The Icelandic Mutation (APP-A673T) Is Protective against Amyloid Pathology In Vivo

Abstract

A previous epidemiological study in Northern Europe showed that the A673T mutation (Icelandic mutation) in the amyloid precursor protein gene (APP) can protect against Alzheimer's disease (AD). While the effect of the A673T mutation on APP processing has been investigated primarily in vitro, its in vivo impact has not been evaluated. This is mainly because most existing AD mouse models carry the Swedish mutation. The Swedish and Icelandic mutations are both located near the β-cleavage site, and each mutation is presumed to have the opposite effect on β-cleavage. Therefore, in the AD mouse models with the Swedish mutation, its effects could compete with the effects of the Icelandic mutation. Here, we introduced the A673T mutation into App knock-in mice devoid of the Swedish mutation (App^G-F mice) to avoid potential deleterious effects of the Swedish mutation and generated App^G-F-A673T mice. APP-A673T significantly downregulated β-cleavage and attenuated the production of Aβ and amyloid pathology in the brains of these animals. The Icelandic mutation also reduced neuroinflammation and neuritic alterations. Both sexes were studied. This is the first successful demonstration of the protective effect of the Icelandic mutation on amyloid pathology in vivo. Our findings indicate that specific inhibition of the APP-BACE1 interaction could be a promising therapeutic approach. Alternatively, the introduction of the disease-protective mutation such as APP-A673T using in vivo genome editing technology could be a novel treatment for individuals at high risk for AD, such as familial AD gene mutation carriers and APOE ε4 carriers.

Keywords: Alzheimer’s disease; App knock-in mice; Icelandic mutation; amyloid pathology; amyloid precursor protein; protective mutation.

Figure 1.

Generation of App^G-F-A673T mice. A, Sequences of wild-type APP and APP with the Swedish or Icelandic mutations around Aβ. The red characters represent the Swedish (KM670/671NL) and Icelandic (A673T) mutations, respectively. B, Exact sequences showing sgRNA (orange line) with the PAM site (blue box) in the mouse App gene. The red characters represent the Icelandic (A673T), Arctic (E693G), and Beyreuther/Iberian (I716F) mutations, respectively. The green characters represent humanized sequences in mouse Aβ (G676R, F681Y, and R684H). C, Schematic illustration of CRISPR-Cas9–mediated genome editing in App^G-F-KI mouse zygotes by microinjection. D, Sanger sequencing results determined the App^G-F/G-F (top panel) and App^{G-F-A673T/G-F-A673T} (bottom panel) genotype, respectively. The boxes indicate the codons encode alanine (GCA, black) and threonine (ACA, magenta), respectively. E, Regional information of potential off-target sites that were identified using Cas-OFFinder (www.rgenome.net/cas-offinder/) and COSMID (https://crispr.bme.gatech.edu/).

Figure 2.

Amyloid pathology of App^G-F-A673T mice. A, Aβ content detected by enzyme-linked immunosorbent assay (ELISA) using the Tris-HCl–soluble fraction (TS) and GuHCl-soluble fraction (GuHCl) of the cortices of App^G-F and App^G-F-A673T mice at 3 months (3 males and 3 females, respectively). # indicates that the value was below the detection limit. Each bar represents the mean ± SEM. B, C, Immunohistochemistry images showing Aβ deposition as indicated by immunostaining with 6E10 antibody against Aβ at 8 months of App^G-F/G-F (B) and App^{G-F-A673T/G-F-A673T} (C) mice. Scale bar, 1 mm. D, E, Quantification of amyloid plaque load in the cortex (D) and hippocampus (E) of App^G-F and App^G-F-A673T mice at 8 months. Scale bar, 1 mm. *p < 0.05, **p < 0.01, and ****p < 0.0001. n = 6 (3 males and 3 females) for each genotype, Student's t test. Please see Extended Data Figures 2-1 and 2-2 for more details.

Figure 3.

APP processing in App^G-F-A673T mice. A, Full-length APP expression in the cortices of WT, App^G-F, and App^G-F-A673T mice at 3 months (3 females). B, Western blot analysis using antibody targeting the C-terminus of APP in the cortex of WT, App^G-F, and App^G-F-A673T mice at 3 months (3 females). mAPP, mature APP; imAPP, immature APP. C–F, Quantification of CTF-β (C), CTF-α (D), CTF-β/CTF-α ratio (E), and AICD (F) of the blot shown in (B). G, Expression of enzymes involved in APP processing. Expression levels of full-length APP protein (A) and enzymes that are involved in APP processing (G) in the brain of App^G-F and App^G-F-A673T mice at 3 months old are quantified in the Western blot. Values of each band were normalized by expression levels of β-actin for APP and GAPDH for enzymes (H–L). **p < 0.01 and ***p < 0.001. One-way ANOVA followed by Tukey's multiple-comparisons test. Please see Extended Data Figure 3-1 for more details.

Figure 4.

Neuroinflammation in App^G-F-A673T mice. A, B, Inflammatory responses in the cortices of App^G-F (A) and App^G-F-A673T mice (B) at 12 months. Antibodies against Aβ (82E1, gray), ionized calcium-binding adapter molecule 1 (Iba1, magenta), and glial fibrillary acidic protein (GFAP, yellow-green) were used, respectively. Scale bar, 500 μm. C, D, Quantification of Iba1 (C) and GFAP (D) signals in the cortex of App^G-F and App^G-F-A673T mice. Those units of the y-axis indicate the area fraction of the cortex. *p < 0.05 and **p < 0.01. n = 10 (5males and 5 females) for App^G-F mice, n = 7 (4males and 3 females) for App^G-F-A673T mice, Student's t test. Please see Extended Data Figure 4-1 for more details.

Figure 5.

Neuritic alterations in App^G-F-A673T mice. Formation of dystrophic neurites (DNs) in the brain of 12-month-old App^G-F (A, D) and App^G-F-A673T (B, E) mice. DNs were detected by antibodies against phosphorylated tau (AT8; A, B) and LAMP1 (D, E); the magenta arrows indicate DNs. Note that most of the AT8 (A, B) and LAMP1 (D, E) signals (arrows) appeared in close proximity to amyloid plaques (green, detected by 6E10 antibody). Quantification of DNs in the cortex (C, F) of App^G-F and App^G-F-A673T mice at 12 months. Scale bar, 100 μm. ***p < 0.001. n = 10 (5males and 5 females) for App^G-F mice, n = 7 (4males and 3 females) for App^G-F-A673T mice, Student's t test. Please see Extended Data Figure 5-1 for more details.