Concomitant Neuronal Tau Deposition and FKBP52 Decrease Is an Early Feature of Different Human and Experimental Tauopathies.

BACKGROUND: Pathological tau proteins constitute neurofibrillary tangles that accumulate in tauopathies including Alzheimer's disease (AD), progressive supranuclear palsy (PSP), and familial frontotemporal lobar degeneration (FTLD-Tau). We previously showed that the FKBP52 immunophilin interacts functionally with tau and strongly decreases in AD brain neurons in correlation with tau deposition. We also reported that FKBP52 co-localizes with autophagy-lysosomal markers and an early pathological tau isoform in AD neurons, suggesting its involvement in autophagic tau clearance. OBJECTIVE: Our objective was to evaluate if differences in neuronal FKBP52 expression levels and subcellular localization might be detected in AD, PSP, familial FTLD-Tau, and in the hTau-P301 S mouse model compared to controls. METHODS: Cell by cell immunohistofluorescence analyses and quantification of FKBP52 were performed on postmortem brain samples of some human tauopathies and on hTau-P301 S mice spinal cords. RESULTS: We describe a similar FKBP52 decrease and its localization with early pathological tau forms in the neuronal autophagy-lysosomal pathway in various tauopathies and hTau-P301 S mice. We find that FKBP52 decreases early during the pathologic process as it occurs in rare neurons with tau deposits in the marginally affected frontal cortex region of AD Braak IV brains and in the spinal cord of symptomless 1-month-old hTau-P301 S mice. CONCLUSION: As FKBP52 plays a significant role in cellular signaling and conceivably in tau clearance, our data support the idea that the prevention of FKBP52 decrease or the restoration of its normal expression at early pathologic stages might represent a new potential therapeutic approach in tauopathies including AD, familial FTLD-Tau, and PSP.

FKBP52 in Neuronal Signaling and Neurodegenerative Diseases: A Microtubule Story.

The FK506-binding protein 52 (FKBP52) belongs to a large family of ubiquitously expressed and highly conserved proteins (FKBPs) that share an FKBP domain and possess Peptidyl-Prolyl Isomerase (PPIase) activity. PPIase activity catalyzes the isomerization of Peptidyl-Prolyl bonds and therefore influences target protein folding and function. FKBP52 is particularly abundant in the nervous system and is partially associated with the microtubule network in different cell types suggesting its implication in microtubule function. Various studies have focused on FKBP52, highlighting its importance in several neuronal microtubule-dependent signaling pathways and its possible implication in neurodegenerative diseases such as tauopathies (i.e., Alzheimer disease) and alpha-synucleinopathies (i.e., Parkinson disease). This review summarizes our current understanding of FKBP52 actions in the microtubule environment, its implication in neuronal signaling and function, its interactions with other members of the FKBPs family and its involvement in neurodegenerative disease.

Decrease of neuronal FKBP4/FKBP52 modulates perinuclear lysosomal positioning and MAPT/Tau behavior during MAPT/Tau-induced proteotoxic stress.

Defects of autophagy-lysosomal protein degradation are thought to contribute to the pathogenesis of several neurodegenerative diseases, and the accumulation of aggregation prone proteins such as MAPT/Tau in Alzheimer disease (AD). We previously showed the localization of the immunophilin FKBP4/FKBP52 in the lysosomal system of healthy human neurons suggesting its possible role in lysosome function. We also showed that decreased FKBP4 levels in AD brain neurons correlate with abnormal MAPT accumulation and aggregation. In this study, we demonstrate that FKBP4 decrease in a human neuronal cell line (SH-SY5Y) and in dorsal root ganglion (DRG) neurons from human MAPTP301S transgenic mice affected the function of the autophagy-lysosomal system under MAPT induced proteotoxic stress conditions. We show that acute MAPT accumulation in SH-SY5Y cells induced perinuclear clustering of lysosomes, triggered FKBP4 localization around the clusters and its colocalization with MAPT and MAP1LC3/LC3-positive autophagic vesicles; a similar FKBP4 localization was detected in some AD brain neurons. We demonstrate that FKBP4 decrease altered lysosomal clustering along with MAPT and MAP1LC3 secretion increase. Although ectopic FKBP4 expression could not induce autophagy under our experimental conditions, it prevented MAPT secretion after MAPT accumulation in SH-SY5Y cells implying a regulatory role of FKBP4 on MAPT secretion. Finally, we observe that FKBP4 deficiency decreased MAP1LC3-II expression and provoked MAPT accumulation during long-term stress in mouse DRG neurons. We hypothesize that the abnormal FKBP4 decrease observed in AD brain neurons might hinder autophagy efficiency and contribute to the progression of the tauopathy by modulating MAPT secretion and accumulation during MAPT pathogenesis.Abbreviations: AD: Alzheimer disease; AKT/protein kinase B: AKT serine/threonine kinase; ALP: Autophagy-lysosomal pathway; ATG: autophagy-related; BafA1: bafilomycin A1; CQ: chloroquine; CTSD: cathepsin D; DIV: days in vitro; DRG: dorsal root ganglion neurons; Dox: doxycycline; DNAJC5: DnaJ heat shock protein family (Hsp40) member C5; EL: empty lentiviral vectors; ENO2/NSE: enolase 2, gamma neuronal; FKBP4/FKBP52: FKBP prolyl isomerase 4; FTLD-Tau: frontotemporal lobar degeneration with Tau pathology; GFP: green fluorescent protein; LAMP1: lysosomal associated membrane protein 1; LDH: lactate dehydrogenase; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; MAPT/Tau: microtubule associated protein tau; MTT: tetrazolium salt; NFTs: neurofibrillary tangles; RPE-1: retinal pigment epithelial cells; shRNA: small-hairpin ribonucleic acid; SQSTM1/p62: sequestosome 1; SD: standard deviation; SEM: standard error of the mean; SH-SY5Y: human neuroblastoma cells; Sh1 or Sh2: Lentiviral shRNA vectors inducing FKBP4 decrease; SH-52GFP: MAPT/Tau-inducible SH-SY5Y cell line constitutively expressing FKBP4-GFP; TUBB3/ßIII tubulin: tubulin beta 3 class III; UPS: ubiquitin-proteasome system.

Bioinspired Hybrid Fluorescent Ligands for the FK1 Domain of FKBP52.

The protein FKBP52 is a steroid hormone receptor coactivator likely involved in neurodegenerative disease. A series of small, water-soluble, bioinspired, pseudopeptidic fluorescent ligands for the FK1 domain of this protein are described. The design is such that engulfing of the ligand in the pocket of this domain is accompanied by hydrogen-bonding of the dansyl chromophore which functions as both an integral part of the ligand and a fluorescent reporter. Binding is concomitant with a significant wavelength shift and an enhancement of the ligand fluorescence signal. Excitation of FK1 domain native tryptophan residues in the presence of bound ligand results in Förster resonance energy transfer. Variation of key ligand residues within the short sequence was undertaken, and the interaction of the resulting library with the protein was measured by techniques including isothermal calorimetry analysis, fluorescence, and FRET quenching, and a range of Kd values were determined. Cocrystallization of a protein ligand complex at 2.30 Å resolution provided detailed information at the atomic scale, while also providing insight into native substrate binding.

Major Differences between the Self-Assembly and Seeding Behavior of Heparin-Induced and in Vitro Phosphorylated Tau and Their Modulation by Potential Inhibitors.

Self-assembly of the microtubule-associated protein tau into neurotoxic oligomers, fibrils, and paired helical filaments, and cell-to-cell spreading of these pathological tau species are critical processes underlying the pathogenesis of Alzheimer's disease and other tauopathies. Modulating the self-assembly process and inhibiting formation and spreading of such toxic species are promising strategies for therapy development. A challenge in investigating tau self-assembly in vitro is that, unlike most amyloidogenic proteins, tau does not aggregate in the absence of posttranslational modifications (PTM), aggregation inducers, or preformed seeds. The most common induction method is addition of polyanions, such as heparin; yet, this artificial system may not represent adequately tau self-assembly in vivo, which is driven by aberrant phosphorylation and other PTMs, potentially leading to in vitro data that do not reflect the behavior of tau and its interaction with modulators in vivo. To tackle these challenges, methods for in vitro phosphorylation of tau to produce aggregation-competent forms recently have been introduced ( Despres et al. ( 2017 ) Proc. Natl. Acad. Sci. U.S.A. , 114 , 9080 - 9085 ). However, the oligomerization, seeding, and interaction with assembly modulators of the different forms of tau have not been studied to date. To address these knowledge gaps, we compared here side-by-side the self-assembly and seeding activity of heparin-induced tau with two forms of in vitro phosphorylated tau and tested how the molecular tweezer CLR01, a negatively charged compound, affected these processes. Tau was phosphorylated by incubation either with activated extracellular signal-regulated kinase 2 or with a whole rat brain extract. Seeding activity was measured using a fluorescence-resonance energy transfer-based biosensor-cell method. We also used solution-state NMR to investigate the binding sites of CLR01 on tau and how they were impacted by phosphorylation. Our systematic structure-activity relationship study demonstrates that heparin-induced tau behaves differently from in vitro phosphorylated tau. The aggregation rates of the different forms are distinct as is the intracellular localization of the induced aggregates, which resemble brain-derived tau strains suggesting that heparin-induced tau and in vitro phosphorylated tau have different conformations, properties, and activities. CLR01 inhibits aggregation and seeding of both heparin-induced and in vitro phosphorylated tau dose-dependently, although heparin induction interferes with the interaction between CLR01 and tau.

Direct Crosstalk Between O-GlcNAcylation and Phosphorylation of Tau Protein Investigated by NMR Spectroscopy.

The formation of intraneuronal fibrillar inclusions of tau protein is associated with several neurodegenerative diseases referred to as tauopathies including Alzheimer's disease (AD). A common feature of these pathologies is hyperphosphorylation of tau, the main component of fibrillar assemblies such as Paired Helical Filaments (PHFs). O-ß-linked N-acetylglucosaminylation (O-GlcNAcylation) is another important posttranslational modification involved in regulation of tau pathophysiology. Among the benefits of O-GlcNAcylation, modulation of tau phosphorylation levels and inhibition of tau aggregation properties have been described while decreased O-GlcNAcylation could be involved in the raise of tau phosphorylation associated with AD. However, the molecular mechanisms at the basis of these observations remain to be defined. In this study, we identify by NMR spectroscopy O-GlcNAc sites in the longest isoform of tau and investigate the direct role of O-GlcNAcylation on tau phosphorylation and conversely, the role of phosphorylation on tau O-GlcNAcylation. We show here by a systematic examination of the quantitative modification patterns by NMR spectroscopy that O-GlcNAcylation does not modify phosphorylation of tau by the kinase activity of ERK2 or a rat brain extract while phosphorylation slightly increases tau O-GlcNAcylation by OGT. Our data suggest that indirect mechanisms act in the reciprocal regulation of tau phosphorylation and O-GlcNAcylation in vivo involving regulation of the enzymes responsible of phosphate and O-GlcNAc dynamics.

Identification of the Tau phosphorylation pattern that drives its aggregation.

Determining the functional relationship between Tau phosphorylation and aggregation has proven a challenge owing to the multiple potential phosphorylation sites and their clustering in the Tau sequence. We use here in vitro kinase assays combined with NMR spectroscopy as an analytical tool to generate well-characterized phosphorylated Tau samples and show that the combined phosphorylation at the Ser202/Thr205/Ser208 sites, together with absence of phosphorylation at the Ser262 site, yields a Tau sample that readily forms fibers, as observed by thioflavin T fluorescence and electron microscopy. On the basis of conformational analysis of synthetic phosphorylated peptides, we show that aggregation of the samples correlates with destabilization of the turn-like structure defined by phosphorylation of Ser202/Thr205.

The Study of Posttranslational Modifications of Tau Protein by Nuclear Magnetic Resonance Spectroscopy: Phosphorylation of Tau Protein by ERK2 Recombinant Kinase and Rat Brain Extract, and Acetylation by Recombinant Creb-Binding Protein.

Nuclear magnetic resonance (NMR) spectroscopy can be used as an analytical tool to investigate posttranslational modifications of protein. NMR is a valuable tool to map the interaction regions of protein partners. Here, we present protocols that have been developed in the course of our studies of the neuronal Tau protein. Tau is found aggregated in the neurons of Alzheimer's disease patients. Development of the disease is accompanied by increased, abnormal phosphorylation and acetylation of Tau. We have used NMR to investigate how these posttranslational modifications of Tau affect the interactions with its partners. We present here detailed protocols of in vitro phosphorylation of Tau by recombinant kinase, ERK2, or kinase activity of rat brain extracts, and acetylation by recombinant Creb-binding protein (CBP) acetyltransferase. The analytical characterization of the modified Tau by NMR spectroscopy is additionally described.

Neuronal Ndrg4 Is Essential for Nodes of Ranvier Organization in Zebrafish.

Axon ensheathment by specialized glial cells is an important process for fast propagation of action potentials. The rapid electrical conduction along myelinated axons is mainly due to its saltatory nature characterized by the accumulation of ion channels at the nodes of Ranvier. However, how these ion channels are transported and anchored along axons is not fully understood. We have identified N-myc downstream-regulated gene 4, ndrg4, as a novel factor that regulates sodium channel clustering in zebrafish. Analysis of chimeric larvae indicates that ndrg4 functions autonomously within neurons for sodium channel clustering at the nodes. Molecular analysis of ndrg4 mutants shows that expression of snap25 and nsf are sharply decreased, revealing a role of ndrg4 in controlling vesicle exocytosis. This uncovers a previously unknown function of ndrg4 in regulating vesicle docking and nodes of Ranvier organization, at least through its ability to finely tune the expression of the t-SNARE/NSF machinery.

A ß-Turn Motif in the Steroid Hormone Receptor's Ligand-Binding Domains Interacts with the Peptidyl-prolyl Isomerase (PPIase) Catalytic Site of the Immunophilin FKBP52.

The immunophilin FKBP52 interacts with nuclear steroid hormone receptors. Studying the crystal structure of human estrogen receptor α (hERα) and using nuclear magnetic resonance, we show here that the short V(364)PGF(367) sequence, which is located within its ligand-binding domain and adopts a type II ß-turn conformation in the protein, binds the peptidyl-prolyl isomerase (PPIase or rotamase) FK1 domain of FKBP52. Interestingly, this turn motif displays strong similarities with the FKBP52 FK1 domain-binding moiety of macrolide immunomodulators such as rapamycin and GPI-1046, an immunophilin ligand with neuroprotective characteristics. An increase in the hydrophobicity of the residue preceding the proline and cyclization of the VPGF peptide strengthen its recognition by the FK1 domain of FKBP52. Replacement of the Pro residue with a dimethylproline also enhances this interaction. Our study not only contributes to a better understanding of how the interaction between the FK1 domain of FKBP52 and steroid hormone receptors most likely works but also opens new avenues for the synthesis of FKBP52 FK1 peptide ligands appropriate for the control of hormone-dependent physiological mechanisms or of the functioning of the Tau protein. Indeed, it has been shown that FKBP52 is involved in the intraneuronal dynamics of the Tau protein.

Caspase-cleaved Tau-D(421) is colocalized with the immunophilin FKBP52 in the autophagy-endolysosomal system of Alzheimer's disease neurons.

Pathologic modifications of the Tau protein leading to neurofibrillary tangle (NFT) formation are a common feature of a wide range of neurodegenerative diseases known as tauopathies, which include Alzheimer's disease (AD). We previously showed that the immunophilin FKBP52 physically and functionally interacts with Tau, and we recently reported that FKBP52 levels are abnormally low in AD patients' brains. To decipher the mechanism of FKBP52 decrease in AD brains, we performed multiple labeling immunohistofluorescence and lysosomal purification using postmortem brain samples of healthy controls (n = 8) and AD (n = 20) patients. Confocal analysis revealed that FKBP52 localizes to the endolysosomal system. We also report FKBP52 colocalization with the truncated Tau-D(421) in the autophagy-endolysosomal system in some AD neurons and that the decrease of FKBP52 correlates with NFT formation. Additional experiments of autophagy inhibition in Tau-inducible SH-SY5Y cells allowed demonstrating FKBP52 release in the extracellular milieu. Our findings point out the possibility that FKBP52 could be abnormally released from NFTs negative neurons in AD brains in correlation with the early pathologic Tau-D(421) neuronal accumulation.

Characterization of Neuronal Tau Protein as a Target of Extracellular Signal-regulated Kinase.

Tau neuronal protein has a central role in neurodegeneration and is implicated in Alzheimer disease development. Abnormal phosphorylation of Tau impairs its interaction with other proteins and is associated with its dysregulation in pathological conditions. Molecular mechanisms leading to hyperphosphorylation of Tau in pathological conditions are unknown. Here, we characterize phosphorylation of Tau by extracellular-regulated kinase (ERK2), a mitogen-activated kinase (MAPK) that responds to extracellular signals. Analysis ofin vitrophosphorylated Tau by activated recombinant ERK2 with nuclear magnetic resonance spectroscopy (NMR) reveals phosphorylation of 15 Ser/Thr sites.In vitrophosphorylation of Tau using rat brain extract and subsequent NMR analysis identifies the same sites. Phosphorylation with rat brain extract is known to transform Tau into an Alzheimer disease-like state. Our results indicate that phosphorylation of Tau by ERK2 alone is sufficient to produce the same characteristics. We further investigate the mechanism of ERK2 phosphorylation of Tau. Kinases are known to recognize their protein substrates not only by their specificity for a targeted Ser or Thr phosphorylation site but also by binding to linear-peptide motifs called docking sites. We identify two main ERK2 docking sites in Tau sequence using NMR. Our results suggest that ERK2 dysregulation in Alzheimer disease could lead to abnormal phosphorylation of Tau resulting in the pathology of the disease.

The FK506-binding protein FKBP52 in vitro induces aggregation of truncated Tau forms with prion-like behavior.

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, Ser(208)-Ser(324)) 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 µM) prevents MT formation generated by Tau-F4 (5 µM) 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.

Immunophilin FKBP52 induces Tau-P301L filamentous assembly in vitro and modulates its activity in a model of tauopathy.

The Tau protein is the major component of intracellular filaments observed in a number of neurodegenerative diseases known as tauopathies. The pathological mutant of Tau containing a proline-to-leucine mutation at position 301 (P301L) leads to severe human tauopathy. Here, we assess the impact of FK506-binding protein with a molecular mass of ∼52 kDa (FKBP52), an immunophilin protein that interacts with physiological Tau, on Tau-P301L activity. We identify a direct interaction of FKBP52 with Tau-P301L and its phosphorylated forms and demonstrate FKBP52's ability to induce the formation of Tau-P301L oligomers. EM analysis shows that Tau-P301L oligomers, induced by FKBP52, can assemble into filaments. In the transgenic zebrafish expressing the human Tau-P301L mutant, FKBP52 knockdown is sufficient to redrive defective axonal outgrowth and branching related to Tau-P301L expression in spinal primary motoneurons. This result correlates with a significant reduction of pT181 pathological phosphorylated Tau and with recovery of the stereotypic escape response behavior. Collectively, FKBP52 appears to be an endogenous candidate that directly interacts with the pathogenic Tau-P301L and modulates its function in vitro and in vivo.

Decrease of the immunophilin FKBP52 accumulation in human brains of Alzheimer's disease and FTDP-17.

Human neurodegenerative diseases characterized by abnormal intraneuronal inclusions of the tau protein, or "tauopathies", include Alzheimer's disease (AD), Pick's disease, progressive supranuclear palsy, corticobasal degeneration as well as fronto-temporal dementia and Parkinsonism linked to chromosome 17 (FTDP-17). Several abnormalities of tau may contribute to the pathological processes, yet the mechanisms involved in tau cellular toxicity remain unclear. Previously, we demonstrated an interaction between various isoforms of tau and the immunophilin FKBP52 (FK506-Binding Protein), suggesting a direct involvement of FKBP52 in tau function. Here we analyze the expression of FKBP52 in human brains of patients with different tauopathies, including AD. Immunohistofluorescence studies carried out on cerebral cortex in different tauopathies reveal that FKBP52 is not sequestered by filamentous tau inclusions while FKBP52 is colocalized with tau in the control case brains. We found that FKBP52 expression level is abnormally low in frontal cortex of AD and FTDP-17 brains, as compared to controls, despite no alteration in the FKBP52 mRNA expression level. The possible involvement of FKBP52 in pathological tau expression/function is discussed.

A role for FKBP52 in Tau protein function.

Tau is a microtubule-associated protein, which is widely expressed in the central nervous system, predominantly in neurons, where it regulates microtubule dynamics, axonal transport, and neurite outgrowth. The aberrant assembly of Tau is the hallmark of several human neurodegenerative diseases, collectively known as tauopathies. They include Alzheimer's disease, Pick's disease, progressive supranuclear palsy, and frontotemporal dementia and parkinsonism linked to chromosome 17. Several abnormalities in Tau, such as hyperphosphorylation and aggregation, alter its function and are central to the pathogenic process. Here, we describe biochemical and functional interactions between FKBP52 and Tau. FKBP52 is a member of the FKBP (FK506-binding protein) family that comprises intracellular protein effectors of immunosuppressive drugs (such as FK506 and rapamycin). We found that FKBP52, which is abundant in brain, binds directly and specifically to Tau, especially in its hyperphosphorylated form. The relevance of this observation was confirmed by the colocalization of both proteins in the distal part of the axons of cortical neurons and by the antagonistic effect of FKBP52 on the ability of Tau to promote microtubule assembly. Overexpression of FKBP52 in differentiated PC12 cells prevented the accumulation of Tau and resulted in reduced neurite length. Taken together, these findings indicate a role for FKBP52 in Tau function and may help to decipher and modulate the events involved in Tau-induced neurodegeneration.

The immunophilin FKBP52 specifically binds to tubulin and prevents microtubule formation.

The FK506 binding protein FKBP52 belongs to the large family of immunophilins and is known as a steroid receptor-associated protein. Previous data suggest that FKBP52 is associated with the motor protein dynein and with the cytoskeleton during mitosis. Here we demonstrate a specific and direct interaction between FKBP52 and tubulin. The region of FKBP52 located between aa 267 and 400, which includes the tetratricopeptide repeat domain, is required for tubulin binding. We provide evidence that FKBP52 prevents tubulin polymerization and that an 84 residue sequence located in the C-terminal part of the molecule (aa 375-458) is necessary and sufficient for its microtubule depolymerization activity. In colocalization experiments in PC12 cells, FKBP52 is associated with tubulin in motile cellular compartments. Furthermore, we suggest that, by using siRNA, a decrease of FKBP52 expression in PC12 cells may lead to differentiated cell phenotype characterized by neurite extensions. Collectively, our data define an unexpected property of FKBP52 as a novel regulator of microtubule dynamics. The possible role of microtubule formation and tubulin binding of other immunophilins such as FKBP12 and FKBP51 is discussed.

Microtubule-associated protein 2 (MAP2) is a neurosteroid receptor.

The neurosteroid pregnenolone (PREG) and its chemically synthesized analog 3beta-methoxypregnenolone (MePREG) bind to microtubule-associated protein 2 (MAP2) and stimulate the polymerization of microtubules. PREG, MePREG, and progesterone (PROG; the physiological immediate metabolite of PREG) significantly enhance neurite outgrowth of nerve growth factor-pretreated PC12 cells. However, PROG, although it binds to MAP2, does not increase the immunostaining of MAP2, contrary to PREG and MePREG. Nocodazole, a microtubule-disrupting agent, induces a major retraction of neurites in control cultures, but pretreatment with PREG/MePREG is protective. Decreasing MAP2 expression by RNA interference does not modify PROG action, but it prevents the stimulatory effects of PREG and MePREG on neurite extension, showing that MAP2 is their specific receptor.

The FKBP-associated protein FAP48 is an antiproliferative molecule and a player in T cell activation that increases IL2 synthesis.

FAP48 was identified and cloned thanks to its interaction with FK506-binding proteins (FKBPs) such as FKBP52 and FKBP12, which belong to the large family of immunophilins that bind the macrolide immunosuppressant drugs FK506 and rapamycin. We have previously shown that FAP48-FKBP complexes are dissociated by FK506 and rapamycin, suggesting that FAP48 is an endogenous ligand of FKBP. The present work describes the biochemical consequences of FAP48 overexpression, induced by the tetracycline analogue doxycycline, in an established cell line derived from Jurkat T cells. We report that overexpression of FAP48 results in the inhibition of cellular proliferation as does the exposure of Jurkat T cells to FK506. We also show that the expression levels of argininosuccinate synthetase and the Myc antagonist Mxi1 are modified by overexpression of FAP48, suggesting that these proteins could be good candidates to mediate the antiproliferative effect of FAP48. FAP48 affects neither the calcineurin-dependent nuclear factor of activated T cells (NFAT)1 nor JNKp38-dependent pathways that mediate immunosuppression by FK506. However, contrary to FK506, which blocks IL2 synthesis, we observed that FAP48-FKBP complexes increase IL2 production, thus revealing a previously uncharacterized aspect of the immunosuppressive mechanism of FK506.