(Stroke. 1995;26:2145-2148.)
© 1995 American Heart Association, Inc.
Articles |
Widespread Appearance of Alz-50 Immunoreactive Neurons in the Human Brain With Cerebral Infarction
From the Department of Neuropathology, Tokyo Institute of Psychiatry (T.U., H.K., K.I.); Department of Neurology, Tokyo Medical and Dental University (T.U., K.T.); Department of Pathology, Tokyo Medical College (K.T.); and Department of Pathology, Tokyo Metropolitan Aoyama Hospital (T.H.) (Japan).
Correspondence to T. Uchihara, MD, PhD, Laboratoire Raymond Escourolle, Service de Neuropathologie, Groupe hospitalier, Pitié-Salpêtrière, 47-83 Boulevard de l'Hôpital, 75651 Paris, Cedex 13, France.
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Abstract |
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Background and Purpose Tau-like immunoreactivity is known to develop in neurons under some experimental conditions simulating ischemia. The purpose of this study is to investigate the expression of tau-like immunoreactivity in the human brain after ischemic insult.
Methods A series of autopsied human brains with or without ischemic lesion were investigated with immunohistochemistry (Alz-50, anti-tau, and anti-ubiquitin) and with silver-staining methods (Gallyas and Bodian methods).
Results Punctate immunoreactivity to Alz-50 was visualized in the cytoplasm not only of the neurons in and around the ischemic lesion but also of the neurons free from classic ischemic changes around the necrosis. Some of the neurons around the ischemic lesion were stained by the Gallyas method. Immunostaining with anti-tau and anti-ubiquitin antibodies and the conventional Bodian method failed to visualize these neurons.
Conclusions The widespread appearance of Alz-50 immunoreactive neurons during the ischemic process signifies that tau-related proteins may be related to ischemic necrosis, but the lack of neurofibrillary tangles morphologically distinguishes ischemic development of tau-related proteins from the neurofibrillary degeneration in Alzheimer's disease.
Key Words: cerebral infarction • neurofibrillary tangles • tau proteins
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Introduction |
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Tau and its related proteins are major components of PHF. They are deposited in the form of A-NFTs in the neurons of the AD brain.1 2 3
However, these proteins are known to also appear in cultured neurons after exposure to glutamate4 5 or in neurons of the animal brain after experimentally induced ischemia.6 7 We therefore examined autopsied human brains with an ischemic lesion to clarify its relation to tau-like IR.
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Methods |
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Autopsied brains from patients with cerebral infarction were used in this study. The time of occurrence of the cerebrovascular accident was determined from the clinical record and/or according to histological findings8 listed in the Table
. Two other brains without neurological disorders
were used as control. Formalin-fixed, paraffin-embedded blocks
including both the area of ischemic necrosis and the
surrounding nonnecrotic area were obtained. We stained
10-µm-thick neighboring sections with the following: (1) cresyl
violet stain; (2) Bodian stain; (3) Gallyas stain pretreated (to
enhance the sensitivity of the staining) with 0.3% potassium
permanganate for 10 minutes followed by 1% oxalic acid for 1 minute9 10 ;
and (4) tau (tau 2,11 mouse monoclonal IgG, 1:1000, Sigma,
and pool 2,12 rabbit IgG, 1:20 000, generously provided
by Dr H. Mori), ubiquitin (rabbit IgG, 1:1000, DAKO), and
Alz-5013 (mouse monoclonal IgM, 1:200, generously provided
by Dr P. Davies) immunohistochemistry.
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Sections were treated with 0.2% hydrogen peroxide for 30 minutes and were then incubated for 2 days at 4°C with the primary antibody diluted with PBS containing 0.3% Triton-X and the corresponding blocking serum. The sections were then incubated with one of the biotinylated secondary antibodies corresponding to the primary antibody (1:1000, biotinylated anti-mouse IgG, biotinylated anti-mouse IgM, or biotinylated anti-rabbit IgG, Vector) for 2 hours. After incubation with avidin-biotin-peroxidase complex (1:1000, ABC-Elite, Vector) for 1 hour at room temperature, peroxidase labeling was visualized by incubating sections with 0.05% Tris-buffered saline (pH 7.6) containing 0.03% 3,3'-diaminobenzidine, 0.00015% hydrogen peroxide, 0.05 mol/L imidazole, and 1.0% nickel sulfate. A deep purple reaction product became visible after 15 to 30 minutes.
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Results |
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In the ischemic lesion, where glial reaction, proliferation of the capillaries, and neuronal depletion are apparent on sections stained by cresyl violet (Fig 1A
), some of
the pyramidal neurons were immunostained by
Alz-50 (Fig 1B). These neurons were scarcely identifiable on sections
stained by cresyl violet. Some of the neurons were weakly stained by a
modification of the Gallyas method (Fig 1C).
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In the nonnecrotic area without glial reaction around the
ischemic lesion, the number and the morphology of neurons were
normal (Fig 2A). In this area also,
pyramidal cells were stained by Alz-50 (Fig 2B). The Alz-50
IR was restricted to the neuronal perikarya and was punctate in
fashion; it was abolished when the primary antibody was replaced by PBS
containing 0.3% Triton-X or by mouse IgM (mouse standard, Tago
Diffu-gen). These neurons were not stained by the modified Gallyas
method.
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Anti-ubiquitin antibody, anti-human tau antibodies (tau 2, pool 2), and the conventional Bodian method failed to visualize these neurons. In the areas far from the ischemic lesion as well as in control cases, no neurons were stained by these antibodies or by silver-staining methods.
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Discussion |
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After experimentally induced ischemia, some neurons are reported to accumulate tau-like or Alz-50 IR6 7 in their cytoplasm in punctate fashion. This is the first report confirming that a similar process takes place in the human brain during ischemia.
Neurons, not containing NFTs, with accumulated tau-related epitopes were described in some restricted parts of the normal human and animal brain,14 15 16 in the nervous system during its development,17 18 and in the AD brain as "pretangle neurons."1 2 3 19 Kato and colleagues20 reported a case of cerebral infarction with abundant NFTs in the nucleus basalis of Meynert in the affected hemisphere. However, it is unusual for A-NFTs to appear in and around the ischemic focus. These examples indicate that tau-related proteins rather frequently accumulate in neurons, such as the increased Alz-50 IR in the ischemic process, but this does not necessarily lead to the formation of NFTs.
As demonstrated by immunoblotting, Alz-50 or anti-tau antibodies (tau 2, pool 2) bind to a recombinant tau molecule or its extracted form from the normal brain,12 21 22 23 while the positive immunohistochemical staining is at most limited to the axon in the fixed normal brain tissue.24 In the AD brain, on the other hand, these antibodies easily immunostain pathological structures such as A-NFTs and neurite reactions.13 19 25 This discrepancy can be partly explained if some of these epitopes in the tissues remain active after fixation only when tau-related proteins are modified into a pathological state (not yet fully specified), as seen in the AD brain.26 In this study the appearance of Alz-50 IR in the fixed brain tissue was accompanied by a positive staining by the Gallyas method, which labels tau, preferentially pathological (PHF) tau.27 These data suggest that tau molecules are pathologically modified during the ischemic process rather than a mere cross-reaction of Alz-50 to molecules other than tau.28
Alz-50 preferentially immunolabels pretangle neurons or intracellular NFTs but not extracellular NFTs which, on the contrary, are labeled by other anti-PHF tau antibodies, Bodian stain, and thioflavine S.1 2 3 19 25 29 SDS-soluble fraction from AD brain containing fewer aggregated fibrils contains more prominent Alz-50 IR than the SDS-insoluble fraction containing more aggregated fibrils such as PHF.30 The Alz-50 immunoreactive epitope on purified bovine tau became selectively active after treatment with transglutaminase without developing other tau epitopes.31 These data are compatible with the hypothesis that Alz-50 immunoreactive epitope becomes more active than other tau epitopes in the early phase of NFT development.13
Although pretangle neurons and some intracellular NFTs stained by Alz-50 in the AD brain are not always immunolabeled by other anti-tau antibodies,1 2 19 29 the absence of IR to anti-tau (tau 2, pool 2) and anti-ubiquitin antibodies in Alz-50 immunoreactive neurons during the ischemic process suggests that the posttranslational modification of tau in these neurons is different from that in AD (for example, phosphorylation, truncation, or ubiquitination).32 33 34 35 In addition, the punctate IR in these ischemic neurons looks like the Alz-50 immunoreactive structures, which are reported to be nonfilamentous, in normal brains14 and is totally different from the diffuse cytoplasmic staining seen in pretangle neurons1 3 19 in the AD brain.
Alz-50 immunoreactive neurons in the ischemic process do not develop NFTs. On the other hand, pretangle neurons in the AD brain may develop A-NFTs, probably after hyperphosphorylation of tau protein.36 It is essential to know what factor or environment is responsible for tau-related proteins being woven into NFTs. Further in vivo or autopsy studies combined with biochemical analyses will be needed to clarify the state of modification of tau-related proteins in various conditions and its relation to the fibrillary structure at the electron microscopic level. Such a study will provide a clue to understanding the key step in the formation of NFTs after tau-related proteins are expressed.
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Selected Abbreviations and Acronyms |
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Received April 3, 1995; revision received August 1, 1995; accepted August 3, 1995.
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References |
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TopAbstractIntroductionMethodsResultsDiscussionReferences |
|---|
1. Bancher C, Brunner C, Lassman H, Budka H, Jellinger K, Wiche G, Seitelberger F, Grundke-Iqbal I, Wisniewski HM. Accumulation of abnormally phosphorylated tau precedes the formation of neurofibrillary tangles in Alzheimer's disease. Brain Res.. 1989;477:90-99. [Medline] [Order article via Infotrieve]
2. Bondareff W, Harrington C, Wischik CM, Hauser DL, Roth M. Immunohistochemical staging of neurofibrillary degeneration in Alzheimer's disease. J Neuropathol Exp Neurol.. 1994;53:158-164. [Medline] [Order article via Infotrieve]
3. Braak E, Braak H, Mandelkow E-M. A sequence of cytoskeleton changes related to the formation of neurofibrillary tangles and neuropil threads. Acta Neuropathol (Berl).. 1994;87:554-567. [Medline] [Order article via Infotrieve]
4. Sindou P, Couratier P, Barthe D, Hugon J. A dose-dependent increase of Tau immunostaining is produced by glutamate toxicity in primary neuronal cultures. Brain Res.. 1992;572:242-246. [Medline] [Order article via Infotrieve]
5. Mattson MP. Antigenic changes similar to those seen in neurofibrillary tangles are elicited by glutamate and Ca2+ influx in cultured hippocampal neurons. Neuron.. 1990;2:105-117.
6. Geddes JW, Schwab C, Craddock S, Wilson JL, Pettigrew LC. Alterations in tau immunostaining in the rat hippocampus following transient cerebral ischemia. J Cereb Blood Flow Metab.. 1994;14:554-564. [Medline] [Order article via Infotrieve]
7. Dewar D, Graham DI, Teasdale GM, McCulloch J. Alz-50 and ubiquitin immunoreactivity is induced by permanent focal cerebral ischaemia in the cat. Acta Neuropathol (Berl).. 1993;86:623-629. [Medline] [Order article via Infotrieve]
8. Chuaqui R, Tapia J. Histologic assessment of the age of recent brain infarcts in man. J Neuropathol Exp Neurol.. 1993;52:481-489. [Medline] [Order article via Infotrieve]
9. Uchihara T, Kondoh H, Ikeda K. Histochemical approach to argyrophilic structures in Alzheimer brain [in Japanese]. Pathol Clin Med.. 1994;12:163-168.
10. Uchihara T, Kondo H, Ikeda K, Kosaka K. Alzheimer-type pathology in melanin-bleached sections of substantia nigra. J Neurol.. 1995;252:485-489.
11. Watanabe N, Takio K, Hasegawa M, Arai T, Titani K, Ihara Y. Tau 2: a probe for Ser conformation in the amino terminus of tau. J Neurochem.. 1992;58:960-966. [Medline] [Order article via Infotrieve]
12. Endoh R, Ogawara M, Iwatsubo T, Nakano I, Mori H. Lack of the carboxyl terminal sequence of tau in ghost tangles of Alzheimer's disease. Brain Res.. 1993;601:164-172. [Medline] [Order article via Infotrieve]
13.
Wolozin BL, Pruchnicki A, Dickson DW, Davies P.
A neuronal antigen in the brains of Alzheimer's
patients. Science.. 1986;232:648-650.
14. Byne W, Mattiace L, Kress Y, Davies P. Alz-50 immunoreactivity in the hypothalamus of the normal and Alzheimer human and the rat. J Comp Neurol.. 1991;306:602-612. [Medline] [Order article via Infotrieve]
15. Liberini P, Piccardo P, Cuello AC. Alz-50 immunoreactivity in the central nervous system of adult rat and primate. Neurosci Lett.. 1993;151:200-204. [Medline] [Order article via Infotrieve]
16. Nelson PT, Marton L, Saper CB. Alz-50 immunohistochemistry in the normal sheep striatum: a light and electron microscope study. Brain Res.. 1993;600:285-297. [Medline] [Order article via Infotrieve]
17. Al-Ghoul WM, Miller MW. Transient expression of Alz-50 immunoreactivity in developing rat neocortex: a marker for naturally occurring neuronal death? Brain Res.. 1989;481:361-367. [Medline] [Order article via Infotrieve]
18. Riederer BM, Binder LI. Differential distribution of tau proteins in developing cat cerebellum. Brain Res Bull.. 1994;33:155-161. [Medline] [Order article via Infotrieve]
19. Hyman BT, Van Hoesen GW, Wolozin BL, Davies P, Kromer LJ, Damasio AR. Alz-50 antibody recognizes Alzheimer-related neuronal changes. Ann Neurol.. 1988;23:371-379. [Medline] [Order article via Infotrieve]
20. Kato T, Hirano A, Katagiri T, Sasaki H, Yamada S. Neurofibrillary tangle formation in the nucleus basalis of Meynert ipsilateral to a massive cerebral infarct. Ann Neurol.. 1988;23:620-623. [Medline] [Order article via Infotrieve]
21.
Ksiezak-Reding H, Davies P, Yen SH. Alz 50, a
monoclonal antibody to Alzheimer's disease antigen,
cross-reacts with tau proteins from bovine and human brain.
J Biol Chem.. 1988;263:7943-7947.
22. Goedert M, Spillantini MG, Jakes R. Localization of the Alz-50 epitope in recombinant human microtubule-associated protein tau. Neurosci Lett.. 1991;126:149-154. [Medline] [Order article via Infotrieve]
23. Ueda K, Masliah E, Saitoh T, Bakalis SL, Scoble H, Kosik KS. Alz-50 recognizes a phosphorylated epitope of tau protein. J Neurosci.. 1990;10:3295-3304. [Abstract]
24. Kowall NW, Kosik KS. Axonal disruption and aberrant localization of tau protein characterize the neuropil pathology of Alzheimer's disease. Ann Neurol.. 1987;22:639-643. [Medline] [Order article via Infotrieve]
25. Love S, Saitoh T, Quijada S, Cole GM, Terry RD. Alz-50, ubiquitin and tau immunoreactivity of neurofibrillary tangles, Pick bodies and Lewy bodies. J Neuropathol Exp Neurol.. 1988;47:393-405. [Medline] [Order article via Infotrieve]
26. Pollock NJ, Wood JG. Differential sensitivity of the microtubule-associated protein, tau, in Alzheimer's disease tissue to formalin fixation. J Histochem Cytochem.. 1988;36:1117-1121. [Abstract]
27. Iqbal K, Braak E, Braak H, Zaidi T, Grundke-Iqbal I. A silver impregnation method for labeling both Alzheimer paired helical filaments and their polypeptide separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Neurobiol Aging.. 1991;12:357-361. [Medline] [Order article via Infotrieve]
28. van de Nes JAP, Sluiter AA, Pool CW, Kamphorst W, Ravid R, Swaab DF. The monoclonal antibody Alz-50, used to reveal cytoskeletal changes, also reacts with a large subpopulation of somatostatin neurons in the normal human hypothalamus and adjoining areas. Brain Res.. 1994;655:97-109. [Medline] [Order article via Infotrieve]
29. Dickson DW, Ksiezak-Reding H, Liu W-K, Davies P, Crowe A, Yen S-HC. Immunohistochemistry of neurofibrillary tangle with antibodies to subregions of tau protein: identification of hidden and cleaved tau epitopes and a new phosphorylation site. Acta Neuropathol (Berl).. 1992;84:596-605. [Medline] [Order article via Infotrieve]
30. Ksiezak-Reding H, Morgan K, Dickson DW. Tau immunoreactivity and SDS solubility of two populations of paired helical filaments that differ in morphology. Brain Res.. 1994;649:185-196. [Medline] [Order article via Infotrieve]
31. Dudek SM, Johnson VW. Transglutaminase catalyses the formation of sodium dodecyl sulfate-insoluble, Alz-50-reactive polymers of tau. J Neurochem.. 1993;61:1159-1162. [Medline] [Order article via Infotrieve]
32. Morishima M, Ihara Y. Posttranslational modifications of tau in paired helical filaments. Dementia.. 1994;5:282-288.
33. Mandelkow E-M, Mandelkow E. Tau as a marker for Alzheimer's disease. Trends Biochem Sci.. 1993;18:480-483. [Medline] [Order article via Infotrieve]
34. Kosik KS. The molecular and cellular biology of tau. Brain Pathol.. 1993;3:39-43. [Medline] [Order article via Infotrieve]
35. Trojanowski JQ, Schmidt ML, Shin R-W, Bramblett T, Rao D, Lee VM-Y. Altered tau and neurofilament proteins in neurodegenerative diseases: diagnostic implications for Alzheimer's disease and Lewy body dementia. Brain Pathol.. 1993;3:45-54. [Medline] [Order article via Infotrieve]
36.
Lee VM-Y, Balin BJ, Otvos L Jr, Trojanowski JQ.
A68: a major subunit of paired helical filaments and derivatated
forms of normal tau. Science.. 1991;251:675-678.
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