Pinning down phosphorylated tau and tauopathies

Biochimica et Biophysica Acta 1739 (2005) 311 – 322 http://www.elsevier.com/locate/bba

Review

Pinning down phosphorylated tau and tauopathies Jormay Lim*, Kun Ping Lu Cancer Biology Program Division of Hematology/Oncology, Department of Medicine, Beth Israel Deaconess Medical Center, Harvard Medical School, 330 Brookline Avenue, NRB 1030K Boston, MA 02215 USA Received 28 April 2004; received in revised form 9 September 2004; accepted 7 October 2004 Available online 22 October 2004

Abstract Neurofibrillary tangles (NFTs) are prominent neuronal lesions in a large subset of neurodegenerative diseases, including Alzheimer’s disease (AD). NFTs are mainly composed of insoluble Tau that is hyperphosphorylated on many serine or threonine residues preceding proline (pSer/Thr–Pro). Tau hyperphosphorylation abolishes its biological function to bind microtubules and promotes microtubule assembly and precedes neurodegeneration. Not much is known about how tau is further regulated following phosphorylation. Notably, we have recently shown that phosphorylated Ser/Thr–Pro motifs exist in two distinct conformations. The conversion between two conformations in some proteins is catalyzed by the prolyl isomerase Pin1. Pin1 binds to tau phosphorylated specifically on the Thr231–Pro site and probably catalyzes cis/trans isomerization of pSer/Thr–Pro motif(s), thereby inducing conformational changes in tau. Such conformational changes can directly restore the ability of phosphorylated Tau to bind microtubules and promote microtubule assembly and/or facilitate tau dephosphorylation by its phosphatase PP2A, as PP2A activity is conformation-specific. Furthermore, Pin1 expression inversely correlates with the predicted neuronal vulnerability in normally aged brain and also with actual neurofibrillary degeneration in AD brain. Moreover, deletion of the gene encoding Pin1 in mice causes progressive age-dependent neuropathy characterized by motor and behavioral deficits, tau hyperphosphorylation, tau filament formation and neuronal degeneration. Distinct from all other mouse models where transgenic overexpression of specific proteins elicits tau-related pathologies, Pin1 is the first protein whose depletion causes age-dependent neurodegeneration and tau pathologies. Thus, Pin1 is pivotal in maintaining normal neuronal function and preventing age-dependent neurodegeneration. This could represent a promising interventive target to prevent neurodegenerative diseases. D 2004 Elsevier B.V. All rights reserved. Keywords: Alzheimer’s disease; Pin1; Peptidyl-prolyl isomerase; Protein phosphorylation; Tau; Tauopathy

1. Introduction To respond to external stimulations and to coordinate internal cellular activities such as cytoskeletal reorganization, vesicular transport, or mitotic division, etc., reversible protein phosphorylation is a rapid regulatory mechanism that is utilized universally. Phosphorylation of serine and threonine residues preceding proline (pSer/Thr–Pro) mediated by a large number of so-called Pro-directed protein kinases is a major signaling mechanism [1–3]. Furthermore, aberrant regulation of Pro-directed phosphorylation can * Corresponding author. Tel.: +1 617 667 4143; fax: +1 617 667 0610. E-mail address: [email protected] (K.P. Lu), [email protected] (J. Lim). 0925-4439/$ - see front matter D 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.bbadis.2004.10.003

cause human diseases [3]. For instance, hyperphosphorylation of the microtubule (MT)-associated protein tau is found in neurofibrillary tangles (NFTs) [4]. NFTs are prominent in many age-dependent neurodegenerative disorders including Alzheimer disease (AD), frontotemporal dementia and parkinsonism linked to chromosome 17 (FTDP-17), Pick disease, progressive supranuclear palsy (PSP) and corticobasal degeneration (CBD) [5–7]. These neurodegenerative diseases are collectively termed tauopathies. Mutations in the gene encoding the tau protein have been identified in FTDP17 patients [5–7]. Furthermore, transgenic overexpression of these tau mutants in mice results in many age-dependent neuropathological changes that resemble those found in FTDP-17 patients [8–14]. These results establish that tau dysfunction can directly result in neurodegeneration.

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Tau has been shown to be regulated by reversible phosphorylation governed by many Pro-directed protein kinases such as cyclin-dependent kinases (CDKs), mitogenactivated protein kinases (MAPKs) and glycogen synthase kinases (GSKs), as well as phosphatases such as PP2A [15– 24]. Ser/Thr phosphorylation has been shown to regulate the biological function of tau presumably through conformational changes [25–28]. However, until recently, little has been known about the nature and regulation of the conformational changes. The recent identification and characterization of a peptidyl–prolyl cis/trans isomerase (PPIase), Pin1, which specifically regulates the conformation of certain Prodirected phosphorylation sites in a subset of proteins, has led to the discovery of a new post-phosphorylation regulatory mechanism [29–33]. In this mechanism, Pin1 binds to and isomerizes specific pSer/Thr–Pro motifs and catalytically induces conformational changes following phosphorylation. Such conformational changes can have profound effects on the function of many Pin1 substrates, thereby playing an important role in many cellular events[1,2]. Interestingly, we have recently shown that tau is a major substrate for Pin1 in neurons [34]. Pin1 targets Tau phosphorylated on the Thr231–Pro motif and directly restores its biological function after Tau is inactivated by hyperphosphorylation. Pin1 can also facilitate tau dephosphorylation because its phosphatase PP2A is strictly conformation-specific [35]. Furthermore, Pin1 expression in the brain inversely correlates with the predicted neuronal vulnerability and actual neurofibrillary degeneration in AD [36]. Moreover, Pin1 is the first gene whose knockout in mice causes progressive age-dependent neuropathy characterized by motor and behavioral deficits, tau hyperphosphorylation, tau filament formation and neuronal degeneration [36]. These results indicate that Pin1 plays a pivotal role in protecting against age-dependent neurodegeneration and provides new insight into the pathogenesis and treatment of AD and related diseases. This review focuses on discussing Pin1 as a novel regulator of phosphorylated tau and its role in the development of human tauopathies. Comprehensive recent reviews on the function and regulation of Pin1 in cell signaling and human diseases are available [2,3].

Tau deletion also potentiates the phenotypes of axon tracts and neuronal layers in mice lacking MAP1B, another major MT-associated protein [41]. In normal brains, Tau proteins are mainly distributed in neuronal axons, regulating cell structure, cell polarity and axonal transport. However, in AD brains, neuronal cytoskeleton is progressively disrupted and replaced by NFTs [42]. NFTs in AD are composed of hyperphosphorylated Tau in two forms both paired helical filaments (PHF) and straight filaments (SF). Tau hyperphosphorylation appears to precede tangle formation and neurodegeneration in AD. Hyperphosphorylated Tau shows defective microtubule binding (Fig. 1), fails to promote microtubule assembly, and self-assembles into NFTs in vitro [4]. In contrast, dephosphorylation in vitro can restore the microtubule-binding function and abolish the aggregation of AD phospho-Tau (pTau) [42]. Taken together, these results indicate that increased tau phosphorylation is a key and early event in the pathogenesis of human tauopathies. The importance of tau has been further supported by recent molecular characterization of familial FTDP-17, a

2. Tau and Tau phosphorylation Tau proteins promote tubulin polymerization and stabilize microtubule structure in vivo [37,38]. This function is crucial for the integrity of the neuronal cytoskeletal networks and the neuronal processes in forming connections with other cells [39]. In vitro, Tau proteins co-assemble with tubulins into microtubules. Embryonic hippocampal cultures isolated from Tau deficient mice showed delayed maturation in terms of axonal and dendritic extensions [40].

Fig. 1. Pin1 binds to phosphorylated Tau. (A) Tau undergoes hyperphosphorylation in brains suffering from neurodegenerative diseases probably due to aberrant kinase or phosphatase activities. The hyperphosphorylated Tau loses its axonal localization in neurons and aggregates in somatodendrites. Binding to microtubules and promoting microtubule assembly are also abolished. (B) Pin1 binds to the pThr231 site of phosphorylated Tau. The PPIase domain may catalyze the cis/trans isomerization of pSer/Thr–Pro motif(s) in Tau and thus change the conformation to favor dephosphorylation or microtubule binding.

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neurodegenerative disease caused by the genetic mutation of the tau gene [43–45]. Tau hyperphosphorylation and NFTs are the hallmarks of this genetic disorder. Furthermore, overexpression of human wild-type Tau or, even more profoundly, FDTP-17 tau mutants in mice causes progressive and age-dependent formation of NFTs [8–10]. Interestingly, different mutations or splice variants of tau have demonstrated different levels and patterns of neuronal loss or axonal degeneration of brain regions and spinal cords. Each model is probably valuable to tackle the multifaceted tauopathies. On the other hand, for unknown reasons, transgenic mice overexpressing a different version of human Tau (namely the smallest isoform, four repeats of microtubule domain isoform, Pro301Leu mutant, Arg406Trp mutant, or Val337Met mutant) all exhibit similar hyperphosphorylation of Tau [8–14]. This raises an obvious question: is there a common mechanism of NFTs formation that leads to neurodegeneration? Thus, Tau hyperphosphorylation might be a converging step in human tauopathies. Tau hyperphosphorylation has been attributed to aberrant regulation of protein kinases and/or phosphatases. However, attempts to identify AD-specific phosphorylation sites on tau have yet to yield conclusive results [46,47]. The most probable candidates are Ser262 and the region around Thr231 and Ser235 [48,49]. Among these sites, Ser262 is identified as a Lys-Xaa-Gly-Ser motif recognized by Par1 kinase (microtubule associated regulating kinase) [23,50,51], whereas pSer/Thr–Pro sites of Thr231 and Ser235 are phosphorylated by CDKs, MAPKs, and/or GSKs [15,17,24,52–54]. These pro-directed kinases also target other pSer/Thr–Pro sites of Tau. A hierarchy of phosphorylation regulation by different kinases has been implicated in the cascade of hyperphosphorylation and certain sites may play an initiative role [52,55]. GSK-3h or CDK-5 has been suggested to induce pretangle or NFT phenotypes, respectively [56–58]. A causative role for kinases upstream of tau in neuronal damage of NFTs was established when Cruz et al. [59] reported that inducible overexpression of p25, a truncated and accumulated version of the CDK-5 activator, in mouse forebrain exhibited tauopathy phenotypes. Consequently, it is important to study whether the hyperphosphorylated Tau could be dephosphorylated or restored to functional forms. Besides the intracellular NFT, the extracellular senile plaques that are mainly composed of Amyloidh (Ah) are also prominent in AD. Ah is released due to the amyloidogenic processing of amyloid protein precursors (APP) [60–62]. APP mutations have been found in the studies of familial AD [63]. The mutations of Presenilin1 (PS1), one of the proteins involved in the APP processing, have also been found in AD [64–68]. Based on murine brain analysis and cell culture manipulation, it is now believed that Tau hyperphosphorylation and NFT formation can be induced by Ah challenge and/or PS1 mutation [69–73].

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3. Pin1: a phosphorylation-specific prolyl isomerase Pro-directed phosphorylation events have been proposed to function through inducing conformational changes in proteins. However, little is known about these conformational changes, and their importance and regulation. Pro residues have the unique property of existing in two completely distinct isomers, cis and trans, in folded proteins. This provides a potential backbone switch in the polypeptide chain that is controlled by cis/trans isomerization about the peptidyl–prolyl bond. This conversion is intrinsically slow and can be catalyzed by cis/trans peptidyl–prolyl isomerases (PPIases) [74]. There are two well-characterized families of PPIases, cyclophilins and FK506 binding proteins (FKBPs), as well as a relatively new family of PPIases, which include parvulin and Pin1 [29,75–79]. Cyclophilins and FKBPs have been shown to be involved in various cell processes, with the most wellknown function being in the immune system where they act as cellular receptors for clinically relevant immunosuppressive drugs [80]. However, the importance of the PPIase activity in these proteins remains elusive. Prolyl isomerization of Ser/Thr–Pro motifs is particularly important because they are the only phosphorylation motifs for a large subfamily of important Pro-directed protein kinases [30,81,82]. Furthermore, phosphorylation not only restrains prolyl isomerization, but also renders the peptide bond resistant to the catalytic action of cyclophilins and FKBPs. In sharp contrast, Pin1 is the only enzyme known so far that can effectively isomerize pSer/Thr–Pro bonds, but not their unphosphorylated counterparts. The human Pin1 gene was originally identified in a combined genetic and biochemical screen for proteins involved in mitotic regulation. Pin1 is highly conserved and is the only essential gene out of a total of 13 known PPIase genes present in the yeast genome [29]. The striking substrate specificity of Pin1 towards certain pSer/Thr–Pro bonds results from its unique two domain structure consisting of an N-terminal WW domain and a C-terminal PPIase domain, which form a bdouble-checkQ mechanism [32,35,83]. The WW domain of Pin1 binds only to specific pSer/Thr–Pro motifs, which are often critical regulatory phosphorylation sites in Pin1 substrates [34,35,84–88]. This WW domain binding targets the Pin1 catalytic domain close to its substrates, where the PPIase domain isomerizes specific pSer/Thr–Pro motifs and catalytically induces conformational changes in proteins [35,85]. Pin1 is ubiquitously expressed in animals and its expression is driven by the E2F transcription factor activated by Neu/Ras induction [33]. Pin1 is mainly localized in the nucleus in HeLa cells, but the actual subcellular localization is versatile and determined by the substrates. Playing an important role in the postphosphorylation protein regulation, the function of Pin1 is inhibited by phosphorylation on its Ser16 residue. It is likely that another site is also phosphorylated by yet

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identified upstream kinases. Pin1 phosphorylation is cell cycle-dependent. Pin1 plays an important role in cell cycle control. Depletion of Pin1 in HeLa cells or the Pin1 homolog in yeast, ESS1, results in mitotic arrest, while overexpression of Pin1 in HeLa cells causes G2 arrest. Recent studies indicate that a Pin1-mediated regulatory mechanism is pivotal for controlling many physiological processes, such as cell cycle progression, transcriptional regulation, RNA processing and cell proliferation and differentiation. Furthermore, deregulation of Pin1 can lead to some human diseases [2,34–36,86,87,89–92]. Notably, Pin1 is not only overexpressed in a large number of human cancers, but also is an excellent prognostic marker in some cancers [86,87,93]. Furthermore, Pin1 overexpression can function as a critical catalyst that amplifies multiple signaling pathways during oncogenesis [33,86,87,89,90]. Significantly, inhibition of Pin1 in cancer cells via multiple approaches triggers apoptosis or suppresses transformed phenotype [29,33,94]. These results suggest that Pin1 may represent a new molecular target for cancer diagnostics and therapeutics.

4. Phosphorylated tau: a major neuronal substrate for Pin1 Pin1 binds to and regulates the function of a defined subset of phosphoproteins, many of which are also recognized by the mitosis- and phosphorylation-specific antibody (mAb) MPM-2. Interestingly, induction of MPM2 epitopes is a prominent common feature of AD, frontotemporal dementia with Parkinsonism linked to chromosome 17 (FTDP-17), Down Syndrome, corticobasal degeneration, progressive supranuclear palsy and Pick’s disease. Furthermore, various studies have previously shown that mitotic events, including Cdc2 kinase, are aberrantly activated in the AD brain [95,96] and the phosphorylation pattern of tau in mitotic cells is strikingly similar to that in the AD brain [95–100]. Indeed, both mitotic and AD tau proteins are recognized by mitosisspecific or AD-specific mAbs that are phosphorylationdependent. These results suggested that Tau might be a Pin1 neuronal substrate. Indeed, we have shown that Pin1 binds to Tau in a phosphorylation-dependent manner and the binding site was mapped to pThr231 of Tau in vitro (Fig. 1) [34]. This phospho-epitope has been proposed to be diagnostic for AD because its levels in the cerebrospinal fluid correlate with the progression of the disease [49,101]. In addition, the mobility shifted pTau was positively probed by both CP9, an antibody specific for pThr231, and TG3, an antibody detecting Alzheimer-specific conformation of pThr231. In AD brain sections, exogenous Pin1 specifically bound to NFTs and neurites, which were also reactive to Pin1 antibody and TG3, indicating endogenous Pin1 also

localized to the pThr231 site. Biochemically, Pin1 was copurified with PHF. Thus, the Pin1 interaction with pTau has been established. Recently, Pin1 binding to pThr212 was reported [102]. However, the authors overlooked that buffers for both the binding reaction and GST-Pin1 preparation must avoid phosphate buffers for efficient substrate binding. This might explain the discrepancy in the dissociation constant of the pThr231 peptide to Pin1 documented; 40 nM reported by Lu et al. [34] and 0.38 mM reported by Smet et al. [102]. Furthermore, Lu et al. characterized the binding extensively using ELISA assays with a panel of phosphorylated Tau peptides covering many of the pSer/ Thr–Pro sites, and using a point mutation approach to test the binding of Pin1 to Thr231Ala, Gly272Leu, and Pro301Leu of full-length Tau. Basically, the Pin1 WW domain only binds to Tau via its pThr231 residue. Point mutants mimicking FTDP-17 bind as efficiently as wild type to Pin1. Therefore, it would be interesting to determine whether overexpression of Pin1 in Tau mutant transgenic mice affects Tau hyperphosphorylation and NFTs development. In a recent study, Pin1 translocation from the soluble fractions to the insoluble fractions was reported in AD brains, while it was mainly present in the soluble fraction in age matched normal brain samples [34]. This observation led to the suggestion that Pin1 might be sequestered to PHF or NFTs in AD neurons and thus not able to perform its job. In this sense, Pin1 protein levels could serve as a determining factor for the susceptibility of neurons to upstream signals in NFTs formation. This idea is supported by the Liou et al.’s [36] findings regarding an inverse correlation between Pin1 expression and predicted vulnerability. Immunohistochemistry analysis performed with AD brain tissue slides revealed that less Pin1 was expressed in CA1 and the subiculum of the hippocampus when compared to the neighboring regions. The same is true for the neurons of the parietal cortex, regions prone to neurofibrillary degeneration in AD. This shows that Pin1 function is important for microtubule binding of Tau. 4.1. Pin1 restores the conformation and biological function of phosphorylated Tau NFTs could probably be a Tau conformational problem given the variability of the point mutants found in FTDP-17 and the information accumulated from the conformational specific antibodies of pathological Tau [28,103–106]. When Tau biochemical function was assessed using an in vitro set up of Taxol-stabilized microtubules and sucrose cushion centrifugation, Tau phosphorylated by Cdc2 showed a marked decrease in microtubule binding when compared to unphosphorylated Tau [34]. The loss of pTau affinity to microtubules could be dramatically rescued by the sole presence of Pin1 molecules, which were detected in pTau/

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microtubule complexes but which did not show any binding to microtubules in the absence of pTau [34]. A similar conclusion was derived when microtubule assembly from tubulin was measured based on turbidity changes detected by spectrophotometer. The ability of Tau to promote tubulin polymerization was abolished after Tau was phosphorylated by Cdc2. Wild-type Pin1, but not the binding defective point mutant, restored the function of pTau. Interestingly, the PPIase catalytic defective mutant reduced but did not completely destroy the effects of Pin1 on pTau [34]. This assay has been criticized for the fact that pTau accumulation could also change the absorbance since Pin1 could probably assist with the aggregation of pTau. We have revisited this issue with an assay that could exclude the interference of accumulated pTau [2]. When fluorescent tubulin was applied in the assay, the end point could be visualized under the fluorescent microscope. As expected, Tau promotes microtubule assembly and pTau loses this function. In this assay, Pin1 restores the function of pTau. Note that in these assays, comparable concentrations of pTau and Pin1 were applied. In the microtubule assembly assays, reduction in Pin1 concentration to one fifth could still produce the same effect. The enzyme/substrate concentration ratio in these assays is unusually high. Thus, whether PPIase catalytic activity is important for in vivo Tau properties in neuronal function remains to be addressed. 4.2. Pin1 facilitates the dephosphorylation of Tau Pin1 binds to pSer/Thr–Pro sites of many MPM-2 antigens, including Tau. The phosphorylation dependent cis/trans-isomerization of pSer/Thr–Pro of many MPM-2 antigens is important for cell cycle control [107–110]. On the other hand, dephosphorylation of the MPM-2 epitopes by phosphatases, notably phosphatase2A (PP2A), also plays an essential role in the regulation of Cdc2 activation and mitosis [111–116]. Interestingly, using metabolically active rat brain slices as a model, Gong et al. [21] showed that down-regulation of PP2A by okadaic acid induced Alzheimer-like hyperphosphorylation of Tau. The inhibition of PP2B by cyclosporin A in the same system did not significantly affect Tau phosphorylation. The role of Pin1 in modulating Tau dephosphorylation has been investigated by our group [35]. We obtained the pure cis isomer of Tau peptide using trans-specific a-chymotrypsin to eliminate the trans isomer [35]. In the absence of any PPIases, it took about 2.5 h to convert the pure cis peptide to equilibrium state, which is about 15% cis. Importantly, the pure cis peptides cannot be dephosphorylated by PP2A [35]. The pThr–Pro peptides were dephosphorylated by PP2A with a rate almost identical to that of trans isomer appearance. The trans isomer-dependent dephosphorylation was neither observed with alkaline phosphatase nor with a non-Procontaining peptide. These experiments indicated that PP2A is a trans conformation-specific phosphatase.

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Indeed, the presence of Pin1 accelerated the dephosphorylation of Tau by PP2A. Thus, Pin1 protein was dissected into truncations of the N-terminal WW domain and the Cterminal PPIase domain. In this in vitro assay, although the WW domain is inactive, the PPIase domain alone was as efficient as the full-length in facilitating the Tau dephosphorylation by PP2A. In accordance with this, the Pin1 Lys63Ala point mutant abolishing catalytic activity ablates the function. The effect of Pin1 on PP2A dephosphorylation was not observed in Tau Thr231Ala mutant. The explanation might be that nonphysiological concentrations of Pin1 variants in this assay can probably reach the substrates without proper substrate targeting of the WW domain. The above results show that PPIase catalytic function is important in providing trans specific substrates for PP2A. This idea was further supported by an in vivo assay of budding yeast in which reciprocal genetic interactions between Pin1 and PP2A were demonstrated [35]. Pin1 was constructed under a GAL promoter that is repressed by glucose and induced by galactose. Induction of Pin1 rescued the mitotic defective of the PP2A temperature-sensitive mutant, and vice versa, PP2A also rescued the Pin1 mutant. In culture media containing both glucose and galactose, i.e., when Pin1 was induced moderately, only full-length Pin 1 rescued the temperature-sensitive mutant. In concurrence with this biochemical assay, excess amounts of the PPIase domain fully induced by galactose media rescued the mutant at nonpermissive temperatures. This observation was supported by a panel of point mutants, in which catalytic activity loss was in perfect correlation with the failure to rescue the mutant [35]. Postmitotic neuronal function in the adult mammalian brain is different from yeast propagation. Although there is a microtubule-associated protein homolog in budding yeast that contains a sequence homologous to the microtubulebinding domain of MAP2, MAP4, and Tau near its Cterminal end [117,118], it is unlikely that Tau and PP2A are involved in mitotic division in neurons. On the contrary, to promote and maintain the microtubule networks for intracellular vesicular transport and intercellular connections, Tau function in the neuronal system might have evolved to require a different role from Pin1 than its homolog in yeast. As a result, it would be exciting if the PPIase activity is also verified by Tau phosphorylation of the mammalian neuronal system.

5. Pin1 inversely correlates with the neuronal vulnerability and degeneration in human brains Pin1 can bind and isomerize the pThr231–Pro motif in Tau, which restores its microtubule function and is required for its dephosphorylation in vitro [34,35]. Furthermore, soluble Pin1 levels are reduced in AD brains [34] and depletion of Pin1 induces mitotic block and apoptosis in

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cancer cells [29]. These results led us to hypothesize that Pin1 might protect against neurodegeneration [2]. However, Holzer et al. [119] reported that in the AD hippocampus, Pin1 expression bwas restricted exclusively to neurons in the CA1 and CA2 pyramidal layer and the subiculumQ, especially to a small subset of tangle-free degenerative neurons, but bwas sparedQ in CA3 and CA4 neurons, and therefore proposed that Pin1 promoted neurodegeneration. Therefore, it is essential to elucidate the neuronal function of Pin1 and obtain genetic evidence indicating a role for Pin1 in AD. As the first step to this end, we examined expression of Pin1 in human brains. In the hippocampus and neocortex, the neurons in specific subregions are more prone to neurofibrillary degeneration than the neighboring subregions, indicating stereotypical patterns of neurodegeneration in AD. To examine the relationship between this stereotypical vulnerability and Pin1 level, immunostaining using Pin1 antibodies was performed [36]. In normal human brains, Pin1 is localized in the neuronal cytoplasm and nucleus. In the hippocampus, expression of Pin1 was relatively higher in CA4, CA3, CA2 and presubiculum, and lower in CA1 and subiculum. In the parietal cortex, expression of Pin1 was relatively higher in layer IIIb-c neurons, and lower in layer V neurons. The subregions with low expression of Pin1 coincide with the subregions that are more susceptible to neurofibrillary degeneration in AD, whereas those containing high Pin1 expression are not, showing an inverse correlation between Pin1 expression and predicted vulnerability [36]. In AD brains, tangle-bearing neurons were enriched in CA1 and subiculum of the hippocampus and in layer V of the parietal cortex [120–123]. Pin1 level is generally low in these regions [36]. Occasionally, high Pin1 immunoreactivity was detected in cytoplasmic granules in a few neurons, mainly in CA1 [36]. This could be explained by a few possibilities. First, Pin1 might be sequestered by strong MPM-2 epitopes in these granules. These neurons are thus deprived of functional Pin1 available to pTau outside the granules. Second, Pin1 level has to be optimum for proper function, since excess or aggregations may also lead to neuronal degeneration. However, aggregated Pin1 may result in loss of enzymatic activity in the presence of substrates. Third, Pin1 could only prevent the pathological development of Tau when neurons are healthy, but could not overcome the abnormality after a certain threshold of Tau hyperphosphorylation has occurred or after Tau aggregation has developed. Although these immunoreactive granules occur in a minor population of neurons in the hippocampus, they can be detected by commercial antibodies, and thus have been addressed by both Holzer et al. [119] and Ramakrishnan et al. [124]. However, it remains to be established whether the interpretations of these two studies indicate that Pin1 plays a role in the pathogenesis of neurofibrillary degeneration, because age-dependent Tauopathy phenotypes have been triggered by the deletion of Pin1 gene in mice.

6. Age-dependent tauopathy phenotypes in Pin1 null mice The findings that Pin1 can regulate the biological function and dephosphorylation of phosphorylated tau in vitro and that Pin1 levels inversely correlate with the neuronal vulnerability and degeneration in human brains suggest that Pin1 might be an important factor in neurodegeneration. To investigate this idea, we examined neuronal phenotypes of Pin1 knockout mice [36]. Pin1 knockout mice show no obvious developmental problem and grow healthily to adulthood [89]. However, they are difficult to breed, which is likely due to germ cell defects in testis and ovary as well as the impaired mammary epithelial expansion [89,92]. Interestingly, retinal degeneration was observed in mice older than a year, while 50% of 4–6-month-old mice underwent mild degeneration [89]. In general, all the phenotypes are subtle in young adults and develop to be more severe when the animals age. Thus, long-term observation was carried out to test the role of Pin1 in Tau regulation [36]. Indeed, Pin1 / mice, but not their wild-type littermates, showed progressive age-dependent motor and behavioural deficits, which included abnormal limb-clasping reflexes, hunched postures, and reduced mobility [36]. These deficits have also been reported in Tau transgenic mice studies [9,125]. This is exciting because no gene deletion model has been reported to cause Tau pathology, thus no endogenous protective mechanism has been proposed. To assess whether these phenotypes were caused by neuronal loss, neurons in various brain regions were quantified. Indeed, neuron number was significantly decreased in the parietal cortex of old, but not young, Pin1 / mice (Fig. 2). A similar neuronal degeneration was found in spinal cords of Pin1 / mice [36]. Neuronal loss could be caused by various reasons since Pin1 targets a panel of cellular substrates. h-Catenin degradation, for example, is likely involved given that the reduction of nuclear h-catenin correlates with Tau hyperphosphorylation in GSK-3h transgenic mice [56]. Nonetheless, it is most likely that pTau regulation is directly impaired in the absence of Pin1. As expected, Tau hyperphosphorylation has been observed in aged Pin1 / mice, with a dramatic mobility shift that is reversed by phosphatase treatment [36]. The retarded forms of pTau in Pin1 / mice were also detected by various phospho-specific or Alzheimer-conformationspecific antibodies, such as AT180 and TG3. The phosphatase activity specific to pThr231 peptide motif was significantly reduced in Pin1 / mice, which is in agreement with Pin1’s role in facilitating PP2A phosphatase activity. In aged Pin1 / mice, immunohistochemical staining of the hippocampus, cortex and spinal cord with specific pTau antibodies showed distinct somatodendritic signals, indicating pathological localization of Tau. The hyperphosphorylation of Tau eventually leads to Tau aggregation and Tau filament formation. This is exactly

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Fig. 2. Pin1 deletion leads to age-dependent neurofibrillary degeneration. Tau proteins are localized to neuronal axons and function in maintaining microtubule cytoskeletal dynamics and related vesicular transport in wildtype mice. Tau functions are intact in young adult Pin1 knockout mice, although whether the dendritic spines and synaptic integrity are affected has not been tested yet. Pin1 may target other neuronal substrates upstream of Tau, which results in the abnormality of neuronal physiology. These dysfunctions are eventually accumulated and revealed as neurofibrillary degeneration in aged animals.

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tau hyperphosphorylation and aggregation in vivo is not clear. We have shown that Pin1 could in vitro bind to pTau and cause a direct restoration of Tau function in microtubule assembly (Fig. 3) [34]. In addition, isomerization of Tau catalyzed by Pin1 facilitates PP2A activity on Tau, thereby reducing the hyperphosphorylation of Tau [35]. Structural results had been presented to suggest that the substrate conformation change is induced by isomerization of pSer/Thr–Pro bonds [32]. Despite the global similarity of Pin1 PPIase to the FKBP-like PPIase, Arg68 and Arg69 of the multivalent anion binding-site are conserved in Pin1, yeast homolog ESS1 and fruit fly homolog Dodo; but not found in other FKBP-like PPIase. Similarly, the structurally superimposed site of Cys113 is a Phe in FKBP-like PPIase. Cys113 is proposed to be important for the transient covalent bond formed between the enzyme and substrate. The mutation of this site to Ala or Ser results in the decrease of catalytic activity by 123- or 20-fold, respectively. Collated structural information and the ability of the PPIase domain to function as efficiently as full-length Pin1 in yeast genetic study strongly suggest substrate conformational changes can be caused by Pin1 catalytic activity. How isomerization of pThr231–Pro232 in Tau could lead to a conformation that regains its microtubule binding and assembling properties remains to be elucidated. Structural studies of Pin1 also revealed that there are two pSer/Thr–Pro binding patches: one in the WW domain and the other in the active site of the PPIase domain [32]. There are two narrow

what happened to Pin1 / mice, as NFT-like Tau filaments decorated by AT180 gold label were isolated from sarkosyl insoluble fractions [36]. The Tau filaments isolated resembled some of the human FTDP-17 mutants [9,11,14,125,126]. Gallyas and thioflavin-staining are two established methods detecting NFTs in AD brains and were thus employed in Pin1 / brains. Indeed, Gallyas and thioflavin-staining were immense in entorhinal cortex and hippocampus, brain regions where impaired neuronal function appears initially in Alzheimer’s disease [36]. In short, Pin1 / mice provide a genetic deletion model as an alternative to overexpression models for future studies of NFTs formation or tauopathy development. To date, this is the only available knockout model for AD related neurodegenerative diseases, which would probably lead to the discovery of one of the intrinsic mechanisms protecting against neurofibrillary degeneration.

7. Molecular mechanisms for Pin1 functions Pin1 deletion in mice results in progressive neurodegeneration resembling Alzheimer’s disease and related neurodegenerative diseases, suggesting Pin1 plays an important role in preventing neurons from undergoing age-dependent neurodegeneration [36]. How Pin1 prevents

Fig. 3. Pin1 roles in restoring phosphorylated Tau function. Pin1 binds to and isomerizes phosphorylated tau, resulting in conformational changes in phosphorylated Tau, which can have two consequences with the same end result. First, these conformational changes can directly restore the ability of phosphorylated tau to bind microtubules and promote their assembly. Second, these conformational changes predispose pTau to dephosphorylation mediated by phosphatases targeted specifically to the trans conformation of pSer/Thr–Pro motifs.

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hydrophobic surfaces encompassing both the WW and PPIase domains connecting the two substrate binding clefts. This might also be important for in vivo substrate binding. It is plausible that Pin1 targeted to pThr231 of pTau via its WW domain allows PPIase to be in close proximity and engage other pSer/Thr–Pro motifs. This could thereby bend pTau into certain conformations that function in microtubule networks. The similarities in the spacing of phosphorylation sites Thr47–Thr68 in Cdc25C and the spacing of Thr212– Thr231 of Tau might indicate the relevant structural factor. Visualizing protein conformational changes induced by pSer/Thr–Pro isomerization may be possible with the recent development of fluorescence resonance energy transfer (FRET) technology. A donor/receptor pair of fluorescent proteins that are fused to different region of Tau may be brought to close proximity within nanometer distances during conformational change. If this assay is successfully set up, Pin1 catalytic effect on Tau conformation could be tested. In addition, Pin1 also facilitates the dephosphorylation of Tau by PP2A (Fig. 3). Pin1 binds to the pThr231 site of Tau and allows the pThr–Pro bond to be isomerized. Meanwhile, Pin1 deletion in the mouse brain leads to hyperphosphorylation of Tau on many sites. However, whether Pin1 binds to pThr231 as a docking mechanism that leads to isomerization of a single site or of multiple pSer/Thr–Pro sites in Tau is still unknown. The NMR structure solved in solution revealed that the flexible linker region connecting the WW domain and the PPIase domain allows both open and closed forms of Pin1 [127]. This flexibility may let the PPIase gain accessibility to other pSer/Thr–Pro sites while the WW domain binds to pThr231. Even if Pin1 is only isomerizing the pThr231– Pro bond, there is probably hierarchical phosphorylation and dephosphorylation occurring in vivo that allows Pin1 to play a determinative role in regulating Tau phosphorylation level. It is possible that the conformational change caused by Pin1 or the dephosphorylation of the pThr231 site facilitated by Pin1 predisposes Tau to dephosphorylation globally. Importantly, it is interesting to ask whether Pin1 is playing a role in preventing the pTau from being over-phosphorylated or if Pin1 could reverse the hyperphosphorylated forms back to the nonpathologic version. If Pin1 overexpression in mice shows an opposite phenotype to Pin1 null mice by rescuing Tauopathies induced by overexpression of tau, tau mutant or tau kinases, the role of Pin1 in rescuing neurons from neurodegeneration would be established. If Pin1 serves a preventive role, but not a restorative role, Pin1 would not be able to function in facilitating the dephosphorylation of hyperphosphorylated Tau prior to exceeding Pin1 expression. The outcome of these experiments would provide valuable preventive or therapeutic insights for neurofibrillary degeneration. Microtubule assembly and vesicle transport through a long axon on microtubule networks are dynamic cellular

events that require flexible/reversible molecular mechanisms; phosphorylation and post-phosphorylation peptidyl– prolyl isomerization may serve this purpose. As an important regulator of neuronal microtubules, we would imagine that the Tau phosphorylation and dephosphorylation cycle is constantly modulated to adapt to ever changing neuronal signals or environments. Similarly, for post-phosphorylation peptidyl–prolyl isomerization of Tau, its equilibrium could be finely adjusted to achieve a suitable rate of microtubule polymerization or vesicle transport of neurotransmitters to be secreted. In turn, through its modulation of Tau function, an optimum level of active Pin1 may be important for preventing Tau pathological development. For instance, the small Gproteins, Rac and Cdc42, cause defective axon outgrowth in both dominant negative and constitutively active versions [128–130]. We have presented that Pin1 deletion leads to Tauopathy phenotypes, while overexpression of Pin1 in transgenic mice may also disturb the equilibrium of Tau phosphorylation instead of protecting neurons from degeneration. This would be an important question to address before a therapeutic strategy is determined. Pin1 acts on many levels of a signaling pathway to achieve a single goal, as has been observed in the case of regulating cyclin D1 protein levels [33,89]. Therefore, it is also plausible that Pin1 targets other upstream regulating factors of Tau phosphorylation. Identification of Pin1 neuronal substrates other than Tau would provide a better understanding of how Pin1 plays a role in preventing Tauopathy phenotypes.

8. Conclusion Hyperphosphorylation of Tau is involved in the pathogenesis of many neurodegenerative diseases. We have reviewed our results concerning Pin1-mediated postphosphorylation protein modulation of Tau. Pin1 binds specifically to phosphorylated Tau via its pThr231 site. Biochemical analysis revealed that the binding causes a cis/trans isomerization of pSer/Thr–Pro motif(s), which leads to conformational changes and predisposition for dephosphorylation of Tau. An inverse correlation observed between Pin1 levels and susceptibility to neurofibrillary degeneration in human brain suggests a potential protective role for Pin1 against phosphorylated Tau aggregation. Pin1 null mice progressively develop phenotypes resembling tauopathies, supporting Pin1 as one of the endogenous molecules preventing neurofibrillary degeneration. In conclusion, a post-phosphorylation modulation of microtubule regulating Tau induced by Pin1, the PPIase specific for pSer/Thr–Pro motifs, plays a pivotal role in protecting aged neurons from neurofibrillary degeneration in the brain. This concept may be beneficial to medical strategies preventing neurodegenerative diseases.

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Acknowledgments We are grateful to Ben Neel, Lew Cantley and Tony Hunter for constructive discussions, to the members of the Lu laboratory for stimulating discussions, especially Lucia Pastorino, Greg Finn and Christina Y. Soohoo for editing the manuscript. J.L. is a Human Frontier Research Program Fellow and K.P.L. is a Pew Scholar and a Leukemia and Lymphoma Society Scholar. Work in the author’s laboratory has been supported by NIH grants CA100301, to X. Z. Z., and GM56230, GM58556, AG17870 and AG22082, to K.P.L.

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