A decrease in Fkbp52 alters autophagosome maturation and A152T-tau clearance in vivo.

The failure of the autophagy-lysosomal pathway to clear the pathogenic forms of Tau exacerbates the pathogenesis of tauopathies. We have previously shown that the immunophilin FKBP52 interacts both physically and functionally with Tau, and that a decrease in FKBP52 protein levels is associated with Tau deposition in affected human brains. We have also shown that FKBP52 is physiologically present within the lysosomal system in healthy human neurons and that a decrease in FKBP52 expression alters perinuclear lysosomal positioning and Tau clearance during Tau-induced proteotoxic stress in vitro. In this study, we generate a zebrafish fkbp4 loss of function mutant and show that axonal retrograde trafficking of Lamp1 vesicles is altered in this mutant. Moreover, using our transgenic HuC::mCherry-EGFP-LC3 line, we demonstrate that the autophagic flux is impaired in fkbp4 mutant embryos, suggesting a role for Fkbp52 in the maturation of autophagic vesicles. Alterations in both axonal transport and autophagic flux are more evident in heterozygous rather than homozygous fkbp4 mutants. Finally, taking advantage of the previously described A152T-Tau transgenic fish, we show that the clearance of pathogenic A152T-Tau mutant proteins is slower in fkbp4 +/- mutants in comparison to fkbp4 +/+ larvae. Altogether, these results indicate that Fkbp52 is required for the normal trafficking and maturation of lysosomes and autophagic vacuoles along axons, and that its decrease is sufficient to hinder the clearance of pathogenic Tau in vivo.

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.

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.

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.

Kinesin-1 regulates microtubule dynamics via a c-Jun N-terminal kinase-dependent mechanism.

In the kinesin family, all the molecular motors that have been implicated in the regulation of microtubule dynamics have been shown to stimulate microtubule depolymerization. Here, we report that kinesin-1 (also known as conventional kinesin or KIF5B) stimulates microtubule elongation and rescues. We show that microtubule-associated kinesin-1 carries the c-Jun N-terminal kinase (JNK) to allow its activation and that microtubule elongation requires JNK activity throughout the microtubule life cycle. We also show that kinesin-1 and JNK promoted microtubule rescues to similar extents. Stimulation of microtubule rescues by the kinesin-1/JNK pathway could not be accounted for by the rescue factor CLIP-170. Indeed only a dual inhibition of kinesin-1/JNK and CLIP-170 completely blocked rescues and led to extensive microtubule loss. We propose that the kinesin-1/JNK signaling pathway is a major regulator of microtubule dynamics in living cells and that it is required with the rescue factor CLIP-170 to allow cells to build their interphase microtubule network.

Tubulin acetylation favors Hsp90 recruitment to microtubules and stimulates the signaling function of the Hsp90 clients Akt/PKB and p53.

Involved in a wide range of cellular processes such as signal transduction, microtubules are highly dynamic polymers that accumulate various post-translational modifications including polyglutamylation, polyglycylation, carboxyterminal cleavage and acetylation, the functions of which just begin to be uncovered. The molecular chaperone Hsp90, which is essential for the folding and activity of numerous client proteins involved in cell proliferation and apoptosis, associates with the microtubule network but the effects of tubulin post-translational modifications on its microtubule binding has not yet been investigated. Herein, we show that both the constitutive (beta) and the inducible (alpha) Hsp90 isoforms bind to microtubules in a way that depends on the level of tubulin acetylation. Tubulin acetylation also stimulates the binding and the signaling function of at least two of its client proteins, the kinase Akt/PKB and the transcription factor p53. This study highlights the role of tubulin acetylation in modulating microtubule-based transport of Hsp90-chaperoned proteins and thus in regulating signaling dynamics in the cytoplasm.