The Hsp90 cochaperone, FKBP51, increases Tau stability and polymerizes microtubules

Abstract

Imbalanced protein load within cells is a critical aspect for most diseases of aging. In particular, the accumulation of proteins into neurotoxic aggregates is a common thread for a host of neurodegenerative diseases. Our previous work demonstrated that age-related changes to the cellular chaperone repertoire contributes to abnormal buildup of the microtubule-associated protein tau that accumulates in a group of diseases termed tauopathies, the most common being Alzheimer's disease. Here, we show that the Hsp90 cochaperone, FK506-binding protein 51 (FKBP51), which possesses both an Hsp90-interacting tetratricopeptide domain and a peptidyl-prolyl cis-trans isomerase (PPIase) domain, prevents tau clearance and regulates its phosphorylation status. Regulation of the latter is dependent on the PPIase activity of FKBP51. FKB51 enhances the association of tau with Hsp90, but the FKBP51/tau interaction is not dependent on Hsp90. In vitro FKBP51 stabilizes microtubules with tau in a reaction depending on the PPIase activity of FKBP51. Based on these new findings, we propose that FKBP51 can use the Hsp90 complex to isomerize tau, altering its phosphorylation pattern and stabilizing microtubules.

Figure 1.

The similar Hsp90 immunophilins, FKBP51 and FKBP52, have divergent effects on tau stability. A, HeLa cells stably overexpressing V5-tagged wild-type human tau were transiently transfected with indicated siRNAs or scrambled negative control siRNA (Ctrl) for 48 h and treated with 1 μm Hsp90 inhibitor 17AAG for 24 h and analyzed by Western. P23 siRNA was used as a positive tau-reducing control. B, Western blot quantification by densitometry from repeated experiments. Levels of tau were calculated from A after GAPDH normalization ± SD. C, Knockdown for each gene was confirmed by Western blot. Ctrl indicates cells transfected with scrambled negative control.

Figure 2.

FKBP51 siRNA potently reduces tau levels in relevant cell models. A, HeLa cells stably overexpressing wild-type human tau were transfected with p23, FKBP51, or scrambled negative control siRNA (Ctrl), and harvested at 0, 6, 24, 48, and 72 h after transfection. Total tau (V5) levels appeared similarly reduced concomitant with p23 or FKBP51 decreases compared to Ctrl. B, Quantitation of p23 and FKBP51 levels after respective siRNA transfection are shown as a percentage of Ctrl siRNA transfected at each time point. C, Quantitation of total tau levels with FKBP51 siRNA (gray squares) and p23 siRNA (black diamonds) are shown to be similar as a percentage of Ctrl at corresponding each time point in hours (Hrs). D, IMR32 cells were transfected with FKBP51, p23, or Ctrl siRNA for 72 h, and lysates were analyzed by Western blot for indicated antibodies.

Figure 3.

FKBP51 increases with age and associates with tau from normal and AD brain. A, Brain lysates from mice harvested at indicated age (in months) were analyzed for FKBP51 levels by Western blot. Detectable levels of FKBP51 were first seen in 5.5-month-old mice and were markedly apparent at 9 months. B, Immunofluorescent staining of FKBP51 (green) and total tau (red) in brain tissue from 9-month-old mice shows very strong colocalization of these two proteins within neuronal axon processes (arrows). C, Higher magnification (100×) of neuronal cells from hippocampus CA1 region of mouse brain, immunostained with FKBP51 antibody (green), tau antibody (red), and nuclei (blue). Merged image shows colocalization of these two proteins. Scale bars, 5 μm. D, Brain tissue lysates from Alzheimer's disease (AD 1 and 2) and age-matched normal (Ctrl 1 and 2) brain tissue lysates were immunoprecipitated with mouse anti-FKBP51 or nonspecific mouse IgG antibodies. Brain lysates (Inputs) and immunoprecipitates were analyzed by Western blot for total tau (Santa Cruz Biotechnology), pS396/S404 tau, FKBP51, and GAPDH antibodies.

Figure 4.

FKBP51 stabilizes tau. A, HeLa cells stably overexpressing V5-tagged wild-type human tau were transiently transfected with FKBP51, FKBP52, or a vector control DNA harvested after 48 h. Western blot shows that FKBP51-transfected cells increase total tau (V5) and phospho-tau (pS212) compared to vector-containing cells. B, HeLa cells stably overexpressing V5-tagged wild-type human tau were transiently transfected with FKBP51, Flag Hsp90, and/or HA-ubiquitin. Co-IP with V5 antibody revealed that FKBP51 overexpression reduces ubiquitination of tau independent of Hsp90. C, HeLa cells stably overexpressing V5-tagged wild-type human tau were transiently transfected with FKBP51 plasmid or FKBP51 siRNA and tau was coimmunoprecipitated from these lysates after 48 h (for overexpression) or 72 h (for siRNA). Binding of endogenous Aha1 and P23, two well characterized Hsp90 cochaperones, was assessed. The association of Aha1 with tau was reduced or increased when FKBP51 was overexpressed or knocked down, respectively. P23 only associated with tau when FKBP51 was knocked down. Hsp90 binding to tau was increased by FKBP51 overexpression, and reduced slightly by FKBP51 knockdown. D, HeLa cells stably overexpressing V5-tagged wild-type human tau were transiently transfected with FKBP51 or empty vector for 48 h and treated with 100 nm okadaic acid (OA; +) or vehicle (−) for 30 min. Co-IP of these lysates with V5 antibody shows increased FKBP51 and Hsp90 binding to tau with OA treatment. Total tau levels were measured with anti-V5.

Figure 5.

Characterization of PPIase-deficient FKBP51 mutant proteins. A, Coomassie Blue staining of recombinant WT, W90A, and F130A FKBP51 purified by Ni-NTA affinity and gel filtration chromatography. B, Column diagram depicting the catalytic efficiency of WT, W90A, and F130A FKBP51 as determined with a protease-coupled enzyme assay. Absolute values are shown.

Figure 6.

FKBP51 PPIase activity is a critical mediator of tau dephosphorylation. A, Recombinant wild type 4R0N tau was incubated with WT or PPIase-dead (F130A) FKBP51 for 5 min in the presence of α-chymotrypsin or buffer for indicated times. Western blot analysis showed that WT FKBP51 abrogated chymotrypsin-mediated tau degradation, while F130A FKBP51 did not. B, Indicated purified recombinant proteins were incubated for 30 min with agitation at 37C and co-IPed with rabbit polyclonal tau (Santa Cruz Biotechnology) antibody or nonspecific rabbit IgG for 1 h at room temperature. Protein alone is indicated as inputs. Total tau was probed for using mouse monoclonal Tau12. C, HeLa cells stably overexpressing V5-tagged wild-type human tau were transiently transfected with vector (−), WT, W90A, or F130A FKBP51 for 48 h. D, Quantitation of Western blot from C shows increases in indicated phospho-tau species following transfection with mutant FKBP51 relative to wild-type FKBP51. Error bars (SD) show variability from replicate experiments.

Figure 7.

PPIase activity is essential for FKBP51 to facilitate microtubule formation and tau-mediated microtubule polymerization is enhanced by FKBP51. In vitro microtubule assembly assays were performed using Xenopus oocyte extracts supplemented with rhodamine-labeled tubulin. A, Panels show tubulin structures in sample buffer only or supplemented with 1 μm recombinant WT FKBP51, 1 μm F130A FKBP51, 1 μm FKBP52, nocodazole, or taxol. B, Microtubule (MT) lengths were measured for each condition (n = number of asters measured per condition) using AxioVision software from Zeiss, and values are indicated in micrometers ± SD. ***p < 0.001. C, Panels show tubulin structures in sample buffer only or supplemented with 2.5 μm tau alone or 2.5 μm tau plus 1 μm FKBP51. Tau alone caused dramatic morphologic changes to microtubules, which were accentuated in the presence of FKBP51.

Figure 8.

FKBP51 works with the Hsp90 complex to recycle tau for microtubule (MT) stabilization. Tau in the cis-conformation stabilizes MTs. Phosphorylation within the MT binding domain by MT affinity regulating kinase 2 or calcium/calmodulin kinase II detaches tau from the microtubules, which favors trans conversion. Hsp90 recognizes the misfolded tau; however, CHIP is unable to ubiquitinate this particular phospho-tau species. FKBP51 is then sequestered to Hsp90, where it isomerizes tau to the cis-conformation, allowing for dephosphorylation and recycling to the microtubule. When PPIase activity is blocked (PPIase def), FKBP51 perpetuates the association of phospho-tau with Hsp90, promoting tau accumulation.