Ординатура / Офтальмология / Английские материалы / Retinal Degeneration Disease_Hollyfield, Anderson, LaVail_1999

thermore, in vivo phagocytic signaling via FAK and MerTK was strongly and transiently stimulated following light onset in wild-type mouse retina but was absent in b5 knockout mouse retina (shown for MerTK in Figure 69.1 b). These data provide conclusive evidence that avb5 integrin signaling regulates rhythmic activation of FAK and MerTK during RPE phagocytosis in the intact retina.

5. PERSPECTIVE

The results discussed here are the first to describe signaling activities by phagocytic receptors in intact retina that precisely correlate temporally with POS shedding and uptake by RPE cells. The late onset retinal dysfunction of the b5 knockout mouse as a consequence of lack of such phagocytic signaling emphasizes the importance of precise temporal regulation of POS phagocytosis by the RPE. In future studies, we will use similar experimental approaches exploring in vivo signaling in normal and mutant animal models to identify further components of the RPE phagocytic mechanism and to unravel their functional interactions.

6. ACKNOWLEDGMENTS

This work was supported by NIH grants EY13295 and EY14184, by a Karl Kirchgessner research grant, and by the Irma T. Hirschl/Monique Weill-Caulier Trust.

7. REFERENCES

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Table 70.1. Photoreceptor retinol dehydrogenases. All listed RDHs are from human, except mouse Rdh11. Accession numbers are as follow: Rdh11 (AF474027); RDH12 (AAH25724); RDH13 (AAH09881); RDH14 (AAH09830); retSDR1 (O75911) and prRDH (AF229845). PR, photoreceptor; IS, inner segment; OS, outer segment; LCA, Leber congenital amaurosis.

tained phototransduction.3 This pathway takes place in photoreceptor and retinal pigment epithelium (RPE) cells and allows the recycling of all-trans-retinal to 11-cis-retinal (see Figure 70.1). When all-trans-retinol is produced in photoreceptors from the reduction of all- trans-retinal, it is transported into the RPE where it is esterified by lecithin retinol acyl transferase (LRAT) and stored as all-trans-retinyl ester. All-trans-retinol is also supplied to the RPE by the choroidal vasculature, entering the RPE, in a receptor-mediated process involving a serum retinol-binding protein/transthyretin complex.4 Retinyl esters stored in the RPE are the substrate for isomerohydrolase (IMH),5 an enzyme proposed to catalyze the concerted hydrolysis of all-trans-retinyl ester and the isomerization to 11-cis-retinol. Oxidation of 11-cis-retinol to 11-cis-retinal by the 11-cis-retinol dehydrogenase RDH5 in the RPE completes the visual cycle. 11-cis-Retinal is transported back to the photoreceptors where it combines with opsin to regenerate photosensitive rhodopsin. The first step of the visual cycle is important because it generates all-trans-retinol, used to replenish the store of retinyl ester in the RPE. However, it is not the only source of all-trans-retinol since circulating all- trans-retinol can be used alternatively.

Reduction of all-trans-retinal is important because all-trans-retinal is a reactive molecule that can form toxic adducts like A2E,6 mediate photodamage,7 bind and activate opsin,8 or inhibit photoreceptor ion channels,9-11 These effects are triggered by light and are theoretically dependent on the rate of all-trans-retinal reduction, which is a slow process that takes tens of minutes in rods.12 As pointed out above, the identity of enzyme(s) catalyzing this reaction in photoreceptors is unknown but the recently cloned photoreceptor RDHs are good candidates.

2. PHOTORECEPTOR RDHs

All six RDHs listed in Table 70.1 belong to the short-chain dehydrogenase/reductases (SDR) family of oxidoreductases. Members of this family are one-domain NAD(P)(H)- dependent enzymes of 250 to 300 amino acid residues. The family is highly divergent, with typically 15%-30% residue identity in pairwise comparisons. The criterion for SDR membership is the occurrence of conserved sequence motifs, arranged in a specific manner.13 In humans, about 60 members of this family have been identified in the genome.

gene among photoreceptor RDHs that has been associated with a retinopathy, the severe early-onset retinal dystrophy Leber Congenital Amaurosis (LCA).19,20 The localization of RDH12 protein in photoreceptors and the molecular mechanism leading to the disease are unknown. However, given its similarity with RDH11, understanding the function of the latter, in vivo, might give some clues regarding RDH12 function.

RetSDR1, predominantly localized in cone outer segments, was first identified by searching an EST database from human retina with a DNA sequence corresponding to a conserved domain among RDHs.21

prRDH, localized in rod and cone outer segments, was identified from a cDNA library from bovine retina that had been simultaneously normalized and subtracted with bovine brain cDNA, in order to enrich it in genes expressed specifically in the retina.2

The subcellular localization of RDHs in photoreceptors is an important indication for their function. RDH14, retSDR1, and prRDH are located in the outer segment of photoreceptors, and therefore are likely candidates for the catalysis of the first step of the visual cycle. On the other hand, Rdh11, RDH13, and possibly RDH12, are located in the inner segment, which suggests a distinct function.

3. CHARACTERIZATION OF RDH11 KNOCKOUT MICE

Rdh11 is the first photoreceptor RDH to be studied in vivo (manuscript submitted for publication). Rdh11 knockout mouse was created by replacing Rdh11 coding sequence with the LacZ reporter gene for expression profiling.23 X-Gal staining of retinal section from Rdh11+/- mice confirmed an active transcription of this gene, only in photoreceptor cells. Rdh11-/- mice appeared normal and fertile, producing litters of normal size.

The visual phenotype of these mice was investigated by electroretinography (ERGs). These experiments revealed that the dark adaptation of knockout mice is delayed by a factor 2.5 to 3 compared to wild types. This result confirms that Rdh11 is involved in vision, more specifically during the process of dark adaptation. After illumination and return to the dark, a number of pathways are activated in photoreceptors to allow their return to the dark adapted state, which is the state of full sensitivity to light.24 This relatively slow process comprises the regeneration of 11-cis-retinal through the visual cycle. However, none of the intermediates of the cycle was significantly changed in the Rdh11 knockout mice during dark adaptation, suggesting that the defect in dark adaptation is not due to a defect in the visual cycle (manuscript submitted for publication).

Rdh11 reduces all-trans-retinal in vitro; therefore, a disruption of Rdh11 is expected to create a delayed clearance of all-trans-retinal in inner segments during dark adaptation. However, it is difficult to demonstrate in vivo, because the portion of all-trans-retinal located in inner segments is small compared to the large amounts released in outer segments during illumination. A local change of all-trans-retinal in inner segments is not expected to significantly change the total amount. Indeed, there is no significant increase of all-trans-retinal amount in Rdh11 knockout mice during dark adaptation, at least when retinoids were extracted from whole eyes (see Figure 70.2).

In vitro experiments using inner segment membrane fractions collected from wild type and Rdh11 knockout retinas would be a more sensitive assay to confirm a decreased rate of all-trans-retinal reduction in Rdh11 knockout mice. If confirmed, such a difference could have significant effects leading to the delay of dark adaptation.

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