Figure 5
From: Huntingtin cleavage product A forms in neurons and is reduced by gamma-secretase inhibitors
Modulators and inhibitors of gamma secretase reduce CpA levels produced from htt171-18Q in primary mouse neurons. (a) Western blot of lysates from neurons treated with Gleevec for 18 hours then infected with lentiPGK-ht171-18Q and cultured an additional 24 hours. Arrows indicates the over-expressed huntingtin fragment (Intact) and the cleavage product, cpA. Blots were re-probed with beta-3-tubulin to control for total neuron protein. (b) Densitometry results from western blot analysis of Gleevec treatment. Levels of cpA were significantly reduced in cultures treated with indicated concentrations compared treatment with DMSO solvent alone. One way ANOVA, p < 0.03, * p < 0.05 Dunnett's Multiple comparison test, n = 3 for DMSO and n = 3 for treatment conditions. (c) Western blot of lysates from neurons infected with lentiPGK-ht171-18Q for 18 hours then treated with LY-411,575 and cultured an additional 24 hours. Arrows indicates the over-expressed huntingtin fragment (Intact) and the cleavage product, cpA. Blots were re-probed with beta-3-tubulin to control for total neuron protein. (d) Densitometry results from western blot analysis of LY-411,575 treatment. Levels of cpA were significantly reduced in cultures treated with indicated concentrations compared treatment with DMSO solvent alone. One way ANOVA, p < 0.003, * p < 0.05 Dunnett's Multiple comparison test, n = 3 for DMSO and n = 3 for treatment conditions. (e) Western blot of lysates from neurons infected with lentiPGK-ht171-18Q for 18 hours then treated with DAPT at 0.01 μM and cultured an additional 24 hours. Arrows indicates the over-expressed huntingtin fragment (Intact) and the cleavage product, cpA. Blots were re-probed with beta-3-tubulin to control for total neuron protein. (f) Densitometry results from western blot analysis of DAPT treatment. Levels of cpA were significantly reduced in cultures treated with indicated concentrations compared treatment with DMSO solvent alone. * p < 0.05, n = 3 for DMSO and n = 3 for treatment conditions, paired t-test.