Phase separation and amyloid formation of TDP-43 C-terminal domain studied by Raman spectroscopy and site-specific p-ethynyl-phenylalanine probes

Authors

• MD Watson
• JC Lee

Abstract

Transactive response DNA-binding protein 43 (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, frontotemporal lobar dementia, and Alzheimer’s disease. TDP-43 forms liquid protein droplets via liquid-liquid phase separation (LLPS), which may be involved in its biological function, but also undergoes amyloid formation, which is characterized by self-catalyzed growth of filamentous structures associated with many neurogenerative diseases. In TDP-43, both processes are driven by the C-terminal domain (TDP-43CTD), and LLPS has been theorized to be on-pathway to amyloidogenesis. Here, we employed Raman spectral imaging to study TDP-43CTD droplet aging by monitoring secondary structural changes revealed by the amide-I and -III bands. To examine the structural differences between droplets and amyloid fibrils at specific sites, an alkyne (C≡C) containing Raman probe—4-ethynylphenylalanine (FCC)—was substituted at aromatic sites via amber codon suppression. Our results indicate that although β-sheet structure is enriched in both hardened droplets and amyloid fibrils, there are key local environmental and tertiary contact differences between the two species. Furthermore, LLPS and amyloidogenesis were shown to be sensitive to even these relatively conservative aromatic-to-aromatic mutations: some mutants accelerated amyloidogenesis without any apparent change in droplet hardening, and one blocked hardening without affecting amyloidogenesis. These results demonstrate that although TDP-43CTD droplet aging is associated with β-sheet development, these structures are distinct from filamentous amyloids. In contrast to the current paradigm, we suggest that TDP-43CTD droplets, rather than being on-pathway to amyloidogenesis, instead represent an alternate β-sheet polymorph.

Scientific Focus Area: Biomedical Engineering and Biophysics

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