Amyotrophic lateral sclerosis-linked mutations increase the viscosity of liquid-like TDP-43 RNP granules in neurons

Proc Natl Acad Sci U S A. 2017 Mar 21;114(12):E2466-E2475. doi: 10.1073/pnas.1614462114. Epub 2017 Mar 6.

Abstract

Ribonucleoprotein (RNP) granules are enriched in specific RNAs and RNA-binding proteins (RBPs) and mediate critical cellular processes. Purified RBPs form liquid droplets in vitro through liquid-liquid phase separation and liquid-like non-membrane-bound structures in cells. Mutations in the human RBPs TAR-DNA binding protein 43 (TDP-43) and RNA-binding protein FUS cause amyotrophic lateral sclerosis (ALS), but the biophysical properties of these proteins have not yet been studied in neurons. Here, we show that TDP-43 RNP granules in axons of rodent primary cortical neurons display liquid-like properties, including fusion with rapid relaxation to circular shape, shear stress-induced deformation, and rapid fluorescence recovery after photobleaching. RNP granules formed from wild-type TDP-43 show distinct biophysical properties depending on axonal location, suggesting maturation to a more stabilized structure is dependent on subcellular context, including local density and aging. Superresolution microscopy demonstrates that the stabilized population of TDP-43 RNP granules in the proximal axon is less circular and shows spiculated edges, whereas more distal granules are both more spherical and more dynamic. RNP granules formed by ALS-linked mutant TDP-43 are more viscous and exhibit disrupted transport dynamics. We propose these altered properties may confer toxic gain of function and reflect differential propensity for pathological transformation.

Keywords: TDP-43; amyotrophic lateral sclerosis; liquid droplets; neurons; ribonucleoprotein granules.

Publication types

  • Research Support, N.I.H., Extramural

MeSH terms

  • Amyotrophic Lateral Sclerosis / genetics*
  • Amyotrophic Lateral Sclerosis / metabolism
  • Animals
  • Axons / chemistry
  • Axons / metabolism
  • Cells, Cultured
  • Cytoplasmic Granules / chemistry
  • Cytoplasmic Granules / genetics
  • Cytoplasmic Granules / metabolism*
  • DNA-Binding Proteins / genetics*
  • DNA-Binding Proteins / metabolism
  • Humans
  • Motor Neurons / chemistry
  • Motor Neurons / metabolism*
  • Mutation
  • Rats
  • Rats, Sprague-Dawley
  • Ribonucleoproteins / genetics
  • Ribonucleoproteins / metabolism*
  • Viscosity

Substances

  • DNA-Binding Proteins
  • Ribonucleoproteins
  • TARDBP protein, human
  • Tardbp protein, rat