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

Abstract

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 frontotemporal dementia (FTD) (C9ALS/FTD). Repeat-associated non-AUG (RAN) translation of repeat-containing C9orf72 RNA results in the production of neurotoxic dipeptide-repeat proteins (DPRs). Here, we developed a high-throughput drug screen for the identification of positive and negative modulators of DPR levels. We found that HSP90 inhibitor geldanamycin and aldosterone antagonist spironolactone reduced DPR levels by promoting protein degradation via the proteasome and autophagy pathways respectively. Surprisingly, cAMP-elevating compounds boosting protein kinase A (PKA) activity increased DPR 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 efficiency of DPRs, while the PKA inhibitor H89 reduced endogenous DPR levels in C9ALS/FTD patient-derived iPSC motor neurons. Together, our results suggest new and druggable pathways modulating DPR levels in C9ALS/FTD.

Recapitulating and reversing human brain ribosomopathy defects via the maladaptive integrated stress response

“…Therefore, we used SNORD118 -mutant human iPSC-derived NPCs and performed cell-based small-molecule screen for reversing proliferation defects in mutant NPCs. On the basis of published studies, we selected those chemical compounds that potentially affect ribosome biogenesis and nucleoli functions, including 2BAct ( 38 ), MCL-186 ( 39 ), bromosporine ( 40 ), nicotinamide ( 41 ), tideglusib ( 42 , 43 ), PFI-1 ( 44 ), YM201636 ( 45 ), cilostazol ( 46 ), leucine ( 47 ), and prostetin ( 48 ). Our initial EdU assay–based cell proliferation screen identified 2BAct, MCL-186, and cilostazol, which significantly increased the proliferation of SNORD118 *5C>G -mutant NPCs (Fig.…”

Enhanced Degradation of Mutant C9ORF72-Derived Toxic Dipeptide Repeat Proteins by 20S Proteasome Activation Results in Restoration of Proteostasis and Neuroprotection

“…It has previously been shown that DPR proteins inhibit the UPS in cell culture. ,, Additional work has shown that increasing protein degradation through the use of a DUB inhibitor, HSP90 inhibitor, or activation of the proteasome through cAMP/protein kinase A-dependent phosphorylation of proteasome subunit PSMD11 counters the toxic effects of the DPR proteins. This suggests that impairment of UPS function contributes to DPR protein toxicity.…”

Aspects of degradation and translation of the expanded C9orf72 hexanucleotide repeat RNA

“…Additionally, in C. elegans model, loss‐of‐function of eukaryotic translation initiation factor 2D decreased poly‐GA and poly‐GR expression level (Sonobe et al, 2021) (Figure 3). Protein kinase A(PKA) enhances poly‐GP expression, and inhibition of PKA by its inhibitor H89 reduces poly‐GP levels in iPSC‐derived motor neurons (Licata et al, 2022). As above, multiple layers of regulations could underlie C9 RAN translation; however, complete picture of C9 RAN translation is yet clear.…”