Figure 3. Mechanism of QC-01–175 clearance of tau is CRL4^CRBN and UPS-dependent.

Neurons were pre-treated for 6 hr with (A) either CRBN ligand excess lenalidomide or tau ligand excess T807, (B) the NAE inhibitor MLN4924, the autophagy inhibitor Baf.A1, or (C) the proteasome inhibitor carfilzomib; followed by 18 hr treatment with QC-01–175 (or negative control QC-03–075), for a total of 24 hr. Total (TAU5) and P-tau S396 levels were analyzed by western blotting. (A–C) Representative blots are shown. (D–F) Densitometry bars represent tau mean intensity values ± SD (n = 3), relative to vehicle-treated samples. Student T-test of QC-01–175 samples relative to vehicle treated, and the remainder bars show p-value of each pre-treatment relative to QC-01–175 to assess rescue of clearance effect (***p<0.001, **p<0.01, *p<0.05, ^nsp>0.05). A152T neurons were differentiated for 6 weeks. Figure 3—figure supplement 1 includes additional specificity controls for A152T, P301L and control neuronal models. The following figure supplement is available for Figure 3.

Figure 3—figure supplement 1. Additional specificity controls for QC-01–175-mediated tau clearance.

(A–C) Western blot and densitometry analysis of total tau, S396 P-tau and CRBN upon 24 hr treatment with QC-01–175 or the negative controls QC-03–075, T807 and a thalidomide analog, lenalidomide (1 μM), in (A) A152T, (B) P301L or (C) control-1 8330–8-RC1 neurons. Cropped lanes correspond to samples not included in this manuscript (Silva and Ferguson, Manuscript in preparation, 2019), but all samples in each set were run in the same gel. Densitometry graph bars represent tau or CRBN mean intensity levels ± SD (n = 3), relative to vehicle-treated samples. (D) QC-01–175 had minimal effect on CRBN upon 24 hr treatment. Student T-test of compound-treated samples relative to vehicle-treated ***p<0.001, **p<0.01, *p<0.05, ^nsp>0.05.