Protein fibrils in nature can enhance amyloid protein A amyloidosis in mice: Cross-seeding as a disease mechanism

Materials and Methods

Animals. Five- to 10-week-old female Naval Medical Research Institute (NMRI) mice obtained from B & K Universal (Sollentuna, Sweden) and age-matched FVB mice of both sexes, bred at the Linköping University Hospital animal facility, were used in the experiments. The mice were kept in cages with steel-wire lids under 12-h dark/12-h light cycles and had free access to water and pellets (type R36, Lactamin, Vadstena, Sweden). The local animal ethics committee in Linköping, Sweden, approved the experimental protocols.

Silk Fibrils. Silk fibrils from the cocoon of B. mori (a gift from Fjärilhuset, Stockholm) were prepared as described (30). Briefly, silk was boiled extensively for 2 h in 2% Na₂CO₃, rinsed, and dried at 60°C. Ten milligrams of treated silk was dissolved in 700 μl of 65% aqueous LiSCN brought to 1% 2-mercaptoethanol. The solution was dialyzed overnight against double-distilled water and brought to 10 ml with double-distilled water (1 mg of protein per ml). The salt concentration was adjusted to 0.9% by using solid NaCl, and the solution was left at 4°C for 4 weeks for fibril formation. The fibril suspension was thoroughly sonicated on ice with Soniprep 150 (Crawley, Sussex, U.K.) before use in animal experiments.

Sup35 Fibrils. Fibrils from the recombinant wild-type prion-determining N-terminal and middle (NM) region of Sup35 and its variant, R₂E₂ NM (31), were kindly provided by Susan Lindquist (Massachusetts Institute of Technology, Cambridge). The fibrils were diluted to a concentration of 15 μg/ml with 0.9% NaCl and sonicated as described before use.

Curli Fibrils. A curli-proficient E. coli K12 strain, YMel, was grown on colonization factor antigen (CFA)-agar plates for 48 h at 28°C, assayed for fibronectin binding (23), heat-inactivated, and suspended to a concentration 7.7 × 10⁸ colony-forming units per ml. Two-hundred-microliter samples of YMel were diluted 10× with 0.9% NaCl and were thoroughly sonicated as described before use in the experiments. Then, curli were purified as described (32). Briefly, bacteria grown as above were treated with RNase A and DNase 1, incubated with lysozyme, and brought to 1% SDS buffer. The material was washed and pelleted by centrifugation. The RNase A, DNase 1, and lysozyme treatment was repeated, and curli were pelleted by centrifugation. The pellet was resuspended in 2 ml of SDS/PAGE sample buffer, boiled, and subjected to electrophoresis for 5 h on an SDS/12% PAGE gel. The curli protein remaining in the wells after electrophoresis (≈25 μg) was recovered, washed, and resuspended in 4 ml of double-distilled water. Then, 500 μl of purified curli was diluted with equal volume of 0.9% NaCl and sonicated as above before use in animal experiments.

Actin Fibrils. Lyophilized actin from rabbit muscle was obtained from Sigma. Actin was dissolved (1 mg/ml) in a G-buffer (33), pH 7.8, containing 5 mM Tris·HCl, 0.1 mM CaCl₂, 0.2 mM DTT, 0.01% sodium azide, and 0.2 mM ATP (Amersham Pharmacia), and stored on ice. Polymerization was started by the addition of 1 mM MgCl₂ to the actin solution at 24°C (33). Actin fibrils (F-actin) were diluted with an equal volume of G-buffer and immediately used in the experiments.

Induction of Amyloid with Silver Nitrate. In the first series of experiments, the time for induction of amyloidosis was estimated for both female and male FVB mice. All mice were injected with 0.5 ml of 1% silver nitrate at the start of the experiments and received injections of 0.1 ml of 1% silver nitrate once a week until the end of the experiments. The mice were divided into groups of three to six and were killed 7, 14, 17, 19, 21, 25, 28, or 58 days after the first silver nitrate injection. Spleens from all mice were collected and, for the demonstration of amyloid, one half of each spleen was crushed between two glass slides, smeared homogeneously over both of the slides, and air-dried in room temperature overnight. The other half was fixed in 4% buffered neutral formaldehyde solution and embedded in paraffin. The time for induction of amyloidosis by repeated s.c. injections of silver nitrate in NMRI mice was estimated in ref. 18.

Enhancement of Amyloid Induction. Amyloid was induced as described elsewhere (18, 19) by i.v. injections of 0.1 ml (100 μg) of silk fibrils, 0.1 ml (1.5 μg) of Sup35 fibrils or its variant, 0.1 ml of sonicated heat-inactivated YMel E. coli, or 0.1 ml of purified curli (≈0.3 μg), followed by s.c. injections of 0.5 ml of 1% silver nitrate. Control groups received i.v. injections of 0.1 ml of 0.15 M NaCl, F-actin (50 μg), or G-buffer, followed by 0.5 ml of 1% silver nitrate exactly as in the experimental groups. Injections of 0.1 ml of 1% silver nitrate were repeated after 7 and 14 days, and the animals in both experimental and control groups were killed 2 days after the last treatment by cervical dislocation in ether narcosis. The spleens were collected, divided into two halves, and treated as described above. In another experiment, one half of spleen was frozen for amyloid protein purification, and the other half was divided in two parts. One of them was smeared between two slides, and the other one was fixed in formaldehyde as described above.

Light Microscopy. Samples from silk, Sup35 variants, purified curli fibril suspensions, YMel E. coli bacteria, and F-actin were dried on glass slides and stained with alkaline Congo red (34). Smears and 10-μm-thick deparaffinized spleen sections were stained with alkaline Congo red and hematoxylin/eosin. The slides were examined blindly in a polarization microscope (BX51, Olympus, Melville, NY) for bright green birefringence, typical of amyloid. Amyloid grading was performed on spleen sections for each mouse separately, as described in ref. 14.

Electron Microscopy. Samples of silk fibrils, Sup35 fibrils, YMel E. coli bacteria, and purified curli were prepared directly after sonication by applying small drops of the suspensions on Form-var-coated nickel grids. Samples of F-actin were prepared immediately after polymerization. The grids were negatively contrasted with 1% uranyl acetate, air-dried, and viewed in a JEOL 1230 electron microscope at 100 kV.

Characterization of Amyloid Deposits. Sections from formalin-fixed spleen samples were labeled with rabbit antiserum (1:400) against mouse protein AA (18, 19). A mouse monoclonal antibody to protein AA (S. Nyström and G.T.W., unpublished data) also was used. Frozen spleen samples from two mice treated with Sup35, three mice treated with silk fibrils, and two mice treated with purified curli were homogenized separately, and amyloid fibrils were extracted as described (18, 19). Fibrils were dissolved in 6 M guanidine·HCl/0.1 M Tris·HCl (pH 8.0) and subjected to size-exclusion chromatography on a Superose 12 HR column (Amersham Pharmacia). Protein material was eluted with 10% formic acid at a flow rate of 0.25 ml/min and monitored at 280 nm. The retarded main protein peaks were dried in a Speed Vac (Savant) and analyzed by MALDI-TOF MS. For this procedure, material was dissolved in 70% acetonitrile/0.3% trifluoroacetic acid and heated to 95°C for 5 min. Equal volumes of sample and α-cyano-4-hydroxycinnamic acid, saturated with 70% acetonitrile/0.3% trifluoroacetic acid, were mixed, and 2-μl aliquots were spotted onto the target plate and analyzed. Reflector mass spectra were recorded by using the instrument setting recommended by the manufacturer. The analysis was carried out by using a mass spectrometer (Voyager-DE Pro, Applied Biosystems).

In further experiments, material from the main retarded protein AA peak from mice that received silk fibrils was dissolved in 0.1 M ammonium carbonate and digested with trypsin (sequencing grade, substrate:enzyme 50:1, Promega), and the material was subjected to MALDI-TOF MS.

Statistical Analysis. For statistical comparison between groups, Fisher's exact test was used with instat 2.01 software (GraphPad, San Diego). P < 0.05 was considered statistically significant.