GSK3β phosphorylation catalyzes the aggregation of tau into Alzheimer's disease-like filaments

Abstract

The pathological deposition of proteins is a hallmark of several devastating neurodegenerative diseases. These pathological deposits comprise aggregates of proteins that adopt distinct structures named strains. However, the molecular factors responsible for the formation of distinct aggregate strains are unknown. Here, we show that the serine/threonine kinase GSK3β catalyzes the aggregation of the protein tau into Alzheimer's disease (AD)-like filaments. We demonstrate that phosphorylation by GSK3β, but not by several other kinases, promotes the aggregation of full-length tau as well as enhances phase separation into gel-like condensate structures. Cryoelectron microscopy further reveals that the fibrils formed by GSK3β-phosphorylated tau adopt a fold comparable to that of paired helical filaments isolated from the brains of AD patients. Our results elucidate the intricate relationship between posttranslational modification and the formation of tau strains in neurodegenerative diseases.

Keywords: Alzheimer's disease; NMR; cryo-EM; phosphorylation; tau.

Fig. 1.

Kinase-specific phosphorylation patterns of tau. (A) Domain diagram of full-length tau. The residues phosphorylated by different kinases and detected by mass spectrometry are indicated with red bars. The lengths of the red ticks are adjusted based on the percentage of phosphorylation calculated by dividing the number of peptides detected containing a particular residue by the total number of peptides detected containing the same residue. The residue-specific percentage of phosphorylation can be found in Dataset S1. The black diamond-headed bars refer to previously reported phosphorylation sites that were not detected due to the absence of peptides in the mass spectrometry experiment. (B–D) Residue-specific intensity changes observed in 2D ¹H-¹⁵N HSQC spectra of tau upon phosphorylation by GSK3β (B), CDK5 (C) and ERK2 (D). I and I₀ are the cross-peak intensities of the phosphorylated and unmodified tau, respectively. The location of serine, threonine, and tyrosine residues is indicated above. (E–G) Signals of the cross-peaks of phosphorylated serine residues in the ¹H-¹⁵N HSQC spectra of phosphorylated tau appeared due to the phosphorylation by GSK3b (E), CDK5 (F), and ERK2 (G). pS404 (pS400) shown in (E) denotes the peak of pS404 shifted due to the presence of pS400. (H) ¹H-¹⁵N HSQC spectra of either unmodified (black) or GSK3β-phosphorylated (orange) C-terminal fragment of tau comprising residues 369 to 441 (referred to as K26). The cross-peaks of the phosphorylated residues and the residues shifted due to nearby phosphorylation are shown in the spectrum. (I) Residue-specific chemical-shift perturbation (CSP) observed in the ¹H-¹⁵N HSQC spectra of K26 upon phosphorylation by GSK3β. The PHF-1 epitope is marked in the amino acid sequence displayed above.

Fig. 2.

Acceleration of tau aggregation by GSK3β phosphorylation. (A) Aggregation kinetics of 25 µM unmodified tau and tau phosphorylated by different kinases. Error bars represent the std of three independently aggregated samples. (B) ThT-intensity span vs. half-time of aggregation (Tm) of unmodified and phosphorylated tau proteins. Error bars represent the std of three independently aggregated samples. (C) Amount of protein aggregated vs. half-time of aggregation (Tm) of unmodified and phosphorylated tau proteins. The amount of aggregated protein was calculated by comparing the intensity of the supernatant (SN) band (after pelleting down the fibrils) to the tau monomer band as shown in SI Appendix, Fig. S5. Error bars represent the std of the half-time of aggregation of three independently aggregated samples. (D) Negative-stain EM of fibrils formed by unmodified and different phosphorylated tau samples. (Scale bars, 200 nm.)

Fig. 3.

GSK3β phosphorylation promotes tau condensation. (A) Domain diagram of 2N4R tau. Residues that undergo phosphorylation in the presence of GSK3β, ERK2, CDK5, and C-Abl are marked with green, black, purple, and dark-yellow colored bars, respectively. S396 and S404, which are phosphorylated by GSK3β, form the epitope that is recognized by the phosphorylation-specific antibody PHF-1. (B) DIC and fluorescence microscopy of the condensates formed by 25 µM GSK3β-phosphorylated tau at room temperature in the aggregation assay buffer (25 mM HEPES, 10 mM KCl, 5 mM MgCl₂, 3 mM TCEP, and 0.01% NaN₃, pH 7.2). A zoomed-in view of the condensate is shown to the Right. The protein was labeled with Alexa Fluor 488 dye. Micrographs are representative of three independent biological replicates. (Scale bar, 10 µm.) (C) Fluorescence recovery after photobleaching (FRAP) experiment of the condensates formed by GSK3β-phosphorylated tau shown in (B). The protein was labeled with Alexa Fluor 488 dye. Error bars represent std of averaged three curves for each time point. Representative micrographs of the condensate before bleaching, after bleaching, and at the end of recovery are displayed to the Right. (Scale bar, 1 µm.) (D) DIC microscopy of the condensates formed by 25 µM ERK2-phosphorylated tau at room temperature in the aggregation assay buffer. A zoomed-in view of the condensate is shown to the Right. Micrographs are representative of three independent biological replicates. (Scale bar, 10 µm.) (E–G) DIC microscopy of 25 µM CDK5-phosphorylated tau (E), C-Abl-phosphorylated tau (F), and unmodified tau (G) at room temperature in the aggregation assay buffer. Micrographs are representative of three independent biological replicates. (Scale bar, 10 µm.)

Fig. 4.

Maturation of tau droplets into gel-like structures upon GSK3β phosphorylation. (A) DIC and fluorescence microscopy of tau droplets induced by the addition of 10% dextran at room temperature in 25 mM HEPES, 10 mM KCl, 5 mM MgCl₂, pH 7.2 buffer. Fluorescently labeled GSK3β partitioned into the droplets. Tau and GSK3β were labeled with Alexa Fluor 594 and Alexa Fluor 488 dye, respectively. Micrographs are representative of three independent biological replicates. (Scale bar, 5 µm.) (B) DIC and fluorescence microscopy of the tau droplets induced by the addition of 10% dextran at room temperature in 25 mM HEPES, 10 mM KCl, 5 mM MgCl₂, pH 7.2 buffer in the presence of 0.02 mg/mL unlabeled GSK3β, and 1 mM ATP. The sample was incubated for 24 h. A zoomed-in view of the condensate formed after 24 h is shown. Tau was labeled with Alexa Fluor 594 dye. Micrographs are representative of three independent biological replicates. (Scale bar, 5 µm.) (C) FRAP experiment of the larger fused condensates of tau in the presence of GSK3β and ATP after incubation for 6 h. Tau was labeled with Alexa Fluor 594 dye. The yellow arrow indicates that the FRAP experiments were performed on the larger condensates formed after incubation for 6 h. Error bars represent std of averaged three curves for each time point. Representative micrographs of the condensate before bleaching, after bleaching, and at the end of recovery are displayed on the Top. (Scale bar, 5 µm.) (D) FRAP experiment of the gel-like condensates of tau in the presence of GSK3β and ATP after incubation for 1 d. Tau was labeled with Alexa Fluor 594 dye. The yellow arrow indicates that the FRAP experiments were performed on the gel-like condensates formed after incubation for 1 d. Error bars represent std of averaged three curves for each time point. Representative micrographs of the condensate before bleaching, after bleaching, and at the end of recovery are displayed on the Top. (Scale bar, 5 µm.)

Fig. 5.

Cryo-EM of GSK3β-phosphorylated tau fibrils. (A) Cryoelectron micrograph of GSK3β-phosphorylated tau fibrils. (B) Reconstruction of a GSK3β phosphorylated tau fibril from low-resolution and big box 2D classes for cross-over estimation. (C) Cross-section of the cryo-EM map of the GSK3β-phosphorylated tau fibril after 3D refinement. (D) Cryo-EM density map of GSK3β phosphorylated tau fibrils. (E) Cryo-EM density map of GSK3β-phosphorylated tau fibrils (cyan surface) compared with the paired helical filament (PHF) from sporadic AD brain (dark blue isomesh; PDB id: 6HRE).