Ординатура / Офтальмология / Английские материалы / Recent Advances in Retinal Degeneration_LaVail, Hollyfield, Anderson _2008

proliferation and differentiation following photoreceptor loss. This in turn may help to refine future approaches in higher vertebrates aimed at enhancing retinal progenitor cell activity for therapeutic purposes.

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v 5 Integrin Receptors at the Apical Surface of the RPE: One Receptor, Two Functions

Emeline F. Nandrot, Yongen Chang, and Silvia C. Finnemann

1 Introduction

Photoreceptors and retinal pigment epithelial (RPE) cells, two adjacent cells types of the outer retina, interact with each other functionally in numerous ways. Maintenance of permanent retinal adhesion and cyclic phagocytosis of shed photoreceptor outer segment fragments (POS) by RPE cells are two forms of these interactions that are crucial for vision. RPE cells form a polarized monolayer and extend apical microvilli that ensheath photoreceptor outer segments. Outer segments consist of stacked membranous disks containing the phototransduction machinery and are permanently renewed. To maintain constant outer segment length photoreceptors eliminate their most aged tips by daily shedding (Young, 1967), which precedes a burst of phagocytosis by the RPE that efficiently clears POS from the subretinal space and recycles many of their components (Young and Bok, 1969). POS shedding and subsequent phagocytosis by RPE cells are critical for photoreceptor cell function and long term survival. Indeed, complete failure to ingest POS by RPE cells from the Royal College of Surgeons (RCS) rat strain causes debris accumulation and rapid photoreceptor degeneration (Mullen and LaVail, 1976; Edwards and Szamier, 1977). Clearing their daily load of POS renders post-mitotic RPE cells the most active phagocytes in the body.

Synchronized POS clearance is tightly regulated and any delay in completing the shedding or digestion process can cause accumulation of autofluorescent lipofuscin inclusion bodies containing a complex mix of proteins and lipids that likely result from incomplete turnover of POS material (Feeney, 1978). In vitro studies have recently shown that lipofuscin components may directly impair RPE function and viability (Finnemann et al., 2002; Schutt et al., 2006). 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.

E.F. Nandrot

Margaret M. Dyson Vision Research Institute, Department of Ophthalmology, Weill Medical College of Cornell University, Box 233, 1300 York Avenue, New York, NY 10021, USA,

Tel: 212-746-2327, Fax: 212-746-8101 e-mail: efn2001@med.cornell.edu

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 cone-dominant species revealed that rods mainly shed their POS within 2 hours after onset of light and cones shed within 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”.

Retinal adhesion is equally essential for vision as daily POS phagocytosis but must be maintained permanently. Different factors such as intraocular pressure and a net fluid transport from retina to RPE contribute to retinal adhesion. Additionally, receptors expressed at the RPE apical surface are thought to adhere to ligands in the interphotoreceptor matrix (IPM), a complex mix of proteins and proteoglycans filling the subretinal space and ensheathing cone and rod POS (Hageman et al., 1995; Hollyfield et al., 1989; Hollyfield, 1999). IPM proteoglycan rearrangement and RPE microvilli collapse are early responses to retinal detachment that, if persistent, result in RPE dedifferentiation and proliferation, POS degeneration and photoreceptor cell death by apoptosis (Cook et al., 1995).

Despite their obvious importance for vision, we still know little about mechanisms that regulate the rhythm of RPE phagocytosis and RPE surface receptors or IPM ligands that mediate retinal adhesion.

2 Studying POS Phagocytosis by RPE Cells In vitro and In vivo

Studies seeking to identify the molecular machinery used by photoreceptors and RPE cells for photoreceptor outer segment renewal greatly benefit from the fact that RPE cells in tissue culture retain their phagocytic activity towards POS. Recording the binding and internalization kinetics of POS by RPE in culture ideally complements the classical microscopic characterization of OS uptake by RPE in vivo. First, OS recognition cannot be studied separately from OS internalization in vivo because shed OS in the subretinal space are juxtaposed to the RPE surface whether or not they are recognized or bound by RPE receptors. Second, far greater numbers of RPE cells can be evaluated in each sample in in vitro assays than in tissue sections. Therefore, small but significant alterations in RPE phagocytic activity may be detected in in vitro assays that may be missed in the light and electron microscopy studies of post-mortem tissues. Third, RPE cells in vitro can be studied following specific manipulation of their phagocytic mechanism by pharmacological compounds, recombinant proteins, protein overexpression or down-regulation, just to name a few. Gainand loss-of-function approaches are well suited to unequivocally identify critical components of the RPE phagocytic machinery. Fourth, in vitro phagocytic challenge of RPE cells allows one to test directly the phagocytic activity of RPE cells towards POS, while altered photoreceptor shedding or IPM may indirectly

including the onset of POS renewal (Finnemann et al., 1997). Stable binding of POS to the cell surface of RPE cells in culture is largely dependent on v 5 integrin receptors (Finnemann et al., 1997). In addition to POS recognition or tethering RPE cells also use v 5 integrin receptors to activate signaling pathways through focal adhesion kinase that are necessary to activate MerTK (Finnemann, 2003; Nandrot et al., 2004). As 5 integrin only dimerizes with v integrin subunits, 5 integrin knockout mice provide the opportunity to study RPE cells that permanently and exclusively lack v 5 receptors (Huang et al., 2000).

To determine whether v 5 integrin plays a role in RPE phagocytosis, we used the technology outlined above to quantify phagocytosis of photoreceptor outer segment fragments (POS) by β5-deficient RPE in vivo and in vitro (Nandrot et al., 2004). Indeed, RPE cells in β5 integrin knockout mice in vivo retain basal levels of phagocytic activity but lack the characteristic burst of phagocytosis upon early morning rod shedding. Moreover, v 5 integrin is essential for POS binding in vitro, as RPE cells derived from 5 integrin knockout mice in primary culture largely fail to bind isolated POS. These data demonstrate that v 5 integrin has a key function in RPE phagocytosis.

Fig. 2 Retinal adhesion is dramatically reduced in 5 knockout mice. (a) After enucleation and lens/cornea removal, retinas are swiftly peeled off opened eyecups creating shearing forces to assess retinal adhesion. In retinas with normal retinal adhesion, apical cellular domains of RPE largely remain attached to the outer surface of the neural retina. (b) Whole-mount of peeled retina, shown outer retina up, demonstrating that 5 knockout retina retains significantly less RPE pigment than wild-type retina. (c) Quantification of RPE pigment in retina peeled at different times of day. 5 knockout retina shows reduced pigment contents and attenuated adhesiveness peak compared to wild-type retina. (d) Representative immunoblots of individual peeled retina confirming the melanin pigment results. Modified from Finnemann and Chang and from Nandrot et al. (2006) with permission from Humana Press Inc. and the American Physiological Society

largely independent of each other since both are defective immediately following the establishment of mature RPE-photoreceptor interactions in 5 knockout mouse retina. We also found that retinal adhesion and POS phagocytosis, each a crucial function for vision, follow independent diurnal rhythms that peak at different times of day.

v 5 integrin can recognize a number of extracellular ligands such as thrombospondin, vitronectin and MFG-E8. It is therefore an intriguing possibility that apical v 5 receptors of the RPE may respond to two different ligands in the subretinal space to either promote POS phagocytic signaling or to mediate retinal adhesion. We are currently exploring candidates to identify the ligand for each of v 5 integrin’s function in the retina in vivo.

Acknowledgments This work was supported by NIH grant EY13295 and the Dyson Foundation. S.C.F. is the recipient of a William and Mary Greeve Scholarship by Research To Prevent Blindness, Inc., and of an Irma T. Hirschl Career Scientist Award.

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Implantation of Mouse Eyes with a Subretinal Microphotodiode Array

Machelle T. Pardue, Tiffany A. Walker, Amanda E. Faulkner, Moon K. Kim, Christopher M. Bonner, and George Y. McLean

1 Introduction

Retinal prosthetics are designed to restore vision in patients with photoreceptor degenerative diseases, such as retinitis pigmentosa (RP) and macular degeneration. Subretinal microphotodiode arrays (MPAs), which response to incident light in a gradient fashion, have been designed to replace degenerating photoreceptors. Such devices have been implanted into rats (Ball et al., 2001), cats (Chow et al., 2001) and humans (Chow et al., 2004). These studies have revealed that implantation of a MPA device is capable of restoring some visual function in patients (Chow et al., 2004) and eliciting a superior colliculus response in normal and degenerating rats (DeMarco et al., 2007). Furthermore, the low level electrical stimulation produced by the MPA device has been shown to have neuroprotective properties (Pardue et al., 2005).

When RCS rats are implanted with an MPA device at the beginning of the degenerative process, photoreceptor function and morphology are preserved (Pardue et al., 2005). Subretinal electrical stimulation may provide protection to the photoreceptors by stimulating the selective expression of FGF-2 in the RCS rat (Ciavatta et al., 2006). While these studies show promise for subretinal electrical stimulation as a treatment of RP, implantation of MPA devices in S334ter rats does not preserve photoreceptors (Walker et al., 2005). We hypothesize that this may be due to the underlying mutations between RCS and S334ter rats. RCS rats have a recessive mutation in a tyrosine kinase gene, Mertk, which results in failed phagocytosis of shed outer segments by the retinal pigment epithelium (Mullen and LaVail, 1976; D’Cruz et al., 2000) while S334ter rats have a rhodopsin mutation which leads to photoreceptor death (Lee et al., 2003).

Rat models with photoreceptor degeneration are few while there are numerous mouse models of RP that have been described (Chang et al., 2002; Dalke and

M.T. Pardue

Rehab R&D, Atlanta VA Medical Center, Decatur, GA 30033, USA; Department of Ophthalmology, Emory University, Atlanta GA 30322, USA, Tel: 404-321-6111 x 7342,

Fax: 404-728-4847

e-mail: mpardue@emory.edu