Luo et al. 10.1073/pnas.0701055104. |
Fig. 6. In tauopathies, Hsp90 maintains the functional stability of abnormal proteins and supports the accumulation of toxic aggregates.
Fig. 7. Degradation of p35 (a and b) and reduction in Tau phosphorylation at putative cdk5 sites (b) and induction of a heat shock response (a) are both consequences of Hsp90 inhibition by PU24FCl. Immunofluorescence staining of cultured cortical neurons treated for 24 h with either vehicle (DMSO) or PU24FCl (10 mM).
Fig. 8. p35 and mTau but not WT Tau are sensitive to Hsp90 inhibition. (a) Western blot analysis of COS-7/Tau and COS-7/TauP301L cells treated with the indicated doses of PU24FCl for 24 h. (b) Western blot analysis of COS-7/Tau and COS-7/TauP301L cells treated with the indicated doses of 17AAG for 24 h. Hsp90 was used as a protein quantification control. Each experiment was conducted in duplicate, and gels presented here are representative runs.
Fig. 9. (a) Both PU24FCl and PU-DZ8 bind with high affinity to Hsp90 as present in the brain of JNPL3 transgenic mice. Binding to Hsp90 was measured as described in SI Materials and Methods. (b) PU-DZ8 reaches pharmacologically relevant concentrations in the brain of JNPL3 mice. One dose of 75 mg/kg PU-DZ8 was administered i.p., and drug levels in brain were analyzed by LC/MS/MS as described in SI Materials and Methods. 17AAG is an Hsp90 inhibitor currently in phase II evaluation in patients with advanced cancers.
Fig. 10. Degradation of p35 and mTau by Hsp90 inhibition is mediated by the proteasome. (a) Western blot analysis of primary embryonic cortical neurons treated for 24 h with vehicle, MG132, pepstatin (PEP), leupeptin (LEU) , E64, or ZVAD in the presence (+) or absence (-) of PU24FCl (20 mM). (b) Western blot analysis of COS-7/TauP301L cells (Upper) or COS-7/Tau cells (Lower) treated for 24 h with vehicle, MG132, or leupeptin in the presence (+) or absence (-) of PU24FCl (20 mM). b-Actin and Hsp90 were used as a protein quantification control. DMSO, vehicle. Each experiment was conducted in triplicate, and gels presented here are representative runs.
Fig. 11. Hsp90 regulates the stability of p35 and mTau. (a) Western blot analysis of primary embryonic cortical neurons treated with cycloheximide (100 mg/ml) in the presence of vehicle or PU24FCl (10 mM) for the indicated times. (b and c) Western blot analysis of COS-7/TauP301L (b) and COS-7/Tau (c) cells treated with cycloheximide (100 mg/ml) in the presence of vehicle or PU24FCl (10 mM) for the indicated times.
Fig. 12. Effects of long-term Hsp90 inhibitor treatment on p35 and mTau. Western blot analysis of soluble (S1) and insoluble (P3) brain fractions extracted from 6-month-old JNPL3 female mice treated for 30 days with 75 mg/kg PU-DZ8 as described in Materials and Methods. Subcortical (s) and cortical (c) brain region are presented. The location of 64-kDa Tau (arrow) is indicated. Hsp90 was used as protein quantification control.
SI Materials and Methods
Reagents.
ZVAD, pepstatin, MG132, leupeptin, and E64 were purchased from Calbiochem (San Diego, CA).
Proteasome Inhibition Studies.
For studies involving inhibitors of protein degradation, compounds were dissolved in DMSO (PU24FCl, ZVAD, pepstatin, and MG132) or water (leupeptin and E64) at a concentration 1,000-fold higher than the final concentration used. Primary cortical cultures were treated with DMSO (-) or 20 mM PU24FCl in the presence of the following inhibitors: 10 mM MG132, 1 mM pepstatin, 75 mM leupeptin, 10 mM E64, or 20 mM ZVAD for 24 h. Cells were collected and sonicated in 2% SDS, and the resulting lysates were analyzed by immunoblot.
Immunofluorescence.
Cultured primary cortical neurons were fixed with 4% formaldehyde, and cells were permeabilized with ice-cold methanol. Fixed monolayers were washed with distilled water and blocked with 5% BSA in PBS solution. After blocking, cells were incubated with the primary antibody followed by incubation with Alexa Fluor 488 and Alexa Fluor 568 secondary antibodies for 1 h at room temperature. Rabbit anti-p35 antibody was used at 1:100, monoclonal anti-Hsp70 antibody was used at 1:200, and phosphorylated Tau was detected with AT8 at 1:50. Images were collected with a Zeiss LSM510 confocal microscope.
Assessment of PU-DZ8 Brain Levels.
Concentrations of compound were determined and quantitated by a multiple reaction monitoring mode using HPLC/MS/MS. A weighed piece of brain was rinsed with saline isotonic solution, dried with gauze, and then homogenized in mobile phase (acetonitrile/0.1% formic acid, 1.2:2.8, vol/vol). Haloperidol was added as the internal standard. PU-DZ8 was extracted in methylene chloride, and the organic layer was separated, speedily dried under vacuum, and reconstituted in the mobile phase. Compound analysis was performed in the API 4000 LC/MS/MS system (Applied Biosystems, Foster City, CA), which was coupled with a Shimadzu LC system and a 96-well plate autosampler. A Gemini C18 column (5 m particle size, 50 ´ 4.6 mm i.d.) was used for the LC separation. The analyte was eluted under an isocratic condition for 4 min at a flow rate of 0.4 ml/min. In vivo experiments were carried out under Institutional Animal Care and Use Committee-approved protocols, and institutional guidelines for the proper and humane use of animals in research were followed.
Binding to JNPL3 Brain Hsp90.
The assay buffer (HFB) contained 20 mM Hepes (K) pH 7.3, 50 mM KCl, 5 mM MgCl2, 20 mM Na2MoO4, and 0.01% Nonidet P-40. Before each use, 0.1 mg/ml bovine gamma globulin (Panvera Corporation, Madison, WI) and 2 mM DTT (Fisher Biotech, Fair Lawn, NJ) were freshly added. GM-cy3B, a specific Hsp90 ligand, was synthesized as reported previously [Moulick K, Clement CC, Aguirre J, Kim J, Kang Y, Felts S, Chiosis G (2006) Bioorg Med Chem Lett 16:4515-4518] and was dissolved in DMSO to form 10 mM solutions. Brains were homogenized in HFB with added protease and phosphatase inhibitors. Saturation curves were recorded, in which GM-cy3B (3 nM) was treated with increasing amounts of brain homogenates. The Hill and Scatchard plot analyses of the experiment were constructed to show that at the low amounts of brain homogenates required to reach saturation, interaction from other cellular material was precluded. The amount of brain homogenate for which >90% of GM-cy3B was Hsp90-bound at equilibrium (24 h) was chosen for the competition study. For the competition experiments, each 96-well plate contained 3 nM GM-cy3B, brain homogenate, and tested inhibitor (initial stock in DMSO) in a final volume of 100 ml. The plate was left on a shaker at 4°C for 24 h, and the fluorescence polarization values in millipoise (1 P = 0.1 Pa×sec) were recorded. EC50 values were determined as the competitor concentrations at which 50% of GM-cy3B was displaced. Fluorescence polarization measurements were performed on an Analyst GT instrument (Molecular Devices, Sunnyvale, CA). For GM-cy3B, an excitation filter at 545 nm and an emission filter at 610-675 nm were used with a dichroic mirror of 565 nm. Measurements were taken in black 96-well microtiter plates (Corning #3650).