Anti-aggregant and pro-degradative molecules decrease mutant ARpolyQ accumulation in SBMA cell models

Spinobulbar muscular atrophy (SBMA) is a neurodegenerative disease caused by an expanded polyglutamine tract (polyQ) within the androgen receptor (AR), characterized by mutant proteins accumulation in motoneurons. We used doxycycline-inducible PC12 cells stably expressing AR.Qs and NSC34 cells transiently expressing AR.Qs, as models of SBMA. The different polyQ pathological length utilized (46 or 112 Qs) conferred different biochemical properties to the mutant AR. We noted that AR.Q112 aggregated into the nucleus while AR.Q46 in the cytosol. In addition, the number and the morphology of ARpolyQ aggregates induced by testosterone (T) or dihydrotestosterone (DHT) were markedly different. Moreover, ARpolyQ bound to the AR antagonist bicalutamide showed a prevalent cytoplasmic and diffused localization, but no aggregates were detectable. It has already been demonstrated that the over expression of several heat shock proteins prevents mutant proteins aggregation. On these bases, we analysed the effects of over-expression of HspB8, a small heat-shock protein (Hsp), which has chaperone activity but is also implicated in autophagy. In fact, we already demonstrated a pro-degradative, autophagy-mediated, effect of HspB8 in Amyotrophic lateral sclerosis (ALS) cell model. We transiently transfected HspB8 in our SBMA cell models, and we analyzed its ability to affect misfolded ARpolyQ levels. We found that the effect of HspB8 on ARpolyQ was highly dependent on the length of the polyQ tract, on the ligand utilized to induce aggregation as well as on its intracellular localization. Beside this molecular approach, we also utilized a chemical one using the disaccharide trehalose and the artificial chaperone cyclodextrin (CD). We found that trehalose inhibited intracellular ARpolyQ aggregate formation, both in NSC34 and PC12 cell models. The aggregation rate was evaluated by filter retardation assays and we noted a remarkable effect of this compound on AR.Qs. CD is a water soluble, non-toxic, cyclic oligosaccharide used as vehicle for drug delivery. In our models, we found that CD decreased the mutant proteins levels (western blot experiments) and the aggregation rate (filter retardation assays). Both molecular and chemical approaches utilized in this study showed pro-degradative and/or anti-aggregation effect on AR.Qs in the two different cell models. These results will serve as the basis to study a possible synergic effect of these molecules and also to extend the analysis to other polyQ diseases. Grants: Telethon - Italy (GGP07063), Fondazione CARIPLO (2008.2307); Fondation Thierry Latran, France.