal degeneration may be caused by light damage during aging, because these six strains of mice are albino and lack a pigmented iris to protect the retina from light during the daily 14 hours of light in the

Figure 48.1 Retinal histological sections in seven wild-derived ARRD strains showing relatively normal retinal structure at relatively younger ages and retinal degeneration at older ages. The strain names are labeled at the top and the ages are labeled within each retinal section. GCL, ganglion cell layer; INL, inner nu-

clear layer; IPL, inner plexiform layer; IS, photoreceptor inner segments; ONL, outer nuclear layer; OPL, outer plexiform layer; OS, photoreceptor outer segments; RPE, retinal pigment epithelial cells.

light-dark cycle of the mouse room. Retinal degeneration can be induced by exposure to excessive doses of light and is used as a model to study photoreceptor apoptosis, the common final pathway of cell loss in human AMD and some forms of inherited retinal degenerations (Rome et al., 1998).

Genetic factors reducing the light damage susceptibility (LDS) of retinal photoreceptors in C57BL/6J mice were postulated in 1987 (LaVail et al., 1987); however, the underlying molecular mechanisms have not yet been identified. Recently, by comparing progeny from a cross between

Figure 48.2 Retinal histological sections in eight laboratory ARRD strains showing relatively normal retinal structure at relatively younger ages and retinal degeneration at older ages. The strain names are labeled at the top and the ages are labeled within each retinal section. GCL, ganglion cell layer; INL, inner nucle-

ar layer; IPL, inner plexiform layer; IS, photoreceptor inner segments; ONL, outer nuclear layer; OPL, outer plexiform layer; OS, photoreceptor outer segments; RPE, retinal pigment epithelial cells.