Methylene blue accelerates liquid-to-gel transition of tau condensates impacting tau function and pathology

Abstract

Preventing tau aggregation is a potential therapeutic strategy in Alzheimer's disease and other tauopathies. Recently, liquid-liquid phase separation has been found to facilitate the formation of pathogenic tau conformations and fibrillar aggregates, although many aspects of the conformational transitions of tau during the phase transition process remain unknown. Here, we demonstrate that the tau aggregation inhibitor methylene blue promotes tau liquid-liquid phase separation and accelerates the liquid-to-gel transition of tau droplets independent of the redox activity of methylene blue. We further show that methylene blue inhibits the conversion of tau droplets into fibrils and reduces the cytotoxicity of tau aggregates. Although gelation slows down the mobility of tau and tubulin, it does not impair microtubule assembly within tau droplets. These findings suggest that methylene blue inhibits tau amyloid fibrillization and accelerates tau droplet gelation via distinct mechanisms, thus providing insights into the activity of tau aggregation inhibitors in the context of phase transition.

Fig. 1. MB facilitates tau phase separation.

a Structure of MB and LMTM. b Turbidity of tau solution measured at 350 nm (OD350) for different tau and MB concentrations, as indicated, in 50 mM Tris (pH 7.4) with 5% PEG8000 or 20 mM HEPES (pH 7.4) without PEG8000. c Representative fluorescence microscopy images of tau droplets with different MB concentrations. The concentrations of tau were 5 μM in 50 mM Tris (pH 7.4) with 5% PEG8000 and 20 μM in 20 mM HEPES (pH 7.4) without PEG8000. Scale bars, 10 μm. d Saturation concentration of tau (C_sat) with different MB concentrations, as indicated, in 50 mM Tris (pH 7.4) with 5% PEG8000 or in 20 mM HEPES (pH 7.4) without PEG8000. e Turbidity of tau solution (5 μM) with different concentrations of MB or DTT-reduced MB, as indicated, in 50 mM Tris (pH 7.4) with 5% PEG8000. f Turbidity of tau solution (5 μM) in the presence of a series of concentrations of MB or LMTM, as indicated, in 50 mM Tris (pH 7.4) with 5% PEG8000. g Design of the tau cysteine-less mutant. Locations of the two N-terminal inserts (N1 and N2) within the N-terminal domain (NTD), the two proline-rich regions (P1 and P2) within the proline-rich domain (PRD), the four microtubule-binding repeats (R1 to R4) within the microtubule-binding domain (MTBD), and the C-terminal domain (CTD) are indicated. h Turbidity of tau solution (5 μM) in the presence of different MB concentrations, as indicated, in 50 mM Tris (pH 7.4) with 5% PEG8000. Data in (d–f, h) are presented as mean values +/− SD of three experiments. Significance levels were determined by unpaired two-sided Student’s t test. NS non-significant. Source data are provided as a Source data file.

Fig. 2. MB facilitates liquid-to-gel transition of WT tau droplets.

a Representative FRAP images of tau droplets formed by 20 μM tau and different concentrations of MB, as indicated, in 20 mM HEPES (pH 7.4) without PEG8000. Scale bars, 5 μm. b Quantified fluorescence intensity of the FRAP experiments. Data are presented as mean values +/− SD of three experiments. c Fusions of two tau droplets measured using optical tweezers in 25 mM HEPES (pH 7.4), 150 mM KCl, 1 mM DTT, 15% PEG6000 and different concentrations of MB, as indicated. To obtain droplets of suitable size for fusion kinetics measurements, PEG was added to facilitate tau droplet formation. d Aspect ratio of the two joined tau droplets as a function of aging time in the presence of different MB concentrations, as indicated. The lines are single exponential fitting of the data points. Refer to Supplementary Table 1 for further details of data fitting. Data are presented as mean values +/− SD of at least three experiments. Source data are provided as a Source data file.

Fig. 3. Influence of MB derivatives on WT tau phase separation.

a Structures of MB derivatives azure A and azure B. b Turbidity of 5 μM tau solution with different concentrations of MB, azure A, and azure B, as indicated, in 50 mM Tris (pH 7.4) with 5% PEG8000. c Normalized FRAP intensity at the end of FRAP experiments. Droplets were formed by 10 μM tau in 50 mM Tris (pH 7.4) with 5% PEG8000 and different concentrations of MB, azure A, and azure B. d Saturation concentration of tau in the presence of different concentrations of MB, azure A, or azure B in 50 mM Tris (pH 7.4) with 5% PEG8000. e FRAP experiments for tau droplets with different concentrations of TMAO, as indicated, in the presence of 1 μM azure A, 1 μM azure B, or 10 μM MB. Tau droplets were formed by 10 μM tau in 50 mM Tris (pH 7.4) with 5% PEG8000. Scale bars, 5 μm. Data in b, c, d are presented as mean values +/− SD of three experiments. Significance levels were determined by unpaired two-sided Student’s t test. NS non-significant. Source data are provided as a Source data file.

Fig. 4. Interactions between tau and MB.

a Turbidity of WT tau (5 μM) measured at different NaCl or 1,6-HD concentrations in 50 mM Tris (pH 7.4) with 5% PEG8000. The concentrations of MB were 0 and 100 μM. Data are presented as mean values +/− SD of three experiments. Significance levels were determined by unpaired two-sided Student’s t-test. NS non-significant. b Design of tau deletion mutants. c Turbidity of tau deletion variants (5 μM) measured at different MB concentrations in 50 mM Tris (pH 7.4) with 5% PEG8000. Data are presented as mean values +/− SD of three experiments. d Overlay of 2D ¹H-¹⁵N SOFAST-HMQC spectra of WT tau (18 μM) with and without 5% PEG8000 or 360 μM MB in 50 mM Tris (pH 7.4); ppm parts per million. e Differential interference contrast (DIC) micrographs of the NMR samples. Scale bars, 20 µm. f Peak amplitude ratios (I/I₀) quantified for tau with 5% PEG8000, 360 μM MB, or 360 μM azure B calculated from the ¹H-¹⁵N SOFAST-HMQC spectra. Residues omitted from the I/I₀ analysis due to lack of reliable peak assignment are designated as gray bars. g The top five docking poses of MB binding to WT tau. The N-terminus of tau is shown as a red ball. The residues which have strong interaction with MB are shown as green balls and sticks. h Tau residue-ligand binding profile calculated using the top five docking poses for MB. Source data are provided as a Source Data file.

Fig. 5. FRET measurements of AF350/AF488-labeled tau variants reveal conformational changes in tau upon MB-induced LLPS.

a Schematic illustration of the fluorescence labeling sites in the four tau variants. b FRET spectra of tau variants in 50 mM Tris (pH 7.4). Black: 10 μM tau (no phase separation); green: 10 μM tau + 100 μM MB (no phase separation); blue: 10 μM tau + 5% PEG8000 (phase separation); red: 10 μM tau + 100 μM MB + 5% PEG8000 (strong phase separation). c Two-photon microscopy images of AF350/AF488-labeled tau_17/244 with or without 100 μM MB in 50 mM Tris (pH 7.4) with 5% PEG8000. Scale bars, 10 μm. d Apparent FRET efficiency of tau droplets measured by the two-photon microscopy images under excitation of AF350 by 700 nm laser. Individual data points represent each droplet. A total of 58 droplets were analyzed. The bars indicate minimum and maximum. The center and bounds of box indicate median and SD, respectively. Significance levels were determined by unpaired two-sided Student’s t test. e The FRET spectra of tau variants in 20 mM HEPES (pH 7.4) without PEG8000. Black: 15 μM tau (weak phase separation); blue: 15 μM tau + 20 μM MB (weak phase separation); red: 15 μM tau and 100 μM MB (strong phase separation). A concentration of 0.2 μM AF350/AF488-labeled tau variants was mixed as dopant in all the above solutions for fluorescence spectra measurements and imaging. Source data are provided as a Source data file.

Fig. 6. MB inhibits tau fibrillization in the context of phase separation.

a Aggregation kinetics of tau monitored by ThT fluorescence at different MB concentrations in 50 mM Tris (pH 7.4) with 7.5% PEG8000 or in 20 mM HEPES (pH 7.4) without PEG8000. Data are presented as mean values +/− SD of three experiments. A.U., arbitrary units. b Representative TEM images of tau aggregates after incubation at 37 °C in 50 mM Tris (pH 7.4) with 7.5% PEG8000 or in 20 mM HEPES (pH 7.4) without PEG8000, in the absence or presence of 100 μM MB. Scale bars for 0 h represent 1 μm and for 18 and 36 h represent 200 nm. c Representative microscopy images of the growth of tau aggregates inside droplets in the absence or presence of 100 μM MB in 50 mM Tris (pH 7.4) with 7.5% PEG8000. Scale bars, 10 μm. Source data are provided as a Source data file.

Fig. 7. Influence of tau droplet gelation on microtubule assembly.

a Representative microscopy images of the growth of microtubules inside the preformed tau droplets with or without 100 μM MB. Scale bars, 10 μm. b Representative FRAP images of tau/tubulin droplets with or without 100 μM MB. Scale bars, 5 μm. c Quantified fluorescence intensity of the FRAP experiments of tau/tubulin droplets with or without 100 μM MB. d Representative FRAP images of tau/tubulin droplets with or without 100 μM MB in the presence of 1 μM DAPI. Scale bars, 5 μm. e Quantified fluorescence intensity of the FRAP experiments of tau/tubulin droplets with or without 100 μM MB in the presence of 1 μM DAPI. f Representative microscopy images showing the co-localization of DAPI and tubulin within tau droplets. Scale bars, 10 μm. g Microtubule assembly kinetics with or without 100 μM MB monitored by DAPI fluorescence. Inset: Influence of MB on the half-time of the DAPI fluorescence. Significance levels were determined by unpaired two-sided Student’s t test. Data in c–g (inset) are presented as mean values +/− SD of three experiments. Source data are provided as a Source data file.

Fig. 8. Illustration of MB-regulated tau phase transition.

a Activity of MB in the phase transition of tau. MB inhibits aggregation of free tau and transition of gelated droplets to aggregates. In contrast, MB promotes LLPS and the liquid-to-gel transition of tau. b Schematic illustration of MB-tau interactions during the phase transition process. MB binds to tau, inducing slight conformational changes in tau. Upon phase separation, tau undergoes significant conformational expansion and MB may bridge interactions between tau molecules.