The protein quality control system in motor neuron degeneration in Spinal and Bulbar Muscular Atrophy

Spinal and bulbar muscular atrophy (SBMA) is an X-linked motoneuron disease caused by an abnormal expansion of a CAG triplet repeat in exon 1 of the androgen receptor (AR) gene. This expansion is translated into an abnormally long polyglutamine tract (polyQ) in the AR protein. The AR is a transcription factor that mediated the biological activities of male sexual hormones. Several evidence suggests that the elongated polyQ tract confers a toxic gain-of-function to the mutant AR. This ARpolyQ toxicity is activated by the AR ligand testosterone (or dihydrotestosterone). Testosterone-dependent toxicity is triggers by the generation of ARpolyQ misfolded species, that in some conditions, tend to aggregate in spinal cord motoneurons and muscle cells. ARpolyQ aggregates are formed in the cytoplasm and nuclei of affected cells, but they toxic role is still largely debated. Instead, there is general agreement on the fact that ARpolyQ toxicity is associated with the nuclear localization of an altered (misfolded) fraction of the protein. Therefore, the prevention of nuclear localization and/or the facilitation of ARpolyQ misfolded fraction degradation may have great beneficial effects against ARpolyQ toxicity. 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. Generally, the aggregates are transported to the microtubule organization center (MTOC) to be engulfed into autophagosomes and destroyed by the autophagic system. 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.