Figure 6.
A novel Gpr52 antagonist E7 reduced mHtt levels. (A) HTT levels in STHdhQ7/Q111 treated for 2 days with different doses of E7 were measured by 2B7/2166 HTRF. Six independently plated and treated wells were tested for each dose of each compound. The plots (mean ± SEM) were fitted with the Boltzmann curve. (B) mHTT levels in Huntington’s disease patient iPSC-derived neurons (Q47) treated with the indicated concentrations of E7 were measured by 2B7/3B5H10 HTRF. The number in each bar represents the number of independently plated and treated wells. (C) mHTT levels in fly head lysates from elav-GAL4 driven transgenic Drosophila expressing full-length human mHTT with 128Q (full-length HTT-Q128) or mHTT-exon1 fragments with 72Q (HTT-exon1-Q72) fed for 6 days with E7 or the DMSO control at the indicated concentrations in the food. mHTT levels were measured by 2B7/3B5H10 HTRF. n indicates the number of independent vials containing 15 virgin female flies in each vial. (D) Mutant Htt levels in the E7 versus vehicle control (DMSO) intracerebroventricularly-injected mouse brain tissues. Mutant Htt levels were measured by 2B7/2166 HTRF. Both the striata and the cortices were tested after 9 days of injection. Six mice in each group were measured. (E) Similar to the E7 treatment in A, but the cells were transfected with the non-targeting (Neg_si) or the Gpr52 siRNA (Gpr52_si) 2 days before E7 treatment. Gpr52 knock-down abolished E7’s effect. (F) The cultured primary striatal neurons from neonatal mice with the indicated genotypes were treated with the indicated concentrations of E7, and their Htt levels were measured by the 2B7/2166 HTRF 2 days after treatment. Six independently plated and treated wells are tested. All bar plots represent mean ± SEM. Statistical analyses were performed by two-tailed unpaired t-tests (D) or one-way ANOVA and Dunnett’s post hoc tests (B, C and F) for indicated comparisons: n.s. = P > 0.1, *P < 0.05; **P < 0.01; ****P < 0.0001.