Functional restoration of mouse Nf1 nonsense alleles in differentiated cultured neurons.

Neurofibromatosis type 1 (NF1), one of the most common autosomal dominant genetic disorders, is caused by mutations in the NF1 gene. NF1 patients have a wide variety of manifestations with a subset at high risk for the development of tumors in the central nervous system (CNS). Nonsense mutations that result in the synthesis of truncated NF1 protein (neurofibromin) are strongly associated with CNS tumors. Therapeutic nonsense suppression with small molecule drugs is a potentially powerful approach to restore the expression of genes harboring nonsense mutations. Ataluren is one such drug that has been shown to restore full-length functional protein in several models of nonsense mutation diseases, as well as in patients with nonsense mutation Duchenne muscular dystrophy. To test ataluren's potential applicability to NF1 nonsense mutations associated with CNS tumors, we generated a homozygous Nf1R683X/R683X-3X-FLAG mouse embryonic stem (mES) cell line which recapitulates an NF1 patient nonsense mutation (c.2041 C > T; p.Arg681X). We differentiated Nf1R683X/R683X-3X-FLAG mES cells into cortical neurons in vitro, treated the cells with ataluren, and demonstrated that ataluren can promote readthrough of the nonsense mutation at codon 683 of Nf1 mRNA in neural cells. The resulting full-length protein is able to reduce the cellular level of hyperactive phosphorylated ERK (pERK), a RAS effector normally suppressed by the NF1 protein.