- Email: [email protected]
Altered conformation of recombinant frontotemporal dementia-17 mutant tau proteins
Neuroscience Letters 260 (1999) 153–156
Altered conformation of recombinant frontotemporal dementia-17 mutant tau proteins Gregory A. Jicha a, Julia M. Rockwood a, Benjamin Berenfeld a, Mike Hutton b, Peter Davies a ,* a
Department of Pathology, Albert Einstein College of Medicine, 1300 Morris Park Avenue, Forcheimer 526, Bronx, NY 10461, USA b Mayo Clinic Jacksonville, Jacksonville, FL 32224, USA Received 3 November 1998; accepted 12 November 1998
Abstract Recently, a series of both non-coding (intronic) and coding (exonic) mutations in the tau gene have been linked to a family of autosomal dominant dementias referred to as frontotemporal dementia-17. While linkage analysis has demonstrated that these mutations segregate with disease in affected families, it is unclear how mutant tau proteins could lead to the degenerative cascade seen in frontotemporal dementia-17. The present study demonstrates that coding mutations of tau seen in frontotemporal dementia-17 exhibit altered physical and structural characteristics as determined by reverse phase high performance liquid chromatography and circular dichroism spectroscopy. These data suggest that the previously identified mutations in the tau gene seen in frontotemporal dementia-17 are not merely benign polymorphisms, but may have functional consequences for microtubule binding, microtubule polymerization, and the abnormal aggregation of tau seen in a variety of neurodegenerative diseases. 1999 Elsevier Science Ireland Ltd. All rights reserved.
Keywords: Frontotemporal dementia; Tau; Conformation
Recently, a series of both non-coding (intronic) and coding (exonic) mutations in the tau gene have been linked to a family of autosomal dominant dementias referred to as frontotemporal dementia-17 [4,7,8]. This family of inherited diseases is characterized by extensive tau pathology, spongiosis, and neuronal loss [8]. While these recent discoveries suggest that alterations in tau may play a central role in the pathogenesis of a variety of tau-related neurodegenerative diseases, it is unclear how mutations in the tau gene can lead to the degenerative cascade seen in frontotemporal dementia-17. While linkage analysis has demonstrated that these mutations segregate with the disease in affected families [4,7,8], it is unclear if they are causative in the disease process or are merely benign polymorphisms that segregate with causative mutations in an as yet undetermined neighboring gene. It has been proposed that the non-coding (intronic) mutations affect the alternative splicing of tau mRNA [4,7,8], favoring excess translation of the four repeat tau isoforms over the three repeat isoforms, it is unclear how this potential imbalance could lead to the neurodegeneration * Corresponding author. Tel.: +1 718 4302053; fax: +1 718 4308541; e-mail: [email protected]
0304-3940/99/$ - see front matter PII: S03 04-3940(98)009 80-X
seen in affected individuals. While definitive answers to these questions await further analysis it should be noted that the coding mutations in the tau gene can be readily analyzed with existing methodologies, and such studies may help to elucidate the role tau plays in the neurodegenerative cascade seen in frontotemporal dementia-17 patients. At present, four coding mutations linked to frontotemporal dementia-17 have been reported. G272V, P301L, and V337M (numbering according to the amino acid sequence of the longest 441 amino acid tau isoform) mutations cluster in and near the microtubule binding repeats in the flexible hinge region characterized by a highly conserved PGGG motif [2]. As such it is possible that these mutations directly influence the tau-microtubule interaction leading to a neurodegenerative state. This hypothesis, however, is weakened by the facts that (1) mice expressing no tau at all exhibit no obvious neuropathology [3] and (2) the autosomal dominant nature of frontotemporal dementia-17 in which patients carry a normal tau allele that could potentially overcome the limitation of an altered gene product [4,7,8]. Another mutation, R406W lies outside the microtubule binding domains and flanking regions and is unlikely
1999 Elsevier Science Ireland Ltd. All rights reserved.
154
G.A. Jicha et al. / Neuroscience Letters 260 (1999) 153–156
to drastically affect microtubule dynamics within the neuron. An alternative hypothesis is that the mutations seen in tau in frontotemporal dementia-17 enhance the abnormal aggregation of tau characteristic of all tau-related neurodegenerative diseases. Such conformational alterations have recently been proposed to underlie the abnormal aggregation of tau seen in AD [5,6]. While much research remains to be done to determine which of these potential mechanisms contribute to the pathogenesis of frontotemporal dementia-17, it should be noted that alterations in the structure of tau could play a crucial role in either or both degenerative mechanisms. In order to determine if structural alterations of tau are induced by these mutations, a series of site directed mutants including A178T (a polymorphism that has been found in a single case of frontotemporal dementia-17 but has not been shown to segregate with disease in the affected family), G272V, P301L, V337M, and R406W were created in a full length tau construct (TauW) and expressed in bacteria. In preparation for studies employing circular dichroism, these recombinant proteins were purified by HPLC using
a reverse phase column. Several of these mutant proteins exhibited altered column retention times under identical conditions (Fig. 1) suggesting that certain amino acid substitutions can have a drastic effect on the physical properties of tau. Additionally it should be noted that although minor variations in column retention times by reverse phase HPLC are characteristic of certain site-directed mutants of tau, the present analysis demonstrates that a 20% increase in acetonitrile concentration is required for elution of the V272G, P301L, V337M, and R406W mutant proteins compared to wild-type tau protein and the A178T mutant protein from a C-8 reverse phase column. This data suggests that either (1) the protein has aggregated (further analysis of these proteins using both reducing and non-reducing SDS-PAGE (data not shown) suggests that this delayed retention time is not due to disulfide dependent dimerization as has been shown for certain tau constructs), or (2) the protein has a drastically altered structure that significantly alters its elution profile perhaps by leading to an increased exposure of hydrophobic residues in extended areas of the molecule. Further analysis of these mutants using circular dichroism
Fig. 1. HPLC profiles of bacterially expressed (A) TauW (htau40 + N-terminal 6xhistidine tag); (B) A178T in TauW; (C) G272V in TauW; (D) P301L in Tau W; (E) V337M in TauW; (F) R406W in TauW. TauW and A178T elute at 27 min in a gradient of 30% acetonitrile while G272V, P301L, V337M, and R406W elute at 38 min in a gradient of 50% acetonitrile. Separations were achieved on a Beckman System Gold 126 using Beckman C8 4.6 mm analytical columns. All detection was at 214 nm. HPLC runs consisted of a 10 min isochratic gradient of 100% (A), followed by a 1.5% addition of (B) per minute for 60 min. Solution (A) contained 100% water and 0.1% TFA, and solution (B) contained 100% acetonitrile and 0.08% TFA.
G.A. Jicha et al. / Neuroscience Letters 260 (1999) 153–156
155
Fig. 2. CD profiles of HPLC purified bacterially expressed (A) TauW (htau40 + N-terminal 6xhistidine tag); (B) A178T in TauW; (C) G272V in TauW; (D) P301L in Tau W; (E) V337M in TauW; (F) R406W in TauW. TauW and A178T exhibit a characteristic random coil profile characterized by a negative absorbance at 198–200 nm whereas G272V, P301L, V337M, and R406W exhibit a predominantly a-helical profile characterized by negative absorbances at 222 and 208 nm. CD spectra of the purified proteins were taken on a Jasco J-720 at room temperature in a 0.5 mm path length cell. Protein concentrations in PBS (pH 7.4) were standardized by Biorad assay at 0.2 mg/ml. Mean residue ellipticity is expressed in degcm2/dmol. Curve smoothing was performed using the algorithm provided by Jasco.
clearly demonstrates that several of these mutant proteins have altered structural features as compared to non-mutated tau (Fig. 2). Specifically, while A178T appears identical to normal tau in HPLC profile and CD spectra, G272V, P301L, V337M, and R406W all exhibit a prolonged column retention time by HPLC analysis and show evidence of enhanced a-helical character by CD spectroscopy. The increase in a-
helical character was paralleled by a concomitant decrease in the random coil structure characteristic of the normal tau protein. The increase in a-helical character seen with the G272V, P301L, and V337M constructs may well be due to a transition from the ‘flexible hinge’ (PGGG) motif to an ahelical motif that allows the a-helical character of the microtubule binding domains [1] to extend unbroken for
156
G.A. Jicha et al. / Neuroscience Letters 260 (1999) 153–156
poral dementia-17 mutations in tau, it clearly demonstrates that certain coding frontotemporal dementia-17 mutations induce structural alterations in tau that may have severe functional consequences. It is highly likely that the structural alterations characteristic of tau in frontotemporal dementia-17 may have drastic effects on microtubule binding, microtubule polymerization, and tau aggregation. Studies are currently underway in our laboratory and others that will directly address these issues. The authors thank E. Nieves for assistance with CD and M. Goedert for providing the htau40 construct. This work was supported by NIMH grant 38623, NIH grant AG06803, and NIH training grant T32GM07288. Fig. 3. Cartoon diagram demonstrating enhanced a-helical character in frontotemporal dementia-17 recombinant tau mutants. Cylinders represent a-helical domains. *Location of mutations in tau.
nearly 70 amino acids instead of the typical 33–35 (Fig. 3). The increased a-helical character seen in the R406W mutant could similarily be due to a coupling of the predicted a-helical domains stretching from amino acids 380–396 and 416–441 [9] (Fig. 3). These transitions might impose rigid restraints on tau allowing the amphipathic a-helical nature of the microtubule binding [1] and C-terminal domains [9] to dominate the overall structure of the molecule leading to an increased exposure of hydrophobic residues laterally down the molecule explaining the delayed retention time seen by HPLC analysis. Previous studies have suggested that elimination of the flexible hinge regions between microtubule binding domains may have deleterious effects on microtubule binding and polymerization [1]. Other studies have suggested that converting the tau molecule to a more rigid state via phosphorylation may promote aggregation. It is possible that the coding mutations in tau seen in frontotemporal dementia-17 may function in an analogous manner. It is interesting that the A178T mutant tau protein gave identical results to non-mutated recombinant tau in this study. This data suggests that this mutation is different from the other coding muatations identified to date. It is possible that the effects of this mutation are reliant on alterations in post-translational modifications of tau such as phosphorylation. The A178T mutation creates a novel proline directed phosphorylation site in an area of the tau molecule flanked by additional proline directed phosphorylation sites previously implicated in the pathogenesis of Alzheimer’s disease. This suggests that modifications of tau other than alterations in primary sequence may be a prerequisite for the degeneration seen in all tau-related diseases. Future studies will attempt to determine if Thr178 is a phosphate acceptor, which neuronal kinase(s) can phosphorylate this site, and if this unique phosphorylation alters the structure and or function of the tau molecule in a manner similar to the other known frontotemporal dementia-17 mutations. While this study does not directly address the potential mechanisms of neurodegeneration caused by frontotem-
[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] Goedert, M. and Jakes, R., Expression of separate isoforms of human tau protein: correlation with tau pattern in brain and effects on tubulin polymerization, Eur. Mol. Biol. Organ. J., 8 (1990) 4225–4230. [3] Harada, A., Oguchi, K., Okabe, S., Kuno, J., Terada, S., Ohshima, T., Sato-Yoshitake, R., Takel, Y., Noda, T. and Hirokawa, N., Altered microtubule organization in small-calibre axons of mice lacking tau protein, Nature, 369 (1994) 488– 491. [4] Hutton, M., Lendon, C.L., Rizzu, P., Baker, M., Froelich, S., Houlden, H., Pickering-Brown, S., Chakraverty, S., Isaacs, A., Grover, A., Hackett, J., Adamson, J., Lincoln, S., Dickson, D., Davies, P., Petersen, R.C., Stevens, M., De Graaff, E., Wauters, E., Van Baren, J., Hillebrand, M., Joose, M., Kwon, J.M., Nowotny, P., Che, L.K., Norton, J., Morris, J.C., Reed, L.A., Trojanowski, J., Basun, H., Lannfelt, L., Neystat, M., Fahn, S., Dark, F., Tannenberg, T., Dodd, P.R., Hayward, N., Kwok, J.B.J., Schofield, P.R., Andreadis, A., Snowden, J., Craufurd, D., Neary, D., Owen, F., Oostra, B.A., Hardy, J., Goate, A., Van Swueten, J., Mann, D., Lynch, T. and Heutink, P., Association of missense and 5′-splice-site mutations in tau with the inherited dementia FTDP-17, Lett. Nature, 393 (1998) 702–705. [5] Jicha, G.A., Bowser, R., Kazam, I.G. and Davies, P., Alz-50 and MC-1, a new monoclonal antibody raised to paired helical filaments, recognize conformational epitopes on recombinant tau, J. Neurosci. Res., 48 (1997) 128–132. [6] Jicha, G.A., Lane, E., Vincent, I., Otvos, L. Jr., Hoffmann, R. and Davies, P., A conformation- and phosphorylation-dependent antibody recognizing the paired helical filaments of Alzheimer’s disease, J. Neurochem., 69 (1997) 2087–2095. [7] Poorkaj, P., Bird, T.D., Wijsman, E., Nemens, E., Garruto, R.M., Anderson, L., Andreadis, A., Wiederholt, W.C., Raskind, M. and Schellenberg, G.D., Tau is a candidate gene for chromosome 17 frontotemporal dementia, Ann. Neurol., 43 (1998) 815–825. [8] Spillantini, M.G., Bird, T.D. and Ghetti, B., Frontotemporal dementia and parkinsonism linked to chromosome 17: a new group of tauopathies, Brain Pathol., 8 (1998) 387–402. [9] Yanagawa, H., Chung, S.-H., Ogawa, Y., Sato, K., ShibataSeki, T., Masai, J. and Ishiguro, K., Protein anatomy: C-tail region of human tau protein as a crucial structural element in Alzheimer’s paired helical filament formation in vitro, Biochemistry, 37 (1998) 1979–1988.
Altered conformation of recombinant frontotemporal dementia-17 mutant tau proteins Gregory A. Jicha a, Julia M. Rockwood a, Benjamin Berenfeld a, Mike Hutton b, Peter Davies a ,* a
Department of Pathology, Albert Einstein College of Medicine, 1300 Morris Park Avenue, Forcheimer 526, Bronx, NY 10461, USA b Mayo Clinic Jacksonville, Jacksonville, FL 32224, USA Received 3 November 1998; accepted 12 November 1998
Abstract Recently, a series of both non-coding (intronic) and coding (exonic) mutations in the tau gene have been linked to a family of autosomal dominant dementias referred to as frontotemporal dementia-17. While linkage analysis has demonstrated that these mutations segregate with disease in affected families, it is unclear how mutant tau proteins could lead to the degenerative cascade seen in frontotemporal dementia-17. The present study demonstrates that coding mutations of tau seen in frontotemporal dementia-17 exhibit altered physical and structural characteristics as determined by reverse phase high performance liquid chromatography and circular dichroism spectroscopy. These data suggest that the previously identified mutations in the tau gene seen in frontotemporal dementia-17 are not merely benign polymorphisms, but may have functional consequences for microtubule binding, microtubule polymerization, and the abnormal aggregation of tau seen in a variety of neurodegenerative diseases. 1999 Elsevier Science Ireland Ltd. All rights reserved.
Keywords: Frontotemporal dementia; Tau; Conformation
Recently, a series of both non-coding (intronic) and coding (exonic) mutations in the tau gene have been linked to a family of autosomal dominant dementias referred to as frontotemporal dementia-17 [4,7,8]. This family of inherited diseases is characterized by extensive tau pathology, spongiosis, and neuronal loss [8]. While these recent discoveries suggest that alterations in tau may play a central role in the pathogenesis of a variety of tau-related neurodegenerative diseases, it is unclear how mutations in the tau gene can lead to the degenerative cascade seen in frontotemporal dementia-17. While linkage analysis has demonstrated that these mutations segregate with the disease in affected families [4,7,8], it is unclear if they are causative in the disease process or are merely benign polymorphisms that segregate with causative mutations in an as yet undetermined neighboring gene. It has been proposed that the non-coding (intronic) mutations affect the alternative splicing of tau mRNA [4,7,8], favoring excess translation of the four repeat tau isoforms over the three repeat isoforms, it is unclear how this potential imbalance could lead to the neurodegeneration * Corresponding author. Tel.: +1 718 4302053; fax: +1 718 4308541; e-mail: [email protected]
0304-3940/99/$ - see front matter PII: S03 04-3940(98)009 80-X
seen in affected individuals. While definitive answers to these questions await further analysis it should be noted that the coding mutations in the tau gene can be readily analyzed with existing methodologies, and such studies may help to elucidate the role tau plays in the neurodegenerative cascade seen in frontotemporal dementia-17 patients. At present, four coding mutations linked to frontotemporal dementia-17 have been reported. G272V, P301L, and V337M (numbering according to the amino acid sequence of the longest 441 amino acid tau isoform) mutations cluster in and near the microtubule binding repeats in the flexible hinge region characterized by a highly conserved PGGG motif [2]. As such it is possible that these mutations directly influence the tau-microtubule interaction leading to a neurodegenerative state. This hypothesis, however, is weakened by the facts that (1) mice expressing no tau at all exhibit no obvious neuropathology [3] and (2) the autosomal dominant nature of frontotemporal dementia-17 in which patients carry a normal tau allele that could potentially overcome the limitation of an altered gene product [4,7,8]. Another mutation, R406W lies outside the microtubule binding domains and flanking regions and is unlikely
1999 Elsevier Science Ireland Ltd. All rights reserved.
154
G.A. Jicha et al. / Neuroscience Letters 260 (1999) 153–156
to drastically affect microtubule dynamics within the neuron. An alternative hypothesis is that the mutations seen in tau in frontotemporal dementia-17 enhance the abnormal aggregation of tau characteristic of all tau-related neurodegenerative diseases. Such conformational alterations have recently been proposed to underlie the abnormal aggregation of tau seen in AD [5,6]. While much research remains to be done to determine which of these potential mechanisms contribute to the pathogenesis of frontotemporal dementia-17, it should be noted that alterations in the structure of tau could play a crucial role in either or both degenerative mechanisms. In order to determine if structural alterations of tau are induced by these mutations, a series of site directed mutants including A178T (a polymorphism that has been found in a single case of frontotemporal dementia-17 but has not been shown to segregate with disease in the affected family), G272V, P301L, V337M, and R406W were created in a full length tau construct (TauW) and expressed in bacteria. In preparation for studies employing circular dichroism, these recombinant proteins were purified by HPLC using
a reverse phase column. Several of these mutant proteins exhibited altered column retention times under identical conditions (Fig. 1) suggesting that certain amino acid substitutions can have a drastic effect on the physical properties of tau. Additionally it should be noted that although minor variations in column retention times by reverse phase HPLC are characteristic of certain site-directed mutants of tau, the present analysis demonstrates that a 20% increase in acetonitrile concentration is required for elution of the V272G, P301L, V337M, and R406W mutant proteins compared to wild-type tau protein and the A178T mutant protein from a C-8 reverse phase column. This data suggests that either (1) the protein has aggregated (further analysis of these proteins using both reducing and non-reducing SDS-PAGE (data not shown) suggests that this delayed retention time is not due to disulfide dependent dimerization as has been shown for certain tau constructs), or (2) the protein has a drastically altered structure that significantly alters its elution profile perhaps by leading to an increased exposure of hydrophobic residues in extended areas of the molecule. Further analysis of these mutants using circular dichroism
Fig. 1. HPLC profiles of bacterially expressed (A) TauW (htau40 + N-terminal 6xhistidine tag); (B) A178T in TauW; (C) G272V in TauW; (D) P301L in Tau W; (E) V337M in TauW; (F) R406W in TauW. TauW and A178T elute at 27 min in a gradient of 30% acetonitrile while G272V, P301L, V337M, and R406W elute at 38 min in a gradient of 50% acetonitrile. Separations were achieved on a Beckman System Gold 126 using Beckman C8 4.6 mm analytical columns. All detection was at 214 nm. HPLC runs consisted of a 10 min isochratic gradient of 100% (A), followed by a 1.5% addition of (B) per minute for 60 min. Solution (A) contained 100% water and 0.1% TFA, and solution (B) contained 100% acetonitrile and 0.08% TFA.
G.A. Jicha et al. / Neuroscience Letters 260 (1999) 153–156
155
Fig. 2. CD profiles of HPLC purified bacterially expressed (A) TauW (htau40 + N-terminal 6xhistidine tag); (B) A178T in TauW; (C) G272V in TauW; (D) P301L in Tau W; (E) V337M in TauW; (F) R406W in TauW. TauW and A178T exhibit a characteristic random coil profile characterized by a negative absorbance at 198–200 nm whereas G272V, P301L, V337M, and R406W exhibit a predominantly a-helical profile characterized by negative absorbances at 222 and 208 nm. CD spectra of the purified proteins were taken on a Jasco J-720 at room temperature in a 0.5 mm path length cell. Protein concentrations in PBS (pH 7.4) were standardized by Biorad assay at 0.2 mg/ml. Mean residue ellipticity is expressed in degcm2/dmol. Curve smoothing was performed using the algorithm provided by Jasco.
clearly demonstrates that several of these mutant proteins have altered structural features as compared to non-mutated tau (Fig. 2). Specifically, while A178T appears identical to normal tau in HPLC profile and CD spectra, G272V, P301L, V337M, and R406W all exhibit a prolonged column retention time by HPLC analysis and show evidence of enhanced a-helical character by CD spectroscopy. The increase in a-
helical character was paralleled by a concomitant decrease in the random coil structure characteristic of the normal tau protein. The increase in a-helical character seen with the G272V, P301L, and V337M constructs may well be due to a transition from the ‘flexible hinge’ (PGGG) motif to an ahelical motif that allows the a-helical character of the microtubule binding domains [1] to extend unbroken for
156
G.A. Jicha et al. / Neuroscience Letters 260 (1999) 153–156
poral dementia-17 mutations in tau, it clearly demonstrates that certain coding frontotemporal dementia-17 mutations induce structural alterations in tau that may have severe functional consequences. It is highly likely that the structural alterations characteristic of tau in frontotemporal dementia-17 may have drastic effects on microtubule binding, microtubule polymerization, and tau aggregation. Studies are currently underway in our laboratory and others that will directly address these issues. The authors thank E. Nieves for assistance with CD and M. Goedert for providing the htau40 construct. This work was supported by NIMH grant 38623, NIH grant AG06803, and NIH training grant T32GM07288. Fig. 3. Cartoon diagram demonstrating enhanced a-helical character in frontotemporal dementia-17 recombinant tau mutants. Cylinders represent a-helical domains. *Location of mutations in tau.
nearly 70 amino acids instead of the typical 33–35 (Fig. 3). The increased a-helical character seen in the R406W mutant could similarily be due to a coupling of the predicted a-helical domains stretching from amino acids 380–396 and 416–441 [9] (Fig. 3). These transitions might impose rigid restraints on tau allowing the amphipathic a-helical nature of the microtubule binding [1] and C-terminal domains [9] to dominate the overall structure of the molecule leading to an increased exposure of hydrophobic residues laterally down the molecule explaining the delayed retention time seen by HPLC analysis. Previous studies have suggested that elimination of the flexible hinge regions between microtubule binding domains may have deleterious effects on microtubule binding and polymerization [1]. Other studies have suggested that converting the tau molecule to a more rigid state via phosphorylation may promote aggregation. It is possible that the coding mutations in tau seen in frontotemporal dementia-17 may function in an analogous manner. It is interesting that the A178T mutant tau protein gave identical results to non-mutated recombinant tau in this study. This data suggests that this mutation is different from the other coding muatations identified to date. It is possible that the effects of this mutation are reliant on alterations in post-translational modifications of tau such as phosphorylation. The A178T mutation creates a novel proline directed phosphorylation site in an area of the tau molecule flanked by additional proline directed phosphorylation sites previously implicated in the pathogenesis of Alzheimer’s disease. This suggests that modifications of tau other than alterations in primary sequence may be a prerequisite for the degeneration seen in all tau-related diseases. Future studies will attempt to determine if Thr178 is a phosphate acceptor, which neuronal kinase(s) can phosphorylate this site, and if this unique phosphorylation alters the structure and or function of the tau molecule in a manner similar to the other known frontotemporal dementia-17 mutations. While this study does not directly address the potential mechanisms of neurodegeneration caused by frontotem-
[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] Goedert, M. and Jakes, R., Expression of separate isoforms of human tau protein: correlation with tau pattern in brain and effects on tubulin polymerization, Eur. Mol. Biol. Organ. J., 8 (1990) 4225–4230. [3] Harada, A., Oguchi, K., Okabe, S., Kuno, J., Terada, S., Ohshima, T., Sato-Yoshitake, R., Takel, Y., Noda, T. and Hirokawa, N., Altered microtubule organization in small-calibre axons of mice lacking tau protein, Nature, 369 (1994) 488– 491. [4] Hutton, M., Lendon, C.L., Rizzu, P., Baker, M., Froelich, S., Houlden, H., Pickering-Brown, S., Chakraverty, S., Isaacs, A., Grover, A., Hackett, J., Adamson, J., Lincoln, S., Dickson, D., Davies, P., Petersen, R.C., Stevens, M., De Graaff, E., Wauters, E., Van Baren, J., Hillebrand, M., Joose, M., Kwon, J.M., Nowotny, P., Che, L.K., Norton, J., Morris, J.C., Reed, L.A., Trojanowski, J., Basun, H., Lannfelt, L., Neystat, M., Fahn, S., Dark, F., Tannenberg, T., Dodd, P.R., Hayward, N., Kwok, J.B.J., Schofield, P.R., Andreadis, A., Snowden, J., Craufurd, D., Neary, D., Owen, F., Oostra, B.A., Hardy, J., Goate, A., Van Swueten, J., Mann, D., Lynch, T. and Heutink, P., Association of missense and 5′-splice-site mutations in tau with the inherited dementia FTDP-17, Lett. Nature, 393 (1998) 702–705. [5] Jicha, G.A., Bowser, R., Kazam, I.G. and Davies, P., Alz-50 and MC-1, a new monoclonal antibody raised to paired helical filaments, recognize conformational epitopes on recombinant tau, J. Neurosci. Res., 48 (1997) 128–132. [6] Jicha, G.A., Lane, E., Vincent, I., Otvos, L. Jr., Hoffmann, R. and Davies, P., A conformation- and phosphorylation-dependent antibody recognizing the paired helical filaments of Alzheimer’s disease, J. Neurochem., 69 (1997) 2087–2095. [7] Poorkaj, P., Bird, T.D., Wijsman, E., Nemens, E., Garruto, R.M., Anderson, L., Andreadis, A., Wiederholt, W.C., Raskind, M. and Schellenberg, G.D., Tau is a candidate gene for chromosome 17 frontotemporal dementia, Ann. Neurol., 43 (1998) 815–825. [8] Spillantini, M.G., Bird, T.D. and Ghetti, B., Frontotemporal dementia and parkinsonism linked to chromosome 17: a new group of tauopathies, Brain Pathol., 8 (1998) 387–402. [9] Yanagawa, H., Chung, S.-H., Ogawa, Y., Sato, K., ShibataSeki, T., Masai, J. and Ishiguro, K., Protein anatomy: C-tail region of human tau protein as a crucial structural element in Alzheimer’s paired helical filament formation in vitro, Biochemistry, 37 (1998) 1979–1988.