Silvia C. Finnemann

Understanding photoreceptor outer segment phagocytosis: Use and utility of RPE cells in culture

RPE cells are the most actively phagocytic cells in the human body. In the eye, RPE cells face rod and cone photoreceptor outer segments at all times but contribute to shedding and clearance phagocytosis of distal outer segment tips only once a day. Analysis of RPE phagocytosis in situ has succeeded in identifying key players of the RPE phagocytic mechanism. Phagocytic processes comprise three distinct phases, recognition/binding, internalization, and digestion, each of which is regulated separately by phagocytes. Studies of phagocytosis by RPE cells in culture allow specifically analyzing and manipulating these distinct phases to identify their molecular mechanisms. Here, we compare similarities and differences of primary, immortalized, and stem cell-derived RPE cells in culture to RPE cells in situ with respect to phagocytic function. We discuss in particular potential pitfalls of RPE cell culture phagocytosis assays. Finally, we point out considerations for phagocytosis assay development for future studies.

Analysis of photoreceptor outer segment phagocytosis by RPE cells in culture.

Retinal pigment epithelial (RPE) cells are among the most actively phagocytic cells in nature. Primary RPE and stable RPE cell lines provide experimental model systems that possess the same phagocytic machinery as RPE in situ. Upon experimental challenge with isolated photoreceptor outer segment fragments (POS), these cells promptly and efficiently recognize, bind, internalize, and digest POS. Here, we describe experimental procedures to isolate POS from porcine eyes and to feed POS to RPE cells in culture. Furthermore, we describe three different and complementary methods to quantify total POS uptake by RPE cells and to discriminate surface-bound from engulfed POS.

Essential diurnal Rac1 activation during retinal phagocytosis requires αvβ5 integrin but not tyrosine kinases focal adhesion kinase or Mer tyrosine kinase

Activation specifically of the GTPase Rac1 in retinal pigment epithelial cells is necessary for F-actin recruitment to and engulfment of photoreceptor debris, a process essential for vision. Rac1 activation requires αvβ integrin and its ligand milk fat globule-EGF8 but, unexpectedly, is independent of tyrosine kinase signaling via focal adhesion kinase and Mer tyrosine kinase.

Dietary antioxidants prevent age-related retinal pigment epithelium actin damage and blindness in mice lacking αvβ5 integrin

In the aging human eye, oxidative damage and accumulation of pro-oxidant lysosomal lipofuscin cause functional decline of the retinal pigment epithelium (RPE), which contributes to age-related macular degeneration. In mice with an RPE-specific phagocytosis defect due to lack of αvβ5 integrin receptors, RPE accumulation of lipofuscin suggests that the age-related blindness we previously described in this model may also result from oxidative stress. Cellular and molecular targets of oxidative stress in the eye remain poorly understood. Here we identify actin among 4-hydroxynonenal (HNE) adducts formed specifically in β5(-/-) RPE but not in neural retina with age. HNE modification directly correlated with loss of resistance of actin to detergent extraction, suggesting cytoskeletal damage in aging RPE. Dietary enrichment with natural antioxidants, grapes or marigold extract containing macular pigments lutein/zeaxanthin, was sufficient to prevent HNE-adduct formation, actin solubility, lipofuscin accumulation, and age-related cone and rod photoreceptor dysfunction in β5(-/-) mice. Acute generation of HNE adducts directly destabilized actin but not tubulin cytoskeletal elements of RPE cells. These findings identify destabilization of the actin cytoskeleton as a consequence of a physiological, sublethal oxidative burden of RPE cells in vivo that is associated with age-related blindness and that can be prevented by consuming an antioxidant-rich diet.

PI 3-kinase independent role for AKT in F-actin regulation during outer segment phagocytosis by RPE cells.

Daily phagocytosis of photoreceptor outer segment fragments (POS) by the retinal pigment epithelium (RPE) is essential for vision. RPE cells use an uptake machinery that is highly similar to the one macrophages use to phagocytose apoptotic cells. In both forms of phagocytosis, particle binding induces phagocyte signaling that is required for F-actin assembly and re-arrangement beneath bound particles. Macrophage binding of apoptotic cells stimulates PI3 kinases (PI3K) and AKT kinases (AKT), which may be downstream of PI3K, and PI3K inhibition decreases engulfment. Here, we used specific inhibitory agents to investigate whether and how PI3K and AKT contribute to RPE phagocytosis. Either PI3K or AKT inhibition eliminated AKT activation by RPE cells in response to POS and increased the numbers of POS bound by RPE cells. Analyzing the quality of bound POS, we found a higher fraction of POS associated with F-actin phagocytic cups and myosin II in RPE receiving AKT inhibitor. In these cells, individual POS also recruited more F-actin and myosin II than POS in control cells. In contrast, PI3K inhibition did not alter frequency of phagocytic cups but individual cups contained less F-actin (but similar levels of myosin II) compared to control cups. Annexin AII, another phagocytic cup protein of RPE cells, associated with bound POS regardless of inhibitor treatment. POS engulfment proceeded normally if cells already carried surface-bound POS when receiving inhibitors. However, PI3K inhibition during POS binding blocked subsequent POS engulfment. In striking contrast, AKT inhibition had no effect on POS engulfment. Taken together, these results suggest distinct regulatory roles of PI3K and AKT during POS phagocytosis by RPE cells.

Dimerization deficiency of enigmatic retinitis pigmentosa-linked rhodopsin mutants

Retinitis pigmentosa (RP) is a blinding disease often associated with mutations in rhodopsin, a light-sensing G protein-coupled receptor and phospholipid scramblase. Most RP-associated mutations affect rhodopsins activity or transport to disc membranes. Intriguingly, some mutations produce apparently normal rhodopsins that nevertheless cause disease. Here we show that three such enigmatic mutations—F45L, V209M and F220C—yield fully functional visual pigments that bind the 11-cis retinal chromophore, activate the G protein transducin, traffic to the light-sensitive photoreceptor compartment and scramble phospholipids. However, tests of scramblase activity show that unlike wild-type rhodopsin that functionally reconstitutes into liposomes as dimers or multimers, F45L, V209M and F220C rhodopsins behave as monomers. This result was confirmed in pull-down experiments. Our data suggest that the photoreceptor pathology associated with expression of these enigmatic RP-associated pigments arises from their unexpected inability to dimerize via transmembrane helices 1 and 5.

Analysis of Photoreceptor Rod Outer Segment Phagocytosis by RPE Cells In Situ

Counting rhodopsin-positive phagosomes residing in the retinal pigment epithelium (RPE) in the eye at different times of day allows a quantitative assessment of engulfment and digestion phases of diurnal RPE phagocytosis, which efficiently clears shed photoreceptor outer segment fragments (POS) from the neural retina. Comparing such activities among age- and background-matched experimental wild-type and mutant mice or rats serves to identify roles for specific proteins in the phagocytic process. Here, we describe experimental procedures for mouse eye harvest, embedding, sectioning, immunofluorescence labeling of rod POS phagosomes in RPE cells in sagittal eye sections, imaging of POS phagosomes in the RPE by laser scanning confocal microscopy, and POS quantification.

Neural Retina and MerTK-Independent Apical Polarity of αvβ5 Integrin Receptors in the Retinal Pigment Epithelium

The apical plasma membrane domain of retinal pigment epithelial (RPE) cells in the eye faces the outer segment portions of rods and cones and the interphotoreceptor matrix in the subretinal space. Two important receptor-mediated interactions between the apical surface of the retinal pigment epithelium (RPE) and adjacent photoreceptors are adhesion ensuring outer segment alignment and diurnal phagocytosis of shed outer segment fragments contributing to outer segment renewal. Both depend on the apical distribution of the integrin family adhesion receptor alphavbeta5 as lack of alphavbeta5 in mice causes weakened retinal adhesion and asynchronous phagocytosis. With age, lack of alphavbeta5 leads to accumulation of harmful lipofuscin in the RPE and to vision loss. Here, we discuss three different possible mechanisms that could generate the exclusive apical distribution of alphavbeta5 integrin receptors in the RPE. (1) alphavbeta5 could be apical in the RPE because RPE attachment to neural retina generally or alphavbeta5 ligands specifically in the subretinal space stabilize apical but not basolateral alphavbeta5 surface receptors. (2) alphavbeta5 could be apical in the RPE because it resides in a complex with other components of the phagocytic machinery that assembles at the apical, phagocytic surface of the RPE. (3) alphavbeta5 could be apical due to mechanisms intrinsic to this receptor protein and specifically to its beta5 integrin subunit.

The Developmental Stage of Adult Human Stem Cell-Derived Retinal Pigment Epithelium Cells Influences Transplant Efficacy for Vision Rescue

Summary Age-related macular degeneration (AMD) is a common cause of central visual loss in the elderly. Retinal pigment epithelial (RPE) cell loss occurs early in the course of AMD and RPE cell transplantation holds promise to slow disease progression. We report that subretinal transplantation of RPE stem cell (RPESC)-derived RPE cells (RPESC-RPE) preserved vision in a rat model of RPE cell dysfunction. Importantly, the stage of differentiation that RPESC-RPE acquired prior to transplantation influenced the efficacy of vision rescue. Whereas cells at all stages of differentiation tested rescued photoreceptor layer morphology, an intermediate stage of RPESC-RPE differentiation obtained after 4 weeks of culture was more consistent at vision rescue than progeny that were differentiated for 2 weeks or 8 weeks of culture. Our results indicate that the developmental stage of RPESC-RPE significantly influences the efficacy of RPE cell replacement, which affects the therapeutic application of these cells for AMD.

Regulation of Phagolysosomal Digestion by Caveolin-1 of the Retinal Pigment Epithelium is Essential for Vision

Caveolin-1 associates with the endo/lysosomal machinery of cells in culture, suggesting that it functions at these organelles independently of its contribution to cell surface caveolae. Here we explored mice lacking caveolin-1 specifically in the retinal pigment epithelium (RPE). The RPE supports neighboring photoreceptors via diurnal phagocytosis of spent photoreceptor outer segment fragments. Like mice lacking caveolin-1 globally, RPECAV1−/− mice developed a normal RPE and neural retina but showed reduced rod photoreceptor light responses, indicating that lack of caveolin-1 affects photoreceptor function in a non-cell-autonomous manner. RPECAV1−/− RPE in situ showed normal particle engulfment but delayed phagosome clearance and reversed diurnal profiles of levels and activities of lysosomal enzymes. Therefore, eliminating caveolin-1 specifically impairs phagolysosomal degradation by the RPE in vivo. Endogenous caveolin-1 was recruited to maturing phagolysosomes in RPE cells in culture. Consistent with these in vivo data, a moderate increase (to ∼2.5-fold) or decrease (by half) of caveolin-1 protein levels in RPE cells in culture was sufficient to accelerate or impair phagolysosomal digestion, respectively. A mutant form of caveolin-1 that fails to reach the cell surface augmented degradation like wild-type caveolin-1. Acidic lysosomal pH and increased protease activity are essential for digestion. We show that halving caveolin-1 protein levels significantly alkalinized lysosomal pH and decreased lysosomal enzyme activities. Taken together, our results reveal a novel role for intracellular caveolin-1 in modulating phagolysosomal function. Moreover, they show, for the first time, that organellar caveolin-1 significantly affects tissue functionality in vivo.