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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- Hamaguchi, T., Eisele, Y.S., Varvel, N.H. et al. Acta Neuropathol (2012) 123: 31. doi:10.1007/s00401-011-0912-1
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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.
KeywordsAlzheimerAmyloidPrionSeedingSenile plaquesTransgenic mouse
The deposition of β-amyloid (Aβ) peptide in senile plaques and cerebral Aβ angiopathy (CAA) can be induced in the brains of β-amyloid precursor protein (APP)-transgenic (tg) mice by the infusion of Aβ-rich brain extracts from patients with Alzheimer’s disease (AD) or from aged APP-tg mice [5, 11, 18, 29]. Mechanistically, the seeding of Aβ deposition resembles the transmission of prion disease in that a misfolded, multimeric form of the disease-associated protein appears to instigate aggregation by corruptive molecular templating [10, 25, 28]. Aβ deposition also has been induced in wild-type marmosets [1, 22], a nonhuman primate that produces human-type sequence Aβ .
Advancing age is the greatest risk factor for developing AD , but how age promotes the development of Aβ plaques and CAA remains uncertain . Previous studies of the seeded induction of Aβ deposition in mice have employed Tg2576 and APP23 mouse models that spontaneously form amyloid plaques and CAA beginning sometime around 8–10 months of age [11, 18]. To unambiguously identify the seeded Aβ deposits in the absence of endogenous and spontaneously generated lesions, these studies typically used relatively short incubation periods of 3–6 months after intracerebral administration [5, 11, 16, 18], and 6–7 months after intraperitoneal administration . After longer periods, distinguishing seeded deposits from those that normally form in these models becomes increasingly problematic, thus hampering the analysis of the impact of advancing age and extended incubation periods on the instigation and spread of Aβ lesions in the brain.
In the present study, we used a genomic, homozygous APP-tg mouse model (R1.40 mice) that begins to exhibit Aβ deposition only in late adulthood  to examine (1) whether seeding is more effective when initiated in older animals, compared to younger animals, and (2) the long-term effects of focally administered Aβ seeds on the evolution and spread of β-amyloid lesions in the brain.
Materials and methods
Male, hemizygous R1.40 APP-tg mice on a B6.129 background were purchased from The Jackson Laboratory (Bar Harbor, ME, USA) and bred with C57BL/6J mice. Homozygous mice were then generated by cross-breeding the hemizygous offspring. R1.40 mice carry a yeast artificial chromosome containing the genomic copy of human APP harboring the K670N-M671L mutation . R1.40 APP-tg mice in the present study were homozygous and were maintained under specific pathogen-free conditions. The experimental procedures were carried out in accordance with the veterinary office regulations of Baden–Württemberg (Germany) and approved by the local Animal Care and Use Committees.
Preparation of brain extracts
Extracts were prepared from 22- to 25-month old, Abeta-depositing APP23 mice (tg extract), or from age-matched, non-transgenic (wild-type [Wt]) mice (Wt extract) as previously described . The forebrains were obtained and frozen on dry ice and stored at −80°C. Samples were homogenized at 10% (w/v) in PBS, vortexed, sonicated 3 × 5 s and centrifuged at 3,000×g for 5 min. The supernatant was used as the seeding agent. Aβ levels were estimated with electrochemiluminescence-linked immunoassay, revealing Aβ concentrations of 10–20 ng/μl.
Stereotaxic injection of brain extracts
Mice were anaesthetized with a mixture of fentanyl (0.05 mg/kg body weight), midazolam (5 mg/kg body weight) and medetomidine (0.50 mg/kg body weight). Bilateral stereotaxic injections of extract were made with a Hamilton syringe into the hippocampus (2.5 μl) and overlying neocortex (1.0 μl). Extract was injected into the hippocampus (AP −2.5 mm, L ±2.0 mm, DV −1.8 mm) over a 2-min period, and the needle was kept in place for an additional 2 min. The needle was raised to the overlying neocortex (AP −2.5 mm, L ±2.0 mm, DV −1.0 mm) and the additional 1 μl of extract was injected over a 1-min period. After 2 more minutes, the needle was slowly withdrawn before suturing. After surgery an antidote (flumazenil 0.5 mg/kg body weight, atipamezole 2.5 mg/kg body weight, naloxone 1.2 mg/kg body weight) was administered. A total of 38 mice were used (see figure captions for details). Two mice were excluded from the analysis because the injections missed the dorsal hippocampus; one mouse was excluded because of hydrocephalus; two mice were excluded due to lack of seeding. The excluded mice were dispersed over the experimental groups.
Tissue processing, histological and stereological analysis
The mice were transcardially perfused with PBS under deep anesthesia (ketamine 400 mg/kg, xylazine 40 mg/kg). Brains were harvested, hemidissected, and the left hemispheres were processed as described . The sections were immunostained using polyclonal antibody CN3 (1:1,000; raised against residues 1–16 of human Aβ)  and visualized using standard immunoperoxidase procedures with the Vectastain Elite ABC Kit (Vector Laboratories, Burlingame, CA, USA).
The areal density of Aβ-immunoreactive lesions was quantitated in random-systematic sets of every 12th section through the neocortex and hippocampus. Pia-associated vessels were not counted. Stereological analysis of CN3-positive staining was performed using a Zeiss microscope equipped with a motorized x–y–z stage coupled to a video-microscopy system and the Stereo Investigator software (MicroBrightField, Inc, Williston, VT, USA) as described .
Adjacent sets of sections were stained with Congo red according to standard protocols and viewed under cross-polarized light and analyzed. The following additional antibodies were used: rabbit polyclonal antibody against cow glial fibrillary acidic protein (GFAP) (Dako, Glostrup, Denmark); rabbit polyclonal antibody against ionized calcium-binding adapter molecule 1 (Iba1) (Wako; Richmond, VA, USA).
Immunoblotting and electrochemiluminescence-linked immunoassay
The right hemisphere of PBS-perfused mice was homogenized at 10% w/v in buffer (50 mM Tris [pH 8.0], 150 mM NaCl, 5 mM EDTA, and Complete Mini protease inhibitor cocktail [Roche Diagnostics GmbH, Penzberg, Germany]). To assess Aβ and APP levels, 4–12% NuPage Bis–Tris mini gels using NuPage LDS sample buffer were used (Invitrogen, Carlsbad, CA, USA), followed by protein transfer onto nitrocellulose membranes and probing with antibody 6E10 (Covance, Princeton, NJ, USA). As an internal control, monoclonal anti-GAPDH antibody (Hy Test Ltd, Turku, Finland) was used. Densitometric values of band intensities were analyzed using ImageJ 1.44 (http://www.rsbweb.nih.gov/ij/).
All values are expressed as means ± SEMs. Statistical analyses were performed using SPSS 16.0 software (SPSS Japan, Tokyo, Japan) and Statview 5.0.1 (SAS Institute Inc., Cary, NC, USA), with the significance threshold set at p ≤ 0.05, two tailed.
Previous studies have shown that brain-derived Aβ aggregates can induce Aβ lesions in the brains of susceptible mice [5, 6, 11, 16, 18, 29]. In the present study, we used the genomic-based R1.40 APP-tg model as host. These animals exhibit endogenous Aβ deposition at approximately 15 months of age , thus allowing for the analysis of extended incubation periods in Aβ extract-inoculated mice. Our results reveal that intracerebral injections of Aβ-containing brain extract also induce Aβ deposition in this model, although the Aβ induction after a 6-month incubation period was less than that seen in APP23 mice after 4 months of incubation [16, 18]. This observation is consistent with the threefold overexpression of human APP in R1.40 mice compared to sevenfold overexpression in APP23 animals [15, 26], and supports the view that the induction of Aβ deposition is dependent on the concentration of Aβ in the inoculum as well its production by the host [8, 18].
We found that a 6-month incubation period induced similar levels of Aβ aggregation in the brains of young (3 months) and older (9 months) R1.40 APP-tg mice, indicating that the aged brain does not provide a more favorable environment for the induction of Aβ deposition once Aβ seeds are present. Our results, while surprising in light of the view that age is a prominent risk factor for amyloid lesions [3, 7, 27], may be attributed to the fact that the brain levels of Aβ are constant in pre-depositing R1.40 mice between 3 and 15 months of age . Unchanging Aβ levels prior to Aβ pathology also have been reported in other AD mouse models [12, 14]. These findings suggest that age may increase the risk of AD by impairing the ability of cells to dispose pathogenic seeds [2, 20]. In addition, it cannot be excluded that a similar experiment in which seeding is compared in a young host versus a host at an age closer to the end of its mean life span would yield different results.
The most striking finding to emerge from the inoculations in R1.40 mice was the presence of Aβ deposits in much of the forebrain after the 12 month incubation period. Previous work has shown that the focal infusion of Aβ seeds into the hippocampus is sufficient to induce Aβ deposition throughout the entire hippocampus and even in connected brain areas; however, induction of lesions in widespread regions of the forebrain has not been previously observed [5, 10]. The mechanism of this extensive spreading of Aβ deposition is not clear, but implies that seeds not only migrate within brain structures and along defined neuronal pathways, but also may disseminate along perivascular fluid drainage channels, by vascular transport, or by simple diffusion through the brain parenchyma . The recent finding that small, soluble Aβ species are particularly potent inducers of β-amyloidosis , similar to the strong infectivity of small, non-fibrillar prion particles , underscores potential mechanistic similarities in the induction and spread of Aβ and PrP aggregates in the brain.
Birefringence under crossed-polarizing filters is indicative of the amyloid-like nature of proteinaceous deposits after staining with the dye Congo red . In seeded R1.40 APP-tg mice, the duration of the incubation period influenced the degree to which the resulting Aβ deposits were congophilic. Whereas the 6-month incubation period, starting at either 3 or 9 months of age, did not result in the induction of congophilic lesions, Congo red-positive plaques were observed after the 12-month incubation period. However, congophilic deposits were only observed near the injection site, indicating that diffuse pathology occurs first and eventually develops into Congo red-positive (i.e., amyloid) plaques. Because the cytological changes associated with Aβ deposition are most strongly associated with amyloid per se  rather than with diffuse deposits, the lack of congophilic amyloid induction after the 6-month incubation period did not allow us to determine whether the amyloid-associated cytopathology (neuritic dystrophy and glial activation) differs between young (9 months) and older (15 months) hosts [3, 7].
From a translational perspective, the present findings support the widely held notion that therapies directed at mitigating the formation of Aβ pathology should begin early in the pathogenic cascade that leads to AD [19, 23]. The exposure to Aβ seeds profoundly influences the onset and degree of β-amyloid pathology in APP-tg mouse models. While there is not yet direct evidence that exogenous seeds are involved in the initiation of AD, it is likely that endogenously generated seeds become increasingly prevalent with age due to the decline of proteostatic processes [3, 20]. The neutralization or removal of endogenous Aβ seeds thus remains a promising but elusive therapeutic goal for interfering with the pathogenesis of AD.
This work was supported by grants from the Competence Network on Degenerative Dementias (BMBF-01GI0705), the BMBF in the frame of ERA-Net NEURON (MIPROTRAN), NIH RR-00165, P50AG025688, the Alzheimer’s Association (NIRG-10-173099) and the CART Foundation. TH is recipient of a postdoctoral fellowship from the Alexander von Humboldt Foundation (Bonn, Germany). We gratefully acknowledge helpful discussions and experimental help of Franziska Langer, Götz Heilbronner, Ulrike Obermüller, Jörg Odenthal, Stephan Kaeser, Michael Hruscha and Andrea Bosch (Tübingen, Germany) and Harry LeVine III (Lexington, KY, USA).
Conflict of interest
The authors declare that they have no conflict of interest.