Expression of tau exon 8 in different species

ELSEVIER

Neuroscience Letters 172 (1994) 167 170

NEUROSClENCE LETTfR$

Expression of tau exon 8 in different species Wei-Ta Chen ~, W a n - K y n g Liu b, Shu-Hui Yen b'* "Department of Physiology, National Yang-Ming Medical College, Shih-Pai, Tuipei, Tuiwan ~'Department qf Pathoh)gy, F-53& Albert Einstein College qf Medicine, 1300 Morris Park Avenue, Bronx, N Y 10461. USA

Received 1 February 1994; Revised version received 24 March 1994; Accepted 24 March 1994

Abstract

A synthetic peptide corresponding to a region encoded by tau gene exon 8 was used to raise an antibody. The antibody, E8, was used to probe normal tau from different species and abnormal tau proteins from subjects with Alzheimer's disease, lmmunoblotting and enzyme-linked immunosorbent assays demonstrated that only bovine tau reacted with the E8 antibody. The E8 immunoreactive tau isoform was estimated to represent less than 2% of the bovine tau. Our results indicate that bovine tau is unique in containing isoforms positive with E8, and that PHF formation does not require the presence of PHF-tau with exon 8. Key words: Bovine tau: PHF-tau; Exon 8 expression

Tau belongs to a group of proteins named microtubule associated proteins, which are capable of promoting microtubule assembly and stabilizing microtubules [2]. Tau proteins consist of several isoforms. The heterogeneity is due to posttranslational modification and alternative gene splicing [3,4,6]. Phosphorylation reduces the ability of tau to promote microtubule assembly [11]. Hyperphosphorylation has been reported to lead to the formation of an abnormal form of tau in the brain of Alzheimer's disease (AD) [16]. This form of tau, PHF-tau, is a major protein component of abnormal filaments in Alzheimer brains. These filaments, regarded as paired helical filaments (PHF), are located in neurons and aggregated in bundles in cell bodies and neurites [1,5,15]. P H F - t a u contains 3~ , times more phosphate than tau fi'om normal adult h u m a n brains, and it is different from normal adult tau in having fewer isoforms [8,9]. Tau proteins derived from brains of different species are different in molecular mass and number of isoforms [2,7]. The amino terminal region of the tau molecule is not identical in different species, whereas the middle and carboxy terminal regions are highly conserved [3,4,6]. The carboxy terminal half of the tau molecule contains 3 or 4 tandem repeats considered to be important for

*Corresponding author. Fax: (1) (718) 892-1720. 0304-3940/94/$7.00 ,~>1994 Elsevier Science Ireland Ltd. All rights reserved S S D I 0304-3940(94)00253-7

binding to tubulin. Bovine tau, according to Himmler et al., is encoded by a gene containing at least 13 exons [4]. Of these 13 exons, exons 2, 3, and 10 are not expressed in the juvenile form of tau. Exon 1 encodes a highly variable region in tau between different species, and exons 5-13 encode conserved regions. By a polymerase chain reaction, a bovine tau c D N A with exon 8 consisting of 54 base pairs (bp) has been reported [4]. It remains unknown whether exon 8 is indeed expressed in bovine tau proteins or tau proteins from other species, and whether only a small fraction or all tau is encoded by transcripts with this exon. Also, an issue of interest is whether P H F - t a u differs from normal human tau in the expression of exon 8. An abnormality in the expression of exon 8 could alter the conformation of the tubulin binding domain of tau, since the first tandem repeat for tubulin binding is encoded by an exon (exon 9) adjacent to exon 8. Our recent findings of P H F - t a u containing proportionally more isoforms with exon 3 than normal tau [13] raises the possibility that abnormal expression of other exons may also be involved in the formation of PHF-tau. To resolve the above issues, a polyclonal antibody to a synthetic peptide corresponding to the region of tau encoded by exon 8 was raised (E8 antibody). This antibody was used to probe tau proteins prepared from bovine, rat, rabbit and human brains, and P H F - t a u from

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Fig. 1. Immunoblots of tau preparations from differentspecies with the E9 antibody. (a) recombinant human tau. Other lanes contained tau proteins prepared from brains of (b) fetal human, (c) bovine, (d) rabbit, and (e) rat. (f) PHF-tau isolated from brains with Alzheimer's disease. Molecular standards of 116, 97, 65, 49 and 29 kDa are marked.

A D brains. For comparison, tau proteins from different species were also tested for reactivities with an antibody (E9 antibody) raised against a synthetic peptide corresponding to a region encoded by exon 9 of tau, which is conserved in tau from different species. E8 and E9 antibodies were raised against synthetic peptides H Q V Q K K P P P A G A K S E R and VAVVRTPPKSPSSAK (amino acid residues 226-240 of the longest human tau isoform) linked to keyhole limpet hemocyanin, respectively [13]. Both antibodies were purified by affinity chromatography with their respective peptides. The tau protein enriched fractions from normal human, aborted fetus (20 weeks of gestation), bovine, rabbit and rat were prepared by homogenizing the brain tissue with 3 volumes of Tris-buffered saline (TBS) containing 2 m M EDTA, 1 m M P M S F and 5 /,tg/ml of leupeptin, pH 7.4. After centrifugation at 27,000 × g for 20 min, solid NaC1 and concentrated fl-mercaptoethanol were added to the supernatant to a final concentration o f 2% each. The solution was boiled at 100°C for 5 min and followed by centrifugation at 27,000 × g for 20 min. The supernatant was collected and 70% perchloric acid (PCA) was added to the supernatant to a final concentration of 2.5%. After standing at 4°C for 10 min, the mixture was re-centrifuged at 100,000 x g for 30 min to remove the acid-insoluble precipitate. The acid-soluble proteins, which remained in the supernatant, were then precipitated with 20% trichloroacetic acid (TCA). The TCA precipitate was dissolved in a small volume of distilled water and re-precipitated by 6 volumes of cold methanol. The methanol precipitate was then dissolved in 20 m M Tris-HC1 p H 7.4 and stored at -20°C until

use. PHF-tau protein was prepared from AD brain tissue by a protocol reported previously [12]. Recombinant tau protein was obtained from expression of a c D N A clone generously supplied by Dr. M. Goedert of MRC [3] that encodes the longest human tau isoform [3]. Protein concentration of different preparations was determined by the micro BCA method (Pierce Co., Rockford, IL). The tau protein prepared from various sources were subjected to SDS-gel electrophoresis, electroblotted to nitrocellulose membrane and probed with both the E8 and the E9 antibodies. The bound antibodies were detected by the avidin-biotin-peroxidase method (VectaStain ABC kit, Vector Laboratories, Burlingame, CA), using 3,3'-diamino-benzindine as chromogen. As shown in Fig. 1, tau proteins from various sources of brains showed immunoreactivity with the E9 antibody. The E9 immunoreactivity was detected mainly on several protein bands migrating between 49 kDa and 68 kDa. The number of E9-positive bands varied between species. The results are consistent with those reported in previous studies, demonstrating that exon 9 is highly conserved in tau from different species [3,4,6]. Also consistent with our previous observations [14] was the detection of E9 immunoreactivity in tau from normal adult human brains (data not shown). Several bands of molecular mass lower than 49 kDa were recognized by the E9 antibody in tau preparations from some species. These proteins are considered to be degraded tau, since they did not react with anti-tau antibodies raised to either the carboxy- or the amino-terminal of tau (regions of tau known to be susceptible to proteolysis), but reacted with other anti-tau antibodies (data not shown). PHF-tau preparations were recognized by the E9 antibody, and the immunoreactive proteins were revealed by immunoblots as three bands of molecular mass 60-68 kDa.

Fig. 2. Immunoblotsof tau preparations from differentspecieswith the E8 antibody (a) recombinant human tau. Other lanes contained tau from brains of (b) bovine, (c) fetal human, (d) rabbit and (e) rat. Lanes (f) and (g) contained PHF-tau isolated from brains with Alzheimer's disease. The protein loading in (g) is higher than that in (f). Molecular standards of 116, 97, 65, 49 and 29 kDa are marked.

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V~-Z Chen et al./Neuroscience Letters 172 (1994) 167 170

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Fig. 3. Enzyme-liked immunosorbent assays of the reactivity of the E8 peptide with the E8 antibody, and the E9 peptide with the E9 antibody. Both the E8 and the E9 antibodies were used at 1 : 50 dilution. The peptide samples in triplicate were plated on a 96-well ELISA plate (VWR Scientific, Bridgeport, N J) overnight, washed with Tris-buffered saline and incubated with E9 or E8 antibody (1 : 50 dilution). The bound immunoglobulins were detected by a VectaStain kit, using 2,2'Azino-di(3-ethyl-benzthioazoline sulfonate) as chromogen.

The reaction of P H F - t a u with E9 antibodies is in agreement with the results obtained in our recent studies [14]. In contrast to the E9 antibody, the E8 antibody reacted only with bovine tau proteins, and not with tau proteins from other species (Fig. 2). In addition, E8 antibody failed to react with PHF-tau. Treatment of electroblots with alkaline phosphatase did not affect the E8 immunoreactivity of bovine tau and tau proteins from other species (data not shown). The absence of E8 immunoreactivity in non-bovine tau was not likely due to loading of an inadequate amount of tau proteins on gel, since increasing of the protein loading did not affect the results (data not shown). Moreover, tau proteins derived from non-bovine brains were as readily detectable by the E9 antibody as bovine tau. These results indicate that exon 8 is uniquely expressed in the bovine tau. Furthermore, the absence of E8 immunoreactivity in PHF-tau suggests that the formation of P H F in Alzheimer's disease is not dependent upon expression of exon 8. The possibility that tau in human or other non-bovine species may be encoded by an exon 8 different from that of bovine is not high, since tau is a highly conserved protein. This issue could be resolved by genomic cloning of tau in different species. Based on immunoblotting, the E9 antibody had a stronger immunoreactivity towards bovine tau than the E8 antibody (compare lane c in Fig. 1 and lane b in Fig.2). To be certain that the differences in E8 and E9 immunoreactivities were not due to differences in antibody titers, we compared the reactivity of the E8 and the E9 antibodies with E8 and E9 peptides, respectively. By enzyme-linked immunosorbent assay (ELISA), the E8 antibody was able to detect E8 peptide at a concentration of 10-s M (Fig. 3). In comparison, the E9 antibody detected E9 peptide at the concentration of 10 -7 M , but not

at 10-8 M. The binding of both antibodies to their respective peptides was with high affinity. The apparent affinity of E8 antibody to E8 peptide is estimated to be 5 x 10 -~ M, and that of E9 antibody to E9 peptide is 6 x 10-7 M. The results suggest that the E8 antibody is about 10 times more sensitive in detecting its antigen than the E9 antibody. This difference is unlikely due to a difference in the absorption of the E8 and E9 peptides to ELISA plates, since both peptides are basic peptides and are comparable in size. The proportion of bovine tau isoforms with and without E8 was determined by ELISA (Fig. 4). At the concentration of 2.0/~g protein/ml, the reaction of bovine tau with E9 antibody had an optical density of 1.0. In comparison, it required a bovine tau sample of protein concentration 100/lg/ml to achieve a similar reactivity with E8 antibody. The results indicate that even in bovine tau exon 8 is expressed in only a small proportion o f t a u molecule. We estimate about 2% of the bovine tau contains isoforms with exon 8 (assuming both the E8 and E9 antibodies have similar sensitivity in detecting tau antigen). Since the E8 antibody is about 10 times more sensitive in detecting antigen than the E9 antibody, the proportion of bovine tau with exon 8 is actually likely to be much less than 2%. These results are consistent with molecular cloning studies in which c D N A of tau with exon 8 was obtained by PCR, but not less sensitive methods. The presence of a low concentration of tau with exon 8 suggests that this form of tau, unlike the E8 negative tau, may not play a significant role in maintaining the structural stability of microtubules. However, we have not ruled out the possibility that the E8 positive tau may localize in a specific region of the bovine brain cell type, or specific subcellular location, which may suggest that it differs from other tau in terms of function. This issue may be resolved by further studies of the expression of E8 positive tau with immunocytochemical and/or in situ hybridization in bovine brain sections. 2.5

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This work was supported by NIH Grant AG01136, and AG04145 (S.-H.Y.) and a grant from Alzheimer's Association/CBS Inc. (W.-K.L.). We thank Drs. D.W. Dickson and W. Lyman for providing Atzheimer and human fetal brain samples, and Ms. F Ruta for the preparation of bovine tau. [1] Braak, H., Braak, E., Grundke-lqbal, I. and lqbal, K., Occurrence of neuropil threads in the senile human brain and in Alzheimer's disease: a third location of paired helical filaments outside of neurofibrillary tangles and neuritic plaques, Neurosci. Lett., 65 (1986) 351-355. [2] Cleveland, D.W., Hwo, H.Y. and Kirshner, M.W., Purification of tau, a microtubule-associated protein that induces assembly of microtubutes from purified tubulin, J. Mol. Biol., 116 (1977) 207 225. [3] Goedert, M., Spillantini, M.G., Jakes, R., Rutherford, R. 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. [4] Himmler, A., Structure of bovine tau gene; Alternatively spliced transcripts generate a protein family, Mol. Cell Biol., 9 (1989) 1389-1396. [5] Kidd, M., Alzheimer's disease-an electron microscopical study, Brain, 87 (1964) 307-320. [6] Kosik, K.S., Orecchio, L.D., Bakalis, S. and Neve, R.L., Developmentally regulated expression of specific tau sequence, Neuron, 2 (1989) 1389-1397. [7] Ksiezak-Reding H., Dickson, D.W. and Yen, S.H., Recognition of tau epitopes by anti-neurofilament antibodies that bind to Alz-

heimer neurofibrillary tangles, Proc. Natl. Acad. Sci. LISA, ~4 (1987) 3410-3414. [8] Ksiezak-Reding, H., Binder, L.I. and Yen, S.H., Atzheimer disease proteins (A68) share epitopes with tau but show distinct biochemical properties, J. Neurosci. Res., 25 (1990)420430. [9] Ksiezak-Reding, H., Liu, W.K. and Yen, S.H., Phosphate analysis and dephosphorylation of modified tau associated with paired helical filaments, Brain Res., 597 (1992) 209 219. [10] Laemmli, U.K., Cleavage of structural protein during assembly of the head of bacteriophage T4, Nature, 227 (1970) 680-685. [11] Lindwall, G. and Cole, R.D., Phosphorylation affects the ability of tau protein to promote microtubule assembly, J. Biol. Chem., 259 (1984) 5301 5305. [12] Liu, W.K., Ksiezak-Reding, H. and Yen, S.H., Abnormal tau proteins from Alzheimer's disease brains, J. Biol. Chem.. 266 (1991) 21723 21727. [13] Liu, W.K., Dickson, D.W. and Yen, S.H., Heterogeneity of tau proteins in Alzheimer's disease: evidence for increased expression of an isoform and preferential distribution of a phosphorylated isoform in neurites, Am. J. Pathol., 142 (1993) 387-394. [14] Liu, W.K., Dickson, D.W. and Yen, S.H., Amino acid residues 226-240 of tau, which encompass the first Lys-Ser-Pro site of tau, are partially phosphorylated in Alzheimer paired helical filamenttau, J. Neurochem., 62 (1994) 1055--1061. [15] Wisnewski, H.M., Terry, R.D and Hirano, A., Neurofibrillary pathology, J. Neuropathol. Exp. Neurol., 29 (1970) 163-176. [16] Yen, S.H., Dickson, D.W., Liu, W.K. and Ksiezak-Reding, H., From microtubule associated protein tau to paired helical filaments and Alzheimer neurofibrillary tangles: an overview. In Ikuta (Ed.), Neuropathology in Brain Research, Elsevier, 1991, pp. 3344.