Figure 6. Polyglutamine expansion increases ataxin-3 affinity for CHIP and results in decreased CHIP levels in SCA3 mice.

(A) Quantitative binding analysis between CHIP or Ub-CHIP and normal or expanded ataxin-3 (Q22 or Q80). Biotinylated CHIP or Ub-CHIP was immobilized to streptavidin-coated sensors and association and dissociation of ataxin-3 (Q22 or Q80) was monitored (K_d =2.2 µM for Q22 to CHIP, K_d =0.39 µM for Q80 to CHIP, and K_d=0.75 µM for Q80 to Ub-CHIP). (B) CHIP levels are decreased in SCA3 Q84.2 transgenic mouse brain. CHIP levels were assessed in whole brain lysates from 4-month-old nontransgenic or Q84.2 hemizygous transgenic mice. CHIP levels are significantly reduced in Q84.2 mice (n=3, *p = 0.0372). Error bars indicate SEM. (C) Immunohistochemistry reveals decreased brain CHIP levels in Q84.2 mice. Brains from Q84.2 hemizygous mice or nontransgenic littermates were sectioned and immunostained with CHIP antibody. DAB immunohistochemistry was carried out simultaneously and identically on sections from nontransgenic and Q84 mice. (D) CHIP levels are not altered in Atxn3 knockout mice. Whole brain lysates from 2 month old wild-type or Atxn3 knockout mice were analyzed by SDS-PAGE and western blot. Error bars indicate SEM.