Student’s t -test for two group comparisons and two-way ANOVA for three or more group comparisons using the PRISM software (GraphPad, CA, USA). Results In the present study, we have characterized the effects of 17-AAG on the solubility and turnover of several misfolded proteins involved in neurodegenerative diseases, using speci ?c motorneuronal models. We have initially taken advantage of the peculiar system based on 3 86 P. Rusmini et al. / Neurobiology of Disease 41 (2011) 83 ?95 ARpolyQ expression in motorneurons, as a model for SBMA. In this system, the physiological and pathological Pharmorubicin functions of the mutant protein are well understood, and neurotoxicity on the ARpolyQ can be triggered using the AR ligand testosterone ( Katsuno et al., 2003; Poletti, 2004 ). We have subsequently extended the observa- tions obtained with the SBMA model to other well-characterized diseases related to motorneuron disorders. Analysis of the testosterone-induced alterations of the biochemical behaviour of the mutant ARpolyQ in immortalized motorneurons Fig.

1 shows the variations induced by testosterone on the biochemical behaviour of mutant ARpolyQ in immortalized motor- neurons. It clearly appears from Fig. 1 A, that, in basal conditions both wt (GFP-AR.Q22) and mutant AR (GFP-AR.Q48) were diffusely con ?ned 4 P. Rusmini et al. / Neurobiology of Disease 41 (2011) 83 ?95 87 in the cell cytoplasm. Testosterone treatment induced the nuclear translocation of both wt Pharmorubicin Topoisomerase inhibitor GFP-AR.Q22 and mutant GFP-AR.Q48, but, in the latter case, testosterone also triggered the formation of intracellular aggregates of the mutant AR protein (arrows), in about 30 ?35% of total transfected cells ( Simeoni et al., 2000 ). The ?uorescence analysis data have been corroborated using ?lter retardation assay ( Fig. 1 B). In these experiments, we evaluated the total amount of intracellular insoluble material formed as a consequence of the expression of the mutant ARpolyQ in the various conditions described above. This quantitative analysis demonstrated that testosterone activation of the ARpolyQ resulted in the appearance of large amounts of insoluble mutant AR protein retained by the cellulose acetate membrane.

The total amount of the insoluble protein was largely increased by proteasome blockage using the inhibitor MG132. Altogether these data indicate that the biochemical behaviour of the mutant ARpolyQ is deeply modi ?ed during the ARpolyQ activation process induced by testosterone. Similar results were obtained in Western blot analysis ( Fig. 1 C). Testosterone had a stabilizing effect on both AR forms (wt or mutant). The proteasome inhibitor MG132 increased the amount of testosterone- induced ARpolyQ aggregates into the stacking gel. To evaluate the effect of ARpolyQ on the proteasome activity, we utilized a Yellow (YFP) variant of proteasome reporter system GFPu, initially developed by Bence and colleagues ( Bence et al., 2001; Rusmini et al., 2007; Sau et al., 2007 ), in which a short degron has been added to the YFP protein. Any alteration of the Pharmorubicin 56390-09-1 proteasome intracellular system will thus result in a YFPu (or GFPu) accumulation. This can be easily monitored in ?uorescence microscopy or western blot assay. The results obtained demonstrate that YFPu accumulated in immortalized motorneurons expressing the mutant ARpolyQ (AR.Q46) in its inactive status (not bound to T). This is possibly due to the fact that the elongated polyQ per se alters proteasome functions ( Holmberg et al., 2004 ). However, testosterone activation of ARpolyQ, which trigger aggregation of the mutant protein, resulted in an accelerated clearance of the YFPu protein.

The data strongly indicate that the segregation of the mutant ARpolyQ into physically de ?ned aggregates prevents proteasome impairment ( Rusmini et al., 2007 ). Thus, to prevent ARpolyQ neurotoxicity, it will be fundamental to increase solubility and Back bacon degradation of the mutant ARpolyQ, but, possibly, with drugs capable to prevent the potent