ained showed that GFPu levels decreased when ARpolyQ forced to aggregate with testosterone treatment, in line with our previous observation in western blot analysis ( Fig. 1 C, see also Fig. 1. Biochemical behaviour of wt and mutant AR in motorneuronal NSC34 cells. Panel A, high resolution ?uorescence microscopy analysis (63) on NSC34 cells transfected with GFP-AR.Q22 or GFP-ARQ.48 in absence ( ?T) or in presence (+ T) of 10 nM of testosterone for 48 h. Nuclei were stained with DAPI (blue). Arrows = mutant Tacrolimus ARpolyQ aggregates. Images were obtained at 63X magni ?cation. (Scale bar = 10 m). The panel shows the mechanisms of aggregation of mutant ARpolyQ induced by its ligand testosterone.
Panel B, Filter retardation assay performed on NSC34 cells transfected with wt AR (AR.Q23) or SBMA mutant (AR.Q46) AR in absence ( ?T) or in presence (+T) of 10 nM of testosterone for 48 h, in basal condition or after treatment with 10 M of MG132 for 24 h. The accumulation of immunoreactive AR on the cellulose acetate membrane indicates the presence of aggregated insoluble species of mutant ARpolyQ. No aggregates were buy Tacrolimus detectable in basal condition in the case of wtAR (AR.Q23) and in untreated ARpolyQ (AR.Q46). Testosterone treatment resulted in a robust increase of ARpolyQ aggregates, which further increased after proteasome blockage with MG132. Panel C, Western blot analysis on cell lysates of NSC34 expressing YFPu and wt AR (AR.Q23) or SBMA mutant (AR.Q46) AR in absence ( ?T) or in presence (+ T) of 10 nM of testosterone for 48 h, in basal condition or after treatment with 10 M of MG132 for 24 h. Actin was utilized to normalize protein loading. It appears that the levels of the monomeric wtAR (AR.Q23) and of mutant ARpolyQ (AR. Q48) are in ?uenced by proteasome inhibition.
On the other hand, mutant ARpolyQ in its soluble status (- T) was able to impair the proteasome system (evaluated by the levels of YFPu accumulated), while testosterone-activated ARpolyQ (AR.Q46), which also forms aggregates, was unable to impair the proteasome, since the YFPu reporter was normally cleared from the cells. Testosterone-induced aggregation of ARpolyQ correlated with proteasome desaturation even in purchase Tacrolimus presence of the proteasome inhibitor MG132. 5 88 P. Rusmini et al. / Neurobiology of Disease 41 (2011) 83 ?95 ( Rusmini et al., 2007 )). As expected, MG132 resulted in a dramatic increase of GFPu accumulation both in absence or in presence of AR ligand testosterone, even if a partial ?de-saturation ?was evident in the NSC34-AR.Q46 treated with testosterone. Autophagic blockage with 3-methyladenine (3-MA) also resulted in a partial accumulation of the GFPu proteasome reporter system. Thus, autophagy inhibition also results in a proteasome impairment ( Rusmini et al., 2010 ).
Finally, 17-AAG treatment has apparently no effects on the total levels of 6 P. Rusmini et al. / Neurobiology of Disease 41 (2011) 83 ?95 89 GFPu, suggesting that 17-AAG mediated removal of mutant ARpolyQ occurs without interfering with the proteasome system. Thus, the inhibition of the autophagic pathway results in the saturation of the proteasome system, even when the ARpolyQ is forced to aggregate by testosterone. It is possible that, testosterone dependent ARpolyQ aggregates may be cleared from the cells by autophagy. Fig. 3 B shows a ?uorescence microscopy analysis performed on samples obtained as described for the cyto ?uorimetric analysis. The two sets of data were very similar and demonstrate that 17-AAG not only prevents aggregate formation, but also prevents GFPu accumulation in cells expressing mutant ARpolyQ (either in basal condition or after testosterone activation of the AR). Therefore, despite the fact that 17-AAG increases ARpolyQ solubility and clearance, the proteasome is not a full breakfast affected by the release of the polyQ peptides. Moreover, even in this assay, autophagy inhibition with 3-MA correlated with a large accumulation of mutant ARpolyQ both in basal conditions and after testo