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

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Abstract
Intraneuronal aggregation of TDP-43 is seen in 97% of all ALS 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 into 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 pre-formed Ddx4N1 droplets, showing dynamic diffusion behavior consistent with liquid-liquid phase transition, but which then aggregated over time. Aggregation of yTDP-43 in droplets also occurred rapidly in response to concentration changes of electrolytes, 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. 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.
Funding Information
  • Canadian Institutes of Health Research
  • Alzheimer Society
  • ALS Society of Canada
  • Weston Brain Institute
  • James Hunter Family ALS Initiative
  • Canadian Consortium of Neurodegeneration and Aging

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