sterone treatment ( Fig. 3 B); this ?nding resembles that obtained with proteasome inhibition with MG132. Since the effects of 17-AAG could be mediated by the activation of the autophagy, induced as a consequence of alterations of the proteasome pathway, we Rapamycin decided to measure all three enzymatic activities associated to the protea- some complex: tryptic, chimotryptic and post-acidic. This analysis has been performed by using speci ?c ?uorescent substrates for each of these enzymatic activities. The results reported in Fig. 3 C con ?rmed that 17-AAG did not modify any of these proteasome activities in presence of the mutant ARpolyQ. Thus 17-AAG should act directly on the autophagic system, and not as a consequence of proteasome blockage. Therefore, we analyzed potential variation of the levels and of the intracellular distribution of the autophagic marker LC3. Real time-PCR analysis showed that 17-AAG, in presence of the mutant ARpolyQ, induced LC3 expression already at 12 h after treatment ( Fig. 4 A), an indication of the activation of the autophagic pathway in response to this drug.

With regards to its intracellular distribution, in basal condition, LC3 protein is diffusely distributed in the cells in the LC3-I form. After autophagy activation, LC3-I becomes Rapamycin 53123-88-9 lipidated and anchored, as LC3-II, to the surface of the newly formed autophago- somes, showing a typical punctate distribution. The results ( Fig. 4 B) show that 17-AAG induced an increase of the punctate distribution of the LC3-II isoform, both in the absence and in the presence of testosterone. These results have been further con ?rmed by analysing the levels of LC3-II protein in western blot ( Fig. 4 C). In fact, LC3-II lipidated protein is dramatically increased in samples expressing ARpolyQ and treated with 17-AAG ( Fig. 4 C, lower inset) either in the absence or in the presence of testosterone. The effects of 17-AAG on testosterone activated ARpolyQ correlated with the appearance of the LC3-II protein. Thus, 17-AAG-induced autophagy may be responsible for the removal of the excess of misfolded protein released after dissociation from the Hsp90. Since it has been shown that the 17-AAG action may be due not only to Hsp90 inhibition but also partially mediated by the up-regulation of heat shock proteins, via the modulation of heat shock factor 1 (HSF1) activation ( Fujikake et al., 2008 ), we analyzed Hsp90 and Hsp70 levels after 17-AAG treatment. The results, Fig. 4 C, con ?rmed that 17-AAG robustly increased the levels of these two chaperones.

Autophagic blockage correlated with the loss of the 17-AAG pro-degradative action on ARpolyQ All together the data reported above indicate that autophagy is an alternative pathway utilized for the ARpolyQ clearance during 17-AAG exposure, and the reason why proteasome saturation is prevented. To con ?rm this mechanism, we analyzed the total levels of ARpolyQ after treatment with 17-AAG, during autophagy blockage. The data buy Rapamycin shown in Fig. 5 A demonstrated that, when the autophagic ?ux is blocked with 3-MA, 17-AAG cannot exert its bene ?cial activity on the turnover of mutant ARpolyQ. However, 3-MA is not a pure autophagic inhibitor; therefore we further con ?rmed the potential involvement of autophagy as mediator of the 17-AAG pro-degradative activity, by silencing one of the most important genes involved in the autophagic pathway: LC3. To this purpose we utilized four different shRNAs recognizing the LC3B mRNA: Real time PCR analysis ( Fig. 5 B) demonstrated that all four constructs abolished LC3 expression when transfected in NSC34 cells.

We compared the effects of 17-AAG on ARpolyQ protein levels in absence or in presence of the shRNA-LC3B (using the most active shRNA-LC3B_1). Western blot corncobs analysis reported in Fig. 5 C clearly showed that LC3B silencing correlated with a signi ?cant reduced potential of 17-AAG to remove mutant ARpolyQ. These data found further support in the immuno ?uorescence microscopy analysis ( Fig. 5 D),