The most common genetic cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) is an expanded G 4 C 2 repeat [(G 4 C 2 ) exp ] in C9ORF72. ALS/FTD-associated toxicity has been traced to the RNA transcribed from the repeat expansion [r(G 4 C 2 ) exp ], which sequesters RNA-binding proteins (RBPs) and undergoes repeat-associated non-ATG (RAN) translation to generate toxic dipeptide repeats. Using in vitro and cell-based assays, we identified a small molecule (4) that selectively bound r(G 4 C 2 ) exp , prevented sequestration of an RBP, and inhibited RAN translation. Indeed, biophysical characterization showed that 4 selectively bound the hairpin form of r(G 4 C 2 ) exp , and nuclear magnetic resonance spectroscopy studies and molecular dynamics simulations defined this molecular recognition event. Cellular imaging revealed that 4 localized to r(G 4 C 2 ) exp cytoplasmic foci, the putative sites of RAN translation. Collectively, these studies highlight that the hairpin structure of r(G 4 C 2 ) exp is a therapeutically relevant target and small molecules that bind it can ameliorate c9ALS/FTD-associated toxicity.

Herein, we describe methods to identify structured regions within disease-causing RNAs and to design lead small molecules that selectively bind these structures to modulate function.

The most common cause of amyotrophic lateral sclerosis and frontotemporal dementia (c9ALS/FTD) is an expanded G 4 C 2 RNA repeat [r(G 4 C 2 ) exp ] in chromosome 9 open reading frame 72 (C9orf72), which elicits pathology through several mechanisms. Here, we developed and characterized a small molecule for targeted degradation of r(G 4 C 2 ) exp . The compound was able to selectively bind r(G 4 C 2 ) exp 's structure and to assemble an endogenous nuclease onto the target, provoking removal of the transcript by native RNA quality control mechanisms. In c9ALS patientderived spinal neurons, the compound selectively degraded the mutant C9orf72 allele with limited off-targets and reduced quantities of toxic dipeptide repeat proteins (DPRs) translated from r(G 4 C 2 ) exp . In vivo work in a rodent model showed that abundance of both the mutant allele harboring the repeat expansion and DPRs were selectively reduced by this compound. These results demonstrate that targeted small-molecule degradation of r(G 4 C 2 ) exp is a strategy for mitigating c9ALS/FTD-associated pathologies and studying disease-associated pathways in preclinical models.

SummaryIt has previously been reported that mouse epiblast stem cell (EpiSC) lines comprise heterogeneous cell populations that are functionally equivalent to cells of either early- or late-stage postimplantation development. So far, the establishment of the embryonic stem cell (ESC) pluripotency gene regulatory network through the widely known chemical inhibition of MEK and GSK3beta has been impractical in late-stage EpiSCs. Here, we show that chemical inhibition of casein kinase 1alpha (CK1alpha) induces the conversion of recalcitrant late-stage EpiSCs into ESC pluripotency. CK1alpha inhibition directly results in the simultaneous activation of the WNT signaling pathway, together with inhibition of the TGFbeta/SMAD2 signaling pathway, mediating the rewiring of the gene regulatory network in favor of an ESC-like state. Our findings uncover a molecular mechanism that links CK1alpha to ESC pluripotency through the direct modulation of WNT and TGFbeta signaling.