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

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

SPECIFIC AIMS

Amyloidosis refers to a group of protein-folding diseases, and amyloid fibrils have exhibited prion-like transmissibility in some kinds of amyloidosis. To understand the mechanism of the propagation of amyloidosis by amyloid fibrils and to develop the methods for preventing it, we here studied the biochemical and biomedical characteristics of the transmission of mouse senile (AApoAII) amyloidosis.

PRINCIPAL FINDINGS

1. AApoAII fibrils are extremely active and can induce amyloidosis following doses less than 1 pg
AApoAII fibrils were isolated from the liver of a R1.P1-Apoa2^c mouse with heavy amyloid deposition. To test the effect of the dose on induction of amyloidosis, AApoAII fibrils were serially diluted in DW and intravenously (i.v.) injected into mice at doses ranging from 10^–10 µg to 100 µg. Mice were killed 2 mo after the injections. The degree of amyloid deposition (amyloid index: AI) increased proportionally to the logarithm of dosage from 10^–1 µg to 100 µg. Slight amyloid deposits were detected in mice injected with doses of AApoAII fibrils between 10^–7 and 10^–2 µg. No amyloid deposits were observed in mice injected with doses less than 10^–8 µg. None of the control mice without AApoAII injection had detectable amyloid deposits. We also performed intraperitoneal (i.p.) and oral injection of AApoAII fibrils in vivo.

These results indicated that 1) AApoAII fibrils are extremely active and can induce amyloidosis following doses less than 1 pg. 2) We can determine the transmission activity of each amyloid fibril semiquantitatively. 3) The magnitude of transmission activity of the amyloid fibrils by the different routes of administration was i.v. i.p. >> oral.

2. AApoAII fibrils resemble prion in the characteristics of disruption and inactivation by physical and chemical methods
Physical and chemical methods were used to disrupt AApoAII fibrils. The degree of disruption was determined by the Thioflavin T (ThT) assay. The fluorescence of AApoAII treated with formalin, RNase, delipidation, freeze-thaw, or urea was 79.3, 75.6, 58.3, 49.0 or 20.2% of untreated control AApoAII. 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 (Fig 1 A).

View larger version (29K): [in this window] [in a new window]   Figure 1. 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 () and 100 () µg in each treatment. Values are means ± SD. Numbers in parentheses represent amyloid positive mice/mice examined.

We i.v. injected mice with 1 µg of amyloid fibrils treated with each disruption method and determined the extent of amyloid deposition. Only a slight decrease in amyloid deposition was observed when AApoAII fibrils were treated with formalin, RNase, or delipidation compared with control fibrils. Injection of AApoAII fibrils treated with freeze-thaw or urea induced much less amyloid deposition. The injection of 1 µg of AApoAII fibrils 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 (Fig 1B ).

We also analyzed the changes in the structure of AApoAII fibrils by Western blotting analysis. 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. 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.

3. We found close correlation between transmissibility and the typical fibril structure of treated AApoAII
Analysis of the morphology of AApoAII fibrils using electron microscopy (EM) 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.

4. AApoAII fibrils exhibit resistance to proteinase K digestion
We investigated the effect of proteinase K digestion of AApoAII fibrils. When AApoAII was digested with proteinase K (1:50), we detected a band of intact apoA-II protein monomer by Western blotting analysis. However, analysis of AApoAII denatured with DMSO and digested with proteinase K revealed only few apoA-II monomers and the apoA-II dimer. Incubation of both natural and denatured AApoAII in proteinase K (1:2) resulted in complete digestion of AApoAII.

5. Some organic compounds disaggregate AApoAII fibrils 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 fibrils at 37°C in the absence of organic compounds. However, the ThT fluorescence decreased after the addition of eight organic compounds in a concentration-dependent manner. The ThT fluorescence did not change after incubation with the other four organic compounds, including erythromycin, chloramphenicol, quercetin, and resveratrol. Further, we observed morphological changes in the fibrils incubated with organic compounds using EM. A lot of intact fibrils were identified in the AApoAII incubated with lincomycin, cephalexin, and benzylpenicillin, but the whole amount of fibrils appeared to be decreased in EM compared with control. The amount of 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.

6. Some organic compounds inactivate AApoAII fibrils and inhibit amyloid deposition in vivo
To evaluate the effects of organic compounds on the transmissible activity of AApoAII, we injected mice with 1 µg of fibrils that had been incubated with 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 inactivation effects on the transmissibility of AApoAII in vivo (Fig. 2 ). 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 amyloid deposition. We found a significant correlation between the disaggregation activity of the organic compounds in vitro and the inhibition of transmission in vivo.

View larger version (23K): [in this window] [in a new window]   Figure 2. Inhibition of transmission of AApoAII fibrils by disaggregation with organic compounds. Amyloid deposition was determined in mice injected with AApoAII fibrils that had been incubated with various organic compounds for 144 h. Value represent means ± SD. *P < 0.05.

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 intramuscular (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. The injection of lincomycin and benzylpenicillin, however, had no significant effect. These findings were consistent with the results of our in vitro experiments.

CONCLUSIONS AND SIGNIFICANCE

Various forms of amyloidosis, except for prion disease, had been thought to be nontransmissible, with a clear boundary existing between transmissible prion disease and general amyloidosis. However, we have described the possibility of the transmission of AApoAII amyloidosis in our previous works. In this study, we characterized the transmissibility of amyloid fibrils in detail, inactivated them by physical or chemical methods, and verified the transmission of AApoAII amyloidosis.

We demonstrated that AApoAII is extremely active and induces amyloidosis following doses of less than 1 pg. In the experiments of physical and chemical disruption of AApoAII fibrils in vitro as well as in vivo, we found (1) complete disruption of the fibril structure and prevention of transmission after treatment with formic acid, guanidine hydrochloride, and autoclaving in 1 N NaOH; (2) partial disruption with 6 M urea, 2 N NaOH, and autoclaving; and (3) no disruption with formalin (4); AApoAII fibrils exhibit resistance to proteinase K digestion. These results suggested strongly that amyloid diseases could be transmitted like prion diseases under certain conditions.

Furthermore, a distinct correlation was obtained between the amounts of amyloid fibrils and their transmissibility, thereby indicating the essential role of fibril conformation for the transmission of amyloidosis. Thus, the inactivation or disruption of the amyloid fibrils should be an important issue for the understanding and prevention of amyloidosis.

We also studied the disaggregation and inactivation of AApoAII by several organic compounds in vitro and found some effects for prevention of transmission in vivo. We could not explain the mechanism of disaggregation by 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. These findings were summarized in Fig. 3 .

View larger version (27K): [in this window] [in a new window]   Figure 3. Schematic diagram of various processes for disruption of AApoAII fibrils. AApoAII amyloidosis has exhibited prion-like transmission. The exogenous AApoAII fibrils can be slightly disaggregated by formalin, delipidation, freeze-thaw, and RNase. The slightly disaggregated fibrils could still induce amyloidosis. Autoclaving, 6 M urea, 2 N NaOH, antibiotics, and NDGA can incompletely disaggregate AApoAII fibrils after which the fibrils induced mild amyloidosis. Autoclaving in 1 N NaOH, formic acid, and guanidine hydrochloride can completely disrupt AApoAII fibrils after which the fibrils did not induce amyloidosis. Accordingly the fibril conformation plays an important role in the transmission of amyloidosis.

FOOTNOTES

To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.05-4890fje

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This Article Abstract Full Text Full Text (PDF) All Versions of this Article: fj.05-4890fjev1 20/7/1012    most recent Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Zhang, H. Articles by Higuchi, K. Search for Related Content PubMed PubMed Citation Articles by Zhang, H. Articles by Higuchi, K.