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Lysosome instability in aged rat brain
Neuroscience Letters, 97 (1989) 215 220
215
Elsevier Scientific Publishers Ireland Ltd.
NSL 05886
Lysosome instability in aged rat brain Yu N a k a m u r a , Masatoshi Takeda, Hideo Suzuki, Hitoshi Morita, Kunitoshi Tada, Shiro Hariguchi and Tsuyoshi Nishimura Department q['Neuropsychiatry, Osaka University Medical School, Osaka (Japan) (Received 6 September 1988; Revised version received 17 October 1988; Accepted 19 October 1988J
Key words: Aging; Cathepsin D; fl-Glucuronidase; Lysosome; L-Leu-methyl ester The study of the age-dependent change in lysosomal enzyme activities of the cerebral tissue showed the significant increase of cathepsin D in the aged rat brain, while those offl-glucuronidase and acid phosphatase remained unchanged. The subcellular distribution study of cathepsin D and fl-glucuronidase revealed the increased activity of these enzymes in the cytosolic fraction from the aged brain. In vitro incubation of the lysosome fraction from the aged rat brain resulted in more leakage of these two enzymes, indicating the instability ot" the lysosome in the aged brain, which resembled the effect of L-Leu-methyl ester to the lysosome.
The activity of lysosomal enzymes is reportedly elevated in pathological conditions, such as Alzheimer's disease [10], multiple sclerosis [1] as well as rabbit brains with experimental neurofibrillary changes after aluminium intoxication [11]. Although the etiology of Alzheimer's disease is unknown, the presenile and senile onset of the disease strongly indicates the relevance of the aging mechanism in pathogenesis of this disease. There is a paper reporting a role of cathepsin D in degradation of microtubule-associated protein 1 (MAPI) and 2 (MAP2) of the aged rat brains, indicating the leakage ofcathepsin D into the cytosolic fraction [7]. In fact, neurofilament triplet proteins, glial fibrillary acidic protein and microtubule proteins are substrates for this enzyme in vitro. This paper reports the change of the activity of three lysosomal enzymes, cathepsin D, fl-glucuronidase, and acid phosphatase, in the aging process. The stability of the lysosome in the aged rat brain is further investigated by studying the subcellular distribution of the two lysosomal matrix enzymes, cathepsin D, and fl-glucuronidase. The leakage of the two enzymes from the lysosomal-mit~chondrial (LM) fraction is studied in the aged brain after incubation in lysosomeinstabilizing conditions [3] and compared with the effect of a lysosome-instabilizing reagent, L-Leu-methyl ester [4, 8].
Corre.s7~ondence." Y. Nakamura, Department of Neuropsychiatry, Osaka Universily Medical School, I-150, Fukushima, Fukushima-ku, Osaka, 553, Japan. 0304-3940/89/$ 03,50 (~) 1989 Elsevier Scientific Publishers Ireland Ltd.
216
The cerebral tissue was excised from 2-, 6-, 18-, and 36-month-old Wistar rats immediately after decapitation (6 rats in each age group), gently homogenized in 3 volumes of 0.25 M sucrose/0.2 M KCI with a Teflon-glass homogenizer (1000 rpm. 3 strokes), and the crude homogenate was centrifuged at 650 g, for 10 min at 4'~C to obtain the supernatant. The supernatant was further centrifuged at 10,000 g for 20 rain at 4°C to obtain the supernatant as the cytosolic fraction. The precipitate was dissolved in 1/5 volume of the crude supernatant with 0.25 M sucrose/0.2 M KC1/40 mM Tris-HCl buffer (pH 7.4) (Buffer A) as the LM fraction [5]. The crude homogenate from 2-, 6-, 18-, and 36-month-old rat brains was dissolved in 4 volumes of Buffer A containing 0.125% Triton X-100, and assayed for the activity of cathepsin D, p-glucuronidase, and acid phosphatase. The assay methods were those reported by Yamamoto [12], Fishman [2], and Kind and King [6], respectively. The cathepsin D and fl-glucuronidase activities were assayed with the cytosolic fraction obtained from the brain of each age group rats, and expressed as the percentage of the total activity. The total activity was obtained by measuring the brain crude homogenate from the corresponding age group rats. The LM fraction from each age group was diluted in 4 volumes of Buffer A containing 0.125% Triton X- 100, pipetted vigorously, incubated for 5 min at 25°C, and centrifuged at 12,000 g for 20 rain at 4°C [5]. The supernatant was assayed for the activities of cathepsin D and/~-glucuronidase as described above and the LM fraction activity was expressed as the percentage of the total activity. The LM fraction from each age group rat brains was diluted in 4 volumes of Buffer A with or without isotonic sucrose, and incubated for 10 min at 0°C, or for 40 min at 40°C [3]. After incubation, the sample was centrifuged at 12,000 g for 20 min and the activity of cathepsin D and fl-glucuronidase in the supernatant was assayed. The percent of Triton X-100 release was calculated by dividing the activities of the supernatant by those of the LM fraction obtained previously. The LM fraction from 2-month-old rats was incubated in the presence of L-Leumethyl ester (0-100 mM) in Buffer A (pH 7.4) for 40 or 80 min at 37°C. The sample was also incubated in Buffer A containing 0.5 mM EGTA/2 mM MgCI2/2 mM ATP for 40 min at 37°C [9] with or without L-Leu-methyl ester. After incubation, the sample was centrifuged at 12,000 g for 20 rain at 4°C. The activities of cathepsin D and p-glucuronidase in the supernatant were assayed and the percent of Triton X-100 release was calculated as described. All results were evaluated by Student's t-test. The brain homogenate prepared from the cerebrum of 2-, 6-, 18-, and 36-monthold rats was assayed for 3 lysosomal enzyme activities, cathepsin D, fl-glucuronidase~ and acid phosphatase. The age-dependent changes of these 3 enzyme levels were expressed in the specific activity per tissue protein (Fig. ! A-C). The level of cathepsin D showed the continuous increase along with aging, with 67% increase in 36-monthold rats. The level of fl-glucuronidase showed the biphasic change with the lowest value in 6-month-old rat brains. The level of acid phosphatase showed essentially no significant fluctuation. Since the amount of protein per wet tissue weight in the homogenate was unchanged throughout the aging process with the value of 127 +_ 18 mg/g tissue wet weight, the increase ofcathepsin D reflects the increase of this enzyme activity in the aged rat brains.
217
The percent activities ofcathepsin D and p-glucuronidase in the LM fraction were plotted (Fig. ID). In the brains of 2- and 6-month-old rats, the LM fraction contained about 35% of the total enzyme activity. On the aged brain, the level of cathepsin D and/:t-glucuronidase in the LM fraction declined to 10% for cathepsin D and 15% for /]-glucuronidase, respectively. On the other hand, the cytosolic fraction showed the continuous increase of these two enzyme activities with aging (Fig. I D). The statistical evaluation confirmed the significant difference in the distribution of these two enzymes in 18- and 36-month-old rats compared with those of 2-month-old rat brains. The lysosome stability was further estimated by measuring the activity ofcathepsin D and/~-glucuronidase in the supernatant after incubating the LM fraction in hypotonic and/or high temperature (37°C) conditions (Fig. 2A, B). After incubation at 0"C for 10 rain in 0.25 M sucrose, the cathepsin D activity in the supernatant from all age group brains remained at the same level. The LM fraction from thc 36-monthold rat brains, however, showed high cathepsin D activity in the supernatant after incubation in 0.25 M sucrose at 37°C, indicating the leakage of the enzyme from the LM fraction to the supernatant. When the LM fraction was incubated in hypotonic conditions, the leakage of cathepsin D was stimulated in 36-month-old rat brains at 3 7 C but not at 0'~C incubation, while the leakage of cathepsin D was essentially of the same level in 2-, 6- and 18-month-old animals. The results above were interpreted
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Fig. I. The age-dependent profiles of the levels of specific activity of cathepsin D (A), fl-glucuronidase (B), and acid phosphatase activity (C) in the crude homogenate from 2-, 6-, 18- and 36-month-old rat brains are plotted. The subcellular distribution of cathepsin D and fl-glucuronidase are plotted as the percentage of the total activity (D). A: cathepsin D activity of the lysosomal mitochondrial fraction; O: cathepsin D activity in the cytosolic fraction: ~ : fl-glucuronidase activity in the lysosomal mitochondrial fraction; and O: ~-glucuronidase activity in the cytosolic fraction. **Significant difference from the levels of 2-month-old rats with P<0.005.
218 (%)
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Fig. 2. The release of the lysosomal enzymes are plotted after in vitro incubation. The activities in the supernatant after incubation are expressed by the percentage of Triton X-100 release. A and B show the change of cathepsin D and fl-glucuronidase, respectively. The incubation conditions are as follows: • : 0.25 M sucrose at 0°C for 10 min: A: 0,05 M sucrose at 0°C for 10 min; O: 0.25 M sucrose at 37°C for 40 min; and ©: 0.05 M sucrose at 37'C for 40 min. Significant difference from the levels of 2-month-old rat brains with *P<0.02 and **P<0.005. The effect of L-Leu-methyl ester on the release of cathepsin D (C) and fl-glucuronidase (D) are plotted. C): at 37"C for 80 min; O: at 37°C for 40 min; and • : in the presence of 0.5 mM EGTA/2 mM MgCI2/2 mM ATP at 37"C for 40 min incubation.
as the decrease o f l y s o s o m e stability in the aged rat brains. The e x p e r i m e n t s with flg l u c u r o n i d a s e activity s h o w e d the s a m e results as those with c a t h e p s i n D, suggesting the l y s o s o m e instability in 3 6 - m o n t h - o l d rat brains. T o s u b s t a n t i a t e the e n z y m e l e a k a g e f r o m the lysosome, a l y s o s o m e - i n s t a b i l i z i n g reagent, L - L e u - m e t h y l ester, was i n c l u d e d into the i n c u b a t i o n c o n d i t i o n s o f the L M fraction f r o m 2 - m o n t h - o l d rat brain. A d d i t i o n o f L - L e u - m e t h y l ester to the i n c u b a t ing s o l u t i o n o f 0.25 M sucrose at 37°C actually s t i m u l a t e d the leakage o f c a t h e p s i n D (Fig. 2C) a n d f l - g l u c u r o n i d a s e (Fig. 2D) at c o n c e n t r a t i o n s higher t h a n 15 m M . T h e results s h o w e d t h a t longer i n c u b a t i o n in L - L e u - m e t h y l ester c a u s e d m o r e l e a k a g e o f c a t h e p s i n D a n d fl-glucuronidase. W h e n the L M fraction was i n c u b a t e d in the presence o f 0.5 m M E G T A , 2 m M MgCI2, a n d 2 m M A T P , the l e a k a g e o f these two enzymes was s u p p r e s s e d even in the presence o f L - L e u - m e t h y l ester. T h e p r e s e n t s t u d y r e p o r t e d the a g e - d e p e n d e n t profile o f the 3 l y s o s o m a l enzymes in 2-, 6-, 18-, a n d 3 6 - m o n t h - o l d rat brains. T h e level o f c a t h e p s i n D increased a l o n g with aging, s h o w i n g 48% increase in 18-month-old, a n d 67% increase in 3 6 - m o n t h - o l d rat brains. T h e d a t a agree with those o f M a t u s et al., r e p o r t i n g the elevated level o f
219 cathepsin D with two-fold increase in 27-month-old rats compared with 3-month-old animals [7]. The level offl-glucuronidase and acid phosphatase showed more or less the same level throughout aging. The observation of high cathepsin D activity with the unchanged level of acid phosphatase in the aged animal coincides with the observation reported in Alzheimer's disease brains [10] and rabbit brains with experimental neurofibrillary changes by aluminium intoxication [11]. The stability of the lysosome in the aged brain was further investigated in this study. The cathepsin D activity in the LM fraction decreased in the aged brains with the concomittant increase of the activity in the cytosolic fraction. The same result was obtained from the study of the distribution offl-glucuronidase activity. These observations can be interpreted as the decrease of lysosome stability in 18-, and 36-month-old rat brains compared with that of 2-month-old rat brains, since the fragile lysosomes can be destructed in the process of preparation resulting in the lower remaining activity in the LM fractions. The stability of the prepared LM fraction of the aged rat brains was measured after incubating in lysosome-instabilizing conditions. The significant leakage of cathepsin D from the LM fraction was observed in the hypotonic high-temperature incubation as well as the isotonic high-temperature incubation in 36-month-old rat brains. The leakage of fl-glucuronidase was slightly different from that of cathepsin D, showing the significant increase of leakage of this enzyme from the LM fraction from 36-month-old rat brains after all of the incubation conditions examined. This may indicate that the LM fractions from 2-, 6- and 18-month-old rats keep the same level of stability, while that from 36-month-old rats shows the instability in spite of the difference in degree of leakage among the enzymes. In order to varify the speculation above, we studied the effect of L-Leu-methyl ester to the prepared LM fraction. Since L-Leu-methyl ester is known to destruct the integrity of the lysosomal membrane [4], the effect of this reagent was compared with the results obtained from the incubation experiments with aged rat brains. When the LM fraction was incubated with L-Leu-methyl ester, it caused the increase of cathepsin D activity in the supernatant in a dose- and time-dependent manner. This fact can be interpreted such that the high cathepsin D activity in the supernatant fraction can be regarded as showing the leakage of this enzyme from the lysosome due to the disintegration of the lysosome membrane. In fact, the inclusion of magnesium and ATP in the incubation solution, which could stimulate the proton pump in the lysosome membrane by activation of Mg-ATPase activity [9], suppressed the leakage of cathepsin D from the LM fraction. The same results were obtained from the experiments with fl-glucuronidase activity as a parameter, strengthening the conclusion deduced from the experiments with cathepsin D. The data presented in this paper suggest the possibility that the lysosome stability is impaired in the aged rat brains, resulting in the leakage of the lysosomal enzymes into the cytoplasm. It is an interesting hypothesis to be verified by further investigations that the high cathepsin D activity in Alzheimer's disease brain is due to the impaired lysosome stability in the diseased brain. Part of this work is supported by a grant from the Ministry of Education of Japan (63570504) and the Sandoz foundation for gerontological research.
220 1 Einstein, E.R., Csejtey, J., Datal, K.B., Adams, C.W.M., Bayliss, O.B. and Hallpike, J.F., Proteolytic activity and basic protein loss in and around multiple sclerosis plaques: combined biochemical and histochemical observations. J. Neurochem., 19 (1972) 653--662. 2 Fishman, W.H. and Green, S., Enzymatic catalysis of glucuronyl transfer, J. Biol. Chem., 225 (1957) 435~452. 3 Gianetto, R. and de Duve, C., Tissue fractionation studies 4. Comparative study of the binding of acid phosphatase, p-glucuronidase and cathepsin by rat-liver particles, Biochem. J., 59 (1955) 433~438. 4 Goldman, R. and Kaplan A., Rupture of rat liver lysosomes mediated by 1-amino acid esters, Biochim. Biophys. Acta, 318 (1973) 205-216. 5 Ignaro, L.J., Effects of anti-inflamatory drugs on the stability of rat liver lysosomes in vitro, Biochem. Pharmac., 20 (1971) 2847 2860. 6 Kind, P.R.N. and King, E.J., Estimation of plasma phophatase by determination ofhydrolysed phenol with amino-antipyrine, J. Clin. Pathol., 7 (1954) 322 326. 7 Matus, A. and Green, G.D.J., Age-related increase in a cathepsin D like protease that degrades brain microtubule-associated proteins, Biochemistry, 26 (1987) 8083-8086. 8 Reeves, J.H., Decker, R.S., Crie, J.S. and Wildenthal, K., Intracellular disruption of heart lysosomes by leucine methyl ester: effect on protein degradation, Proc. Natl. Acad. Sci. U.S.A., 78 (1981) 4426 4429. 9 Schneider, D.L., The acidification rate of liver lysosomes in vitro: a role for the membranous ATPase as a proton pump, Biochem. Biophys. Res. Commun., 87 (1979) 559-565. 10 Takeda, M., Tada, K., Hariguchi, S. and Nishimura, T., Change of cathepsin D activities in vinblastine-injected rabbit brain and Alzheimer's disease brain, Neurochem. Res., 11 (1986) 117 (abstract). 11 Suzuki, H., Takeda, M., Nakamura, Y., Tada, K., Hariguchi, S., and Nishimura, T., Activities of lysosomal enzymes in rabbit brain with experimental neurofibrillary changes, Neurosci. Lett., 89 (1988) 234-239. 12 Yamamoto, K., Katsuda, N. and Kato, K., Affinity purification and properties of cathepsin E like acid protease from rat spleen, Eur. J. Biochem., 92 (1978) 499-508.
215
Elsevier Scientific Publishers Ireland Ltd.
NSL 05886
Lysosome instability in aged rat brain Yu N a k a m u r a , Masatoshi Takeda, Hideo Suzuki, Hitoshi Morita, Kunitoshi Tada, Shiro Hariguchi and Tsuyoshi Nishimura Department q['Neuropsychiatry, Osaka University Medical School, Osaka (Japan) (Received 6 September 1988; Revised version received 17 October 1988; Accepted 19 October 1988J
Key words: Aging; Cathepsin D; fl-Glucuronidase; Lysosome; L-Leu-methyl ester The study of the age-dependent change in lysosomal enzyme activities of the cerebral tissue showed the significant increase of cathepsin D in the aged rat brain, while those offl-glucuronidase and acid phosphatase remained unchanged. The subcellular distribution study of cathepsin D and fl-glucuronidase revealed the increased activity of these enzymes in the cytosolic fraction from the aged brain. In vitro incubation of the lysosome fraction from the aged rat brain resulted in more leakage of these two enzymes, indicating the instability ot" the lysosome in the aged brain, which resembled the effect of L-Leu-methyl ester to the lysosome.
The activity of lysosomal enzymes is reportedly elevated in pathological conditions, such as Alzheimer's disease [10], multiple sclerosis [1] as well as rabbit brains with experimental neurofibrillary changes after aluminium intoxication [11]. Although the etiology of Alzheimer's disease is unknown, the presenile and senile onset of the disease strongly indicates the relevance of the aging mechanism in pathogenesis of this disease. There is a paper reporting a role of cathepsin D in degradation of microtubule-associated protein 1 (MAPI) and 2 (MAP2) of the aged rat brains, indicating the leakage ofcathepsin D into the cytosolic fraction [7]. In fact, neurofilament triplet proteins, glial fibrillary acidic protein and microtubule proteins are substrates for this enzyme in vitro. This paper reports the change of the activity of three lysosomal enzymes, cathepsin D, fl-glucuronidase, and acid phosphatase, in the aging process. The stability of the lysosome in the aged rat brain is further investigated by studying the subcellular distribution of the two lysosomal matrix enzymes, cathepsin D, and fl-glucuronidase. The leakage of the two enzymes from the lysosomal-mit~chondrial (LM) fraction is studied in the aged brain after incubation in lysosomeinstabilizing conditions [3] and compared with the effect of a lysosome-instabilizing reagent, L-Leu-methyl ester [4, 8].
Corre.s7~ondence." Y. Nakamura, Department of Neuropsychiatry, Osaka Universily Medical School, I-150, Fukushima, Fukushima-ku, Osaka, 553, Japan. 0304-3940/89/$ 03,50 (~) 1989 Elsevier Scientific Publishers Ireland Ltd.
216
The cerebral tissue was excised from 2-, 6-, 18-, and 36-month-old Wistar rats immediately after decapitation (6 rats in each age group), gently homogenized in 3 volumes of 0.25 M sucrose/0.2 M KCI with a Teflon-glass homogenizer (1000 rpm. 3 strokes), and the crude homogenate was centrifuged at 650 g, for 10 min at 4'~C to obtain the supernatant. The supernatant was further centrifuged at 10,000 g for 20 rain at 4°C to obtain the supernatant as the cytosolic fraction. The precipitate was dissolved in 1/5 volume of the crude supernatant with 0.25 M sucrose/0.2 M KC1/40 mM Tris-HCl buffer (pH 7.4) (Buffer A) as the LM fraction [5]. The crude homogenate from 2-, 6-, 18-, and 36-month-old rat brains was dissolved in 4 volumes of Buffer A containing 0.125% Triton X-100, and assayed for the activity of cathepsin D, p-glucuronidase, and acid phosphatase. The assay methods were those reported by Yamamoto [12], Fishman [2], and Kind and King [6], respectively. The cathepsin D and fl-glucuronidase activities were assayed with the cytosolic fraction obtained from the brain of each age group rats, and expressed as the percentage of the total activity. The total activity was obtained by measuring the brain crude homogenate from the corresponding age group rats. The LM fraction from each age group was diluted in 4 volumes of Buffer A containing 0.125% Triton X- 100, pipetted vigorously, incubated for 5 min at 25°C, and centrifuged at 12,000 g for 20 rain at 4°C [5]. The supernatant was assayed for the activities of cathepsin D and/~-glucuronidase as described above and the LM fraction activity was expressed as the percentage of the total activity. The LM fraction from each age group rat brains was diluted in 4 volumes of Buffer A with or without isotonic sucrose, and incubated for 10 min at 0°C, or for 40 min at 40°C [3]. After incubation, the sample was centrifuged at 12,000 g for 20 min and the activity of cathepsin D and fl-glucuronidase in the supernatant was assayed. The percent of Triton X-100 release was calculated by dividing the activities of the supernatant by those of the LM fraction obtained previously. The LM fraction from 2-month-old rats was incubated in the presence of L-Leumethyl ester (0-100 mM) in Buffer A (pH 7.4) for 40 or 80 min at 37°C. The sample was also incubated in Buffer A containing 0.5 mM EGTA/2 mM MgCI2/2 mM ATP for 40 min at 37°C [9] with or without L-Leu-methyl ester. After incubation, the sample was centrifuged at 12,000 g for 20 rain at 4°C. The activities of cathepsin D and p-glucuronidase in the supernatant were assayed and the percent of Triton X-100 release was calculated as described. All results were evaluated by Student's t-test. The brain homogenate prepared from the cerebrum of 2-, 6-, 18-, and 36-monthold rats was assayed for 3 lysosomal enzyme activities, cathepsin D, fl-glucuronidase~ and acid phosphatase. The age-dependent changes of these 3 enzyme levels were expressed in the specific activity per tissue protein (Fig. ! A-C). The level of cathepsin D showed the continuous increase along with aging, with 67% increase in 36-monthold rats. The level of fl-glucuronidase showed the biphasic change with the lowest value in 6-month-old rat brains. The level of acid phosphatase showed essentially no significant fluctuation. Since the amount of protein per wet tissue weight in the homogenate was unchanged throughout the aging process with the value of 127 +_ 18 mg/g tissue wet weight, the increase ofcathepsin D reflects the increase of this enzyme activity in the aged rat brains.
217
The percent activities ofcathepsin D and p-glucuronidase in the LM fraction were plotted (Fig. ID). In the brains of 2- and 6-month-old rats, the LM fraction contained about 35% of the total enzyme activity. On the aged brain, the level of cathepsin D and/:t-glucuronidase in the LM fraction declined to 10% for cathepsin D and 15% for /]-glucuronidase, respectively. On the other hand, the cytosolic fraction showed the continuous increase of these two enzyme activities with aging (Fig. I D). The statistical evaluation confirmed the significant difference in the distribution of these two enzymes in 18- and 36-month-old rats compared with those of 2-month-old rat brains. The lysosome stability was further estimated by measuring the activity ofcathepsin D and/~-glucuronidase in the supernatant after incubating the LM fraction in hypotonic and/or high temperature (37°C) conditions (Fig. 2A, B). After incubation at 0"C for 10 rain in 0.25 M sucrose, the cathepsin D activity in the supernatant from all age group brains remained at the same level. The LM fraction from thc 36-monthold rat brains, however, showed high cathepsin D activity in the supernatant after incubation in 0.25 M sucrose at 37°C, indicating the leakage of the enzyme from the LM fraction to the supernatant. When the LM fraction was incubated in hypotonic conditions, the leakage of cathepsin D was stimulated in 36-month-old rat brains at 3 7 C but not at 0'~C incubation, while the leakage of cathepsin D was essentially of the same level in 2-, 6- and 18-month-old animals. The results above were interpreted
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Fig. I. The age-dependent profiles of the levels of specific activity of cathepsin D (A), fl-glucuronidase (B), and acid phosphatase activity (C) in the crude homogenate from 2-, 6-, 18- and 36-month-old rat brains are plotted. The subcellular distribution of cathepsin D and fl-glucuronidase are plotted as the percentage of the total activity (D). A: cathepsin D activity of the lysosomal mitochondrial fraction; O: cathepsin D activity in the cytosolic fraction: ~ : fl-glucuronidase activity in the lysosomal mitochondrial fraction; and O: ~-glucuronidase activity in the cytosolic fraction. **Significant difference from the levels of 2-month-old rats with P<0.005.
218 (%)
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postnatal age(month ) n = 6
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75 100 (raM)
Fig. 2. The release of the lysosomal enzymes are plotted after in vitro incubation. The activities in the supernatant after incubation are expressed by the percentage of Triton X-100 release. A and B show the change of cathepsin D and fl-glucuronidase, respectively. The incubation conditions are as follows: • : 0.25 M sucrose at 0°C for 10 min: A: 0,05 M sucrose at 0°C for 10 min; O: 0.25 M sucrose at 37°C for 40 min; and ©: 0.05 M sucrose at 37'C for 40 min. Significant difference from the levels of 2-month-old rat brains with *P<0.02 and **P<0.005. The effect of L-Leu-methyl ester on the release of cathepsin D (C) and fl-glucuronidase (D) are plotted. C): at 37"C for 80 min; O: at 37°C for 40 min; and • : in the presence of 0.5 mM EGTA/2 mM MgCI2/2 mM ATP at 37"C for 40 min incubation.
as the decrease o f l y s o s o m e stability in the aged rat brains. The e x p e r i m e n t s with flg l u c u r o n i d a s e activity s h o w e d the s a m e results as those with c a t h e p s i n D, suggesting the l y s o s o m e instability in 3 6 - m o n t h - o l d rat brains. T o s u b s t a n t i a t e the e n z y m e l e a k a g e f r o m the lysosome, a l y s o s o m e - i n s t a b i l i z i n g reagent, L - L e u - m e t h y l ester, was i n c l u d e d into the i n c u b a t i o n c o n d i t i o n s o f the L M fraction f r o m 2 - m o n t h - o l d rat brain. A d d i t i o n o f L - L e u - m e t h y l ester to the i n c u b a t ing s o l u t i o n o f 0.25 M sucrose at 37°C actually s t i m u l a t e d the leakage o f c a t h e p s i n D (Fig. 2C) a n d f l - g l u c u r o n i d a s e (Fig. 2D) at c o n c e n t r a t i o n s higher t h a n 15 m M . T h e results s h o w e d t h a t longer i n c u b a t i o n in L - L e u - m e t h y l ester c a u s e d m o r e l e a k a g e o f c a t h e p s i n D a n d fl-glucuronidase. W h e n the L M fraction was i n c u b a t e d in the presence o f 0.5 m M E G T A , 2 m M MgCI2, a n d 2 m M A T P , the l e a k a g e o f these two enzymes was s u p p r e s s e d even in the presence o f L - L e u - m e t h y l ester. T h e p r e s e n t s t u d y r e p o r t e d the a g e - d e p e n d e n t profile o f the 3 l y s o s o m a l enzymes in 2-, 6-, 18-, a n d 3 6 - m o n t h - o l d rat brains. T h e level o f c a t h e p s i n D increased a l o n g with aging, s h o w i n g 48% increase in 18-month-old, a n d 67% increase in 3 6 - m o n t h - o l d rat brains. T h e d a t a agree with those o f M a t u s et al., r e p o r t i n g the elevated level o f
219 cathepsin D with two-fold increase in 27-month-old rats compared with 3-month-old animals [7]. The level offl-glucuronidase and acid phosphatase showed more or less the same level throughout aging. The observation of high cathepsin D activity with the unchanged level of acid phosphatase in the aged animal coincides with the observation reported in Alzheimer's disease brains [10] and rabbit brains with experimental neurofibrillary changes by aluminium intoxication [11]. The stability of the lysosome in the aged brain was further investigated in this study. The cathepsin D activity in the LM fraction decreased in the aged brains with the concomittant increase of the activity in the cytosolic fraction. The same result was obtained from the study of the distribution offl-glucuronidase activity. These observations can be interpreted as the decrease of lysosome stability in 18-, and 36-month-old rat brains compared with that of 2-month-old rat brains, since the fragile lysosomes can be destructed in the process of preparation resulting in the lower remaining activity in the LM fractions. The stability of the prepared LM fraction of the aged rat brains was measured after incubating in lysosome-instabilizing conditions. The significant leakage of cathepsin D from the LM fraction was observed in the hypotonic high-temperature incubation as well as the isotonic high-temperature incubation in 36-month-old rat brains. The leakage of fl-glucuronidase was slightly different from that of cathepsin D, showing the significant increase of leakage of this enzyme from the LM fraction from 36-month-old rat brains after all of the incubation conditions examined. This may indicate that the LM fractions from 2-, 6- and 18-month-old rats keep the same level of stability, while that from 36-month-old rats shows the instability in spite of the difference in degree of leakage among the enzymes. In order to varify the speculation above, we studied the effect of L-Leu-methyl ester to the prepared LM fraction. Since L-Leu-methyl ester is known to destruct the integrity of the lysosomal membrane [4], the effect of this reagent was compared with the results obtained from the incubation experiments with aged rat brains. When the LM fraction was incubated with L-Leu-methyl ester, it caused the increase of cathepsin D activity in the supernatant in a dose- and time-dependent manner. This fact can be interpreted such that the high cathepsin D activity in the supernatant fraction can be regarded as showing the leakage of this enzyme from the lysosome due to the disintegration of the lysosome membrane. In fact, the inclusion of magnesium and ATP in the incubation solution, which could stimulate the proton pump in the lysosome membrane by activation of Mg-ATPase activity [9], suppressed the leakage of cathepsin D from the LM fraction. The same results were obtained from the experiments with fl-glucuronidase activity as a parameter, strengthening the conclusion deduced from the experiments with cathepsin D. The data presented in this paper suggest the possibility that the lysosome stability is impaired in the aged rat brains, resulting in the leakage of the lysosomal enzymes into the cytoplasm. It is an interesting hypothesis to be verified by further investigations that the high cathepsin D activity in Alzheimer's disease brain is due to the impaired lysosome stability in the diseased brain. Part of this work is supported by a grant from the Ministry of Education of Japan (63570504) and the Sandoz foundation for gerontological research.
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