Tissue Distribution, Biochemical Properties, and Transmission of Mouse Type A AApoAII Amyloid Fibrils
2004; Elsevier BV; Volume: 164; Issue: 5 Linguagem: Inglês
10.1016/s0002-9440(10)63718-2
ISSN1525-2191
AutoresTatsumi Korenaga, Xiaoying Fu, Yanming Xing, Takatoshi Matsusita, Kazunao Kuramoto, Seigo Syumiya, Kazuhiro Hasegawa, Hironobu Naiki, Masaki Ueno, Tokuhiro Ishihara, Masanori Hosokawa, Masayuki Mori, Keiichi Higuchi,
Tópico(s)Alzheimer's disease research and treatments
ResumoIn mouse strains with the amyloidogenic apolipoprotein A-II (ApoA-II) gene (Apoa2c), the type C ApoA-II protein (APOAIIC) associates to form amyloid fibrils AApoAII(C) that lead to development of early onset and systemic amyloidosis with characteristic heavy amyloid deposits in the liver and spleen. We found age-associated heavy deposition of amyloid fibrils [AApoAII(A)] composed of type A ApoA-II protein (APOAIIA) in BDF1 and C57BL/6 mice reared at one of our institutes. AApoAII(A) fibrils were deposited in the intestine, lungs, tongue, and stomach but not in the liver or spleen. AApoAII(A) fibrils were isolated, and morphological, biochemical, and structural characteristics distinct from those seen in AApoAII(C) and mouse AA amyloid fibrils were found. Transmission electron and atomic force microscopy showed that the majority of isolated AApoAII(A) amyloid fibrils featured fine, protofibril-like shapes. AApoAII(A) fibrils have a much weaker affinity for thioflavine T than for AApoAII(C), whereas APOAIIA protein contains less of the β-pleated sheet structure than does APOAIIC. The injection of AApoAII(A) fibrils induced amyloid deposition in C57BL/6 and DBA2 mice (Apoa2a) as well as in R1.P1-Apoa2c mice (Apoa2c), but AApoAII(A) induced more severe amyloidosis in Apoa2a strains than in the Apoa2c strain. It was found that AApoAII(A) fibrils isolated from mice with mildly amyloidogenic APOAIIA protein have distinct characteristics. Induction of amyloidosis by heterologous amyloid fibrils clearly showed interactions between amyloid protein monomers and fibrils having different primary structures. In mouse strains with the amyloidogenic apolipoprotein A-II (ApoA-II) gene (Apoa2c), the type C ApoA-II protein (APOAIIC) associates to form amyloid fibrils AApoAII(C) that lead to development of early onset and systemic amyloidosis with characteristic heavy amyloid deposits in the liver and spleen. We found age-associated heavy deposition of amyloid fibrils [AApoAII(A)] composed of type A ApoA-II protein (APOAIIA) in BDF1 and C57BL/6 mice reared at one of our institutes. AApoAII(A) fibrils were deposited in the intestine, lungs, tongue, and stomach but not in the liver or spleen. AApoAII(A) fibrils were isolated, and morphological, biochemical, and structural characteristics distinct from those seen in AApoAII(C) and mouse AA amyloid fibrils were found. Transmission electron and atomic force microscopy showed that the majority of isolated AApoAII(A) amyloid fibrils featured fine, protofibril-like shapes. AApoAII(A) fibrils have a much weaker affinity for thioflavine T than for AApoAII(C), whereas APOAIIA protein contains less of the β-pleated sheet structure than does APOAIIC. The injection of AApoAII(A) fibrils induced amyloid deposition in C57BL/6 and DBA2 mice (Apoa2a) as well as in R1.P1-Apoa2c mice (Apoa2c), but AApoAII(A) induced more severe amyloidosis in Apoa2a strains than in the Apoa2c strain. It was found that AApoAII(A) fibrils isolated from mice with mildly amyloidogenic APOAIIA protein have distinct characteristics. Induction of amyloidosis by heterologous amyloid fibrils clearly showed interactions between amyloid protein monomers and fibrils having different primary structures. Amyloidosis is a structural disorder of proteins in which proteins that are normally soluble are deposited in tissues as abnormally ordered, insoluble amyloid fibrils made up of β-pleated sheets.1Westermark P The pathogenesis of amyloidosis: understanding general principles.Am J Pathol. 1998; 152: 1125-1127PubMed Google Scholar Several serious human diseases such as Alzheimer's disease, type II diabetes, prion diseases, and familial amyloid polyneuropathy are associated with amyloid fibril deposition.2Booth DR Sunde M Bellotti V Robinson CV Hutchinson WL Fraser PE Hawkins PN Dobson CM Radford SE Blake CC Pepys MB Instability, unfolding and aggregation of human lysozyme variants underlying amyloid fibrillogenesis.Nature. 1997; 385: 787-793Crossref PubMed Scopus (983) Google Scholar, 3Glenner GG Amyloid deposits and amyloidosis: the beta-fibrilloses.N Engl J Med. 1980; 302: 1283-1292Crossref PubMed Scopus (1310) Google Scholar Thus, to devise means of preventing amyloidosis, it may be essential to study the properties of amyloid proteins and the mechanisms for fibril formation. Many factors such as aging, the primary sequences and mutations of amyloid proteins, the genetic background of patients, and epigenetic factors, as well as the means of amyloid fibril transmission, may influence fibril formation.4Xing Y Higuchi K Amyloid fibril proteins.Mech Ageing Dev. 2002; 123: 1625-1636Crossref PubMed Scopus (61) Google Scholar In mice, spontaneous senile amyloidosis has been found in many strains.5Higuchi K Hosokawa M Takeda T Senescence-accelerated mouse.Methods Enzymol. 1999; 309: 674-686Crossref PubMed Scopus (22) Google Scholar We isolated a unique amyloid fibril protein from the liver of the SAMP1 mouse strain and found that apolipoprotein A-II (ApoA-II), the second most abundant apoprotein of serum high-density lipoprotein, is deposited in the form of amyloid fibrils (AApoAII). These fibrils are deposited systemically, but not in the brain.6Higuchi K Yonezu T Kogishi K Matsumura A Takeshita S Kohono A Matsushita T Hosokawa M Takeda T Purification and characterization of a senile amyloid-related antigenic substance (apoSASSAM) from mouse serum. apoSASSAM is an apoA-II apolipoprotein of mouse high density lipoproteins.J Biol Chem. 1986; 261: 12834-12840Abstract Full Text PDF PubMed Google Scholar, 7Higuchi K Matsumura A Honma A Takeshita S Hashimoto K Hosokawa M Yasuhira K Takeda T Systemic senile amyloid in senescence-accelerated mice. A unique fibril protein demonstrated in tissues from various organs by the unlabeled immunoperoxidase method.Lab Invest. 1983; 48: 231-240PubMed Google Scholar Three alleles (Apoa2a, Apoa2b, and Apoa2c) of the ApoA-II gene encode three variants of the ApoA-II protein (APOAIIA, APOAIIB, and APOAIIC) (Table 1).8Higuchi K Kitagawa K Naiki H Hanada K Hosokawa M Takeda T Polymorphism of apolipoprotein A-II (apoA-II) among inbred strains of mice. Relationship between the molecular type of apoA-II and mouse senile amyloidosis.Biochem J. 1991; 279: 427-433Crossref PubMed Scopus (91) Google Scholar Mice such as SAMP1, SAMP2, SAMP10, A/J, SJL/J, and SM/J, which have an APOAIIC protein with a glutamine at position 5, show a high incidence of severe senile amyloidosis, whereas the strains with a very low incidence of amyloidosis produce APOAIIB with a proline at position 5. Severe AApoAII amyloidosis is linked to the Apoa2c allele.8Higuchi K Kitagawa K Naiki H Hanada K Hosokawa M Takeda T Polymorphism of apolipoprotein A-II (apoA-II) among inbred strains of mice. Relationship between the molecular type of apoA-II and mouse senile amyloidosis.Biochem J. 1991; 279: 427-433Crossref PubMed Scopus (91) Google Scholar, 9Naiki H Higuchi K Shimada A Takeda T Nakakuki K Genetic analysis of murine senile amyloidosis.Lab Invest. 1993; 68: 332-337PubMed Google Scholar, 10Higuchi K Wang J Kitagawa K Matsushita T Kogishi K Naiki H Kitado H Hosokawa M Accelerated senile amyloidosis induced by amyloidogenic Apoa2 gene shortens the life span of mice but does not accelerate the rate of senescence.J Gerontol A Biol Sci Med Sci. 1996; 51A: B295-B302Crossref Scopus (21) Google Scholar The C57BL/6, AKR/J, and DBA/2 strains encode an APOAIIA protein with Pro5 and Met26, and mild tissue-specific amyloidosis has been reported in these strains.8Higuchi K Kitagawa K Naiki H Hanada K Hosokawa M Takeda T Polymorphism of apolipoprotein A-II (apoA-II) among inbred strains of mice. Relationship between the molecular type of apoA-II and mouse senile amyloidosis.Biochem J. 1991; 279: 427-433Crossref PubMed Scopus (91) Google Scholar Aged C57BL/Ka mice were found to have a high incidence of gastrointestinal AApoAII amyloidosis.11HogenEsch H Niewold TA Higuchi K Tooten PC Gruys E Radl J Gastrointestinal AAPOAII and systemic AA-amyloidosis in aged C57BL/Ka mice. Amyloid-type dependent effect of long-term immunosuppressive treatment.Virchows Arch B Cell Pathol Incl Mol Pathol. 1993; 64: 37-43Crossref PubMed Scopus (15) Google Scholar AApoAII amyloidosis was observed in the ileum, lung, and heart of CD-1 Swiss mice,12Gruys E Tooten PJC Kuijpers MHM Swiss mice used for toxicity studies. Pulmonary amyloid indicates AApoAII.Lab Anim. 1996; 30: 28-34Crossref PubMed Scopus (20) Google Scholar and amyloid deposition was also seen in the renal glomeruli of NSY mice.13Shimizu K Morita H Niwa T Maeda K Shibata M Higuchi K Takeda T Spontaneous amyloidosis in senile NSY mice.Acta Pathol Jpn. 1993; 43: 215-221PubMed Google Scholar A better understanding of the mechanisms linking these unique tissue distribution patterns and the different degrees of deposition severity with the primary structures of ApoA-II proteins may shed light on amyloid fibrillogenesis.Table 1Mice Strains and Amyloid Proteins Used in this InvestigationMouse strainApoA-II alleleApoA-II proteinAmino acid substitutionAmyloid fibrilsAmyloidosisBDF1, C57BL/6J, DBA/2Apoa2aAPOAIIA5 Pro, 26 Val, 38 ValAApoAII(A)MildR1.P1-ApoA2c, SAMP1Apoa2cAPOAIIC5 Gln, 26 Ala, 38 MetAApoAII(C)SevereB6RCF1Apoa2a/cAPOAIIA/APOAIICAApoAII(A) Open table in a new tab Prion, an abnormal form (PrPSC) of the host cellular prion protein (PrPC), is responsible for transmissible spongiform encephalopathies (TSE), which include scrapie in sheep, bovine spongiform encephalopathy, and human Creutzfeldt-Jakob disease.14Prusiner SB Prion diseases and the BSE crisis.Science. 1991; 252: 1515-1522Crossref PubMed Scopus (1749) Google Scholar, 15Pan KM Baldwin M Nguyen J Gasset M Serban A Groth D Mehlhorn I Huang Z Fletterick RJ Cohen FE Prusiner SB Conversion of alpha-helices into β-sheets features in the formation of the scrapie prion proteins.Proc Natl Acad Sci USA. 1993; 90: 10962-10966Crossref PubMed Scopus (2083) Google Scholar In TSE, the prion causes a conformational change of PrPC to PrPSc and a detectable phenotype or disease in the affected individual. Recent studies with yeast have broadened the definition of prion from a proteinaceous infectious agent of TSE to a set of self-propagative proteins or protein-based genetic elements.16Wickner RB Edskes HK Maddelein ML Taylor KL Moriyama H Prions of yeast and fungi. Proteins as genetic material.J Biol Chem. 1999; 274: 555-558Crossref PubMed Scopus (66) Google Scholar We described the prion-like transmission of AApoAII amyloidosis in reports of our previous studies. In the first of these, intravenous injection of AApoAII(C) fibrils markedly accelerated amyloid deposition in young R1.P1-Apoa2C mice.17Higuchi K Kogishi K Wang J Chen X Chiba T Matsushita T Hoshii Y Kawano H Ishihara T Yokota T Hosokawa M Fibrilization in mouse senile amyloidosis is fibril conformation-dependent.Lab Invest. 1998; 78: 1535-1542PubMed Google Scholar In the second study, an in vitro experiment demonstrated that the extension of AApoAII(C) proceeds as a result of the linking of soluble APOAIIC to the ends of existing fibrils.18Naiki H Higuchi K Nakakuki K Takeda T Kinetic analysis of amyloid fibril polymerization in vitro.Lab Invest. 1991; 65: 104-110PubMed Google Scholar In the third study, we fed young R1.P1-Apoa2C mice with AApoAII(C) fibrils, or reared the young mice in the same cage with old R1.P1-Apoa2C mice that had severe amyloid deposits. All of these mice developed amyloid deposits.19Xing Y Nakamura A Chiba T Kogishi K Matsushita T Li F Guo Z Hosokawa M Mori M Higuchi K Transmission of mouse senile amyloidosis.Lab Invest. 2001; 81: 493-499Crossref PubMed Scopus (92) Google Scholar Lastly, we found that AApoAII(C) induced a conformational change in the less-amyloidogenic APOAIIB to a different amyloid fibril structure, and that this protein could also induce amyloidosis in the less-amyloidogenic SAMR1 strain.20Xing Y Nakamura A Korenaga T Guo Z Yao J Fu X Matsushita T Kogishi K Hosokawa M Kametani F Mori M Higuchi K Induction of protein conformational change in mouse senile amyloidosis.J Biol Chem. 2002; 277: 33164-33169Crossref PubMed Scopus (49) Google Scholar In the study presented here we detected early onset and heavy AApoAII(A) amyloid deposition in BDF1 and C57BL/6 mice, and were able to isolate enough amyloid fibrils for biochemical and morphological characterization, as well as for studies on transmission of amyloidosis. The AApoAII(A) fibrils proved to have unique features and provided new insights into the process of amyloidogenesis. BDF1 are hybrid mice obtained by breeding female C57BL/6CrSlc (C57BL/6) mice with male DBA/2CrSlc (DBA/2) mice. BDF1 and C57BL/6 mice were purchased from Japan SLC, Inc. (Hamamatsu, Japan) and kept at the Tokyo Metropolitan Institute of Gerontology. They were raised under specific pathogen-free conditions at 24°C with a light-controlled regimen (12 hours light/dark cycle). A commercial diet (CRF-1; Oriental Yeast Co., Ltd., Tokyo, Japan) and sterile water were available ad libitum. BDF1 mice at the ages of 4, 9, 14, 19, 25, and 31 months and C57BL/6 mice at 12 and 24 months of age were killed by cardiac puncture under diethyl ether anesthesia. Tissues from the whole body were fixed in 10% neutral buffered formalin, embedded in paraffin, and cut into 4-μm sections for hematoxylin and eosin, Congo-red, and immunohistochemical staining.7Higuchi K Matsumura A Honma A Takeshita S Hashimoto K Hosokawa M Yasuhira K Takeda T Systemic senile amyloid in senescence-accelerated mice. A unique fibril protein demonstrated in tissues from various organs by the unlabeled immunoperoxidase method.Lab Invest. 1983; 48: 231-240PubMed Google Scholar The intestines and livers were stored at −70°C for biochemical analysis. Animal studies were conducted in accordance with guidelines for the use of laboratory animals of the Tokyo Metropolitan Institute of Gerontology. AApoAII amyloidosis was induced in 2-month-old female R1.P1-Apoa2c, C57BL/6, DBA/2, and B6RCF1 mice by injection of amyloid fibrils (Table 1). A congenic strain, R1.P1-Apoa2c, features the amyloidogenic ApoA-II gene (Apoa2c) of the SAMP1 strain on the genetic background of the less-amyloidogenic SAMR1 strain.21Higuchi K Naiki H Kitagawa K Kitado H Kogishi K Matsusita T Takeda T Apolipoprotein A-II gene and development of amyloidosis and senescence in a congenic strain of mice carrying amyloidogenic ApoA-II.Lab Invest. 1995; 72: 75-82PubMed Google Scholar R1.P1-Apoa2c mice were maintained through sister-brother mating at the Division of Laboratory Animal Research, Research Center for Human and Environmental Science, Shinshu University. F1 hybrid mice B6RCF1 were obtained by breeding female C57BL/6 mice with male R1.P1-Apoa2c mice. AApoAII(A) amyloid fibrils were isolated from the BDF1 intestines and AApoAII(C) fibrils were obtained from the livers of aged R1.P1-Apoa2c mice. These fibrils were then suspended in pure water and samples (0.1 mg), prepared freshly without freezing, were injected intravenously after sonication.17Higuchi K Kogishi K Wang J Chen X Chiba T Matsushita T Hoshii Y Kawano H Ishihara T Yokota T Hosokawa M Fibrilization in mouse senile amyloidosis is fibril conformation-dependent.Lab Invest. 1998; 78: 1535-1542PubMed Google Scholar Mice strains and injected amyloid fibrils used in this study are summarized in Table 1. The mice were reared under specific pathogen-free conditions at 24°C with a light controlled regimen (12 hours light/dark cycle). A commercial diet (MF; Oriental Yeast, Co., Ltd.) and tap water were available ad libitum. Animal studies were conducted in accordance with the guidelines for the use of laboratory animals of Shinshu University School of Medicine. Three and 6 months after injection of amyloid fibrils, the mice were killed as described above. Depositions of amyloid fibrils were identified by their green birefringence in Congo Red-stained sections, visible with polarizing microscopy. The amyloid fibril proteins, AApoAII and AA, were identified immunohistochemically with the avidin-biotinylated horseradish peroxidase complex method using specific antiserum against murine AApoAII and AA.7Higuchi K Matsumura A Honma A Takeshita S Hashimoto K Hosokawa M Yasuhira K Takeda T Systemic senile amyloid in senescence-accelerated mice. A unique fibril protein demonstrated in tissues from various organs by the unlabeled immunoperoxidase method.Lab Invest. 1983; 48: 231-240PubMed Google Scholar SAP and ApoE were also identified immunohistochemically using specific antiserum against murine SAP (Calbiochem-Novabiochem Co., San Diego, CA) and ApoE (CorTex Biochem, Inc., San Leandro, CA). The intensities of the AApoAII amyloid depositions were determined semiquantitatively according to the amyloid index (AI). The AI represented the mean value of the amyloid deposition scores, graded from 0 to 4, for the seven major tissues (liver, spleen, tongue, heart, intestine, stomach, and skin) that were stained with Congo Red, as described previously.19Xing Y Nakamura A Chiba T Kogishi K Matsushita T Li F Guo Z Hosokawa M Mori M Higuchi K Transmission of mouse senile amyloidosis.Lab Invest. 2001; 81: 493-499Crossref PubMed Scopus (92) Google Scholar, 20Xing Y Nakamura A Korenaga T Guo Z Yao J Fu X Matsushita T Kogishi K Hosokawa M Kametani F Mori M Higuchi K Induction of protein conformational change in mouse senile amyloidosis.J Biol Chem. 2002; 277: 33164-33169Crossref PubMed Scopus (49) Google Scholar Tissues were examined by two independent observers who were blinded to the experimental protocol. The method for in situ electron microscopic observations of amyloid fibrils has been described previously.22Inoue S Kuroiwa M Tan R Kisilevsky RA A high resolution ultrastructural comparison of isolated and in situ murine AA amyloid fibrils.Amyloid. 1998; 5: 99-110Crossref PubMed Scopus (28) Google Scholar Small blocks (1 mm3) of the intestine and tongue from aged BDF1 mice (29 months) were fixed with 3% glutaraldehyde in 0.1 mol/L sodium cacodylate (pH 7.4) for 3 hours at room temperature and postfixed with osmium tetraoxide in the same buffer for 1.5 hours at 4°C. En block staining was performed with 1.0% uranyl acetate in 70% ethanol for 40 minutes at 4°C. Thin sections of tissues embedded in Epon were placed on a copper grid and examined at an accelerating voltage of 70 kV under a Hitachi H-7000 electron microscope (Hitachi Ltd., Tokyo, Japan). The amyloid fibril fraction was isolated as a suspension in water from the livers and intestines of 20-month-old R1.P1-Apoa2c mice and from the intestine, lungs, tongue, and stomach of 31-month-old BDF1 mice.19Xing Y Nakamura A Chiba T Kogishi K Matsushita T Li F Guo Z Hosokawa M Mori M Higuchi K Transmission of mouse senile amyloidosis.Lab Invest. 2001; 81: 493-499Crossref PubMed Scopus (92) Google Scholar These fibrils were then used for biochemical and morphological analyses. AA fibrils were extracted from the livers of 24-month-old C57BL/6 mice with severe inflammation. Tris-Tricine/sodium dodecyl sulfate-polyacrylamide gel electrophoresis was performed on 16.5% acrylamide gels.23Schagger H 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 (10503) Google Scholar Proteins were visualized by means of Coomassie Brilliant Blue R250. The duplicated part of the gel was transferred electrophoretically to a polyvinylidene difluoride membrane (Bio-Rad Laboratories, Hercules, CA). Proteins that reacted with anti-AApoAII and anti-AA antibody were visualized by using biotinylated pig anti-rabbit immunoglobulin, avidin combined with horseradish peroxidase, and 3, 3′-diaminobenzidine.24Fu L Matsuyama I Chiba T Xing Y Korenaga T Guo Z Nakayama J Mori M Higuchi K Extrahepatic expression of apolipoprotein A-II in mouse tissues: possible contribution to mouse senile amyloidosis.J Histochem Cytochem. 2001; 49: 739-748Crossref PubMed Scopus (15) Google Scholar A suspension of AApoAII(A), AApoAII(C), and AA amyloid fibrils (0.1 to 1.0 mg/ml) in 5 μl of water, stained negatively with 5 μl of 2% sodium phosphotungstate (pH 7.2), was spread on a carbon-stabilized Formvar-membrane (Okenshoji Co., Ltd., Tokyo, Japan).25Naiki H Higuchi K Hosokawa M Takeda T Fluorometric determination of amyloid fibrils in vitro using the fluorescent dye thioflavin T.Anal Biochem. 1989; 177: 244-249Crossref PubMed Scopus (1003) Google Scholar These samples were then examined under a JEM1200 transmission electron microscope (JEOL Ltd., Tokyo, Japan) with acceleration at 80 kV. For AFM observations an SPI-3800N with DF40 cantilevers (Seiko Instruments, Chiba, Japan) and a spring constant of 40 N/m was used. The 5-μl water suspensions of amyloid fibrils (0.3 mg/ml) were spread on the surface of freshly cleaved mica and excess water was removed with a gentle stream of air. An RF-1500 spectrofluorometer (Shimazu Corp., Kyoto, Japan) was used for fluorescence spectroscopy at room temperature. In all experiments, the size of the reaction mixture was 1.0 ml and the reactions were performed in polypropylene tubes (1.5 ml) at room temperature. The incubation mixture contained 50 mmol/L glycine-NaOH buffer (pH 9.0), 5 μg of amyloid fibrils, and various concentrations of thioflavine T (ThT) (Wako Pure Chemical Industries, Ltd., Osaka, Japan). Five-μl aliquots from each of the reaction tubes were used for fluorescence spectroscopy. In all studies, emission spectra at 482 nm were obtained.25Naiki H Higuchi K Hosokawa M Takeda T Fluorometric determination of amyloid fibrils in vitro using the fluorescent dye thioflavin T.Anal Biochem. 1989; 177: 244-249Crossref PubMed Scopus (1003) Google Scholar Both APOAIIA and APOAIIC monomers were obtained from amyloid fibrils by means of six molar urea polyacrylamide gel electrophoreses.18Naiki H Higuchi K Nakakuki K Takeda T Kinetic analysis of amyloid fibril polymerization in vitro.Lab Invest. 1991; 65: 104-110PubMed Google Scholar Each type of monomer (20 μg) was dissolved in phosphate buffer (50 mmol/L, pH 7.5). The circular dichroism (CD) spectrum of each protein solution was recorded on a Jasco 725 spectrometer (JASCO Corp., Tokyo, Japan) at 25°C, and at 0.2-nm intervals between wavelengths of 194 and 250 nm. Reaction mixtures (40 μl) contained 25 mmol/L phosphate buffer at pH 7.5, 100 μmol/L (35 μg) APOAIIA or APOAIIC monomer purified from high-density lipoprotein of C57BL/6 and R1.P1-Apoa2c mice,5Higuchi K Hosokawa M Takeda T Senescence-accelerated mouse.Methods Enzymol. 1999; 309: 674-686Crossref PubMed Scopus (22) Google Scholar 3 μg of sonicated AApoAII(A) or AApoAII(C) fibrils, and 300 μmol/L urea. For the inhibition analysis, various amounts of APOAIIA monomers were added to the reaction mixtures, which were prepared at 4°C. Reactions were performed in tubes at 37°C three times. Five-μl aliquots from each reaction tube were used for fluorescence spectroscopy.25Naiki H Higuchi K Hosokawa M Takeda T Fluorometric determination of amyloid fibrils in vitro using the fluorescent dye thioflavin T.Anal Biochem. 1989; 177: 244-249Crossref PubMed Scopus (1003) Google Scholar Significant differences in the values of AIs among the various groups of mice were examined using the Mann-Whitney U-test. Sequence analysis of ApoA-II cDNA revealed that both BDF1 and C57BL/6 have the Apoa2a allele of the ApoA-II gene (data not shown). The intensity of amyloid deposition in BDF1 mice increased with age (Figure 1), while no amyloid deposits were seen in young mice (4 months of age), and only slight depositions in the tongue, intestine, and stomach were seen in mice at the age of 9 months. In the aged mice (25- and 31-month-old), however, the grade of amyloid deposition in the intestine, tongue, stomach, skin, and heart was nearly 4, which means corresponding to severe and systemic deposition, although no deposits were observed in the livers and spleens of these mice. The amyloid deposit stained positively with anti-AApoAII antiserum and negatively with the anti-AA antiserum (Figure 2; A to C). The villi in the intestines of aged BDF1 mice were filled with amyloid deposits, whereas other tissues such as tongue, heart, and stomach also contained severe AApoAII deposition (Figure 2; D to F). The amyloid deposit in the villi and other tissues of aged BDF1 stained positively with antiserum against mouse SAP and ApoE (Figure 2; G to I). In situ TEM showed amyloid fibril-like components ∼8 nm wide in the intestines and tongues of aged BDF1 mice (Figure 3).Figure 3In situ TEM of amyloid deposition in aged BDF1 mice. AApoAII(A) amyloid fibrils were detected in the intestine (A) and tongue (B) of an aged (29 months) BDF1 mouse. Gently curved, ∼8-nm-wide entities were observed. Scale bar, 100 nm.View Large Image Figure ViewerDownload Hi-res image Download (PPT) The degrees of amyloid deposition in the C57BL/6 mice at the ages of 12 and 24 months are summarized in Figure 1. Aged C57BL/6 (24 months) mice also showed severe amyloid deposition in the intestine, stomach, and tongue. In two 24-month-old C57BL/6 mice, AA fibrils were deposited in the spleen and liver (data not shown). AA fibrils were extracted from all of these tissues. Amyloid fibril fractions were extracted from various tissues of aged BDF1 and R1.P1-Apoa2c mice and analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. A dominant single band having a molecular weight of ∼8.6 kd was detected (Figure 4A), which was consistent with the size of the ApoA-II monomer. Additional bands in the molecular weight range 10 ∼ 18 kd were observed in the lung fraction. Western blotting of the amyloid fibril fractions extracted from the intestines of the BDF1 mice with anti-AApoAII or anti-mouse AA antibody produced two bands reacting with the anti-AApoAII antibody (Figure 4B). The same results were obtained for the amyloid fractions from the other tissues (data not shown). These bands indicated the presence of the ApoA-II monomer (molecular weight, ∼8.6 kd) and dimer (molecular weight, ∼18 kd). No specific band was stained with the anti-mouse AA antibody. The amyloid fraction from the intestine of the BDF1 mice was not stained with anti-ApoE or anti-transthyretin antiserum (Santa Cruz Biotechnology, Inc., Santa Cruz, CA) (data not shown). TEM images of AApoAII(C) fibrils showed the typical helical structure of amyloid fibrils (Figure 5A), which were 10 nm in diameter, rigid, and nonbranching. Mouse AA also showed typical amyloid fibrils that were nonbranched, 8-nm-wide fibril structures (Figure 5B). On the other hand, typical fibril images were rarely obtained in the case of AApoAII(A) fibrils, but fibrils with a spherical shape (Figure 5C) and short fibril-like materials with a width of ∼5 nm were observed (Figure 5D). AFM images of amyloid fibrils showed typical amyloid fibrils of AApoAII(C) (height up to 10 nm) and mouse AA (height up to 8 nm) (Figure 5, E and F), whereas fine and small fibrils (height up to 2 nm) and very small amounts of larger and longer fibrils (height up to 7 nm) were found in the amyloid fibril fractions from BDF1 mice (Figure 5, G and H). Morphological differences observed among AApoAII(A), AApoAII(C), and AA fibrils suggested that the biochemical properties of these amyloid fibrils may be different. ThT binds to amyloid fibrils and emits fluorescence at a specific wavelength. The binding constant Kb was defined as the concentration of ThT that produced half of the maximum emission intensity at 482 nm. AApoAII(C) from the R1.P1-Apoa2c liver indicated a strong affinity for ThT (kb = 106 nmol/L) (Figure 6), as did AApoAII(C) from the R1.P1-Apoa2c intestine (kb = 35 nmol/L). On the other hand, AApoAII(A) from the BDF1 intestine showed a much weaker affinity (kb = 4.2 μmol/L). AA fibrils were characterized by a typical amyloid fibril structure, but the affinity was much weaker (kb = 5.6 μmol/L) than that of AApoAII(C). The affinity of Congo Red for AApoAII(A) fibrils in vitro was weaker than that for AApoAII(C) fibrils (data not shown). To evaluate the conformational differences between the two types of ApoA-II monomers, a CD analysis was performed using ApoA-II monomers at neutral pH. These monomers had been used for polymerization reactions of amyloid fibrils in vitro (Figure 7). In the case of the APOAIIA monomer, the spectrum exhibited only a negative ellipticity with a negative peak at 201 nm. This indicates that this protein has a nonhelical, non-β-strand secondary structure. On the other hand, the spectrum of APOAIIC exhibited a trough at a higher wavelength, demonstrating that APOAIIC has a more extensive β-sheet structure than does APOAIIA. To examine fibril formation in vitro, AApoAII(C) and AApoAII(A) fibrils and 100 μmol/L of two types of ApoA-II monomers were incubated at 37°C in phosphate buffer (pH 7.5). AApoAII(C) fibrils act as seeds for AAPOAIIC monomers, resulting in extension of the amyloid fibrils (Figure 8A). However, fibril extension in vitro was not detected in the reaction of APOAIIA monomer with AApoAII(A) fibrils (data not shown). Fibril extension of AApoAII(C) in reaction with APOAIIC was inhibited by the APOAIIA monomer. AApoAII(C) fibrils and 100 μmol/L APOAIIC monomer were incubated with various concentrations of the APOAIIA monomer. Fibril formation was significantly reduced with an increase in APOAIIA monomer concentration (Figure 8B). Equivalent amounts of APOAIIA (100 μmol/L) inhibited fibril formation by 80%. Sonicated AApoAII(A) or AApoAII(C) amyloid fibrils (100 μg) were injected intravenously into 2-month-old R1.P1-Apoa2c,
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