Clearance and transport of misfolded protein responsible for motor neuron diseases (MNDs)

Mutated proteins responsible for motor neuron diseases such as as: mutant SOD1, TDP-43, FUS, VCP, OPTN, and recently C9ORF72 RAN translated poly di-peptide in familial and sporadic ALS amyotrophic lateral sclerosis (ALS) and mutant androgen receptor (ARpolyQ) in spinal and bulbar muscular atrophy (SBMA) misfolds and aggregates into cytoplasm or nuclei of motor neuron. The protein quality control (PQC) system maintains protein homeostasis by re-folding (by chaperone) or by degradation (by autophagy or proteasome) of misfolded proteins. This prevents their toxicity. Here the trafficking of mutated misfolded proteins represents a key point to control their aggregation and degradation. In NSC34 cells, we evaluated the role of dynein retrograde transport on the PQC. Using RT-qPCR, WB and IF analysis we observed that dynein down-regulation altered SQSTM1/p62 and LC3 localization. Similarly, dynein ATPase activity inhibition by EHNA compound counteracted the induction of SQSTM1/p62 and LC3 levels by trehalose treatment. In addition, dynein inhibition increased selectively BAG1 mRNA involved in misfolded protein degradation via proteasome. Indeed, dynein inhibition clearly reduced the PBS insoluble fraction of mutated misfolded proteins (SOD1-G93A, TDP-43 C and ARpolyQ) in filter retardation assay (FRA) also when autophagy was blocked while its effects was counteracted by proteasome inhibition. In addition, BAG1 overexpression reduced aggregation of misfolded species and BAG1 down-regulation blocked the EHNA effects. Interestingly, we observed that C9ORF72 poly-GP insoluble species were processed by autophagy via HSPB8 facilitation and also in this particular model dynein inhibition counteracted the formation of misfolded protein insoluble species. Collectively, these data showed that when autophagy alteration occurs misfolded proteins can be re-routed to proteasome for degradation by BAG1