Ординатура / Офтальмология / Английские материалы / Retinal Degenerations biology, diagnostics, and therapeutics_Tombran-Tink, Barnstable_2007

Fig. 7. Fluorescence microscopy of retinal cryosections from wild-type (A,D) and retinoschisin-deficient mice 5 mo after subretinal delivery of a serotype 5 AAV vector containing the wild-type human RS1 cDNA driven by the mouse opsin promoter (AAV5-mOPs-RS1) to the right eye of 15-d-old mice (B,E). The left eyes of the Rs1h–/Y mice were not injected and served as internal controls (C,F). (A–C) Sections were labeled with RS1 3R10 anti-RS1 monoclonal antibody (69). (D–F) sections were stained with DAPI (blue) and imaged with DIC microscopy. Note the wild-type like expression pattern of RS1 and the recovery of the retinal structures in the treated animals. The arrows point to schisis cavities in the untreated animals.

OPL (Fig. 7A,B). The INL and to a lesser degree the IPL was also labeled. As expected, no immunostaining was observed in the uninjected left control retina (Fig. 7C). It is interesting to note that in the treated eyes retinoschisin can be found at physical distances as far away from the site of secretion (i.e., photoreceptors) as the IPL. This may indicate that secreted retinoschisin is able to spread through the various retinal layers. In agreement with this, we also observed an impressive lateral movement of secreted retinoschisin away from the site of injection in all animals analyzed.

The expression of RS1 coincided with a marked improvement in the structural organization of the retinal layers as visualized in differential interference contrast images merged with DAPI nuclear stain (Fig. 7D–F). The treated retina was organized into characteristic layers with a distinct separation of the INL and ONL and an absence of gaps between bipolar cells. An increased thickness of the ONL, indicative of enhanced photoreceptor survival, was also seen in the treated retina (Fig. 7E). In contrast, the untreated eye showed the known manifestations of advanced disease (Fig. 7F).

Recently, Zeng and associates (73) reported on a similar approach to treat retinoschisin-deficient mice, although with some minor variations. Instead of the human

gene, they delivered an AAV construct containing the orthologous mouse Rs1h cDNA under the control of a cytomegalovirus (CMV) promoter. By intraocular instead of subretinal injections, a serotype 2 AAV vector was administered to adult knockout mice of 13 wk of age. Similar to our results, their preliminary data indicate retinoschisin expression in all retinal layers. So far, no data are available documenting the effects of the treatment on the morphology of the retinal layers or on photoreceptor cell survival. Despite the late delivery of retinoschisin at an advanced stage of disease progression, ERG recordings nevertheless showed a reversal of the electronegative a-wave and restoration of the normal positive b-wave (73). The latter findings may be encouraging for the future design of gene therapy protocols aimed at adult RS patients with advanced pathology.

CONCLUSIONS AND FUTURE DIRECTIONS

Although a relatively rare Mendelian condition, RS is an important disease to study, not only to achieve a better understanding of retinal physiology in the normal and diseased eye but also more importantly to define targeted treatment options for the patient. From the early descriptions of the clinical features of RS more than 100 yr ago, advances in molecular research over the last 10 yr have given us tremendous insight into RS pathology not without the promise for novel therapeutic avenues. The gene mutated in RS codes for retinoschisin, a protein secreted as a disulfide-linked oligomeric complex from the photoreceptors, to a minor degree from bipolar cells and possibly other neuronal cells of the retina. Retinoschisin is firmly associated with membrane surfaces owing to a highly conserved discoidin motif, which is known in other proteins to mediate cell adhesion/aggregation properties. Particularly, high concentrations of RS1 are found along the entire length of the photoreceptor inner segment membranes. Most bipolar cell types also markedly bind retinoschisin. Müller cells, however, long thought to play a crucial role in RS pathology, appear devoid of retinoschisin strongly arguing against a pivotal part of this cell type in disease etiology. Rather, the characteristic retinal expression pattern of retinoschisin brings primary pathology of photoreceptor and bipolar cells more into focus.

Analysis of the molecular pathology of disease-associated RS1 mutations has suggested that the mutant gene product is either absent or nonfunctional in patients with RS, leading to a complete absence of retinoschisin in males. Consequently, retinoschisindeficient mice closely mimic human RS pathology by forming cystic structures within the inner retina and revealing a characteristic electronegative ERG waveform pattern. In addition, a striking reduction of rod and, to a greater extent, cone photoreceptor cells is seen in several week old mutant animals. In postnatal retinal development, retinoschisin deficiency results in destabilization of the retinal organisation and decreased cohesion of cell structures but appears also critical for the formation and maintenance of retinal synapses in the outer and inner plexiform layers.

The disease mechanism in RS suggests that replacement of the normal gene product in the retina may provide an adequate therapeutic approach to improve the outcome for patients with RS. Toward this end, gene delivery to the retinoschisin-deficient mouse retina via AAV particles has successfully been attempted. The findings are most promising and provide proof-of-concept for the feasibility of protein replacement in RS. AAV-mediated RS1 gene therapy lies ahead of us as an optional treatment for patients with RS.

ACKNOWLEDGMENTS

The authors wish to thank Andreas Janssen and Andrea Gehrig (Institute of Human Genetics, University of Würzburg, Germany) for their help with the figures and Robert S. Molday for his support and his critical comments to the manuscript.

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has been linked to Usher 3. Thus, so far, Usher syndrome can be divided further into eleven different genetic loci, for which eight of the genes have been identified (Table 1). It should be noted that mutations in these genes do not always result in Usher syndrome. Cases of nonsyndromic deafness have been linked to mutations in the Usher 1B, 1C and 1D genes (11–15). Conversely, mutations in USH2A and USH3A can cause autosomal recessive RP without reported hearing loss (16–18).

Usher 1 and 2 are the most common forms of Usher syndrome, with Usher 3 contributing to a large proportion of cases only in isolated areas, such as Finland (19) and Birmingham, UK (20).

VISUAL IMPAIRMENT IN USHER SYNDROME

The visual loss in Usher syndrome begins with deterioration of peripheral and night vision, as in other forms of RP. Measurements of the kinetics of visual field loss in patients with Usher 2 were determined to be comparable to those of nonsyndromic RP (21). Among the different types of Usher syndrome, some differences in visual impairment have been identified, although such differences are much less evident than differences in vestibular and auditory dysfunction. Fishman’s group has observed that the visual loss with respect to age is significantly greater in Usher 1 than in Usher 2, on the grounds of visual acuity and visual field (22,23), and according to the probability of developing foveal lesions (24). In a multifocal electroretinogram (ERG) analysis (25), it was found that the ERG amplitude of Usher 1, Usher 2, and nonsyndromic patients with RP was reduced to a similar extent and in the same pattern across the retina (i.e., more reduced in the periphery). However, the waveform of the ERG differed between patients with Usher 1 and Usher 2. The time to the peak of the response (latency or implicit time) was unaffected in patients with Usher 1, but this time was significantly longer in patients with Usher 2 or nonsyndromic RP.

The finding of distinguishing characteristics between Usher 1 and 2 has not extended to differences among different genotypes of the same type of Usher syndrome. No significant difference in visual acuity, visual field, ERG amplitude, and ERG implicit

time was detected between Usher 1B and other Usher 1 genotypes (25,26). Similarly, no distinguishing characteristics between Usher 2A and 2C were found, using psychophysical, electrophysiological, and retinal imaging analyses (27).

RETINAL FUNCTION OF USHER PROTEINS

The function of proteins, involved in phototransduction and related events, is best understood in the photoreceptor and retinal pigment epithelial (RPE) cells. Although the cell biology of the photoreceptor and RPE cells—these cells are among the most specialized cells in our bodies—is clearly important, considerably less is known about the proteins involved in their structural organization. Yet, hair cells of the inner ear and the photoreceptor and RPE cells of the retina are more similar structurally than they are functionally. In particular, all possess regions of amplified plasma membrane: the stereocilia in the hair cells, the apical microvilli of the RPE cells, and the disk membranes that make up the photoreceptor outer segments. Not surprisingly, then, the Usher proteins appear to be more related to cell structure than function.

The proteins encoded by the known Usher genes are listed in Table 1, their distributions in the retina are shown in Fig. 1, and their structural organization is depicted in Figs. 2 and 3. MYO7A was predicted to encode an unconventional myosin, i.e., a molecular motor that uses energy from adenosine triphosphate (ATP) hydrolysis to move along actin filaments. Direct experiments have now shown this to be the case; myosin VIIa is a bona fide actin-based motor (28,29). The USH1C gene generates a number of different isoforms, belonging to three different classes of harmonin. The isoforms each contain two or three PDZ domains (modular protein interaction domains), so that harmonin is predicted to be a scaffolding protein (30). The Usher 1D and 1F genes are both predicted to encode cadherins (15,31–33). The Usher 1G protein, sans, has no indicated function (34). Usherin, encoded by USH2A, is an extracellular matrix protein that binds type IV collagen (35). The Usher 2B gene is not known, although a likely candidate is that encoding the sodium bicarbonate cotransporter, NBC3. NBC3 is at the Usher 2B locus, and mice lacking NBC3 undergo degeneration of the retina and inner ear (36). VLGR1 appears to be a G protein-coupled receptor with a large N-terminal region (37). Lastly, the only reported Usher 3 gene encodes clarin1, which has been speculated to function in synaptic shaping and maintenance, based on loose homology with a protein known to function in this manner in the cerebellum (38).

The precise retinal functions of these proteins are largely unknown. It has been proposed that many of the proteins might function together in a common cellular mechanism. Such a unifying hypothesis is attractive and, certainly, the similarities of clinical phenotype within the different types of Usher syndrome suggest that the mutated genes of each type might affect a common cellular mechanism. Experimental evidence to support this notion has come from studies indicating that some of the Usher 1 proteins can interact with each other.

In 1956, it was reported that a genetic interaction in ear function was evident from crossing shaker1 mice with waltzer mice (39). We now know that shaker1 mice carry mutations in Myo7a (40,41), and waltzer mice carry mutations in Cdh23 (42), so that this result indicates that these two Usher 1 proteins might interact. More recently, in studying the retinas of shaker1 and waltzer mutant crosses, no interaction was found by ERG analysis (43). However, after approx 12 mo, double homozygous mutants were