Drusen, choroidal neovascularization, and retinal pigment epithelium dysfunction in SOD1-deficient mice: A model of age-related macular degeneration Yutaka Imamura*†, Setsuko Noda‡, Kouhei Hashizume*, Kei Shinoda*§, Mineko Yamaguchi¶, Satoshi Uchiyama¶, Takahiko Shimizu¶, Yutaka Mizushimaʈ, Takuji Shirasawa¶, and Kazuo Tsubota*†** *Department of Ophthalmology, Keio University School of Medicine, 35-Shinanomachi, Shinjuku-ku, Tokyo 160-8582, Japan; ‡Department of Nursing, Tokai University School of Health Science, Boseidai, Isehara, Kanagawa 259-1193, Japan; §Laboratory of Visual Physiology, National Institute of Sensory Organs, National Hospital Organization, Tokyo Medical Center, 2-5-1-Higashigaoka, Meguro-ku, Tokyo 152-8902, Japan; ¶Research Team for Molecular Biomarkers, Tokyo Metropolitan Institute of Gerontology, Itabashi-ku, Tokyo 173-0015, Japan; ʈDDS Institute, Jikei University School of Medicine, 3-25-8 Nishi-shinbashi, Minato-ku, Tokyo 105-8461, Japan; and **Department of Ophthalmology, Tokyo Dental College, 5-11-13 Sugano, Ichikawa, Chiba 272-8513, Japan Edited by Jeremy Nathans, Johns Hopkins University School of Medicine, Baltimore, MD, and approved June 9, 2006 (received for review March 19, 2006) Oxidative stress has long been linked to the pathogenesis of evidence suggests that cumulative oxidative damage may be neurodegenerative diseases; however, whether it is a cause or responsible for AMD (18, 19); however, a causative link has not merely a consequence of the degenerative process is still unknown. been definitively demonstrated (18). We show that mice deficient in Cu, Zn-superoxide dismutase To determine whether there is a causative role of oxidative (SOD1) have features typical of age-related macular degeneration stress in the pathogenesis of AMD, we first focused on the in humans. Investigations of senescent Sod1؊/؊ mice of different age-related changes in the eyes of mice deficient in oxidative ages showed that the older animals had drusen, thickened Bruch’s stress scavengers. One of the main antioxidant systems in the membrane, and choroidal neovascularization. The number of retina is made up of three superoxide dismutase (SOD) isoen- ؊ ؊ drusen increased with age, and exposure of young Sod1 / mice zymes that catalyze superoxide radical dismutation (20). Cu, to excess light induced drusen. The retinal pigment epithelial cells Zn-SOD (SOD1) exists in the cytosol, Mn-SOD (SOD2) in ؊ ؊ of Sod1 / mice showed oxidative damage, and their ␤-catenin- the mitochondrial matrix, and extracellular SOD (SOD3) in the mediated cellular integrity was disrupted, suggesting that oxida- tissue interstitium as the secretory form (21). Because the tive stress may affect the junctional proteins necessary for the amount and activity of SOD1 are highest among the three barrier integrity of the retinal pigment epithelium. These obser- isoenzymes in the human retina (21), it seemed reasonable to vations strongly suggest that oxidative stress may play a causative hypothesize that the lack of SOD1 would accelerate age-related role in age-related retinal degeneration, and our findings provide pathological changes in the human retina. evidence for the free radical theory of aging. In addition, these We have investigated the age-related changes of the retinas of ؊/؊ results demonstrate that the Sod1 mouse is a valuable animal Sod1Ϫ/Ϫ mice and found that these mice have many of the key model to study human age-related macular degeneration. elements of human AMD including drusen, thickened Bruch’s membrane, and CNV. Moreover, the RPE cells of these mice ͉ animal model superoxide dismutase show signs of oxidative damage, and their junctional integrities are damaged with the disruption of ␤-catenin-mediated cell ge-related macular degeneration (AMD) is the leading adhesions. Thus, our results suggest a causative role of oxidative Acause of legal blindness in humans in developed countries stress in the pathogenesis of retinal degeneration and demon- (1–5). AMD is characterized by a progressive degeneration of strate the critical role of SOD1 in protecting the RPE from the macula, usually bilateral, leading to a severe decrease in age-related degeneration. vision and a central scotoma. The decrease in vision results either from retinal degeneration, called geographic atrophy (dry Results or nonexudative AMD), or from the secondary effects of Drusen in Senescent Sod1؊/؊ Mice. Fundus examinations were choroidal neovascularization (CNV; wet or exudative AMD). An performed on 60 Sod1Ϫ/Ϫ mice (age, 3–18 months) and 50 early sign of AMD is the appearance of drusen, which are age-matched wild-type mice. The fundi of Sod1Ϫ/Ϫ mice (n ϭ 25) extracellular deposits that accumulate below the retinal pigment under 7 months of age appeared normal and indistinguishable epithelium (RPE) and are known to be risk factors for devel- from those of wild-type mice (n ϭ 25). Mice older than 7 months oping CNV (6–8). showed white to slightly yellowish retinal deposits that appeared Mouse models of AMD that manifest some of the features of to be similar to drusen in humans (Fig. 1A). The drusen were human AMD have recently begun to appear (9–14); however, hyperfluorescent during fluorescein angiography, as in humans most of these mice have only some of the characteristics of (Fig. 1B), and the numbers increased with age (Fig. 1C). Drusen human AMD (15). The severity of AMD in humans progresses Ϫ Ϫ were detected in 31 of 36 eyes (86%) of Sod1 / mice older than with increasing age, finally resulting in extensive degeneration of 10 months. In contrast, wild-type mice and heterozygous mice the retina. Therefore, animal models that mimic the complex and progressive characteristics of AMD are needed to investigate the pathogenesis of AMD. Conflict of interest statement: No conflicts declared. Oxidative stress, which refers to cellular or molecular damage This paper was submitted directly (Track II) to the PNAS office. caused by reactive oxygen species (ROS), has been implicated in Freely available online through the PNAS open access option. many age-related diseases and aging itself (16, 17). ROS include Abbreviations: AMD, age-related macular degeneration; CNV, choroidal neovasculariza- free radicals, hydrogen peroxide, and singlet oxygen and are tion; RPE, retinal pigment epithelium; SOD, superoxide dismutase; C5, complement 5; CML, often the by-products of oxygen metabolism. The retina is carboxymethyl lysine. particularly susceptible to oxidative stress because of its high †To whom correspondence may be addressed. E-mail: [email protected] or consumption of oxygen, high concentration of polyunsaturated [email protected]. fatty acids, and exposure to light (18). A growing body of © 2006 by The National Academy of Sciences of the USA 11282–11287 ͉ PNAS ͉ July 25, 2006 ͉ vol. 103 ͉ no. 30 www.pnas.org͞cgi͞doi͞10.1073͞pnas.0602131103 Downloaded by guest on October 2, 2021 Fig. 1. Senescent Sod1Ϫ/Ϫ mice showing drusen. (A) Fundus photograph from a 15-month-old Sod1Ϫ/Ϫ mouse. White and slightly yellowish deposits are evident (arrows). (B) Fluorescein angiogram showing hyperfluorescent areas corresponding to the sites of the drusen (arrows). (C) Plot of the number of drusen in Sod1Ϫ/Ϫ mice at 3–18 months of age, showing an increase in the number of drusen with age. Values for Sod1Ϫ/Ϫ are plotted in red, and those for Sod1ϩ/ϩ are plotted Ϫ Ϫ in blue. (D) Dome-shaped deposit (arrow) between the RPE (*) and choroid (**) in a 12-month-old Sod1 / mouse. (E) Electron micrograph of drusen (arrow) from a 12-month-old Sod1Ϫ/Ϫ mouse. Heterogeneous materials separated by a septum (arrowhead) are observed. The drusen appears to contain debris-like material of outer segments. BM, Bruch’s membrane. (F) Immunohistochemistry with markers for vitronectin (Vn), CD46, C5, TIMP3, CML, and Ig. Drusen and͞or their surrounding tissues from 12-month-old Sod1Ϫ/Ϫ mice (Right) are positive for vitronectin, CD46, C5, TIMP3, CML, and Ig in contrast to the results for age-matched wild-type mice (Left). *, RPE; **, choroid; arrow, positive signal; arrowhead, positive signal of CML in RPE cell layer. (G) Fundus changes in a 5-month-old Sod1Ϫ/Ϫ mouse after continuous light exposure for 8 weeks (white fluorescent light of 10,000 lux). Drusen are present (circles and arrows). (H) The number of drusen increases with the length of light exposure in Sod1Ϫ/Ϫ mice, as compared with the minimal appearance of drusen in wild-type mice. Values for Sod1Ϫ/Ϫ are plotted in red, and those for Sod1ϩ/ϩ are plotted in blue. (Scale bars: D and F,50␮m; E,2␮m.) (Sod1ϩ/Ϫ) showed very few, if any, drusen (Figs. 2A and 3 A, C, complements, and complement-related genes may play a role in and E). the pathogenesis of AMD in humans (26–29). Light microscopic examination of histological sections of the retinas of a 12-month-old Sod1Ϫ/Ϫ mouse showed dome-shaped Induction of Drusen by Exposure to Light. Because exposure to light deposits between the RPE and Bruch’s membrane (Fig. 1D). (30, 31) and smoking (32) have been reported in epidemiological Heterogeneous materials were detected beneath the RPE on studies to be risk factors for AMD in humans, we attempted to Bruch’s membrane by electron microscopy (Fig. 1E). induce drusen in young Sod1Ϫ/Ϫ mice by light exposure. Young Markers of drusen, including vitronectin, a fluid-phase regu- Sod1Ϫ/Ϫ mice (age, 5 months) have normal retinal architecture lator binding the terminal component complex to regulate the and do not have any drusen. However, after constant white control complex-mediated lysis (22); CD46, a membrane-bound fluorescent light exposure (10,000 lux, equal to outdoor sunlight) regulator that facilitates inactivation of activated complement for 24 h per day, drusen appeared (Fig. 1G), and their numbers components (23); complement 5 (C5); tissue inhibitor of metal- increased with the duration of the light exposure (Fig. 1H) loproteinases 3 (TIMP3), a potent local inhibitor of matrix metalloproteinase regulating the rate of Bruch’s membrane Degeneration of RPE and Thickened Bruch’s Membrane in Sod1؊/؊ turnover (24); carboxymethyl lysine (CML); and Igs were de- Mice.