Motoneuron and muscle-selective removal of ALS-related misfolded proteins

Background: Amyotrophic lateral sclerosis (ALS) occurs in clinically indistinguishable sporadic (sALS) or familial (fALS) forms. Most of the fALS-related mutant proteins identified so far, such as mutant SOD1, TDP-43, FUS, are prone to misfold; also the product of the mutant C9ORF72 gene aberrantly codes for small highly hydrophobic dipeptides. Both misfolded proteins and hydrophobic peptides accumu- late into insoluble proteinaceous material inside motor neurons. This material must be cleared away from cells with the assistance of the molecular chaperones. Chaperones may act on aberrant proteins either by assisting their refolding or by directing them to degradation through the proteasome (UPS) or the autophagic system. Results: Motor neurons are very sensitive to misfolded protein toxicity, but other cell types, such as astrocytes, oligodendrocytes, and muscle cells could also be affected by their presence. Notably, muscle-restricted expression of mutant SOD1 (mutSOD1), responsible for some fALS, induces muscle atrophy and motor neuron death. We found that several genes are altered in the skeletal muscle of mutSOD1 mice. In fact, we observed upregulation of specific muscle genes, such as MyoD, myogenin, and also of several components of cell response to proteotoxicity (atrogin-1, HspB8, Bag1, and Bag3). Similar changes were found to occur in cultured ALS myoblasts. We then compared the potential mutSOD1 toxicity in motor neuron (NSC34) and muscle (C2C12) cells. Initially, we found that muscle ALS models possess much higher chymotryptic proteasome activity and autophagy power than motor neuron ALS models. The mutSOD1 molecular behaviour was also very different. MutSOD1 clearance was much higher in muscle than in motor neurons, and the mis- folded protein formed aggregates and impaired proteasome only in motor neurons. The motor neuronal cells were also more sensitive to superoxide-induced oxidative stress. In muscle cells, mutSOD1 remained soluble even after protea- some inhibition, possibly because of high mutSOD1 autophagic clearance. Finally, N-terminal TDP-43 fragment accumulated in NSC34, but not in C2C12 cells. In the case of TDP-43, proteasome inhibition resulted in a large accu- mulation of both wt and N-terminal fragment of TDP-43. Discussion: Therefore, our results suggest that muscle cells differentially manage misfolded mutSOD1 and TDP-43 and their toxicity in muscle may not directly depend on aggregation. Acknowledgements: Italian Ministry of Health (Conv- Mondino/UNIMI); Università di Milano; RegioneLombar- dia; Fondation Thierry Latran, France; AFM-France.