Targeting of nonlipidated, aggregated apoE with antibodies inhibits amyloid accumulation

Abstract

The apolipoprotein E E4 allele of the APOE gene is the strongest genetic factor for late-onset Alzheimer disease (LOAD). There is compelling evidence that apoE influences Alzheimer disease (AD) in large part by affecting amyloid β (Aβ) aggregation and clearance; however, the molecular mechanism underlying these findings remains largely unknown. Herein, we tested whether anti-human apoE antibodies can decrease Aβ pathology in mice producing both human Aβ and apoE4, and investigated the mechanism underlying these effects. We utilized APPPS1-21 mice crossed to apoE4-knockin mice expressing human apoE4 (APPPS1-21/APOE4). We discovered an anti-human apoE antibody, anti-human apoE 4 (HAE-4), that specifically recognizes human apoE4 and apoE3 and preferentially binds nonlipidated, aggregated apoE over the lipidated apoE found in circulation. HAE-4 also binds to apoE in amyloid plaques in unfixed brain sections and in living APPPS1-21/APOE4 mice. When delivered centrally or by peripheral injection, HAE-4 reduced Aβ deposition in APPPS1-21/APOE4 mice. Using adeno-associated virus to express 2 different full-length anti-apoE antibodies in the brain, we found that HAE antibodies decreased amyloid accumulation, which was dependent on Fcγ receptor function. These data support the hypothesis that a primary mechanism for apoE-mediated plaque formation may be a result of apoE aggregation, as preferentially targeting apoE aggregates with therapeutic antibodies reduces Aβ pathology and may represent a selective approach to treat AD.

Keywords: Alzheimer’s disease; Neuroscience.

Figure 1. Characterization of HAE series anti–apoE antibodies.

(A–D) Antibody binding to plates coated with recombinant apoE2, apoE3, or apoE4. Serially titrated HAE antibodies were incubated and binding was detected with HRP anti–mouse IgG antibody. (E–G) SPR was used to detect 3-fold serially diluted apoE antibody (starting at 100 nM for HAE-2 and 1000 nM for HAE-1 and HAE-4) binding to biotinylated recombinant apoE4 captured on a streptavidin chip. Samples were injected at a flow rate of 30 μl/min.

Figure 2. Effects of intraperitoneally administered anti–apoE antibodies on amyloid pathology in APPPS1-21/APOE4 mice.

At the age of 2 months, the mice were injected i.p. weekly with 50 mg/kg antibodies (all female, n = 10–13/group). The mice were sacrificed at the age of 3.5 months and the Aβ pathology in the brain was assessed using histology and biochemical approaches. (A) Representative Aβ staining using HJ3.4 (left panel) and representative X-34 staining for fibrillar plaques (right panel). Scale bar = 1 mm. (B) Quantification of HJ3.4 staining. (C) Quantification of X-34 staining. (D) Aβ₄₀ and (E) Aβ₄₂ was measured by ELISA in insoluble fractions of tissue lysates (Guan = Guanidine) by ELISA. One-way ANOVA followed by Tukey’s t test was performed to compare different groups shown in B–E. Data are mean ± SEM. *P < 0.05, **P < 0.01.

Figure 3. Binding profile of HAE-1, HAE-2, and HAE-4 with lipidated apoE.

(A and B) Antibody binding to mouse plasma from EKO, APOE2, APOE3, and APOE4 mice was coated onto the plates. Titrations of chi–HAE-2 and chi–HAE-4 were incubated and antibody that was captured was detected with HRP-goat anti–human IgG antibody. (C and D) HAE-2 or HAE-4 was immobilized on the plate followed by the addition of plasma from mice with different genotypes. apoE, captured from the plasma, was detected with HRP-goat polyclonal anti-apoE. (E) Plasma inhibition of anti-apoE binding to immobilized recombinant apoE4. HAE-1 (50 nM), HAE-2 (4 nM), and HAE-4 (50 nM) were preincubated with serially diluted plasma from APOE4-KI mice and then added to plates coated with recombinant apoE4. The HAE antibodies bound to the plates were detected with HRP-goat anti–mouse IgG antibodies. (F) Plasma antibody concentrations of HAE-4 or control IgG following i.p. injection into APOE4-KI or EKO mice. HAE-4 was dosed at 2 mg/kg, 10 mg/kg, and 50 mg/kg and plasma samples were collected by submandibular puncture. Control murine IgG2a (msIgG2a) was anti-Her2 and dosed at 10 mg/kg. Quantification of dosed antibodies in plasma was by antigen-capture ELISA using coated recombinant apoE4 to detect HAE-4, with recombinant Her2 used to detect the control IgG.

Figure 4. HAE-1 and HAE-4 staining of amyloid plaques in unfixed mouse brain sections and specificity for heat-induced aggregates of apoE4.

(A) Unfixed frozen brain sections from APPPS1-21/APOE4 or APPPS1-21/EKO mice were stained with anti–Aβ antibody HJ3.4 and anti–apoE antibodies HAE-1 and HAE-4. Scale bar = 400 μm. (B) Binding of HAE-1, HAE-2, and HAE-4 to untreated recombinant apoE4 (untreated) or apoE4 that had been incubated at 40°C for 24 hours (40°C). (C) Incubation of apoE4 at 40°C for 24 hours results in the formation of aggregates recovered in the pellet fraction following ultracentrifugation at 186,000 g for 1 hour. Lane 1, untreated apoE4. Lane 2, apoE4 that had been incubated at 40°C for 24 hours. Supernatant (S) and pellet (P) from ultracentrifugation were resolved on SDS-PAGE and stained by Coomassie blue. (D) Binding of HAE-4 to different preparations of apoE4 immobilized at the same concentration (0.5 μg/ml) on the ELISA plate. Sup of untreated: supernatant fraction of untreated apoE4 from ultracentrifugation. Sup/Pel of 24 hours 40°C: supernatant/pellet fraction of apoE4 incubated at 40°C for 24 hours. (E) Binding of HAE-4 to untreated apoE4 and apoE4 that had been incubated at 40°C before and after denaturation by 1% SDS or 4 M guanidine HCl.

Figure 5. Binding of HAE-1, HAE-4, and control antibody to human apoE4 in the brains of living mice.

(A) Control IgG2ab (n = 7), HAE-1 (n = 5), and HAE-4 (n = 6) conjugated with Alexa 594 were applied directly onto the surface of the brain in living APPPS1-21/APOE4 mice that were 6 months of age, and antibody localization was observed using 2-photon microscopy. Amyloid was labeled using methoxy-X04. The signal from Alexa 594 and methoxy-X04 was merged (MERGE) to show the colocalization of antibodies and plaques. (B) Control human IgG (n = 2) or chi–HAE-4 at 50 mg/kg body weight was injected i.p. in 1 dose (0 hour, n = 3) or 2 doses (0 and 48 hour, n = 3). APPPS1-21/APOE4 mice were sacrificed 48 hours after final injection. The antibodies in the brain were detected by biotinylated rabbit anti–human IgG followed by DAB. Left panel, bar = 1 mm. Right panel, high-power image of the indicated areas shown in the left panel; bar = 300 μm.

Figure 6. Reduction of plaques by HAE-1 and HAE-4 requires effector function.

(A) At the age of 4 months, the APPPS1-21/APOE4 mice received 4 i.p. injections of 50 mg/kg of antibodies every 3 days. The mice were sacrificed 24 hours after the final injection and the fibrillar plaques were stained with X-34 and the activated microglia was stained with CD45. The ratio of percentage of area covered by CD45 staining/percentage of area covered by X-34 staining was quantified (equal numbers of male and female mice, n = 8–9/group). (B–E) APPPS1-21/APOE4 mice were injected at day P0 with AAV 2/8 into the lateral ventricle (equal numbers of male and female mice, n = 17–25/group). AAV 2/8 is able to express and secrete full-length HAE-1 and HAE-4 antibodies as well as the same constructs with a D265A mutation in the Fc domain (HAE-1Δ and HAE-4Δ). At the age of 3.5 months, the Aβ plaques (B) were stained with antibody HJ3.4, the fibrillar plaques were stained with X-34 (C), and the insoluble Aβ₄₂ (D) and Aβ₄₀ (E) were measured by ELISA. One-way ANOVA followed by Tukey’s t test was performed to compare different groups shown in A–E. Data are mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.