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Early-onset and Robust Cerebral Microvascular Accumulation of Amyloid β-Protein in Transgenic Mice Expressing Low Levels of a Vasculotropic Dutch/Iowa Mutant Form of Amyloid β-Protein Precursor*
Frank P. Smith Laboratories for Neurosurgery, Department of Neurosurgery and Division of Neurovascular Biology, University of Rochester Medical Center, Rochester, New York 14642
Frank P. Smith Laboratories for Neurosurgery, Department of Neurosurgery and Division of Neurovascular Biology, University of Rochester Medical Center, Rochester, New York 14642
To whom correspondence should be addressed: Dept. of Medicine, Health Sciences Center, T-15/081, Stony Brook University, Stony Brook, NY 11794-8153. Tel.: 631-444-1661; Fax: 631-444-7518;
* This work was supported by National Institutes of Health Grants NS36645 (to W. E. V. N.), NS34467 (to B. V. Z.), and AG16233 (to B. V. Z. and W. E. V. N.) and by Alzheimer's Association Grant IIRG-02-3995 (to W. E. V. N.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Cerebrovascular deposition of amyloid β-protein (Aβ) is a common pathological feature of Alzheimer's disease and related disorders. In particular, the Dutch E22Q and Iowa D23N mutations in Aβ cause familial cerebrovascular amyloidosis with abundant diffuse amyloid plaque deposits. Both of these charge-altering mutations enhance the fibrillogenic and pathogenic properties of Aβ in vitro. Here, we describe the generation of several transgenic mouse lines (Tg-SwDI) expressing human neuronal Aβ precursor protein (AβPP) harboring the Swedish K670N/M671L and vasculotropic Dutch/Iowa E693Q/D694N mutations under the control of the mouse Thy1.2 promoter. Tg-SwDI mice expressed transgenic human AβPP only in the brain, but at levels below those of endogenous mouse AβPP. Despite the paucity of human AβPP expression, quantitative enzyme-linked immunosorbent assay measurements revealed that Tg-SwDI mice developed early-onset and robust accumulation of Aβ in the brain with high association with isolated cerebral microvessels. Tg-SwDI mice exhibited striking perivascular/vascular Aβ deposits that markedly increased with age. The vascular Aβ accumulations were fibrillar, exhibiting strong thioflavin S staining, and occasionally presented signs of microhemorrhage. In addition, numerous largely diffuse, plaque-like structures were observed starting at 3 months of age. In vivo transport studies demonstrated that Dutch/Iowa mutant Aβ was more readily retained in the brain compared with wild-type Aβ. These results with Tg-SwDI mice demonstrate that overexpression of human AβPP is not required for early-onset and robust accumulation of both vascular and parenchymal Aβ in mouse brain.
The progressive accumulation of amyloid β-protein (Aβ)
). The Aβ peptide is derived from the Aβ precursor protein (AβPP), a type I integral membrane protein, through sequential proteolytic processing mediated by β- and γ-secretase activities (
). Several mutations that are linked to familial forms of early-onset Alzheimer's disease have been identified in the AβPP gene. These mutations tend to cluster around the β- and γ-secretase cleavage sites within AβPP and lead to increased production of total Aβ or a preferential increase in the levels of the longer, more pathogenic Aβ42 peptide (
). On the other hand, several mutations in the AβPP gene that reside within residues 21–23 of Aβ and that give rise to familial forms of cerebral amyloid angiopathy (CAA) have been found. The first recognized of these was the Dutch E22Q mutation, which is associated with diffuse Aβ deposition in the neuropil and severe CAA, leading to recurrent and often fatal hemorrhagic episodes at mid-life (
). More recently, the Iowa D23N mutation in Aβ was identified in a cohort presenting with late-onset dementia accompanied by severe CAA with numerous small cortical hemorrhages, cortical and subcortical infarcts, and neurofibrillary tangles (
) showed, that compared with wild-type Aβ, the Dutch E22Q and Iowa D23N mutant Aβ peptides exhibit enhanced fibrillogenic and pathogenic properties in cultured cerebrovascular cells used as in vitro models for CAA. Moreover, an experimental Aβ peptide containing the Dutch and Iowa mutations together (E22Q/D23N) possesses even more robust fibrillogenic and pathogenic properties in vitro compared with either single mutation alone (
). These combined findings suggest that the loss of negative charges and gain of pathogenicity in Aβ associated with the Dutch and Iowa mutations may directly correlate with the accumulation of Aβ in the brain, particularly around the cerebral vasculature.
To further investigate this in vivo, we generated transgenic mice expressing human Swedish, Dutch, and Iowa triple-mutant AβPP (Tg-SwDI) in brain that produce Dutch/Iowa E22Q/D23N double-mutant Aβ. Although these transgenic mice were found to express human AβPP at levels below those of endogenous mouse AβPP, three independent Tg-SwDI mouse lines developed strikingly similar early-onset and robust accumulation of Aβ in the brain with high association with the cerebral microvasculature. Importantly, these results demonstrate that overexpression of human AβPP is not necessary for the development of Aβ pathology in mouse brain. Furthermore, functional studies revealed, that compared with wild-type Aβ, Dutch/Iowa mutant Aβ was poorly cleared from mouse brain into the circulation. These findings suggest that the observed clearance deficit of Dutch/Iowa mutant Aβ likely contributes to its strong accumulation in and around cerebral blood vessels and in the parenchyma of the Tg-SwDI mice and possibly in patients with either the Dutch or Iowa familial CAA mutations.
EXPERIMENTAL PROCEDURES
Vector Construction and Generation of Transgenic Mouse Lines—A pcDNA3 vector containing 2.1 kb of human AβPP (isoform 770) cDNA was used to introduce mutations Swedish K670N/M671L, Dutch E693Q, and Iowa D694N using the QuikChange kit (Stratagene, La Jolla, CA). The AβPP770-SwDI cDNA was amplified by PCR using primers containing the NheI 5′-linker and SacII 3′-linker. The PCR product was digested and subcloned between exons II and IV of a Thy1.2 expression cassette (a gift from Dr. F. LaFerla, University of California, Irvine, CA) using NheI and SacII restriction sites. The completed construct was entirely sequenced to confirm its integrity. The 9-kb transgene was liberated by NotI/PvuI digestion, purified, and microinjected into pronuclei of C57Bl/6 single cell embryos at the Stony Brook Transgenic Mouse Facility. Three founder transgenic mice were identified by Southern blot analysis of tail DNA. Transgenic offspring from each line were determined by PCR analysis of tail DNA using the following primers specific for human AβPP: 5′-CCTGATTGATACCAAGGAAGGCATCCTG-3′ and 5′-GTCATCATCGGCTTCTTCTTCTTCCACC-3′ (generating a 500-bp product). All subsequent analyses were performed with heterozygous transgenic mice.
Immunoblot Quantitation of AβPP—Mouse forebrain, distinct mouse brain regions, or various peripheral tissues were homogenized in 10 volumes of 50 mm Tris-HCl (pH 7.5) containing 150 mm NaCl, 1% SDS, 0.5% Nonidet P-40, 5 mm EDTA, and proteinase inhibitor mixture (Roche Applied Science). The tissue homogenates were clarified by centrifugation at 14,000 × g for 10 min. Protein concentrations of the resulting supernatants were determined using the BCA protein assay Kit (Pierce). The levels of AβPP in the tissue homogenate samples were determined by performing quantitative immunoblotting as described (
). Briefly, 35 μg of total protein from each sample were electrophoresed on SDS-10% polyacrylamide gels, and the proteins were transferred onto Hybond nitrocellulose membranes (Amersham Biosciences). Unoccupied sites on the membranes were blocked overnight with 5% nonfat milk in phosphate-buffered saline with 0.05% Tween 20. The membranes were probed either with monoclonal antibody P2-1, which is specific for human AβPP (
), or with monoclonal antibody 22C11 (Chemicon International, Inc., Temecula, CA), which is specific for mouse and human AβPP, and then incubated with a secondary peroxidase-coupled sheep anti-mouse IgG antibody at a dilution of 1:1000. The peroxidase activity on the membranes was detected using Super-signal Dura West (Pierce). Bands corresponding to AβPP were measured using a VersaDoc 3000 imaging system (Bio-Rad) with the manufacturer's Quantity One software and compared with standard curves generated from known quantities of purified AβPP.
Enzyme-linked Immunosorbent Assay (ELISA) Quantitation of Aβ Peptides—Soluble pools of Aβ40 and Aβ42 were determined by specific ELISAs of carbonate-extracted mouse forebrain tissue; and subsequently, the insoluble Aβ40 and Aβ42 levels were determined by ELISA of guanidine lysates of the insoluble pellets resulting from the carbonate-extracted brain tissue (
). Total vascular Aβ40 and Aβ42 levels were measured in guanidine lysates of the brain microvessels isolated from Tg-SwDI mice. In the sandwich ELISAs, Aβ40 and Aβ42 were captured using their respective carboxyl terminus-specific antibodies m2G3 and m21F12, and biotinylated antibody m3D6, specific for human Aβ, was used for detection (
). Because antibody m3D6 recognizes an epitope in the first five amino acids of Aβ, this ensured that the sandwich ELISA was measuring amino-terminally intact Aβ peptides.
Immunohistochemical Analysis—Mice were killed at specific ages, and the brains were removed and, in most cases, bisected in the mid-sagittal plane. One hemisphere was snap-frozen and used for the protein analyses described above. The other hemisphere was placed in 70% ethanol overnight and subjected to increasing sequential dehydration in ethanol, followed by xylene treatment and embedding in paraffin. Sections were cut from mouse brain hemispheres in the sagittal plane at 5 μm using a microtome, placed in a flotation water bath at 45 °C, and then picked on glass slides. Paraffin was removed from the sections by washing with xylene, and the tissue sections were rehydrated in decreasing concentrations of ethanol. Antigen retrieval was performed by treating the tissue sections with proteinase K (0.2 mg/ml) for 10 min at 22 °C. Primary antibodies were detected with horseradish peroxidase-conjugated or alkaline phosphatase-conjugated secondary antibodies and visualized either with a stable diaminobenzidine solution (Invitrogen) or with the fast red substrate system (Spring Bioscience, Fremont, CA), respectively, as substrate. Sections were counterstained with hematoxylin. Thioflavin S staining for fibrillar amyloid was performed as described (
), and rabbit polyclonal antibody to collagen type IV (Research Diagnostics Inc., Flanders, NJ). The percent of Aβ-associated blood vessels in the frontotemporal cortex, thalamic, and subiculum regions was determined in four mice at each of the specified ages using stereological principles as described (
). After radiolabeling, the preparations were subjected to HPLC to separate the monoiodinated non-oxidized forms of Aβ (which is the tracer we used) from di-iodinated Aβ, unlabeled non-oxidized Aβ, and oxidized Aβ species. The content of material in the peaks eluted by HPLC was determined by matrix-assisted laser desorption ionization time-of-flight mass spectrometry to ensure the purity of the radiolabeled species. These analyses confirmed that oxidized species of 125I-labeled wild-type Aβ40 and Dutch/Iowa mutant Aβ40 were not present in the preparations. At specific activities between 55 and 85 μCi/μg, the radiolabeled peptides were stabilized using ethanol as a quenching system and kept for up to 96 h. Prior to infusion into animals, we performed HPLC purification of the tracer to ensure use of monomeric Aβ species.
Brain Clearance Model—Central nervous system clearance of 125I-labeled wild-type Aβ40 or Dutch/Iowa mutant Aβ40 was determined simultaneously with [14C]inulin (a metabolically inert reference marker) in male C57Bl/6 wild-type mice (8–10 weeks old) as described (
). A stainless steel guide cannula was implanted stereotaxically into the right caudate putamen of anesthetized mice (60 mg/kg sodium pentobarbital administered intraperitoneally), and 0.5 μl of tracer fluid containing 125I-labeled wild-type Aβ40 or Dutch/Iowa mutant Aβ40 (1–120 nm) were injected over 5 min along with [14C]inulin using the Ultra Micropump (World Precision Instruments, Inc., Sarasota, FL). Radioactivity analysis was performed within 30 min.
Tissue Sampling and Radioactivity Analysis—Brains were sampled and prepared for radioactivity analysis. Degradation of 125I-labeled Aβ40 peptides was initially studied by trichloroacetic acid precipitation. Previous studies with 125I-labeled Aβ40 demonstrated an excellent correlation between the trichloroacetic acid and HPLC methods (
). Brain samples were mixed with trichloroacetic acid (10% final concentration) and centrifuged at 14,000 × g for 8–10 min at 4 °C, and the radioactivity in the precipitate, water, and chloroform fractions was determined in a γ-counter. The integrity of 125I-labeled wild-type Aβ or Dutch/Iowa mutant Aβ injected into the brain was ≥99% as determined by trichloroacetic acid analysis. Degradation of 125I-labeled Aβ peptides in the brain was further studied by HPLC and SDS-PAGE analyses. Following intracerebral injections of 125I-labeled Aβ, brain tissue was homogenized in phosphate-buffered saline containing proteinase inhibitors (0.5 mm phenylmethylsulfonyl fluoride, 1 μg/ml leupeptin, and 1 mmp-aminobenzamidine) and centrifuged at 100,000 × g for 1 h at 4 °C. The supernatant was then lyophilized. The resulting material was dissolved in 0.005% trifluoroacetic acid (pH 2.0) in water before injection onto a Vydac C4 column (Separations Group, Hesperia, CA). Separation was achieved with a 30-min linear gradient of 25–83% acetonitrile in 0.1% trifluoroacetic acid at a flow rate of 1 ml/min as described (
). Under these conditions, the Aβ standards eluted between 29.1 and 31.2 min for the wild-type Aβ40 and Dutch/Iowa mutant Aβ40 peptides. The eluted fractions were collected and counted. The integrity of 125I-labeled Aβ peptides injected into the brain was >98% as determined by HPLC analysis, confirming the results of trichloroacetic acid analysis. For SDS-PAGE analysis, trichloroacetic acid-precipitated samples were resuspended in 1% SDS, vortexed, incubated at 55 °C for 5 min, neutralized, boiled for 3 min, homogenized, and analyzed by electrophoresis on 10% Tris/Tricine gels, followed by fluorography. Lyophilized HPLC fractions were resuspended in sample buffer, neutralized, boiled, and electrophoresed as we reported previously (
Calculations—The percentage radioactivity remaining in the brain after microinjection was determined from the following equation: % recovery in brain = 100 × (Nb/Ni), where Nb is the radioactivity remaining in the brain at the end of the experiment, and Ni is the radioactivity injected into the brain. In all calculations, the dpm values for [14C]inulin and the cpm values for trichloroacetic acid-precipitable 125I radioactivity reflecting the intact peptide were used. Inulin was studied as a reference marker that is neither transported across the blood-brain barrier nor retained by the brain and therefore reflects the rate of transport via passive diffusion of the interstitial fluid (interstitial fluid) bulk flow (
). In the case of Aβ, there are two possible physiological pathways of elimination: direct transport across the blood-brain barrier into the bloodstream and elimination via interstitial fluid bulk flow into the cerebrospinal fluid and cervical lymphatics (
). To assess the effects of these CAA-associated Aβ mutations in vivo, we generated transgenic mice expressing the human AβPP770 isoform harboring the Swedish, Dutch, and Iowa mutations in neurons of the central nervous system under the control of the mouse Thy1.2 promoter (Fig. 1). The Swedish K670N/M671L mutation was included in the AβPP transgene to enhance β-secretase processing and production of Aβ (
). The adjacent Dutch E693Q and Iowa D694N mutations were included in the human AβPP transgene since we previously showed that the presence of both these mutations in Aβ markedly enhances the in vitro cerebrovascular pathogenic properties of Aβ compared with either single mutation (
). The transgenic mice were generated by microinjection of the AβPP770-SwDI construct into oocytes in a pure C57Bl/6 background. The presence of the transgene was confirmed by PCR analysis. All mice used in the subsequent characterization studies were heterozygous for the human AβPP transgene.
Fig. 1Schematic of the mouse Thy1.2 promoter-transgenic human SwDI mutant AβPP construct. The 9-kb transgene construct used to produce the transgenic mice was composed of cDNA from human AβPP770, containing the Swedish K670N/M671L, Dutch E693Q, and Iowa D694N mutations, subcloned between exons II and IV of a Thy1.2 expression cassette.
AβPP Expression and Aβ Production in Tg-SwDI/B Mice— The first transgenic mouse line generated (designated Tg-SwDI/B) showed expression of human AβPP in the brain (Fig. 2A). Although human AβPP expression was observed in the cortex, hippocampus, and brain stem, much lower levels were observed in the cerebellum, with no detectable expression in other non-neural tissues (Fig. 2B). However, analysis using a monoclonal antibody that detects both endogenous mouse AβPP and transgenic human AβPP unexpectedly revealed that expression of transgenic human AβPP was modest, and it was estimated by quantitative image analysis to be only at <50% the level of endogenous mouse AβPP (Fig. 2C). Consistent with the low level expression of transgenic human AβPP, young Tg-SwDI/B mice (≈2 months old) exhibited very low levels of total Aβ40 and Aβ42 in the brain, with the predominant species being the shorter Aβ40 peptide (Fig. 2D).
Fig. 2Analysis of transgenic human AβPP expression and Aβ levels in Tg-SwDI/B mice. Quantitative immunoblotting was performed as described under “Experimental Procedures.” A, immunoblot analysis of human AβPP expression in total brain homogenates from Tg-SwDI/B (lane 1) and wild-type (lane 2) mice. B, immunoblot analysis of human AβPP expression in tissue homogenates prepared from different brain regions and peripheral tissues of Tg-SwDI/B mouse. Sk, skeletal. C, immunoblot analysis of endogenous mouse AβPP and transgenic (Tg) human AβPP in wild-type mouse brain (lane 1) and Tg-SwDI/B mouse brain (lane 2) homogenates. D, the levels of total Aβ40 (lane 1) and total Aβ42 (lane 2) determined in 2-month-old Tg-SwDI/B mouse forebrains by ELISA measurements as described under “Experimental Procedures.” The data presented are the means ± S.D. of triplicate measurements in three mice.
Over the course of 1 year, the expression of transgenic human AβPP protein remained consistently low and was estimated to be ≈33 ± 4 ng/mg of total brain protein based on comparative quantitative immunoblot measurements against known concentrations of purified human AβPP (Fig. 3A). Despite the continuous paucity of transgenic human AβPP expression, quantitative ELISA analysis revealed a progressive and robust accumulation of insoluble Aβ40 and Aβ42 in the Tg-SwDI/B mice (Fig. 3, B and C, respectively). The increase in insoluble Aβ peptides first appeared at 3 months and markedly increased by 12 months. The levels of soluble Aβ40 and Aβ42 increased several-fold through 12 months (Fig. 3, D and E, respectively), but composed a minor fraction compared with the amount of accumulating insoluble Aβ peptides (Fig. 3, B and C). In each case of insoluble and soluble Aβ peptides, the Aβ40 levels were ≈10-fold higher than the Aβ42 levels. The antibodies used to detect Aβ in Tg-SwDI/B mice were human-specific, i.e. did not recognize mouse Aβ, and were directed to the first five amino acids of Aβ, indicating that they possess an intact amino terminus (
). Furthermore, a monoclonal antibody directed against the mid-region of wild-type Aβ did not recognize either synthetic Dutch/Iowa Aβ40 or Aβ in Tg-SwDI/B mouse brain (data not shown). These findings indicate that, despite low expression of transgenic human SwDI mutant AβPP, the resulting Dutch/Iowa mutant Aβ peptides exhibit a striking early and robust accumulation in mouse brain.
Fig. 3Quantitation of temporal expression of AβPP and temporal accumulation of Aβ peptides in Tg-SwDI/B mouse brain.A, shown are the results from immunoblot analysis of the temporal expression of transgenic human AβPP in Tg-SwDI/B mouse forebrain tissue homogenates. B–E, the temporal accumulation of insoluble (B and C) and soluble (D and E) Aβ40 and Aβ42 was determined by sequential carbonate and guanidine extraction, respectively, from mouse forebrains as described under “Experimental Procedures.” The data presented are the means ± S.D. from six to eight Tg-SwDI/B mice/time point.
Early and Progressive Vascular Aβ Deposition in Tg-SwDI/B Mice—Since the Dutch and Iowa mutations are associated with familial disorders that develop prominent cerebrovascular amyloid deposition, we were particularly interested in the level of brain vascular accumulation of Aβ in the Tg-SwDI/B mice. Quantitative ELISA measurement of vascular Aβ showed that, at 12 months, the levels of Aβ40 and Aβ42 were 12- and 14-fold higher, respectively, in brain microvessels isolated from Tg-SwDI/B mice forebrains than in the whole forebrain tissue homogenates (Fig. 4B). At ∼3 months of age, the levels of Aβ40 and Aβ42 were similarly significantly higher in brain microvessels than in the whole forebrains of Tg-SwDI/B mice (Fig. 4A), but the absolute levels were much lower compared with the 12-month-old mice, as expected. Quantitative regional analysis of Tg-SwDI/B mice forebrain tissue showed that vascular Aβ in the thalamus and subiculum was highly prominent and significantly increased with age; 45–55% of the vessels were affected in these regions at 12 months (Fig. 4C).
Fig. 4Quantitation of the temporal and regional accumulation of vascular Aβ in Tg-SwDI/B mouse brain. The Aβ40 and Aβ42 concentrations were determined in total forebrain homogenates (black bars) and forebrain isolated microvessels (gray bars) from 3-month-old (A) and 12-month-old (B) Tg-SwDI/B mice. The data presented are the means ± S.D. from four mice at each age. *, p < 0.05; **, p < 0.001. The vascular Aβ profiles in the frontotemporal (F/T) cortex, thalamus, and subiculum in 6-month-old (black bars) and 12-month-old (gray bars) Tg-SwDI/B mice were quantitated as described under “Experimental Procedures” (C). The data presented are the mean ± S.D. from four mice at each age. *, p < 0.001; **, p < 0.05.
Moreover, immunohistochemical analysis revealed that, starting at ∼6 months and increasing with age, numerous Aβ accumulations in and around microvessels, particularly in the thalamic and subiculum regions of the brain (Fig. 5, A and C, respectively), were observed in the Tg-SwDI/B mice. The microvascular Aβ accumulations were mainly fibrillar, displaying strong thioflavin S staining (Fig. 5B). Although much less common, some arterioles in these regions showed strong vascular and perivascular Aβ deposition (Fig. 5, D and E). Other blood vessel-rich regions, including the hippocampal fissure, consistently exhibited large accumulations of perivascular Aβ (Fig. 5F). Occasionally, evidence of microhemorrhage was observed as assessed by hemosiderin staining adjacent to microvessels with Aβ deposits (Fig. 5G).
Fig. 5Immunohistochemical analysis of cerebrovascular Aβ accumulation in Tg-SwDI/B mouse brain. Tg-SwDI/B mouse brain sections from 12-month-old animals were double-immunostained for Aβ (brown) and collagen type IV (red) or stained with thioflavin S to identify fibrillar deposits as described under “Experimental Procedures.” A, double immunostaining showing prominent microvascular Aβ deposits in the thalamus; B, thioflavin S staining of vascular Aβ deposits in the thalamus; C, double immunostaining showing prominent microvascular Aβ deposits in the subiculum; D and E, double immunostaining showing vascular and perivascular Aβ deposits in larger cerebral vessels in the thalamus; F, double immunostaining of Aβ accumulations in the blood vessel-rich hippocampal fissure; G, Prussian blue staining revealing previous microhemorrhage in the thalamus; H, adjacent double-immunostained tissue section showing microvascular Aβ deposits at the site of previous microhemorrhage. Scale bars = 50 μm.
In addition to vascular amyloid deposits, immunohistochemical analysis revealed the presence of Aβ plaque-like deposits in the brains of Tg-SwDI/B mice beginning at ∼3 months of age (Fig. 6, A and D). These deposits initially appeared in the regions of the subiculum, hippocampus, and cortex. As the Tg-SwDI/B mice aged to 6 months, the Aβ plaque-like deposits became more numerous and appeared in the olfactory bulb and thalamic region as well (Fig. 6, B and E). By 12 months of age, the Tg-SwDI/B mice showed Aβ deposition throughout most of forebrain (Fig. 6, C and F). In contrast to the fibrillar vascular Aβ deposits, the overwhelming majority of the parenchymal Aβ accumulations presented as diffuse plaque-like deposits (Fig. 6G), consistent with the parenchymal Aβ deposits that are present in patients with the Dutch and Iowa disorders (
). Occasionally, Aβ plaque-like deposits that had a more compact structure and that stained with thioflavin S were observed in the brain, suggesting that they were fibrillar (Fig. 6, H and I). However, it is notable that these more compact, thioflavin S-positive Aβ deposits were very rare and randomly found and did not appreciably increase with age in the Tg-SwDI/B mice.
Fig. 6Immunohistochemical analysis of parenchymal Aβ accumulation in Tg-SwDI/B mouse brain.A–C, Tg-SwDI/B mouse brain sections (3, 6, and 12 months old, respectively) immunostained for Aβ as described under “Experimental Procedures.” Ce, cerebellum; Hc, hippocampus; Mc, motor cortex; Ob, olfactory bulb; Sb, subiculum; Th, thalamus. Scale bar = 1 mm. D–F, higher magnifications of Aβ deposits in the motor cortex of Tg-SwDI/B mouse brain (3, 6, and 12 months old, respectively). Scale bar = 50 μm. G and H, immunostaining of diffuse and compact Aβ plaque-like deposits, respectively. I, thioflavin S staining of compact fibrillar Aβ deposits. Scale bar = 50 μm.
Consistent Robust Accumulation of Dutch/Iowa Mutant Aβ in Independent Tg-SwDI Mouse Lines—We generated two additional Tg-SwDI mouse lines to confirm the unique properties of Dutch/Iowa mutant Aβ in the brain observed in the initial findings presented above with Tg-SwDI/B mice. Tg-SwDI/A and Tg-SwDI/B mice were found to express similar low levels of transgenic human AβPP protein, whereas Tg-SwDI/C mice expressed nearly twice the amount of human AβPP in the brain compared with the former two lines (Fig. 7, A and B). Nevertheless, the levels of human AβPP in Tg-SwDI/C mice were still no higher than those of endogenous mouse AβPP in the brain. ELISA measurement of Aβ40 and Aβ42 levels at 6 months of age showed that Tg-SwDI/A and Tg-SwDI/B mice, which expressed similar levels of human AβPP, accumulated similar levels of soluble and insoluble Aβ40 and Aβ42 (Fig. 7, C and D). However, Tg-SwDI/C mice, which expressed nearly twice the levels of human AβPP compared with the other two Tg-SwDI lines, accumulated nearly four times the amount of soluble and insoluble Aβ40 and Aβ42 in the brain (Fig. 7, C and D).
Fig. 7Quantitation of AβPP expression and Aβ accumulation in different Tg-SwDI mouse lines. Shown are the results of immunoblot analysis of transgenic human AβPP expression in 3-month-old Tg-SwDI mouse forebrain tissue homogenates (A). Lanes 1 and 2, Tg-SwDI/A mice; lanes 3 and 4, Tg-SwDI/B mice; lanes 5 and 6, Tg-SwDI/C mice. The levels of transgenic human AβPP expression in Tg-SwDI mouse forebrain tissue homogenates were determined by quantitative immunoblotting as described under “Experimental Procedures” (B). Lane 1, Tg-SwDI/A mice; lane 2, Tg-SwDI/B mice; lane 3, Tg-SwDI/C mice. The data presented are the means ± S.D. from four mice in each line. *, p < 0.01. The accumulation of insoluble (C) and soluble (D)Aβ40 and Aβ42 was determined by sequential carbonate and guanidine extraction, respectively, from 6-month-old mouse forebrains from the different Tg-SwDI lines as described under “Experimental Procedures.” The data presented are the means ± S.D. from four to six mice in each Tg-SwDI line. *, p < 0.05; **, p < 0.01.
Immunohistochemical analysis for Aβ showed that, at 1 year of age, all three Tg-SwDI lines developed strong accumulation of Aβ in the brain microvasculature (Fig. 8, A–C) and largely diffuse, plaque-like deposits in the parenchyma (Fig. 8, D–F), with similar regional deposition in the brain (Fig. 8, G–I). Together, these findings demonstrate that, despite the low levels of transgenic human AβPP expression, the early-onset and robust accumulation of Dutch/Iowa mutant Aβ in the cerebral microvasculature and brain parenchyma is a unique and consistent phenotype in independent Tg-SwDI lines.
Fig. 8Immunohistochemical analysis of Aβ accumulation in Tg-SwDI mouse brains. Mouse brain sections from 12-month-old animals of three independent Tg-SwDI lines were double-immunostained for Aβ (brown) and collagen type IV (red) to identify Aβ deposits as described under “Experimental Procedures.” A–C, double immunostaining showing prominent microvascular Aβ deposits in the thalamic region in each Tg-SwDI line. Scale bar = 50 μm. D–F, immunostaining showing diffuse Aβ deposits in the motor cortex in each Tg-SwDI line. Scale bar = 50 μm. G–I, immunostaining showing similar regional Aβ accumulation in each Tg-SwDI line. Scale bar = 1 mm.
Increased Brain Retention of Dutch/Iowa Mutant Aβ —We next determined whether the early and robust accumulation of Dutch/Iowa mutant Aβ at perivascular/vascular sites and in the parenchyma in Tg-SwDI mice could be attributed to reduced elimination of this mutant form of Aβ in the brain. To test this, we compared the retention of Dutch/Iowa mutant Aβ40 and wild-type Aβ40 peptides from mouse brain after intracerebral microinjections of radiolabeled peptides at different carrier concentrations (
). In these experiments, we focused on Aβ40 peptides since this is largely the predominant form that accumulates in the Tg-SwDI mice (Figs. 3, 4, and 7). Retention was estimated within the first 30 min after intracerebral injections of peptides, as described previously for mice and squirrel monkeys (
), the retention of Dutch/Iowa mutant Aβ40 was ≈10-fold greater than that of wild-type Aβ40 (Fig. 9A). At the higher concentration of 12 nm, corresponding to the pathophysiological Aβ40 cerebrospinal/brain interstitial fluid levels in transgenic mice (
), wild-type Aβ40 exhibited reduced elimination compared with that at the lower concentration of 1 nm, consistent with a saturable nature of the Aβ brain clearance mechanism in vivo (
). However, the retention of Dutch/Iowa mutant Aβ40 at 12 nm was still significantly higher by ≈2.5-fold compared with wild-type Aβ40 at 12 nm (Fig. 9A). At 120 nm, Dutch/Iowa mutant Aβ40 was almost completely retained in the brain, whereas wild-type Aβ40 still exhibited measurable disappearance, but approached its elimination limit close to 120 nm (
). The interstitial fluid drainage assessed by elimination of the simultaneously infused reference marker inulin in the presence of either Aβ peptide was not significantly affected (Fig. 9B). These results suggest that the differences in retention between the two peptides are unlikely to reflect differences in removal through cerebrospinal/brain interstitial fluid bulk flow and may therefore reflect primarily significantly reduced clearance of Dutch/Iowa mutant Aβ from the brain into the bloodstream.
Fig. 9Deficient clearance of Dutch/Iowa mutant Aβ40 peptides from mouse brain.A, retention of 125I-labeled wild-type Aβ40 (WT; black bars) and Dutch/Iowa mutant Aβ40 (DI; open bars) in the brain at different peptide concentrations. B, slow elimination of simultaneously infused [14C]inulin in the experiments shown in A indicating that inulin clearance was not affected by either of the Aβ peptides. The data presented are the means ± S.D. from three to eight separate measurements for each concentration. *, p < 0.001; **, p < 0.01.
Aβ deposition in the cerebral vasculature and plaque-like structures are prominent pathological features of Alzheimer's disease and several rare familial CAA disorders (
). Investigation into the pathogenic effects of Aβ in Alzheimer's disease has been bolstered by the generation of several transgenic mouse models that express human forms of AβPP in the brain and that develop age-dependent Aβ deposits in the central nervous system (
). Generally, the successful mouse models to date have relied on marked overexpression of human AβPP containing one or more mutations flanking the β- and γ-secretase processing sites associated with familial forms of Alzheimer's disease (
). Mutations at these key processing sites in the AβPP transgene were included to promote total production of Aβ peptides and/or to increase the production of the longer, more pathogenic Aβ42 isoform. Most of these transgenic mouse lines primarily accumulate Aβ plaque-like deposits, although several lines have been found to start developing CAA after 12 months of age (
). However, in each of these mouse models, AβPP overexpression is required to produce and deposit human wild-type Aβ peptides. Here, we have described the generation and initial characterization of transgenic mice that express, in the brain, very low levels of human AβPP containing Dutch/Iowa CAA double mutations that produce vasculotropic mutant Aβ model peptides.
To generate the Tg-SwDI mice, we used the mouse Thy1.2 promoter, which provides neuronal expression of the transgene in the central nervous system. Although the use of this promoter is somewhat limiting in that it provides for only a neuronal source of AβPP and Aβ, it was previously used to successfully generate two independent transgenic mouse lines overexpressing Swedish K670N/M671L mutant AβPP and producing wild-type Aβ peptides that developed extensive fibrillar plaque deposits in the brain and some cerebrovascular amyloid after 12 months (
). In our transgenic AβPP cDNA construct, we similarly included the tandem Swedish mutations to help promote Aβ production (Fig. 1). However, we also incorporated the tandem Dutch and Iowa mutations (E693Q/D694N) in AβPP, resulting in the production of Dutch/Iowa CAA double mutant Aβ peptides. Our earlier in vitro studies showed that Dutch/Iowa mutant Aβ, with the loss of two negative charges at positions 22 and 23, possesses more robust fibrillogenic and pathogenic properties (
). Therefore, the present Tg-SwDI/B mice provide a novel in vivo model to investigate the central nervous system activities of this experimental peptide with amplified fibrillogenic and pathogenic properties compared with either CAA single mutant form of Aβ.
Analysis of human AβPP expression in the Tg-SwDI/B mouse line showed restriction to the brain, consistent with neuronal expression of the Thy1.2 promoter. However, further comparative analysis indicated that transgenic human AβPP was modestly expressed at levels actually below those of endogenous mouse AβPP. Furthermore, the initial levels of Aβ peptides in very young 2-month-old Tg-SwDI/B mice were quite low, consistent with the low transgenic human AβPP expression. At first glance, this was disappointing since previous successes in generating Aβ pathology in earlier transgenic mouse models relied on marked overexpression (as high as 7-fold) of transgenic human AβPP (
). Therefore, it was surprising to find that, as the Tg-SwDI/B mice continued to age, they accumulated large amounts of human insoluble mutant Aβ40 and Aβ42 peptides. In fact, Aβ accumulation in Tg-SwDI/B mice was much earlier in onset and more robust than that reported for the commonly used Tg-2576 mouse line, which highly overexpresses human AβPP (
). The Tg-SwDI/B mice expressed much lower levels of human AβPP than reported for other AβPP-expressing transgenic mice, indicating that the presence of the tandem charge-altering Dutch/Iowa mutations within the Aβ peptides produced in the present model has a striking effect on the accumulation of Aβ in mouse brain.
Although the ELISA measurements of total forebrain homogenates clearly showed robust accumulation of Aβ in Tg-SwDI/B mouse brain, they did not reveal in which structures this accumulation was most pronounced. The subsequent compartmental quantitative ELISA analysis showed a 12–14-fold higher association of Aβ with isolated brain microvessels compared with whole forebrain tissue at 3 and 12 months, under-scoring the vasculotropic nature of the Dutch/Iowa mutant Aβ peptides. Furthermore, immunohistochemical analysis confirmed that Tg-SwDI/B mice developed progressive and extensive Aβ deposits in cerebral blood vessels, most notably in the microvasculature. It is noteworthy that, in the regions of the subiculum and thalamus, with high numbers of microvessels with deposited Aβ (Fig. 4C), Aβ was found to congregate in and closely around the microvessels, with little deposition in the parenchymal tissues between the vessels (Fig. 5, A–C). This observation emphasizes the highly vasculotropic nature of Dutch/Iowa mutant Aβ peptides in these brain regions. Although other human AβPP-expressing transgenic mice develop cerebrovascular Aβ deposition, this has been found to generally occur in mice older than 12 months of age, with predominant accumulation in meningeal vessels (
). On the other hand, the cerebrovascular Aβ deposits in Tg-SwDI/B mice developed much earlier (i.e. starting at 6 months) and were abundant in the microvasculature of the thalamic and subiculum regions. Although there was much less involvement of the meningeal and cortical vessels in Tg-SwDI mice, the extent of cortical vascular Aβ (i.e. ≈4%) was still appreciably higher than in other AβPP-expressing transgenic mice (
). The finding that the vascular Aβ deposits were largely fibrillar (showing strong thioflavin S staining) is consistent with the nature of the cerebrovascular Aβ deposits found in patients with either the Dutch- or Iowa-type CAA disorder (
). Occasionally, evidence of prior microhemorrhage was observed in 12-month-old Tg-SwDI/B mice using Prussian blue iron staining to identify residual hemosiderin adjacent to amyloid-laden microvessels. Although these events were rare at 12 months, it is anticipated that they will become more common as the Tg-SwDI/B mice age.
Tg-SwDI/B mice showed the appearance of Aβ plaque-like deposits at an early 3 months of age, with a progressive and widespread accumulation by 1 year. Similar to the early onset of microvascular Aβ deposits, the accumulation of Aβ plaque-like structures markedly precedes the development of deposits in other Aβ-depositing, human AβPP-expressing transgenic mice. Since Dutch/Iowa mutant Aβ strongly assembles into fibrils in vitro (
), it was interesting to find that the overwhelming majority of the Aβ plaque-like deposits were of the diffuse type, whereas fibrillar, thioflavin S-staining plaques were extremely rare. However, this is highly consistent with the pathological findings of largely diffuse Aβ plaques in patients with either the Dutch- or Iowa-type CAA disorder (
). This suggests that the enhanced fibrillogenic properties of Dutch and Iowa mutant Aβ observed in vitro do not directly translate in vivo to the parenchymal Aβ deposits in the brain. Perhaps other Aβ-interacting molecules in the brain, which likely facilitate or impede parenchymal fibril formation, possess different specificity for wild-type and Dutch and Iowa mutant Aβ peptides.
Two additional Tg-SwDI mouse lines subsequently generated, again with levels of human AβPP expression at or below those of endogenous mouse AβPP, exhibited early-onset and robust accumulation of Aβ peptides in the brain similar to that found in Tg-SwDI/B mice. A dosage effect was observed where Tg-SwDI/C mice, which expressed approximately twice the amount of human AβPP compared with Tg-SwDI/A and Tg-SwDI/B mice, accumulated significantly more Aβ in the brain compared with the latter two transgenic lines. However, all three Tg-SwDI lines showed consistent Aβ accumulations as strong microvascular deposits and diffuse plaque-like structures. These findings, from three independent Tg-SwDI mouse lines, clearly demonstrate the unique behavior of Dutch/Iowa mutant Aβ peptides in mouse brain. Importantly, this novel and reproducible transgenic mouse paradigm demonstrates that vast overexpression of human AβPP is not required for the accumulation of Aβ in mouse brain. This suggests that Tg-SwDI mice provide a unique in vivo model to study Aβ accumulation in the brain without the confounding issue of abnormally high expression of human AβPP that is commonly found in other AβPP-expressing transgenic mice.
The mechanisms responsible for cerebrovascular accumulation of Aβ remain unclear, although recent studies suggest that they may involve ineffective transport of Aβ out of the central nervous system and into the circulation (
). Therefore, it was interesting to find that, in addition to Aβ deposition in the microvessels, there was a pronounced accumulation of Aβ around the immediate vicinity of the vessels and in blood vessel-rich regions such as the hippocampal fissure (Fig. 4F). This suggests that the deficient clearance of Dutch/Iowa mutant Aβ from the central nervous system at the cerebral vasculature may account for this striking accumulation around cerebral blood vessels and also in the parenchyma. Accordingly, in vivo clearance studies of 125I-labeled Aβ peptides at varying carrier concentrations suggested that, compared with wild-type Aβ40, Dutch/Iowa mutant Aβ40 was retained in the brain (Fig. 9). This is similar to the finding of Monro et al. (
) in guinea pigs that demonstrated decreased elimination of Dutch mutant Aβ from the cerebrospinal fluid. It has been suggested the blood-brain barrier removes Aβ40 from the brain largely via low density lipoprotein receptor-related protein-1 (
). Thus, further studies are needed to clarify whether retention of Dutch/Iowa mutant Aβ reflects its reduced affinity for low density lipoprotein receptor-related protein-1-mediated clearance.
Recent work indicated that bidirectional transport of Aβ across the blood-brain barrier is the dominant pathway regulating the concentrations of Aβ in the central nervous system under physiological and pathophysiological conditions (
), may all promote clearance of central nervous system-derived Aβ into the bloodstream as shown in different types of transgenic mice expressing AβPP. Several physiological and pharmacological modifiers of Aβ transport clearance systems have been described, including insulin-like growth factor I (
). The present data suggest that the observed retention of Dutch/Iowa mutant Aβ in the brain likely contributes to its striking accumulation around cerebral blood vessels and in the parenchyma of the Tg-SwDI mice. Although not a precise model of Dutch- or Iowa-type familial CAA, Tg-SwDI mice mimic many of the features of these disorders and provide a novel and useful experimental paradigm to investigate the effects of these vasculotropic mutations in vivo and potential therapies for CAA.
Acknowledgments
We thank Dr. Ron DeMattos and Lilly Research Laboratories for generously providing the antibody reagents for performing the Aβ40 and Aβ42 ELISA measurements.
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