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Detection of β-Amyloid Peptide Aggregation Using DNA Electrophoresis
Analytical Biochemistry 284, 401– 405 (2000) doi:10.1006/abio.2000.4719, available online at http://www.idealibrary.com on
Detection of -Amyloid Peptide Aggregation Using DNA Electrophoresis 1 Bong Whan Ahn, 2 Dong Up Song, Young Do Jung, Kee Oh Chay, Min A Chung, Sung Yeul Yang, and Boo Ahn Shin Chonnam National University Research Institute of Medical Sciences, Hakdong 5, Donggu, Kwangju 501-190, Korea
Received May 15, 2000
DNA could readily associate with the aggregated forms of the -amyloid peptides (1– 40) and (25–35), giving rise to a shift in the electrophoretic mobility of DNA. As a result, DNA was retained at the top of a 1% agarose gel. In contrast, the electrophoretic mobility of DNA was little influenced by the monomeric forms of (1– 40) and (25–30). DNA from different sources such as phage, Escherichia coli plasmid, and human gene showed similar results. However, the electrophoretic mobility of RNA was shifted by the monomeric (1– 40) and (25–35) as well as by the aggregated (1– 40) and (25–35). The association of DNA with the aggregated -amyloid peptides could occur at pH 4 –9. The inhibitory action of hemin on -amyloid aggregation could be confirmed using the DNA mobility shift assay. These results indicate that the DNA mobility shift assay is useful for kinetic study of -amyloid aggregation as well as for testing of agents that might modulate -amyloid aggregation. © 2000
and eventually to senile dementia. -AP can aggregate under physiological conditions to form fibrils consisting of antiparallel -pleated sheets (2, 3). Many studies have shown that -amyloid aggregates are neurotoxic (4 –10). Therefore, agents that can inhibit the aggregation of -AP may be of therapeutic benefit. Also, an assay system that detects the aggregated -AP but not the nonaggregated -AP is useful for identifying such agents. Polyanionic compounds such as Congo red, glycosaminoglycans, and proteoglycans can associate with -AP, modulating the aggregation or neurotoxicity of -AP (11–13). Nucleic acids are polyanionic at physiological pH. In a study with nucleic acids for effects on aggregation and neurotoxicity of -AP, we have found that DNA associates preferentially with the aggregated -AP to nonaggregated forms. In this paper, an assay system that can detect the aggregation of -AP employing a DNA probe is presented.
Academic Press
Key Words: -amyloid peptide; aggregation; DNA mobility.
Alzheimer’s disease is a neurodegenerative disease characterized by senile plaques, composed primarily of -amyloid peptides (-APs) 3 (1). Although the precise role of -AP in the pathogenesis of Alzheimer’s disease is not clear, aggregation of -AP has been suggested to be a critical event that can lead to neuronal damage 1 This study was supported by a grant from the Korea Health 21 R&D Project, Ministry of Health and Welfare, Republic of Korea (HMP-98-MS-0001). 2 To whom correspondence should be addressed. Fax: 82-62-2238321. E-mail: [email protected]. 3 Abbreviations used: -AP, -amyloid peptide; DMSO, dimethyl sulfoxide.
0003-2697/00 $35.00 Copyright © 2000 by Academic Press All rights of reproduction in any form reserved.
MATERIALS AND METHODS
(1– 40), (25–35), phage DNA, yeast tRNA phe, and hemin were purchased from Sigma Chemical Co. A constructed yeast plasmid, pSJ101-hRAD50, which contains a human RAD50 gene insert, was used as a preparation of plasmid and human DNAs after cutting with HindIII to yield a plasmid DNA piece of 9.0 kb and two human DNA pieces of 4.3 and 1.8 kb. Tissue RNA was extracted from a 3-month-old rat liver using acid guanidinium thiocyanate/phenol/chloroform as described by Chomczynski and Sacci (14). Monomeric and aggregated forms of (1– 40) and (25–35) were prepared by dissolving in dimethyl sulfoxide (DMSO) at 8 mg/ml and aging in water at 4 mM at 37°C for 5 days, respectively. These conditions have been shown in previous studies to result in predominantly soluble monomeric peptide and predominantly aggregated peptide, respectively (15, 16). For association reaction with nu401
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FIG. 1. Effects on DNA electrophoretic mobility of (1– 40) and (25–35) that had been aged in H 2O or prepared in DMSO. (A, B) One hundred micromolar (1– 40) (A) or (25–35) (B) was incubated at 37°C for 30 min with 1.5 g/ml DNA or HindIII digest of pSJ101-hRAD50 in 40 l of TBE buffer containing 10% (v/v) DMSO. After incubation, the mixtures were electrophoresed on 1.0% agarose gels for 1 h. Lane 1, DNA alone; lane 2, DNA incubated with -AP aged in H 2O; lane 3, DNA incubated with -AP prepared in DMSO; lane 4, pSJ101-hRAD50 digest alone; lane 5, pSJ101-hRAD50 digest incubated with -AP aged in H 2O; lane 6, pSJ101-hRAD50 digest incubated with -AP prepared in DMSO. The migration of DNA size markers is shown on the right margins. (C) SDS–PAGE analysis of (25–35) and (1– 40) aged in H 2O or prepared in DMSO. Lane 1, (25–35) aged in H 2O; lane 2, (25–35) prepared in DMSO; lane 3, (1– 40) aged in H 2O; lane 4, (1– 40) prepared in DMSO. The migration of marker proteins is shown on the right margin. The monomeric (25–35) in lanes 1 and 2 was suggested to be lost from the gel during staining procedures.
cleic acids, -AP was incubated at 37°C with 1.5 g/ml DNA or RNA, 20 mM buffer, and additives in a total volume of 40 l. At the selected time of incubation, the mixture was added with sample buffer and electrophoresed. In a standard assay to detect the aggregated -AP, -AP was incubated at 37°C for 30 min with 1.5 g/ml DNA in 40 l of TBE buffer (20 mM Tris– borate/1 mM EDTA, pH 8.0) containing 10% (v/v) DMSO. DNA samples were electrophoresed on a 1.0% agarose gel at 5 V/cm for 1 h and visualized by ethidium bromide (EtBr, 0.5 g/ml) staining (17). RNA samples were electrophoresed on a 1.2% agarose gel containing 2.2 M formaldehyde and Mops buffer (20 mM Mops/5 mM sodium acetate/1 mM EDTA, pH 7.0) at 5 V/cm for 1 h and visualized by EtBr staining (18). After electrophoresis, the gels were photographed with a Polaroid camera (Spectronics CH-810), and the fluorescence densities of DNA or RNA bands on the pictures were analyzed using a digital image analyzer (Fuji FLA-3000R). SDS–PAGE for -AP was carried out as described by Laemmli (19). Briefly, -AP was dissolved in sample buffer and electrophoresed on a 16% acrylamide gel under reducing conditions. RESULTS AND DISCUSSION
Effects of different forms of -AP on the electrophoretic mobility of nucleic acids were examined. As shown in Figs. 1A and 1B, incubation of phage DNA, Escherichia coli plasmid DNA, and human DNA with aggregated (1– 40) and (25–35) preparations resulted in shifts in the electrophoretic mobility of the DNAs. All the tested DNA bands were shifted to the
top of the gel, indicating that DNA formed high-molecular-weight complexes with the aggregated -APs. On the other hand, the electrophoretic mobilities of the
FIG. 2. Effects on RNA electrophoretic mobility of (25–35) and (1– 40) that had been aged in H2O or prepared in DMSO. One hundred micromolar (25–35) or (1– 40) was incubated at 37°C for 30 min with 1.5 g/ml rat liver RNA (A) or yeast tRNA (B) in 40 l of Mops buffer containing 10% (v/v) DMSO. After incubation, the mixtures were electrophoresed on 1.2% agarose gels for 1 h. Lane 1, RNA alone; lane 2, RNA incubated with (25–35) aged in H2O; lane 3, RNA incubated with (25–35) prepared in DMSO; lane 4, RNA incubated with (1– 40) aged in H 2O; lane 5, RNA incubated with (1– 40) prepared in DMSO.
DETECTION OF -AMYLOID AGGREGATION USING DNA ELECTROPHORESIS
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FIG. 3. Concentration-dependent effects of aggregated (1– 40) and (25–35) on DNA electrophoretic mobility. (A, B) One hundred micromolar (1– 40) (A) or (25–35) (B) composed of different proportions of two preparations that had been aged in H 2O or prepared in DMSO was incubated at 37°C for 30 min with 1.5 g/ml DNA in 40 l of TBE buffer containing 10% (v/v) DMSO. After incubation, the mixtures were electrophoresed on 1.0% agarose gels for 1 h. (C) DNA remaining at the original location on the gels (unassociated DNA) was quantitated by densitometry. Data represent the means ⫾ SD from triplicate experiments.
DNAs were little influenced by incubation of DNA with monomeric (1– 40) and (25–35). To confirm the monomeric and aggregated states of peptides dissolved in DMSO and aged in H 2O, respectively, aliquots of each peptide preparation were subjected to SDS–PAGE/ Coomassie blue staining (Fig. 1C). The results showed that significant amounts of the (1– 40) and (25–35) preparations that had been aged in H 2O existed as high-molecular-weight aggregates, whereas such aggregates were not detected in the (1– 40) and (25–35) preparations dissolved in DMSO. These results are similar to those shown in a previous study (15). However, results from experiments with RNA were quite different from those with DNA. As shown in Fig. 2, (1– 40) and (25–35) dissolved in DMSO as well as (1– 40) and (25–35) aged in H 2O could cause shifts in the electrophoretic mobility of tissue RNA and purified tRNA. The RNA bands were shifted to the top of the gel, indicating that RNA formed high-molecular-
weight complexes with both the monomeric and aggregated forms of -APs. The reason why DNA and RNA showed very different results in these experiments is not clear. But, two possibilities can be suggested: First, RNA may have higher affinities for -AP than DNA and bind many molecules of monomeric -AP to form high-molecular-weight complexes. Second, RNA may induce aggregation of -AP. Then, the aggregated -AP can readily associate with RNA. The above results suggested that DNA but not RNA could be used as a probe for identifying aggregated -AP in mixtures of monomeric and aggregated -AP. Therefore, we tested the DNA mobility shift assay with DNA for mixtures composed of different proportions of monomeric and aggregated -AP preparations (Fig. 3). As the proportions of aggregated (1– 40) and (25– 35) increased, the density of the DNA band remaining at the original location on the gels (unassociated DNA) was decreasing and the densities of the DNA
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FIG. 4. Time course of association between aggregated -AP and DNA. One hundred micromolar (1– 40) or (25–35) that had been aged in H 2O was incubated at 37°C for the indicated time with 1.5 g/ml DNA in 40 l of TBE buffer containing 10% (v/v) DMSO. After incubation, the mixtures were electrophoresed on 1.0% agarose gels for 1 h, and unassociated DNA was quantitated by densitometry. Data represent the means ⫾ SD from triplicate experiments.
bands at the top of the gels (associated DNA) were increasing. As shown in Fig. 3C, the density of unassociated DNA decreased almost linearly with the increment of the aggregated -AP concentration. In contrast to the case of the unassociated DNA band, it was difficult to quantitate the density of associated DNA bands because the shape and number of the bands at the top of gels were quite variable. Therefore, we recommend measuring the change in the density of unassociated DNA for the DNA mobility shift assay of -AP aggregation. Effects of a few reaction conditions on association between DNA and aggregated -AP were examined. Figure 4 shows that the association between DNA and aggregated -AP occurred quite rapidly. The association reaction was complete in less than 30 min for
FIG. 5. Effects of DMSO on association between aggregated -AP and DNA. One hundred micromolar (1– 40) or (25–35) that had been aged in H 2O was incubated at 37°C for 30 min with 1.5 g/ml DNA in 40 l of TBE buffer containing the indicated concentration (v/v) of DMSO. After incubation, the mixtures were electrophoresed on 1.0% agarose gels for 1 h, and unassociated DNA was quantitated by densitometry. Data represent the means ⫾ SD from triplicate experiments.
FIG. 6. pH-dependent association between aggregated -AP and DNA. One hundred micromolar (1– 40) (A) or (25–35) (B) that had been aged in H 2O was incubated at 37°C for 30 min with 1.5 g/ml DNA in 40 l of 20 mM buffer of the indicated pH containing 10% (v/v) DMSO. After incubation, the mixtures were electrophoresed on 1.0% agarose gels for 1 h. The buffers used were as follows: pH 4 – 6, sodium acetate; pH 7–10, Tris–HCl.
both (1– 40) and (25–35). DMSO, which was used as a vehicle for preparation of monomeric -AP, showed little effect on the association between DNA and aggregated -AP at 0 –20% (v/v) (Fig. 5). The association reaction could occur over a wide range of pH, at least pH 4 –9 for (1– 40) and pH 4 –10 for (25–35) (Fig. 6). Therefore, we employed the following reaction conditions in the standard assay to detect the aggregated -AP: specimens containing -AP were incubated at 37°C for 30 min with 1.5 g/ml DNA, 20 mM TBE buffer (pH 8.0), and 10% DMSO. Addition of 10% DMSO was required to prevent aggregation of monomeric -AP, which might occur during incubation, particularly in the case of (25–35). TBE buffer was chosen because the same buffer would also be used in the subsequent DNA electrophoresis step. We applied the DNA mobility shift assay for testing the action of inhibitors of -amyloid aggregation. Hemin is known to be an inhibitor of -amyloid aggregation (20). Under the conditions of Fig. 7, the amount of unassociated DNA decreased progressively with incubation time in the absence of hemin although the rates of decrease were different for (1– 40) and (25–
DETECTION OF -AMYLOID AGGREGATION USING DNA ELECTROPHORESIS
FIG. 7. Inhibitory effect of hemin on -AP aggregation as determined by DNA mobility shift assay. One hundred micromolar (1– 40) or (25–35) that had been prepared in DMSO was incubated at 37°C for the indicated period with 1.5 g/ml DNA in 40 l of TBE buffer containing 10% (v/v) DMSO in the presence or absence of 100 M hemin. After incubation, the mixtures were electrophoresed on 1.0% agarose gels for 1 h, and unassociated DNA was quantitated by densitometry. Data represent the means ⫾ SD from triplicate experiments.
35). However, in the presence of 100 M hemin, the amount of unassociated DNA remained unchanged with incubation time for both (1– 40) and (25–35). These results indicate that the DNA mobility shift assay described in the present paper is applicable for detection of -amyloid aggregation and identifying agents that modulate -amyloid aggregation. Because this assay employs relatively simple procedures and equipment, the assay may be very useful for screening inhibitors of -amyloid aggregation. REFERENCES 1. Selkoe, D. J. (1991) The molecular pathology of Alzheimer’s disease. Neuron 6, 487– 498. 2. Simmons, L. K., May, P. C., Tomaselli, K. J., Rydel, R. E., Fuson, K. S., Brigham, E. F., Wright, S., Lieberburg, I., Becker, G. W., and Brems, D. N. (1994) Secondary structure of amyloid beta peptide correlates with neurotoxic activity in vitro. Mol. Pharmacol. 45, 373–379. 3. Burdick, D., Soreghan, B., Kwon, M., Kosmoski, J., Knauer, M., Henschen, A., Yates, J., Cotman, C., and Glabe, C. (1992) Assembly and aggregation properties of synthetic Alzheimer’s A4/ beta amyloid peptide analogs. J. Biol. Chem. 267, 546 –554. 4. Pike, C. J., Burdick, D., Walencewicz, A. J., Glabe, C. G., and Cotman, C. W. (1993) Neurodegeneration induced by beta-amyloid peptides in vitro: The role of peptide assembly state. J. Neurosci. 13, 1676 –1687. 5. Takadera, T., Sakura, N., Mohri, T., and Hashimoto, T. (1993) Toxic effect of a beta-amyloid peptide (beta 22–35) on the hippocampal neuron and its prevention. Neurosci. Lett. 161, 41– 44.
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6. Ueda, K., Fukui, Y., and Kageyama, H. (1994) Amyloid beta protein-induced neuronal cell death: Neurotoxic properties of aggregated amyloid beta protein. Brain Res. 639, 240 – 244. 7. Delobette, S., Privat, A., and Maurice, T. (1997) In vitro aggregation facilitates beta-amyloid peptide-(25–35)-induced amnesia in the rat. Eur. J. Pharmacol. 319, 1– 4. 8. Giovannelli, L., Scali, C., Faussone-Pellegrini, M. S., Pepeu, G., and Casamenti, F. (1998) Long-term changes in the aggregation state and toxic effects of beta-amyloid injected into the rat brain. Neuroscience 87, 349 –357. 9. Hirakura, Y., Satoh, Y., Hirashima, N., Suzuki, T., Kagan, B. L., and Kirino, Y. (1998) Membrane perturbation by the neurotoxic Alzheimer amyloid fragment beta 25–35 requires aggregation and beta-sheet formation. Biochem. Mol. Biol. Int. 46, 787–794. 10. Fukuda, H., Shimizu, T., Nakajima, M., Mori, H., and Shirasawa, T. (1999) Synthesis, aggregation, and neurotoxicity of the Alzheimer’s Abeta1– 42 amyloid peptide and its isoaspartyl isomers. Bioorg. Med. Chem. Lett. 9, 953–956. 11. Lorenzo, A., and Yankner, B. A. (1994) -Amyloid neurotoxicity requires fibril formation and is inhibited by Congo red. Proc. Natl. Acad. Sci. USA 91, 12243–12247. 12. Pollack, S. J., Sadler, I. I. J., Hawtin, S. R., Tailor, V. J., and Shearman, M. S. (1995) Sulfated glycosaminoglycans and dyes attenuate the neurotoxic effects of -amyloid in rat PC12 cells. Neurosci. Lett. 184, 113–116. 13. Iversen, L. L., Mortishire-Smith, R. J., Pollack, S. J., and Shearman, M. S. (1995) The toxicity in vitro of -amyloid protein. Biochem. J. 311, 1–16. 14. Chomczynski, P., and Sacci, N. (1987) Single-step method of RNA isolation by acid guanidinium thiocyanate–phenol– chloroform extraction. Anal. Biochem. 162, 156 –159. 15. Boland, K., Behrens, M., Choi, D., Manias, K., and Perlmutter, D. H. (1996) The serpin-enzyme complex receptor recognizes soluble, nontoxic amyloid-peptide but not aggregated, cytotoxic amyloid-peptide. J. Biol. Chem. 271, 18032–18044. 16. Pike, C. J., Burdick, D., Walencewicz, A. J., Glabe, C. G., and Cotman, C. W. (1993) Neurodegeneration induced by -amyloid peptides in vitro: The role of peptide assembly state. J. Neurosci. 13, 1676 –1687. 17. Sambrook, J., Fritsch, E. F., and Maniatis, T. (1989) in Molecular Cloning: A Laboratory Manual, pp. 6.3– 6.19, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY. 18. Sambrook, J., Fritsch, E. F., and Maniatis, T. (1989) in Molecular Cloning: A Laboratory Manual, pp. 7.43–7.45, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY. 19. Laemmli, U. K. (1970) Cleavage of the structural proteins during the assembly of the head of the bacteriophage T4. Nature 277, 680 – 685. 20. Howlett, D., Cutler, P., Heales, S., and Camilleri, P. (1997) Hemin and related porphyrins inhibit beta-amyloid aggregation. FEBS Lett. 417, 249 –251.
Detection of -Amyloid Peptide Aggregation Using DNA Electrophoresis 1 Bong Whan Ahn, 2 Dong Up Song, Young Do Jung, Kee Oh Chay, Min A Chung, Sung Yeul Yang, and Boo Ahn Shin Chonnam National University Research Institute of Medical Sciences, Hakdong 5, Donggu, Kwangju 501-190, Korea
Received May 15, 2000
DNA could readily associate with the aggregated forms of the -amyloid peptides (1– 40) and (25–35), giving rise to a shift in the electrophoretic mobility of DNA. As a result, DNA was retained at the top of a 1% agarose gel. In contrast, the electrophoretic mobility of DNA was little influenced by the monomeric forms of (1– 40) and (25–30). DNA from different sources such as phage, Escherichia coli plasmid, and human gene showed similar results. However, the electrophoretic mobility of RNA was shifted by the monomeric (1– 40) and (25–35) as well as by the aggregated (1– 40) and (25–35). The association of DNA with the aggregated -amyloid peptides could occur at pH 4 –9. The inhibitory action of hemin on -amyloid aggregation could be confirmed using the DNA mobility shift assay. These results indicate that the DNA mobility shift assay is useful for kinetic study of -amyloid aggregation as well as for testing of agents that might modulate -amyloid aggregation. © 2000
and eventually to senile dementia. -AP can aggregate under physiological conditions to form fibrils consisting of antiparallel -pleated sheets (2, 3). Many studies have shown that -amyloid aggregates are neurotoxic (4 –10). Therefore, agents that can inhibit the aggregation of -AP may be of therapeutic benefit. Also, an assay system that detects the aggregated -AP but not the nonaggregated -AP is useful for identifying such agents. Polyanionic compounds such as Congo red, glycosaminoglycans, and proteoglycans can associate with -AP, modulating the aggregation or neurotoxicity of -AP (11–13). Nucleic acids are polyanionic at physiological pH. In a study with nucleic acids for effects on aggregation and neurotoxicity of -AP, we have found that DNA associates preferentially with the aggregated -AP to nonaggregated forms. In this paper, an assay system that can detect the aggregation of -AP employing a DNA probe is presented.
Academic Press
Key Words: -amyloid peptide; aggregation; DNA mobility.
Alzheimer’s disease is a neurodegenerative disease characterized by senile plaques, composed primarily of -amyloid peptides (-APs) 3 (1). Although the precise role of -AP in the pathogenesis of Alzheimer’s disease is not clear, aggregation of -AP has been suggested to be a critical event that can lead to neuronal damage 1 This study was supported by a grant from the Korea Health 21 R&D Project, Ministry of Health and Welfare, Republic of Korea (HMP-98-MS-0001). 2 To whom correspondence should be addressed. Fax: 82-62-2238321. E-mail: [email protected]. 3 Abbreviations used: -AP, -amyloid peptide; DMSO, dimethyl sulfoxide.
0003-2697/00 $35.00 Copyright © 2000 by Academic Press All rights of reproduction in any form reserved.
MATERIALS AND METHODS
(1– 40), (25–35), phage DNA, yeast tRNA phe, and hemin were purchased from Sigma Chemical Co. A constructed yeast plasmid, pSJ101-hRAD50, which contains a human RAD50 gene insert, was used as a preparation of plasmid and human DNAs after cutting with HindIII to yield a plasmid DNA piece of 9.0 kb and two human DNA pieces of 4.3 and 1.8 kb. Tissue RNA was extracted from a 3-month-old rat liver using acid guanidinium thiocyanate/phenol/chloroform as described by Chomczynski and Sacci (14). Monomeric and aggregated forms of (1– 40) and (25–35) were prepared by dissolving in dimethyl sulfoxide (DMSO) at 8 mg/ml and aging in water at 4 mM at 37°C for 5 days, respectively. These conditions have been shown in previous studies to result in predominantly soluble monomeric peptide and predominantly aggregated peptide, respectively (15, 16). For association reaction with nu401
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FIG. 1. Effects on DNA electrophoretic mobility of (1– 40) and (25–35) that had been aged in H 2O or prepared in DMSO. (A, B) One hundred micromolar (1– 40) (A) or (25–35) (B) was incubated at 37°C for 30 min with 1.5 g/ml DNA or HindIII digest of pSJ101-hRAD50 in 40 l of TBE buffer containing 10% (v/v) DMSO. After incubation, the mixtures were electrophoresed on 1.0% agarose gels for 1 h. Lane 1, DNA alone; lane 2, DNA incubated with -AP aged in H 2O; lane 3, DNA incubated with -AP prepared in DMSO; lane 4, pSJ101-hRAD50 digest alone; lane 5, pSJ101-hRAD50 digest incubated with -AP aged in H 2O; lane 6, pSJ101-hRAD50 digest incubated with -AP prepared in DMSO. The migration of DNA size markers is shown on the right margins. (C) SDS–PAGE analysis of (25–35) and (1– 40) aged in H 2O or prepared in DMSO. Lane 1, (25–35) aged in H 2O; lane 2, (25–35) prepared in DMSO; lane 3, (1– 40) aged in H 2O; lane 4, (1– 40) prepared in DMSO. The migration of marker proteins is shown on the right margin. The monomeric (25–35) in lanes 1 and 2 was suggested to be lost from the gel during staining procedures.
cleic acids, -AP was incubated at 37°C with 1.5 g/ml DNA or RNA, 20 mM buffer, and additives in a total volume of 40 l. At the selected time of incubation, the mixture was added with sample buffer and electrophoresed. In a standard assay to detect the aggregated -AP, -AP was incubated at 37°C for 30 min with 1.5 g/ml DNA in 40 l of TBE buffer (20 mM Tris– borate/1 mM EDTA, pH 8.0) containing 10% (v/v) DMSO. DNA samples were electrophoresed on a 1.0% agarose gel at 5 V/cm for 1 h and visualized by ethidium bromide (EtBr, 0.5 g/ml) staining (17). RNA samples were electrophoresed on a 1.2% agarose gel containing 2.2 M formaldehyde and Mops buffer (20 mM Mops/5 mM sodium acetate/1 mM EDTA, pH 7.0) at 5 V/cm for 1 h and visualized by EtBr staining (18). After electrophoresis, the gels were photographed with a Polaroid camera (Spectronics CH-810), and the fluorescence densities of DNA or RNA bands on the pictures were analyzed using a digital image analyzer (Fuji FLA-3000R). SDS–PAGE for -AP was carried out as described by Laemmli (19). Briefly, -AP was dissolved in sample buffer and electrophoresed on a 16% acrylamide gel under reducing conditions. RESULTS AND DISCUSSION
Effects of different forms of -AP on the electrophoretic mobility of nucleic acids were examined. As shown in Figs. 1A and 1B, incubation of phage DNA, Escherichia coli plasmid DNA, and human DNA with aggregated (1– 40) and (25–35) preparations resulted in shifts in the electrophoretic mobility of the DNAs. All the tested DNA bands were shifted to the
top of the gel, indicating that DNA formed high-molecular-weight complexes with the aggregated -APs. On the other hand, the electrophoretic mobilities of the
FIG. 2. Effects on RNA electrophoretic mobility of (25–35) and (1– 40) that had been aged in H2O or prepared in DMSO. One hundred micromolar (25–35) or (1– 40) was incubated at 37°C for 30 min with 1.5 g/ml rat liver RNA (A) or yeast tRNA (B) in 40 l of Mops buffer containing 10% (v/v) DMSO. After incubation, the mixtures were electrophoresed on 1.2% agarose gels for 1 h. Lane 1, RNA alone; lane 2, RNA incubated with (25–35) aged in H2O; lane 3, RNA incubated with (25–35) prepared in DMSO; lane 4, RNA incubated with (1– 40) aged in H 2O; lane 5, RNA incubated with (1– 40) prepared in DMSO.
DETECTION OF -AMYLOID AGGREGATION USING DNA ELECTROPHORESIS
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FIG. 3. Concentration-dependent effects of aggregated (1– 40) and (25–35) on DNA electrophoretic mobility. (A, B) One hundred micromolar (1– 40) (A) or (25–35) (B) composed of different proportions of two preparations that had been aged in H 2O or prepared in DMSO was incubated at 37°C for 30 min with 1.5 g/ml DNA in 40 l of TBE buffer containing 10% (v/v) DMSO. After incubation, the mixtures were electrophoresed on 1.0% agarose gels for 1 h. (C) DNA remaining at the original location on the gels (unassociated DNA) was quantitated by densitometry. Data represent the means ⫾ SD from triplicate experiments.
DNAs were little influenced by incubation of DNA with monomeric (1– 40) and (25–35). To confirm the monomeric and aggregated states of peptides dissolved in DMSO and aged in H 2O, respectively, aliquots of each peptide preparation were subjected to SDS–PAGE/ Coomassie blue staining (Fig. 1C). The results showed that significant amounts of the (1– 40) and (25–35) preparations that had been aged in H 2O existed as high-molecular-weight aggregates, whereas such aggregates were not detected in the (1– 40) and (25–35) preparations dissolved in DMSO. These results are similar to those shown in a previous study (15). However, results from experiments with RNA were quite different from those with DNA. As shown in Fig. 2, (1– 40) and (25–35) dissolved in DMSO as well as (1– 40) and (25–35) aged in H 2O could cause shifts in the electrophoretic mobility of tissue RNA and purified tRNA. The RNA bands were shifted to the top of the gel, indicating that RNA formed high-molecular-
weight complexes with both the monomeric and aggregated forms of -APs. The reason why DNA and RNA showed very different results in these experiments is not clear. But, two possibilities can be suggested: First, RNA may have higher affinities for -AP than DNA and bind many molecules of monomeric -AP to form high-molecular-weight complexes. Second, RNA may induce aggregation of -AP. Then, the aggregated -AP can readily associate with RNA. The above results suggested that DNA but not RNA could be used as a probe for identifying aggregated -AP in mixtures of monomeric and aggregated -AP. Therefore, we tested the DNA mobility shift assay with DNA for mixtures composed of different proportions of monomeric and aggregated -AP preparations (Fig. 3). As the proportions of aggregated (1– 40) and (25– 35) increased, the density of the DNA band remaining at the original location on the gels (unassociated DNA) was decreasing and the densities of the DNA
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FIG. 4. Time course of association between aggregated -AP and DNA. One hundred micromolar (1– 40) or (25–35) that had been aged in H 2O was incubated at 37°C for the indicated time with 1.5 g/ml DNA in 40 l of TBE buffer containing 10% (v/v) DMSO. After incubation, the mixtures were electrophoresed on 1.0% agarose gels for 1 h, and unassociated DNA was quantitated by densitometry. Data represent the means ⫾ SD from triplicate experiments.
bands at the top of the gels (associated DNA) were increasing. As shown in Fig. 3C, the density of unassociated DNA decreased almost linearly with the increment of the aggregated -AP concentration. In contrast to the case of the unassociated DNA band, it was difficult to quantitate the density of associated DNA bands because the shape and number of the bands at the top of gels were quite variable. Therefore, we recommend measuring the change in the density of unassociated DNA for the DNA mobility shift assay of -AP aggregation. Effects of a few reaction conditions on association between DNA and aggregated -AP were examined. Figure 4 shows that the association between DNA and aggregated -AP occurred quite rapidly. The association reaction was complete in less than 30 min for
FIG. 5. Effects of DMSO on association between aggregated -AP and DNA. One hundred micromolar (1– 40) or (25–35) that had been aged in H 2O was incubated at 37°C for 30 min with 1.5 g/ml DNA in 40 l of TBE buffer containing the indicated concentration (v/v) of DMSO. After incubation, the mixtures were electrophoresed on 1.0% agarose gels for 1 h, and unassociated DNA was quantitated by densitometry. Data represent the means ⫾ SD from triplicate experiments.
FIG. 6. pH-dependent association between aggregated -AP and DNA. One hundred micromolar (1– 40) (A) or (25–35) (B) that had been aged in H 2O was incubated at 37°C for 30 min with 1.5 g/ml DNA in 40 l of 20 mM buffer of the indicated pH containing 10% (v/v) DMSO. After incubation, the mixtures were electrophoresed on 1.0% agarose gels for 1 h. The buffers used were as follows: pH 4 – 6, sodium acetate; pH 7–10, Tris–HCl.
both (1– 40) and (25–35). DMSO, which was used as a vehicle for preparation of monomeric -AP, showed little effect on the association between DNA and aggregated -AP at 0 –20% (v/v) (Fig. 5). The association reaction could occur over a wide range of pH, at least pH 4 –9 for (1– 40) and pH 4 –10 for (25–35) (Fig. 6). Therefore, we employed the following reaction conditions in the standard assay to detect the aggregated -AP: specimens containing -AP were incubated at 37°C for 30 min with 1.5 g/ml DNA, 20 mM TBE buffer (pH 8.0), and 10% DMSO. Addition of 10% DMSO was required to prevent aggregation of monomeric -AP, which might occur during incubation, particularly in the case of (25–35). TBE buffer was chosen because the same buffer would also be used in the subsequent DNA electrophoresis step. We applied the DNA mobility shift assay for testing the action of inhibitors of -amyloid aggregation. Hemin is known to be an inhibitor of -amyloid aggregation (20). Under the conditions of Fig. 7, the amount of unassociated DNA decreased progressively with incubation time in the absence of hemin although the rates of decrease were different for (1– 40) and (25–
DETECTION OF -AMYLOID AGGREGATION USING DNA ELECTROPHORESIS
FIG. 7. Inhibitory effect of hemin on -AP aggregation as determined by DNA mobility shift assay. One hundred micromolar (1– 40) or (25–35) that had been prepared in DMSO was incubated at 37°C for the indicated period with 1.5 g/ml DNA in 40 l of TBE buffer containing 10% (v/v) DMSO in the presence or absence of 100 M hemin. After incubation, the mixtures were electrophoresed on 1.0% agarose gels for 1 h, and unassociated DNA was quantitated by densitometry. Data represent the means ⫾ SD from triplicate experiments.
35). However, in the presence of 100 M hemin, the amount of unassociated DNA remained unchanged with incubation time for both (1– 40) and (25–35). These results indicate that the DNA mobility shift assay described in the present paper is applicable for detection of -amyloid aggregation and identifying agents that modulate -amyloid aggregation. Because this assay employs relatively simple procedures and equipment, the assay may be very useful for screening inhibitors of -amyloid aggregation. REFERENCES 1. Selkoe, D. J. (1991) The molecular pathology of Alzheimer’s disease. Neuron 6, 487– 498. 2. Simmons, L. K., May, P. C., Tomaselli, K. J., Rydel, R. E., Fuson, K. S., Brigham, E. F., Wright, S., Lieberburg, I., Becker, G. W., and Brems, D. N. (1994) Secondary structure of amyloid beta peptide correlates with neurotoxic activity in vitro. Mol. Pharmacol. 45, 373–379. 3. Burdick, D., Soreghan, B., Kwon, M., Kosmoski, J., Knauer, M., Henschen, A., Yates, J., Cotman, C., and Glabe, C. (1992) Assembly and aggregation properties of synthetic Alzheimer’s A4/ beta amyloid peptide analogs. J. Biol. Chem. 267, 546 –554. 4. Pike, C. J., Burdick, D., Walencewicz, A. J., Glabe, C. G., and Cotman, C. W. (1993) Neurodegeneration induced by beta-amyloid peptides in vitro: The role of peptide assembly state. J. Neurosci. 13, 1676 –1687. 5. Takadera, T., Sakura, N., Mohri, T., and Hashimoto, T. (1993) Toxic effect of a beta-amyloid peptide (beta 22–35) on the hippocampal neuron and its prevention. Neurosci. Lett. 161, 41– 44.
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