Interplay between phase separation and amyloid formation: a Raman microscopy study of the TDP-43 C-terminal domain

Authors

• M Bono
• A Behensky
• J Gopalakrishnan
• N Fernando
• HY Shih

Abstract

The transactive response DNA binding protein of 43 kD (TDP-43) is an important regulator of RNA processing, but cytoplasmic accumulation of TDP-43 is associated with several neurodegenerative diseases including amyotrophic lateral sclerosis (ALS), frontotemporal lobar dementia (FTLD), and Alzheimers disease (AD). TDP-43 has been shown to form liquid protein droplets via liquid-liquid phase separation (LLPS)—which may be involved in biological function—as well as amyloid formation, which is characterized by the self-catalyzed growth of filamentous structures associated with a number of distinct proteins and diseases. Both processes are known to be driven by the C-terminal domain (TDP-43(CTD)), and LLPS has been theorized to be on-pathway to amyloidogenesis, with liquid droplets promoting amyloid nucleation. Further, TDP-43(CTD) droplets have been shown to gelate over time, which we have shown to be associated with an increase in amyloid-like β-sheet content via Raman microscopy. In order to examine the structural differences between droplets and amyloid fibrils, an alkyne (C≡C) containing Raman probe—4-ethynylphenylalanine (Fcc)—was substituted at aromatic sites throughout TDP-43(CTD) via Amber codon suppression. Analysis of the amide I, III and Fcc C≡C stretching bands revealed that although β-sheet structure is enriched in both gelated droplets and amyloid fibrils, there are key local environmental and tertiary contact differences between the two species. These results demonstrate that although TDP-43(CTD) gelation is associated with development of β-sheet structure, these structures are distinct from filamentous amyloids. This suggests that TDP-43(CTD) droplet gelation, rather than being on-pathway to amyloidogenesis, instead represents a trapped, non-productive β-sheet structure.

Scientific Focus Area: Biomedical Engineering and Biophysics

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