Visual function and retinal pigmented epithelium (RPE) histology are protected in anti-Aβ40/42–treated APOE4-HFC mice. (A) Scotopic ERG flash responses. Stimulus response curves of b-wave amplitudes. Baseline ERGs obtained from normal APOE4-ND controls (black, ND) and affected APOE4-HFC vehicle-treated controls (green, HFC). b-Wave amplitudes are fully preserved in APOE4-HFC mice that received weekly 3 mg/kg i.p. anti-Aβ40/42 antibody injections (red, HFC-anti Aβ40/42), with no significant difference from APOE4-ND controls. Data are expressed as mean ± SEM. (B–F) Light microscopic images showing the outer segment/RPE/choroid interface in toluidine blue-stained sections from eyes of aged normal and HFC-fed APOE4 mice after immunotherapy. The morphologies of the RPE in the normal APOE4-ND control mice (B) and APOE4-HFC mice injected with anti-Aβ40/42 (3 mg/kg; D) seem normal, whereas RPE in the vehicle- (C), anti-Aβ40– (E), and Aβ42- (F) treated APOE4-HFC mice exhibits damage, including large vacuoles (asterisks in C), hypo- and hyperpigmentation (arrowheads in C), and basal deposits and amorphous debris (arrows in C). (Scale bar: 10 μm.) (G–I) EMs of the interface between RPE and Bruch's membrane in APOE4 mice. RPE basal infoldings (left bracket) and Bruch's membrane thickness (right bracket) appear normal in the anti-Aβ40/42–treated eye (I) compared with the normal APOE4-ND control mice (G). In contrast, in the HFC-fed APOE4 mice (H), Bruch's membrane is thicker, and there are thick basal deposits with electron dense material among the basal infoldings (arrowheads in H). (Scale bar: 1 μm.) OS, outer segments; CC, choriocapillaris; BrM, Bruch's membrane.

Morphometric analysis of APOE4 RPE flat mounts. (A–C) Confocal fluorescence images of flat mounts of the central RPE from (A) a normal 80-wk-old APOE4-ND mouse, (B) an age-matched affected APOE4-HFC mouse, and (C) an anti-Aβ40/42–treated APOE4-HFC mouse stained with Hoechst 33342 (blue) and anti–ZO-1 (green) and imaged RPE apical side up with the neural retina removed. (A) Anti–ZO-1 labeling of tight junctions reveals typical hexagonal shape of RPE cells, which are also largely binucleate in normal APOE4-ND mice. In APOE4-HFC mice (B), there are many more enlarged and multinucleate cells, whereas normal RPE morphology is largely maintained in the anti-Aβ40/42–treated APOE4-HFC mice (C). (Scale bar: 100 μm.) (D) Graph of cell area of 22,495 cells from 23 images of the central RPE from 17 mice in the three groups represented in A–C. The solid red line represents the normalized distribution of cell areas, which include areas of normal cells and abnormally large cells. Normal mouse RPE cells may have one or two nuclei. Positing that the normal cells with one nucleus and two nuclei would each have a normal distribution with a different mean and variance, the area distribution of the normal cells can be modeled by a double Gaussian curve. The dashed blue line represents the theoretical distribution of the normal cell area when this double Gaussian model was fitted to the data. Next, based on this model, the theoretical minimum size of the abnormally large cells was defined. The threshold of abnormally large cells was selected as the smallest area, with predicted distribution of less than 1 of 22,496 cells. (E) Morphometric analysis confirmed that there was a statistically significant increase in the percent of abnormally large RPE cells per unit area in vehicle-treated APOE4-HFC animals compared with normal APOE4-ND control mice (Student t test; ***P < 0.00001). In contrast, the percent of abnormally large cells in the anti-Aβ40/42–treated APOE4-HFC mice was reduced to near normal levels that were not statistically distinguishable from those levels of control APOE4-ND mice (P = 0.1136). Error bars represent SD (n.s., not significant).

Human RPE flat mounts. (A–C) Confocal fluorescence images of RPE flat mounts from equivalent perimacular regions (RPE basal side up). (A) Seventy-six year old (Y) Caucasian males with no history of ocular disease are shown; (B and C) 85 Y Caucasian females with clinically documented dry AMD in an area with multiple binucleate RPE cells (arrowheads in B) and vacuoles (arrows in B) and an area with a trinucleate RPE cell (asterisk in C) is shown. (D–F) A–C are segmented to highlight cell borders and nuclei using DOCAP. (Scale bar: 50 μm.)

Aβ levels are reduced in sub-RPE deposits of anti-Aβ40/42–treated APOE4-HFC mouse eyes compared with those eyes of vehicle-treated APOE4-HFC mice. Confocal immunofluorescence images depicting Aβ (red) and ApoE (green) detected in (A–C) normal diet APOE4 mice (ND), (D–F) HFC-fed APOE4 mice (HFC), and (G–I) immunotherapy-treated APOE4-HFC mice (HFC–anti-Aβ40/42). Aβ immunolocalizes within the choroid (Ch) and in ApoE-positive basal deposits (arrowheads in F). RPE autofluorescence (particulate red fluorescence) stemming from lipofuscin accumulation exhibited normal animal to animal variation. Cell nuclei are stained with Hoechst 33342 (blue). BrM, Bruch's membrane. (Scale bar: 20 μm.)

Deposition of complement C3 fragments (C3b/iC3b/C3c) in APOE4 mouse eyes and circulating in plasma. (A–C) Confocal immunofluorescence images depicting immunolocalization of C3b/iC3b/C3c- (red) and ApoE- (green) positive basal deposits (arrows in B) of 75-wk-old (A) normal control APOE4-ND (ND), (B) affected APOE4-HFC (HFC), and (C) anti-Aβ40/42–treated APOE4-HFC (HFC–anti-Aβ40/42) mice. C3 fragments are found in small, sub-RPE patches (arrowheads in B). These patches are diminished in anti-Aβ40/42–treated APOE4-HFC and normal control APOE4-ND mice compared with affected APOE4-HFC animals. BrM, Bruch's membrane; Ch, choroid; RPE, retinal pigment epithelium. (Scale bar: 20 μm.) (D) Levels of circulating C3a/C3a desArg were measured by ELISA in the plasma of normal control APOE4-ND (ND), affected APOE4-HFC (HFC), and anti-Aβ40/42–treated APOE4-HFC (HFC–anti-Aβ40/42) mice. There is a statistically significant increase in C3a plasma levels in both groups of the HFC-fed APOE4 mice compared with the ND-fed controls (Student t test, **P < 0.01). Black horizontal bars indicate average C3a levels in each group.

Visual function is protected in anti-Aβ40/42–treated APOE4-HFC mice in a dose-dependent fashion that correlates with plasma levels of Aβ. (A) Dose-response study of ERG b-wave recovery in APOE4-HFC mice treated with three different doses of the anti-Aβ40/42 antibody. Baseline b-wave amplitudes are a function of flash intensity obtained from APOE4-ND controls (black, ND) and APOE4-HFC controls (green, HFC). As shown in Fig. 1A, b-wave amplitudes in the 3-mg/kg anti-Aβ40/42 antibody-treated APOE4-HFC animals (red, HFC 3 mg/kg) were fully preserved. In contrast, b-wave amplitudes in the 0.3- and 0.03-mg/kg anti-Aβ40/42 antibody-treated APOE4-HFC mice (olive green, HFC 0.3 mg/kg; blue, HFC 0.03 mg/kg, respectively) decreased to the same level as vehicle-treated APOE4-HFC animals (data are expressed as mean ± SEM). (B and C) Dose-dependent increase in plasma levels of Aβ and anti-Aβ40/42. Weekly i.p. injections of anti-Aβ40/42 at 0 (vehicle), 0.03, 0.3, and 3 mg/kg produced a statistically significant, dose-dependent increase in the plasma levels of total (bound and unbound) Aβ (B) that correlated with a dose-dependent increase in the concentrations of plasma anti-Aβ40/42 antibodies (C). Significant difference indicating a dose-dependent elevation of plasma (Aβ and anti-Aβ40/42) after increasing dose of antibody was shown by one-way ANOVA (P < 0.0001) and the posttest for linear trend (i.e., dose-dependent change), which was significant at P < 0.0001. In addition, there is a statistically significant increase in total Aβ plasma levels in response to the HFC diet compared with ND (*P < 0.05; B). Error bars represent SD.

Visual function and RPE histology are protected in anti-Aβ40/42–treated APOE4-HFC mice. (A) Scotopic electroretinogram flash responses show the stimulus response curves of b-wave amplitudes. Baseline electroretinograms were obtained from APOE4-ND controls (black) and affected APOE4-HFC vehicle-treated controls (green). b-Wave amplitudes were fully preserved in APOE4-HFC mice that received weekly 3 mg/kg i.p. anti-Aβ40/42 antibody injections (red), with no significant difference from APOE4-ND controls. (B–D) Confocal fluorescence images showing flat mounts of the central RPE from (B) a normal 80-wk APOE4-ND mouse, (C) an age-matched affected APOE4-HFC mouse, and (D) an anti-Aβ40/42–treated APOE4-HFC mouse stained with Hoechst 33342 (cyan) to counterstain RPE nuclei and labeled with antizona occludens 1 (ZO-1, green) imaged RPE apical side up with the neural retina removed. (B) Anti ZO-1 labeling of tight junctions (green) reveals typical hexagonal shape of RPE cells, which are also largely binucleate (cyan) in normal APOE4-ND mice. In APOE4-HFC mice (C), there are many more enlarged and multinucleate cells, whereas normal RPE morphology is largely maintained in the anti-Aβ40/42–treated APOE4-HFC mice (D). (Scale bar: 50 μm.)