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Hippocampal pathology in diffuse Lewy body disease using ubiquitin immunohistochemistry
Journal of Neurological Sciences 149 (1997) 165–169
Hippocampal pathology in diffuse Lewy body disease using ubiquitin immunohistochemistry Eizo Iseki*, Feng Li, Toshinari Odawara, Kenji Kosaka Department of Psychiatry, Yokohama City University School of Medicine, 3 -9 Fukuura, Kanazawa-ku, Yokohama 236, Japan Received 16 September 1996; revised 20 December 1996; accepted 12 January 1997
Abstract Various ubiquitin-positive structures in the hippocampus in diffuse Lewy body disease (n512) and non-demented aged subjects (n53) were investigated immunohistochemically. These structures were composed of ubiquitin-positive granular structures (UPG), ubiquitinpositive neuritic structures (UPN), spheroidal structures, neuritic plaques, neurofibrillary tangles (NFT), Lewy bodies (LB) and ubiquitin-positive neurons. UPG, UPN, spheroidal structures and neuritic plaques were distributed with special reference to the hippocampal pathway assumed in this study. This pathway was thought to run along the stratum oriens, mostly perforating the stratum pyramidale at many sites of the subiculum and CA1–3, and to end partly in the CA2–3 and the subiculum of the uncus (UPG or UPN). After perforating the stratum pyramidale and giving off terminal branches (ubiquitin-positive neurons or neuritic plaques with degenerative neurites), it was thought to run along the stratum radiatum and continue along the stratum moleculare of the dentate gyrus, forming synapses with the apical dendrites from the stratum pyramidale and the stratum granulosum (spheroidal structures or neuritic plaques). These findings suggest that many of the ubiquitin-positive structures may be caused by degeneration of terminal or distal axons of this pathway. NFT and LB also had a somewhat orderly distribution with reference to this pathway. 1997 Elsevier Science Ireland Ltd. Keywords: Hippocampus; Perforant pathway; Ubiquitin; Diffuse Lewy body disease
1. Introduction Diffuse Lewy body disease (DLBD) is pathologically characterized by the widespread occurrence of Lewy bodies (LB) in the central nervous system (Kosaka et al., 1984). DLBD is classified into two forms; a common form, with many senile changes, and a pure form, with no or few senile changes (Kosaka, 1990). The common form of DLBD is also referred to as ‘‘senile dementia of Lewy body type’’ (Perry et al., 1990) or ‘‘the Lewy body variant of Alzheimer’s disease’’ (Hansen et al., 1990). There have been some reports, however, suggesting different pathogenesis between DLBD and Alzheimer-type dementia (ATD). Dickson et al. (1991) reported that *Corresponding author: Tel.: 181-45-787-2667. Fax: 181-45-7832540
ubiquitin-immunoreactive neurites were observed in the CA2–3 region of the hippocampus in DLBD cases. Immunoelectron micrographs of these neurites were suggestive of distal axons. These neurites were partially immunostained with a neurofilament antibody but not with antibodies to tau, paired helical filaments or tyrosine hydroxylase (Dickson et al., 1994). The constant association and immunological similarity between these neurites and cortical LB suggest that both structures may share a common pathogenesis (Dickson et al., 1994; Kim et al., 1995). Dickson et al. (1990) found ubiquitin-immunoreactive granules in the neuropil of the entorhinal cortex and amygdala in non-demented aged subjects. We showed that most of these ubiquitin-positive granular structures (UPG) might be derived from degenerating terminal axons of the large multipolar neurons in layer pre-a of the lateral part
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of the entorhinal cortex and the large pyramidal neurons in layers IIIc and V of the transentorhinal cortex, and found that UPG disappeared in both ATD and DLBD cases, accompanying some ubiquitin-positive neuritic structures (UPN) similar to the CA2–3 neurites in only DLBD cases (Iseki et al., 1996). In this study, we investigated various ubiquitin-positive structures, including UPG and UPN, in the hippocampus in DLBD cases. We suggest that many of these structures may be caused by degeneration of the hippocampal pathway assumed in this study.
2. Materials and methods We studied 12 pathologically verified DLBD cases (10 common form and two pure form cases) and three nondemented aged cases (Table 1). Cerebral hemispheres were fixed in 4% paraformaldehyde in 0.1 M phosphate buffer (pH 7.4), sliced in two coronal planes through the mammilary body and the lateral geniculate body, embedded in paraffin and cut into 8 mm-thick sections. The sections ¨ were stained using the hematoxylin–eosin (HE), Kluver– Barrera (KB), methenamine–silver (MS) and Gallyas– Braak (GB) methods for pathological examination, and were immunostained with anti-ubiquitin antibody (MAB1510; monoclonal, mouse, 1:8000 dilution; Chemicon, USA). MAB1510 immunolabelling was detected using the avidin-biotinylated HRP complex (ABC) method (Elite Kit, Vector, USA) and visualized with 3,39diaminobenzidine tetrahydrochloride (DAB). In addition, the sections were double-immunostained with MAB1510 and tau-2 (monoclonal, mouse, 1:8000; Sigma, St. Louis, MO, USA) or anti-A 4 protein antibody (polyclonal, rabbit, 1:5000; a gift from Dr. T. Ishii) to examine the relationship between ubiquitin-positive structures and neurofibrillary tangles (NFT) or amyloid deposits, respectively. Immunolabelling was detected using the ABC method and the ABC alkaline phosphatase method (ABC-AP Kit, Vector) and visualized with DAB and fast blue, respectively. Anti-A 4 protein antibody was used after formic acid treatment of the sections. The regional classification of the hippocampus followed the description by Duvernoy (1988).
Fig. 1. A and B. Schema of the hippocampus following Duvernoy (1988). (A) The level of the mammilary body; (B) the level of the lateral geniculate body. AM5amygdala, UN5uncus, SB5subiculum, EC5 entorhinal cortex. The cross represents the location of spheroidal structures. Gray shaded areas represent the distribution of ubiquitin-positive neuritic structures (UPN) with ubiquitin-positive granular structures (UPG), and faint-line shaded areas represent neuritic plaques, ubiquitinpositive degenerative neurons, neurofibrillary tangles (NFT) or Lewy bodies (LB).
3. Results The distribution of the ubiquitin-positive structures in the hippocampus is schematically shown in Fig. 1A–B. Solid and dotted lines demonstrate the hippocampal pathway assumed in this study. Solid lines run along the
Table 1 Summary of patients’ characteristics in the examined cases Number
Mean age (years)
10
74.7
4.0
1161
Pure form DLBD cases
2
64.0
14.0
1230
Non-demented aged cases
3
81.3
2
1067
Common form DLBD cases
DLBD5diffuse Lewy body disease; BW5body weight.
Mean duration (years)
Mean BW (g)
E. Iseki et al. / Journal of Neurological Sciences 149 (1997) 165 – 169
stratum radiatum and the stratum moleculare of the dentate gyrus. Dotted lines run along the stratum oriens and perforate the stratum pyramidale at many sites. The ubiquitin-positive structures were composed of UPG, UPN, spheroidal structures, neuritic plaques, NFT, LB and ubiquitin-positive neurons. UPG appeared to be identical with those of the entorhinal and transentorhinal cortices. UPN frequently had continuity with UPG. Spheroidal structures were found singly or in clusters. Neuritic plaques had ubiquitin-positive degenerative neurites as well as MS- and A 4 -positive amyloid deposits. NFT were positively stained not only with ubiquitin but also with GB and tau-2. LB were defined as ubiquitin-positive inclusions other than NFT. Ubiquitin-positive neurons with diffusely weak cytoplasmic staining included both degenerative neurons and immature NFT- or LB-containing neurons. These ubiquitin-positive structures were not evenly, but were orderly, distributed in the hippocampus. In the three non-demented aged cases, some-to-many UPG were found in the neuropil of the stratum pyramidale of the CA2–3 and the subiculum of the uncus (Fig. 2). Some UPG were also scattered in the neuropil or clustered around the pyramidal neurons, predominantly in the CA1 and subiculum (Fig. 3). These cases showed a few NFT and many UPG, but no amyloid deposits, in the entorhinal and transentorhinal cortices. In the two pure form DLBD cases, some-to-many UPN with UPG were found in the CA2–3 and the subiculum of the uncus (Fig. 4). These UPN were distributed from the stratum oriens to the stratum pyramidale. Some spheroidal structures were arranged in the stratum lacunosum-moleculare along solid lines (Fig. 5). Some-to-many LB or ubiquitin-positive neurons were predominantly found in the CAl and CA3–4 as well as the uncus (Fig. 6). These cases showed some NFT, some UPG with UPN and mild spongiform change, but no amyloid deposits, in the entorhinal and transentorhinal cortices. In the 10 common form DLBD cases, some-to-many UPN with UPG were found in the CA2–3 (Fig. 7) and the subiculum of the uncus (Fig. 8). Some spheroidal structures or neuritic plaques were arranged in the stratum lacunosum-moleculare and in the outer portion of the stratum moleculare of the dentate gyrus along solid lines (Fig. 9). Some to many neuritic plaques, NFT and LB were found in the CA1–4 and subiculum as well as the uncus, although their numbers varied in each case. Three structures had a somewhat different distribution. Neuritic plaques and NFT were predominantly found in the CA1 and subiculum, while LB were frequently found in the CAl and CA3–4 as well as the uncus. In addition, there were some ubiquitin-positive degenerative neurons surrounded by UPG or degenerative neurites, predominantly in the CA1 and subiculum (Fig. 10). These cases showed someto-many NFT, some UPG with UPN, moderate-to-severe spongiform change and many amyloid deposits in the entorhinal and transentorhinal cortices.
167
4. Discussion Anti-ubiquitin antibody recognizes not only neuronal inclusions, such as NFT and LB, but also degenerative neurites, including distal or terminal axons (Dickson et al., 1990; Iseki et al., 1996). Therefore, ubiquitin immunohistochemistry is useful for tracing the degeneration of projections at their distal portion. In DLBD, unlike ATD, the degeneration of distal or terminal axons can be visualized by the occurrence of UPG, UPN, spheroids or spongiform vacuoles, because the neurons of origin of those axons may degenerate more rapidly in DLBD than in ATD (Iseki et al., 1995, 1996, 1997). In a previous paper (Iseki et al., 1996), we showed that UPG were predominantly distributed among layer pre-a of the lateral part of the entorhinal cortex and in layers II–IIIIab of the transentorhinal cortex, and in the intermediate areas of the accessory basal and basal amygdaloid nuclei. We suggested that cortical UPG were derived from degenerating terminals of the recurrent collateral ascending axons of the large multipolar neurons in layer pre-a and the large pyramidal neurons in layers III C and V and amygdaloid UPG from those of their projecting axons. In this study, many UPG were found in the CA2–3 and the subiculum of the uncus in non-demented aged cases. Many UPN, in continuity with UPG, were observed in the same areas in pure- and common form DLBD cases. This finding suggests that these UPG and UPN are caused by degeneration of terminal and distal axons, respectively, of the same projections. Some UPG were also scattered in the neuropil or clustered around the pyramidal neurons, predominantly in the CA1 and subiculum in non-demented aged cases. In common form DLBD cases, there were some ubiquitinpositive neurons surrounded by UPG or degenerative neurites and some neuritic plaques in the same areas, suggesting that these degenerative neurites are caused by degeneration of the same terminal or distal axons as for UPG. Spheroidal structures or neuritic plaques were arranged in the stratum lacunosum-moleculare and in the outer portion of the stratum moleculare of the dentate gyrus along solid lines in pure and common form DLBD cases. These spheroidal structures or neuritic plaques appear to represent the degeneration of terminal axons of the pathway that forms synapses with the apical dendrites from the stratum pyramidale and the stratum granulosum. The perforant pathway originates in the lateral part of the entorhinal cortex and ends in the stratum moleculare of the dentate gyrus (Duvernoy, 1988; Hyman et al., 1988). However, the intrahippocampal routes of this pathway are insufficiently known. In ATD, pathological studies of the perforant pathway showed NFT in the neurons of its origin and neuritic plaques in its terminal zone (Hyman et al., 1986, 1988; Casanova et al., 1990; Senut et al., 1991;
168
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Fig. 2. UPG in the neuropil of the CA2–3. A non-demented aged case. Anti-ubiquitin immunostaining. Fig. 3. UPG clustered around the pyramidal neurons in the CA1. A non-demented aged case. Anti-ubiquitin immunostaining. Fig. 4. Some UPN (arrows) with UPG in the CA2–3. A pure form DLBD case. Anti-ubiquitin immunostaining. Fig. 5. Some spheroidal structures in the stratum lacunosum-moleculare. A pure form DLBD case. Anti-ubiquitin immunostaining. Fig. 6. Some LB (arrows) and UPN (arrowheads) in the subiculum of the uncus. A pure form DLBD case. Anti-ubiquitin immunostaining. Fig. 7. Some UPN (arrows) with UPG in the CA2–3. A common form DLBD case. Anti-ubiquitin immunostaining. Fig. 8. Numerous UPN with UPG in the subiculum of the uncus. A common form DLBD case. Anti-ubiquitin immunostaining. Fig. 9. Neuritic plaques accompanied by UPG or ubiquitin-positive degenerative neurites (brown; arrowheads) and A 4 -positive amyloid deposits (blue; arrows) in the stratum moleculare of the dentate gyrus. A common form DLBD case. Double immunostaining with anti-ubiquitin and anti-A 4 antibodies. Fig. 10. Ubiquitin-positive degenerative neurons (arrows) surrounded by UPG or ubiquitin-positive degenerative neurites (brown) and tau-2-positive NFT (blue; arrowheads) in the CA1. A common form DLBD case. Double immunostaining with anti-ubiquitin and anti-tau-2 antibodies.
E. Iseki et al. / Journal of Neurological Sciences 149 (1997) 165 – 169
Lippa et al., 1992). Mizutani and Kasahara (1995) suggested that the perforant pathway ran along the stratum lacunosum-moleculare to end in the dentate gyrus after perforating the subiculum. The hippocampal pathway assumed in this study was thought to run along the stratum oriens, mostly perforating the stratum pyramidale, not only at the subiculum but also at the CA1–3, and to end partly in the CA2–3 and the subiculum of the uncus. After perforating the stratum pyramidale and giving off terminal branches, it was thought to run along the stratum radiatum and continue along the stratum moleculare of the dentate gyrus. The above-described ubiquitin-positive structures, which may be derived from terminal or distal axons, can be explained by degeneration of this pathway. This concept is supported by the report that similar structures in the lateral part of the entorhinal cortex, the transentorhinal cortex and the amygdala in DLBD may be caused by degeneration of terminal or distal axons from the neurons of origin of the perforant pathway (Iseki et al., 1996). Consequently, it seems appropriate to consider this pathway as the alternative perforant pathway. In this study, the distribution of neuronal inclusions such as NFT and LB also appeared to be related to the same pathway, although their pathogenic mechanism remains unknown. Further study will be necessary to examine whether this pathway can explain the hippocampal pathologies of ATD and other neurodegenerative disorders.
Acknowledgments This study was partly supported by Grants-in-Aid for scientific research from the Ministry of Education, Science and Culture.
References Casanova, M.F., Stevens, J.R. and Kleinman, J.E. (1990) Astrocytosis in the molecular layer of the dentate gyrus: a study in Alzheimer’s disease and schizophrenia. Psychiatry Res., 35: 149–166. Dickson, D.W., Wertkin, A., Kress, Y., Ksiezak-Reding, H. and Yen, S.-H. (1990) Ubiquitin immunoreactive structures in normal human brains. Distribution and developmental aspects. Lab. Invest., 63: 87–99. Dickson, D.W., Ruan, D., Crystal, H., Mark, M.H., Davies, P., Kress, Y. and Yen, S.-H. (1991) Hippocampal degeneration differentiates diffuse Lewy body disease (DLBD) from Alzheimer’s disease: Light and
169
electron microscopic immunocytochemistry of CA2–3 neurites specific to DLBD. Neurology, 41: 1402–1409. Dickson, D.W., Schmidt, M.L., Lee, V.M.-Y., Zhao, M.-L., Yen, S.-H. and Trojanowski, J.Q. (1994) Immunoreactivity profile of hippocampal CA2–3 neurites in diffuse Lewy body disease. Acta Neuropathol., 87: 269–276. Duvernoy, H.M. (1988) The human hippocampus. In: An Atlas of Applied Anatomy, Bergmann, Munich, pp. 1–159. Hansen, L.A., Salmon, D., Galasco, D. Masliah, E., Katzman, R., DeTeresa, R., Thal, L., Pay, M.M., Hofstetter, R., Klauber, M., Rice, V., Butters, N. and Alford, M. (1990) The Lewy body variant of Alzheimer’s disease. A clinical and pathological entity. Neurology, 40: 1–8. Hyman, B.T., Van Hoesen, G.W., Kromer, L.J. and Damasio, A.R. (1986) Perforant pathway changes and the memory impairment of Alzheimer’s disease. Ann. Neurol., 20: 472–481. Hyman, B.T., Kromer, L.J. and Van Hoesen, G.W. (1988) A direct demonstration of the perforant pathway terminal zone in Alzheimer’s disease using the monoclonal antibody Alz-50. Brain Res., 450: 392–397. Iseki, E., Odawara, T., Suzuki, K., Kosaka, K., Akiyama, H. and Ikeda, K. (1995) A pathological study of Lewy bodies and senile changes in the amygdala in diffuse Lewy body disease. Neuropathology, 15: 112–116. Iseki, E., Odawara, T., Li, F., Kosaka, K., Nishimura, T., Akiyama, H. and Ikeda, K. (1996) Age-related ubiquitin-positive granular structures in non-demented subjects and neurodegenerative disorders. J. Neurol. Sci., 142: 25–29. Iseki, E., Li, F. and Kosaka, K. (1997) Close relationship between spongiform change and ubiquitin-positive granular structures in diffuse Lewy body disease. J. Neurol. Sci. (in press). Kim, H., Gearing, M. and Mirra, S.S. (1995) Ubiquitin-positive CA2 / 3 neurites in hippocampus coexist with cortical Lewy bodies. Neurology, 45: 1768–1770. Kosaka, K., Yoshimura, M. and Budka, H. (1984) Diffuse type of Lewy body disease: progressive dementia with abundant cortical Lewy bodies and senile changes of varying degree—A new disease? Clin. Neuropathol., 3: 185–192. Kosaka, K. (1990) Diffuse Lewy body disease in Japan. J. Neurol., 237: 197–204. Lippa, C.F., Hamos, J.E., Pulaski-Salo, D., DeGennaro, L.J. and Drachman, D.A. (1992) Alzheimer’s disease and aging: effects on perforant pathway perikarya and synapses. Neurobiol. Aging, 13: 405–411. Mizutani, T. and Kasahara, M. (1995) Degeneration of the intrahippocampal routes of the perforant and alveolar pathways in senile dementia of Alzheimer type. Neurosci. Lett., 184: 141–144. Perry, R.H., Irving, D., Blessed, G., Fairbairn, A. and Perry, E.K. (1990) Senile dementia of the Lewy body type. A clinically and neuropathologically distinct form of Lewy body dementia in the elderly. J. Neurol., 95: 119–139. Senut, M.C., Roudier, M., Davous, P., Fallet-Bianco, P., Lamour, Y. (1991) Senile dementia of the Alzheimer type: is there a correlation between entorhinal cortex and dentate gyrus lesions? Acta Neuropathol., 82: 306–315.
Hippocampal pathology in diffuse Lewy body disease using ubiquitin immunohistochemistry Eizo Iseki*, Feng Li, Toshinari Odawara, Kenji Kosaka Department of Psychiatry, Yokohama City University School of Medicine, 3 -9 Fukuura, Kanazawa-ku, Yokohama 236, Japan Received 16 September 1996; revised 20 December 1996; accepted 12 January 1997
Abstract Various ubiquitin-positive structures in the hippocampus in diffuse Lewy body disease (n512) and non-demented aged subjects (n53) were investigated immunohistochemically. These structures were composed of ubiquitin-positive granular structures (UPG), ubiquitinpositive neuritic structures (UPN), spheroidal structures, neuritic plaques, neurofibrillary tangles (NFT), Lewy bodies (LB) and ubiquitin-positive neurons. UPG, UPN, spheroidal structures and neuritic plaques were distributed with special reference to the hippocampal pathway assumed in this study. This pathway was thought to run along the stratum oriens, mostly perforating the stratum pyramidale at many sites of the subiculum and CA1–3, and to end partly in the CA2–3 and the subiculum of the uncus (UPG or UPN). After perforating the stratum pyramidale and giving off terminal branches (ubiquitin-positive neurons or neuritic plaques with degenerative neurites), it was thought to run along the stratum radiatum and continue along the stratum moleculare of the dentate gyrus, forming synapses with the apical dendrites from the stratum pyramidale and the stratum granulosum (spheroidal structures or neuritic plaques). These findings suggest that many of the ubiquitin-positive structures may be caused by degeneration of terminal or distal axons of this pathway. NFT and LB also had a somewhat orderly distribution with reference to this pathway. 1997 Elsevier Science Ireland Ltd. Keywords: Hippocampus; Perforant pathway; Ubiquitin; Diffuse Lewy body disease
1. Introduction Diffuse Lewy body disease (DLBD) is pathologically characterized by the widespread occurrence of Lewy bodies (LB) in the central nervous system (Kosaka et al., 1984). DLBD is classified into two forms; a common form, with many senile changes, and a pure form, with no or few senile changes (Kosaka, 1990). The common form of DLBD is also referred to as ‘‘senile dementia of Lewy body type’’ (Perry et al., 1990) or ‘‘the Lewy body variant of Alzheimer’s disease’’ (Hansen et al., 1990). There have been some reports, however, suggesting different pathogenesis between DLBD and Alzheimer-type dementia (ATD). Dickson et al. (1991) reported that *Corresponding author: Tel.: 181-45-787-2667. Fax: 181-45-7832540
ubiquitin-immunoreactive neurites were observed in the CA2–3 region of the hippocampus in DLBD cases. Immunoelectron micrographs of these neurites were suggestive of distal axons. These neurites were partially immunostained with a neurofilament antibody but not with antibodies to tau, paired helical filaments or tyrosine hydroxylase (Dickson et al., 1994). The constant association and immunological similarity between these neurites and cortical LB suggest that both structures may share a common pathogenesis (Dickson et al., 1994; Kim et al., 1995). Dickson et al. (1990) found ubiquitin-immunoreactive granules in the neuropil of the entorhinal cortex and amygdala in non-demented aged subjects. We showed that most of these ubiquitin-positive granular structures (UPG) might be derived from degenerating terminal axons of the large multipolar neurons in layer pre-a of the lateral part
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166
E. Iseki et al. / Journal of Neurological Sciences 149 (1997) 165 – 169
of the entorhinal cortex and the large pyramidal neurons in layers IIIc and V of the transentorhinal cortex, and found that UPG disappeared in both ATD and DLBD cases, accompanying some ubiquitin-positive neuritic structures (UPN) similar to the CA2–3 neurites in only DLBD cases (Iseki et al., 1996). In this study, we investigated various ubiquitin-positive structures, including UPG and UPN, in the hippocampus in DLBD cases. We suggest that many of these structures may be caused by degeneration of the hippocampal pathway assumed in this study.
2. Materials and methods We studied 12 pathologically verified DLBD cases (10 common form and two pure form cases) and three nondemented aged cases (Table 1). Cerebral hemispheres were fixed in 4% paraformaldehyde in 0.1 M phosphate buffer (pH 7.4), sliced in two coronal planes through the mammilary body and the lateral geniculate body, embedded in paraffin and cut into 8 mm-thick sections. The sections ¨ were stained using the hematoxylin–eosin (HE), Kluver– Barrera (KB), methenamine–silver (MS) and Gallyas– Braak (GB) methods for pathological examination, and were immunostained with anti-ubiquitin antibody (MAB1510; monoclonal, mouse, 1:8000 dilution; Chemicon, USA). MAB1510 immunolabelling was detected using the avidin-biotinylated HRP complex (ABC) method (Elite Kit, Vector, USA) and visualized with 3,39diaminobenzidine tetrahydrochloride (DAB). In addition, the sections were double-immunostained with MAB1510 and tau-2 (monoclonal, mouse, 1:8000; Sigma, St. Louis, MO, USA) or anti-A 4 protein antibody (polyclonal, rabbit, 1:5000; a gift from Dr. T. Ishii) to examine the relationship between ubiquitin-positive structures and neurofibrillary tangles (NFT) or amyloid deposits, respectively. Immunolabelling was detected using the ABC method and the ABC alkaline phosphatase method (ABC-AP Kit, Vector) and visualized with DAB and fast blue, respectively. Anti-A 4 protein antibody was used after formic acid treatment of the sections. The regional classification of the hippocampus followed the description by Duvernoy (1988).
Fig. 1. A and B. Schema of the hippocampus following Duvernoy (1988). (A) The level of the mammilary body; (B) the level of the lateral geniculate body. AM5amygdala, UN5uncus, SB5subiculum, EC5 entorhinal cortex. The cross represents the location of spheroidal structures. Gray shaded areas represent the distribution of ubiquitin-positive neuritic structures (UPN) with ubiquitin-positive granular structures (UPG), and faint-line shaded areas represent neuritic plaques, ubiquitinpositive degenerative neurons, neurofibrillary tangles (NFT) or Lewy bodies (LB).
3. Results The distribution of the ubiquitin-positive structures in the hippocampus is schematically shown in Fig. 1A–B. Solid and dotted lines demonstrate the hippocampal pathway assumed in this study. Solid lines run along the
Table 1 Summary of patients’ characteristics in the examined cases Number
Mean age (years)
10
74.7
4.0
1161
Pure form DLBD cases
2
64.0
14.0
1230
Non-demented aged cases
3
81.3
2
1067
Common form DLBD cases
DLBD5diffuse Lewy body disease; BW5body weight.
Mean duration (years)
Mean BW (g)
E. Iseki et al. / Journal of Neurological Sciences 149 (1997) 165 – 169
stratum radiatum and the stratum moleculare of the dentate gyrus. Dotted lines run along the stratum oriens and perforate the stratum pyramidale at many sites. The ubiquitin-positive structures were composed of UPG, UPN, spheroidal structures, neuritic plaques, NFT, LB and ubiquitin-positive neurons. UPG appeared to be identical with those of the entorhinal and transentorhinal cortices. UPN frequently had continuity with UPG. Spheroidal structures were found singly or in clusters. Neuritic plaques had ubiquitin-positive degenerative neurites as well as MS- and A 4 -positive amyloid deposits. NFT were positively stained not only with ubiquitin but also with GB and tau-2. LB were defined as ubiquitin-positive inclusions other than NFT. Ubiquitin-positive neurons with diffusely weak cytoplasmic staining included both degenerative neurons and immature NFT- or LB-containing neurons. These ubiquitin-positive structures were not evenly, but were orderly, distributed in the hippocampus. In the three non-demented aged cases, some-to-many UPG were found in the neuropil of the stratum pyramidale of the CA2–3 and the subiculum of the uncus (Fig. 2). Some UPG were also scattered in the neuropil or clustered around the pyramidal neurons, predominantly in the CA1 and subiculum (Fig. 3). These cases showed a few NFT and many UPG, but no amyloid deposits, in the entorhinal and transentorhinal cortices. In the two pure form DLBD cases, some-to-many UPN with UPG were found in the CA2–3 and the subiculum of the uncus (Fig. 4). These UPN were distributed from the stratum oriens to the stratum pyramidale. Some spheroidal structures were arranged in the stratum lacunosum-moleculare along solid lines (Fig. 5). Some-to-many LB or ubiquitin-positive neurons were predominantly found in the CAl and CA3–4 as well as the uncus (Fig. 6). These cases showed some NFT, some UPG with UPN and mild spongiform change, but no amyloid deposits, in the entorhinal and transentorhinal cortices. In the 10 common form DLBD cases, some-to-many UPN with UPG were found in the CA2–3 (Fig. 7) and the subiculum of the uncus (Fig. 8). Some spheroidal structures or neuritic plaques were arranged in the stratum lacunosum-moleculare and in the outer portion of the stratum moleculare of the dentate gyrus along solid lines (Fig. 9). Some to many neuritic plaques, NFT and LB were found in the CA1–4 and subiculum as well as the uncus, although their numbers varied in each case. Three structures had a somewhat different distribution. Neuritic plaques and NFT were predominantly found in the CA1 and subiculum, while LB were frequently found in the CAl and CA3–4 as well as the uncus. In addition, there were some ubiquitin-positive degenerative neurons surrounded by UPG or degenerative neurites, predominantly in the CA1 and subiculum (Fig. 10). These cases showed someto-many NFT, some UPG with UPN, moderate-to-severe spongiform change and many amyloid deposits in the entorhinal and transentorhinal cortices.
167
4. Discussion Anti-ubiquitin antibody recognizes not only neuronal inclusions, such as NFT and LB, but also degenerative neurites, including distal or terminal axons (Dickson et al., 1990; Iseki et al., 1996). Therefore, ubiquitin immunohistochemistry is useful for tracing the degeneration of projections at their distal portion. In DLBD, unlike ATD, the degeneration of distal or terminal axons can be visualized by the occurrence of UPG, UPN, spheroids or spongiform vacuoles, because the neurons of origin of those axons may degenerate more rapidly in DLBD than in ATD (Iseki et al., 1995, 1996, 1997). In a previous paper (Iseki et al., 1996), we showed that UPG were predominantly distributed among layer pre-a of the lateral part of the entorhinal cortex and in layers II–IIIIab of the transentorhinal cortex, and in the intermediate areas of the accessory basal and basal amygdaloid nuclei. We suggested that cortical UPG were derived from degenerating terminals of the recurrent collateral ascending axons of the large multipolar neurons in layer pre-a and the large pyramidal neurons in layers III C and V and amygdaloid UPG from those of their projecting axons. In this study, many UPG were found in the CA2–3 and the subiculum of the uncus in non-demented aged cases. Many UPN, in continuity with UPG, were observed in the same areas in pure- and common form DLBD cases. This finding suggests that these UPG and UPN are caused by degeneration of terminal and distal axons, respectively, of the same projections. Some UPG were also scattered in the neuropil or clustered around the pyramidal neurons, predominantly in the CA1 and subiculum in non-demented aged cases. In common form DLBD cases, there were some ubiquitinpositive neurons surrounded by UPG or degenerative neurites and some neuritic plaques in the same areas, suggesting that these degenerative neurites are caused by degeneration of the same terminal or distal axons as for UPG. Spheroidal structures or neuritic plaques were arranged in the stratum lacunosum-moleculare and in the outer portion of the stratum moleculare of the dentate gyrus along solid lines in pure and common form DLBD cases. These spheroidal structures or neuritic plaques appear to represent the degeneration of terminal axons of the pathway that forms synapses with the apical dendrites from the stratum pyramidale and the stratum granulosum. The perforant pathway originates in the lateral part of the entorhinal cortex and ends in the stratum moleculare of the dentate gyrus (Duvernoy, 1988; Hyman et al., 1988). However, the intrahippocampal routes of this pathway are insufficiently known. In ATD, pathological studies of the perforant pathway showed NFT in the neurons of its origin and neuritic plaques in its terminal zone (Hyman et al., 1986, 1988; Casanova et al., 1990; Senut et al., 1991;
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Fig. 2. UPG in the neuropil of the CA2–3. A non-demented aged case. Anti-ubiquitin immunostaining. Fig. 3. UPG clustered around the pyramidal neurons in the CA1. A non-demented aged case. Anti-ubiquitin immunostaining. Fig. 4. Some UPN (arrows) with UPG in the CA2–3. A pure form DLBD case. Anti-ubiquitin immunostaining. Fig. 5. Some spheroidal structures in the stratum lacunosum-moleculare. A pure form DLBD case. Anti-ubiquitin immunostaining. Fig. 6. Some LB (arrows) and UPN (arrowheads) in the subiculum of the uncus. A pure form DLBD case. Anti-ubiquitin immunostaining. Fig. 7. Some UPN (arrows) with UPG in the CA2–3. A common form DLBD case. Anti-ubiquitin immunostaining. Fig. 8. Numerous UPN with UPG in the subiculum of the uncus. A common form DLBD case. Anti-ubiquitin immunostaining. Fig. 9. Neuritic plaques accompanied by UPG or ubiquitin-positive degenerative neurites (brown; arrowheads) and A 4 -positive amyloid deposits (blue; arrows) in the stratum moleculare of the dentate gyrus. A common form DLBD case. Double immunostaining with anti-ubiquitin and anti-A 4 antibodies. Fig. 10. Ubiquitin-positive degenerative neurons (arrows) surrounded by UPG or ubiquitin-positive degenerative neurites (brown) and tau-2-positive NFT (blue; arrowheads) in the CA1. A common form DLBD case. Double immunostaining with anti-ubiquitin and anti-tau-2 antibodies.
E. Iseki et al. / Journal of Neurological Sciences 149 (1997) 165 – 169
Lippa et al., 1992). Mizutani and Kasahara (1995) suggested that the perforant pathway ran along the stratum lacunosum-moleculare to end in the dentate gyrus after perforating the subiculum. The hippocampal pathway assumed in this study was thought to run along the stratum oriens, mostly perforating the stratum pyramidale, not only at the subiculum but also at the CA1–3, and to end partly in the CA2–3 and the subiculum of the uncus. After perforating the stratum pyramidale and giving off terminal branches, it was thought to run along the stratum radiatum and continue along the stratum moleculare of the dentate gyrus. The above-described ubiquitin-positive structures, which may be derived from terminal or distal axons, can be explained by degeneration of this pathway. This concept is supported by the report that similar structures in the lateral part of the entorhinal cortex, the transentorhinal cortex and the amygdala in DLBD may be caused by degeneration of terminal or distal axons from the neurons of origin of the perforant pathway (Iseki et al., 1996). Consequently, it seems appropriate to consider this pathway as the alternative perforant pathway. In this study, the distribution of neuronal inclusions such as NFT and LB also appeared to be related to the same pathway, although their pathogenic mechanism remains unknown. Further study will be necessary to examine whether this pathway can explain the hippocampal pathologies of ATD and other neurodegenerative disorders.
Acknowledgments This study was partly supported by Grants-in-Aid for scientific research from the Ministry of Education, Science and Culture.
References Casanova, M.F., Stevens, J.R. and Kleinman, J.E. (1990) Astrocytosis in the molecular layer of the dentate gyrus: a study in Alzheimer’s disease and schizophrenia. Psychiatry Res., 35: 149–166. Dickson, D.W., Wertkin, A., Kress, Y., Ksiezak-Reding, H. and Yen, S.-H. (1990) Ubiquitin immunoreactive structures in normal human brains. Distribution and developmental aspects. Lab. Invest., 63: 87–99. Dickson, D.W., Ruan, D., Crystal, H., Mark, M.H., Davies, P., Kress, Y. and Yen, S.-H. (1991) Hippocampal degeneration differentiates diffuse Lewy body disease (DLBD) from Alzheimer’s disease: Light and
169
electron microscopic immunocytochemistry of CA2–3 neurites specific to DLBD. Neurology, 41: 1402–1409. Dickson, D.W., Schmidt, M.L., Lee, V.M.-Y., Zhao, M.-L., Yen, S.-H. and Trojanowski, J.Q. (1994) Immunoreactivity profile of hippocampal CA2–3 neurites in diffuse Lewy body disease. Acta Neuropathol., 87: 269–276. Duvernoy, H.M. (1988) The human hippocampus. In: An Atlas of Applied Anatomy, Bergmann, Munich, pp. 1–159. Hansen, L.A., Salmon, D., Galasco, D. Masliah, E., Katzman, R., DeTeresa, R., Thal, L., Pay, M.M., Hofstetter, R., Klauber, M., Rice, V., Butters, N. and Alford, M. (1990) The Lewy body variant of Alzheimer’s disease. A clinical and pathological entity. Neurology, 40: 1–8. Hyman, B.T., Van Hoesen, G.W., Kromer, L.J. and Damasio, A.R. (1986) Perforant pathway changes and the memory impairment of Alzheimer’s disease. Ann. Neurol., 20: 472–481. Hyman, B.T., Kromer, L.J. and Van Hoesen, G.W. (1988) A direct demonstration of the perforant pathway terminal zone in Alzheimer’s disease using the monoclonal antibody Alz-50. Brain Res., 450: 392–397. Iseki, E., Odawara, T., Suzuki, K., Kosaka, K., Akiyama, H. and Ikeda, K. (1995) A pathological study of Lewy bodies and senile changes in the amygdala in diffuse Lewy body disease. Neuropathology, 15: 112–116. Iseki, E., Odawara, T., Li, F., Kosaka, K., Nishimura, T., Akiyama, H. and Ikeda, K. (1996) Age-related ubiquitin-positive granular structures in non-demented subjects and neurodegenerative disorders. J. Neurol. Sci., 142: 25–29. Iseki, E., Li, F. and Kosaka, K. (1997) Close relationship between spongiform change and ubiquitin-positive granular structures in diffuse Lewy body disease. J. Neurol. Sci. (in press). Kim, H., Gearing, M. and Mirra, S.S. (1995) Ubiquitin-positive CA2 / 3 neurites in hippocampus coexist with cortical Lewy bodies. Neurology, 45: 1768–1770. Kosaka, K., Yoshimura, M. and Budka, H. (1984) Diffuse type of Lewy body disease: progressive dementia with abundant cortical Lewy bodies and senile changes of varying degree—A new disease? Clin. Neuropathol., 3: 185–192. Kosaka, K. (1990) Diffuse Lewy body disease in Japan. J. Neurol., 237: 197–204. Lippa, C.F., Hamos, J.E., Pulaski-Salo, D., DeGennaro, L.J. and Drachman, D.A. (1992) Alzheimer’s disease and aging: effects on perforant pathway perikarya and synapses. Neurobiol. Aging, 13: 405–411. Mizutani, T. and Kasahara, M. (1995) Degeneration of the intrahippocampal routes of the perforant and alveolar pathways in senile dementia of Alzheimer type. Neurosci. Lett., 184: 141–144. Perry, R.H., Irving, D., Blessed, G., Fairbairn, A. and Perry, E.K. (1990) Senile dementia of the Lewy body type. A clinically and neuropathologically distinct form of Lewy body dementia in the elderly. J. Neurol., 95: 119–139. Senut, M.C., Roudier, M., Davous, P., Fallet-Bianco, P., Lamour, Y. (1991) Senile dementia of the Alzheimer type: is there a correlation between entorhinal cortex and dentate gyrus lesions? Acta Neuropathol., 82: 306–315.