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

fuscin storage bodies contain a complex mix of proteins and lipids and likely results from incomplete turnover of POS material (Feeney, 1978). In vitro studies have recently shown that lipofuscin components may directly impair RPE function and viability (Holz et al., 1999; Finnemann et al., 2002). These data suggest that defective digestion of POS by RPE cells may contribute to development or progression of age-related retinal diseases such as age-related macular degeneration.

Outer segment renewal in higher vertebrates is synchronized by circadian rhythms influenced by the daily dark-light cycle (Goldman et al., 1980). Animal studies in rodor conedominant species revealed that rods mainly shed their POS ~2 hours after onset of light and cones shed ~2 hours after dusk (LaVail, 1976; Young, 1977). The increase in the number of phagosomes present in RPE cells at these two time points suggests a peak in phagocytic activity every 12 or 24 hours for RPE cells depending on whether they serve rods, cones or both. No untimely phagocytosis has been observed so far, suggesting that RPE cells may downregulate their phagocytic activity if not “on duty”. Currently, mechanisms that regulate RPE phagocytosis are only poorly understood.

2. THE PHAGOCYTIC MACHINERY OF THE RPE

Three plasma membrane receptors have been shown to fullfill distinct roles in RPE phagocytosis. These are the Mer tyrosine kinase receptor MerTK (D’Cruz et al., 2000; Nandrot et al., 2000), the scavenger receptor CD36 (Ryeom et al., 1996), and the adhesion receptor avb5 integrin (Finnemann et al., 1997; Miceli et al., 1997; Lin and Clegg, 1998). MerTK is involved in the internalization step of phagocytosis (Edwards and Szamier, 1977). MerTK deficient macrophages have a reduced capacity to phagocytose apoptotic cells confirming that OS phagocytosis by the RPE is a mechanism similar to the macrophage clearance mechanism for apoptotic cells (Finnemann and Rodriguez-Boulan, 1999; Scott et al., 2001). In vitro experiments have shown that CD36 ligation regulates the rate of POS internalization suggesting a role for CD36 in phagocytic signaling (Finnemann and Silverstein, 2001).

avb5 expression at the apical surface of rodent RPE in vivo coincides with postnatal establishment of mature interactions between photoreceptors and RPE including the onset of POS renewal (Ratto et al., 1991; Finnemann et al., 1997). Stable binding of POS to the cell surface of RPE cells in culture is largely dependent on avb5 integrin receptors (Finnemann et al., 1997). In addition to POS recognition or tethering RPE cells also use avb5 integrin receptors to activate signaling pathways through focal adhesion kinase that are necessary to activate MerTK (Finnemann, 2003; Nandrot et al., 2004; and see chapter by Finnemann and Nandrot in this volume). As b5 integrin only dimerizes with av integrin subunits, b5 integrin knockout mice provide the opportunity to study RPE cells that permanently and exclusively lack avb5 receptors.

3. IMPACT OF LACK OF avb5 INTEGRIN ON RPE FUNCTION

b5 knockout mice are viable and fertile, and have normal lifespan (Huang et al., 2000). When we first examined their retinal tissues we did not find gross anatomical abnormalities in b5 null mice regardless of age (Nandrot et al., 2004). We then set out to compare wild-

Figure 18.1. b5 integrin deficient RPE cells display defects in POS phagocytosis. (a) After 1 to 3 hours of POS phagocytic challenge in vitro, b5-/- cells in primary culture phagocytosed less OS than b5+/+ cells. (b) Phagosomes were counted on cross sections of b5+/+ and b5-/- eyecups at different time-points of the 24-hour phagocytic cycle. The phagocytic peak detected in b5+/+ mice 2 hours after light onset is absent in b5-/- mice. Modified from Nandrot et al. (2004), J. Exp. Med. 200:1539-1545 by copyright permission of The Rockefeller University Press.

type and b5 knockout RPE phagocytic function both in vitro and in vivo. Finally, we investigated vision and RPE cell ultrastructure as a function of age in b5 knockout and wild-type control mice.

To test POS phagocytosis in vitro we isolated and maintained in primary culture RPE cells from b5 null and wild-type control animals. Strikingly, b5 null RPE cells failed to efficiently take up OS. This decrease was not due to slower uptake kinetics as uptake by b5 null cells remained low at all times of phagocytic stimulation (Figure 18.1a).

To check POS phagocytosis in vivo we counted phagosomes present in RPE cells of 4 week old wild-type and b5 null mice at different time points of the 24-hour period. As expected, we detected a peak in the number of phagosomes in wild-type retina ~2 hours after the light onset occuring at 6 AM in our facility (Figure 18.1b) (LaVail, 1976). This peak was lost in b5 null retina in which we counted equal numbers of phagosomes at all time points tested. However, phagosome counts in b5 null retina were similar or higher than phagosome counts in wild-type retina outside of the phagocytosis peak. Thus, phagocytosis in b5 knockout retina is not abolished but loses its temporal regulation. Absence of avb5 integrin eliminates the burst of phagocytic activity required for synchronized POS engulfment.

These results show that POS phagocytosis by RPE cells is impaired in 1-month-old b5 null mice. However, b5 null mice develop impaired vision only at a much older age. Scotopic electroretinograms (ERGs) recorded in 1-year-old animals showed a strongly attenuated response in b5 null compared to wild-type mice (Figure 18.2a). The reduction of retinal responses to light stimuli was progressive from 4 months of age in b5 null animals whereas responses did not vary greatly for wild-type animals (Figure 18.2b).

Finally, we detected excessive autofluorescent lipofuscin deposits in RPE of 1-year-old b5 null mice by wide-field fluorescence microscopy (Nandrot et al., 2004). These lipofuscin granules appeared as dense opaque bodies on ultrathin sections examined by electron

Figure 18.2. Age-related retinal changes in b5-/- mice. (a) At 1 year of age, scotopic ERG responses are greatly reduced in b5-/- mice. (b) ERGs were recorded for wild-type ( ) and b5-/- ( ) mice between the ages of 4 and 12 months. Both a- and b-waves declined in b5-/- mice older than 4 months. RPE cells of 1-year-old wild-type

(c) and b5-/- (d) mice were examined by electron microscopy on ultrathin sections. Electron dense inclusion bodies were detected in b5-/- RPE in larger numbers than in wild-type RPE. Modified from Nandrot et al. (2004), J. Exp. Med. 200:1539-1545 by copyright permission of The Rockefeller University Press.

microscopy. We observed few of these inclusion bodies in wild-type RPE (Figure 18.2c), whereas they were present in large numbers in b5 null RPE (Figure 18.2d).

4. PERSPECTIVE

Our results show that lack of avb5 integrin receptors eliminates the rhythm of POS phagocytosis in the retina. With age, mice lacking avb5 integrin progressively lose vision and their RPE accumulates lipofuscin granules. Strikingly, even with an early phagocytosis defect b5 null mice only develop late onset pathology. Our findings suggest that constant instead of rhythmic POS phagocytosis by RPE cells may impair POS digestion causing gradual accumulation of autofluorescent compounds as lipofuscin.

Taken together these data emphasize the importance of timely regulation of RPE phagocytosis. avb5 integrin activity synchronizes this daily process that is essential for vision (Nandrot et al., 2004). The age-related changes we observed in retinas of b5 knockout mice share some characteristics of retinas of humans with age-related macular degeneration. The b5 knockout mouse strain may thus provide a useful animal model to study lipofuscin buildup with age and gene therapies to delay or reverse its accumulation.

5. 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.

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expression produced earlier signs of retinal degeneration and more severe disruption of photoreceptor morphology (Naash et al., 2004). Furthermore, the rod a-wave amplitudes and the amounts of photosensitive pigment were substantially reduced at early ages. Thus, these findings suggest a failure of G90D opsin to efficiently complex with the chromophore to form a light-sensitive rhodopsin in the rod outer segments (ROS). Due to the significant loss of rod sensitivity observed in these G90D retinas; this study addresses the effect of such a desensitized rod environment on the light-dependent translocation of transducin alpha subunit (Ta). Studies have shown that upon illumination, a substantial proportion of Ta found to rapidly move out of the ROS and return only in the dark (Whelan and McGinnis, 1988). The process of Ta movement out of the ROS seems to be mediated by the activation of transducin by photo-isomerized rhodopsin, while the reverse movement depends on the deactivation of the transduction cascade (Hardie, 2003).

2. MATERIALS AND METHODS

2.1. Generation of G90D Transgenic Mice

A 15 kb mouse opsin genomic fragment was used to generate the G90D line. This fragment contains 6.0 kb of the promoter region, all the introns and exons, and 3.5 kb of the 3¢ untranslated region containing all of the multiple polyadenylation signals. Mutations were introduced by site directed mutagenesis in PCR reactions as described in previous work (Naash et al., 2004). Ten potential founders were identified and eight of them passed the transgene to their offspring. Transgenic mice were mated to rhodopsin knockout mice (-/-) (Lem et al., 1999) to express the transgene in different backgrounds of wild-type (wt) opsin (i.e., +/+, +/-, or -/-). All mice studied here were maintained in the breeding colony under cyclic light (12L:12D) conditions; cage illumination was ~7 foot-candles during the light cycle. Light-adapted animals were taken after 4 hours of being in their light cycle while dark-adapted animals were kept in the dark for at least 4-6 hours. All procedures were approved by the local Institutional Animal Care and Use Committees and adhered to the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research.

2.2. Histology and Immunohistochemistry

For histology using light microscopy, animals were sacrificed, and eyes were fixed in Davidson’s fixative (95% ethanol, 10% neutral buffered formalin, glacial acetic acid, and distilled water) overnight at 4°C. The eyes were then washed 3 times in PBS and stored in 70% ethanol prior to serial dehydration and embedment in paraffin. Eyes were sectioned (6- to 8-mm thickness) on a standard microtome and stained with hematoxylin/eosin.

For immunohistochemistry, animals were sacrificed and eyes were fixed in 0.1 M phosphate buffer (pH 7.4) containing 4% formaldehyde. Tissues were incubated overnight with 10% sucrose in 0.1 M phosphate buffer (pH 7.4) and then stored in 30% sucrose phosphate buffer. The eyes were embedded and processed as described previously (Ding et al., 2004; Nour et al., 2004). The sections were then incubated overnight at 4°C with either 1D4 (a generous gift of Dr. Robert Molday) or with a polyclonal antibody to Ta (purchased from Santa Cruz Biotechnology) at 1 : 100 dilution. Three retinal sections per slide were incu-

Figure 19.2. (A). Structural analysis at the light level of one month old wt and G90D retinas on all rhodopsin genetic backgrounds. (B). Immunofluorescence of frozen sections of the same animals used in panel A labeled with 1D4. Immunofluorescence was confined almost exclusively to the ROS and rod inner segments (RIS). The fluorescent intensity appeared to be greatest in the ROS. No labeling was detected in the outer nuclear layer (ONL). Scale bar: 25 mm.

which indicates that G90D opsin has been properly localized and integrated into the ROS disc membranes.

3.2. Light-Dependent Translocation of Transducin Alpha in the G90D Retinas

In wt mouse retinas, light triggers the translocation of transducin subunits from the outer segment in the dark, to the inner segment in the light. This process is dependent on photoisomerization of rhodopsin (Hardie, 2003). This experiment, tested whether the desensitization state of the G90D retinas interferes with the translocation of Ta in rod cells. Comparisons of the Ta antibody staining patterns of darkand light-adapted wt and G90D retinas revealed that the majority of Ta resides in the ROS of dark-adapted G90D retinas from all genetic backgrounds, whereas in the light-adapted state Ta is found throughout the G90D photoreceptor layer, including the RIS and the synapse, similar to the distribution pattern of Ta in non-transgenic controls. These results suggest that Ta redistribution is not effected by the desensitization state imposed by the G90D mutation. Ta translocation was also obvious in the G90D retinas on the rhodopsin null background (Fig. 19.3K&L). Since Ta level in the rhodopsin null retinas is significantly low, its light-dependent translocation in the absence of rhodopsin was not apparent (Fig. 19.3I&J).

Figure 19.3. Sub cellular distribution of Ta in rod cells in darkand light-adapted wt and G90D retina from all rhodopsin genetic backgrounds (+/+, +/-, and -/-). The patterns of immuno-staining in sections taken from darkand light-adapted wt and G90D retinas are similar suggesting that the desensitization state of the G90D rods has no effect on the translocation of transducin. Scale bars: 25 mm; (insets).

4. DISCUSSION

Light-dependent translocation of phototransduction proteins between the inner and outer segments of photoreceptor cells has been thought to play an important role in the adaptation of these cells to light (Whelan and McGinnis, 1988; Zhang et al., 2003; Elias et al., 2004; Lee and Montell, 2004). However, the mechanism by which simultaneous translocation of these proteins remains unknown. Studies have demonstrated that some of these movements occur within minutes following the onset of light (Elias et al., 2004). It has been proposed that the major function of these translocations is to provide protection of photoreceptors in bright light rather than increased vision in dim light (Elias et al., 2004).

The present study tested the light-dependent trafficking of Ta in transgenic retinas carrying the G90D mutation in the opsin gene, a mutation that has been shown to cause a form of CSNB in humans (Sieving et al., 1995), constitutive activation of opsin in COS cells (Rao et al., 1994), and a persistent state of light adaptation (“dark light”) in transgenic mice (Sieving et al., 2001; Naash et al., 2004). Since photoreceptor degeneration is directly related to the levels of transgene expression due to opsin over-expression (Naash et al., 2004), a transgenic line was used in this study that expresses G90D opsin at a level equivalent to that of wt. Although no signs of photoreceptor degeneration was seen in this line at early ages (Fig. 19.2A), retinal morphological changes were evident in older animals on different rhodopsin backgrounds (Naash et al., 2004). These changes are attributed to the presence of the G90D opsin rather than opsinoverexpression (Tan et al., 2001). As shown in Figure 19.3, the majority of Ta resides in ROS in dark-adapted wt and G90D retinas. In the

light-adapted state of the G90D retina, Ta is found throughout the photoreceptor layer, including the RIS and the synapse, similar to the distribution of Ta in wt mouse retinas. These results suggest that Ta redistribution is independent of the activation status of rhodopsin, an observation that was previously reported (Mendez et al., 2003).

5. ACKNOWLEDGMENT

This research was funded by National Eye Institute (NEI) grant EY-10609 (MIN), Department of Ophthalmology NEI Core grant (EY-01792), the Foundation Fighting Blindness (MIN). Dr. Naash is a recipient of the Research to Prevent Blindness James S. Adams Scholar award.

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