©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
Inhibition of Amyloid Protein Aggregation and Neurotoxicity by Rifampicin
ITS POSSIBLE FUNCTION AS A HYDROXYL RADICAL SCAVENGER (*)

(Received for publication, November 2, 1995; and in revised form, December 28, 1995)

Takami Tomiyama (§) Akira Shoji Ken-ichiro Kataoka Yorimasa Suwa Satoshi Asano Hideshi Kaneko Noriaki Endo

From the Teijin Institute for Biomedical Research, 4-3-2 Asahigaoka, Hino, Tokyo 191, Japan

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
REFERENCES

ABSTRACT

Aggregation of physiologically produced soluble amyloid beta protein (Abeta) to insoluble, neurotoxic fibrils is a crucial step in the pathogenesis of Alzheimer's disease. Aggregation studies with synthetic Abeta1-40 peptide by the thioflavin T fluorescence assay and electron microscopy and cytotoxicity assays using rat pheochromocytoma PC12 cells showed that an antibiotic, rifampicin, and its derivatives, which possess a naphthohydroquinone or naphthoquinone structure, inhibited Abeta1-40 aggregation and neurotoxicity in a concentration-dependent manner. Hydroquinone, p-benzoquinone, and 1,4dihydroxynaphthalene, which represent partial structures of the aromatic chromophore of rifampicin derivatives, also inhibited Abeta1-40 aggregation and neurotoxicity at comparable molar concentrations to rifampicin. Electron spin resonance spectrometric analysis revealed that the inhibitory activities of those agents correlated with their radical-scavenging ability on hydroxyl free radical, which was shown to be generated in cell-free incubation of Abeta1-40 peptide. These results suggest that at least one mechanism of rifampicin-mediated inhibition of Abeta aggregation and neurotoxicity involves scavenging of free radicals and that rifampicin and/or appropriate hydroxyl radical scavengers may have therapeutic potential for Alzheimer's disease.


INTRODUCTION

Amyloid beta protein (Abeta), (^1)a 39-43 amino acid peptide, is a primary component of the amyloid that is deposited in the brains of patients with Alzheimer's disease (AD). Abeta is physiologically produced as a soluble form by enzymatic cleavage of the larger precursor, termed amyloid precursor protein (1, 2, 3) . Soluble Abeta is not toxic and its physiological function is not known; however, it has been shown that aggregation of Abeta to insoluble fibrils causes neurotoxic change of the peptide(4, 5, 6) . Therefore, inhibition of this process would seem to be an effective therapeutic strategy for AD.

The mechanisms of Abeta aggregation and neurotoxicity are not completely known. Recently, it was suggested that free radical generation may be involved in the processes of Abeta aggregation and/or neurotoxicity (7, 8, 9) . Those hypotheses imply that appropriate radical scavengers could inhibit Abeta aggregation and/or neurotoxicity.

It was previously reported that non-demented elderly leprosy patients showed an unusual absence of senile plaques in their brains compared with age-matched controls(10) . Although that finding itself is still a matter of controversy(11) , we surmised that some drug being used for leprosy might be preventing Abeta aggregation, resulting in the absence of amyloid deposition. Thus, we tested two well known anti-leprosy drugs, dapsone and rifampicin, and found that rifampicin inhibited Abeta1-40 aggregation and neurotoxicity in vitro(12) . Rifampicin is a semisynthetic derivative of the rifamycins, a class of antibiotics that are fermentation products of Nocardia mediterranei (for a review, see (13) ). The common structure of rifamycins is a naphthohydroquinone or naphthoquinone chromophore spanned by an aliphatic ansa chain. Taken together with the above free radical hypotheses, this structural feature of rifampicin suggests that this drug may function as a radical scavenger with its naphthohydroquinone ring in inhibiting Abeta aggregation and neurotoxicity.

In the present study, we confirmed the published finding that free radicals are generated in cell-free incubation of Abeta1-40 peptide (7) and show that at least the hydroxyl radical is involved in this process. Also, we show that the inhibitory activities of rifampicin and its derivatives against Abeta aggregation and neurotoxicity correlate with their radical-scavenging ability on hydroxyl radical, which function arises from their naphthohydroquinone or naphthoquinone structure. These results implicate therapeutic potential of rifampicin for AD and provide useful information for developing new compounds for the treatment of AD.


MATERIALS AND METHODS

Agents and Peptides

Rifampicin and rifamycin SV are commercially available (Sigma), while other rifampicin derivatives were synthesized from rifamycin SV in our laboratory according to the methods of Kump and Chen (U.S. Patent 5 003 070, 1991), which are summarized in Fig. 1. All other agents were obtained from Tokyo Kasei Kogyo Co. (Japan), but three natural radical scavengers (alpha-tocopherol, ascorbic acid, and beta-carotene) were purchased from Sigma. Abeta1-40 peptide was synthesized by ordinary solid-phase methods with Fmoc (N-(9-fluorenyl)methoxycarbonyl) amino acids (Applied Biosystems Inc.) using a 431A peptide synthesizer (Applied Biosystems Inc.) and purified by reverse-phase high performance liquid chromatography using a Cosmosil 5C(4)-AR-300 column (Nacalai Tesque Inc., Japan).


Figure 1: Structures and preparation of rifampicin derivatives. a, MnO(2), CH(2)Cl(2), room temperature, 15 min; b, 1) N-(2,4,6-trimethylbenzyl)piperazine, dioxane, 70 °C, 3 h; 2) ascorbic acid, room temperature, 30 min; c, 1) MnO(2), CH(2)Cl(2), room temperature, 15 min; 2) ClCOC(CH(3))(3), pyridine, room temperature, 30 min; 3) Zn powder, tetrahydrofuran, 1 N HCl, room temperature, 15 min; d, ClCOC(CH(3))(3), pyridine, 50 °C, 30 min; e, CH(3)OCH(2)CH(2)OH, reflux, 130 °C, 5 h; f, 1) H(2), Pd/C, EtOH, room temperature, 3 days; 2) ascorbic acid, room temperature, 30 min.



Aggregation Studies

Peptide aggregation was measured by the thioflavin T (ThT) fluorescence assay, in which the fluorescence intensity reflects the degree of aggregation(14) . All agents to be tested were dissolved in dimethyl sulfoxide (Me(2)SO) at various concentrations. The solubility of beta-carotene in Me(2)SO was very low, so suspensions of beta-carotene were used. The Abeta1-40 peptide was solubilized in double deionized water at a concentration of 40 µM and dispensed into Eppendorf tubes (50 µl/tube). One µl of each test agent solution was added to the tubes in triplicate, and then the peptide solutions were mixed with an equal volume of 2 times phosphate-buffered saline solution. Control peptide solutions containing 1% Me(2)SO without any test agent were also prepared. The solutions were incubated at 37 °C for 7 days, and then the ThT fluorescence assay was performed using a FP-770 spectrofluorometer (Jasco, Japan), as described previously(12) . Fibril formation by Abeta1-40 peptide was also examined by electron microscopy using a H-7100 electron microscope (Hitachi, Japan), as described previously(12) . No bacterial contamination was observed in the peptide solutions even after a 7-day incubation.

Cytotoxicity Assays

The cytotoxic effects of Abeta were assessed by 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) reduction with rat pheochromocytoma PC12 cells(15) . The PC12 cells were prepared as described previously(12) . The peptide solutions incubated with each test agent for 7 days were added to the cell culture at a final peptide concentration of 2 µM. After a 2-day incubation, the MTT assay was performed as described previously(15) .

Electron Spin Resonance (ESR) Spectrometry

The Abeta1-40 peptide was dissolved in double deionized water to a concentration of 1 mg/ml in the presence of 50 mMN-tert-butyl-alpha-phenylnitrone (PBN) (Sigma). One of the solutions was mixed with a 1/10 volume of Me(2)SO. The solutions were transferred into 75-µl aqueous quartz ESR flat cells and incubated at room temperature. After various incubation times, the ESR spectra were determined using a JES-FE2XG ESR spectrometer (JEOL, Japan) under conditions of a magnetic field of 329.0 ± 5 millitesla, a magnetic power of 20 milliwatts, 9.225 GHz, a response of 1 s, a temperature of 25 °C, an amplitude of 5,000, a sweep time of 16 min, and a modulation of 100 kHz times 0.08 millitesla. To evaluate the radical-quenching ability of agents, the hydroxyl radical generated by the Fenton reaction (16) was used. All agents to be tested were dissolved in acetonitrile at various concentrations. One-hundred microliters of 2 mM FeSO(4) solution was dispensed into Eppendorf tubes, and then 20 µl of agent, 100 µl of 2 mM H(2)O(2), and 2 µl of 5 mM 5,5-dimethyl-1-pyrroline-N-oxide (DMPO) (Aldrich) were sequentially added to the tubes. The ESR spectra were determined at 3 min after the addition of DMPO under the same conditions as described above, except for using an amplitude of 1,000 and a sweep time of 4 min.


RESULTS

To identify the structure required for the inhibitory activities of rifampicin, we synthesized a panel of rifampicin derivatives (Fig. 1) and tested them for effects on Abeta1-40 aggregation and neurotoxicity (Table 1). Fig. 2shows the time course of aggregation of Abeta1-40 peptide without any test agent. Rifampicin inhibited Abeta aggregation and neurotoxicity in a concentration-dependent manner. This inhibitory effect of rifampicin against Abeta aggregation was confirmed by electron microscopy ( Table 1and Fig. 3). A control peptide solution showed apparent amyloid-like fibrils of Abeta1-40, while very few fibrils were observed in the presence of 100 µM rifampicin. Rifamycin SV and rifamycin S, which possess a naphthohydroquinone and naphthoquinone ring, respectively, also inhibited Abeta aggregation and neurotoxicity, without any functional group at position C-3 of their aromatic ring. From these observations and the free radical hypotheses(7, 8, 9) , we speculated that the active structure of rifampicin responsible for the inhibition of Abeta's activities might be the naphthohydroquinone (or naphthoquinone) ring. In support of this, hydroquinone, p-benzoquinone, and 1,4-dihydroxynaphthalene, which represent partial structures of the aromatic chromophore of rifampicin derivatives, inhibited Abeta aggregation and neurotoxicity at comparable molar concentrations to rifampicin. The hydroxyl group at position C-1 of the naphthohydroquinone ring must be essential for the inhibitory activities of rifampicin, because substitution of the hydroxyl group at this position, or cyclization between the hydroxyl group at position C-1 and the carbon at position C-15 of the ansa chain, considerably reduced the activities, as demonstrated with RFM-007 and RFM-008, respectively. In addition, p-methoxyphenol also showed only weak inhibition, suggesting that both of the hydroxyl groups at positions C-1 and C-4 are equally required for the inhibitory activities. On the contrary, the hydroxyl group at position C-8 and the double bonds of the ansa chain, which are essential for the antibacterial activity of rifampicin(13) , were not necessary for the inhibitory activities against Abeta aggregation and neurotoxicity, as shown with RFM-005 and RFM-030, respectively. As well, the functional group at position C-3, which modulates the antibacterial activity of rifampicin probably by influencing transport of the drug molecule through the bacterial wall and membrane(13) , appeared not to affect the anti-Abeta activities, as shown with rifamycin SV and RFM-002. Thus, we concluded that the inhibitory activities of rifampicin against Abeta aggregation and neurotoxicity arise from its naphthohydroquinone (or naphthoquinone) structure.




Figure 2: Time course of Abeta1-40 peptide aggregation. Abeta1-40 peptides at 20 µM in phosphate-buffered saline were incubated at 37 °C. Peptide aggregation was monitored by the ThT fluorescence assay. Each point represents the mean ± S.D. for triplicate determinations.




Figure 3: Electron micrographs of Abeta1-40 peptide aged in the presence of test agents. Abeta1-40 peptides at 20 µM were incubated at 37 °C for 7 days in the presence of 100 µM test agents. The control peptide solution containing 1% Me(2)SO showed apparent fibrils of Abeta1-40 peptide (A). Rifampicin (B), hydroquinone (C), and 1,4-dihydroxynaphthalene (D) inhibited the fibril formation, whereas RFM-008 (E) and p-methoxyphenol (F) did not. The scale bar is 0.2 µm.



As a reference for evaluating the activity of rifampicin, three natural radical scavengers, alpha-tocopherol (vitamin E), ascorbic acid (vitamin C), and beta-carotene (provitamin A), were also examined. These vitamins were all effective in inhibiting Abeta aggregation, but their activities were weaker than that of rifampicin. Thus, 10-100-fold higher concentrations were necessary for the vitamins to achieve the same degree of inhibition of Abeta aggregation as shown by rifampicin.

As mentioned already, the naphthohydroquinone (or naphthoquinone) structure of rifampicin is speculated to function as a radical scavenger. Thus, using ESR spectrometry, we examined the ability of rifampicin and the related agents to quench free radicals (Fig. 4). Initially we confirmed the published finding that free radicals are generated in cell-free incubation of Abeta1-40 peptide(7) . With PBN as a spin trapping agent, an Abeta1-40 solution showed an obvious three-line spectrum (alpha(N) = 17.1 G) after 3-day incubation. When the peptide was incubated in the presence of Me(2)SO, it showed a different ESR spectrum (alpha(N) = 17.2 G and alpha(N) = 16.0 G, alpha^H = 3.5 G) partly due to new species of free radicals. Since Me(2)SO is known to react with the hydroxyl radical to produce the methyl radical(17) , and actually, the ESR spectrum of hydroxyl radical in the presence of Me(2)SO (alpha(N) = 16.4 G, alpha^H = 3.6 G) corresponded to some portion of the ESR spectrum of an Abeta1-40 solution containing Me(2)SO, the above ESR result suggests that at least the hydroxyl radical is involved in the radical-generating process in Abeta solution. Based on this observation and because of the simplicity of the radical-generating system, we focused our experiments on the radical-quenching ability of agents in relation to the hydroxyl radical generated by the Fenton reaction(16) . With DMPO instead of PBN, a typical 1:2:2:1 four-line spectrum (alpha(N) = alpha^H = 14.9 G) due to hydroxyl radical (18) was detected in the control solution. As expected from the molecular structure, rifampicin quenched the hydroxyl radical in a concentration-dependent manner. The two analogs, hydroquinone and 1,4-dihydroxynaphthalene, also diminished the hydroxyl radical, while RFM-008 and p-methoxyphenol showed no quenching ability at concentrations up to 1 mM. These results suggest that the inhibitory activities of the agents against Abeta aggregation and neurotoxicity correlate with their radical-quenching ability on hydroxyl radical and that at least one mechanism by which rifampicin and its analogs inhibit Abeta aggregation and neurotoxicity involves scavenging of free radicals.


Figure 4: ESR spectra of Abeta1-40 peptide and hydroxyl radical in the presence of test agents. A, ESR spectrum with PBN of free radicals generated in cell-free incubation of Abeta1-40 peptide in the absence (a) or presence (b) of Me(2)SO. c, ESR spectrum with PBN of hydroxyl radical generated by the Fenton reaction in the presence of Me(2)SO. B, ESR spectra with DMPO of hydroxyl radical generated by the Fenton reaction in the presence of test agents. The control solution without any test agent showed a 1:2:2:1 four-line spectrum (a). Rifampicin (b), hydroquinone (c), and 1,4-dihydroxynaphthalene (d) quenched free radicals, whereas RFM-008 (e) and p-methoxyphenol (f) did not.




DISCUSSION

Neuronal degeneration is a significant pathological feature of AD brains, and the toxicity of Abeta has been implicated in the neuronal damage. Although the molecular mechanisms of Abeta neurotoxicity are not completely known, there is general agreement that the neurotoxicity of Abeta correlates with its state of aggregation(4, 5) . Furthermore, it was shown that fibril formation by Abeta is definitely necessary for its neurotoxicity(6) . These observations suggest that drugs that inhibit Abeta aggregation may be able to protect neurons from Abeta toxicity and hence may have therapeutic potential for AD. Here we have shown that rifampicin and its derivatives, which inhibit Abeta aggregation, also inhibit Abeta neurotoxicity.

It was recently proposed that the Abeta1-40 peptide, in aqueous solution, spontaneously fragments into free radical peptides, which may react with one another to generate covalently bonded aggregates and may also attack nerve cell membranes to induce neuronal degeneration(7) . We confirmed that free radicals are generated in cell-free incubation of Abeta1-40 peptide, and our findings that radical scavengers inhibit Abeta aggregation and neurotoxicity may support this hypothesis. However, the possibility cannot be ruled out that free radicals are generated independently of Abeta peptide and then trapped and stabilized by the peptide to be detected in ESR analysis. Oxidation of Abeta by free radicals was shown to cause peptide aggregation, which was prevented by radical scavengers(8) .

It was also demonstrated that Abeta induces increased intracellular H(2)O(2) accumulation, which may cause oxidative damage on neurons probably via hydroxyl radical generation(9) . A number of antioxidants and the H(2)O(2)-degrading enzyme, catalase, protected cells from H(2)O(2) accumulation and also Abeta neurotoxicity(9) . Our findings that hydroxyl radical scavengers inhibited Abeta neurotoxicity may also support this model. They did not refer to any relationship between Abeta aggregation and Abeta-induced H(2)O(2) production. It may be that aggregated Abeta has more potent activity to induce H(2)O(2) production than soluble Abeta.

In addition to AD, there are numerous other human amyloidoses. Although those amyloids contain different proteins, all amyloidogenic peptides are characterized by the antiparallel beta-sheet conformation(19) . It was recently shown that these amyloidogenic peptides may share a common cytotoxic mechanism(20, 21) . For example, three amyloidogenic peptides, i.e. amylin, calcitonin, and atrial natriuretic peptide, are all toxic to clonal and primary neurons and increase the intracellular H(2)O(2) level(20) . The cytotoxicity of these peptides is suggested to be mediated through a free radical pathway indistinguishable from that of Abeta(20) . These observations imply that rifampicin could also inhibit the cytotoxicity of other amyloidogenic peptides besides Abeta and that agents that inhibit amyloid fibril formation or cytotoxicity may have therapeutic potential for several different amyloidoses.

In summary, we have shown that rifampicin and its derivatives inhibit Abeta aggregation and neurotoxicity, and their inhibitory activities are attributed to the naphthohydroquinone or naphthoquinone structure, which possibly functions as a radical scavenger. Although the ansa chain appears not to be essential for the inhibitory activities, its lipophilicity may contribute to transport of the drug molecule into the brain in vivo(22) . Our data presented here provide useful information for investigating the mechanisms of Abeta aggregation and neurotoxicity and developing new compounds for the treatment of AD.


FOOTNOTES

*
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed. Tel.: 81-425-86-8135; Fax: 81-425-87-5516.

(^1)
The abbreviations used are: Abeta, amyloid beta protein; AD, Alzheimer's disease; ThT, thioflavin T; Me(2)SO, dimethyl sulfoxide; MTT, 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide; PBN, N-tert-butyl-alpha-phenylnitrone; DMPO, 5,5-dimethyl-1-pyrroline-N-oxide.


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©1996 by The American Society for Biochemistry and Molecular Biology, Inc.