FKBP52-TAU INTERACTION AS A NOVEL THERAPEUTICAL TARGET FOR TREATING THE NEUROLOGICAL DISORDERS INVOLVING TAU DYSFUNCTION

Abstract

The invention relates generally to neuroprotection and repair in neurological disorders involving Tau dysfunction (including Alzheimer's disease). The invention describes AND INCLUDES a direct interaction between proteins FKBP52 and Tau. More particularly, the invention relates to a method for screening a drug for the prevention and treatment of neurological disorders involving Tau dysfunction comprising the following steps: a) determining the ability of a candidate compound, to modulate the interaction between a Tau polypeptide and a FKBP52 polypeptide and b) selecting positively the candidate compound that modulates said interaction. The present invention finally relates to diagnostic, prognostic, and monitoring assays of neurological disorders involving Tau dysfunction.

Claims

1-10. (canceled)

11. A method of treating mammalian eukaryotic cells comprising the administration to said mammalian eukaryotic cells of an effective amount of a compound which enhances the interaction between pathologic Tau and FKBP52 proteins, so as to prevent the dysfunction of Tau protein.

12. The method according to claim 11, wherein the dysfunction of Tau in protein mammalian eukaryotic cells, is characterized by an aggregation or accumulation of said Tau protein associated with neurological disorders from the group consisting of Alzheimer's disease, frontotemporal dementia, progressive supranuclear palsy, corticobasal degeneration and frontotemporal lobar degeneration.

13. The method according to claim 12, wherein the neurological disorder is Alzheimer's disease.

14. The method according to claim 11, further comprising performing a screening method to select the compound which comprises the steps of: a) determining the ability of a candidate compound to enhance the interaction between Tau and FKBP52 proteins, and b) selecting positively the candidate compound if the candidate compound enhances said interaction.

15. A method of preventing the accumulation or aggregation of Tau protein in mammalian eukaryotic cells comprising the administration to said mammalian eukaryotic cells of an effective amount of a compound which enhances the interaction between pathologic Tau and FKBP52 proteins.

16. A method according to claim 5 further comprising the step of selecting said compound by performing the steps of: a) determining the ability of a candidate compound to enhance the interaction between Tau and FKBP52 proteins, and b) selecting positively the candidate compound if the candidate compound enhances said interaction.

Description

FIGURES

[0153] FIG. 1: FKBP52 in the brain. Twenty g of cytosol proteins from different adult rat brain regions were analyzed by Western blotting using anti-FKBP52 antibody 761. Actin served as the loading control.

[0154] FIGS. 2A-D: Association between FKBP52 and Tau proteins. A) GST Pull-down assay: Immunoblot (IB) for Tau showing the binding of soluble microtubule extract proteins incubated with GST-tagged FKBP52, or GST alone as control. B) Co-immunoprecipitation assay: A soluble microtubule extract was subjected to immunoprecipitation (IP) with immunopurified anti-FKBP52 antibody, or pre-immune serum used as control. The supernatants (S) and precipitates (P) were immunoblotted with anti-Tau antibody (clone DC25). C) The ability of Tau proteins to bind FKBP52 directly was monitored by dot blot assay. Different amounts of recombinant Tau (hT40) were spotted onto nitrocellulose membranes and then assayed for bound FKBP52 (0.5 g) using anti-FKBP52 antibody. 5 g GST spotted onto nitrocellulose membrane were used as the control. D) Quantitation: 100% corresponds to 0.5 g of FKBP52 loading before milk saturation, and 0% corresponds to 0.5 g of FKBP52 loading after milk saturation. The background is defined as the signal when GST was loaded instead of hT40. The level of FKBP52 captured by hT40 was calculated after subtraction of background.

[0155] FIGS. 3A-C: Relevance of Tau phosphorylation for its interaction with FKBP52. A) Recombinant Tau (hT40), P-Tau and HP-Tau were analyzed by SDS-Page. Phosphorylation and hyperphosphorylation of HT40 resulted in a marked reduction in the gel mobility of recombinant Tau as shown on an immunoblot (IB), with anti-Tau antibody (clone DC25). B) Dot blot assay with 2.2 g of HP-Tau (1), P-Tau (2), pure recombinant hT40 (3), to which had been added, just before spotting, the same amount of cytosol as used to generate P-Tau (4) or HP-Tau (5). Dot 6 refers to the GST (5 g) load.

[0156] FIGS. 4A-B: Colocalization of FKBP52 and TAU in primary cortical neurons and PC12 cells. A) Immunofluorescence staining of primary cortical neurons and PC12 cells treated with 50 nM NGF for 5 days. Double staining for Tau and FKBP52 was performed after cytosol extraction to reveal cytoskeletal association. Arrows indicate preferential colocalization of both proteins in the distal part of the nerve cell axon and at the extremity of PC12 cell neurites.

[0157] B) Confocal images of primary cortical neurons. Double staining was performed as in (A). Analysis of 0.5 m slices confirms the preferential colocalization in the distal part of the axon (see arrowheads)

[0158] FIGS. 5A-G: Effect of FKBP52 on tubulin polymerization induced by recombinant Tau isoforms. Tubulin polymerization was performed by switching the samples from 4 C. to 37 C. and the change in turbidity was monitored at 345 nm for 15 min. Tubulin (1 mg/ml) purified from rat brain was incubated in the absence (.diamond-solid.) or presence of 1.7 M (231 g for HT40) different human Tau isoforms without FKBP52 () or with 3.5 M (55 g) FKBP52 (.box-tangle-solidup.) Tau isoforms differ from each other by the number of repeats in the microtubule binding domain and insertions in the N-terminal. The labelling of tau isoforms uses the published nomenclature (28). A: ht40, B: ht39, C: hT37. D: hT34. E: hT24, F: hT23, G: This control experiment was carried out as in (A), except that 3.51 M GST (.box-tangle-solidup.) was used instead of FKBP52.

[0159] FIGS. 6A-F: Effect of FKBP52 overexpression on Tau accumulation and neurite outgrowth A) PC12 cells treated or not with NGF (50 nM) in the absence or presence of 1 g/ml Dox (doxycycline). Ten g of total protein extracts were analyzed for FKBP52 levels by western blot using anti-FKBP52 antibody 761. B) The FKBP52 level was determined as in (A) in H7C2 cells treated or not with Dox. The use of rabbit FKBP52 as the exogenous protein explains the small difference in gel mobility with the endogenous rat protein. C) H7C2 and D) PC12 cells were treated or not with NGF for 5 days, in the presence or absence of Dox for one week; 50 g of extracts was subjected to SDS-PAGE and immunoblotted with anti-Tau (antibody clone DC25). Actin was used as the loading control. E) Representative H7C2 cells in the presence of NGF (50 nM) with or without Dox. F) Neurite length was quantified from random photographs (see materials and methods). Similar results were obtained in 3 separate experiments. **: P<0.01 (Student-Newman-Keuls test, Anova).

[0160] FIGS. 7A-B: centrifugal sedimentation and western blot analysis of Tau polymerization: (A) in oxidative conditions: Ox (B) in reducing conditions: Red. S. Supernatants, P, Pellets.

[0161] FIG. 8: Centrifugal sedimentation and western blot analysis of Tau polymerization in oxidative conditions: recombinant Tau at 0.5 mg/ml 1: alone, 2: with FKBP52, 3: with GST. S, Supernatants, P. Pellets.

[0162] FIGS. 9A-F: Expression level of FKBP52 in Alzheimer disease and control brains. Western blot analysis of FKBP52 in soluble homogenate fraction from normal and Alzheimer disease. GAPDH was used as loading control. Intensities of the chemoluminescence were quantified with Image-J software. (A), soluble fractions 1-5; (B), soluble fractions 6-12; (C) soluble data combined; (D) insoluble fractions 1-5; (E), insoluble fractions 6-12; (F) insoluble data combined.

[0163] FIGS. 10A-B: Expression level of mRNA FKBP52 in Alzheimer disease and controls. Quantification of mRNA FKBP52 was carried out after normalisation with mRNA GAPDH. (A) individual samples; (B) combined data.

EXAMPLE 1: A ROLE FOR FKBP52 IN TAU PROTEIN FUNCTION

[0164] Materials and Methods

[0165] Antibodies and Reagents:

[0166] Anti-Tau mAB (clone DC25) and anti-Tau mAB (Tau5) was from Sigma and Calbiochem respectively. Anti-FKBP52 pAB 761 was as described (9). GTP was from Sigma and doxycycline was from Clontech.

[0167] Preparation of Tubulin and Microtubule Assembly Assay:

[0168] Male adult Sprague-Dawley rats (body weight 250 g) were obtained from Janvier (Le Genest-St.-Isle, France). They were killed by decapitation, according to institutional guidelines, and whole brains were used immediately to prepare tubulin as described (9). Microtubule assembly assays were performed as in (9).

[0169] Protein Purification and Overexpression of Different Tau Isoforms and FKBP5Z Protein:

[0170] The six isoforms of human brain Tau were expressed in E. coli from clones hT40, hT39, hT37, hT34, hT24, hT23 and purified as described (28). Full length FKBP52 was affinity purified as in (23). For the Tubulin polymerization assay. FKBP52 bound to glutathione-sepharose beads (GE Healthcare) were cleaved overnight at 4 C. with 2 units of thrombin (GE Healthcare) and dialyzed against buffer L (0.1 M Mes, 1 mM EGTA, 1 mM MgCL.sub.2, 0.1 mM EDTA) with 10% glycerol, and complemented before use to 1 mM GTP and 1 mM DTT and 10 M PPACK (a potent irreversible inhibitor of thrombin) (Biomol).

[0171] Phosphorylation and Hyperphosphorylation of Recombinant Tau:

[0172] Rat brain extract was used as the source of kinase activity, as described (18). Briefly, recombinant hT40 was incubated with cytosol of adult rat brain, in the presence or absence of okadaic acid, to give HP-Tau (hyperphosphorylated-Tau) and P-Tau (phosphorylated-Tau), respectively.

[0173] Protein Binding Assays:

[0174] GST-pull down assay: 100 l of glutathione-Sepharose beads preloaded with 1 nmol GST-FKBP52 or 1 nmol GST, were washed 4 times with 500 l of buffer A (buffer L complemented with 1 mM DTT and 1 mM GTP) and then resuspended in the same buffer containing protein microtubule extract. The proteins were analyzed for the presence of Tau isoforms by SDS/PAGE western blot analysis using antibody anti-Tau (clone DC25) diluted 1/1000.

[0175] Coimmunoprecipitation Assay:

[0176] It was carried out with 1 mg cytosol microtubule extract as described (12).

[0177] Dot Blot Assay:

[0178] 100 l of buffer A containing different amounts of hT40 were applied to a nitrocellulose membrane, blocked with 5% non-fat milk in phosphate-buffered saline (PBS) containing 0.1% Tween 20 (PBS-T) at room temperature, washed with PBS-T and buffer A, followed by 2 h incubation at room temperature with 100 l of buffer A containing 0.5 g recombinant FKBP52. The membranes were washed with buffer IP (50 mM Tris (pH 7.5)/150 mM NaCl/2 mM MgCl.sub.2/0.1% Brij97 (Sigma)/10% glycerol/protease inhibitors) and with PBS-T. After blocking with milk in PBS-T, the membranes were incubated with anti-FKBP52 761 antibody. The presence of FKBP52 was revealed by ECL. Quantitation was performed with Quantity-one software using Chemidoc XRS fitted with a 16 bit CCD camera (Biorad).

[0179] Cell Line and Stable DNA Transfection:

[0180] Generation of H7C2 Cells:

[0181] The cDNA encoding rabbit FKBP52 was inserted into the HindIII and AccI restriction sites of the pTRE2 vector (Clontech) in order to give pTRE2-FKBP52. Transfection of 100 g of pTR2-FKBP52 and 10 g of pTK-hygromicin was carried out in a commercially available PC12 Tet-on cell line (Clontech) that expresses the reverse tetracycline controlled transactivator, using Lipofectamine (Invitrogen). Stably transfected cells were selected with 100 g/ml hygromycin and screened individually.

[0182] Cell Culture:

[0183] PC12 cells and H7C2 cells were grown in DMEM containing 10% (v/v) horse serum and 5% (v/v) FBS (Invitrogen) at 37 C. in 90% O.sub.2/10% CO.sub.2. The differentiated neuronal phenotype of cells grown on plastic dishes coated with 10 g/ml poly(L)-lysine (Sigma), was induced by adding nerve growth factors (NGF) (Invitrogen) for 5 days. Primary cultures from cerebral cortex of embryonic day 17rat foetuses were carried out. Dissociated cells were plated (50,000 cells per ml) on glass coverslips coated with poly(L-ornithine) and cultured in a defined medium in 95% O.sub.2/5% CO.sub.2 at 37 C.

[0184] Immunocytochemistry:

[0185] Cells were grown on glass coverslips precoated in 12-well tissue culture plates. Primary cells and PC12 cells were incubated for 2.5 min and 3 min respectively, in PEM buffer (80 mM PIPES, 1 mM MgCl.sub.2, 2 mM EGTA, pH6.9) with 0.05% Triton, rinsed with warm Triton-free PEM, fixed for 5 min with methanol at 20 C. and incubated with affinity-purified anti-FKBP52 761 (1/1000), and anti-Tau5 (1/100). Anti-rabbit Alexa Fluor 488-conjugated (Invitrogen), anti-mouse FITC-conjugated or Cy3 red-conjugated (GE-Healthcare) antibodies were used at 1/500 and 1/1000, respectively. The coverslips were examined by epifluorescence using a Zeiss axioplan 2 microscope with, either a 63 objective or by confocal microscopy (Zeiss, Thornwood, N.Y., USA)

[0186] Quantitation of Neurite Outgrowth:

[0187] Random field photographs of PC12 and H7C2 cells, in each of three wells, were analyzed with Neuron J software. The average neurite length was determined by measuring the longest neurites of at least 200 cells randomly selected.

[0188] Results

[0189] Tau FKBP52 Association:

[0190] FKBP52 is widely distributed in the brain as shown by Western blots of cytosolic proteins from several brain areas (FIG. 1). In order To investigate whether MAPs may be involved in the effect of FKBP52 on microtubule stability (9), GST pull-down assays were carried out incubating GST-FKBP52 bound to sepharose beads with microtubules cytosol prepared from adult rat brain. Specifically bound proteins were analyzed by immunoblotting using antibodies directed against MAP1b, MAP2 and Tau. In these experimental conditions, no immunoreactivity was observed for MAP1b or MAP2, but Tau immunoreactivity was present (FIG. 2A). In rat brain homogenates, Tau appears as multiple bands representing different splice isoforms with various degrees of phosphorylation. Several Tau species were also found in pull-down experiments of rat brain cytosol microtubules using GST-FKBP52. Tau immunoreactivity was not detected in controls using purified GST (FIG. 2A). To confirm the specificity of this association, microtubules of adult rat brain cytosol were immunoprecipitated with a polyclonal antibody against FKBP52. Immunoprecipitates were analyzed by Western blotting with a monoclonal Tau antibody. Tau co-immunoprecipitated with FKBP52 but not with a preimmune serum (FIG. 2B). Thus, Tau and FKBP52 form a complex in rat brain. These experiments do not address whether the binding of Tau to FKBP52 is direct or whether it involves additional factors. To investigate this, recombinant Tau (hT40, the longest isoform, expressed in E. coli and purified) was spotted onto nitrocellulose and incubated with purified recombinant FKBP52. Then, proteins sequestered by Tau were detected with a polyclonal antibody against FKBP52. As shown on FIG. 2C, FKBP52 was retained in a dose dependent manner by Tau, but not by the GST. These findings indicate a direct interaction between FKBP52 and Tau.

[0191] Effect of Tau Phosphorylation on its Interaction with FKBP52:

[0192] To define whether the phosphorylation of Tau modulates its association with FKBP52, dot blot experiments were performed using recombinant phosphorylated Tau (P-Tau) and hyperphosphorylated Tau (HP-Tau) (18) (FIG. 3A). The specificity of the interaction between FKBP52 and P-Tau or HP-Tau was determined by experiments using as the bait either these phosphorylated ht40, non phosphorylated hT40, or hT40 to which had been added, just prior to spotting, the same amount of cytosol protein as used to obtain phosphorylated or hyperphosphorylated hT40. As shown in FIG. 3B, the amount of FKBP52 recruited by Tau depends on its phosphorylation state. 73% (7) of FKBP52 was retained by 2.2 g HP-Tau, whereas only 3.5% and 6.6% were respectively retained by the same amount of hT40 and by hT40 in the presence of cytosolic proteins used as controls. When P-Tau was used as bait, only 41% (15) of FKBP52 was captured. This difference in binding between HP-Tau and P-Tau to FKBP52 may be explained by the different degree of Tau phosphorylation at specific sites or by the global amount of Tau phosphorylation, or by a combination of both mechanisms (FIG. 3A). In any case, these results underline the importance of Tau phosphorylation for its binding to FKBP52.

[0193] Colocallzation of Tau and FKBP52 in Primary Cortical Neurons and PC12 Cells:

[0194] The subcellular localization of Tau and FKBP52 was examined by immunofluorescence experiments with rat primary cortical neurons. After 6 days of culture, and mild extraction selectively removing unbound cytosolic proteins while retaining proteins associated with the cytoskeleton, double staining was performed on neurons with monoclonal antibody Tau5 and affinity-purified polyclonal anti-FKBP52 antibody. In agreement with an earlier report (19), Tau was concentrated in the distal portion of axons and at the growth cone neck, where a strong accumulation of FKBP52 was also observed (FIG. 4). Colocalization and accumulation of FKBP52 and Tau were also found at the growth cones of PC12 cells (FIG. 4). Very recently it has been reported that Tau is selectively enriched at axonal tips and that this may be due to its specific anchoring (20). Our results suggest that FKBP52 may be involved in the trapping of Tau, and thereby able to influence its subcellular distribution.

[0195] FKBP52 Inhibits Tubulin Polymerization Induced by Tau In Vitro:

[0196] To demonstrate a functional interaction between Tau and FKBP52, a microtubule kinetic assay was set up. In experiments with purified rat brain tubulin alone, no microtubule was formed, whereas in experiments with recombinant human Tau isoforms, an increased absorbance reflecting microtubule assembly was observed (FIG. 5). However, when Tau was added to the tubulin in the presence of purified recombinant FKBP52, formation of microtubules was prevented, whereas GST was ineffective. Similar results were obtained with the 6 isoforms of human Tau (FIG. 5). We concluded that FKBP52 inhibits the promotion of microtubule assembly by Tau.

[0197] FKBP52 Prevents Tau Accumulation and Neurite Outgrowth in PC12 Cells:

[0198] An FKBP52-inducible expression system based on a tetracycline-responsive element allowing the generation of a stably transformed PC12 cell line was used (21) to determine a cellular role for FKBP52. Among clones which were positively tested, one clone, so called H7C2, was selected and used to study the effects of FKBP52 overexpression on PC12 cells, and to further investigate the possible relationship between FKBP52 and Tau. Under basal conditions H7C2 cells expressed endogenous FKBP52, and treatment with Dox (doxycycline) resulted in a marked increase of recombinant FKBP52 protein expression (FIG. 6A). FKBP52 induction in H7C2 cells was about 4 fold after 5 days of Dox treatment.

[0199] The effect of FKBP52 on the accumulation of Tau was examined next. The amount of Tau protein was determined by Western blotting of extracts from cultures of either PC12 cells or H7C2 cells, treated or not with NGF (50 nM) for 5 days with or without Dox. In PC12 cells, FKBP52 expression was unchanged after treatment with NGF (FIG. 6B). As expected, in both PC12 and H7C2 cells an increase in Tau was observed after NGF treatment. However, when H7C2 cells were exposed to Dox in addition to NGF, thus overexpressing FKBP52, no additional accumulation of Tau protein occurred. An increase in Tau protein was still observed in PC12 cells treated with NGF and Dox, ruling out the possibility that Dox was responsible for the lack of decrease in Tau (FIG. 6C). In conclusion, FKBP52 prevented the accumulation of Tau induced by NGF in PC12 cells.

[0200] Since one role of Tau is to stimulate neurite outgrowth (12), we investigated the consequence of FKBP52 overexpression on neurite length in both PC12 and H7C2 cells. In the absence of NGF, no neurite outgrowth was observed in H7C2 cells, whether or not they were treated with Dox for a week. However in H7C2 cells treated with 50 nM NGF and Dox, a 40% (+7) decrease in neurite length, compared to control (H7C2 not treated with Dox) was observed (FIG. 6D). The same effect of Dox on neurite length was observed in H7C2 cells treated with 10 or 20 nM NGF. That Dox by itself was involved in the process of neurite outgrowth could be ruled out, since no difference in neurite length between Dox-treated and untreated PC12 cells was observed. The inhibition of neurite outgrowth resulting from FKBP52 overexpression is in agreement with our previous report showing that the loss of FKBP52 in PC12 cells results in the formation of neurite extensions (9). The FKBP52 effect on neurite length could be explained by the binding of Tau to FKBP52, removing Tau from microtubules. In addition the prevention of Tau accumulation by overexpression of FKBP52 is consistent with the decrease of neurite length and evokes a potential role of this immunophilin in Tau function.

[0201] Discussion

[0202] This newly discovered anti-Tau activity of FKBP52 leads us to re-examine the functions of this protein, which was originally identified and cloned as modulator of hormone steroid receptors (8, 22). FKPB52 is a multimodular protein, which includes a peptidyl prolyl isomerase (rotamase) segment, the function of which is blocked by FK506 (23), rapamycin and some related non-immunosuppressive derivatives. There is a noteworthy structural similarity between FKBP52 and Pin1: both proteins have a peptidyl-prolyl isomerase (PPIase) activity and a specific protein-protein interaction domain (7). Since the Pin1 PPIase activity restores the function of phosphorylated Tau protein in a model of Alzheimer's disease (7), the interaction observed between Tau and FKBP52 may have implications for the pathogenesis of the tauopathies, including Alzheimer's disease. It must be remembered that, unlike FKBP12 (24) FKBP52 does not bind calcineurin (25), and thus FKBP52 does not mediate the immunosuppressant capacity of FK506. Therefore, the pharmacological modulation of the rotamase activity of FKBP52 by non-immunosuppressive FK506/rapamycin derivatives may offer a novel approach for preventing/reducing the pathogenic effects of misfolded Tau.

[0203] In addition to its peptidyl prolyl isomerase activity, FKBP52 serves as a molecular chaperone. This activity depends on its tetratricopeptide repeat domain (26) to which the molecular chaperone HSP90 and other proteins bind. It has already been noted that chaperone co-chaperone protein complexes play a critical role in neurodegenerative diseases characterized by Tau accumulation (27). We now report that FKBP52 could decrease the function/accumulation of Tau, and therefore suggest its possible involvement in these described cochaperone systems (27).

[0204] Our results establish a role of FKBP52 in Tau function. The interaction described in this report rapidly deserves to be studied further, since effective pharmacological targeting of FKBP52 is likely to become a reality in the near future.

EXAMPLE 2: EFFECT OF FKBPS2 ON OLIGOMERIZATION OF TAU IN VITRO

[0205] Aggregates of Tau protein are characteristic of multiple neurodegenerative diseases. (Delacourte A and Bu L, 2000). Studies of conditions for Tau aggregation in vitro have led to different experimental systems including oxidative conditions, inductions by polyanions such as heparin, and by fatty acids such as arachidonic acid (Barghorn S and Mandelkow E, 2002).

[0206] To investigate the effect of FKBP52 on Tau oligomerization we monitored by sedimentation assay the ability of recombinant Tau protein (here using the longest isoform) to polymerise in oxidative conditions in presence or absence of recombinant FKBP52. After dialysis of Tau (0.5 mg/ml) overnight at 4 C. in Tris 10 mM pH7.4 and centrifugation at 14000 rpm, quantitative western blotting was used to monitor the polymerization of Tau protein. In this condition sedimentation analysis revealed the presence of Tau in pellet fraction contrary to the experiment carried out in presence of DTT where no detectable pelletable polymers of Tau could be obtained (FIG. 7). This experiment means that Tau in oxidative conditions self polymerise and confirms, as already reported, the importance of cysteines in first step of Tau oligomerization process (Schweers et al 1995).

[0207] The effects of coincubating, in oxidative medium, Tau and FKBP52 on Tau oligomerization process were monitored. As shown on FIG. 8 no detectable presence of Tau in pellet fraction could be observed when dialysis were performed in equimolecular presence of FKBP52 where a pelletable polymers of Tau could be observed when coincubation was carried out in presence of GST used as control.

[0208] However when sedimentation analysis experiments were realized in varying the concentration of FKBP52 (1 to 0.1 mg/ml) where the concentration of Tau was constant (0.5 mg/ml) the presence of FKBP52 at 0.1 or at 0.25 mg/ml results in increasing Tau polymers in pellet where the presence of equimolecular or two times quantities of FKBP52 versus Tau prevent the presence of pelletable polymers of Tau (FIG. 9). These results suggest a dual role for FKBP52: firstly: a FKBP52 conformational activity on Tau, possibly involving peptidyl prolyl isomerase activity of FKBP52, leading Tau in a favourable proaggregative form; secondly; FKBP52 could protecte by possible steric hindrance the ability of Tau to oligomerize via the formation of disulfide bridges.

EXAMPLE 3: DOWN REGULATION OF FKBP52 PROTEIN IN BRAINS FROM PATIENTS WITH ALZHEIMER DISEASE

[0209] Human brain pre frontal cortex, obtained thanks to the access of National BrainBank (GIE NeuroCEB), were homogenized in 5 volumes of buffer containing Tris 10 mM. Saccharose 0.32M, DTT 1 mM and protease inhibitor cocktail. The homogenates were centrifugated at 14000 RPM at 4 C. for 5 min. The supernatant was used as soluble fraction. The pellet was solubilised by homogenization in the same conditions and used as insoluble fraction. FKBP52 levels in the soluble and insoluble fractions were analyzed by western blotting. The FKBP52 level in soluble and insoluble fraction represents respectively 80%0/and 20% of total FKBP52;

[0210] In the aim of elucidate if FKBP52 protein expression level might be altered in patient brain we have compared its expression in the pre frontal cortex of normal control (n=3) and patients with Alzheimer disease (n=12). As shown on FIG. 10 marked reduction of FKBP52 in Alzheimer disease brains is observed in soluble fraction. After normalization with GAPDH (Glyceraldehyde 3-phosphatedehydrogenase) expression levels of FKBP52 found in soluble fraction of Alzheimer disease brains represent 35% (24) compared to the controls. No modification of expression protein level of FKBP52 could be detectable in insoluble fraction; this last observation suggests that FKBP52 is not trapped in aggregates of Tau.

[0211] Subsequently the mRNA expression level of FKBP52 gene was analyzed by real time quantitative RT-PCR (QPCR) in prefrontal cortex of human brain disease and controls. As shown on FIG. 11 no modification of FKBP52 mRNA level is observed in brain diseases versus control. These results indicate that the decreasing of FKBP52 protein expression in the patient brain is due to a post transcriptional mechanism.

REFERENCES

[0212] Throughout this application, various references describe the state of the art to which this invention pertains. The disclosures of these references are hereby incorporated by reference into the present disclosure.

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