Liquid–Liquid Phase Separation Enhances TDP-43 LCD Aggregation but Delays Seeded Aggregation

Abstract

Aggregates of TAR DNA-binding protein (TDP-43) are a hallmark of several neurodegenerative disorders, including amyotrophic lateral sclerosis (ALS). Although TDP-43 aggregates are an undisputed pathological species at the end stage of these diseases, the molecular changes underlying the initiation of aggregation are not fully understood. The aim of this study was to investigate how phase separation affects self-aggregation and aggregation seeded by pre-formed aggregates of either the low-complexity domain (LCD) or its short aggregation-promoting regions (APRs). By systematically varying the physicochemical conditions, we observed that liquid–liquid phase separation (LLPS) promotes spontaneous aggregation. However, we noticed less efficient seeded aggregation in phase separating conditions. By analyzing a broad range of conditions using the Hofmeister series of buffers, we confirmed that stabilizing hydrophobic interactions prevail over destabilizing electrostatic forces. RNA affected the cooperativity between LLPS and aggregation in a “reentrant” fashion, having the strongest positive effect at intermediate concentrations. Altogether, we conclude that conditions which favor LLPS enhance the subsequent aggregation of the TDP-43 LCD with complex dependence, but also negatively affect seeding kinetics.

Watching liquid droplets of TDP-43CTD age by Raman spectroscopy

“…However, all droplets examined here solidified. Interestingly, seeded aggregation of TDP-43 CTD was shown to be delayed under LLPS conditions ( 21 ), suggestive of underlying structural incompatibility of the aggregates. This then could explain the observed differences in ThT activities of the aggregates in droplets and fibrils, reflecting fibril polymorphism as reported in other amyloids ( 22 ).…”

Protein Aggregation Landscape in Neurodegenerative Diseases: Clinical Relevance and Future Applications

“…Moreover, residues from 328 to 333 may form steric zippers characteristic of amyloids. Finally, the presence of aromatic residues in the IDRs allows the possibility of forming low-complexity aromatic-rich kinked segments, instrumental in the formation of MLOs [146]. Indeed, the ability of TDP-43 to perform LLPS and segregate into SGs is one of its best-characterized features [144].…”

“…TDP-43 is shuttled into the nucleus due to its nuclear localization signal, where it participates in a wide range of RNA metabolic steps [147]. Under stressful conditions, TDP-43 is exported from the nucleus to the cytoplasm, where it undergoes LLPS through its kinked helical C-terminal structure [146]. However, metabolic stress induced by lactate exposure revealed the ability of TDP-43 to coalesce into micronuclei in cultured cells [148].…”

Transcription Regulators and Membraneless Organelles Challenges to Investigate Them

“…Alternation of the material properties represents a common principle underlying neurodegeneration. Many proteins (e.g., FUS, TDP-43, TIA1, tau, α-synuclein) associated with Parkinson’s disease or amyotrophic lateral sclerosis (ALS) also undergo LLPS and are constituents of different types of MLOs [ 9 , 10 , 73 , 74 , 75 ]. It suggests that the liquid-to-solid phase transition can be enhanced within the liquid phase.…”