Abstract Details

The receptor of activated C-kinase 1 (RACK1) is a well-conserved scaffold protein with >100 recognized activities.  Co-aggregation of RACK1 with TDP-43 inclusions has been observed in sporadic ALS suggesting that it may be part of a pathogenic interactome involving the 2 proteins.  While RACK1 knock-out is embryonic lethal in mice, we hypothesized that modulation of RACK1 through knockdown in mature cells and D. melanogaster expressing pathogenic TDP-43 may provide a functional benefit.

Determine whether knockdown of RACK1 in mature cells may be beneficial against TDP-43 proteinopathy in cultured cells and in a living organism.

HEK293T cells were transfected with HA-tagged plasmids encoding wild-type or nuclear localization signal defective mutant dNLS-TDP43. RACK1 knockdown was achieved by transfecting a pool of 3 target-specific siRNA plasmids against human RACK1. Cytoplasmic aggregates of TDP-43 were visualized by immunocytochemistry (ICC) and global protein translation was measured in a Surface Sensing Translation (SUnSET) assay where cells were incubated with puromycin followed by detection of newly synthesized proteins using an anti-puromycin antibody.

The fly UAS-Gal4 expression system was used to achieve expression of hTDP-43^WT or an ALS mutation hTDP-43^Q331K and RACK1-RNAi in retinal (GMR-driven) neurons or motor (D42-driven) neurons.

ICC analysis showed that RACK1 knockdown in HEK293T cells significantly reduced aggregation of dNLS-TDP-43 accompanied by nuclear repatriation in a sub-population of transfected cells. SUnSET analysis showed that RACK1 knockdown significantly restored global translational suppression induced by wild-type TDP-43 over-expression and mutant dNLS TDP-43. RACK1 knockdown in transgenic flies expressing hTDP-43^WT or hTDP-43^Q331K alleviated retinal neuronal degeneration and improved locomotion. No degeneration in control knockdown flies was observed.

Our results suggest that RACK1 knockdown represents a viable approach to alleviate the detrimental effects of TDP-43 proteinopathy without apparent adverse effects.