PROTEIN MISFOLDING IN KENNEDY¿S DISEASE AND IN RELATED MOTOR NEURON DISEASES (MNDS)

Motor neuron diseases, like spinobulbar muscular atrophy (SBMA) and amyotrophic lateral sclerosis (ALS) are characterized by the presence of inclusions or aggregates of proteinaceous materials. In SBMA, inclusions are formed by testosterone dependent aggregates of mutant androgen receptor (AR) with an elongated polyglutamine tract (ARpolyQ), while in ALS inclusions contain several aggregated proteins including TDP43, ubiquilin, optineurin. Exceptions are familial ALS forms linked to superoxide dismutase 1 (SOD1) mutations, to mutated TDP43 and to C9ORF72 poly-dipeptides (DPRs), in which aggregates are mainly composed of mutant SOD1, mutant TDP43 or DPRs, respectively. In general, protein aggregation is due to generation of aberrant protein conformations (misfolding) combined to a failure, in neuronal cells, of the protein quality control (PQC) system, which may be insufficient to correctly remove the misfolded proteins. In other target tissue, such as the muscles, a different physiological PQC regulation may be helpful to remove misfolded proteins related to MNDs. The PQC system requires the activities of chaperones, degradative systems ubiquitin- proteasome (UPS) and autophagy. 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. In this thesis, I have investigated the implications of protein misfolding in SBMA and in ALS. Taking advantage of a comparative analysis of misfolded proteins response in skeletal muscle and in spinal cord of SMBA mice, we proved that autophagy is dramatically perturbed in muscles. Indeed, we found the up-regulation of most autophagic markers (Beclin-1, ATG10, p62/SQSTM1, LC3). In addition, the chaperon small Heat Shock Protein B8 (HSPB8) and its co-chaperone BCL2-Associated Athanogene 3 (BAG3), required for autophagy, were robustly up-regulated together with other specific HSPB8 interactors (HSPB2 and HSPB3). Interestingly, the BAG3:BAG1 ratio, increased in muscle, suggesting preferential misfolded proteins routing to autophagy rather than to proteasome. Misfolded proteins, recognized by HSPB8-BAG3 complex, are actively transport by dynein to MTOC to be inserted in autophagosome and degraded by autophagy, Then, we analysed the role of dynein mediate transport in the autophagic removal of misfolded proteins. In immortalized motoneuronal NSC34 cells, we found that the reduction of dynein protein levels, obtained using a specific siRNA, resulted in autophagy inhibition and in unexpected testosterone dependent ARpolyQ aggregates reduction. Also, we found that pharmacological dynein inhibition, with erythro-9-(2- Hydroxy-3-nonyl) adenine hydrochloride (EHNA), in NSC34 cells expressing ARpolyQ, mutant SOD1, truncated TDP43 form or C9ORF72 DPRs, induced a great reduction of mutant protein aggregates, even in presence of an autophagy inhibitor (3-MA), but not of a proteasome inhibitor (MG132). By performing fractionation studies we found that EHNA increased the ARpolyQ levels in PBS and Triton-X100 fractions. Surprisingly, we found that ENHA effects were paralleled by an increased expression of BAG1, a co- chaperone which routes misfolded proteins to UPS, but not of BAG3 suggesting the prevalence of UPS functions. Indeed, when dynein activity was blocked, BAG3:BAG1 ratio was decreased, thus in favour of BAG1 expression, suggesting the involvement of the pro-degradative activity of BAG1 on ARpolyQ aggregates. Collectively, these data show that mutant ARpolyQ induces a potent autophagic response in muscle cells. This may be useful to evaluate the SBMA progression. In parallel, dynein blockage perturbs autophagy and modifies the response of PQC system to misfolded protein. This results in reduced aggregation of MNDs-related misfolded proteins, a phenomenon that may occurs via an increase in their solubility and the induction of UPS functions