Neuroscience Letters, 146 (1992) 135-138
135
© 1992 Elsevier Scientific Publishers Ireland Ltd. All rights reserved 0304-3940/92/$ 05.00
NSL 09053
Regional accumulation of amyloid fl/A4 protein precursor in the gerbil brain following transient cerebral ischemia Hideaki Wakita, Hidekazu Tomimoto, Ichiro Akiguchi, Katsunori Ohnishi, Shinichi N a k a m u r a and Jun Kimura Deparment of Neurology, Kyoto University Hospital, Kyoto (Japan) (Received 21 April 1992; Revised version received 31 July 1992; Accepted 3 August 1992)
Key words: Amyloid fl/A4 protein precursor; Transient cerebral ischemia; Neuronal viability Alterations offl/A4 amyloid protein precursor (APP) were investigated immunohistochemically in the gerbil brain after transient global ischemia and subsequent reperfusion. Marked accumulation of this protein peaking at 24 h occurred in the neurons of the CA3 and paramedian region of the hippocampus as well as layers III, V and VI of the cerebral cortex. On the contrary, the accumulation was not observed in the neurons of the CA1 region. These results indicate that distribution of APP is altered depending on tissue viabilities after cerebral ischemia.
fl/A4 protein is a core protein of brain amyloid which accumulates in the brains of patients with Alzheimer's disease (AD) [4]. The amino acid sequence of this protein resides within at least 4 distinct precursor proteins (amyloid fl/A4 protein precursors (APP)) which are produced by alternative splicing of the amyloid gene [5, 8]. Regulation of these protein precursors is of special interest, since recent studies revealed that brain damage causes abnormal expression of APP and deposition offl/A4 protein [6, 11, 13]. These data prompted us to test possible alterations of APP level and regional correlation with neuronal viabilities using the bilateral carotid occlusion model in the gerbil, in which the regional evolution of neuronal death has been well characterized. Male Mongolian gerbils (50-60 g) were used. Under inhalation anesthesia with ether, both the common carotid arteries were exposed and temporarily occluded for 5 or 15 min with aneurysmal clips. After 30 min, 3, 12, 24 h and 3 and 7 days following ischemia, the animals were deeply anesthetized with sodium pentobarbital and perfused transcardially with 0.01 M phosphate buffered saline (PBS), followed by a fixative containing 4% paraformaldehyde and 0.2% picric acid in 0.1 M phosphate buffer (PB, pH 7.4). Four animals were used for each ischemic and postischemic period. As non-ischemic controls, 4 animals were sacrificed just after the identical Correspondence: H. Tomimoto, Department of Neurology, Kyoto University Hospital, Kyoto 606, Japan.
surgical procedure without clipping. Brains were immersed for 12 h in 4% paraformaldehyde in 0.1 M PB (pH 7.4), and stored in 15% sucrose in 0.1 M PB (pH 7.4). Serial sections (20 pm thick) were cut in a cryostat and reacted with a monoclonal antibody recognizing an epitope located between amino acids 60 and 100 in the N-terminal part [10, 16] (Boehringer Mannheim; 1:4,000) or polyclonal antiserum raised against recombinant amino-terminal 592 residues of APP fusion protein (APP592, a generous gift from Dr. Y. Tokushima, Asahi Chemical Industry Co. Ltd., 1:40,000) for 24 h in a free floating state. Specificity of staining with the polyclonal antiserum was established by disappearance of specific staining with the antiserum preabsorbed with 0.2/IM of recombinant amino-terminal 592 residues of APP fusion protein or non-immunized rabbit serum. The sections were subsequently incubated with an appropriate secondary antibody (1:200) for 1 h and avidin-biotin-peroxidase complex solution (l:100) for 1 h. After each incubation, the sections were washed for 15 min with 0.01 M PBS containing 0.3% Triton X-100. Finally, immunoreaction products were visualized with a mixed solution of 0.02% 3,3'-diaminobenzidine tetrahydrochloride, 0.3% nickel ammonium sulfate and 0.005% H 2 0 2 in 0.05 M Tris buffer (pH 7.6). The relative percentage of the intensely immunoreactive neurons to the whole neurons was estimated by counting the number of cells in 0.4 mm 2. Weakly APP-immunoreactive neurons were widely distributed in sham operated animals. Immunoreactivity
136
appeared as granular deposits in the periphery of neuronal perikaya (Figs. la,d and 2a,d). After 12 h reperfusion, intense labeling of neuronal perikarya and proximal dendrites were observed in the non-pyramidal neurons of layer VI and partly the pyramidal neurons of layer V of the cerebral cortex. In the hippocampus, the perikarya and dendrites of pyramidal neurons in the paramedian and CA3 regions were intensely labeled as well as their efferent axons in the stratum oriens of these regions. At 24 h reperfusion, the number of intensely labeled cells peaked and some of the pyramidal neurons in layer III were also labeled (Figs. le and 2b,e). The per-
centage of intensely labeled pyramidal neurons was 65.6+1.7% in the paramedian region and 72.1+1.0% in the CA3a and CA3b regions of the hippocampus at 24 h reperfusion (mean+S.D.). They decreased over time (Fig. 1f and 2c,f), with only a few left after reperfusion for 7 days. On the other hand, the intensely immunoreactive neurons were not observed in vulnerable areas such as the pyramidal cell layer of the CA 1 region after 24 h (Fig. lb), and later (Fig. lc). These results were variable in the animals subjected to 5 min ischemia, but constant after ischemia for 15 min. The two antisera gave identical results, with the polyclonal antiserum producing more
Fig. 1. Alterations of immunohistochcmical staining for APP in the CA1 (a-c) and CA3 (d-f) regions of the hippocampus after ischemia for 5 rain. Pyramidal neurons in the CAl and CA3 regions showed granular staining for APP in controls (a,d). Most pyramidal neurons and their efferent axons in the CA3 region were intensely labeled after 24 h (e). After 3 days, a few pyramidal neurons were intensely labeled (f). In contrast, the intensely labeled neurons were not observed in the CAI region after 24 h (b) and 3 days (c). SO, stratum oriens; SP, stratum pyramidale. Bars = 30/zm.
137
Fig. 2. Immunohistochemical staining for APP in the frontoparietal cortex after ischemia for 5 min. The figures in the bottom row depict higher magnifications of those in the top. In control animals, neuronal perikarya were weakly labeled (a,d). After 24 h, Golgi-like figures were observed in the non-pyramidalneurons of layer VI and partly in the pyramidal neurons of layers III and V (b,e). After 3 days, the number of these neurons showed a marked decrease (c,f). Bars = 200/.tm (a-c), 30/zm (d-f). intense staining than the monoclonal one. T h r o u g h o u t each period of reperfusion, no glial cells were labeled. Our results have shown changes of APP expression depending on regional differences of neuronal viability after cerebral ischemia [7, 17]. A major unanswered question concerns whether A P P staining increase in specific neurons is due to up-regulation of synthesis or focal accumulation by suppression of degradation and/or axonal transport. A Northern blot analysis showed no alterations of A P P m R N A in the CA1 and cerebral cortex in the same model as the one used in the present study [1]. Nevertheless, this does not
preclude increased synthesis, since accumulation of A P P was localized to narrow areas. The regions o f A P P accumulation partly correspond to those o f heat-shock protein induction [14, 15] and regions relatively resistant to ischemic insults. In conjunction with the data suggesting a heat-shock control element in the A P P gene [12] and neurotrophic effect of APP [3], our results may indicate that accumulation of this protein is a response to neuronal stress. On the other hand, A P P deposition in the swollen axons suggests an alternative possibility that A P P is accumulated in the neuronal perikarya as a result of disturbed axonal transport [9]. To address this ques-
138
tion, in situ hybridization study of APP mRNA seems to be required. Recently Abe et al. reported induction of Kunitz-type protease inhibitor (KPI) domain containing APP (APP751/770) mRNA 24 h after focal cerebral ischemia [2]. In accordance with this observation, delayed expression of KPI domain containing APP has been revealed in astroglia after kainate injection [6]. Since the same monoclonal antiserum has labeled glial cells in this setting, the lack of staining in glial cells in the present investigation may be attributable to a relatively short reperfusion period. Obviously, further studies over an extended period are required to verify the possible expression of APP in glial cells. We are grateful to Dr. Yanagihara of Osaka University for his useful advice. 1 Abe, K., Tanzi, R.E. and Kogure, K., Induction of HSP 70 mRNA after transient ischemia in gerbil brain, Neurosci. Lett., 125 (1991) 166-168. 2 Abe, K., Tanzi, R.E. and Kogure, K., Selective induction of Kunitztype protease inhibitor domain containing amyloid precursor protein mRNA after persistent focal ischemia in rat cerebral cortex, Neurosci. Lett., 125 (1991) 172-174. 3 Araki, W., Kitaguchi, N., Tokushima, Y., Ishii, K., Aratake, H., Shimohama, S., Nakamura, S. and Kimura, J., Trophic effect of fl-amyloid precursor protein on cerebral cortical neurons in culture, Biochem. Biophys. Res. Commun., 181 (1991) 265-271. 4 Glenner, G.G. and Wang, C.W., Alzheimer's disease: initial report of the purification and characterization of a novel cerebrovascular amyroid protein, Biochem. Biophys. Res. Commun., 120 (1984) 885-890. 5 Kang, J., Lemaire, H.G., Unterbeck, A., Salbaum, J.M., Masters, C.L., Grzeshik, K.H., Multhaup, G., Beyreuther, K. and MullerHill, B., The precursor of Alzheimer's disease amyloid A4 protein resembles a cell-surface receptor, Nature, 325 (1987) 733-736.
6 Kawarabayashi, T., Shoji, M., Harigaya, Y., Yamaguchi, H. and Hirai, S., Expression of APP in the early stage of brain damage. Brain Res., 563 (1991) 334-338. 7 Kirino, T., Delayed neuronal death in the gerbil hippocampus following ischemia, Brain Res., 239 (1982) 57-69. 8 Kitaguchi, N., Takahashi, Y., Tokushima, Y., Shiojiri, S. and Ito, H., Novel precursor of Alzheimer's disease amyloid protein shows protease inhibitor activity, Nature, 331 (1988) 530-532. 9 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's disease undergoes fast anterograde axonal transport, Proc. Natl. Acad. Sci. USA, 87 (1990) 1561-1565. 10 Martin, L.J., Sisodia, S.S., Koo, E.D., Cork, L.C., Dellovade, T.L., Weidemann, A., Beyreuther, K., Masters, C. and Price, D.L., Amyloid precursor protein in aged nonhuman primates, Proc. Natl. Acad. Sci. USA, 88 (1991) 1461-1465. 11 Roberts, G.W., Gentleman, S.M., Lynch, A. and Graham, D.I., r/A4 amyloid protein deposition in brain after head trauma, Lancet, 338 (1991) 1422-1423. 12 Salbaum, J.M., Weidemann, A., Lemaire, H., Masters, C.L. and Beyreuther, K., The promotor of Alzheimer's disease amyloid A4 precursor gene, EMBO J., 7 (1988) 2807-2813. 13 Siman, R., Card, J.P., Nelson, R.B. and Davis, L.G., Expression of fl-amyloid precursor protein in reactive astrocytes following neuronal damage, Neuron, 3 (1989) 275-285. 14 Takemoto, O., Tomimoto, H., and Yanagihara, T., Induction of C-fos and C-jun gene products and heat shock protein after transient cerebral ischemia in gerbils, Stroke, 22 ( 1991) 131 (Abstract.). 15 Vass, K., Welch, W.J., Nowak Jr., T.S., Localization of 70-kDa stress protein induction in gerbil brain after ischemia, Acta Neuropathol., 77 (1988) 128-135. 16 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. 17 Yanagihara, T,, Yoshimine, T., Morimoto, K., Yamamoto, K. and Homburger, H.A., Immunohistochemical investigation of cerebral ischemia in gerbils, J. Neuropathol. Exp. Neurol., 44 (1985) 204215.
135
© 1992 Elsevier Scientific Publishers Ireland Ltd. All rights reserved 0304-3940/92/$ 05.00
NSL 09053
Regional accumulation of amyloid fl/A4 protein precursor in the gerbil brain following transient cerebral ischemia Hideaki Wakita, Hidekazu Tomimoto, Ichiro Akiguchi, Katsunori Ohnishi, Shinichi N a k a m u r a and Jun Kimura Deparment of Neurology, Kyoto University Hospital, Kyoto (Japan) (Received 21 April 1992; Revised version received 31 July 1992; Accepted 3 August 1992)
Key words: Amyloid fl/A4 protein precursor; Transient cerebral ischemia; Neuronal viability Alterations offl/A4 amyloid protein precursor (APP) were investigated immunohistochemically in the gerbil brain after transient global ischemia and subsequent reperfusion. Marked accumulation of this protein peaking at 24 h occurred in the neurons of the CA3 and paramedian region of the hippocampus as well as layers III, V and VI of the cerebral cortex. On the contrary, the accumulation was not observed in the neurons of the CA1 region. These results indicate that distribution of APP is altered depending on tissue viabilities after cerebral ischemia.
fl/A4 protein is a core protein of brain amyloid which accumulates in the brains of patients with Alzheimer's disease (AD) [4]. The amino acid sequence of this protein resides within at least 4 distinct precursor proteins (amyloid fl/A4 protein precursors (APP)) which are produced by alternative splicing of the amyloid gene [5, 8]. Regulation of these protein precursors is of special interest, since recent studies revealed that brain damage causes abnormal expression of APP and deposition offl/A4 protein [6, 11, 13]. These data prompted us to test possible alterations of APP level and regional correlation with neuronal viabilities using the bilateral carotid occlusion model in the gerbil, in which the regional evolution of neuronal death has been well characterized. Male Mongolian gerbils (50-60 g) were used. Under inhalation anesthesia with ether, both the common carotid arteries were exposed and temporarily occluded for 5 or 15 min with aneurysmal clips. After 30 min, 3, 12, 24 h and 3 and 7 days following ischemia, the animals were deeply anesthetized with sodium pentobarbital and perfused transcardially with 0.01 M phosphate buffered saline (PBS), followed by a fixative containing 4% paraformaldehyde and 0.2% picric acid in 0.1 M phosphate buffer (PB, pH 7.4). Four animals were used for each ischemic and postischemic period. As non-ischemic controls, 4 animals were sacrificed just after the identical Correspondence: H. Tomimoto, Department of Neurology, Kyoto University Hospital, Kyoto 606, Japan.
surgical procedure without clipping. Brains were immersed for 12 h in 4% paraformaldehyde in 0.1 M PB (pH 7.4), and stored in 15% sucrose in 0.1 M PB (pH 7.4). Serial sections (20 pm thick) were cut in a cryostat and reacted with a monoclonal antibody recognizing an epitope located between amino acids 60 and 100 in the N-terminal part [10, 16] (Boehringer Mannheim; 1:4,000) or polyclonal antiserum raised against recombinant amino-terminal 592 residues of APP fusion protein (APP592, a generous gift from Dr. Y. Tokushima, Asahi Chemical Industry Co. Ltd., 1:40,000) for 24 h in a free floating state. Specificity of staining with the polyclonal antiserum was established by disappearance of specific staining with the antiserum preabsorbed with 0.2/IM of recombinant amino-terminal 592 residues of APP fusion protein or non-immunized rabbit serum. The sections were subsequently incubated with an appropriate secondary antibody (1:200) for 1 h and avidin-biotin-peroxidase complex solution (l:100) for 1 h. After each incubation, the sections were washed for 15 min with 0.01 M PBS containing 0.3% Triton X-100. Finally, immunoreaction products were visualized with a mixed solution of 0.02% 3,3'-diaminobenzidine tetrahydrochloride, 0.3% nickel ammonium sulfate and 0.005% H 2 0 2 in 0.05 M Tris buffer (pH 7.6). The relative percentage of the intensely immunoreactive neurons to the whole neurons was estimated by counting the number of cells in 0.4 mm 2. Weakly APP-immunoreactive neurons were widely distributed in sham operated animals. Immunoreactivity
136
appeared as granular deposits in the periphery of neuronal perikaya (Figs. la,d and 2a,d). After 12 h reperfusion, intense labeling of neuronal perikarya and proximal dendrites were observed in the non-pyramidal neurons of layer VI and partly the pyramidal neurons of layer V of the cerebral cortex. In the hippocampus, the perikarya and dendrites of pyramidal neurons in the paramedian and CA3 regions were intensely labeled as well as their efferent axons in the stratum oriens of these regions. At 24 h reperfusion, the number of intensely labeled cells peaked and some of the pyramidal neurons in layer III were also labeled (Figs. le and 2b,e). The per-
centage of intensely labeled pyramidal neurons was 65.6+1.7% in the paramedian region and 72.1+1.0% in the CA3a and CA3b regions of the hippocampus at 24 h reperfusion (mean+S.D.). They decreased over time (Fig. 1f and 2c,f), with only a few left after reperfusion for 7 days. On the other hand, the intensely immunoreactive neurons were not observed in vulnerable areas such as the pyramidal cell layer of the CA 1 region after 24 h (Fig. lb), and later (Fig. lc). These results were variable in the animals subjected to 5 min ischemia, but constant after ischemia for 15 min. The two antisera gave identical results, with the polyclonal antiserum producing more
Fig. 1. Alterations of immunohistochcmical staining for APP in the CA1 (a-c) and CA3 (d-f) regions of the hippocampus after ischemia for 5 rain. Pyramidal neurons in the CAl and CA3 regions showed granular staining for APP in controls (a,d). Most pyramidal neurons and their efferent axons in the CA3 region were intensely labeled after 24 h (e). After 3 days, a few pyramidal neurons were intensely labeled (f). In contrast, the intensely labeled neurons were not observed in the CAI region after 24 h (b) and 3 days (c). SO, stratum oriens; SP, stratum pyramidale. Bars = 30/zm.
137
Fig. 2. Immunohistochemical staining for APP in the frontoparietal cortex after ischemia for 5 min. The figures in the bottom row depict higher magnifications of those in the top. In control animals, neuronal perikarya were weakly labeled (a,d). After 24 h, Golgi-like figures were observed in the non-pyramidalneurons of layer VI and partly in the pyramidal neurons of layers III and V (b,e). After 3 days, the number of these neurons showed a marked decrease (c,f). Bars = 200/.tm (a-c), 30/zm (d-f). intense staining than the monoclonal one. T h r o u g h o u t each period of reperfusion, no glial cells were labeled. Our results have shown changes of APP expression depending on regional differences of neuronal viability after cerebral ischemia [7, 17]. A major unanswered question concerns whether A P P staining increase in specific neurons is due to up-regulation of synthesis or focal accumulation by suppression of degradation and/or axonal transport. A Northern blot analysis showed no alterations of A P P m R N A in the CA1 and cerebral cortex in the same model as the one used in the present study [1]. Nevertheless, this does not
preclude increased synthesis, since accumulation of A P P was localized to narrow areas. The regions o f A P P accumulation partly correspond to those o f heat-shock protein induction [14, 15] and regions relatively resistant to ischemic insults. In conjunction with the data suggesting a heat-shock control element in the A P P gene [12] and neurotrophic effect of APP [3], our results may indicate that accumulation of this protein is a response to neuronal stress. On the other hand, A P P deposition in the swollen axons suggests an alternative possibility that A P P is accumulated in the neuronal perikarya as a result of disturbed axonal transport [9]. To address this ques-
138
tion, in situ hybridization study of APP mRNA seems to be required. Recently Abe et al. reported induction of Kunitz-type protease inhibitor (KPI) domain containing APP (APP751/770) mRNA 24 h after focal cerebral ischemia [2]. In accordance with this observation, delayed expression of KPI domain containing APP has been revealed in astroglia after kainate injection [6]. Since the same monoclonal antiserum has labeled glial cells in this setting, the lack of staining in glial cells in the present investigation may be attributable to a relatively short reperfusion period. Obviously, further studies over an extended period are required to verify the possible expression of APP in glial cells. We are grateful to Dr. Yanagihara of Osaka University for his useful advice. 1 Abe, K., Tanzi, R.E. and Kogure, K., Induction of HSP 70 mRNA after transient ischemia in gerbil brain, Neurosci. Lett., 125 (1991) 166-168. 2 Abe, K., Tanzi, R.E. and Kogure, K., Selective induction of Kunitztype protease inhibitor domain containing amyloid precursor protein mRNA after persistent focal ischemia in rat cerebral cortex, Neurosci. Lett., 125 (1991) 172-174. 3 Araki, W., Kitaguchi, N., Tokushima, Y., Ishii, K., Aratake, H., Shimohama, S., Nakamura, S. and Kimura, J., Trophic effect of fl-amyloid precursor protein on cerebral cortical neurons in culture, Biochem. Biophys. Res. Commun., 181 (1991) 265-271. 4 Glenner, G.G. and Wang, C.W., Alzheimer's disease: initial report of the purification and characterization of a novel cerebrovascular amyroid protein, Biochem. Biophys. Res. Commun., 120 (1984) 885-890. 5 Kang, J., Lemaire, H.G., Unterbeck, A., Salbaum, J.M., Masters, C.L., Grzeshik, K.H., Multhaup, G., Beyreuther, K. and MullerHill, B., The precursor of Alzheimer's disease amyloid A4 protein resembles a cell-surface receptor, Nature, 325 (1987) 733-736.
6 Kawarabayashi, T., Shoji, M., Harigaya, Y., Yamaguchi, H. and Hirai, S., Expression of APP in the early stage of brain damage. Brain Res., 563 (1991) 334-338. 7 Kirino, T., Delayed neuronal death in the gerbil hippocampus following ischemia, Brain Res., 239 (1982) 57-69. 8 Kitaguchi, N., Takahashi, Y., Tokushima, Y., Shiojiri, S. and Ito, H., Novel precursor of Alzheimer's disease amyloid protein shows protease inhibitor activity, Nature, 331 (1988) 530-532. 9 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's disease undergoes fast anterograde axonal transport, Proc. Natl. Acad. Sci. USA, 87 (1990) 1561-1565. 10 Martin, L.J., Sisodia, S.S., Koo, E.D., Cork, L.C., Dellovade, T.L., Weidemann, A., Beyreuther, K., Masters, C. and Price, D.L., Amyloid precursor protein in aged nonhuman primates, Proc. Natl. Acad. Sci. USA, 88 (1991) 1461-1465. 11 Roberts, G.W., Gentleman, S.M., Lynch, A. and Graham, D.I., r/A4 amyloid protein deposition in brain after head trauma, Lancet, 338 (1991) 1422-1423. 12 Salbaum, J.M., Weidemann, A., Lemaire, H., Masters, C.L. and Beyreuther, K., The promotor of Alzheimer's disease amyloid A4 precursor gene, EMBO J., 7 (1988) 2807-2813. 13 Siman, R., Card, J.P., Nelson, R.B. and Davis, L.G., Expression of fl-amyloid precursor protein in reactive astrocytes following neuronal damage, Neuron, 3 (1989) 275-285. 14 Takemoto, O., Tomimoto, H., and Yanagihara, T., Induction of C-fos and C-jun gene products and heat shock protein after transient cerebral ischemia in gerbils, Stroke, 22 ( 1991) 131 (Abstract.). 15 Vass, K., Welch, W.J., Nowak Jr., T.S., Localization of 70-kDa stress protein induction in gerbil brain after ischemia, Acta Neuropathol., 77 (1988) 128-135. 16 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. 17 Yanagihara, T,, Yoshimine, T., Morimoto, K., Yamamoto, K. and Homburger, H.A., Immunohistochemical investigation of cerebral ischemia in gerbils, J. Neuropathol. Exp. Neurol., 44 (1985) 204215.
