To test if RACK1 knockdown in models of TDP-43 proteinopathy might be safe and effective.

The Receptor of Activated C-Kinase 1 (RACK1) is a ribosomal protein that co-aggregates with pathogenic TDP-43 cytoplasmic inclusions characteristic of ALS and FTLD-TDP, participating in consolidation of TDP-43 aggregation and impaired global protein translation.

TDP-43 proteinopathy was modeled by transfection of misfolding HA-tagged dNLS-TDP-43 in HEK293T cells, in which morphology and localization of cytoplasmic aggregates were detected by HA-reactive antibodies, and global protein translation was measured by Surface Sensing of Translation (SUnSET). Drosophila melanogaster expression system (UAS-Gal4) was used to target expression of hTDP-43^WT or hTDP-43^Q331K to retinal or motor neurons.

No detectable changes in HEK293T cell morphology or viability were generated by siRNA knockdown of endogenous human RACK1. In cells transfected with dNLS-TDP-43, RACK1 knockdown significantly reduced average aggregate area and average aggregate size per transfected cell, surprisingly accompanied by significantly increased nuclear localization of this TDP-43 nuclear localization mutant.  RACK1 knockdown also restored global translational suppression induced by dNLS-TDP-43.   In D. melanogaster flies expressing hTDP-43^WT or ALS-associated mutant hTDP-43^Q331K, RACK1 RNAi knockdown alleviated retinal degeneration and improved locomotion. No degeneration was observed in flies upon RACK1 knockdown alone.

Our data suggest that RACK1 knockdown is well-tolerated in cultured cells and fly neurons in vivo. It alleviates TDP-43 aggregation as well as associated global translational suppression in cultured cells. The significant amelioration of neurodegeneration by RACK1 knockdown in flies supports its therapeutic potential for treating TDP-43 proteinopathy in sporadic ALS and FTLD-TDP.