title: Suppression of galactocerebrosidase premature termination codon and rescue of galactocerebrosidase activity in twitcher cells.
abstract: Krabbe's disease (KD) is a degenerative lysosomal storage disease resulting from deficiency of β-galactocerebrosidase activity. Over 100 mutations are known to cause the disease, and these usually occur in compound heterozygote patterns. In affected patients, nonsense mutations leading to a nonfunctional enzyme are often found associated with other mutations. The twitcher mouse is a naturally occurring model of KD, containing in β-galactocerebrosidase a premature stop codon, W339X. Recent studies have shown that selected compounds may induce the ribosomal bypass of premature stop codons without affecting the normal termination codons. The rescue of β-galactocerebrosidase activity induced by treatment with premature termination codon (PTC) 124, a well-characterized compound known to induce ribosomal read-through, was investigated on oligodendrocytes prepared from twitcher mice and on human fibroblasts from patients bearing nonsense mutations. The effectiveness of the nonsense-mediated mRNA decay (NMD) inhibitor 1 (NMDI1), a newly identified inhibitor of NMD, was also tested. Incubation of these cell lines with PTC124 and NMDI1 increased the levels of mRNA and rescued galactocerebrosidase enzymatic activity in a dose-dependent manner. The low but sustained expression of β-galactocerebrosidase in oligodendrocytes was sufficient to improve the morphology of the differentiated cells. Our in vitro approach provides the basis for further investigation of ribosomal read-through as an alternative therapeutic strategy to ameliorate the quality of life in selected KD patients. © 2016 Wiley Periodicals, Inc.
authors.affiliation: Department of Molecular and Developmental Medicine, Siena University, Siena, Italy. luddi@unisi.it.
authors.forename: Alice
authors.initials: A
authors.lastname: Luddi
authors.affiliation: Department of Molecular and Developmental Medicine, Siena University, Siena, Italy.
authors.forename: Laura
authors.initials: L
authors.lastname: Crifasi
authors.affiliation: Department of Molecular and Developmental Medicine, Siena University, Siena, Italy.
authors.forename: Angela
authors.initials: A
authors.lastname: Capaldo
authors.affiliation: Department of Molecular and Developmental Medicine, Siena University, Siena, Italy.
authors.forename: Paola
authors.initials: P
authors.lastname: Piomboni
authors.affiliation: Department of Molecular and Developmental Medicine, Siena University, Siena, Italy.
authors.forename: Elvira
authors.initials: E
authors.lastname: Costantino-Ceccarini
mesh_terms: D000818:Animals; D000831:Animals, Newborn; D002461:Cell Line, Transformed; D018388:Codon, Terminator / Q000235:genetics* / Q000378:metabolism; D004195:Disease Models, Animal; D004305:Dose-Response Relationship, Drug; D005347:Fibroblasts / Q000187:drug effects / Q000201:enzymology; D005698:Galactosylceramidase / Q000172:deficiency* / Q000235:genetics*; D005699:Galactosylceramides / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D007965:Leukodystrophy, Globoid Cell / Q000473:pathology*; D051379:Mice; D008810:Mice, Inbred C57BL; D008817:Mice, Mutant Strains; D059365:Nonsense Mediated mRNA Decay / Q000187:drug effects; D009836:Oligodendroglia / Q000187:drug effects / Q000201:enzymology; D010069:Oxadiazoles / Q000494:pharmacology; D012333:RNA, Messenger / Q000378:metabolism
chemical_list: D018388:Codon, Terminator; D005699:Galactosylceramides; D010069:Oxadiazoles; D012333:RNA, Messenger; C002454:galactocerebroside; D005698:Galactosylceramidase; C515878:ataluren
keywords: Krabbe's disease; NMD; NMDI1; PTC124; lysosomal storage disease; oligodendrocytes; premature termination codon; twitcher