Fig. 4: CHIR99021 treatment restores the CAST-calpain pathway in HD models.

a HdhQ111 cells were treated with vehicle (Veh; DMSO), CHIR99021 or BRD3731 (3 µM each) for 2 days. Total cell lysates from HdhQ111 and HdhQ7 cells were harvested and label-free tandem mass spectrometry (LC-MS/MS) analyses performed. The numbers of changed proteins in CHIR99021- or BRD3731-treated cells are shown. The 173 proteins altered by BRD3731 were removed from total altered proteins in CHIR99021-treated HdhQ111 striatal cells. b The biological functions of the 109 proteins from the pool marked asterisk (*) in a were analyzed. Heat map analysis of enriched proteins in proteolysis pathways. Data are analyzed by one-sided Fisher’s exact test. c Total protein lysates were harvested from postmortem caudate putamen of normal subjects (NS) (n = 3) and patients with HD (n = 6) and subjected to western blotting. Normal subject and patient information is listed in Table 1. Quantification of the relative abundance of CAST, cleaved-α-spectrin, and enolase is shown. d Total protein lysates were harvested from the striatum of 12-week-old R6/2 and WT littermates treated with vehicle or CHIR99021 and subjected to western blotting with the indicated antibodies. *Cleaved α-spectrin. Histograms show the quantification of relative calpain (n = 5), CAST (n = 7), and cleaved α-spectrin (n = 7) levels in the indicated groups. e Total protein lysates were harvested from the striatum of YAC128 and WT littermates treated with vehicle or CHIR99021 and subjected to western blotting with the indicated antibodies. Histograms show the quantification of relative calpain (n = 5), CAST (n = 8), and cleaved α-spectrin (n = 4) levels in the indicated groups. f In vitro calpain activity was assayed in the presence or absence of CHIR99021 (n = 4). Z-LLY-PMK was used as a selective calpain inhibitor. All values are reported as mean ± SEM and compared using one-way ANOVA with Tukey’s post hoc test in f, Dunnett’s post hoc test in d, e, and the unpaired Student’s t test (two-tailed) in c. Data are representative of at least three independent experiments. Exact p values are shown in the figures.