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Published as doi: 10.1096/fj.05-4890fje.
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(The FASEB Journal. 2006;20:1012-1014.)
© 2006 FASEB

Transmissibility of mouse AApoAII amyloid fibrils: inactivation by physical and chemical methods

Huanyu Zhang, Jinko Sawashita, Xiaoying Fu, Tatsumi Korenaga, Jingmin Yan, Masayuki Mori and Keiichi Higuchi1

Department of Aging Biology, Institute on Aging and Adaptation, Shinshu University Graduate School of Medicine, Matsumoto, Japan

1Correspondence: Department of Aging Biology, Institute on Aging and Adaptation, Shinshu University Graduate School of Medicine, Asahi 3–1-1, Matsumoto 390-8621, Japan. E-mail: khiguchi{at}sch.md.shinshu-u.ac.jp

ABSTRACT

AApoAII amyloid fibrils have exhibited prion-like transmissibility in mouse senile amyloidosis. We have demonstrated that AApoAII is extremely active and can induce amyloidosis following doses less than 1 pg. We tested physical and chemical methods to disrupt AApoAII fibrils in vitro as determined by thioflavin T binding and electron microscopy (EM) as well as inactivating the transmissibility of AApoAII fibrils in vivo. Complete disruption of AApoAII fibrils was achieved by treatment with formic acid, 6 M guanidine hydrochloride, and autoclaving in an alkaline solution. Injection of these disrupted AApoAII fibrils did not induce amyloidosis in mice. Disaggregation with 6 M urea, autoclaving, and alkaline solution was incomplete, and injection of these AApoAII fibrils induced mild amyloidosis. Treatment with formalin, delipidation, freeze-thaw, and RNase did not have any major effect. A distinct correlation was obtained between the amounts of amyloid fibrils and the transmissibility of amyloid fibrils, thereby indicating the essential role of fibril conformation for transmission of amyloidosis. We also studied the inactivation of AApoAII fibrils by several organic compounds in vitro and in vivo. AApoAII amyloidosis provides a valuable system for studying factors that may prevent transmission of amyloid disease as well as potential novel therapies.


Key Words: amyloidosis • disruption • organic compounds

AMYLOIDOSIS REFERS TO a group of protein-folding diseases (1) . Currently, more than 25 amyloid diseases have been identified (2) . Various innocuous and soluble proteins polymerize to insoluble amyloid fibrils in several serious human diseases, such as Alzheimer’s disease, type II diabetes, familial amyloid polyneuropathy, and prion diseases (3) . Although the diverse amyloid proteins have unrelated sequences, drastic structural changes of the amyloid proteins from the normal form to the unique ß-sheet rich fibril is the most important event in amyloid diseases (4) . Systemic AApoAII amyloidosis was first identified in mice (5) and was found in humans with the kidneys being predominantly affected (6) . ApoA-II is the second most abundant apolipoprotein in plasma high-density lipoprotein. In mice, apoA-II polymerization to amyloid fibrils (AApoAII), with subsequent deposition in the whole body except the brain, was found to be universally present in aged mice of various strains (7) . Three variants of apoA-II (types A, B, and C) with different amino acid substitutions at four positions are present in inbred strains of mice. Senescence-accelerated prone (SAMP1) mice have a C-type apoA-II (APOAIIC, Gln5, and Ala38) and spontaneously exhibit a high incidence of AApoAII amyloidosis with aging. Senescence-accelerated resistant (SAMR1) mice with wild-type B apoA-II (APOAIIB, Pro5, and Val38) exhibit few, if any, signs of AApoAII amyloidosis. R1.P1-Apoa2c mice are a congenic strain with the amyloidogenic c allele of the apoA-II gene from the SAMP1 strain on the genetic background of the SAMR1 strain (8) . This congenic strain spontaneously exhibits a high incidence of amyloidosis and severe amyloid deposition with aging (9) .

Nucleation-dependent polymerization is postulated to be a model that explains well the kinetics of fibrilization of amyloid proteins in Alzheimer’s disease, scrapie, and mouse AApoAII amyloidosis (10 11 12) . Among them, prion diseases such as transmissible spongiform encephalopathy (TSE), including scrapie in sheep, bovine spongiform encephalopathy (BSE), and human Creutzfeldt-Jakob disease (13) , are highly infectious. In TSE, prion (PrPSc) is an abnormal form of the host cellular prion protein (PrPC) and induces the conformational change of PrPC to PrPSc, resulting in a detectable phenotype or disease in the affected individual. Recent studies with yeast prion proteins have broadened the definition of prion from the proteinaceous infectious agent of TSE to infectious protein-based genetic elements (14) . Various forms of amyloidosis, except for prion disease, had been thought to be nontransmissible (15) , with a clear boundary existing between transmissible prion disease and general amyloidosis.

However, we have described the prion-like transmission of AApoAII amyloidosis in our previous work. Intravenous (i.v.) injection of AApoAII(C) fibrils markedly accelerated amyloid deposition in young R1.P1-Apoa2c mice (12) . The transmission of amyloid fibrils via the gastrointestinal tract and propagation from mice exhibiting severe amyloid deposition to amyloid negative mice were suggested (16 , 17) . Furthermore, prion-like transmission has also been revealed in studies of inflammation-associated experimental (AA) amyloidosis (18 , 19) . Intracerebral injection of brain homogenate from a patient with Alzheimer’s disease into marmoset monkeys induced the formation of amyloid ß plaques in these primates (20) . These results suggested that amyloid diseases could be transmitted like prion disease under certain conditions (21) . Thus, the inhibition of transmission of amyloid diseases is an important issue for understanding and preventing amyloidosis.

Because nucleation-dependent polymerization is evident in the kinetics of fibrilization of several kinds of amyloid proteins, seeding by amyloid fibrils was thought to be a key factor for the transmission of amyloidosis. Thus, the disruption of amyloid fibrils would be predicted to significantly inhibit transmission. The inhibition of transmission in prion diseases by inactivation of infectious materials using physical and chemical methods has been extensively investigated (22 , 23) and has provided insight into methods to control infection. In this study, we investigated physical and chemical methods to disrupt the structure of the amyloid fibrils in order to inhibit the transmission of AApoAII amyloidosis in a murine model. Meanwhile, the ability of many organic compounds to inactivate several kinds of amyloid fibrils has recently been investigated (24 25 26) . For example, tetracycline altered the chemico-physical properties of PrPSc from variant Creutzfeldt-Jakob disease (vCJD) and BSE and reduced prion infectivity (27) . Rifampicin inhibited the aggregation and neurotoxicity of Aß protein (28) . Nordihydroguaiaretic acid (NDGA) potently degraded preformed Aß amyloid fibrils in vitro (29) . In this work we have used several antibiotics and polyphenols to alter the conformation of AApoAII fibrils and prevent transmission in the R1.P1-Apoa2c model.

Our results suggested that the specific fibril structure might be a prerequisite for the transmissibility of amyloid fibrils.

MATERIALS AND METHODS

Animals
R1.P1-Apoa2c mice were raised in the Division of Laboratory Animal Research, Department of Life Science, Research Center for Human and Environmental Sciences, Shinshu University, Matsumoto, Japan. Mice were maintained under specific pathogen-free conditions at 24 ± 2°C with a light-controlled regimen (12 h light/dark cycles). A commercial diet (MF; Oriental Yeast, Tokyo, Japan) and tap water were available ad libitum. Mice were sacrificed by cardiac puncture under diethyl ether anesthesia. All experiments were performed with the consent of the Animal Care and Use Committee of Shinshu University School of Medicine.

Isolation of amyloid fibrils
AApoAII fibrils were isolated as described previously (30) from the liver of an 8-month-old mouse, which had severe amyloid deposition induced by an i.v. injection of AApoAII fibrils. The amyloid-laden liver (2.0 g) was homogenized in 20 ml of ice-cold 0.15 M NaCl with an ultradispenser for 30 s at 30-s intervals 3x. The mixture was centrifuged at 4 x 104 g for 20 min at 4°C, after which the supernatant was discarded. These operations were repeated 10–14x before the supernatant had an optical absorbance <0.3 at 280 nM. The pellet of the final centrifugation was rinsed with 20 ml of distilled water (DW) by 30 s of homogenization and was centrifuged at 4 x 104 g for 20 min after which the supernatant was discarded. The amyloid fibril fraction was extracted in 20 ml of DW by homogenization 30 s and centrifuged at 3 x 104 g for 20 min at 4°C. This extraction procedure was repeated 3–6x. Isolated amyloid fibrils in the supernatant were further purified as the pellet by ultracentrifugation at 105 g for 1 h at 4°C.

Isolated amyloid fibrils were resuspended in DW at a concentration of 1.0 mg/ml. We placed 1 ml of this mixture into a 1.5 ml Eppendorf tube and sonicated on ice for 30 s with an ultrasonic homogenizer VP-5S (Tietech Co., Ltd., Tokyo, Japan) at maximum power. This procedure was repeated 5x at 30-s intervals. Sonicated AApoAII samples were used immediately.

Detection of amyloid deposition
Each organ of the whole body was fixed in 10% neutral buffered formalin, embedded in paraffin, and cut into serial 4 µm sections. Amyloid deposition was identified by green birefringence in tissue sections stained with Congo red and examined under polarizing microscopy. Amyloid deposition was also immunohistochemically stained by the avidin-biotinylated horseradish peroxidase complex method using antiapoA-II antiserum as the primary antibody. The intensity of the AApoAII deposition was determined semiquantitatively using the amyloid index (AI). The AI is the average of the degree of amyloid deposition graded from 0 to 4 in the 7 organs examined (liver, spleen, skin, heart, stomach, small intestine, and tongue) in sections stained with Congo red, as described previously (12 , 31) . Two blinded observers, who had no information regarding the tissue, graded and averaged the AI independently for each mouse.

Detection of AApoAII fibrils in vitro
The amounts of AApoAII fibrils were assayed by the fluorometric method (32) with thioflavin T (ThT) and a spectrofluorophotometer RF-5300PC (Shimadzu Co., Kyoto, Japan). The assay vol was 1.0 ml, with excitation at 450 nm and emission at 482 nm. The excitation and emission slits were set at 5 and 10 nm, respectively. The reaction mixture contained 250 nM ThT and 50 mM glycine-NaOH buffer, pH 9.0.

Western blotting analysis of AApoAII fibrils
We boiled 1µg of AApoAII samples in sample buffer (2% SDS, 12% glycerol, 62.5 mM Tris pH 6.8, 10% 2-mercaptoethanol, 0.02% bromphenol blue) and separated by 16.5% Tris-tricine SDS-PAGE (PAGE). In addition, AApoAII samples in another sample buffer (12% glycerol, 62.5 mM Tris pH 6.8, 0.02% bromphenol blue) were separated by PAGE on 5–15% Tris acetate gels in 0.01% SDS, 25 mM Tris pH 8.3, and 192 mM glycine. Proteins in the gels were transferred electrophoretically to polyvinylidine difluoride membranes. Proteins on the membranes were reacted with rabbit antimouse apoA-II antiserum (1:3000), followed by peroxidase-conjugated goat IgG against rabbit immunoglobulin (1:1000). Immunoreactive proteins were visualized with enhanced chemiluminescence reagents (Amersham Bioscience, Buckinghamshire, UK).

In vivo assay of transmission activity of AApoAII fibrils
The transmissibility of AApoAII fibrils was determined in vivo. We prepared a series of concentrations to test the transmission activity of AApoAII fibrils from doses of 10–10 µg to 100 µg. Mice were divided into 13 experimental groups with 4–6 animals in each group. Each mouse received a single i.v. injection of 0.1 ml of AApoAII suspended in DW. Control animals were injected with 0.1 ml of DW. We also analyzed the transmission activity of AApoAII fibrils by oral and intraperitoneal (i.p.) injection. All animals were sacrificed 2 mo after the injection. The intensity of amyloid deposition was determined using the AI.

Disruption and inactivation of AApoAII fibrils by physical and chemical treatments
AApoAII fibrils (5 ng/µl) were treated with 88% formic acid (8 h), 6 M guanidine hydrochloride (24 h), 6 M urea (144 h), 2 N NaOH (1 h), 10% formalin (24 h), or RNase (72 h) (Funakoshi Co., Tokyo, Japan) at room temperature. AApoAII fibrils were autoclaved (121°C) for 1, 2, or 3 h or exposed to 1 N NaOH for 0.5 h. We also determined the effect of delipidation with diethyl ether and ethanol or freeze-thaw (5 cycles), repeated freeze at –70°C for 1 h and thaw at room temperature for 1 h. The disruption of AApoAII fibrils was determined with the ThT binding assay, EM, and Western blotting analysis.

Treated AApoAII fibrils (1 and 100 µg) were injected i.v. into mice. Formic acid, guanidine hydrochloride and urea were removed from the treated AApoAII fibrils by dialyzing against DW for 6 h prior to injection. AApoAII fibrils treated with formalin, delipidation, and RNase were washed 3 times with DW after centrifugation of amyloid fibrils (1 h at 105 g). AApoAII fibrils treated with NaOH was neutralized with HCl and dialyzed against DW prior to injection. AApoAII fibrils treated with autoclaving and freeze-thaw were directly injected into mice. The control mice were injected with 1 µg of AApoAII fibrils alone. After 2 mo, the mice were sacrificed to determine the intensity of amyloid deposition.

The proteinase K treatment of AApoAII fibrils
To investigate the effect of proteinase K (37 U/mg, Sigma Aldrich, St. Louis, MD) digestion on AApoAII fibrils, two experiments were performed. First, 5 ng/µl AApoAII fibrils were incubated with 0, 0.004, 0.02, 0.1, 0.5, and 2.5 ng/µl proteinase K at 37°C for 1 h and then subjected to ThT fluorescence spectroscopy. Second, AApoAII fibrils with or without denaturation in 100% dimethyl sulfoxide (DMSO) for 24 h at room temperature were incubated with 0, 0.1, and 2.5 ng/µl proteinase K for 1 h at 37°C. Digested AApoAII fibrils were separated by 16.5% Tris-tricine SDS-PAGE and detected as described previously.

Disaggregation and inactivation of AApoAII by organic compounds
AApoAII fibrils were treated with various antibiotics and polyphenols and assayed by the ThT method and EM. The reaction mixtures, containing 5 ng/µl AApoAII fibrils and 0, 100, 500, or 1000 µM organic compounds (the concentration of polymyxin B was 0, 1, 5, or 10 µM) in 50 mM phosphate buffer (pH 7.5) and 100 mM NaCl, were prepared on ice and then incubated at 37°C. Some organic compounds were first dissolved in DMSO and diluted such that the final concentration of DMSO in the reaction mixture was <1%. We determined that 1% DMSO was ineffective for the disaggregation of AApoAII fibrils. At each of the designated incubation times (0 to 144 h), the reaction was stopped by cooling on ice and subjected to the ThT fluorescence assay. We confirmed that these organic compounds did not affect ThT fluorescence at the diluted concentration (data not shown). AApoAII fibrils incubated with organic compounds for 144 h were investigated by EM.

Inhibition of amyloid deposits by organic compounds in vivo
First, 1 µg of AApoAII fibrils was incubated with organic compounds for 144 h and then i.v. injected into mice. The concentration of various organic compounds was 1000 µM (concentration of polymyxin B was 10 µM). The control mice were injected with 1 µg of AApoAII fibrils treated with 50 mM phosphate buffer (pH 7.5) and 100 mM NaCl for 144 h.

Second, 1 µg of AApoAII fibrils was injected i.v. into mice. In addition, NDGA, tetracycline, rifampicin, cephalexin, polymyxin B, or lincomycin was introduced into the stomach of mice by oral gavage while streptomycin or benzylpenicillin was administered by intramuscular (i.m.) injection. The various organic compounds were injected once daily for 6 d. The doses were administered in two ways, i.e., 1 mmol/mouse within 6 d and 33–66 mg/kg/day for various organic compounds. Control mice were injected i.v. with 1 µg of AApoAII fibrils alone. After 2 mo, all mice were sacrificed and the extent of amyloid deposition was determined.

Electron microscopy
We mixed 10 µl of the various treated fibrils solutions (0.4 mg/ml) and 10 µl of 2% phosphotungstic acid (pH 7.0). Half of the carbon-coated plastic grid (Ouken, Tokyo, Japan) was immersed in this mixture for 1 min. The negatively stained samples were observed with a JEOL 1200 EX electron microscope (JEOL, Tokyo, Japan) operated at 80 kV.

Statistic analysis
We used the StatView software package (Abacus Concepts, Berkley, CA) to analyze the data. Because the AI is a nonlinear index, the AI of different groups of mice was compared by using the nonparametric Mann-Whitney U-test.

RESULTS

Induction of amyloidosis by various injection doses and routes of administration of AApoAII fibrils
To test the effect of the AApoAII dose on induction of amyloidosis, the sample was serially diluted in DW and i.v. injected into mice at doses ranging from 10–10 µg to 100 µg. The degree of amyloid deposition increased proportionally to the logarithm of AApoAII dosage from 10–1 µg to 100 µg (Fig. 1 A, B). Slight amyloid deposits were detected in mice injected with doses of AApoAII fibrils between 10–7 and 10–2 µg. Some mice did not develop amyloidosis following doses of AApoAII fibrils less than 10–3 µg while no amyloid deposits were observed in mice injected with doses less than 10–8 µg. None of the control mice had detectable AApoAII deposits. From these results, we decided to inject 1 µg of treated AApoAII fibrils in subsequent experiments quantifying transmissibility.


Figure 1
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  		Figure 1. Induction of amyloid deposition by various injection doses and routes of administration of AApoAII fibrils. A) AApoAII deposition in R1.P1-Apoa2c mice 2 mo after i.v. injection of doses of AApoAII fibrils between 10–10 and 100 µg. Values are means ± SD. B) Amyloid deposition in the tongue of mice was detected by green birefringence in Congo red-stained sections under polarized microscopy (a–e) and immunohistochemical staining (f–j); 1 µg (a and f), 10 µg (b and g), and 100 µg (c and h) AApoAII without treatment and AApoAII (1 µg) treated with formic acid (d and i) and 6 M urea (e and j) were i.v. injected into mice. The grade of amyloid deposition in the tongue is 2, 3, 4, 0, and 1 in a, b, c, d, and e, respectively. Arrows show the AApoAII deposits in tissues. Scale bar equals 100 µm. C) AApoAII amyloid deposition in R1.P1-Apoa2c mice after 2 mo following i.v. ({square}), i.p. ([GRAPHIC]), and oral ({blacksquare}) administration of AApoAII fibrils. Values are means ± SD. Numbers in parentheses represent amyloid positive mice/mice examined.

Intraperitoneal injection of 10 µg AApoAII fibrils induced significant amyloid deposits with the AI being comparable with i.v. injection (Fig. 1C ). However, 10–2 µg AApoAII induced very weak amyloid deposits while 10–5 µg AApoAII did not induce amyloid deposits. Although oral administration of doses of 1, 10, or 100 µg AApoAII induced amyloidosis, the intensity of amyloid deposition was much lower than that induced by i.v. injection. These results indicated that the magnitude of transmission activity of the amyloid fibrils by the different routes of administration was i.v. ≥ i.p. >> oral.

Disruption and inactivation of AApoAII fibrils by physical and chemical methods
Physical and chemical methods were used to disrupt AApoAII fibrils. We observed that all methods could disrupt AApoAII fibrils but the methods exhibited significant variability in the degree of disruption determined by the ThT binding assay. The final fluorescence of AApoAII treated with formalin, RNase, delipidation, freeze-thaw or urea was 79.3, 75.6, 58.3, 49.0, and 20.2% of untreated control AApoAII (Fig 2 A). Meanwhile, the fluorescence of AApoAII treated with autoclaving, 2 N NaOH, guanidine hydrochloride, autoclaving in 1 N NaOH, or formic acid was markedly decreased compared with that of control.


Figure 2
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  		Figure 2. Disaggregation and inactivation of AApoAII fibrils by physical and chemical methods. A) AApoAII fibrils were measured by fluorescence of ThT after treatment with physical and chemical methods. Values represent means ± SD from three determinations. B) The degree of amyloid deposition (AI) in mice induced by amyloid fibrils treated with various physical and chemical methods was determined in vivo. Doses of injected AApoAII fibrils were 1 ({blacksquare}) and 100 ({square}) µg in each treatment. Values are means ± SD. Numbers in parentheses represent amyloid positive mice/mice examined. C) PAGE analysis of the structure of AApoAII fibrils treated with physical and chemical methods. a. AApoAII fibrils treated with physical and chemical methods were analyzed by Western blotting analysis with 16.5% Tris-tricine SDS-PAGE. b. AApoAII fibrils treated with physical and chemical methods were analyzed by Western blotting analysis with 5–15% Tris acetate PAGE. AApoAII oligomers are indicated on the left and MW standards (kDa) are shown on the right. D) AApoAII fibrils treated with various physical and chemical methods were observed by EM. Amyloid fibrils were treated with a. DW, b. 10% formalin, c. delipidation, d. freeze-thaw, e. 6 M urea, f. autoclaving for 3 h, g. 2 N NaOH at room temperature for 1 h, h. 6 M guanidine hydrochloride, i. autoclaving for 0.5 h in 1 N NaOH, and j. 88% formic acid. Arrows, arrowheads, and * indicate intact AApoAII fibrils, swollen small fibrils, and denatured aggregates without a fibril structure, respectively. The scale bars are 50 nm.

We i.v. injected mice with 1 µg of AApoAII fibrils treated with each disruption method and determined the extent of amyloid deposition (Figs. 2B and 1B ). Only a slight decrease in amyloid deposition was observed when AApoAII fibrils were treated with formalin, RNase, or delipidation prior to injection. Injection of AApoAII fibrils treated with freeze-thaw or urea induced much less amyloid deposition compared with control fibrils. The injection of 1 µg of AApoAII fibrils that had been treated with autoclaving, 2 N NaOH, guanidine hydrochloride, autoclaving in 1 N NaOH, or formic acid did not induce amyloid deposition in any mouse. Mice injected with 100 µg of treated fibrils exhibited amyloid deposition unless the fibrils had been treated with autoclaving in 1 N NaOH and formic acid solutions.

We also analyzed the changes in the structure of AApoAII fibrils by Western blotting analysis (Fig. 2C ). SDS-PAGE analysis revealed bands of monomer, dimer and trimer, and smears of larger oligomers in the AApoAII treated with formalin, RNase, delipidation, freeze-thaw, or urea. The smears of oligomers were greatly reduced in AApoAII treated with autoclaving, 2 N NaOH, guanidine hydrochloride, or formic acid; no band in AApoAII degraded by autoclaving in 1 N NaOH. Treated AApoAII fibrils were subjected to PAGE without further denaturation in 5–15% Tris acetate gels. We found monomeric to pentameric and large polymers in AApoAII fibrils treated without or with formalin, RNase, delipidation, or freeze-thaw. The ladders of oligomers larger than pentamer were greatly reduced in AApoAII treated with urea, autoclaving, or 2 N NaOH. AApoAII treated with guanidine hydrochloride or formic acid revealed increasing of monomer to pentamer. AApoAII fibrils were degraded completely by autoclaving in 1 N NaOH.

Analysis of the morphology of AApoAII fibrils using EM (Fig. 2D ) confirmed the results of our in vivo and in vitro experiments. We found abundant and intact amyloid fibrils in the solution of AApoAII treated with formalin, RNase, delipidation, or freeze-thaw. In contrast, we found only small amounts and denatured aggregates of amyloid fibrils in the solution treated with urea, autoclaving or 2 N NaOH, respectively. We could not find any fibrillar structures but did find denatured aggregates without a fibril structure in the solution treated with guanidine hydrochloride or autoclaving in 1 N NaOH or formic acid.

Proteinase K digestion of AApoAII fibrils
We investigated the effect of proteinase K digestion of AApoAII fibrils. AApoAII fibrils were digested with an increasing amount of proteinase K, and the remaining fibril structure was quantified by the ThT method (Fig. 3 A). When the ratio of proteinase K to AApoAII fibrils was 1:50 (enzyme:substrate), only 29.4% of amyloid fibrils was digested. However, over 80% of amyloid fibrils was digested when the ratio of proteinase K to AApoAII fibrils was 1:2. When AApoAII was digested with proteinase K (1:50), we detected a band of intact apoA-II protein monomer by Western blotting analysis (Fig. 3B ). However, analysis of AApoAII denatured with DMSO and digested with proteinase K revealed only few apoA-II monomer and the apoA-II dimer. Incubation of both natural and denatured AApoAII in proteinase K (1:2) resulted in complete digestion of AApoAII.


Figure 3
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  		Figure 3. Digestion of AApoAII fibrils by proteinase K. A) AApoAII fibrils were digested with different dilutions of proteinase K for 1 h at 37°C. The degree of digestion of amyloid fibrils was measured by fluorescence of ThT. Values represent means ± SD from three determinations. B) Western blotting analysis of AApoAII digested by proteinase K. AApoAII fibrils were treated with DW or DMSO overnight. When 20 ng proteinase K was incubated with 1 µg of AApoAII treated with DW (1:50), apoA-II dimer decreased but the monomer did not change. However, when 20 ng proteinase K was incubated with 1 µg of AApoAII treated with DMSO (1:50), both the apoA-II dimer and monomer disappeared. When 500 ng proteinase K was incubated with 1 µg of AApoAII treated with DW or DMSO (1:2), AApoAII was almost completely digested. The amount of AApoAII in each well is 1 µg.

Disaggregation of AApoAII by organic compounds in vitro
We used 12 organic compounds to disrupt AApoAII fibrils in vitro. The organic compounds included NDGA, tetracycline, benzylpenicillin, rifampicin, streptomycin, polymyxin B, cephalexin, lincomycin, chloramphenicol, erythromycin, resveratrol, and quercetin. The ThT fluorescence was almost unchanged during the incubation of AApoAII fibrils at 37°C in the absence of organic compounds. However, the ThT fluorescence decreased after the addition of eight different organic compounds in a concentration-dependent manner. After AApoAII was incubated in 100, 500, and 1000 µM NDGA for 72 h, the fluorescence of AApoAII was reduced to 25.1, 3.1, and 1.5% of the initial fluorescence, respectively (Fig. 4 ). Similarly, the fluorescence of AApoAII incubated with 100, 500, and 1000 µM tetracycline for 144 h was 11.6, 8.9, and 5.4% of the initial fluorescence, respectively. When AApoAII was incubated with 100 µM rifampicin, streptomycin and 1 µM polymyxin B, the fluorescence of ThT at time 0 was already decreased to 69.3, 56.6, and 58.8% of the fluorescence in the absence of organic compounds, respectively. At 144 h, the fluorescence of AApoAII incubated with 1000 µM rifampicin, streptomycin and 10 µM polymyxin B were decreased to 0, 6.6, and 12% of control fluorescence, respectively. When AApoAII was incubated with 1000 µM benzylpenicillin, cephalexin, and lincomycin for 144 h, the decrease in the fluorescence was less marked and the fluorescence of AApoAII was 27.1, 28.9, and 42.1% of the initial fluorescence, respectively. The ThT fluorescence did not change after incubation with the other four organic compounds, including erythromycin, chloramphenicol, quercetin and resveratrol.


Figure 4
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  		Figure 4. Disaggregation of AApoAII fibrils by various organic compounds. Kinetics of disaggregation was measured using the ThT binding assay. AApoAII (5 ng/µl) fibrils were incubated with various organic compounds at the concentration of 0 ({square}), 100 ({blacklozenge}), 500 ({circ}), and 1000 ({blacktriangleup}) µM or 0 ({square}), 1 ({blacklozenge}), 5 ({circ}), and 10 ({blacktriangleup}) µM for polymyxin B. NDGA and tetracycline did not change the fluorescence of ThT at time 0, but the fluorescence decreased rapidly in a dose-dependent manner. The fluorescence of ThT at time 0 was already decreased and then decreased further in the presence of organic compounds, including rifampicin, streptomycin, and polymyxin B. When AApoAII was incubated for 144 h with organic compounds, including benzylpenicillin, cephalexin, and lincomycin, considerable fluorescence of ThT remained. The fluorescence of ThT did not change after treatment with chloramphenicol, erythromycin, resveratrol, and quercetin.

Inactivation of AApoAII and inhibition of amyloid deposition by organic compounds in vivo
To evaluate the effects of organic compounds on the transmissible activity of AApoAII, we injected mice with 1 µg of AApoAII fibrils that had been incubated with organic compounds for 144 h. Because chloramphenicol, erythromycin, resveratrol, and quercetin did not disaggregate AApoAII effectively in vitro, we did not include them in the in vivo studies. In accordance with the in vitro results, eight organic compounds exhibited different extents of inactivation effects on the transmissibility of AApoAII in vivo (Fig. 5 A). Lincomycin did not have a significant inactivation effect, while the other seven organic compounds did exhibit inactivation effects. Streptomycin and polymyxin B showed the strongest inhibition effects with one of three mice injected with AApoAII treated with these compounds exhibiting no evidence of amyloid deposition. We found a significant correlation between the disaggregation activity of the organic compounds in vitro and the inhibition of transmission in vivo (Fig. 5B ). Further, we observed morphological changes in the AApoAII fibrils incubated with organic compounds using EM (Fig. 5C ). A lot of intact AApoAII fibrils were identified in the AApoAII incubated with lincomycin, cephalexin, and benzylpenicillin, but the whole amount of AApoAII fibrils appeared to be decreased in EM compared with control. The amount of AApoAII fibrils was obviously decreased, and swollen small fibrils or denatured aggregates were observed in the solution incubated with rifampicin, NDGA, tetracycline, streptomycin, and polymyxin B. These findings were consistent with the results of in vitro experiments.


Figure 5
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  		Figure 5. Inhibition of transmission of AApoAII fibrils by disaggregation with organic compounds. A) Amyloid deposition was determined in mice injected with AApoAII fibrils that had been incubated with various organic compounds for 144 h. Values represent means ± SD. *P < 0.05. B) The relationship between amyloid index in vivo and fluorescence of ThT in vitro is shown. Symbols represent NDGA ({lozenge}), tetracycline (•), rifampicin ({blacklozenge}), streptomycin ({circ}), polymyxin B ({blacktriangleup}), benzylpenicillin ({triangleup}), cephalexin ({square}), lincomycin ({blacksquare}), and control (⊚). C) The disruption effects of various organic compounds on the structure of AApoAII fibrils were observed using EM. Arrows, arrowheads, and * indicate intact AApoAII fibrils, swollen small fibrils, and denatured aggregates without a fibril structure, respectively. The scale bars were 50 nm.

We further evaluated the inhibitory effects of these organic compounds on amyloid deposition in vivo. AApoAII fibrils (1 µg) were i.v. injected into mice and organic compounds were administered orally or by i.m. injection once a day for 6 d. Six organic compounds (tetracycline, rifampicin, streptomycin, polymyxin B, cephalexin, and NDGA) exhibited a slight but statistically significant effect on the deposition of amyloidosis (Table 1 ). The injection of lincomycin and benzylpenicillin, however, had no significant effect.


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  		Table 1. Prevention effects of organic compounds against the transmission of AApoAII fibrils. AApoAII fibrils (1 µg) were injected into the veins of mice, and organic compounds were injected once a day into the stomach or muscle for 6 days. Mice were sacrificed after 2 months, and amyloid deposition was determined.

DISCUSSION

In this study, we verified the transmissibility of AApoAII fibrils reported in our previous work (12 , 16) . First, we injected R1.P1-Apoa2c mice with the amyloidogenic Apoa2c gene with serially diluted amyloid fibrils and determined amyloid deposition after 2 mo using the AI as a parameter. We could determine semiquantitatively the transmissibility of the fibrils since the degree of amyloid deposition increased proportionally to the logarithm of AApoAII dosage from 10–1 µg to 100 µg (Fig. 1A ). Comparison of the AI in mice receiving i.v. or oral amyloid fibrils indicated that the transmissibility of amyloid fibrils was reduced more than 1000-fold following oral administration. Also, we detected amyloid deposits in mice injected with AApoAII at a dose <1 pg of amyloid fibrils. This result was similar to experimental AA amyloidosis as i.v. injection <1 ng of AA fibrils induced amyloid deposition in mice (19) . In prion disease, wire contaminated with serial dilutions of brain homogenates from hamsters terminally ill with scrapie was implanted into the brains of recipients and a relationship between the infectivity titer (end point at 105.6 LD50) and the transmission rate was established (22) . These results suggested that AApoAII was extremely active and comparable with AA fibrils and prion.

Physical and chemical methods have been used to prevent various transmissible diseases. In our experiments, treatment with 88% formic acid, 6 M guanidine hydrochloride, and autoclaving in 1 N NaOH completely disaggregated or degraded and inactivated the effect of AApoAII fibrils both in vitro and in vivo. Formic acid has been used to dissolve the Aß-peptide and several kinds of amyloid fibrils and prevent aggregation in biological and biophysical studies (33) . Exposing the scrapie amyloid protein (PrP27–30) to formic acid inactivated scrapie infectivity (34) . These experiments suggested that formic acid could disaggregate the structure of amyloid fibrils completely. NaOH solution has been used to decontaminate equipment exposed to patients with Creutzfeldt-Jakob diseases (35) . Recent studies have demonstrated that standard chemical decontamination methods with 2 N NaOH without autoclave reduced prion infectivity but not completely (22) . Inactivation of prion, AApoAII, and AA fibrils by dissociation of fibrils with 6 M guanidine hydrochloride has been reported (12 , 36) . In many experiments, 6 M urea has been used to disrupt amyloid fibrils including prion (37) and Aß (38) . Despite this, we could detect the fibril structure of AApoAII fibrils by ThT binding and EM after 144 h treatment with 6 M urea, and the transmitted treated fibrils induced amyloid deposition. In prion studies, autoclaving 1 h at 121°C reduced the titer of scrapie in brain homogenates by ~7.5 log LD50/g of brain homogenate but left 2.5 log LD50/g of residual infectivity (39) . Similarly, AApoAII fibrils treated by autoclaving for 3 h were not completely disaggregated or inactivated both in vitro and in vivo.

Multiple freeze/thaw cycles were particularly detrimental for detection of Aß fibrils (40 , 41) . The partial disaggregation and inactivation by multiple freeze/thaw cycles was also observed for AApoAII fibrils. Amplification of PrPres in vitro was abolished by treatment with purified RNase A (42) , but AApoAII fibrils were only slightly disaggregated by RNase. It has been reported that steel instruments may retain CJD infectivity even after 10% formalin treatment (43) and formalin exhibited the weakest disaggregating action on AApoAII fibrils in this study.

Although recent research has shown that soluble oligomers of amyloidogenic proteins were the primary pathogenic effectors in all kinds of amyloidosis (44 , 45) , other research indicates that amyloid fibrils are also considered to be an important cause of amyloid diseases (46 47 48) . To understand the propagation and pathogenesis of amyloidosis, the relationship among the structure of amyloid fibrils, binding to ThT, and transmissibility has been discussed (21 , 49) . Here, we found close correlation between ThT fluorescence and transmissibility of amyloid fibrils treated by physical and chemical methods. Western blotting analysis revealed that the amyloid fibril fractions contain monomer, dimer, larger oligomers, and fibrils which did not enter PAGE gels. The treatments with autoclaving, guanidine hydrochloride, and formic acid, which inactivated the transmissibility, disaggregated amyloid fibrils almost completely and monomer to pentamer increased because large oligomers and fibrils were disaggregated. Furthermore, we did not find amyloid fibrils treated with guanidine hydrochloride, autoclaving in 1 N NaOH, and formic acid under EM. From these findings, we postulate that the typical amyloid structure is critically important for transmissibility.

Many experiments have shown that PrPSc proteins are resistant to proteinase K digestion (50) . We verified that AApoAII exhibited resistance to proteinase digestion but that AApoAII denatured in DMSO was more susceptible to proteinase digestion. These results verified the similarity in the physical and chemical properties as well as the transmissibility of AApoAII fibrils and PrPSc.

Tetracycline, a well-known antibiotic, can disrupt many kinds of amyloid fibrils, including prion protein, Aß fibrils (51) , and transthyretin amyloid fibrils (52) . Rifampicin and its derivatives, which possess a naphthohydroquinone or naphthoquinone structure, inhibited Aß (1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40) aggregation and neurotoxicity in a concentration-dependent manner with the mechanism possibly involving scavenging of free radicals (28) . NDGA could disrupt Aß fibrils into aggregates that were larger than monomers or oligomers and did not bind ThT (29 , 53) . NDGA was also shown not to disaggregate Aß (1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40) but to inhibit fibril growth in other experiments (54) . Prevention of formation and destabilization of Aß fibril by several kinds of polyphenols has been reported (55) . According to these results, we investigated the disaggregation and inactivation of AApoAII fibrils by antibiotics and polyphenols. We found similar disaggregation effects of tetracycline, rifampicin, and NDGA in vitro as has been found in other forms of amyloidosis. These organic compounds also inactivated the transmission of AApoAII fibrils in mice. We found various disaggregation and inactivation effects for all kinds of organic compounds, except erythromycin, chloramphenicol, resveratrol, and quercetin. The ability of these organic compounds to disaggregate and inactivate not only AApoAII fibrils but also other amyloid fibrils implies the recognition of a common motif present in amyloid fibrils.

We could not explain the mechanism of inactivation by various kinds of organic compounds until now. But these organic compounds present safe toxicological profiles, and we think that these organic compounds will be useful for the development of preventive and therapeutic methods for various amyloid diseases. In our organic compounds experiments, we administered organic compounds for 6 d to the mice injected with AApoAII fibrils and examined the effects of organic compounds after 2 mo. We observed a significant reduction in amyloid deposition in mice given several organic compounds exhibiting disaggregation/inactivating effects in vitro. The effects, however, were relatively small. We did not observe any reduction in amyloid deposition compared with controls when organic compounds were administered for 6 d to the mice that had been injected with AApoAII fibrils 2 mo previously (data not shown). Further research is required to determine factors that may improve the effects of these organic compounds on the transmission and progression of amyloidosis.

ACKNOWLEDGMENTS

This work was supported in part by Grants-in-Aid for Priority Areas (15032217, 17028018) and Scientific Research (B) (17390111) from the Ministry of Education, Culture, Sports, Science and Technology of Japan and by a grant from the Ministry of Health, Labor and Welfare of Japan.

Received for publication September 12, 2005. Accepted for publication December 6, 2005.

REFERENCES

  1. Westermark, P. (1998) The pathogenesis of amyloidosis: understanding general principles. Am. J. Pathol. 152,1125-1127[Medline]
  2. Westermark, P., Benson, M. D., Buxbaum, J. N., Cohen, A. S., Frangione, B., Ikeda, S., Masters, C. L., Merlini, G., Saraiva, M. J., Sipe, J. D. (2002) Amyloid fibril protein nomenclature—2002. Amyloid. 9,197-200[Medline]
  3. Booth, D. R., Sunde, M., Bellotti, V., Robinson, C. V., Hutchinson, W. L., Fraser, P. E., Hawkins, P. N., Dobson, C. M., Radford, S. E., Blake, C. C., Pepys, M. B. (1997) Instability, unfolding and aggregation of human lysozyme variants underlying amyloid fibrillogenesis. Nature 385,787-793[CrossRef][Medline]
  4. Glenner, G. G. (1980) Amyloid deposits and amyloidosis. The beta-fibrilloses. N. Engl. J. Med. 302,1283-1292[Medline]
  5. Higuchi, K., Yonezu, T., Kogishi, K., Matsumura, A., Takeshita, S., Higuchi, K., Kohno, A., Matsushita, M., Hosokawa, M., Takeda, T. (1986) 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. 261,12834-12840[Abstract/Free Full Text]
  6. Benson, M. D., Liepnieks, J. J., Yazaki, M., Yamashita, T., Hamidi Asl, K., Guenther, B., Kluve-Beckerman, B. (2001) A new human hereditary amyloidosis: the result of a stop-codon mutation in the apolipoprotein AII gene. Genomics 72,272-277[CrossRef][Medline]
  7. Yonezu, T., Higuchi, K., Tsunasawa, S., Takagi, S., Sakiyama, F., Takeda, T. (1986) High homology is present in the primary structures between murine senile amyloid protein (ASSAM) and human apolipoprotein A-II. FEBS Lett. 203,149-152[CrossRef][Medline]
  8. Higuchi, K., Kitado, H., Kitagawa, K., Kogishi, K., Naiki, H., Takeda, T. (1993) Development of congenic strains of mice carrying amyloidogenic apolipoprotein A-II (Apoa2c). Apoa2c reduces the plasma level and the size of high density lipoprotein. FEBS Lett. 317,207-210[CrossRef][Medline]
  9. Higuchi, K., Naiki, H., Kitagawa, K., Kitado, H., Kogishi, K., Matsushita, T., Takeda, T. (1995) Apolipoprotein A-II gene and development of amyloidosis and senescence in a congenic strain of mice carrying amyloidogenic ApoA-II. Lab. Invest. 72,75-82
  10. Jarrett, J. T., Lansbury, P. T., Jr (1993) Seeding "one-dimensional crystallization" of amyloid: a pathogenic mechanism in Alzheimer’s disease and scrapie?. Cell 73,1055-1058[CrossRef][Medline]
  11. Bessen, R. A., Kocisko, D. A., Raymond, G. J., Nandan, S., Lansbury, P. T., Caughey, B. (1995) Non-genetic propagation of strain-specific properties of scrapie prion protein. Nature 375,698-700[CrossRef][Medline]
  12. Higuchi, K., Kogishi, K., Wang, J., Chen, X., Chiba, T., Matsushita, T., Hoshii, Y., Kawano, H., Ishihara, T., Yokota, T., Hosokawa, M. (1998) Fibrilization in mouse senile amyloidosis is fibril conformation-dependent. Lab. Invest. 78,1535-1542
  13. Caughey, B. (2000) Transmissible spongiform encephalopathies, amyloidoses and yeast prions: common threads?. Nat. Med. 6,751-754[CrossRef][Medline]
  14. Wickner, R. B., Edskes, H. K., Maddelein, M. L., Taylor, K. L., Moriyama, H. (1999) Prion of yeast and fungi. Proteins as genetic material. J. Biol. Chem. 274,555-558[Free Full Text]
  15. Chesebro, B. (1998) BSE and prions: uncertainties about the agent. Science 279,42-43[Free Full Text]
  16. Xing, Y., Nakamura, A., Chiba, T., Kogishi, K., Matsushita, T., Fu, L., Guo, Z., Hosokawa, M., Mori, M., Higuchi, K. (2001) Transmission of mouse senile amyloidosis. Lab. Invest. 81,493-499
  17. Fu, X., Korenaga, T., Fu, L., Xing, Y., Guo, Z., Matsushita, T., Hosokawa, M., Naiki, H., Baba, S., Kawata, Y., Ikeda, S., Ishihara, T., Mori, M., Higuchi, K. (2004) Induction of AApoAII amyloidosis by various heterogeneous amyloid fibrils. FEBS Lett. 563,179-184[CrossRef][Medline]
  18. Cui, D., Kawano, H., Takahashi, M., Hoshii, Y., Setoguchi, M., Gondo, T., Ishihara, T. (2002) Acceleration of murine AA amyloidosis by oral administration of amyloid fibrils extracted from different species. Pathol. Int. 52,40-45[CrossRef][Medline]
  19. Lundmark, K., Westermark, G. T., Nystrom, S., Murphy, C. L., Solomon, A., Westermark, P. (2002) Transmissibility of systemic amyloidosis by a prion-like mechanism. Proc. Natl. Acad. Sci. USA 99,6979-6984[Abstract/Free Full Text]
  20. Baker, H. F., Ridley, R. M., Duchen, L. W., Crow, T. J., Bruton, C. J. (1994) Induction of beta (A4)-amyloid in primates by injection of Alzheimer’s disease brain homogenate. Comparison with transmission of spongiform encephalopathy. Mol. Neurobiol. 8,25-39[Medline]
  21. Sigurdsson, E. M., Wisniewski, T., Frangione, B. (2002) Infectivity of amyloid diseases. Trends. Mol. Med. 8,411-413[CrossRef][Medline]
  22. Fichet, G., Comoy, E., Duval, C., Antloga, K., Dehen, C., Charbonnier, A., McDonnell, G., Brown, P., Lasmezas, C. I., Deslys, J. P. (2004) Novel methods for disinfection of prion-contaminated medical devices. Lancet 364,521-526[CrossRef][Medline]
  23. McDonnell, G., Burke, P. (2003) The challenge of prion decontamination. Clin. Infect. Dis. 36,1152-1154[CrossRef][Medline]
  24. Merlini, G., Ascari, E., Amboldi, N., Bellotti, V., Arbustini, E., Perfetti, V., Ferrari, M., Zorzoli, I., Marinone, M. G., Garini, P., Diegoli, M., Trizio, D., Ballinari, D. (1995) Interaction of the anthracycline 4'-indo-4'-deoxydoxorubicin with amyloid fibrils: inhibition of amyloidogenesis. Proc. Natl. Acad. Sci. USA 92,2959-2963[Abstract/Free Full Text]
  25. Eriksen, J. L., Sagi, S. A., Smith, T. E., Weggen, S., Das, P., Mclendon, D. C., Ozols, V. V., Jessing, K. W., Zavitz, K. H., Koo, E. H., Golde, T. E. (2003) NSAIDs and enantiomers of flurbiprofen target gamma-secretase and lower Abeta 42 in vivo. J. Clin. Invest. 112,440-449[CrossRef][Medline]
  26. May, B. C., Fafarman, A. T., Hong, S. B., Rogers, M., Deady, L. W., Prusiner, S. B., Cohen, F. E. (2003) Potent inhibition of scrapie prion replication in cultured cells by bis-acridines. Proc. Natl. Acad. Sci. USA 100,3416-3421[Abstract/Free Full Text]
  27. Forloni, G., Iussich, S., Awan, T., Colombo, L., Angeretti, N., Girola, L., Bertani, I., Poli, G., Caramelli, M., Grazia Bruzzone, M., Farina, L., Limido, L., Rossi, G., Giaccone, G., Ironside, J. W., Bugiani, O., Salmona, M., Tagliavini, F. (2002) Tetracyclines affect prion infectivity. Proc. Natl. Acad. Sci. USA 99,10,849-10,854[Abstract/Free Full Text]
  28. Tomiyama, T., Shoji, A., Kataoka, K., Suwa, Y., Asano, S., Kaneko, H., Endo, N. (1996) Inhibition of amyloid beta protein aggregation and neurotoxicity by rifampicin. Its possible function as a hydroxyl radical scavenger. J. Biol. Chem. 271,6839-6844[Abstract/Free Full Text]
  29. Ono, K., Hasegawa, K., Yoshiike, Y., Takashima, A., Yamada, M., Naiki, H. (2002) Nordihydroguaiaretic acid potently breaks down pre-formed Alzheimer’s beta-amyloid fibrils in vitro. J. Neurochem. 81,434-440[CrossRef][Medline]
  30. Pras, M., Zucker-Franklin, D., Rimon, A., Franklin, E. C. (1969) physical, chemical, and ultrastructural studies of water-soluble human amyloid fibrils. Comparative analyses of nine amyloid preparations. J. Exp. Med. 130,777-796[Abstract]
  31. Xing, Y., Nakamura, A., Korenaga, T., Guo, Z., Yao, J., Fu, X., Matsushita, T., Kogishi, K., Hosokawa, M., Kametani, F., Mori, M., Higuchi, K. (2002) Induction of protein conformational change in mouse senile amyloidosis. J. Biol. Chem. 277,33,164-33,169[Abstract/Free Full Text]
  32. Chiba, T., Kogishi, K., Wang, J., Xia, C., Matsushita, T., Miyazaki, J., Saito, I., Hosokawa, M., Higuchi, K. (1999) Mouse senile amyloid deposition is suppressed by adenovirus-mediated overexpression of amyloid-resistant apolipoprotein A-II. Am. J. Pathol. 155,1319-1326[Abstract/Free Full Text]
  33. Klunk, W. E., Pettegrew, J. W. (1990) Alzheimer’s beta-amyloid protein is covalently modified when dissolved in formic acid. J. Neurochem. 54,2050-2056[CrossRef][Medline]
  34. Safar, J., Roller, P. P., Gajdusek, D. C., Gibbs, C. J., Jr (1993) Thermal stability and conformational transitions of scrapie amyloid (prion) protein correlate with infectivity. Protein. Sci. 2,2206-2216[Abstract]
  35. Brown, P., Rohwer, R. G., Gajdusek, D. C. (1984) Sodium hydroxide decontamination of Creutzfeldt-Jakob disease virus. N. Engl. J. Med. 310,727[Medline]
  36. Safar, J., Roller, P. P., Gajdusek, D. C., Gibbs, C. J., Jr (1993) Conformational transitions, dissociation and unfolding of scrapie amyloid (prion) protein. J. Biol. Chem. 268,20276-20284[Abstract/Free Full Text]
  37. Fischer, M. B., Roeckl, C., Parizek, P., Schwarz, H. P., Aguzzi, A. (2000) Binding of disease-associated prion protein to plasminogen. Nature 408,479-483[CrossRef][Medline]
  38. Botelho, M. G., Gralle, M., Oliveira, C. L., Torriani, I., Ferreira, S. T. (2003) Folding and stability of the extracellular domain of the human amyloid precursor protein. J. Biol. Chem. 278,34,259-34,267[Abstract/Free Full Text]
  39. Brown, P., Rohwer, R. G., Green, E. M., Gajdusek, D. C. (1982) Effect of chemicals, heat, and histopathologic processing on high-infectivity hamster-adapted scrapie virus. J. Infect. Dis. 145,683-687[Medline]
  40. Southwick, P. C., Yamagata, S. K., Echols, C. L., Jr, Higson, G. J., Neynaber, S. A., Parson, R. E., Munroe, W. A. (1996) Assessment of amyloid beta protein in cerebrospinal fluid as an aid in the diagnosis of Alzheimer’s disease. J. Neurochem. 66,259-265[Medline]
  41. Vanderstichele, H., Van Kerschaver, E., Hesse, C., Davidsson, P., Buyse, M. A., Andreasen, N., Minthon, L., Wallin, A., Blennow, K., Vanmechelen, E. (2000) Standardization of measurement of beta-amyloid(1–42) in cerebrospinal fluid and plasma. Amyloid 7,245-258[Medline]
  42. Deleault, N. R., Lucassen, R. W., Supattapone, S. (2003) RNA molecules stimulate prion protein conversion. Nature 425,717-720[CrossRef][Medline]
  43. Zobeley, E., Flechsig, E., Cozzio, A., Enari, M., Weissmann, C. (1999) Infectivity of scrapie prions bound to a stainless steel surface. Mol. Med. 5,240-243[Medline]
  44. Silveira, J. R., Raymond, G. J., Hughson, A. G., Race, R. E., Sim, V. L., Hayes, S. F., Caughey, B. (2005) The most infectious prion protein particles. Nature 437,257-261[CrossRef][Medline]
  45. Bitan, G., Fradinger, E. A., Spring, S. M., Teplow, D. B. (2005) Neurotoxic protein oligomers-what you see is not always what you get. Amyloid 12,88-95[Medline]
  46. Stefani, M., Dobson, C. M. (2003) Protein aggregation and aggregate toxicity: new insights into protein folding, misfolding diseases and biological evolution. J. Mol. Med. 81,678-699[CrossRef][Medline]
  47. Zerovnik, E. (2002) Amyloid-fibril formation. Proposed mechanisms and relevance to conformational disease. Eur. J. Biochem. 269,3362-3371[Medline]
  48. Bucciantini, M., Giannoni, E., Chiti, F., Baroni, F., Formigli, L., Zurdo, J., Taddei, N., Ramponi, G., Dobson, C. M., Stefani, M. (2002) Inherent toxicity of aggregates implies a common mechanism for protein misfolding diseases. Nature 416,507-511[CrossRef][Medline]
  49. Ban, T., Hamada, D., Hasegawa, K., Naiki, H., Goto, Y. (2003) Direct observation of amyloid fibril growth monitored by thioflavin T fluorescence. J. Biol. Chem. 278,16462-16465[Abstract/Free Full Text]
  50. Prusiner, S. B. (1998) Prions. Proc. Natl. Acad. Sci. USA 95,13,363-13,383[Abstract/Free Full Text]
  51. Forloni, G., Colombo, L., Girola, L., Tagliavini, F., Salmona, M. (2001) Anti-amyloidogenic activity of tetracyclines: studies in vitro. FEBS Lett. 487,404-407[CrossRef][Medline]
  52. Cardoso, I., Merlini, G., Saraiva, M. J. (2003) 4'-iodo-4'-deoxydoxorubicin and tetracyclines disrupt transthyretin amyloid fibrils in vitro producing noncytotoxic species: screening for TTR fibril disrupters. FASEB J. 17,803-809[Abstract/Free Full Text]
  53. Naiki, H., Hasegawa, K., Yamaguchi, I., Nakamura, H., Geiyo, F., Nakakuki, K. (1998) Apolipoprotein E and antioxidants have different mechanisms of inhibiting Alzheimer’sedimentary coeffcient beta-amyloid fibril formation in vitro. Biochemistry 37,17,882-17,889[CrossRef][Medline]
  54. Moss, M. A., Varvel, N. H., Nichols, M. R., Reed, D. K., Rosenberry, T. L. (2004) Nordihydroguaiaretic acid does not disaggregate beta-amyloid (1–40) protofibrils but does inhibit growth arising from direct protofibril association. Mol. Pharmacol. 66,592-600[Abstract/Free Full Text]
  55. Ono, K., Yoshiike, Y., Takashima, A., Hasegawa, K., Naiki, H., Yamada, M. (2003) Potent anti-amyloidogenic and fibril-destabilizing effects of polyphenols in vitro: implications for the prevention and therapeutics of Alzheimer’s disease. J. Neurochem. 87,172-181[CrossRef][Medline]



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