The role of protein quality control system in SBMA

Motor neuron diseases, like the spinobulbar muscular atrophy (SBMA) are characterized by the presence of inclusions o aggregates of proteinaceous materials. In SBMA, aggregates contain mutant androgen receptors (AR) with an elongated polyglutamine tract (ARpolyQ), responsible for disease, the ubiquitin, chaperones and several other components of the protein quality control (PQC) system. In general, protein aggregation is due to generation of aberrant protein conformations (misfolding). Thus, in neuronal cells, the PQC system may be insufficient to correctly remove the misfolded proteins.The PQC system requires the activities of efficient chaperones and of the degradative systems ubiquitin-proteasome (UPS) and autophagy. Recently, we demonstrated that the ubiquitin proteasome system (UPS) degrades a large fraction of misfolded ARpolyQ. However, an excess of ARpolyQ may overwhelm the UPS, escaping the degradation and thus tend to accumulate in aggregates. These aggregates are useful to confine the toxic protein in a physically defined subcellular compartment, thus reducing the potential toxicity of undegraded misfolded ARpolyQ. We found that when UPS is impaired in motoneurons, cells may responds to misfolded species by activating the expression of a small HSP, HSPB8, which facilitate the autophagic removal of misfolded species, restoring a proper autophagic flux. The HSPB8 acts in conjunction with a co-chaperone BAG3, which is delivered by dynein in a complex with HSC70-CHIP to the microtubules organization center where autophagosomes are assembled. Here, CHIP ubiquitinated misfolded protein substrates allowing their recognition by p62 and clearance from the motoneurons. Generally, the aggregates are transported to the microtubule organization center (MTOC) to be engulfed into autophagosomes and destroyed by the autophagic system. After misfolded protein recognition by chaperones, the dynein motor complex plays a crucial role to efficiently remove these species via autophagy transporting them to autophagosome and assisting autophagosome-lysosome fusion. When an insufficient pool of specific chaperones is present, the autophagic flux may be blocked and this lead to the formation of insoluble inclusions. In fact, we found that the silencing of HspB8, a member of the small heat shock protein family, correlated with a massive accumulation of misfolded ARpolyQ in immortalized motoneuronal cells. Conversely, HspB8 overexpression facilitates the autophagic removal of misfolded aggregating species of ARpolyQ. HspB8 is highly induced in surviving motoneurons of patients affected by motoneuron diseases and HspB8 participates in the stress response aimed at cell protection. HspB8 does not induce autophagy (p62 and LC3 expression, two key autophagic molecules), but it prevents p62 bodies formation, restoring a fully functional autophagic flux. Notably, we found that, trehalose, a natural disaccharide know to be a potent autophagy stimulator, induces HspB8 expression suggesting that HspB8 could be one of the molecular mediators of the pro-autophagic activity of trehalose. Therefore, based on the evidence that testosterone triggers nuclear toxicity by inducing AR nuclear translocation, and misfolded species accumulate because of an impaired autophagic flux, we hypothesize that prevention of ARpolyQ nuclear localization, combined with an increased ARpolyQ cytoplasmic clearance should reduce its toxicity. We thus used the antiandrogen Bicalutamide (Casodex®), which slows down AR activation and nuclear translocation, in combination with trehalose, and we found that in motoneurons the two compounds synergically reduced ARpolyQ insoluble forms, with an efficiency higher than the one observed in single treatments. This effect was also present on insoluble species of AR with a very long polyQ (Q112) tract, capable to generate nuclear aggregates into the cell nuclei. Therefore, the combinatory use of Bicalutamide and trehalose greatly facilitate ARpolyQ clearance, and might be a promising approach to be tested in vivo in animal models of SBMA.