Acetylcholinesterase-Aβ Complexes Are More Toxic than Aβ Fibrils in Rat Hippocampus
2004; Elsevier BV; Volume: 164; Issue: 6 Linguagem: Inglês
10.1016/s0002-9440(10)63774-1
ISSN1525-2191
AutoresAriel E. Reyes, Marcelo A. Chacón, Margarita C. Dinamarca, Waldo Cerpa, Carlos Morgan, Nibaldo C. Inestrosa,
Tópico(s)Computational Drug Discovery Methods
ResumoNeuropathological changes generated by human amyloid-β peptide (Aβ) fibrils and Aβ-acetylcholinesterase (Aβ-AChE) complexes were compared in rat hippocampus in vivo. Results showed that Aβ-AChE complexes trigger a more dramatic response in situ than Aβ fibrils alone as characterized by the following features observed 8 weeks after treatment: 1) amyloid deposits were larger than those produced in the absence of AChE. In fact, AChE strongly stimulates rat Aβ aggregation in vitro as shown by turbidity measurements, Congo Red binding, as well as electron microscopy, suggesting that Aβ-AChE deposits observed in vivo probably recruited endogenous Aβ peptide; 2) the appearance of laminin expressing neurons surrounding Aβ-AChE deposits (such deposits are resistant to disaggregation by laminin in vitro); 3) an extensive astrocytosis revealed by both glial fibrillary acidic protein immunoreactivity and number counting of reactive hypertrophic astrocytes; and 4) a stronger neuronal cell loss in comparison with Aβ-injected animals. We conclude that the hippocampal injection of Aβ-AChE complexes results in the appearance of some features reminiscent of Alzheimer-like lesions in rat brain. Our studies are consistent with the notion that Aβ-AChE complexes are more toxic than Aβ fibrils and that AChE triggered some of the neurodegenerative changes observed in Alzheimer's disease brains. Neuropathological changes generated by human amyloid-β peptide (Aβ) fibrils and Aβ-acetylcholinesterase (Aβ-AChE) complexes were compared in rat hippocampus in vivo. Results showed that Aβ-AChE complexes trigger a more dramatic response in situ than Aβ fibrils alone as characterized by the following features observed 8 weeks after treatment: 1) amyloid deposits were larger than those produced in the absence of AChE. In fact, AChE strongly stimulates rat Aβ aggregation in vitro as shown by turbidity measurements, Congo Red binding, as well as electron microscopy, suggesting that Aβ-AChE deposits observed in vivo probably recruited endogenous Aβ peptide; 2) the appearance of laminin expressing neurons surrounding Aβ-AChE deposits (such deposits are resistant to disaggregation by laminin in vitro); 3) an extensive astrocytosis revealed by both glial fibrillary acidic protein immunoreactivity and number counting of reactive hypertrophic astrocytes; and 4) a stronger neuronal cell loss in comparison with Aβ-injected animals. We conclude that the hippocampal injection of Aβ-AChE complexes results in the appearance of some features reminiscent of Alzheimer-like lesions in rat brain. Our studies are consistent with the notion that Aβ-AChE complexes are more toxic than Aβ fibrils and that AChE triggered some of the neurodegenerative changes observed in Alzheimer's disease brains. Alzheimer's disease (AD) is one of the most common neurodegenerative dementias, characterized by a progressive decline of cognitive functions and psychomotor abilities.1Salmon DP Bondi MW Neuropsychology of Alzheimer disease.in: Terry RD Katzman R Bick KL Sisodia SS Alzheimer Disease. 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A comparative study in the hippocampus and entorhinal cortex.Acta Neuropathol. 1990; 80: 624-628Crossref PubMed Scopus (107) Google Scholar among others. 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5: 1063-1071Abstract Full Text Full Text PDF PubMed Scopus (123) Google Scholar, 19Beeri R Le Novere N Mervis R Huberman T Grauer E Changeux JP Soreq H Enhanced hemicholinium binding and attenuated dendrite branching in cognitively impaired acetylcholinesterase-transgenic mice.J Neurochem. 1997; 69: 2441-2451Crossref PubMed Scopus (88) Google Scholar, 20Sternfeld M Shoham S Klein O Flores-Flores C Evron T Idelson GH Kitsberg D Patrick JW Soreq H Excess "read-through" acetylcholinesterase attenuates but the "synaptic" variant intensifies neurodeterioration correlates.Proc Natl Acad Sci USA. 2000; 97: 8647-8652Crossref PubMed Scopus (115) Google Scholar On the other hand, experiments in our laboratory have demonstrated that AChE promotes Aβ peptide fibrils assembly in vitro21Inestrosa NC Alvarez A Perez CA Moreno RD Vicente M Linker C Casanueva OI Soto C Garrido J Acetylcholinesterase accelerates assembly of amyloid-β-peptides into Alzheimer's fibrils: possible role of the peripheral site of the enzyme.Neuron. 1996; 16: 881-891Abstract Full Text Full Text PDF PubMed Scopus (1012) Google Scholar and biochemical, physical, and morphological data strongly suggest that an amyloid-AChE complex is formed when AChE accelerates the assembly of Aβ peptides.22De Ferrari GV Canales MA Shin I Weiner LM Silman I Inestrosa NC A structural motif of acetylcholinesterase that promotes amyloid β-peptide fibril formation.Biochemistry. 2001; 40: 10447-10457Crossref PubMed Scopus (377) Google Scholar Such complexes were both Th-T fluorescence-positive and AChE activity-positive.23Alvarez A Opazo C Alarcón R Garrido J Inestrosa NC Acetylcholinesterase promotes the aggregation of amyloid-β-peptide fragments by forming a complex with the growing fibrils.J Mol Biol. 1997; 272: 348-361Crossref PubMed Scopus (277) Google Scholar More important those complexes were more toxic for primary cultured chick retina neurons and PC12 cells than Aβ fibrils lacking AChE.24Alvarez A Alarcon R Opazo C Campos EO Muñoz FJ Calderón FH Dajas F Gentry MK Doctor BP De Mello FG Inestrosa NC Stable complexes involving acetylcholinesterase and amyloid-β peptide change the biochemical properties of the enzyme and increase the neurotoxicity of Alzheimer's fibrils.J Neurosci. 1998; 18: 3213-3223Crossref PubMed Google Scholar Interestingly, the toxicity of the Aβ-AChE complex was also dependent on the AChE concentration present in the complexes.25Muñoz FJ Inestrosa NC Neurotoxicity of acetylcholinesterase-amyloid β-peptide aggregates is dependent on the type of Aβ peptide and the AChE concentration present in the complexes.FEBS Lett. 1999; 450: 205-209Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar, 26Muñoz FJ Opazo C Gil-Gómez G Tapia G Fernández V Valverde MA Inestrosa NC Vitamin E but not 17β-estradiol protect against vascular toxicity induced by β-amyloid wild-type and the Dutch amyloid variant.J Neurosci. 2002; 22: 3081-3089PubMed Google Scholar All these evidences are consistent with the possibility that local increments of AChE, as those present in AD brains,12Geula C Mesulam M Special properties of cholinesterases in the cerebral cortex of Alzheimer's disease.Brain Res. 1989; 498: 185-189Crossref PubMed Scopus (132) Google Scholar should play a role in the neurodegeneration and cognitive deterioration observed in AD.14Kasa P Rakonczay Z Gulya K The cholinergic system in Alzheimer's disease.Prog Neurobiol. 1997; 52: 511-535Crossref PubMed Scopus (364) Google Scholar, 27Inestrosa NC Roberts WL Marshall TL Rosenberry TL Acetylcholinesterase from bovine caudate nucleus is attached to membranes by a novel subunit distinct from those of acetylcholinesterases in other tissues.J Biol Chem. 1987; 262: 4441-4444Abstract Full Text PDF PubMed Google Scholar Our hypothesis implies that AChE plays a role in the pathological changes observed in AD by promoting amyloid formation and stability, astrocytosis, and neurotoxicity. We decided, therefore, to evaluate and to compare in vivo the neurotoxicity of both Aβ fibrils and Aβ-AChE complexes. To perform these studies, we injected Aβ fibrils and Aβ-AChE complexes bilaterally in the rat dorsal hippocampus and the resulting neuropathological changes were studied. Results indicate that rats injected with Aβ-AChE complexes in the hippocampus form amyloid deposits that seem bigger in size than those observed in matched controls injected with the same amount of Aβ peptide, suggesting that endogenous rat Aβ may became incorporated into the amyloid deposits. The injection of Aβ-AChE complexes also results in the appearance of Alzheimer-like lesions in rat brain, and in all cases, the toxicity of the Aβ-AChE complex depends on the amount of enzyme present in the complexes. Aβ peptide corresponding to residues 1 to 40 of the human wild-type sequence (Aβ1-40) was obtained from Bachem (Torrance, CA) and the rat Aβ1-40 peptide was obtained from Genemed Biotechnologies, Inc. (San Francisco, CA). Tetrameric G4 AChE form (sedimentation coefficient, 10.7 S) was purified from bovine caudate nucleus, using acridine affinity chromatography as previously described.27Inestrosa NC Roberts WL Marshall TL Rosenberry TL Acetylcholinesterase from bovine caudate nucleus is attached to membranes by a novel subunit distinct from those of acetylcholinesterases in other tissues.J Biol Chem. 1987; 262: 4441-4444Abstract Full Text PDF PubMed Google Scholar Both specific activity (6000 U/mg protein) and staining intensity after sodium dodecyl sulfate-polyacrylamide gel electrophoresis28Laemmli UK Cleavage of structural proteins during the assembly of the head of bacteriophage T4.Nature. 1970; 227: 680-685Crossref PubMed Scopus (208244) Google Scholar were used to verify purity. AChE activity was determined by the method of Ellman and colleagues.29Ellman GK Courtney KD Andress V Featherstone RM A new rapid colorimetric determination of acetylcholinesterase activity.Biochem Pharmacol. 1961; 1: 88-95Crossref Scopus (22159) Google Scholar Aβ fibrils were formed in a turbidity assay as previously described.23Alvarez A Opazo C Alarcón R Garrido J Inestrosa NC Acetylcholinesterase promotes the aggregation of amyloid-β-peptide fragments by forming a complex with the growing fibrils.J Mol Biol. 1997; 272: 348-361Crossref PubMed Scopus (277) Google Scholar, 24Alvarez A Alarcon R Opazo C Campos EO Muñoz FJ Calderón FH Dajas F Gentry MK Doctor BP De Mello FG Inestrosa NC Stable complexes involving acetylcholinesterase and amyloid-β peptide change the biochemical properties of the enzyme and increase the neurotoxicity of Alzheimer's fibrils.J Neurosci. 1998; 18: 3213-3223Crossref PubMed Google Scholar Briefly, Aβ peptide stock solution was prepared by dissolving freeze-dried aliquots of Aβ1-40 in dimethyl sulfoxide (DMSO) at 15 mg/ml (3.5 mmol/L). An aliquot of this stock solution equivalent to 70 nmol of Aβ peptide was added to aqueous buffer (725 μl total volume; 0.1 mol/L Tris-HCl, pH 7.4). For the aggregation assay in the presence of AChE, an identical aliquot of the stock solution (70 nmol) was added to a buffer containing AChE (100 nmol/L) at a final molar ratio of AβμAChE = 1000:1. The solutions were stirred continuously (1350 rpm) at room temperature for 48 hours and then left at 4°C for another 48 hours. Aggregation was measured by turbidity at 400 nm against a buffer blank. Amyloid fibrils obtained were characterized by Congo Red (CR) binding. Fibril aliquots were added to a solution containing 25 μmol/L CR solution, 100 mmol/L phosphate buffer (pH 7.4), and 150 mmol/L NaCl in a final volume of 960 μl, and incubated for 30 minutes at room temperature.23Alvarez A Opazo C Alarcón R Garrido J Inestrosa NC Acetylcholinesterase promotes the aggregation of amyloid-β-peptide fragments by forming a complex with the growing fibrils.J Mol Biol. 1997; 272: 348-361Crossref PubMed Scopus (277) Google Scholar Absorbance was measured at 480 nm and 540 nm, and CR binding was determined by CR (concentration) = (A540/25,295) − (A480/46,306).30Klunk WE Pettegrew JW Abraham DJ Quantitative evaluation of Congo red binding to amyloid-like proteins with a β-pleated sheet conformation.J Histochem Cytochem. 1989; 37: 1273-1281Crossref PubMed Scopus (573) Google Scholar One hundred μmol/L of Aβ fibril suspensions were formed as described above either in the absence or the presence of 100 nmol/L AChE purified from bovine brain. Then, mouse laminin from Englebreth-Holm-Swarm tumor (Sigma L-2020 or Gibco catalog no. 23017-015; in 20 mmol/L Tris-HCl, 150 mmol/L NaCl, pH 7.2) was added to the fibril suspensions at a final laminin concentration as indicated in the legend of Figure 6. Th-T measurements were taken from 4-μl sample aliquots (added to 500 μl of 50 mmol/L phosphate buffer, pH 6.0, containing 15 μl of 0.1 mmol/L Th-T) at indicated incubation time points by monitoring fluorescence at λEx = 450 nm and λEm = 485 nm as described previously.21Inestrosa NC Alvarez A Perez CA Moreno RD Vicente M Linker C Casanueva OI Soto C Garrido J Acetylcholinesterase accelerates assembly of amyloid-β-peptides into Alzheimer's fibrils: possible role of the peripheral site of the enzyme.Neuron. 1996; 16: 881-891Abstract Full Text Full Text PDF PubMed Scopus (1012) Google Scholar, 23Alvarez A Opazo C Alarcón R Garrido J Inestrosa NC Acetylcholinesterase promotes the aggregation of amyloid-β-peptide fragments by forming a complex with the growing fibrils.J Mol Biol. 1997; 272: 348-361Crossref PubMed Scopus (277) Google Scholar Assembled fibrils were washed four times with phosphate-buffered saline (PBS) at 14,000 rpm for 30 minutes to remove soluble peptide and AChE. Pellets were finally resuspended in artificial cerebrospinal fluid (ACF) at a concentration of 2.3 μg/μl Aβ peptide. Aliquots were mixed with a denaturing buffer and subjected to Tris-Tricine sodium dodecyl sulfate-polyacrylamide gel electrophoresis31Schagger H von Jagow G Tricine-sodium dodecyl sulfate-polyacrylamide gel electrophoresis for the separation of proteins in the range from 1 to 100 kDa.Anal Biochem. 1987; 166: 368-379Crossref PubMed Scopus (10538) Google Scholar to quantify Aβ peptide present into the fibrils by densitometry scanning. An Aβ peptide stock of known concentration was used as standard. Data was processed by a GS365W program from Hoeffer Scientific Instruments (San Francisco, CA). Before the injection treatment, we followed amyloid formation by turbidity at 400 nm and CR binding. Similar methods were used during Aβ fibril formation. The final products were analyzed using CR fibril staining under polarized light as well as by protease resistance assay (data not shown). All these assays did not show any apparent difference in the characteristic of the final amyloid product used for intracerebral injections. The amyloid fibrils formed in turbidity assays were examined by electron microscopy. The fibrils were placed on Formvar carbon-coated 300-mesh nickel grids and negatively stained with 1% phosphotungstic acid solution for 1 minute. Grids were examined under a Phillips EM-300 electron microscope at 60 kV.21Inestrosa NC Alvarez A Perez CA Moreno RD Vicente M Linker C Casanueva OI Soto C Garrido J Acetylcholinesterase accelerates assembly of amyloid-β-peptides into Alzheimer's fibrils: possible role of the peripheral site of the enzyme.Neuron. 1996; 16: 881-891Abstract Full Text Full Text PDF PubMed Scopus (1012) Google Scholar Male Sprague-Dawley rats (280 to 320 g; 3 months old) were anesthetized with Equitesin (2.5 ml/kg), and injected bilaterally into the upper leaf of dentate gyrus in dorsal hippocampus (−3.5 mm anteroposterior, ±2.0 mm medial-lateral, −2.7 mm dorsal-ventral from the dura, according to bregma)32Paxinos G Watson C The Rat Brain in Stereotaxic Coordinates ed 2. Academic Press, New York1986Google Scholar stereotaxically with a 10-μl Hamilton syringe with 27-gauge stainless steel needle. The injection is medial to granule layer and ventral to hippocampal fissure. The animals were injected bilaterally with 3 μl (at a rate of 0.5 μl/minute) of in vitro assembled Aβ fibrils formed in the presence (n = 9) or absence of AChE (n = 9), which is equivalent to 7 μg of Aβ peptide or Aβ-AChE complex into each hippocampus. Control animals were injected with an identical volume of ACF (n = 6). Two (n = 3) and eight (n = 6) weeks later, animals were fixed by intracardiac perfusion to perform histochemical procedures (see below). Sprague-Dawley rats (280 to 320 g) were anesthetized with Equitesin (2.5 mg/kg, i.p.) and then rats were placed on a stereotaxic apparatus. These animals were with implanted a 27-gauge double cannula at the dorsal hippocampus under the coordinates mentioned above. Through a polyethylene hose, each cannula was connected to a 200-μl Alzet 2002 miniosmotic pump (Alza Corp., Mountain View, CA), which releases its content to a rate of 0.5 μl/hour. The pumps were placed under the skin back the head toward the rat shoulder. The pumps were charged with both soluble Aβ1-40 peptide (0.75 μg/μl) and with AChE (0.272 μg/μl), final concentration in PBS to molar ratio of 1:200 (total volume, 200 μl). Fourteen days after this procedure, in the case of Figure 5, the procedure take 4 weeks, rats were fixed by perfusion with 4% paraformaldehyde and coronal brain sections were conducted for immunohistochemical analysis. Animals were anesthetized with Equitesin (2.5 ml/kg. i.p.) and injected with heparin (4 USP/kg, i.p.) to inhibit blood coagulation before perfusion. To study the evolution of amyloid deposits, rats were fixed at day 2 and 8 weeks after injection to visualize the evolution of amyloid deposits. Then they were perfused through the heart with perfusion buffer33Côté SL Ribeiro-Da-Silva A Cuello AC Cuello AC Immunocytochemistry II. John Wiley & Sons, New York1993Google Scholar containing 0.1% sodium nitrite, followed by fixation with 4% paraformaldehyde in 0.1 mol/L phosphate buffer (PB) for 30 minutes. Brains were removed from the skulls and postfixed in the same fixative for 3 hours at room temperature, followed by 10% sucrose in PB at 4°C overnight. After fixation, brains were coded to ensure unbiased processing and analysis. The brains were then cut into 50-μm coronal sections with a cryostat (Leitz 1900) at −20°C, from bregma −1.8 mm to bregma −4.8 mm.32Paxinos G Watson C The Rat Brain in Stereotaxic Coordinates ed 2. Academic Press, New York1986Google Scholar Sections from the same brain were divided into six groups for analysis by the following procedures: Nissl staining (0.3% cresyl violet); immunohistochemical staining for glial fibrillary acidic protein (GFAP), laminin, and Aβ peptide; and CR and Th-S for specific amyloid staining. Free-floating immunohistochemical procedure was performed as previously described.34Elghetany MT Saleem A Methods for staining amyloid in tissues: a review.Stain Technol. 1988; 63: 201-212Crossref PubMed Scopus (141) Google Scholar Washing and dilution of immunoreagents was performed with 0.01 mol/L PBS with 0.2% Triton X-100 (PBS+T) throughout the experiments, and two PBS+T washes were performed after incubation with each antibody incubation. All three immunohistochemical procedures for each sample were performed simultaneously. Sections were pretreated with 0.3% H2O2 for 30 minutes to reduce endogenous peroxidase activity followed by treatment with 5% normal goat serum (DAKO, Carpinteria, CA) at room temperature for 1 hour to avoid nonspecific binding. GFAP, laminin, and Aβ detection was performed using the following antibodies: rabbit anti-GFAP polyclonal antibody (1:500) (DAKO), rabbit anti-laminin (1:60), and rabbit anti-Aβ1-40 (1:100) (Sigma Chemical Co., St. Louis, MO) incubated overnight at 4°C. A horseradish peroxidase-conjugated goat anti-rabbit IgG second antibody was used in all three cases (1:600), incubated for 1 hour at room temperature. The staining was developed by incubation for 15 minutes with 0.6% diaminobenzidine followed by addition of H2O2 and incubation for 4 minutes. After immunostaining, all sections were mounted on gelatin-coated glass, air-dried, dehydrated in ascending concentrations of ethanol, cleared with xylene, and coverslipped with Canada balsam (Merck, Darmstadt, Germany). Mounted sections were defatted in xylene and hydrated in ethyl alcohol and water series. Nissl staining (cresyl violet) was performed as previously described.33Côté SL Ribeiro-Da-Silva A Cuello AC Cuello AC Immunocytochemistry II. John Wiley & Sons, New York1993Google Scholar CR staining using alkaline CR methods was performed as previously described.35Puchtler H Sweat F Levine M On the binding of Congo red by amyloid.J Histochem Cytochem. 1961; 10: 355-364Crossref Google Scholar Th-S staining was performed as described by Elghetany and Saleem.34Elghetany MT Saleem A Methods for staining amyloid in tissues: a review.Stain Technol. 1988; 63: 201-212Crossref PubMed Scopus (141) Google Scholar All histological and immunohistochemical images were acquired from a Zeiss Axioplan microscope with a 35-mm camera system. To analyze all plaque-like deposits in one field, ×10 and ×20 optical magnifications were used. The pictures were digitalized in a Nikon Coolscan III Scanner, and each image was routed into a PC-compatible microcomputer via Adobe-Photoshop program (5.5). Once digitalized, the images were analyzed with Scion Image public domain software (Scion Image for Windows β 4.02, on the internet at http://www.scioncorp.com). Custom macro subroutine was used to calculate positively stained areas, of both immunopositives staining for Aβ Th-S-fluorescence and CR-positive deposits. The same slides were analyzed single blind by two different investigators to ensure that trends were independent of individual bias. Serial coronal sections of the hippocampus were performed between the coordinates in an anteroposterior location to the injection site. The parameter measured was the positive area stained in six 50-μm-thick sections per rat per assay. Results from this analysis revealed the average of total area of the deposit. Analysis of number of cells, GFAP and laminin staining area, and GFAP intensity were measured with SigmaScan Pro software. Data from the image analysis macros were exported to a Sigma Plot file for statistical analysis. Results were expressed as mean ± SE. Statistical significance was determined by one-way analysis of variance. A specific antibody against the Aβ peptide revealed successful injection of Aβ fibrils into the dorsal hippocampus. Anti-Aβ immunohistochemistry showed clear accumulation of Aβ-positive deposits after intrahippocampal injection of Aβ fibrils assembled with and without AChE. These deposits were observed in animals after 2 (Figure 1, A and B) and 8 weeks (Figure 2, D and G). Significant differences in the size of the deposits between both treatment groups were observed after 8 weeks of treatment using three different staining methods, anti-Aβ antibody, thioflavine S, and CR (Figure 2J). In fact, animals examined at 2 weeks showed similar size of the Aβ immunopositive areas with both injection treatments (Figure 1G), however, when animals were examined after 8 weeks of injection, a significant increase in the size of Aβ-immunopositive area was observed in the case of animals injected with Aβ-AChE complexes. This was evident after performing a digital quantification of the hippocampal areas stained with the anti-Aβ antibody, which indicated almost a threefold increase of amyloid load in animals co-injected with Aβ-AChE complexes compared with Aβ (Figure 2J). Control animals injected with an unrelated peptide sequence derived from the β-APP gave no specific amyloid staining (data not shown).Figure 2Aβ deposits and amyloid detection in situ in animals 8 weeks after injection. Anti-Aβ immunostaining of a coronal section from animals injected into hippocampus with ACF, Aβ fibrils, and Aβ-AChE complexes, Th-S fluorescence of the corresponding sections, and CR staining under polarized light. D, E, and F: Staining of sections from animals injected with Aβ fibrils that maintain immunopositive Aβ deposits (D), amyloid-positive as shown by Th-S (E)- and CR (F)-specific staining. G, H, I: The brain slides from animals injected with Aβ-AChE complexes are also immunopositive for Aβ deposits using an anti-Aβ antibody; however these deposits are larger than those observed in rats injected with Aβ fibrils (G). These Aβ deposits are amyloid-positive as shown by Th-S fluorescence (H) and CR (I) staining. Note that the amyloid deposits obtained with Aβ-AChE complexes stained with CR, present birefringent deposits and Maltese cross-positive pattern (I, inset), in contrast, rats injected with Aβ fibrils did not present Maltese crosses (F, inset). J: The graph corresponds to a digital quantification (SigmaScan Pro) of the amyloid deposits shown above (see Figure 1G and Material and Methods). Results were expressed as mean ± SE. Statistical significance was determined by one-way analysis of variance; *, P < 0.05 was regarded as statistically significant. Scale bars: 100 μm (A–E, G, H); 50 μm (F, I).View Large Image Figure ViewerDownload Hi-res image Download (PPT) To verify that Aβ deposits were in fact amyloid-positive, we performed two well-known specific and sensitive staining procedures: Th-S-fluorescence and polarized light microscopy after CR staining under polarized light. We observed that extracellular deposits positive for an antibody against the Aβ peptide were indeed positive for Th-S-fluorescence (a 2.5-fold increase, Figure 2J), in a similar way to very dense amyloid cores observed in AD lesions36Dickson DW Farlo J Davies P Crystal H Fuld P Yen SH A
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