A Genome-Scale RNA–Interference Screen Identifies RRAS Signaling as a Pathologic Feature of Huntington's Disease

Abstract

A genome-scale RNAi screen was performed in a mammalian cell-based assay to identify modifiers of mutant huntingtin toxicity. Ontology analysis of suppressor data identified processes previously implicated in Huntington's disease, including proteolysis, glutamate excitotoxicity, and mitochondrial dysfunction. In addition to established mechanisms, the screen identified multiple components of the RRAS signaling pathway as loss-of-function suppressors of mutant huntingtin toxicity in human and mouse cell models. Loss-of-function in orthologous RRAS pathway members also suppressed motor dysfunction in a Drosophila model of Huntington's disease. Abnormal activation of RRAS and a down-stream effector, RAF1, was observed in cellular models and a mouse model of Huntington's disease. We also observe co-localization of RRAS and mutant huntingtin in cells and in mouse striatum, suggesting that activation of R-Ras may occur through protein interaction. These data indicate that mutant huntingtin exerts a pathogenic effect on this pathway that can be corrected at multiple intervention points including RRAS, FNTA/B, PIN1, and PLK1. Consistent with these results, chemical inhibition of farnesyltransferase can also suppress mutant huntingtin toxicity. These data suggest that pharmacological inhibition of RRAS signaling may confer therapeutic benefit in Huntington's disease.

R-Ras contributes to LTP and contextual discrimination

“…Semaphorins are best known for their role in promoting the collapse of growth cones of axons during development of synapses, and suppression of R-Ras activity is a required step in this process (Oinuma et al, 2004a, Oinuma et al, 2004b, Oinuma et al, 2010). R-Ras has also been detected in an shRNA screen for genes that contribute to neuronal toxicity associated with Huntington’s disease (Miller et al, 2012). The results presented here are the first to identify a normal role for R-Ras in a specific form of animal behavior, contextual discrimination, that may be impaired in certain neurodegenerative disorders, and a form of synaptic plasticity that contributes to it.…”

A Large Scale Huntingtin Protein Interaction Network Implicates Rho GTPase Signaling Pathways in Huntington Disease

“…pDEST40-LacZ was used as a negative control. Entry clones containing HTT N-terminal fragments 1-558 (with 23Q or 135Q) fused to a C-terminal GFP tag have been described previously (26). LR reaction was performed with pLenti CMV Puro DEST vector (w118-1, Addgene); integrity of clones and length of CAG repeats were verified by sequencing at all steps.…”

Hunting for the mutant without the MAP(K)

“…To date, several groups have performed unbiased RNA interference (RNAi) screens to identify candidate molecules whose modulation may alter the development and progression of HD, however, these screens have been primarily performed in models that rely on the exogenous overexpression of polyQ expanded mHTT [5][6][7][8][9]. Although these previous reports have successfully identified a diverse set of pharmacologically targetable molecules capable of reducing mHTT aggregation and its associated toxicity in cellular and animal models of HD, these target pathways may not directly apply to the endogenous expression of pathological mHTT, especially in the context of HDrelevant human cell types.…”