Rodent Aβ Modulates the Solubility and Distribution of Amyloid Deposits in Transgenic Mice
2007; Elsevier BV; Volume: 282; Issue: 31 Linguagem: Inglês
10.1074/jbc.m611050200
ISSN1083-351X
AutoresJoanna L. Jankowsky, Linda H. Younkin, Victoria Gonzales, Daniel J. Fadale, Hilda H. Slunt, Henry A. Lester, Steven G. Younkin, David Borchelt,
Tópico(s)Computational Drug Discovery Methods
ResumoThe amino acid sequence of amyloid precursor protein (APP) is highly conserved, and age-related Aβ aggregates have been described in a variety of vertebrate animals, with the notable exception of mice and rats. Three amino acid substitutions distinguish mouse and human Aβ that might contribute to their differing properties in vivo. To examine the amyloidogenic potential of mouse Aβ, we studied several lines of transgenic mice overexpressing wild-type mouse amyloid precursor protein (moAPP) either alone or in conjunction with mutant PS1 (PS1dE9). Neither overexpression of moAPP alone nor co-expression with PS1dE9 caused mice to develop Alzheimer-type amyloid pathology by 24 months of age. We further tested whether mouse Aβ could accelerate the deposition of human Aβ by crossing the moAPP transgenic mice to a bigenic line expressing human APPswe with PS1dE9. The triple transgenic animals (moAPP × APPswe/PS1dE9) produced 20% more Aβ but formed amyloid deposits no faster and to no greater extent than APPswe/PS1dE9 siblings. Instead, the additional mouse Aβ increased the detergent solubility of accumulated amyloid and exacerbated amyloid deposition in the vasculature. These findings suggest that, although mouse Aβ does not influence the rate of amyloid formation, the incorporation of Aβ peptides with differing sequences alters the solubility and localization of the resulting aggregates. The amino acid sequence of amyloid precursor protein (APP) is highly conserved, and age-related Aβ aggregates have been described in a variety of vertebrate animals, with the notable exception of mice and rats. Three amino acid substitutions distinguish mouse and human Aβ that might contribute to their differing properties in vivo. To examine the amyloidogenic potential of mouse Aβ, we studied several lines of transgenic mice overexpressing wild-type mouse amyloid precursor protein (moAPP) either alone or in conjunction with mutant PS1 (PS1dE9). Neither overexpression of moAPP alone nor co-expression with PS1dE9 caused mice to develop Alzheimer-type amyloid pathology by 24 months of age. We further tested whether mouse Aβ could accelerate the deposition of human Aβ by crossing the moAPP transgenic mice to a bigenic line expressing human APPswe with PS1dE9. The triple transgenic animals (moAPP × APPswe/PS1dE9) produced 20% more Aβ but formed amyloid deposits no faster and to no greater extent than APPswe/PS1dE9 siblings. Instead, the additional mouse Aβ increased the detergent solubility of accumulated amyloid and exacerbated amyloid deposition in the vasculature. These findings suggest that, although mouse Aβ does not influence the rate of amyloid formation, the incorporation of Aβ peptides with differing sequences alters the solubility and localization of the resulting aggregates. Although genetically engineered mice have become a commonly used tool for Alzheimer disease research, wild-type rats and mice do not innately develop age-associated amyloid pathology (1Shivers B.D. Hilbich C. Multhaup G. Salbaum M. Beyreuther K. Seeburg P.H. EMBO J. 1988; 7: 1365-1370Crossref PubMed Scopus (388) Google Scholar). Many other animals, including dogs, bears, and primates, display amyloid lesions similar to those of Alzheimer disease in humans (2Selkoe D.J. Bell D.S. Podlisny M.B. Price D.L. Cork L.C. Science. 1987; 235: 873-877Crossref PubMed Scopus (376) Google Scholar, 3Struble R.G. Price Jr., D.L. Cork L.C. Price D.L. Brain Res. 1985; 361: 267-275Crossref PubMed Scopus (150) Google Scholar, 4Wisniewski H.M. Ghetti B. Terry R.D. J. Neuropathol. Exp. 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The amyloid precursor protein (APP), 3The abbreviations used are: APP, amyloid precursor protein; Aβ, amyloid-β; BACE, β-APP cleaving enzyme; PrP, prion protein promoter; PS1, presenilin-1; dE9, PS1 encoding the exon-9 deletion mutation; DEA, diethylamine; ELISA, enzyme-linked immunosorbent assay; FA, formic acid; FAD, familial Alzheimer disease; hu, human; mo, mouse; ro, rodent; mo/hu, chimeric mouse/human; swe, APP encoding the Swedish mutation; wt, wild type; NTg, non-transgenic; PBS, phosphate-buffered saline; MES, 4-morpholineethanesulfonic acid; Tricine, N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine; TBS, Tris-buffered saline; ANOVA, analysis of variance; BisTris, 2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)-propane-1,3-diol; CHAPS, 3-[{3-cholamidopropyl)dimethylammonio}]-1-propanesulfonic acid. from which Aβ is derived, is well conserved, and >96% of the amino acid sequence is identical between mouse, rat, monkey, and human (1Shivers B.D. Hilbich C. Multhaup G. Salbaum M. 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The replacement of His-13 for Arg in rodent Aβ disrupts a metal coordination site, which renders the rodent peptide much less prone to zinc-induced aggregation in vitro (18Yang D.S. McLaurin J. Qin K. Westaway D. Fraser P.E. Eur. J. Biochem. 2000; 267: 6692-6698Crossref PubMed Scopus (113) Google Scholar, 19Liu S.T. Howlett G. Barrow C.J. Biochemistry. 1999; 38: 9373-9378Crossref PubMed Scopus (192) Google Scholar, 20Bush A.I. Pettingell W.H. Multhaup G. d Paradis M. Vonsattel J.P Gusella J.F Beyreuther K. Masters C.L. Tanzi R.E. Science. 1994; 265: 1464-1467Crossref PubMed Scopus (1411) Google Scholar). The amino acid sequence of Aβ also affects interactions with APP-processing enzymes (21De Strooper B. Simons M. Multhaup G. Van Leuven F. Beyreuther K. Dotti C.G. EMBO J. 1995; 14: 4932-4938Crossref PubMed Scopus (162) Google Scholar). BACE1 is responsible for releasing the N terminus of Aβ from APP at either of two cleavage sites within the peptide sequence: +1 or +11. 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To date, only one study has addressed the effects of elevating murine Aβ in vivo by overexpressing wild-type moAPP (30Hsiao K.K. Borchelt D.R. Olson K. Johannsdottir R. Kitt C. Yunis W. Xu S. Eckman C. Younkin S. Price D. Iadecola C. Clark H.B. Carlson G. Neuron. 1995; 15: 1203-1218Abstract Full Text PDF PubMed Scopus (482) Google Scholar). This study examined animals only 3.5-4 months of age and, therefore, did not test whether overexpression of moAPP would induce late onset amyloid formation. A complicating factor in this initial study was the FVB background strain used to generate the transgenic lines. Transgenic mice of this strain are prone to premature death when human APP is expressed via the hamster prion protein promoter (PrP) vector (30Hsiao K.K. Borchelt D.R. Olson K. Johannsdottir R. Kitt C. Yunis W. Xu S. Eckman C. Younkin S. Price D. Iadecola C. Clark H.B. Carlson G. Neuron. 1995; 15: 1203-1218Abstract Full Text PDF PubMed Scopus (482) Google Scholar). We have subsequently moved one of these original moAPP transgenic lines out of the FVB background by crossing onto a hybrid C3H/HeJ × C57BL/6J strain for more than five generations. This strategy eliminated the premature death of animals, allowing us to examine the potential for amyloid formation in aged moAPP transgenic mice. We report here that neither overexpression of moAPP alone, nor co-expression with the human presenilin-1 exon-9 deletion variant (PS1dE9 (11Jankowsky J.L. Fadale D.J. Anderson J. Xu G.M. Gonzales V. Jenkins N.A. Copeland N.G. Lee M.K. Younkin L.H. Wagner S.L. Younkin S.G. Borchelt D.R. Hum. Mol. Genet. 2004; 13: 159-170Crossref PubMed Scopus (1143) Google Scholar, 31Lesuisse C. Xu G. Anderson J. Wong M. Jankowsky J. Holtz G. Gonzalez V. Wong P.C. Price D.L. Tang F. Wagner S. Borchelt D.R. Hum. Mol. Genet. 2001; 10: 2525-2537Crossref PubMed Scopus (47) Google Scholar)), resulted in amyloid composed only from mouse Aβ. We further demonstrate that mouse Aβ does not accelerate the deposition of human Aβ in transgenic mice overexpressing both peptides, but reveal that high levels of mouse peptide alter the solubility of the resulting Aβ aggregates and increase the prevalence of vascular deposits. We further find that Aβ11-40/42, predicted to be the predominant form of Aβ produced from mouse APP, does not appear to be a major co-depositing peptide. These findings provide insight into the potential role of specific Aβ sequences in modulating the solubility and distribution of amyloid deposits in rodent models. Generation of Transgenic Mice—All transgenic mice used in this study have been described and fully characterized in earlier publications. All transgenes were expressed under control of the mouse prion protein promoter (MoPrP.Xho), which drives high protein expression in neurons and astrocytes of the central nervous system (31Lesuisse C. Xu G. Anderson J. Wong M. Jankowsky J. Holtz G. Gonzalez V. Wong P.C. Price D.L. Tang F. Wagner S. Borchelt D.R. Hum. Mol. Genet. 2001; 10: 2525-2537Crossref PubMed Scopus (47) Google Scholar, 32Borchelt D.R. Davis J. Fischer M. Lee M.K. Slunt H.H. Ratovitsky T. Regard J. Copeland N.G. Jenkins N.A. Sisodia S.S. Price D.L. Genet Anal. 1996; 13: 159-163Crossref PubMed Scopus (303) Google Scholar). Line S-9, expressing human PS1 harboring the FAD exon-9 deletion (PS1dE9), is described in Lee et al. (33Lee M.K. Borchelt D.R. Kim G. Thinakaran G. Slunt H.H. Ratovitski T. Martin L.J. Kittur A. Gandy S. Levey A.I. Jenkins N. Copeland N. Price D.L. Sisodia S.S. Nat. Med. 1997; 3: 756-760Crossref PubMed Scopus (130) Google Scholar). Line 1874, expressing wild-type mouse APP (moAPPwt), is described in Hsiao et al. (30Hsiao K.K. Borchelt D.R. Olson K. Johannsdottir R. Kitt C. Yunis W. Xu S. Eckman C. Younkin S. Price D. Iadecola C. Clark H.B. Carlson G. Neuron. 1995; 15: 1203-1218Abstract Full Text PDF PubMed Scopus (482) Google Scholar). Line 85, co-expressing human PS1dE9 and mouse/human (mo/hu) chimeric APP695 (humanized Aβ domain) harboring the Swedish (K594M/N595L) mutation, is described in Jankowsky et al. (11Jankowsky J.L. Fadale D.J. Anderson J. Xu G.M. Gonzales V. Jenkins N.A. Copeland N.G. Lee M.K. Younkin L.H. Wagner S.L. Younkin S.G. Borchelt D.R. Hum. Mol. Genet. 2004; 13: 159-170Crossref PubMed Scopus (1143) Google Scholar). Unlike lines S-9 and 1874, line 85 was created by co-injecting two transgenes, each driven by their own prion promoter element. The two transgenes co-integrated and co-segregate as a single locus (34Jankowsky J.L. Slunt H.H. Ratovitski T. Jenkins N.A. Copeland N.G. Borchelt D.R. Biomol. Eng. 2001; 17: 157-165Crossref PubMed Scopus (616) Google Scholar). Lines 85 and S-9 have been deposited with Jackson Laboratories (Bar Harbor, ME) for distribution (Stock numbers 004462 and 005866, respectively). After these experiments were completed, line 1874 was lost through accidental mistyping of breeding stock. Line 85 and line S-9 animals used in this study were maintained on a hybrid background by backcrossing to C3HeJ × C57BL/6J F1 animals obtained from Jackson Laboratories. Line 1874 was backcrossed to C57BL/6J for two generations after it was originally generated on the FVB background. After discovering premature lethality in the offspring, the line was crossed back to the hybrid C3HeJ × C57BL/6J F1 strain for two additional generations, which restored normal longevity to the line. Offspring from the second C3/B6 backcross were used as breeders to generate the cohorts described in this study. Offspring were genotyped for the presence of the transgene by PCR amplification of genomic DNA extracted from 1-cm tail clippings as described previously (34Jankowsky J.L. Slunt H.H. Ratovitski T. Jenkins N.A. Copeland N.G. Borchelt D.R. Biomol. Eng. 2001; 17: 157-165Crossref PubMed Scopus (616) Google Scholar). Reactions contained three primers, one antisense primer matching sequence within the vector that is also present in mouse genomic PrP (5′: GTG GAT ACC CCC TCC CCC AGC CTA GAC C), one sense primer specific for the transgene cDNA (PS1: 5′: CAG GTG GTG GAG CAA GAT G, huAPP: 5′: CCG AGA TCT CTG AAG TGA AGA TGG ATG, moAPP: 5′: CCT TCA GGA TTT GAA GTC CGC), and a second sense primer specific for the genomic PrP coding region, which has been removed from the MoPrP vector (5′: CCT CTT TGT GAC TAT GTG GAC TGA TGT CGG). All reactions give a 750-bp product from the endogenous PrP gene as a control for DNA integrity and successful amplification; transgene-positive samples have an additional band at 400 bp (huAPP), 350 bp (moAPP) or 1.3 kb (PS1). Animals were housed in microisolator cages with free access to food and water. All procedures involving animals were approved by the Johns Hopkins University Institutional Animal Care and Use Committee. Western Blotting—Mice of each genotype (NTg and 1874, n = 3-5; lines 85 and 1874 × 85, n = 10-15) were harvested at 8 months of age for assessment of amyloid pathology and APP/Aβ biochemistry. One-half of the brain was immersed in 4% paraformaldehyde and used for histology as described below. The remaining hemisphere was frozen on dry ice and prepared as a 20% homogenate that was used for Western blotting, filter trap assay, and ELISA. Frozen hemi-brain samples were sonicated in 5 volumes of 1× PBS containing 5 mm EDTA and 1× protease inhibitor mixture (Mammalian cell mix, Sigma). Homogenates were further diluted 1:1 with additional PBS/EDTA/protease inhibitor and centrifuged at high speed for 10 min, and the supernatant was used for analysis. Approximately 50 μg of protein homogenate per sample (5 μg for 22C11) was loaded onto 4-12% BisTris Novex gels (Invitrogen) and electrophoresed at 175 V for 1.5-2 hin1× MES buffer (Invitrogen). Proteins were transferred for 1 h at 100 V to 0.45-μm Optitran nitrocellulose (Schleicher and Schuell, Keene, NH) in 1× NuPAGE transfer buffer made with 10% methanol and 1% antioxidant solution (Invitrogen). Blots were blocked in PBS containing 5% nonfat dry milk powder for 30-60 min at room temperature. After blocking, blots were incubated with primary antibody for either 3 h at room temperature or overnight at 4 °C. The following primary antibodies and dilutions were used: 6E10 mouse anti-human Aβ monoclonal (Signet Laboratories, Dedham, MA) 1:2000; rabbit anti-rodent APP purified polyclonal antibody (AB5571P, Chemicon, Temecula, CA) 1:2000; 22C11 mouse anti-APP N terminus monoclonal, kind gift of Drs. Konrad Beyreuther and Andreas Weidemann, 1:2000 (35Weidemann A. Konig G. Bunke D. Fischer P. Salbaum J.M. Masters C.L. Beyreuther K. Cell. 1989; 57: 115-126Abstract Full Text PDF PubMed Scopus (1038) Google Scholar); m/hSOD1 rabbit anti-SOD1 polyclonal, 1:4000 (36Borchelt D.R. Lee M.K. Slunt H.S. Guarnieri M. Xu Z.S. Wong P.C. Brown Jr., R.H. Price D.L. Sisodia S.S. Cleveland D.W. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 8292-8296Crossref PubMed Scopus (533) Google Scholar). After incubation with primary antibody, the blots were washed several times with PBS containing 0.1% Tween 20, and then incubated with either goat anti-rabbit IgG or goat anti-mouse IgG conjugated to horseradish peroxidase (Jackson ImmunoResearch, West Grove, PA) diluted 1:2500 to 1:5000 in blocking solution. After washing several times in PBS containing 0.1% Tween 20, blots were developed with enhanced chemiluminescence reagent (ECL Plus, Amersham Biosciences/GE Biosciences) and exposed to film. Intensity of immunostaining was quantified from digitally scanned films with ImageJ by first inverting to create a negative image and then measuring the integrated density of each band. Background values calculated from a blank portion of the gel were subtracted manually from each sample before assessing the average signal intensity for the genotype. Aβ Immunoprecipitation—50 μl of the 20% PBS homogenate described above was diluted 10-fold in radioimmunoprecipitation assay buffer (0.2% SDS, 0.5% Nonidet P-40, 0.5% deoxycholate, 5 mm EDTA, in 1× PBS) and boiled for 10 min. After cooling, protease inhibitors were added, and the solution was incubated overnight at 4 °C with 2 μl of purified 4G8 (Signet Laboratories). The antibody was recovered with protein A-agarose beads (1 h at 4 °C), and nonspecific binding was removed by several washes with additional radioimmunoprecipitation assay buffer at 4 °C. The beads were heated to 95 °C for 5 min in 2× Tricine-SDS sample buffer, and the entire reaction was loaded onto 10-20% Tricine gels (Bio-Rad). Gels were pre-run for 10 min prior to loading, and then run in 15-min voltage steps of 25, 50, and 100, before running the gel to completion at 150 V. Protein was transferred to 0.45-μm Optitran nitrocellulose (Schleicher and Schuell) in 1× Tris-glycine/20% methanol/0.1% SDS, after which the blot was boiled 5 min in 1× PBS and blocked in 2% ECL Advance blocking reagent (Amersham Biosciences/GE Biosciences)/1× TBS/0.1% Tween-20. Blots were incubated overnight at room temperature with 4G8 diluted 1:10,000 in Advance block with 0.1% sodium azide. After washing several times in blocking reagent, blots were incubated for 2-3 h at room temperature with peroxidase-conjugated anti-mouse IgG diluted 1:20,000 in block. Blots were washed thoroughly with TBS/0.1% Tween-20, developed with ECL Advance (Amersham Biosciences/GE Biosciences), and exposed to film ∼1 h after developing with ECL. To demonstrate that 4G8 was capable of binding Aβ11-42, several reactions were run using 15 μl of 20% homogenate spiked with 10-50 ng of synthetic human Aβ11-42 (kindly provided by Dr. David Teplow, UCLA). Aβ ELISA: Steady-state Levels (7-24 Weeks of Age)—Brain tissue used for ELISA was harvested from female mice prior to the onset of amyloid pathology (lines 1874, 85, and 1874 × 85: 7-10 weeks of age; line 1874 × S-9: 9-24 weeks of age). Frozen mouse hemi-brains (n = 4-6 per genotype) were extracted by sonication in 0.2% diethylamine (DEA)/50 mm NaCl at a concentration of 100 mg/ml. After centrifugation at 100,000 × g for 1 h at 4 °C, the supernatant was removed and saved as the DEA extract. The pellet was then sonicated in 70% formic acid (FA) diluted in water, using a volume equal to the original volume of DEA. After centrifugation at 100,000 × g for 1 h at 4°C, the supernatant was removed and saved as the FA extract. The DEA extracts were neutralized by adding a 1/10 volume of 0.5 m Tris-HCl, pH 6.8. The FA extracts were neutralized and prepared for ELISA by diluting 1:20 in 1 m Tris-phosphate buffer, pH 11. The samples were then assayed by sandwich ELISA as described below. Aβ ELISA: Amyloid Solubility (8 Months of Age)—An aliquot of PBS 20% homogenate generated for Western analysis described above was subjected to a three-step sequential extraction using PBS, 2% SDS, and 70% formic acid (NTg and line 1874, n = 4; lines 85 and 1874 × 85, n = 8). At each step, the sample was sonicated in appropriate buffer and centrifuged at 100,000 × g for 30 min at 4 °C. The supernatant was removed for analysis, and the pellet was sonicated in an equal volume of the next solution in sequence. The 2% SDS extracts were diluted at least 1:40 in EC buffer (0.02 m sodium phosphate buffer, pH 7.0, 2 mm EDTA, 400 mm NaCl, 0.2% bovine serum albumin, 0.05% CHAPS, 0.4% BlockAce (Dainippon Pharmaceuticals), 0.05% NaN3), prior to testing to bring the SDS concentration below 0.05%; the FA extracts were neutralized with 1 m Tris-phosphate buffer, pH 11, and then diluted with EC buffer prior to testing. Brain extracts were measured by sandwich ELISA as described previously (37Suzuki N. Cheung T.T. Cai X.D. Odaka A. Otvos Jr., L. Eckman C. Golde T.E. Younkin S.G. Science. 1994; 264: 1336-1340Crossref PubMed Scopus (1358) Google Scholar, 38Gravina S.A. Ho L. Eckman C.B. Long K.E. Otvos Jr., L. Younkin L.H. Suzuki N. Younkin S.G. J. Biol. Chem. 1995; 270: 7013-7016Abstract Full Text Full Text PDF PubMed Scopus (593) Google Scholar, 39Kawarabayashi T. Younkin L.H. Saido T.C. Shoji M. Ashe K.H. Younkin S.G. J. Neurosci. 2001; 21: 372-381Crossref PubMed Google Scholar). Human Aβ was measured in each fraction using BAN50 for capture (epitope Aβ1-16) and BA27 and BC05 for detection (Aβ40 and Aβ42, respectively). Total Aβ (mouse plus human) was measured in each fraction using BNT77 for capture (epitope Aβ 11-28) and BA27 and BC05 for detection. Although BNT77 recognizes both mouse and human Aβ 1-x and 11-x, it does not bind α-secretase processed APP (40Asami-Odaka A. Ishibashi Y. Kikuchi T. Kitada C. Suzuki N. Biochemistry. 1995; 34: 10272-10278Crossref PubMed Scopus (153) Google Scholar), and measurements with BNT77 therefore do not include p3. All values were calculated as picomoles per g based on the initial weight of brain tissue. Filter Trap Assay—An aliquot of 20% PBS protein homogenate from each 8-month-old animal was partially solubilized by the addition of SDS to a final concentration of 1%. Serial 1:1 dilutions were made with 1× PBS/1% SDS, and 90 μl of each dilution was then vacuum-filtered through a pre-wet 0.22-μm cellulose acetate membrane (OE66, Schleicher and Schuell, Keene, NH). Each well was washed several times with PBS, after which blots were blocked for an hour in 1× TBS plus 5% nonfat dry milk powder. Blots were then incubated at 4 °C overnight with polyclonal anti-Aβ peptide antibody (71-5800, Zymed Laboratories) diluted 1:600 in blocking solution. After washing the blots several times in 1× TBS/0.1% Tween 20, the membrane was incubated for 1 h with an IRDye 800-conjugated goat anti-rabbit IgG secondary antibody (Rockland Immunochemicals, Gilbertsville, PA) diluted 1:5000 in blocking solution. The membranes were again washed three times with 1× TBS/0.1% Tween 20, given a final rinse in 1× TBS, and then imaged with an Odyssey fluorescence imager (LI-COR, Lincoln, NE). Staining intensity for each well was quantified using Odyssey analysis software, from which the linear range of the dilution series was determined and used for all genotype comparisons. Histology—Brains from lines 1874, 85, and 1874 × 85 were harvested for histological analysis at 4 months (n = 3-4 per genotype) and at 8 months (n = 5-14 per genotype) of age. Mice were euthanized by ether inhalation, and the brain was removed for analysis. One half was used for biochemical analysis described above; the remaining hemisphere was used for histology. After immersion in 4% paraformaldehyde/1× PBS for 48 h at 4 °C, the fixed hemi-brains were transferred to PBS, dehydrated in an alcohol series, treated with cedar wood oil and methylsalicylate, and embedded in paraffin for sectioning. Hirano Silver Stain—Silver impregnation histology was performed on 10-μm paraffin-embedded sections by Hirano’s modification of the Bielschowsky method (41Hirano A. Zimmermann H.M. Arch. Neurol. 1962; 6: 114-122Crossref PubMed Scopus (37) Google Scholar, 42Yamamoto T. Hirano A. Neuropathol. Appl. Neurobiol. 1986; 12: 3-9Crossref PubMed Scopus (308) Google Scholar). Briefly, sections were deparaffinized through xylene and alcohols into tap water before being placed into fresh 20% silver nitrate solution for 20 min. After washing thoroughly with distilled water, slides were immersed in 20% silver nitrate solution titrated with fresh ammonium hydroxide. After 20 min, slides were washed with ammonia water before being individually developed with 100 μl of developer (20 ml of 37% formaldehyde, 100 ml of distilled water, 50 μl of concentrated nitric acid, and 0.5 g of citric acid) added to 50 ml of titrated silver nitrate solution. Slides were then rinsed in tap water, fixed in 5% sodium thiosulfate, and dehydrated through alcohols and xylene. Campbell-Switzer Silver Stain—A detailed protocol for this stain was kindly provided by Dr. Bob Switzer of NeuroScience Associates. Thioflavine-S Staining—Following deparaffinization of sections through xylene and alcohols, amyloid impregnation with thioflavine-S was performed according to the Guntern modification of the standard protocol. Slides were washed twice in distilled water, then immersed for 5 min in a 0.25% potassium permanganate solution, followed by 5 min in a 1% potassium metabisulfate/1% oxalic acid solution. After this preparation, slides were placed into a filtered aqueous 0.02% thioflavine-S solution (Chroma-Gesellschaft, Schmid GmbH and Co., Kongen, Germany) for 8 min. Excess stain was removed by two brief rinses in 80% ethanol, then two in distilled water, after which slides were finished in aqueous mounting medi
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