Glial tau-positive structures lack the sequence encoded by exon 3 of the tau protein gene

Neuroscience Letters 224 (1997) 169–172

Glial tau-positive structures lack the sequence encoded by exon 3 of the tau protein gene Toru Nishimura a, Kenji Ikeda a ,*, Haruhiko Akiyama a, Tetsuaki Arai a, Hiromi Kondo a, Masayasu Okochi a, Yoshiko Furiya a, Hiroshi Mori a, Tatsuro Oda b, Masanori Kato c, Eizo Iseki d a

Tokyo Institute of Psychiatry, 2-1-8 Kamikitazawa, Setagaya-ku, Tokyo 156, Japan b National Hospital of Shimofusa Sanatorium, Chiba 266, Japan c Soga Hospital, Odawara 250-02, Japan d Department of Psychiatry, Yokohama City University School of Medicine, Yokohama 236, Japan Received 10 December 1996; revised version received 18 February 1997; accepted 18 February 1997

Abstract Tau protein comprises six distinct isoforms defined by the presence or absence of sequences encoded by alternatively spliced exon 2, 3 and 10. We have investigated immunohistochemically the expression of exon 3-derived fragment (E-3) of tau protein in brains of patients with Alzheimer’s disease (AD) and other neurodegenerative diseases in which the abnormal accumulation of tau protein takes place. In AD, a subset of neurofibrillary tangles, neuropil threads and dystrophic neurites in senile plaques were stained positively with an anti-E-3 antibody. In sharp contrast, glial tau-positive structures, such as astrocytic plaques and oligodendroglial coiled bodies, were negative for E3 in all cases examined in this study. This is the first report to discriminate tau-positive inclusions in glial cells from those in neurons at the molecular level.  1997 Elsevier Science Ireland Ltd. Keywords: Alternative splicing; Astrocyte; Oligodendroglia; Coiled body; Tangle

Microtubule-associated protein tau promotes the assembly and stability of microtubules in cells of the nervous system. In human brain, alternative splicing of tau mRNA gives rise to the expression of six distinct isoforms of tau protein [4]. The carboxy terminal region has either three or four imperfect sequence repeats of 31 or 32 amino acids which mediate the binding of tau to microtubules [1,6,7]. Three variations also exist in the amino terminal region which contains a 29 amino acid insert encoded by exon 2, a 58 amino acid insert encoded by exon 2 and 3, or no insert, making up in total six isoforms [4]. So far, no clear function has yet been attributed to the amino terminal inserts, although the presence or absence of these inserts are developmentally regulated [11]. In Alzheimer’s disease (AD) brain, all six isoforms have been shown to be involved in the formation of paired helical filaments (PHF) [5], a major * Corresponding author. Tel.: +81 3 33045701; fax: +81 3 33298035; e-mail: [email protected]

component of neurofibrillary tangles (NFTs), neuropil threads and dystrophic neurites in senile plaques (SPs). Tau-positive abnormal structures also occur in glial cells in a number of neurodegenerative diseases [2,3,14]. These include progressive supranuclear palsy (PSP) and corticobasal degeneration (CBD), both of which cause dementia in elderly patients. In this study, we have investigated immunohistochemically the presence of exon 3-derived fragment (E-3) of tau protein in brains of patients with AD and in other diseases with glial tau-positive structures. Twenty-one brains were employed in this study. The diagnoses were AD in 16 patients (67–88 years of age), PSP in one (66 years of age), CBD in one (67 years of age) and Pick’s disease in one (67 years of age). Two came from patients (51 and 83 years of age) with an yet unclassified neurodegenerative disease where massive oligodendroglial tau-positive inclusions appeared. The clinicopathological features of these two cases will be reported elsewhere. Diagnoses were made in every case by routine

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neuropathological examination. At autopsy, small blocks of brain were dissected from the hippocampus, middle temporal gyrus, precentral gyrus, striatum, and pons. The detailed procedure for immunohistochemistry has been described elsewhere [13]. For single staining, primary antibody labeling was detected using the avidin-biotinylated HRP complex (ABC) system (Vector Lab, Burlingame, CA, USA) coupled to a diaminobenzidine (DAB) reaction intensified with nickel ammonium sulfate to yield a dark purple precipitate. For double staining, primary antibody labeling in the second cycle was detected using Tris-aminophenylmethane (TAPM; Nakalai Tesque, Japan) as a chromogen to yield a pink precipitate [8]. The anti-tau primary antibodies used in this study were: Alz-50 (mouse IgM, generous gift of Dr. P. Davies) [15]; TF-11 (mouse IgG) [12]; and anti-E-3 (rabbit antiserum). Alz-50 recognizes the amino terminal region of tau. The epitope recognized by TF-11 is located between residues 188 and 222 of tau [12]. The anti-E-3 antiserum was raised against a synthetic peptide corresponding to the 14 amino acid residues (CVTAPLVDEGAPGK) of the sequence encoded by exon 3 of the tau protein. Specificity of the anti-E3 antibody was confirmed with competition studies on both immunoblotting and immunohistochemistry. The soluble fraction of human brain homogenates was boiled and the precipitates subjected to immunoblotting with the anti-E3 antibody. Fig. 1 illustrates the resultant 57–69 kDa bands which are comparable

Fig. 1. Immunoblotting of a sample from control human brain tissue with anti-E-3 antibody. Lane 1 shows the intense bands comparable in molecular weight with tau proteins containing the exon-3 derived fragment. These intense bands disappeared in lane 2 where 1 mg/ml of the antigen peptide was added to the primary antibody solution.

in molecular weight with tau proteins containing E-3 (lane 1). The bands disappeared by adding 1 mg/ml of the antigen peptide to the primary antibody solution (Fig. 1, lane 2). In tissue sections, positive staining was completely abolished by addition of the same concentration of antigen peptide to the primary antibody solution (data not shown). In AD brain, a subset of NFTs, neuropil threads and dystrophic neurites in SPs were stained positively with the antiE-3 antibody (Fig. 2A). E-3 positive NFTs were fewer than TF-11 positive ones. In addition to NFTs, anti-E-3 labeled granular structures in the cytoplasm of some neurons (Fig. 2A, arrow). Fig. 2B illustrates double immunostaining with anti-E-3 (dark purple) and TF-11 (pink), in which the E-3 negative but TF-11 positive structures are colored pink. E-3 positive NFTs and dystrophic neurites were also stained with TF-11. But a number of TF-11 positive structures were negative or stained faintly for E-3 (Fig. 2B, arrowheads). E-3 labeling of some NFTs was often granular (Fig. 2B, arrow). In double immunostaining with anti-E-3 and Alz-50, E-3 positive granules were seen occasionally in neurons with granular labeling with Alz-50 (Fig. 2C, arrow). Some NFTs were entirely stained for E-3 (Fig. 2C, arrowhead) while only parts of others were positive. The vast majority of extracellular NFTs (ghost tangles) were negative with all anti-tau antibodies used in this study. Fig. 2D–F illustrate double immunostaining of PSP brain with anti-E-3 (dark purple) and TF-11 (pink). The globose type of NFTs, which are considered to be characteristic in PSP, were E-3 positive (Fig. 2D, arrow). All astrocytic plaques [14] were TF-11 positive but E-3 negative (Fig. 2E,F). In CBD brain, astrocytic plaques were more frequent [2] and were stained positively with TF-11 (Fig. 2G) and Alz50 (Fig. 2H). They were again negative for E-3 (Fig. 2G,H). A subset of neurons which contained granules positive for TF-11 and Alz-50 were also labeled for E-3 (Fig. 2G,H, arrows). Other TF-11 and Alz-50 positive structures with thread-like or coil-like profiles which occurred abundantly in the basal ganglia and brain stem of CBD and less frequently in PSP [9] were negative for E-3. In the brains of the two patients with an yet unclassified neurodegenerative disease, numerous oligodendroglial coiled bodies [10] were present and stained positively with TF-11 (Fig. 2I,J) and Alz-50. They were negative for E-3 (Fig. 2I,J). In these cases, a number of neurons were positive with Alz-50 and TF-11. Such neurons were often positive for E-3 (Fig. 2I,J, arrows). Pick bodies in a patient with Pick’s disease were not stained with the anti-E-3 antibody (data not shown). Immunohistochemical staining for tau protein reveals such fibrous structures as NFTs, neuropil threads and dystrophic neurites. It also labels neuronal granular structures which are referred to as pretangles. The antibody to E-3 stained all forms of these neuronal tau abnormalities except for Pick bodies. A subpopulation of these structures were negative for E-3, suggesting that the expression of tau isoforms varies from neuron to neuron. A number of tau-posi-

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tive inclusions also appear in astrocytes and oligodendroglia in brains of patients with PSP, CBD, and some other neurodegenerative diseases [2,3,14]. It is noteworthy that, in

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contrast to neurons, none of these glial tau-positive structures were stained positively for E-3. We speculate that, in glial cells, E-3 containing isoforms are either expressed at

Fig. 2. Single (A) and double (B–J) immunostaining for E-3 and other tau epitopes. (A) The entorhinal cortex from an AD patient stained for E-3. NFTs, neuropil threads, and dystrophic neurites in SPs are positive for E-3. Granular E-3 immunoreactivity is seen in the neuronal cytoplasm (arrow). (B–J) Double immunostaining for E-3 (dark purple) and TF-11 (pink, B,D–G,I,J) or Alz-50 (pink; (C,H)). (B) The AD hippocampus. NFTs and dystrophic neurites in SPs are often doubly labeled. Granular E-3 immunoreactivity is seen in association with TF-11 positive NFTs (arrow). (C) The hippocampus of another AD case. Some NFTs are uniformly stained for E-3 (arrowhead). E-3 positive granules are also seen within the Alz-50 positive neurons (arrow). (D) The temporal cortex from a PSP patient. Globose-type NFTs are doubly labeled for E-3 and TF-11 (arrow). (E) The frontal cortex from a PSP patient. Astrocytic plaques are E-3 negative but TF-11 positive (arrowheads). Note that E-3 positive neurons are seen in the same section (arrow). (F) A high-power photomicrograph of the astrocytic plaque in (E). There is no E-3 immunoreactivity. (G) The parietal cortex from a CBD patient. Astrocytic plaques are negative for E-3 (arrowheads), while some neurons are positive for TF-11 (arrow). (H) The parietal cortex from a CBD patient. Astrocytic plaques are negative for E-3 but positive for Alz-50 (arrowheads). Some neurons are stained intensely for E-3 (arrow). (I) The parietal white matter from a patient with an unclassified neurodegenerative disease. Many oligodendroglial tau positive inclusions are seen but are negative for E-3 (arrowheads). Note that some neurons are stained positively for E-3 (arrow). (J) The hippocampus from another patient with the unclassified disease. TF-11 positive oligodendroglial coiled inclusions are E-3 negative (arrowheads). Some NFTs are positive for both E-3 and TF-11 (arrows).Bar, (A–D,F–I)50 mm; (E,J) 100 mm.

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very low levels or are not involved in tau pathology. Feany et al. reported that any cytoskeletal lesions in CBD were negative for E-3 while NFTs in AD and PSP were positive [3]. These authors argued that the absence of E-3 in tau pathology discriminated CBD from PSP. However, the results of our study suggests that the difference might be cell type-specific rather than disease-specific. This research was supported by a research grant for dementia from the Ministry of Health and Welfare, Japan. We are grateful to Dr. Peter Davies for providing the Alz-50 antibody, and Dr. Kuniaki Tsuchiya (Tokyo Medical College, Japan) for providing brain tissue. [1] Butner, K.A. and Kirschner, M.W., Tau protein binds to microtubules through a flexible array of distributed weak sites, J. Cell. Biol., 115 (1991) 717–730. [2] Feany, M.B. and Dickson, D.W., Widespread cytoskeletal pathology characterizes corticobasal degeneration, Am. J. Pathol., 146 (1995) 1388–1396. [3] Feany, M.B., Ksiezak-Reding, H., Liu, W.-K., Vincent, I., Yen, S.H.C. and Dickson, D.W., Epitope expression and hyperphosphorylation of tau protein in corticobasal degeneration: differentiation from progressive supranuclear palsy, Acta Neuropathol., 90 (1995) 37–43. [4] Goedert, M., Spillantini, M.G., Jakes, R., Rutherford, D. and Crowther, R.A., Multiple isoforms of human microtubule-associated protein tau: sequences and localization in neurofibrillary tangles of Alzheimer’s disease, Neuron, 3 (1989) 519–526. [5] Goedert, M., Spillantini, M.G., Cairns, N.J. and Crowther, R.A., Tau proteins of Alzheimer paired helical filaments: abnormal phosphorylation of all six brain isoforms, Neuron, 8 (1992) 159–168. [6] Gustke, N., Trinczek, B., Biernat, J., Mandelkow, E.M. and Mandelkow, E., Domains of tau protein and interactions with microtubules, Biochemistry, 33 (1994) 9511–9522.

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