Astrocytic APOE4 removal confers cerebrovascular protection despite increased cerebral amyloid angiopathy

Abstract

Background: Alzheimer Disease (AD) and cerebral amyloid angiopathy (CAA) are both characterized by amyloid-β (Aβ) accumulation in the brain, although Aβ deposits mostly in the brain parenchyma in AD and in the cerebrovasculature in CAA. The presence of CAA can exacerbate clinical outcomes of AD patients by promoting spontaneous intracerebral hemorrhage and ischemia leading to CAA-associated cognitive decline. Genetically, AD and CAA share the ε4 allele of the apolipoprotein E (APOE) gene as the strongest genetic risk factor. Although tremendous efforts have focused on uncovering the role of APOE4 on parenchymal plaque pathogenesis in AD, mechanistic studies investigating the role of APOE4 on CAA are still lacking. Here, we addressed whether abolishing APOE4 generated by astrocytes, the major producers of APOE, is sufficient to ameliorate CAA and CAA-associated vessel damage.

Methods: We generated transgenic mice that deposited both CAA and plaques in which APOE4 expression can be selectively suppressed in astrocytes. At 2-months-of-age, a timepoint preceding CAA and plaque formation, APOE4 was removed from astrocytes of 5XFAD APOE4 knock-in mice. Mice were assessed at 10-months-of-age for Aβ plaque and CAA pathology, gliosis, and vascular integrity.

Results: Reducing the levels of APOE4 in astrocytes shifted the deposition of fibrillar Aβ from the brain parenchyma to the cerebrovasculature. However, despite increased CAA, astrocytic APOE4 removal reduced overall Aβ-mediated gliosis and also led to increased cerebrovascular integrity and function in vessels containing CAA.

Conclusion: In a mouse model of CAA, the reduction of APOE4 derived specifically from astrocytes, despite increased fibrillar Aβ deposition in the vasculature, is sufficient to reduce Aβ-mediated gliosis and cerebrovascular dysfunction.

Keywords: APOE; Amyloid-β; Astrocyte; Cerebral amyloid angiopathy; Cerebrovasculature.

Fig. 1

Tamoxifen-induced Cre recombination reduces both APOE mRNA and protein. a Schematic timeline of tamoxifen treatment (5 daily injections at 200 mg/kg) in 2-month-old 5XFAD (line 7031) x APOE4^flox/flox (5X+AL-) or 5XFAD (line 7031) x APOE4^flox/flox x Aldhl1l-Cre/ERT2 (5X+AL+) mice, assessed at 10-months of age. b Relative expression of APOE mRNA normalized to beta-actin in cortex. c, d PBS-soluble and guanidine-HCL-soluble (“insoluble”) APOE protein concentrations assessed by ELISA from cortex. Data expressed as mean ± SD, student’s t-test (two-sided) performed for all statistical analyses except (b), where Welch’s t-test was performed. ∆ = males, ○ = females. ***P < 0.001, ****P < 0.0001. No other statistical comparisons are significant unless indicated

Fig. 2

Removal of astrocytic APOE4 before amyloid deposition shifts Aβ distribution from plaques to the cerebrovasculature. a–d, X34 staining for fibrillar plaques/CAA (a) with percent area coverage of total X34 (b), CAA (c), and amyloid plaques (d) in the cortex overlying the hippocampus of 10-month-old 5X+AL- or 5X+AL+ mice after astrocytic APOE4 removal at 2-months-of-age. e, Proportion of CAA in total X34⁺ staining. f–i, Aβ immunoreactivity (Aβ-IR) (f) with percent area coverage of total Aβ-IR (g), vascular Aβ-IR (h), and plaques (i) in the cortex overlying the hippocampus. j Proportion of vascular Aβ in total Aβ-IR. Scalebar: 300 µm. Data expressed as mean ± SD, unpaired student’s t-test (two-sided) performed for all statistical analyses except (b), where Welch’s t-test was performed. ∆ = males, ○ = females. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. No other statistical comparisons are significant unless indicated

Fig. 3

Astrocytic APOE4 regulates the distribution of Aβ₄₀ and Aβ₄₂ deposition in plaques, parenchymal vessels, and leptomeningeal vessels. a–d, PBS-soluble and guanidine-HCL-soluble (“insoluble”) Aβ₄₀ and Aβ₄₂ protein concentrations assessed by ELISA from cortex. e–h Aβ₄₀ immunoreactivity (IR) (e) with percent area coverage of total Aβ₄₀ (f), total vascular Aβ₄₀ (g), and Aβ₄₀ in plaques (h). i Proportion of vascular Aβ₄₀ in total Aβ₄₀ IR. j, k Aβ₄₀ IR restricted to pial vessels (j) and penetrating vessels (k). (l) Proportion of vascular Aβ₄₀ in penetrating vessels out of total vascular Aβ₄₀. m–p, Aβ₄₂ immunoreactivity (IR) (m) with percent area coverage of total Aβ₄₂ (n), total vascular Aβ₄₂ (o), and Aβ₄₂ in plaques (p). q Proportion of vascular Aβ₄₂ in total Aβ₄₂ IR. r, s, Aβ₄₂ IR restricted to pial vessels (r) and penetrating vessels (s). t Proportion of vascular Aβ₄₂ in penetrating vessels out of total vascular Aβ₄₂. Blue arrow = vascular Aβ. Purple arrows = plaques. Scalebar: 500 µm. Data expressed as mean ± SD, student’s t-test (two-sided) performed for all statistical analyses except (d), where Welch’s t-test was performed. ∆ = males, ○ = females. *P < 0.05, *P < 0.01, ***P < 0.001, ****P < 0.0001. No other statistical comparisons are significant unless indicated

Fig. 4

Depletion of APOE in astrocytes diminishes certain disease-associated glial signatures. a–e, GFAP⁺ astrocytic staining (a) and quantification of area coverage (b) in the cortex of 10-month-old 5X+AL- or 5X+AL+ mice. Scalebar: 300 µm. Representative images (c) and quantification of the number of astrocytes surrounding CAA (d) or plaques (e). Scalebar: 20 µm. Each point represents the number of astrocytes surrounding a single CAA or plaque normalized to that CAA or plaque area from n = 8–10 mice per group. f–j, IBA1⁺ microglial coverage (f) and quantification of area coverage (g) in the cortex overlying the hippocampus. Scalebar: 300 µm. Per plaque analysis of number of microglia clustering CAA (h, i) or plaques (j). Scalebar: 20 µm. Each point represents the number of microglia surrounding a single CAA or plaque normalized to that CAA or plaque area from n = 8–10 mice per group. k, l, Relative mRNA expression of homeostatic and disease-associated astrocytic (k) and microglial genes (l). Data imputed for Clec7a mRNA (column 10) but not used in statistical analysis. Each column represents an individual mouse. Data expressed as mean ± SD, student’s t-test (two-sided) performed for all statistical analyses except (d), (e), (k – S100β), and (l – Cst7, Cst3, Itgax, Spp1) where Welch’s t-test was performed. ∆ = males, ○ = females. *P < 0.05, **P < 0.01. No other statistical comparisons are significant unless indicated

Fig. 5

Astrocytic APOE removal protects against dystrophic neurites. a–d, LAMP1 staining for dystrophic neurites (a) with quantification of the percentage of total LAMP1 in the cortex (b), percent area coverage of LAMP1 staining around CAA normalized to percent area covered by X34⁺ CAA (c), and percent area coverage of LAMP1 staining around plaques normalized to percent area covered by X34⁺ plaques (d). Scalebar: 200 µm. Purple arrowheads: LAMP1 around plaques. Blue arrowheads: LAMP1 around CAA. Data expressed as mean ± SD, student’s t-test (two-sided) performed for all statistical analyses except (b) and (c), where Welch’s t-test was performed. ∆ = males, ○ = females. *P < 0.05, **P < 0.01, ****P < 0.0001. No other statistical comparisons are significant unless indicated

Fig. 6

Vascular degeneration is ameliorated despite elevated CAA. a, b Staining for the blood-derived protein fibrinogen (a) with quantification of percent area covered (b) in the cortex of 10-month-old 5X+AL- or 5X+AL+ mice. Yellow arrow: fibrinogen staining. Scalebar: 100 µm. c, d, Average microhemorrhage number (c) and size (d) via Prussian blue staining. e, f, In vivo assessment of leptomeningeal function measured by percent vasodilatory change from baseline after topical application of vascular smooth muscle cell-dependent molecule (SNAP). Purple arrowheads: Increased dilation. Blue arrowheads: No change in dilation. Data from 9 vessels in 4 mice (5X+AL-) and 6 vessels in 3 mice (5X+AL+). Scalebar: 15 µm. SNAP = S-Nitroso-N-acetylpenicillamine. Data expressed as mean ± SD, student’s t-test (two-sided) performed for all statistical analyses except (c) and (d), where Welch’s t-test was performed. ∆ = males, ○ = females. *P < 0.05, ***P < 0.001. No other statistical comparisons are significant unless indicated