Cell Culture Model That Mimics Drusen Formation and Triggers Complement Activation Associated with Age-Related Macular Degeneration

Cell culture model that mimics drusen formation and triggers complement activation associated with age-related macular degeneration

Lincoln V. Johnsona,1, David L. Foresta, Christopher D. Bannaa, Carolyn M. Radekea, Michelle A. Maloneya, Jane Hub, Christine N. Spencera, Aimee M. Walkera, Marlene S. Tsiea, Dean Bokc, Monte J. Radekea, and Don H. Andersona

aCenter for the Study of Macular Degeneration, Neuroscience Research Institute, University of California, Santa Barbara, CA 93106; and bDepartment of Ophthalmology and cDepartment of Neurobiology, Brain Research Institute, Jules Stein Eye Institute, The David Geffen School of Medicine, University of California, Los Angeles, CA 90095

Edited* by Jeremy Nathans, The Johns Hopkins University, Baltimore, MD, and approved September 9, 2011 (received for review June 15, 2011) We introduce a human retinal pigmented epithelial (RPE) cell-culture genesis, as well as the relevant molecular interactions leading to model that mimics several key aspects of early stage age-related drusen deposition, have not yet been fully elucidated. macular degeneration (AMD). These include accumulation of sub-RPE During the past decade, compelling evidence has emerged deposits that contain molecular constituents of human drusen, and implicating the immune system—and the complement system in activation of complement leading to formation of deposit-associated particular—in drusen biogenesis and AMD (15, 20, 21). A terminal complement complexes. Abundant sub-RPE deposits that number of the proteins detected in drusen are either comple- are rich in apolipoprotein E (APOE), a prominent drusen constituent, ment components or related molecules. Importantly, variations are formed by RPE cells grown on porous supports. Exposure to in several complement-related genes have been shown to be human serum results in selective, deposit-associated accumulation of highly significant risk factors for the development of AMD (20, additional known drusen components, including vitronectin, clus- 21). Taken together, these findings are consistent with the gen- terin, and serum amyloid P, thus suggesting that specificprotein– eral conclusion that chronic local inflammation at the RPE/ protein interactions contribute to the accretion of plasma proteins Bruch’s membrane interface contributes to drusen formation during drusen formation. Serum exposure also leads to complement and to AMD pathogenesis (12, 14, 22, 23). activation, as evidenced by the generation of C5b-9 immunoreactive Despite these significant advances, the identity of the mole- terminal complement complexes in association with APOE-contain- cules responsible for triggering activation of the complement ing deposits. Ultrastructural analyses reveal two morphologically cascade, as well as the downstream molecular interactions that distinct forms of deposits: One consisting of membrane-bounded promote AMD pathology, remain elusive. This is due, in part, to multivescicular material, and the other of nonmembrane-bounded the dearth of animal and cell-culture models that reproduce the particle conglomerates. Collectively, these results suggest that dru- most salient pathologic features of AMD under controlled ex- sen formation involves the accumulation of sub-RPE material rich in perimental conditions. We introduce here an RPE cell culture APOE, a prominent biosynthetic product of the RPE, which interacts model that mimics numerous aspects of AMD pathology ob- with a select group of drusen-associated plasma proteins. Activation served in humans, including accumulation of sub-RPE deposits of the complement cascade appears to be mediated via the classical containing known constituents of drusen, activation of the com- pathway by the binding of C1q to ligands in APOE-rich deposits, plement system, and deposition of terminal complement com- triggering direct activation of complement by C1q, deposition of plexes. This system provides a unique experimental platform that terminal complement complexes and inflammatory sequelae. This model system will facilitate the analysis of molecular and cellular will facilitate dissection of the cellular and molecular events that lead to drusen formation and contribute to AMD pathogenesis. aspects of AMD pathogenesis, and the testing of new therapeutic CELL BIOLOGY agents for its treatment. Results Examination of differentiated cultures of primary human RPE ge-related macular degeneration (AMD) is characterized in cells grown on porous supports led to the identification of a pop- Aits early stages by the presence of extracellular deposits, ulation of globular extracellular deposits that accumulate within known as drusen, that accumulate between the basal surface of the pores of the support material. Initially, we identified these sub- the retinal pigmented epithelium (RPE) and Bruch’s membrane, RPE deposits based on their immunoreactivity for apolipoprotein an extracellular matrix complex that separates the neural retina E (APOE) (Fig. 1). Antibodies to several other apolipoproteins, from the capillary network in the choroid. Early electron mi- including apolipoproteins A and B, did not show similar immu- croscopic studies suggested that drusen formation may be noreactivity. APOE is a circulating lipid transport protein, a a consequence of degeneration of the RPE (1–3), initiated by ubiquitous component of human ocular drusen (24–26), and an membranous debris shed from its basal surface (4, 5). These abundant biosynthetic product of RPE cells (16, 24). The APOE early morphological observations have since been confirmed by deposits are typically spheroid, sometimes tubular in shape, a number of more recent studies (6–13). Contemporary investigations of the molecular composition of drusen have provided additional insights into their biogenesis. Author contributions: L.V.J., D.L.F., C.D.B., C.M.R., M.A.M., J.H., D.B., M.J.R., and D.H.A. Immunohistochemical and proteomic studies show that drusen designed research; L.V.J., D.L.F., C.D.B., C.M.R., M.A.M., J.H., C.N.S., A.M.W., and M.S.T. performed research; L.V.J., D.L.F., C.D.B., C.M.R., M.A.M., J.H., C.N.S., A.M.W., M.S.T., D.B., contain a variety of protein and lipid components (14, 15). Among M.J.R., and D.H.A. analyzed data; and L.V.J. and D.H.A. wrote the paper. these are several plasma proteins, the presence of which implies The authors declare no conflict of interest. a systemic contribution to their genesis. Although the primary *This Direct Submission article had a prearranged editor. biosynthetic source for most of these circulating molecules is the Freely available online through the PNAS open access option. liver, a number of them are also known to be synthesized locally by 1To whom correspondence should be addressed. E-mail: [email protected]. – RPE cells (15 19). The respective contributions of RPE-derived This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. and plasma-derived molecules to the process of drusen bio- 1073/pnas.1109703108/-/DCSupplemental.

www.pnas.org/cgi/doi/10.1073/pnas.1109703108 PNAS | November 8, 2011 | vol. 108 | no. 45 | 18277–18282 Downloaded by guest on September 24, 2021 Fig. 1. APOE-containing basal deposits are elaborated by human RPE cells in vitro. Confocal immunofluorescence images of sectioned (A and B) and flat- mounted (C) RPE cell cultures on porous supports. Spherical to amorphous deposits that are immunoreactive for APOE (green, arrows) accumulate within the support matrix. These deposits are nonuniformly distributed with areas of high deposit density (arrows in A and C) interspersed among areas with few to no detectable deposits (asterisk in A). Immunoreactivity for GPNMB (red in A), an abundant RPE membrane protein, is localized to RPE cell surfaces and to particulate sub-RPE material (arrowheads in A) that is distinct from the APOE deposits. Blue, nuclear (“N” in B) DAPI fluorescence in A and B. (Scale bars, 10 μminA and C;5μminB.)

ranging from 0.5 to 3 μm in diameter. They accumulate within the assayed immunocytochemically for C5b-9 show no evidence of porous culture matrix and are most concentrated within 20 μmofthe immunoreactivity (Fig. S1), indicating that neither components of basal surface of the RPE. The deposits are nonuniformly distributed the medium nor the support matrix are responsible for comple- along the substratum, with areas of higher density interspersed with ment activation and deposition of C5b-9 complexes. areas where the deposits are relatively sparse (Fig. 1). This sporadic By electron microscopy, two morphologically distinct deposit distribution of sub-RPE material is consistent with the irregular subtypes are identified: nonmembrane-bounded conglomerates pattern of drusen development in the eye. The accumulation of of small osmophilic particles, and membrane-bounded, multi- APOE from bovine serum that is a constituent of the RPE cell- vesicular structures (Fig. 3). These two subtypes are sometimes culture medium does not appear to contribute to the APOE im- segregated, and sometimes comingled within deposits. munoreactivity because cell culture inserts without cells incubated in We assessed the relative contributions of the different com- bovine serum-containing medium and assayed immunocytochemi- plement activation pathways to deposit-associated formation of cally for APOE show no evidence of immunoreactivity (Fig. S1). C5b-9 terminal complement complexes by exposing cultures to The APOE-containing deposits also show comparatively low human sera depleted of specific components required for acti- levels of immunoreactivity for two other known drusen con- vation of the classical or alternative pathways (Fig. 4 A–F). Ini- stituents, clusterin (17, 18, 22) and vitronectin (7, 27), that have tially, we confirmed that C5b-9 immunoreactivity is abolished in been reported to be biosynthetic products of the RPE. However, the presence of serum depleted of C5, a required element of the following exposure to human serum, immunoreactivity for both terminal complement pathway. When cultures are exposed to clusterin and vitronectin is apparent within these deposits (Fig. serum depleted of Factor B, a component required for activation 2). Serum exposure also facilitates the accumulation of a third of the alternative pathway, no significant alteration in C5b-9 plasma protein and known drusen constituent, serum amyloid P labeling levels is detected compared with whole serum controls. component (APCS) (28). Both vitronectin and APCS are also In contrast, when cultures are exposed to serum depleted of C1q, detected in a few deposits with minimal or no APOE immuno- a component required for formation of the C1 complex and reactivity (Fig. 2). In contrast, haptoglobin, another highly activation of the classical pathway, C5b-9 immunoreactivity as- abundant plasma protein, but one that is not typically found in sociated with sub-RPE deposits is dramatically reduced. Re- drusen, is not detected (Fig. S2), suggesting that there is a se- plenishment of the depleted serum with purified C1q restores lective accumulation of plasma-derived proteins in sub-RPE deposits. The presence of both RPE- and plasma-derived pro- C5b-9 immunoreactivity. The presence of C1q in association with APOE-containing deposits following serum exposure was con- teins in these sub-RPE deposits is consistent with the composi- fi H–I tional profile of human drusen. rmed immunocytochemically (Fig. 4 ), indicating that mo- Strikingly, exposure of the cultures to human serum also enables lecular components of these deposits have both C1q-binding and activation of the complement system, as demonstrated by the de- complement-activating properties. Exposure of cells to human position of C5b-9 terminal complement complexes in association serum previously treated with cobra venom factor, in order to with the sub-RPE deposits. Anti–C5b-9 immunoreactivity is as- irreversibly activate the C3 convertase and generate abundant sociated with particulate material that often codistributes with soluble C5b-9 complexes (30), results in markedly reduced levels APOE-containing deposits (Fig. 3), but the APOE and C5b-9 of C5b-9 immunoreactivity (Fig. S3). This finding indicates immunoreactivity patterns are not strictly colocalized, indicating that the association of preformed, soluble C5b-9 complexes with that they represent spatially distinct elements of the deposits. sub-RPE deposit material does not contribute significantly to Notably, C5b-9 immunoreactivity is associated only with the basal deposit-associated C5b-9 immunoreactivity. Classical pathway deposits, and not with the surfaces of viable RPE cells. Consistent activation is most often triggered by binding of C1q to antigen- with this observation, these deposits are not typically labeled with antibody complexes. Therefore, we also assessed the ability of an antibody directed against Glycoprotein NMB (GPNMB), an human serum depleted in IgG to provoke C5b-9 formation. IgG abundant RPE surface protein (29) (Fig. 1A). Cell-culture inserts depletion has no detectable effect on deposit-associated C5b-9 without cells incubated in bovine serum-containing medium and immunoreactivity levels compared with whole serum controls

18278 | www.pnas.org/cgi/doi/10.1073/pnas.1109703108 Johnson et al. Downloaded by guest on September 24, 2021 CELL BIOLOGY

Fig. 2. Association of serum proteins with APOE deposits. Double-label confocal immunoﬂuorescence images showing localization of APOE (red) and vitronectin (green in A–F), clusterin (green in G–L), or serum amyloid P component (APCS, green in M–R) in sub-RPE deposits with or without exposure to human serum (± SERUM). RPE nuclei are indicated by blue DAPI ﬂuorescence. Deposit-associated APOE immunoreactivity is present without human serum exposure (A, G, M); comparatively weak immunoreactivity is detected for vitronectin (B) and clusterin (H), but not for APCS (N). Following exposure to 10% human serum for 24 h, APOE immunoreactivity associated with sub-RPE deposits is marginally increased (C, I, O), whereas there is substantially increased deposit-associated immunoreactivity for vitronectin (D), clusterin (J), and APCS (P). Merged images (E, F, K, L, Q, R) show striking overlap of immunoreactivity (yellow), indicating that serum vitronectin, clusterin, and APCS, all known drusen constituents, selectively associate with APOE-containing sub-RPE deposits. Almost all APOE deposits show evidence of colocalization with these serum proteins; however, there are examples where there is minimal colocalization of vitronectin (arrowhead in F) and some where APCS immunoreactivity appears to be largely independent of APOE (arrowhead in R). (Scale bars, 10 μminA, applicable to A–E, G–K,andM–Q;3.5μminF, applicable also to L and R.)

(Fig. S4), thus suggesting that activation of the classical pathway a permeable support produce extracellular sub-RPE deposits occurs via an antibody-independent mechanism. that accumulate within the pores of the supporting material over the course of several weeks in culture. Immunocytochemical Discussion analyses show that these sub-RPE deposits contain some of the In this study, we introduce an RPE cell-culture model system most abundant protein constituents of human drusen. One of that mimics facets of drusen formation and complement activa- these, APOE, is a ubiquitous drusen-associated protein that is tion in vivo. Highly differentiated human RPE cells grown on synthesized and secreted by the RPE (24). Others, including

Johnson et al. PNAS | November 8, 2011 | vol. 108 | no. 45 | 18279 Downloaded by guest on September 24, 2021 Fig. 3. Complement activation by sub-RPE deposits/ultrastructure. (A) Confocal image showing APOE immunofluorescence (green) associated with spheroid to globular sub-RPE deposits and C5b-9 immunofluorescence (red) associated with particulate deposit material that is frequently apposed to the surfaces of the APOE deposits (arrows). Blue, nuclear DAPI fluorescence. (B) Electron microscopic image illustrating the membrane-bounded, multivesicular (arrow) and dense, aggregated (arrowhead) forms of sub-RPE deposits identified ultrastructurally. The deposits occupy anastomotic space among processes that make up the support matrix (M). (Scale bars, 10 μminA, 400 nm in B.)

vitronectin, clusterin, and APCS, selectively accumulate in APOE- in vivo. The membrane-bounded form has similarities to the RPE- containing deposits when exposed to human serum. Haptoglobin, derived, membranous blebs described originally by both Burns another abundant plasma protein, does not show similar accumu- and Feeny-Burns (4) and Ishibashi et al. (5) in human tissue lation. Taken together, these findings suggest that specificprotein– sections. This form would appear to be the most likely site of protein interactions between RPE-derived and plasma-derived deposit-associated MAC deposition and C5b-9 immunolabeling, proteins are involved in the formation of these in vitro deposits, just since formation of the MAC requires membrane insertion. On the as they appear to be in the process of drusen biogenesis in vivo. other hand, the particles/vesicles within the enclosing membrane Another population of sub-RPE deposits formed in vitro are the have dimensions similar to the lipoprotein particles previously target of complement activation and formation of the terminal described as components of drusen and other sub-RPE deposits complement complex [i.e., the membrane attack complex (MAC): (9, 26, 33–38). At this point, we have not determined whether a cytolytic, macromolecular complex consisting of the C5b frag- APOE immunoreactivity is associated with one or both sub- ment, C6, C7, C8, and multiple C9 molecules (C5b-9n)]. When cul- structural forms of sub-RPE deposits, and we did not attempt to tures are incubated with human serum, a complete source of com- identify the lipid moieties associated with APOE. Further analy- plement system components, C5b-9 immunoreactivity is detected in ses using protocols designed for enhanced preservation of lipids association with particulate deposits that are distinct from, but in will be required to more accurately characterize the nature of lipid close proximity to, the more globular APOE-containing deposits. materials associated with the sub-RPE deposits and expanded, C5b-9 immunoreactivity is not detected on intact RPE cell mem- unbiased proteomic profiling will be required to reveal the entire branes, most likely because of the robust expression of membrane- spectrum of proteins comprising the deposits. associated complement inhibitors by the RPE (15, 31, 32). RPE cells express transcripts for APOE at levels comparable The substructural morphology of the in vitro deposits (Fig. 3B) to those in the liver and brain, the two most abundant bio- is consistent with the fine structure of sub-RPE deposits identified synthetic sources of APOE (24). APOE is secreted in consider-

Fig. 4. Deposit-associated complement activation is C1q-dependent. Immunoreactivity for deposit-associated terminal complement complexes (C5b-9 neoepitope, red in A–G). In the absence of human serum exposure, C5b-9 immunoreactivity (A) is indistinguishable from the secondary antibody control (B), confirming the serum-dependence of C5b-9 formation. Abundant C5b-9 immunoreactivity is detected following exposure to complete human serum (C), but only background levels of fluorescence are detected following exposure to sera depleted of C5 (D), or C1q (F). The markedly reduced C5b-9 immunoreactivity in the presence of C1q-depleted serum (F) implicates classical pathway activation, whereas the presence of C5b-9 immunoreactivity on exposure to Factor B- depleted serum (E), at levels similar to that of whole serum (C), indicates a lack of significant contribution by alternative pathway activation. Restoration of complement activation by reintroduction of C1q into C1q-depleted serum (G)confirms dependence on C1q for deposit-associated complement activation. Immunoreactivity for C1q (red in I and J) and APOE (green in H and J) colocalize in many APOE-containing sub-RPE deposits following serum exposure (arrowheads in J). C1q also is associated with basal regions of RPE cells (asterisks in I and J). Blue, nuclear DAPI fluorescence. (Scale bars, 10 μminA and applicable to A–G;10μm in H and applicable to H–J.)

18280 | www.pnas.org/cgi/doi/10.1073/pnas.1109703108 Johnson et al. Downloaded by guest on September 24, 2021 able amounts by RPE cells both apically and basally, and has were cultured on porous supports as opposed to tissue-culture been implicated in lipid trafficking, facilitating the efflux of lipids plastic. In the in vitro system we describe here, the macromo- from the RPE, and their transit across Bruch’s membrane to the lecular material shed at the basal RPE surface tends to accu- choroidal vasculature (16). In human RPE/choroid tissue, we mulate in the pores of the culture support. The size and shape of previously documented the presence of APOE-immunoreactive the deposits are likely to be influenced by the physical dimen- structures of similar size and morphology to those we observe sions of the substrate pores and, to some extent, this porous in vitro (24), thus suggesting that the basal shedding of APOE- meshwork may also act to retain deposit material, limiting its containing material also occurs in situ. Drusen-associated im- diffusion into the basal medium compartment. munoreactivity for apolipoproteins A-1 and B has been shown in In situ, it is probable that a mechanism exists for the removal other studies (37, 38); however, we did not observe immunore- of basally shed RPE debris because, early in life, it does not activity for these moieties in the deposits. These results may be typically accumulate in the sub-RPE compartment. However, related to antibody-dependent issues and/or the nature of the disruption of the normal clearance mechanism, perhaps as a re- cell culture system employed. sult of age-related changes in phagocytic activity, immune func- Although AMD risk-conferring gene polymorphisms have ’ fi tion, and/or the structure and composition of Bruch s membrane, been identi ed for complement Factor H, the major negative would be predicted to lead to the accumulation of sub-RPE regulator of the alternative complement pathway, the alternative debris and eventually to the formation of human drusen. If this pathway does not appear to be the primary activation pathway in model is correct, the use of experimental substrates with bio- this system. Serum depletion of Factor B, a complement com- chemical and diffusional properties that mimic the lipid-rich, ponent required for alternative pathway activation, does not hydrophobic environment that characterizes the aged Bruch’s diminish deposition of C5b-9 complexes. However, when cul- membrane might result in the buildup of deposit aggregates that tures are incubated in serum depleted of C1q, a pattern-recog- more closely resemble human drusen. Alternatively, interactions nition molecule required for classical pathway activation, C5b-9 immunoreactivity levels are dramatically reduced. Supplemen- among drusen-associated proteins and lipids that are not present tation with purified C1q restores C5b-9 immunoreactivity. These in our in vitro system may be required for the establishment of findings implicate the classical pathway as the principal activa- characteristic drusen morphology. tion pathway, and suggest that it may be mediated directly via It is noteworthy that complement-activating sub-RPE debris is C1q binding. Because the alternative pathway acts as an ampli- formed by cultured human RPE cells that are not exposed to fication loop for the classical pathway and can be stimulated by shed photoreceptor outer segments and, thus, are not impacted classical pathway activation (39), this conclusion is not in- by the metabolic derivatives of membrane lipids that have been consistent with a role for the alternative pathway in AMD. Given proposed as complement-activating and immunogenic elements this interplay between the classical and alternative pathways, in AMD (48, 49). However, such factors, in conjunction with some diminution of C5b-9 deposition might have been expected aging, environmental influences, and genetic predisposition in the absence of Factor B. However, high concentrations of could play a role in promoting the susceptibility of RPE cells complement Factor H in the human serum may act to effectively to directed or bystander complement attack in vivo. We do not repress the alternative pathway in this in vitro system. In vivo, find evidence for RPE-directed complement attack (i.e., C5b-9 where local Factor H concentrations are likely lower, dysregu- complexes associated with the RPE cell surface), most likely lation of the amplification loop of the alternative pathway because the cells are chronologically young (derived from fetal stemming from Factor H dysfunction would be predicted to ex- sources), express membrane-associated negative regulators of acerbate complement-mediated inflammation with pathologic complement including CD46, CD55, and CD59 (15, 31, 32), and consequences (40). have not been affected by senescence or exposed to environ- The classical pathway is typically triggered by binding of C1q mental “stressors.” to the Fc fragment of IgG or IgM in clusters of antigen-antibody Importantly, the cell-based system we have used in these complexes on the surfaces of microbial pathogens. When ex- studies recapitulates some of the key pathologic processes at play CELL BIOLOGY posed to serum depleted of IgG, C5b-9 immunoreactivity levels in AMD. As such, this system should facilitate rigorous experi- are unaffected, thus suggesting that activation is not triggered by mental analysis of the molecular basis of drusen formation and an antibody-mediated mechanism. However, the classical path- complement activation in AMD, the evaluation of known AMD way can also be activated by direct binding of C1q to a variety of risk factors at cellular and molecular levels, and the testing of β other molecules, such as C-reactive protein, APCS, amyloid- , novel therapeutic strategies for the treatment of AMD in its fi and modi ed LDL cholesterol (41, 42). In addition, C1q can earliest stages. bind apoptotic cells and cell membrane blebs with consequent activation of the classical complement pathway (43–45). Col- Methods lectively, these findings are consistent with the general conclu- RPE Cell Culture. RPE cells were derived from human fetal eyes (Advanced sion that one or more C1q-binding molecules are the most likely Bioscience Resources) as described by Hu and Bok (50). Early passage (p1–p2) triggers of the complement cascade in this system and perhaps in cells were seeded onto laminin-coated porous supports (Millipore Millicell- the eye as well. The identification of C1q in association with HA Culture Plate Inserts, PIHA 01250) and cultured in “Miller medium” (51). APOE-containing deposits following serum exposure suggests One- to 3-mo postseeding, cultures were exposed to complement-compe- that they contain a molecular ligand for C1q, and thus a potential tent human sera, human sera depleted of C1q, C5, or Factor B (Table S1), or activator of the complement cascade. Membranous deposits that human serum depleted of IgG by absorption with protein A/G coupled to lie in close proximity to the APOE/C1q-containing deposits agarose beads (Pierce) (Fig. S4) at 10% (vol/vol) in serum-free Miller medium × fi would then be predicted to provide the surface required for for 24 h. The specimens were then rinsed in PBS (3 , 15 min), xed for 10 min MAC formation. Further analyses will be required to identify in 4% paraformaldehyde in PBS, and stored in 0.4% paraformaldehyde pending sectioning and immunohistochemical analyses. specific C1q binding partners that may be responsible. Previous studies of sub-RPE deposit formation in vitro used Immunohistochemistry. Confocal laser scanning immunofluorescence imaging ARPE-19 cells grown on impermeable tissue-culture plastic. was performed as described previously (52) on 100-μm vibratome sections or Under these conditions, sub-RPE deposits with drusen-like prop- flat mounts of fixed RPE cell cultures. Flat mounts were bleached (Melanin erties were not apparent, nor were known drusen constituents Bleach Kit; Polysciences) for 10 min to allow visualization of sub-RPE deposits identified (46, 47). Interestingly, Amin et al. (46) did note an that would otherwise be obscured by RPE pigment. Antibodies utilized are increase in total membranous and condensed deposits when cells listed in Table S2.

Johnson et al. PNAS | November 8, 2011 | vol. 108 | no. 45 | 18281 Downloaded by guest on September 24, 2021 Electron Microscopy. RPE cultures were ﬁxed in 2% glutaraldehyde and 2% ACKNOWLEDGMENTS. We thank Drs. Catherine Bowes Rickman and formaldehyde, postﬁxed in 2% osmium tetroxide, dehydrated in a graded Gregory S. Hageman for insightful comments on the manuscript. This work ethanol series up to 100% and embedded in LX-112 resin (Ladd Research). was supported by Grant EY R24 EY017404 from the National Eye Institute Thin sections were stained with 2% uranyl acetate and lead citrate and and by generous benefactors of the Center for the Study of Macular imaged on a JEOL JEM-1230 transmission electron microscope. Degeneration at the University of California, Santa Barbara.

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