effective against the oligomeric species formed by the truncated TDP-43. Panel D, Filter retardation assay performed on NSC34 expressing FLAG-tagged FL TDP-43 or  C-TDP43 in basal condition or after the treatment with different doses of 17-AAG ([17-AAG] 110 or 165 nM) for 48 h. The histogram represents a quanti ?cation of the ?lter retardation assay (from three different replicates). 17-AAG treatment was not effective against the insoluble species formed by the truncated TDP-43 retained on the cellulose acetate membrane. The results indicated that solubility and turnover of the truncated Fingolimod TDP-43 was not affected by this drug. Notably, Hsp70 and Hsp40 have been found in ARpolyQ inclusions and their over-expression has been shown to prevent AR aggregation ( Kobayashi et al., 2000; Takeuchi et al., 2002 ). A possible explanation for the different effects of 17-AAG on ARpolyQ and of mutant SOD1 or TDP-43 might be related to this alternative mechanism of action of 17-AAG involving Hsp90 inhibi- tion.

No evidences have been reported that SOD1 and TDP-43 require Hsp90 to reach their stable active conformation. On the contrary, it is known that the AR in its inactive pre-folded status is associated to a cytoplasmic multi-heteromeric complex and is directly bound to two Fig. 5. Inhibition of the autophagic pathway reduces the 17-AAG mediated clearance of mutant ARpolyQ in a motorneuronal SBMA model. Panel A, ?ow cyto Fingolimod 162359-56-0 ?uorimetric analysis performed on NSC34 expressing DsRed monomer and GFP-AR.Q48 in absence ( ?T) or in presence (+ T) of 10 nM of testosterone. Cells were analyzed in basal condition or after the treatment with 165 nM 17-AAG, 10 mM of 3-MA, the 17-AAG and the 3-MA co-treatment for 24 h (* p b 0.05 vs. testosterone untreated controls; ** p b 0.01 vs. testosterone untreated controls;  p b 0.05 vs. GFP-AR.Q48-T;  p b 0.01 vs. GFP-AR.Q48-T;  p b 0.01 vs. GFP-AR.Q48 + T). Pharmacological inhibition of the autophagic pathway resulted in the loss of the pro- degradative action of 17-AAG on mutant ARpolyQ. Panel B, Real-time PCR on LC3B mRNA expression levels on NSC34 expressing four shRNA against LC3B or shRNA scrambled control (** p b 0.01 vs. shRNA scrambled control). All four shRNAs against LC3B were found to be very active in reducing the intracellular expression levels of the LC3B mRNA. Panel C, Western blot analysis on cell lysates of NSC34 expressing AR.Q46 and co-transfected with shRNA against LC3B or shRNA scrambled control in absence ( ?T) or in presence (+ T) of 10 nM of testosterone for 48 h.

Cells were analyzed in basal condition or after treatment with 165 nM 17-AAG for 48 h. Actin was used to normalize protein loading. The histogram represents a quantitative evaluation of AR.Q46 protein level carried out by densitometric scanning of the blots (from four different replicates) ( p b 0.05 vs. AR.Q46-T; * p b 0.05 vs. AR. Q46 + T; # p b 0.05 vs. AR.Q46-T + 17-AAG;  vs. AR.Q46 + T + 17-AAG). In LC3-silenced cells, 17-AAG pro-degradative effects on ARpolyQ were markedly reduced, con ?rming that 17-AAG requires the normal autophagic activity to exert its action on mutant ARpolyQ in Fingolimod Src-bcr-Abl inhibitor motorneuronal SBMA model. Panel D, Immuno ?uorescence analysis on NSC34 cells transfected with GFP-shRNA_LC3 (or shRNA scrambled control) and AR.Q46 (red) in presence of 10 nM of testosterone. Cells were analyzed in basal condition or after the treatment with 17-AAG for 24 h.

Nuclei were stained with DAPI (blue). Images were obtained at 40X magni ?cation. (Scale bar = 16  m). 17-AAG treatment resulted in a marked reduction of AR.Q46 oral cavity aggregation and of the total AR.Q46 immuno ?uorescence per cells. The silencing of LC3 expression correlated with the loss of the pro-degradative effects of 17-AAG on AR. Q46, indicating that autophagy is required to mediate the action of 17-AAG on mutant ARpolyQ. 11 94 P. Rusmini et al. / Neurobiology of Disease 41 (2011) 83 ?95 Hsp90 molecules and one Hsp70 ( Poletti, 2004; Poletti et al., 2005 ). The AR act