RACK1 Knockdown is a Potential Therapeutic Target in ALS and FTLD-TDP (P1-6.003)

Abstract

<h3>Objective:</h3> To test if RACK1 knockdown in models of TDP-43 proteinopathy might be safe and effective. <h3>Background:</h3> 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. <h3>Design/Methods:</h3> 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. <h3>Results:</h3> 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. <h3>Conclusions:</h3> 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. <b>Disclosure:</b> Dr. Cashman has received personal compensation for serving as an employee of ProMIS Neurosciences. Dr. Cashman has received personal compensation in the range of $5,000-$9,999 for serving on a Scientific Advisory or Data Safety Monitoring board for Mitsubishi-Tanabe. Dr. Cashman has stock in ProMIS Neurosciences. The institution of Dr. Cashman has received research support from ProMIS Neurosciences. Dr. Cashman has received intellectual property interests from a discovery or technology relating to health care. Dr. Cashman has a non-compensated relationship as a BoD memeber with ALS Society of BC that is relevant to AAN interests or activities. Beibei Zhao has received intellectual property interests from a discovery or technology relating to health care. Dr. Cowan has nothing to disclose. Miss Saraph has nothing to disclose. Mr. Christy has nothing to disclose. Ms. Coutts has nothing to disclose. Dr. Kaplan has received personal compensation for serving as an employee of ProMIS Neurosciences. Dr. Kaplan has stock in ProMIS Neurosciences. Dr. Kaplan has received intellectual property interests from a discovery or technology relating to health care.