The effect of the protein quality control system on TDP43 accumulation in motoneuronal and muscle models of ALS

Recent studies have shown muscle involvement at onset and progression of ALS. We focused our attention on TDP43 which is mislocalized and aggregates in the cytoplasm. The protein quality control (PQC) system is responsible for the correct protein homeostasis. The chaperones maintain proteins in the correct folding. If this system fails chaperones drives misfolded proteins to the degradative systems: ubiquitin-proteasome system (UPS) and autophagy. We characterized PQC system behavior in presence of TDP43-full length or TDP43-25, a pathological fragment present in familial ALS cases. Initially, we compared the activity of the PQC system in motoneurons NSC34 and muscle C2C12 cells. By RTq PCR, Western Blot and Immunofluorescence analyses of key protein involved in the degradative systems (Bag3, Bag1, p62 and LC3) we found that muscle cells have a more activated autophagic system than motorneurons. Then, we studied the behavior of the PQC system in presence of TDP43 full length or TDP43-25 fragment. We pharmacologically inhibited degradative systems and by Filter Trap Assay and Western Blot analysis we observed that both TDP43 species are primarily degraded trough UPS. Immunofluorescence analysis showed that UPS inhibition increased TDP43 levels and induced accumulation of the truncated protein in the cytoplasm. We also investigated chaperones involvement in the removal of toxic proteins. We have already shown that overexpression of the small heat shock protein B8 (sHSPB8) reduces TDP43 aggregation in motoneurons. Surprisingly, we noted that HSPB8 overexpression had no effect on TDP43 aggregation. Silencing HSPB8 in muscle cells produced no effect on the aggregation of both proteins. In conclusion, we demonstrated that PQC system has a key role in the removal of pathological protein. We also demonstrated that degradative systems could be differentially activated in motoneuronal or muscle cells. GRANTS: Regione Lombardia; AFM-TELETHON; FONDAZIONE TELETHON; FONDAZIONE CARIPLO; FONDAZIONE ARISLA; Ministero della Sanità.