APOE immunotherapy reduces cerebral amyloid angiopathy and amyloid plaques while improving cerebrovascular function

Abstract

The ε4 allele of the apolipoprotein E (APOE) gene is the strongest genetic risk factor for late-onset Alzheimer's disease (AD) and greatly influences the development of amyloid-β (Aβ) pathology. Our current study investigated the potential therapeutic effects of the anti-human APOE antibody HAE-4, which selectively recognizes human APOE that is co-deposited with Aβ in cerebral amyloid angiopathy (CAA) and parenchymal amyloid pathology. In addition, we tested whether HAE-4 provoked brain hemorrhages, a component of amyloid-related imaging abnormalities (ARIA). ARIA is an adverse effect secondary to treatment with anti-Aβ antibodies that can occur in blood vessels with CAA. We used 5XFAD mice expressing human APOE4 ^+/+ (5XE4) that have prominent CAA and parenchymal plaque pathology to assess the efficacy of HAE-4 compared to an Aβ antibody that removes parenchymal Aβ but increases ARIA in humans. In chronically treated 5XE4 mice, HAE-4 reduced Aβ deposition including CAA compared to a control antibody, whereas the anti-Aβ antibody had no effect on CAA. Furthermore, the anti-Aβ antibody exacerbated microhemorrhage severity, which highly correlated with reactive astrocytes surrounding CAA. In contrast, HAE-4 did not stimulate microhemorrhages and instead rescued CAA-induced cerebrovascular dysfunction in leptomeningeal arteries in vivo. HAE-4 not only reduced amyloid but also dampened reactive microglial, astrocytic, and proinflammatory-associated genes in the cortex. These results suggest that targeting APOE in the core of both CAA and plaques could ameliorate amyloid pathology while protecting cerebrovascular integrity and function.

Fig. 1:. HAE-4 reduces parenchymal Aβ plaques and CAA in 5XE4 mice.

A, Schematic timeline of antibody treatment in 5XFAD (line 7031) x APOE4^+/+ (5XE4) mice with CAA assessed at 10-months-old. B–E, Representative immunostainings of HJ3.4B for mixed pan-Aβ pathology (B) and quantification of percent area in cortex of total Aβ (C), parenchymal Aβ plaques (D), and CAA (E). Control IgG, n = 13; HAE-4, n = 14, chi-Adu, n = 12. Scale bar = 750 μm. F–I, Thioflavin S (ThioS) staining for insoluble, fibrillar plaques (F) with quantification for area covered by total amyloid (G), parenchymal plaques (H), and CAA (I) pathology in overlaying cortex (Control IgG, n = 12; HAE-4, n = 14, chi-Adu, n = 12). Scale bar = 750 μm. J, K, Insoluble Aβ₄₀ (J) or Aβ₄₂ (K) protein concentrations from bulk cortical tissue lysate homogenized in guanidine measured by ELISA and normalized to total protein concentration in cortex (Control IgG, n = 13; HAE-4, n = 14, chi-Adu, n = 12). L–O, Forebrain vessel isolation paradigm using dextran gradient centrifugation of a separate 5XE4 cohort treated with antibodies (L; Control IgG, n = 13; HAE-4, n = 12, chi-Adu, n = 13). Isolated brain vasculature fluorescently stained for X34⁺ fibrillar CAA, endothelial marker CD31, and nuclei marker TOPRO3. Scale bar = 150 μm. (M). Insoluble Aβ₄₀ (N) and Aβ₄₂ (O) protein concentrations assessed by ELISA from forebrain-extracted vessels sonicated in guanidine-HCL and normalized to total protein concentration. Control = Control IgG. Chi-Adu = Chimeric Aducanumab. Data expressed as mean ± SEM, one-way ANOVA with Tukey’s post hoc test (two-sided) performed for all statistical analyses except Kruskal-Wallis test with Dunn’s multiple comparisons test (two-sided) for parenchymal Aβ/ThioS, and Aβ₄₀ analysis (D, H, J). *P < 0.05, **P < 0.01. No other statistical comparisons are significant unless indicated.

Fig. 2:. HAE-4 selectively binds dense core fibrillar plaques whereas chi-Adu recognizes both dense core and diffuse Aβ plaques.

A, B, Triple co-staining of X34, HAE-4 (A), and chi-Adu (B) in unfixed, cortical tissue of a 22-month-old 5XE4 male mouse for plaque-binding profile of antibodies to either APOE (HAE-4) or Aβ (chi-Adu). Left panel in A and B: parenchymal plaque. Right panel: CAA. Scale bar = 50 μm. C–H, Human autopsy brain tissue from patients (n = 1 per group) including CAA (C, D), AD only (E, F), or no pathology (G, H) stained with X34, HJ3.4 (pan-Aβ), HAE-4, or chi-Adu. Scale bar = 50 μm. Chi-Adu = Chimeric Aducanumab.

Fig. 3:. Chi-Adu exacerbates CAA-associated brain microhemorrhages whereas HAE-4 does not.

A, Dual labeling of ThioS⁺ fibrillar CAA and Prussian blue for hemosiderin deposits revealed CAA-associated microhemorrhages (right panel: microhemorrhage digitally converted to yellow) in 10-month-old 5XE4 mice. Scale bar = 100 μm. B, C, Prussian blue staining analysis for microhemorrhage frequency (B) and size (C) in 5XE4 mice dosed weekly for 8 weeks with antibody treatment (50 mg/kg, i.p.; Control IgG, n = 11; HAE-4, n = 13, chi-Adu, n = 12). One-way ANOVA with Tukey’s post hoc test (two-sided). D, Number of microhemorrhages binned in 100 μm² increments for frequency/size distribution. Control = Control IgG. Chi-Adu = Chimeric Aducanumab. Data expressed as mean ± SEM, two-way ANOVA with Tukey’s post hoc test (two-sided). *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. No other statistical comparisons are significant unless indicated.

Fig. 4:. HAE-4 restores vascular function to CAA-laden leptomeningeal arteries in vivo.

A, Schematic of antibody treatment and experimental design in 12-month-old 5XE4 mice for assessment of pial vascular function using endothelial-dependent ACH, vascular smooth muscle cell-dependent SNAP, and CO₂-induced hypercapnia. B, Representative images of X34⁺ CAA on vessel segment at baseline or after ACH exposure. C–E, Percent vasodilatory change calculated at baseline and after ACH (C, Control IgG, n = 7; HAE-4, n = 8, chi-Adu, n = 7, Non Tg, n = 4), SNAP (D, Control IgG, n = 7; HAE-4, n = 8, chi-Adu, n = 6, Non Tg, n = 4), or hypercapnia (E, Control IgG, n = 6; HAE-4, n = 8, chi-Adu, n = 6, Non Tg, n = 4). Scale bar = 25 μm. ACH = Acetylcholine. SNAP = S-Nitroso-N-acetyl-DL-penicillamine. Control = Control IgG. Chi-Adu = Chimeric Aducanumab. Non Tg = Non-transgenic. Green arrowheads indicate changes in percent dilation from baseline. Data expressed as mean ± SEM, one-way ANOVA comparison with Tukey’s post hoc test (two-sided) between treatment groups in vessels with less or more than 50% CAA coverage unless otherwise stated. *P < 0.05, **P < 0.01. Non-transgenic (n = 4) versus control IgG comparisons: ACH, P < 0.001; SNAP, P < 0.05; Hypercapnia, P < 0.05. Non-transgenic (n = 4) versus chi-Adu comparisons: ACH, P < 0.001; SNAP, P = 0.097. No other statistical comparisons are significant unless indicated or stated.

Fig. 5:. Strong glial response after acute HAE-4 and chi-Adu peripheral administration.

A, Schematic design of 10.5-month-old 5XE4 mice injected once every 3 days for 4 times and assessed at 11 months-of-age. B–D, Quantification of total X34⁺ (B), parenchymal (C), and CAA (D) fibrillar plaques (Control IgG, n = 7; HAE-4, n = 7, chi-Adu, n = 5). E, F, CD45 (activated microglia) staining and quantification in cortex (Control IgG, n = 7 mice; HAE-4, n = 7 mice, chi-Adu, n = 5 mice). G, Heatmap analysis of bulk cortical microglial, astrocytic, and pro-inflammatory cytokine gene expression pattern by qPCR (Control IgG, n = 6; HAE-4, n = 7, chi-Adu, n = 5). “*” denotes statistical significance for HAE-4 versus control (*P < 0.05, **P < 0.01, ***P < 0.001); “+” for chi-Adu versus control (⁺P < 0.05, ⁺⁺⁺P < 0.001).; “#” for HAE-4 versus chi-Adu (^#P < 0.05). Parench = Parenchymal. Control = Control IgG. Chi-Adu = Chimeric Aducanumab. Data expressed as mean ± SEM, one-way ANOVA with Tukey’s post hoc test (two-sided). *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. No other statistical comparisons are significant unless indicated.

Fig. 6:. HAE-4 and chi-Adu treatments stimulate differential glial responses to Aβ plaques and CAA.

A, Relative expression of microglial, astrocytic, and pro-inflammatory transcripts from bulk cortex of antibody-treated 10-month-old 5XE4 mice (50 mg/kg, weekly i.p. for 8 weeks; Control IgG, n = 13; HAE-4, n = 13, chi-Adu, n = 12) measured via qPCR, clustered by antibody treatment, and normalized to control IgG gene expression. “*” denotes statistical significance for HAE-4 versus control (*P < 0.05, **P < 0.01); “#” for HAE-4 versus chi-Adu (^#P < 0.05). Genes analyzed using one-way ANOVA with Dunnett’s multiple comparisons test: S100β, GFAP, Vim, IL1α. One-way ANOVA with Tukey’s post hoc test (two-sided) unless otherwise noted. B–C, Representative images of co-staining using X34 for fibrillar plaques and Iba1 for microglia (B) or GFAP for astrocytes (C) in cortex. D–G, Percent area of Iba1⁺ microglia colocalized with total X34 (D), parenchymal (E), or CAA plaques (F), normalized to respective percent area of plaque load (Iba1⁺X34⁺/X34⁺). Microglial colocalization with CAA correlated to microhemorrhage number per section (G; Pearson correlation: r = 0.279, R² = 0.078, P = 0.405, control IgG (n = 11); r = 0.329, R² = 0.108, P = 0.297, HAE-4 (n = 12); r = 0.232, R² = 0.054, P = 0.468, chi-Adu (n = 12)). H–K, Percent colocalization of GFAP⁺ astrocytes and total X34 (H), parenchymal (I), or CAA plaques (J), normalized to respective percent area of amyloid load (GFAP⁺X34⁺/X34⁺). Correlation between colocalized astrocyte/CAA and microhemorrhage number per section (K; Pearson correlation: r = 0.105, R² = 0.011, P = 0.759, control IgG (n = 11); r = 0.561, R² = 0.315, P = 0.058, HAE-4 (n = 12); r = 0.748, R² = 0.560, P = 0.005, chi-Adu (n = 12)). Large panel: Scale bar = 50 μm. Small panel: Scale bar = 50 μm. Parench = Parenchyma. Control = Control IgG. Chi-Adu = Chimeric Aducanumab. Data expressed as mean ± SEM, one-way ANOVA with Tukey’s post hoc test (two-sided) for all group comparisons except Kruskal-Wallis test with Dunn’s multiple comparisons test (two-sided) for data not normally distributed (F, H, and I). *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. No other statistical comparisons are significant unless indicated.