- Email: [email protected]
Enhancement of MTT, a tetrazolium salt, exocytosis by amyloid β-protein and chloroquine in cultured rat astrocytes
Neuroscience Letters 266 (1999) 129±132
Enhancement of MTT, a tetrazolium salt, exocytosis by amyloid b -protein and chloroquine in cultured rat astrocytes Ichiro Isobe, Makoto Michikawa, Katsuhiko Yanagisawa* Department of Dementia Research, National Institute for Longevity Sciences, 36-3 Gengo, Morioka, Obu 474±8522, Japan Received 12 March 1999; received in revised form 22 March 1999; accepted 22 March 1999
Abstract The effect of amyloid b -protein (Ab ) on the cellular reducing activity has been a controversial issue. We determined the cellular reducing activity in cultured astrocytes using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and 2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium (WST-8) reduction assays following Ab treatment. MTT reduction was inhibited whereas WST-8 reduction was unaffected by the Ab treatment. Furthermore, the early extracellular appearance of MTT formazan, a reduced product of MTT, was observed in association with the rapid disappearance of intracellular formazan granules. Notably, similar results were obtained in cultures treated with chloroquine, a perturbant of membrane traf®cking. Our results suggest that MTT formazan exocytosis is enhanced in a similar manner by Ab and chloroquine and that this biological activity of Ab may underlie the pathogenesis of Alzheimer's disease. q 1999 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Alzheimer's disease; b -Amyloid; Astrocyte; 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; Chloroquine
In cell culture models to elucidate the cellular toxicity of amyloid b -protein (Ab ), 3-(4,5-dimethylthiazol-2-yl)-2,5diphenyltetrazolium bromide (MTT) is widely used. Although the cellular compartment responsible for MTT reduction remains to be determined, it is generally believed that in living cells the reduction of MTT to a formazan product re¯ects the pyridine nucleotide redox state of the cells [13,14] and that inhibition of MTT reduction by Ab is an early indicator of the Ab -induced impairment of the cellular reducing activity [6,13,14]. However, it has recently been reported that Ab inhibits cellular MTT reduction by enhancing MTT formazan exocytosis rather than by inhibiting MTT reduction directly [1,8]. In this study, we determined the cellular reducing activity in cultured astrocytes following Ab treatment using two kinds of tetrazolium salts in colorimetric assays; MTT and 2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium (WST-8). We also used chloroquine, which is known to facilitate some types of cellular exocytosis (e.g. lysosomal enzyme exocytosis (3]), to investigate whether this drug could induce the early appearance of extracellular MTT formazan similar to Ab . * Corresponding author. Tel.: 181-562-44-5651 ext. 834; fax: 181-562-44-6594. E-mail address: [email protected] (K. Yanagisawa)
Fig. 1. Effect of Ab on cellular reducing activity in astrocyteenriched cultures. Astrocytes, cultured in 48-well plates, were incubated with Ab 1±40 at the concentrations indicated for 3 h. The MTT reduction assay and the WST-8 reduction assay were performed as described in the text. Each column represents the mean value of three wells in a single experiment with the SEM. The experiments were repeated three times using the same procedure and yielded similar results. Analysis of variance (ANOVA) followed by Bonferroni's multiple comparison test was employed for evaluation of the signi®cance of differences in values among more than two groups, *P , 0:01:
0304-3940/99/$ - see front matter q 1999 Elsevier Science Ireland Ltd. All rights reserved. PII: S03 04 - 394 0( 9 9) 00 28 2- 7
130
I. Isobe et al. / Neuroscience Letters 266 (1999) 129±132
Astrocyte-enriched cultures were obtained from embryonic day 18 Sprague±Dawley rat embryos as described below. Brie¯y, isolated cortices of the fetuses were minced and incubated with 0.25% trypsin and 0.02% DNaseI in PBS (Ca 21 and Mg 21-free) for 20 min at 378C. Single cells, obtained by triturating the tissue with a Pasteur pipette, were cultured in Dulbecco's modi®ed Eagle's medium containing 10% fetal bovine serum. After 2±3 weeks, the cells on the astrocytic monolayer were removed by shaking the ¯asks vigorously at 200 rev./min for 17 h. The remaining monolayer cells were trypsinized (0.1%) and reseeded on plates of appropriate sizes at a density of 5 £ 104 /cm 2. For the treatment of the culture with Ab , synthetic Ab 1±40 (Peptide Institute) was dissolved in dimethyl sulfoxide
(DMSO) at a concentration of 8 mM, and diluted with culture medium immediately before use. The MTT reduction assay was performed according to the methods of Mosmann [10]. After preincubation of astrocytes with Ab or other agents, a 5 mg/ml MTT (Sigma) stock solution in PBS was added to each culture to give a ®nal MTT concentration of 0.5 mg/ml in the culture medium. After 2 h of incubation, the reduced formazans were solubilized with 0.01 M HCl in isopropanol and the absorbances were measured. WST-8 is a tetrazolium salt, which is reduced by cellular reducing systems coupled with NADH [10]. The WST-8 assay was performed according to the manufacturer's instructions (Seikagaku, Japan). After preincubation with Ab or other agents, a solution contain-
Fig. 2. Effect of Ab and chloroquine on the appearance of needle-like MTT formazan crystals on the cell surface in astrocyte-enriched cultures. Astrocytes cultured in 24-well plates were incubated without (A,D,G) or with 10 mM Ab 1±40 (B,E,H) or 50 mM chloroquine (C,F,I) for 3 h, and then MTT was added. Photographs were taken 10 (A±C), 30 (D±F) and 60 min (G±I) after the addition of MTT. Scale bar, 15 mm.
I. Isobe et al. / Neuroscience Letters 266 (1999) 129±132
ing WST-8 and 1-methoxy-5-methyl phenazinium methylsulfate (1-methoxy PMS; an electron-coupling agent) was added to the culture media (®nal concentrations: 0.5 mM WST-8 and 0.02 mM PMS). After 2 h of incubation, the absorbances were measured. Ab treatment caused inhibition of MTT reduction in a dose-dependent manner, whereas WST-8 reduction was not suppressed at all but rather was increased to a small extent (Fig. 1). Calcein AM staining, an indicator of cell viability, was also unaffected following Ab treatment (data not shown), suggesting that Ab does not impair cell viability or cellular reducing activity. On microscopic examination, we found that the extent of MTT formazan granular deposition inside the cells in Ab -treated cultures was comparable to that in control cultures up to 10 min of incubation with MTT (Fig. 2A,B). In the Ab -treated cultures, however, incubation with MTT resulted in the marked appearance of needle-like formazan crystals on the cell surface within 30 min, in association with the rapid disappearance of intracellular formazan granules (Fig. 2E,H). In contrast, in the control cultures, even after 60 min incubation, only about 40% of the astrocytes revealed needle-like formazan crystals on their cell surfaces, and the intracellular formazan granules were still seen prominently (Fig. 2D,G). In the experiment using chloroquine, intracellular formazan granules were generated, as they were in the controls (Fig. 2C); notably, the early extracellular appearance of MTT formazan in association with the rapid disappearance of intracellular formazan granules, was observed microscopically (Fig. 2F,I). Furthermore, chloroquine treatment of cultured astrocytes also caused an apparent inhibition of MTT reduction whereas WST-8 reduction was not affected at all (Fig. 3A). Chloroquine did not affect calcein AM staining (data not shown). These results corresponded precisely to those observed with Ab treatment. To investigate whether the effect of chloroquine on the enhancement of exocytosis was simply due to its inhibition of acidi®cation in endosomes and lysosomes, we incubated the cultures with ba®lomycin A1, a vacuolar H 1-ATPase inhibitor, which speci®cally inhibits acidi®cation without perturbing the formation of intracellular organelles [5]. As shown in Fig. 3, in the cultures incubated with ba®lomycin A1, no inhibition of reduction of MTT or WST-8 was observed (Fig. 3B) and no early extracellular appearance of MTT formazan was observed (data not shown). As both MTT and WST-8 are chie¯y reduced by pyridine nucleotide redox systems [4,13,14] and WST-8 is signi®cantly more sensitive than MTT [4], these results suggest that the inhibition of MTT reduction by Ab is not likely to be a re¯ection of the impairment of cellular reducing activity, at least not in the case of short-term incubation with Ab . Careful microscopic observations revealed that Ab did not abolish the intracellular formation of reduced MTT formazan granules at the initial stage of incubation with MTT, and that it induced an early, marked appearance of extracellular formazan in association with a
131
Fig. 3. Effect of chloroquine (A) and ba®lomycin A1 (B) on MTT reduction and WST-8 reduction in astrocyte-enriched cultures. Astrocytes cultured in 48-well plates were incubated with chloroquine for 3 h or with ba®lomycin for 45 min at the concentrations indicated. The MTT reduction assay and the WST-8 reduction assay were performed as described in materials and methods. Each column represents the mean value of three wells in a single experiment with the SEM. The experiments were repeated twice using the same procedure and yielded similar results. Statistical analysis was performed as described in the legend for Fig. 1. *P , 0:01.
rapid disappearance of the intracellular formazan granules. These results were consistent with the recent reports [1,8] and suggest that Ab does not impair the intracellular MTT reduction activity, and that enhancement of exocytosis of MTT formazan, which is thought to cause the early appearance of extracellular formazan [9], is closely related to the inhibition of MTT reduction. The results of the experiments with chloroquine support this idea, that is, inhibition of MTT reduction by chloroquine also occurred in association with enhancement of MTT formazan exocytosis without inhibition of WST-8 reduction. Furthermore, it has been reported that genistein, a blocker of MTT formazan exocytosis, completely abolishes the inhibition of cellular MTT reduction by Ab [8]. Here we report, for the ®rst time, that similar results were obtained in cultures treated with chloroquine. It was previously reported that chloroquine appears to induce cell biological changes, similar to those observed in Alzheimer's disease (AD) brains as follows: ®rst, chloroquine causes an increase in the number of clathrin molecules apposed to the
132
I. Isobe et al. / Neuroscience Letters 266 (1999) 129±132
plasma membrane [7]. In AD brains, the number of clathrincoated vesicles and coated pits at the plasma membrane is also increased in amyloid-associated cells [12]. Secondly, chloroquine induces a myopathy in experimental animals which shares some immunopathological features with AD [11,15]. Thus, it is well worth considering how chloroquine induces the enhanced exocytosis of MTT formazan. The majority of effects of chloroquine on cellular functions are believed to be due to the drug's inhibitory effect on the acidi®cation of intracellular organelles [7]. The negative results of ba®lomycin A1 treatment, however, suggest that enhanced exocytosis of MTT formazan by chloroquine is caused by mechanism(s) other than the impaired acidi®cation of vesicular organelles. Several lines of evidence suggest that Ab and chloroquine affect an aspect of cellular membrane traf®cking in endosome/lysosome systems [3,7,16] and that endosomal and lysosomal abnormalities may be early markers of neuronal dysfunction in AD brains [2]. In addition, intracellular MTT formazan granules are reported to partially colocalize with endosomes or lysosomes [9]. These ®ndings, coupled with our results, suggest that Ab -induced enhancement of exocytosis of MTT formazan may be caused by alteration of membrane traf®cking in endosomal/lysosomal systems, and that the consequent changes in cellular function(s) could lead to AD. This study was supported by a research grant for Longevity Sciences (8A-1) and Brain Research Science from the Ministry of Health and Welfare and by CREST (Core Research for Evolutional Science and Technology), Japan. The authors thank Y. Hanai for assistance in the preparation of this manuscript. [1] Abe, K. and Saito, H., Amyloid b protein inhibits cellular MTT reduction not by suppression of mitochondrial succinate dehydrogenase but by acceleration of MTT formazan exocytosis in cultured rat cortical astrocytes. Neurosci. Res., 31 (1998) 295±305. [2] Cataldo, A.M., Hamilton, D.J., Barnett, J.L., Paskevich, P.A. and Nixon, R.A., Properties of the endosomal-lysosomal system in the human central nervous system: disturbances mark most neurons in populations at risk to degenerate in Alzheimer's disease. J. Neurosci., 16 (1996) 186±199. [3] Claus, V., Jahraus, A., Tjelle, T., Berg, T., Kirschke, H., Faulstich, H. and Grif®ths, G., Lysosomal enzyme traf®cking between phagosomes, endosomes, and lysosomes in
[4]
[5]
[6] [7]
[8]
[9]
[10] [11] [12] [13]
[14]
[15]
[16]
J774 macrophages. Enrichment of cathepsin H in early endosomes. J. Biol. Chem., 273 (1998) 9842±9851. Ishiyama, M., Miyazono, Y. and Sasamoto, K., A highly water-soluble disulfonated tetrazolium salt as a chromogenic indicator for NADH as well as cell viability. Talanta, 44 (1997) 1299±1305. Johnson, L.S., Dunn, K.W., Pytowski, B. and McGraw, T.E., Endosome acidi®cation and receptor traf®cking: ba®lomycin A1slows receptorexternalization by a mechanism involving the receptor's internalization motif. Mol. Biol. Cell, 4 (1993) 1251±1266. Kato, M., Saito, H. and Abe, K., Nanomolar amyloid b protein-induced inhibition of cellular redox activity in cultured astrocytes. J. Neurochem., 68 (1997) 1889±1895. Lippincott-Schwartz, J. and Fambrough, D.M., Cycling of the integral membrane glycoprotein, LEP100, between plasma membrane and lysosomes: kinetic and morphological analysis. Cell, 49 (1987) 669±677. Liu, Y. and Schubert, D., Cytotoxic amyloid peptides inhibit cellular 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) reduction by enhancing MTT formazan exocytosis. J. Neurochem., 69 (1997) 2285±2293. Liu, Y., Peterson, D.A., Kimura, H. and Schubert, D., Mechanism of cellular 3-(4,5-dimethylthiazol-2-yl)-2,5diphenyltetrazolium bromide (MTT) reduction. J. Neurochem., 69 (1997) 581±593. Mosmann, M.P., Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays. J. Immunol. Methods, 65 (1983) 55±63. Murakami, N., Ihara, Y. and Nonaka, I., Chloroquine treated rat: a possible model for Alzheimer's disease. Muscle Nerve, 18 (1995) 123±125. Roher, B.A., Gray, E.G. and Paula-Barbosa, M., Alzheimer's disease: coated vesicles, coated pits and the amyloidrelated cell. Proc. R. Soc. Lond., 232 (1988) 367±373. Shearman, M.S., Hawtin, S.R. and Tailor, V.J., The intracellular component of cellular 3-(4,5-dimethylthiazol-2-yl)-2, 5diphenyltetrazolium bromide (MTT) reduction is speci®cally inhibited by b-amyloid peptides. J. Neurochem., 65 (1995) 218±227. Shearman, M.S., Ragan, C.I. and Iversen, L.L., Inhibition of PC12 cell redox activity is a speci®c, early indicator of the mechanism of b -amyloid-mediated cell death. Proc. Natl. Acad. Sci. USA, 91 (1994) 1470±1474. Tsuzuki, K., Fukatu, R., Takamaru, Y., Yoshida, T., Mafune, N., Kobayashi, K., Fujii, N. and Takahata, N., Co-localization of amyloid-associated proteins with amyloid beta in rat soleus muscle in chloroquine-induced myopathy: a possible model for amyloid b formation in Alzheimer's disease. Brain. Res., 699 (1995) 260±265. Yang, A.J., Chandswangbhuvana, D., Margol, L. and Glabe, C.G., Loss of endosomal/lysosomal membrane impermeability is an early event in amyloid Ab1-42 pathogenesis. J. Neurosci. Res., 52 (1998) 691±698.
Enhancement of MTT, a tetrazolium salt, exocytosis by amyloid b -protein and chloroquine in cultured rat astrocytes Ichiro Isobe, Makoto Michikawa, Katsuhiko Yanagisawa* Department of Dementia Research, National Institute for Longevity Sciences, 36-3 Gengo, Morioka, Obu 474±8522, Japan Received 12 March 1999; received in revised form 22 March 1999; accepted 22 March 1999
Abstract The effect of amyloid b -protein (Ab ) on the cellular reducing activity has been a controversial issue. We determined the cellular reducing activity in cultured astrocytes using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and 2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium (WST-8) reduction assays following Ab treatment. MTT reduction was inhibited whereas WST-8 reduction was unaffected by the Ab treatment. Furthermore, the early extracellular appearance of MTT formazan, a reduced product of MTT, was observed in association with the rapid disappearance of intracellular formazan granules. Notably, similar results were obtained in cultures treated with chloroquine, a perturbant of membrane traf®cking. Our results suggest that MTT formazan exocytosis is enhanced in a similar manner by Ab and chloroquine and that this biological activity of Ab may underlie the pathogenesis of Alzheimer's disease. q 1999 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Alzheimer's disease; b -Amyloid; Astrocyte; 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; Chloroquine
In cell culture models to elucidate the cellular toxicity of amyloid b -protein (Ab ), 3-(4,5-dimethylthiazol-2-yl)-2,5diphenyltetrazolium bromide (MTT) is widely used. Although the cellular compartment responsible for MTT reduction remains to be determined, it is generally believed that in living cells the reduction of MTT to a formazan product re¯ects the pyridine nucleotide redox state of the cells [13,14] and that inhibition of MTT reduction by Ab is an early indicator of the Ab -induced impairment of the cellular reducing activity [6,13,14]. However, it has recently been reported that Ab inhibits cellular MTT reduction by enhancing MTT formazan exocytosis rather than by inhibiting MTT reduction directly [1,8]. In this study, we determined the cellular reducing activity in cultured astrocytes following Ab treatment using two kinds of tetrazolium salts in colorimetric assays; MTT and 2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium (WST-8). We also used chloroquine, which is known to facilitate some types of cellular exocytosis (e.g. lysosomal enzyme exocytosis (3]), to investigate whether this drug could induce the early appearance of extracellular MTT formazan similar to Ab . * Corresponding author. Tel.: 181-562-44-5651 ext. 834; fax: 181-562-44-6594. E-mail address: [email protected] (K. Yanagisawa)
Fig. 1. Effect of Ab on cellular reducing activity in astrocyteenriched cultures. Astrocytes, cultured in 48-well plates, were incubated with Ab 1±40 at the concentrations indicated for 3 h. The MTT reduction assay and the WST-8 reduction assay were performed as described in the text. Each column represents the mean value of three wells in a single experiment with the SEM. The experiments were repeated three times using the same procedure and yielded similar results. Analysis of variance (ANOVA) followed by Bonferroni's multiple comparison test was employed for evaluation of the signi®cance of differences in values among more than two groups, *P , 0:01:
0304-3940/99/$ - see front matter q 1999 Elsevier Science Ireland Ltd. All rights reserved. PII: S03 04 - 394 0( 9 9) 00 28 2- 7
130
I. Isobe et al. / Neuroscience Letters 266 (1999) 129±132
Astrocyte-enriched cultures were obtained from embryonic day 18 Sprague±Dawley rat embryos as described below. Brie¯y, isolated cortices of the fetuses were minced and incubated with 0.25% trypsin and 0.02% DNaseI in PBS (Ca 21 and Mg 21-free) for 20 min at 378C. Single cells, obtained by triturating the tissue with a Pasteur pipette, were cultured in Dulbecco's modi®ed Eagle's medium containing 10% fetal bovine serum. After 2±3 weeks, the cells on the astrocytic monolayer were removed by shaking the ¯asks vigorously at 200 rev./min for 17 h. The remaining monolayer cells were trypsinized (0.1%) and reseeded on plates of appropriate sizes at a density of 5 £ 104 /cm 2. For the treatment of the culture with Ab , synthetic Ab 1±40 (Peptide Institute) was dissolved in dimethyl sulfoxide
(DMSO) at a concentration of 8 mM, and diluted with culture medium immediately before use. The MTT reduction assay was performed according to the methods of Mosmann [10]. After preincubation of astrocytes with Ab or other agents, a 5 mg/ml MTT (Sigma) stock solution in PBS was added to each culture to give a ®nal MTT concentration of 0.5 mg/ml in the culture medium. After 2 h of incubation, the reduced formazans were solubilized with 0.01 M HCl in isopropanol and the absorbances were measured. WST-8 is a tetrazolium salt, which is reduced by cellular reducing systems coupled with NADH [10]. The WST-8 assay was performed according to the manufacturer's instructions (Seikagaku, Japan). After preincubation with Ab or other agents, a solution contain-
Fig. 2. Effect of Ab and chloroquine on the appearance of needle-like MTT formazan crystals on the cell surface in astrocyte-enriched cultures. Astrocytes cultured in 24-well plates were incubated without (A,D,G) or with 10 mM Ab 1±40 (B,E,H) or 50 mM chloroquine (C,F,I) for 3 h, and then MTT was added. Photographs were taken 10 (A±C), 30 (D±F) and 60 min (G±I) after the addition of MTT. Scale bar, 15 mm.
I. Isobe et al. / Neuroscience Letters 266 (1999) 129±132
ing WST-8 and 1-methoxy-5-methyl phenazinium methylsulfate (1-methoxy PMS; an electron-coupling agent) was added to the culture media (®nal concentrations: 0.5 mM WST-8 and 0.02 mM PMS). After 2 h of incubation, the absorbances were measured. Ab treatment caused inhibition of MTT reduction in a dose-dependent manner, whereas WST-8 reduction was not suppressed at all but rather was increased to a small extent (Fig. 1). Calcein AM staining, an indicator of cell viability, was also unaffected following Ab treatment (data not shown), suggesting that Ab does not impair cell viability or cellular reducing activity. On microscopic examination, we found that the extent of MTT formazan granular deposition inside the cells in Ab -treated cultures was comparable to that in control cultures up to 10 min of incubation with MTT (Fig. 2A,B). In the Ab -treated cultures, however, incubation with MTT resulted in the marked appearance of needle-like formazan crystals on the cell surface within 30 min, in association with the rapid disappearance of intracellular formazan granules (Fig. 2E,H). In contrast, in the control cultures, even after 60 min incubation, only about 40% of the astrocytes revealed needle-like formazan crystals on their cell surfaces, and the intracellular formazan granules were still seen prominently (Fig. 2D,G). In the experiment using chloroquine, intracellular formazan granules were generated, as they were in the controls (Fig. 2C); notably, the early extracellular appearance of MTT formazan in association with the rapid disappearance of intracellular formazan granules, was observed microscopically (Fig. 2F,I). Furthermore, chloroquine treatment of cultured astrocytes also caused an apparent inhibition of MTT reduction whereas WST-8 reduction was not affected at all (Fig. 3A). Chloroquine did not affect calcein AM staining (data not shown). These results corresponded precisely to those observed with Ab treatment. To investigate whether the effect of chloroquine on the enhancement of exocytosis was simply due to its inhibition of acidi®cation in endosomes and lysosomes, we incubated the cultures with ba®lomycin A1, a vacuolar H 1-ATPase inhibitor, which speci®cally inhibits acidi®cation without perturbing the formation of intracellular organelles [5]. As shown in Fig. 3, in the cultures incubated with ba®lomycin A1, no inhibition of reduction of MTT or WST-8 was observed (Fig. 3B) and no early extracellular appearance of MTT formazan was observed (data not shown). As both MTT and WST-8 are chie¯y reduced by pyridine nucleotide redox systems [4,13,14] and WST-8 is signi®cantly more sensitive than MTT [4], these results suggest that the inhibition of MTT reduction by Ab is not likely to be a re¯ection of the impairment of cellular reducing activity, at least not in the case of short-term incubation with Ab . Careful microscopic observations revealed that Ab did not abolish the intracellular formation of reduced MTT formazan granules at the initial stage of incubation with MTT, and that it induced an early, marked appearance of extracellular formazan in association with a
131
Fig. 3. Effect of chloroquine (A) and ba®lomycin A1 (B) on MTT reduction and WST-8 reduction in astrocyte-enriched cultures. Astrocytes cultured in 48-well plates were incubated with chloroquine for 3 h or with ba®lomycin for 45 min at the concentrations indicated. The MTT reduction assay and the WST-8 reduction assay were performed as described in materials and methods. Each column represents the mean value of three wells in a single experiment with the SEM. The experiments were repeated twice using the same procedure and yielded similar results. Statistical analysis was performed as described in the legend for Fig. 1. *P , 0:01.
rapid disappearance of the intracellular formazan granules. These results were consistent with the recent reports [1,8] and suggest that Ab does not impair the intracellular MTT reduction activity, and that enhancement of exocytosis of MTT formazan, which is thought to cause the early appearance of extracellular formazan [9], is closely related to the inhibition of MTT reduction. The results of the experiments with chloroquine support this idea, that is, inhibition of MTT reduction by chloroquine also occurred in association with enhancement of MTT formazan exocytosis without inhibition of WST-8 reduction. Furthermore, it has been reported that genistein, a blocker of MTT formazan exocytosis, completely abolishes the inhibition of cellular MTT reduction by Ab [8]. Here we report, for the ®rst time, that similar results were obtained in cultures treated with chloroquine. It was previously reported that chloroquine appears to induce cell biological changes, similar to those observed in Alzheimer's disease (AD) brains as follows: ®rst, chloroquine causes an increase in the number of clathrin molecules apposed to the
132
I. Isobe et al. / Neuroscience Letters 266 (1999) 129±132
plasma membrane [7]. In AD brains, the number of clathrincoated vesicles and coated pits at the plasma membrane is also increased in amyloid-associated cells [12]. Secondly, chloroquine induces a myopathy in experimental animals which shares some immunopathological features with AD [11,15]. Thus, it is well worth considering how chloroquine induces the enhanced exocytosis of MTT formazan. The majority of effects of chloroquine on cellular functions are believed to be due to the drug's inhibitory effect on the acidi®cation of intracellular organelles [7]. The negative results of ba®lomycin A1 treatment, however, suggest that enhanced exocytosis of MTT formazan by chloroquine is caused by mechanism(s) other than the impaired acidi®cation of vesicular organelles. Several lines of evidence suggest that Ab and chloroquine affect an aspect of cellular membrane traf®cking in endosome/lysosome systems [3,7,16] and that endosomal and lysosomal abnormalities may be early markers of neuronal dysfunction in AD brains [2]. In addition, intracellular MTT formazan granules are reported to partially colocalize with endosomes or lysosomes [9]. These ®ndings, coupled with our results, suggest that Ab -induced enhancement of exocytosis of MTT formazan may be caused by alteration of membrane traf®cking in endosomal/lysosomal systems, and that the consequent changes in cellular function(s) could lead to AD. This study was supported by a research grant for Longevity Sciences (8A-1) and Brain Research Science from the Ministry of Health and Welfare and by CREST (Core Research for Evolutional Science and Technology), Japan. The authors thank Y. Hanai for assistance in the preparation of this manuscript. [1] Abe, K. and Saito, H., Amyloid b protein inhibits cellular MTT reduction not by suppression of mitochondrial succinate dehydrogenase but by acceleration of MTT formazan exocytosis in cultured rat cortical astrocytes. Neurosci. Res., 31 (1998) 295±305. [2] Cataldo, A.M., Hamilton, D.J., Barnett, J.L., Paskevich, P.A. and Nixon, R.A., Properties of the endosomal-lysosomal system in the human central nervous system: disturbances mark most neurons in populations at risk to degenerate in Alzheimer's disease. J. Neurosci., 16 (1996) 186±199. [3] Claus, V., Jahraus, A., Tjelle, T., Berg, T., Kirschke, H., Faulstich, H. and Grif®ths, G., Lysosomal enzyme traf®cking between phagosomes, endosomes, and lysosomes in
[4]
[5]
[6] [7]
[8]
[9]
[10] [11] [12] [13]
[14]
[15]
[16]
J774 macrophages. Enrichment of cathepsin H in early endosomes. J. Biol. Chem., 273 (1998) 9842±9851. Ishiyama, M., Miyazono, Y. and Sasamoto, K., A highly water-soluble disulfonated tetrazolium salt as a chromogenic indicator for NADH as well as cell viability. Talanta, 44 (1997) 1299±1305. Johnson, L.S., Dunn, K.W., Pytowski, B. and McGraw, T.E., Endosome acidi®cation and receptor traf®cking: ba®lomycin A1slows receptorexternalization by a mechanism involving the receptor's internalization motif. Mol. Biol. Cell, 4 (1993) 1251±1266. Kato, M., Saito, H. and Abe, K., Nanomolar amyloid b protein-induced inhibition of cellular redox activity in cultured astrocytes. J. Neurochem., 68 (1997) 1889±1895. Lippincott-Schwartz, J. and Fambrough, D.M., Cycling of the integral membrane glycoprotein, LEP100, between plasma membrane and lysosomes: kinetic and morphological analysis. Cell, 49 (1987) 669±677. Liu, Y. and Schubert, D., Cytotoxic amyloid peptides inhibit cellular 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) reduction by enhancing MTT formazan exocytosis. J. Neurochem., 69 (1997) 2285±2293. Liu, Y., Peterson, D.A., Kimura, H. and Schubert, D., Mechanism of cellular 3-(4,5-dimethylthiazol-2-yl)-2,5diphenyltetrazolium bromide (MTT) reduction. J. Neurochem., 69 (1997) 581±593. Mosmann, M.P., Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays. J. Immunol. Methods, 65 (1983) 55±63. Murakami, N., Ihara, Y. and Nonaka, I., Chloroquine treated rat: a possible model for Alzheimer's disease. Muscle Nerve, 18 (1995) 123±125. Roher, B.A., Gray, E.G. and Paula-Barbosa, M., Alzheimer's disease: coated vesicles, coated pits and the amyloidrelated cell. Proc. R. Soc. Lond., 232 (1988) 367±373. Shearman, M.S., Hawtin, S.R. and Tailor, V.J., The intracellular component of cellular 3-(4,5-dimethylthiazol-2-yl)-2, 5diphenyltetrazolium bromide (MTT) reduction is speci®cally inhibited by b-amyloid peptides. J. Neurochem., 65 (1995) 218±227. Shearman, M.S., Ragan, C.I. and Iversen, L.L., Inhibition of PC12 cell redox activity is a speci®c, early indicator of the mechanism of b -amyloid-mediated cell death. Proc. Natl. Acad. Sci. USA, 91 (1994) 1470±1474. Tsuzuki, K., Fukatu, R., Takamaru, Y., Yoshida, T., Mafune, N., Kobayashi, K., Fujii, N. and Takahata, N., Co-localization of amyloid-associated proteins with amyloid beta in rat soleus muscle in chloroquine-induced myopathy: a possible model for amyloid b formation in Alzheimer's disease. Brain. Res., 699 (1995) 260±265. Yang, A.J., Chandswangbhuvana, D., Margol, L. and Glabe, C.G., Loss of endosomal/lysosomal membrane impermeability is an early event in amyloid Ab1-42 pathogenesis. J. Neurosci. Res., 52 (1998) 691±698.