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. 2019 Apr 19;294(16):6306-6317.
doi: 10.1074/jbc.RA118.007222. Epub 2019 Feb 27.

The Role of Liquid-Liquid Phase Separation in Aggregation of the TDP-43 Low-Complexity Domain

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Free PMC article

The Role of Liquid-Liquid Phase Separation in Aggregation of the TDP-43 Low-Complexity Domain

W Michael Babinchak et al. J Biol Chem. .
Free PMC article

Abstract

Pathological aggregation of the transactive response DNA-binding protein of 43 kDa (TDP-43) is associated with several neurodegenerative disorders, including ALS, frontotemporal dementia, chronic traumatic encephalopathy, and Alzheimer's disease. TDP-43 aggregation appears to be largely driven by its low-complexity domain (LCD), which also has a high propensity to undergo liquid-liquid phase separation (LLPS). However, the mechanism of TDP-43 LCD pathological aggregation and, most importantly, the relationship between the aggregation process and LLPS remains largely unknown. Here, we show that amyloid formation by the LCD is controlled by electrostatic repulsion. We also demonstrate that the liquid droplet environment strongly accelerates LCD fibrillation and that its aggregation under LLPS conditions involves several distinct events, culminating in rapid assembly of fibrillar aggregates that emanate from within mature liquid droplets. These combined results strongly suggest that LLPS may play a major role in pathological TDP-43 aggregation, contributing to pathogenesis in neurodegenerative diseases.

Keywords: TAR DNA-binding protein 43 (TDP-43) (TARDBP); amyloid; amyotrophic lateral sclerosis (ALS) (Lou Gehrig disease); fibrillation; intrinsically disordered protein; liquid-liquid phase separation; neurodegeneration; protein aggregation.

Conflict of interest statement

The authors declare that they have no conflicts of interest with the contents of this article

Figures

Figure 1.
Figure 1.
Liquid–liquid phase separation of the TDP-43 LCD. A, representative fluorescence microscopy images of 20 μm LCD at pH 4, 6, and 7 with varying salt concentrations at 25 °C using a 200:1 ratio of unlabeled to Alexa Fluor 488–labeled protein. B, LLPS of TDP-43 LCD as monitored by turbidity (A600) at varying pH and salt concentrations, 25 °C. Protein concentration, 20 μm. C, LLPS as monitored by turbidity at decreasing protein concentrations in the presence of varying amounts of salt. Buffer conditions, 10 mm potassium phosphate, pH 6. D, turbidity as a measure of LLPS at 25 and 37 °C with increasing salt concentrations. Protein concentration, 20 μm; buffer conditions, 10 mm potassium phosphate, pH 6. E, turbidity measured with increasing concentration of PEG-10,000 and various concentrations of NaCl. Protein concentration, 20 μm; buffer conditions, 10 mm potassium phosphate, pH 6, 25 °C. F, LLPS as monitored by turbidity as a function of protein concentration. Buffer conditions, 10 mm potassium phosphate, pH 7.3, 150 mm NaCl, 10% PEG-10,000, 37 °C. G, representative bright-field images corresponding to conditions described in F. Scale bar, 5 μm. H, LLPS as monitored by turbidity as a function of protein concentration. Buffer conditions, 10 mm potassium phosphate, pH 7.3, 150 mm KCl, 10% PEG-10,000, 37 °C, with and without 1 mm DTT. I, representative bright-field images corresponding to conditions in H. Scale bar, 5 μm. Error bars represent S.D.
Figure 2.
Figure 2.
Rapid oligomerization of TDP-43 in the context of LLPS. A, representative EPR spectra of TDP-43 LCD (20 μm) spin-labeled at position 280 (black) and 333 (red). Spectra were recorded at pH 4 in the absence of NaCl. B, the mobility parameter (inverse of the central line width, ΔH0−1) for residues probed by SDSL at pH 4 and 6, both in the absence of NaCl. C, spin-normalized EPR spectra for TDP-43 LCD labeled at residue 327 at 0 (black) and 150 mm NaCl (blue). Protein concentration, 20 μm. D, scaling of spectra shown in C to the same intensity of the central line clearly reveals the appearance in the presence of 150 mm NaCl of a second, broad component with intensity, I2, that is distinct from the first, sharp component with intensity, I1. Inset, the second component can be even better visualized via spectral subtraction of the first component. E, intensity ratios (I2/I1) of the broad-to-sharp components (as defined in D) for residues probed by SDSL in the absence (gray) and presence of 150 mm NaCl (blue) at pH 6 (left; LLPS was observed in the presence of salt) and pH 4 (right; no LLPS under either condition). F, size distribution of TDP-43 LCD at pH 4 with and without 150 mm NaCl. Protein concentrations, 100 μm at 25 °C.
Figure 3.
Figure 3.
TDP-43 LCD fibrillation in the absence of LLPS. A, atomic force microscopy image of fibrillar aggregates formed from 20 μm protein in the absence of NaCl at 25 °C. B, representative ThT fluorescence intensity trace for 5 μm TDP-43 LCD incubated without NaCl at pH 6, 25 °C (no LLPS). C, lag times derived from ThT fluorescence traces for LCD incubated in the absence of salt at pH 6, 25 °C, at varying protein concentrations. Error bars represent S.D. D, representative ThT fluorescence kinetic traces for 20 μm LCD incubated at pH 4 with various salt concentrations at 25 °C (each trace shown represents an average of three experiments). RFU, relative fluorescence units.
Figure 4.
Figure 4.
Formation of TDP-43 amyloid fibrils within liquid droplets. A, representative fluorescence microscopy images for FRAP experiments on individual droplets (left) and beaded droplets (right) prepared from 50 μm LCD and 100 nm Alexa Fluor 488–labeled LCD in pH 7 buffer with 300 mm NaCl. Scale bar, 5 μm. B, fluorescence recovery traces after photobleaching for experiments illustrated in A. Each trace represents an average of at least three droplets, and error bars represent standard deviation. C, atomic force microscopy images of TDP-43 LCD droplets deposited and dried on mica at various time points during incubation at 25 °C under LLPS conditions in a buffer containing 300 mm NaCl at pH 4 (upper images) or pH 6 (lower images). Protein concentration, 20 μm. White scale bars correspond to 400 nm. D, representative atomic force microscopy images of fibrils after prolonged (6-day) incubation of TDP-43 LCD under LLPS conditions at pH 4, 6, or 7 in the presence of 300 mm NaCl. Scale bars, 400 nm.
Figure 5.
Figure 5.
LLPS strongly accelerates fibrillation of TDP-43 LCD. A, ThT fluorescence traces for 20 μm LCD incubated at 25 °C in pH 6 buffer with various concentrations of NaCl. B, C, and D, lag phase time from ThT kinetic traces at pH 4 (B), 6 (C), and 7 (D) at various concentrations of NaCl. Protein concentration, 20 μm. E, lag phase time of fibrillation as a function of initial turbidity depicting data accumulated in this study at conditions of different pH and salt concentrations. Data in B–E represent averages from experiments using at least three separately prepared batches of protein. Protein concentration, 20 μm. Error bars represent S.D.
Figure 6.
Figure 6.
LLPS intrinsically promotes TDP-43 LCD fibrillation independently of electrostatic effects. Turbidity (A) and fibrillation lag phase time (B) for 20 μm protein at 25 °C in the presence or absence of 5% 1,6-hexanediol. Buffer conditions, pH 4, 200 mm NaCl (control, no LLPS with and without 5% 1,6-hexanediol) or pH 6, 200 mm NaCl (LLPS in the absence of 5% 1,6-hexanediol only). Turbidity (C) and fibrillation lag phase time (D) for 20 μm protein at either 25 or 37 °C. Buffer conditions, pH 4, 200 mm NaCl (control, no LLPS at both temperatures) or pH 6, 200 mm NaCl (LLPS at 25 °C only) (no 1,6-hexanediol was used). Error bars represent S.D.

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