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

Intrinsically disordered regions (IDRs) in proteins can undergo liquid-liquid phase separation (LLPS) for functional assembly, but this increases the chance of forming disease-associated amyloid fibrils. Not all amyloid fibrils form through LLPS however, and the importance of LLPS relative to other pathways in fibril formation remains unclear. We investigated this question in TDP-43, a motor neuron disease and dementia-causing protein that undergoes LLPS, using thioflavin T (ThT) fluorescence, NMR, transmission electron microscopy (TEM), and wide-angle X-ray scattering (WAXS) experiments. Using a fluorescence probe modified from ThT strategically designed for targeting protein assembly rather than β-sheets and supported by TEM images, we propose that the biphasic ThT signals observed under LLPS-favoring conditions are due to the presence of amorphous aggregates. These aggregates represent an intermediate state that diverges from the direct pathway to β-sheet-dominant fibrils. Under non-LLPS conditions in contrast (at low pH or at physiological conditions in a construct with key LLPS residues removed), the protein forms a hydrogel. Real-time WAXS data, ThT signals, and TEM images collectively demonstrate that the gelation process circumvents LLPS and yet still results in the formation of fibril-like structural networks. We suggest that the IDR of TDP-43 forms disease-causing amyloid fibrils regardless of the formation pathway. Our findings shed light on why both LLPS-promoting and LLPS-inhibiting mutants are found in TDP-43-related diseases.