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Accumulation of amyloid β-protein precursor (APP) in Purkinje cells and increase of amino-terminal fragments of APP in cerebrum and cerebellum of aged rat brain
BRAIN RESEARCH ELSEVIER
Brain Research 643 (1994) 319-323
Short Communication
Accumulation of amyloid/3-protein precursor (APP) in Purkinje cells and increase of amino-terminal fragments of APP in cerebrum and cerebellum of aged rat brain Yu Nakamura a , b Masatoshi Takeda a.., Hisayoshi Niigawa c, Fuyuki Kametani Shiro Hariguchi a Isao Yoshida b, Shogo Kitajima b Tsuyoshi Nishimura a
d
a Department of Neuropsychiatry, Osaka University Medical School, 2-2 Yamadaoka, Suita-shi, Osaka, 553, Japan, b Department of Neuropsychiatry, Nissei Hospital, 6-3-8 Itachibori, Nishi-ku, Osaka, 550, Japan, c Sayama Hospital, lwamuro, Sayama-shi, Osaka, 589, Japan, d Department of Molecular Biology, Tokyo Institute of Psychiatry, 2-1-8 Kamikitazawa, Setagaya-ku, Tokyo, 156, Japan
(Accepted 18 January 1994)
Abstract
In aged rat brain, amyloid /3-protein precursor (APP) is accumulated in dendrites and cell bodies of Purkinje cells as full-length or truncated APP, because dendrites and cell bodies are positively stained by antibodies against both the amino- and carboxy-termini of APP. Western blot analysis of homogenates of brains of aged and young rats showed no apparent differences except for an increase in amino-terminal fragments in cerebrum and cerebellum of aged rat. These results indicate that the expression, transport or metabolism of APP in specific regions of brains may be affected by the aging process. Key words: Amyloid fl-protein precursor; Aging; Purkinje cell
Amyloid /3-protein, which appears in plaques, is considered to be closely involved in the pathology of Alzheimer's disease, and its precursor, amyloid /3-protein precursor (APP), may also be involved because APP is also deposited in plaques or dystrophic neurites [2,4]. Although the expression and metabolism of APP have been intensively studied to elucidate the pathogenesis of Alzheimer's disease, the roles of this protein are still unknown. Expression of APP is increased by neurotoxins, such as kinate or ibotenic acid [10,14], and APP has recently been reported to have neuroprotective effects [7], indicating that APP might play a role of neuroprotection against damage and stress. On the other hand, the aging process has been believed to be a major pathological factor of Alzheimer's disease. Therefore, the influence of the aging process on expression and metabolism of APP in brain tissue should be investigated from the viewpoint that both the aging process and APP may be indispensable for the patho-
* Corresponding author. Fax: (81) (6) 879-3059. 0006-8993/94/$07.00 © 1994 Elsevier Science B.V. All rights reserved SSDI 0006-8993(94)00093-R
genesis of Alzheimer's disease. In this report the distribution of APP in aged and young rat brain was immunohistochemically studied and the various brain regions analyzed by Western blot. Three- and 36-month-old Wistar rats (JCL strain, each group consisting of 4 rats) were perfused and fixed with 10% formalin/phosphate-buffered saline and placed in 20% sucrose for 1 day. 15 ~m-thick free-floating sections prepared with a cryostat were stained using a Vectastain Kit in 10% normal goat serum/Tris-buffered saline [17] using 22Cll (Boehringer-Mannheim; its epitope lies between residues 60-100 of APP) [15], R17 (raised against residues 1-24 of fl-amyloid protein) [8] and R37 (raised against residues 687-695 of APP) [3]. For Western blot analysis, the brains of three aged and young rats, sacrificed under pentobarbital anesthesia, were immediately separated into cerebrum, striatum, hippocampus, pons and cerebellum on ice, homogenated with 2 vols. wet wt. of 10 M urea/ 10 mM bis-Tris-HC1, pH 6.5, using a polytron [12]. The homogenates were centrifuged at 100,000 x g for 39 min at 4°C, and the resulting super-
320
Y. Nakamura et aL / Brain Research 643 (1994) 319-323
natants were analyzed by Western blot using the Protoblot system (Promega). By H E staining there was no apparent difference between aged and young rats throughout the brain except for a slight decrease in thickness of cortexes of cerebrum and cerebellum. By immunohistochemistry, using the antibodies against APP, an obvious difference was found only in the cerebellum, with no apparent findings in the other regions (data not shown). In the molecular layer of cerebellum of aged rat, a granular structure was clearly stained by 22Cll, R17 and R37 (Fig. 1). Some cell bodies of Purkinje cells of aged rat were simultaneously strongly stained by all the antibodies, whereas those of young rat were weakly or negatively stained (Fig. 1). Investigating the sections minutely, the granular structure in the molecular layer revealed dendrites of Purkinje cells (Fig. 2). There were no irregular dendrites of Purkinje cells in aged rats compared with dystrophic neurites found in brains with Alzheimer's disease. On the other hand, axons of
Purkinje cells were not positively stained, and torpedo-like regional axonal swelling of Purkinje cells was not seen. On Western blot analysis of APP, an increase in amino-terminal fragments (approximately 20-30 kDa) was found in cerebellum of aged rats, whereas the amounts of full-length and truncated APP were not different (Fig. 3A). However, antibody R37 against the carboxy-terminus of APP showed no differences (Fig. 3B), even when the homogenates were analyzed using 16% Tris-Tricine gel (data not shown). Of the other parts examined, only homogenates of cerebrum of aged rats showed an increase in amino-terminal fragments of APP compared with that of young rats (Fig. 4A), while the homogenates of hippocampus, striatum and pons showed no difference (Fig. 4BCD). In cerebrum, hippocampus, straitum and pons, R37 showed no difference, as also seen in cerebellum (data not shown). In spite of the strong staining of free-floating sections, R17 only weakly reacted with APP on Western blot
Fig. 1. Irnmunohistochemical study of young (A-C) and aged (D-F) rat cerebellum (low magnification) using 22Cll (1 /xg/ml, A,D), R37 (1,000 × of antiserum, B,E) and R17 (1,000 × of antiserum, C,F). Bar = 200 ~zm.
321
Y. Nakamura et al. / Brain Research 643 (1994) 319-323
Fig. 2. Immunohistochemical study of cerebellthe aged rat (high magnification) using 22Cll (1 /zg/ml, A,D), R37 (1,000 x , B,E) and R17 (1,000 × , C,F). Bar = 50/xm.
KDa Y 0 200=" ,~ , ~ ;
Y 0
YO
Y 0
Y 0
YO
Y 0 .
.
.
Y 0 YO .
.
.
.
.
.
.
.
.
.
.
.
t
-
67" j ~ 5
30D" 20" f,
-~
: i
A
~.
!
B
C
D
E
F
G
H
d
Fig. 3. Western blot analysis of aged (O) and young (Y) rat cerebellum. The homogenates were subjected to 10% SDS-PAGE, and transferred onto a nitrocellulose membrane. The membranes were stained in 3% normal goat serum/Tris-buffered saline by 22Cll (1 /,g/ml, A), R37 (1,000 x of antiserum, B), anti-MAP2 (1 ~g/ml, C), anti-MAP1B (1/zg/ml, D), CKHC9 (5 x (medium), E), anti-neurofilament-H (1/zg/ml, F), anti-neurofilament-L ( 1 / , g / m l , G), SY-38 (1/*g/ml, H) and LCB.2(1/zg/ml, J) using Protoblot system. The asterisk in A points to an increase in amino-terminal fragments (20-30 kDa) of APP in aged rat cerebellum.
322
YO
A
Y. Nakamura et al. / Brain Research 643 (1994) 319-323
YO
B
Y 0
C
YO
D
Fig. 4. Amino-terminal fragments (20-30 kDa) of APP in cerebrum (A), hippocampus (B), striatum (C) and pons (D) of aged (O) and young (Y) rats, visualized by the antibody, 22Cll (1 ~g/ml), using the Protoblot system.
analysis (data not shown). Other antibodies, e.g. against MAP2 (Amersham), MAP1B (Amersham), kinesin (CKHC9, provided by Toyoshima, I.), neurofilament-H (Amersham), neurofilament-L [11], synaptophysin (Boehringer-Mannheim), and neuron-specific clathrin light chain b (LCB.2; provided by F.M. Brodsky) [16] showed no difference between aged and young rats on Western blot analysis (Fig. 3). This immunohistochemical study showed that fulllength or truncated APP accumulates in cell bodies and dendrites of Purkinje cells of aged rat since they were positively stained by antibodies against the amino-terminus, /3-region and carboxy-terminus of APP. It has been reported that APP is transported along the fast axonal transport system [6], and that this can be interfered with by ligation of axons [6], colchicine [13], ibotenic acid [10] and IDPN [5]. Considering these studies, the findings described herein suggest the possibility that APP might be accumulated partly as a result of an impairment of axonal transport during the aging process. We have recently reported an abnormal distribution of clathrin in brain with Alzheimer's disease, indicating impairment of slow axonal transport [12]. The aging process may affect axonal transport [9], leading to the impairment of APP transport. Recently, in aged rat brain, APP was found in swollen neurites [5]. Although we found neither the typical swollen neurites nor torpedo-like axonal swelling of Purkinje cells in this study, our results and the other report may indicate that quite similar incidents occur with aging. The difference between the two reports may be due to differences in the strains and the feeding conditions used. In this study, age-dependent changes of APP were further investigated by Western blot which showed no apparent differences between aged and young rat throughout the brain except for an increase in aminoterminal fragments in cerebrum and cerebellum of aged rats. In other regions, the amino-terminal fragments were present even in brain of young rat, indicating regional differences of brain aging. The increase in amino-terminal fragments of APP in the specific regions suggests that the metabolism of APP might be
affected by the aging process. Although Western blot analysis indicated that the total amount of APP did not change extensively with aging, it is possible that the amount of APP might only be increased in Purkinje cells, since the amounts of APP of Purkinje ceils were so small that they would not affect the total amounts of APP in whole cerebellum. Because increased expression of APP was seen in senescent cultured fibroblasts [1], an increase in APP expression might be found in specific neurons of aged brain. On the other hand, Western blot analysis has demonstrated that MAP2, MAP1B, kinesin, neurofilament-H/L, synaptophysin, and clathrin light chain are not affected by the aging process, indicating that the changes in metabolism of APP are more sensitive to the aging process than those of other proteins related to axonal transport. All these findings suggest that the aging process may affect the expression, transport or metabolism of APP. It would contribute to elucidating the pathogenesis of Alzheimer's disease to investigate the effect of aging on APP metabolism further. [1] Adler, M.J., Coronel, C., Shelton, E., Seegmiller, J.E. and Dewji, N.N., Increased gene expression of Alzheimer disease /3-amyloid precursor protein in senescent-cultured fibroblasts, Proc. Natl. Acad. Sci. USA, 88 (1991) 16-20. [2] Cras, P., Kawai, M., Lowery, D., Gonzalez-Dewhit, P., Greenberg, B. and Perry, G., Senile plaque neurites in Alzheimer disease accumulate amyloid precursor protein, Proc. Natl. Acad. Sci. USA, 88 (1991) 7552-7556. [3] Ishii, H., Karnetani, F., Haga, S. and Sato, M., The immunohistochemical demonstration of subsequences of the precursor of the amyloid A4 protein in senile plaques in senile plaques in Alzheimer's disease, Neuropathol. Appl. NeurobioL, 15 (1989) 135-147. [4] Joachim, C., Games, D., Morris, J., Ward, P., Frenkel, D. and Selkoe, D., Antibodies to non-/3 regions of the /3-amyloid precursor protein detect a subset of senile plaques, Am. J. Pathol., 138 (1991) 373-384. [5] Kawarabayashi, T., Shoji, M., Yamaguchi, H., Tanaka, M., Harigaya, Y., Ishiguro, K. and Hirai, S., Amyloid /3 protein precursor accumulates in swollen neurites throughout rat brain with aging, Neurosci. Lett., 153 (1993) 73-76. [6] Koo, E.H., Sisodia, S.S., Archer, D.R., Martin, L.J., Weidemann, A., Beyreuther, K., Fischer, P., Masters, C.L. and Price, D.L., Precursor of amyloid protein in Alzheimer disease undergoes fast anterograde axonal transport, Proc. Natl. Acad. Sci. USA, 87 (1990) 1561-1565. [7] Mattson, M.P., Cheng, B., Culwell, A.R., Esch, F.S., Lieberburg, I. and Rydel, R.E., Evidence for excitoprotective and interneuronal calcium-regulating roles for secreted forms of the /3amyloid precursor protein, Neuron, 10 (1993) 243-254. [8] McGreer, P.L., Akiyama, H., Kawamata, T., Yamada, T., Walker, D.G. and Ishii, T., Immunohistochemical localization of /3-amyloid precursor protein sequences in Alzheimer and normal brain tissue by light and electron microscopy, J. Neurosci. Res., 31 (1992) 428-442. [9] McQuarrie, I.G., Brady, S.T. and Lasek, R.J., Retardation in slow axonal transport of cytoskeletal elements during maturation and aging, NeurobioL Aging, 10 (1989) 359-365. [10] Nakamura, Y., Takeda, M., Niigawa, H., Hariguchi, S. and Nishimura, T., Amyloid /3 protein precursor deposition in rat
Y. Nakamura et aL / B r a i n Research 643 (1994) 319-323
hippocampus lesioned by ibotenic acid injection, Neurosci. Lett., 136 (1992) 95-98. [11] Nakamura, Y., Takeda, M., Aimoto, S., Hariguchi, S., Kitajima, S. and Nishimura, T., Acceleration of bovine neurofilament L assembly by deprivation of acidic tail domain, Eur. J. Biochern., 212 (1993) 565-571. [12] Nakamura, Y., Takeda, M., Yoshimi, K., Hattori, H., Hariguchi, S., Kitajima, S., Hashimoto, S. and Nishimura, T., Involvement of clathrin light chains in the pathology of Alzheimer's disease, Acta NeuropathoL, 84 (1994) 23-31. [13] Shigematsu, K. and McGeer, P.L., Accumulation of amyloid precursor protein in neurons after intraventricular injection of colhicine, Am. J. PathoL, 140 (1992) 787-794. [14] Siman, R., Card, J.P., Nelson, R.B. and Davis L.G., Expression
323
of /3-amyloid precursor protein in reactive astrocytes following neuronal damage, Neuron, 3 (1989) 275-285. [15] Weidemann, A., Konig, G., Bunke, D., Fischer, P., Salbaum, J.M., Masters, C.L. and Beyreuther, K., Identification, biogenesis, and localization of precursors of Alzheimer's disease A4 amyloid protein, Cell, 57 (1989) 115-126. [16] Wong, D.H., Ignatius, M.J., Parosky, G., Parham, P., Trojanowski, J.Q. and Brodsky, F.M., Neuron-specific expression of high-molecular-weight clathrin light chain, J. Neurosci., 10 (1990) 3025-3031. [17] Yoshimi, K., Takeda, M., Nishimura, T., Kudo, T., Nakamura, Y., Tada, K. and Iwata, N., An immunohistochemical study of MAP2 and clathrin in gerbil hippocampus after cerebral ischemia, Brain Res., 560 (1991) 149-158.
Brain Research 643 (1994) 319-323
Short Communication
Accumulation of amyloid/3-protein precursor (APP) in Purkinje cells and increase of amino-terminal fragments of APP in cerebrum and cerebellum of aged rat brain Yu Nakamura a , b Masatoshi Takeda a.., Hisayoshi Niigawa c, Fuyuki Kametani Shiro Hariguchi a Isao Yoshida b, Shogo Kitajima b Tsuyoshi Nishimura a
d
a Department of Neuropsychiatry, Osaka University Medical School, 2-2 Yamadaoka, Suita-shi, Osaka, 553, Japan, b Department of Neuropsychiatry, Nissei Hospital, 6-3-8 Itachibori, Nishi-ku, Osaka, 550, Japan, c Sayama Hospital, lwamuro, Sayama-shi, Osaka, 589, Japan, d Department of Molecular Biology, Tokyo Institute of Psychiatry, 2-1-8 Kamikitazawa, Setagaya-ku, Tokyo, 156, Japan
(Accepted 18 January 1994)
Abstract
In aged rat brain, amyloid /3-protein precursor (APP) is accumulated in dendrites and cell bodies of Purkinje cells as full-length or truncated APP, because dendrites and cell bodies are positively stained by antibodies against both the amino- and carboxy-termini of APP. Western blot analysis of homogenates of brains of aged and young rats showed no apparent differences except for an increase in amino-terminal fragments in cerebrum and cerebellum of aged rat. These results indicate that the expression, transport or metabolism of APP in specific regions of brains may be affected by the aging process. Key words: Amyloid fl-protein precursor; Aging; Purkinje cell
Amyloid /3-protein, which appears in plaques, is considered to be closely involved in the pathology of Alzheimer's disease, and its precursor, amyloid /3-protein precursor (APP), may also be involved because APP is also deposited in plaques or dystrophic neurites [2,4]. Although the expression and metabolism of APP have been intensively studied to elucidate the pathogenesis of Alzheimer's disease, the roles of this protein are still unknown. Expression of APP is increased by neurotoxins, such as kinate or ibotenic acid [10,14], and APP has recently been reported to have neuroprotective effects [7], indicating that APP might play a role of neuroprotection against damage and stress. On the other hand, the aging process has been believed to be a major pathological factor of Alzheimer's disease. Therefore, the influence of the aging process on expression and metabolism of APP in brain tissue should be investigated from the viewpoint that both the aging process and APP may be indispensable for the patho-
* Corresponding author. Fax: (81) (6) 879-3059. 0006-8993/94/$07.00 © 1994 Elsevier Science B.V. All rights reserved SSDI 0006-8993(94)00093-R
genesis of Alzheimer's disease. In this report the distribution of APP in aged and young rat brain was immunohistochemically studied and the various brain regions analyzed by Western blot. Three- and 36-month-old Wistar rats (JCL strain, each group consisting of 4 rats) were perfused and fixed with 10% formalin/phosphate-buffered saline and placed in 20% sucrose for 1 day. 15 ~m-thick free-floating sections prepared with a cryostat were stained using a Vectastain Kit in 10% normal goat serum/Tris-buffered saline [17] using 22Cll (Boehringer-Mannheim; its epitope lies between residues 60-100 of APP) [15], R17 (raised against residues 1-24 of fl-amyloid protein) [8] and R37 (raised against residues 687-695 of APP) [3]. For Western blot analysis, the brains of three aged and young rats, sacrificed under pentobarbital anesthesia, were immediately separated into cerebrum, striatum, hippocampus, pons and cerebellum on ice, homogenated with 2 vols. wet wt. of 10 M urea/ 10 mM bis-Tris-HC1, pH 6.5, using a polytron [12]. The homogenates were centrifuged at 100,000 x g for 39 min at 4°C, and the resulting super-
320
Y. Nakamura et aL / Brain Research 643 (1994) 319-323
natants were analyzed by Western blot using the Protoblot system (Promega). By H E staining there was no apparent difference between aged and young rats throughout the brain except for a slight decrease in thickness of cortexes of cerebrum and cerebellum. By immunohistochemistry, using the antibodies against APP, an obvious difference was found only in the cerebellum, with no apparent findings in the other regions (data not shown). In the molecular layer of cerebellum of aged rat, a granular structure was clearly stained by 22Cll, R17 and R37 (Fig. 1). Some cell bodies of Purkinje cells of aged rat were simultaneously strongly stained by all the antibodies, whereas those of young rat were weakly or negatively stained (Fig. 1). Investigating the sections minutely, the granular structure in the molecular layer revealed dendrites of Purkinje cells (Fig. 2). There were no irregular dendrites of Purkinje cells in aged rats compared with dystrophic neurites found in brains with Alzheimer's disease. On the other hand, axons of
Purkinje cells were not positively stained, and torpedo-like regional axonal swelling of Purkinje cells was not seen. On Western blot analysis of APP, an increase in amino-terminal fragments (approximately 20-30 kDa) was found in cerebellum of aged rats, whereas the amounts of full-length and truncated APP were not different (Fig. 3A). However, antibody R37 against the carboxy-terminus of APP showed no differences (Fig. 3B), even when the homogenates were analyzed using 16% Tris-Tricine gel (data not shown). Of the other parts examined, only homogenates of cerebrum of aged rats showed an increase in amino-terminal fragments of APP compared with that of young rats (Fig. 4A), while the homogenates of hippocampus, striatum and pons showed no difference (Fig. 4BCD). In cerebrum, hippocampus, straitum and pons, R37 showed no difference, as also seen in cerebellum (data not shown). In spite of the strong staining of free-floating sections, R17 only weakly reacted with APP on Western blot
Fig. 1. Irnmunohistochemical study of young (A-C) and aged (D-F) rat cerebellum (low magnification) using 22Cll (1 /xg/ml, A,D), R37 (1,000 × of antiserum, B,E) and R17 (1,000 × of antiserum, C,F). Bar = 200 ~zm.
321
Y. Nakamura et al. / Brain Research 643 (1994) 319-323
Fig. 2. Immunohistochemical study of cerebellthe aged rat (high magnification) using 22Cll (1 /zg/ml, A,D), R37 (1,000 x , B,E) and R17 (1,000 × , C,F). Bar = 50/xm.
KDa Y 0 200=" ,~ , ~ ;
Y 0
YO
Y 0
Y 0
YO
Y 0 .
.
.
Y 0 YO .
.
.
.
.
.
.
.
.
.
.
.
t
-
67" j ~ 5
30D" 20" f,
-~
: i
A
~.
!
B
C
D
E
F
G
H
d
Fig. 3. Western blot analysis of aged (O) and young (Y) rat cerebellum. The homogenates were subjected to 10% SDS-PAGE, and transferred onto a nitrocellulose membrane. The membranes were stained in 3% normal goat serum/Tris-buffered saline by 22Cll (1 /,g/ml, A), R37 (1,000 x of antiserum, B), anti-MAP2 (1 ~g/ml, C), anti-MAP1B (1/zg/ml, D), CKHC9 (5 x (medium), E), anti-neurofilament-H (1/zg/ml, F), anti-neurofilament-L ( 1 / , g / m l , G), SY-38 (1/*g/ml, H) and LCB.2(1/zg/ml, J) using Protoblot system. The asterisk in A points to an increase in amino-terminal fragments (20-30 kDa) of APP in aged rat cerebellum.
322
YO
A
Y. Nakamura et al. / Brain Research 643 (1994) 319-323
YO
B
Y 0
C
YO
D
Fig. 4. Amino-terminal fragments (20-30 kDa) of APP in cerebrum (A), hippocampus (B), striatum (C) and pons (D) of aged (O) and young (Y) rats, visualized by the antibody, 22Cll (1 ~g/ml), using the Protoblot system.
analysis (data not shown). Other antibodies, e.g. against MAP2 (Amersham), MAP1B (Amersham), kinesin (CKHC9, provided by Toyoshima, I.), neurofilament-H (Amersham), neurofilament-L [11], synaptophysin (Boehringer-Mannheim), and neuron-specific clathrin light chain b (LCB.2; provided by F.M. Brodsky) [16] showed no difference between aged and young rats on Western blot analysis (Fig. 3). This immunohistochemical study showed that fulllength or truncated APP accumulates in cell bodies and dendrites of Purkinje cells of aged rat since they were positively stained by antibodies against the amino-terminus, /3-region and carboxy-terminus of APP. It has been reported that APP is transported along the fast axonal transport system [6], and that this can be interfered with by ligation of axons [6], colchicine [13], ibotenic acid [10] and IDPN [5]. Considering these studies, the findings described herein suggest the possibility that APP might be accumulated partly as a result of an impairment of axonal transport during the aging process. We have recently reported an abnormal distribution of clathrin in brain with Alzheimer's disease, indicating impairment of slow axonal transport [12]. The aging process may affect axonal transport [9], leading to the impairment of APP transport. Recently, in aged rat brain, APP was found in swollen neurites [5]. Although we found neither the typical swollen neurites nor torpedo-like axonal swelling of Purkinje cells in this study, our results and the other report may indicate that quite similar incidents occur with aging. The difference between the two reports may be due to differences in the strains and the feeding conditions used. In this study, age-dependent changes of APP were further investigated by Western blot which showed no apparent differences between aged and young rat throughout the brain except for an increase in aminoterminal fragments in cerebrum and cerebellum of aged rats. In other regions, the amino-terminal fragments were present even in brain of young rat, indicating regional differences of brain aging. The increase in amino-terminal fragments of APP in the specific regions suggests that the metabolism of APP might be
affected by the aging process. Although Western blot analysis indicated that the total amount of APP did not change extensively with aging, it is possible that the amount of APP might only be increased in Purkinje cells, since the amounts of APP of Purkinje ceils were so small that they would not affect the total amounts of APP in whole cerebellum. Because increased expression of APP was seen in senescent cultured fibroblasts [1], an increase in APP expression might be found in specific neurons of aged brain. On the other hand, Western blot analysis has demonstrated that MAP2, MAP1B, kinesin, neurofilament-H/L, synaptophysin, and clathrin light chain are not affected by the aging process, indicating that the changes in metabolism of APP are more sensitive to the aging process than those of other proteins related to axonal transport. All these findings suggest that the aging process may affect the expression, transport or metabolism of APP. It would contribute to elucidating the pathogenesis of Alzheimer's disease to investigate the effect of aging on APP metabolism further. [1] Adler, M.J., Coronel, C., Shelton, E., Seegmiller, J.E. and Dewji, N.N., Increased gene expression of Alzheimer disease /3-amyloid precursor protein in senescent-cultured fibroblasts, Proc. Natl. Acad. Sci. USA, 88 (1991) 16-20. [2] Cras, P., Kawai, M., Lowery, D., Gonzalez-Dewhit, P., Greenberg, B. and Perry, G., Senile plaque neurites in Alzheimer disease accumulate amyloid precursor protein, Proc. Natl. Acad. Sci. USA, 88 (1991) 7552-7556. [3] Ishii, H., Karnetani, F., Haga, S. and Sato, M., The immunohistochemical demonstration of subsequences of the precursor of the amyloid A4 protein in senile plaques in senile plaques in Alzheimer's disease, Neuropathol. Appl. NeurobioL, 15 (1989) 135-147. [4] Joachim, C., Games, D., Morris, J., Ward, P., Frenkel, D. and Selkoe, D., Antibodies to non-/3 regions of the /3-amyloid precursor protein detect a subset of senile plaques, Am. J. Pathol., 138 (1991) 373-384. [5] Kawarabayashi, T., Shoji, M., Yamaguchi, H., Tanaka, M., Harigaya, Y., Ishiguro, K. and Hirai, S., Amyloid /3 protein precursor accumulates in swollen neurites throughout rat brain with aging, Neurosci. Lett., 153 (1993) 73-76. [6] Koo, E.H., Sisodia, S.S., Archer, D.R., Martin, L.J., Weidemann, A., Beyreuther, K., Fischer, P., Masters, C.L. and Price, D.L., Precursor of amyloid protein in Alzheimer disease undergoes fast anterograde axonal transport, Proc. Natl. Acad. Sci. USA, 87 (1990) 1561-1565. [7] Mattson, M.P., Cheng, B., Culwell, A.R., Esch, F.S., Lieberburg, I. and Rydel, R.E., Evidence for excitoprotective and interneuronal calcium-regulating roles for secreted forms of the /3amyloid precursor protein, Neuron, 10 (1993) 243-254. [8] McGreer, P.L., Akiyama, H., Kawamata, T., Yamada, T., Walker, D.G. and Ishii, T., Immunohistochemical localization of /3-amyloid precursor protein sequences in Alzheimer and normal brain tissue by light and electron microscopy, J. Neurosci. Res., 31 (1992) 428-442. [9] McQuarrie, I.G., Brady, S.T. and Lasek, R.J., Retardation in slow axonal transport of cytoskeletal elements during maturation and aging, NeurobioL Aging, 10 (1989) 359-365. [10] Nakamura, Y., Takeda, M., Niigawa, H., Hariguchi, S. and Nishimura, T., Amyloid /3 protein precursor deposition in rat
Y. Nakamura et aL / B r a i n Research 643 (1994) 319-323
hippocampus lesioned by ibotenic acid injection, Neurosci. Lett., 136 (1992) 95-98. [11] Nakamura, Y., Takeda, M., Aimoto, S., Hariguchi, S., Kitajima, S. and Nishimura, T., Acceleration of bovine neurofilament L assembly by deprivation of acidic tail domain, Eur. J. Biochern., 212 (1993) 565-571. [12] Nakamura, Y., Takeda, M., Yoshimi, K., Hattori, H., Hariguchi, S., Kitajima, S., Hashimoto, S. and Nishimura, T., Involvement of clathrin light chains in the pathology of Alzheimer's disease, Acta NeuropathoL, 84 (1994) 23-31. [13] Shigematsu, K. and McGeer, P.L., Accumulation of amyloid precursor protein in neurons after intraventricular injection of colhicine, Am. J. PathoL, 140 (1992) 787-794. [14] Siman, R., Card, J.P., Nelson, R.B. and Davis L.G., Expression
323
of /3-amyloid precursor protein in reactive astrocytes following neuronal damage, Neuron, 3 (1989) 275-285. [15] Weidemann, A., Konig, G., Bunke, D., Fischer, P., Salbaum, J.M., Masters, C.L. and Beyreuther, K., Identification, biogenesis, and localization of precursors of Alzheimer's disease A4 amyloid protein, Cell, 57 (1989) 115-126. [16] Wong, D.H., Ignatius, M.J., Parosky, G., Parham, P., Trojanowski, J.Q. and Brodsky, F.M., Neuron-specific expression of high-molecular-weight clathrin light chain, J. Neurosci., 10 (1990) 3025-3031. [17] Yoshimi, K., Takeda, M., Nishimura, T., Kudo, T., Nakamura, Y., Tada, K. and Iwata, N., An immunohistochemical study of MAP2 and clathrin in gerbil hippocampus after cerebral ischemia, Brain Res., 560 (1991) 149-158.