Targeted Dephosphorylation of Tau by Phosphorylation Targeting Chimeras (PhosTACs) as a Therapeutic Modality

Abstract

Microtubule-associated protein tau is essential for microtubule assembly and stabilization. Hyperphosphorylation of the microtubule-associated protein tau plays an important pathological role in the development of Alzheimer's disease and other tauopathies. In vivo studies using kinase inhibitors suggest that reducing tau phosphorylation levels has therapeutic potential; however, such approaches showed limited benefits. We sought to further develop our phosphorylation targeting chimera (PhosTAC) technology to specifically induce tau dephosphorylation. Herein, we use small molecule-based PhosTACs to recruit tau to PP2A, a native tau phosphatase. PhosTACs induced the formation of a stable ternary complex, leading to rapid, efficient, and sustained tau dephosphorylation, which also correlated with the enhanced downregulation of tau protein. Mass spectrometry data validated that PhosTACs downregulated multiple phosphorylation sites of tau. We believe that PhosTAC possesses several advantages over current strategies to modulate tau phosphorylation and represents a new avenue for disease-modifying therapies for tauopathies.

Figure 1.

Design of Tau-targeting Phosphorylation TArgeting Chimeras (PhosTACs) A, scheme of PhosTAC action mechanism. B, Design of inducible tau expression construct. C, fluorescent microscopy image of tau-expressing Hela cells after doxycycline induction. D, validation of tau expression and tau phosphorylation in Hela cells. Tau-expressing Hela cells were treated with or without doxycycline for 24h. Induced cells were then treated with gefitinib (200 nM), wortmannin (1 uM), CK2i (1 uM), and rapamycin (500 nM), respectively for another 24 h. Cell lysates were collected and analyzed by WB using indicated antibodies. E, structures of PhosTAC7 and PhosTAC7F.

Figure 2.

PhosTAC7 dephosphorylates Tau in a PP2A-dependent manner. A. PhosTAC induced stable ternary complex with tau and PP2A A subunit and C subunit. Tau/FKBP12(F36V)-PP2A A HeLa cells were treated with doxycycline (dox) for 24 h and then incubated with PhosTACs (1μM, 24h) and lysed for HaloTrap pulldown and Western blot using indicated antibodies. B. PhosTAC7 but not the inactive PhosTAC7F induced tau dephosphorylation. Tau/FKBP12(F36V)-PP2A A HeLa cells were treated with dox for 24 h, followed by treatment with indicated concentrations of PhosTAC7, PhosTAC7F, DMSO, or rapamycin (0.5μM) for 24 h. Cell lysates were collected and analyzed by western blot using indicated antibodies. Data were quantified from the phosphorylated or total 2N4R** tau species with two replicates and summarized as mean and standard deviation. C. PhosTAC7 induced tau dephosphorylation via PP2A. Tau/FKBP12(F36V)-PP2A A HeLa cells (dox-induction for 24 h) were treated with indicated concentrations of PhosTAC7, PhosTAC7F, or PhosTAC7 – OA (20 nM, 24 h) cotreatment. Cell lysates were collected 24 h after treatment and analyzed by western blot using indicated antibodies.

Figure 3.

Tau2–8 dephosphorylates Tau. A. Structures of Tau2–8. B. Tau2–8 induced Tau dephosphorylation. Tau/FKBP12(F36V)-PP2A A HeLa cells were treated with dox for 24 h, followed by treatment with indicated concentrations of Tau2–4, Tau2–6 or Tau2–8. Cell lysates were collected 24 h after treatment and analyzed by WB using indicated antibodies. Data were quantified from the phosphorylated or total 2N4R** Tau species with two replicates and summarized as mean ± sd. C. Tau2–8 induced tau dephosphorylation via PP2A. Tau/FKBP12(F36V)-PP2A A HeLa cells were treated with dox for 24 h, followed by treatment with indicated concentrations of Tau2–8 with or without OA (20 nM) cotreatment. Cell lysates were collected 18 h after treatment and analyzed by WB using indicated antibodies. D. PP2A Pro179 is critical for PhosTAC-mediated dephosphorylation. Tau/FKBP12(F36V)-PP2A A (P179R) HeLa cells were treated with dox for 24 h, followed by treatment with indicated concentrations of PhosTAC7, PhosTAC7F, or Tau2–8. Cell lysates were collected 24 h after treatment and analyzed by WB using indicated antibodies.

Figure 4.

Rapid and long-lasting effects of Tau-PhosTACs. A. PhosTACs induced rapid tau dephosphorylation. Doxycycline induced Tau/FKBP12(F36V)-PP2A A HeLa cells (dox-induction for 24 h) were treated with PhosTAC7 (1μM) or Tau2–8 (1 μM). Cell lysates were collected after treatment of indicated time and analyzed by WB using indicated antibodies. Data were quantified from two biological samples and summarized as mean and standard deviation. B. PhosTACs induced long-lasting tau dephosphorylation. Flow chart of experiment design was shown at top right panel. Doxycycline induced Tau/FKBP12(F36V)-PP2A A HeLa cells were treated with PhosTAC7 (0.5μM), Tau2–8 (10 μM) or rapamycin (0.5 μM) for 24h, the media were then changed to remove any treatment. Cell lysates were collected after indicated time and analyzed by WB using indicated antibodies. Data were quantified from two biological samples and summarized as mean and standard deviation.

Figure 5.

Proteomic approaches validated tau dephosphorylation and PP2A enrichment by PhosTACs. A-C. Validation of tau dephosphorylation on tau pT181, pT231, and pS202 by PhosTACs. HaloTag fusion tau and its interactome were pulled down by HaloTrap after PhosTAC7 or Tau2–8 treatment. The eluted tau and interacting proteins were analyzed by mass spectrometry. Data were collected from three biological samples for each condition. D. Heatmap of tau phosphorylation level of 24 sites measured by mass spectrometry. E. Heatmap of enrichment level for PP2A C subunit (PPP2CA), PP2A B55 subunit (PPP2R2A), PP2A A subunit (PPP2R1A) and Halotag7 (tau) proteins. F. Heatmap of enrichment level for tubulin beta chain (TUBB), tubulin alpha-1B chain (TUBA1B), tubulin beta-2A chain (TUBB2A) and tubulin beta-4B chain (TUBB4B) proteins.

Figure 6.

The biological effects on Tau protein expression by PhosTAC. A. PhosTAC induced dephosphorylation correlated with accelerated tau protein down regulation. Tau/FKBP12(F36V)-PP2A A HeLa cells were treated with dox for 24 h, after which dox was removed and cells were treated with DMSO or PhosTAC7 (1μM) for the indicated times. Tau levels were assessed by mClover mean fluorescence intensity by flow cytometry. Data were quantified from two biological samples and summarized as mean ± standard deviation. B. PhosTAC-mediated tau degradation correlated with phosphatase and proteasome activity. Doxycycline induced Tau/FKBP12(F36V)-PP2A A HeLa cells were treated with DMSO, PhosTAC7 (1μM), PhosTAC7F (1μM) for 2 days, then treated with OA (10 nM), MG132 (10 μM) or Bafilomycin A1 (500 nM) for 24h. Tau protein levels was monitored by measuring mClover fluorescence intensity with flow cytometry. Data were quantified from two biological samples and summarized as mean and standard deviation, t tests were performed with Prism 9.