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
2004; Elsevier BV; Volume: 279; Issue: 19 Linguagem: Inglês
10.1074/jbc.m312946200
ISSN1083-351X
AutoresJudianne Davis, Feng Xu, Rashid Deane, Galina Romanov, Mary Lou Previti, Kelly Zeigler, Berislav V. Zloković, William E. Van Nostrand,
Tópico(s)Amyloidosis: Diagnosis, Treatment, Outcomes
ResumoCerebrovascular 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. 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) 1The abbreviations used are: Aβ, amyloid β-protein; AβPP, amyloid β-protein precursor; CAA, cerebral amyloid angiopathy; Tg-SwDI, transgenic mouse expressing human Swedish, Dutch, and Iowa triple mutant AβPP; ELISA, enzyme-linked immunosorbent assay; HPLC, high pressure liquid chromatography; Tricine, N-[2-hydroxy-1, 1-bis(hydroxymethyl)ethyl]glycine. 1The abbreviations used are: Aβ, amyloid β-protein; AβPP, amyloid β-protein precursor; CAA, cerebral amyloid angiopathy; Tg-SwDI, transgenic mouse expressing human Swedish, Dutch, and Iowa triple mutant AβPP; ELISA, enzyme-linked immunosorbent assay; HPLC, high pressure liquid chromatography; Tricine, N-[2-hydroxy-1, 1-bis(hydroxymethyl)ethyl]glycine. in senile plaques and the cerebral vasculature is a prominent feature of Alzheimer's disease and several related disorders (1Selkoe D.J. Physiol. Rev. 2001; 81: 741-766Crossref PubMed Scopus (5121) Google Scholar, 2Vinters H.V. Farag E.S. Adv. Neurol. 2003; 92: 105-112PubMed Google Scholar). 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 (1Selkoe D.J. Physiol. Rev. 2001; 81: 741-766Crossref PubMed Scopus (5121) Google Scholar, 3Selkoe D.J. J. Clin. Investig. 2002; 110: 1375-1381Crossref PubMed Scopus (211) Google Scholar). 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 (1Selkoe D.J. Physiol. Rev. 2001; 81: 741-766Crossref PubMed Scopus (5121) Google Scholar, 3Selkoe D.J. J. Clin. Investig. 2002; 110: 1375-1381Crossref PubMed Scopus (211) Google Scholar, 4Citron M. Oltersdorf T. Haass C. McConlogue L. Hung A.Y. Seubert P. Vigo-Pelfrey C. Lieberberg I. Selkoe D.J. Nature. 1992; 360: 672-674Crossref PubMed Scopus (1518) Google Scholar). 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 (5Levy E. Carman M.D. Fernandez-Madrid I.J. Power M.D. Lieberburg I. van Duinen S.G. Bots G.T.A.M. Luyendijk W. Frangione B. Science. 1990; 248: 1124-1126Crossref PubMed Scopus (1150) Google Scholar, 6Van Broeckhoven C. Haan J. Bakker E. Hardy J.A. Van Hul W. Wehnert A. Vegter Van der Vlis M. Roos R.A. Science. 1990; 248: 1120-1122Crossref PubMed Scopus (357) Google Scholar, 7Wattendorff A.R. Frangione B. Luyendijk W. Bots G.T.A.M. J. Neurol. Neurosurg. Psychiatry. 1995; 59: 699-705Crossref Scopus (93) Google Scholar). 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 (8Grabowski T.J. Cho H.S. Vonsattel J.P.G. Rebeck G.W. Greenberg S.M. Ann. Neurol. 2001; 49 (J. P. G.): 697-705Crossref PubMed Scopus (428) Google Scholar). The reason as to why mutations in this region within Aβ lead preferentially to a strong accumulation of cerebrovascular amyloid remains unclear.It is noteworthy that both the Dutch E22Q and Iowa D23N mutations result in loss of a negative charge in the Aβ peptide. In previous studies, we (9Davis J. Van Nostrand W.E. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 2996-3000Crossref PubMed Scopus (159) Google Scholar, 11Van Nostrand W.E. Melchor J.P. Cho H.S. Greenberg S.M. Rebeck G.W. J. Biol. Chem. 2001; 276: 32860-32866Abstract Full Text Full Text PDF PubMed Scopus (203) Google Scholar) and others (10Miravalle L. Tokuda T. Chiarle R. Giaccone G. Bugiani O. Tagliavini F. Frangione B. Ghiso J. J. Biol. Chem. 2000; 275: 27110-27116Abstract Full Text Full Text PDF PubMed Google Scholar) 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 (11Van Nostrand W.E. Melchor J.P. Cho H.S. Greenberg S.M. Rebeck G.W. J. Biol. Chem. 2001; 276: 32860-32866Abstract Full Text Full Text PDF PubMed Scopus (203) Google Scholar). 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 PROCEDURESVector 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 (11Van Nostrand W.E. Melchor J.P. Cho H.S. Greenberg S.M. Rebeck G.W. J. Biol. Chem. 2001; 276: 32860-32866Abstract Full Text Full Text PDF PubMed Scopus (203) Google Scholar). 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 (12Van Nostrand W.E. Wagner S.L. Suzuki M. Choi B.H. Farrow J.S. Geddes J.W. Cotman C.W. Cunningham D.D. Nature. 1989; 341: 546-549Crossref PubMed Scopus (347) Google Scholar), 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 (13DeMattos R.B. O'Dell M.A. Parsadanian M. Taylor J.W. Harmony J.A.K. Bales K.R. Paul S.M. Aronow B.J. Holtzman D.M. Proc. Natl. Acad. Sci. U. S. A. 2002; 99 (J. A. K.): 10843-10848Crossref PubMed Scopus (271) Google Scholar, 14Johnson-Wood K. Lee M. Motter R. Hu K. Gordon G. Barbour R. Khan K. Gordon M. Tan H. Games D. Lieberburg I. Schenk D. Seubert P. McConlogue L. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 1550-1555Crossref PubMed Scopus (579) Google Scholar). Brain microvessels were isolated from mouse forebrains as described (15Zlokovic B.V. Mackic J.B. Wang L. McComb J.G. McDonough A. J. Biol. Chem. 1993; 268: 8019-8025Abstract Full Text PDF PubMed Google Scholar). 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 (13DeMattos R.B. O'Dell M.A. Parsadanian M. Taylor J.W. Harmony J.A.K. Bales K.R. Paul S.M. Aronow B.J. Holtzman D.M. Proc. Natl. Acad. Sci. U. S. A. 2002; 99 (J. A. K.): 10843-10848Crossref PubMed Scopus (271) Google Scholar). 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 (16Dickson D.W. Wertkin A. Mattiace L.A. Fier E. Kress Y. Davies P. Yen S.H. Acta Neuropathol. 1990; 79: 486-493Crossref PubMed Scopus (91) Google Scholar). Prussian blue iron staining was performed to detect hemosiderin to reveal signs of previous microhemorrhage as described (17Gomori G. Am. J. Pathol. 1936; 12: 655-663PubMed Google Scholar, 18Winkler D.T. Bondolfi L. Herzig M.C. Jann L. Calhoun M.E. Wiederhold K.H. Tolnay M. Staufenbiel M. Jucker M. J. Neurosci. 2001; 21: 1619-1627Crossref PubMed Google Scholar). The following antibodies were used for immunohistochemical analysis: monoclonal antibody 66.1, which recognizes residues 1–5 of human Aβ (19Deane R. Yan S. Submamaryan R.K. LaRue B. Jovanovic S. Hogg E. Welch D. Manness L. Lin C. Yu J. Zhu H. Ghiso J. Frangione B. Stern A. Schmidt A.M. Armstrong D.L. Arnold B. Liliensiek B. Nawroth P. Hofman F. Kindy M. Stern D. Zlokovic B. Nat. Med. 2003; 9: 907-913Crossref PubMed Scopus (1121) Google Scholar), 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 (19Deane R. Yan S. Submamaryan R.K. LaRue B. Jovanovic S. Hogg E. Welch D. Manness L. Lin C. Yu J. Zhu H. Ghiso J. Frangione B. Stern A. Schmidt A.M. Armstrong D.L. Arnold B. Liliensiek B. Nawroth P. Hofman F. Kindy M. Stern D. Zlokovic B. Nat. Med. 2003; 9: 907-913Crossref PubMed Scopus (1121) Google Scholar).Radioiodination of Synthetic Aβ Peptides—Radioiodination was carried out using the lactoperoxidase method (20Thorell J.I. Johansson B.G. Biochim. Biophys. Acta. 1971; 251: 363-366Crossref PubMed Scopus (1069) Google Scholar). 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 (15Zlokovic B.V. Mackic J.B. Wang L. McComb J.G. McDonough A. J. Biol. Chem. 1993; 268: 8019-8025Abstract Full Text PDF PubMed Google Scholar). 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 (15Zlokovic B.V. Mackic J.B. Wang L. McComb J.G. McDonough A. J. Biol. Chem. 1993; 268: 8019-8025Abstract Full Text PDF PubMed Google Scholar, 19Deane R. Yan S. Submamaryan R.K. LaRue B. Jovanovic S. Hogg E. Welch D. Manness L. Lin C. Yu J. Zhu H. Ghiso J. Frangione B. Stern A. Schmidt A.M. Armstrong D.L. Arnold B. Liliensiek B. Nawroth P. Hofman F. Kindy M. Stern D. Zlokovic B. Nat. Med. 2003; 9: 907-913Crossref PubMed Scopus (1121) Google Scholar, 21Shibata M. Yamada S. Kumar S.R. Calero M. Bading J. Frangione B. Holtzman D.M. Miller C.A. Strickland D.K. Ghiso J. Zlokovic B.V. J. Clin. Investig. 2000; 106: 1489-1499Crossref PubMed Scopus (1104) Google Scholar). 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 mm p-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 (15Zlokovic B.V. Mackic J.B. Wang L. McComb J.G. McDonough A. J. Biol. Chem. 1993; 268: 8019-8025Abstract Full Text PDF PubMed Google Scholar). 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 (15Zlokovic B.V. Mackic J.B. Wang L. McComb J.G. McDonough A. J. Biol. Chem. 1993; 268: 8019-8025Abstract Full Text PDF PubMed Google Scholar, 19Deane R. Yan S. Submamaryan R.K. LaRue B. Jovanovic S. Hogg E. Welch D. Manness L. Lin C. Yu J. Zhu H. Ghiso J. Frangione B. Stern A. Schmidt A.M. Armstrong D.L. Arnold B. Liliensiek B. Nawroth P. Hofman F. Kindy M. Stern D. Zlokovic B. Nat. Med. 2003; 9: 907-913Crossref PubMed Scopus (1121) Google Scholar).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 (15Zlokovic B.V. Mackic J.B. Wang L. McComb J.G. McDonough A. J. Biol. Chem. 1993; 268: 8019-8025Abstract Full Text PDF PubMed Google Scholar, 19Deane R. Yan S. Submamaryan R.K. LaRue B. Jovanovic S. Hogg E. Welch D. Manness L. Lin C. Yu J. Zhu H. Ghiso J. Frangione B. Stern A. Schmidt A.M. Armstrong D.L. Arnold B. Liliensiek B. Nawroth P. Hofman F. Kindy M. Stern D. Zlokovic B. Nat. Med. 2003; 9: 907-913Crossref PubMed Scopus (1121) Google Scholar). 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 (15Zlokovic B.V. Mackic J.B. Wang L. McComb J.G. McDonough A. J. Biol. Chem. 1993; 268: 8019-8025Abstract Full Text PDF PubMed Google Scholar). All calculations were based on 30-min experiments.Statistical Analysis—Data were analyzed by multifactorial analysis of variance, Student's t test, and Dunnett's t test.RESULTSGeneration of Tg-SwDI Mice Expressing Vasculotropic Mutant AβPP—The E22Q and D23N mutations within Aβ primarily manifest as strong cerebrovascular amyloid-depositing disorders (5Levy E. Carman M.D. Fernandez-Madrid I.J. Power M.D. Lieberburg I. van Duinen S.G. Bots G.T.A.M. Luyendijk W. Frangione B. Science. 1990; 248: 1124-1126Crossref PubMed Scopus (1150) Google Scholar, 6Van Broeckhoven C. Haan J. Bakker E. Hardy J.A. Van Hul W. Wehnert A. Vegter Van der Vlis M. Roos R.A. Science. 1990; 248: 1120-1122Crossref PubMed Scopus (357) Google Scholar, 7Wattendorff A.R. Frangione B. Luyendijk W. Bots G.T.A.M. J. Neurol. Neurosurg. Psychiatry. 1995; 59: 699-705Crossref Scopus (93) Google Scholar, 8Grabowski T.J. Cho H.S. Vonsattel J.P.G. Rebeck G.W. Greenberg S.M. Ann. Neurol. 2001; 49 (J. P. G.): 697-705Crossref PubMed Scopus (428) Google Scholar). 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 (4Citron M. Oltersdorf T. Haass C. McConlogue L. Hung A.Y. Seubert P. Vigo-Pelfrey C. Lieberberg I. Selkoe D.J. Nature. 1992; 360: 672-674Crossref PubMed Scopus (1518) Google Scholar, 22Mullan M. Crawford F. Axelman K. Houlden H. Lilius L. Winblad B. Lannfelt L. Nat. Genet. 1992; 1: 345-347Crossref PubMed Scopus (1168) Google Scholar). 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 (11Van Nostrand W.E. Melchor J.P. Cho H.S. Greenberg S.M. Rebeck G.W. J. Biol. Chem. 2001; 276: 32860-32866Abstract Full Text Full Text PDF PubMed Scopus (203) Google Scholar). 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.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.View Large Image Figure ViewerDownload (PPT)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 (13DeMattos R.B. O'Dell M.A. Parsadanian M. Taylor J.W. Harmony J.A.K. Bales K.R. Paul S.M. Aronow B.
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