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.

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.

Regulation of lipid droplet dynamics in Saccharomyces cerevisiae depends on the Rab7-like Ypt7p, HOPS complex and V1-ATPase.

It has now been clearly shown that lipid droplets (LDs) play a dynamic role in the cell. This was reinforced by LD proteomics which suggest that a significant number of trafficking proteins are associated with this organelle. Using microscopy, we showed that LDs partly co-localize with the vacuole in S. cerevisiae. Immunoblot experiments confirmed the association of the vacuolar Rab GTPase Rab7-like Ypt7p with LDs. We observed an increase in fatty acid content and LD number in ypt7Δ mutant and also changes in LD morphology and intra LD fusions, revealing a direct role for Ypt7p in LD dynamics. Using co-immunoprecipitation, we isolated potential Ypt7p partners including, Vma13p, the H subunit of the V1 part of the vacuolar (H+) ATPase (V-ATPase). Deletion of the VMA13 gene, as well as deletion of three other subunits of the V1 part of the V-ATPase, also increased the cell fatty acid content and LD number. Mutants of the Homotypic fusion and vacuole protein sorting (HOPS) complex showed similar phenotypes. Here, we demonstrated that LD dynamics and membrane trafficking between the vacuole and LDs are regulated by the Rab7-like Ypt7p and are impaired when the HOPS complex and the V1 domain of the V-ATPase are defective.