Cell Motility and the Cytoskeleton 15199-203 (1990)

Views and Reviews Tau Protein: An Update on Structure and Function Gloria Lee Program in Neuroscience, Harvard Medical School, and Center for Neurologic Diseases, Brigham and Women‘s Hospital, Boston, Massachusetts

Tau protein was among the first microtubule-associated proteins (MAPs) to be identified through the cycled assembly purification of microtubule protein. Following its purification, which revealed extensive tau protein heterogeneity, in vitro studies documented its ability to promote microtubule assembly and immunocytochemical data described its association with microtubules in vivo. However, as other MAPs had been similarly purified from brain and other cell types and exhibited similar properties, investigators asked why there were so many MAPs and whether there were functional differences between them in cells. Although the revelations 1) that microtubules are dynamic structures in the cell and 2) that many types of tubulin exist have changed the context in which these questions are asked, the questions persist. Although tau has been shown to stabilize microtubules when introduced into a cell by microinjection and to affect the frequency of transition between the growing and shrinking phases of dynamic microtubules in vitro, it is quite probable that MAP2 will also display these properties, given the sequence homologies between these proteins (see below). Therefore, a unique role for tau protein in cellular development remains to be shown. The isolation of cDNA clones for tau protein has led to much new information on the structure, function, and developmental expression of tau isoforms. Tau protein sequences have now been determined from mouse, human, bovine, and rat, and conservation between species is high. The tau protein family within each species is generated from alternatively spliced transcripts originating from one gene [Himmler, 19891. The figure shows a schematic of 13 isoforms of tau protein using the tau gene exon numbers described by the bovine genomic data [Himmler, 19891. Homology between species is greatest in the carboxy terminal half (> 97% in exons 9 , 10, 11, 12, and 13); numerous single amino acid changes 0 1990 Wiley-Liss, Inc.

between species are located in the amino terminal third (approximately 15% of the residues are different). The 1* exon in human differs from exon 1 in other species by an intercalation of 11 residues; the striped exon in mouse represents an alternative carboxy terminus. Clones isolated by Himmler [ 19891, Kanai et al. [ 19891, Kosik et al. [ 19891, and Goedert et al. [1989a,b] originated from adult brain libraries, whereas the clone isolated by Goedert et al. 119891 originated from fetal brain. Mouse clones were isolated from 6 day post-natal brain. Other forms beyond those listed here are likely to exist, based on cDNA and genomic data. These include adult bovine isoforms with an alternate amino terminus (exon 6) or an alternate extended carboxy terminus (exon 14) [Himmler, 19891, a fetal rat isoform with exon 10 deleted [Kosik et al., 19891, and an adult rat isofonn with exon 3 deleted [Kanai et al., 19891. The most striking feature of the primary structure of tau protein as predicted from cDNA clones is a stretch of 31 or 32 residues that is imperfectly repeated three or four times in the carboxy terminal half of the molecule. Each repeat resides in a different exon (9, 10, 11, or 12) and the similarity between the repeats is greatest in the 18 residue stretch located at the carboxy proximal side of the repeat. The suggestion that the repeats constitute microtubule binding units has been tested with in vitro synthesized tau protein fragments [Himmler et al., 1989; Lee et al., 19891 and synthetic peptides [Aizawa et al., 1989; Ennulat et al., 19891. Microtubule polymerization assays using synthetic one-repeat peptides has shown

Accepted December 6, 1989. Address reprint requests to Gloria Lee, Center for Neurologic Diseases, Brigham and Women’s Hospital, 75 Francis Street, Boston, MA 02115.

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that such peptides are capable of promoting assembly and that the microtubules formed are normal polymers (unlike those induced by nonspecific polycations) . However, the molar quantity of peptide required relative to tubulin is about 100 times that required of full-length tau protein. This difference may be due to the reiteration of the repeat in the full-length protein since the efficiency of binding of in vitro-synthesized tau fragments to taxolstabilized microtubules increases as the number of repeats present increases [Lee et al., 19891. However, although isolated individual repeat units do interact with microtubules, it is also likely that full activity is dependent on conformation that may be influenced by neighboring regions. Although tau is thought to be elongated and rod-shaped, there may still be interactions between various sections along the rod, for instance, the stretching and contracting of tau protein, as suggested by im-

ages of tau paracrystals [Lichtenberg et al., 19881. Additional study will be required to clarify the role of the repeat unit in microtubule elongation and nucleation both in vitro and in vivo. An additional function that has been ascribed to tau protein is that of bundling microtubules. Kanai et al. 119891 made this observation in vivo after having expressed tau cDNA in fibroblast cells by DNA transfection. The domain of tau protein engaged in this function has been identified by Lewis et al. [1989]. They show that the carboxy terminal end of tau contains a short hydrophobic “zipper” sequence that mediates crosslinking between tau molecules, thereby bringing microtubules together. Their earlier work had also revealed that MAP2 and the three-repeat isoform of tau share homology in the carboxy terminal end [Lewis et al., 19881; MAP2 contains three repeats, and regions beyond

Tau Protein: Update on Structure and Function

the repeat area are also similar (of 185 residues at the carboxy terminal end, 124 are identical to tau sequence). The “zipper” motif is conserved, and, indeed, MAP2 also bundles microtubules [Lewis et al., 19891. One interesting difference is that a three-repeat MAP2 molecule is able to bundle much more efficiently than is a threerepeat tau protein; the results of Kanai et al. [ 19891 were obtained with a four-repeat tau isoform. A 190 kd microtubule-associated protein from bovine adrenal gland [Aizawa et al., 19891 has also been found to contain the repeat unit. At this point, it is not known how much additional homology exists among this MAP, tau, and MAP2. However, as more sequence information is obtained for microtubule-associated proteins from a variety of sources, it will be interesting to see the extent to which tau’s exons 9, 10, 11, 12, and 13 have been conserved for other microtubule-associated proteins. MAP2 has not been found to employ an exon 10 [Lewis et al., 19881. Since developmental changes in tau protein size and activity have been previously described, it is not surprising that differences in clones isolated from fetal and adult brain libraries have been found. Additional .tests have been performed on brain RNA in order to characterize the observed differences. Exon 2 and exon 3 , which each encode an insert of 29 amino acids near the amino terminal end of tau, were found exclusively in human adult brain, with mRNAs containing exon 2 alone being more prevalent than the combination of 2 plus 3 [Goedert et al., 1989bJ. Similarly, exon 10, which encodes the second repeat unit of four, was not expressed in fetal human brain; in the adult, mRNAs with exon 10 (the four-repeat form) was estimated to be one-third the amount of the three-repeat form (exon 10 deleted) [Goedert et al., 1989b1. In the rat brain, exon 2 and exon 10 are also adult specific [Kosik et al., 19891. However, exon 10 deleted forms (three-repeat form) were not present in adult rat brain, suggesting that the three-repeat form in rat is fetal specific. Kosik et al. [1989] also examined human RNA and failed to find three-repeat tau in the adult. Although this result does not agree with that obtained by Goedert et al. [1989a,b], the source of disparity could be the tissue used for RNA preparation. In situ hybridization studies have shown that although many areas of the adult brain express both the three- and fourrepeat form, one area of the hippocampus expresses only the three-repeat form [Goedert et al., 1989al. It is probable that these glimpses of tau temporal and spatial specificity only touch the “tip of the iceberg” and that the pattern of tau gene expression in neuronal cells may be quite varied. It also suggests that each tau isoform is regulated so as to appear at specific times in specific places and that each tau isoform has a specific function. The integration of the structural, functional, and

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developmental data on tau protein is most apparent when one considers the role of tau protein in neuronal cell development. In the embryo, the tau isoform with exons 2, 3, and 10 deleted is expressed. Functionally speaking, this isoform has a lower efficiency of microtubule binding and a lower microtubule bundling capability than does its adult counterpart, the exon 10 containing (fourrepeat) isoform. Its function in immature neurons is undoubtedly related to the promotion of microtubule assembly in growing neuritic processes. One day old neuronal cultures possess processes that are exploratory and unstable; a mature tau isoform that would stabilize and cross-link microtubules at this stage might not be appropriate. However, as the neuronal culture ages, by 7 days, neurites gain permanency and the cell is polarized with dendritic and axonal processes being established. At this point, tau has become localized to the axonal compartment of the neuron where the adult isoform could have a role in the stabilization and organization of axonal microtubules. The localization of tau to the axonal compartment was first reported using the monoclonal antibody tau1 . Although a subsequent investigation suggested that tau protein could also be detected by tau1 in the somatodendritic compartment following phosphatase treatment of the tissue [Papasozomenos and Binder, 19871, the initial result localizing tau to axons has been confirmed by other investigators using a variety of polyclonal and monoclonal antibodies [Brion et al., 1988; Kosik and Finch, 1987; Trojanowski et al., 19891. The mechanism of this intracellular compartmentalization has yet to be elucidated. Although it has been reported that immature tau isoforms are able to localize to the axon [Brion et al., 19881, several reports to the contrary also exist [Dotti et al., 19871. This spatial specificity sets tau apart from MAP2, which has been found exclusively in the cell body and dendrites. Besides this difference in microtubule-associated proteins, a difference in the polarity of the microtubules in the axon and the dendrites also exists. Baas et al. [1988] have found that dendrites contain microtubules with equal numbers oriented plus ends toward the growth cone and plus ends toward the cell body; axonal microtubules are uniformly oriented with all plus ends toward the growth cone. The relationship between this organization of axonal and dendritic microtubules and their associated proteins has yet to be shown, Major questions to be answered are 1) What mechanism(s) results in the localization of tau to the axon and MAP2 to the dendrite? 2) Do all isoforms of tau localize to the axon? and, most importantly, 3) Does tau protein have a role in axonal function, beyond that of microtubule stabilization? The fate of tau protein in an aging, degenerating neuron appears to be related to neurofibrillary tangles,

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which are composed of intraneuronal 10 nm filaments forming paired helical filaments (PHF). These filaments are a pathological indication of Alzheimer’s disease and have long evaded biochemical analyses because of their insolubility. The initial identification of tau protein as a component of PHF was based on the cross reactivity of tau antibodies with PHF and of PHF antibodies with tau protein. Peptide sequence analysis of PHF solubilized with the aid of proteolytic digestion has now removed any doubt that tau is a component of PHF [Wischik et al., 1988a; Kondo et al., 19891. Although peptide sequences have not indicated that the amino terminal half of tau is a part of PHF, monoclonal antibodies with epitopes spanning that entire region do react with neurofibrillary tangles [Goedert et al., 1989b; Kosik et al., 19881, suggesting that the entire molecule is present in PHF. Moreover, antisera specific for various tau isoforms also react, suggesting that multiple isoforms are incorporated into neurofibrillary tangles [Goedert et al., 1989b1. However, it is likely that the carboxy half of tau protein is more closely associated to the PHF core since the amino terminal half becomes undetectable after SDS extraction or proteolytic digestion of PHF. Although Wischik et al. [1988a] have found more than one tau isoform in their PHF protein sequence data, Mori et al. [ 19891 have suggested that only the three-repeat tau isoform is found in PHF. This discrepancy could be due to a difference in their PHF fragment isolation protocols. The exact nature of tau protein found in PHF is not clear. Anti-tau antibodies recognize PHF less efficiently than do anti-PHF sera, perhaps suggesting that specifically modified tau epitopes may exist in PHF. Separation of PHF fragments on HPLC has revealed several tauimmunoreactive peaks chromatographing anomolously [Kondo et al., 19891; these investigators have indirect evidence identifying phosphorylation as the modification that differentiates PHF-associated tau from normal tau. Earlier immunocytochemical data had also suggested that phosphorylated tau is a component of PHF. Evidence for the presence of modified tau protein in PHF has also been obtained with Alz50, a monoclonal antibody raised against Alzheimer brain homogenate. Besides labeling neurofibrillary tangles and dystrophic neurites in Alzheimer brain, Alz50 immunoprecipitates proteins specific to diseased brain that also react with tau antibodies but appear larger than normal brain tau protein [Nukina et al., 19881; Alz.50 will also recognize normal brain tau (Ksiezek-Reding et al., 19881. The nature of the molecular interactions responsible for tau protein’s association with PHF are unknown. Tau protein may be self-assembling or co-assembling with other components to form PHF. Tau protein constitutes an estimated 10% of PHF structure [Wischik et al., 1988b], and the remaining core material has yet to be

elucidated. Since neurofibrillary tangles appear in the somatodendritic compartment, one wonders whether PHF-associated tau was formerly bound to axonal microtubules or whether it was newly synthesized material that could not be sorted to the axon. (Incidentally, no differences in tau mRNA levels have been detected in Alzheimer’s diseased brains relative to normal brains [Goedert et al., 19881). One might also ask how the microtubules in these degenerating neurons disappeared and whether this disappearance preceded or followed the formation of PHF. In addition, if PHF-associated tau is abnormally phosphorylated, one would like to know whether this event is related to the pathogenesis of Alzheimer’s disease. Undoubtedly, there are many steps between the unraveling of the cell’s cytoskeletal structure to the formation of neurofibrillary tangles. Finally, to conclude, there are additional questions that need to be asked, but for which there are no clues as to what the answers will be. What is the function of the amino terminal half of tau protein and what is the importance of exons 2 and 3 that are adult specific and contain limited homology to neurofilament M protein? Also, tau protein can nucleate microtubules-Is this related to binding and/or bundling and, if not, will a new structural motif(s) be required for this activity? REFERENCES Aizawa, H., Kawasaki, H., Murofushi, H., Kotani, S . , Suzuki, K . , and Sakai, H. (1989): A common amino acid sequence in 190kDa microtubule-associated protein and tau for the promotion of microtubule assembly. J. Biol. Chem. 264:5885-5890. Baas, P.W., Deitch, J.S., Black, M.M., and Banker, G.A. (1988): Polarity orientation of microtubules in hippocampal neurons: Uniformity in the axon and nonuniformity in the dendrite. Proc. Natl. Acad. Sci. U.S.A. 85:8335-8339. Brion, J.P., Guilleminot, J., Couchi, D., Flament-Durand, J., and Nunez. J. (1988): Both adult and juvenile tau microtubuleassociated proteins are axon specific in the developing and adult rat cerebellum. Neuroscience 25: 139-146. Dotti, C.G., Banker, G.A., and Binder, L.I. (1987): The expression and distribution of the microtubule-associated proteins tau and microtubule-associated protein 2 in hippocampal neurons in the rat in situ and in cell culture. Neuroscience 23:121-130. Ennulat, D.J., Liem, R.K.H., Hashim, G.A., and Shelanski, M.L. (1989): Two separate 18-amino acid domains of tau promote the polymerization of tubulin. J. Biol. Chem. 2645327-5330. Goedert, M., Wischik, C.M., Crowther, R.A., Walker, J.E., and Klug, A . (1988): Cloning and sequencing of the cDNA encoding a core protein of the paired helical filament of Alzheimer disease. Identification as the microtubule-associated protein tau. Proc. Natl. Acad. Sci. U.S.A. 85:4051-4055. Goedert, M., Spillantini, M.G., Potier, M.C., Ulrich, J., and Crowther, R.A. (1989a): Cloning and sequencing of the cDNA encoding an isoform of microtubule-associated protein tau containing four tandem repeats: Differential expression of tau protein mRNAs in human brain. EMBO J. 8:393-399. Goedert, M., Spillantini, M.G., Jakes, R., Rutherford, D., and Crowther, R.A. (1989b): Multiple isoforms of human micro-

Tau Protein: Update on Structure and Function tubule-associated protein tau: Sequences and localization in neurofibrillary tangles in Alzheimer’s disease. Neuron 3:5 19-526. Himmler, A. (1989): Structure of the bovine tau gene: Alternatively spliced transcripts generate a protein family. Mol. Cell. Bid. 9: 1389-1396. Himmler, A., Drechsel, D., Kirschner, M. W., and Martin, D.W., Jr. (1989): Tau consists of a set of proteins with repeated C-terminal microtubule-binding domains and variable N-terminal domains. Mol. Cell. Biol. 9:1381-1388. Kanai, Y., Takemura, R., Oshima, T., Mori, H., Ihara, Y., Yanagisawa, M., Masaki, T., and Hirokawa, N. (1989): Expression of multiple tau isoforms and microtubule bundle formation in fibroblasts transfected with a single tau cDNA. J. Cell Biol. 109:1173-1184. Kondo, J., Honda, T., Hiroshi, M., Hamada, Y . , Miura, R., Ogawara, M., and Ihara, Y. (1989): The carboxyl third of tau is tightly bound to paired helical filaments. Neuron 1327-834. Kosik, K.S., and Finch, E.A. (1987): MAP 2 and tau segregate into dendritic and axonal domains after the elaboration of morphologically distinct neurites: An immunocytochemical study of cultured rat cerebrum. J. Neurosci. 7:3142-3153. Kosik, K.S., Orecchio, L.D., Binder, L., Trojanowski, J.Q., Lee, V.M.-Y., and Lee, G. (1988): Epitopes that span the tau molecule are shared with paired helical filaments. Neuron 15317825. Kosik, K.S., Orecchio, L.D., Bakalis, S . , and Neve, R.L. (1989): Developmentally regulated expression of specific tau sequences. Neuron 2: 1389-1397. Ksiezak-Reding, H., Davies, P., and Yen, S.-H. (1988): Alz50, a monoclonal antibody to Alzheimer’s disease antigen, crossreacts with t proteins from bovine and normal human brain. J. Biol. Chem. 263:7Y43-7947. Lee, G., Cowan, N., and Kirschner, M. (1988): The primary structure and heterogeneity of tau protein from mouse brain. Science 239~285-288.

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Lee, G., Neve, R.L., and Kosik, K.S. (1989): The microtubule binding domain of tau protein. Neuron 2:1615-1624. Lewis, S.A., Wang, D., and Cowan, N.J. (1988): Microtubule-associated protein MAP2 shares a microtubule binding motif with tau protein. Science 242:Y36-939. Lewis, S.A., Ivanov, I.E., Lee, (3.-H., and Cowan, N.J. (1989): Organization of microtubules in dendrites and axons is determined by a short hydrophobic zipper in microtubule-associated proteins MAP2 and tau. Nature 342:498-505. Lichtenberg, B., Mandelkow, E.-M., Hagestedt, T., and Mandelkow, E. (1988): Structure and elasticity of microtubule-associated protein tau. Nature 334:359-362. Mori, H., Hamada, Y., Kawaguchi, M., Honda, T., Kondo, J., and Ihara, Y. (1989): A distinct form of tau is selectively incorporated into Alzheimer’s paired helical filaments. Biochem. Biophys. Res. Commun. 159:1221-1226. Nukina, N., Kosik, K.S., and Selkoe, D.J. (1988): The monoclonal antibody, Alz50, recognizes tau proteins in Alzheimer’s disease brain. Neurosci. Lett. 87:240-246. Papasozomenos, S.C., and Binder, L.I. (1987): Phosphorylation determines two distinct species of tau in the central nervous system. Cell Motil. Cytoskeleton 8:210-226. Trojanowski, J.Q., Schuck, T., Schmidt, L., and Lee, V.M.-Y. (1989): Distribution of tau proteins in the normal human central and peripheral nervous system. J. Histochem. Cytochem. 37: 209 -2 15. Wischik, C.M., Novak, M., Thogersen, H.C., Edwards, P.C., Runswick, M.J., Jakes, R., Walker, J.E., Milstein, C., Roth, M., and Klug, A. (1988a): Isolation of a fragment of tau derived from the core of the paired helical filament of Alzheimer disease. Proc. Natl. Acad. Sci. U.S.A. 85:4506-4510. Wischick, C.M., Novak, M., Edwards, P.C., Klug, A., Tichelaar, W., and Crowther, R.A. (1988b): Structural characterization of the core of the paired helical filament of Alzheimer disease. Proc. Natl. Acad. Sci. U.S.A. 85:4884-4888.

Tau protein: an update on structure and function.

Cell Motility and the Cytoskeleton 15199-203 (1990) Views and Reviews Tau Protein: An Update on Structure and Function Gloria Lee Program in Neurosci...
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