COMPOSTI AD AZIONE ANTI-AGGREGANTE ESERCITANO UN EFFETTO BENEFICO SU MODELLI CELLULARI DI ATROFIA MUSCOLARE SPINALE BULBARE

Spinobulbar Muscular Atrophy (SBMA) is a rare inherited X-linked neurodegenerative disease caused by an abnormal expansion of the CAG repeat, encoding for the poly-glutamine tract (polyGln), in the N-terminal region of the Androgen Receptor (AR) gene. This mutation leads the protein to form intranuclear aggregates in the spinal cord and brainstem and cause the death of those specific cell populations. AR belongs to the nuclear hormone receptors superfamily and it works as a transcriptional factor after binding its specific ligand (testosterone or dihidrotestosterone) in the carboxyl-terminal ligand-binding domain (LBD). The interaction with the ligand has a central role in the aggregation process and converts the AR to an active conformation in which the transactivation domains are exposed and the AR can interact with transcriptional cofactors, bind the DNA on specific genes’ promoter and start the transcription. One of the fundamental events that occur during the AR activation is the close association between two transcriptional regions in a single protein, the AF2 domain (within the LBD) and the AF1 (a FXXLF motif in the N-terminal domain, NTD) in a N/C interaction. Previous studies suggest this interaction is one of the initial component of both the ligand-regulated AR function and the aggregation process of the mutant SBMA protein. In this study we tested in a PC12 cell model of SBMA (112 polyGln repeats AR expressed through an inducible system) six compounds, AR antagonist or partial agonists analogs, defined as SARMs (Selective AR Modulator), known to prevent the N/C interaction but to permit the nuclear receptor transcriptional activity. The treatment of our cells with this molecules indeed prevents the aggregation of the mutant AR [coupled to a slight reduction of protein levels] in absence of DHT and reduce the aggregation in presence of DHT, confirming that the N/C interaction is a central event in the aggregation process in SBMA, and making the SARMs possible candidates in the SBMA therapy. Moreover, other experiments performed in our lab on different SBMA cell models (NSC34 transiently expressing ARQ46)showed that the cell quality control system was impaired, causing proteasome saturation and ineffective protein clearance. In addition. it has already been demonstrated that the over expression of several Heat shock proteins (Hsps) prevents aggregation of mutant proteins addressing them to a rapid degradation. We, therefore, tested the effect of the artificial chaperone cyclodextrin (CD) on ARpolyQ. CD is a water soluble, non-toxic, cyclic oligosaccharide used as vehicle for drug delivery. CD is also validated as artificial chaperone for protein refolding in vitro, preventing interactions between aberrantly exposed amino acids and avoiding the formation of incorrect secondary structures. In our models, we found that CD decreased the mutant proteins levels (western blot experiments) and aggregation rate (filter retardation assays, immunocytochemistry analyses). Moreover, using a proteasome activity reporter (YFPu), we also observed that CD was effective in desaturating the proteasome, even in presence of the proteasome inhibitor MG132. CD may, therefore, behave like a natural chaperone and enhance the clearance of mutant proteins. Eventually, CD may desaturate the proteasome machinery or enhance alternative degradation pathways (i.e. autophagy). This non-toxic compound might, thus, represent a potential therapeutic for diseases characterized by insoluble protein aggregation.