TDP-43 Amyloid Fibril Formation via Phase Separation-Related and -Unrelated Pathways

Area of Science:

• Biochemistry
• Molecular Biology
• Neuroscience

Background:

• Intrinsically disordered regions (IDRs) in proteins can drive liquid-liquid phase separation (LLPS).
• LLPS is linked to functional protein assembly but also to disease-associated amyloid fibril formation.
• The relative importance of LLPS versus other pathways in fibril formation is not fully understood.

Purpose of the Study:

• To investigate the role of LLPS in the formation of amyloid fibrils by TDP-43, a protein implicated in neurodegenerative diseases.
• To differentiate between LLPS-mediated and non-LLPS-mediated fibril formation pathways for TDP-43.

Main Methods:

• Utilized thioflavin T (ThT) fluorescence assays with a modified probe.
• Employed Nuclear Magnetic Resonance (NMR) spectroscopy.
• Conducted transmission electron microscopy (TEM) and wide-angle X-ray scattering (WAXS) experiments.

Main Results:

• Under LLPS-favoring conditions, biphasic ThT signals suggest amorphous aggregates as intermediates, diverging from direct fibril formation.
• Under non-LLPS conditions (low pH or modified TDP-43 construct), gelation occurred, forming fibril-like networks.
• Both LLPS-dependent and LLPS-independent pathways lead to fibril-like structures.

Conclusions:

• TDP-43 intrinsically disordered regions form disease-causing amyloid fibrils irrespective of the specific formation pathway (LLPS or gelation).
• Findings explain the presence of both LLPS-promoting and LLPS-inhibiting mutations in TDP-43-related diseases.
• Understanding these pathways is crucial for neurodegenerative disease research.