The FASEB Journal • Research Communication

The FK506-binding protein FKBP52 in vitro induces aggregation of truncated Tau forms with prion-like behavior Julien Giustiniani,* Kevin Guillemeau,* Omar Dounane,* Elodie Sardin,* Isabelle Huvent,† Alain Schmitt,‡,§,{ Malika Hamdane,k,# Luc Bu´ee,k,# Isabelle Landrieu,† Guy Lippens,† Etienne Emile Baulieu,*,1,2 and B´eatrice Chambraud*,1,2 *Institut National de la Sant´e et de la Recherche M´edicale, Unit´e Mixte de Recherche 1195, Universit´e Paris XI, Le Kremlin Bicˆetre, France; †Centre National de la Recherche Scientifique, Universit´e de Lille 1, Unit´e Mixte de Recherche 8576, Villeneuve-d’Ascq, France; ‡Institut National de la Sant´e et de la Recherche M´edicale, Unit´e 1016, Institut Cochin, Paris, France; §Centre National de la Recherche Scientifique, Unit´e Mixte de Recherche 8104, Paris, France; {Universit´e Paris Descartes, Sorbonne Paris Cit´e, France; kInstitut National de la Sant´e et de la Recherche M´edicale, Unit´e Mixte de Recherche 1172, Centre de Recherche Jean-Pierre Aubert, Lille, France; and #University of Lille, School of Medicine, Lille, France ABSTRACT Tauopathies, including Alzheimer’s disease (AD), are neurodegenerative diseases associated with the pathologic aggregation of human brain Tau protein. Neuronal Tau is involved in microtubule (MT) formation and stabilization. We showed previously that the immunophilin FK506-binding protein of MW ∼52 kDa (FKBP52) interferes with this function of full-length Tau and provokes aggregation of a disease-related mutant of Tau. To dissect the molecular interaction between recombinant human FKBP52 and Tau, here, we study the effect of FKBP52 on a functional Tau fragment (Tau-F4, Ser208-Ser324) containing part of the proline- rich region and MT-binding repeats. Therefore, we perform MT assembly and light-scattering assays, blue native PAGE analysis, electron microscopy, and Tau seeding experiments in SH-SY5Y human neuroblastoma cells. We show that FKBP52 (6 mM) prevents MT formation generated by Tau-F4 (5 mM) and induces Tau-F4 oligomerization and aggregation. Electron microscopy analyses show granular oligomers and filaments of Tau-F4 after short-time FKBP52 incubation. We demonstrate that the terminal parts of Tau interfere with the effects of FKBP52. Finally, we find that FKBP52-induced Tau-F4 oligomers cannot only generate in vitro, direct conformational changes in full-length Tau and that their uptake into neuronal cells can equally lead to aggregation of wild-type endogenous Tau. This suggests a potential prion-like property of these particular Tau-F4 aggregates. Collectively, our results strengthen the hypothesis of FKBP52 involvement in the Tau pathogenicity process.—Giustiniani, J., Guillemeau, K., Dounane, O., Sardin, E., Huvent, I., Schmitt, A., Hamdane, M., Bue´ e, L., Landrieu, I.,

Abbreviations: 3D, 3-dimensional; AD, Alzheimer’s disease, ANR, Agence Nationale de la Recherche; BN-PAGE, blue native PAGE; FKBP, FK506-binding protein; FTDP17, frontotemporal dementia with Parkinsonism linked to chromosome 17; GST, glutathione S-transferase; L buffer, 0.1 M MES, 1 mM EGTA, 1 mM MgCl2, 0.1 mM EDTA, pH 6.2, 1 mM DTT; (continued on next page)

0892-6638/15/0029-3171 © FASEB

Lippens, G., Baulieu, E. E., Chambraud, B. The FK506binding protein FKBP52 in vitro induces aggregation of truncated Tau forms with prion-like behavior. FASEB J. 29, 3171–3181 (2015). www.fasebj.org Key Words: microtubule • immunophilin • Tau assembly • seeding TAU IS A MICROTUBULE (MT)-ASSOCIATED protein that is physiologically found under a highly soluble form; however, Tau is mainly found aggregated in several neurodegenerative diseases called tauopathies, including Alzheimer’s disease (AD) (1). Tau aggregation is a multistep process that involves transient, small oligomeric species and can evolve into filaments (2). In AD brains, most of these filaments present a twisted appearance and are commonly termed “paired helical filaments” (PHFs), which are the major source for the formation of larger Tau aggregates, also called neurofibrillary tangles (3). PHFs include hyperphosphorylated and truncated forms of Tau (4). The importance of a truncated form of Tau in disease progression has been underlined by the identification of various sizes of Tau species, ranging from 10 kDa to higher molecular mass, in aggregates of human AD brains (5). On the other hand, recent studies have demonstrated that injection of brain homogenates containing mutant Tau aggregates in the brain of transgenic mice expressing wild-type human Tau can transmit Tau pathology, suggesting a prion-like spread of Tau (6). Moreover, it has been reported that in cultured neuronal cells, extracellular-misfolding Tau is capable of

1

These authors contributed equally to this work. Correspondence: INSERM, Unit´e Mixte de Recherche 1195, Universit´e Paris XI, Le Kremlin Bicˆetre, France. E-mail: beatrice. [email protected] (B.C.); [email protected] (E.E.B.) doi: 10.1096/fj.14-268243 This article includes supplemental data. Please visit http:// www.fasebj.org to obtain this information. 2

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crossing cellular membranes and propagates fibrilization of intracellular Tau (7–9). One of Tau’s major functions involves the stabilization of the MT network. The binding of Tau to tubulin is provided by the MT-binding repeats (MTBRs) in combination with the adjacent proline-rich flanking domain (10, 11). Recently, Fauquant et al. (12) identified a functional fragment of Tau (Tau-F4, Ser208-Ser324), consisting of adjacent parts of a proline-rich region and 3 repeats of the MTBRs; it binds more tightly to stabilized MTs than full-length Tau and is sufficient by itself to polymerize tubulin into MTs. This Tau fragment includes 2 short motifs, present in the MTBR, that are prone to form a b-structure and that are crucial for paired helical formation (13). The present study deals with the interaction of Tau-F4 and the FK506-binding protein of MW ;52 kDa (FKBP52). FKBP52 belongs to the immunophilin family and is an intracellular protein-binding immunosuppressant drug, such as FK506 and rapamycin (14, 15). This protein, particularly abundant in the nervous system, harbors in its N-terminal domain a peptidyl-prolyl cis/trans isomerase (PPiase) activity, inhibited by FK506 binding (16). Involved in target protein folding and function, FKBP52 prevents in vitro MT formation by its interaction with tubulin and Tau (17, 18). We have shown previously that levels of FKBP52 protein are decreased dramatically in the brains of patients with AD or with frontotemporal dementia with Parkinsonism linked to chromosome 17 (FTDP-17), a tauopathy caused by Tau mutations (19). Recently, we have demonstrated in vitro that FKBP52 is able to induce the oligomerization and assembly into filaments of a pathologic mutant Tau-P301L, identified in FTDP-17 (20). Here, we show that FKBP52 prevents MT formation induced by Tau-F4 in vitro and activates the oligomerization of this fragment. We demonstrate that the C-terminal part or the N-terminal projection domain of wild-type Tau interferes with its oligomerization and further filament formation induced by FKBP52. Finally, we describe a prionlike aptitude of FKBP52-induced Tau-F4 oligomers in SHSY5Y cells. In summary, our data underline the importance of FKBP52 in the Tau pathogenicity process.

Protein purification Purified tubulin was isolated from the brain of 50 male, adult Sprague-Dawley rats, as previously described (18). Recombinant, wild-type, full-length Tau protein was expressed in Escherichia coli from clone hT40 and clone hT40-ΔC and purified as described (21). Recombinant Tau-F4 and -F1 were expressed in E. coli and purified as described (12); at the end of purification, the material was applied to a NAP10 column (GE Healthcare, Pittsburgh, PA, USA), equilibrated in 0.1 M MES, 1 mM EGTA, 1 mM MgCl2, 0.1 mM EDTA, pH 6.2, 1 mM DTT (L buffer). Full-length human FKBP12 or FKBP52 and glutathione S-transferase (GST) expressed in E. coli were prepared as described previously (16, 20). Purified GST, FKBP12, and FKBP52 proteins were then dialyzed against L buffer with 10% glycerol. For all proteins, 1 mM DTT was added before use, and 1 mM GTP was added for tubulin polymerization assay. Light-scattering assay Recombinant Tau-F4 (5 mM) was incubated with tubulin (10 mM), with or without FKBP52 (6 mM) in L buffer, complemented with 1 mM GTP for 30 min at 37°C. Variation in optical density indicating MT formation was monitored by use of a SpectraMax M2 spectrometer (Molecular Devices, Sunnyvale, CA, USA). The same approach was used to analyze recombinant full-length Tau or truncated forms (5 mM), incubated with FKBP52 (6 mM) without tubulin. Spin-down assays and Western blot analysis After protein incubation for 30 min at 37°C in tubulin polymerization assays or experiments with no tubulin, the samples were collected and centrifuged at 14,000 rpm during 10 min. The supernatant and pellet, corresponding, respectively, to the soluble and insoluble fractions, were analyzed by SDS-PAGE. Western blots were carried out with appropriate antibodies [FKBP52, mouse monoclonal EC1 (1:1000; Enzo Life Sciences, Farmingdale, NY, USA); Tau, rabbit polyclonal K9JA (1:5000; Dako, Glostrup, Denmark); tubulin, mouse monoclonal DM1A (1:1000; Sigma-Aldrich, St. Louis, MO, USA); and GST, mouse monoclonal 3G10/1B3 (1:1000; Abcam, Cambridge, MA, USA)].

Blue native PAGE

MATERIALS AND METHODS 1

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PCR was carried out by use of as template wild-type Tau full-length cDNA (hT40) cloned into vector pRK172, and the sequences of primers follow: forward 59 ATA CAT ATG GCT GAG CCC C 39 and reverse 59 CGT GAA TTC TCA ATT TCC TCC GCC AGG GAC 39. The corresponding PCR product was digested with restriction enzymes NdeI and EcoRI before subcloning into pRK172. After amplification, the plasmid was verified by sequence analysis (GenoScreen, Lille, France).

Native gel electrophoresis analyses were performed by 4–16% blue native (BN) PAGE (Invitrogen, Carlsbad, CA, USA), as described previously (20, 22). Western blots were carried out with the primary antibodies [FKBP52, mouse monoclonal EC1 (1:1000; Enzo Life Sciences), and a rabbit polyclonal 761 (1: 1000); and Tau, rabbit polyclonal Tau K9JA (1:5000; Dako)]. Species-specific, peroxidase-conjugated secondary antibodies were subsequently used to obtain ECL (Pierce, Life Technologies, Rockford, IL, USA). Images were recorded with the GeneGnome5 (Syngene, Frederick, MD, USA). Semidenaturing detergent agarose gel electrophoresis

(continued from previous page) MT, microtubule; MTBR, microtubule-binding repeat; PHF, paired helical filament; PPiase, peptidyl-prolyl cis/trans isomerase; SBBB, Sea Block blocking buffer; SDD-AGE, semidenaturing detergent agarose gel electrophoresis; TEM, transmission electron microscopy

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Tau-hT40 (5 mM) mix with FKBP52 (6 mM) or Tau-hT40 (20 mM) mix with heparin (5 mM) was incubated up to 72 h at 37°C and analyzed by semidenaturing detergent agarose gel electrophoresis (SDD-AGE), as described previously (23). Tau-hT40 (2 mg) was loaded on 1.8% agarose gel, and Tau rabbit polyclonal K9JA (1:5000; Dako) was used for Western blotting. The presence of

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GIUSTINIANI ET AL.

Tau was revealed by ECL (Pierce, Life Technologies). Images were recorded by use of the GeneGnome5 (Syngene). Purification of Tau-F4 oligomers After incubation of 25 mM Tau-F4 with 5 mM FKBP52 at 37°C for 30 min in buffer L, the mix was loaded on a Sephadex G75 column, and 20 fractions of 500 ml were collected. Two major peaks at optical density 280 nm were observed, and subsequently, 10 ml each fraction were analyzed by Western blotting by use of Tau5 and EC1 antibodies. Fractions 14–18 present only Tau-F4 oligomers without FKBP52. These samples (100 ml) were then incubated with 5 mM hT40 at 37°C up to 48 h. Tau hT40 (500 ng) was analyzed by 4–16% BN-PAGE gels (Invitrogen) by use of Tau 46 mouse mAb recognizing the C-terminal part of Tau hT40 (1/1000; Abcam). The presence of Tau was revealed by ECL (Pierce, Life Technologies). Quantifications were carried out by use of the GeneTools software (Syngene).

PHF-1 (1/1000; gift from P. Davies, Albert Einstein College of Medicine, Bronx, NY, USA)—for 1 h at 37°C, followed by staining with Alexa Fluor 488-conjugated secondary antibody (Life Technologies) for 45 min at 37°C. Nuclei were stained with DAPI. The coverslips were examined by epifluorescence by use of a Leica SP8 confocal microscope. Three-dimensional (3D) projection from Z-stack imaging of an infected cell was obtained by use of ImageJ FIJI, 3D Viewer software. Statistical analysis Quantification of intracellular endogenous Tau aggregates, .1 mm2 in size, was performed in 3 independent experiments. Only cells harboring intracellular exogenous Tau were counted (150 infected cells)/experiment. Values reported are means (SD). Comparisons between 2 groups of samples were performed by use of Student’s t test. Values of P , 0.05 were considered statistically significant. The software Statistica was used for statistical analysis.

Electron microscopy

RESULTS

After incubation of 5 mM Tau proteins, with or without 6 mM FKBP52, 10 ml of the sample was placed on a Formvar 400 mesh hexagonal grid for 30 s. The sample on the grid was stained with uranyl acetate 2% for 1 min. At each step, excess stain was removed by blotting the edge of the grid with filter paper. Grids were observed with a Jeol 1011 transmission electron microscope. Acquisitions were performed with a an Erlangshen chargecoupled device camera (Gatan, Pleasanton, CA, USA) by use of Digital Micrograph software.

FKBP52 modulates the solubility of Tau-F4 in vitro

Labeling of Tau-F4 with cyanine Tau-F4 proteins were labeled with Cy5.5 by use of Amersham FluoroLink monofunctional dye labeling kit (GE Healthcare), according to the previously described protocol (20, 24). Separation of protein from free dye was performed by gel filtration by use of Sephadex G-50 coarse (GE Healthcare). Cell culture and uptake of Tau aggregates Neuroblastoma SH-SY5Y cells were cultivated as described previously (25). Cells were grown on poly-D-lysine-coated glass coverslips for microscopy and were plated at 5 3 104 cells/well in a 12-well tissue-culture plate. On the next day, cells were treated with doxycycline for 15 h to induce Tau hT34 overexpression. The isoform Tau hT34 contains 4 repeats in the MT domain, and the 2nd insertion in the N terminus is lacking. Cells were washed 3 times with PBS and then incubated with fresh medium. Subsequently, samples of purified, Cy5.5-labeled Tau-F4 (5 mM), incubated alone or with FKBP52 (6 mM) for 30 min at 37°C, were added to fresh cell medium for 24 h incubation. The average of cells infected was determined by measuring at least 100 redlabeled cells randomly selected in 3 separate experiments. Immunocytofluorescence and confocal microscopy analyses Cells were washed 2 times with PBS containing 0.025% trypsin for 1 min and then fixed with methanol for 5 min at 220°C. After blocking with Sea Block blocking buffer (SBBB; 1/10; Pierce, Life Technologies) in PBS for 1 h at room temperature, cells were incubated in PBS SBBB (1/10) with the following primary antibodies—rabbit polyclonal anti-Tau K9JA (1/1000) and mouse monoclonal anti-Tau Tau13 (1/1000; Abcam); and MC1 and

FKBP52 INDUCES TRUNCATED TAU AGGREGATION IN VITRO

MT assembly kinetics were performed to investigate whether FKBP52 is able to prevent in vitro the tubulin polymerization induced by Tau-F4, as demonstrated previously for the 6 different Tau isoforms (18). Similar to the case of full-length Tau hT40, incubation of Tau-F4 with tubulin at 37°C leads to an increase of absorbance that reflects MT formation. However, whereas in the presence of FKBP52, the assembly function of full-length Tau hT40 is blocked completely, the absorbance is only reduced to ;45% for Tau-F4/tubulin (Fig. 1B). To evaluate the level of MTs formed in these conditions, the different samples were collected after 30 min at 37°C and subjected to a spin-down assay. The supernatant and pellet fractions, containing soluble and polymerized tubulin, respectively, were analyzed by Western blotting. As expected, no tubulin could be detected in the pellet when incubated alone, whereas it was found in the pellet in presence of Tau-F4 (Fig. 1C). When FKBP52 was added to the mixed Tau-F4/tubulin sample, no tubulin could be detected in the pellet fraction, meaning that FKBP52 does fully prevent MT assembly induced by Tau-F4 (Fig. 1C). Surprisingly, Tau-F4 was predominantly present in the pellet fraction in spite of the absence of MT, and this result may be correlated to the significant, residual turbidity observed in this case (Fig. 1B, C). To examine if Tau-F4 insolubility is exclusively a result of the presence of FKBP52, we incubated Tau-F4 and FKBP52 without tubulin. The mixture of the 2 proteins induced an increase of absorbance comparable with that observed previously, whereas no turbidity was measured when Tau-F4 or FKBP52 was incubated alone or with GST (Fig. 1D). Spin-down assay reveals that Tau-F4, when incubated with FKBP52 and not GST, is mainly detected in the pellet fraction, showing that FKBP52 is responsible for the insolubility of Tau-F4 in vitro (Fig. 1E). FKBP52 induces the oligomerization of Tau-F4 in vitro To analyze the physical state of the Tau-F4 protein obtained after incubation with FKBP52, we characterized these proteins by native gel electrophoresis. Fixed 3173

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Figure 1. FKBP52 inhibits Tau-F4 function in vitro and promotes its insolubility. A) Schematic representation of full-length Tau (hT40 and hT34) and Tau fragments (F1, F4, and hT40ΔC). Blue boxes represent the proline-rich domains (PR1 and PR2) of Tau. and the orange boxes consist of the MTBRs (R1–R4). The b-promoting regions, at the beginning of R2 and R3, are represented by yellow boxes. B) Tubulin polymerization assay. Tubulin (Tub; 10 mM), purified from rat brain, was incubated alone or in the presence of Tau-F4 (F4; 5 mM), without or with FKBP52 (6 mM). Optical density was monitored at 345 nm for 30 min at 37°C. C) Spin-down assays. Each of the samples obtained in B was centrifuged to give supernatant (S) and pellet (P) fractions. Western blot analysis of these fractions was performed by use of appropriate, specific antibodies. D) Light scattering. The turbidity detected for 30 min at 37°C was measured for each sample, as indicated on the figure: Tau-F4 (5 mM), FKBP52 (6 mM), and GST (6 mM) used as controls. E) Each of the samples obtained in D was analyzed by spin-down assay, as described in C.

quantities of Tau-F4 were mixed with increasing amounts of FKBP52, incubated for 30 min at 37°C, and then subjected to BN-PAGE. Western blot analyses with use of an anti-Tau antibody showed higher molecular weight bands in a FKBP52 concentration-dependent manner, whereas no modification occurred upon exposure of Tau-F4 to GST used as a control (Fig. 2). In addition, the detection of FKBP52 by use of 2 different antibodies with the same samples did not reveal any modifications in the migration profile, suggesting that the new bands of Tau-F4 detected correspond to different oligomeric states of Tau rather than to the formation of a stable complex between FKBP52 and Tau-F4. However, the Tau-F4 oligomerization, induced by FKBP52, could not be reversed by the addition of FK506 (until 30 mM), and we could not detect any effect of FKBP12 on Tau-F4 oligomerization (data not shown). Altogether, our results suggest that FKBP52 but not FKBP12 can generate in vitro conformational changes of the truncated Tau-F4 leading to its oligomerization.

30 min at 37°C. TEM examination allowed us to detect spheres showing heterogeneous size with diameters of 26.5 6 7.4 nm (n = 100), ranging between 15 and 40 nm (Fig. 3A). Some of these spherical objects were assembled linearly into prefilamentous structures (arrowheads). These particular shapes of Tau have already been described for the longest isoform hT40 after incubation with heparin (26, 27). Electron micrographs also reveal the presence of various thin Tau-F4 filaments. Some of them are long filaments of .1 mm, alternating in width between 7.9 6 1.2 nm and 12.75 6 1.8 nm (n = 12 different fibrils), and show a mean periodicity of 40.6 6 5.9 nm (n = 12), reminiscent of PHF-like structures (Fig. 3B, left, arrows). We also detected some thin filaments found in bundles (Fig. 3B, right), similarly to the bundled structures of TauF4 obtained after its incubation with heparin (28). No particular structure was observed when FKBP52 or Tau-F4 was incubated alone (Fig. 3C). Therefore, these data show that FKBP52 can induce in vitro the formation of granular oligomers and filaments of Tau-F4.

FKBP52 induces the formation of granular oligomers and filaments of Tau-F4

The N- or C-terminal part of wild-type Tau interferes with its oligomerization and filament formation induced by FKBP52

As FKBP52 is able to induce Tau-F4 oligomerization in vitro, we wondered if higher-order aggregates can be formed in these conditions. Transmission electron microscopy (TEM) was used to determine the structure of Tau-F4 oligomers obtained after incubation with FKBP52 for 3174

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To investigate whether FKBP52 is able to induce in vitro oligomerization of longer fragment of Tau, we repeated these experiments with the longest isoform of Tau (hT40) and a truncated Tau fragment F1 (Tau-F1, Gly164-Leu441)

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WB: Tau

WB: FKBP52

* * *

GST (μM) 10 F4 (μM) 5 5 FKBP52 (μM) - -

- - - 5 5 5 5 2 4 6 10

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Figure 2. FKBP52 induces in vitro Tau-F4 oligomerization. The ability of FKBP52 to promote Tau-F4 (F4) conformational changes in vitro was analyzed by BN-PAGE (4/16%). Tau-F4 incubation with increasing concentration of FKBP52 or GST used as control at 37°C was revealed by Western blotting (WB) by use of specific antibodies. Novel Tau oligomers appear along with the increase in FKBP52 concentration, as indicated by asterisks (*).

devoid of its N-terminal projection domain (12) (see constructs in Fig. 1A). Incubation of FKBP52 with Tau-F1 leads to an increase of absorbance similar to that observed with Tau-F4, whereas no turbidity is detectable when FKBP52 is incubated with Tau hT40. Tau or FKBP52 proteins, incubated separately with GST, did not show any effect (Fig. 4A). Spin-down assay of these samples reveals that Tau-F1, incubated with FKBP52 but not GST, is detected mainly in the pellet fraction, as observed for Tau-F4, whereas Tau

hT40 remains in the supernatant (Fig. 4B). BN-PAGE analysis reveals additional bands observed in the Tau-F1/ FKBP52 and Tau hT40/FKBP52 samples that have different electrophoretic mobility from those of isolated proteins and are not present when the same samples were analyzed and detected with an anti-FKBP52 antibody; this suggests that a structural modification of Tau-F1 and Tau hT40 could be induced by FKBP52 (Fig. 4C). With the use of TEM, we analyzed the structure of Tau-F1 and Tau hT40, obtained after their incubation with FKBP52. As demonstrated for Tau-F4, we detected the presence of granular oligomers of Tau-F1, isolated or assembled linearly into a prefilamentous structure, with a similar heterogeneous size of diameters and also some fibers that resemble those of Tau-F4 filaments (Fig. 5A). As shown by electron micrographs, only rare granules of Tau hT40 can be observed after incubation with FKBP52, and we failed to detect fibers (Fig. 5B). To rule out the possibility that incubation time was a limiting condition for FKBP52 to promote oligomerization of Tau hT40, we therefore performed experiments up to 72 h of incubation. The samples were analyzed by BN-PAGE and SDD-AGE, which allows detection of large-size polymers. The migration profile stays unchanged between the control and Tau hT40 mix with FKBP52, even after 72 h of incubation (Supplemental Fig. 1). Therefore, the structural modification of Tau-F1 induced by FKBP52 is sufficient to induce the formation of larger oligomers, contrary to full-length Tau. In the same way, the effect of FKBP52 on hT40-ΔC (Met1-Asn368) was checked by use of a light-scattering assay (Fig. 6A) and BNPAGE analysis (Fig. 6B). As shown in Fig. 6, the C-terminal truncation Tau mutant facilitates the effect of FKBP52 on Tau oligomerization. We conclude that the N- and Cterminal part of Tau hT40 can obstruct the FKBP52induced oligomerization. Truncation of Tau-F1, hT40-ΔC.

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FKBP52 INDUCES TRUNCATED TAU AGGREGATION IN VITRO

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Figure 3. FKBP52 induces assembly of Tau-F4 into granular oligomers and filaments. Electron micrographs of Tau-F4 after its incubation with FKBP52 for 30 min at 37°C. A) Granular Tau-F4. Some of these spherical objects are assembled linearly into prefilamentous structures (arrowheads). B) Fibrillar Tau-F4 structures showing a periodicity in their width (left, arrows). Filaments of Tau-F4 can assemble into bundles (right). C) No particular structure was observed when FKBP52 and Tau-F4 were incubated alone.

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Figure 4. Effects of FKBP52 on Tau-F1 and wild-type Tau hT40. A) Light-scattering assay. The turbidity detected for 30 min at 37°C was measured for each sample, as indicated in the figure: Tau-F4 (F4; 5 mM), Tau-F1 (F1; 5 mM), hT40 (5 mM), FKBP52 (6 mM), and GST (used as control; 6 mM). B) Spin-down assays. Each of the samples obtained in A was centrifuged to give supernatant and pellet fractions. Western blot analysis of these fractions was performed by use of specific antibodies. C) BN-PAGE analysis. The same amount of sample was loaded on a 4–16% BN-PAGE and analyzed by Western blotting by use of specific antibodies.

or Tau-F4 enhances the capacity of FKBP52 to induce oligomers and fibers in the fragments. FKBP52-induced Tau-F4 oligomers generate in vitro conformational changes of Tau and seed the aggregation of endogenous Tau in SH-SY5Y cells To check in vitro the ability of Tau-F4 oligomers induced by FKBP52 to generate a direct conformational change of Tau 3176

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hT40, Tau-F4 oligomers have been separated from FKBP52 by gel chromatography and then incubated with Tau hT40 up to 48 h before Western blot analysis, and a timedependent, gradual decrease of Tau hT40 after incubation with Tau-F4 oligomers was observed. The level of hT40 found after 8 h incubation was 50% compared with the control (sample without incubation and considered as 100%), and only 4% of hT40 was detected after 48 h incubation (Fig. 7A). The progressive decrease of Tau hT40 monomers suggested the formation of higher-order oligomers, which are too large to enter in the gel. No changes in Tau hT40 were observed when it was incubated alone or in presence of Tau-F4 monomer (Fig. 7B). These observations suggested that Tau-F4 oligomers induced by FKBP52 directly can modify the hT40 conformation. Therefore, in the light of the emerging concept of prionlike transmissibility and propagation of Tau in cells (7–9), which seems to be potentiated by the oligomeric status of Tau, we wondered whether the FKBP52-induced Tau-F4 oligomers could manifest the same pathologic properties in cultured neuronal cells. To track this protein in cells, TauF4 was labeled with a far-red fluorescent cyanine dye (TauF4-Cy5.5). As shown by BN-PAGE analysis, FKBP52 was also able to produce Tau-F4-Cy5.5 oligomers, indicating that this fluorescent dye did not interfere with Tau-F4 aggregation (Supplemental Fig. 2). Cell incubation of FKBP52, Tau-F4-Cy5.5, or FKBP52-induced Tau-F4-Cy5.5 oligomers (F4-Cy5.5/FKBP52), respectively, was carried out in neuroblastoma SH-SY5Y cells that over express isoform Tau hT34 (1N, 4R) after doxycycline induction (as inducible Tau hT34 cannot be discriminated from endogenous Tau in cells; henceforth, both are termed endogenous Tau). After washing the cells with trypsin to digest extracellular Tau deposits, confocal immunofluorescence analysis was performed by use of a pan-Tau antibody (K9JA), which is directed against the MTBR of Tau and detects exogenous and endogenous Tau (green). Far-red fluorescence in cells clearly showed that Tau-F4-Cy5.5 (Fig. 8A, middle; arrowheads) and F4-Cy5.5/FKBP52 oligomers (Fig. 8A, right; arrows) are capable of entering cells. Red fluorescence labeling in cells was 35.39% (63.2) and 36.20% (63.7) for cells incubated, respectively, with Tau-F4-CY5.5 alone or with FKBP52, showing that FKBP52 does not interfere with the Tau-F4-Cy5.5 transport into cells. Moreover, no modification of endogenous Tau staining was observed after cell incubation with FKBP52 (Fig. 8A). Large, intracellular accumulations of endogenous Tau colocalized with F4-Cy5.5/FKBP52 oligomers (Fig. 8A, right; arrows) but very rarely with exogenous Tau-F4-Cy5.5 (Fig. 8A, B). We observed significant, 6-fold more endogenous Tau aggregates in F4-Cy5.5/FKBP52 oligomer-infected cells (6.1 6 0.6%) compared with Tau-F4-Cy5.5-infected cells (1.1 6 0.4%; n = 3; 150 infected cells counted/experiment; P , 0.001; Fig. 8F). Rotation of 60° and 90° around the vertical axis of a 3D projection-zoomed cell indicated that internalized F4-Cy5.5/FKBP52 oligomers were in the same plane as the nucleus, further confirming their intracellular localization (Fig. 8B and Supplemental Movie 1). Tau-13 antibody, which is directed against the N terminus of Tau and consequently, only labels endogenous Tau, also recognized this Tau accumulation, supporting that exogenous F4-Cy5.5/FKBP52 oligomers can seed the aggregation ofendogenous wild-type Tau (Fig. 8C). This endogenous

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Tau accumulation was also detected by MC1, a conformation-dependent antibody that recognizes an early, pathologic Tau conformation (29). This observation suggests that F4-Cy5.5/FKBP52 oligomers can induce conformational changes of endogenous Tau (Fig. 8D and Supplemental Movie 2). In addition, we observed accumulation of red labeling (F4-Cy5.5/FKBP52 oligomers), which did not colocalize with endogenous Tau, detected by MC1, suggesting that only some F4-Cy5.5/FKBP52 oligomers encode catalyzing, conformational information (Fig. 8D and Supplemental Movie 2). It is noteworthy that large inclusions also were detected by PHF-1, which is a phosphodependent, anti-Tau antibody (Fig. 8E). The recognition site of PHF-1 (pSer396/pSer404) is missed in Tau-F4 fragment, suggesting that the labeling observed was consistent with the fact that F4-Cy5.5/FKBP52 oligomers could induce phosphorylation of endogenous Tau. However, we cannot rule out the possibility that phosphorylation of endogenous Tau could be a consequence of Tau accumulation in cells. Collectively, these observations demonstrate an ability of FKBP52 to generate in vitro-truncated Tau oligomers with prion-like properties. DISCUSSION In recent years, Tau has been shown to be cleaved by different proteolytic enzymes, for example, caspases, at both their N and C termini (30). The resulting Tau fragments accumulate in tangles of AD neurons (31). A recent study has demonstrated in vivo that caspase activation precedes and leads to tangles in living Tau transgenic mice, suggesting that Tau truncation may be an early change occurring in Tau pathology (32). In addition, different Tau species with an intact N terminus, intact C terminus, and various double-truncated Tau proteins with molecular mass ranging from 10 kDa to high molecular-mass aggregates have been identified in AD brains and in a transgenic rat model for AD (5). These different studies show that Tau is a substrate for many proteases in AD brain, therefore, permitting consideration that truncated Tau fragments used in our study are hence pathologically relevant. Results FKBP52 INDUCES TRUNCATED TAU AGGREGATION IN VITRO

Figure 5. Structure analysis of Tau-F1 and Tau hT40 by TEM. Electron micrographs of Tau proteins (Tau-F1 and Tau hT40) after their incubation with FKBP52 for 30 min at 37°C. A) Structures of Tau-F1 (granular oligomers (left) and fibers (right). B) Electron micrographs show only rare granules of Tau hT40 (left) but no fibers (right).

reported here demonstrate a functional interaction between FKBP52 and truncated forms of Tau, not only in terms of inhibition of MT assembly but also in the ability of FKBP52 to induce oligomerization of these Tau fragments. FKBP52 directly induces Tau fragment aggregation The interaction of truncated forms of Tau (Tau-F4, Ser208Ser324; Tau-F1, Gly164-Leu441) and FKBP52 quickly induces the oligomerization and filament formation of these Tau fragments, as demonstrated by native gels electrophoresis and TEM analysis. Given that no complex of FKBP52 with Tau fragments could be detected in the native gel experiments, we conclude that the short-lived interaction between both proteins initiates a conformational change of Tau-F4 and Tau F1 that leads to their oligomerizations and filament formations in vitro. TEM studies allowed us to detect spherical oligomers and some filaments of Tau-F4 and Tau-F1 after FKBP52 incubation. Similar granular Tau oligomers, also called spherical nucleation units, have been purified from the human frontal cortex of patients with AD (26) and could also be obtained in vitro after the incubation of wild-type hT40 with heparin (27, 33). Here, the periodicity of these Tau-truncated filaments is 2 times smaller compared with the classic PHFs detected in AD brain neurons (34); this observation may be a result of the size of the Tau fragments studied or their different conformations and assemblies orchestrated by FKBP52. Our observations are in agreement with recent studies showing in vitro thin filament formation by heparin of Tau hT40 or a 3-repeat Tau fragment named K19, with approximately similar thickness and periodicity (35). The absence of a projection domain in Tau-F4 and Tau-F1 could explain the observation of bundled filaments, already described for Tau-F4 after its incubation with heparin (28). It is noteworthy that FK506 fails to reverse the FKBP52 effect on Tau-F4 oligomerization, and in addition, no modification on Tau-F4 structures could be detected when experiments were carried out with FKBP12. These results suggest that FKBP52 PPiase activity is not sufficient or necessary to explain the Tau-F4 oligomerization induced by FKBP52. It 3177

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FKBP52 interaction. It is noteworthy that we have recently shown that FKBP52 is able to induce the oligomerization of the pathogenic Tau-P301L protein (20). This single mutation in full-length Tau reduces its ability to interact with MTs (38) and stimulates Tau aggregation into filaments (39, 40). It has been reported that this mutation enhances local b-structure, promoting its aggregation (41); this enhanced, local b-structure in this Tau mutant may make the P301L Tau mutant more prone to aggregation by FKBP52. In the same way, the removal of the N- or C-terminal part of Tau could also interfere intrinsically with Tau structure, leading these Tau mutants, deleted from the N- or C-terminal part, more prone to aggregation by FKBP52. Tau-F4 oligomers induced by FKBP52 show prionlike properties

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Figure 6. Effects of FKBP52 on hT40-ΔC. A) Light-scattering assay. The turbidity detected for 30 min at 37°C was measured for each samples as indicated on the figure. Tau-F4 (5 mM), hT40-ΔC (5 mM), FKBP52 (6 mM), and GST (6 mM). B) Western blotting analysis. The same amount of sample was loaded on a 4–16% BN-PAGE and analyzed by Western blotting by use of anti-Tau antibody (K9JA).

has been proposed recently that FKBP12, a small FKBP, prevents the in vitro aggregation of an oligopeptide, corresponding to the R3 region of the MTBR (R3, 31aa) (36) through its PPiase activity. This discrepancy may be explained by the presence of other several functional domains of FKBP52 and reflects the complexity of the different immunophilins regarding their function on Tau. N- or C-terminal truncation greatly facilitates the effect of FKBP52 on Tau polymerization In our conditions, we failed to detect an effect of FKBP52 on the aggregation of full-length Tau hT40, whereas a distinct FKBP52 activity was demonstrated with the N-terminaltruncated Tau-F1 fragment or a Tau fragment in which the C-terminal part was removed (hTau-ΔC). The simultaneous presence of N and C termini of hT40 interfering with the FKBP52-induced oligomerization would be in agreement with the “paper clip” model proposing that Tau forms in vitro a double hairpin, whereby N and C termini are folded into the proximity of the core domain (MTBR) (37). However, the paper clip conformation of Tau is only transient in solution, so we cannot rule out, at least for Tau-F1, that the net-negative charge of the Tau N-terminal fragment near physiologic pH may be sufficient to interfere with the 3178

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Evidence indicates that Tau oligomers, induced in vitro by polyanions as arachidonic acid or heparin, display prionlike properties, whereby Tau encodes self-catalyzing, conformational information that propagates indefinitely (7, 8, 42). In this study, we have shown that FKBP52-induced TauF4 oligomers can lead to direct Tau hT40 conformational changes, and we report that these oligomers were also able to enter neuronal cells and could propagate the aggregation of wild-type, endogenous Tau, demonstrating that FKBP52 can generate in vitro particular oligomeric structures of Tau-F4 that display prion-like aptitude. This last observation suggests a potential implication of FKBP52 in pathologic Tau spreading in neuronal cells. As seen in cells, Tau-F4-Cy5.5 oligomers induced by FKBP52 do not always appear to promote endogenous Tau aggregation. This observation may be explained by a structural heterogeneity of these Tau-F4 oligomers, whereby only some of them can promote Tau aggregation. It is also possible that some Tau-

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Figure 7. Time-dependent decrease of Tau hT40 monomers after its incubation with FKBP52-induced Tau-F4 oligomers. Purified Tau-F4 oligomers induced by FKBP52 (A) or Tau-F4 monomers used as control (B) were mixed with Tau hT40 at 37°C. Samples collected at different times, as indicated on the figure, were analyzed by BN-PAGE (4/16%) and revealed by Western blotting by use of Tau46 antibody. Quantifications of Tau hT40 monomers are represented by histograms.

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Figure 8. Intracellular aggregates of endogenous Tau can be triggered by exogenous Tau-F4 oligomers induced by FKBP52 in SHSY5Y cells overexpressing Tau. A) SH-SY5Y cells incubated with FKBP52, Cy5.5-labeled Tau-F4 (far-red), or mixed FKBP52/Cy5.5labeled Tau-F4, as indicated in Materials and Methods, were analyzed by confocal immunofluorescence. Endogenous Tau is detected by a pan Tau antibody (K9JA; green). Far-red fluorescence shows that Tau-F4-Cy5.5 (arrowheads) and F4-Cy5.5/FKBP52 oligomers (arrows) are intracellular. B) Yellow box in A. Zoom and 3D projection of an infected cell by the uptake of F4-Cy5.5/ FKBP52 oligomers. Rotation about the vertical axis of 60° and 90° of the cell indicates that internalized F4-Cy5.5/FKBP52 oligomers are present within the endogenous Tau cluster and in the same plane as the nucleus. C–E) The same analysis was, respectively, performed with Tau13 antibody, which is directed against the N terminus of Tau, with a conformation-dependent antibody MC1 showing an early pathologic conformation of Tau and with a phospho-dependent anti-Tau mAb PHF-1. F) Quantification of large clustering of endogenous Tau (.1 mm2 in size), showing 6-fold more Tau accumulations in F4-Cy5.5/FKBP52 oligomer-infected cells compared with Tau-F4-Cy5.5-infected cells (n = 3; 150 infected cells counted/experiment; t test; *P , 0.001). In all images with cell labeling, nucleus was stained with DAPI (blue). Scale bars, 10 mm.

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F4 Cy5.5 oligomers may be neutralized and accumulated, thanks to a cell-defense response, therefore precluding aggregation of endogenous Tau. In our conditions, we also detect that the uptake of monomeric Tau-F4-Cy5.5 in cells can propagate the aggregation of endogenous Tau. This observation is in agreement with recent studies proposing that a fragment of Tau, called K18 (corresponding to the MTBR of Tau), can enter the cells and accumulates in endosomal compartments where it forms aggregate seeds that can induce the aggregation of endogenous Tau (43). Here, we show that FKBP52 can form seeds of Tau-F4 aggregates in vitro that potentiate the propagation of endogenous Tau aggregation in cells. In conclusion, we show for the first time that granular oligomers and filamentous structures of N-terminaltruncated Tau (F4 and F1) can be obtained in vitro by the activity of FKBP52. With the knowledge that Tau is particularly found accumulated and aggregated in multiple truncated forms in AD (44), notably, in N-terminaltruncated forms (5, 45), we speculate that FKBP52, in this pathologic context, may participate in Tau oligomerization and pathogenicity in neurons in vivo. On the other hand, we observe that Tau oligomers induced by FKBP52 behave, in cells that overexpress Tau, similar to a magnet that attracts excess Tau, thereby forming large aggregates. It has been postulated that large aggregates could be inert or even protective for cells (46, 47); therefore, the possibility of a toxic or protective nature of FKBP52-induced Tau-F4 aggregates still needs to be investigated. In any case, our data support the idea that FKBP52 may play a role in Tau pathogenicity and also designate FKBP52 as a new, potential therapeutic target in tauopathies. The authors thank Dr. M. Tawk and Dr. G. Meduri (Institut National de la Sant´e et de la Recherche M´edicale, Unit´e 788, Le Kremlin-Bicˆetre, France) for critical reading and discussion, E. Bourrin for technical help with confocal imaging and analyses, and A. Kamah (Centre Nationale de la Recherche Scientifique Universit´e de Lille 1, Unit´e Mixte de Reccherche 8576, Villeneuve-d’Ascq, France) for technical help for pilot experiments. The authors also thank Dr. P. Davies (Albert Einstein College of Medicine, Bronx, NY, USA) for the generous gift of MC1 and PHF-1 antibodies. E.S., O.D., and K.G. were funded by Fondation Vivre Longtemps, Institut M´erieux, and Institut Baulieu. J.G. is supported by Agence Nationale de la Recherche [ANR; Program Maladie d’Alzheimer et Maladies Apparent´ees (MALZ): Tau Association with FKBP52 (TAF)]. The work has been supported jointly by ANR and by Laboratoire d’Excellence D´eveloppement de Strat´egies Innovantes pour une Approche Transdisciplinaire de la Maladie d’Alzheimer (LABEX DISTALZ) grants. The authors declare no competing financial interests.

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38. Hong, M., Zhukareva, V., Vogelsberg-Ragaglia, V., Wszolek, Z., Reed, L., Miller, B. I., Geschwind, D. H., Bird, T. D., McKeel, D., Goate, A., Morris, J. C., Wilhelmsen, K. C., Schellenberg, G. D., Trojanowski, J. Q., and Lee, V. M. (1998) Mutation-specific functional impairments in distinct tau isoforms of hereditary FTDP-17. Science 282, 1914–1917 39. Goedert, M., Jakes, R., and Crowther, R. A. (1999) Effects of frontotemporal dementia FTDP-17 mutations on heparin-induced assembly of tau filaments. FEBS Lett. 450, 306–311 40. Nacharaju, P., Lewis, J., Easson, C., Yen, S., Hackett, J., Hutton, M., and Yen, S. H. (1999) Accelerated filament formation from tau protein with specific FTDP-17 missense mutations. FEBS Lett. 447, 195–199 41. Von Bergen, M., Barghorn, S., Li, L., Marx, A., Biernat, J., Mandelkow, E. M., and Mandelkow, E. (2001) Mutations of tau protein in frontotemporal dementia promote aggregation of paired helical filaments by enhancing local beta-structure. J. Biol. Chem. 276, 48165–48174 42. Sanders, D. W., Kaufman, S. K., DeVos, S. L., Sharma, A. M., Mirbaha, H., Li, A., Barker, S. J., Foley, A. C., Thorpe, J. R., Serpell, L. C., Miller, T. M., Grinberg, L. T., Seeley, W. W., and Diamond, M. I. (2014) Distinct tau prion strains propagate in cells and mice and define different tauopathies. Neuron 82, 1271–1288 43. Michel, C. H., Kumar, S., Pinotsi, D., Tunnacliffe, A., St George-Hyslop, P., Mandelkow, E., Mandelkow, E. M., Kaminski, C. F., and Kaminski Schierle, G. S. (2014) Extracellular monomeric tau protein is sufficient to initiate the spread of tau protein pathology. J. Biol. Chem. 289, 956–967 44. Wang, Y., Garg, S., Mandelkow, E. M., and Mandelkow, E. (2010) Proteolytic processing of tau. Biochem. Soc. Trans. 38, 955–961 45. Nieto, A., Correas, I., L´opez-Ot´ın, C., and Avila, J. (1991) Tau-related protein present in paired helical filaments has a decreased tubulin binding capacity as compared with microtubule-associated protein tau. Biochim. Biophys. Acta 1096, 197–204 46. Bretteville, A., and Planel, E. (2008) Tau aggregates: toxic, inert, or protective species? J. Alzheimers Dis. 14, 431–436 47. Santacruz, K., Lewis, J., Spires, T., Paulson, J., Kotilinek, L., Ingelsson, M., Guimaraes, A., DeTure, M., Ramsden, M., McGowan, E., Forster, C., Yue, M., Orne, J., Janus, C., Mariash, A., Kuskowski, M., Hyman, B., Hutton, M., and Ashe, K. H. (2005) Tau suppression in a neurodegenerative mouse model improves memory function. Science 309, 476–481 Received for publication December 3, 2014. Accepted for publication March 31, 2015.

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