Physiologically Important Electrolytes as Regulators of TDP-43 Aggregation and Droplet-Phase Behavior

Abstract

Intraneuronal aggregation of TDP-43 is seen in 97% of all amyotrophic lateral sclerosis cases and occurs by a poorly understood mechanism. We developed a simple in vitro model system for the study of full-length TDP-43 aggregation in solution and in protein droplets. We found that soluble, YFP-tagged full-length TDP-43 (yTDP-43) dimers can be produced by refolding in low-salt HEPES buffer; these solutions are stable for several weeks. We found that physiological electrolytes induced reversible aggregation of yTDP-43 into 10–50 nm tufted particles, without amyloid characteristics. The order of aggregation induction potency was K+ < Na+ < Mg2+ < Ca2+, which is the reverse of the Hofmeister series. The kinetics of aggregation were fit to a single-step model, and the apparent rate of aggregation was affected by yTDP-43 and NaCl concentrations. While yTDP-43 alone did not form stable liquid droplets, it partitioned into preformed Ddx4N1 droplets, showing dynamic diffusion behavior consistent with liquid–liquid phase transition, but then aggregated over time. Aggregation of yTDP-43 in droplets also occurred rapidly in response to changes in electrolyte concentrations, mirroring solution behavior. This was accompanied by changes to droplet localization and solvent exchange. Exposure to extracellular-like electrolyte conditions caused rapid aggregation at the droplet periphery. The aggregation behavior of yTDP-43 is controlled by ion-specific effects that occur at physiological concentrations, suggesting a mechanistic role for local electrolyte concentrations in TDP-43 proteinopathies.

Structural Insights Into TDP-43 and Effects of Post-translational Modifications

“…The TDP-43 N-terminal domain has been shown to form dimers and higher-order oligomers both in vitro and in the cell (Shiina et al, 2010;Chang et al, 2012;Wang et al, 2013;Zhang et al, 2013;Afroz et al, 2017;Jiang et al, 2017;Mompeán et al, 2017;Sun et al, 2018; with a single dissociation constant of approximately 70 µM, indicating absence of cooperativity in binding of subunits . Unlike pathological aggregates, which are hyperphosphorylated and ubiquitinated (Arai et al, 2006;Hasegawa et al, 2008), reversible formation of TDP-43 polymers through the NTD has been shown to be required for splicing activity (Afroz et al, 2017;Jiang et al, 2017;Mompeán et al, 2017; and to contribute to phase separation via liquid-droplet formation (Afroz et al, 2017;, thought to contribute to formation of cytoplasmic stress granules (SGs) (Molliex et al, 2015).…”

TDP-43 and Phosphorylated TDP-43 Levels in Paired Plasma and CSF Samples in Amyotrophic Lateral Sclerosis

“…Recent studies showed some evidence of physiological electrolytes induced reversible aggregation of YFP-tagged full-length TDP-43 (yTDP-43) in vitro model system. The order of aggregation induction potency was K+ < Na+ < Mg2+ < Ca2+( 39 ). The EDTA-plasma tubes cause less aggregation of TDP-43 than polypropylene-CSF tubes, which is consistent with the results of higher levels of TDP-43 in the plasma but little in the CSF.…”

Differences in interaction lead to the formation of different types of insulin amyloid

“…This process is driven by temporary interactions, such as electrostatic, hydrophobic, hydrogen bonding, π-π, and cation-π interactions. It has been reported that LLPS occurs during the formation of amyloid aggregates of various proteins, such as α-synuclein 13 , tau 14 , islet amyloid polypeptide 15 , and TDP-43 16 , 17 . The amyloid formation process in the droplet (e.g., nucleation and maturation) differs depending on the protein.…”