Readthrough of nonsense mutations in Rett syndrome: evaluation of novel aminoglycosides and generation of a new mouse model - PubMed (original) (raw)

Readthrough of nonsense mutations in Rett syndrome: evaluation of novel aminoglycosides and generation of a new mouse model

Cornelia Brendel et al. J Mol Med (Berl). 2011 Apr.

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Abstract

Thirty-five percent of patients with Rett syndrome carry nonsense mutations in the MECP2 gene. We have recently shown in transfected HeLa cells that readthrough of nonsense mutations in the MECP2 gene can be achieved by treatment with gentamicin and geneticin. This study was performed to test if readthrough can also be achieved in cells endogenously expressing mutant MeCP2 and to evaluate potentially more effective readthrough compounds. A mouse model was generated carrying the R168X mutation in the MECP2 gene. Transfected HeLa cells expressing mutated MeCP2 fusion proteins and mouse ear fibroblasts isolated from the new mouse model were treated with gentamicin and the novel aminoglycosides NB30, NB54, and NB84. The localization of the readthrough product was tested by immunofluorescence. Readthrough of the R168X mutation in mouse ear fibroblasts using gentamicin was detected but at lower level than in HeLa cells. As expected, the readthrough product, full-length Mecp2 protein, was located in the nucleus. NB54 and NB84 induced readthrough more effectively than gentamicin, while NB30 was less effective. Readthrough of nonsense mutations can be achieved not only in transfected HeLa cells but also in fibroblasts of the newly generated Mecp2(R168X) mouse model. NB54 and NB84 were more effective than gentamicin and are therefore promising candidates for readthrough therapy in Rett syndrome patients.

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Figures

Fig. 1

Fig. 1

NB54- and NB84-mediated suppression of premature stop mutations. Western blot analysis of protein samples prepared from aminoglycoside-treated and untreated transiently transfected HeLa cells, using a monoclonal anti-FLAG antibody or a specific antibody detecting the C terminus of MeCP2. Thirty microgram of protein extract was loaded. Arrows denote the full-length FLAG-MeCP2 protein as well as the truncated FLAG-MeCP2 isoforms. As a loading control, GAPDH was detected in all samples. The cells were treated for 24 h with 500 μg/ml of gentamicin (a), NB30 (a), NB54 (a–b), or NB84 (b). The effect of 24-h drug treatment was quantified by densitometric western blot analysis from at least three independent experiments (c)

Fig. 2

Fig. 2

Nuclear localization of readthrough full-length MeCP2-FLAG fusion protein in drug-treated HeLa cells. Immunofluorescence of HeLa cells transfected with MeCP2-WT-FLAG- (a–c) compared to MeCP2-R168X-FLAG-expressing HeLa cells cultured under untreated (d–f) and drug-treated (g–i 500 μg/ml NB54; j–l 500 μg/ml NB84) conditions. Treatment was performed for 24 h. Localization of FLAG fusion proteins (left column) was visualized by using a monoclonal anti-FLAG antibody and immunofluorescence microscopy. Because the fusion protein was C-terminally FLAG-tagged, only full-length MeCP2 proteins were detected. The nuclei were counterstained with DAPI (middle column). Right column MeCP2-WT-FLAG expression as well as treated MeCP2-294-FLAG overlaps with DAPI staining, indicating a nuclear localization. Scale bars are 50 μm

Fig. 3

Fig. 3

Gene targeting and Flp-mediated recombination at the Mecp2 locus. The diagram shows the wild-type Mecp2 genomic locus, the targeting vector, and the targeted locus before and after Flp-mediated recombination. The 5′ and 3′ regions of homology are shown in light gray. Small arrows indicate primers used to confirm gene targeting and Flp-mediated recombination, and to screen the genotype (a). Homologous recombination was verified by nested PCR of genomic DNA of the selected ES cell clones; primers 5/6 and 7/8 were as shown in a. The 1.9-kb band is from the mutated allele. The primer sets were designed not to amplify the wild-type Mecp2 gene. E5, E7, E32, and E38 are names of ES clones, while c and b indicate a plasmid DNA control demonstrating the mutant PCR fragment and blank. Primer set 9 and 10 were used for the genotyping PCR after FLP recombination (c). The 313-bp band represents the mutant allele (Hem hemizygous male, Het heterozygous female) containing the remaining FRT site, while the 265-bp band represents the wild-type allele (WT wild type). To confirm the presence of Mecp2 mRNA in the brain of two wild-type (WT (+/Y)) and two R168X (−/Y) mutant male mice, total RNA was extracted from brain of male wild-type and mutant mice and used for cDNA synthesis and RT-PCR (d). Western blot analysis (e) and immunofluorescent staining (f) demonstrated the absence of wild-type MeCP2 protein in nuclear extracts of hemizygous brains (Sup supernatant, Nuc nuclear extract) and in nuclei of mouse ear fibroblast using an antibody recognizing the N terminus of MeCP2. Note that no additional truncated protein was detected

Fig. 4

Fig. 4

Drug-mediated re-expression of endogenous MeCP2. Western blot analysis of nuclear extracts prepared from aminoglycoside-treated and untreated mouse ear fibroblasts generated from wild-type and Mecp2 168 male mice: Immunoreactivity was detected by using a monoclonal anti-FLAG antibody and a specific antibody detecting the C terminus of MeCP2. Thirty microgram of protein extract was loaded into each lane. Arrows denote the full-length endogenous MeCP2 protein. The cells were treated for 4 days with 0–800 μg/ml of gentamicin, NB54, or NB84 (a). The effect of drug treatment was quantified by densitometric developed western blot analysis from at least three independent experiments (b)

Fig. 5

Fig. 5

Nuclear localization of endogenous Mecp2 in drug-treated mouse fibroblasts. Immunofluorescent staining of mouse ear fibroblasts generated from wild-type and Mecp2 168 male mice. Cells were treated 4 days with 800 μg/ml gentamicin (i–l), NB54 (m–p), and NB84 (q–t), respectively. Localization of endogenous MeCP2 protein (first column) was visualized by using a specific MeCP2 antibody detecting the C-terminal part of the protein and immunofluorescent microscopy. The nuclei and the cytoskeleton of the cells were counterstained with DAPI (second column) and tubulin (third column). All three stainings are merged (last column). No MeCP2-specific staining was detected in untreated fibroblasts (e–h). Scale bars are 50 μm

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References

    1. Rett A. On a unusual brain atrophy syndrome in hyperammonemia in childhood. Wien Med Wochenschr. 1946;116:723–726. - PubMed
    1. Hagberg B. Rett syndrome: clinical peculiarities and biological mysteries. Acta Paediatr. 1995;84:971–976. doi: 10.1111/j.1651-2227.1995.tb13809.x. - DOI - PubMed
    1. Amir RE, Van den Veyver IB, Wan M, Tran CQ, Francke U, Zoghbi HY. Rett syndrome is caused by mutations in X-linked MECP2, encoding methyl-CpG-binding protein 2. Nat Genet. 1999;23:185–188. doi: 10.1038/13810. - DOI - PubMed
    1. D’Esposito M, Quaderi NA, Ciccodicola A, Bruni P, Esposito T, D’Urso M, Brown SD. Isolation, physical mapping, and northern analysis of the X-linked human gene encoding methyl CpG-binding protein, MECP2. Mamm Genome. 1996;7:533–535. doi: 10.1007/s003359900157. - DOI - PubMed
    1. Guy J, Hendrich B, Holmes M, Martin JE, Bird A. A mouse Mecp2-null mutation causes neurological symptoms that mimic Rett syndrome. Nat Genet. 2001;27:322–326. doi: 10.1038/85899. - DOI - PubMed

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