Hirano bodies contain tau protein

Brain Research, 403 (1987) 337-340 Elsevier

337

BRE 22025

Hirano bodies contain tau protein Pamela G. Galloway 1, George Perry 2, Kenneth S. Kosik 3 and Pierluigi Gambetti 2 1Department of Pathology, Cleveland Metropolitan General Hospital, 3395 Scranton Road, Cleveland, OH 44109 (U.S.A.), 2Division of Neuropathology, Institute of Pathology, Case Western Reserve University, Cleveland, 0H44106 (U.S.A.)and 3Department of Neurology, Harvard Medical School, Boston, MA 02115 (U.S.A.) (Accepted 30 September 1986)

Key words: Hirano body; Tau protein; Alzheimer's disease; Cytoskeleton; Neurofibrillary tangle; Paired helical filament; Immunocytochemistry

Hirano bodies are intraneuronal inclusions Whose frequency increases with age and Alzheimer's disease. These paracrystalline inclusions have been shown previously using immunocytochemistry to share epitopes with actin, tropomyosin, a-actinin and vinculin. Hirano bodies have not previously been demonstrated to share components with neurofibrillary tangles, another intraneuronal inclusion characteristic of Alzheimer's disease. In this study, we show that Hirano bodies bind antibodies to the microtubule-associated protein tau, a component of Alzheimer neurofibrillary tangles.

A l z h e i m e r ' s disease is a form of d e m e n t i a characterized by neuronal inclusions containing components of the cytoskeleton 2,13,25. O n e of these inclusions, the H i r a n o b o d y , is most c o m m o n l y seen in neurons of the S o m m e r sector of the h i p p o c a m p u s 16 and in the underlying stratum lacunosum 14, and m o r e rarely in the spinal cord 28, cerebellum s, and cerebral cortex 27. T h e y are located primarily in dendrites and axons, but can also be seen in the p e r i k a r y o n 29. I m m u n o c y t o c h e m i c a l studies have shown that Hirano bodies share epitopes with actin 1°-12,14 and actin-associated proteins a°-12 including t r o p o m y o s i n , a-actinin and vinculin. These findings suggest that H i r a n o bodies are derived primarily from neuronal microfilaments, since actin is the core protein and m a j o r c o m p o n e n t of microfilaments 6. In contrast, the neurofibrillary tangle and its main constituent, the paired helical filament ( P H F ) 2°, do not show immunologic cross-reactivity with c o m p o n e n t s of microfilaments 12. Polyclonal antisera raised against sodium dodecylsulfate extracted p a i r e d helical filaments usually show immunoreactivity with the microtubule-associated protein, tau 1s'22,25. A n t i b o d i e s

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Fig. 1. Characterization of tau monoclonal antibody (5E2). Lanes a-c are Coomassie-stained gel of heat-stable brain MAPs from mature rat (a), fetal rat (b), and fetal human (c). Lanes d-f are the same samples immunoblotted with 5E2. Molecular weight (MW) standards in ascending order are lysozyme (Mr 14,000), soybean trypsin inhibitor (Mr 22,000), carbonic anhydrase (Mr 30,000), ovalbumin (Mr 43,000), bovine serum albumin (Mr 68,000), phosphorylase b (Mr 92,000).

Correspondence: P.G. Galloway, Department of Pathology, Cleveland Metropolitan General Hospital, 3395 Scranton Road, Cleveland OH 44109, U.S.A. 0006-8993/87/$03.50 © 1987 Elsevier Science Publishers B.V. (Biomedical Division)

338 against the 200 kilodalton (kDa) and 145 k D a neurofilament subunits 1'3'26 and microtubule associated protein 2 ( M A P 2 ) (ref. 18), as well as tau protein are immunoreactive with neurofibrillary tangles 1,4,18,

21,25,33.

To d e t e r m i n e a possible relationship b e t w e e n

neurofibrillary tangles and H i r a n o ~odies, H i r a n o bodies were i m m u n o s t a i n e d with a m o n o c l o n a l (5E2) and a polyclonal a n t i b o d y 7 to tau protein.

Fig. 2. A and B: section of human hippocampus from a patient with Alzheimer's disease, showing Hirano bodies stained with the monoclonal tau antibody (1:200) (A) and the polyclonal tau antibody (1:200) (B) (small arrowheads). Other Hirano bodies are not immunostained but are demonstrated by the eosin counterstain (large arrowheads). Also present in both fields are immunostained neurofibrillarytangles, x200.

339 Sections of human hippocampus from patients with Alzheimer's disease containing large numbers of Hirano bodies were prepared from tissue fixed in 10% formalin in 0.1 M phosphate, pH 7.0, and embedded in paraffin. Tissue was sectioned 8/~m thick and immunostained by the peroxidase-antiperoxidase technique 3°. Diaminobenzidine (Sigma Chemicals, St. Louis, MO) (0.75 mg/ml) was used as cosubstrate with H20 2 (0.015%) in 0.05 M Tris-HC1, pH 7.6. Development times were generally less than two minutes. Sections were counterstained with hematoxylin eosin to be viewed at 200x to 1000x. The primary antibodies were a polyclonal antibody and a monoclonal antibody to tau protein. The polyclonal antibody has been characterized as previously described 7. The immunogen for the monoclonal antibody was the heat-stable fraction of taxol-stabilized microtubules prepared 31 from a frozen human fetal brain (20-week gestation) (Fig. 1). In mature brain, the two dominant components of this preparation are MAP2 and tau; however, at 20 weeks human gestation MAP2 is present in only trace amounts and tau protein alone represents the dominant component. Tau heterogeneity is reduced at this early developmental time point; only two major tau bands are detected by Coomassie staining, in contrast to the mature forms which have multiple bands in various species 5'7'9. This immunogen (1 mg) was injected into a BALB/c mouse and booster injections were given at 4 and 6 weeks. Fusion of the spleen cells with NSI mouse myeloma cells was done 19. Supernatants were screened colorimetrically 23 by binding 0.2-2.0 g of the antigen to a Flexible Assay Plate (Falcon). Positive clones were analyzed by immunoblotting heatstable microtubule fractions from both adult rat and fetal human brain (Fig. 1). Of approximately 300 clones positive on the initial screen, only one was specific for the tau bands on the immunoblot. This clone, designated 5E2, was subcloned by the limited dilution technique 19. Hirano bodies were stained by both tau antibodies (Fig. 2A,B). In previous studies using antibodies to actin and actin-associated proteins, all Hirano bodies

in a section of hippocampus were immunostained. In contrast, in these sections only about 20% of Hirano bodies were stained by the tau antibodies. When either mouse or rabbit non-immune serum or an unrelated hybridoma supernatant was used as primary antibody, Hirano bodies were not immunostained. Immunostaining was removed by adsorption of antibody with 200 j~g/ml of bovine tau protein 24. Tau is a microtubule-associated protein 5 which can also serve as an actin cross-linking protein 6'32. Two other proteins which have been shown to be present in Hirano bodies 12, a-actinin and vinculin, are crosslinking proteins3:; thus, pathologic cross-linking of actin filaments may be related to Hirano body formation. Recent studies indicate that tau is an antigenic component of Alzheimer neurofibrillary tangles 4'18'22'25'33. Rabbits immunized with SDS-extracted PHF fractions frequently developed antibodies to tau. On Western blots, the PHF and tau antibodies reacted identically with all forms of tau from mature and fetal rat brain 22. In another study is, tau protein was purified from bovine brain using several methods, and all tau preparations reacted with antibodies to paired helical filaments. As yet, it is not clear what role tau protein plays in both Hirano body and neurofibrillary tangle formation. Although it is possible that neurofibrillary tangles derive largely from a derangement of neurofilaments and microtubules, while Hirano bodies result predominantly from an alteration of microfilaments, our findings suggest that the components contributing to formation of these structures are not mutually exclusive.

The authors thank Dr. Mark Kirschner for providing the polyclonal antibody. The authors also thank Ms. Madelyn Weiss, Mrs. Debra Moscalink and Mrs. Karen Boyda for helping prepare the manuscript, Mr. Vince Messina for photographic assistance, and Ms. Michelle Onorato for performing the adsorption.

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