title: Antisense oligonucleotide-mediated exon skipping as a strategy to reduce proteolytic cleavage of ataxin-3.
abstract: Spinocerebellar ataxia type-3 (SCA3) is a neurodegenerative disorder caused by a polyglutamine repeat expansion in the ataxin-3 protein. Cleavage of mutant ataxin-3 by proteolytic enzymes yields ataxin-3 fragments containing the polyglutamine stretch. These shorter ataxin-3 fragments are thought to be involved in SCA3 pathogenesis due to their increased cellular toxicity and their involvement in formation of the characteristic neuronal aggregates. As a strategy to prevent formation of toxic cleavage fragments, we investigated an antisense oligonucleotide-mediated modification of the ataxin-3 pre-mRNA through exon skipping of exon 8 and 9, resulting in the removal of a central 88 amino acid region of the ataxin-3 protein. This removed protein region contains several predicted cleavage sites and two ubiquitin-interacting motifs. In contrast to unmodified mutant ataxin-3, the internally truncated ataxin-3 protein did not give rise to potentially toxic cleavage fragments when incubated with caspases. In vitro experiments did not show cellular toxicity of the modified ataxin-3 protein. However, the modified protein was incapable of binding poly-ubiquitin chains, which may interfere with its normal deubiquitinating function. Low exon skipping efficiencies combined with reduction in important ataxin-3 protein functions suggest that skipping of exon 8 and 9 is not a viable therapeutic option for SCA3.
authors.affiliation: Department of Human Genetics, Leiden University Medical Center, Albinusdreef 2, 2333ZA Leiden, The Netherlands.
authors.forename: Lodewijk J A
authors.initials: LJ
authors.lastname: Toonen
authors.affiliation: Department of Human Genetics, Leiden University Medical Center, Albinusdreef 2, 2333ZA Leiden, The Netherlands.
authors.forename: Iris
authors.initials: I
authors.lastname: Schmidt
authors.affiliation: Department of Human Genetics, Leiden University Medical Center, Albinusdreef 2, 2333ZA Leiden, The Netherlands.
authors.forename: Martijn S
authors.initials: MS
authors.lastname: Luijsterburg
authors.affiliation: Department of Human Genetics, Leiden University Medical Center, Albinusdreef 2, 2333ZA Leiden, The Netherlands.
authors.forename: Haico
authors.initials: H
authors.lastname: van Attikum
authors.affiliation: Department of Human Genetics, Leiden University Medical Center, Albinusdreef 2, 2333ZA Leiden, The Netherlands.
authors.forename: Willeke M C
authors.initials: WM
authors.lastname: van Roon-Mom
mesh_terms: D000067699:Ataxin-3 / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D001665:Binding Sites / Q000235:genetics; D002154:Calpain / Q000378:metabolism; D002460:Cell Line; D053903:DNA Breaks, Double-Stranded; D005091:Exons / Q000235:genetics; D006801:Humans; D017827:Machado-Joseph Disease / Q000235:genetics / Q000378:metabolism / Q000628:therapy; D050505:Mutant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D016376:Oligonucleotides, Antisense / Q000235:genetics; D025821:Polyubiquitin / Q000378:metabolism; D059748:Proteolysis; D012322:RNA Precursors / Q000235:genetics / Q000378:metabolism; D012097:Repressor Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
chemical_list: D050505:Mutant Proteins; D016376:Oligonucleotides, Antisense; D012322:RNA Precursors; D012097:Repressor Proteins; D025821:Polyubiquitin; C092341:ATXN3 protein, human; D000067699:Ataxin-3; D002154:Calpain; C569870:CAPN2 protein, human