The prolyl isomerase Pin1 restores the function of Alzheimer ... - Nature

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Acknowledgements. This work was supported by NIH. We thank J. Lisman for encouraging our search for theta; J. Lisman and O. Jensen for helpful discussions during the course of this research; and E. Tulving, E. Marder, L. Abbott and E. Menschik for helpful comments on a previous version of this manuscript. We acknowledge the cooperation of colleagues in the Children's Hospital Epilepsy program including P. M. Black, B. Bourgeois, F. Duffy and L. Kull. Finally, we thank the patients and their families for their participation and support. Correspondence and requests for materials should be addressed to M.J.K. (e-mail: [email protected]).

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4. Bland, B. H. The physiology and pharmacology of hippocampal formation theta rhythms. Prog. Neurobiol. 26, 1±54 (1986). 5. Stewart, M. & Fox, S. E. Hippocampal theta activity in monkeys. Brain Res. 538, 59±63 (1991). 6. Huerta, P. T. & Lisman, J. E. Heightened synaptic plasticity of hippocampal CA1 neurons during a cholinergically induced rhythmic state. Nature 364, 723±725 (1993). 7. Buzsaki, G. Two-stage model of memory trace formation: role for ``noisy'' brain states. Neuroscience 31, 551±570 (1989). 8. Sohal, V. S. & Hasselmo, M. E. GABA(B) modulation improves sequence disambiguation in computational models of hippocampal region CA3. Hippocampus 8, 171±193 (1998). 9. Buzsaki, G. The hippocampo-neocortical dialogue. Cereb. Cortex 6, 81±92 (1996). 10. Lisman, J. E. & Idiart, M. A. Storage of 7+/-2 short-term memories in oscillatory subcycles. Science 267, 1512±1515 (1995). 11. Buzsaki, G. Memory consolidation during sleep: a neurophysiological perspective. J. Sleep Res. 7, 17± 23 (1998). 12. Jensen, O. & Lisman, J. E. An oscillatory short-term memory buffer model can account for data on the Sternberg task. J. Neurosci. 18, 10688±10699 (1998). 13. Borst, J. G., Leung, L. W. & MacFabe, D. F. Electrical activity of the cingulate cortex. II. Cholinergic modulation. Brain Res. 407, 81±93 (1987). 14. Slawinska, U. & Kasicki, S. Theta-like rhythm in depth EEG activity of hypothalamic areas during spontaneous or electrically induced locomotion in the rat. Brain Res. 678, 117±126 (1995). 15. Routtenberg, A. & Taub, F. Hippocampus and superior colliculus: congruent EEG activity demonstrated by a simple measure. Behav. Biol. 8, 801±805 (1973). 16. Halgren, E., Babb, T. L. & Crandall, P. H. Human hippocampal formation EEG desynchronizes during attentiveness and movement. Electroencephalograph. Clin. Neurophysiol. 44, 778±781 (1978). 17. Arnolds, D. E. A. T., Lopes Da Silva, F. H., Aitink, J. W., Kamp, A. & Boeijinga, P. The spectral properties of hippocampal EEG related to behaviour in man. Electroencephalogr. Clin. Neurophysiol. 50, 324±328 (1980). 18. Tesche, C. D. Non-invasive detection of ongoing neuronal population activity in normal human hippocampus. Brain Res. 749, 53±60 (1997). 19. Aguirre, G. K., Detre, J. A., Alsop, D. C. & D'Esposito, M. The parahippocampus subserves topographical learning in man. Cerebral Cortex 6, 823±829 (1996). 20. Maguire, E. A. et al. Knowing where and getting there: a human navigation network. Science 280, 921± 924 (1998). 21. Epstein, R. & Kanwisher, N. A cortical representation of the local visual environment. Nature 392, 598±601 (1998). 22. Maguire, E. A., Frackowiak, S. J. & Frith, C. D. Learning to ®nd your way: a role for the human hippocampal formation. Proc. R. Soc. Lond. B 263, 1745±1750 (1996). 23. Maguire, E. A., Frackowiak, S. J. & Frith, C. J. Recalling routes around London: activation of the right hippocampus in taxi drivers. J. Neurosci. 17, 7103±7110 (1997). 24. Sperling, M. R. Clinical challenges in invasive monitoring in epilepsy surgery. Epilepsia (Suppl.) 38, S6±S12 (1997). 25. Talairach, J. & Tournoux, P. Co-planar Stereotaxic Atlas of the Human Brain (Verlag, Stuttgart, 1988). 26. Tulving, E. Elements of Episodic Memory (Oxford Univ. Press, New York, 1983). 27. Grossmann, A. & Morlet, J. Mathematics + Physics Vol. 1 (World Scienti®c, Singapore, 1985).

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45 Figure 1 Interaction of Pin1 with pTau and Alzheimer's disease tau, but not with pTauT231A. a, In vitro translated tau and its mutant were incubated with interphase (I), mitotic extracts (M) or mitotic extracts followed by dephosphorylation with phosphatase (M CIP), and then separated on SDS±PAGE directly (input) or after puri®cation with GST (GST) or GST±Pin1 (Pin1) beads. b, Tau was phosphorylated by I or M extracts and immunoblotted with monoclonal antibodies with different speci®city: CP27, all tau forms; CP9, pT231 tau; TG3, an Alzheimerspeci®c conformation of pT231 tau. c, Beads containing GST±Pin1 or GST were incubated with normal brain (NB) or Alzheimer's disease (AD) brain extracts, or

The prolyl isomerase Pin1 restores the function of Alzheimer-associated phosphorylated tau protein Pei-Jung Lu*, Gerburg Wulf*, Xiao Zhen Zhou*, Peter Davies² & Kun Ping Lu* * Cancer Biology Program, Division of Hematology/Oncology, Department of Medicine, Beth Israel Deaconess Medical Center and Harvard Medical School, Boston, Massachusetts 02215, USA ² Department of Pathology, Albert Einstein College of Medicine, Bronx, New York 10461, USA .........................................................................................................................

One of the neuropathological hallmarks of Alzheimer's disease is the neuro®brillary tangle, which contains paired helical ®laments (PHFs) composed of the microtubule-associated protein tau1,2. Tau is hyperphosphorylated in PHFs3±5, and phosphorylation of tau abolishes its ability to bind microtubules and promote microtubule assembly6,7. Restoring the function of phosphorylated tau might prevent or reverse PHF formation in Alzheimer's disease. Phosphorylation on a serine or threonine that precedes proline (pS/T±P) alters the rate of prolyl isomerization and creates a binding site for the WW domain of the prolyl isomerase Pin1 (refs 8±14). Pin1 speci®cally isomerizes pS/T±P bonds and 784

PHFs, and bead-associated proteins were immunoblotted with CP27. Lane 1, GST AD; lane 2, PHFs; lane 3, GST-Pin1 PHFs; lane 4, GST-Pin1 NB; lane 5, GST-Pin1 AD. d, PHFs were puri®ed from AD brain extracts and immunoblotted with Pin1 antibodies. Pin1 present in normal brain extracts was used as a control.

regulates the function of mitotic phosphoproteins8±10,12. Here we show that Pin1 binds to only one pT±P motif in tau and copuri®es with PHFs, resulting in depletion of soluble Pin1 in the brains of Alzheimer's disease patients. Pin1 can restore the ability of phosphorylated tau to bind microtubules and promote microtubule assembly in vitro. As depletion of Pin1 induces mitotic arrest and apoptotic cell death8, sequestration of Pin1 into PHFs may contribute to neuronal death. These ®ndings provide a new insight into the pathogenesis of Alzheimer's disease. Pin1 is a highly conserved and essential mitotic regulator10,12±15. It contains an amino-terminal WWdomain, a phosphoserine-binding module interacting with speci®c pS/T±P motifs13, and a unique carboxy-terminal prolyl isomerase domain that speci®cally isomerizes pS/T±P bonds10. Pin1 binds and regulates the function of a subset of mitotic phosphoproteins, most of which are also recognized by MPM-2, a mitosis- and phosphorylation-speci®c monoclonal antibody10,12±15. As tau is an MPM-2 antigen that is phosphorylated on multiple S/T±P motifs during mitosis16, we tested whether Pin1 binds tau. Pin1 bound tau only after mitosisspeci®c phosphorylation, and the binding was abolished by dephosphorylation (Fig. 1a). Thus, Pin1 binds tau in a mitosis-speci®c and phosphorylation-dependent manner, as shown for other binding proteins12. Mitotic events are aberrantly activated in the brains

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NATURE | VOL 399 | 24 JUNE 1999 | www.nature.com

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Figure 3 Depletion of soluble Pin1 in AD brains. a, Brain tissues were homogenized and soluble protein was analysed by immunoblotting with antibodies against GSK3b, Cdc2 and Pin1. Lanes 1±3, three representative normal individuals; lanes 4±6, three representative Alzheimer's disease patients. b, Soluble proteins isolated from normal or AD brain extracts were immunoprecipitated using Pin1 antibodies in the absence (-) or presence (+) of excess Pin1 antigen, followed by immunoblotting analysis with Pin1 antibodies. c, Homogenized lysates (T) of normal and AD brain tissues were separated into soluble (S) and insoluble (P) fractions by centrifugation, and immunoblotted with Pin1 antibodies.

Figure 2 Localization of Pin1 in normal and AD brains and competition of Pin1 binding by pT231 antibody. a±d, Rehydrated ®xed sections were incubated with 0.5 mM Pin1 or buffer control, followed by immunostaining with Pin1 antibodies or TG3. a, NB Pin1; b, AD buffer; c, AD Pin1; d, AD section stained with TG3. e±h, Sections from normal and AD brains were ®rst subjected to an antigenretrieval procedure, followed by immunostaining with Pin1 antibodies or TG3. e, Pin1-speci®c antibodies were depleted before immunostaining; f, normal brains stained with Pin1 antibodies; g, AD brain stained with Pin1 antibodies; h, AD brain stained with TG3. Sections were counter-stained with haematoxylin and eosin. Inserts show higher magni®cation pictures. i±k, Rehydrated ®xed sections were incubated with buffer control (i), 0.5 mM Pin1 (j) or ®rst CP9 antibody and then Pin1 (k), followed by immunostaining with Pin1 antibodies.

of patients with Alzheimer's disease (AD), including re-expression of Cdc2 kinase and cyclin B17±19. The phosphorylation patterns of tau in mitotic cells and AD brains are strikingly similar17,18,20,21. We also con®rmed that mitotically phosphorylated tau (pTau) was recognized by AD-speci®c, phosphorylation-dependent tau monoclonal antibodies, including CP9, TG3 and PHF1 (Fig. 1b)17,18,20,21. These results indicate that the common S/T±P motifs of tau are phosphorylated in normal mitotic cells and in AD brains. We therefore considered that Pin1 might bind and regulate the function of tau in the AD brain. We ®rst investigated whether Pin1 binds tau in AD brain extracts by using a glutathione S-transferase (GST)±Pin1 pulldown assay12. Glutathione beads containing GST or GST±Pin1 were incubated with normal or AD brain extracts, or puri®ed PHFs22. Pin1-binding proteins were then analysed by immunoblotting with the antibody CP27, which recognizes all isoforms of tau. Figure 1c shows that GST±Pin1, but not GST, bound tau in AD extracts or PHFs. In contrast, Pin1 did not bind any tau in extracts from age-matched NATURE | VOL 399 | 24 JUNE 1999 | www.nature.com

normal brains (Fig. 1c). To examine whether Pin1 forms a complex with PHFs in vivo, PHFs were puri®ed from AD brains using a series of procedures, including immunoaf®nity chromatography22, and then analysed by immunoblotting with anti-Pin1 antibodies. Pin1 was detected in PHFs puri®ed from all six AD brains examined (Fig. 1d, data not shown). These results indicate that Pin1 binds PHFs in Alzheimer's disease brains. To con®rm that Pin1 has speci®c af®nity only for PHFs, we incubated brain sections with recombinant Pin1 and then stained the sections with af®nity-puri®ed Pin1 antibodies to localize the bound Pin1. If no Pin1 was added, no immunoreactive signal was observed (Fig. 2b), indicating that Pin1 antibodies do not recognize endogenous Pin1 under the conditions used. However, if normal and AD brain sections were incubated with Pin1 protein, robust Pin1 binding was detected in the cytoplasm of neurons in AD brain sections (Fig. 2c) but not in normal brain (Fig. 2a). Pin1 speci®cally bound to neuro®brillary tangles and neurites (Fig. 2c), which were also detected by staining with TG3 (Fig. 2d), a monoclonal antibody recognizing the AD-speci®c conformation of tau phosphorylated on Thr 231 (ref. 23). Thus, exogenous Pin1 speci®cally binds the neuro®brillary tangles in neurons. Given that Pin1 has a high af®nity for the tangles and co-puri®es with PHFs, it is critical to show that Pin1 binds PHFs in vivo. We ®rst subjected the brain sections to an antigen-retrieval procedure and saw strong immunoreactivity with the Pin1 antibodies both in normal and AD brain sections (Fig. 2f, g). To ensure that these signals represented Pin1, the Pin1-speci®c antibodies were ®rst depleted using GST±Pin1 beads and then used for immunostaining. The Pin1-depleted antibodies showed no speci®c immunoreactivity (Fig. 3e). Very different patterns of Pin1 localization were seen in normal and AD brain sections, Pin1 was mainly localized in the nuclei of neurons in normal brain and was also detected in neuronal


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Table 1 Identi®cation of the Pin1-binding site in tau Pin1

Phosphorylation of tau on Thr 231 (pT231 tau) has been well documented in Alzheimer's disease brains and can be recognized by antibodies including CP9 (refs 16, 18, 21, 23). To test whether Pin1 interacts with pT231-tau, we used GST±Pin1 beads to isolate tau from Alzheimer's disease brain extracts or PHFs, and then detected Thr 231 phosphorylation using CP9. Tau isolated by Pin1 beads was strongly immunoreactive with CP9 (Fig. 4b), indicating that Pin1 binds pT231 tau and that this binding does not induce dephosphorylation of pT231. As T231 is readily phosphorylated by Cdc2 kinase in vitro17,18,23, we examined whether Pin1 binds tau phosphorylated by Cdc2. Pin1 and its WW domain, but not its isomerase domain, bound Cdc2-pTau (Fig. 4c). These results support the idea that Pin1 binds pT231 tau through its WW domain. To show that pT231 is both necessary and suf®cient for mediating the interaction between Pin1 and tau, we tested whether three different pT231-speci®c antibodies, CP9, CP17 and TG3, compete with Pin1 for binding to PHFs in brain sections. Each of the antibodies strongly competed with Pin1 for binding to PHFs (Fig. 2i±k and data not shown), showing that pT231 is needed for Pin1 to bind PHFs. We next replaced Thr 231 with the non-phosphorylatable alanine

Binding (OD at 405 nm)

nuclei in AD brains (Fig. 2f, g), consistent with reports that Pin1 is primarily localized in the nucleus of HeLa cells8. However, in AD brains, robust Pin1 immunostaining was also found in the cytoplasm of neurons, speci®cally at the tangle structure that was also recognized by TG3 (ref. 23) (Fig. 2g, h). Therefore, both exogenous and endogenous Pin1 speci®cally localize to the neuro®brillary tangles in Alzheimer's disease brains. Binding of Pin1 to PHFs might trap Pin1 in the tangles, leading to depletion of soluble Pin1 in neurons. To test this possibility, we compared the amounts of Pin1 and two tau kinases, GSK3b and Cdc2, in the soluble fraction of AD and normal brain extracts. When compared with age-matched normal brains, GSK3b was only slightly reduced (40 6 11%, n 6) (Fig. 3a, top), but Cdc2 levels were signi®cantly increased by ,5 fold in diseased brains (547 6 87%, n 6) (Fig. 3a, middle). These ®ndings are consistent with previous ®ndings that Cdc2, but not GSK3b, is abnormally elevated in the brain in Alzheimer's disease18. Levels of soluble Pin1 in all diseased brains examined were much lower than those in normal brains (Fig. 3a, bottom), being reduced by an average of ,5 fold (22:4 6 3:4%, n 6). This Pin1 decrease was further con®rmed by immunoprecipitation analysis (Fig. 3b). If Pin1 is trapped in the tangles, most of the Pin1 in diseased brains would be in the insoluble fraction. As shown in Fig. 3c, although the total levels of Pin1 were not signi®cantly different, most Pin1 was detected in the soluble fraction in normal brain, but in the insoluble fraction in AD brain (Fig. 3c). Together with the results on Pin1 localization, these data indicate that Pin1 is relocalized to the neuro®brillary tangles in Alzheimer's disease, resulting in depletion of soluble Pin1. Pin1 binds mitotic phosphoproteins through its N-terminal WW domain13. To identify the Pin1 binding site on tau, we synthesized a series of phosphorylated and non-phosphorylated peptides that covered most known tau phosphorylation sites, and then tested their ability to bind Pin1 using the ELISA (enzyme-linked immunosorbent) assay23 (Table 1). Although little or no binding was detected between Pin1 and most phosphopeptides, Pin1 exhibited speci®c and high-af®nity binding to one tau peptide containing pT231 (Table 1), with a dissociation constant of ,40 nM (Fig. 4a). In contrast, no binding was observed between Pin1 and the nonphosphorylated tau (Fig. 4a), demonstrating that T231 phosphorylation is required for Pin1 binding of tau. To determine whether the WW domain of Pin1 is responsible for binding, the mutant Pin1Y23A was used, which contains a single alanine substitution at the critical Tyr 23 in the WW domain, resulting in a loss of the phosphoserine-binding activity13. Pin1Y23A showed much less binding to pT231 peptide (Table 1). The residual binding might be due to binding of the pT231 peptide to the much lower af®nity isomerase domain of Pin113. These results indicate that the WW domain mediates Pin1 binding to the pT231 sequence of tau.

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Pin1

DAGLKEpSPLQTPTE TRIPAKpTPPAPKT GYSSPGpSPGITPGSR SRSRTPpSLPTPPT KVSVVRTPPKSPS KVSVVRpTPPKSPS VRTPPKpSPSSAKSR VQSKIGpSLDNITH GSLDNIpTHVPGGG TSPPIHLpSNVSSTG PRHLSNVpSSTGSIDMV PRHLSNVSpSTGSIDMV NVSSTGpSIDMVDS SIDMVDpSPQLATL

(pS46) (pT175) (pS202) (pS214) (T231) (pT231) (pS235) (pS356) (pT361) (pS409) (pS412) (pS413) (pS416) (ps422)

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Figure 4 Characterization of the Pin1±tau interaction. a, Different concentrations of Pin1 were incubated with a ®xed concentration of T231 or pT231 tau peptide (top) or vice versa (bottom), followed by ELISA assay with Pin1 antibodies. b, Beads containing GST or GST±Pin1 were incubated with PHFs, or normal or Alzheimer's disease brain extracts, followed by immunoblotting analysis with CP9. c, Tau was phosphorylated by Cdc2 kinase in the presence of [g32P]ATP, and then subjected to GST pulldown assay with Pin1 or its mutants. WW and PPIase are the N-terminal WW domain and C-terminal catalytic domain, respectively. d, Tau and its mutants were phosphorylated by cyclin B/Cdc2, and then either separated on SDS±PAGE directly (input) or after af®nity puri®cation using GST± Pin1 beads (Pin1).



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(tauT231A) to rule out the possibility that Pin1 binds tau through other pS/T sequences. When incubated with either mitotic extracts or puri®ed Cdc2 kinase, tauT231A was still phosphorylated (Figs 1a, 4d). However, Pin1 almost completely failed to bind tauT231A that was phosphorylated by either method (Figs 1a, 4d). In contrast, two other tau mutations, G272L and P301L, which are found in patients with hereditary forms of fronto-temporal dementia (FTDP-17)24±26, did not affect Pin1 binding of pTau (Fig. 4d). Thus, pT231 is the only Pin1-binding site in tau. In addition, phosphorylation of tauT231A by Cdc2 was signi®cantly reduced. This result con®rms previous ®ndings that T231 is an important Cdc2 phosphorylation site in tau, and is also consistent with Pin1 binding mitotically pTau (Fig. 1a) and being sequestered on to PHFs in AD brains, where Cdc2 is upregulated (Fig. 3). a

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Figure 5 Restoration of function of pTau by Pin1, but not its mutants or cyclophilin. a, Taxol-stabilized microtubules were incubated with non-phosphorylated or Cdc2-pTau in the presence or absence of Pin1 or directly incubated with Pin1. A sucrose cushion was underlain and the bound tau was separated by centrifugation, followed by immunoblotting analysis with the tau antibody CP27 or Pin1 antibodies. The membranes were also stained with Coomassie blue, as shown in the lower panel. b, Top rate of microtubule assembly promoted by tau in the presence and absence of Pin1, or by Cdc2-pTau. Control, tubulin only; Tau, tubulin + tau; pTau, tubulin + tau phosphorylated by Cdc2; Tau + Pin1, tubulin + tau + Pin1; Pin1, tubulin + Pin1. Bottom: rate of microtubule assembly promoted by Cdc2-pTau with or without of Pin1, its mutants or cyclophilin (Cyp;), as indicated. NATURE | VOL 399 | 24 JUNE 1999 | www.nature.com

The high-af®nity interaction between Pin1 and pTau indicates that Pin1 might affect the biological activity of pTau. When it is phosphorylated by many protein kinases, including Cdc2, tau loses its ability to bind microtubules and promote their assembly6,7,27,28. To examine whether Pin1 affects the ability of pTau to bind microtubules, we generated pTau in vitro using puri®ed Cdc2 (refs 17, 18), and determined its ability to bind Taxol-stabilized microtubules with or without Pin1. Although Cdc2 phosphorylation disrupted the ability of tau to bind microtubules; the binding was fully restored by preincubation with Pin1 (Fig. 5a). Furthermore, Pin1 was detected in the fraction of tau-bound microtubules (Fig. 5a). However, no Pin1 was detected in the microtubule fraction if pTau was not added (Fig. 5a), indicating that Pin1 does not bind microtubules directly. Thus, Pin1 binds pTau and restores its ability to bind microtubules. We next assessed the effect of Pin1 on the ability of pTau to promote microtubule assembly by using light-scattering assays27,28. The rate of the turbidity change was minimal without tau, but was markedly increased if tau was added (Fig. 5b, top). This increase was almost abolished if tau was phosphorylated by Cdc2 (Fig. 5b), con®rming that phosphorylation of tau by Cdc2 disrupts its ability to promote microtubule assembly6,7,28. Importantly, Pin1 fully restored the ability of Cdc2-phosphorylated tau to promote microtubule assembly in a dose-dependent manner, although Pin1 had no effect on non-phosphorylated tau (Fig. 5b, bottom, data not shown). To evaluate the speci®city of the effect of Pin1, we ®rst used the isomerase cyclophilin and the pT231-speci®c antibody CP9 in the assay. Neither had any detectable effects on pTau (Fig. 5b, bottom, and data not shown). Next, we introduced point mutations into Pin1 speci®cally to affect its phosphoprotein-binding or isomerase activity. The substitution of Tyr 23 with alanine in the WW domain does not affect the isomerase activity, but disrupts the ability of Pin1 to bind phosphoproteins13 including pTau (Fig. 4c). This Y23A mutation also abolished the ability of Pin1 to restore the function of pTau (Fig. 5b, bottom), indicating an essential role for Pin1 binding. Conversely, introducing alanine into Lys 63, an active-site residue conserved in all Pin1-related isomerases14, did not affect binding to pTau (Fig. 4c), but did reduce isomerase activity to about 10% of the wild-type level, as assayed with an in vitro peptide substrate10. This mutation also signi®cantly reduced the ability of Pin1 to restore promotion of microtubule assembly by pTau (Fig. 5b, bottom), indicating a role for the isomerase activity in regulating tau function. These data show that Pin1 not only binds pTau, but also restores its biological activity. Tau stabilizes the internal microtubular structure of neurons1,2. The importance of tau is demonstrated by ®ndings that tau mutations cause the inherited dementia FTDP-17 (refs 24±26). Tau is hyperphosphorylated in FTDP-17, as in Alzheimer's disease29. Furthermore, some FTDP-17 mutations also disrupt the ability of tau to bind microtubules and promote their assembly30, demonstrating a critical role for the tau±microtubule interaction. Pin1 binds Alzheimer's disease-associated tau and restores the ability of pTau to bind microtubules and promote their assembly. Pin1 binds pS/T±P motifs and isomerizes pS/T±P peptide bonds10,12,13. The initial mutational analyses have revealed that each of these activities is critical for regulating tau function. This is consistent with the fact that both the WW domain and the isomerase domain are essential for Pin1 function in vivo8,13. Although further work is needed to elucidate the exact role of the isomerase activity, it is possible that Pin1 binds and somehow alters the conformation of pS/T±P motifs in tau to regulate its biological function. As depletion of Pin1 induces mitotic arrest and apoptosis8, Pin1 may be required to prevent abnormal activation of mitotic events in neurons and to control the function of phosphoproteins, such as tau. Abnormal activation of mitotic events would result in hyperphosphorylation of tau, which binds and sequesters Pin1, as seen in Alzheimer's disease brains17±19. This may have two consequences.


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letters to nature First, hyperphosphorylation of tau may create more binding sites than the capacity of the available Pin1, as indicated by extra binding sites in PHFs for exogeneous Pin1. This hyperphosphorylated tau cannot bind microtubules, and subsequently forms PHFs, affecting neuronal function1,2. On the other hand, sequestration of Pin1 in PHFs depletes soluble Pin1, which itself might also have a deleterious effect on neurons that already contain mitotic phosphoproteins. Therefore, both depletion of Pin1 and formation of PHFs might contribute to neuronal loss in Alzheimer's disease. As the aggregates of hyperphosphorylated tau are also a common neuropathological feature of several other neuronal degenerative diseases, such as FTDP-17 (ref. 29), Pin1 may also be involved in these diseases. Future studies on the role of Pin1 in these neurodegenerative diseases, including the identi®cation of possible Pin1 mutations, would help us to understand their aetiology. In addition, the prolyl isomerase Pin1 might be used to derive new therapies for Alzheimer's and related neurodegenerative diseases. M .........................................................................................................................

Methods

Production and phosphorylation of tau. The longest tau isoform and its

mutants, generated by PCR mutagenesis10,12, were either synthesized by in vitro transcription and translation in the presence of 35S-Met, or produced in bacteria as N-terminal His-tagged proteins, followed by puri®cation on NTA±Ni columns, as described10,12. To generate the interphase- and mitosisspeci®c phosphorylated form, we incubated tau proteins with Xenopus interphase and mitotic extracts, respectively12. To prepare Cdc2 pTau, we incubated puri®ed recombinant tau proteins with puri®ed cyclin B/Cdc2 (UBI) for 6±12 h at room temperature in a buffer containing 500 mM cold ATP, plus trace [32P]ATP in some experiments13,18. Determination of Pin1±tau interaction and Pin1 levels. Recombinant and mutant Pin1 proteins were produced as N-terminal GST or His-tagged fusion proteins, as described12,13. Pin1 binding to tau peptides was assayed using ELISA, as described23, which gave lower Kd values than those obtained using a ¯uorescence-polarization binding assay13. The interaction between Pin1 and tau or its mutants was determined using a GST±Pin1 pulldown assay, as described12. PHFs were puri®ed by a series of procedures, including immunoaf®nity chromatography with an anti-tau monoclonal antibody22, and Pin1 and tau antibodies (CP9, CP17, CP27, TG3 and PHF1) have been described12,23. To determine levels of Pin1, brain tissues were sliced, cut into ®ne pieces and homogenized in buffer A (50 mM HEPES, pH 7.4, 150 mM NaCl, 10% glycerol, 1% Triton X-100, 5 mM MgCl2, 1 mM EGTA, 1 mM DTT, 100 mM NaF, 2 mM Na3VO4 and various protease inhibitors). The homogenates were centrifuged at 100,000g at 4 8C for 30 min and the supernatants were directly used for immunoprecipitations or immunoblotting analysis with Pin1 antibodies12, or stored in aliquots at -80 8C before assays. The pellets were boiled in SDS-sample buffer repeatedly before subjecting to immunoblotting analysis. All Alzheimer's disease cases used were clinically diagnosed with senile dementia of the Alzheimer's type and con®rmed histopathologically to have plaques and tangles, whereas control brains were from age-matched normal individuals17,18,23. Immunocytochemistry. To detect the localization of exogenously added Pin1, 50 mm sections were cut from formalin-®xed frontal cortex or hippocampus of human brains, and endogenous peroxidase activity was blocked with H2O2, followed by incubation with Pin1 at 0.5 mM. For antibody competition experiments, monoclonal antibodies were added onto brain sections 1 h before adding Pin1. After extensive washing, sections were incubated with anti-Pin1 antibodies that had been puri®ed using GST±Pin1 glutathione beads, and visualized by the immunoperoxidase staining protocol, as described23. To localize endogenous Pin1, the ®xed brain sections were ®rst microwaved in an antigen-retrieval buffer (Biogenex), as described by the manufacturer, before undergoing immunostaining procedures. Microtubule binding and assembly. The ability of tau to bind microtubules was determined as described6,28. Brie¯y, microtubules were assembled from puri®ed bovine tubulin (cytoskeleton) and stabilized by Taxol. The nonphosphorylated or Cdc2-phosphorylated recombinant tau (0.1 mg ml-1) was incubated with Pin1 (0.1 mg ml-1) at 35 8C for 5 min before adding to the microtubules. Bound tau was isolated by centrifugation (50,000g) at 25 8C for 20 min, followed by immunoblotting analysis using CP27 and Pin1 antibodies. 788

The ability of tau to promote microtubule assembly was determined using light-scattering assays27,28, with some modi®cations. Brie¯y, microtubule assembly was initiated by incubating tubulin (2 mg ml-1) with or without tau (0.05 mg ml-1) in 80 mM PIPES, pH 6.8, 1 mM EGTA, 1 mM MgCl2, 1 mM GTP, 20% glycerol at 35 8C for 2 min. The mixture was then transferred to a 100 ml cuvette and the rate of microtubule assembly was monitored at room temperature by following the turbidity increase at 350 nm. To examine the effect of added proteins, tau or Cdc2-pTau (0.05 mg ml-1) was preincubated with Pin1, its mutants, CP9 or cyclophilin (Sigma) (0.05 mg ml-1) at 35 8C for 5 min before the microtubule assembly assays. To determine stoichiometric amounts, Pin1 concentrations were reduced from 0.05 mg ml-1 to 0.01 mg ml-1, and similar results were obtained. Each experiment was repeated at least three times, with similar results. Results using GST±Pin1 or His±Pin1 were similar, indicating that the N-terminal tags have no effect on the microtubule assembly assayed. Received 7 April; accepted 4 May 1999. 1. Kosik, K. S., Qiu, W. Q. & Greenberg, S. Cellular signaling pathways and cytoskeletal organization. Ann. NY Acad. Sci. 777, 114±120 (1996). 2. Spillantini, M. G. & Goedert, M. Tau protein pathology in neurodegenerative diseases. Trends Neurosci. 21, 428±433 (1998). 3. Lee, V. M., Balin, B. J., Otvos, L. Jr & Trojanowski, J. Q. A68: a major subunit of paired helical ®laments and derivatized forms of normal Tau. Science 251, 675±678 (1991). 4. Goedert, M., Spillantini, M. G., Cairns, N. J. & Crowther, R. A. Tau proteins of Alzheimer paired helical ®laments: abnormal phosphorylation of all six brain isoforms. Neuron. 8, 159±168 (1992). 5. Greenberg, S. G., Davies, P., Schein, J. D. & Binder, L. I. Hydro¯uoric acid-treated tau PPHF proteins display the same biochemical properties as normal tau. J. Biol. Chem. 267, 564±569 (1992). 6. Bramblett, G. T. et al. Abnormal tau phosphorylation at Ser396 in Alzheimer's disease recapitulates development and contributes to reduced microtubule binding. Neuron 10, 1089±1099 (1993). 7. Yoshida, H. & Ihara, Y. Tau in paired helical ®laments is functionally distinct from fetal tau: assembly incompetence of paired helical ®lament-tau. J. Neurochem. 61, 1183±1186 (1993). 8. Lu, K. P., Hanes, S. D. & Hunter, T. A human peptidyl-prolyl isomerase essential for regulation of mitosis. Nature 380, 544±547 (1996). 9. Ranganathan, R., Lu, K. P., Hunter, T. & Noel, J. P. Structural and functional analysis of the mitotic peptidyl-prolyl isomerase Pin1 suggests that substrate recognition is phosphorylation dependent. Cell 89, 875±886 (1997). 10. Yaffe, M. B. et al. Sequence-speci®c and phosphorylation-dependent proline isomerization: A potential mitotic regulatory mechanism. Science 278, 1957±1960 (1997). 11. Schutkowski, M. et al. Role of phosphorylation in determining the backbone dynamics of the serine/ threonine-proline motif and Pin1 substrate recognition. Biochemistry 37, 5566±5575 (1998). 12. Shen, M., Stukenberg, P. T., Kirschner, M. W. & Lu, K. P. The essential mitotic peptidyl-prolyl isomerase Pin1 binds and regulates mitosis-speci®c phosphoproteins. Genes Dev. 12, 706±720 (1998). 13. Lu, P. J., Zhou, X. Z., Shen, M. & Lu, K. P. Function of WW domains as phosphoserine or phosphothreonine-binding modules. Science 283, 1325±1328 (1999). 14. Lu, K. P. Phosphorylation-dependent prolyl isomerization: a novel cell cycle regulatory mechanism. Prog. Cell Cycle Res. (in the press). 15. Crenshaw, D. G., Yang, J., Means, A. R. & Kornbluth, S. The mitotic peptidyl-prolyl isomerase, Pin1, interacts with Cdc25 and Plx1. EMBO J. 17, 1315±1327 (1998). 16. Illenberger, S. et al. The endogenous and cell cycle-dependent phosphorylation of tau protein in living cells: implications for Alzheimer's disease. Mol. Biol. Cell 9, 1495±1512 (1998). 17. Vincent, I., Rosado, M. & Davies, P. Mitotic mechanisms in Alzheimer's disease? J. Cell Biol. 132, 413± 425 (1996). 18. Vincent, I., Jicha, G., Rosado, M. & Dickson, D. W. Aberrant expression of mitotic cdc2/cyclin B1 kinase in degenerating neurons of Alzheimer's disease brain. J. Neurosci. 17, 3588±3598 (1997). 19. Nagy, Z., Esiri, M. M. & Smith, A. D. Expression of cell division markers in the hippocampus in Alzheimer's disease and other neurodegenerative conditions. Acta Neuropathol. (Berl.) 93, 294±300 (1997). 20. Kondratick, C. M. & Vandre, D. D. Alzheimer's disease neuro®brillary tangles contain mitosis-speci®c phosphoepitopes. J. Neurochem. 67, 2405±2416 (1996). 21. Preuss, U. & Mandelkow, E. M. Mitotic phosphorylation of tau protein in neuronal cell lines resembles phosphorylation in Alzheimer's disease. Eur. J. Cell Biol. 76, 176±184 (1998). 22. Vincent, I. J. & Davies, P. A protein kinase associated with paired helical ®laments in Alzheimer disease. Proc. Natl Acad. Sci. USA 89, 2878±2882 (1992). 23. Jicha, G. A. et al. A conformation- and phosphorylation-dependent antibody recognizing the paired helical ®laments of Alzheimer's disease. J. Neurochem. 69, 2087±2095 (1997). 24. Hutton, M. et al. Association of missense and 59-splice-site mutations in tau with the inherited dementia FTDP-17. Nature 393, 702±705 (1998). 25. Clark, L. N. et al. Pathogenic implications of mutations in the tau gene in palido-ponto-nigral degeneration and related neurodegenerative disorders linked to chromosome 17. Proc. Natl Acad. Sci. USA 95, 13103±13107 (1998). 26. Spillantini, M. G. et al. Mutation in the tau gene in familial multiple system tauopathy with presenile dementia. Proc. Natl Acad. Sci. USA 95, 7737±7741 (1998). 27. Iqbal, K., Zaidi, T., Bancher, C. & Grundke-Iqbal, I. Alzheimer paired helical ®laments. Restoration of the biological activity by dephosphorylation. FEBS Lett. 349, 104±108 (1994). 28. Alonso, A. C., Zaidi, T., Grundke-Iqbal, I. & Iqbal, K. Role of abnormally phosphorylated tau in the breakdown of microtubules in Alzheimer disease. Proc. Natl Acad. Sci. USA 91, 5562±5566 (1994). 29. Spillantini, M. G., Bird, T. D. & Ghetti, B. Frontotemporal dementia and Parkinsonism linked to chromosome 17: a new group of tauopathies. Brain Pathol. 8, 387±402 (1998). 30. Hong, M. et al. Mutation-speci®c functional impairments in distinct tau isoforms of hereditary FTDP-17. Science 282, 1914±1917 (1998). Acknowledgements. We thank B. Neel, L. Cantley, C. S. Chen, G. Lee and X. D. Fu for constructive comments. P.-J.L. and G.W. are fellows of Leukemia Society of America and DOE Breast Cancer Research Program, respectively. K.P.L. is a Pew Scholar and a Leukemia Society of America Scholar. This study was supported by NIH grants to P.D. and K.P.L. Correspondence and requests for materials should be addressed to K.P.L. (e-mail: klu@caregroup. harvard.edu).

