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Originally published In Press as doi:10.1074/jbc.M601849200 on May 3, 2006

J. Biol. Chem., Vol. 281, Issue 28, 19296-19304, July 14, 2006
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The Peptidylprolyl cis/trans-Isomerase Pin1 Modulates Stress-induced Dephosphorylation of Tau in Neurons

IMPLICATION IN A PATHOLOGICAL MECHANISM RELATED TO ALZHEIMER DISEASE*

Marie-Christine Galas, Pierre Dourlen, Séverine Bégard, Kunié Ando, David Blum, Malika Hamdane, and Luc Buée1

From the INSERM U815, Institut de Médecine Prédictive et Recherche Thérapeutique, 59045 Lille Cedex and Université Lille 2, School of Medicine, Place de Verdun, 59000 Lille, France

Received for publication, February 27, 2006 , and in revised form, May 2, 2006.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Deregulation of Tau phosphorylation is a key question in Alzheimer disease pathogenesis. Recently, Pin1, a peptidylprolyl cis/trans-isomerase, was proposed to be a new modulator in Tau phosphorylation in Alzheimer disease. In vitro, Pin1 was reported to present a high affinity for both Thr(P)-231, a crucial site for microtubule binding, and Thr(P)-212. In fact, Pin1 may facilitate Thr(P)-231 dephosphorylation by protein phosphatase 2A through trans isomerization of the Thr(P)-Pro peptide bound. However, whether Pin1 binding to Tau leads to isomerization of a single site or of multiple Ser/Thr(P)-Pro sites in vivo is still unknown. In the present study, Pin1 involvement was investigated in stress-induced Tau dephosphorylation with protein phosphatase 2A activation. Both oxidative (H2O2) and heat stresses induced hypophosphorylation of a large set of phospho-Tau epitopes in primary cortical cultures. In both cases, juglone, a Pin1 pharmacological inhibitor, partially prevented dephosphorylation of Tau at Thr-231 among a set of phosphoepitopes tested. Moreover, Pin1 is physiologically found in neurons and partially co-localized with Tau. Furthermore, in Pin1-deficient neuronal primary cultures, H2O2 stress-induced Tau dephosphorylation at Thr(P)-231 was significantly lower than in wild type neurons. Finally, Pin1 transfection in Pin1-deficient neuronal cell cultures allowed for rescuing the effect of H2O2 stress-induced Tau dephosphorylation, whereas a Pin1 catalytic mutant did not. This is the first demonstration of an in situ Pin1 involvement in a differential Tau dephosphorylation on the full-length multiphosphorylated substrate.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
In Alzheimer disease (AD)2 brain, Tau is the main component of the paired helical filaments that compose neurofibrillary tangles, a hallmark of the disease. The phosphoprotein Tau is a key regulator of microtubule dynamics. The functions of Tau are regulated by site-specific phosphorylation events (1). Among the numerous phosphorylation sites in Tau, Thr-231 plays a critical regulatory role because its phosphorylation greatly diminishes the ability of Tau to bind and stabilize microtubules in the cell (2-4). In neurofibrillary tangles, Tau is hyperphosphorylated on many serine or threonine residues preceding proline (Ser/Thr(P)-Pro). Among them, Thr-212 and Thr-231 are Tau conformation-dependent epitopes clearly identified as abnormally phosphorylated in AD (5, 6).

A complex set of modulators tightly regulates the dynamic phosphorylation state of Tau. The phosphorylation state of a protein reflects the balance between activation and inhibition of kinases, phosphatases, and partners able to support their activity through conformational changes. A deregulation of these mechanisms is a central point in the etiopathology of AD and other neurodegenerative pathologies (for review Ref. 1). Thus, characterization of proteins that modulate Tau phosphorylation-dephosphorylation processes is essential for the understanding of the pathological events. The peptidylprolyl isomerase Pin1 catalyzes the cis/trans isomerization in a subset of proteins on Ser(P)/Thr(P) residues preceding proline. Although Pin1 has been widely studied in the cell cycle and cancer, little is known about its role in the brain. Recent data suggested that Pin1 might be involved in AD pathogenesis, because it is found in neurofibrillary tangles (7). In vitro studies showed that Pin1 was able to bind Tau through Thr(P)-231 (7). Nevertheless, further studies on Pin1 knock-out mice suggested that there was an inverse correlation between Pin1 expression and neurofibrillary degeneration in AD (8). Recently, NMR studies reported a high affinity for both Thr-231 and Thr-212 of Tau on Pin1 (9, 10). Deregulation of Pin1 activity, expression or localization might induce an imbalance in the phosphorylation-dephosphorylation state at specific epitopes inducing a disturbance in the interactions between Tau and its target proteins. In fact, Pin1 may facilitate Tau dephosphorylation by protein phosphatase 2A (PP2A) (11). However, the mechanisms underlying the functional involvement of Pin1 in Tau deregulation are still unknown.

Oxidative stress, an early event in Alzheimer disease occurring prior to cytopathology, induces rapid hypophosphorylation of Tau epitopes (12). This Tau dephosphorylation is likely to be mediated through PP2A activation (13). Such PP2A activation leading to Tau dephosphorylation is also observed in other stress conditions such as heat shock (13, 14). In the present study, the potential involvement of Pin1 in Tau dephosphorylation was assessed in primary neuronal cell cultures using both oxidative and heat stresses and a pharmacological Pin1 inhibitor, juglone (15). The role of Pin1 was further investigated in Pin1-deficient cultures obtained from Pin1 knock-out mice embryos. In these studies, we demonstrated that Pin1 plays an active role in stress-induced Tau dephosphorylation, preferentially at Thr-231 epitope. This is the first demonstration of an in situ Pin1 involvement in a differential Tau dephosphorylation.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Primary Embryonic Neuronal Culture—Rat primary cortical neurons were prepared from 17-18-day-old Wistar rat embryos as follows. Briefly, brain and meninges were removed. Cortex was carefully dissected out and mechanically dissociated in culture medium by triturating with a polished Pasteur pipette. Once dissociated and after blue trypan counting, cells were plated at a density of 2.5 x 104 cells/cm2 in poly-D-lysine and laminin-coated 24-well plates or Labtech (Becton Dickinson). For dissociation, plating, and maintenance, we used Neurobasal medium supplemented with 2% B27 containing 200 mM glutamine and 1% antibiotic-antimycotic agent (Invitrogen). Medium was replenished every 3 days and 24 h before cell treatment.

Pin1 Knock-out Primary Embryonic Neuronal Culture—For knock-out studies, primary cortical neurons were prepared from 15-17-day-old mice embryos. Pin1-deficient neurons were obtained from embryos derived from heterozygous pairings. Homozygous pairings are not possible because homozygous male are sterile. Each embryo was dissected and plated individually. In each experiment, neurons acquired from wild type littermate embryos were used as controls. The genotype of each embryo was determined using the remaining tissue and primers as described in Fujimori et al. (16).

Pin1 cDNAs—Pin1 cDNA was obtained from Prof. P. Davies (AECOM, Bronx, NY). It was subcloned in a pcDNA4 plasmid (Invitrogen). Site-directed mutagenesis was made by PCR using the QuikChange system from Stratagene. Ser-115, a residue located close to the juglone targeted Cys-113, was mutated into glutamate. It is a consensus residue among peptidylprolyl isomerases and breaks one of the helixes of the catalytic pocket (17). Pin1 S115E sequence primers were sense, 5'-CAGCGACTGCAGCGAAGCCAAGGCCAGG-3' and anti-sense, 5'-CCTGGCCTTGGCTTCGCTGCAGTCGCT-3'. These three pcDNA4 plasmids (empty, Pin1, and [S115E]Pin1) were used for further transfections.

Transfection of Primary Embryonic Culture—Primary cortical cultures were transfected 7 days after plating with Pin1 cDNA plasmids using a modified Ca2+-phosphate transfection method (18). Briefly, culture medium was replaced by prewarmed Dulbecco's modified Eagle's medium and preserved. Both the original culture medium and cells were returned to a 5% CO2 incubator. In the meantime, the DNA/Ca2+-phosphate precipitate was prepared using ProFection Mammalian Transfection System (Promega). Per well (24-well plate), 3 µg of cDNA and 3.1 µl of 2 M CaCl2 adjusted with H2O to a total volume of 25 µl (solution A) were mixed to 25 µl of 2 x Hanks' balanced salt (solution B). Solution A was added by ~1/8 volume to solution B while gently mixing (600 rpm). The mixed solution was kept at -20 °C for 5 min to prime the precipitate and then at room temperature for 15 min. The DNA/Ca2+-phosphate suspension solution was added dropwise to each well. Cells were incubated in presence of the precipitate for 30 min in a 5% CO2 culture incubator at 37 °C. After incubation, transfection medium was replaced by 10% CO2-equilibrated Dulbecco's modified Eagle's medium and incubated for 20 min in a 5% CO2 incubator. Dulbecco's modified Eagle's medium was replaced by the original culture medium, and cells were returned to 5% CO2 incubator.

Cell Treatment—At 10 days in vitro, cells were preincubated for 2 h with 1 µM juglone and/or treated with 1 mM H2O2 for the indicated time, or exposed to 44 °C in a 5% CO2 incubator for 30 min. Thirty percent stock hydrogen peroxide (Prolabo) was dissolved in 0.2 M phosphate-buffered saline (pH 7.4). Thirty-one µM stock okadaic acid (Sigma) was dissolved in ethanol.

COS Cell Culture—COS cells were grown as described previously (19). Transient transfection was performed using ExGen (Euromedex, France), and cell lysates were analyzed after 24 h (2, 19, 20).

Antibodies—AD2 is directed against phosphorylated Ser-396 and Ser-404, Tau1 recognizes Tau only when serines 195, 198, 199, and 202 are not phosphorylated, AT180 (Pierce Perbio) recognizes phosphorylated Thr-231, and anti-Tau (pT212) (BIOSOURCE) recognizes Thr(P)-212. Phosphorylation-independent antibodies M19G and anti-Tau-carboxyl-terminal (Tau-C-ter) are well characterized antisera, directed against the first 19 amino acids and the last 15 amino acids of Tau sequence, respectively (1, 2, 19, 20).

The anti-beta-actin antibody was obtained from Sigma, anti-synaptophysin and anti-Pin1 were from Santa-Cruz (H-123), anti-neuronal nuclei was from Chemicon (MAB377), anti-cyclin D was from Upstate%20Biotechnology">Upstate Biotechnology, and anti-phospho-Pin1 (Ser-16) was from Cell Signaling.

Electrophoresis and Immunoblotting—Equal volumes of cells harvested in Nu-Page sample buffer (Invitrogen) were denaturated at 100 °C during 5 min, loaded on 4-12% NuPAGE Novex gels (Invitrogen), and transferred to nitrocellulose. Membranes were blocked in Tris-buffered saline, pH 8, 0.05% Tween 20 with 5% skim milk or bovine serum albumin, and incubated with primary antibody. Membranes were incubated with horse-radish peroxidase-labeled secondary antibody (goat anti-rabbit or anti-mouse IgGs, Sigma), and bands were visualized by chemiluminescence (ECL, Amersham Biosciences). Western blots were quantified by densitometry as described previously. Phosphorylated and total Tau (detected by M19G antibody) levels were normalized to beta-actin (used as an internal loading control) for each blot (2). We previously verified that actin was not cleaved under the different treatments. Phosphorylated Tau levels were further normalized to total Tau to measure the exact phosphorylation state.

Immunocytochemistry—Cell cultures were fixed in cold 2% paraformaldehyde, 0.05% glutaraldehyde, 1 mM MgCl2, 1 mM EGTA, and 30% glycerol in 70 mM PIPES, pH 6.8, for1hat room temperature. Permeabilization was carried out in 0.2% Triton X-100 in phosphate-buffered saline for 2 min. Cells were treated with NaBH4 (10 mg/ml) for 7 min. After a 30-min saturation in 4% bovine serum albumin, double immunostainings were carried out using anti-Pin1 and AT180 antibodies. Pin1 staining was revealed with a goat anti-rabbit IgG (H+L) antibody coupled to Alexa Fluor®488 and AT180 with a goat anti-mouse IgG (H+L) antibody coupled to Alexa Fluor®568 (Molecular Probes). Slides were analyzed by a Leica TCS NT confocal laser scanning microscope.


Figure 1
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  		FIGURE 1.
Effect of juglone on H2O2-induced dephosphorylation of Tau. A, Western blots analysis using phospho-dependent (AT180, anti-pTau212, AD2, Tau1) and phospho-independent (M19G, anti-TauCter) Tau antibodies. Cortical cultures were treated with 1 mM H2O2 for 1 h in the absence or presence of 1 µM juglone as described under "Experimental Procedures." For all Tau phosphorylation epitopes examined, H2O2 rapidly decreased phosphorylated Tau. Juglone pretreatment prevented H2O2-induced Tau dephosphorylation only at the epitope Thr-231 (AT180). Arrow indicates the 50-kDa molecular mass marker. B, densitometric analysis of Western blots using anti-total Tau and anti-phospho-Tau. Results are expressed as percentage of control. Data shown are the mean ± S.E. of three different experiments. *, p < 0.001 versus H2O2-treated cells.

 


Figure 2
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  		FIGURE 2.
Effect of juglone on heat shock-induced dephosphorylation of Tau. A, Western blots analysis using phospho-dependent (AT180, anti-pTau212, AD2, Tau1) and phospho-independent (M19G, anti-TauCter) Tau antibodies. Cortical cultures were heated to 44 °C for 30 min in absence or presence of 1 µM juglone as described under "Experimental Procedures." Heat shock-induced dephosphorylation for all Tau epitopes was examined. Juglone pretreatment prevented heat shock-induced Tau dephosphorylation only at the epitope Thr231 (AT180). B, densitometric analysis of Western blots using anti-total Tau and anti-phospho-Tau. Results are expressed as percentage of control. Data shown are the mean ± S.E. of three different experiments. *, p < 0.001 versus heat shock-treated cells.

 
Analysis and Statistics—Results were expressed as means ± S.E. of at least three independent experiments. Statistics were performed by one-way analysis of variance followed by post-hoc Newman-Keuls.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Juglone Partially Reverses Oxidative Stress-induced Dephosphorylation of Thr(P)-231 Tau—As Tau is extensively phosphorylated in fetal neurons, primary neuronal cultures provide an appropriate model to study mechanisms regulating Tau dephosphorylation. H2O2 is a highly toxic product of oxidative stress mainly because of its free diffusion through the cells. Oxidative stress induced by hydrogen peroxide has been previously reported to induce hypophosphorylation of Tau through PP2A (12, 13, 21). Cortical cultures were exposed to 1 mM H2O2 for 1 h. Western blots (Fig. 1A) revealed with the anti-phosphorylated Tau antibodies AT180, anti-Tau (pT212), and AD2 showed that H2O2 induced a decrease in phosphorylated Tau at threonine 231, threonine 212, and serines 396 and 404 (using the numbering convention of the longest human isoform of Tau), respectively. H2O2-induced dephosphorylation was confirmed by immunoblotting with the Tau1 antibody, which recognizes Tau only when it is not phosphorylated on serine residues 195, 198, 199, and 202. Dephosphorylated Tau detected by Tau1 increased after treatment with H2O2. Furthermore, H2O2 cell exposure induced a change of electrophoretic mobility and a decrease of total Tau detected by M19G antibody (20 ± 7%) and anti-Tau-C-ter (15 ± 3%), reflecting a global dephosphorylation and a partial degradation of Tau.

To assess the involvement of Pin1 in stress-induced dephosphorylation of Tau, juglone was used to inhibit its activity (15, 22). Neuronal cultures were preincubated for 2 h with 1 µM juglone prior H2O2 addition (Fig. 1A). We checked that 1 µM juglone did not alter either cell viability as assessed by MTS assay or the basal phosphorylation state of the various Tau epitopes tested (not shown).

Juglone pretreatment before H2O2 addition did not modify the levels of phosphorylated Tau epitopes Thr(P)-212-Tau, AD2, and Tau1. Conversely, inhibition of Pin1 by juglone partially prevented dephosphorylation of Tau at threonine 231 (27 ± 6%) as seen on Western blot revealed with the phospho-dependent Tau antibody AT180. Furthermore, as we reported above, H2O2 addition induced a partial decrease in total Tau as previously described (23, 24). Under the same treatments, no significant changes were observed by immunoblotting for the neuron-specific marker NeuN and for the synaptic vesicle component synaptophysin indicating that there is no loss of cellular integrity (data not shown). These data suggest that Pin1 participates to H2O2-induced Tau dephosphorylation at threonine 231 under conditions that dephosphorylate a large set of phospho-Tau epitopes.


Figure 3
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  		FIGURE 3.
Pin1 phosphorylation at serine 16. A, Western blots using anti-total Pin1 and anti-Ser(P)-16-Pin1. Cortical cultures were treated with 1 mM H2O2 for 1 h. A low basal level of Ser(P)-16-Pin1 was observed. H2O2 treatment did not modify total Pin1 level but induced a decrease of phosphorylation on serine 16. 50 nM okadaic acid was used to increase basal phosphorylation. B, densitometric analysis of Western blots using anti-total Pin1 and anti-Ser(P)-16-Pin1. Results are expressed as percentage of control. Data shown are the mean ± S.E. of three different experiments. *, p < 0.001 versus control cells.

 


Figure 4
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  		FIGURE 4.
Detection of Pin1 in neuronal cultures from wild type and Pin1-deficient embryos. A, representative gel analysis of the PCR product of the two genotypes of embryos. B, Pin1 was detected by Western blots in cultures from wild type or Pin1-deficient embryos.

 
To extend the analysis of Pin1 involvement in stress mechanisms with PP2A activation, the juglone effect was tested on heat shock-induced Tau dephosphorylation. Previous studies described how heat shock caused rapid dephosphorylation of Tau in rats (14) and in cultured oligodendrocytes (13). In cortical cultures, heat shock (30 min at 44 °C) partially dephosphorylated Tau at serines 195, 198, 199, 202, 396, 404, and threonine 212 and 231. Juglone, like in H2O2 treatment, was only able to partially and selectively prevent heat shock-induced Tau dephosphorylation at Thr-231 (Fig. 2). These results suggest that heat shock-induced Tau dephosphorylation involves Pin1 at threonine 231. Thus, Pin1 participation to Tau dephosphorylation may be a common mechanism to various stresses.

Modulation of Pin1 Activation State through Its Phosphorylation on Serine 16—Because the direct rotamase activity of Pin1 could only be visualized directly by NMR spectroscopy, we first analyzed phosphorylation on serine 16, which is located inside the WW domain, and recently reported to inhibit Pin1 binding to its substrates (25). Ser(P)-16 was analyzed in basal conditions and under H2O2 and/or okadaic acid treatment (at a 50 nM concentration specifically inhibiting PP2A) (Fig. 3).

Total Pin1 levels were not modified under H2O2 and/or okadaic acid treatments. Phosphoserine-16-Pin1 antibody only slightly detected Pin1 protein in control conditions indicating that Pin1 could bind to its substrates, in our culture conditions. H2O2 further decreased Pin1 Ser-16 phosphorylation, suggesting that Pin1 has a better ability to bind its substrates in stress-induced conditions. Conversely, the phosphatase inhibitor okadaic acid induced a strong phosphorylation of Pin1 suggesting that dephosphorylation of phosphoserine 16 would be mediated, at least partly, by PP2A (Fig. 3).

Oxidative Stress-induced Dephosphorylation of Tau in Pin1-deficient Cultures—To challenge our results obtained with juglone inhibition, we therefore tested whether Pin1 deficiency would modify the response to the most efficient stress-induced Tau dephosphorylation, H2O2. We compared the effect of H2O2 on AT180, Thr(P)-212-Tau, AD2, Tau1, M19G, and anti-Tau-C-ter epitopes in Pin1 and Pin1-deficient cortical neuronal cultures. We first verified by genotyping and immunoblotting the absence of Pin1 in cells cultured from knock-out Pin1 embryos (Fig. 4). Regarding the effects of H2O2 on Tau dephosphorylation, data were similar in rat and mice primary neuronal cell cultures (Fig. 1 versus Fig. 5, panels wt). H2O2-induced dephosphorylation of Tau revealed with anti-Thr(P)-212-Tau, AD2, Tau 1, M19G, and anti-Tau-C-ter remained unchanged in Pin1-deficient neuronal cultures as compared with wild type cultures. Interestingly, significant protection to H2O2 treatment in Pin1-deficient neuronal cultures was observed only using the anti-phosphorylated Tau antibody AT180 (Fig. 5). This provides further evidence that Pin1 is specifically involved in Thr(P)-231 Tau dephosphorylation.


Figure 5
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  		FIGURE 5.
Pin1 deficiency prevents H2O2-induced dephosphorylation of Tau at Thr-231 site. Western blots analysis using phospho-dependent (A, AT180; B, anti-pTau212; C, AD2; D, Tau1) and phospho-independent (E, M19G; F, anti-TauCter) Tau antibodies. Cortical cultures were prepared from individual wild type (WT)or Pin1-deficient embryos and treated with 1 mM H2O2. Tau dephosphorylation was significantly reduced at epitope Thr-231 (A, AT180) in H2O2-treated Pin1-deficient cultures compared with wild type. At the opposite, no difference was observed for the other phospho-Tau epitopes tested and for total Tau. The densitometric analysis was represented for each epitope tested. Each data point represents combined values from four separate embryos. *, p < 0.01 versus H2O2-treated wild type cells. Arrows indicate the 50-kDa molecular mass marker.

 
Thr(P)-231 Tau Distribution in Wild Type or Pin1-deficient Cortical Neurons—Confocal microscopy using anti-Pin1 antibody revealed that Pin1 localizes both to the nucleus and the cellular body in cortical neurons (Fig. 6). A strong presence of Pin1 was also observed in neurites and varicosities. On parallel, AT180 mainly stained axons and cellular body. A double staining with both antibodies (merge) revealed partial colocalization between anti-Pin1 and AT180 antibodies to cellular bodies and neurites. These data correlate a possible Pin1 and Thr(P)-231 Tau colocalization, facilitating the access of Pin1 to Tau and specific isomerization of the Thr(P)-231-proline peptide bond under dephosphorylation conditions. In Pin1-deficient neurons, a similar AT180 immunoreactivity was found (Fig. 6, bottom panel).

Rescue of H2O2-induced Dephosphorylation of Tau in Pin1-deficient Neurons—To learn the precise role of the Pin1 prolyl isomerase activity in H2O2-induced Tau dephosphorylation, Pin1-deficient cultures were transfected at 7 days in vitro with cDNA coding for wild type Pin1 or a catalytic mutant where Ser-115 was replaced by a glutamic acid, S115E. Three days after transfection, we observed by Western blotting a strong expression of both wild type and Glu-115-mutated Pin1 as detected with anti-Pin1 antibody (Fig. 7A). These results proved the high efficiency of transfection of the modified Ca2+-phosphate method used. Thus, the effect of wild type Pin1 and Glu-115 mutant overexpression on H2O2-induced dephosphorylation of Tau in Pin1-deficient neurons was tested by immunoblotting.

First, to test the efficiency of wild type Pin1 and catalytic mutant Glu-115 activities, the effect of their overexpression on cyclin D levels were analyzed in COS cells and Pin1-deficient cortical cultures (Fig. 7A). To assess the efficiency of both constructs, the amounts of cyclin D, which was shown as stabilized at both mRNA and protein levels by Pin1, was quantified by immunoblotting (26-28). A cyclin D increase was observed in both cultures overexpressing Pin1. Conversely, such cyclin D1 increase was not visualized with the Glu-115 mutant overexpression (Fig. 7B).

Three days after transfection, Pin1-deficient cultures were treated by H2O2 for 30 min to mildly dephosphorylate Tau. We compared H2O2 treatment effect on AT180, Thr(P)-212-Tau, AD2, Tau1, and M19G epitopes in Pin1-deficient cortical neuronal culture transfected with no coding cDNA, wild type, or Glu-115 mutated-Pin1 (Fig. 8A). No effect of wild type or Glu-115-mutated Pin1 overexpression was observed for the phosphorylated Tau epitopes Thr(P)-212-Tau, AD2, and Tau1. However, wild type Pin1 overexpression increased Tau dephosphorylation only using the anti-phosphorylated Tau antibody AT180. The catalytic Glu-115 mutant was not as effective because its overexpression partially prevented the increased dephosphorylation of Tau (Fig. 8B).

H2O2-induced dephosphorylation of Tau at the Thr-231 site in transfected Pin1-deficient cultures was illustrated by immunofluorescence studies and analyzed by confocal microscopy (Fig. 9). Overexpression of either wild type Pin1 or the Glu-115 mutant induced a strong staining detected by anti-Pin1 antibody in transfected cells. In cells exhibiting a clear neuronal morphology, an AT180 immunoreactivity was observed. Then, we looked for cells with similar morphology that exhibited Pin1 immunoreactivity. A weak AT180 staining was found in cells transfected with Pin1, whereas AT180 immunoreactivity in cells transfected with the Pin1 mutant Glu-115 was similar to the one observed in non-transfected cells. Altogether, these observations further strengthen the role of the catalytic domain of Pin1 prolyl isomerase in the H2O2-induced dephosphorylation of Tau at Thr-231 site.


Figure 6
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  		FIGURE 6.
Thr(P)-231-Tau localization in wild type and Pin1-deficient cortical cultures. Double immunostaining of wild type (WT) and Pin1-deficient cortical cultures for Thr(P)-231-Tau (AT180) and total Pin1. Thr(P)-231 was localized to cell bodies and axons. Pin1 was found strongly in nucleus but also in cellular bodies and neurites. Absence of Pin1 did not alter Thr(P)-231-Tau localization in Pin1-deficient cells. KO, knock-out.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
Tau phosphorylation is likely to be a main actor in neurofibrillary degeneration. However, the deregulation of Tau phosphorylation is still a key question in AD pathogenesis. Protein phosphorylation is regulated through a balance of kinases and phosphatases. Recently, glycogen synthase kinase-3beta (GSK3beta) inhibitors were shown to slow down neurofibrillary degeneration in a Tau transgenic model, suggesting that GSK3beta is a main kinase involved in Tau phosphorylation (29). Conversely, recent data also indicated that the lack of phosphatase activity in AD brains might lead to Tau hyperphosphorylation (30). Finally, a new regulator of phosphorylation was also involved, the peptidylprolyl cis/trans-isomerase Pin1. Many phosphorylation sites defined as pathological are phospho-Ser/Thr followed by Pro. The conformation of this peptide bond is crucial for the action of phosphatases. PP2A is only able to dephosphorylate Ser(P)/Thr(P)-Pro when it is in a trans conformation but not in a cis conformation (11). The interest in Pin1 as a key actor in neurofibrillary degeneration is supported by the fact that Pin1 knock-out mice develop neurofibrillary tangles (7, 8).

Pin1 was reported to present a high affinity for both the Thr(P)-231 and Thr(P)-212 of Tau protein (7, 9, 10). However, whether binding of Pin1 to Tau leads to isomerization of a single site or of multiple Ser(P)/Thr(P)-Pro sites in vivo is still unknown (31). Moreover, the enzyme activity of Pin1 is dependent on the level of phosphorylation of its substrate. For instance, Tau peptide with a unique phosphorylation site (Thr(P)-212) is a better substrate than multiphosphorylated peptides. Indeed, the WW domain of Pin1 may act as a negative regulator of the cis/trans isomerization activity when Pin1 substrates are multiphosphorylated (31). Thus, it is necessary to determine the effect of Pin1 in vivo on phosphorylated full-length Tau protein. Six Ser/Thr-Pro residues (Ser-199, Ser-202, Thr-212, Thr-231, Ser-396, and Ser-404) dephosphorylated by PP2A were studied to cover the entire Tau molecule (32).


Figure 7
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  		FIGURE 7.
Characterization of Pin1 and Glu-115 (E115) mutant overexpression in COS cells and Pin1-deficient cultures. A, Western blots analysis using anti-total Pin1 and anti-cyclin D antibodies of COS cells and Pin1-deficient cortical cultures transfected with cDNA coding for Pin1 or the catalytic Pin1 mutant Glu-115. Pin1 and Glu-115 mutant were strongly expressed in both cell types. Pin1 overexpression induced a cyclin D increase in COS cells and Pin1-deficient cultures. Overexpression of Glu-115 mutant fully prevented cyclin D increase in COS cells and partially in Pin1-deficient cultures. KO, knock-out. B, densitometric analysis of Western blots using anti-total Pin1 and anti-cyclin D antibodies. Results are expressed as percentage of control. Data shown are the mean ± S.E. of three different experiments. *, p < 0.01 Pin1 versus control cells; **, p < 0.05 Glu-115 versus Pin1; ***, p = 0.001 Pin1 versus control cells.

 
Finally, further demonstration is needed to highlight Pin1 and Tau relationships in dephosphorylation mechanisms. Two stresses leading to PP2A activation were used to assess the involvement of Pin1 in Tau dephosphorylation.


Figure 8
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  		FIGURE 8.
Pin1 overexpression increases H2O2-induced dephosphorylation of Tau at the Thr-231 site in transfected Pin1-deficient cultures. A, Western blots analysis using phospho-dependent (AT180, anti-pTau212, AD2, Tau1) and phospho-independent (M19G, anti-TauCter) Tau antibodies of Pin1-deficient cortical cultures transfected with cDNA coding for Pin1 or the catalytic Pin1 mutant Glu-115 (E115). Cortical cultures were treated with 1 mM H2O2 for 30 min at 37 °C. Pin1 overexpression increased H2O2-induced Tau dephosphorylation only at epitope Thr-231 (AT180). Mutation of the catalytic site partially prevented Pin1 effect on H2O2-induced Thr(P)-231 dephosphorylation. B, densitometric analysis of Western blots using anti-total Tau and anti-phospho-Tau. Results are expressed as percentage of control. Data shown are the mean ± S.E. of three different experiments. *, p < 0.001 H2O2-treated Pin1 overexpressing cells versus H2O2-treated control cells; **, p = 0.001 H2O2-treated Glu-115 versus H2O2-treated Pin1 overexpressing cells.

 


Figure 9
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  		FIGURE 9.
Modulation of Thr(P)-231 in transfected Pin1-deficient neurons. Double immunostaining of Pin1-deficient neurons overexpressing Pin1 or the catalytic mutant Glu-115 (E115) for Thr(P)-231 Tau (AT180) and total Pin1. Pin1 but not Glu-115 overexpression strongly decreases Thr(P)-231 staining. -, non-transfected Pin1-deficient neuronal cell culture.

 
First, the present data indicate that Pin1 preferentially modulates stress-induced dephosphorylation of Tau on Thr-231. Juglone, a pharmacological Pin1 inhibitor was used to inactivate rotamase activity. Pin1 inhibition led to a decreased effect of both oxidative and heat stresses induced dephosphorylation of Thr(P)-231 Tau without affecting dephosphorylation of other sites. Nevertheless, juglone inhibits numerous parvulins as well as other enzymes comprising Cys residues in their catalytic domains (15, 22, 33-35). Other pharmacological Pin1 inhibitors are available (36-39), but Pin1-deficient cells are likely to be the most useful tools. We have set up an original approach by developing primary neuronal cultures from Pin1 knock-out mice.

Thus, the pharmacological effect of juglone observed in Tau dephosphorylation experiments in wild type neurons was then compared with similar work in Pin1 knock-out neurons. Pin1 deficiency led to a decreased effect of H2O2-induced dephosphorylation of Thr(P)-231 Tau without affecting dephosphorylation of other sites. It further supports that lack of Pin1 activity leads to a deficient Tau dephosphorylation at Thr(P)-231 under stress conditions. Nevertheless, we cannot speak about an exclusive selectivity of Pin1 isomerase activity for Thr(P)-231 because only 6 of the 17 Ser(P)/Thr(P)-Pro sites present on Tau were investigated. However, our results allow for demonstrating that Pin1 does not modulate H2O2-induced dephosphorylation of Thr(P)-212 Tau despite recent NMR results showing an even higher affinity of Pin1 for Thr(P)-212 than for Thr(P)-231 Tau (10). Anyhow, this does not preclude that Pin1 may not isomerize Thr(P)-212 Tau in cells under different dephosphorylating conditions or in specific types of neurons or brain area.

Neither juglone nor Pin1 deficiency completely abolished the H2O2-induced effect, indicating that Thr(P)-231 may be dephosphorylated without Pin1. It is not surprising because only a small percentage of the peptide bound is in a cis conformation. Most of it is already in a trans conformation. This could be because of a direct, although slower, isomerization of Thr(P)-231-Pro peptide bond, or possibly another isomerase enzyme.

Pin1 is constituted of two structural domains, a C-terminal catalytic and an anchoring N-terminal WW domain. Phosphorylation of the WW domain on Ser-16 abolishes its capacity to interact with phosphoepitopes (25). Our results indicate that in control conditions, a majority of Pin1 is in a favorable state to bind to its substrates. Stress further increases Pin1 ability to bind Tau through dephosphorylation of Ser(P)-16 WW domain of Pin1. So, stress-induced dephosphorylation of Ser(P)-16-Pin1 increases its potential to bind Tau and isomerase Thr(P)-231 site. The transfection of either functional Pin1 or a catalytic defective mutant in Pin1-deficient primary neuronal cultures further supports the role of the rotamase activity in the stress-induced Tau dephosphorylation process.

In differentiated cultured neurons, Pin1 was localized not only in the nucleus but also in the cytoplasm and the neurite network (40). Histochemical data previously reported cytoplasmic and nuclear Pin1 in the brain (8). On one hand, the partial co-localization of Pin1 with Thr(P)-231-Tau illustrates their close vicinity in the neuron, making possible a rapid interaction between both proteins. On the other hand, Thr(P)-231 Tau localization is likely to be Pin1-independent, as no change was observed in Pin1-deficient neurons.

As Thr-231 is a crucial site for the microtubule binding activity of Tau, in the brain, reduction or absence of Pin1 isomerase activity would be able to significantly modify the response of Tau to pathological assault. As oxidative stress is involved early in AD, Pin1 alteration might be a very preliminary request to initiate neurofibrillary tangles and, consequently, restoration of Pin1 activity may represent an attractive target for future therapeutics drugs.


   FOOTNOTES
 
* This work was supported by CNRS, Inserm, and grants from GIS Longévité and the European Community (APOPIS, LSHM-CT-2003-503330). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

1 To whom correspondence should be addressed. Tel.: 33-320-622074; Fax: 33-3-20-62-20-79; E-mail: buee{at}lille.inserm.fr.

2 The abbreviations used are: AD, Alzheimer disease; PP2A, protein phosphatase type 2A; Pin1, peptidylprolyl cis/trans-isomerase (EC 5.2.1.8); juglone, 5-hydroxy-1,4-naphthoquinone; PIPES, 1,4-piperazinediethanesulfonic acid. Back


   ACKNOWLEDGMENTS
 
We thank Pr. Uchida for providing Pin1 knock-out mice and Martial Flactif for his technical expertise in confocal microscopy.



   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
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