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, 123 (1), 31-7

The Presence of Aβ Seeds, and Not Age Per Se, Is Critical to the Initiation of Aβ Deposition in the Brain

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The Presence of Aβ Seeds, and Not Age Per Se, Is Critical to the Initiation of Aβ Deposition in the Brain

Tsuyoshi Hamaguchi et al. Acta Neuropathol.

Abstract

The deposition of the β-amyloid (Aβ) peptide in senile plaques and cerebral Aβ-amyloid angiopathy can be seeded in β-amyloid precursor protein (APP)-transgenic mice by the intracerebral infusion of brain extracts containing aggregated Aβ. Previous studies of seeded β-amyloid induction have used relatively short incubation periods to dissociate seeded β-amyloid induction from endogenous β-amyloid deposition of the host, thus precluding the analysis of the impact of age and extended incubation periods on the instigation and spread of Aβ lesions in brain. In the present study using R1.40 APP-transgenic mice (which do not develop endogenous Aβ deposition up to 15 months of age) we show that: (1) seeding at 9 months of age does not induce more Aβ deposition than seeding at 3 months of age, provided that the incubation period (6 months) is the same; and (2) very long-term (12 months) incubation after a focal application of the seed results in the emergence of Aβ deposits throughout the forebrain. These findings indicate that the presence of Aβ seeds, and not the age of the host per se, is critical to the initiation of Aβ aggregation in the brain, and that Aβ deposition, actuated in one brain area, eventually spreads throughout the brain.

Conflict of interest statement

Conflict of Interest The authors declare that they have no conflict of interest.

Figures

Fig. 1
Fig. 1. Steady-state levels of APP and Aβ are similar in pre-depositing R1.40 mice
(a) Human APP and Aβ levels in the brains of R1.40 mice at multiple ages. (3 months [n=6; 3 male, 3 female], 9 months [n=6; 3 male, 3 female] and 15 months [n=6; 3 male, 3 female]). Shown are immunoblots, using antibody 6E10, of three mice/age group; GAPDH served as a loading control. (b) Quantification of protein levels in all mice revealed no difference among the groups (one-way ANOVA for APP and Aβ; both p>0.05). Three samples in each age group were applied to 1 gel, and 2 gels were used in total. Densitometric values of band intensities were analysed using ImageJ 1.44, and percentages relative to the amount in 3month-old mice were calculated from the average value at 3 months of age. (c) No Aβ deposition was found in the brains of 3-and 9-month-old R1.40 mice by immunohistochemical analysis, while in two of six 15-month-old mice, infrequent Aβ deposits were present in the frontal cortex. Scale bar = 500 μm.
Fig. 2
Fig. 2. Study design
Group 1 was intracerebrally seeded at 3 months of age, and analysed at 9 months; Group 2 was seeded at 9 months of age and analysed at 15 months; Group 3 was seeded at 3 months of age and analysed at 15 months.
Fig. 3
Fig. 3. Older mice and younger mice show similar levels of induced Aβ deposition. However, long incubation periods allow for generation of widespread Aβ aggregation and glial cell response
(a) Induction of Aβ deposition was evident in all three groups of R1.40 mice that were intracerebrally seeded with the Tg extract, but not with the Wt extract. While the β-amyloid induction was similar in groups 1 and 2, Aβ load was greatest in Group 3. Adjacent sections were double-stained with Congo red and GFAP or Iba-1. While in groups 1 and 2, all induced β-amyloid was Congo red-negative, group 3 showed at least some Congo red-positive deposits surrounded by darkly stained and hypertrophic GFAP-positive astrocytes (insert, left) and Iba1-positive microglia (insert, right). (b) Quantitative stereological analysis. Group 1 (WT extract: n=5 [3 female, 2 male], Tg extract: n=7 [4 female, 3 male]); Group 2 (WT extract injected: n=6 [2 female, 4 male], Tg extract: n=6 [2 female, 4 male]); Group 3 (WT extract: n=5 [2 female, 3 male], Tg extract: n=4 [3 female, 1 male]). 3-way ANOVA for Gender × Group × Extract revealed significant main effects for Group and Extract (p<0.001) but no significant effect of gender (p>0.05). There was a significant Group × Extract interaction (F[2,21]=89.23; p<0.001) and subsequent Scheffé post hoc analyses were used to pinpoint group differences (*; p<0.05, **; p<0.01, ***; p<0.001). Scale bar = 500 μm and 10 μm.
Fig. 4
Fig. 4. Longer incubation times result in extensive spreading of Aβ deposition in the forebrain
(a) Twelve months after intracerebral injections of the Tg extract, copious Aβ was deposited in the brain (shown are coronal sections of the mouse presented in Fig. 3; the coordinates from bregma A–P are indicated). Note that Aβ deposition was widespread throughout the neocortex and hippocampus, although Tg extract was injected only locally into the hippocampus and overlying cortex at AP −2.5mm. (b) Aβ load in each section from anterior to posterior is shown for the 12-month incubation mice (group 3) and compared to the 6-month incubation mice (group 1 &2) (section 1 to section 11; approximate location from bregma A–P is as follows, section 1= AP +2.2mm, section 5=AP +0.1mm, section 8= AP −1.8mm, section 11=AP −3.3mm). Note that the induced Aβ is highest around the injection site (corresponding to sections 9&10) in all groups, and that the Aβ deposits have spread throughout the brain, including the most anterior parts. Scale bar = 500 μm.

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