Translational readthrough by the aminoglycoside geneticin (G418) modulates SMN stability in vitro and improves motor function in SMA mice in vivo

• Published in: Human molecular genetics Vol. 18; no. 7; pp. 1310 - 1322
• Main Authors: Heier, Christopher R., DiDonato, Christine J.
• Format: Journal Article
• Language: English
• Published: Oxford Oxford University Press 01.04.2009; Oxford Publishing Limited (England)
• Subjects: Aminoglycosides - administration & dosage; Aminoglycosides - pharmacology; Aminoglycosides - therapeutic use; Animals; Base Sequence; Biological and medical sciences; Cell Line; Codon, Terminator; Exons - genetics; Fibroblasts - drug effects; Fibroblasts - metabolism; Fundamental and applied biological sciences. Psychology; Genetics of eukaryotes. Biological and molecular evolution; Gentamicins - administration & dosage; Gentamicins - pharmacology; Gentamicins - therapeutic use; Humans; Mice; Molecular and cellular biology; Molecular Sequence Data; Motor Activity - drug effects; Muscular Atrophy, Spinal - drug therapy; Muscular Atrophy, Spinal - metabolism; Muscular Atrophy, Spinal - physiopathology; Protein Biosynthesis - drug effects; Protein Stability - drug effects; Survival of Motor Neuron 1 Protein - genetics; Survival of Motor Neuron 1 Protein - metabolism; In vivo; Vertebrata; Mammalia; Stability; Mouse; Aminoglycoside; Rodentia; Central nervous system disease; Genetics; In vitro
• ISSN: 0964-6906; 1460-2083
• DOI: 10.1093/hmg/ddp030

Proximal spinal muscular atrophy (SMA) is a neuromuscular disorder for which there is no available therapy. SMA is caused by loss or mutation of the survival motor neuron 1 gene, SMN1, with retention of a nearly identical copy gene, SMN2. In contrast to SMN1, most SMN2 transcripts lack exon 7. This alternatively spliced transcript, Δ7-SMN, encodes a truncated protein that is rapidly degraded. Inhibiting this degradation may be of therapeutic value for the treatment of SMA. Recently aminoglycosides, which decrease translational fidelity to promote readthrough of termination codons, were shown to increase SMN levels in patient cell lines. Amid uncertainty concerning the role of SMN's C-terminus, the potential of translational readthrough as a therapeutic mechanism for SMA is unclear. Here, we used stable cell lines to demonstrate the SMN C-terminus modulates protein stability in a sequence-independent manner that is reproducible by translational readthrough. Geneticin (G418) was then identified as a potent inducer of the Δ7-SMN target sequence in vitro through a novel quantitative assay amenable to high throughput screens. Subsequent treatment of patient cell lines demonstrated that G418 increases SMN levels and is a potential lead compound. Furthermore, treatment of SMA mice with G418 increased both SMN protein and mouse motor function. Chronic administration, however, was associated with toxicity that may have prevented the detection of a survival benefit. Collectively, these results substantiate a sequence independent role of SMN's C-terminus in protein stability and provide the first in vivo evidence supporting translational readthrough as a therapeutic strategy for the treatment of SMA.