The role of liquid–liquid phase separation in aggregation of the TDP-43 low-complexity domain

Prottusha Sarkar,Witold K. Surewicz,Jin-Kyu Choi,W. Michael Babinchak,Krystyna Surewicz,Raza Haider,Benjamin K. Dumm

doi:10.1074/jbc.ra118.007222

Prottusha Sarkar, Witold K. Surewicz

Open Access

https://doi.org/10.1074/jbc.ra118.007222

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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.

Highlights

Pathological aggregation of the transactive response DNAbinding protein of 43 kDa (TDP-43) is associated with several neurodegenerative disorders, including ALS, frontotemporal dementia, chronic traumatic encephalopathy, and Alzheimer’s disease
Proteinaceous inclusions containing the transactive response DNA-binding protein of 43 kDa (TDP-43)3 are a pathological hallmark of sporadic ALS [1, 2] and are observed in numerous other neurodegenerative disorders associated with protein misfolding, including frontotemporal lobar degeneration with ubiquitin-positive inclusions [1, 3], Alzheimer’s disease (4 –6), chronic traumatic encephalopathy [7], and cerebral age-related TDP-43 with sclerosis [8]
We found that the propensity of the TDP-43 low-complexity domain (LCD) to undergo liquid–liquid phase separation (LLPS) is strongly pH-dependent, requiring higher salt concentrations as pH is decreased (Fig. 1B)

Summary

ARTICLE cro

The role of liquid–liquid phase separation in aggregation of the TDP-43 low-complexity domain. Proteinaceous inclusions containing the transactive response DNA-binding protein of 43 kDa (TDP-43) are a pathological hallmark of sporadic ALS [1, 2] and are observed in numerous other neurodegenerative disorders associated with protein misfolding, including frontotemporal lobar degeneration with ubiquitin-positive inclusions [1, 3], Alzheimer’s disease (4 –6), chronic traumatic encephalopathy [7], and cerebral age-related TDP-43 with sclerosis [8]. The key finding of these studies is that LLPS conditions per se strongly promote TDP-43 amyloid fibril formation and that self-assembly of the protein is heavily regulated by These observations in vitro provide a foundation for understanding the role of LLPS in the formation in vivo of pathological TDP-43 aggregates within the context of neurodegenerative diseases

Results

Discussion

Experimental procedures

Fluorescence microscopy and FRAP measurements

Atomic force microscopy imaging