which showed that LC3 silencing resulted in the complete loss of the pro-degradative action of 17-AAG. In fact, 17-AAG treatment resulted in a marked reduction of ARpolyQ (AR.Q46) aggregation, as well as in a consistent decrease of the total AR.Q46 immuno ?uorescence per cells (see also Fig. 2 B); LC3 silencing correlated with the Idarubicin re-appearance of several ARpolyQ aggregates and a restoration of high levels of the ARpolyQ immunoreactivity even in immortalized motorneurons treated with 17-AAG. This indicates that a functional autophagic pathway is required to mediate the 17-AAG pro-degradative action on the misfolded mutant ARpolyQ. Effects of the 17-AAG on other misfolded proteins involved in motorneuronal diseases The results showed above demonstrate that 17-AAG removed mutant ARpolyQ, without interfering with the proteasome, but possibly by activating an autophagic mediated degradation. The AR protein is a classical client of the Hsp90 ( Poletti, 2004 ), the chaperone Fig. 2. Effects of 17-AAG on mutant ARpolyQ aggregation, solubility and degradation in a motorneuronal SBMA model. Panel A, Cell viability assay on immortalized motorneuron NSC34 treated with different doses of 17-AAG. Left panel, the histogram represents a quanti ?cation of the cell viability assay after treatment with 330 nM of 17-AAG ([17-AAG] 330 nM vs.

untreated *** p b 0.001). Right panel , the histogram represents a quanti ?cation of the cell viability assay after treatment with increasing concentration of 17-AAG (*** p b 0.001 vs. untreated). It appears that 17-AAG induced the clearance both of ARpolyQ monomeric forms and ARpolyQ aggregates. Panel B, High resolution ?uorescence microscopy analysis (63) on NSC34 cells transfected with GFP-AR.Q48 in presence (+T) of 10 nM of testosterone in basal condition or after treatment with 165  M of 17-AAG for 48 h. Nuclei were stained with DAPI (blue). Images were obtained at 63 magni ?cation. (Scale bar Idarubicin 57852-57-0  10  m). The immuno ?uorescent signal of the GFP tagged ARpolyQ is easily detectable in absence of 17-AAG, but it becomes very low after treatment with 17-AAG, con ?rming that 17-AAG induces the removal of mutant ARpolyQ from immortalized motorneurons. Panel C, Evaluation of the aggregation properties of ARpolyQ using ?uorescence microscopy analysis performed on NSC34 cells expressing DsRed monomer and GFP-AR.Q48 in presence (+T) of 10 nM of testosterone in basal condition or after treatment with 165  M of 17-AAG for 48 h. Left panel, total number of GFP-AR.Q48 positive cells/DsRed monomer positive cells (** p b 0.01 vs. GFP-AR.Q48 + T).

Right panel, evaluation of the number of GFP-AR.Q48 positive cells bearing aggregates/DsRed monomer positive cells (** p b 0.01 vs. GFP-AR.Q48+ T). Both the total number of ARpolyQ aggregates and the number of cells containing ARpolyQ buy Idarubicin aggregates are reduced after 17-AAG treatment. Panel D, Western blot analysis on cell lysates of NSC34 expressing ARpolyQ in absence ( ?T) or in presence (+T) of 10 nM of testosterone for 48 h, in basal condition or after treatment with different doses of 17-AAG for 48 h ([17-AAG] 110 and 165 nM). Actin was used to normalize protein loading. 17-AAG, not only decreased ARpolyQ aggregation, but also increased ARpolyQ turnover. Panel E, Filter retardation assay performed on NSC34 cells transfected with ARpolyQ (AR.Q46) in absence ( ?T) or in presence (+T) of 10 nM of testosterone for 48 h, in basal condition or after treatment with different doses of 17-AAG for 48 h ([17-AAG] 110 and 165 nM). The histogram was obtained from dots optical densities of experiments performed in triplicate. (* p b 0.05 vs. AR.Q46 + T). 17-AAG treatment resulted in the removal of ARpolyQ insoluble species retained on the cellulose acetate membrane.

Panel F, Flow cyto ?uorimetric analysis performed on NSC34 expressing cinnamon GFP-AR.Q48 and DsRed monomer in absence ( ?T) or in presence (+T) of 10 nM of testosterone in basal condition or after the treatment with 165 nM 17-AAG for 48 h