RETINAL ADHESION

Physiology of Retinal Adhesion

Efficient exchange of substrates between POSs, IPM, and RPE requires maximal alignment and interdigitation. The apical surface of the RPE, including microvilli, tightly adheres to IPM components and possibly photoreceptors in the healthy retina, maintaining this tissue organization. The importance of retinal adhesion to retinal function becomes obvious in the drastic consequences of retinal detachment. Persistent retinal detachment directly results in RPE dedifferentiation and proliferation, POS degeneration, and photoreceptor cell death [13]. Furthermore, retinal detachment causes irreversible changes in inner retinal neurons. For a comprehensive recent review of these effects of retinal detachment on retinal cells, please see [14]. In the context of RPE–photoreceptor interactions, it is particularly relevant that IPM proteoglycan rearrangement and RPE microvilli collapse are early, reversible responses to retinal detachment. A variety of physiological activities contribute to retinal adhesion. These include active fluid transport, intraocular pressure, and osmotic pressure gradients. In addition, apical surface receptors of the RPE adhere to specific IPM ligands [15].

The actin-binding protein ezrin is an important and abundant structural component of the apical microvilli of the RPE [16]. Ezrin expression is both necessary and sufficient for RPE microvilli extension [17, 18]. At the light microscopic level, ezrin distribution in the mammalian retina does not change with illumination or time of day [19]. This suggests that apical RPE microvilli extension and interdigitate in between POSs is constant in mammalian retina. In contrast, the strength of retinal adhesion as measured by RPE attachment to peeled-off neural retina (see Molecular Mechanisms of Retinal Adhesion and Fig. 2) increases by 58% between 2 and 3.5 h after light onset in mice entrained to a 12-h light/12-h dark cycle [19]. Retinal adhesion increases on time even in constant darkness in previously entrained mice. This suggests that murine retinal adhesion does not respond to light but may be regulated by circadian rhythms. In contrast, light onset directly increases retinal adhesion in Xenopus laevis retina [20]. Finally, strength of retinal adhesion decreases with age in mouse retina such that resistance of RPE to shear stress in 12-month-old mice is only 45% compared to the resistance in 1-month- old retina in animals of the same genotype [19]. The molecular basis of this change in RPE–photoreceptor interaction remains to be explored.

Molecular Mechanisms of Retinal Adhesion

Studying the molecular mechanisms of retinal adhesion in the healthy retina is hampered by the lack of experimental model systems. Because of the numerous participating components and their complicated and highly organized architecture, neither direct nor indirect, IPM-mediated adhesive interactions of RPE and POSs have been reconstituted ex vivo. However, Endo and colleagues in 1988 developed and characterized an assay testing strength of RPE–retinal adhesion in enucleated rabbit eyes [21] (Fig. 2). These investigators established that RPE adhesion directly correlates with the amount of RPE pigment fractionating with the neural retina when it is peeled from an open eyecup. Using this assay, Endo and colleagues found that postmortem, retinal adhesion in rabbit

Fig. 2. Mechanical separation of retinal pigment epithelium (RPE) and neural retina allows quantification of retinal adhesion. In a modification of a protocol by Endo and colleagues [21], mice are sacrificed by CO2 asphyxiation. Lens and cornea are swiftly removed from each enucleated eyeball (A) in HEPES-buffered Hank’s saline solution containing Ca2+ and Mg2+ at room temperature, conditions that preserve retinal adhesion. After transferring an individual eyecup to an empty plastic dish, a single radial cut is performed toward the optic nerve, flattening the eyecup retina facing up. B The neural retina is then peeled off with forceps from one side of the cut to the other. C The isolated retina reveals attached apical portions of RPE cells that contain melanin pigment. D Bright-field microscopy of a whole-mounted peeled-off retina with exposed outer retinal surface demonstrates the extent of RPE retrieval with retina harvested from 2-month-old wild-type mice 2 h after light onset. Scale bar 100 m (Printed with permission from [19].)

retina rapidly decreases if tissues are chilled on ice or incubated in Ca2+- and Mg2+-free buffer solution. This suggests that receptors involved in retinal adhesion require physiological temperature and the presence of divalent cations for ligand binding.

Focusing on the role of glycosylated IPM components on retinal adhesion, Yao and colleagues subsequently found that subretinal injections of chondroitinase ABC, neuraminidase, and testicular hyaluronidase temporarily reduce retinal adhesion, suggesting that regulation of retinal adhesion in vivo may involve changes in glycosylation [22]. A similar experimental approach showed that the drug cytochalasin D weakens

retinal adhesion in a concentration-dependent manner if applied to the subretinal space, suggesting a role for actin microfilaments in RPE–photoreceptor interactions [23].

While some progress has been made in characterizing the complex mixture of glycoproteins and proteoglycans molecules that comprise the IPM, we know l ittle about RPE surface receptors that participate in retinal adhesion. The homotypic neural-cell adhesion molecule (N-CAM) localizes to the apical surface of RPE and to outer segments, suggesting that it may mediate direct interactions between the two cell types [24]. However, this hypothesis remains to be tested directly.

Most recently, the increasing availability of mutant animal models has facilitated study of the contributions of individual molecules to retinal adhesion. For example, vitiligo mice, which carry a mutation in the microphthalmia transcription factor gene [25], display early onset retinal detachment likely due to a primary defect in retinal adhesion [26]. Expression of specific adhesive receptors or ligands that participate in retinal adhesion may thus be reduced in vitiligo mice. However, this possibility has not yet been investigated.

The cell–substrate adhesion receptor αvβ5 integrin is the only receptor of the large integrin family that localizes to the apical surface of the RPE [27–29]. The apical surface of the RPE is the sole site of αvβ5 integrin expression in the retina. Localization of αvβ5 integrin at the RPE’s phagocytic surface is conserved between rats, mice, and humans [27, 28]. Comparing strength of retinal adhesion between β5 integrin knockout mice and strainand age-matched, wild-type mice served to directly assess whether lack of apical αvβ5 integrin altered retinal adhesion. In a modification of the protocol of Endo and colleagues (Fig. 2), retina samples were solubilized in detergent buffer following mechanical peeling off the eyecup. This method of preparation allowed quantifying both RPE pigment and RPE and retinal proteins in each sample by spectroscopy and by immunoblotting, respectively. Importantly, melanin quantification of neural retina samples correlates very closely with partitioning of the RPE-specific cytoplasmic protein RPE65 and the RPE apical microvilli marker protein ezrin with the neural retina, confirming that RPE fractionation with neural retina accurately reflects RPE–retinal adhesion.

As shown in Fig. 3, the experiments demonstrated greatly reduced pigment as well as proteins RPE65 and ezrin retrieved with the ripped-off neural retina at all times of day in β5 integrin knockout mice compared to strainand age-matched wild-type mice. Thus, retinal adhesion is severely weakened in β5 integrin knockout mice. In addition, lack of αvβ5 greatly attenuates the synchronized daily fluctuation of retinal adhesion (Fig. 3B). Thus, αvβ5 integrin receptors contribute to retinal adhesion at all times and have a major role in maximizing retinal adhesion 3.5 h after light onset. β5 integrin knockout retina contain wild-type levels of other receptor proteins of the RPE thought to be relevant for retinal adhesion, N-CAM and integrin subunits other than β5 integrin’s partner subunit αv. This suggests that lack of αvβ5 integrin receptors decreases retinal adhesion directly. These experiments identified αvβ5 integrin as the first RPE surface receptor directly implicated in retinal adhesion.

In summary, the recent identification of the role of αvβ5 integrin in retinal adhesion has confirmed the long-standing hypothesis that specific apical plasma membrane receptors of the RPE adhere to IPM components or outer segment surface receptors via cell–substrate or cell–cell interactions, respectively. Given the repertoire of extracellular glycoproteins

Fig. 3. Decreased strength and attenuated diurnal peak of retinal adhesion in β5 integrin knockout mice. A Bright-field microscopy shows exposed outer retinal surface of peeled-off retinas harvested from 2-month-old mice at 8 a.m. β5 integrin knockout retina retains significantly less retinal pigment epithelium (RPE) pigment compared to wild-type retina. Scale 100 µm B Immunoblots of lysates prepared from individual peeled-off neural retina samples harvested from 2-month-old mice at indicated times of day show increased amounts of the RPE marker protein RPE65 in samples harvested at 9.30 a.m. compared with other time points. Ezrin, MerTK (tyrosine kinase Mer), and IRBP (interphotoreceptor retinol-binding protein) did not change significantly. Notably, RPE65 levels in β5 integrin knockout retina samples also increased between 9:30 a.m. and 8 a.m. but remained far below levels of RPE65 in wild-type retina samples at all times of day. (Modified with permission from [19].)

that serve as ligands of αvβ5 and related integrins in other tissues, it appears likely that there are specific ligands for αvβ5 integrin in the IPM that bridge the RPE–retina interface. These ligands, as well as RPE receptors that presumably contribute to retinal adhesion in addition to αvβ5, remain to be identified.

Significance of Retinal Adhesion for Retinal Function

Long-term retinal detachment causes outer segment degeneration [30] and, subsequently, apoptotic cell death of photoreceptors [13], proving that proper retinal attachment is critical for vision. Despite their obvious importance for photoreceptor survival and hence vision, we still know little about RPE surface receptors for IPM ligands that mediate