C9orf72 ALS/FTD dipeptide repeat protein levels are reduced by small molecules that inhibit PKA or enhance protein degradation

Intronic GGGGCC (G4C2) hexanucleotide repeat expansion within the human C9orf72 gene represents the most common cause of familial forms of amyotrophic lateral sclerosis (ALS) and fronto-temporal dementia (FTD) (C9ALS/FTD). Repeat-associated non-AUG (RAN) translation of repeat-containing C9orf72 RNA results in the production of neurotoxic dipeptide-repeat (DPR) proteins. DPR proteins misfold and aggregate into cytoplasm or nuclei of motor neuron. Here they can alter the proteotoxic response machinery. The protein quality control (PQC) system maintains protein homeostasis by re-folding (by chaperone) or by degradation (by autophagy or proteasome) of misfolded proteins to counteract proteotoxicity. We developed a high-throughput drug screen for the identification of positive and negative modulators of DPR levels. In NSC34 cells we evaluated the role of the selected compound in the regulation of the two main degradative pathways of PQC. Using RT-qPCR, WB and IF analysis, we observed that none of the compounds were able to modulate TFEB, SQSTM1/p62, and LC3 expression and localization. Nevertheless, the reduction of DPR levels observed in cells treated with geldanamycin (an HSP90 inhibitor) and with spironolactone (an aldosterone antagonist) is counteracted by autophagy and proteasome inhibitor suggesting that these compounds promote DPR proteins degradation via the proteasome and autophagy pathways respectively. Surprisingly, cAMP-elevating compounds boosting protein kinase A (PKA) activity increased DPR protein levels. Inhibition of PKA activity, by both pharmacological and genetic approaches, reduced DPR levels in cells and rescued pathological phenotypes in a Drosophila model of C9ALS/FTD. Moreover, knockdown of PKA-catalytic subunits correlated with reduced translation e ciency of DPRs, while the PKA inhibitor H89 reduced endogenous DPR protein levels in C9ALS/FTD patient- derived iPSC motor neurons. Together, our results suggest new and druggable pathways modulating DPR protein levels in C9ALS/FTD.