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Apolipoprotein E immunoreactivity in cerebral amyloid deposits and neurofibrillary tangles in Alzheimer's disease and kuru plaque amyloid in Creutzfeldt-Jakob disease
Brain Research, 541 (1991) 163-166 Elsevier
163
BRES 24533
Apolipoprotein E immunoreactivity in cerebral amyloid deposits and neurofibrillary tangles in Alzheimer's disease and kuru plaque amyloid in Creutzfeldt-Jakob disease Yoshio Namba 1'3, Masanori Tomonaga 1, Hiroshi Kawasaki 2, Eiichi Otomo 2 and Kazuhiko Ikeda 3 1Department of Neuropathology, Institute of Brain Research, Facultyof Medicine, University of Tokyo, Tokyo (Japan), 2Yokufukai Geriatric Hospital, Tokyo (Japan) and ~Departmentof Ultrastructure and Histochemistry, Psychiatric Research Institute of Tokyo, Tokyo (Japan) (Accepted 6 November 1990)
Key words: Apolipoprotein E; Amyloid; Senile plaque; Neurofibrillary tangle; Alzheimer's disease; Creutzfeld-Jakob disease; Immunohistochemistry
During the course of our immunohistochemical studies on the change of lipids in Alzheimer's disease brains by using antibody to apolipoprotein E, a protein having a special relevance to nervous tissue, we unexpectedly found that apo E immunoreactivity was associated with amyioid in both senile plaques and cerebral vessels and neurofibrillary tangles. The immunoreactivity was also found in amyloid of kuru plaques in Creutzfeldt-Jakob disease. Pretreatment of the sections with formic acid greatly enhanced immunoreactivity of senile and kuru plaques to antibody to apo E.
Apolipoprotein E (apo E) is one of almost dozen protein constituents of plasma lipoproteins that serve various functions, including maintenance of the structure of the lipoprotein particles and regulation of the metabolism of several different lipoproteins (for review see ref. 16). Apo E is unique, however, among lipoproteins in that it has special relevance to nervous tissue: unlike other apolipoproteins that are mainly produced in the liver, apo E is produced also by Schwann cells 2 in the peripheral nervous system (PNS) and astrocytes 2'17'19 and oligodendrocytes 23 in the central nervous system (CNS), and it comprises the major apolipoprotein in cerebrospinal fluid 2°'21. In both CNS and PNS, the protein is postulated to be involved in the mobilization and redistribution of lipids in repair, growth and maintenance of myelin and axonal membranes during development or after injury 16. In the brains of patients with Alzheimer's disease (AD), there is a widespread degeneration of neurons, which could lead to secondary axonal destruction. Also, primary myelinopathy has been implicated in this disease condition 7. We therefore conducted immunohistochemical studies of A D brains using antibodies to apo E. The present report deals with an unexpected and novel finding that apo E immunoreactivity was present in neurofibrillary tangles and amyloid deposits in both
senile plaques and cerebral vessels in A D brains. Immunoreactivity was also found to be associated with amyloid deposits of kuru plaques in the brains of patients with Creutzfeldt-Jakob disease (CJD). Brains were obtained at autopsy from 5 patients with AD. Each parietal portion was snap frozen and stored at -80 °C. Sections were cut on a cryostat, fixed in cold acetone for 15 min and subjected to immunohistochemistry using biotinylated secondary antibody and streptavidin system 8. The rest of the brains were fixed in formalin and processed for standard neuropathological examination. Formalin-fixed, paraffin-embedded sections of these A D brains, together with similarly processed brain sections of patients with cerebral amyloid angiopathy, Down syndrome (DS) and CJD in the collection of our neuropathological department were also used in the present study. The primary antibodies to apo E were a goat polyclonal antibody to human apo E (Medix Biotech, CA) diluted 1:1000 and a mouse monoclonal antibody to rabbit apo E (a generous gift from Dr. Yamada, University of Tokyo, Tokyo) diluted 1:100. All the biotinylated secondary antibodies, i.e. horse anti-goat IgG antibody, goat anti-rabbit IgG antibody and rabbit anti-mouse IgG antibody, were purchased from Vector (Burlingame). Peroxidase conju-
Correspondence: Y. Namba, Department of Ultrastructure and Histochemistry, Psychiatric Research Institute of Tokyo, 2-1-8, Kamikitazawa, Setagayaku, Tokyo 156, Japan. 0006-8993/91/$03.50 ~ 1991 Elsevier Science Publishers B.V. (Biomedical Division)
164 gated streptavidin was purchased from Zymed (San Francisco, CA). Adjacent sections were immunostained with monoclonal antibody to amyloid fl-protein (provided by Dr. Allsop, Psychiatric Research Institute of Tokyo, Tokyo, Japan) t to visualize fl-protein amyloid or rabbit antibody to human r-protein (provided by Dr. Ihara, Tokyo Metropolitan Gerontological Institute, Tokyo) t° to visualize neurofibrillary tangles and neuropil threads. In addition to immunostaining, adjacent sections were stained with Congo red or Bielschowsky silver stain to confirm amyloid or neurofibrillary tangles. In cryostat sections of AD brains, antibody to human apo E stained smooth muscle cells of cerebral vessels in both parenchyma and meninges and reactive astrocytes that were abundant in these brains (data not shown). The localization of apo E immunoreactivity in these cell types in CNS is consistent with the previous reports on rat brain 2 and young adult human brain 17. However, in addition to these staining, strong immunostaining was observed in abnormal structures occurring in AD brains; senile plaques and neurofibrillary tangles together with scattered neuropil threads (Fig. 1A). In senile plaques, apo E immunoreactivity was found in core amyloid and peripheral dystrophic neurites. Both extracellular and intracellular tangles (Fig. 1, inset) were immunostained. These structures were stained with antibody to r-protein in an adjacent section (Fig. 1B). A monoclonal antibody to rabbit apo E also immunostained all of these ADassociated abnormal structures (data not shown). In sections of formalin-fixed, paraffin-embedded materials from patients with AD, senile plaques were
positively stained with polyclonal antibody to apo E (Fig. 2A), although immunoreactive plaques were very much reduced in number when compared with those in cryostat sections. Neuropil threads and intracellular tangles were not immunostained; however, a few of the extracellular tangles were stained. In sections of patients with cerebral amyloid angiopathy, amyloid deposits in vessel walls were strongly stained with antibody to apo E (Fig. 2C). These deposits were positively stained by Congo red in an adjacent section. In brain sections of patients with Creutzfeld-Jakob disease, a few kuru plaques were stained with the antibody. Monoclonal antibody to apo E did not immunostain any of these AD-associated structures, kuru plaques as well as astrocytes and vascular smooth muscle cells in these deparaffinized sections. Since pretreatment of the deparaffinized sections with formic acid has been known to enhance immunoreactivity of senile plaques and kuru plaques to antibodies to fl-protein and PrP (prion protein) 12, respectively, we examined whether this is the case for apo E immunoreactivity. Pretreatment of the sections with 90% formic acid for 15 rain brought about a widespread appearance of apo E immunoreactive diffuse plaques in DS brain sections (Fig. 2B) and kuru plaques in CJD brain sections (Fig. 2D). No enhancement was observed for amyloid deposits in vascular walls. In this report we showed a novel finding that polyclonal antibody to human apo E immunostained cerebral amyloid deposits in both senile plaques and kuru plaques, cerebrovascular amyloid and neurofibrillary tangles. This finding, together with the observation that a monoclonal
Fig. 1. Cryostat brain sections of a patient with AD. A: immunostaining for apo E; inset: an intraceUular tangle. B: immunostaining for r-protein. Counterstained with hematoxylin. A,B: x 120; inset x360. Note senile plaques (arrowheads) and neurofibriilary tangles (arrows).
165
Fig. 2. Formalin-fixed, paraffin-embedded brain sections immunostained for apo E. A: a senile plaque in AD. B: diffuse plaques in DS. C: amyloid deposits in vascular wail in a case with cerebral amyioid angiopathy. D: cerebellar kuru plaques in CJD. Counterstained with hematoxylin. A,C: not pretreated, B,D: pretreated with formic acid. A,D, x360; B, x90; C, x180.
antibody to rabbit apo E also immunostained all of the AD-associated structures strongly suggests that apo E is associated with various kinds of cerebral amyloid deposits and neurofibrillary tangles. Cerebral amyloids are thus far classified into 3 types based upon the difference of chief constituent3; amyioid with fl-protein in senile plaques and cerebral vessels, kuru plaque amyloid with PrP protein and a special type of vascular wall amyloid with a variant of cystatin C in a rare condition of hereditary cerebral hemorrhage with amyloidosis of Icelandic type. However, other molecules are also associated with amyloid deposits, irrespective of chief constituent. Among these, human amyloid P or amyloid P component (SAP) is well known to be associated with almost all types of amyloid deposits in both cerebral and systemic amyloidosis 4'5'H'22. Interestingly, SAP was very recently demonstrated also to be associated with neurofibrillary tangles 4"5'11. Therefore, apo E may share features with SAP with respect to the association with various cerebral amyloids and tangles. Whether or not apo E is present in amyloid deposits in systemic amyloidosis is being studied.
SAP in cerebral amyloids has been thought to be derived from circulation across the damaged blood-brain barrier 11, since the protein is only synthesized in the liver. This may hold true also for apo E. However, the expression of apo E mRNA in the brain has been reported 6. Moreover, previous studies have demonstrated apo E immunoreactivity in reactive astrocytes and smooth muscle cells of the vessels in rat and young adult human brains 3'17. This finding was confirmed by the present study in AD brains. Therefore, in addition to apo E from circulation, apo E produced by astrocytes and smooth muscle cells could contribute to the source for its association with amyloid deposits and extracellular tangles. As for cerebrovascular amyloid, apo E derived from smooth muscle cells may well take a major part. Thus, amyloid formation in vessel walls may possibly differ from amyloid formation in senile plaques as far as the source of apo E is concerned. Apo E immunoreactivity was also detected in intracellular tangles as well as in extracellular tangles. Although neurons do not synthesize apo E, it has been suggested that they incorporate serum proteins 15. Furthermore, apo E has been demonstrated
166 to be internalized through specific receptor(s) at neurites or growth cone in the cultured neuronal cells9. These mechanisms could provide neurons with apo E, leading to the association with intracellular tangles. In any event, it is of interest to see whether apo E content is elevated in the brains of A D patients when c o m p a r e d to the age-matched controls. A p o E immunoreactivity was observed not only in typical plaques but also in so-called diffuse plaques. A l t h o u g h whether diffuse plaques represent the early stage of typical plaques remains unsettled, it has been postulated that fl-protein present in this type of plaque forms p r e a m y l o i d that lacks amyloid fibrils24. The association of apo E immunoreactivity with diffuse plaques raises the possibility that apo E may also be involved in the p r e a m y l o i d formation. A l t h o u g h there is no direct information about the nature of possible association of apo E with amyloid deposits, the observation that p r e t r e a t m e n t of the sec-
tions with formic acid e n h a n c e d immunoreactivity for apo E of senile plaques and kuru plaques m a y suggest that apo E is tightly linked with these kinds of amyloid deposits, since the mechanism of the e n h a n c e m e n t is considered to be due to the d e n a t u r a t i o n of polymers of amyloid c o m p o n e n t . It is of interest to note that certain serum apolipoproteins, i.e. serum amyloid A 14, h u m a n apo A I TM and murine apo AI113, are m a j o r components
We wish to thank Drs. Yamada, AUsop and Ihara for their generous gifts of antibodies. We also thank Mr. K. Kato and Mr. A. Kagiwada for their photographic works. This study was supported in part by a Grant-in-Aid for Scientific Research from the Ministry of Education, Science and Culture, Japan.
1 Allsop, D., Landon, M., Kidd, M., Lowe, J.S., Reynolds, G.P. and Gardner, A., Monoclonal antibodies raised against a subsequence of senile plaque core protein react with plaque cores, plaque periphery and cerebrovascular amyloid in Alzheimer's disease, Neurosci. Lett., 68 (1986) 252-256. 2 Boyles, J.K., Pitas, R.E., Wilson, E., Mahley, R.W. and Taylor, J.M., Apolipoprotein E association with astrocytic gila of the central nervous system and with non-myelinating glia of the peripheral nervous system, J. Clin. Invest., 82 (1985) 1501-1513. 3 Castano, E.M. and Frangione, B., Human amyioidosis, Alzheimer disease and related disorders, Lab. Invest., 58 (1988) 122-132. 4 Coria, F., Castano, E.N., Preili, E, Larrondo-Lillo, M., Van Duinen, S., Shelanski, M.L. and Frangione, B., Isolation and characterization of amyloid component from Alzheimer's disease and other types of cerebral amyloidosis, Lab. Invest., 58 (1988) 454-458. 5 Duong, T., Pommier, E.C. and Scheibel, A.B., Immunodetection of the amyloid P component in Alzheimer's disease, Acta Neuropathol., 78 (1989) 429-437. 6 Elshourbagy, N.A., Liao, W.S., Mahley, R.W. and Taylor, J.M., Apolipoprotein E mRNA is abundant in the brain and adrenals, as well as in the liver, and is present in other peripheral tissues of rats and marmosets, Proc. Natl. Acad. Sci. U.S.A., 82 (1985) 203-207. 7 Englund, E., Brun, A. and Ailing, C., White matter change in dementia of Alzheimer type, Brain, 111 (1988) 1425-1439. 8 Hsu, S.M., Raine, L. and Fanger, H., Use of avidin-biotinperoxidase complex (ABC) in immunoperoxidase techniques: a comparison with unlabeled antibody (PAP) procedure, J. H~tochem. Cytochem., 29 (1981) 577-580. 9 Ignatius, M.J., Smooter, E.M., Pitas, R.E. and Mahley, R.W., Lipoprotein uptake by growth cones in vitro, Science, 236 (1986) 959-962. 10 Ihara, Y., Massive somatodendritic sprouting of cortical neurons in Alzheimer's disease, Brain Research, 459 (1988) 138-144. 11 Karalia, R.N. and Grahovac, I., Serum amyloid P immunoreactivity in hippocampal tangles, plaques and vessles: implications for leakage across the blood-brain barrier in Alzheimer's disease, Brain Research, 516 (1990) 349-353. 12 Kitamoto, T., Ogomori, K., Tateishi, J. and Prusiner, S.B., Formic acid pretreatment enhances immunostaining of cerebral
and systemic amyloids, Lab. Invest., 57 (1987) 230-236. 13 Kunisada, T., Higuchi, K., Aota, S., Takeda, T, and Yamagishi, H., Molecular cloning and nucleotide sequence of cDNA for murine senile amyloid protein: nucleotide substitutions found in apolipoprotein A-II cDNA of senescence accelerated mouse (SAM), Nucl. Acids Res., 14 (1989) 5729-5740. 14 Levin, M., Franklin, E.C., Frangione, B. and Pras, M., The amino acid sequence of a major non-immunoglobulin component of some amyloid fibrils, J. Clin. Invest., 51 (1982) 2773-2776. 15 Liu, H.M., Atack, J.R. and Rapoport, S.I., Immunohistochemical localization of intracellular plasma proteins in the central nervous system, Acta Neuropathol., 78 (1989) 16-21. 16 Mahley, K.W., Apolipoprotein E; cholesterol transport protein with expanding role in cell biology, Science, 240 (1988) 622-630. 17 Murakami, M., Ushio, Y., Morino, Y., Ohta, T. and Matsukado, Y., Immunohistochemical localization of apolipoprotein E in human glial neoplasms, J. Clin. Invest., 82 (1988) 177-188. 18 Nichols, W.C., Dwulet, F., Liepnieks, J. and Benson, M.D., Variant apolipoprotein AI as a major constituent of a human hereditary amyloid, Biochem. Biophys. Res. Commun., 156 (1988) 762-768. 19 Pitas, R.E., Boyles, J.K., Lee, S.H., Foss, D. and Mahley, R.W., Astrocytes synthesize apolipoprotein E and metabolize apolipoprotein E-containing lipoproteins, Biochim. biophys. Acta, 917 (1987) t48-161. 20 Pitas, R.E., Boyles, J.K., Lee, S.H., Hui, D. and Weisgraber, K.H., Lipoproteins and their receptors in the central nervous system, J. Biol. Chem., 262 (1987) 14352-14360. 21 Roheim, P.S., Carey, M., Forte, T. and Vega, G.L., Apolipoproteins in human cerebrospinal fluid, Proc. Natl. Acad. Sci. U.S.A., 76 (1979) 4646-4649. 22 Shirahama, M., Skinner, M. and Cohen, A.S., Immunocytochemical identification of amyloid in formalin-fixed paraffin sections, Histochemistry, 72 (1981) 161-171. 23 Stoll, G., Mueller, H.W., Trapp, B.D. and Griffin, J.W., Oligodendrocytes but not astrocytes express apolipoprotein E after injury of rat optic nerve, Glia, 2 (1989) 170-176. 24 Yamaguchi, H., Nakazato, Y., Hirai, S., Shoji, M. and Harigaya, Y., Electron micrograph of diffuse plaques: initial stage of senile plaque (ormation in the Alzheimer brain, Am. J. Pathol., 135 (1989) 593-597.
of amyloid deposits in o t h e r forms of amyloidosis. Therefore, apo E might be among the c o m p o n e n t s of cerebral amyloid deposits, rather than a loosely attached molecule. D e t e c t i o n of apo E in isolated cerebrovascular amyloid is now underway.
163
BRES 24533
Apolipoprotein E immunoreactivity in cerebral amyloid deposits and neurofibrillary tangles in Alzheimer's disease and kuru plaque amyloid in Creutzfeldt-Jakob disease Yoshio Namba 1'3, Masanori Tomonaga 1, Hiroshi Kawasaki 2, Eiichi Otomo 2 and Kazuhiko Ikeda 3 1Department of Neuropathology, Institute of Brain Research, Facultyof Medicine, University of Tokyo, Tokyo (Japan), 2Yokufukai Geriatric Hospital, Tokyo (Japan) and ~Departmentof Ultrastructure and Histochemistry, Psychiatric Research Institute of Tokyo, Tokyo (Japan) (Accepted 6 November 1990)
Key words: Apolipoprotein E; Amyloid; Senile plaque; Neurofibrillary tangle; Alzheimer's disease; Creutzfeld-Jakob disease; Immunohistochemistry
During the course of our immunohistochemical studies on the change of lipids in Alzheimer's disease brains by using antibody to apolipoprotein E, a protein having a special relevance to nervous tissue, we unexpectedly found that apo E immunoreactivity was associated with amyioid in both senile plaques and cerebral vessels and neurofibrillary tangles. The immunoreactivity was also found in amyloid of kuru plaques in Creutzfeldt-Jakob disease. Pretreatment of the sections with formic acid greatly enhanced immunoreactivity of senile and kuru plaques to antibody to apo E.
Apolipoprotein E (apo E) is one of almost dozen protein constituents of plasma lipoproteins that serve various functions, including maintenance of the structure of the lipoprotein particles and regulation of the metabolism of several different lipoproteins (for review see ref. 16). Apo E is unique, however, among lipoproteins in that it has special relevance to nervous tissue: unlike other apolipoproteins that are mainly produced in the liver, apo E is produced also by Schwann cells 2 in the peripheral nervous system (PNS) and astrocytes 2'17'19 and oligodendrocytes 23 in the central nervous system (CNS), and it comprises the major apolipoprotein in cerebrospinal fluid 2°'21. In both CNS and PNS, the protein is postulated to be involved in the mobilization and redistribution of lipids in repair, growth and maintenance of myelin and axonal membranes during development or after injury 16. In the brains of patients with Alzheimer's disease (AD), there is a widespread degeneration of neurons, which could lead to secondary axonal destruction. Also, primary myelinopathy has been implicated in this disease condition 7. We therefore conducted immunohistochemical studies of A D brains using antibodies to apo E. The present report deals with an unexpected and novel finding that apo E immunoreactivity was present in neurofibrillary tangles and amyloid deposits in both
senile plaques and cerebral vessels in A D brains. Immunoreactivity was also found to be associated with amyloid deposits of kuru plaques in the brains of patients with Creutzfeldt-Jakob disease (CJD). Brains were obtained at autopsy from 5 patients with AD. Each parietal portion was snap frozen and stored at -80 °C. Sections were cut on a cryostat, fixed in cold acetone for 15 min and subjected to immunohistochemistry using biotinylated secondary antibody and streptavidin system 8. The rest of the brains were fixed in formalin and processed for standard neuropathological examination. Formalin-fixed, paraffin-embedded sections of these A D brains, together with similarly processed brain sections of patients with cerebral amyloid angiopathy, Down syndrome (DS) and CJD in the collection of our neuropathological department were also used in the present study. The primary antibodies to apo E were a goat polyclonal antibody to human apo E (Medix Biotech, CA) diluted 1:1000 and a mouse monoclonal antibody to rabbit apo E (a generous gift from Dr. Yamada, University of Tokyo, Tokyo) diluted 1:100. All the biotinylated secondary antibodies, i.e. horse anti-goat IgG antibody, goat anti-rabbit IgG antibody and rabbit anti-mouse IgG antibody, were purchased from Vector (Burlingame). Peroxidase conju-
Correspondence: Y. Namba, Department of Ultrastructure and Histochemistry, Psychiatric Research Institute of Tokyo, 2-1-8, Kamikitazawa, Setagayaku, Tokyo 156, Japan. 0006-8993/91/$03.50 ~ 1991 Elsevier Science Publishers B.V. (Biomedical Division)
164 gated streptavidin was purchased from Zymed (San Francisco, CA). Adjacent sections were immunostained with monoclonal antibody to amyloid fl-protein (provided by Dr. Allsop, Psychiatric Research Institute of Tokyo, Tokyo, Japan) t to visualize fl-protein amyloid or rabbit antibody to human r-protein (provided by Dr. Ihara, Tokyo Metropolitan Gerontological Institute, Tokyo) t° to visualize neurofibrillary tangles and neuropil threads. In addition to immunostaining, adjacent sections were stained with Congo red or Bielschowsky silver stain to confirm amyloid or neurofibrillary tangles. In cryostat sections of AD brains, antibody to human apo E stained smooth muscle cells of cerebral vessels in both parenchyma and meninges and reactive astrocytes that were abundant in these brains (data not shown). The localization of apo E immunoreactivity in these cell types in CNS is consistent with the previous reports on rat brain 2 and young adult human brain 17. However, in addition to these staining, strong immunostaining was observed in abnormal structures occurring in AD brains; senile plaques and neurofibrillary tangles together with scattered neuropil threads (Fig. 1A). In senile plaques, apo E immunoreactivity was found in core amyloid and peripheral dystrophic neurites. Both extracellular and intracellular tangles (Fig. 1, inset) were immunostained. These structures were stained with antibody to r-protein in an adjacent section (Fig. 1B). A monoclonal antibody to rabbit apo E also immunostained all of these ADassociated abnormal structures (data not shown). In sections of formalin-fixed, paraffin-embedded materials from patients with AD, senile plaques were
positively stained with polyclonal antibody to apo E (Fig. 2A), although immunoreactive plaques were very much reduced in number when compared with those in cryostat sections. Neuropil threads and intracellular tangles were not immunostained; however, a few of the extracellular tangles were stained. In sections of patients with cerebral amyloid angiopathy, amyloid deposits in vessel walls were strongly stained with antibody to apo E (Fig. 2C). These deposits were positively stained by Congo red in an adjacent section. In brain sections of patients with Creutzfeld-Jakob disease, a few kuru plaques were stained with the antibody. Monoclonal antibody to apo E did not immunostain any of these AD-associated structures, kuru plaques as well as astrocytes and vascular smooth muscle cells in these deparaffinized sections. Since pretreatment of the deparaffinized sections with formic acid has been known to enhance immunoreactivity of senile plaques and kuru plaques to antibodies to fl-protein and PrP (prion protein) 12, respectively, we examined whether this is the case for apo E immunoreactivity. Pretreatment of the sections with 90% formic acid for 15 rain brought about a widespread appearance of apo E immunoreactive diffuse plaques in DS brain sections (Fig. 2B) and kuru plaques in CJD brain sections (Fig. 2D). No enhancement was observed for amyloid deposits in vascular walls. In this report we showed a novel finding that polyclonal antibody to human apo E immunostained cerebral amyloid deposits in both senile plaques and kuru plaques, cerebrovascular amyloid and neurofibrillary tangles. This finding, together with the observation that a monoclonal
Fig. 1. Cryostat brain sections of a patient with AD. A: immunostaining for apo E; inset: an intraceUular tangle. B: immunostaining for r-protein. Counterstained with hematoxylin. A,B: x 120; inset x360. Note senile plaques (arrowheads) and neurofibriilary tangles (arrows).
165
Fig. 2. Formalin-fixed, paraffin-embedded brain sections immunostained for apo E. A: a senile plaque in AD. B: diffuse plaques in DS. C: amyloid deposits in vascular wail in a case with cerebral amyioid angiopathy. D: cerebellar kuru plaques in CJD. Counterstained with hematoxylin. A,C: not pretreated, B,D: pretreated with formic acid. A,D, x360; B, x90; C, x180.
antibody to rabbit apo E also immunostained all of the AD-associated structures strongly suggests that apo E is associated with various kinds of cerebral amyloid deposits and neurofibrillary tangles. Cerebral amyloids are thus far classified into 3 types based upon the difference of chief constituent3; amyioid with fl-protein in senile plaques and cerebral vessels, kuru plaque amyloid with PrP protein and a special type of vascular wall amyloid with a variant of cystatin C in a rare condition of hereditary cerebral hemorrhage with amyloidosis of Icelandic type. However, other molecules are also associated with amyloid deposits, irrespective of chief constituent. Among these, human amyloid P or amyloid P component (SAP) is well known to be associated with almost all types of amyloid deposits in both cerebral and systemic amyloidosis 4'5'H'22. Interestingly, SAP was very recently demonstrated also to be associated with neurofibrillary tangles 4"5'11. Therefore, apo E may share features with SAP with respect to the association with various cerebral amyloids and tangles. Whether or not apo E is present in amyloid deposits in systemic amyloidosis is being studied.
SAP in cerebral amyloids has been thought to be derived from circulation across the damaged blood-brain barrier 11, since the protein is only synthesized in the liver. This may hold true also for apo E. However, the expression of apo E mRNA in the brain has been reported 6. Moreover, previous studies have demonstrated apo E immunoreactivity in reactive astrocytes and smooth muscle cells of the vessels in rat and young adult human brains 3'17. This finding was confirmed by the present study in AD brains. Therefore, in addition to apo E from circulation, apo E produced by astrocytes and smooth muscle cells could contribute to the source for its association with amyloid deposits and extracellular tangles. As for cerebrovascular amyloid, apo E derived from smooth muscle cells may well take a major part. Thus, amyloid formation in vessel walls may possibly differ from amyloid formation in senile plaques as far as the source of apo E is concerned. Apo E immunoreactivity was also detected in intracellular tangles as well as in extracellular tangles. Although neurons do not synthesize apo E, it has been suggested that they incorporate serum proteins 15. Furthermore, apo E has been demonstrated
166 to be internalized through specific receptor(s) at neurites or growth cone in the cultured neuronal cells9. These mechanisms could provide neurons with apo E, leading to the association with intracellular tangles. In any event, it is of interest to see whether apo E content is elevated in the brains of A D patients when c o m p a r e d to the age-matched controls. A p o E immunoreactivity was observed not only in typical plaques but also in so-called diffuse plaques. A l t h o u g h whether diffuse plaques represent the early stage of typical plaques remains unsettled, it has been postulated that fl-protein present in this type of plaque forms p r e a m y l o i d that lacks amyloid fibrils24. The association of apo E immunoreactivity with diffuse plaques raises the possibility that apo E may also be involved in the p r e a m y l o i d formation. A l t h o u g h there is no direct information about the nature of possible association of apo E with amyloid deposits, the observation that p r e t r e a t m e n t of the sec-
tions with formic acid e n h a n c e d immunoreactivity for apo E of senile plaques and kuru plaques m a y suggest that apo E is tightly linked with these kinds of amyloid deposits, since the mechanism of the e n h a n c e m e n t is considered to be due to the d e n a t u r a t i o n of polymers of amyloid c o m p o n e n t . It is of interest to note that certain serum apolipoproteins, i.e. serum amyloid A 14, h u m a n apo A I TM and murine apo AI113, are m a j o r components
We wish to thank Drs. Yamada, AUsop and Ihara for their generous gifts of antibodies. We also thank Mr. K. Kato and Mr. A. Kagiwada for their photographic works. This study was supported in part by a Grant-in-Aid for Scientific Research from the Ministry of Education, Science and Culture, Japan.
1 Allsop, D., Landon, M., Kidd, M., Lowe, J.S., Reynolds, G.P. and Gardner, A., Monoclonal antibodies raised against a subsequence of senile plaque core protein react with plaque cores, plaque periphery and cerebrovascular amyloid in Alzheimer's disease, Neurosci. Lett., 68 (1986) 252-256. 2 Boyles, J.K., Pitas, R.E., Wilson, E., Mahley, R.W. and Taylor, J.M., Apolipoprotein E association with astrocytic gila of the central nervous system and with non-myelinating glia of the peripheral nervous system, J. Clin. Invest., 82 (1985) 1501-1513. 3 Castano, E.M. and Frangione, B., Human amyioidosis, Alzheimer disease and related disorders, Lab. Invest., 58 (1988) 122-132. 4 Coria, F., Castano, E.N., Preili, E, Larrondo-Lillo, M., Van Duinen, S., Shelanski, M.L. and Frangione, B., Isolation and characterization of amyloid component from Alzheimer's disease and other types of cerebral amyloidosis, Lab. Invest., 58 (1988) 454-458. 5 Duong, T., Pommier, E.C. and Scheibel, A.B., Immunodetection of the amyloid P component in Alzheimer's disease, Acta Neuropathol., 78 (1989) 429-437. 6 Elshourbagy, N.A., Liao, W.S., Mahley, R.W. and Taylor, J.M., Apolipoprotein E mRNA is abundant in the brain and adrenals, as well as in the liver, and is present in other peripheral tissues of rats and marmosets, Proc. Natl. Acad. Sci. U.S.A., 82 (1985) 203-207. 7 Englund, E., Brun, A. and Ailing, C., White matter change in dementia of Alzheimer type, Brain, 111 (1988) 1425-1439. 8 Hsu, S.M., Raine, L. and Fanger, H., Use of avidin-biotinperoxidase complex (ABC) in immunoperoxidase techniques: a comparison with unlabeled antibody (PAP) procedure, J. H~tochem. Cytochem., 29 (1981) 577-580. 9 Ignatius, M.J., Smooter, E.M., Pitas, R.E. and Mahley, R.W., Lipoprotein uptake by growth cones in vitro, Science, 236 (1986) 959-962. 10 Ihara, Y., Massive somatodendritic sprouting of cortical neurons in Alzheimer's disease, Brain Research, 459 (1988) 138-144. 11 Karalia, R.N. and Grahovac, I., Serum amyloid P immunoreactivity in hippocampal tangles, plaques and vessles: implications for leakage across the blood-brain barrier in Alzheimer's disease, Brain Research, 516 (1990) 349-353. 12 Kitamoto, T., Ogomori, K., Tateishi, J. and Prusiner, S.B., Formic acid pretreatment enhances immunostaining of cerebral
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of amyloid deposits in o t h e r forms of amyloidosis. Therefore, apo E might be among the c o m p o n e n t s of cerebral amyloid deposits, rather than a loosely attached molecule. D e t e c t i o n of apo E in isolated cerebrovascular amyloid is now underway.